SOMATIC EMBRYOGENESIS IN WOODY PLANTS

FORESTRY SCIENCES
Volume67

The titles published in this series are listed at the end of this volume.
Somatic Embryogenesis
in Woody Plants
Volume 6

Edited by

S. MOHAN JAIN
Plant Breeding and Genetics Section,
FAO/IAEA Division,
International Atomic Energy Agency,
Vienna, Austria

PRAMOD K. GUPTA
Weyerhaeuser Inc., Tacoma,
Washington, U.S.A.

and

RONALD J. NEWTON
Department of Biology,
East Carolina University Greenville,
North Carolina, U.S.A.

''
~·

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A C.l.P. Catalogue record for this book is available from the Library of Congress.

ISBN 978-90-481-5508-8 ISBN 978-94-017-3030-3 (eBook)

DOI 10.1007/978-94-017-3030-3

Printed on acid~free paper

All Rights Reserved

© 2000 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2000
Softcover reprint of the hardcover I st edition 2000
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
including photocopying, recording or by any information storage and
retrieval system, without written permission from the copyright owner.
 Contents
General preface vii

Preface ix

Acknowledgements xi

Section A

Dedication : Harry Waris, a pioneer in somatic embryogenesis- Liisa Simola

1) Historical insights into some contemporary problems in somatic 17

embryogenesis -A. D. Krikorian

2) Current status of thin cell layer method for the induction of organogenesis or
somatic embryogenesis in woody trees- K. Iran Thanh Van and Bui Van Le 51

3) Somatic embryogenesis in tropical fruit trees- N. Akhtar, N. Kumari, S. Pandey, 93

H. Ara, M. Singh, U. Jaiswal, V.S. Jaiswal and S.M .Jain

4) Somatic embryogenesis in fruit and forest trees of arid zone - R. Raj Bhansali 141
and Manjit Singh

5) Status ofsomatic embryogenesis in Indian forest trees- P. S Rao, P. 169

Suprassana, T. R. Ganapathi and V.A. Bapat

6) Somatic embryogenesis research on fruit trees in India - R. Nadgauda, G. 193

Mathur and S. Gogte

7) Applications of somatic embryogenesis for the improvement of tropical fruit 215

trees- N. Akhtar and S. Mohan Jain

Section B

8) Somatic embryogenesis in oil palm- A. Rival 249

9) Applied and basic studies on somatic embryogenesis in hazelnut (Corylus

aveUana L.)- R. Rodriguez, B. Berres, M. Luz Centeno, M. Rovira, A. Rodriguez 291
and L. Radojevic

10) Somatic embryogenesis in Pistachio (Pistacia vera L.)- A. Onay and C.E. Jeffree 361

11) Somatic embryogenesis in pecan (Carya iUinoinensis)- H. Y. Wetzstein, B. S. 391

Jeyaretnam, W.A. Vendrame and A.P.M. Rodriguez

12) Somatic embryogenesis in Iongan (Dimocarpus Iongan Lour.)- Z. Lai, C. Chen, 415
L. Zeng and Z Chen
vi

13) Somatic embryogenesis induction in Tamarillo (Cyphomandra betacea)- M.L. 433

Lopes, M.R. Ferreira, J.M. Carloto, G.S. Cruz and J.M. Canhoto

14) Somatic embryogenesis in ArtJUcaria angustifolia (Bert) O.Ktze- M.P.Guerra, 457

V. Silveira, A.L.W. dos Santos, L. V. Astarita and R.O. Nodari

15) Somatic and gametic embryogenesis in Quercus suber L - M. A. Bueno, A. 479

Gomez and J.A. Manzanera

16) Somatic embryogenesis in Aspidosperma polyneuron MulL Arg- L.L.F. Ribas, 509
M.P. Guerra, F. Zanette and L. Kulchetscki

17) Somatic embryogenesis in a leguminous tree-Acacia senegal (L.) Willd. 539

S. Shahana and S.C. Gupta

18) Somatic embryogenesis in cypress (Cupressus sempervirens L)- M. Lambardi 553

19) Somatic embryogenesis in rattan (Calamus spp.)- D.K.S. Goh, 0. Monteuuis 569
andM-C.Bon

20) Somatic embryogenesis in jojoba (Simmontlsia chiensis) - V. Agrawal, S. 587

Prakash and S.C. Gupta

21) Somatic embryogenesis in Aegle marmelos (L) corr., a medicinal tree - S. 605
Arwnugam and M. V. Rao

22) Environmental and biochemical factors controlling the in vitro emergence of

somatic embryos European spindle tree (Euonymus europaeus L)- L.M. 657
Bonneau

23) Somatic Embryogenesis in Quercus acutissima- Kim Yong-Wook 671

Section C

24) Cryostorage of Citrus embryogenic cultures - R.M. Perez 687

25) Cryopreservation of embryogenic cultures of conifers- H.M. Haggman, T.S. 707

Aronen and L.A. Ryynanen

26) Cryopreservation of embryogenic calli of Heavea brasiliensis 729

-F. Engelman and H. Etienne
 General Preface

The quality of human life has been maintained and enhanced for generations by
the use of trees and their products. In recent years, ever rising human population
growth has put a tremendous pressure on trees and tree products; growing
awareness of the potential of previously unexploited tree resources; and environ-
mental pollution have both accelerated the development of new technologies for
tree propagation, breeding and improvement. Biotechnology of trees may be the
answer to solve the problems which can not be solved by conventional breeding
methods. The combination of biotechnology and conventional methods such as
plant propagation and breeding may be a novel approach to improving and
multiplying a large number of the trees and woody plants.

So far, plant tissue culture technology has largely been exploited by commercial
companies in propagation of ornamentals, especially foliage house plants. Gene-
rally, tissue culture of woody plants has been recalcitrant. However, limited
success has been achieved in tissue culture of angiosperm and gymnosperm
woody plants. A number of recent reports on somatic embryogenesis in woody
plants such as Norway spruce (Picea abies), Loblolly pine (Pinus taedb),
Sandalwood (Santalum album), Citrus, mango (Mangifera indica), etc., offer a
ray of hope of: a) inexpensive clonal propagation for large-scale production of
plants or "emblings" or somatic seedlings; b) protoplast work; c)
cryopreservation; d) genetic transformation; and e) synthetic or artificial or
manufactured seed production. In the future, with the basic biology for better
understanding of the genetic control somatic embryogenesis and in embryo
development and maturation with the help of molecular biologists, it may be
possible for us to have a better control over the induction of somatic
embryogenesis. For cost effective large-scale production of elite trees, robotics
and automation technology will interface with somatic embryogenesis in 21st
century.

The rapid progress of somatic embryogenesis and its prospects for potential
applications to improving woody plants prompted us to edit this book initially in
three volumes, and now additional two more volumes. Moreover, most of the
research information in this field on woody plants is scattered in national and
international meeting proceedings, refereed journals, biotechnology books etc.
There is a lack of availability of a comprehensive work on somatic embryogenesis
in woody plants including both angiosperms and gymnosperms. We were all
convinced that such a treatise was needed and would be extremely useful to
researchers and students.

In our earlier endeavor, we attempted to bring all the research information on

somatic embryogenesis in woody plants in three volumes. Recent tremendous
Vll
viii

progress on this subject prompted us to bring out two additional volumes. In

Volume 4, we have three sections, and in Section A contains the review articles
including studies of Norway spruce embryo development and cell biology;
proliferative somatic embryogenesis in woody species; somatic embryo germina-
tion and desiccation tolerance in conifers; performance of somatic seedlings;
apoptosis during early somatic embryogenesis; water relation parameters in
somatic embryos; image analysis of somatic embryos; somatic embryogenesis of
woody legumes; cold storage and cryopreservation of embryogenic cultures; and
commercialization of somatic embryogenesis. Sections B and C contain mostly
selected angiosperm and gymnosperm woody plants, respectively. Volume 5 deals
with mainly angiosperm woody plants including tropical and arid zone fruits;
transformation of conifer and fruit embryogenic cultures. We have also encour-
aged authors to incorporate their recent data in their manuscripts. These
volumes are designed as the key reference works, providing detailed information
on all aspects of somatic embryogenesis for beginners as well as experienced
researchers. The invited authors are well known in the somatic embryogenesis
research and they belong to academic institutes, universities, and industries.

All the manuscripts have been critically reviewed by two persons, and revised
accordingly. The final revised manuscripts were submitted as camera-ready to
publication, and editors had no opportunity to make any further corrections.
Therefore, editors presumed that authors checked thoroughly their manuscripts
before sending camera-ready manuscripts, and don't bear any responsibility for
any mistake, if any, in book volumes 4 and 5.

S. Mohan Jain
P.K. Gupta
R.J. Newton
 Preface
This volume is dedicated to the memory of Prof. Harry Waris, Helsinki, Finland, who
did pioneer work on somatic embryogenesis during the time when Steward and others
were actively engaged in this area. His student Prof. Liisa Simola, University of
Helsinki, Finland has written the dedication to her teacher Prof. Waris.

This book contains 25 chapters and is divided into three sections. In Section A, seven
chapters are included such as somatic embryogenesis (SE) history, thin cell layer for
somatic embryogenesis induction in woody trees, SE in tropical fruit trees, SE in fruit
and forest arid trees, status of SE in Indian forest trees, SE research in fruit trees in
India, and applications of SE for the improvement of tropical fruit trees. Section B
deals with 16 chapters, and they are: SE in oil palm, hazelnut (Corylus ave/lana L.),
pistachio (Pistacia vera. L.), Araucaria angustifolia, Quercus suber, Aspidosperma
poljmeuron, Acacia senegal, Simmondsia chiensis, Cupressus sempervirens, pecan
(Carya illinoinensis), rattan (Calamus spp.), tamarillo (Cyphomandra betacea), Iongan
(Dimocarpus Iongan Lor.), Aegle marmelos, and Euonymus europaeus. Section C has
three chapters on cryo-storage in citrus, conifers and rubber.

We are thankful to all: a) contributory authors for their co-operation in submitting

manuscripts well in time, and b) reviewers for spending their valuable time in reviewing
the manuscripts.

ix
 Acknowledgement

My thanks are due to my friends Prof. Ronald J. Newton and Dr. Pramod K.
Gupta, co-editors of this book, for their promptness in responding to me
whenever I needed their help. They were helpful in reviewing the manuscripts. It
has been my great pleasure to work with Ron and Pramod on this project, and
certainly we have formed an excellent and highly efficient team.

I would like to acknowledge with great appreciation Drs. S.M. Attree, Erica
Benson, J.M. Bonga, Peter Bozhkov, J.M. Canhoto, P.J. Charest, A. David,
Pramod Gupta, K. Ishii, Jules Janick, R.E. Litz, S.A. Merkle, R.J. Newton, J. K.
Norgaard, David Thompson, A.M. Vieitez, for critically reviewing the manus-
cripts promptly, and to all the contributory authors for sending their manus-
cripts well in time. However, we had some anxious moments due to delay in some
manuscripts.

I wish to express my thanks to my colleague Eija Pehu of our Plant Production

Department for her help and assistance.

Also, with great love and affection, I want to thank my daughters Sarita and
Sonia, and my wife Marja-Liisa for their unceasing patience and understandipg
while I was working on these volumes.

Finally, I express my deepest sense of appreciation to Mr. Adrian Plaizier of

Kluwer Academic Publishers, The Netherlands, for giving us the opportunity to
work on this book project. Adrian has always been cooperative and helpful, and
gave me useful advice.

S. Mohan Jain
Book Project Leader

XI
PROFESSOR HARRY WARIS
 HARRY WARIS, A PIONEER IN SOMATIC EMBRYOGENESIS

Liisa Kaarina Simola

Division ofPlant Physiology, Department ofBiosciences
PL 56 {Viikinkaari 9), FIN-00014 University ofHelsinki, Finland

1. SchooUng and University education

2. Professor and rector at the Turku University
3 Professor at the University of Helsinki
3.1. SOMATIC EMBRYOGENESIS
3.2. NEOMORPHS
3.3. DEVELOPMENTAL BOT ANY
4. Conclusion
5. Acknowledgements
6. References

1. Schooling and University education

Hany Waris was born on the 5th of May, 1893 in the family of a physician Eliel
Waren in Saarijfuvi, Central Finland. His mother Elina (born Ignatius) belonged to
a well-known academic family. His early education was ·in a public school (The
Finnish Normal Lyceum) in Helsinki. Many of his pupils continued with their
higher education and well placed at higher positions in the Finnish society.

Hany Waren (since 1935,Waris) began to study biology, especially botany in 1911
at the University of Helsinki and received the Master's degree in 1916 (Kallio
1975, Autio 1997). Professor Fredrik Elfving, the head of the Botanical Institute,
introduced obligatory laboratory courses in plant physiology for higher studies in
botany. He inspired many students to study plant physiology the Ph.D.
progrannnes. Spore plants including microfungi and lichens were among the
groups studied. The Ph.D. thesis of Waren on axenic cultures of lichen gonidia
(Reinkulturen von Flechtengonidien) was published in 1920, and was regarded as
pioneer work in lichenology (Ahmadjian 1967, Tschennak-Woess 1988). This
theme was central at the Institute because Professor Elfving was in disagreement
with Dr. Simon Schwendener (1872) that lichens are double organisms (Collander
1965). Waren was able to cultivate the algal component of several lichens (1920).
Although his results supported Schwendener' s views, he succeeded in maintaining
good relationship with his supervisor.

Waren began his career as a botanist at the Finnish Society ofPeatland Cultivation
(1919-1927). His task was to evaluate the bonity of peatland for agriculture.
During this period, he acquired a wide knowledge of Nordic vegetation. He was
able to identify not only difficult Sphagnum species but also many vascular plants
growing under wet conditions. This knowledge he applied later in several
experiments dealing with whole seed plants cultivated under aseptic conditions. He
published several phytosociological papers on peatland vegetation by taking into
consideration the chemical composition of the soil (1924, 1926a, b).

S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 1-16.
© 2000 Kluwer Academic Publishers.
2

2. Professor and Rector at tbeTtrku University

Dr. Waren moved to Turku and became Professor of Botany in 1927, and for many
years worked as the only botanist at Turku University. He received a Rockefeller
Foundation fellowship ( 1929-1930) to work at the Pflanzenphysiologisches Institut
der Deutschen Karls-Universitiit zu Prague. He devoted attention mainly to the
developmental physiology of Micrasterias-algae. Desmids had already interested
Professor Elfving ( 1881 ), who had described several new species. Designing of
new experimental technique and detailed microscopic obseiVations of the cell
development gave new insight into the subject (Waren 1926a, 1933, Waris 1936,
1939).

Professor Waris was elected as the Vice-Rector (1941-1944) of Turku University,

and seiVed as Rector from 1945 to 1948 (Turun Yliopiston Vuosikitjat 1939-
1942, 1945-1948). The great responsibilites and the poor economic situation of
the universities during and after the Second World War needed most of his
attention which interrupted his research.. During the war, young men were called
to seiVe in the anny while women and older men participated in civil seiVice.
Consequently, a considerable drop in active students and teachers at the
universities was seen. However, some courses in plant anatomy and physiology
were arranged in Turku. Waris made an excursion to East Karelia and collected
living plant material for the Botanical Garden in Turku. After 1947, he was
honoured and given the full membership of the Finnish Academy of Sciences.

The new Scandinavian Society of Plant Physiology was established in 1949, and
apparently inspired Waris to continue cytophysiological studies on Micrasterias
(Waris 1950a, b, 1951). In 1950, he had already established the idea of the
cytoplasmic framework. A corresponding structure was, however, not seen in
electron microscopic studies. At that time, the fixation methods did not preseiVe
microtubules, and the cytoskeleton had not been discovered. After the Second
World War, he began to publish his results in English mainly in the new
Scandinavian journal of plant physiology, Physiologia Plantarurn.

3. Professor at the University of Helsinki

Waris continued intensive work (Waris 1953, 1956) on morphogenesis of

Micrasterias at the Helsinki University (1953-1972). He obtained new
equipments, especially microscopes and continued to supeiVise the Ph.D. thesis of
Paavo Kallio in Turku concerning morphogenesis of Micrasterias. Kallio was the
only one having Micrasterias as the topic of research and their collaboration
continued for a long time and led to a deeper understanding of the morphogenesis
of desmids (Waris & Kallio 1957, 1964, 1972). As a member of the Finnish
Academy of Sciences, Waris also bad execellent opportunities to present his
new results to a good audience and to publish his scientific results in the
biological series (Ser. Biologica) of Annates Academiae Scientiarum Fennica.
 3
Waris was elected as the president of the Scandinavian Society of Plant Physiology
(1961-1964).

3.1. SOMATIC EMBRYOGENESIS

In Helsinki, Waris was in close contact with plant biochemists having a special
interest in nitrogen metabolism, and started to investigate aseptic cultures of
angiosperms (Waris 1957a, b, 1958a, Miettinen & Waris 1958). There was an
early short report on the ability of higher plants to use amino acids as nitrogen
sources (Virtanen & Linkola 1946), but some of the compounds tested affected
their morphogenesis, as well. The frenching phenomenon of tobacco shoots as a
result of an exogenously added amino acid (e.g., L-isoleucine 100 mgll), or as a
result of mineral nutrient deficiency leading to an increased level of some amino
acids in the leaves, arose interest among scientists, and the effect of amino acids
was even compared with plant honnones (Steinberg 1947, 1949, Steinberg et al.
1950). Since root exudates may contain amino acids, and plants are able to use
some amino acids also as nitrogen sources, the practical applications stimulated
research in several laboratories. Particularly, the excised root tips oftomato (White
1937) and the roots of Pisum sativum seemed relatively sensitive to various amino
acids (Fries 1951). Complex media were found suitable for normal plant
development in aseptic culture, but the addition of single amino acids at relatively
low concentrations or their combination could stimulate or inhibit a certain phase
of plant organ development(Audus & Quastel 1947, Sanders & Burkholder 1948,
Street et al. 1960).

The developmental changes in angiosperms caused by addition of exogenous

amino acid to the defined nutrient medium led to new thinking on the metabolic
changes regulating morphogenesis. Waris presented his work on aseptic cultures
of Oenanthe aquatica (Fig. I) on 20th of March 1957 in the meeting of Societas
Biochemica and Biophysica et Microbiologiae Fenniae, and published his data on
the effect of glycine in Suomen Kemistilehti, as a short communication It is only a
single printed page in English (Waris 1957a). They demonstrated first a normal
appearance of plants but ceased to grow within 3-4 months and became morbid
(Fig. 2). Waris pointed out that 'a number of fresh green plants had grown in the
flask containing the original, almost dead plant. They were originated from minute
grains formed by some root tips of the original plant'. He continues: 'The further
reproduction of the new type growth took place from cell groups that were
spontaneously detached from colourless outgrowths formed by the green leaves,
which indicates that the epidermis was responsible for the reproduction. After
being freed, or even earlier, the cell groups developed colourless "embryos" that
later became green and assumed a shape resembling normal seedlings, though
more than two "cotyledons" were sometimes formed'.

The publication forum was rather modest but very rapid. The Nobel Laureate Prof.
Artturi I. Virtanen was the chairman of this young society and also used Suomen
Kemistilehti to report results of the effect of some amino acids and amines on the
4
growth and the form of the pea plant (Virtanen & Linkola 1957). Virtanen, who
had published an earlier note in Nature (Virtanen & Linkola 1946), used this

u,~~~~~ !PJ~,m.;
st;;,~

t' cn.e .

Figure: I A herbarium specimen of Oenanthe aquatica 0.43 x.

 5

journal as the forum also for his later results on amino acids as the sole nitrogen
sources of higher plants (Valle & Virtanen 1960). Later on Waris could
concentrate more on detailed analysis of the phenomena behind the changes in the
morphogenesis. Virtanen's young colleague Dr. Jonna K Miettinen (see below)
became Waris's collaborator and did paper chromato-graphic analyses on
"neomorphs" (Miettinen & Waris 1958).

Waris refers to Virtanen and Linkola's (1946) and Steinberg's (1947, 1949)
papers in his article "Kukkakasvin kemiallisesti aikaansaatu muodonmuutos" (A
chemically-induced change in the morphogenesis of a flowering plant) which was
the lecture given on the 8th of November, 1957, at the meeting of the Finnish
Academy of Sciences. This Finnish paper is illustrated with six good figures, five
of them showing development of young seedling-like structures in aseptic cultures
of Oenanthe aquatica (Fig. 3) 0. /achenalii, and

Ii ~ ! ; ll : I i

Figure. 2. Oenanthe aquatica. A normal seedling, developed in a nutrient solution containing glycine
(0.1 %), which ceased its growth, turned brown, and was near to death, but formed small gemma at the
end of a couple of lateral roots, which detached and developed into individs with throughout a changed
form. A clearly visible gemma in the side root is pointing upwards 2.3 x.(Waris !957b).. Figure. 3.
6
Oenanthe aquatica. Plants developed from gemma and resembling seedlings. In the leaves of the
"plantlet" in the middle are bulging cell groups, which are able to develop into new individs (Waris
1957b). Figure. 4. Oenanthe lachenalii. An embryogenic germ, one end truncate, the other
attenuated 20 x.(Waris 1962).

Daucus carota (Waris 1957b). The first object had been 0. aquatica (Fig. 1),
which is a rare umbelliferous plant growing in shallow water in southern Finland.
The compound morphology of leaves, rapid growth, and adaptation of the lowest
leaves to development under water made this species a promising experimental
system, although the size of the plant, with a shoot up to one meter long in nature,
was far from an ideal laboratory object. Waris (1957b) reported that his
experiments were initiated from seeds in November 1955. Due to the lack of good
climate chambers, the plants also received sucrose (1%) in their aseptic nutrient
medium, Some of the cultures were supplemented with 0.1 or 0.4% glycine, and
the plants developed like those serving as controls, for 3-4 months. After this, their
growth ceased, they turned brown, and seemed to be near death. Waris writes
about his situation as follows: 'At this stage my amino acid experiments seemed to
have failed, but it happened, however, that this failing was the beginning of greater
success than I had dared to hope. The success was due to the fact, that I did not
cast away these dying cultures' (Waris 1957b).

It is quite clear that Waris understood that he had found an interesting new
phenomenon in the reproduction of flowering plants. In the Finnish text Waris first
uses the term "itujyvanen", corresponding to the Latin term gemma, used for
cryptogams (algae, bryophytes, and pteridophytes) to describe a small reproductive
unit having variable form and origin (Tirri et al. 1993).

3.2. NEOMORPHS

It was somewhat difficult to describe the development ofVallisneria-like seedlings

of Oenanthe aquatica; Waris began by calling them "neomorphs" and introduced
the word "neomorphosis" into the plant physiological literature (Miettinen &
Waris 1958, Waris 1959, 1962). These terms did not obtain full acceptance among
some plant physiologists (Lang 1961, Allsopp 1965), but as a matter of fact Lang
considered the phenomenon itself very interesting, and Allsopp included a nice
group of photos on Waris's experiments (1959) in his article.

Waris answered to Lang's objections (1961) in the introduction of his paper in

Physiologia Plantarum (1962) as follows: 'In fact the "neomorphs" of Oenanthe
aquatica include seedling-like forms comparable to the heart-stage seedlings but
also "thalloid" forms which cannot be described in terms of normal morphology.
Moreover, the seedling-like "neomorphs" have a new type of leaf which is
comparable though not quite similar to the cotyledons'. The term "neomorph"
was, however, accepted by Johri and Sehgal (1965). In their ovary cultures of
Anethum graveolens (Umbelliferae Apiacere) many embryo-like structures were
observed, and they developed further very variably, some of them resembling
 7
neomorphs of 0. aquatica (Miettinen & Waris 1958). Some of the cultures 'give
rise to normal Anethum plants, and the authors noticed that neomorphs can be
used by plant breeders for culturing a large population ofseedlings with desired
characters.

Effects of alanine, arginine, glycine, and leucine were tested with 0. aquatica and
0. lachenalii as objects, and also lower concentrations were used (Waris 1959,
1962). He found that in 0. lachenalii it was possible with these compounds to
induce both reversible and irreversible morphogenetic effects (Fig. 4). A
reversible morphogenetic effect was induced with glycine when supplied at
moderate concentration (0.003 M). From their shoots the seedlings formed
embryonic germs which grew into juvenile plantlets suggestive of heart-stage
seedlings but having larger leaves than the cotyledons and capable of proliferation
(Waris 1962). These juvenile plantlets in turn reproduced in the same way and
were able to develop into normal mature plants both in the presence and absence
of glycine.

An irreversible morphogenetic effect was induced in 0. lachenalii when it

developed from seed in a nutrient medium supplemented with L-leucine at a
critical concentration (about 0.01 M) (Waris 1962). After a period of morbidity
lasting several months, callus-like nodules became detached from the shoot apices
of some seedlings and gave rise to plantlets of a new type, showing very reduced
differentiation The new neomorphic plantlets were capable of vegeGitive
reproduction and retained their characteristic features even in normal nutrient
solutions for an indefinite period of time. They were distinctly different from the
neomorphs ofO. aquatica described previously (Waris 1957b, 1959).

The neomorphs of 0. aquatica contained a higher level of free amino acids and a
new amino acid and a peptide which both remained unidentified. Their level of free
sugars was lower than in the normal plants (Miettinen & Waris 1958). These
results suggested that neomorphosis was due to competitive antagonism between
glycine and one or more other amino acids. Glycine may inhibit special enzymatic
reactions and in this way synthesis of compounds which are necessary to the
normal development.

The developmental physiology of seed plants was quite a new field of interest to
Waris. The short list of references in the first larger paper concerning
neomorphism (Miettinen & Waris 1958) shows not only that this was a new trend
in plant physiology, but also that the field was new for him. In later publications
(Waris 1959, 1962, 1967) it emerges that Waris was familiar with the trends in
developmental physiology and especially with the role of amino acids. Waris did
not try to compete with Steward's and Reinert's groups in describing the first
stages of somatic embryogenesis. He was more interested in the factors behind
morphogenesis and in cytological studies, which were relatively popular in tissue
culture research. An increased chromosome number was found in irreversible
neomorphosis of 0. lachenalii, although whether in the form of polyploidy or
8
polysomy remained open (Waris 1962).

In a large study (Waris 1967) in which several species of seed plants and in
addition to amino acids also purines and pyrimidines were tested, irreversible
neomorphosis was found in 6 wnbelliferous species, and reversible neomorphosis
was shown by dwarf shoots of Samolus valerandi (Primulacere, Fig. 5) and by the
vegetative reproduction of various umbelliferous plants. In the presence of nitrate,
as much as 0.0 1M L-alanine allowed normal growth of Cicuta virosa, while as the
only source of nitrogen, as low a concentration as 0.001 M of this amino acid was
sufficient to induce abnormal growth. All species tested, e.g., Nasturtium
o.fjicinale (Brassicacere), showed, however, no remarkable changes in these
experiments. This paper contains 81 figures, and the figure legends help the reader
to understand the wide variation in the offspring. The terms "embryoids",
"embryonic" germs, and "thalloid individuals" are used to describe the
morphology. Waris uses three pages for the general features and terminology of the
abnormal forms. It was concluded that the morphogenetic effectiveness of
compounds capable of inducing morphogenesis depends on their interfering with
protein synthesis, and the irreversible neomorphosis, in addition, that hereditary
factors are affected.

Figure. 5. Samolus valerandi. Nonnal and dwarf shoots, derived from a piece of dwarf shoot cushion
and grown in nonnal solution. Dwarf shoots were obtained by glycine treatment and the two types of
shoots derived from a piece of the dwarf shoot cushion. 0.66 x.(Waris 1967).

The terms "neomorph" and "neomorphosis" were accepted by Wardlaw (1965,

1968), and he cites Waris's experiments relatively widely. Wardlaw also chose a
neomorph grown in a medium containing only a low level of arginine (0.00063
M). The appearance of embryo-like structures is included in the text, and the first
small report (Waris 1957a) is found in the list of references. Neomorphs were
mentioned by Steward (1968) quite briefly in connection with totipotency in his
 9
famous book Growth and Organization in Plants. He was apparently not so much
interested in defined nutrient media, because the media for tissue cultures were
usually supplemented with coconut milk (10%). Thus Waris's results did not
remain unknown, but they were not widely cited (Krikorian & Simola 1999).

Possibly encouraged by Wardlaw's kind attitude, Waris (1967) presents the

following background and defence: 'These terms have been criticized by Lang
(1961) and Allsopp (1965) but adopted by Wardlaw (1965). The reason for the
objection has been that the "neomorphs" represent arrests and retardations rather
than fundamental alterations of the normal course of development (Allsopp 1965)
and that new morphological characteristics, implied by the term "neomorph", are
not involved (Lang 1961). Thus the applicability of the terms depends upon what
is regarded as new and fundamental. The use of the former attribute seems to be a
matter of taste, whereas the latter may require some further comment. If a
morphological change is both great and irreversible, appearing in successive
cultures for an indefinite period of time, it must, in the author's opinion, be
regarded as fundamental. Such a case is represented by the "neomorph" of
Oenanthe /achenalii induced with leucine (Waris 1962), and further cases will be
described in the "present paper" '.

Among the species experimented on, the wnbellifous held a unique position, due
to the fact that they alone, and all of them, responded to the chemical compounds
with vegetative reproduction (Waris 1967). Daucus carota seemed to show an
excellent regenerative and morphogenetic response (Waris 1957a, b, Steward et
al. 1958, Reinert 1959a, b,), and this plant has been used in numerous
experiments, especially as a model system of somatic embryogenesis. It is quite
interesting that adventive embryos may develop from the epidermal cells of the
hypocotyl ofRanuncu/us sceleratus, as well (Konar & Nataraja 1965).

Waris used a complete mineral nutrient solution for several seed plants including
D. carota (Waris 1957a, b, 1958a, 1962, 1967). This medium had a good balance
of biogenic elements (Waris 1958a) and was supplemented only with sucrose and
the amino acid tested. Reinert's group used a defined medium for carrot tissue
cultures, and embryogenesis was observed if auxin was omitted (Reinert 1959b,
Reinert et al. 1966). In Steward's experiments (1958) the nutrient solution
contained a high level of coconut milk. However, Halperin (1964, 1966) has
pointed out that in fact, neither coconut milk nor any other similar nutrient
complex is required for embryogenesis in carrot cell cultures.
The reason for the limited use of the terms "neomorph" and "neomorphosis" may
be due to the fact that Waris did not have any near colleague among established
plant physiologists who had shared his enthusiasm in this field and continued this
type of experiment. He was near the age of retirement or already retired (1960)
when he made the most important experiments and he continued to study both
Micrasterias and neomorphs. The amino acid pool of the experimental organism
was rarely known, and concentrations used in several early experiments were non-
physiological. The aim was to find the right amino acid composition and design a
10

defined nutrient medium. Growth promotion

was observed in many experiments with amino acid supplementation (e.g., root
tips of tomato, White 1937; embryos of Datura, Sanders & Burkholder 1948;
callus of Ginkgo, Tulecke 1960). Morphogenetic effects were prominent in leaves
of Nicotiana and Chrysanthemum, for instance (Steinberg 1949, Woltz 1963), and
in tissue culture of carrot, but its later passages did not respond to these substances
(Syono 1965).

3.3. DEVELOPMENTAL BOTANY

Waris did not attract very many young students at the Helsinki University due to
their lack of interest in labomtory works. The plant physiological teaching
labomtory in Helsinki was very poorly equipped, and even did not have normal
labomtory benches (Fig. 7) but tables which were used for studying the herbarium
specimens.

In autumn 1958, the author attended Waris's lectures in developmental plant

physiology (Fig. 6) and participated in a smalllabomtory course in this field. We
did not quite understand the secrets behind morphogenesis of the curious forms of
Micrasterias, but later on some of us realized that cell physiological research had
not yet made enough progress for better understanding. Neomorphosis was quite a
mysterious new phenomenon, and Waris made no attempts to attmct young
physiologists to these studies. To the Swedish Dr. Tage Eriksson, working with
tissue cultures, Waris had kindly pointed out a scientist having such project ought
already to have a permanent job (Eriksson, personal communication).

The author herself, as a Ph.D. student and post doc., was quite convinced that the
amino acid pool of the species, especially the mre non-protein amino acids, may be
one of the endogenous factors influencing the morphogenesis of a plant (Simola
1968b). The centre ofboth the morphological and chemical evolution of the genus
Lathyrus lies in the Meditermnean region (Simola 1966, 1968a). However,
Professor H. E. Street (personal communication 1968) presented an objection to
the role of amino acids as factors influencing normal morphogenesis. On the
contmry, many toxic effects of exogenous amino acids on plants were pointed by
Street.

Waris's research was supported by the Finnish Cultuml Foundation and by the
National Research Council for the Sciences. So he had possibilities to buy
chemicals and small equipment for his experiments and was able to continue
scientific work as Professor Emeritus (Fig. 8). Ms Aune GranO, M. Sc., was for
some years his part-time skilful assistant. The author had the pleasure to be of
help in seveml small practical tasks (1967-1969). Gamma mdiation was used in a
new series of mutagenesis, and permanent and tempomry changes were induced
by these mys onMicrasterias algae (Waris & Rouhiainen 1970). Seveml mutant
lines were cultured in the new illumination stand designed by the labomtory staff
on the celebmtion of Harry Waris's 75 birthday.
 11

Figure 6. Professori Harry Waris demonstrating effects of gibberellins to students, 1958.. Figure. 7.
Liisa Simola as a young student working in the old teaching laboratory, Practicum Physiologicum,
1958. Figure. 8. Harry Waris and his wife Martta (nee Oesch) on an excursion after the conference of
the Scandinavian Society of Plant Physiology, 1967, in Norway.
12
The idea of plasma-structural continuity in Micrasterias had already been
presented as a theory of cytoplasmic framework in 1950. This means that the basis
of the symmetry resides in the cytoplasm and is transmitted during cell division
from parent to new half-cells even in the case of enucleation The main results
concerning the effect of enucleation on this object have been presented in a lengthy
summary (Waris & Kallio 1972). Many Micrasterias species originating from
Waris's experiments are still growing at Turku and Helsinki Universities.

Waris's last series of experiments on the effect of different sucrose levels (up to 10
or 20%) with and without gibberellin on aseptic cultures of seed plants suggested
that some species (e.g.,Asteracantha Jongifolia and Plantago major) were able to
tolerate osmotic stress better if the nutrient medium was supplemented with
gibberellic acid (Waris et al. 1972). In fact, even plants flowering in the large S-
tube Edenmeyers (Simola & Mikkila 1972, Simola 1978), as well as smaller
flasks supplemented with amino acids and inoculated with callus cultures, were
quite common (Salonen & Simola 1977, Salonen 1980, Simola & Santanen 1990)
during the following years in the Practicum Physiologicum. Although Waris had
already left his laboratory, interest in the effects of amino acids on plant
development cqntinued at the Department of Botany, University of Helsinki. The
author had the honor of dedicating an electron microscope study of Atropa
belladonna callus culture and intact plant to Professor Harry Waris on her
teacher's 80th birthday celebration in early May 1973 (Simola 1973) about half a
year before his death on 22nd November 1973 in Helsinki.

4. Conclusion

It is remarkable that one plant physiologist could make such valuable and lasting
primary observations in so many fields of botany. Waris's thesis (1920) on lichen
gonidia has been considered an outstanding study on the axenic culture of lichen
alga, describing the isolation and culture techniques of these algae and their
utilization of different nitrogen sources (Ahmadjian 1967). Largely through
Waris's work, it has been realized that with the genus Micrasterias
(Desmidiaceae) it may be possible to explore why cells adopt their characteristic
shape (Brook 1981). These algae are unique material for the study of the role of
the nucleus and cytoplasm in determining the morphology of the developing
Micrasterias semicell. The inorganic nutrient medium (MY) designed by Waris
(1953) for desmids is continuously used in physiology and experimental algology,
 13
and reference is made to his works (Meindl1993). The same amino acids, which
were tested with lichen gonidia, were then applied to aseptic cultures of seed
plants. The first observations of somatic embryogenesis were reported in the
wnbellifer Oenanthe aquatica, and as a matter of fact they pre-date both Steward
and Reinert (Waris 1957a. b). Although it is difficult to give full credit to- one
person to the well-docwnented discovery of somatic embryogenesis (cf. Vasil &
Vasil 1972, Gautheret 1983, Halperin 1995), it can be pointed out that in the
history of somatic embryogenesis in vitro, Harry Waris also deserves a pioneer's
place (Krikorian & Simola 1999).

5. Acknowledgements

I wish to thank Eliel Waris, MD, for the loan of the collection of publications by
·his father Professor Harry Waris, and Kuvasiskot for permission to print the
photographs in the beginning of the volwne. I am grateful to the Finnish Academy
of Sciences for permission to use Figs. 2-5. My thanks are also due to Tuuli
Timonen, Ph.D, for photographing the hetbarium specimen and to Maija-Liisa
Salonen, Ph.D, for helpful discussion and careful reading of the manuscript Mrs
Sirlcka Siillinen has been of valuable help in ordering nwnerous papers for my use
from different libraries.

6. References

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Blaisdell Publishing Company, Waltham, M. A 152 pp.
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Audus, L. J. & J. H. Quastel, 1947. Toxic effects of amino acids and amines on seedling growth.
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Autio, V.-M. (ed.), 1997. Professorimatrikkeli 1918-1996 = Professorsmatrikel 1918-1996. Helsingin
yliopisto, Gummerus, Helsinki. ISBN 951-45-7818-X.
Brook, A J., 1981. The biology of desmids. University of California Press, Berkeley, CA 276 pp.
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Callander, R., 1965. The history ofbotany in Finland, 1828-1918.With an appendix on forest science
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Elfving, F., 1881. Anteckningar om finska desmidieer. Acta Societatis pro Fauna et Flora fennica 2 (2):
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Fries, N., 1951. The influence of amino acids on growth and lateral root formation in cotyledonless pea
seedlings. Experientia 7:378.
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Halperin, W., 1964. Morphogenetic studies with partially synchronized cultures of carrot embryos.
Science 146:408-410.
Halperin, W., 1966. Single cell, coconut milk and embryogenesis in vitro. Science 153:1287-1288.
Halperin, W., 1995. ln vitro embryogenesis: Some historical issues and unresolved problems. ln:
Thorpe, T. (ed.) In vitro embryogenesis in plants. Kluwer Academic Publishers, Dordrecht,
pp. 1-16. ISBN 0-7923-3149-4.
Johri, B. M. & C. B. Sehgal, 1965. In vitro production of neomorphs in Anethum graveolens. L.
Nature 205:1337.
Kallio, P., 1975. Harry Ilmari Waris. Suomalainen Tiedeakatemia. Esitelmiit ja Poytiikirjat 1975:73-
77.
Konar, R.N. & K. Nataraja, 1965. Experimental studies inRanunculus sceleratus L. Development of
embryos from the stem epidermis. Phytomorphology 105: 348-355.
14
Krikorian, A D. & L. K. Simola, 1999. Totipotency, somatic embryogenesis, and Harry Waris (1893-
1973). Physiologia Plantarum 105:348-355.
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Aspekte). BerichtilberdieJahre 1957-1960. FortschrittederBotanik 23:312-345.
Meindl, U., 1993. Micrasterias cells as a model system for research on morphogenesis. Microbiolo-
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Miettinen, J. K. & H. Waris, 1958. A chemical study on the neomorphosis induced by glycine in
Oenanthe aquatica. Physiologia Plantarum II: 193-199.
Reinert, J., 1959a. Untersuchungen ilber die Morphogenese an Gewebekulturen. Berichte der
Deutschen Botanischen Gesellschaft 71 : 15.
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an Gewebekulturen aus Karotten. Planta 53:318-333.
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Kulturformen von Umbelliferen. Planta 68:375-378.
Salonen, M.-L., 1980. Glutamate- and aspartate-derived amino acids as nitrogen sources for the callus
of Atropa belladonna L. Annales Botanici Fennici 17:357-378.
Salonen, M.-L. & L. K. Simola, 1977. Dipeptides and amino acids as nitrogen sources for the callus of
Atropa belladonna. Physiologia Plantarum 41:55-58.
Sanders, M. E. & P. R. Burkholder, 1948. Influence of amino acids on growth of Datura embryos in
culture. Proceedings of the National Academy of Sciences 34:516-5 26.
Schwendener, S., 1872. Erorterungen zur Gonidienfrage III. Flora 15:225-234.
Simola, L. K., 1966. Chemical and morphological characteristics of some endemic species of Lathyrus.
Suomen Kemistilehti B 39:148-150.
Simola, L. K., 1968a. Comparative studies on number ofleaflets, venation and epidermal structure in
the genusLathyrus. Canadian Journal Botany 46:71-84.
Simola, L. K., 1968b. Comparative studies on the amino acid pools of three Lathyrus species. Acta
Botanica Fennica 81:1-62.
Simola, L. K., 1973. Development of chloroplasts in intact Atropa belladonna and in stem callus
cultures during greening and leaf differentiation. Annales Academia: Scientiarum Fennicae,
Ser. IV Bioi. 196: 10 pp.
Simola, L. K., 1978. The effect of several amino acids and some inorganic nitrogen sources on the
growth of Drosera rotundifolia in long- and short-day conditions. Zeitschrift fiir Pflan-
zenphysiologie 90:61-68.
Simola, L. K. & T. Mikkilii, 1972. The effect of gibberellic acid and CCC on the anatomy and
morphogenesis of Coreopsis tinctoriaL. Annales Botanici Fennici 9:1-19.
Simola, L. K. & A Santanen, 1990. Improvement of nutrient medium for growth and embryogenesis of
megagametophyte and embryo callus lines of Picea abies. Physiologia Plantarum 80:27-35.
Steinberg, R. A, 1947. Growth responses to organic compounds by tobacco seedlings in aseptic
culture. Journal of Agricultural Research 75:81-92.
Steinberg, R. A, 1949. Symptoms of amino acid action on tobacco seedlings in aseptic culture. Journal
of Agricultural Research 78:733-741.
Steinberg, R. A, Bowling, J. D. and J. E. McMurtrey, Jr., 1950. Accumulation of free amino acids as a
chemical basis for morphological symptoms in tobacco manifesting frenching and mineral
deficiency symptoms. Plant Physiology 25:279-288.
Steward, F. C., 1968. Growth and organization in plants. Addison-Wesley, Reading, MA 564 pp.
Steward, F. C., Mapes, M. 0. & K. Mears, 1958. Growth and organized development of cultured cells
II: Organization in cultures grown from freely suspended cells. American Journal of Botany
45:705-708.
Street, H. E., Hughes, J. C. & J. S. Lewis, 1960. Studies on the growth ofexcised roots. X. Individual
amino acids and acid-hydrolysed casein as nitrogen sources for the growth of excised tomato
roots. New Phytologist. 59: 273-287.
Syono, K., 1965. Changes in organ forming capacity of carrot root calluses during subcultures. Plant et
Cell Physiology 6:403-419.
Tirri, R., Lehtonen, J., Lemmetyinen, R., Pihakaski, S. & P. Portin, 1993. Biologian sanakilja [The
biological dictionary], Otava, Keuruu, 607 pp, ISBN 951-11390-9.
Tscherrnack-Woess, E., 1988. The algal partner. In: M. Galun: CRC handbook of lichenology. Vol.
1:39-92. CRC Press, Inc. Boca Raton. Florida: ISBN 0-8493-3581-7.
Tulecke, W., 1960. Arginine-requiring strains of tissue obtained from Ginkgo pollen. Plant Physiology
35:19-24.
Turun Yliopiston Vuosikirja 1939-1942 [Year book of Turku University 1939-1942]. Polytypos
 15

Turku, 1943, 114 pp. Turun Yliopiston Vuosikirja 1943-1948 [Year book of Turku
University 1943-1948]. Polytypos Turku, 1949, 123 pp.
Valle, E. & A I. Virtanen, 1960. On the uptake and assimilation ofD- and L-amino acids by pea and
barley plants. Suomen Kemistilehti 33B:201-204.
Vasil, I. K. & V. Vasil, 1972. Totipotency and embryogenesis in plant cell and tissue cultures. In Vitro
8:117-127.
Virtanen, A I. & H. Linkola, 1946. Organic nitrogen compounds as nitrogen nutrition for higher
plants. Nature 158:515.
Virtanen, A I. & H. Linkola, 1957. On the effect of amino acids and arnines on the growth and the
form of the pea plant. Suomen Kemistilehti 30B:220-221.
Wardlaw, C. W., 1965. Organization and evolution in plants. Longmans, London, 499 pp.
Wardlaw, C. W., 1968. Morphogenesis in plants. A contemporary study. "Mattwen & Co. LTD".
London, 451 pp.
Waren, H., 1920. Reinkulturen von Flechtengonidien. bfversigt af Finska Vetenskaps-Societetens
Forhandlingar 61 A(14): 79 pp +plates IX.
Waren, H., 1924. Untersuchungen iiber die botanische Entwicklung der Moore mit Beriicksichtigung
der chemischen Zusammensetzung des Torfes. Suomen Suoviljely-yhdistys. Tieteellisiil
julkaisuja. Wissenschaftliche Veroffentlichungen des Finnischen Moorkulturvereins 5:1-95.
Waren, H., 1926a. Nahrungsphysiologische Versuche anMicrasterias. Societas Scientiarum Fennica.
Comrnentationes Biologiae II (8}: 42 pp.
Waren, H., 1926b. Untersuchungen iiber Sphagnumreiche Pflanzengesellschaften der Moore Finnlands
unter Beriicksichtigung der soziologischen Bedeutung der einzelnen Arten. Acta Societatis pro
Fauna et Flora Fennica 55 (8): 133 pp +plates IX.
Waren, H., 1933. Ober die Rolle des Calciurns im Leben der Zelle auf Grund von Versuchen am
Micrasterias. Planta 19 (1): 45 pp.
Waris (Waren), 1936. H. Ober des Calciumbediirfuis der niederen Algen. Planta 25:460-470.
Waris, H., 1939. Ober den Antagonismus von Wasserstoffionen und Metallkationen bei Micrasterias.
Acta Botanica Fennica 24, 36 pp.
Waris, H., 1950a. Cytophysiological studies onMicrasterias. I. Nuclear and cell division. Physiologia
Plantarum 3:1-16.
Waris, H., 1950b. Cytophysiological studies on Micrasterias. II. The cytoplasmic framework and its
mutation. Physiologia Plantarum 3: 236-246.
Waris, H., 1951. Cytophysiological studies on Micrasterias. III. Factors influencing the development
of enucleate cells. Physiologia Plantarum 4:387-409.
Waris, H., 1953. The significance for algae of chelating substances in the nutrient solutions.
Physiologia Plantarum 6:538-543.
Waris, H., 1956. Cytophysiological studies on Micrasterias. IV. The effects of acids upon the nuclear
aspect and the resistance of the cell. Physiologia Plantarum 9:82-101.
Waris, H., 1957a. A striking morphogenetic effect of amino acid in seed plant. Suomen Kemistilehti
30:121.
Waris, H., 1957b. Kukkakasvin kemiallisesti aikaansaatu muodonmuutos [A chemical-induced change
in the morphogenesis of a flowering plant]. Suomalainen Tiedeakatemia. Esitelmilt ja
Poytiikirjat 1957, 117-127.
Waris, H., 1958a. Simple devices for aseptic culture of seed plants. Physiologia Plantarum 11:627-630.
Waris, H., 1958b. Splitting of the nucleus by centrifuging in Micrasterias. Annates Academia:
Scientiarum Fennicae, Ser. A IV Bioi. 40: 20 pp +plates XII.
Waris, H., 1959. Neomorphosis in seed plants induced by amino acids I. Oenanthe aquatica.
Physiologia Plantarum 12:753-766.
Waris, H., 1962. Neomorphosis in seed plants induced by amino acids II. Oenanthe lachenalii.
Physiologia Plantarum 15:736-752.
Waris, H., 1967. Morphological changes in seed plants induced with amino acids, purines and
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Waris, H. & P. Kallio, 1957. Morphogenetic effects of chemical agents and nucleo-cytoplasmic
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+plates IV.
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Waris, H. & P. Kallio, 1972. Effects of enucleation on Micrasterias. Biology and Radiobiology of
Anucleate Systems. II. Plant Cells., pp. 137-144.
Waris, H. & I. Rouhiainen, 1970. Permanent and temporary morphological changes in Micrasterias
induced by gamma rays. Annales Academia: Scientiarum Fennicae, Ser. A IV, Bioi. 167: 13
16
pp.
Waris, H., L. K. Simola & A Grano, 1972. Aseptic cultures of seed plants at various sucrose
concentrations with and without gibberellin. Annates Academiae Scientiarum Fennicae, Ser. A
IV Bioi. 188: 12 pp.
White, Ph. R., 1937. Amino acids in the nutrition of excised tomato roots. Plant Physiology 12:793-
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Woltz, S. S., 1963. Growth-modifYing and antimetabolite effects of amino acids on Chrysanthemum.
Plant Physiology 38:93-99.
1. Historical Insights into Some Contemporary Problems in Somatic Embryogenesis

A. D. Krikorian
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook
Stony Brook, New York 11794-5215 USA Preferred email: adkrikorian@carthlink.net
or, alternatively akrikorian@notes.e<:.sunysb.edu
Preferred mailing address for correspondence associated with the manuscnpt
P. 0. Box 404, Port Jefferson, NY 11777, USA Telephone: 526 473-7016

Contents
1. Introduction
2. Plant Molecular Biology and Embryogenesis
3. Towards a 'Taxonomy' ofthe Events in Embryogenic Cultures
3.1 How do Somatic Embryos Arise?
3 .l.l Indirect vs. Direct Processes
3.1.2 The Indirect Process
3 .1.3 The Direct Process
3.1.4 Can Indirect and Direct Modes Exist in the Same Culture?
4. The On-Going Events in an Embryogenic Suspension
4.1 The 'Old' Perspective
4.1.1 Single and Free Cells
5. The 'New' or 'Reconstrocted' P~ective
5 .I Daylily as a Model
5.2 The PEM Concept Needs Modification
5.3 Analysis ofEmbryogenicity of Suspension Components
5.4 Past Failures
5.5 Liquid vs. Semi-Solid Culture Environments
5.6 Regeneration under Limiting Conditions
5.6.1 Poor Morphology
5. 7 The 'Auxoph]10n'
5.8 'Neomorphs'
6. Optimization of Regeneration by Auxin Removal, Transferral to Setru-Solid and Use of Other Modifications
6.1 Plating Protocols
6.2 Agar Quality
6.2.1 Nature of the Inhibitor(s) in Agar
6.3 pH and Buffering
6.4 Low pH and PGSPs
7. Daylily 'Pre-Shoots' Are Somatic Embryos
8. Early Events in Generation of Embryogenic Daylily Suspensions
8.1 Extreme Strategies for Production of Embryogenic Suspe_nsions
9. Site oflnitial Response in Daylily
9.1. Enlbryoids and Embryomas
10. Somatic Embryos and Somaclonal Variation
11. Conclusions
12. Acknowledgmeots
13. References

1. Introduction

Structures now commonly referred to by tissue culture workers as somatic embryos were first discovered in
cultures of cultivated carrot and related umbellifers in the late 1950s (Waris, 1957; Steward et a!., 1958;
Reinert 1959a and b). Many have since investigated the nature of somatic embryogenesis and aimed to
develop its potential for applied uses like clonal micropropagation (see Krikorian, 1982; Halperin 1995;
Minocha and Minocha, 1995; Neumann, 1995; Krikorian and Simola, 1999 and refs. there cited for some early
history). Even so, the establishment and subsequent management of embryogenic systems remains empiric
although there are some general underlying principles ( cf. e.g. Bajaj, 1995; Thorpe, 1995 and all the volumes
in this series).

Soon after the discovery of somatic embryogenesis it was asked how and when somatic cells became
embryogenic. The expression 'determined' was not widely used (the animal developmental biologist Radom
popularized the term in 1965) but had it been, one might well have asked the question this way. When does
the process which initiates this specific pathway of development by singling it out from among various
possibilities for which a cellular system is competent occur? F. C. Steward, one of the discoverers of somatic
embryogenesis, believed that special chemical stimuli, such as those found in embryo sac fluids like coconut
milk [ more accurately, coconut water], caused 'free' cultured cells to embark upon an embryogenic pathway.
Carrots and coconuts figured heavily in his outlook for quite a few years (cf. Steward, 1968; Steward et al.,
1975). While the claimed requirement for coconut milk was shown to be incorrect (Halperin, 1969, 1970), a
residue of that vie\\}loint has endured. The legacy is that somatic embryogenesis is a complex process

17
S.M. Jain. PK Gupta and R..l. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6. 17-49.
© 2000 Kluwer Academic Publishers.
18

involving a series of well-orchestrated, albeit plastic, events which are set in motion and modulated bv a
variety of growth regulators (see. e.g. Steward and Krikorian, 1971; Ammirato, 1983, 1986; Krikorian, 1995a
and refs. there cited).

Somatic embryogenesis implies that an egg cell which might supply maternal factors is not required in
programming embryo development. Thus, the potential to express an egg-cell gene elq>ression program would
reside in all nucleated somatic cells and could be ex-pressed under appropriate conditions. The formation of an
embryo in the absence of a seed would also seem to suggest that the process does not require signals derived
from the seed. That does not imply, however, that it would not be possible for the necessary signal(s) to be
supplied by components of the growth medium or even the physical aspects of the culture environment.
Therein lies the problem of determination in somatic embryogenesis. But there is another aspect to the
problem that one has to deal with, not just determination. It is not a casual activity to manage the embryogenic
cells after they are determined if one is to study or use them for practical ends.

It is a truism that every scientific discipline has its cache of carefully nurtured myths. A myth espoused by
many plant biologists is that an isolated somatic cell can readily yield in vitro an embryo morphologically
similar, if not identical, to a zygotic embryo. Tissue culturists will appreciate how inaccurate the word
'readily' is. Another myth is that cultured cells are amenable to study like single-celled microorganisms. But
cultured plant cells very rarely if ever grow as single cells (cf. e.g. Street and Henshaw, 1963; Hall, 1991;
Krikorian, 1996b). Yet another myth is that aseptic culture conditions eliminate variables by providing a
rigorously controlled, defined environment. But the truth is that cultured cells are e:\-posed to many adverse
physical, chemical and environmental stresses. How extensive and varied these may be is only now being
more fully appreciated (Krikorian, 1996a). It is a fact that plants continually gather and process information
from a wide range of environmental variables which elicit specific or more general responses. However, it is
also true that environmental signals may be harmful and, to be teleological, the plant's response is then to
minimize damage. But cultured cells can show to a remarkable extent the same capabilities of sensing when in
isolation, as in 'tissue culture', not just when they are part of a whole organism. When the level and basis of
this sensing and response to stress signals become better known (see e.g. Havel and Durzan 1996), one will be
able to develop strategies to either minimize, eliminate or even exploit them.

A recurring theme throughout this chapter will be that one needs to optimize repeatedly if one is to exploit
fully the somatic embryogenic process. Pre-occupation with 'minor details' has enabled a more sharpened
focus on the events of cell culture and somatic embryogenesis to emerge. It is hoped that the 'reordering' of
important events and the 'reconstructed' and expanded perspective to be presented will be salutary because
cells from suspensions are still being used for somatic embryogenesis studies for which they are unsuited (cf.
e.g. de Vries et al., 1988; Aleith and Richter, 1990; Komamine et al., 1990, 1992; Pennell et al., 1992;Toonen
et al., 1994; Sato et al., 1995; Wang et al., 1996; Toonen and De Vries, 1997). In some ways this is hard to
understand because long ago it was pointed out that "auxin-plus and auxin-minus cultures are not comparable
to "callus" and "embryo populations ... " (Anunirato, 1983 p. 84). But what I will say is not only of theoretical
significance. It should enable investigators to view species traditionally considered as "recalcitrant" in new
light. They may not be as recalcitrant as some believe. Conditions for working with woody species may need
to be optimized using strategies slight different from
more tractable ones.

Kohlenbach (1978, 1982, 1985) seems to have been the first to raise the point in print that established
embryogenic cultures are already determined. But definitive 'proof' was lacking. And as will become clear,
his concept of what hormone does is off the mark and this confused the issue. Even so, Kohlenbach correctly
repeated the view that systems "based on stock cultures of Daucus in the form of embryogenic clumps or
embryogenic cell suspensions, grown in the presence of auxin ... cannot serve to analyze induction, it can only
serve to analyze the release of the already induced embryogenic stage .. .lnduction may have occurred long ago,
possibly years ago and as a rare event, concerning only a single cell undergoing embryogenic differentiation,
surrounded by a great number of cells not involved. This makes it impossible to trace induction back to its
very beginning.... "(cf. e.g. Kohlenbach, 1985 pp. 104-106). " Some investigators have been either unaware of
his views or knew of them and went on to ignore them, or simply felt that they were not substantiated by
enough evidence. There are many papers where investigators have alluded to this fact about embryogenic
cultures but many have equivocated on the matter or made inconsistent statements. Equally important, it
simply has not been a major .issue for most working with embryogenic cultures. Phrases like 'determination',
or 'totipotent' or 'pluripotent' 'stem lines' that are now routine among animal developmental biologists have
only relatively recently garnered the attention of plant biologists.
 19

What follows will hopefully not only be historically interesting but will hopefully have heuristic- ~ue.
Certainly. there is no need to subscribe to the fatalistic view disappointingly e"1Jressed several years ago by two
well-respected researchers that "callus and cell suspension cultures ... are being phased out; cell phenomena are
being studied in planta using molecular probes .... callus and cell suspensions ... become reduced in importance
to source materials for certain enzymes, chemicals and e"1JCrimental material for special aspects of
differentiation. Changes in goals requires changes in treatment of methodology of plant tissue culture"
(Constabel and Shyluk 1994 p. 3). My prediction is that to the contrary, the coming period will be the
beginning of a new golden age in plant cell culture, both for herbaceous and woody species.

2. Plant Molecular Biology and Embryogenesis

Tracking the expression of a specific gene(s) to identify specifics of somatic cells undergoing embryogenesis is
sure to become a reality. The point at which a cell becomes what might be called a 'zygote-equivalent', could
then be learned. And the factor(s) and signal(s) responsible for triggering any purported event could then be
determined by analysis of the control of these genes. But too little is now known about the activity or
regulation of genes that control the critical morphogenetic events necessary to the establishment of plant body
form. Certainly not much is known how regulatory information is partitioned to activate different sets of genes
within a plant embryo, be it zygotic or somatic. It was shown quite a few years ago that embryo-specific
proteins occurred in both zygotic and non-zygotic embryos (see e.g. Crouch, 1982; Perez-Grau and Goldberg,
1989 for some early attempts to gain some insights.)

Transcription factors that have been identified and characterized in plants thus far have, however, been shown
to contain DNA-binding domains like those described in other eukaryotes. The first characterized plant
transcription factors demonstrated their regulatory function in flower and leaf development. More recently,
some progress ·has been made in isolating genes encoding regulatory factors expressed during zygotic
embryogenesis. Although a few have an implicated regulatory function in late embryogenesis, an unequivocal
association with early embryo pattern formation or commitment to embryogenesis has not yet been made.
Thus, genetic markers which could identify cells at very early points in their commitment to embryo
development are not yet, or may just be becoming available. To illustrate this point, a few relevant examples
may be presented.

Early events in embryogenesis probably involve homeotic genes. These genes are master regulatory genes
which specify cell identity and control development through their action as transcription factors. Two
important classes of these genes found in both plants and animals are homeobox and MADS-box genes. The
homeobox genes share a common 180 bp sequence element (the homeobox) that encodes the 60 aa
homeodomain which represents the DNA binding domain. Members of the MADS-box family contain a
highly conserved 55-60 aa motif (the MADS domain) that functions as both a DNA-binding domain and
dimerization domain. In plants, the AGAMOUS gene of Arabidopsis thaliana is a homeotic gene involved in
stamen and carpel development (Ma et al., 1991; Shiraishi et al., 1993). The Deficiens (DEF) gene from
Antirrhinum is also a floral homeotic gene (Sommer et al., 1991). MCMI in the yeast Saccharomyces
cerevisiae and SRF in humans are also in this family. Thus, the shared region of similarity is named MADS
box for MCMI, AGAMOUS, DEF A, and SRF. More than 28 members of the MADS box gene family have
now been identified in Arabidopsis (Riechmann and Meyerowitz, 1997) and undoubtedly more will be found.
Although the majority so far are floral structure specific, one of the most divergent members of the family
AGLl5 (AGAMOUS-like 15), is the only member so far as I know that is preferentially expressed during
zygotic and somatic embryogenesis (Perry et al., 1999). This gene is hypothesized to participate in the
regulation of programs active during early embryo development. However, AGLI5 is not very useful for
investigating somatic embryogenesis because it is not embryo-specific, it is expressed too late for establishing
when cultured embryogenic cells would become determined (its onset is at or before the octant stage) and its
developmental role is essentially undefined. An Arabidopsis homeobox gene has been identified that is
described as the earliest (the only?) molecular marker for apical-basal, as well as radial, pattern formation.
ATML1 (for Arabidopsis thaliana meristem L1 layer) was isolated as the Arabidopsis homologue of
Phalaenopsis 039 after screening anArabidopsis floral bud eDNA library (Lu et al., 1996). Phalaenopsis 039
is expressed early in ovule primordium differentiation and may be involved in the commitment to ovule
development. The ATML1 gene is first expressed in the apical cell (but not the basal cell) after the first
asymmetric division of the zygote and apparently continues to be expressed in all proembryo cells until the
eight-cell stage. In the 16-cell proembryo, the ATMLI gene shows a distinct pattern of expression, with its
mRNA becoming restricted to the protoderm. In the torpedo stage of embryo development, ATMLl mRNA
disappeared altogether but reappeared later only in the L 1 layer of the shoot apical meristem in the mature
embryo. Ultimately, the gene was expressed in the protoderm of the ovule primordia, floral organ primordia,
20

the inner and outer integuments and also was ex-pressed in the endosperm. Thus it may be that ATMI would
be useful in establishing when determination occurs in a somatic embryo even as its developmental role is
undefined. However, again because it is not embryo-specific, some confusion could arise since expression
would be expected in apical meristems that ntight form on/in cultures undergoing organogenesis. Also, the
exlJression of ATMI, while early may not distinguish apical polaril)' at a first asymmetric division because a
somatic embryo has no suspensor. There is no convincing evidence that an apical:basal cell relationship from
which a suspensor forms occurs in somatic embryos (more later on this point). Thus, if one considers early
ontogenetic differences, there is more than a little doubt whether ATMI is expressed early in somatic
embryogenesis.

Other types of nontinally "embryo-specific" transcription factors have also been recently reported. One is the
A rabidopsis gene FEU containing a Cys3His zinc finger domain. This is ex-pressed throughout the embryo
from the globular to late cotyledon stage (Li and Thomas, 1998). PEII may function primarily in the apical
domain of the zygotic embryo but for reasons that have been already given above such transcription factors
would not be very useful in determining when somatic embryos become determined.

In any case, cellular and molecular studies of the early stages of zygotic embryo development (and by extension
somatic embryogenesis) are fraught with many challenges. Cells constituting embryos are small and relatively
few in number. Moreover, they are usually surrounded by endosperm (there are some endosperm-less species
however) and are covered by ovular tissues which later will become the seed coat. Direct application of
modern molecular techniques to the study of somatic embryogenesis is still impossible in many cases since the
process is still not sufficiently refined in the species most studied at the molecular level, namely Arabidopsis
tha/iana.

In short, modern cell biological and molecular studies have so far failed to add significantly to the
identification of the initial stage of the zygotic or somatic embryogenic process (cf. e.g. Choi et al., 1987;
Wilde et al., 1988; Aleith and Richter, 1990; Kiyosue et al., 1993; Sato et. al., 1995).

3. Towards a 'Taxonomy' ofthe Events in Embryogenic Cultures

3. I How Do Somatic Embryos Arise?

3.1.1 Indirect vs. Direct Processes

It has been suggested that somatic embryos arise by either of two modes, an indirect one and an indirect one
and/or an indirect one. In either instance the culture procedure would involve two phases. The phase in which
a primary explant is treated in an appropriate inducing medium is the "induction phase", and the phase in
which the somatic embryo develops, often referred to as the "regeneration phase". In laboratory practice, the
inducing medium nonnally contains a growth regulator of the auxin type such as 2,4-D, and regeneration is
fostered or associated with a drastic reduction or complete removal of the auxin.

3.I. 2 The Indirect Process

Somatic embryogenesis in suspensions is almost universally considered to occur indirectly (cf. e.g. Haccius,
1978; Amntirato, 1987; deVries et al., 1988; Komamine et al., 1990, 1992; Dubois et. al., 1991). The
ex1ensive work of Komantine and his co-workers may perhaps be cited as a paramount example (e.g.
Komarnine et al., 1992). The assertion is that competent single but quiescent 'differentiated' cells in an
explant re-enter the cell division cycle and produce undifferentiated 'callus'. An exogenous auxin is needed
for the cell(s) to de-differentiate and make callus. (Recall that 'callus' by definition consists only of
undifferentiated growth.) Over time, and dependent on the presence of auxin, cells with embryogenic potential
and that are competent to respond to further effects of auxin are said to arise. These cells form a cell-cluster or
mass (referred to as an Embryogenic Cluster-abbreviated EC, or a Pro-Embryogenic Mass, hereafter
abbreviated as a PEM or PEMs, plural). When the auxin is removed or its level is reduced, a cell (or cells)
from certain parts of this cluster develops (or develop) into a somatic embryo. The significant point in this
indirect model is that during growth in auxin-containing medium the ECs or PEMs gain the ability to form
somatic embryos at the point when the hormone is removed or its level is reduced (Komarnine et al.,
1990, 1992).

The origin of the cells that develop into PEMs has not been rigorously determined but it has been suggested
that they arise from either or both of the following pathways (Toonen ct al., 1994): (I) cells are formed de novo
 21

from pre,~ously non-embryogenic single cells or cell clusters (e.g. de Vries et al., 1988); or (2) cells arise from
a cell type(s) susceptible to auxins like 2,4-D, or naturally-occurring equivalents present in the eA'Jilant. This
ceiii}'JIC would then develop into a self-perpetuating sub-population of the embryogenic cells and reJRain so as
long as the culture is embryogenic. In either case cultures would contain both embryogenic and non-
embryogenic cells. These processes of de-differentiation and re-detennination would thus be continuously
occurring and thus the earliest events which transform somatic cells into embryogenic cells are always
occurring.

In my laboratory, work with carrot suspensions eventually enabled a fine-tuning of the vocabulaiy associated
with somatic embryogenesis. We resurrected the designation 'pre-globular stage proembryos' (PGSPs), and
slightly later we even modified it to 'pre-globular stage embryos' (PGSEs), to refer to what we earlier
imprecisely called 'embryogenically competent cells'. This was because we knew they somehow represented
very early stage somatic embryos and could develop directly into later stages (see e.g. Smith and Krikorian,
1990). How equivalent they were morphologically and developmentally to PEMs was never seriously
considered since I never personally cared for the term PEM. (It just did not mean much to me. Every time I
made an attempt to track down what a proembryo was supposed to be, it turned out to be a futile exercise since
even classical embryologists seemed indifferent to defining it consistently-see e.g. Schnarf, 1929 p. 395.)

If one takes the trouble to read the papers from this laboratory in which PGSPs from carrot were described, one
will note that there is ambiguity in some of our statements. Embryogenically competent cultured cells were
incorrectly referred to as "unorganized cells that can develop into somatic embryos." At odds with this was
that we emphasized that PGSPs did not progress through a filamentous or well-defined proembryo stage.
Instead, they developed directly into globular stage embryos (Smith and Krikorian, 1990). Photographs of
sections of late globular and early heart-stage somatic embryos were pro~ded. Although we made no special
mention of it, careful examination of some of those photographs will reveal that those embryos are comprised
of looser cell associations than one might normally expect in a zygotic (or even somatic) globular stage or heart
stage dicotyledonous embryo. In fact, they look as if they could crumble (see Fig. 13 of Smith and Krikorian,
1990). (We shall see later that this is exactly what happens if the conditions for regeneration are not optimal.)
For me it meant that even at as late a developmental stage as late globular/early heart, the constituent cellular
associations are not necessarily permanent. That constituted an important clue to an improved understanding
of the possible relationships among cells of a developing somatic embryo. Admittedly, understanding the
details of how a somatic embryo develops does not necessarily improve merely by adoption of a new term. In
fact, jargon that purports to facilitate scientific communication such as proembryo, PEM, PGSP etc. all come
with their historical baggage. (I'll intelject here that several anonymous ~ewers said they "hated "the word
PGSP" and wished it could be dropped from the literature. Ob~ously we argued that PGSP at least made an
attempt to get the idea across that a developmental stage directly preceding the globular embryo was being
talked about. As such we tried to get the point across to readers that they were already somatic embryos so far
as we were concerned.

3 .1.3 The Direct Process

In the case of direct somatic embryogenesis, a somatic embryo develops without interveliing undifferentiated
'callus'. This mode is often encountered when the primary explant is an immature wounded zygotic embryo or
some tissue related to reproduction like pollen or even nucellus (see Litz, 1989). In the direct process all the
embryogenic cells can theoretically develop into somatic embryos. Thus the process is tenninal since no
undeveloped cells remain to yield additional embryos unlike the indetenninate indirect mode. Herein lies yet
another part of the problem. Historically, the first cultures yielding somatic embryos did not contain
exogenous auxin other than the tiny amounts in coconut water. Mention was made that F. C. Steward
attributed embryogenicity of carrot cultures to coconut milk in the medium. The work of Halperin (1965)
ushered in the era when 2,4-D became the growth regulator of choice for somatic embryogenesis. Out of
several auxins tried 2,4-D was the only one that gave a response worth pursuing (Halperin, 1965). Hence all
his papers reflect work carried out using 2,4-D (cf. e.g. Halperin, 1966, 1970). Thereafter, 2,4-D found its way
into many protocols (see e.g. Amrnirato, 1983; Carman, 1990; Dudits et al., 1991; Thorpe, 1995; Bajaj, 1995
and refs. there cited). It is of some interest that the Cornell Laboratory at that time was routinely using NAA
(cf. Steward, et al., 1964).

Although 2,4-D is by no means the only auxin that 'works' it certainly ended up being the one most widely
used and its used persists even today (cf. e.g. Krikorian, 1995a). And, although the addition of an auxin or
auxin-equivalent to a culture medium usually increases the frequency of somatic embryogenesis, it will be
recognized that this does not constitute evidence for a requirement for exogenous auxin to bring about
determination (Choi et al., 1987; Bogre et al., 1990; Gray and Purohit, 1991; Zimmerman•. 1993; Fransz et al.,
1993). Exogenous auxin is not necessarily required for somatic embryogenic induction (cf. Smith and
Krikorian, 1988, 1989 and refs. there cited; Harada et al., 1990).

However there is one crucial consequence of inappropriate auxin use that is often taken for granted and hence
has not figured much into any analysis of somatic embryogenesis. Too low a level of exogenous auxin in an
'embryogenic culture' soon results in its inability to be sustainable through serial subculture. An exception to
this statement is of course· when heterotrophic embryogenic cultures are autotrophic for auxin through
habituation (Smith and Krikorian, 1989).

3.1.4 Can Indirect and Direct Modes Exist in the Same Culture?

Cultures have been described in which both indirect and direct processes seem to occur (cf. e.g. Hanning and
Conger, 1982; Gray and Conger, 1985; Grieb eta!., 1997). A hypothesis to resolve this and ou'ler conflicting
observations concerning somatic embryo induction and origination was first advanced by Sharp et a!. ( 1980)
and further elaborated by Evans eta!. (1981) and Sharp eta!. (1982). From experiences largely gained from
explanted coffee leaves, these workers suggested that direct embryogenesis proceeds from cells already
determined for embryogenic development prior to explantation. So-called Pre-Embryogenic Determined Cells
(PDECs) only required growth regulators or favorable conditions to allow their entry into cell division and
ex1Jression of embryogenesis. By contrast, indirect embryogenesis nominally required re-determination of
differentiated cells, callus proliferation and the acquisition or development of the embryogenically determined
state. Also, exogenous growth regulators were said to be required for such Induced Embryogenically
Determined Cells (IEDCs) not only for re-entry into mitosis but also for the determination of the embryogenic
state.

4. The On-Going Events in an Embryogenic Suspension

4.1 The 'Old' Perspective

4 .1.1 Single and Free Cells

It seems reasonable that following single cells from embryogenic suspension cultures should establish how
somatic embryos develop. This is especially so since historically there was controversy about single cells
producing somatic embryos. This matter was laid to rest for most people when an apparently single cell was
followed into a somatic embryo first by Backs-Hiisemann and Reinert, 1970; Reinert eta!., 1971) and then by
Nomura and Komarnine
(1995).

That earliest reports that single cells could yield somatic embryos came to be taken by some that somatic cells
always derived from single cells. Obviously, 'Can do' versus 'always do' is no small detail. It also seems
likely that production of somatic embryos from pollen (androgenesis) (see. e.g. Kyo and Harada, 1985;
Sangwan and Sangwan, 1987; Reynolds, 1993) and egg cells (gynogenesis) (Raghavan, 1994, 1997) also
strengthened the conception that it was indeed the single cells that develop into somatic embryos. It may also
be that investigators who were unaware of early controversies concerning mode of development from single
cells thought that obtaining embryos from 'single' cells was easier than was actually the case. Some even
made the mistake of equating single cells with 'free' cells or cells in isolation.

'Free' cells from suspensions, or single cells in cultured explants can develop into somatic embryos (cf. e.g.
Nomura and Komarnine, 1995; Sato et al., 1995 and Gray and Conger, 1985 respectively). That the frequency
of somatic embryo formation from single cells has invariably been very low has not, however, figured heavily
in discussions as to how or when determination occurs, or whether absolutely free cells or only cells in
physiological isolation are 'really' embryogenic. (Surprisingly, stomatal guard cells can be embryogenic (Hall
et a!., 1996). I say 'surprisingly' since it has long been held that highly differentiated cells are not good
sources of starting material for cell cultures in general, much less for cells that can be embryogenic.
Nevertheless, it is difficult to ignore the fact that guard cells nominally show no plasmodesmata! connections
and are thus in physiological isolation.)

This discussion of single, free cells may seem academic because as F. C. Steward always used to quip whenever
we heatedly discussed the issue that "Even a fertilized egg does not make an embryo directly!" True, but I still
maintain that the issue is a very real one. If difficult -to-identifY internal divisions always precede the initiation
of embryogenesis from a supposedly single cell, then it could mean that the timing of one or more of those
 23

dhisions constitutes the point of detennination. That would mean that embryogenesis would be indirect even
though it appeared direct. Another possibility is that a reduction di\ision followed by endoreduplication is
involved (cf. e.g. Nuti Ronchi et al., 1992 and Cannan, 1997 for some novel perspectives). Obviously a cell
that has been killed and sectioned and shows no internal divisions provides no irrefutable C\idencc that it
represents the 'nonn' in an embryogenic culture (e.g. Halperin and Jensen. 1967; Street and Withers. 1974).
There is simply too much going on in a liquid culture to get reliable samples without e":traordinary measures.
Nevertheless, it seems that there should be a way of circumventing this problem. (We shall return to this point
later.)

The relationship of 'single' cells and embryogenesis from, or in an established suspension is generally assumed
to be an indirect one. Even a quick look at the many published pictures of suspension aliquots, whether of cells
or embryos, reveal their extensive heterogeneity. While many appreciate that cells with dense cytoplasm are
those most likely to produce embryos, they also know that there may be many others that appear equally dense
that do not develop. The same holds for clusters of cells. All this suggests that induction may not yet have
occurred in some cells or cell clusters. Again to generalize, most have appreciated that cultures may also
contain cells that are much more elongated than others. These might also divide and develop. But they usually
are not the 'most embryogenic' cells (cf. e.g. Steward et al., 1964, 1975). Few experienced investigators today
would have selected for study the single embryogenic cell followed by Backs-Hiisemann and Reinert (1970;
Reinert et al., 1971). It is simply too elongated. In any case, few seem have been concerned with precisely
how all this apparent heterogeneity comes into being.

A recurring theme of this overview will be that embryogenic cells, whether free or in clusters, maintained or
regenerated under limiting, i.e. unoptimized conditions, give the appearance that embryos are being produced
indirectly. In retrospect, few would have imagined the extent of optimization that is necessary to disclose that
embryos always develop directly. One might even say that it was because culture environment has been Jess
than optimal for a number of reasons that I shall now address in greater detail that we came to the conclusion
in my laboratory that development is direct.

5. The 'New' or 'Reconstructed' Perspective

5.1 Day lily as a Model

It may initially seem peculiar that the daylily, a herbaceous, albeit perennial monocotyledon, Hemerocal/is
(Liliaceae), should figure in any discussion on somatic embryogenesis in a Volume in a series dedicated to the
process in woody plants. But the connection is not trivial. Monocotyledons like woody species have
historically been considered more or less recalcitrant to culture (Hunault, 1979; Cronauer and Krikorian, 1987;
Nambisan and Chopra, 1992). In retrospect there is no good explanation why this should be so but there is no
longer any reason to believe that monocotyledons are no less embryogenic than other plants (Ahloowalia and
Maretzki, 1983; Ammirato, 1983; Lu and Vasil, 1985; Ammirato 1986). And one can extend this to woody
plants. The volumes in this series indicates how far the work has come. In the early 1970s I decided to use
daylilies for my monocotyledonous culture work because daylily was then, and still is, the most widely used
perennial herbaceous plant in the USA. I also reasoned that it could provide insights for dealing with woody
perennial monocotyledons with a history of being very recalcitrant such as oil and coconut palm and bamboos
(see Krikorian, 1989, 1994).

Daylily suspensions have proven amenable to being rendered embryogenically unifonn, and staying so for very
long periods (order of at least several years), and slow enough in their growth rate to allow certain events in
somatic embryo development to be identified and studied (Krikorian and Kann, 1981; Krikorian et al., 1995).

The induction/maintenance phase was gradually optimized and re-optimized via medium design and
establishment and implementation of a precise transfer protocol using carefully sieved fractions so 'free' cells
and aggregates of the smallest possible dimension could be obtained. Similarly, the regeneration phase was
modified far beyond the more usual simple removal of auxin and/or adjustment of salts in the medium.

Optimized regeneration in daylily from cells has now been shown to be dependent not only on fastidiously
grown cells, but on a number of other factors. For instance, we often use a buffered but not necessarily totally
honnone-free medium in whlch the salts have been specially tailored, both for regeneration and maintenance.
We use a combination of both liquid and semi-solid environments. For regeneration we use a medium made
semi-solid medium \lith rigorously-washed agar. This in tum is overlaid with a rigorously washed activated
charcoal-impregnated filter paper (# 508 Schleicher and Schuell, Keene, NH). The charcoal paper is in tum
24

overlaid with a layer of washed dialysis membrane with particular pore dimensions (Spectra/Par Molecular
Porous Membrane, 6,000-8,000 molecular weight cut off. 32 mm diameter. 50 mm flat \\idth; 30-50 Fm thick
before autoclaving, Spectrum Medical Industries. Los Angeles. CA. USA).

5.2 The PEM Concept Needs Modification

All of our work supports the view that embryogenic cultures are already dctennined at the primary culture
stage, well before an embryogenic culture is perpetuated through subculture. While the bulk of our evidence
derives from daylily I do not doubt that what will be said is applicable in principle to all plants. Maintenance
culture conditions perpetuate a detennined state in the form of a proliferative collection of initials capable of
developing into somatic embryos. In liquid maintenance culture, suspensions are limited in their development
and so are constantly undergoing a 'forced regenerative polyembryony'. Gupta et al., (1987) have termed the
equivalent of this in gymnospermous cultures as 'somatic polyembryogenesis'. The initials normally do not
develop beyond a few divisions. Then, the newly formed cells 'separate'. In effect, cells within a very young
developing embryo are shifted out of their normal position. This leads to a detachment or fragmentation of the
units that comprise the embryo and in tum produces yet another embryogenic unit, which again may detach. A
repetitive embryogenesis is thus fostered and variously-sized polyembryonic fragments are produced. The
number and size of these embryogenic units varies with the species and the nature of the maintenance medium.
(More about this later.) (See Krikorian, 1996b for a discussion of the designation 'units' as it applies to cell
clusters in vitro. The term was borrowed from yeast geneticists, who interestingly enough, no longer use it.)

'Development', 'regeneration', 'advancement', call the process what you will, from the embryogenic units or
fragments thus ends up being dependent on a pennissive environment. So far as embryogenic suspensions are
concerned, somatic embryos are formed ouly as a consequence of the direct development of pre-existing
somatic embryo initials, be they in fragments or more rarely, in the form of free cells. PEMs do not exist
according to their more usual, classical, definition. Nothing has to occur in or to a cell in an entity called a
PEM to enable it (them) to become embryogenic. They already are somatic embryos, albeit in different degrees
of development and vigor. 'Classical' PEMs could be more accurately described as clusters of 'zygote
equivalents' or 'budding somatic embryo initials' each with a different capacity for further development. They
could equally well be called 'polyembryonic fragments'. Admittedly these terms are not brief but they project
the idea that they are already detennined and are not undifferentiated cells. This is not just a matter of
semantics. The term PEM comes with a lot of historical counter-productive baggage. It may well be umealistic
to suggest dropping the term it is a fact that established terms tend to remain in the scientific vocabulary even
if they are imprecise. The very term "tissue culture" persists because of widespread use despite the fact that it
has long been recognized that it is not strictly correct since they rarely are derived from or are comprised of
specific tissues (Bailey, 1943). But the connotation of a PEM should surely change. Just like a change in
charge on an iron molecule going from Fe++ to Fe+++, the valency must change in \~ew of our now improved
understanding of the events.

5.3 Analysis ofEmbryogenicity ofDaylily Suspension Components

To avoid the pitfall that many early investigators fell into by utilizing aliquots removed from a culture, and
then piecing together a presumed sequence of events, at Stony Brook the technique of following cells
immobilized in Gelrite was adopted. This allowed the relationship between developing somatic embryos to
specific cells and various cellular associations or fragments to be established.

Healthy daylily suspensions consist of cellular fragments or clusters with an irregular appearance. Larger
fragments differ only by size and cell number. There are far more fragments than single cells and we call them
'finely granular'. The cells are small, more or less isodiametric and densely cytoplasmic. This is especially
true of the most recently divided cells. Most absolutely free, single cells show some vacuolation. Highly
vacuolated cells are characteristically non-responsive.

The regenerative potential of all fractions separated into fairly narrow size ranges has been evaluated and re-
evaluated many times over the years. The developmental fate of each cell in some 1,054 fragments of varying
size was followed. These consisted of 579 cells clearly interpretable as absolutely single, 121 single units
which were multinucleate (more about these later), 351 two-celled fragments, 201 three-celled fragments, 115
four-celled fragments and 266 fragments or units containing more than four cells. In all cases, free cells and
cells contained in fragments formed somatic embryos directly. Although segmentation of the earliest stage
somatic embryos appeared to occur \ia internal divisions, observations of many developing embryos suggested
that their constituent cells were tightly adherent and partitioned by thin walls. This accounts for some 'single'
 25

embryo initials appearing multi-nucleated when in fact very thin walls separate the nuclei and their
surrounding cytoplasm. The very small cells merely have not expanded. According to the indirect model,
somatic embryo development from these small fragments or units would proceed from either the least
determined elements of the culture, or if determination had occurred. from the youngest stages of development.
If the process was indirect and from single cells. cluster formation would precede somatic embryo formation.
Also, somatic embryos arising from the small groups would be expected to arise from cenain cell(s) of the
clusters. None of this has been observed in our immobilized cultures.

In all of our evaluations, somatic embryos develop from all fractions. However, as the polyembryonic
fragments decrease in size, their ability to divide or develop diminishes dramatically. This has been
particularly true of isolated single cells. Halperin (1966) speculated long ago that the apparent inability of
single cells to give rise to embryos is probably due to the fact that few such cells di\~de in liquid suspensions.
While isolated single cells can give rise to somatic embryos, very few actually do (Toonen et al., 1994; Toonen
and deVries, 1997). Cultures can be prepared by filtration, settling, concentration .and re-sie,~ng and
combining cells from several equivalent flasks. But when absolutely single cells prepared without special
procedures such as concentration and filtration are evaluated, growth and ultimate somatic embryo
development is characteristically low. Initials either lose their identity when not part of a larger budding mass
of embryo initials, or they do not receive adequate nutrients or requisite growth factors. Equally possible is
that they cannot withstand insults meted out to them through a hostile culture en~ronrnent. The free cells are
simply too delicate and the e1_1~ronrnent too harsh. Conditioned medium has not been useful in my laboratory.

Even so, the high regeneration from our daylily cultures contrasts sharply to embryogenic cultures reported by
investigators using a very diverse range of species. Many embryogenic systems seem to contain relatively few
polyembryonic fragments. Typically they comprise a very small percent of the total number of cells (see e.g. de
Vries et al., 1988). One could argue that the indirect model has gained its greatest credibility from
observations on cells from cultures that are inadequately optimized. The ability to identify efficiently lack of
optimization and to target means to optimize will go a long way towards enabling somatic embryogenesis to
progress to a fully mature technology. I will only mention here in passing that the empiric de~ce of selecting
somatic embryos in carrot by their 'pearly white' appearance on the primary explant permits rapid
establishment of embryogenic cultures (cf. Krikorian, 1989 p. 127; Krikorian and Smith, 1992 p. 10; this
'trick' was pointed out to me many years ago by P. V. Anunirato when he was a graduate student). This is
because cells that are not already determined as somatic embryos are in large measure excluded from the
subsequent culture process . 'Husbanding' of such initials, although a slow process can lead to cultures with
high performance and stability over many years. Daylily does not give a signal as obvious as the 'pearly
white' somatic embryos of carrot, but one can with experience identify and select the earliest responses. The
prompt identification of the initial embryogenic response thus becomes critical and failure to do so has many
negative consequences since protracted exposure to unoptimized conditions leads to failure.

5 .4 Past Failures

Failure of earlier work in this laboratory on carrot (cf. e.g. Krikorian, 1982) and that of many others (Gray et
al., 1984) to disclose direct development from suspension components is in retrospect almost cenainly
attributable to use of inadequately optimized regeneration en~ronrnents. Growth regulators may have been
used at concentrations higher than necessary. We just did not know what fastidious optimization meant. In
the few cases of direct embryogenesis reported from single cells, an understanding of the nature of the cell(s)
being followed relative to development into a somatic embryo seems to be lacking. At best it has been under-
appreciated since no efforts to integrate such observations into a more accurate depiction of the process exists
(cf. e.g. Conger et al., 1983; Miura and Tabata 1986; Toonen et al.,1994; Wang et al., 1996; Toonen and de
Vries, 1997).

Maintenance under any sub-optimal condition, especially in liquid, is antagonistic to regeneration and thus any
attempt to follow direct advancement of initials is frustrated. Cultures are not only generally less responsive,
but they contain many somatic embryo initials that quickly lose the capacity to develop. But their proliferation
as true callus is not restrained. As and when this occurs, as it ine\~tably does in a culture being grown in
maintenance medium, somatic embryos develop only from those initials which still have the \~gor to do so.
Stated another way, largely non-embryogenic 'masses', albeit originated from determined initials, only contain
some 'embryogenic' initials. The smaller the components of an embryogenic culture, the more adversely
affected by maintenance conditions they are. Undoubtedly, non-optimized conditions will exacerbate this and
development preferentially occurs from larger more vigorous, or 'protected' units.
26

But somatic embryo initials may still develop even in a maintenance medium non-<lptimized from the
standpoint of being "too regenerative". These are distorted, developmentally-limited embryos. Development
probably proceeds from the most recently budded, least 'embryogenically degraded' or 'insulted' or 'stressed'
initials. These are the ones that are most unrestricted by the non-permissive conditions that exist during
relatively protracted maintenance culture. This observation ·explains' why it is that some somatic embryos
develop in staling maintenance medium. The 'non-permissive medium' becomes more permissive as hormone
levels become depleted, and certain nutrient relationships change, especially those involving nitrogen (see
later). A few considerably more 'normal' secondary embryos can develop in depleted maintenance medium, or
just after a transfer to regeneration medium. This may explain the kind of development described by Haccius
as indirect (Haccius, 1978). In that much-cited paper, embryo development from a 'callus derived'
embryogenic growth is described - this is her Proembryonal Cell Complex (Haccius, 1978). The 'cell
complex' can be explained equally well as a secondary embryo formed from among a group of somatic embryo
initials most of which had lost considerable vigor in a staled medium. Incidentally, expressions like
'rudimentary proembryonal cell complex' have found favor in some quarters and Halperin (1966) earlier had
referred to this exact kind of growth as a PEM. The matter of a suspensor also becomes clearer. Somatic
embryo initials that have budded and 'failed' can give the appearance of a suspensor (Haccius, 1965;
Kohlenbach, 1978) from which the embryo is developed. The question of a suspensor in pollen-derived
embryos was never as bothersome since embryogenesis there often occurs without extensiye budding (see e.g.
Sangwan and Sangwan, 1987; Palmer and Keller, 1997 and refs. there cited).

5.5. Liquid vs. Semi-Solid Culture Environments

When carrot 'embryogenesis principles' were applied by us to daylily, regeneration in hormone-free

regeneration which was otherwise optimized as to salts and pH could not be achieved. However, regeneration
did occur on this same medium when it was made semi-solid. Unlike carrot, neither could liquid-generated
cultures that developed on semi-solid medium develop when plated in it, even in a very thin layer. This made
it impossible to follow development of specific initials.

I have emphasized that when cells equivalent to those followed in regeneration medium are observed in
maintenance medium, direct development is limited and perturbed. Newly produced cells that would normally
compiise a developing embryo get physically displaced from their normal position. Such cells become the
'new' initials that 'attempt' reconstitution of the perturbed somatic embryo. However, being in a non-
permissive environment, once again the process repeats itself. Only rarely has unperturbed 'unitized'
development of such day lily cells been observed to progress beyond the several-cell stage.

Small cell groups form in maintenance medium into the larger ones typical of those in suspensions of a similar
age. Staining the nuclei of isolated cells and very small fragments with DAPI shows that same cell division
patterns consistent with early embryo development exist during maintenance growth. Similar activity in
single, isolated somatic embryo initials emphasizes that the same process is, in fact, occurring in clusters of
varying size. The presence of multinucleate somatic embryos in maintenance culture serves to support further
our interpretation that the entire suspension consists of clusters of buds in limited stages of development.

Multiple nuclei in seemingly 'single' cells emphasizes the ease of misinterpreting early stage somatic embryos
as 'undifferentiated single cells'. The underlying polyembryogenic process generally fosters the existence of
very compact multicellular embryos (very early globular stage) virtually the same size as single cells. The
possibility of such 'contamination' is important when evaluating claims of success in obtaining somatic
embryos from 'apparently' single cells. (It also means that sieving cells through screens becomes demanding
as well.) Initials from larger units (even a two-cell embryo fragment) divide or develop at a much higher
frequency than free, single cell initials. Our experience explains why free, single cells are such a small
component of suspensions and why they respond with such low frequency unless heroic optimization measures
are undertaken.

5.6 Regeneration under Limiting Conditions

5.6.1 Poor Morphology

Regeneration of embryos in liquid culture is desirable for practical reasons but liquid-regeneration in many
species is difficult to achieve. When it is, it is often associated with developmental abnormalities. The
common empirical practice of utilizing semi-solid medium for regeneration for at least part of the process can
be traced back to the first reports of the in vitro formation of somatic embryos in carrot (Steward et a!., 1958;
Steward ct al., 1964; Margara and Bouniols, 1967). Shoots seemingly did not form until units were removed
from liquid and placed on semi-solid medium. The interpretation at the time, now know to be erroneous. was
that some sort of gravity-related signal was necessary for shoot production. Since then, it has been noted that
development is almost always improved when semi-solid medium is used throughout or a semi-solid phase is
incorporated into liquid schemes (Cousson and Tran Thanh Van, 1981; Tran Thanh Van, 1981; Krikorian.
1982; Daguin and Letouze,l988; Cronauer-Mitra and Krikorian. 1988; Etienne et al., 1993; Alvard et al..
1993; Dumet et al .. 1994; D"uval et al., 1995; Teisson and A1vard. 1995; Krikorian, 1996c; Cabasson et al ..
1997; Tahardi, 1999). Comparison of daylily with carrot and other systems we have worked with indicates
that daylily is more sensitive and does not 'like' liquid culture. The woody plant literature presents a range of
sensitivities as well.

5. 7 The 'Auxoph)ton'

Having said that many embryogenic systems do not 'like' liquid culture, a slight detour may be in order here.
This is because of the distinctive way we grow cells in liquid at Stony Brook. After World War II Steward and
Caplin designed an apparatus, shortly afterwards named an 'auxoph)ton', to rotate specially designed culture
tubes around a horizontal axis. The carefully contrived tumbling action imposed on the vessel contents permits
alternate bathing in liquid nutrient and exposure to air (see Steward et al., 1952). Cells are less o:-;ygen-limited
and water-logged than those that are submerged in liquid as in Erlenmeyer flasks on a rotary or reciprocal
shaker. The 'auxophyton' is not like the roller tube apparatus that animal culture workers generally use for
large scale culture of attached cells. These date back to a suggestion by the animal tissue culture pioneer
Alexis Carrel in 1913 and cells are continuously bathed in fluid (Gey, 1933).

Variations of the 'auxophyton' were soon adopted for it was appreciated that aeration was increased even as
cells were less waterlogged. Growth was much enhanced over that normally achieved on semi-solid media.
But all the imitations did not necessarily produce an equivalent apparatus. For example, in their early cell
culture work Riker and his group at Wisconsin used a "drum aerator that was "a modification of the well
known rotary tube cultures and auxophyton described by Caplin and Steward (I 949)" (Muir et al., 1958).
Their roller apparatus held culture flasks horizontally but tissues were continuously bathed. Roller tubes used
in orchid micropropagation likewise permit continuous bathing of explants (Arditti and Krikorian, 1996).

I have been surprised to see the 'auxophyton' being used in various laboratories throughout the world but its
use is largely used by his former students and associates. Although I have never validated that use of an
'auxophyton' along with its distinctive 'nipple' culture flask yields finer and more responsive embryogenic
suspensions, I suspect that this could be shown. I believe that the 'auxophyton' has contributed much to our
being able to grow and maintain cells fastidiously.

Liquid versus semi-solid in the context of an indirect model has, in the past, suggested that transferral to semi-
solid medium as a required treatment to somehow foster or even trigger development from embryogenic
clusters. It was even speculated years ago that 'sloshing' around in a culture flask could provide competent
cells a stimulus to somatic embryogenesis. Indeed, close examination of cells at the air-liquid interface of
culture vessels indicates that selective culturing that most workers ignore, not just those involving somatic
embryogenesis, can occur (Ball and Joshi, 1965). Semi-solid medium can thus be a permissive modification.
If development in liquid was not absolutely restrictive, somatic embryos so derived would provide further
evidence of a permissive process. Thus, daylily regeneration in liquid was not only desired for practical
reasons but because it would further support that embryogenic cultures are comprised of already-determined
polyembryonic fragments.

It has taken time to learn how to 'quality control' the factors important to permissiveness. When done ex post
facto, comparisons can be made if they are appropriately modified. Daylily somatic embryos can, in fact, be
regenerated in liquid but while the quality of the embryos is acceptable, they are not of the highest quality.
Our early attempts to generate readily identifiable somatic embryos in liquid failed because the proper pH was
not maintained through buffering. Even so, development is perturbed when initials develop in liquid under
even the best of optimization protocols. Cotyledon and shoot development is generally diminished but root
development is pronounced. Epidermal development is perturbed and the apical depressions representing the
site of the future shoot are only minimally developed in liquid-derived embryos, even when they are nominally
'mature'. When serially cultured in liquid, virtually all late-stage globular embryos develop into 'rooty'
embryos. In some, apical depressions do become evident after further growth and elongation.

By contrast, normal embryos always develop from equivalent initials on optimized semi-solid regeneration
28
medium. A prominent depression at the shoot pole is always apparent. The embi)'OS have a well-defined
rounded or oblong shape, a high quality epidermis and form a cotyledon typified by an overarching and
expansion of a ridge-like gro\\th around the apical meristem. As the plumule begins to emerge it is visible as a
slight bulge in the center of the apical depression. As is the case in normal zygotic embryos of daylily. shoot
emergence always precedes root.

5.8 'Neomorphs'

The liquid-derived 'rooty' somatic embryos of day lily are very similar if not identical to what we referred to as
'neomorphs'(Krikorian and Kann, 1981). There is a large 'literature' on neomorphs but it has not been
specifically referred to as soch. It reflects the many instances in which poor quality embryos have been
obtained. The reports have all been phenomenological and mostly ignored. One common type of abnormality
involves the lack of apical meristerns. This is especially so in the case of the stem tip or, if present, in the
branches of regenerated structures (see e.g. Wadhi and Mohan Ram, 1964; Durzan et al., 1973; Durzan, 1982,
1984; Narayanaswamy and George, 1974; Favre, 1977; Seckinger et al. 1979; Price and Smith, 1979; Nakano
and Maeda, 1979; Kononowicz et al., 1984; Jarrett et al., 1985; Li et al., 1985; Chee and Cantliffe. 1988;
Padmaja et al., 1990).

Other manifestations of aberrancy other than the absence of shoot or root apices, the so-called "half embryos,
i.e. root or shoot primordia" of Thorpe (1995 p. 32), include fasciation, multiplicity of structures including
diverse kinds of organ fusions, adnations and twinning or production of even higher multiples of either or both
ends of the primary axis such as polycotyledony and multiple-rootedness (cf. e.g. Ammirato, 1987). Any of
these kinds of abnormalities, when present to a greater or lesser extent, qualify to be called neomorphs (see e.g.
Waris, 1959; Johri and Sehgal, 1965; Narayanaswamy and George, 1974; Krikorian and Kann, 1981;
Christianson et al., 1983; Rangaswamy, 1986; Smith and Krikorian, 1991; Litz and Gray, 1992 p. 4). For me
'neomorphs' also include structures that are embryos so severely "vitrified" or "hyper-hydric" as to be aberrant.
These may or may not be true developmental 'dead-ends'.

"Neomorph" was originally applied by Waris in 1957 to refer to experimentally-induced morphological off-
types from cells sloughed off seedlings grown for prolonged periods in liquid culture containing non-
physiological levels of various amino acids (see Waris, 1962; Krikorian and Simola, 1999). Neomorphosis was
defined as a "fundamental morphological change, without any irreversible change of the genome .... " I
adopted the term neomorph in an attempt to codify in vitro-generated embryonal forms of daylily that seemed
terminally aberrant and unable to yield plantlets directly (Krikorian and Kann, 1981; Smith and Krikorian,
1991). It was, however, possible to obtain plantlets from them provided fresh cultures were re-established.

Our neomorph work also emphasized that unlike the case of the in vitro glycine- and other amino acid-
challenged seedlings of the umbellifer Oenanthe aquatica which Waris and Miettinen worked with (Miettinen
and Waris,l958; Waris, 1959, 1962), there could be a direct influence of basal nutrient medium components
other than amino acid or hormones on morphogenesis of cultured cells (Krikorian and Kann, 1981;
Christianson et al., 1983; Kevers et al., 1984). Moreover, the reversibility of the "neomorphosis" showed that
epigenetic changes could occur with or without exogenously added growth regulators (Halperin, 1966;
Krikorian and Kann, 1981; Smith and Krikorian, 1991; Nickle and Yeung, 1993).

Over the years, we have encountered various kinds of neomorphs in embryogenic carrot, daylily, banana and
plantains, Para rubber, citrus and opium poppy cultures --in every embryogenic system we have worked with.
Although they usually comprised a small percentage of undesirable growth forms among otherwise successful
regenerants, their presence has provided clues in our attempts to dissect the embryogenic process. In our first
embryogenic cultures of plantain they were predominant (Cronauer and Krikorian, 1983).

When we first encountered organized structures in daylily suspensions, I reserved judgement as to their being
embryos and ambiguously called them 'embryonal forms'. We decided that we would merely refer to them as
'pre-shoots', admittedly a very unorthodox term but nevertheless a convenient one (Krikorian and Kann, 1981;
Krikorian et al., 1986, 1988b). 'Pre-shoots' and some neomorphs were obtained after an involved culture
process using hormones in the regeneration medium. As Waris had implied in his definition, the 'neomorphic
cells' were not permanently altered genetically since upon re-establishment of fresh 'callus' cultures on semi-
solid medium, normal plants could be obtained.

The identity of rooty forms as aberrant somatic embryos has now been demonstrated by several strategies by
Weidenfeld in this laboratory. The most convincing confirmation of 'pre-shoots' as somatic embryos has been
 29

through 'rescue' of liquid-derived structures on semi-solid medium. If before transfer to semi-solid

regeneration medium, somatic embryo development in liquid has occurred for a period equal to that required
for mature somatic embryos to develop after plating on optimized semi-solid regeneration medium
(approximately 20 days), most somatic embryos develop normally. They are globular and vary in size but some
have already developed into 'rooty' structures. Thus material with varying levels of root development is
conveniently available for testing. And others which val)' in terms of the development of areas distal to the
root (the shoot apical end) can be tested as well. These latter forms presumably have the best chance for
continued development. After semi-solid 'rescue', practically all of the globular, liquid-derived embryos
develop into rooty forms that 'attempt' to and in some cases 'succeed' in yielding plantlets. These plantlets
develop, however, somewhat abnormally. 'Rooty' forms can produce shoots but they are not robust. Even so,
the percentage ofliquid-derived late stage (globular) somatic embryos that give rise to plantlets is still vel)' low
(some 2-4%) compared to control embryos which develop on semi-solid medium (order of 80 to 90% or even
higher).

Significantly, structures with only root development (in laboratory jargon the 'no heads') are generally not able
to form plantlets after transfer to semi-solid. Rescue of 'rooty' embryos which can retain embryo structure
yield plantlets with small normally-positioned shoots. In one series of experiments, some sixteen percent of
the best-developed rooty forms and 10% of the more prevalent types formed plantlets after semi-solid rescue.
E>.1ensive studies have not been carried out to establish relative vigor of the forms in terms of ability to yield
plants in soil because the primary objective of this kind of experimentation has been to confirm the identity of
the liquid-derived embryos. But it must be recalled that most of the plantlets which form from liquid-derived
embryos have poor initial shoot development.

In one kind of experiment, efforts were made to see whether increased osmoticum in the form of higher sucrose
levels (up to 7.0% w/v) could simulate in liquid the favorable water relations that exist during semi-solid
regeneration. Significant improvements of the type found in some systems (see e.g Ammirato and Steward,
1971; Leva and Muleo,1993; Etienne et al., 1993) were not found.

The ability of growth regulators to overcome the limited shoot development in liquid medium even though it is
formulated for regeneration was evaluated. Zeatin has long been appreciated for its ability to stimulate shoot
development (see e.g. Anunirato, 1977). Abscisic acid can be useful in managing embryogenic systems since it
fosters maturation and can reduce abnormalities (Anunirato, 1983). In one series of experiment, both were
added, singly and in combination to liquid regeneration medium for a period corresponding to the time needed
for a somatic embryo initial to develop into a late stage somatic embryo (approximately 20 days). The
regulators were then removed for an additional 20 days of growth. If high fidelity somatic embryos and/or
plantlets did not form by the end of the second liquid period, the structures so formed were transferred to semi-
solid medium with the intent of 'rescuing' them. Not unexpectedly, in zeatin cultures, there was more embryo
structure distal to the root pole. This resulted in larger rounded or 'tear-drop shaped' structures which are
produced as the shoot emerges from the rounded body of a somatic embryo in the area directly opposite the
well-developed root. Embryos without shoots also show greening in the 'head' end, i.e. the presumptive shoot
apex area. Greening of the leaves in embryos that form shoots is apparent. Common types are those with
rounded bodies which are larger than the controls and those with small tear-drop shaped axes. Normal
production of adventitious roots typical of those in normal daylily somatic (and zygotic as the primal)· root in
daylily is ephemeral) embryos commouly occurs leading to complete plantlets. They are also more normal in
the status of their epidermis, more vigorous and sometimes have multiple shoots. Embryos developed in ABA
are still largely rooty forms, similar to those obtained in hormone-free controls. They are, however,
characteristically affected by ABA to the extent that elongation occurs distal to the rooty structure, i.e. at the
'incipient shoot end'. Plantlet recovery after semi-solid 'rescue' has routinely been some 10% of the common
'rooty' forms taken from the control (i.e. the 'no hormone') cultures, approximately 20% from the ABA
cultures and approximately 40% from the zeatin cultures, as opposed to the more normal 80 to 90% 'controls'
from liquid-derived embryo initials cultured on semi-solid medium.

One explanation for rooty development may be as follows. Schiavone (1988) showed that root elongation
which occurred after bean and torpedo stage carrot somatic embryos were cut at the bean and torpedo stage
into apical and basal halves was controlled by either the polar transport of auxin or the accumulation of auxin
at the root end. In carrot, polar transport of auxin apparently occurs as early as the globular stage (Schiavone
and Cook, 1987). Poor shoot and/or vascular development in liquid-derived daylily embryos might be
similarly based.

6. Optimization of Regeneration by Auxin Removal, Transferraf to Semi-Solid and Use of Other

30

Modifications

We have seen that our working hypothesis has been that any modifications required to achieve or optimize
regeneration must act permissively, and lack of adequate optimization results in poor performance both in
numbers and morphological quality. And that semi-solid medium for regeneration can be a permissive
modification. The value of adjusting the salts, maintaining an optimal pH through buffering with MES and use
of activated charcoal-impregnated filter paper and dialysis membrane additions have also been investigated
singly and in combination in my laboratory over many years. Embryogenic initials are predictably influenced
by controlling factors like pH, media salts and the presence and absence of inhibitors. These modifications do
not induce or trigger the production of somatic embryos from classical PEMs.

All this derives from results from an assay which scores the number of suspension fragments that yield somatic
embryos under various environmental conditions. Thus, additional support that daylily cultures are uniformly
composed of polyembryonic fragments derives from the high responses scored when regeneration conditions
are optimal. This, in turn, validates that the cultures are uniform and highly responsive. The conclusion is
that only role of auxin in established embryogenic cultures is that of a growth factor fostering growth of
determined, very immature embryos. Removal or reduction of auxin is only one of many possible and
important permissive modifications. Indirect evidence of this will be presented here relative to effects of low
pH on hormone-free cultures under maintenance conditions (Smith and Krikorian, 1991).

The empirically-derived optimized conditions for daylily regeneration are a composite, refinement and
extension of modifications initially developed to improve carrot somatic embryo regeneration (see Smith and
Krikorian, 1990). The 'final' protocol consists of inoculating onto a dialysis membrane, which in turn is
placed on an activated charcoal impregnated filter paper directly in contact with a buffered, washed agar-
solidified regeneration medium (usually in 100 x 15 mm plastic petri dishes). Regeneration medium is a
buffered hormone-free variant of the maintenance medium but the concentration of many of the components
(most notably nitrogen) is reduced. A comparison of the maintenance culture medium (MS medium) and the
regenerative culture medium (DS medium) is shown in Table 1.

Table 1. Comparison of the components of the media used for 'induction' and 'maintenance' of daylily
suspension cultures (MS medium) with the medium used for somatic embryo 'regeneration' (DS medium).
The maintenance/induction medium is comprised of the salts and vitamins of Murashige and Skoog
(Murashige and Skoog, 1962) and contains the additions specified below the table. The regenerative medium
contains DS 5a nitrogen-free salts (Smith and Krikorian, 1989) plus 0.5 mM KNO,·, 5 mM NH.,Cl and the
additions specified at the end of the Table. Both media contain 100 FM FeEDTA (madefrom 100 FM [35.4
mgll] Na 2EDTA H20 and 100 FM [27.8 mgll) FeSO, 7 H2 0 as in Singh and Krikorian, 1980).

Medium component MS (mM) DS 5a (mM)

NH.,T 20.60 5.00

No,· 39.40 0.50

ca- 3.00 1.00

K+ 20.06 .005 (2-5 mM when pH adjusted)

Mg++ 1.50 1.00

S04" 1.73 1.21

Na+ 0.20 0.70

EDTA. 0.10 0.10

H2P04 1.25 2.00

cr 6.00 3.00

Mn++ 0.10 0.10

Fe++ 0.10 0.10

 31

Bo3- 0.10 0.10

co- 0.0001 0.0001
cu- 0.0001 0.0001
zn- O.oJ 0.01

r 0.005 0.005

Moo.- 0.001 0.001

..
Other addiuons:
MS hormone addition= 2,4-D 2mg/l ; kinetin lmg/1
MS vitamin addition = .3 mg/1 glycine, .05mgll nicotinic acid, .01 mg/1 thiamine HCl, .01 mg/1 pyridoxine
HCI.
DS vitamin addition= .01 thiamine HCl
DS buffer= lOmM MES.
lOOmg/1 inositol is added to the MS medium.
Carbon source = 3. 0 % sucrose.
pH= 5.8 adjusted

Rigorous washing of the agar is as critical to success as the nse of acti:vated charcoal paper and of dialysis
membrane. Along the way we also adopted yet another procedure, long in nse by microbiologist but rarely
mentioned in protocols by tissue culturists, involving the 'curing' of petri dishes. 'Curing' allows them to
harden and to lose moisture. The extent of 'curing' can have a very significant effect on regeneration of
daylily, and no doubt on many systems.

Results of experiments carried out repeatedly over many years emphasize the roles of the opturuzations. The
benefits attributable to each modification now correlates very well with the observations that led to their
adoption in the first instance. For instance, the inoculation technique utilized at the outset of our work was as
follows. Acceptable dilutions of sieved suspension fractions from which the inoculum were prepared had been
found to be in the 1:30 - 1:10 settled mass/volume range. A one ml plastic disposable pipet was used to
inoculate the smaller fractions and a wide mouth 10 ml glass pipet was used to inoculate larger fractions. The
inoculum was applied in the form of drops. The volume plated (usually 0.2-0.3 ml), the height and angle of
release, and the change in unit concentration determined by their settling before plating all contributed
significantly to the art of consistently getting a good response.

Routine dialysis membrane use began only after noting much-improved growth on them. Porous membrane
had initially been adopted to facilitate transferring, recovering and weighing the growth associated with
somatic embryo development. Growth on membrane also enabled chromosome studies on specific units to be
carried out with greater ease. To improve overall embryo quality, activated charcoal was introduced in the
form of activated charcoal-impregnated filter papers since powdered charcoal added to the medium for this
purpose was not very effective (see e.g. Smith and Krikorian, 1990). At that time the utility was thought likely
to reside in its ability to adsoJb inhibitory compounds released by the cultured cells. This seemed a reasonable
assumption because the medium was presumed to be inhibitor-free due to rigorous washing of its components.
The possibility that inhibitors arose during autoclaving was not considered a realistic possibility since similar
results were obtained when filter-sterilized medium was used. All the evidence pointed to the value of
activated charcoal and membrane medium additions as countermeasures to some agar-associated inhibitor(s).

6.1. Plating Protocols

In the bioassay control dishes are normally inoculated with samples prepared from a 100 to 200 (> 73 Fm #
140 Fm) fraction. The reasons why this fraction has been preferred are many. A 'low dilution' (1:40 settled
cell volume/medium) and a 'high dilution' (order of 1:1000 settled cell volume/medium) stock can be used in
the form of only 0.2 mi. For daylily, this dilution and volume are much more important than one might
suppose. Positive scoring is based on the appearance of easily recognizable somatic embryos which as judged
from experience have potential for further growth and germination. The scoring is more or less lenient. This is
because gradations in morphology cannot be easily scored without considerable labor. If the assay is to be
rendered useful for broad studies, it has to have some flexibility and broad trends that can be validated are what
one is looking for at the outset of work with a new system.
32

Reasonable responses are counted such that the quantitative score is as objective as possible. Responses from
the 'low dilution' dishes have been mostly only used for general quantitative assessment. Responses from the
'high dilution' are carefully counted to quantify the effects of various environmental conditions on somatic
embryo regeneration. The 'high dilution' dishes are prepared such that some 100 to 200 fragments are
contained in a 0.2 ml inoculum. The number offragments in each high dilution inoculum are determined by
directly counting the fragments in five 0.2 ml samples as viewed under an invened microscope. The 100 to
200 size fragments are too large and too sticky to be counted using a hemocytometer. Dishes are streak-
inoculated (unless a membrane is present in which case inoculum is allowed to spread out and distribute itself
naturally). The inoculum is normally spread in a rectangle of similar dimensions to the membrane.
Suspension fragments are thus fully and individually exposed to available medium. Also, inoculum applied in
this manner prevents a density-inhibited response.

When inoculum is plated in the form of drops, the fragments aggregate and pile up such that development is
always adversely effected. This has long been encountered in our studies but it is now better appreciated that
this is due to the progression of many initials in a limited space. When the response is density-inhibited in this
way, somatic embryos undergo repetitive budding and form masses of small, very poorly-formed embryos.
Development of multiple initials contained in each fragment inevitably results in an unavoidable density
inhibition. Thus, the commonly encountered production of multiple embryos is due to the effects of the 'forced
regenerative polyembryony'. Another imponant effect of a drop inoculum is that the embryo initials aggregate
and pile up. The initials contained in such fragments that achieve some separation from the agar surface
preferentially progress. Thus, the advantages of drop plating on agar become better appreciated. If the
inoculum density is too high, so much so that it leads to a physical elevation of the somatic embryo initials,
distinctions between growth conditions may be especially difficult to observe. If examined carefully some
variation in quality is always apparent.

6.2 Agar Quality

Agar has historically been the preferred gelling agent for semi-solid tissue culture media. In this laboratory,
agar has long been the preferred agent to regenerate somatic embryos. Most routine culture work has generally
used a food and pharmaceutical grade agar as specified in the U. S. National Formulary (the N.F.). This
reasoilably-priced agar is routinely washed (10 times in 40 volumes of deionized water and 10 times in
deionized, glass-distilled water) and used at 1.2% w/v. This agar has been purchased in relatively large lots
and its qnality after washing has always been judged at least equal to, and usually better than, much more
expensive agars (including those supposedly of the highest purity and hence which are very costly). Also,
petri dishes made with the same lot of high qnality agar have been routinely included as a positive control for
the many batches of laboratory washed agar that were used. This kind of work has shown through bioassay
that when one compares regeneration dishes containing 1.2% (w/v) laboratory washed N.F. grade agar, N.F.
agar plus a membrane and N.F. agar plus a washed activated charcoal-impregnated filter paper and a
membrane, the dishes with all the modifications give the best results.

Comparisons between washed and unwashed N.F. agar have shown that both are extremely inhibitory to small
somatic embryo initials and even high grade Noble agar is very inhibitory when assessed in our assay. Embryo
quality on agar is diminished. Many are irregularly shaped and the shoot apical region is poorly developed.
Level and quality of respoilse are improved substantially when a dialysis membrane restricts contact of the
inoculum with the agar surface. But compared to a fully optimized plate containing a charcoal paper and
membrane, the embryos are less distinctly contoured and not as many show clear signs of advanced
development.

The conclusion is that inhibition from the agar is not fully ameliorated in such cases. In support of this is that
application of a dialysis membrane to an unwashed N .F. agar surface does not significantly 'rescue'
responsiveness. But activated charcoal-impregnated paper and membrane additions do yield high rates of
rescue. This situation has obtained in all tests aimed at identifYing the optimal conditions for high quality
responses. In other experiments it has been shown that response rates greater than 90% can be obtained with
powdered charcoal. We were initially surprised to see that the addition of a charcoal paper and a membrane
can dramatically rescue embryo responsiveness to unwashed N.F. agar. Although the quality of the embryos
are almost as good as the same condition with washed agar, to choose this condition, thereby eliminating the
tedious task of washing and drying agar, would be to risk toxicity. There may be, moreover, problems with
subsequent germination are encountered when agar is used without the modifications just described.

Comparisons between washed and unwashed 'Noble' agar without additions suggested that our standard
 33

washing was not completely cleaning the agar. In fact, the washed Noble agar gives slightly less positive
response than the unwashed Noble suggesting the presence of a gro'l'th promoting factor in the unwashed.
Also. when agars were first compared, it seemed that it might be better to use high grade, high cost washed
Noble agar. We have since convinced ourselves that the embryo quality on Noble agar, even with all the
optimizations described, is somewhat less than that with our cheaper washed N.F. agar. Activated charcoal
thus adsorbs an inhibitor(s) present in the agar and the use of dialysis membrane acts as a micro-porous
support which distances embryo initials from the agar.

Significant 'rescue' also occurs when the petri dishes are outfitted with a charcoal filter paper but no
membrane. However, the rescue is significantly less and more variable than that achieved with a charcoal
paper and a membrane. (The effects of using powdered charcoal and charcoal filter papers will be discussed
shortly.) To determine if the inhibition we detected was common to all agars, a wide range of commercial
agars and agaroses were tested using the bioassay assay for regenerative responsiveness. It will be known that
agarose is a purer form of agar and is neutrally charged. It was theorized that agarose may be cleaner and that
inhibition by agar may be due to the negativity charged sulphate groups present on some of its components. In
addition, Gelrite the common non-agar gelling agent used so ex1ensively in our laboratory for other kinds of
work, and in that of many others was tested. (Gelrite is a microbial gum, a fermentation product of
Pseudomonas species, Shungu et al., 1983).

Most of the agars and agaroses were tested unwashed and after they were extensively washed. Although
washing improves the responses on some, no responses have been higher than on washed N.F. agar. Thus, all
agars and agaroses are inhibitory using our scoring measures. Gelrite, however, does not inhibit
responsiveness. The response is high and similar to that on agar dishes containing a charcoal paper and a
membrane. But, although the responses are good, the embryos are inevitably very hyperhydric and show all
the objectionable characteristics encountered in liquid-derived somatic embryos.

Agar was not anticipated as being anywhere near as inhibitory as it has turned out to be when compared to
Gelrite. I was aware of situations where Gelrite was supposedly pivotal to success, Huang and Chi ( 1988) but I
attributed the results to unwashed 'control' agar. Because of our washing agar and also because our dishes
were normally drop-inoculated with a relatively highly concentrated inoculum, we never anticipated such great
differences. In addition, since development of daylily in liquid had not been achieved at the high level that
was ultimately achieved, there was little hint that this single change in the medium could mean that agar was
inhibitory. Incidentally, Gelrite does not inhibit growth while all the seaweed-origin extracts we have tested
such as agar, various agaroses, alginates and carrageenans have proven inhibitory to regeneration even after
extensive washing. Gelatin, which of course of animal origin is, likewise inhibitory.

6.2.1 Nature of the Inhibitor(s) in Agar

Efforts have been made to determine the amount of agar required to inhibit regeneration and thus gain some
insight into the nature of the inhibitor(s). For instance, somatic embryos have been regenerated on dishes in
which the gelling agent consisted of increasing amounts of washed N.F. grade agar added to 0.2% wlv Gelrite.
Also, the nature of the suspected inhibitor was investigated by determining whether the inhibitor(s) is
retrievable in water released after the freeze-thawing of rigorously washed N.F. grade agar. The additions of
washed N.F. grade agar to the Gelrite dishes yield decreased responsiveness in a dose-dependent fashion.
Responsiveness declines sharply when as little as 0.1% w/v unwashed N.F. agar is added. In the experiments
where extract is added to Gelrite, decreased responses are also encountered in a dose-dependent fashion.
Equally interesting is that the morphological 'profile' and extent of embryogenic response is similar to that
obtained when the quantity of agar extracted is compared to the amount of agar that is added. Embryos that do
develop are smaller, less developed and give the appearance of sloughing off material, that is the response is
not 'clean'.

Such experiments disclose why regenerative responses are poorer after inoculation on an agar-solidified
medium which is either too 'wet' due to its preparation (not allowed to cure), or too 'wet' due to an excess
volume added in the inoculum when compared to 'drier', and hence more optimized controls. A likely
e:>.."Jllanation for 'sub-standard' regeneration on a 'wet' agar surface is the enhanced e:>.."JlOSure of the
embryogenic initials to a soluble inhibitor. The use of activated charcoal-impregnated filter papers and/or
membranes also counteracts the use of plating techniques that lead to wet dishes.

To provide further evidence that confirms that an inhibitor is present in the agar we have reasoned that a low
concentration of washed powdered activated charcoal should enhance rescue. Improved responses do indeed
34

occur when the semi-solid agar medium contains powdered activated charcoal. The lowest concentration of
this charcoal required to eliminate inhibition (tested without a membrane) is somewhere between 0.01% and
0.1% w/v. Although high responses can be obtained on media modified with the powdered activated charcoal.
direct contact with it perturbs development and fosters repetitive cycling of developing embryos. This has also
been noted when powdered charcoal is used instead of charcoal-impregnated papers to improve regeneration
in carrot (see e.g. Smith and Krikorian, 1990).

The response with the commercially available, activated charcoal-impregnated filter paper used in the Stony
Brook laboratory to optimize regeneration has been unique in that while the responses are not as high nor the
quality as good as when charcoal filter paper and a dialysis membrane are used together, the detrimental
effects of the charcoal are much reduced. Presumably this has something to do with the undisclosed
procedures used to make the product, which incidentally is used to monitor pollutants in water supplies.
Whatever the features of the activated charcoal-impregnated paper may be, these apparently serve to limit
exposure of the embryogenic initials to the charcoal. At any rate, overall the activated charcoal-impregnated
paper is not as effective in absorbing the inhibitor as is powdered charcoal.

One difference that we have looked into is that finely powdered activated charcoal is present during
autoclaving. To eliminate deleterious effects of direct contact with powdered charcoal, tests have been made in
which a membrane has been added to the regeneration dishes containing washed, powdered, activated
charcoal. Then, the responses are even higher than when the charcoal filter paper and membrane are used
(generally > 90%). The adverse effects of direct contact with charcoal are virtually eliminated. Higher
responses here may also be attributed to greater contact with the medium.

To support further the hypothesis that the primary benefit of charcoal and membrane additions to regeneration
media are to diminish the effects of an agar-associated or -derived inhibitor, more effort has been made to
clean the N.F. grade agar beyond our long-routine process of exhaustive washing with water. Agar has been
prepared by 'bleaching' with 10% (w/v) calcium hypochlorite for l h. The hypochlorite treatment is carried
out after 10 washes with deionized water. After 'bleaching', the now 'white' agar is washed with lO washes of
deionized water and 10 washes of glass distilled water. Bleaching of agar significantly, but still far from
completely, reduces its inhibitory effect on the development of somatic embryo initials. The positive effect is
most dramatic when the cultured material is in an absolutely optimal state to progress at mid-culture. This has
been determined by repeating this kind of experiment with inoculum taken at various points during the
maintenance culture period. Thus, it turns out that the status of the cells at different periods after maintenance
transfer is at least one factor that contributes to the kind of variability observed by virtually all workers between
and among 'tissue culture' experiments. We now, however, have a much better understanding of why a
considerably greater variability is encountered in the least optimized environments. This also emphasizes that
although one tries to repeat experiments under the very same conditions, it is not simple to achieve the exact
same conditions.

6.3 pH and Buffering

In daylily, and in the other systems that have been used in this laboratory, the regeneration medium has
inevitably been a buffered but 'weaker' variant of the maintenance formulation. The form and concentration of
nitrogen (Smith and Krikorian, 1989) has been shown to be the most significant and important adjustment so
far as the salts are concerned (see Table 1). In daylily, reduction of potassium from 20 mM to 2 mM, reduction
of calcium from 3.0 mM to 1.0 mM, and an increase in phosphorus from 1.25 mM to 2.0 mM are beneficial
but not absolutely required. Our carrot work (and to a lesser extent with daylily) has shown that vitamins or
inositol do not affect regeneration whatever. As part of the more extensive work with daylily a range of
nitrogen concentrations and forms has been tested in the bioassay. Because responsiveness has routinely been
'fair to good' for most of the tested nitrogen values, the numbers of responses obtained did not at the outset of
adequately describe the effects of these treatments. Most differences were better described qualitatively. The
form of nitrogen and the concentration (mM) is represented as a ratio between nitrate and ammonium,
specifically KN03 :NR,CI. In maintenance medium, the nitrogen is N = 40:20. If only the results indicating
very low response were considered, the poor conditions would beN= 40:0 and 40:20. When the quality of the
response was considered N = 5:0, 10:0, 20:0 must be included. Thus, the least permissive conditions are those
in which nitrogen is 'all' nitrate, or too 'high' in nitrate. The more finely tuned nitrogen relationships, in the
context of the other DS medium salts (see Table 1), are N = 0.5:5, 0:5, 2.5:2.5, 5:5, and 10:10. When dishes
made with buffered MS salts (N = 40:20) are tested, embryo production and quality are much better than DS
medium with identical nitrogen but there is a tendency to 'callus'.
 35

Somatic embryos have repeatedly been regenerated in hormone-free maintenance medium (MS) with its pH
adjusted and pH buffered to 5.8 and in regeneration medium (DS medium) pH buffered to 5.8 to demonstrate
the importance of salt correction and medium buffering for optimal regeneration. The effects of medium salts
and pH are more dramatic in liquid although similar effects are encountered in semi-solid medium. Embryo
regeneration is improved when the maintenance salts (MS salts) are adjusted to the regeneration medium salts
(DS salts) and when the pH is stabilized. The progression of embryo initials in hormone-free maintenance
medium is hardly discernible when these two factors are not corrected. Initials do not develop vel)' far in this
environment; even the stage at which development is arrested is imperfect. Growth is 'loose', and for lack of a
better descriptor 'bumpy'. A regenerative response of this type is described may also be described as
'callusey'. Regeneration is much improved when the maintenance medium is buffered but the embryo quality
is still not good. Embryos are translucent, rather hyperhydric, and still have a callus-like appearance. In
buffered liqu.id regeneration (DS) medium, initials develop into typical liquid-derived embryos but they are
larger, densely and opaquely white, and are not 'callusey'. Liqu.id embryos still have a disordered epidermis
and develop mainly into 'rooty' embryos. Hence, the embryos that are most likely to develop normally into
plantlets are those produced on the semi-solid regeneration medium.

The importance of pH for regeneration (DS medium) cannot be over-emphasized. Regeneration on variously
buffered agar dishes to which a dialysis membrane is added (recall that agar alone inhibits growth and because
activated charcoal, which is a component of fully optimized dishes, raises the pH) leads to some interesting
results. Response is only fair on agar with only a membrane addition. The pH optimum for embryo number
and quality is pH 5.8. Morphologically recognizable embryos which can germinate form on dishes below pH
5.5 (4.5-5.5) and to some extent at pH 5.5 but they are far less developed and are irregular. Also, as
development is generally perturbed, embryos show secondary embryogenesis. Low pH fosters budding and
new buds adopt the same habit. It is precisely in this environment that low pH hormone-free maintenance
cultures can be established (Smith and Krikorian, 1990, 1991). At low pH there is little, but still adequate
growth, and very little organization. Responses decline beyond pH 5.8 but at pH 6.0 and 6.5 some of the
initials advance in an uncrowded environment. This reduction in density-inhibition results in the development
of larger embryos. Responsiveness and quality continue to decline at pH 7.0 and 7.5 and embryo development
becomes very limited.

6.4 L6w pH and PGSPs

Because stock maintenance is labor-intensive we tried in my laboratory to maintain some less-used cultures by
transferring aliquots from embryogenic suspensions to the surface of a low pH, hortl)one-free semi-solid
medium. The idea was that this harsh culture environment would allow selective culturing of cells that were
determined for somatic embryogenesis (Smith and Krikorian, 1991). Such cells/cell clusters were in the form
ofPGSPs (PGSEs). Upon raising pH to 5.8, the process whereby embryos emanated from them was thought to
continue. (Although years earlier Steward had adopted the device of partially replacing the sucrose in a
medium with poorly metabolized sugar alcohols such as sorbitol and mannitol to "poise" cultures, that is to get
them to a state where they would dramatically develop when the sucrose was restored, physiologically poorly
metabolized metabolites to 'poise' cnltures - see Steward et al., 1975 - pH was not considered). A more
precise view of the role of low pH in maintenance now is one in which pre-determined embryos of varying size
and complexity are perpetuated by, and multiplied in, sub-optimal conditions (low pH). The embryo initials on
semi-solid medium at low pH are developmentally identical to those which normally exist in maintenance
suspension culture, albeit the somatic embryos at low pH are in very slightly but still perceptible, more
advanced stages.

Routine day lily maintenance and subculture in liqu.id involves a drop in pH by day three to between pH 4.0 and
4.5. Although hormone is mainly responsible for the proliferative continuation of the culture 'maintained',
low pH also contributes. That identical 'maintenance cultures' can be maintained by low pH in the absence of
auxin is further proof that auxin's role in 'maintenance' is to stimulate multiplication of already determined
but yet very immature, earliest stage(s) if you will, somatic embryos. This low pH effect prevents development
and thus overrides development. Auxin thus becomes only one factor of the non-permissive maintenance
environment which actually leads to the creation of the suspension culture (or a proliferating embryogenic
culture on semi-solid medium as well).

7. Daylily 'Pre-Shoots' Are Somatic Embryos

Evidence for 'true' somatic embryos was lacking when we first encountered organized structures from our
daylily suspensions. We did thereafter encounter some 'morphologically correct' somatic embryos but these
36

derived from complicated protocols. The connection between the embryos so generated and the means
whereby plantlets were formed in liquid was not apparent. Development entirely in liquid was by structures
referred to as 'pre-shoots' (Krikorian and Kann 1981; Krikorian et al., 1986, 1988b). These were, in fact, the
first plantlets produced en masse from monocotyledonous cell suspensions. "Neomorphs' and 'rooty' embryos
in liquid culture emironments have been described, and it was in 'pre-shoot' cultures that daylily'neomorphs'
formed. While not produced in great numbers, but they collld not be comfortably ignored. The only way then
apparent to deal with them on a practical level was to eliminate them selectively in procedures geared to
further gTO\\th on agar medium prior to gro\\th in soil. Alternatively, we spread 'pre-shoot' cultures onto semi-
solid media to enable them to grow more normally thus much reducing 'neomorph' development (Krikorian,
1982; Krikorian et al., 1988b).

My first reaction to 'neomorphs' was that they reflected 'leakiness' in the developmental program. Some of
the 'morphogenetically competent masses', were expressing an alternative morphological pathway. Most
important was that shoot production from 'pre-shoots predominated over root formation from neomorphic
rooty forms. Even today, with our more complete understanding of somatic embryogenesis, daylily plantlet
regeneration via the original 'pre-shoot' protocol remains the most efficient way to propagate in liquid. It is
considerably less labor-intensive and hence more economically attractive than those protocols using the
modifications described so e~:tensively here. Even so, in an effort to be consistent with the characterization of
suspensions and the roles of the regenerative modifications given here in an historical context, it will now be
demonstrated why 'pre-shoots' are indeed somatic embryos.

The developmental origin of 'pre-shoots' was not considered in our original reports. It seemed most likely that
'pre-shoots' arose somehow in a way consistent with the indirect model of somatic embryogenesis. We
assumed that somatic embryos came from PEMs or embryogenic cell clusters but exactly how was a mystery
(Krikorian et al., 1986, 1988b). The origin of the embryonal forms or 'pre-shoots' seemed related to, but was
significantly different from embryos. Somatic embryos of monocotyledons had not yet been produced in liquid
and there was no literature to make comparisons. (Work on grasses was then emerging but structures such as
coleoptiles are lacking in non-graminaceous genera, Vasil and Vasil, 1982.) Although I tried to read as much
as I could on zygotic embryos of monocotyledons, there were too many inconsistencies between them and our
'pre-shoots'. I decided not to clutter up the literature any more than it already was with unproven contentions
of somatic embryogenesis. It is interesting that some were early cominced that our 'pre-shoots' were embryos.
Our daylily work was cited to support the view that somatic embryos of monocotyledons grown in suspension
offered opportunities for efficient clonal multiplication. Or, they cited it as a system requiring considered
sequential treatment to elicit an embryogenic response. Indeed, I even wondered whether an alternate
morphological pathway existed, partly adventitious partly embryogenic. Sections made of the developing pre-
shoots were not easy to interpret at the cellular level and a definitive understanding remained elusive (see
Villanti, 1983).

It has long been appreciated that the embryogenic potential can be detected, although" in many cases not
optimized, when auxin is removed from the maintenance medium or its level is reduced. Most regeneration
protocols involve removal or reduction in auxin. Any cytokinin component is also usually removed or reduced.
Work from this laboratory showed that embryogenic cultures of carrot could be obtained merely by wounding.
This observation was not original to us. Indeed, embryogenesis had been achieved in the absence of exogenous
auxin or any other exogenous gro\\th regulator for that matter (see Smith and Krikorian, 1988 fro refs.) but
they had been more or less mentioned merely in passing in otherwise fairly comprehensive reviews. They were
often viewed as oddities.

When daylily was first tested for embryogenicity, the logical thing was to use methods that 'worked' for carrot
(see e.g. Steward et al., 1975; Krikorian and Smith, 1992) but no clear-<:ut embryos were obtainable in liquid
or semisolid medium after hormone removal/reduction from the maintenance medium. The embryogenic
potential of our daylily cultures was only convincingly demonstrated after the various culture modifications
that were so effective in the case of carrot (Smith and Krikorian, 1989; Smith and Krikorian, 1990) were
finally applied to daylily (Smith and Krikorian, 1991 ). It was incidental but helpful that the daylily cultures
on which this was first done were hormone-autotrophic.

Prior to unequivocal regeneration of embryos, plantlet formation via the 'pre-shoots' was achieved (and still
can be) as follows. The culture is grown on MS plus coconut water at 10% vlv and 2,4-D 2 mg/1 ( kinetin 2
mg/1 serves as a substitute for coconut water). After three weeks gro\\th, cells are filtered, concentrated and
inoculated into the same medium but lacking 2,4-D. After three weeks in 'minus-2,4-D', another transfer is
effected into a medium with reduced salts (Schenk and Hildebrandt, 1972 suffices) and grown for an additional
 37

3 weeks. At this point 'pre-shoot' structures are recognizable. They are then transferred to the same medium
containing half-strength salts and exposed to the light. (All the initial operations are carried out in total
darkness since light is inhibitOIJ to the maintenance process. The reason for this has never been investigated
in detail but my guess is that light fosters shoot growth and hence the units become larger and thus more
difficult to retain in earlier stages of development through 'crumbling'. The less friable they are, as in light.
the harder it is to get fine material for subculture.) From this point onwards, plantlet formation sufficient for
growth in soil can take as long as 6 months. The process can, however, be hastened if the medium is made
semi-solid from the first step following removal from maintenance medium).

In our more recent work; plantlets have been regenerated via the 'pre-shoot' protocol with one procedural
alteration. To derive more 'singularized' or 'unitized' embryos, sieving is carried out. Smaller units allow
more easily interpretable responses, and so a 100 to 200 (> 73 Fm # 140 Fm) suspension fraction rather than
the entire suspension has been used to re-examine 'pre-shoot' development Regenerants were normally in the
final medium for 17 weeks prior to observation. Under these conditions, embryos are easy to identify and some
may even be 'germinating'. The fate of the initials is certain and has even been studied with scanning electron
microscopy. The epidermis of these embryos ('pre-shoots') is disordered, however, typical of liquid-derived
embryos.

Similarly, apical depressions and presumptive shoots are not apparent although there are some hints of these in
the form of more opaque 'centers'. Thus even when 'singularized' structures are observed, lack of superficial
but typical details confounds their identification as embryos. Characterization of these ·structures has been
further complicated over the years by the presence of extended regenerative masses. To some degree, most of
the structures are in this form and singular masses can only imagined to have separated from them. We now
appreciate that these extended masses derive from developed somatic embryo initials contained in the
suspension fragments used to initiate this process. But they do not become detached or separated. This is
observable in virtually all somatic embryo regeneration systems I am familiar with. However, in this process
the extent that natural separation occurs as development proceeds is much reduced. Also, these multiple
structures are more numerous when very coarsely filtered suspensions are used for 'pre-shoots'.

Furthermore, as shoots and/or roots emerge from the obscured somatic embryos in these masses, regenerative
forms' develop that can readily lead to misinterpreting the events as organogenic, albeit somewhat atypic.
Thus, after the formation of the shoots, the appearance of roots from singular or multiple structures readily
lends itself to the interpretation of having been initiated adventitiously in the mass. In structures that end up
having multiple shoots, this event occurs often. It is especially prevalent when shoots develop from embryos
that are part of unseparated masses. There is a fair body of literature on production of multiple shoots from
somatic embryos in the presence of cytokinin but its precise nature has not been understood (see e.g.
Ammirato, 1977; Polisetty et al., 1997).

Re-examination of the morphogenetic relationship between embryos and 'pre-shoots' has thus been shown as
products of the same developmental process. As was the case when improved conventional liquid regeneration
was achieved, the first step in this protocol was a 3 week growth period in the presence of a cytokinin with the
auxin component removed. During this time, somatic embryo initials develop into globular stage embryos.
Thus, in the 'pre-shoot' liquid regeneration protocol, many embryo initials advance without losing their ability
to form a shoot. Overall, the 'pre-shoots' formed in liquid are from initials which benefit first from the
presence of the cytokinin. They subsequently benefit from the presence of higher nutrient levels in the
regeneration medium. This serves to enhance their size since they undergo more cell division. A much-
needed perspective emerges on the long recognized fact that somatic embryos at the same level of
morphological development can vary tremendously in size!

Plantlets formed via 'pre-shoots' in liquid still present disadvantages. The process is long and involved, rooty
and multiple-shooted embryos accompany the process, that is many multiple, non-'singularized', plantlets
form from embryonic clumps that have not separated. In an ornamental like daylily the multiplicity is not a
particular problem. Some might view it as an advantage because rapid multiplication allows early separation
in soil. But encapsulation of propagules and management in 'synthetic' seeds becomes more complicated (see
e.g. Gray and Purohit, 1991; Redenbaugh, 1993). Finally, basic scientific investigation is limited by use of the
'pre-shoot' protocol since one would be adding a perturbed developmental process to the analysis of an already
difficult enough-to-interpret developmental process.

8. Early Events in Generation of Embryogenic Daylily Suspensions

38

Identification of the key events in embryogenic cell suspension production would seem to require study of the
process from its inception through all subsequent stages of development. But the bulk of a now vast literature
is replete with reports from study of cells already proliferated in culture. Even today, one most often starts off
with an embryogenic culture initiated on semi-solid medium and then transfers it to liquid. Some
investigators (those with faith in the indirect pathway?) put priman· explants in liquid medium in anticipation
that totipotent cells will be 'induced' into an embryogenic state, even as it would be on semi-solid. In my
laboratory, embryogenic cultures of various species have been initiated directly in liquid but they take
considerable effort in 'cleaning' them up. There are many cells that are not embryogenic and much true callus
in them, and they have to be gotten rid of through repetitive density separation and/or filtration (see Krikorian,
I 989). Few studies have focused on precisely identifying the primary response in the primary explant,
whether it be initially ex-posed to semi-solid or liquid environment. Limitations of technique(s) allow one to
question nominal 'primary' responses. They may represent post-determination events (Margara and Bouniols,
1967; Schaefer et al., 1988; Dubois et al., 1990, 1991). What is urgently needed is a technique or probe of
some sort to identify events that precede or even accompany the first division associated with the embryogenic
response. This would go far towards establishing whether primary somatic embryo origin is from a single cell,
or an apparently single cell comprised of thin walled uninucleate cells that have arisen by internal division of a
single cells. Or whether true embryogenicity occurs after additional divisions from such structures.

Historically, whenever an embryogenic suspension has been achieved, its derivation typically has been
described as from competent callus, derived in tum, from a wide range of primary ex"J)lants. In daylily,
suspensions which can be manipulated to give rise to 'pre-shoots' and eventually plants were derived from
ex"J)lanted ovaries from unopened flower buds, vegetative shoot tips, leaves, leaf bases, floral stalks (scapes),
and even petals. This focus on 'callus' from specific locations supposedly being more 'competent' than others
is best explained by my motivation to achieve and maintain an 'open-ended' process or supply of
morphogenetically responsive cells as quickly as possible (Steward et al., 1975; Krikorian·et a!., 1986, 1988b;
Krikorian, I 989). It was critical that no plantlet production occur at the maintenance culture stage. Otherwise,
it was reasoned, no continuous supply of 'morphogenetically competent' material from which to derive
plantlets would be available (Krikorian et al., 1986). In retrospect, trying to balance basic and applied research
served to delay our understanding of the cell biology and 'taxonomy'.

In a word, the 'true' embryogenicity of such suspensions was not appreciated as early as it might have been
because I was concerned with the (bio)technology rather than the biology. Similarly, concern with the idea
that embryos had to grow into plants (see e.g. Ammirato, 1983 p. 83, a definition clearly more horticultural
than developmental) delayed identification of neomorphs as embryos. Moreover, most work was done with
cultures derived from ovary bases and shoot tips (Krikorian and Kann, 1981, Smith and Krikorian, 1991)
because I thought that they yielded the highest quality cultures. Ultimately, shoot tips were used most often as
a source of embryogenic cultures because they are available year round. But the fact is that the perceived better
performance of shoot tip ex'jllants was due to our only minimal optimization of the culture conditions when
other explants were tested. We were far from fine-tuning the conditions for supposedly intransigent cultures
as early in the culture process as could have been done. No difference exists in potential of any of these
ex"J)lants. Ex1Jlants of different origins and species simply need to be dealt with when they 'need' to be dealt
with. That means they must be examined frequently, and their performance assessed. The earlier the
intervention the better. Parenthetically this implies that adoption of the device of a 'consensus medium', that
can quickly screen and thereby nominally accommodate explants from many different sources, is not a prudent
strategy if one has a valid reason for initiating embryogenic studies to begin with. I would argue that very few
explants better examined and tended can give quicker results. Pre-occupation with large sample sizes to get
statistics may seem 'scientific' but it is counter-productive in initiating a somatic embryn culture system de
novo.

8. I Extreme Strategies for Production of Embryogenic Suspensions

The earliest events involved in the development of daylily embryogenic suspensions have been re-investigated
by examining initial tissue origin and the degree of organization and morphology of the primary response.

Ex1JCrienced investigators reading this Chapter will be familiar with the fact that some cultures have a
considerably more friable growth habit than others. The primary response on a daylily explant is
characteristically compact, very nodular and a hardness much like the flesh of a fresh cucumber. These
growths can be multiplied by repeated subdivision. Our strategy to obtain embryogenic cells from these has
varied. Use of an auxin like 2,4-D to foster more friable growth has proven to be marginal. And, it may be
interjected here, this problem of compact, nodular growth, especially at the earliest stages of culture, is in my
 39

ex-perience not uncommon in monocotyledons. In daylily, the compact, nodular grO\\th has been so
intransigent to 'classical' manipulation that more drastic measures have had to be adopted. (It may be
interjected here that the kind of darkening so commonly encountered in woody species upon wowunding can be
minimized by a number of techniques-Krikorian, 1989). The responsive tissue has been reduced into
increasingly smaller cell masses by sequentially cutting them, forcing them through stainless steel sieves, and
most often, by judicious use of a combination of the two procedures. Some modifications of the medium
components have proven helpful as well (Krikorian et al., 1995).

Prior to the full development of the culture conditions and protocols for manipulating daylily tissue into
embryogenic suspensions, organized development was obtainable from nodular growths. Plantlets were
described as forming via adventitious shoots and roots when the hormone levels were reduced. But others in
the same explant were not adventitious but rather were derived from pre-existing axillary buds carried over in
the culture process from excised shoots and leaf ' bases'. Growth in hormone-containing medium for a period
of transfers was considered integral to this process as indeed was growth on a hormone-containing ('inducing')
regeneration medium. Thus, initial growths were considered 'callus' and regeneration was generally viewed as
'organogenic', i.e. adventitious. Primary responses very similar to that seen in daylily were encountered in
banana shoot tip explants as well (Cronauer and Krikorian, 1984, 1987). In the case of banana we referred to
it as a 'calloid' mode of growth since it seemed to be neither true callus nor an organ culture. Nyman et al.,
(1983) had earlier used 'calloid' to describe such growths in taro cultures. A defining feature of a 'calloid' is
that some form of epidermis is present. Curtis and Nichol (1948) were among the earliest to use the word
'calloid' in connection with their work on orchid protocorms derived from aseptically cultured seeds.

Although the type of nodular growth like that encountered in daylily has normally been described by many
investigators as a 'callus', some are beginning to draw special attention to the fact that organized tissues may
be involved (see e.g. Teng, 1997). Significantly, when this kind of growth is interpreted as a callus, any
encountered production of somatic embryos is usually identified with superficial cells. Such proliferations of
embryo-yielding cells have been described as deriving from a superficial cambium-like zone or the equivalent
(Springer et a!. ,1979; Ho and Vasil, 1983; Lu and Vasil, 1985). Nevertheless, in the vast majority of
published cases, the primary response is described simply as one of production of an 'embryogenic callus'.
This kind of 'embryogenic callus' typically shows nests of meristematic cells; sometimes they are more broadly
distributed but still more or less discrete. Nodules or lobes have also been described as forming from localized
superficial proliferations of broad zone responses (Vasil and Vasil, 1982). Somatic embryos are commonly
seen at the periphery of this kind of growth. As has been the case in growths that are rather nodular, the
accuracy of referring to less structured initial responses from explants as callus has been questioned (see e.g.
Cure and Mott, 1978; Springer eta!., 1979; Swamy and Krishnamurthy, 1981). But the key point is that
embryos that arise from any form of 'callus' attached to the explant have been presumed, whether consciously
or not, to arise after re-determination of cells in accord with the indirect mode of somatic embryogenesis.

When somatic embryos are produced from embryogenic suspensions (embryogenic 'callus' is subculturable)
the indirect process is assumed to continue. In many cases, shoots and/or roots develop from
'morphogenetically competent callus' and these structures are described as arising via organogenesis. It has,
however, been suggested that such development could derive from an embryogenic process (Ammirato, 1983;
Wicart et al., 1984). The view that callus that forms on induced explants can be partially 'organized' has not
figured so far as I know in any discussions of the indirect scheme of somatic embryogenesis.

Some generalities may be made concerning the primary embryogenic response, however. In all cases that I
am familiar with, responding cells quickly become densely cytoplasmic and there is an increase in the nucleus
to cell area ratio (cf. e.g Vasil and Vasil, I982; Barciela and Vieitez, 1993). Responding cells in almost all
cases (excepting some cases such as pollen) are parenchyma cells in close association with the vasculature (e.g.
Nwankwo and Krikorian, 1983; Ho and Vasil 1983; Alizadeh and Mantell, 1991; Lee et al., 1997) and/or
epidermal/subepidermal cells (Lu and Vasil, 1985; SchMer eta!., 1988; Barciela and Vieitez,1993). In many
cases both single and multiple cell embryo origins are described (Vasil and Vasil,! 982; Hanrung and Conger,
1982; Gray et al., 1984; Wang and Janick, 1986; Kim and Janick, 1989; Quinn et al., 1989; Figuera and
Janick, 1995; Lee et. a!, 1997 and refs. there cited). Similarly, evidence for single cell origin has most often
been derived from observations of internal segmenting divisions in single cells (see e.g. Reinert, 1959b;
Norreel and Nitsch, 1968; Vasil and Vasil, 1982; Ho and Vasil, 1983; Lu and Vasil, 1985 and refs. there cited)
and observations of a few-celled 'proembryos' with common end walls ( Lu and Vasil, 1985; SchMer et al.,
1988; Barciela and Vieitez, 1993).

Broad multiple cell origins have generally been explained as suspensors (Ho and Vasil,1983) and as somehow
40

related to development from multicellular proembryonal complexes (Vasil and Vasil, 1982). Often the
primary response has been identified as direct (i.e. embryos are formed without an intervening callus). There
are many examples where these responses appear very· similar if not identical to the nodular responses
discussed above and to the response seen from daylily (e.g. Dubois et al. '1990; Dubois et al.. 1991; Hilbert et
al., 1992; Lee et al.. 1997).

The similarity of all types of apparent callus to direct somatic embryogenesis should be emphasized. Another
feature common to descriptions of the primary· response is that apparently equivalent cells form embrvos
directly and indirectly after they form embryogenic callus (see e.g. Sharp et al., 1980; Evans et al., 1981; S~rp
et al., 1982; Hanning and Conger, 1982; Gray et al., 1984; Vasil and Vasil, 1982; Lu and Vasil, 1985; Dubois
et al., 1991).

This personalized historical overview and fresh reconstruction of the somatic embryogenic process has
emphasized that once an embryogenic culture is established, there is no undifferentiated or unorganized
'callus' capable of somatic embryogenesis. There is no need to invoke the concept of a PEM. We were closer
to an accurate depiction, but still not a perfect one when we adopted the phrase 'pre-globular stage embryos'.
If the 'reconstruction' given is correct, no true callus would ever exist in this process. The entire process
would be direct and the primary response would be a somatic embryo initial that due to the non-permissive
nature of the maintenance culture environment would yield an 'embryoma' or conceivably a 'calloid'. The
term 'embryoma' has not been used very much by botanists but E. F. Smith the plant pathologist who first
studied crown gall tumors in detail used it to describe embryonic cells induced by the bacterium (Smith, 1917).
An embryogenic suspension would then be produced from secondary embryos that would arise and be
proliferated as polyembryonic fragments. It would also be consistent if the primary response were what might
be termed an 'organoid' (or 'meristemoid'?) wherein somatic embryo initials would be directly formed and
similarly propagated. If true 'callus' was involved, it would be of an extremely brief duration and involve a
few cell divisions at most. It would be highly localized. Such a scenario would not significantly affect the
characterization of the events given in this overview. It is interesting indeed how a word like 'callus', so
widely used in tissue culture can have so many connotations.

9. Site of the Initial Response in Daylily

When shoot tip explants of day lily are examined in the course of initiating suspensions the primary response is
located near the vasculature of leaves and leaf bases. The latter are unavoidably included in small stem tip
cxplants because of the compressed shoot tip morphology. All stem tip explants and even isolated leaves have
responded similarly so there is no doubt as to the following generalizations. Leaves have been cultured and
examined at critical periods in suspension culture formation. Excised ovary bases or petals have not been re-
examined but I would predict that the same events occur. Not surprisingly, the response is a synchronous.
New responses are constantly emanating from the vascular region throughout the course of such
experimentation. Cleared and Feulgen-stained explants show the response to originate from single
parenchyma cells in the peri-vascular region. No responses have been visually detected at 2 weeks and there
have been no responses in control explants exposed to hormone-free medium. It takes about 8 weeks for some
40% of the explants to respond. The earliest growth can develop into globular structures, but in most cases a
tightly grouped assemblage of small cells is first formed. Varying degrees of lateral growth establishes the
base of the assemblage which then produces a stalk of cells that connect the globular growth to the vasculature
(compare this with Williams and Maheshwaran, 1986).

The initial response proliferates into a nodular mass, many of whose components may be compared to globular
stage embryos complete with epidermis. Development of high quality somatic embryos is never encountered in
a medium with high levels of growth regulator as in our induction/maintenance medium (see Table 1). It also
seems that in this kind of hormone-containing medium environment the potential for embryo initial
development is high, provided responsive cells are in the explant. What occurs may be likened to embryo
initial development in large suspension fragments. Similar conditions apparently exist when embryos
preferentially develop from initials that are deep within the protected masses of 'clumped' 'totipotent'
protoplasts (i.e. wall-less initials) prepared from competent suspensions (Fitter and Krikorian. 1981). Those
'buried' in a mass of cells become 'buffered' from exposure to adverse conditions at the periphery, regenerate
their walls, divide and develop (Krikorian et al., !988a). That goes far to explain why, in some systems, it may
seem easier to identify embryogenic cells within a mass, rather than at the periphery (see e.g. Reinert, 1959b;
Kononowicz et al., 1984; Wang and Janick, 1986; Kim and Janick, 1989; Quinn et al., 1989). And it may
e"-plain why we were only able to regenerate daylily plants from protoplasts provided they derived from
vigorous embryogenic suspensions, i.e. preparations full of healthy somatic embryo initials that could better
 41

tolerate being stripped of their walls (Fitter and Krikorian, 1981, 1983; Krikorian et al., 1990). Other
examples could be cited such as 'meristemoids' in thin layers as well (Tran Thanh van, 1981; Compton and
Veilleux, 1992)

We have seen that daylily somatic embryo initials in suspensions are particularly perturbed when auxin is
relatively high. Auxin might well be described as a 'necessary evil' in embryogenic culture establishment and
maintenance. Long ago Gautheret warned that auxins could be toxic to plant tissues in vitro even at levels
much lower (order of 10 -& M) than usually used today in growing somatic embryos. Gautheret was even
prescient enough to recognize that synthetic auxins could somehow uncouple the processes of proliferation and
organization (Gautheret, 1947). Even so, synthetic auxins were adopted as the auxins of choice for somatic
embryogenesis for a variety of reasons, not the least of which was that they could withstand in situ oxidation,
autoclaving and they were inexpensive (Gautheret, 1955). Parenthetically, Gautheret also recognized years
ago that one frequently risked cells and tissues becoming 'waterlogged' if one provided too wet an
environment. It is also of interest from a historical perspective that there are ongoing attempts to encourage
adoption of the term 'hyperhydricity' in place of 'vitrification' (Debergh et al., 1981, 1992). Paupardin and
Gautheret (1954) used both the noun and the adjectival form hyperhydric many years ago. The French would
say: Plus ca change, plus c'est Ia meme chose!

Nodules can be readily separated from cultured day lily leaves only after some 4 weeks of e>.:posure to medium
and tested on hormone-free regeneration medium. Dissection from the leaf would be necessary for earlier
testing, and hence has not been carried out extensively for fear that wounding responses might cloud
interpretation. Shoots and/or roots can form from the nodular masses. Any plantlets that are recovered seem
to have their origin from shoots that have rooted. It is important to emphasize that suspensions identical to
those derived from shoot tip explants can be initiated from the vascular nodules. It has not been possible from
the work done so far to state conclusively that the masses are tightly associated somatic embryos. That we can
establish equivalent suspensions strengthens but does not prove that the embryogenic units in suspension and
the nodular units initially produced at the primary explant stage are exactly the same, whatever the initial
origin or explant form, and in whatever state they are maintained in. They differ developmentally only to the
extent that they are separated in time and space.

In addition to studying development in explanted leaves, shoot tip explants as they give rise to suspension
material have been followed. Representative components were sampled and tested on hormone-free
regeneration medium as the cultures became fine suspensions. Testing has normally begun at 12 weeks and
has continued until unequivocal somatic embryo formation can be observed. Each culture followed has derived
from a single shoot tip. Testing has routinely been done for many lines or early isolates to evaluate quality of
embryogenic response and hence to determine if continued culturing is warranted. This constitutes a pre-
screening and can save enormous amounts of wasted effort. The results in general have been as follows. At 12
weeks cultures yield growths from which shoots can develop, and these in tum can root and form plantlets.
Alternatively, they can make some somatic embryos. Yet, some of the responses sampled at 12 weeks are in
the form of perturl>ed shoot structures, made abnormal by the culture conditions. These are reminiscent of
growths from banana calloid mentioned above (see Cronauer and Krikorian, 1987). As the culture process
continues, the regenerative responses seem to come from parts of the culture which were increasingly
undergoing conversion to growths comprised of somatic embryo initials. By 19 weeks, responses generally
appear to be uniformly and unequivocally in the form of cells capable of yielding somatic embryos. There are
no responses whatever from any culture component that might be considered undifferentiated, parenchymatous
callus. Thus, relative to established suspensions, any 'real' callus, if there is any at all, must be sustained as
such for a very short period. Equally significant is that as any structures identifiable as somatic embryos
become evident, any nodular growth mode has gone from firm to friable, the latter being replete with somatic
embryo initials in the form of polyembryonic fragments.
Everything presented supports the conclusion that in an embryogenic daylily suspension, and I do not hesitate
to state that this same process accounts for all suspensions that can produce somatic embryos, herl>aceous plant
or woody plant, monocotyledon or dicotyledonous or gymnospermous, derives from varying levels of budding
of already determined somatic embryo initials undergoing dissociation from, and long after, the primary
response. After the embryo initials form from the initial mass(es), the embryogenicity is in place and is
perpetuated because of the culture protocol. It can be argued that these groups of already-determined cells
should be called 'polyembryonic fragments' or 'polyembryonic masses' or 'groups of somatic embryo initials',
or even 'groups of zygote equivalents'. No auxin-dependent, undifferentiated, unorganized growth of cells
needs to occur in order to establish embryogenic competence.

9. I Embryoids and Embryomas

42

The evidence is that the primary nodule of daylily has a 'calloid' or 'embryoma' character. It is not
undifferentiated. One could perhaps choose to call it an 'embryoid' but that would invite confusion. This is
because the term 'embryoid' was first invented in part to differentiate somatic tissue-derived embryos from
zygote-derived ones (Steward et al., 1961). But another reason was that earliest somatic embryos did not have
the highest morphological fidelity (see also Vasil and Vasil, 1972; Swamy and Krishnamurthy, 1981:
Rangaswamy, 1986). While arguments could be made to support the interpretation that the primary response
in daylily explants is a directly-formed somatic embryo, we cannot be definitive on this important point. It
may simply be that the physical constraints associated with the site of determination serve to spatially modify
and otherwise perturb what would have been clear-cut somatic embryo development. Perhaps it is unrealistic
to ex-pect 'morphologically perfect' somatic embryos to form in a cramped, limiting space. Perhaps the ex1ent
of friability peculiar to a species becomes pivotal to early identification of an embryo. It would be here that a
molecular approach to the nature of the intercellular substances in embryogenic cultures that are more friable
than others can resolve the matter.

Thus, it remains uncertain whether the initial embryogenic response from a primary explant o daylily is a
direct one. If it were certain, all the phenotypic variation and apparent timing of responsiveness in all
morphogenetically competent systems that I am aware of could be accounted for. Any variation would be
attributable to when after induction responses were viewed, the growth conditions obtaining at the lime the
observations were made, the location (i.e. the degree of physical restriction) of the responding cells, and finally
but to a lesser e;xtent, the species studied. I say species 'to a lesser extent' with the following qualification.
The tenacity with which cells retain their adhesiveness via the middle lamella most likely has a genetic basis.
Attempts were made some years ago to relate degree of embryogenicity with changes in cell wall composition
(see e.g. Wright and Northcote, 1973; Turnham and Northcote, 1982). Unequivocal resolution of the question
of if and when the primary response involves direct somatic embryo formation might be facilitated by study of
the earliest response in a plant that has looser cell association (see Kohlenbach, 1982, I 985). In that way any
physical constraints due to compactness of closely associated cells near the initial responders might be
minimized if not eliminated. Whatever the ultimate resolution of this question turns out to be there seems no
doubt that an established, embryogenic suspension culture is already determined and hence cannot be used to
study events associated with determination. This is so whether the primary response is a morphologically
difficult-to-recognize, yet directly-formed somatic embryo, or what might be termed an 'organoid' or advanced
'meristemoid', or a very short-lived callus. Nothing said here undermines any of these options.

10. Somatic Embryos and Somaclonal Variation

Few reliable generalizations may be made on the e;xtent of tolerable digression from normal karyology that can
exist without compromising regeneration. Even so, both semi-solid 'callus' and suspensions often show much
greater nuclear variability and karyotype spectrum than plants regenerated from them. This implies that there
may be selective regeneration from cells with more 'normal' genotypes (cf. Bayliss, 1980; Larkin and
Scowcroft, 1981; Scowcroft, 1981; Krikorian et al., 1983; Orton, 1984; D'Amato,1985; Vasil, 1986 for
valuable historical background).

Bits of evidence were indeed presented years ago that phenotypic and genotypic stability encountered in plants
derived from embryogenic cultures is greater than that from organogenic cultures (cf. e.g. Swedlund and Vasil,
1985; Wakhlu and Bajwa, 1987). Historically, this had its origin in the view that adventitious buds are
multicellular and hence potentially chimera! in origin (cf. D'Arnato, 1978 and refs. cited therein), whereas
somatic embryos by definition are unicellular in origin (cf. Polito et al., 1989). Caution in generalization is
needed here, however, since adventitious buds may be of unicellular origin (cf. Broertjes and van Harten, I985;
van Harten, 1998). The point has also been raised above that somatic embryo initials may have discrete,
'subtle' internal divisions.

It has been known for a long time that aneuploids may be encountered among certain agronomic crop varieties,
and despite genetic imbalance can survive under field conditions. While most aneuploids show reduced vigor,
others show few if any departures from normal euploid phenotype (cf. e.g. John and Lewis, 1968; Khush,
1973; Bingham and McCoy, 1986). Virtually all of the literature dealing with aneuploid regeneration relates
to identification of aneuploid plants as a subpopulation within a larger group that included normal plants as
well as plants with various levels of chromosomal off-type. A classic exception to this observation is the
production via organogenesis of a completely aneuploid (and phenotypically aberrant as well) population of
tobacco plants from a 20 year-old and habituated/transformed culture by Sacristan and Melchers (1969).
 43

The possibility of achieving true clonal fidelity has been demonstrated in day lily time and time again (Chen
and Goeden-Kallemeyn, 1979; Krikorian et al., 1986; Griesbach, 1989) provided certain precautions are
followed during culture. But understanding the role of empiric precautions have until recently been based on
concepts that derived more from rationalized supposition rather than from extended experimentation, and so it
is with many other species, woody and non-woody. Even so, each of the in vitro schemes developed for day lily
involves generation, selection, proliferation and manipulation of totipotent cells. All this implies that there is
potential for the generation of off-types (cf. e.g. D'Amato, 1978. 1985; Larkin and Scowcroft, 1981, Scowcroft.
1985; Karp and Maddock, 1984; Karp and Bright, 1985; Karp, 1989; Walbot and Cullis, 1985; Jain et al.,
1998 and refs. cited therein for examples from many species both herbaceous and woody ).

Results from my laboratory with daylily using methods ranging from stem tip culture, embryogenesis from
suspensions and recovery of plants from protoplasts prepared from embryogenic suspensions shows there is
progressive and increasing departure from the normal genotype and phenotype with decrease in unit size and
level of pre-existing development in the vilro-derived propagule (Krikorian eta!., 1982, i.e. protoplast-derived
plants show the greatest number of off-types (Fitter and Krikorian, 1988); plants that derive from stem tips and
embryos from 'large' multicellular embryo initials/fragments/units show the least tendency to deviate from the
original clonal type (Krikorian eta!., !988b). Suspension culture-generated materials are 'intermediate' in
their tendency to remain genotypically stable (Krikorian et a!., 1988b; Griesbach, 1989) to the e:>."tant that it
depends on the size of the fragments containing the somatic embryo initials yielding the final embryos.
Suspensions of somatic embryo initials have been maintained for over six years at a time (they probably could
have been maintained longer) that are genotypically stable provided inocula for maintaining cultures are
retained within specific range of size (Krikorian et a!., in preparation), and a specific size fraction is used for
regeneration. Instability occurs and is sustained through subculture when units selected for culture
maintenance are 'small'. This 'size' principle surely holds true for many species, woody and herbaceous, and
must have an underlying principle that we need not concern ourselves with here. This is not to say that
somaclonal variation is due exclusively to size of unit from which regeneration proceeds. It is but one, and an
important one at that, of many possibilities (cf. Lee and Phillips, 1988; Phillips et al., 1994; van Harten,
1998; Jain et al., 1998 and references cited therein). The observation on size is significant in that it helps
explain why various investigators get different results. Methods are critical (see Krikorian, 1999).

Surprisingly few kinds of chromosomal variants have been encountered in our extensive daylily work.
'Normal' diploid daylily has 22 somatic chromosomes. Tetraploid plants have been obtained from both
embryogenic suspensions and their protoplasts from them. Tetraploids 'cells' were extant in the cultures from
which they derived. A few aneuploid plants have also been encountered. One genotype has a karyotype of 2n-l
and was initially derived from a protoplast culture (cf. Fitter and Krikorian, 1988); two others (both trisomies,
2n+ 1, but readily distinguishable karyologically from one another) derived from suspension culture-generated
plants. Yet another, from embryogenic suspension is 2n=25. Phenotypically they vary insignificantly. Of
greatest significance is that few off-types have been found when rigorous management schemes are put into
place. It seems unlikely that daylily is more 'stable' than other species. Our work on plantain has shown that
starting material is critical since some clones are chimeras and chimera dissection can occur through tissue
culture but even here there are strategies to minimize if not eliminate variation due to this cause (cf. Krikorian
eta!., 1999.)

11. Conclusions

Somatic embryogenesis has yet to evolve from an art to exact science. The hope is that refinements and
improvements in the theory behind this process will eventually permit us to manipulate with greater insight
and less empirically the many and varied physical, chemical and environmental parameters affecting the
process. One may predict with some confidence that all this will one day be precisely expressed quantitatively
as numbers, and will place a new face on laboratory procedures. Not only should methods become more
elegant, but the time required to achieve a manageable embryogenic culture should be drastically reduced. I
predict that the principles presented in this overview using daylily as the main model will be applicable to all
plants, including woody plants. One can predict that commercialization using somatic embryos will be a reality
in woody species. That somatic embryogenesis remains very inadequately defined at the molecular level is no
doubt due to the fact that the basic cell biology, the 'taxonomy' of embryogenic systems if you will, has not
been unequivocally placed in a conte:>."t amenable to objective experimentation. Success has largely been
limited by the study of processes and events in 'models' that are developmentally far past their initial
embryogenic commitment. Indeed, the models themselves have not yet been adequately defined to render them
fully useful for such studies. An attempt has been made here to draw together a number of facts and
perspectives into a consistent whole. Now that a defensible and consistent 'taxonomy' is closer to hand, both
44

the routine use of existing techniques and further quantum leaps in various kinds of scientific technology itself
should eventually have a major impact in this field.

12. Acknowledgments

I thank NASA for supporting my tissue culture work over many years. Dr. Thora W. Halstead in particular
had the foresight to encourage somatic embryogenesis investigations. The contributions of various students
and associates and technicians who have worked on somatic embryogenesis in this laboratory is also gratefully
acknowledged. These include, in alphabetical order, Sandra Cronauer, F. Ronald Dutcher, Mindy S. Fitter,
Robert P. Kann, Elizabeth T. Kim, Mary E. Scott and David L. Smith. The more recent work of Joel
Weidenfeld deserves to be singled out since he has been the last to work on this problem in my laboratory. His
investigations have provided valuable insights and allowed fuller assemblage of the work into a harmonious
whole. I say 'last' rather than 'latest' since I am retired feeling confident that definitive molecular work can
now be undertaken by a new generation. I also thank the senior editor of this series for his kind help in
editing and formatting this chapter for the press.

13. References

Ahloowalia, B.S. and Maretzki, A (1983) .Plant regeneration via somatic embryogenesis in sugarcane. Plant Cell Reports 2, 21-25
Aleith, F. and Richter, G. (1990) Gene expression during the induction of somatic embryogenesis in carrot cell suspensions. Planta 183, 17-24.
Alizadeh., S. and Mantell, S. H. (1991) Early cellular events during direct somatic embryogenesis in cotyledon explants of Solanum av1cuiare
Forst. Ann. Bot. 67, 257-263.
Alvard, D., COte, F. and Teisson, C. (1993) Comparison of methods of liquid culture for banana micropropagation. effects of temporal)'
immen;ion. Plant Cell Tus. Org. Cult. 32,55-60.
Anunirato, P.V. (1977) Hormonal control over somatic embryo development from cultured cells ofcaraway.Plant Physiol.. 59, 579·586.
Ammirato, P. V. (1983) Embryogenesis, in D. A Evans, W. R. Sharp, P. V. Anunirato, andY. Yamada(eds.),Handbook o[PlantCe/1
Culture, Macmillan, New York, Vol! pp. 82-123.
Ammirato, P.V. (1986) Carrot, in DA Evans, W.R. Sharp and P.V. Ammirato (eds.),Handbook ofPlant Cell Culture, Macmillan, New York,
Vol. 4 pp. 457-499.
Ammirato, P. V. (1987) Organizational events during somatic embryogenesis, in C. E. Green, D. A Somers, W. P. Hackett and D. D. Biesboer
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 2. Current status of Thin Cell Layer method for the induction of organogenesis or
somatic embryogenesis
K. Tran Thanh Van and Bui Van Le
Institut de Biotechnologie des Plantes, UMR 8168-Universite de Paris-sud, 91405
Orsay Cedex, France; e-mail: Tran@ qcd.th.u-psud.fr

Contents
1. Introduction
2. The Thin Cell Layer (TCL) method
2.1. CONCEPT
2.2. PROCEDURES
2.3. FOUR TCL MODEL SYSTEMS
3. Application of TCL method for the induction of organogenesis and/ or somatic
embryogenesis on woody and non-woody species.
3.1. WOODY SPECIES
3.2. NON WOODY SPECIES
4. Biochemical/ Molecular markers of morphogenesis
5. Reflection on regeneration and prospects
6. Concluding remarks

Abbreviations: TCL: Thin Cell Layer; tTCL: transverse Thin Cell Layer; CPPU,
forchlofenuron: N-(2-chloro-4-pyridyl)-N' -phenylurea; TDZ, thidiazuron: N-phenyl-
N' -1 ,2,3-thiadiazol-5-ylurea; BA: 6-benzyladenine; IBA: Indole-3-butyric acid; NAA:
Naphthaleneacetic acid, K: kinetin, 2,4-D: 2,4-dichlorophenoxyacetic acid; picloram: 4-
amino-3,5,6-trichloropicolinic acid; 2iP: N 6-isopentyladenine; dicamba: dichloro-o-
anisic acid; PES: Pseudo-embryo-structure.
51
S.M. Jain. P.K. Gupta and R.J. Newton (eds.). Somatic Embryogenesis in Woody Plants. Volume 6, 51-92.
© 2000 Kluwer Academic Publishers.
52
1. Introduction

The regeneration of woody and non-woody species via organogenesis and! or somatic
embryogenesis is of both fundamental and applied interests. Several excellent reviews
have been written on somatic embryogenesis and organogenesis (Ammirato 1983,
Nomura and Komamine 1985, Brown and Thorpe 1986, Carman 1990, Terzi and
Loschiavo 1990, Brown eta!. 1995, Dunstan eta!. 1995, Jain eta!. 1995, Jain 1998,
Jain and Ishii 1998).
Up till now, work on regeneration tended to be conducted on non-woody species
and among them, the dicots were more frequently studied than the monocots.
According to the literature, dicots species responded better to in vitro manipulation.
Therefore, most of the species recalcitrant to regeneration are among the monocots and
woody species. "Proper conditions" have been successful in inducing somatic
embryogenesis on about 180 species of herbaceous dicot belonging to 37 out of 357
families covering approximatively 18,295 species (Brown et a!. 1995). As for the
woody species, somatic embryogenesis has been reported on only approximately 150
species (Dunstan et a!. 1995). Even in the case of easy-to-regenerate species, we only
know "how" to obtain regeneration, but we lack a basic understanding of the
mechanisms which control regeneration. In order to analyse these mechanisms, we need
developmental mutant (s) or, since there are very few mutants, model systems where
regeneration can be controlled.
Woody species, and in view of the urgent need for reforestation and bioremediation,
particularly tree-forest species require more work because of: i) the reluctance to
regeneration, and our limited knowledge of the basic mechanisms of cell division
induction, cell signalling and cell determination in the process of regeneration on
species with a long life cycle; ii) the importance to the ecosystem of plants and
especially of woody species and iii) deforestation by man and/ or by environmental
abiotic and biotic stresses (salinity, water stress including flooding, pollution by heavy
metals, herbicides, salts).
Somatic embryogenesis and organogenesis are among the most striking processes in
biology. In plants, cells which have already fulfilled their ontogenic program of
differentiation - the one inscribed in their genome and its environment within the
integrated organism - can be reprogrammed under certain circumstances, in order to
build new morphogenic patterns and functions. The most classically described are
callus, roots, shoot buds and somatic embryos; the latter two patterns can give rise to
new individuals while bypassing meiosis and sexual reproduction.
It is not unclear when a cell or a group of cells reprogram its or their own ontogenic
program for building a bipolar structure (i.e. somatic embryo) or a caulinary-or root
meristem or a callus.
Based on studies of the development of Arabidopsis embryo mutants affected in
pattern formation, a caulinary-or root meristem regenerated in vitro can be considered
as one predominantly developing pole of a bipolar structure. For example, the
Arabidopsis gnom mutant embryo has no root and no cotyledon (Mayer et al. 1991).
However, in contrast to the neoformed shoot/ root which establishes vascular
connection with the surrounding tissue, somatic embryos during their genesis "isolated"
themselves or were "isolated" from the surrounding tissues: successive cell divisions
take place inside a cell - whose cell wall thickens during this process - resulting in an
 53
epidermized nodule of embryonic cells, the body of the future somatic embryo. Cell
wall thickening and callose deposition mark a change in cell-cell interaction and
constitute a selected barrier from the surrounding cells. By its structural (callose, cutine
synthesis) and functional changes, the cell wall also constitutes a special matrix for the
future somatic embryo. In contrast to the neoformed shoots or roots, somatic embryos,
once mature, can be detached from the original explant. The cell-to-cell or cell-matrix
interrelation is different in organogenesis and in somatic embryogenesis. These
relations need to be characterised at the physical, physiological and molecular levels.
In the very first phase of somatic embryogenic or organogenic processes, a cell or a
group of cells undergoes mitosis which can lead to the following structures: a) a friable
callus composed of large vacuolated cells, b) epidermized nodules made of
meristematic cells from which will arise the embryos or the shoot/ root meristem, c)
direct somatic embryos, and d) direct bud-, root- or flower meristems. If the conditions
are not favourable, the second pattern can revert back to the first one.
Friable callus can either remain friable or develop new structures as in the
following. Among the large vacuolated cells of the callus, some cells will not undergo
their cell wall extension and from mitosis to mitosis, remain small and become
isodiametric and meristematic. These clumps of cells will form an epidermal structure.
This differentiation signals an abrupt change in their "behaviour", i.e. their interaction
with the surrounding cells.
The key control of the induction of friable callus or embryo/ organ formation lies in
the balance of cell division and cell extension, each (but especially the latter) involving
the important role of cell wall components and enzymes.

The following questions still remain unsolved:

. What are the signals for: a) the onset of cell division and specialised type of cell
division, and b) the selection of cell( s) for a given differentiation pattern. ·
. According to the literature on the regeneration of non-woody (Bhaskaran and Smith
1990, Krishnaraj and Vasil 1995, Brown et al. 1995) and woody species (Tautorus et al.
1991, Dunstan et al. 1995) the following factors can trigger these putative signals: a)
growth regulators, b) environmental conditions, c) the physiological stage of the donor
plant and the nature of the inoculum and d). above all, undeciphered genetic parameters.
To these, other factors should be included such as the importance of excision, wounding
and the size of the inoculum.
Most of the work on the regeneration of woody species used, as inoculum, tissues
surrounding or derived from immature/ mature embryos and inflorescences, the latter
being contiguous with (or in the vicinity of) embryonic tissues; they can be considered
as "close" to the embryogenic state. Presumably these cells/ tissues have retained or
regained their ability to divide at a frequency that maintains the daughter cells in
isodiametric shape and a meristematic state. Almost all work done on woody species
reported on somatic embryogenesis. Although somatic embryos (because they originate
in most cases from a single cell) are a good system for genetic transformation, they do
have several disadvantages for propagation. They can be blocked at this stage and
conversion into normal plants can be prevented, otherwise they will go through the
juvenile phase well known in woody species. Furthermore, by so using products of
gametes fusion i.e- immature or mature embryos, the regeneration of plants conform to
the original elite one can not be expected as it is the case with regeneration from mature
54
organs of this elite individual. Moreover, transfer to a nursery, or to field conditions,
somatic seedlings require utmost care for a better survival rate than that from zygotic
seedlings, although the seed viability of certain tropical species can be low.
Apart from the specific purpose of transformation, artificial manufactured seed
production (Redenbaugh and Walker, 1990; Attre and Fowke 1993) or cryopreservation
of embryonic lines for cloning forestry (Park et al. 1998), it would be more useful to
obtain also neoformation of adult shoots from mature vegetative organs (such as leaves,
stem tissues, lateral branches, bracts, floral stalk, inflorescences) which have the
advantage of being more available or accessible in woody species than the immature/
mature zygotic embryos.

In this chapter, we discuss some aspects of the current status of the Thin Cell Layer
method to study organogenesis/ somatic embryogenesis in woody and non-woody
species. We also report on work of other authors using a thin inoculum (of reduced
size) called thin section, cross-section, micro-cross-sections. We also include data on
small pieces of leaf lamina which can be considered as thin inoculum, because they are
composed of a few cell layers.

2. The Thin Cell Layer (TCL) method

2.1. THE THIN CELL LAYER (TCL) CONCEPT

Within an organism, growth and development events occur according to a temporal and
spatial expression of the genome. From the embryo to the adult stage, these events are
intertwined temporally and spatially in different organs, tissues and cells. We have
shown that developmental processes can be altered by changes in organ/ tissue/ cell
correlation (Le Kiem Ngoc-Tran ThanhVan 1965, Tran Thanh Van 1980, 1981, Tran
Thanh Van et al. 1974a) and that these correlations result in inhibitory processes at the
whole organism level. The existence of such an inhibitory network can be demonstrated
by induced flowering in these species which flower only after several years of
vegetative growth (as in orchids) or only in meristerns which never flower such as
terminal bud of Geum urbanum (Tran Thanh Van 1974a, 1974b, Tran Thanh Van and
Trinh 1978).
This inhibitory network led us (Tran Thanh Van 1973a, 1973b) to develop Thin Cell
Layer concept: reprogramming the cell(s) by excising cell/ tissues into layer(s) from the
surrounding tissues and organs in order to remove them from the tissue/ cell/ organ
correlation network. Excision and wounding are important parameters in activating
synthesis of cell wall enzymes which synthesize new types of cell wall polysaccharides
and/ or release specific oligosacharides, some of them play a role in cell signalling
processes for inducing plant growth and development (Tran Thanh Van et al. 1985a,
Tran Thanh Van and Mutaftschiew 1990, Mohnen et al. 1990).
As a consequence of their small size, more cells within a TCL become
morphologically responsive due to their close contact with:
a) wounded cells, sites for the synthesis of new cell wall constituents and release of
oligosaccharides,
b) nutrients and other "morphogenic" factors (growth substances).
 55
The ratio of morphogenetically responsive cells to non-responsive cells is higher
than in classical explant such as stem or leaf fragment which are relatively more
voluminous and show a strong polarity in the regeneration. They also bring to the
culture medium - that we previously formulated with well defmed compounds - their
endogenous unknown substances in significant quantities, in contrast to thin explants
which do not, in general, exhibit polarity and are more medium dependent. With thin
explants, however, the osmotic pressure of the culture medium as well as the molecule
flow in and out of the cells need to be considered.

2.2. THE TCL PROCEDURES

The TCLs are explants of small size. They are excised: i) longitudinally and composed
of only one or a few tissue types: epidermis, subepidermis, cortex, cambium, pith ,
endodermis; ii) transversally and, therefore, are composed of several tissue types, which
generally are difficult to separate from each other by dissection, for example: leaf
lamina, leaf vein, root. Longitudinal TCLs are 0.5 or 1 mm wide and 5-10 mm long;
they include one to several cell layers. Transverse TCLs (tTCL) also designated as Thin
sections (TS) of varying thicknesses from 0.1 mm to 4-5 mm cut (by hand or if thinner,
by microtome) through different types of organ: stem, root, leaf (lamina, vein, tendril),
zygotic/ somatic embryo (immature, mature), endosperm, scutellum, cotyledon,
embryonic axis, protocorm, floral organs (ovary, style, stigma, anther, filament, petal,
sepal), seeds, integuments, scale, rhizome, tuber, etc. Thickness and length of the Thin
Cell Layers, the composition of the culture medium, especially of macroelements and
growth substance concentration as well as the pH variation of the culture medium
during the time course, the sequence of light/ darkness are parameters to be defined for
each material. As TCL explants can be a monolayer or a multilayer (with a reduced
number of cell layers), the macromineral composition could be a factor for success.
Culture media such as Murashige and Skoog (1962), Gamborg et al. (1968) in either
full or reduced strength can be used for TCL.
The microelements of Murashige and Skoog (MS), Gamborg or Schenk and
Hildebrandt (SH) (1972) could be assayed. In most work, there is no clear rationale in
selecting the culture medium formulation.
If the above mentioned culture media do not produce positive results, we suggest the
following formulation for macroelements designed for TCLs that we named by TCL 1
and TCL 2 medium, to be used in full or half strength. TCL 1 and TCL 2 differ from each
other mainly by the level of reduced nitrogen, the rapid absorption (Cousson and Tran
Thanh Van 1992) of which can cause a rapid variation in pH of the culture medium and,
therefore, have an impact on in vitro morphogenesis (Cousson et al. 1989).
56
TABLE 1. Composition of" TCL 111 and 11 TCL 2 11 culture medium

a) Macroe/ements

Macroelements TCL 1 (mg 1"1) TCL2 (mg 1" 1)

KN03 2214 2214

NH4N03 1237
CaCI 2,2 H20 355 184
MgS04,7Hp 328 303
KH 2P04 210
NH4H2P04 204 150
(NH4)2 P04 140

b) Microelements (mg 1" 1): ofMS, Gamborg et al. (1968) or SH medium

c) Vitamins: Myo-inositol (100 mg 1" 1), thiamin HCl (100 mg l- 1) and other vitamins of
MS or Gamborg or SH medium.
d) Carbohydrates: Sucrose (g 1"1): 10-30 or>, for embryogenic callus to differentiate
embryos. Could be complemented by mannitol for osmotic pressure
e) Growth substances (JlM): 2,4-D, NAA, BA, Kinetin, zeatin, 2iP: 0.1-10; dicamba
(0.001-10); pichloram; TDZ or CPPU: 0.01-10
f) Amino acids (proline, asparagine, casein hydrolysate), coconut water.
g) pH: 5.8 or different according to the material used; pH buffer can be used if pH has
to be maintained during culture.
h) Gelifiant agents: gelose (ethylene emission could be variable depending upon the
source), or phytagel.
i) Containers: Petri dishes 1, 2, 3 em height, test tubes, jars, multiwell (of different
diameters) plates ... please note that the gaseous atmosphere (ethylene, C02 ,
alcohol. .. ) surrounding the inoculum is crucial for the success, therefore the degree
of hermeticity/ gaz exchange depends on the mode of covering the containers. It is
important to adapt the volume of the container to the density of inoculum.
j) Environmental conditions: Light of different sources (for example light emitting
diodes), sequence dark/ light.
k) Sectionning: Caution, dehydratation ofTCL.
1) See for details of the TCL method (Tran Thanh Van and Gendy 1993b).

The concept, results, applications and perspectives of longitudinal and transverse Thin
Cell Layer methods were published (Tran Thanh Van 1973a, 1980, 1981, 1991, Tran
Thanh Van and Trinh 1990, Tran Thanh Van and Gendy 1993b, Tran Thanh Van et al.
1974a, 1974b, 1985a, 1990b, Richard et al. 1991, Gendy et al. 1996, Bui Van Le 1997,
Bui Van Le et al. 1997, 1998a, 1998b, 1998c, 1998d, 1998e). These methods were
successfully applied to woody species (Lee-Stadlemann et al. 1989, Jullien and Tran
Thanh Van 1994, Goh et al. 1994, Pedroso and Pais 1995a, 1995b) and non-woody
species (monocots or dicots) (Van den Ende et a1, 1984a, 1984b, Klimaszewska and
Keller 1985, Charest et al. 1988, Detrez et al. 1988, Pelissier et al. 1990, Gill et al.
1992, Becher et al. 1992, Compton and Veilleux 1992, Golds et al. 1993, Pihakaski-
 57
Maunsbach et al. 1993, Jehan eta!. 1994, Creemers-Molenaar et al. 1994, Goh et al.
1995, Lakshmanan et al. 1995, Kieffer et al. 1995, Okole and Schulz 1996).
In the following part, we present four experimental model systems and the
applications of TCL systems in woody and non-woody species for the induction of
organogenesis and somatic embryogenesis.

2.3. FOUR TCL MODEL SYSTEMS

The four experimental model systems are: a) the longitudinal TCLs of Nicotiana
tabacum, with the control of all patterns of morphogenesis, b) the longitudinal mono/
multilayer of Torenia fournieri with the cell/ tissue correlation, c) the transverse tTCLs
of Iris pallida and d) the transverse tTCLs of Digitaria sanguinalis with the control of
somatic embryogenesis and the genesis of a new pattern, the pseudo-embryo-caulinary-
structures (PECS).

2.3.1. Longitudinal TCL: multiprogramable patterns in Nicotiana tabacum

Tobacco TCL are small explants, 1mm wide x 5 mm long with 3 to 6 layers of
epidermal and subepidermal cells excised from floral branches of flowering plants
bearing 10-15 green fruits. They comprise only differentiated cells.
They are re-programmed to differentiate directly on the surface of the epidermis,
approximately 30 to 50 functional flowers per TCL, at its proximal pole (towards the
stem of the donor plant) (Figures 1-4, Plate 1; Figure 1, Plate 2). Alternatively, they can
be re-programmed to differentiate directly and separately the following morphogenetic
patterns: roots (10 to 20 roots per TCL (Figures 2, 3, Plate 2), vegetative buds (500 to
800 with no polarity (Figure 4, Plate 2), in contrast to flower program), somatic
embryos, however, blocked in their further development, callus without organogenesis
(Tran Thanh Van 1977). Smaller TCL of 1 mm2 formed flower without polarity. There
is no critical size (at the macroscopic level) for flower differentiation.
All these organs were differentiated on 100 % TCL, from the same layer, the
subepidermal layer (in which cell division was arrested before the excision of TCLs
from the donor plant). The time interval was very short; 10-12 days for flower and
vegetative programs, 16-18 days for root program. Such a rapid and direct
programming of selected organ versus callogenesis raised the problem of the difference
in the nature of cell division itself in the subepidermal cells. TCL is a unique system in
which the subepidermal cells can be reprogrammed to express different morphogenetic
patterns separately or combined. Their control was obtained through a balance of
carbohydrates, auxin and cytokinin; and for a given balance, the morphogenetic control
can be modulated by the pH of the culture medium (Cousson et al. 1989), the nature of
cytokinins (Tran Thanh Van 1991) and oligosaccharides supply (Tran Thanh Van et al.
1985a).
This model allows to study cytological, physiological, biochemical and molecular
changes occured at early stages of each morphogenetic programme (Tran Thanh Van et
al. 1990a, 1990b, Meeks-Wagner et al. 1989, Kay and Basile 1987, Thorpe et al. 1978,
Tiburcio et al. 1989, Richard eta!. 1991, Tran Thanh Van 1991). To assess the specific
changes for a given morphogenetic programme, a comparison between all these pure
morphogenetic programmes will allow us to select the specific markers. The model's
58
advantage over morphogenetic mutants -if any- resides in the fact that the studied
programme, (a flower programme for example) is not mixed with other programmes
such as root, stem, leaf, which are mixed at the whole plant level. The functions of all
these programmes normally found in whole plants, are inextricably mixed, thereby,
resulting in a network which cannot be dissected at any of the levels mentioned above.
Once the morphogenetic markers are identified, they can be localised with confocal
scanning microscopy (for example thaumatine antibodies, Richard et al. 1991 and
unpublished results) at the precise cell site, the subepidermal layer in this case and their
expression tested in in vivo system, on normal plant (Meeks-Wagner et al. 1989). With
this model, one can assess the specificity of inducing factors.

2.3 .1.1. Influence of cytokinins and oligosaccharides on flower induction in Nicotiana

tabacum TCL

The key-factors such as light intensity and quality, a balance of sugar (Cousson and
Tran Thanh Van 1983), indole butyric acid (IBA) or cytokinin concentration were
essential for the floral differentiation (Tran Thanh Van 1991). A pure flower program
(100% TCL form only flowers) was obtained only when a cytokinin, kinetin was used.
With other cytokinins such as 6-benzyladenin (BA), zeatin, N6-isopentyladenine (2iP),
are used, a mixed program of flowers and vegetative buds was obtained. One of our
working hypothesis was based on a possible difference in catabolism between kinetin
versus other cytokinins. We, therefore, compared the floral and vegetative
differentiation using different concentrations of kinetin, zeatin and a substituted form of
zeatin, the dihydrozeatin, adenine and their ribosides (Tran Thanh Van 1991).
Morphological responses were recorded and endogenous levels of cytokinins were
measured: a significantly higher level of endogenous cytokinins was found in TCLs
treated either with kinetin or dihydrozeatin; in these cases only, flowers were obtained
in 100% TCLs. The TCL system demonstrates the effect on differentiation by different
aminopurine derived cytokinins and led to the study of phenyl-urea derived cytokinin-
like compounds on tabacco (Tran Thanh Van and Trinh 1990) and in other plants (Ahn
et al. 1996, Bui Van Le et al. 1998a, 1998c, 1998e). Cell wall oligosaccharides shift the
flower program into vegetative bud program (Tran Thanh Van et al. 1985a, 1990b).
This suggests that some oligosaccharides, presumably released by growth regulators
treatment ofTCL (Tran Thanh Van et al. 1985a) or by pH (Cousson et al. 1989, Tran
Thanh Van and Mutaftschiev 1990), can act as signalling molecules. Marfa et al. (1991)
showed the active oligogalacturonides having less than 14 sugars. We have shown that
synthetic tetra and penta oligomers xyloglucan were active at the concentration of 10' 11
M in the induction of cell division of tobacco TCL and on wheat coleoptile elongation
(in collaboration with P. Sinai, unpublished results).
The implication of oligosaccharides in the differentiation of flowers, vegetative
buds, roots or callus suggests that cell wall hydrolases could be involved in
morphogenetic differentiation. Gene(s) identified as being expressed in early stages of
flower differentiation were the ones which presumably encode the following proteins:
basic ~ 1-3 glucanase, basic chitinase, a cell wall protein, extensine and osmotine
(Neale et al. 1990, Meeks-Wagner et al. 1989). Chitinase, glucanase and osmotine were
found to be activated also in plants resisting to pathogen attack (Legrand et al. 1987).
They were then described as "pathogen related proteins". This apparent similarity has
 59
made some authors doubt about the direct involvement of these proteins in flower
differentiation (Compton and Veilleux 1992). In fact, it has been shown. that these
proteins are also expressed in a reproductive organ, the pistil of healthy tobacco (Lotan
et al. 1989). However, Neale et al. ( 1990) have only determined gene sequences; the
presence of corresponding proteins and their functions have not yet been established.
Because of the homology between the FB7-2 gene family and thaumatin, the synthesis
ofthaumatin-like proteins was analysed by immunodetection and in situ localisation on:
a) TCL during flower-, vegetative bud and root differentiation, b) apical and root
meristems of plants derived from seeds at vegetative, prefloral and floral stages, and c)
petal, sepal, anther and ovary. The thaumatin-like proteins of 42 and 47 KD were
present only in flower TCL, floral meristem, anther and ovary (Richard et al. 1991 ).
Although they share homology with osmotin, a 26 KD protein found in water stressed
tobacco roots (King et al. 1986), the difference in their cellular localisation (in the
vacuole for osmotin and in the cytoplasm for thaumatin-like protein) suggests different
function. In the literature, it so happens that names were often given before the function
was identified.
The same situation can be applied to "chitinase" for which the same gene(s) is
activated in healthy plants without having any chitin as substrate. "Heat shock proteins"
constitute another example: they are also found in circumstances other than heat shock.

2.3.1.2. Preinduction or induction

Tobacco, a day neutral species, flower program can only be induced on TCLs excised
from floral branches and not from the base of the stem (Tran Thanh Van 1973a, 1973b).
This suggests that the TCL are preinduced to form flower. However, the transcripts of
genes identified in TCL-flower-program were not expressed in theTCLs at the time of
their excision from the donor plant, indicating that they were not preinduced for floral
differentiation. Furthermore, other morphogenic programs, (root, shoot buds, callus,
embryo and not exclusively flower) can be separately induced as well from the same
cell layer. These considerations suggest strongly that the differentiation of organs on
TCLs, in this case, flower, results from an activation of gene(s) during in vitro TCL
culture in specific conditions and not from a preinduced phase "inherited" from a
particular physiological stage of the donor plant.
In conclusion, with this model system, all patterns of morphogenesis displayed by a
plant can be induced either separately or in combination, each with a well defined
reversible/ irreversible phase. Other TCL systems were also programmable: Petunia
TCL formed floral shoots (instead of flowers) or vegetative buds or root (Mulin and
Tran Thanh Van, 1989), flowers were formed from cotyledon in Torenia which
exhibited a new pattern, the unicellular hair as found on Begonia TCL (Tran Thanh Van
et al. 1974a).
60
2.3.2. Comparison of morphogenic expression of monolayer, multilayer and stem of
Torenia fournieri

The following explants were cultured on the same medium containing indole acetic acid
(IAA) 1 J.lM and kinetin 1 J.lM: i) stem fragment of 10 mm long, 2.5 mm wide and 2
mm thick, ii) epidermis, iii) cortical layers, without epidermis, iv) epidermis and
cortical layers and v) epidermis peeled off the stem fragment and replaced, on the
subjacent cortical layers either directly or on an agar layer placed upon the subjacent
cortical layers. The stem fragment directly formed only shoot buds on the epidermis,
and their distribution was correlated with the localisation of vascular bundles i.e. close
to the two longitudinal borders of the explant. Their density decreased acropetally. The
explant (humber iii) composed of one cortical cells formed only root. This
morphogenetic potential was inhibited in the context of the stem fragment, under the
above mentioned conditions.
Explants (iv) and (v) formed shoot buds on the epidermis without polarity and root
formation while explants (i) did not respond. This suggests that the potential of shoot
buds formation of the epidermis required a contact with the cortical cells and that the
putative "contact" molecules were water soluble.
The existence of inter-tissue correlation, demonstrated for the first time on Torenia
TCL system (Chlyah et al. 1975), was confirmed by other authors on Helianthus
(Pelissier et al. 1990) and Beta vulgaris (Detrez et al. 1988).
The monolayer system also offered the possibility of direct observation of the whole
explant, from time 0 (Figure 1, Plate 3) to the first anticlinal cell division called cell
division center, to the periclinal ones (Figure 2-3, Plate 3), and the formation of domes,
the future bud primordia (Figure 4, Plate 3). The distribution of these centers, their
progression during time as well as their correlation with hair or stomata cells, were
analysed (Tran Thanh Van et al. 1974a). The first anticlinal cell division occurred 48 h
after culture. There were approximatively 10 cell division centers on day 3 and 200 on
day 6, with 50 to 100 daughter cells for each center. The temporal and spatial
observation of the entire epidermal cell population showed that: a) the epidermal cells
surrounding the guard-cells can divide (not the guard-cells), b) cell division did not
occur only in these cells, but also in other epidermal cells, c) cell division did not
happen in the basal hair cell as shown on Begonia epidermis, and d) a bud can be
initiated from divisions arising from one or two epidermal cells.
This is a good system for the in situ localization of specific morphogenetic markers.

2.3.3. Transverse (tTCL) oflris pallida leaves

Thin sections (0.3-0.5) mm made across a mature shoot (comprising 5-6 leaves), and
from the base towards the apex were cultured, the 5-6 tTCL leaf bases maintained
together or separated. The somatic embryogenic response was only observed in the first
case, on the tTCL of the young leaf bases (as it was observed on Bambusa tTCLs).
When cultured separately, each tTCL turned necrotic in the conditions tested. This
suggests that there could be a release of signalling factors from wounded leaf bases at
different stages of development and there could be a threshold reached only when the
tTCLs were maintained together.
 61
The cell division occurred on the responsive tTCL, formed directly epidermized
nodules with a deep yellow colour (Figure 1, Plate 4) on which somatic embryos were
initiated directly (Figures 2-4, Plate 4) and formed plantlets and normal plants (Schricke
et al. 1988). ·

2.3.4. Transverse TCL (tTCL) of Digitaria sanguinalis nodes and shoot tip for the
induction of somatic embryos and pseudo-embryo-structures (PES)

Digitaria sanguinalis is a C4 monocot which is used as a food crop in Central Europe

and India. Unlike three previous TCL model systems which comprise only
differentiated tissues, in this system, tTCLs (0.2-0.4 rom thick, 1 rom in diameter) were
cut across the stem from the base to the shoot tip of a 4-week-old-plantlet. Depending
on the level of their excision on the stem, some tTCL comprised only a thin section of a
mature leaf sheath surrounding the stem; other sections included in addition either
young stem or young leaves, leaf primordia and meristematic tissues. They were
cultured on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (5 or 10 J.!M)
with or without kinetin (I J.lM) for somatic embryogenesis (Bui Van Le 1997, Bui Van
Le et al. 1997) or a combination of 2,4-D (1 J.lM) and BA (10 J.lM) to induce a new
pattern, the pseudo-embryo structures (PES) (Bui Van Le et al. 1998b). The response,
of three events (friable callus, somatic embryogenesis and a new pattern PES) followed
an increasing gradient, from the base to the apex. For a given level of excision on the
stem, the TCL of the outer leaf, or sheath (chronologically more advanced than the
inner tTCL), differentiated only friable callus, regardless of the culture medium used
(Figure 1, Plate 5), the tTCL of the inner, therefore, younger leaf form direct somatic
embryos (Figure 1, Plate 5) one week after culture. Secondary embryogenesis occurred
concomitantly. From one tTCL, up to 250 somatic embryos can be obtained. These
embryos were developmentally blocked at the scutellum-cotyledonary stage - an
elongated white opaque embryo body with its scutellum fused to its cotyledon. Transfer
on a medium without growth substances or containing different doses of kinetin was
inefficient, while supply of BA (10 J.lM) or Zeatin (10 J.lM) or a combination of both,
without 2,4-D, allowed germination of the somatic embryos 2 weeks after tTCL culture.
We have shown that the reduction of 2,4-D to 1 J.lM (instead of 5 or 10 J.lM) and
addition ofBA (5 or 10 J.lM) with a sequence of dark (7 days)/ light from the beginning
of culture of the tTCL, these differentiated together with somatic embryos (similar to
the ones described above), new structures, the pseudo-embryos structures (PES). From
7 to 10 days, 40% tTCL formed dark green PES (Figure 2, Plate 5) which, by day 14,
developed directly into whole plants (Figure 4, Plate 5), which matured into normal
fertile plants forming mature seeds by day 35. From day 14, 100 % tTCl formed PES,
their progeny was tested for conformity during 2 generations (F1 and F2). These PES
presented a large meristem (250 to 3,000 J.lm) surrounded by a truncated coleoptile
(Figure 3, Plate 5). According to literature data, the truncated structure was often
associated with the presence of 2,4-D. However in our study, somatic embryos were
also induced by 2,4-D without truncated coleoptile.
The large meristem divided into several meristems, each meristem formed two or
three ring shaped leaves followed by elongated leaves with circular section and small
internodes before developing adult type leaves with flat lamina and trichomes (Figure 3,
Plate 5). Therefore, after a short embryonic phase, adult flowering plants can be
62
obtained directly without subculturing. One percent of plants flowered early without
transfer to soil.
The Digitaria tTCL system has several advantages: a) the possibility to obtain
somatic embryos, PES and friable callus allows us to conduct the analysis of the
mechanisms controlling these patterns of motiJhogenesis; b) PES, as most somatic
embryos, presumably originated from one cell and developed directly and rapidly into
mature fertile plants without callus phase and subculture, provide good systems for
genetic transformation. Gene(s) transfer was performed on Digitaria tTCLs and
transgenic plants obtained (Bui Van Le 1997, Bui Van Le et al. 1998d).

3. Application of TCL method for the induction of organogenesis and/ or somatic

embryogenesis on woody and non-woody species.

3.1. WOODY SPECIES

Woody species differ from most non-woody species with their accumulated
lignification over years, their morphology and long life cycle. However we consider, as
did previous authors (Tulecke 1987; Warm 1988) as woody species, Manihot esculenta
and vines such as Psophocarpus tetragonolobus.
We present organogenesis and somatic embryogenesis on TCL or thin sections
without reporting the culture media composition used (except in some cases); this can
be found in the original work (see references). Unless otherwise stated, the culture
conditions used were: 100 11mol m- 2 s-l for ligt intensity and 16 h photoperiod, 24 °C.

3.1.1. Organogenesis

3 .1.1.1. Poncirus trifoliata commonly used as rootstock

tTCL explants (0.2-0.3 mm) were excised from the shoot tips and internodes of one-
year-old-plants and cultured on a medium containing BA (10 J..LM) and TDZ (1 J..LM)
formed directly bud primordia from the parenchyma and medullary cells (20-30 buds
per tTCL after one week, nearly 100 shoot buds after 4 weeks). Rooted plants of 10 em
height were obtained after 6 weeks . TCL, excised longitudinally along the internode
also formed direct shoots (Figures 1-2, Plate 6) (in collaboration with T. Ha Nguyen
and A. Hong Le). A rapid true-to-type and high frequency regeneration method was
designed. The same method was applied with success on tTCL of Citrus aurantium
(Hoang et al. 1998).
 63
3.1.1.2 Psophocarpus tetragonolobus, a widely distributed tropical legume with high
resistance to pest and a good adaptation to poor soil.

Longitudinal TCLs were induced directly to form vegetative buds, compact callus,
friable callus or roots. Buds were obtained after 3 to 5 weeks dependant on the age of
the donor plant - 2 or 5 weeks, respectively (Trinh et al. 1981, Tran Thanh Van et al.
1986). Synthesis of a glycoprotein of 29 KD was detected in friable callus, compact
callus and organogenic TCL forming bud or root. However, for friable callus, the level
of glycoprotein released in the culture medium was higher than the intracellular level.
The reverse was found in compact callus and organogenic TCL (Meimeth et al. 1982
and Tran Thanh Van unpublished results).
The possible role of glycoproteins could be related to the one described in carrot
somatic embryogenic cell mutant (De Jong et al. 1993a, 1993b). They could also be
involved in cell surface signalling process; in Brassica, the arabinogalactan protein
(AGP) sequential and transient expression was correlated with development (Pennell et
al. 1991).

3 .1.1.3 Conifers

For most of the woody species and specially conifers, true-to-type cloning of mature
trees requires rejuvenation by grafting or serial cutting. Longitudinal TCLs excised
from the shoot tip of adult elite trees of Pseudotsuga menziesii, Sequoiadendron
giganteum, Sequoia sempervirens regenerated shoots without a callus phase. In
Sequoia, fertiles cones were obtained from tTCL rejuvenated shoot internodes which
developed spontaneously from the base of the main stem (Tran Thanh Van et al.
1985b). Monteuuis (1991) showed that meristem excised from a 100-year-old
Sequoiadendron giganteum shoot during bud break, cultured in vitro regenerated
juvenile lines which remained juvenile during 3 years.

3.1.1.4. Vitis

tTCLs 0.3 mm thick excised from stem of Vitis vinifera 7-day-old seedling formed
directly shoots. Direct and high frequency in regeneration of mature shoot buds was
obtained on medium containing naphthaleneacetic acid (NAA) and BA (in collaboration
with Hoang, Nhan, Do, Le Tran, Le and Tran Thanh Van unpublished results).
Shoot bud differentiation leads to viable true-to-type plants in constrast to somatic
embryogenesis, which especially in Vitis rupestris, showed abnormalities in 97 %
somatic embryos (Faure 1989). In Vitis longii, anthers and ovaries derived embryogenic
callus gave rise to secondary somatic embryos on a medium deprived of growth
substances (Gray and Mortensen 1987). Their morphology was slightly altered,
however, they germinated after addition of BA 1 f.!M in the culture medium, and these
results are similar to the ones observed in Digitaria.

3.1.1.5. Other reports

64
3.1.1.5.1 Populus

Shoot regeneration frequency was compared between: a) thin section or "micro-cross

sections" (of different thicknesses from 100, 200, 300, 400, 500 Jlm) made through a
leaf mid-vein, and b) different widths: less than 1 mm, 1-2 mm or larger than 3 mm for
thin sections of 400 Jlm. They were cultured on a medium containing BA 0.2 mg/1 and
NAA 0.01 mg/1 (Lee-Stadlemann et al. 1989). The results showed that thin sections
were more effective (25 times greater) than 1 em explant, the optimal thicknesses were
300, 400, 500; thin sections produced buds even when they were randomly oriented on
the medium. In contrast to larger explants, the initial explant orientation (pole proximal/
distal or abaxial/ adaxial) was not critical. The efficiency of bud regeneration on micro
cross-sections of 1-2 mm long was 3 times higher as compared to 3 mm long, and two
times higher when compared with less than 1 mm long. These results demonstrated the
importance of the explant size.

3.1.1.5.2 Garcinia mangostana

High frequency of direct shoot bud regeneration were obtained on thin transverse 3 mm
sections made across the leaf vein. The shoot buds developed at the distal pole of each
thin section cultured in the presence of BA (20 JlM). They formed roots and plantlets
after transfer to indole-3-byturic acid (IBA). The number of shoots was 50 times higher
on thin transverse sections than halfleaf explants (Goh et al. 1994).

3.1.1.5.3. Pinus radiata

Cotyledon sections of 3-5 mm long neoformed shoot primordia from single cells of the
first subepidermal cell layer which underwent cell division 3 days after culture, in the
presence of BA 25 11M (Villalobo et al. 1985). They mentioned that organogenesis
occured on each cotyledon tTCL, more intensely on the border where the cotyledon is
thinner. Shoots were obtained after 3 weeks of culture. The structures organized directly
from meristematic centers similar to those described for the monolayer of Torenia
(Chlyah et al. 1975).

3.1.2. Somatic embryogenesis

3 .1.2 .1 Bambusa glaucescens

Bambusa, a woody monocot, has been used for inducing somatic embryogenesis by the
TCL method. tTCL from young leaves sectioned through a bud from the base to the
apex were cultured on a mediuin containing 2,4-D (18 JlM). They formed after five
weeks in the dark, friable and mucilaginous callus or compact and epidermized
embryonic nodules, depending on the horizontal and vertical gradient: the inner tTCLs
leaf and the tTCLs of the basal part of this leaf were the most responsive for somatic
embryogenesis (Jullien and Tran Thanh Van 1994). The pale yellow nodules were
similar to the embryonic nodules differentiated on leaf tTCL of Iris pallida (see next
section, non-woody species). Flavonoi:ds were related to auxin transport. Polysaccharide
mucilaginous secretion was activated by 2,4-D in embryonic maize cells (Everett et al.
 65
1985), and often associated with somatic embryogenesis of Dactylis glomerata (Hahne
et al. 1988) and Pennisetum americanum (Vasil and Vasill982).

The embryonic nodules of Bambusa formed embryoids (embryo-like structures)

which failed to develop any further under the described experimental conditions. Their
protein pattern presented two bands which were absent in friable callus and in plant root
samples. Somatic embryos of Dactylis glomerata showed 11 specific polypeptides
(Hahne et al. 1988).

Somatic embryogenesis with subsequent plant development and in vitro flowering

were obtained in Bambusa glaucescens, Dendrocalamus giganteus and Dendrocalamus
strictus after a callus phase initiated on excised zygotic embryos or nodal segments of
10-day-old in vitro-grown seedlings (Rout and Das 1994).

3.1.2.2. Other reports

3.1.2.2.1 Cocos nucifera

" Fine transverse slices (0.2-0.5 mm thick)" of young inflorescences of different sizes
were used to induce nodular structures which developed later into shoot-like structures.
It was mentioned that "reducing the thickness of the transverse slices minimized
browning: 32 %browning in 1 mm slices and 11 % in 0.5 mm slices" (Sugimura and
Salvana 1989). Other authors reported that smaller explants have a better survival rate
than larger explants (Blake 1983). Gupta et al. (1984) regenerated plants by using
zygotic and somatic embryos.

3.1.2.2.2 Elaeis guineensis

Inflorescence sections of 1-2 mm produced, on a medium containing 500 ~M 2,4-D,

and 5 % (w/v) activated charcoal, hard yellow callus formed which differentiated later
into normal somatic embryos and plantlets (Teixeira et al. 1994).

3.1.2.2.3 Hardwickia binata, a tropical leguminous timber tree

Small segments of cotyledons ( 5 x 8 mm) without embryonic axis formed a greenish-

yellow embryonic nodular callus which resulted in somatic embryos and plantlets (Das
et al. 1995).

3.1.2.2.4 Manihot esculenta

Immature leaves of 3-6 mm size from 15 genotypes were excised and cultured in the
presence of different concentrations of 2,4-D. Somatic embryos developed directly on
the explants with 4-6 mg/12,4-D. Secondary embryogenesis was induced and long term
culture established and maintained up to 18 months. Plantlets developed from primary
and secondary embryos after addition of 0.1 mg/1 BA, 1 mg/1 GA 3 and lowering 2,4-D
concentration to 0.01 mg/1. Differences in response were reported with genotypes:
66
usually up to 15-20 plantlets emerged on one explant, 2 or 3 for the most reluctant
genotype (Szabados et al. 1987).

3.2. NON WOODY SPECIES

The TCL method was initially developed for non-woody species and extended to the
recalcitrant species such as monocot and woody species.
As this book deals with woody species, we will only report briefly the results
pertaining to organogenesis/ somatic embryogenesis on a few selected non-woody
species, in order to emphasize the common features of organogenetic/ embryogenic
expression at the tissue level when using the TCL method.
In complement to data presented in Table 2, some observations and results are
commented in the following part.

3 .2.1. Organogenesis

3.2.1.1. Amaranthus edulis

The 0.2-0.4 mm transverse thin cell layers (tTCL) excised from the apical and subapical
zone of Amaranthus seedlings neoformed directly on the epidermal surface bud
primordia after one week and shoots appeared after two weeks (Figure 5, Plate 3) on a
medium containing only TDZ 3 f..LM. Buds developed into normal plants after transfer to
a rooting medium containing either IBA (10 f..LM) or TDZ (0.1 f..LM) combined with BA
(10 f..LM) (Bui Van Le et al. 1998a). Besides the use of its seeds for food- its lysin rich
protein content: 13.6-18 % is higher than in cereals- this species presents a double
advantage: a) it is one of the rare dicots with the C4 photosynthetic type and b) mutants
deficient in phosphoenolpyruvate carboxylase, PEPC, a key enzyme of C02 fixation,
have been isolated (Dever et al. 1995). It is an excellent model for PEPC gene
complementation studies (in collaboration with J. Vidal and P.J.Lea).
Embryoid-like structures were obtained through the vein of leaf disc of A.
hypochondriacus cultured on 2,4-D, addition of coconut water to NAA (2 mg/1) and BA
(0.2 mg/1) initiated callus followed by buds (Flores and Teutonico 1986).

3.2.1.2 Other species

. Dicot species

a! Brassica napus. The TCL excised from the stem of Brassica napus formed directly
vegetative buds without an intermediate callus phase (in collaboration with Ian de la
Roche 1982 unpublished results). These results, confirmed later by Klirnaszewska and
Keller (1985), were applied to transformation using the 35S GUS vector (Charest et al.
1988, Pua et al. 1987; 1989).

b/ Vigna unguiculata. TCL explants (0.3 mm wide, 1 em long) were compared to

embryonic axis half explants for their ability to form a multishoot from a cotyledonary
node. It was found that buds were formed at a higher frequency (32 buds per explant)
 67
on the TCL than on the embryonic axis halves (23 buds). Furthermore, this last type of
explant formed a voluminous callus. The presence of callus may have an incidence on
the low survival rate of regenerated plants due to the presence of tracheids
differentiated between the regenerated shoot and root (in collaboration with Y.Zuily, M.
Cruz de Carvalho and A. Thu Pham).

c/ Saintpaulia ionantha. The 0.3-0.5 mm tTCL excised from mature leaf petiole, leaf
vein and lamina sections(# 5 mm2 ) of Saintpaulia ionantha formed directly vegetative
buds from the epidermic or parenchyma cells for leaf lamina. Up to 150 buds can be
obtained on one tTCL and over 30.000 plants could be produced from a leaf petiole.
They evolved into mature shoots which formed adventitious roots. The concentration of
TDZ (1 f!M) had a direct influence on the density of shoot neoformation (Figures 3-4,
Plate 6). This direct regeneration can lead to true-to-type shoots (In collaboration with
D. Hai Nguyen, K. Hoan Le, A. Hong Le). Other workers using 2 mm petiole cross-
sections cultured on 0.1 mg/1 NAA and 0.01 mg/1 BA (Bilkey et al. 1978) obtained
shoots after an intermediate callus.

d/ Beta vulgaris. The 0.3-0.5 mm tTCLs of leaf petioles neoformed directly buds from
the subepidermal layers. Detrez et al. (1988) used thin cell layer strips isolated from
different tissues of petiole (at the transition between petiole and leaflamina), a) dorsal
half petiole with vascular tissues, b) epidermal layer, c) sub-epidermal layers, d) the
latter two combined. Only the type (d) of explant formed viable shoots, the others were
not morphogenic including the leaf petiole with vascular tissues, i.e. comprising
cambial cells .

. Monocot species

Orchids. Among monocot, some orchids such as Phaleanopsis and at a lesser degree
Paphiopedilum remained recalcitrant to regeneration.
Phaleanopsis young leaf lamina explants (# 4 mm2 ) and tTCLs of young florals
stalks can be induced to form directly protocorms along the wounded edges of the
lamina and on the surface of the tTCL. Thin slices of these protocorms gave rise to
more protocorms. This method have allowed us since 1971 (unpublished results) to
multiply selected adult Phaleanopsis plants without destroying them by reverting the
floral meristems on the young floral stalk to vegetative stage. This reversion can be
obtained by a treatment at 27 oc. We have shown that the induction to flowering was
obtained after a treatment at 20°C-22°C during 8 to 12 weeks (Tran Thanh Van 1974a;
1974b).
The TCL method has allowed us to obtain direct regeneration of mature vegetative
shoot instead of protocorms on Rhynchostylis gigantea (Bui Van Le et al. 1998c) and
several Paphiopedilium (auroreum, holdeni, maluki, in collaboration with M. Tanaka)
using NAA, BA and CPPU or TDZ. Results on the last species are being extended to
Paphiopedilium delenati, an endemic orchid species from Vietnam.

3.2.1.3. Other reports

68
a/ Aranda. The 0.6-0. 7 mm thick tTCLs obtained by transverse sectioning of a single
shoot tip (6-7 mm) neoformed, on the same culture medium, 13.6 protocorms per tTCL
instead of 2.7 from a shoot tip. Up to 80.000 plantlets were produced in a year,
compared to 11.000 by the conventional shoot tip method. Thin sections showed a
higher frequency of regeneration (Lakshmanan et al. 1995).

b/ Musa (Banana and Plantain genotypes). Thin Cell Layer or micro-cross sections
(from 50 J.lm -using a microtome- to 500 J.lm) of banana and plantain leaf base were
used for callus and shoot bud induction. As in Populus cross sections, 400 J.lm was the
optimum thickness: an average of 15 plantlets can be obtained, compared to the 4-5
from standard protocols. With thinner micro-cross sections (50-300 J.lm), 1-2 shoots
were formed. This may be due to the smaller number of intact cells and/ or the
composition of the medium, especially with regard to the mineral concentration which
must be adapted to the TCL method (see suggested composition of the culture medium
for thin sections). The authors recommended the TCL method as a useful alternative to
standard protocols (shoot meristem, cell or protoplast culture) (Okole and Schulz 1996)
because of its direct and high frequency bud differentiation.

3.2.2. Somatic embryogenesis

3.2.2.1. Iris pallida, Sorghum, Oryza sativa, Panax ginseng

a/ Iris pallida, Sorghum, Oryza sativa. The tTCL method was applied to monocot
species: Iris pal/ida, Sorghum, rice where somatic embryos were obtained on mature
leaf tTCLs of Iris pal/ida (Schricke et al. 1988), young leaf tTCLs of Sorghum (Gendy
et al. 1996), and rice (Hoang et al. 1997) and scutellum tTCLs of rice. Plantlets and
fertile normal plants were regenerated. Pretreatment by 2,4-D (10 J.lM) during rice seed
germination followed by culture of tTCL excised on one-week-old seedlings resulted in
differentiation of pseudo-embryo structures (PES) similar to the ones reported on
Digitaria (Bui Van Le et al. 1998b) on a medium containing BA (1 J.lM) in addition to
2,4-D (in collaboration with AT Bui, NT Do My, A. Hong Le).

In Sorghum, the explant size was shown to be critical for somatic embryogenesis: 5
mm long explants were compared to tTCL of 1 mm, 0.5 and 0.3 mm thickness. No
morphogenetic response was obtained with 5 mm explants; 84 % of 0.3 mm tTCL and
72% of0.5 mm tTCL formed somatic embryos respectively (Gendy et al. 1996).

b/ Panax ginseng. tTCLs derived from Panax ginseng cotyledons (Ahnet al. 1996) and
longitudinal TCL from Soja biloxi (unpublished results) cotyledon formed somatic
embryos and plantlets (on a medium containing 2,4-D, BA and TDZ) which can be
grown into mature plants. Pre-treatment of seedlings by TDZ enhanced somatic
embryogenesis.

3.2.2.2. Other reports

a/ Helianthus annuus. As previously performed both on TCL mono/ multilayer of

Torenia stem (Chlyah et al. 1975) and TCL of sugar beet leaf (Detrez et al. 1988),
 69
different tissues of hypocotyl Helianthus annuus were compared for their
embryogenesis and callogenesis capacities: a) 2 em long segments of hypocotyl, b)
hypocotyl without epidermis, c) monolayer of epidermis d) subepidermal layer, e)
epidermis plus parenchyma layers similar to the tobacco or Torenia TCL. They were
cultured on a medium containing NAA 1 mg/1, BA 1 mg/1, and coconut water 20 ml/1.
The epidermal monolayer, the subsepidermal layers and the hypocotyl without
epidermis were not embryogenic. Only the TCLs comprising the epidermis plus
parenchyma layer and the hypocotyl segments were embryonic; the first type was more
embryonic than the sec;ond one. These results, similar to those obtained on Torenia,
confirm the existence of inter-tissue correlation and the release from inhibition exerted
by inner tissues on superficial tissues. They also confirmed our observation on tobacco
TCL where the subepidermal layer expressed its capacity to form flowers ( 10-12 days
after culture) once released from the inner tissues. Compared to tobacco stem
fragments, such a capacity was only expressed in cambial cells which formed 2-3
flowers after a callus phase 45 days after culture (Chouard and Aghion 1961).
The first somatic embryos differentiated on Helianthus TCL gave rise to secondary
embryos which developed into normal fertile plants.

b/ Hordeum vulgare, Zea mays. Thin cross sections derived from barley scutellum
formed somatic embryos with a gradient from the base to the top (Becher et al. 1992).
Sections of young plantlets leaves of Zea mays (3 to 4 mm long) formed embryogenic
callus on thin sections excised from the leaf base and direct somatic embryos on thin
sections from the leaf tip (Conger et al. 1987). In both cases, somatic embryogenesis
was expressed according to a gradient of the donor organ, whereas on the thin section
no polarity was observed.
 -..J
TABLE 2. Morphogenetic response in thin cell layer cxplants of non-woody species 0

Species Size ofTCL Organ source Plant growth regulators Morphogenetic References
response

Dicot:

Amaranthus edulis *0.2-0.4 mm Stem 3 ~M TDZ Bud Bui Van Le et al. 1998a

Begonia rex Epidermal monolayer Leaf vein Bud, Root, Chlyah and Tran Thanh Van 1974, Tran

Unicellular hair Thanh Van et al. 1974a

*0.3 mm Petiol Bud Bui Van Le and Tran Thanh Van

(unpublished results)

Beta vulgaris *0.3-0.5 mm Petiol 3 mg/1 NAA + 3 mg/1 BA Bud Detrez et al. 1988

Brassica napus 1-5 mm, 61ayers Leaf vein Klimaszewka and Keller 1985

Chrysanthemum morifolium *0.3 mm Petal, Stem Tran Thanh Van et al. (unpublished results)

Cichorum intybus witloof Epidermal monolayer Leaf Bud Liebert and Tran Thanh Van 1972

'Zoom"

Helianthus annuus 1 mmx !Omm Hypocotyl I mg/1 NAA + I mg/1 BA Somatic embryos Pelissier et al. 1990

Nautilocalyx lynchei 1 mmx lOmm Leaf vein Bud, Root Tran Thanh Van and Drira 1970

Nicotiana tabacum See pages 7-10

*: tTCL
Panax ginseng *0.2mm Cotyledon 5J.1M 2,4-D+ O.IJ.lM BA Somatic embryos Ahn et al. 1996

+ 0.1 J.1M Zeatin

Petunia hybrida lmmxiOmm Inflorescences I J.1M IAA + I J.1M Ki Flower, Bud Mulin and Tran Thanh Van 1989

Saintpaulia ionantha *0.5mm Leaf-petiol and IJ.1MTDZ Bud Bui Van Le et al. (unpublished results)

inflorescences

Soja biloxi - - Jullien and Wyndaele 1992

Torenia fournieri - - - See page 10

Vigna unguiculata 0.5-1 mm Cotyledonary 10J.1MTDZ Bud Bui Van Le et al. (unpublished results)

node

Monocot:

Aranda *0.6-0.7 mm Shoot tip 2.75J.1MNAA Protocorm Lakshrnanan et al. 1995

Digitaria sanguinalis - - - See pages 11-12

Echinochloa colona 3-5mm Leaf 4.44 J.1M BA + 4.64 J.1M Somatic embryos Sarnantary et al. 1997

Ki + 8.05 J.1M NAA

Heliconia psittacorum *0.8-1 mm Shoot tip 40- 80 J.1M 2,4-D Bud Goh et al. 1995

Hordeum vulgare *1 mm-2mm Scutellum 10 J.1M 2,4-D Somatic embryos Becher et al. 1992

Iris pal/ida *0.3 -0.5 mm Leaf Schricke et al. 1988

Lilium longiflorum *0.3-0.5 mm Pseudo-bulblet 1-3J.1MCPPU Bud Bui Van Le et al. 1998 c

*: tTCL
-...]
 -....]
Musa and Plantain *0.4mm Shoot tip IO!!MBA Bud Okole and Schulz 1996 N

Oryza sativa *0.3-0.5 mm Leaf 10 11M 2,4-D + 5!!M Somatic embryos Hoang et al. 1997

NAA+ IO!!M BA

Stem, leaf 10 11M 2,4-D + I 11M BA Bud Bui Van Le et al. (unpublished results)

Paphiopedilium auroreum, *0.3-0.5 mm Leaf, floral - Tran Thanh Van, Bui Van Le and M.

P. holdeni, P. Maluki. P. stalk Tanaka (unpublished results)

delenati

Pha/aenopsis amabilis *0.3-0.5 mm Leaf, floral 5!!MNAA+20 !!MBA Tran Thanh Van et al. 1974a

stalk

Rhynchostylis gigantea *0.3-0.5 mm Shoot tip 3 11M BA + 3 11M TDZ Bud primordia Bui Van Le et al. 1998c

Sorghum bicolor *0.3 mm Epicotyl 3 11M 2,4-D + 300 11M Somatic embryos Gendy et al. 1996

dicamba or 0.1 11M K

Zea mays *3-4 mm Leaf 10 11M 2,4-D Somatic embryos Conger et al. 1987

* :tTCL
 73
4. Biochemical/ Molecular markers of morphogenesis

The analysis of mechanisms which control somatic embryogenesis/ organogenesis in

plants requires: a) developmental mutants or b) model system(s) on which the
morphogenesis can be strictly programmed.
Although the carrot embryogenic suspension cell system does not cover
morphogenetic patterns other than somatic embryogenesis, the embryogenic carrot
mutant cell lines, as well as the tobacco TCL, emphasize the importance of cell wall
composition and function. The subepidermal cells of tobacco TCL undergo changes in
their shape before the onset of cell division (Nassogne et al. 1985); activation of cell
wall enzymes (chitinases, glucanases) was detected (Gendy 1991). Furthermore, it has
been reported that 32 KD protein, a glycosylate acidic endochitinase excreted by the
embryogenic carrot suspension cells, can rescue the thermosensitive carrot mutant cell
line tsll arrested at the globular stage. Transgenic carrot plants bearing the acidic
chitinase encoding gene, now available, could be a useful tool for studying the
incidence of this gene in somatic embryogenesis/ organogenesis of the hypocotyl
explant (Gilbert et al. 1996).
It was thought that the EP2 lipid transfer protein gene expressed by carrot
embryogenic cells is involved in cutin synthesis (Sterk et al. 1991 ).
Another protein involved in carrot somatic embryogenesis is a cationic peroxidase
isoenzyme of 38 KD which catalyses the cross linking of glycoproteins and wall
polysaccharides phenolic side chains and therefore could inhibit cell wall expansion. In
tobacco TCLs, we have shown that isoperoxidase activity changed with the
morphogenic program. The cationic peroxidase activity was higher in the root and in
the flower program, compared to the callus and the vegetative bud program (the
"embryoid" program was not analysed), and the pattern of peroxidase zymogram of
callus was different from the three other morphogenic programs (Thorpe et al. 1978).
Kay and Basile (1987) analysed in soluble and bound fractions of peroxidases
tobacco TCL and found a correlation between histological changes and differentiation
of morphogenetic program: 3 to 6 peroxydase isozymes correlated with cell division
and differentiation of tracheids and 7 others to callus formation.
Other markers of morphogenesis such as polyamines (Appelbaum et al. 1988; Bagni
et al. 1993; Tran Thanh Van and Gendy 1993a) were identified on tobacco TCL.
Molecular markers of TCL morphogenesis were reviewed previously (Tran Thanh
Van 1991; Tran Thanh Van and Gendy 1993a).
The strict control of a pure morphogenetic program on tobacco TCL system allowed
us to better identify of specific markers for a given morphogenetic program.
As for gene expression during zygotic embryogenesis in woody species most of the
data were related to embryo development and not embryo initiation; for example late
embryogeny abundant (LEA) genes cloned in cotton (Baker et al. 1989) were thought to
encode the hydrophilic class of proteins which protect the embryo during seed
desiccation (Huyhes and Galau 1989). Other genes encoding proteins involved in
storage protein and in oil biosynthesis and storage, displayed similar patterns of
expression during the maturation stage (Dunstan et al. 1995, Dong and Dunstan 1996,
Dong et al. 1996).
74
On Picea abies embryogenic cell lines, a correlation between specific extracellular
protein and embryo morphology (group A with dense embryogenic region and group B,
less embryogenic) was reported (Mo et al. 1996). These 28, 66 and 85 KD proteins
were thought to be chitinase and peroxidases involved in somatic embryogenesis of
Daucus carota (de Vries et al. 1988).
On Picea glauca 28 cDNAs were isolated from cotyledonary embryos by
differential screening against immature embryos and non embryonic tissue. With the
exception of 12 eDNA clones with no known homology and some other clones
homologues to conifer storage protein genes, others eDNA clones were lea like (Dong
and Dunstan 1996). However their transcripts were observed at stage 1 of embryos,
which were uncommun for lea like proteins. Two eDNA (PgEMB5 and 25) were ABA
responsive and non-lea-like. They were induced after suspension cultured embryogenic
tissues were fed with ABA (Dong and Dunstan 1997).
Developmental and abscisic acid gene regulation during zygotic and somatic
embryogenesis has been reported by Hatzopoulos (1993). Early biochemical and
molecular markers ofTCL morphogenesis were reviewed previously (Tran Thanh Van
1991, Tran Thanh Van and Gendy 1993a, Compton and Veilleux 1992)

5. Reflection on regeneration and prospects

In much of reported works, there is a considerable variation in the culture media used
and many of the factors used empirically as "inducing factors" lack specificity. During
in vitro culture, the composition of the culture medium becomes more complicated, its
pH changes as does cell permeability, since the inoculum interacts with the medium.
Because the target cells (which recognise the morphogenetic signal(s) the nature of
which are unknown) as well as the responsive cells are not identified, it is better to have
an inoculum as small as possible. In this regard, isolated cells would be a good system
provided they are more homogenuous and also other morphogenetic programs with
somatic embryos be induced. However, their simplicity is only transcient since isolated
cells develop clumps of cells or callus; while in a cell layer system, cell-to-cell
correlation is maintained although tissue to tissue correlation can be altered.
The choice made by the cell(s) between somatic embryogenesis and organogenesis
constitutes another problem. An interesting example was described by Tabei et al.
(1991) on melon where low concentrations of 2,4-D resulted in caulogenesis and at
high concentrations induced somatic embryogenesis. On Digitaria and rice tTCL, a new
pattern, the pseudo-embryogenic-structures (PES), develops rapidly into adult plants
and this can be induced directly without subculture. We have shown that the acquisition
of embryogenic potential versus PES depends on the ratio between 2,4-D and BA.
Bergervoet et al. (1989) reported that the use of NAA instead of 2,4-D shifts
embryogenesis to organogenesis in cucumber. Osmotic pressure can induce
caulogenesis on tobacco callus (Ha Ngoc and Tran Thanh Van 1979) or can direct
organogenesis/ embryogenesis (Ozias-Akins and Vasil1982).
The TCL system, a simplified system which can mimic an entire plant, is at present
a good system for the study of fundamental and applied aspects of regeneration since: a)
there is a high frequency of direct and rapid regeneration of embryo/ organ in various
woody and non-woody species, b) their reduced size allows a maximal exposure of
responsive cells and wounded cells to the "inducing" factors and foreign DNA in
 75
transformation process. The gene(s) to be inserted can be introduced by Agroinfection
(enhanced by wounding), by biolistic particles or even by electroporation. Furthermore,
the putative transformed cells/ tissues are more directly exposed to selecting agents,
thus reducing the number of false resistants plants.
On very thin sections, direct observation of cytological, histological, biochemical
and molecular changes by in situ localisation techniques is possible, for example:
Populus micro cross-section 50 f.!m (Lee-Stadlemann et a!. 1989), monolayer of
Toreniafournieri (Chlyah eta!. 1975). On Camelliajaponica (Pedroso and Pais 1995a,
1995b ), a woody species where different morphogenetic potentials were expressed on
specific areas of the same leaf, thin sections (50-500 f.!m)made on these specific area
allow one to: a) follow in vivo changes in different elements and ions, in cell wall
properties and composition during the course of the corresponding morphogenetic
program: embryogenesis, callus, root, bud; b) determine the characteristics of the cell
wall of cells which were situated precisely at the frontier of two zones expressing
different morphogenetic potential; and c) reprogram them by changes in callose/ cutine
or other cell wall compounds synthesis by an over expression of specific cell wall
enzymes. Furthermore, transgenic plants harbouring sense/ antisense genes of interest
could be very useful, since it has been reported that cell wall changes were important
markers in the embryogenic/ non embryogenic process in this interesting experimental
model.
Lastly, with the TCL method which requires less space, less culture medium volume
(15 f.!l per TCL ), and small containers, more combinations of factors can be analysed at
the same time.
With regard to woody species, thin sections could be made through out-growth buds
or axillary buds induced to growth in vitro after breaking their dormancy. TCL can also
be made on these out-growth buds after several cycles of in vitro culture. We have
shown in Petunia TCL that flowering can be induced to differentiate directly after
several cycles of in vitro culture (Mulin and Tran Thanh Van 1989). The regeneration
success is a limiting factor to plant improvement either by breeding or genetic
transformation.
Genes of interest cloned in non-woody species could be used for the genetic
transformation of woody species, for example, to cite only a few, the Arabidopsis LFY
gene (Schultz and Haughn 1991) for early flowering, when over-expressed in the Aspen
tree (5 months instead of 8-20 years) (Weigel and Nilsson 1995), wax biosynthesis
genes (CERl-6) {Aarts eta!. 1995, Negruk eta!. 1996) involved in epidermis structure,
cell wall enzymes genes: ~ 1-3 glucanase, chitinase, some peroxidases involved in cell
wall structure and auxin catabolism as well as genes involved in ethylene synthesis. But
more importantly than for the herbaceous species, woody species have specific
problems that we must learn to solve for environment safety because of their long life
cycle, their important production of pollen and the uncontrolled cross pollination.
However, because of the irreplaceable role played by woody species on the earth
(photosynthesis, production of biomass, bioremediation) and because of their
recalcitrance to regeneration, more effort should be devoted to the analysis of the
mechanisms controlling regeneration in woody species, in order to improve them either
by breeding or transformation.
76
6. Concluding remarks

By using TCL, direct and high frequency organogenesis/ somatic embryogenesis can
also be obtained in woody species. Among other cell(s), the one(s) selected for building
an organised structure will change the cell to cell relation by: a) remodelling its
membrane properties/ permeability through the distribution of its surface molecules
(such as the arabinogalactan protein, AGP), and b) thickening its cell wall. It was often
described for normal somatic embryogenesis to occur that an asymmetric division takes
place leading to two inequal daughter cells (one cytoplasm-rich, the other more
vacuolated) and that the position as well as the orientation of the cell plate is
determinant. The differentiation of an epidermis (with callose, cutine) indicates that
organised structures are being differentiated. These observations point to the
importance of structural and functional changes -in relation to the treatment by the
putative "inducing factors"- in the cell wall compartment (including its ionic
environment), and the plasmodesmata. The cell wall is a dynamic interface between the
cell and its environment.
Inoculum size, together with excision and wounding, are also important factors to be
analysed. We suggest that, on the basis of a limited combination of culture medium,
growth substances and environmental conditions, the inoculum size parameter should
be assayed, since only then can failure be attributed to the genotype parameter.
Empirically, we are tempted to put under genotype all other undefmed and, therefore,
uncontrolled parameters. Less empirically however, superficial cells such as epidermal
or subepidermal cells can be reprogrammed in order to differentiate flower or root
which normally originate only from perivascular tissues.
We have learnt with the TCL method that: a) new morphogenetic potentials which
have up to now been masked by the ontogenic differentiation process, can be revealed
and controlled; and b) instead of being limited by regeneration (i.e. generation de novo
of a previously existing structure), one can reprogram differentiated cell(s) into
multiprogrammable patterns according to a spatial and temporal sequence determined
by the investigator (and not by the ontogenetic program). A wider application of TCL
method on woody species would help to overcome their apparent recalcitrance to
regeneration.

Acknowledgements: The authors are thankful to scientists of our group, especially Dien
N., H. Chlyah, A. Chlyah-Arnason, H. Trinh, A. Cousson, T. Do My, C. Gendy, M.
Jeanneau, L. Richard, E. Sadik and to other groups for their contribution in developing
the TCL method. Dr L. Celnikier is deeply acknowledged for reading the manuscript.
 77
PLATE 1: Tobacco TCL
Figure 1: A tobacco TCl at time 0 (x 6).
Figure 2: Scanning electron micrograph of five flower primordia differentiated directly on the surface (arrow)
of the TCL (x 75).
Figure 3: Flowers (#50) developed on one tobacco TCL at day 14 (x 4).
Figure 4: Flower program: Homogenuous response of flower differentiation on 20 TCL explants (x 2).

PLATE 2: Tobacco TCL (continued)

Figure 1: Full size functional flower developed (among# 50) at day 25 (x 4.5).
Figure 2: Roots neoformed directly on a TCL at day 18 (x 8).
Figure 3: Root program: Homogenous response of root differentiation on 20 TCL explants on liquid medium
(x 1).
Figure 4: Vegetative bud program:# 500-700 vegetative buds on one TCL (x 5.5).

PLATE 3: Torenia TCL (Figures 1-4), Amaranthus edulis (Figure 5).

Figure 1: TCL epidermis monolayer before inoculation (1, 2, 3: cells surrounding the stomata guard cells, cb:
-basal cell of glandular hair).
Figure 2: TCL epidermis 48 hours after culture: anticlinal and periclinal cell divisions (arrows).
Figure 3: Close up of figure 2.
Figure 4: Future bud primordia (arrows) arising directly on the epidermis.
Figure 5: Direct neoformation of shoot buds on a tTCL of Amaranthus on a medium containing TDZ 3 !!M.
Stage 14 days (x 25).

PLATE 4: Iris pal/ida tTCLs

Figure 1: Yellow epidermized nodules differentiated on tTCL of Iris pal/ida young leaf bases (x 4.4 ).
Figure 2: Differentiation of clumps of embryos and isolated embryos from the epidermized nodules (x 6).
Figure 3: Close up of Figure 2 (x 12).
Figure 4: Somatic embryos and plantlets (arrow) (x 6).

PLATE 5: Digitaria. sanguinalis tTCL

Figure 1: tTCL cultured on 10 !!M 2,4-D. Friable callus initiated from the tTCL of the outer leaf surrounding
white opaque somatic embryos differentiated from the tTCL of the inner and younger leaf (x 30).
Figure 2: tTCL cultured on 1 !!M 2,4-D and 10 !!MBA: Formation of dark green pseudo embryo structures
(PES) from the inner leaftTCL (x 30).
Figure 3: Scanning electron micrograph of a PES (x 150).
Figure 4: Conversion of green PES into plant without subculture (x 15).

PLATE 6. Poncirus trifolia}a (Figures 1-2), Saintpaulia inonantha (Figures 3-4).

Figure 1: Differentiation of shoot buds on a tTCL (x 4).
Figure 2: Scanning electron micrograph of shoot buds formed on a tTCL (x 100).
Figure 3: Scanning electron micrograph showing the extrusion through the leaf epidermis of an epidermized
structure and a bud primordia (x 100).
Figure 4: Shoot buds differentiated directly without a callus phase on a leaflamina (x 20).
78

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 3. SOMATIC EMBRYOGENESIS IN TROPICAL FRUIT TREES

1NasimAkhtar, 2Nishi Kumari, 2Shashi Pandey, 2HussainAra, 2Madhulika Singh,

2 UmaJaiswa/, 2 VijayS. Jaiswa/ and 3Shri. M Jain
1IIT-BREF Biotek, Indian Institute ofTechnology, KHARAGPUR- 721 302 (WB), India. Phone & Fax: (091)

03222 77890, E-mail : nasim@hijli.iitkgp.ernet.in. 1 Laboratory of Morphogenesis, Centre of Advanced

Study in Botany, Department of Botany, Banaras Hindu University, VARANASI-221 005, India. E-mail:
E"or! Bookmark not rkfUJed 3Plant Breeding and Genetics Section, FAO!JAEA Joint Division, International
Atomic Energy Agency (IAEA), Wagramer Strasse 5, PO Box 100, A-1400, Vienna, Austria; Phone: 43 1 2600
21623; FAX: 431 26007; E-mail: SMJain@iaea.org

Contents:

1. Introduction.
2. Recent Progress in Tropical Fruit Tree Somatic Embryogenesis.
3. Induction of Somatic Embryogenesis.
4. Development of Somatic Embryos.
5. Morphological Stages and Developmental Asynchrony.
6. Factors Controlling Induction of Somatic Embryogenesis.
7. Maintenance of Somatic Embryogenesis.
8. Maturation and Germination of Somatic Embryos.
9. Soil Transfer, Acclimatization and Growth of Somatic Plants.
10. Genetic Stability of Plants Regenerated Through Somatic Embryogenesis.
11. Current Limitations and Future Prospects.
12. Concluding Remarks.
13. Acknowledgements.
14. References.

1. Introduction:

Food and shelter are two basic needs of life. Since the earliest times, humans have
enjoyed eating fresh fruits, as indicated by the frequent mention of fruits throughout
the recorded history. The cultivation of fruits such as datepalm and pomegranate, has
been dated back to 7000 to 3500 BC, respectively (Singh, 1985). In today's world fruit
play a vital role in our diet due to its nutritional importance (Nagy eta/., 1990). Many
fruit species are trees and with a few exceptions, e.g. banana, pineapple and
strawberry, are woody. Genetic improvement of fruit crops by conventional plant
breeding achieved through the selection of superior genotypes from genetically variable
population derived from sexual recombination and seedling propagation (Bose, 1985).
Tree architecture, floral morphology, prolonged juvenile period and irregularities in
93
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 93-140.
© 2000 Kluwer Academic Publishers.
94
bearing habit limits the improvement of fruit trees through traditional breeding
methods (Ni.ijar, 1977). Since most important horticultural characteristic, conferred by
complexes of genes, the genetic integrity of many important fruit tree cultivars has
been maintained by means of vegetative propagc:ttion. Using the conventional methods
of vegetative propagation such as, cutting, layering, and grafting, substantial number
of plants are produced. However, many tropical fruits and spice trees (e.g. clove) can
not be propagated asexually (Litz, 1984b). Due to the increasing significance of
tropical fruits for food and export income (Underhill, 1993), there has been a recent
expansion of efforts and resources devoted to tropical fruit cultivar improvement. It is
almost imposible to keep pace with the increasing demand of tropical fruits only
through the conventional methods of plant propagation. For improvement of tropical
and subtropical fruits by somatic cell genetics, a highly efficient regeneration system is
a prerequisite (Litz and Jaiswal, 1991). Many programs are attempting to integrate
emerging biotechnologies with conventional breeding methods to facilitate and
expedite the fruit tree cultivars improvement (Litz, 1997). These include in vitro
culture and micropropagation (Grosser, 1994), production of homozygous parental
lines by chromosome doubling of haploids (Zhang and Lespinasse, 1992), regeneration
of interspecific and intergeneric hybrids for root stock development (Gmitter et a/.,
1992; Ochatt eta/., 1992), production of artificial seeds (Akhtar and Jaiswal, 1994;
Bajaj, 1995a, b, Gray, 1987a, c; Redenbaugh, 1993), in vitro selection of somaclonal
variation having enhanced diseased resistance (Hammerschlag, 1992; Jain et a/.,
1997), genetic transformation for specific horticultural traits (Gradner, 1993; Oliveira
eta/., 1996)
Currently, over one thousand plant species have been regenerated de novo via
organogenesis and somatic embryogenesis (Bajaj, 1995a, b; Bhojwani and Dhawan,
1990, 1995; Evans eta/. 1983; Hammerschlag and Litz, 1992; Jain eta/., 1995a, b, c).
Somatic embryogenesis has several distinct advantages over organogenesis (Ammirato,
1983; Litz and Gray, 1992; Terzi and LoSchiavo, 1990; Thorpe, 1988, 1990). Initially,
micropropagation of several temperate fruit and nut crops dominated the field and has
made great progress (Hammerschlag, 1986a, b; Hansman and Owens de Nova, 1986;
Hutchinson and Zimmerman, 1987 and Zimmerman, 1985, 1986, 1991). The
regeneration of tropical fruit trees through in vitro technique was first reprted by
Maheshwari and Rangaswami (1958). Infact Stevenson (1956) preceeded in attempting
the induction of somatic embryogenesis in citrus. Efficient regeneration through
induction of somatic embryogenesis, for many of the important tropical fruit species is
a relatively recent phenomenon. Much more progress has been made with tropical and
subtropical fruits particularly in banana, citrus, mango, papaya etc. in the present
decade (Hammerschlag and Litz, 1992).
The purpose of this review is to contemporary interpretation of the process of
somatic embryogenesis while emphasizing achievement involving tropical fruit crop
species. Attention has been focused on the influence of factors such as genotype,
explant, media composition, growth regulators, carbohydrate nutrition and treatment
periods and the interactions of some of these factors on the induction of somatic
embryogenesis, maitenance of embryogenic cultures, development, maturation and
 95
germination of somatic embryos as well as transplantation of somatic seedlings in the
author's laboratory or derived from experimental data published by other authors.

2. Recent Progress In Tropical Fruit Tree Somatic Embryogenesis:

The present understanding of somatic embryogenesis has arisen from several different
experimental approaches using various explants in a number of plants. A partial list of
tropical fruit tree somatic embryogenesis is presented in table 1. Several excellent
reviews has been rwritten on various aspects of the subject in the recent past by
Ammirato (1983), Bajaj, (1995a, b), de Jong eta/. (1993), Dudits eta/. (1991), Emons
(1994), Jain eta/. (1995), Litz (1985), Litz and Gray (1992), Litz eta/. (1985), Merkle
eta/. (1990), Raghavan (1983, 1986), Sharp eta/. (1980), Tautorus eta/. (1991), Terzi
and LoSchiavo (1990), Thorpe (1988), Tisserat eta/. (1979), Tulecke (1987), Wann
(1988), Zimmerman (1993).

3. Induction of Somatic Embryogenesis:

Redetermination of a cell, i.e. dedifferentiation, enable it to respond to certain

environmental or cultural stimuli. In other words, the cell acquires competence and
can be triggered to express eother an organogenic or embryogenic potential. This
change in the commitment of a cell is properly referred to as an inductive event (Litz
and Gray, 1992). Backs-Huseman and Reinert (1970) provided evidence that somatic
embryogenesis is initiated in suspension cultures when vacuolated cells under go an
unequal division to form an embryogenic and a parenchymatous cell. Kohlenbach
(1978) suggested that stimulation of densely cytoplasmic cells from a population of
rapidly dividing vacuolated parenchymatous cells is equivalent to induction. The
temporal separation of acquisition of competence and induction has been demonstrated
by Ammirato (1985), who reported that carrot petioles can form callus following a 3-
day pulse with 2,4-D however, 4-day pulse is necessary in order to induce an
embryogenic callus. According to Thorpe (1988) for all cases of organized
development in vitro, there has been an interplay between the explant, the culture
media and the culture conditions. To achieve optimum response, understanding the
interaction of these factors is essential.

4. Development of Somatic Embryos:

Somatic embryo development have always been assumed to have a single cell origin
however, differentiation of a single isolated cell into somatic embryos has not yet been
demonstrated (Raghvan, 1986). Williams and Maheswaran (1986) suggested that
somatic embryos could arise either from single cell or from a group of cell. With some
96
species histological studies have confirmed the probable single cell origin of somatic
embryos when they developed directly either from an explant or as secondary embryos
from the surface of older somatic embryos. There are distinct histomorphological
differences between embryogenic and non-embryogenic cell (Lee et al., 1997;
Sannasgala et al., 1990; Schumann et a/., 1995; Schwendiman et al., 1988, 1990;
Tisserat and De Manson, 1980). Embryogenic cells are small and isodiametric and are
metabolically very active, with intense RNA synthesis, dense cytoplasm,
microvacuolated, large nuclei, prominent nucleoli and are filled with numerous free
ribosomes, mitochondria, plastids, endoplasmic reticulum and starch grains. Non-
embryogenic cells are large, highly vacuolated with thin strands of cytoplasm, low
number of ribosome mitochondria and little endoplasmic reticulum and have thicker
cell wall (Ammirato, 1987; Emons, 1994; Raghvan, 1983; Thorpe, 1988). Somatic
embryogenesis in cherry was associated with considerably higher proportion of linoleic
acid in both neutral lipid, and phosphatidylcholine together with high proportion of
palmitic acid and phosphatidylethanolamine and phosphatidylinositol in comparision
to non-embryogenic cultures (Reidiboym and Grenier, 1999). Development of somatic
embryos has been observed either directly from explants or indirectly from the
intermediate callus (Wann, 1988). Embryogenesis from zygotic embryos in guava (Fig.
1A) (Akhtar, 1997), mango (Ara, 1998; Pandey, 1998) and Sterculia (Fig. IB)
(Pandey, 1998) are induced directly without the intermediate callus phase (Fig. 1).
Somatic embryos in mango (Ara, 1998; Pandey, 1998) are also observed indirectly
from the intervening proembryogenic calli induced from nucellus. Whereas, in
Terminalia arjuna (Nishi, 1997) intervening calli induced from leaf discs explants
resulted in the formation of proembryogenic globular structures. The development of
somatic embryos occurs directly from the surface cells of these proembryogenic
globular structures (Fig 1C). Furthermore, the epidermal and subepidermal layer of
zygotic embryo contributed to the development of somatic embryos in Sterculia (Fig
1B) and Psidium (Fig 1A) respectively. Among large number of epidermal and
subepidermal cells or the cells of callus mass, only a few of the cells are competent to
differentiate and develop into somatic embryos. Hence, during somatic embryogenesis
large number of cells are lost in contrast to zygotic embryogenesis in these species and
in other systems (de Jong et a/., 1993; Emons, 1994; Zimmerman, 1993). Similarly
differentiation of several cell layers of graded staining property in the ground tissue
(Fig. 1A) might be due to the formation of internal gradation and the differential
activity of cells in response to the exogenously applied factors (growth regulators).
Rodriguez and Wetzstein (1998) have found that both NAA and 2,4-D enhanced cell
division activity in the subepidermal cell layers. Further, differences were observed in
mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus
growth and embryo morphology. Callus induced with NAA had embryogenic regions
composed of homogeneous, isodiametric, meristematic cells. In contrast, tissue induced
on media with 2,4-D had more intense and heterogeneous regions of cell division.
Proliferating cell regions were composed of meristematic cell interspesed with callus
and involved more extensive regions of mesophylls (Rodriguez and Wetzstein, 1998).
The epidermal cells have been reported to develop into somatic embryos directly from
 97

Figure 1: Histological sections showing the ontogeny of developing somatic embryos in tropical fruit and tree
species. A. Cross section of zygotic embryo explant of Psidium guajava showing a developing globular somatic
embryo and the gradation in staining behaviour of various layers (EP= Division product of epidermal layer
transformed into callus tissues; SB= Division product of subepidermal layer directly contributing to somatic
embryo formation; SS= Suspensor cells connecting somatic embryo and the subepidermal progenator cell(s).
(bar= 114 fUll). B. A portion of cross section of zygotic embryo explant of Sterculia alata showing stages of
globular somatic embryo development from the epidermal cells (bar= 57 fUll). C. A portion of cross section of
proembryogenic globular structure showing the development of globular somatic embryo from the outer most
layer in Terminalia arjuna (bar=57 fUll).
98

the explant like cotyledons in date palm (Sundersan eta/., 1993), Feijoa (Cruz eta/.,
1990), and papaya (Fitch and Manshardt, 1990). In certain fruits such as mango
(DeWald et a/., 1989a, b; Litz et a/., 1982, 1984, 1993; Monsalud et a/., 1995),
pomegranate (RajBhansali, 1990), and Euphoria Iongan (Litz, 1988) embryogenesis
was reported either from the calli, or through the proliferation of the proembryogenic
masses (PEMs).

5. Morphological Stages and Developmental Asynchrony:

Asynchrony in development is a common feature noted in almost all the published

reports on somatic embryogenesis. In general, development of globular or heart stage
somatic embryos occured during 2-10 weeks of culture initiation. Development of
advanced and mature stages of somatic embryos requires another 2-4 weeks in culture
depending upon the species. As a consequence several distinct morphological stages of
somatic embryos observed developing from the explant surface at a given point of time.
In guava, seven discernible morphological stages have been recognized on the same
explant surface even after 10 weeks of culture initiation (Fig. 2A, B). These stages in
guava are: (i) globular stage: (50-200 J..l.m), (ii) Early heart stage, (iii) Late heart stage
(100-300 J..l.m), (iv) Early cotyledonary stage (200-700 J..l.m), (v) Late cotyledonary stage
(500-1000 J..l.m), (vi) Short torpedo stage (1.0-1.5 mm) and (vii) Elongated torpedo
stage (2.0-3.0 mm). In mango, cotyledons are very large (4-7 em long), and continue to
grow even during maturation and germination. Similarly criterion has been used for
the differentiation of somatic embryos in mango (Fig. 2C-E), Dendrocalamus strictus
(Fig. 2F), Terminalia arjuna (Fig. 2G-H) and Sterculia alata (Fig. 2 I-L). All these
stages of somatic embryos differ in their state of maturation with respect to size.

6. Factors controlling Induction of Somatic Embryogenesis:

6.1. EXPLANTS:

Exudation of phenolic compounds in the culture medium turns media brown leading to
the death of explants, is a serious problem in many recalcitrant plants particularly in
trees. The zygotic embryos are free of phenolic exudation in guava (Akhtar, 1997) and
Sterculia (Pandey, 1998) and served as the best starting explant material for induction
of somatic embryogenesis. Addition of antioxidant compounds which adsorb the
phenolics such as polyvinyl pyrolidone (PVP) (Babbar and Gupta, 1986a, b), activated
charcoal (Table 1) and ascorbic acid have been beneficial to reduce the adverse effect
of phenolic compounds. But these compounds were not successful in controlling media
browing and establishment of cultures in guava and Sterculia. Zygotic embryo has
 99

Figure 2: Developing somatic embryos in tropical fruit and tree species. A. Early stages (G-globular; H-heart; C-
cotyledonary} somatic embryos developing from zygotic embryos explant of guava (bal=SOO f.UII). B. Advanced
stages (ST-slwrt torpedo; ET- eloll8ated torpedo) somatic embryos of guava (bar= I mm). C. Globular somatic
embryos of mango separated from proembryogenic calli (bar=SOO f.UIIl' D. Heart (H) and early cotyledonary (C)
stages sornat.U; embry011 of IMII8'I ~SOO f.UII). E. Maturing cotyledonary stages somatic embryos of mango
(bar=l mm). F. Developing somatic embryos (G-globuar; S-scutelar; M- mature scutelar) of Dendrocalamus
srictus (bar= I mm). G. Globular (G.E.) somatic embryos of Terminal/a arjuna (bar=SOO f.UII). H. A mature
cotyledonary stage somatic embryo ofT. arjuna (bar= I mm). I. Globular somatic embryos of Sterculia a/ata
(bar=250 1-iffi). J. An early heart stage somatic embryo of S. alata (bar=250 1-iffi). K. Maturing cotyledonary
stages somatic embryos of S. alata (bar=l mm). L. Mature cotyledonary (C) and torpedo (T) stages somatic
embryos ofS. alata (bar=l nun).
100
been the starting explant material for the induction of somatic embryogenesis in many
recalcitrant species like feijoa (Cruz et a/., 1990), banana (Cronauer-Mitra and
Krikorian, 1988; Escalant and Teisson, 1988), Cocos (Magnaval et al., 1997), coconut
palm (Gupta eta/., 1984), datepalm (Reuveni,1979; Reynolds and Murashige, 1979;
Tisserat, 1979; Zaid and Tisserat, 1984), mango (Ara, 1998; DeWald et al., 1989a, b;
Litz et al., 1982, 1984, 1993; Pandey, 1998; Pliego-Alfaro eta/., 1996a, b), Musa (Lee
eta/., 1997; Sannasgala eta/., 1990), papaya (Dhir and Yadava, 1995), pomegranate
(RajBhansali, 1990) and Prunus (De March et a/., 1993; Garin et a/., 1997).
Embryogenic cultures have been induced from the nucellus of mango (Ara, 1998;
DeWald eta/., 1989a, b; Litz et al., 1982, 1984, 1993; Monsalud eta/., 1995; Pandey,
1998; Pliego-Alfaro eta/., 1996a, b), Citrus, Jaboticaba, Carica and Syzygium etc.
(Table 1). In Terminalia arjuna (Nishi, 1997) embryogenic callus were induced from
leaf discs, as other explants failed due to excessive phenolic exudation. Other explants
such as ovules, shoot tips, basal leaf sheath, rhizome, seedlings, peduncle, cell
suspension and protoplast have been successfully utilized for induction of somatic
embryogenesis in various species (Table 1).

6.2. CULTURE MEDIA:

A number of media are being used for the induction of embryogenic cultures (Table 1).
However, over 70% of successful cases have reported the use of Murashige and
Skoog's (MS) basal salts (Murashige and Skoog, 1962) or the derivatives thereof
(Ammirato, 1983; Litz and Gray, 1992; Thorpe, 1988). These media contain high level
of ammonia and nitrate salts. Often the ratio of nitrate and ammonia in the medium is
important for induction (Niedz, 1993, 1994). Nitrogen in the form of amino acid such
as glutamine has been shown to enhance the process in mango and papaya (Table 1).
Other amino acids have been ineffective for the use as nitrogen source except for the
aspargine used in Carica (Dhir and Yadava, 1995) and cystein in Musa (Lee et a/.,
1997). Sharma eta/. (1984) found that phosphate and thiamine in the medium have
regulatory effect on induction in Phoenix dactylifera. Polyamines in the nutrient
medium can have a stimulatory effect on the induction of somatic embryogenesis
(Minocha and Minocha, 1995). Addition ofpolyamines (putresine and spermine) to the
culture media promoted the somatic embryogenesis in coconut and papaya (Adkin et
al., 1998). The complex organic nutrients of undefined composition such as coconut
water, casein hydrolysate, malt extract etc., have also been used and have been
demonstrated to affect the induction in several species (Table 1). The importance of
reduced nitrogen, certain amino acids, amides as well as potassium and phosphate has
been discussed in some details by Tisserat eta!., (1979) and Ammirato (1983).

6.3. CARBOHYDRATE NUTRITION:

Sucrose is the most commonly used carbon source in the culture medium. However, in
some cases galactose (Cabasson eta!., 1997), lactose (Jumin and Nato, 1995), maltose
(Dhir and Yadava, 1995), glucose and fructose (Canhoto and Cruz, 1994) and
mannitol (Kunitake eta/., 1991) have enhanced induction as well as the production of
 101

morphologically normal somatic embryos when used in combination with sucrose in

the medium. Complex organic addenda of undefined composition (Table 1) have often
been beneficial to enhance the induction and development of somatic embryos in many
species. In guava (Akhtar, 1997), other sugars were ineffective when used as a sole
carbon source for induction of somatic embryogenesis. Further, when added in
combination with sucrose in the culture medium, the induction rate of somatic
embryogenesis reduced considerably in the presence of glucose, maltose and lactose,
while fructose, sorbitol and manito) were found inhibitory for induction of somatic
embryogenesis (Akhtar; 1997). However, mannitol and sorbitol had a stimulatory effect
on somatic embryogenesis in otl;ler species (Litz, 1986). In some species, maltose was
effective both in the presence and absence of sucrose (Strickland et a!., 1987).
Similarly glycerol was found stimulatory for induction of somatic embryogenesis in
Citrus (Ben-Hayyim and Neumann, 1983).
6.4. PLANT GROWTH REGULATORS:

Plant growth regulators (PGRs) are indispensable for the induction of somatic
embryogenesis. However, the type and concentration of PGR(s) vary with the species
(Table 1). The process of induction of somatic embryogenesis in different plant species
shows three different patterns of sensitivity to PGRs: 1) Presence of growth regulator(s)
continues during all the phases of somatic embryo development. 2) growth regulators
are necessary during the induction phase, however, they need to be excluded from the
medium as development of advanced stages is sensitive to them. 3) the induction of
somatic embryogenesis undoubtedly occurred in the presence of growth regulator(s),
but the development of somatic embryos beyond globular stage is insensitive to them.
Induction of somatic embryogenesis in tropical fruit trees has been initiated
following explanting onto medium containing 2,4-D or other synthetic auxins, NAA,
dicamba (DIC), or picloram (PIC). An auxin, usually 2,4-D and NAA has been used in
over 50% and 25% of cases, respectively (Evans eta!., 1983; Litz and Gray, 1992).
There has been cytological and histological differences between cell division acitivity
of proliferating embryogenic cells and calli and in the morphology of developing
somatic embryos induced on different auxins (Rodriguez and Wetzstein, 1998).
Although cytokinin, have sometimes been incorporated in the induction medium, they
are probably not critical for success (Table 1). Sometimes low auxin concentration or a
sequential transfer of cultures to gradually reduced concentration is also beneficial
(Ammirato, 1983; RajBhansali eta!., 199la, b). However, a combination of auxin and
cytokinin found beneficial to achieve most efficient induction in some species (Cruz et
al., 1990; Nishi, 1997). Gibberellin affect the rate of embryo development, but has no
effect on frequency of embryogenesis (Sharp et a/., 1980). Abscisic acid has been
reported to affect either the initiation of embryo development or lower the rate of
embryo development, plays a special role in somatic embryo maturation and
germination (Ammirato, 1974, 1983, 1987; Sharp et a!., 1980). Ethylene was
ineffective to induce somatic embryogenesis. Tisserat and Murashige (1977) have
demonstrated the inhibition of embryogenesis by some growth factors, ethylene,
ethanol, Ethophon etc. Litz et a/. (1993),have reprted that somatic embryogenesi is
102
inhibited in the cultured nucellus of polyembryogenic ("Paris") and monoembryonic
("Keitt") mango by 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate
precursor of ethylene, however, inhibition was more pronounced for the
polyembryogenic type nucellus over the range of ACC concentration used. Results of
these studies indicated that inductive (monoembryonic mango) and permissive
(polyembryonic mango) somatic embryogenesis are distinguishable on the basis of
their response to ethylene stimulation and inhibition. Inductive somatic embryogenesis,
i.e. acquisition of embryogenic competence or totipotency is inhibited by ethylene
suppression in mango. Permissve somatic embryogenesis from pre-embryogenic-
dtermined cells in mango nucellus, by contrast, is inhibited by the stimulation of
ethylene production. Further, the sensitivity of explant tissue varies with types and
concentration of growth regulator(s), the genotype, the nutritional status and
physiological age of explant, the exposure time of explant with a particular growth
regulator. Some of these factors have been studied in different interactive combination
for some species (see following sections). Simlarly, increase in ethylene concentration
reduces callus growth and somatic embryogenesis in coconu and papaya. However,
addition of aminoethoxyvinylglycine (AVG) and silver thiosulfate (STS) improvrd
somatic embryogenesis by 100% and 67% respectively (Adkin et al., 1998).

6.5. TREATMENT PERIOD:

The duration of explant exposure to a growth regulator is an important factor for

-.....
Figure 3: Interaction of explant treatment period and 2,4-D concentration on relative efficiency (EE) of
embryogenesis in guava (Akhtar et al. 1997). Ten week post anthesis zygotic embryos were treated for 0, 2, 4, 6,
8 10,12, 14, 18, 28,38 and 60 days with different concentrations (0.0, 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0 and 5.0
mgll) of 2,4-D containing (induction) medium and subsequently transferred to hormone free (development)
medium. The MS basal medium supplemented with 3% sucrose in both induction and development phase. Note
an eight days treatment of zygotic embryos with 0.5 mg!l of2,4-D showed maximum (highest peak) efficiency of
embryogenesis.
 103
induction as well as for the production of normal somatic embryos. In guava (Akhtar,
1997), the continuous exposure (60 days) of zygotic embryos showed high efficiency of
embryogenesis in the presence of 0.01 mg/1 2,4-D, BA, KIN and thidiazuron (TDZ);
1.0 mg/1 IAA, NAA and 3.0 mg/1 IBA. When explants were transferred to growth
regulator free medium after exposure to growth regulator for different time intervals, a
minimum of 8-days time period was required for inducing somatic embryogenesis with
the higher efficiency irrespective of the type of growth regulator used (Fig. 3). Auxins
proved to be the highly efficient whencompared with cytokinins and TDZ. Most
efficient embryogenesis was induced with 2,4-D treatment.
Such interactive effects have not been observed in many fruit species.
However, in mango, (Ladet al., 1997), have observed the temporal effect of 2,4-D on
induction of somatic embryogenesis directly from the nucellus explant. Among 0, 7,
14, 21, 28, 35, 42, 49, 56 and 63-days of pulse treatment, a minimum of 7-14 days
pulse with 2,4-D was necessary for induction of somatic embryogenesis. Optimum
level of induction was achieved with 28-days of pulse treatment, while embryogenesis
was continued but in the least efficiency, with the maximum of 56-days pulse treatment
with 2,4-D. Similar temporal effect of growth regulators was also reported by Ara
(1998) and Pandey (1998) in mango nucellar and zygotic embryo culture. In Feijoa
(Canhoto and Cruz, 1994, 1996; Cruz et a/., 1990), induction of somatic
embryogenesis was reported in continuous presence of 2,4-D as well as with a
minimum of 5-day pulse treatment of zygotic embryos with 2,4-D. Somatic
embryogenesis has also been reported by the continuous presence of PGRs in papaya
(Fitch and Manshardt, 1990) and pomegranate (RajBhansali, 1990). In Prunus avium
L. (De March et a/., 1993) a minimum 10 days were required for the induction of
somatic embryogenesis. In Elaeis guineensis different treatment periods enhanced the
production of somatic embryos (de Touchet eta!., 1990, 1991). In Terminalia arjuna
embryogenic calli was initiated within one week of culture and about 3 weeks were
needed for callus proliferation prior to the development of somatic embryos on
hormone free medium (Nishi, 1997). In Cocos nucifera L. (Verdeil eta!., 1994,Verdeil
and Buffard-Morel, 1995), a gradual reduction in 2,4-D concentration induced
formation of normal somatic embryos.

6.6. INTERACTION OF EXPLANTS AGE AND SUCROSE:

Often the age (Wetzstein eta!., 1989) or maturation stage of the explants (Karunaratne
eta!., 1991) is an important factor for the induction of somatic embryogenesis. Hence,
the optimization of developmental or physiological age of the explant is a prime factor
for inducing efficient somatic embryogenesis in new species. Further the basic
nutritional (sucrose/carbohydrate) requirement of any tissue varies considerably with
the developmental age (Monnier, 1990). Moreover, sensitivity of a tissue to growth
regulator(s), particularly 2,4-D, is much dependent on sucrose concentration in the
104
culture medium (Thompson and Thorpe, 1987). Akhtar (1997) studied the interaction
of explant age and sucrose concentration during induction and development phase in
guava. When zygotic embryos of different ages (7 -14 weeks post anthesis) are cultured
under the optimal conditions (8 day treatment with 0.5 mgll 2,4-D) and the induction
medium was supplemented with various concentrations of sucrose (0-30%) and only
3% sucrose in the development medium, 10-week-old zygotic embryo explants showed
highest efficiency of embryogenesis in the presence of 10% sucrose during induction
(Fig. 4). Further, enhancement in the induction of somatic embryogenesis was
achieved with a 5% sucrose in the culture media during induction and development
phase (Akhtar, 1997). Similarly, somatic embryo development could be stimulated
with sucrose and tissue age in Citrus (Kochba eta/., 1974). High sucrose also induce
stress environment in the culturemedium and inhibits precocious germination of
somatic embryos (Thompson and Thorpe, 1987). Zygotic embryos older than 10-week
required high concentration (10-15%) for the induction of somatic embryogenesis.
Although the induction of somatic embryogenesis was less efficient from zygotic
embryos of younger than 10-weeks, they required even higher concentration (15-20%)
of sucrose in guava {Akhtar, 1997) (Fig. 4). Hence, a high sucrose concentration (4-
6%) at the induction and developmental phase has always been beneficial for an
enhanced production of somatic embryos in many plant species.

Figure 4: Interaction of explant age and sucrose concentration on relative efficiency (EE) of embryogenesis in
guava (Akhtar et a/.1997). Zygotic embryos of 7, 8, 9, 10, 12, 14 weeks post antbesis were treated for 8 days
with 0.5 mg/1 of2,4-D in MS- basal induction medium supplemented with 0, 1, 3, 5, 10, 15, 20, 25 and 30%
sucrose and subsequently transferred to 2,4-D free development medium supplemented with only 3% sucrose.
Note that 10-week old zygotic embryos showed enhanced efficiency of embryogenesis when induction medium
contained 10% sucrose as compared to 3% sucrose containing medium as shown in figure 3.

6.7. INTERACTION OF 2,4-D AND SUCROSE:

 105
There has been several- fold increase in the induction process when 2,4-D and sucrose
are used in guava somatic embrygenesis (Akhtar, 1997). Efficiency of embryogenesis
enhanced by 8 and 5 fold with 1.0 mg/1 2,4-D and 10% sucrose in the induction and
3% sucrose during the development phase (Fig. 5). However, the best induction of
somatic embryogenesis was noted with 1 mg/1 2,4-D and 5% sucrose during both
induction and development phase (Akhtar, 1997).
The developmental stage of explant is also an important factor to influence
the induction of somatic embryogenesis Mangifera (Ara, 1998; Pandey, 1998). In
guava, addition of any of the growth regulator such as BA, KIN, TDZ, IAA, IBA or
NAA either singly or in pairs in any combination negate the effect of 2,4-D (Akhtar,
1997). Similarly different combinations of auxins and cytokinins enhanced the
induction of somatic embryogenesis in many other tropical fruit species (Beloualy,
1991; Canhoto and Cruz, 1994, 1996; Cruz eta!., 1990; Chen eta/., 1987; Jordan and
Velozo, 1996; Magnaval et at., 1995, 1997; Mandai eta/., 1995; RajBhansali, 1990).
Hence, it is evident from the foregoing discussion on guava (Akhtar, 1997),
that any variation in the optimal conditions, i.e. concentration of 2,4-D (or the growth
regulators), treatment period, sucrose concentration and the explant age would greatly
affect the frequency and intensity as well as development of convertible somatic
embryos and thereby the efficiency of somatic embryogenesis. Similar finding has also
been confirmed in Citrus recently by Carimi et at. (1998).

Figure 5: Interaction of 2,4-D and sucrose concentration on relative efficiency (EE) of embryogenesis in guava
(Akhtar eta/., 1997). Ten week old zygotic embryos were treated for 8 days with various concentrations (0.0,
0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0 and 5.0 mg/1) of2,4-D in MS- basal induction medium supplemented with 0, 1,
3, 5, 10, 15, 20, 25 and 30% sucrose and subsequently transferred to 2,4-D free development medium
supplemented with only 3% sucrose). It is clear from the figure that in the presence of 10% sucrose in the
induction medium and 3% sucrose in the development medium, 1.0 mg/1 of 2,4-D showed about 8 and 5 times
enhanced efficiency of embryogenesis as compared to figure 3 and 4 respectively.
106
6.8. OSMOTIC STRESS:

Osmotic stress can enhance and sometimes even induce somatic embryogenesis
(Etienne et al., 1993; Kamada et a/., 1989; Litz, 1986; Mahon et a/., 1996). The
osmotic
stress could also account for the observed effect of high sucrose in the medium (Pliego-
Alfaro, 1996a, b). The salt and water stress provided by adding NaCl and polyethylene
glycol (PEG) in the medium are unable to induce somatic embryogenesis in the
absence of 2,4-D but slightly enhance the potential of 2,4-D in guava (Akhtar, 1997).
Other stress caused by sodium hypochlorite treatment of explant (seeds) has the
potential for induction of embryogenesis even in the absence of 2,4-D in the medium
(Akhtar, 1997). But the treatment reduced the potential of 2,4-D when applied in
combination. Nutritional stresses in the medium, have marked effect on induction of
somatic embryogenesis in kiwifruit (Oliveira and Pais, 1991, 1992). Similarly,
nutritional and low oxygen stresses provided in the form of much diluted strength of
liquid medium (half to one eighth strength of original MS medium composition) and
with explant submerged in the liquid phase (non-shaking condition) has very little
potential for induction of somatic embryogenesis in the absence of exogenously
supplied growth regulator in guava (Akhtar, 1997). A low sucrose concentration in the
medium along with these stresses has no stimulatory effect indicating thereby, that
sucrose in the medium acts mainly as carbon source and only to a lesser extent as an
osmoticum.
6.9. MEDIUM STRENGTH:

Strength of the basic medium composition has been found to affect the induction of
somatic embryogenesis. In guava (Akhtar, 1997}, half strength MS basal medium
supplemented with 10% sucrose and 2.0 mg/1 2,4-D increased the potential of somatic
embrygenesis. The diluted strength of medium decreased the induction potential of
somatic embryogenesis. The effect of media composition and strength on induction of
somatic embryogenesis has been demonstrated in many species, e.g. Actinidia (Oliveira
and Pais, 1992), Carica (Fitch and Manshardt, 1990), Citrus (Gill et a/., 1995;
Gmitter et a/., 1992; Kochba and Spiegel-Roy, 1973), Cocos (Gupta et a/., 1984),
mango (Ara, 1998; DeWald eta/., 1989a, b; Monsalud et al., 1995; Pandey 1998},
Myrciaria (Litz 1984a), Prunus (Scorza et a/., 1990). Similarly, Veramendi and
Navarro (1996) studied the effect of composition of nutrient medium, sucrose
concentration and other physical factors on induction of somatic embryogenesis in
datepalm.

6.10. PHYSICAL STATE OF MEDIUM:

Somatic embryogenesis more oftenly induced on semi-solid medium. However, state of

the medium (semisolid or liquid) is often being important for the maintenance and
 107

proliferation of embryogenic cultures. In mango (Ara, 1998; DeWald eta/., 1989a, b;

Litz et a/., 1982, 1984, 1993; Monsalud et a/., 1995; Pandey, 1998), initial
inductionwas achieved on agar solidified medium while, proembryogenic cultures
proliferate more rapidly in the liquid medium. They further observed· that the
requirement for the different physical state of the medium to induce somatic
embryogenesis varies with genotypic and is cultivar specific. In guava (Akhtar, 1997)
and Feijoa (Cruz et a/., 1990), agar solidified medium supports the induction of
somatic embryogenesis efficiently, while in liquid medium much reduced level of
induction was observed. The medium type and state enhanced the somatic
embryogenesis induction in Carica (Fitch 1993; Litz and Conover, 1982, 1983), Citrus
(Ashi eta/., 1985; Grosser and Gmitter, 1990a, b), Feijoa (Canhoto and Cruz, 1994),
Musa (Marroquin eta/., 1993), Phoenix (Veramendi and Novarro, 1996) and Prunus
(Burgos and Ledbetter, 1993). In Termina/ia arjuna (Nishi, 1997), proembryogenic
cultures were most successfully induced and proliferated on agar solidified culture
medium.

6.11. EXPLANTING SEASON:

Very little information is available on the effect of explanting seasons on induction of

somatic embryogenesis. Further, the developmental age and the morphological stage of
explant are not the same factors determining the induction as discussed earlier. The
morphological stage of zygotic embryo observed in 10-week-old fruit during favorable
season, could also be achieved quite earlier during the off-seasons in case of guava
(Akhtar, 1997). But the embryogenic results of the different explanting seasons
(fruiting seasons) in guava when processed for time series analysis showed
insignificant effect on the induction of somatic embryogenesis (Akhtar, 1997). In T.
arjuna varying degree of embryogenic calli were produced when leaf disc explant of
different seasons were inoculated, but maximum embryo development occurred from
the fresh flushes of leaves during the month of April-May (Nishi, 1997). In Cocos
nucifera L. (Magnaval et al., 1995), effect of various sampling dates has been noted on
amino acid composition of embryogenic calli. Litz and Conover (1981a, b) observed
some effect of season on induction of somatic embryogenesis in Carica. Hence, the
physiological age and the developmental stage of the explant, their nutritional status
and relation to the environmental factors are need to be critically determined for
inducing somatic embryogenesis.

6.12. TEMPERATURE:

The effect of temperature on the induction process has not been studied well in details.
However, low temperature of the explant treatment can enhance the induction of
somatic embryogenesis in Carica (Litz and Conover, 1981a, b) and in other woody
plants (Tulecke and McGranahan, 1985). When guava cultures (Akhtar, 1997) were
incubated at different temperatures, somatic embryogenesis could be seen between 15-
35 °C, with best induction occured at 25±2 °C. It is also observed that fairly constant
108

temperature during the incubation proved beneficial. Any large vanatton in

temperature (ambient ± 5 °C} during the incubation drastically reduced the efficiency
of the process. Earlier, Babbar and Gupta (l986a, b) applied prechilling treatment to
anthers of guava and were unable to induce embryogenesis.

6.13. CULTURE ILLUMINATION:

Other cultural environments also influence the induction of somatic embryogenesis.

The process has been reported under a variety of light and dark regimes. In general
darkness proved better particularly during the induction phase. In mango, since there
was an excessive phenolic exudation from the explant sources, the cultures were
incubated in complete darkness during induction, development and the proliferation of
somatic embryos (Ara, 1998; DeWald eta/., 1989a, b; Lad et a!., 1997; Litz et a!.,
1982, 1984, 1993; Mathews and Litz, 1992; Monsalud et a!., 1995; Pandey, 1998;
Pliego-Alfaro et a/., 1996a, b). However, in guava, condition of culture illumination
has no significant effect on the induction of somatic embryogenesis (Akhtar, 1997).
The intensity of light has been shown important in Citrus (Nito and Iwarnasa, 1990;
Vardi et a/., 1986), Iongan (Litz, 1988), mango (Jana et a/., 1994), peach (Raj
Bhansali eta!., 1990, 199la, b) and pomegranate (Raj Bhansali, 1990).

6.14. GENOTYPE:

Genotype has strong influence on the induction of somatic embryogenesis in tropical

fruit trees and other plant species. Different genotypes of Psidium guajava L. showed
variation in frequency, intensity and efficiency of somatic embryogenesis as well as
maturation and germination of somatic embryos (Akhtar, 1997). Variation in somatic
embryogenesis among various genotypes of different species have been reported by
Ara, 1998; Carimi et a/., 1998; Cheliak and Klimaszeswka, 1991; DeWald et a/.,
1989a, b; Jana eta!., 1994; Komatsuda and Ohyama, 1988; Komatsuda et a/., 1991;
Litz eta!., 1982, 1984, 1993, 1998; Monsalud eta/., 1995; Pliego-Alfaro eta!., 1996a,
b; Tetu eta/., 1990; Vardi eta/., 1982; Witjaksono and Litz, 1997). In Carica (Litz
and Conover, 1981a), variation in somatic embryo production was related to different
sex types.

7. Maintenance of Somatic Embryogenesis:

High concentrations of auxin(s) often lead to abnormal somatic embryo morphology

after a long induction period (Cruz eta/., 1990; Dublin eta/., 1991), poor germination
and development of secondary somatic embryos (Ara, 1998; DeWald eta!., 1989a, b;
Litz et a!., 1984; Pandey, 1998). Secondary embryogenesis, generally, occurs when
primary somatic embryos fail to mature normally and give rise to successive cycles of
embryogenesis, most commonly, from the epidermal cells of cotyledons or the
hypocotyl (Merkle eta/., 1990). William and Maheshwaran (1986), reported the loss of
 109
integrated control results in the secondary embryogenesis as budding from the
epidermal cells of mature and germinating somatic embryos. This process has often
been categorized as the anomalous development and has been considered as one of the
major problems in controlling somatic embryo maturation and germination. However,
when it is prevented or controlled and/or stimulated at will, it can be served for embryo
cloning through maintenance of recurrent cycle of embryogenesis that will greatly
facilitates mass propagation, metabolite production and genetic transformation. The
maintenance of such recurrent cycle of embryogenesis is termed as repetitive,
accessory, proliferative or secondary embryogenesis (Merkle eta/., 1990) and has been
successfully utilized for rapid multiplication of peach and nectarine (Raj Bhansali et
a/., 1990).
In standard embryogenic systems the unit of subculture is globular
_proembryogenic masses (PEMs), and are maintained most successfully in liquid
suspension culture which proliferate by repetitive budding of PEMs. In addition to
carrot, this has been described in Citrus (Neidz, 1993; Ohgawara eta/., 1991; Saito et
a/., 1991), papaya (Litz and Conover, 1983), Juglans regia (Polito eta/., 1989), mango
(Ara 1998; DeWald et a/., 1989a, b; Litz et a/., 1982, 1984; Pandey, 1998), peach
(RajBhansali, 1991a, b), Musa spp. (Marroquin et al., 1993), oil palm (de Touchet et
a/., 1991). In liquid suspension culture, rapidly dividing, proembryogenic cells are
continuously shed from large proembryogenic masses which can then repeat the
process. Embryogenic mango cultures are maintained on the semisolid medium and in
the liquid suspension cultures containing 2,4-D. Initially, proembryogenic masses are
maintained on semisolid medium and required to subcultur at 4-5 day intervals in
order to avoid blackening (not the necrosis) of the tissue (Ara, 1998; Litz eta/., 1993;
Pandey 1998). Embryogenic cultures of most of the cultivars is successfully maintained
in the liquid medium containing growth regulators (Ara, 1998; DeWald eta/., 1989a,
b; Litz eta/., 1993, 1995; Mathews and Litz, 1992). Enhanced growth rate has been
achieved in the liquid medium and considerable differences are observed in subculture
frequency among the cultivars (Ara, 1998; Litz et al., 1993, 1995; Pandey, 1998).
Suspension cultures consist of proliferating globular proembryos and with the
increasing time in suspension, these proembryogenic globular masses enlarge and
eventually lose their ability to form individual somatic embryos. Somatic embryo
development beyond the globular or early heart stage is inhibited by 2,4-D (Ara, 1998;
DeWald eta/., 1989a, b; Pandey, 1998) in the semisolid medium. For further recovery
of somatic embryos, embryogenic suspension cultures are sieved through fractionation
fabrics and subcultured in the liquid or semisolid medium devoid of auxins. Subculture
onto the semisolid medium without 2,4-D is found deleterious if embryogenic cultures
are maintained in liquid suspension (Litz et a/., 1993). Whereas, Ara (1998) and
Pandey (1998) found equally good response in liquid and semisolid medium without
2,4-D even if cultures were maintained in suspension. Moreover, hyperhydric somatic
embryo development has also been noted in the liquid medium as compared to
semisolid medium (Ara, 1998; Monsalud eta/., 1995; Pandey, 1998).
Similarly, the maintenance of proliferating Citrus embryogenic cultures are
more efficient in suspension culture medium containing 2,4-D or a mixture of auxin
110

and cytokinins (Gmitter et a/., 1990; Kochba and Spiegel-Roy, 1977; Saito et a/.,
1991 ), consisting entirely of globular proembryos at various stages of development
(Button et a/., 1974; Cabasson et a/., 1997). Sometimes, maintenance of
embryogenesis is achieved by initial exposure of explant or proembryogenic masses to
a very high auxin concentration (40.0 mg/1 2,4-D), followed by maintenance of
recurrent systems using a lower levels of auxin (5.0 mg/1 2,4-D) which prevents the
transition from proembryogenic to embryogenic development (Blake, 1990; Finer and
Nagasawa, 1988; Karunaratne, 1989; Verdeil eta/., 1994, Verdeil and Buffard-Morel,
1995). In oilpalm, embryogenic induction is observed on nodular calli (Schwendiman
et a/., 1988, 1990) and fast growing callus (Ahee et a/., 1981; Hanower and
Pannetier, 1982; Smith and Thomas, 1973) in the presence of auxin concentration
usually lower than that used during the callogenesis. Auxins like 2,4-D and NAA can
be employed separately and cytokinin may be added in the medium at low
concentration (Wooi, 1990). These treatments resulted in developing polyembryonic
cultures with a group of meristematic cells and somatic embryos at various stages of
development (Duval eta/., 1995). In T. arjuna (Nishi, 1997), embryogenesis was
most successfully maintained through the subculture of proembryogenic structures
along with calluses strictly on the semisolid medium. In guava embryogenic potential
was maintained through the subculturing of somatic embryo using as explanting unit to
subsequent generation starting as the new cycle of induction (Akhtar, 1996, Akhtar
and Jaiswal, 1994). The potential for recurrent cycle of embryogenesis varies
considerably with different stages of somatic embryos in this species (Akhtar, 1997).

8. Maturation and Germination of Somatic Embryos:

Despite their morphological similarities with zygotic embryos, somatic embryos are not
anatomically and biochemically identical (Schumann et a/., 1995) and germinate
irregularly and inefficiently. Zygotic embryo undergo a period of developmental rest
during seed maturation and germination. Bewley (1997) and Bewley and Black (1994)
distinguished two broad categories of arrested growth i.e. quiescence and dormancy.
Quiescence refers to all types of reversible developmental arrest and can be reversed by
water soaking. Dormancy requires factors in addition to water e.g. cold or heat
treatment for the resumption of growth (Gray, 1987b). Accordingly, seed and somatic
embryos are categorized into two groups viz. orthodox and recalcitrant depending upon
their ability to dehydrate (Litz and Gray, 1992). Orthodox embryos characteristically
undergo maturation drying during seed development and can tolerate dehydration as
low as 5% moisture content (Roberts, 1973). On the other hand, recalcitrant embryos
can not tolerate maturation drying (Chin, 1988; Roberts, 1973) and can not survive
storage at low temperature (King and Roberts, 1979). In recalcitrant embryo,
maturation, germination and seedling development represent a single continuous
process and do not have a period of developmental arrest. Dehydration of orthodox
embryos imposes a period of developmental arrest that can last for many years, making
it easier to store such seeds for a longer period that is typical of seeds of most
 111

temperate and a few tropical fruit trees. Mature recalcitrant embryos can not survive
even partial dehydration and cannot be stored for more than a few days or at most a
few week. The typical recalcitrant seeds are generally very large and contains very
little solid endosperm. The embryo is also large and fleshy with high water contents
and requires several months to reach maturity. Many species of tropical fruits and
forest trees have recalcitrant seeds (Chin, 1988; Roberts, 1979; Redenbaugh, 1990).
Somatic embryos must have functional shoot and root apices capable of
meristematic growth and germinate similar to that of zygotic embryo. Currently, the
germination rate of somatic embryos into plantlets or somatic seedlings is very poor in
most species and is a problem for the commercial utilization. The control of somatic
embryo maturation has been the most critical and difficult process in trees species (Litz
et a/., 1998). The process of somatic embryo development has shown a continuum
wherein subsequent stages followed one after the other and germinate immediately,
while various stages simply represent temporary developmental halt. However,
somatic embryos are arrested at the early stages of development, without achieving the
normal morphology, leading to maturation and as a result asynchrony observed.
Typically, developmental abnormalities such as fasciation and fusion of two or more
somatic embryos can occur when cell division in meristematic areas occurs prior to
differentiation of the shoot apex and cotyledons. If cell division continue to be
stimulated somewhat later when the cotyledonary primordia are initiated,
polycotyledonary, fused cotyledons etc. can result (Ammirato, 1987). Induced resting
in somatic embryos that would otherwise germinate rapidly would be considered as
secondary dormancy (Bewley, 1997; Bewley and Black, 1994). However, if somatic
embryos enter a resting phase after a normal drying step which is missing in
embryogenic culture systems, this type of rest should be categorized as quiescence
(Merkle eta/., 1990). For some species, efficient conversion to plantlet also requires
the imposition of temporary desiccation before germination (Dumet eta/., 1993; Gray,
1989).

8.1. EFFECT OF PGRS ON MATURATION AND GERMINATION OF SOMATIC

EMBRYOS:

Generally high auxin concentrations are used in the primary culture for the induction
of somatic embryogenesis. High auxin level can inhibit development and growth of the
shoot meristem (Gorst et a/., 1987; Murlidharan and Mascarenhas, 1987; Parrott,
1991; Parrott eta/., 1988). At low auxin levels shoot meristem formation is generally
achieved after the initiation of cotyledons so that under inappropriate culture condition,
germination can occur prematurely to give weak and nonviable plantlets (Merkle and
Wieko, 1989). In coconut palm, the most essential factors in the expression of embryo
maturation is the gradual drop in 2,4-D concentration (Blake, 1990; Karunaratne,
1989; Verdeil et a/., 1994, Verdeil and Buffard-Morel, 1995). If the drop in auxin
concentration is too rapid, the maturation of embryonic structure usually leads to
incomplete or abnormal forms. However, auxins are not essential for somatic embryo
maturation in other species. Somatic embryo maturation in Mangifera indica L. is
112

enabled only by the subculture of proembryogenic masses to 2,4-D free medium.

Somatic embryos at heart stage need to be subcultured on semisolid medium from
liquid suspension culture to achieve normal development and proper maturation, (Ara,
1998; DeWald et a/., 1989a, b; Jana et a/., 1994; Litz et a/., 1982, 1984, 1993;
Monsa1ud et a/., 1995; Pandey, 1998; Pleigo-Alfaro et a/., 1996a, b). Further,
maturation and germination are achieved by sequential transfer of somatic embryos
onto medium containing 20% (v/v) coconut water and reduced sucrose concentration
(DeWald eta/., 1989a, b). However, Ara (1998) and Pandey (1998) observed cultivar
and explant dependent maturation of mango somatic embryos. Cytokinins can be
important for maturation and have been demonstrated to influence cotyledon
development (Ammirato and Steward, 1971) and development of shoot apex
(Kavathekar, et .al., 1978). In mango, BA and kinetin can stimulate cotyledon
formation in 'Hindi' after 30-days, while in absence of cytokinins a minimum of 60-70
days was required for cotyledon development (Litz eta/., 1995).
During embryo development in orthodox seeds, the endogenous ABA level
increases, reaching a maximum of 1-lOJ..LM approximately midway through growth,
and preceeding maturation and germination (Hetherington and Quatrano, 1991). It is
believed that ABA prevent precocious germination of maturing embryo during seed
development and in someway is involved with the acquisition of desiccation tolerance
(Kermode, 1990). Although dehydration is known to stimulate the production of ABA
in excised embryo and plant tissues, increased ABA level in orthodox seed preceed
water loss and are not a result of water stress. The involvement of ABA in recalcitrant
embryo develoment is less clear. The development and maturation of recalcitrant
embryo has not been thought to be inflenced by ABA in developing Theobroma cacao
L. recalcitrant zygotic embryos reaches a maximum early in the maturation phase of
development, resembling the pattern in orthodox embryos.
Ammirato ( 197 4) has shown that ABA prevented precocious germination of
somatic embryos of caraway (Carum carvi) in suspension. In addition, ABA promoted
the development of fully differentiated cotyledons and suppressed the production of
multiple embryos in carrot culture (Ammirato, 1983). In mango, different weeks of
pulse with ABA supressed the nucellar embryo germination at every concentration
tested. In 4-week pulses with ABA at 250-500J..LM concentration resulted in a
germination rate of 15% and 5% respectively compared to 95% of the control nucellar
embryo germination in absence of ABA. All the other ABA concentration (750-
1750J..LM) almost completely suppressed the germination of shoot development. ABA
pulses of 8-weeks suppressed nucellar embryo germination in all treatment compared
to control. (Pliegi-Alfaro eta/., 1996a, b).
The role of ABA in initiating the accumulation of storage reserves has not
been ruled out especially as the initiation of reserve food coincides with the highest
level of endogenous ABA (Huges and Galan, 1989; Galan eta/., 1990). Further, it has
been demonstrated that maturation of somatic embryos initiated with the accumulation
of reserve food, reduced metabolism and the loss of water from cell sap (Morcillo et
al., 1998). ABA levels are highest during early maturation of dicotyledonous embryos,
and induce the accumulation of a group of hydrophilic proteins to act as desiccation
 113
protectants (Rajasekaran et a/., 1982). Bornman (1993) and Janick et a/. (1993)
suggested that ABA increases endogenous levels of storage proteins and fatty acids in
somatic embryos. An increase in lipids and saturated fatty acids is associated with high
ABA levels in cacao embryos (Pence, 1991, 1992). The deposition of high levels of
starch could also be involved during maturation of somatic embryos (Fujii eta!., 1993).
In guava (Akhtar, 1997), somatic embryos mature simultaneously with their
developmental progression from globular to elongated torpedo stage and germinate
readily upon subculturing on the fresh medium of similar or modified composition.
The period of somatic embryo in development medium after initial exposure to 8-days
with 2,4-D is important for production of mature somatic embryos in guava (Akhtar,
1997). An 8-week development period required for production of convertible somatic
embryos. The inclusion of dormancy breaking hormone e.g. cytokinins (BA, KIN) and
gibberellin (GA3 ) in all the possible combinations permutations into the medium did
not improve the germination of old somatic embryos. Adenine sulphate and GA 3 have
been reported to enhance rooting of Citrus somatic embryos (Kochba et a/., 1974).
Somatic embryos of papaya, its hybrid and relatives, germinated rapidly in the absence
of auxin or on media containing relatively low concentration of auxin and cytokinin
(Fitch, 1995). In the absence of maturation inducing compounds like cytokinin or
ABA, de Bruijne et al. (1974) were unable to culture embryos beyond the mature
cotyledonary stage. Media devoid of growth regulators are widely used for germination
(Litz and Conover, 1980, 1983; Litz, 1986; Manshardt and Wenslaff, 1989). However,
in other reports one or more growth regulators are supplemented in the medium to
enhance germination (Chen, 1988a, b; Chen eta/., 1987; Jordan eta/., 1982; Litz and
Conover 1982; Yie and Liaw, 1977).

8.2. EFFECT OF MEDIUM OSMOLARITY ON MATURATION AND

GERMINATION OF SOMATIC EMBRYOS:

Normally embryo development medium contains high sucrose (3-6%). The optimum
concentration {5-6%) of sucrose has been used for somatic embryogenesis in Citrus
(Murashige and Tucker, 1969); guava (Akhtar, 1997); mango (DeWald et al., 1989a)
and papaya (Fitch, 1993; Litz and Conover, 1983). Malt extract in Citrus (Jumin and
Nito, 1995, 1996; Oh et a!., 1992) and in many other species (Table l) have been
beneficial for somatic embryo maturation. Progressively increasing levels of sucrose
upto 40% have been used for some species to achieve maturation (Lee and Thomas,
1985; Janick, 1986) which has resulted into osmotic desiccation of somatic embryos
and better germination. Carman (1988) has reported that gradual reduction in osmotic
potential through desiccation of mature somatic embryos to improve germination in
wheat. Sucrose and PEG improved the quality of somatic embryos in guava (Akhtar,
1997; Jaiswal and Akhtar, 1993, 1994). High osmolarity and ABA stimulates embryo
maturation of rubber (Etienne et a/., 1993), but normal seedling morphology have
occured only with osmotic treatment. In contrast, Fujii et a!. (1989) observed better
maturation and plantlet recovery with ABA alone. Treatment of guava somatic
embryos with ABA (O.l-1.0 mgll) and high sucrose (5%) in full strength medium has
114

shown improved maturation and germination upon dehydration and storage beyond 8-
weeks (Akhtar, 1997). Maturation of mango somatic embryos was achieved on media
rich in organic composition (half strength B5 major salt with MS minor elements and
organic components of maintenance and embryogenesis media, 40mg/l sucrose, 400
mg/1 glutamine and 20% (v/v) filter sterilized coconut water), because of long
maturation period (i.e. 120-160 days) and extremely large size of somatic embryos (3-
6cm) (DeWald eta/., 1989a, b). Interaction of 5.0 J.lM ABA, 0-5% mannitol and 4-8
week pulse treatment produced highest number of normal somatic embryos in mango
(Pleigo-Alfaro et a/., 1996a). They further reported that an 8 week pulse treatment
with 50 and 100 J.lM ABA and 5-10% mannitol in the medium have completely
suppressed the precocious germination of mango nucellar somatic embryos (Pieigo-
Alfaro eta/., 1996b). Mango somatic embryos germinated precociously after osmotic
stress unless 100 J.lM ABA is also present (Monsalud eta/., 1995).

8.3. EFFECT OF CULTURE CONDITIONS ON MATURATION AND

GERMINATION OF SOMATIC EMBRYOS:

Agar solidified medium supports better germination rate when compared with liquid
germination medium in guava (Akhtar, 1997) and T. arjuna (Nishi, 1997) somatic
embryos. The medium type and strength and sucrose concentration played an
interactive role on germination of guava somatic embryos. Half strength MS- basal
medium amended with 3% sucrose shows better germination than the full strength
medium with similar or higher sucrose concentration (Akhtar, 1997). Germination of
Musa somatic embryos can be improved by using a double layer media system (liquid
basal medium overlies solidified medium supplemented with ZEA and activated
charcoal (Novak eta/., 1989). Maturation and germination in mango somatic embryos
are continuous, and there is no period of developmental arrest. Germination in mango
is characterized by rapid elongation of hypocotyl and greening of cotyledons of somatic
embryos under light condition (Litz et a/., 1993, 1995). Cultures maintained in
complete darkness throughout the maturation period in order to achieve normal
maturation and to prevent the precocious germination of mango somatic embryos.
Fully mature somatic embryos are roughly measured between 4-7 em in length with
large fleshy and white cotyledons with a well define embryonic axis. After
enlargement, somatic embryos are transferred to light (50-70 J.lEm-2 s- 1) as a result, the
hypocotyl enlarge and a taproot emerges and cotyledons quickly turn green. Shortly
after a shoot develops, the root system usually fails to ramify quickly (Mathews and
Litz, 1992; Litz eta/., 1995). Escalant eta/. (1994) have found that when embryogenic
cultures (with translucent embryogenic callus bearing an average of 150 embryos) were
placed in the upper compartment of specially modified filter unit and immersed (1
min. every 6 hrs) temporarily in MP medium with Morel's Vitamin, 2.2 j.lM picloram
and 0.87 M sucrose (pH 5.8) allowed several fold enhanced production of mature
somatic embryos in Musa spp. Similarly temporary immersion (1 min. every 4 hrs) of
Citrus embryogenic cultures enabled cotyledon and protoderm development and
embryo were morphologically identical to nucellar embryo. Further, secondary embryo
 115
development was reduced and there was enhanced synchronization of plantlet
regeneration (Cabasson et a/., 1997). Temperature also affects the maturation of
somatic embryos to some extent. As described arlier, guava somatic embryos developed
well at 15-35 °C (Akhtar, 1997). Somatic embryos, developed in complete darkness at
lower temperature (15 and 20 °C) remain physiologically immature (translucent milky
white with high water content) as compared to those developed in darkness at 25 °C.
Such somatic embryos are developmentally mature, possess well defined shoot and root
meristems and converted to normal plantlets upon transfer to germination medium
under the light. On the other hand somatic embryos, developed at high temperature (30
and 35 °C), are changed to solid milky white earlier than those develped at 25 °C
irrespective of developmental stage. They converted into normal plantlets in
germination medium. These changes in the behaviour of somatic embryos can not be
attributed to the temperature or light conditions, rather, dehydration of the medium
exerting water stress at high temperature might be leading to early maturation. In
mango (Pliego-Alfaro eta/., 1996a), germination of somatic embryos and shoot growth
were completely inhibited by 4 week to 7.5 and 15 °C, whereas, they germinated at 22-
37.5 °C. Shoot and root elongation were stimulated by heat treatment of 30 and 37.5
°C, respectively. Somatic embryos exposed to 45 °C did not survive. Similarly, Paristol
(1988) found that somatic embryos failed to germinate at 5 °C. Although maturation
of nucellar embryos has been achieved in a number of plant species following
manipulation of culture condition, the germination and plantlet formation is still low.
A more thorough understanding of the control of maturation and germination in
zygotic embryos is required to achieve high efficiency of conversion of somatic
embryos.

9. Soil Transfer, Acclimatization and Growth of Somatic Plants:

In vitro grown shoots and plantlets are continuously exposed to a unique

microenvironment that has been selected to provide minimal stress and nearly all the
optimal conditions for plant multiplication. Plantlets grown within culture vessels
under low light, aseptic conditions, on a medium containing sucrose and nutrients for
heterotrophic growth under high relative humidity. In vitro grown plantlets have
reduced development of cuticular wax and abnormal stomatal function (Wetzstein and
Somer, 1983). All of these factors contribute to difficulties in plantlets and shoots to
survive the environmental conditions when directly transferred in the green house or
field (Preece and Sutter, 1991). Kozai (1988, 1991) has successfully micropropagated
plants by growing plants photoautotrophically. This is accomplished by elevating C02
in the culture vessel, raising light intensity and growing the plantlet on the medium
devoid of sugars. The condition has resulted in an increased growth and survival rate
when the plantlets are transferred to ex vitro condition (Kozai, 1988).
In guava (Akhtar, 1997), somatic plantlets could not survive on non-sterile
soil mixture because of heavy fungal contamination. Therefore, growth of the plantlets
for about 2- week in full strength MS basal medium with 3% sucrose was essential
116
prior to soil transfer and acclimatization. The plantlets grown in liquid medium
required initially a more porous potting mixture of sand, garden soil and compost in a
ratio of 5:1:2. Once the root system was adjusted to this primary mixture, transfer of
plantlets to a mixture with elevated proportions of soil and compost was required. The
plantlets were kept covered with either glass beakers or polythene bags during
hardening in the primary mixture at room temperature. Almost 100% guava somatic
regenerants survived following soil transfer and acclimatization (Akhtar, 1997).
However, the survival percentage in most other tropical trees has been very low. For
example, about 50% survival is reported in Euphoria Iongan (Litz, 1988) and only a
small percentage in Feijoa (Cruz eta/., 1990).
In mango, the plantlet conversion and survival frequencies have been very low
under the standard light intensity condition (Litz eta/., 1982, 1984; DeWald et a/.,
1989b). Litz et a/. (1993, 1995), have observed the extensive root development and
vigorous plantlet growth through the use of protocol developed by Kozai (1988, 1991),
involving the exposure of germinating mango somatic embryos at 25 °C to controlled
high levels of C0 2 (20,000 ppm in a nitrogen carrier) under high light intensity (180
Jlmolm-2s-\ Recently, Ara (1998) and Pandey (1998) found 50-60% survial of
plantlets regenerated from mature cotyledonary somatic embryos. Somatic plantlets
with growing pinkish brown tap root with pink-white tips along with 1-2 leaves and 3-
4 em long shoots, acclimatized most successfully and survived well upon transfer to
soil (Pandey, 1998). Irrigation of pots with 1/lOth diluted B5 major and MS minor
salts after a week interval benefited the healthy growth of plantlets at rooin
temperature (DeWald eta/., 1989b; Pandey, 1998). Covering of plantlets with a glass
beaker or a polyethylene bag is essential for about 2-4 month for maximum survival
(Pandey, 1998). Ara eta/., (1998a) succeeded in achieving 89.71% survival rate upon
transfer into soil and acclimatization of microcuttings of somatic plantlets of mango.
Similarly, in papaya (1-2 em tall) cuttings of somatic plantlets could be efficiently
rooted in MS-medium containing 1-4 mg/1 IBA (De Winnaar, 1988; Litz and Conover,
1979, 1981a; Miller and Drew, 1990; Reuveni eta/., 1990) or 5.0 mg/1 IAA (Yie and
Liaw, 1977). Drew eta/. (1991) showed enhanced rooting by the addition of 0.4mg/l
riboflavin to culture medium containing 2 mgll rnA In most cases, once new roots
were established, the plants grew rapidly in greenhouse as well as in the field upon
acclimatization. Citrus somatic plantlets with 2-3 mature leaves that developed from
embryoid in the presence of GA3 (Hidaka and Omura, 1989; Kobayashi et a/., 1984;
Kunitake eta/., 1991; Ling eta/., 1990) or in the presence of IAA (Beloualy, 1991;
Vardi and Galun, 1989) transferred to the pots in the greenhouse, after acclimatization,
have highest survival frequency (Kunitake and Mii, 1995). In oilpalm, the
polyembryogenic cultures produce new somatic embryos on hormone free medium, and
the most advanced embryos developed into shoots. When shoots have reached a
sufficient size (5-7 em), they are separated normally and rooted on the medium
containing NAA and sometimes with GA3 (Nwanko and Krikorian, 1983). Plantlets
are acclimatized under controlled condition in order to avoid hydric stress, which is a
common for all plants produced in vitro. Wooi, (1990) and Wooi et a/. (1982)
maintained an acclimatization procedure ensuring 90% success with rooted plantlets
 117
placed under a plastic frame. Whereas, Duval et a/. (1995) have not found rooting
during hardening even after auxin treatment of shoots. After 4~weeks of
acclimatization, the plantlets are transferred to the prenursecy and treated like
seedlings before their transfer to field. In T. arjuna (Nishi, 1997), survival of plantlets
to soil was dependent on the hardening process. Plantlets when transferred to sterile
sand with MS liquid medium without sucrose showed higher survival rate (>75%). The
hardening process required longer period (5-8 weeks) during primacy acclimatization
step under the controlled condition of light and humidity. Further work is needed for
synchronized root and shoot development as well as improved acclimatization protocol
to decrease stress and increase growth and survival.

10. Genetic Stability of Somatic Seedlings:

For any clonal propagation system, genetic stability of regenerated plants is highly
desirable. Genetic variability in cell culture may lead to somaclonal variation in
regenerated plants which is beneficial and sometimes highly desirable for crop
improvement through breeding. Variability in somatic seedling has been exploited for
improvement of some species (see chapter Application of somatic embryogenesis for
improvement of tropical fruit trees by the authors). However, somatic embryos derived
plants in several Citrus species have been reported to be genetically stable with respect
of morphology, cytology and physiology (Kobayashi et a/., 1984; Vardi and Spiegel-
Roy, 1982; Vardi et al., 1982). The isozyme banding pattern of three enzymes
(glutamate oxaloacetate transaminase, peroxidase and phosphoglucose mutase) as well
as gas chromatographic pattern of leaf oil and chromosome number in ell).btyogenic
calli did not show any variation among somatic embryo derived plants of 'Valencia'
orange and calamondin (C. madurensis Laour.) (Kunitake and Mii, 1995). Moreover,
there were no differences among plants regenerated from nucellar seedling or mature
trees. Molecular control of genetic stability is reported for willow (Salix sp.) and
datepalm (Phoenix dactylifera) regenerated through somatic embryogenesis (Daguin
and Letouze, 1988, 1997).

11. Current Limitations and Future Prospects:

Only a small number of fruits and tree species have been regenerated via somatic
embryogenesis. Certainly, the most challenging task in fruit trees is the induction of
somatic embryogenesis, in species where it has not been observed or occurs at a low
frequency. The full potential of somatic embryogenesis as a mean of propagating fruit
trees is also limited by the fact that it can only be induced from embryonic and juvenile
explants of limited number of species. Therefore, it is not a preferred explant for true-
to-type cloning of phenotypically superior fruit trees. This is further complicated by the
long life cycle and inconsistency in seed production. Further research should therefore,
be aimed at screening for totipotent cells from mature donar explants of proven fruit
118

species. Moreover, exhaustive and systematic efforts are required in order to achieve
the somatic embryogenic induction in new fruit species. Another problem is the
selection of appropriate developmental stage and the physiological age of explant, and
this should be worked out in an interactive manner along with other factors e.g.
nutritional requirement and sensitivity to a growth regulator.
For many systems the process of somatic embryogenesis is not well defined.
The frequency of induction and the number of somatic embryos are the important
parameters but unable to give any information about the proportion of physiologically
and developmentally mature and convertible somatic embryos. Asynchrony is a serious
concern and requires further improvement of induction process. The protocol described
by Fujimura and Komamine (1979) Merkle et al. (1990) and Wang and Phillips (1984)
for synchronization of the process should be taken into account.
Maturation of somatic embryos has always been a problem in tree species
particularly with recalcitrant seeds. Further research are required for the design of and
the conditions for the growth and continuous production of developmentally uniform
somatic embryos. Without an understanding of molecular basis of the mode of
hormone action, studies on somatic embryogenesis will remain purely empirical.
Characterization of the components involved in the signal transduction in hormone
treated cells and isolation of cell cycle genes will result in significant progress in
research into embryogenesis.

12. Concluding Remarks:

Many of the important parameters that inflence the induction of somatic embryogenic
cultures from various explant sources, the maintenance of these cultures, development,
maturation and gerimnation of somatic embryos and regeneration of plants, limitation
of the techniques and future use for crop improvement programme with special
reference to tropical and subtropical fruit trees have been described. Somatic cell
genetics of fruit crops is contingent upon the efficient and reliable regeneration of
plants fron cell and tissue culturefrom clonal selection. The regeneration of fruit tree
species through somatic embryogenesis (Table 1) has paved the way for biotechnology,
in these species (see chapter on Application of somatic embryogenesis for improvement
of fruit trees by the authors in this volume). With the progress made inthe present and
the last decade, one can anticipate substantial progress in the regeneration of clonal
selection of many fruit crop species at commercial proportion in near future. Although
induction, development, amturation and germination of somatic embryos is being
controlled to some extent, high grequency regeneration, synchronous development of
embryos and transplantation to the field is not very successful. The advances must be
accompanied by better understanding of the factors that influence embryogenesis i.e.
genitype, explant type (age and stage of development), preconditioning, medium
composition (particularly phytohormones) and other physical and cultural conditions.
An interaction of most of these factors must be considered in order to explore
embryogenesis in a new species, genotype or the explant tissue. During the recent years
 119
the progress in basic and fundamental aspects of somatic entbryogenesis (Bajaj, 1995a)
encourages more intensified and multidirectional research aided with computer and
robotics and the molecular biological approaches, to study the factors responsible for
developmental control of somatic embryogenesis. Coupled with these approaches there
is certain optimization that sooner or later it would become possible to regenerate
almost all species via somatic embryogenesis with the judicial selection of genotype,
explant, media composition and physical and cultural conditions.

13. Acknowledgements:

The financial support to the Laboratory of Morphogenesis, Centre of Advanced Study

in Botany, Banaras Hindu University, Varanasi, provided by various agencies like
University Grant Commision, Council of Scientific and Industrial Research, Central
Silk Board, Department of Biotechnology, Department of Science and Technology,
Government of India is gratefully acknowledged.

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 Table 1: Tropical and Subtropical Fruit Trees Regenerated through Somatic Embryogenesis. w
N
Plant Name Generic Name Explants Processes Medium GR Adjuvents & Other References
Factors
Avocado Persea americana cv. ze Induction development, MS PIC, 2ip AC Mooney and Van-Staden,
Hass plantlets 1986
Persea americana cv. ze Induction MS 2,4-D AC Pliego-Aifaro and
Hass Murashige, 1988
Persea americana Mill ze Induction, plantlets MS,B5 PIC Genotype Witjaksono and Litz,
1997
Persea americana Mill ye, nu, Induction, development, MS,B5 PIC Mannitol, glutamine Witjaksono et al., 1998
prot. plantlets medium osmolarity
and strength
Apricot Prunus armeniaca L. cv ze Induction, germination WP -- Explant stage, Burgos and Ledbetter,
Castleton medium type 1993

Prunus subhirtella rt Induction, Transformation, MS NAA, IBABAP GUS expression, da Camara Machado et
autumno rosa Plantlets Northern Blot al., 1995
Prunus avium ze, se Indirect induction - BAP,KIN - Garin et al., 1997
NAA, IBA
Prunus granatum L. pt Induction, plantlets MS IAA, KIN, BAP - Natrajan and Neelambika,
1996
Banana& Musa sapientum fl induction MP, MS, Morel PIC - Escalant eta/., 1994
Plantain Vit.
Musasp. fl Induction, development, MS BAP,IAA AC, ME, medium Ganapathi et a/.,1999
maturation, germination strength
plant formation
Musa spp. (AAB jl, ci Induction regeneration - - - Grapin et al., 1996
genome)
Musa spp. (AAB) fl, sc Induction, plantlets -- -- - Grapin et al., 1998
Musa sapientum rh, sc Induction, histology MS 2,4-D, PIC CH, cystein, AA Lee et al., 1997
Musa acuminata se, sc Induction, development, MS, Morel PIC,BAP, IAA medium state Marroquin et al., 1993
plant formation Vit.
Musa spp. (AAB} sc, prot. Induction, development BAP,IAA Darkness, nurse Matsumoto et al., 1998
culture
Musa acuminata -- Plant regeneration MS 2,4-D, NAA, - Navarro et al., 1997
IAA, KIN, BAP
 Musa (AA, AAB, ABB) If, rt Induction, development, MS DIC, ZEA - Novaketal., 1989
plant formation
Cherry Prunus avium ze Induction MS 2,4-D,NAA, CH, treatment period De March et al., 1993
IAA, KIN, ZEA
Prunus avium ze Induction, development Ms NAA, KIN, BAP, Maltose Reidiboym et al., 1998
ABA
Prunus avium ze Lipid and fatty acid MS NAA, KIN, BAP, -- Reidiboym et al., 1999
comparision in embryogenic ABA
and non-embryogenic callus
Citrus Citrus sinensis Obs. nu prot. whole plant MT BAP,GA3 ME, adenine, Ashietal., 1985
sucrose, medium
state
Citrus sp. nu, hy Induction, development, MT GA, ME Belkoura et al., 199 5
plantlets
Citrus aurantium, ci Induction, globular MT IAA, GA3 BAP,KIN, NAA ME, OR combination Beloualy, 1991
Poncirus trifoliata, embryoid, shoot buds
Carrizo c1trange rooting, plantlets

Citrus trifoliata ze Induction, plantlet MT 2,4-D, BAP, NaCl tolerance, Beloulaly and
formation, NAA Bounharnont, 1992
Citrus jambhiri nu Induction, development MT - -- Ben-Hayyim and
Neumann, 1983
Citrus sp. ou Induction, development - - -- Button and Bronman,
1971
Citrus deliciosa nu, sc Induction, development, MT GA, ME, sorbitol, Cabasson et al., 1997
plantlets galactose
Citrus limon (L.) Burm. sy Induction, development, MS 2,4-D, BAP ME Carimi et al., 1994
plantlets
Citrus sinensis sy Induction, development, MS BAP ME Carimi et al., 1995
Citrus paradisi Citrus plantlets
aurantium Citrus
deliciosa
Citrus sinensis (L.) Osb. sy, sm, ou Induction, development, MS KIN,BAP ME Carimi et al., 1999
germination, plantlet
Citrus reticulata If, ep, ct, Induction MS NAA, KIN medium strength Gillet al., ~995
rt
Citru sinensis. ou Induction, development MT 2,4-D, BAP ME Gmitter et al., 1987
Citrus paradisi, .......
w
w
Citrus limon >-'
w
Citrus sinensis x Citrus es Induction, development, MT -- - Gmitter et al., 1990 .j:::..
paradisi hybrid plantlets, triploid hybrid
Citrus clemintina nu Induction, development MS - - Gmitter et al., 1991
Citrus sp. es Induction, development -- -- - Gmitter et al., 1992
Citrus sinensis x prot. Induction, protoplasts -- -- medium state Grosser and Gmitter,
Citropsis gilleliana fusion, intergeneric hybrid 1990a, b, c
Citrus microcarpa x If, prot. Protoplasts fusion, -- -- - Grosser et at., 1996
Citrus aurantium cybridization, Induction,
Citrus reticulata x Citrus development, plantlets,
sinensis Citrus Isozyme analysis, RFLP
limonx Citrus
sinensis
Citrus sinensis, ze, prot. Induction, plantlets MS KIN, ZEA, GA3 ME Hidaka and Kajiura, 1988
Citrus yuko,
Citrus reticulata
Murraya panniculata ze, hp, Induction MT BAP, 2,4-D, GA. ME, lactose Jumin and Nito, 199 5
prot.
Citrus schweinforthii prot, ci Induction, protoplast to MT BAP,KIN ME Jumin and Nito, 1996
plant protocols
Citrus sinensis Obs. var If, nu,sl Somatic embryo induction, MT GA3 PEG Kobayashi et al., 1991
brasiliensis Tanaka, 'F.N. protoplast fusion,
Washington' chloroplast DNA analysis
Citrus sp. ci Induction, development - - -- Kochba and Spiegel-Roy,
1973
Citrus spp. nu Induction, development MS -- - Kochba et al., 1972, 1982
Citrus unshiu Marc. -- Induction , germination MT GA, Adenine, sucrose, Kunitake et al., 1991
mannitol
Citrus microcarpa nu Induction, proembryo WH -- CH Maheswari and
Rangaswamy, 1958
Citrus limon nu Induction, development MS -- -- Navarro et al., 1985
Citrus sinensis L. prot. Induction, germination MS NAA Calcium alginate, Neidz, 1993
sucrose
Citrus sinensis L. ci,prot. Induction to germinetion MS BAP Nitrate, Ammonia Neidz, 1994
ratio
Citrus unshiu Marc. ov Induction, development, MS NAA, KIN, GA3 Light intensity Nito and Iwamasa, 1990
plantlets
Citrus Junos Sieb et. ou Induction MT BAP, 2,4-D ME Oh eta!., 1992
 Tanaka
Citrus sinensis x Citrus if, prot. Induction, protoplasts - - -- Ohgawara et al., 1989
paradisi fusion, somastic hybrid
Citrus sinensis Obs. cv. nu,prot. Induction, plantlet MT -- PEG other organic Ohgawara eta/., 1991
Bahia regenaration solvent
Citrus sudachi x Citrus nu, sc, Induction, development, MT GR glucose, ME, Saito et al., 1991
aurantifo/ias prot. electrofusion, AS
Citrus sinensis Obs. nu Induction, freezing, MT -- glycerol, liquid Sakai et al., 1991
Cryopreservation nitrogen,
Citrus spp. prot. Induction, development - -- - Vardi and Spiegel-Roy,
1982 Kobayashi eta/.,
1983
Ohgawara eta/., 1985
Grosser et al., 1988a,
1988b
Microcotrus x Citrus ou prot. protoplasts fusion, somatic MT - light intensity Vardi et al., 1986
cu/tivar hybrid regeneration,
Citrusjambhiri Lush ci,prot. Genetic transformation, -- -- PEG Vardi eta/., 1990
Induction, development,
plantlets regeneration
Citrus sinensis ou Induction MS KIN CH Waithaca and Obukosia,
1988
Feijoa Feijoa sellowiana ct, ze induction, MS 2,4-D Treatment period Canhoto and Cruz , 1996
histodifferentiation
Feij·oa sellowiana ct. ze Induction MS 2,4-D, KIN, GA1 fructose, glucose, Canhoto and Cruz, 1994
maltose, sucrose,
medium state
Feij'oa sellowiana ze Induction, development, MS 2,4-D, NAA Treatment period Cruz et al., 1990
maturation, plantlet IBA, KIN
Guava Psidium guajava L ze Induction, development, MS 2,4-D and other Carbohydrates, PEG, Akhtar, 1996, 1997;
maturation, germination, GRs NaCl, , Light Akhtar & Jaiswal, 1994,
encapsulation & synthetic intensity, 1995
seed production temperature, breeding Jaiswal and Akhtar, 1993,
mechanism, NaOCl, 1994
seasons, genotypes
Psidium guajava L. an Induction MS,B5 BAP, 2,4-D, KIN PVP, sucrose, Babbar and Gupta,
prechilling 1986a, b
Psidium guajava L. ze Induction, development MS 2ip, BAP, KIN, - Ramirez and Salazar,
w
Vl
-
 ribozeatin, ZEA, 1998 w
Jaboticaba Myriciaria cauliflora nu,ou Induction, germination MS 2,4-D, BAP Medium strength Litz, 1984a 0\
Kiwifruit Actinidia deliciosa var if, prot. Induction MS ZEA Medium strength, Oliveira and Pais, 1992
Deliciosa cv Hyward nutrient stress,
planting orientation
Litchi Litchi chinensis cv. ye, SC, Induction, development - -- -- Yuetal., 1996
Xiaofanzhi prto.
Longan Euphoria longan lf Induction MS KIN, 2,4-D Light intensity Litz, 1988
Mango Mangifera indica L. cv nu Induction, development, MS,B5 2,4-D, GA3, IAA, Medium strength, Ara et al., 1998
Amrapali germination, plantlets IBA, NAA sucrose, medium
state, glutamine and
rooting in
microcutting of
somatic plants
Mangifera indica nu Maturation, germination MS 2,4-D, KIN, BAP Glutamine, AA, CH, DeWaldetal., 1989b
cw
Mangifera indica nu Induction , development MS 2,4-D, KIN, BAP Glutamine, AA, CH, DeWals et al., !989a.
cw
Mangifera indicanu cv nu Induction, maturation, MS 2,4-D,GA3, AC,CW,CH, Jana et al., 1994
AJphanso,Mundarn, germination ABA glutamine, light
Baneshan intensity
Mangifera indica cv. nu ·Induction development, MS,B5 2,4-D, KIN, BAP Glutamine, treatment Ladet al., 1997
'Carabao' maturation period
Mangifera indica nu Induction MS BAP Glutamine, AA,CW Litz et al., 1982
Mangifera indica nu,ze Induction MS 2,4D, 2ip NAA, Glutamine, AA, CW, Litz et al., 1984
BAP, ME
Mangifera indica nu Development, maturation, MS -- Polyamin ethylene Litz et al., 1993
germination and etc.
biochemical studies
Mangifera indica nu Hyperhydrocity, MS,B5 2,4-D, BAP, AC, CW, glutamine, Monsalud et al., 1995
Development, maturation, ABA medium state,
germination medium strength
Mangifera indica nu Osmolarity, dessication, MS,B5 ABA -- Pliego-AJfaro et al.,
development, maturation 1996a
Mangifera indica nu Osmolarity, dessication, MS,B5 ABA -- Pliego-Alfaro et al.,
development, maturation 1996b
Mangifera indica nu Induction, development, MS,B5 2,4-D, KIN, GA3 AC, CW, CH, Thomas, 1999
maturation, germination, glutamine, medium
 plantlets strength
Mulberry Morus indica an Androgenic calli, MS BAP, KIN, NAA PVPP, Cold Jain et al., 1996
embryogenesis, rhizogenesis pretreatment
Palm Cocos nucifera Induction, development, -- -- Ethylene, AVG, STS Adkin eta/., 1998
inhition of embryogenesis
Cocos nucifera If. rc Induction - -- - Branton and Blake,
l983a, b
Cocos nucifera ze Induction - -- Explant type, Guptaetal., 1984
medium composition
Cocos nucifera If Induction, germination - -- Time of explanting, Karunaratne eta/., 1991
leaf maturity
Cocos nucifera If. ci Induction -- -- - Pannetier and Buffard-
Morel, 1982
Cocos nucifera If. in Induction, germination -- -- - Verdeil and Buffard-
Morel, 1995
Cocos nucifera L. -- Amino acid composition of -- - Various treatments, Magnaval eta/., 1995
embryogenic calli sampling date
Cocos nucifera L. -- Induction MS,NT, BAP, 2,4-D Fe-EDT A, glucose, Magnaval et al., 1997
Morel Vit. AA,ME,AC
Cocos nucifera L. inf Induction, development MS 2,4-D AC, gradual Verdeil eta/., 1994
reduction of2,4-D
concentration
Elaeis guineensis Jacq. sc Induction, development, MS 2,4-D, BAP AS, AC, Glucose, deToucheteta/., 1991
germination, plantlets treatment period
Elaeis guineensis Jacq. Induction, photosynthetic -- 2,4-D -- Rival et a/., 1997
parameters in plantlets
Elaeis guineensis Jacq. -- Somaclonal variation, - -- - Rival eta/., 1998
RAPD
Elaeis guineensis Jacq. Inf Induction, development MS 2,4-D AC Teixeira eta/., 1994
Phoenix dacty/ifera L. sh,ls, ax Induction, development, MS 2,4-D, 2ip, BAP, - Azra eta/., 1997
plantlets GA3
Phoenix dactylifera L. st, inf Induction, development, MS 2,4-D, 2ip AC Bhaskaran and Smith,
plantlets 1992
Phoenix dacty/ifera L. st Induction -- -- - Gabr and Tisserat, 1985
Phoenix dactylifera L. ou Induction
, MS PIC, DIC, KIN - Omar and Novak, 1990

Phoenix dactylifera L. ze Induction -- -- -- Reuveni, 1979

Phoenix dactylifera L. ze Induction -- -- -- Reynold and Murashige, ......
.....,
--:)
 1979 (.;.>
Phoenix dactylifera L st Induction, development MS 2ip,BAP Phosphate, thiamine 00
Phoenix dactylifera L If Induction -- -- --
Sharma eta!., 1984
Sharma eta/., 1980
-
Phoenix dactylifera L If, buds Induction, plantlets MS 2,4-D, BAP, AC Sharma et a/., 1986
NAA
Phoenix dactylifera L st Induction, development, MS NAA, 2ip, KIN AC, AA, glutamine Sundersan et a/., 1993
plantlets
Phoenix dactylifera L st Induction , Histological MS 2,4-D -- Tisserat and De Manson,
analysis 1980
Phoenix dactylifera L st Induction MS 2,4-D AC Tisserat eta/., 1981
Phoenix dactylifera L st,lf Induction, development MS 2,4-D, 2ip AC,medium Veramendi and Navarro,
strength, medium 1996
state
Phoenix dactylifera L ze Induction -- -- - Zaid and Tisserat, 1984
Phoenix dactyliferai L ci Induction -- -- - Daguin and Letouze,
1988
Phoenix sylvestris Roxb. st Induction, plantlets MS 2,4-D,BAP AC Sharma et al., 1988
Papaya Carica papaya -- Induction, development, -- Ethylene AVG,STS Adk.in eta!., 1998
inhibition of embryogenesis
Carica papaya sl,pt Induction MS,WH 2ip,NAA -- de Bruijne et al., 1974
Carica papaya sl, st Induction, development, -- -- -- Yie and Liaw, 1977
germination
Carica papaya pd, sc Induction MS -- AC Litz and Conover, 1980
Carica papaya ou Induction, polyebryony WH -- Sex type, season, Litz and Conover, 1981a,
other factors b
Carica papaya nu Induction, maturation, WH NAA,BAP CW, High sucrose, Litz and Conover, 1982
germination glutamine, medium
state
Carica papaya nu Induction, maturation, WH NAA,BAP CW, High sucrose, , Litz and Conover, 1983
germination glutamine, medium
state
Carica papaya L. cv hp, sl Induction, development, MS 2,4-D BAP, KIN Glutamine, sucrose, Fitch, 1993
Kapoho, Sunrise, Sunset, maturation, germination medium state
Waimanalo
Carica papaya x ze, sc, Induction, proliferation, MS ABA -- Chen and Chen, 1992
Carica cauliflora prot. development

Carica papaya L. sh. st, If, Induction MS NAA, KIN GR combination, AS Chen eta/., 1987
 ct rt GA,
Carica papaya L. ou Induction MS 2,4-D Genotype, glutamine, Litz, 1986
NaCl, mannitol
Carica papaya L. ze Induction, maturation, MS 2,4-D Glutamine, medium Fitch and Manshardt,
germination, plantlets strength 1990
Carica papaya L. ze Induction, encapsulation MS ABA Sucrose , agarose, Castillo eta/., 1998
sodium alginate,
CaCh
Carica papaya L. -- Transformation, somatic -- -- Osmotic stress Mahon eta/., 1996
embryo production
Carica papaya L. ze Transformation of somatic MS 2,4-D Resistance to kan. Cheng eta/., 1996
embryos
Carica papaya L. buds Induction and plantlets NN NAA, IAA, BAP, Combination ofGR Jordan and Velozo, 1996
formation TDZ
Carica papaya L. ze Induction MS 2,4-D Aspargine, maltose, Dhir and Yadav, 1995
glutamine
Carica papaya L. sc, ze Transformation, somatic - - - Cai et a/.,1999
embryogenesis, transgenic
plant
Carica papaya L. sc, ze Transformation, somatic -- - -- Gonsalves eta/., 1998
embryogenesis, transgenic
plant
Carica papaya ou, ze, hy Induction, development -- 2,4-D -- Suksa et aL, 1998
Peach Prunus persia L. ct, ze Induction, development, MS 2,4-D, BAP, CH, Glutamine, light Raj Bhansali eta/., 199la
plantlet KIN intensity, treatment
period
Prunus persia L. ct, sc Induction, development, MS 2,4-D, BAP, KIN Ca(N03)2 CH, 2,4-D Raj Bhansali eta/., 199lb
plantlet gradual reduction,
medium strength,
slutarnine
Prunus persiQ L. ct, se Induction, multiplication MS 2,4-D, NAA, CH, AC, light Raj Bhansali et a/.. 1990
BAP,KIN intensity, treatment
period
Prunus persia L. If, ze Induction, development, MS BAP,NAA CH,medium Scorza et al., 1990
plantlet transformation, strength, neomycin
phosphotransferase,
Pomegranate Punica granatum ze Induction, plantlet MS -- CH, light intensity, RajBhansali, 1990
Glutamine, Inositol,
w
\0
-
 sucrose, .......
Rose and Syzygium spp. Nu Induction MS 2,4-D Litz, l984b 0
"""'
Malay apple
Syzygium spp. Nu Induction MS 2,4-D Litz, 1985

Media:
WP =Lloyd and McCown (1980) MS= Murashige & Skoog (1962) WH= White (1963)
B5= Garnborg et a!. ( 1968) MT= Murashige & Tucker (1969)

Explants:
an= anther hp = hypocotyl ou =ovule rt =root sm =style
ax = axillary bud inf= inflorescence ov= ovary sc = suspension cell st =stem
ci =calli it = integuments prot. = protoplasts culture sy= style
ct = cotyledons If= leaf pt = prtiole se = secondary embryo ye = young embryo
es = endospenn Is = leaf sheath rc = rachillae sh = shoot tips ze = zygotic embryo
fl =flower nu =nucellus rh =rhizome sl = seedling

Adjuvents:
AA = Ascorbic acid
AS = Adenine sulphate
AC= Activated charcoal
CH = Casein hydrolysate
CW= Coconut Water PVP= Polyvinyl pyrolinidone STS= silver thiosulfate
ME= Malt extract NaCI= Sodium chloride
Ca(N03) 2=Calcium nitrate NaOCI= Sodium hypochlorite
PEG= Polyethylene glycol AVG= Aminoethoxyvinylglycine

Growth Regulators:
2ip= N6-(2-isopentenyl)aminopurine 2,4-D = 2,4- dichlorophenoxyacetic acid ABA= Abscisic acid
ZEA= Zeatin IAA= Indole 3 acetic acid GA3= Gibberellic acid
DIC= Dicarnba IBA= Indole 3 butyric acid BAP= 6- benzyl aminopurine
KIN= Kinetin NAA= Naphthalene acetic acid
PIC= Piclorarn NOA= Naphthoxy acetic acid
4. SOMATIC EMBRYOGENESIS IN FRUIT AND FOREST TREES
OF ARID ZONE

R. Raj Bhansali and Manjit Singh

Division of Plant Sciences and Biotechnology
Central Arid Zone Research Institute
Jodhpur-342 003, India
Chapter contents-
!. Introduction
.1.1 In vitro_concept
1.2 Embryogenesis
2. Arid woody perennials
2.1 Fruit trees
2.1.1 Citrus
2.1.2 Date palm
2.1.3 Ber
2.1.4 Pomegranate
2.1.5 Bael
2.2 Agro-forestry trees
3. General protocols
3.1 Induction
3.2 Establishment
3.3 Maturation
3.4 Germination
4.,Critical factors
4.1 Explants
4.2 Selection procedure
4.3 Quantification
4.4 Media requirements
4.5 Growth regulators
4.6 Activated charcoal
4.7 Caroohydrates
4.8 Environmental conditions
5. Uniformity
6. Conclusion and future prospectus
7. References

1. INTRODUCTION
The application of tissue culture methods to the propagation of plants has dramatically altered the way
plants are routinely propagated. As tissue culture technology continues to expand and improve, these
techniques will be applied to the growing list of plant species. Most of the species that are presently being
propagated on a large-scale through tissue culture are herbaceous, ornamental, fruit and forest trees. This
technology provides the opportunity to produce in large number uniform, disease-free arid zone hardy
plants where lack of method for vegetative propagation, uniformity and/ or virus infestation had been
previously a serious problem. Tissue culture propagation is not likely to be applied in the near future to
crops that are easily and satisfactorily propagated through other conventional methods. But there are a large
number of species, many of which are important agronomically and horticultural crops, that are reproduce
asexually and could get benefit from this technology. Unfortunately, conventional vegetative propagation
practices can lead to the spread of plant pathogens, resulting in the loss of yield and quality. But, through

141
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 141-167.
© 2000 Kluwer Academic Publishers.
142

tissue culture, methods for producing pathogen-free stock plants can successfully be generated and
maintained in vitro, which ultimately increase the productivity on cultivation.

1.1/n vitro concept:

Currently, three in vitro methods are employed for micropropagation of arid zone woody
perennials. Firstly, clonal propagation generally involves placing shoot tips and/ or axillary buds on
multiplication medium to induce axillary shoot development. These axillary shoots are recycled on the
multiplication medium until the desired level of production is achieved. Shoot tips are then rooted,
hardened and transplanted in soil. This method is very labour-intensive due to a number of individual
manual manipulations involved and low multiplication rate. Second method is adventitious bud formation, a
form of organogenesis. In this case adventitious shoots can be induced directly on the original explants, then
rooted, harden-off and transplanted in soil. This method requires less manipulation than shoot tip culture but
requires a large number of explant sources depending upon the multiplication rate. The adventitious buds
from callus cultures have also the similar problem. In both systems, multiplication rate can be up to an order
of higher magnitude than from axillary bud multiplication. In number of cases the number of individual
manipulation is less, bringing down the cost per unit.

Finally, the third method is somatic embryogenesis. The initiation and development of embryos
from somatic tissues in plant tissue cultures was first observed by Steward eta!., 1958 and Reinert (1958,
1959) in cultures of carrot tissues, since then there has been a significant progress and numerous reviews
(Evans eta!., 1981; Amrnirato, 1983; Vasil, 1985; Lutz eta!., 1985; Tulecke, 1987; Monier, 1990; Kurtz et
a!, 1991; Liu eta!., 1993; Jain eta!., 1995, 1999; Meinke, 1995; Dodeman eta!., 1997). Consequently, this
review will mainly emphasize the work on somatic embryogenesis of arid zone fruits including agro-forestry
trees.

1.2 Embryogenesis:
In angiosperms, double fertilization generates the embryos and endosperm simultaneously, this
process leads to form viable seeds. Plant development from zygotic embryos involves embryogenesis up to
the cotyledonary stage and the maturation followed by germination. During the development of an embryo,
it passes through globular, oblong, heart and torpedo and cotyledonary stages and eventually to the mature
de,hydrated embryo, passes through a sequence of 20 different stages, based on studies conducted on
Arabidopsis tha/iana (Jurgens and Mayer, 1992). Seeds may also be developed without fertilization
(apomixis), means from the somatic cells of female gametophyte (megaspore mother cells by mitosis) and
sporophytic tissues (nucellus or integument of ovules). Isolated somatic or gametic cells (microspore) can
also induce embryos.

Adventive or asexual or somatic embryogenesis is the development of bipolar embryos from

somatic cells or tissues. Developmental stages are mostly similar to normal embryogenesis generated from
fusion of gametes, without having any vascular connection. Precisely, how adventive embryos arise from
the above tissues has been the subject of numerous studies. Although individual cells are totipotent and
carry all the genetic templates necessary for the development of the whole plant, isolated single cells do not
generally transformed into embryos by repeated divisions. Externally applied stimuli are necessary for the
transition of somatic cell to competent embryogenic cell to produce embryo (Toonen eta!., 1994). Somatic
embryos which are either initiated on callus or directly on explant tissues from superficial clumps of cells
associated with highly vacuolated cells which are non-embryogenic. Such observations have been made on
the cellular structures of embryogenic clumps of cells in callus derived from immature zygotic embryos of
Punica granatum and Azadirachta indica woody trees of arid zone (Raj Bhansali, 1990; Raj Bhansali et a!.,
1995).

Dense cytoplasmic contents, large starch grains, and relatively large nucleus with darkly stained
nucleolus characterize the embryo forming cells. Staining reagents indicated that these embryogenic cells
have high concentrations of proteins and RNA (Fig. 1). Each pro-embryogenic cell is capable of passing
through the sequential changes and attains stages of embryo formation (i.e., globular, heart shape and
torpedo shape) (Fig. 2). Two critical events are involved in early programming of this process: 1) induction
of cytodifferentiation of the pro-embryogenic cells, and 2) unfolding of developmental sequences by these
 143

pro-embryogenic cells (Kohlenbach, 1978). Sometimes embryo development may be blocked by an

imbalance of nutrients in the culture medium particularly high auxins and or nitrogen sources. For normal
development of somatic embryos, generally two different types of media are required: 1) Medium for
initiation of embryogenic cells, and 2) complete development of embryos.

Sharp et al., (1980) described two routes to somatic embryogenesis. The first is embryogenesis
where embryos initiate directly from tissues in absence of callus. This occurs through "pre-embryogenic
determined cells" (PEDC) where cells are already committed to embryogenic development. The second is
indirect embryogenesis where some cells or callus proliferation is required. This occurrence is in response
of differentiated or non-embryogenic cells or "induced embryogenic determined cells" (IEDC). Later on
these routes were more appropriately named as direct and indirect somatic embryogenesis in tissue cultures
(Sharp et a!., 1984). Direct embryogenesis is referred to the development of embryos directly from explant
tissues, while indirect embryogenesis, developed from callus cells or tissues or suspension cultures. The
indirect method is highly repetitive, therefore, also called as "Repetitive Somatic Embryogenesis" (RSE).
This method is most widely used for most of plant species (Ammirato, 1983; Gupta and Durzan, 1985; Jain
eta!., 1988 & 1989; Raj Bhansali and Kaul, 1991)

Genetic gains of arid zone woody plant species can now be captured and amplified through
adopting biotechnology of both types of somatic embryogenesis. It offers an inexpensive, large-scale
propagation system for superior genotypes. It has a potential to produce of large number of somatic
embryos (60,000 embryos per liter medium) (Ammirato 1983). Presence of root and shoot meristems, easily
scale up and transfer, encapsulation, long-term storage, packaging, direct delivery system and genetic
manipulation are other benefits of this technology.

Both zygotic and somatic embryogenesis are complex phenomena (Meinke, 1995). Early
developing embryos are of globular shape, whereas the mature embryo is a bilaterally symmetrical structure
along the apical-basal axis and has two cotyledons in dicotyledonary plants. The sequence of developmental
events is an asymmetric cell division, cell polarity and formation of mature embryos in dicots.

Somatic or asexual embryos can be differentiated directly or indirectly into secondary

elllbryogenesis from cells, organ or intermediate callus of a plant. Most often somatic embryos will further
proliferate to yield secondary embryos (Fig. 3). These embryos are easily separable and may be grown to
complete new individual plants and established in soil. Somatic embryogenesis has now become one of the
most potential in vitro methods for efficient plant regeneration in many of arid zone taxa, since literally
million of bipolar embryos and complete plants could be produced from a relatively small amount of
cultured cells or tissues.

It is estimated that approximately 1,000 date palm embryos could be obtained from 200-mg
embryogenic cultures per vessels. Forty per cent of embryos germinated into normal plantlets (Bhaskaran
and Smith, 1992). Conventionally, date palm is propagated through offshoots produced from mother palms.
But, the offshoot production is very limited in number (10-25) in whole life span. Therefore planting
material of superior cultivars are highly expensive. Looking to this constraint biotechnology of date palm
somatic embryogenesis alongwith synthetic seed technology could be a boon in boosting production of elite
planting material of date palm. Significant research efforts continue to be directed towards the utilization of
somatic embryogenesis in vitro pathway to produce artificial seeds of economically important via coating or
encapsulation of somatic embryos (Lutz eta!., 1985; Redenbaugh et al., 1986: Gupta et al., 1991). Now,
additional research is required in area of encapsulation, storage and conversion into normal plants. The
major limitation of somatic embryogenesis as routine regeneration method for commercial plant production
is that very few arid zone species have been adequately characterized concerning the development of
commercially feasible protocols.

Although the list of arid zone plant species that can induce somatic embryogenesis is short,
however, few species can regenerate complete plants and is not restricted to few taxa. The potentialities
exist in cultured cells from any plant provided appropriate stimuli and conditions, could be fostered to
144

Fig, 2 :Various stages of somatic embryogenesis in pomegranate (Punica granalum)

(!)-- Embryo forming ceU

- Embryogenic clumps

/ !"-

y --Heart stage

' v
,I
"'"'
1
'ol

- - Torpedo stage

Germinated
somatic
seedling

induce embryos. At this stage molecular and genetic stimuli aimed at identifying the mechanisms under
lying the sequence of events during embryogenesis is fragmentary, but techniques are available to
understand the somatic embryogenesis pathway. How does an undifferentiated mass of cells that
comprising an early embryo take on a pattern to develop complete mature embryo having bilateral
symmetry? This question remains one of the most basic in developmental biology so far evidences have
proven elusive, particularly in the plants (Bryant and Cuming, 1993; Chasan, 1993; Liu et al., 1993;
Dodeman et al., 1997; Krishnamurthy, 1999).
 145

Identification of embryos in callus tissues of pomegranate through staining technique.

Proliferation of somatic embryos through secondary embryogenesis

Induction of embryogenic calli from nodal explant tissues of citrus.

146

2. ARID WOODY PERENNIALS

The most common economically important fruit and agro-forestry trees are listed in Table-!. The
induction of somatic embryogenesis in arid zone perennial plants has been reported in Citrus sinensis
(Chaturvedi and Mitra, 1974), C. aurantifolia (Raj Bhansali and Arya, 1978b, 1979), Punica granatum
(Raj Bhansali, 1990), Aegle marmelos (Islam eta!., 1996 a & b), Zizyphus spp.,(Raj Bhansali, 1989; Sung
and Song, 1992; Islam eta!., 1994), Phoenix dactylifera (Sharma eta!., 1984, 1986; Raj Bhansali et al.,
1988), and agro-forestry tree species: Azadiracta indica (Raj Bhansali et a!., 1995; Murthy and Saxena,
1998), Eucalyptus citriodora (Muralidharan and Mascarenhas, 1987), Dalbergia latifolia) (Rai, 1993), D.
sissoo (Das eta!., 1997), Acacia nilotica (Garget a!., 1996), Hardwickia binata (Das et al., 1995).

Somatic embryogenesis has been induced in more than 8 families, genera, species and numerous
tree cultivars of arid zone (Table 2). Among characteristics of woody perennials, which make them more
intractable for the induction of somatic embryogenesis are: I) lack of juvenile phase or short seasonal time
period for active growth stages in mature trees, when plant parts are available for initiation of cultures 2)
tissues are hardy for initiation of cultures and take long period for establishment and regeneration 3)
production of toxic phenolics from explants during starting of tissue culture 4) induction of somatic
embryogenesis from most of mature arid zone tree explant tissues are difficult.

Table 1. Common fruits and agro-forestry trees of arid and semi-arid regions in India

Plant family Plant species Common name

Fruit trees
Annonaceae Annona squamosa Custard apple
Capparaceae Capparis decidua Ker
Euphorbiaceae Emblica officina/is Aonla
Myrtaceae Syzygium cumini Jamun
Punicaceae Punica granatum Pomegranate
Rhamnaceae Zizyphus mauritiana Ber
Zizyphus rotundifolia Bordi
Zizyphus nummularia Jharberi
Rutaceae Citrus sinensis Sweet lime
Citrus aurantifolia Kaghji lime
Aegle marmelos Bael
Tiliaceae Grewia subnaequalis Phalsa
Agro-forestry tress
Bignoniaceae Tecomella undulata Rohida
Buxaceae Sirnmondsia chinensis Jojoba
Combretaceae Anogeissus pendula Dhawra
Commelinaceae Commiphora wightii Guggal
(Burseraceae)
Legurninosae Acacia tortilis Israeli babul
Acacia senegal Kumat
Acacia nilotica Babul
Albizia lebbeck Siris
Dalbergia sissoo Shishum
Dalbergia latifolia Rose wood
Hardwickia binata Anjan
Prosopis cineraria Khejari
Prosopis juliflora Vilayati babul

Meliaceae Azadirachta indica Ncem

Myrtaceae Eucalyptus camadulensis Safeda
Salvadoraceae Salvadora oleoides MithaJal
Salvadora persica Khara Jal

However, long time span is single advantage of perennial trees for obtaining appropriate tissues from
mature trees in raising tissue cultures. The potential for developing new improved tree species from
 147

suspension culture, protoplast fusion product(s), high frequency somatic embryogenic lines from wide
crosses, disease resistant and free plants, somaclonal variation and genetically engineered for superior
genetic gains are well justified in fruit and forest trees of arid zone.

2.1 Fruit trees:

Arid and semi arid zone fruit plants have mostly been studied for in vitro micropropagation in
India include: Phoenix dactylifera, Punica granatum, Annona squamosa, Zizyphus mauritiana, Z
rotundifolia, Z nummularia, various Citrus species, Aegle marmelos, Emblica officina/is, Capparis
decidua, Syzygium cumini and Grewia subnaequalis etc. However, somatic embryogenesis method has been
used in few genera (Table.2). We will describe somatic embryogenesis in these genera.

2.1.1 Citrus
Citrus fruits particularly Citrus sinensis, C. reticu/ata, C. paradisi and C. aurantifolia are grown profitably
in arid tracts having irrigation facilities (Chandra et al., 1994). They are the most important citrus species
and cultivars of tropical fruits. The major areas of citrus cultivation in arid and semi-arid zones are in
.Punjab (Ferozpur, Faridkot and Bhatinda districts). In Rajasthan, Ganganagar is well kinetinown for
Kinnow followed by sweet oranges are cultivated at large scale. Kotha, Jhalawar, Swai Modhopur and
Jodhpur are other potential districts for citrus cultivation. In Haryana, Sirsa and Hisar districts are major
citrus growing regions.

Grafting buds of scion cultivars onto the seedlings of rootstocks generally propagates citrus. It is
also possible to raise true-to-type seedlings due to occurrence of polyembryony in citrus species. In kaghji
nimbu (C. aurantifolia), propagation through seeds is a commercial method. Besides grafting, cutting and

Table 2. Somatic embryogenesis in cultures of fruit and forest tree species of arid zone

PLANT EXPLANT GROWTH RESPOSE REFERENCE

Fruit Trees
Rutaceae
Aegle numnelos zygotic embryos SE&PL Arumugam and Rao, 1996.
with cotyledons Islam et al., 1996

cftrus nucellus/callus SE&PL Rangan et al., 1968, 1969

Bitters et al., 1970
Navarro and Juarez, 1977
styles SE&PL Canni et al., 1995
ovules SE Parathasarthy, 1993

Citrus aurantifalia seed callus SE&PL Raj Bhansali and Arya, 1978 a,b&c
Chaturvedi and Sharma, 1985

Citrus sinensis seed callus SE&PL Raj Bhansali and Arya, 1978 c,
Ohgawara et al., 1989
Pal mae/
Aracaceae
Phoenix dactylifera ovule/embryo/ SE&PL Reynold and Murashige, 1979
axillary bud/ Tissert, 1987
shoot tip/ Shannaet al., 1984&1986
inflorescence/ Mater, 1986
suspension ·culture Raj Bhansali et al., 1988
Raj Bhansa!i and Kaul, 1991
Bhaskaran and Smith, 1992
Punicaceae
Punica granatum zygotic embryo SE&PL Raj Bhansali, 1990
with cotyledonary tissues/petal Nataraja and Neelarnbica, 1996
Rhamnaceae
Zizyphus jujuba immature cotyledons/ SE&PL Sung and Song, 1992
seedling explant Mitrofanova et al., 1997
Raj Bhansali, 1989
Kabir et al., 1994
Zizyphus mauritiana Seedling SE&PL
148

Forest Trees
Burseraceae
Commiphora wightii immature embryos SE Singh eta!., 1997
Buxaceae ovule SE&PL Lee et al., 1982
Sirnmondsia chinensis

Leguminosae
Acacia nilotica immature endospenn SE & Triploid plant Garget al .• 1996

Albi;zia Iebbeck hypocotyl SE Gharyal and Maheshwari, 1981

Dalbergia sissoo callus/immature seed SE Das eta!., 1997

Dalbergia latifolia immature zygotic embryos SE&PL Rao and Laxrni Sita, 1996

Hardwickia binata immature cotyledonary explant SE Das eta!., 1995

Meliaceae
Auulirachta indica callus/seedling explant/zygotic SE&PL Raj Bhansali et a1.,1995
embryos Murthy and Saxena, 1998
Myrtaceae
Eucalyptus citriodora embryo with SE Muralidharan and Mascarenhas.
cotyledons 1987
Eucalyptus camadulensis zygotic embryos SE Muralidharan et al., 1989
Eucalyptus grandis embryos with cotyledons SE Wattet al .• 1991
Eucalyptus tereticornis seed SE Gill et al .• 1994
SE= somatic embryos PL=Plantlets

air layering methods have also been used but are not very much satisfactory. Tissue culture methods have
been used for meeting various aims and objectives for micropropagation (organogenesis and somatic
embryogenesis), embryo rescue, embryo culture, meristem culture etc.

Somatic embryogenesis: Many citrus species are polyembryogenic and adventive embryos are usually
produced in vivo from nucellar tissues. Somatic embryogenesis has been reported in cultures of
adventitious embryos, styles, isolated nucelli, fertilized and unfertilized ovules and directly from
gorminating seeds or callus raised from these tissues (Rangan et al., 1968, 1969; Bitters et al., 1970;
Navarro and Juarez, 1977; Raj Bhansali and Arya, 1978c; Carmi et a!., 1995; Srivastava et al., 1997).
Regeneration of plants via somatic embryogenesis has also been reported from the protoplasts isolated from
embryogenic callus of six plant species related to citrus (Jumin and Nito, I 996)

Raj Bhansali and Arya (1978c) found that embryogenic calli and somatic embryos were initiated
from seedling explants of C. sinensis and C. aurantifolia (Fig. 4). Mitra and Chaturvedi (1972) also
obtained similar results in unfertilized ovules of polyembryonic citrus species. Embryogenic citrus
suspension cultures have been obtained generally from embryogenic calli grown on semi-solid medium
(Kochba et al., 1978; Harms and Potrykus, 1980). Factors responsible for in vitro somatic embryogenesis
from under developed ovules of mature fruits has been reported (Moore 1985; Parthasarthy and Nagaraju,
1992 &1994).

There have been few Reports on the induction of embryogenic callus from mono-embryogenic
citrus species. Embryogenic calli have been induced from other explant sources of different citrus species.
Embryogenesis as well as organogenesis simultaneously or separately occurs in stem segment callus from
sweet orange, lime and sweet lime. Triploid somatic embryos and plantlets have been obtained from
pummelo endosperm callus (Wang and Chang, 1978). Androgenic plants have been produced from
uninucleate pollen grains at tetrad stage by inducing embryoids (Chaturvedi and Sharma, 1985). Raj
Bhansali and Arya (1978c) found a direct relationship between polyembryony and somatic embryogel)esis
in citrus genus. Polyembryonic species are more prolific than Jess embryonic plants. There are only a few
reports on the embryogenesis in somatic citrus cells that are neither nucellar nor ovular in origin (Ongawara
et al., 1988; Nito and Iwamasa, 1990; Tusa et al., 1990). As embryos and calli obtained from cultured
ovules are derived solely from zygotic and nucellar embryos already present in the explants, a genetic
 149

'contamination' of the callus obtained from the ovules may be occurred. In comparison to these explant
tissues, the genome of embryos regenerated from styles and other somatic plant parts cannot be influenced
by the presence of the zygotic embryo, and should be genetically identical to the mother plants from which
flowers or somatic tissues had been collected. In this way somatic embryos from the styles of citrus cultivars
tested produced 15,000 embryos within 4 months of culture, which subsequently germinated and grew into
plantlets at a high frequency (Carimi et al., 1995). More recently a few plants are regenerated from leaf
protoplasts of 'Marsh' grapefruit (Ohgawara eta!., 1989) and lemon (Tusa et al., 1990) co-cultivated with
protoplasts isolated from embryogenic habituated nucellus derived callus. Nita and lwamasa (1990) induced
the formation ofeight embryoids from juice vesicle callus of Satsuma. Fortunately at this stage, somatic
embryogenesis in many citrus species has been reported from embryonic and somatic tissues of non-ovular
in origin for rapid micropropagation.

2. 1.2 Date palm

Date palm (Phoenix dactylifera L.) is the oldest among the cultivated fruit trees. In India, it is
believed that date palm was introduced by the soldiers of Alexander in the 4th century in the Indus valley.
The date palm cultivation by planting regular orchard in Rajasthan was first started by then the ruler of
erstwhile Bikaner state, Maharaja Ganga Singh. He planted the suckers of date palm cultivars e.g. Halawy,
Khadrawy and Zahidi at Siriganganagar.

The date palm requires specific climatic conditions, as per an Arabic saying "Date palm should be
grown with its feet in running water and its head in fire". It requires prolonged hot dry summers and rain-
free area particularly at the time of fruit ripening for attaining Pind stage (July-August period) (Chandra et
a!., 1994). Western Rajasthan comprising Jaiselmer, Barmer, Bikaner and Jodhpur districts are ideally
suited for date palm cultivation and it has the vast potential. The date palm varieties including Halawy,
Shamran, Khadrawy, Medjool, Barbee, Zagloul, Hayani, Zahidi, Khalas and Sewi are important in reference
to requirement of heat summation unit to reach colour-turning stage (early Doka and Pind stage). These
varieties have been found suitable in the states of Rajasthan, Gujarat, Haryana and Punjab (Manohar and
Chandra, 1995).

Date palm is commercially propagated from the suckers (offshoots). It can also be propagated from
seeds but being dioceous in nature, it produces male and female plants on separate palrns. Therefore
propagation through suckers is preferred ovllr seeds. An offshoot is a lateral branch, which develops from
the base of above on the trunk of a tree. Basal offshoots have usually well developed root system as
compared to aerial suckers. Date palm produces 10 to 25 suckers during whole lifetime (Chandra et a!.,
1994). According to Pareek and Nath (1996) 8-20 offshoots of 8-15 kg size can be obtained from each
mother palm during its 41h-101h year and none thereafter. Vegetative propagation has been reported by the
several workers through tissue culture methods (Oppehenmer and Reuveni, 1972; Tisserat, 1984; Raj
Bhansali and Kaul, 1991).

Somatic embryogenesis: Asexual embryogenesis has been obtained from excised zygotic embryos (Arnmar
and Benbadis, 1977, Reynold and Murashige, 1979) and also from somatic tissues (Tisserat eta!., 1979;
Mater, 1986; Sharma et al., 1984,1986; Raj Bhansali, eta!., 1988, Dass eta!., 1989; Bhaskaran and Smith,
1992; Sudhersan et a!., 1993). During establishment of embryogenic callus growth and development in
vitro, the-date palm explants release excessive browning substances which is a great problem. (Zaid, 1987).
These substances (phenols) have profound physiological effects on the establishment and growth of
embryogenic callus. Browning of explant tissues and the tissue culture medium is assumed to be due to the
oxidation of polyphenols and formation of quinones, which are highly reactive and toxic to the tissues (Raj
Bhansali and Singh, 1982). The inhibitory effects may result from the bonding of phenols with proteins and
their subsequent oxidation to the quinones.

Murashige (1974) has suggested the presoaking of explants in ascorbic acid and citric acid
solutions and adding them to the culture medium for curtailing the oxidation of the phenols. Incorporation
of polyvinyl pyrrolidone, cysteine-HCl and ascorbic acid also helped in minimizing browning problem in
150

number of plant species including sugarcane and date palm (Raj Bhansali and Singh 1982; Dass et al.,
1989). Zaid and Tisserat (1983) soaked the date palm explants in antioxidant solution (150 mg/1 citric acid
and 100 mg/1 ascorbic acid) prior to surface sterilization treatment. Raj Bhansali and Kaul (1991) have also
used these antioxidant solutions for 30 minutes incubation in cold storage (0 °C). Further nutritionally
balance media having 3gll activated charcoal have checked significantly the browning problems of date
palm. Raj Bhansali et al., (1988) found that shoot tips and lateral bud cultures grow successfully on
frequent transfers (after a short period of incubation 7 to 15 days) them onto the fresh media.

Table 3. Date palm explants and their somatic embryogenesis

Explant source Availability Somatic embryogenesis Remark
potential

Apical tip One per tree/ Very high Sacrifice whole plant or offshoot
offshoot
Inflorescence Abundant Immature-High Dependent on
Mature-Low flowering time
Lateral Several Variable Sacrifice the plant to
bud Young-High obtain
Old-Low
Zygotic Abundant Variable Not clonal, specific
embryo Immature-High genotype

Date palm somatic embryogenesis has been developed by various workers (Rhiss et al., 1979;
Drira, 1983; Sharma et al., 1986; Raj Bhansali, et al., 1988; Sudhersan et al., 1993) (Fig. 5). Free-living
plants have been established in the field raised through tissue culture at Haryana Agriculture University,
Hisar and in other Institutes (Fig. 6). The repetitive somatic embryogenesis (RSE) process developed by us
is highly efficient in raising date palm tissue culture in certain varieties.

Suspension culture: Various workers have also tried to establish suspension culture in date palm friable
callus for rapid embryogenesis (Sharma eta!., 1986; Bhaskaran and Smith, 1992). The pro-embryo masses
developed into embryos after passing through several sequential and distinct embryo development phases.
Hyndreds of embryos could be developed from suspension culture within 3 weeks. These embryos were
developed after one month to promote further growth. Approximately 1000 embryos could be obtained
from 200 mg embryogenic friable callus cultured per vessel (Bhaskaran and Smith, 1992). These embryos
(up to 40 o/o) germinated into normal plantlets on plating on solid medium. They could be transferred to the
soil. The somatic embryogenesis in date palm will lead to cryopreservating and encapsulation of embryos
for long-term storage and shipment for export.

2.1.3 Ber

Ber (Zizyphus mauritiana) is a hardiest cultivated fruit tree in north Indian plains. Ber fruits have
been in use since ancient times. It is distributed all over the warm arid and semi-arid regions due to the
drought and heat tolerant. It is now becoming more and more popular due to low price and having high
nutritional value and rich in vitamin C, A and B complex (Chandra et al., 1994). Ber leaves are rich source
of proteins and mineral matter, which provide a nutritious fodder for the animals. The major grafted ber
growing states are Haryana, Punjab, Uttar Pradesh, Rajasthan, Gujarat, Madhya Pradesh, Bihar,
Maharashtra, Andhra Pradesh and Tamil Nadu. Seedling her trees are also grow widely in arid and semi-
arid areas (Pareek, 1997).

Ber belongs to family Rharnnaceae and genus Zizyphus. There are two prominent species of her
viz., Chinese ber (Z. jujuba) and India ber (Z. mauritiana). Multiplications of true-to-type of superior
cultivars with good fruit quality and higher yield are important factors for profitable cultivation. The
popular method of propagation is by l-or T-shield, ring and patch budding. The most common commercial
method is by the T -shield budding because it is easier to adopt and having 87 to 97% success (Chandara et
a!., I 994 ). Rootstocks viz .. , Z. rotundifolia, Z. nummularia, Z. oenop/ia, Z. rugosa and Z. xylocarpa can be
used but Z. rotundifolia has most vigorous rootstock giving maximum plant height, spread, collar diameter
 ,, ..•
151

., ~--· ·-·
5. Somatic embryos in date palm callus tissues originated from apical shoot tip.

6. Large number of somatic seedlings developed from somatic embryos of date palm.

7a&b. Development of somatic embryos and regeneration of ber somatic seedlings.

152

and canopy area (Chaqndara eta!., 1994; Pareek, 1997). Micropropagation through proliferation of axillary
bud have been reported in various species of Zizyphus species (Goyal and Arya, 1985; Rathore eta!., 1992;
Kabir eta!., 1994; Mathur eta!., 1995; Raj Bhansali, 1998).

The suitable cultivars in ber are: Gola suited to very dry condition, Kaithali, to slightly higher
rainfall, and Umran to most moderate climatic conditions. The cultivars Dharakhi-1, Dharakhi-2, Villaiti
and seedless have shown resistance to powdery mildew at Rahuri and whereas Illachi, Jhajjar, Katha, Phal,
Safed Rothak and Sanaur-5 at Hisar (Pareek, 1997).

Somatic embryogenesis: Asexual embryogenesis in Z mauritiana was induced in seedling explant tissues
on Murashige and Skoog medium (Murashige and Skoog, 1962) containing 2.5 mgn 2,4-
dichlorophenoxyacetic acid, 0.5 mgn benzylaminopurine and 0.25 mgn kinetin in darkness initially for 2-3
subcultures at 26° C (Raj Bhansali, 1989 and Kabir eta!., 1994). The cellular embryos were in various
stages of development but recovery of plantlets was very rare, on 2,4-dichlorophenoxyacetic acid
containing medium. However, Sung and Song (1992) has reported induction of somatic embryogenesis in
Chinese jujube (Zizyphus jujuba Miller) from immature cotyledons and seedling parts on MS containing 2.5
mgn 2,4-dichlorophenoxyacetic acid, benzylaminopurine and 50% sucrose and complete regeneration on 1
mgn zeatin and 0.01 mgn 2,4-dichlorophenoxyacetic acid. Embryos developed into rooted plantlets on
medium supplemented with 2-5 mgn benzylaminopurine without auxin under 16 hr photoperiod (Fig.
7a&b). Toxin resistant cell lines against Fusarium oxysporum and Colleototricum species were also
developed in Z. rotundifolia rootstock (Raj Bhansali, 1993b, 1999). Mitrofanova et a! (1997) also induced
somatic embryogenesis and complete regeneration of plants in Z jujuba (Chinese date) on half strength MS
having I mgn 2,4-dichlorophenoxyacetic acid through direct somatic embryogenesis. The plantlets were
regenerated on modified Pi erik medium (Pi erik, 1971) without growth regulators and acclimatized in vitro
conditions.

2.1. 4 Pomegranate:

Pomegranate (Punica granatum) is a favourite table fruit and is popularly kinetinown as Anar or
Dalima or Dadima in India. It came to India from Middle East via Persia or Afghanistan presumable in the
fiJ;St century. The tree is large shrub or small tree having cylindrical branches with spines acclimatized very
well in the tropical and sub-tropical regions. The fruit is very nutritious and refreshing. The edible portion
is the seed aril containing mineral matter particularly phosphorus and iron, besides thiamine, riboflavin and
vitamin C. It is also valued highly for secondary metabolites, which are of pharmacological interest.

It belongs to family Punicaceae and genus Punica. Pomegranate cultivation became commercially
important in Maharashtra, Gujarat and Rajasthan, Andhra Pradesh, Uttar Pradesh, Karnataka and Tamil
Nadu. The prominent commercial cultivars are Ganesh and Musket in Maharashtra, Basin seedless in
Karnataka, Dholka in Gujarat and Jalore seedless in Rajasthan. Jodhpur-Red has occupied considerable area
in Rajasthan but its seeds are hard. Plants can be easily multiplied by cuttings or by air layering. Hard
wood cuttings taken from fully matured wood of one-year-old plant are most suitable for vegetative
propagation. Dipping of cutting 200 ppm indoleacetic acid has been found helpful to induce rooting with
profuse root system. Tissue culture methods have also been used for micropropagation of various varieties
of pomegranate by employing apical and nodal buds, leaf and seed explant tissues (Omura et al., 1987 a &
b; Jaidka and Mehra 1986).

Somatic embryogenesis: It has been reported in seed explant and petal tissue (Raj Bhansali 1990; Nataraja
and Neelambica, 1996). Embryogenic cell lines can be selected by using staining techniques developed by
Raj Bhansali et al. 1990 for peach somatic embryogenesis by using acetocarmine (Sharma and Sharma,
1972) or Alexander stain (Alexander, 1980)(Fig. I).

Embryos produced roots after 8 weeks on MS medium containing 5 mgn indoleacetic acid or
naphthaleneacetic acid. However, shoots were developed when embryogenic cultures were placed on
medium having 5 mgn benzylaminopurine or 2.5 mgn kinetin. Embryos rapidly proliferated into secondary
 153

embryos in the presence 5 mg/1 benzylaminopurine and 0.1 mg/1 indoleacetic acid (Raj Bhansali, 1990)
(Fig. 8). Complete plantlets were obtained on half strength MS medium containing 1 mg/1 indoleacetic acid
or indolebutyric acid. Large number of fully developed plantlets could be produced by using nodal bud
segments or via somatic embryogenesis in number of pomegranate species including ornamental types.
Undoubtedly, the in vitro technique would facilitate rapid multiplication of elite varieties in a short time
than the conventional propagation by cutting besides the genetic manipulation at cellular level.

2.1.5 Bael

Bael (Aegle marmelos Linn.) Corr. is a hardy fruit tree and can be grown in arid climate. It
belongs to family Rutaceae. The tree is deciduous and medium sized having characteristic trifoliate leaves,
which are divided into three leaflets. Bael tree has both mythological (commonly grown in temple or
religious compounds) and medicinal significance (good for stomach ailments, diarrhoea and dysentery).
Ripe fruit serves as a tonic, laxative and good for heart and brain. Fruit has high nutritive value having
riboflavin, carotene, niacin and vitamin C.

It is easily grown in poor soil even in acidic, alkaline and stony soils having pH range from 5 to 10.
There are no standard names of bael cultivars. They are kinetinown by name of place such as Mirzapuri,
Rampuri, Kaghji Gonda, Kaghji Etawah, Deoria large, Basti No. 1 etc. Common method of propagation of
bael is through seeds, which are sown in the month of June and seedlings become ready for transplantation
after a year. The seedling trees are not true-to-type. Plants can also be propagated by means of budding,
grafting and air layering. Budding (T-hudding, patch budding, chip budding) is most popular for raising
saplings for plantation in new orchards. Micropropagation has been quite successful for rapid multiplication
of various selected varieties (Arya eta!., 1981; Hossain eta!., 1993a; Arumugham and Rao 1996; Hazarika
et al., 1996; Ajitkumar and Seeni, 1998).

Somatic embryogenesis: Asexual embryogenesis and regeneration of plantlets.have been reported in seeds
obtained from unripe fruits of 20-years-old A. marmelos tree (Islam et al., 1996b). In this case embryonic
callus has been established from zygotic embryos on 2,4-dichlorophenoxyacetic acid and
benzylaminopurine (1J.LM) containing medium. Higher concentration of benzylaminopurine above I J.lm
showed inhibitory effect on the induction of somatic embryos. However, futher refinement of of in vitro
technique is requred before the somatic embryogenesis technology can be applied to commercial bael
culti vars on large-scale.

2.2 Agro-forestry trees:

The forest vegetation specially agro-forestry tress such as Prosopis cineraria, (khejari), P. julijlora
(vilayati babul), Tecomella undulata (rohida), Acacia nilotica (babul), A. senegal (kumat), A. toni/is
(Israeli babul), Anogeissus pendula (dhau), Caligonum polygonoides (phog), Salvadora oleoides (mitha
jal), S. persica (khara jal) A<.adirachta indica (neem), Dalbergia sissoo (shishum), Eucalyptus
camadulensis (safeda) and Maytenus emerginata are multipurpose trees of arid zone of India (Paroda,
1979; Goodin and Northington, 1985; Bhandari, 1990). These trees are drought and heat resistant biomass
producers under arid climate. These woody perennials are well adopted in arid environment and
ecofriendly. They are valuable to stabilize the sandy soil and provide fodder, food, fuel and timber beside
various industrial byproducts to support the life system of arid ecosystem (Faroda et al., 1997).

Tissue culture methods h!lve been employed by various workers for rapid multiplication of
selected elite trees as they are mostly slow growing and non-availability of vegetative propagation method
(Raj Bhansali, 1995). Efforts have been made to develop a quick method for clonal propagation through
organogenesis and proliferation of axillary buds by using in vitro techniques (Arya et al., 1981; Raj
Bhansali, 1998, 1999). Nodal buds of mature trees of P. cineraria (Nandwani and Ramawat, 1993), T.
undulata (Rathore et al., 1992; Raj Bhansali, 1993a) Azadiracta indica (Eeswara et al., 1998; Roy et al.
1994; Gill et al., 1994), Acacia tortilis (Nandwani, 1995). Somatic Embryogenesis is another potential
tissue culture pathway for mass cloning of elite genotypes as it has various advantages over organogenesis
besides easy manipulation by using protoplast and molecular techniques for the improvement of arid zone
!54

8. Embryogenic calli having larae number of cellular somatic embryos induced in

pomegranate cultures.·

9a&b. Large number of cellular somatic embryos and regeneration of plant-lets of

Azadirachta indica

10. Development of pro-embryogenic clusters from mixture of old and new embryogenic
centres in suspension cultures
 !55

plant species. Somatic embryogenesis of agro-forestry trees potentially offers alternative and efficient
system for plant multiplication. It has been achieved in number of dicotyledenous and monocotyledenous
angiosperms. However, adventive embryogenesis has been r~ported for relatively fewer arid zone woody
plant species, including leguminous trees appear to be recalcitrant to in vitro culture and regeneration of
plants. Thus success rate is rather low in inducing somatic embryogenesis and plant regeneration has been
achieved in very few genera of arid zone woody plant species. A comprehensive list is given in table 2.

Medha eta!., (1993) reported somatic embryogenesis and plant regeneration in Azadirachta indica
which was obtained in intervening callus phase. Direct embryo development on immature seed explant
tissues has been reported on MS medium containing benzylaminopurine I mg/1 and naphaleneacetic acidD
or 2,4-dichlorophenoxyacetic acid 0.25 mg/1 (Raj Bhansali eta!., 1995) (Fig. 9a&b). Thidiazuron (TDZ, I-
50 J.lM) has been found successful in initiation of somatic embryos in mature seeds (Murthy and Saxena,
1998). Cell suspension cultures were also established with TDZ and large cell clumps were formed within
2-3 weeks. Development of complete plantlets and continuous growth in greenhouse environment has been
achie~ed (Murthy and Saxena, 1998). Immature endosperm of Acacia nilotica developed into nodular callus
on MD medium supplemented with 2,4-dichlorophenoxyacetic acid, benzylaminopurine and casein
hydrolysate (Garget a!, 1996). After third passage in dark, callus induced somatic embryos, which were
triploids in nature. The triploids are desirable, as it is likely to promote vegetative growth instead of seed
production. The A. nilotica has an inherent character of profusely seedling tree.

Several species of Dalbergia are main source of rose wood timber. It is in great demand in Indian
timber industry because quality of wood is commercially important. Micropropagation by somatic
embryogenesis has been used to speed up the planting material of superior planting stock. Das et a!., 1997
has reported induction of somatic embryogenesis in callus derived·from 40-year-old semi-mature zygotic
embryos of D. sissoo on MS medium containing 0.46-1.16 J.1.M kinetin, 6.78-9.04 J.1.M 2,4-
dichlorophenoxyacetic acid. The light green embryos germinated on Yz strength MS salts and supplemented
with 0.5 mg/1 ABA and 2% sucrose. Rao and Laxmi Sita, 1997 have reported direct regeneration if somatic
embryos from immature zygotic embryos of D. latifolia. Preculture on high 2,4-dichlorophenoxyacetic acid
for 4 weeks induced direct somatic embryogenesis. Developed embryos produced plantlets on MS medium
with 0.5-1.0 mg/1 benzylaminopurine.

Hardwickia binata is a hardy leguminous tree. It successfully grows in dry tracks of arid and semi-
arid areas. The wood is commercially important for construction and ornamental work. Somatic
embryogenesis has been achieved in callus culture derived from immature cotyledonary explants of H.
binata on MS containing 2900 mgll KN03 supplemented with 4.64 J.1.M kinetin and 5.37 J.1.M
naphaleneacetic acid (Das et a!., 1995). Embryos proliferated quickly after transfer to MS basal medium
supplemented with 2052.6 J.!.M, L-glutamine and 0.084 J.!.M, Gibberellic acid (GA3). Maturation of embryos
was achieved on half strength MS basal medium supplemented with 1.23 J.1.M indolebutyric acid and 2%
sucrose.

3. GENERAL PROTOCOL
3.1 Induction:
Growth initiation for embryo induction is better on solidified medium contammg (2,4-
dichlorophenoxyacetic acid, 1-5 mg/1). The embryogenesis occurs directly on the explants or induced callus
from them within 3-4 subcultures by initially incubating in dark phase.
156

Table 4. General protocol for somatic embryogenesis and plant regeneration

Stage/process Factors
I. Selection of material · Genetic base, zygotic tissues, leaf, petal, shoot tip and stage of development
2. Conditions Ught, dark, low temperature treattnent
3.Media Conditioning. inductive, sequence, frequency of transfer, charcoal, complex
addenda, growth regulators
4. Somatic Direct, adventive, repetitive, indirect, suspension of cells, protoplasts, screening
embryogenesis
5. Somatic embryos Maturation, selection, donnancy, cold treattnent, abnonnalities, growth requirement
6. Gennination Primary root and shoot, cocyledon, expansion and greening, epico!)ll growth, leaves
7. Propagation in potlsoil Transfer to non-sterile environment, dilute nutrient medium, day length, ambient
humidi!)l, fungicides
8. Acclimatization Gradual leaf development in ambient temperature and humidity, hardiness to
atmospheric fluctuations
9. Evaluation Test for clonal or variant characters, selection and screening of plants

3..2 Establishment:

1. Embryogenic cultures should be removed from flask or tube with sterile forceps and transferred to
petridish containing filter paper. Discard the agar medium and browning parts of callus and inoculate a
fragment of about 0.25-2g approx. in 100-250 ml Erlenmeyer flask containing 50 mlliquid or 30 ml
semi-solid (agar) medium. Smaller Erlenmeyer flask can be used, but the volume of liquid medium in
relation to the size of flask must be kept, for adequate aeration. About 20 % of the volume of the flask
can be used for medium for suspension culture. These flasks are placed on gyratory or orbital
incubating shakers and agitated at 100-150 rpm for aeration and growth of suspension culture.
2. When plant material is first placed on medium, there is an initial leg period prior to cell division. This
is then after 2 or 3 sub-cultures followed by an exponential rise in growth of cultures. Finally, the cells
in suspension culture enter in a stationery phase. In order to maintain viability of the cultures, the cell
should be sub-cultured at the bignning of the stationery phase. Usually it is reached within 2-3 weeks.
The suspension has to be transferred to fresh medium at regular intervals within this period alongwith a
minimum density to maintain embryogenic potentiality.
3. To subculture embryogenic suspension, wait several minutes until cells are settled at the bottom of the
flask and decant almost all the medium. Re-suspend the suspension by gently rotating the flask and
transfer 1/4 of entire population to fresh medium by using micropipet.
4. A population of cells I callus tissues show a wide range of pro-embryogenic stages visible under the
microscope. It is a mixture of single cells, pro-embryogenic clusters and new embryogenic centres and
old embryos (Fig. 10). To obtain certain degree of uniformity, it is necessary to sieve the inoculum
before transferring to fresh medium.

The pro-embryo suspension is passed through a 200 J..tm sieve. and the 100 Jlm sieve. In second
sieve, differentiated embryos from late globular stage are retained and may be microscopically examined,
while pro-embryonic cells pass through. Cell suspension, which has passed the sieves, is settled, decant
most of the medium to obtain a suspension with high density of embryogenic cells.

3.3 Maturation:
The globular stage embryo inoculum is transferred to a medium having same composition but
either reduced level of auxin and L-glutamine or completely lacking of both. Cells quickly exhibit theif
embryogenic potentiality. The different developing stages of somatic embryos appear if initial inoculum has
not had uniform embrygenic cells. They attain mature stage within 2 wk. These embryos can be dispersed,
in a petridish on an agar medium without auxin, but the cytokinin (0.1-1.0 mg/1 of benzylarninopurine or
iso-pentenylarnino purine or zeatin).

3.4 Germination:
Mature embryos are then placed in the tubes either on Filter Paper Bridge or on semi-solid medium
with low concentration of sucrose (5 mg/1). To stimulate the formation of leafy shoot apices, development,
 157

and the tube must be well illuminated. The germinated seedlings having 5-7 leaves and well developed roots
are placed into the pots with a mixture of peat, soil and vermiculite for subsequent development and making
autotrophic to regenerated plantlets. After their transfer, plantlets have to be protected from desiccation by
covering the pots with plastic film for 2-3 wk.

4. CRITICAL FACTORS
4.1 Explants:
The establishment of callus growth and subsequently in somatic embryogenesis induction has been
achieved in many woody plant species of arid zone. The nature of explant sources is an important factor on
which somatic embryogenesis initiates from a cell or a group of cells or directly from explant tissue
depends. This refers to the conditions under which the source of plant was grown and stage of development
of plant part from which explant tissues were obtained. As a matter of fact, juvenile tissues of many woody
plant species appear to be the most suitable for the induction of somatic embryogenesis in most of woody
perennials of arid and semi arid areas (Raj Bhansali. 1999).

Plant regeneration has been successfully accomplished from various explant tissues such as
immature zygotic embryos, cotyledons and hypocotyls. Young seedlings, young shoot tips from mature
plants, petals, inflorescence, ovular tissues and embryos grown in vitro. Most viable plant cells can be
induced to undergo mitosis during cultures of these tissues. In citrus tissue (Rangan et al., 1968, 1969;
Chaturvedi and Mitra, 1974; Raj Bhansali and Arya, 1978c), ber and Chinese jujube (Raj Bhansali, 1989;
Islam et al., 1994; Mitrofanova, 1997), pomegranate (Raj Bhansali, 1990; Nataraja and Neelambica, 1996),
bael (Islam et al., 1996) and date palm (Ammer and Benbadis, 1977; Reynold and Murashige, 1979; Tissert
et al., 1979; Mater, 1986; Sharma et al, 1984, 1986; Raj Bhansali eta!., 1988; Neem (Raj Bhansali, 1995)
induction and regeneration of somatic embryogenesis have been reported. For given a species or a variety, a
particular type of explant tissues may be necessary for successful plant regeneration, e.g., embryogenic
tissues are most suitable for many Zizyphus spp., Punica granatum, A. indica and Aegle marmelos, whereas
young shoot tip and inflorescence of mature Phoenix dactylifera. Explants from both mature and immature
organs can be regenerated into somatic embryos directly or through callus on an appropriate culture
medium prior to plant regeneration. Even size and age of explant may be critical. The increased cell number
in-callus or explants of greater volume increases the probability of obtaining viable cultures.

The physiological state of the plant from which explant is taken, is also extremely important. The
season in which material is removed has an important role in making suitableness of explant material for
raising cultures (Tulecke, 1987; Tooten and de Varies, 1994). Within certain species of Citrus, Zizyphus,
Punica, Phoenix, different genotypes have shown varying potentialities for somatic embryogenesis (Raj
Bhansali, 1995). The polyembrygenic species are more potential for inducing somatic embryogenesis in
vitro, than mono-embryogenic species (Maheshwari and Rangaswamy, 1958; Raj Bhansali and Arya,
1978c; Litz, 1984). The integrated physiology of whole plant also controls the embryogenic potential of
each in the plants. Thick walled cells or inducible cells may be separated from neighbouring from non-
embryogenic cells (Raj Bhansali, 1990). Among the polyembryonic cultivars of Mangifera, there are
differences in degree of polyembryony. Generally, mango cultivars having the hoghest level of
polyembryony respond better than less polyembryonic cultivars (Litz, 1984) Among the tissues used to
induce somatic embryos are the cotyledons, ovules, leaf, megagametophyte, zygotic embryos, hypocotyl
cells, suspension, anther, pollen, endosperm and protoplast, apical tips (meristems) (Ammmirato, 1983;
Tulecke, 1987).

4.2 Selection procedure:

The induction of somatic embryogenic cultures depends on the optimum age, proper
developmental stage of explant tissues, effective medium and providing suitable cultural environmental
conditions. Embryogenic cultures could be induced by proper conditioning of cells or tissues prior to
transfer on inductive medium where embryogenic centers may be developed for establishing embryogenic
cell lines. Proliferating callus tissues having characteristics of embryogenic cells (small-round, thick walled
and having dense cytoplasm and high degree of organization) may be selected by staining technique (Gupta
!58

and Durzan, !985; Raj Bhansali eta!., 1990) for embryogenic cell lines from non-embryogenic cells or
tissues. Feulgen cytophotometry was used to confirm the triploid nature of embryos developed from
endosperm culture of A. nilotica (Garg et a!., 1996). Alternatively, explants may directly be directed to
induce somatic embryogenesis by selection of globular, nodular and embryoids structures. Homogenicity of
cell clusters for embryogenesis is important for further synchronization of embryogenesis (Osuga and
Komamine, 1994). At cellular stage somatic embryos, generally occur from globular to the torpedo,
cotyledonary and mature bipolar structures. Using specific nutritionally balance media, developmental
synchronization of cellular and mature embryos could be regulated (Osuga and Komamine, I 994). A whole
plant from bipolar somatic embryos could be developed on another specific media.

4.3 Quantification:
In embryogenic callus masses, quantification of cellular embryos is difficult because of lack of
homogeneity of cell composition or developmental stage of embryos. Controlling cell cycle, sieving and
separating embryogenic and non-embryogenic cell population or clusters, by using abscisic acid (ABA) to
accumulate mature organized embryos are important steps towards effective quantification of embryos.
Ammirato (1983) quantified the production of embryos i.e., 60,000 embryos from I litre medium. Similarly,
in 200 mg of callus tissues of date palm 1000 somatic embryos were induced and estimated by Sudhershan
et a!., 1993. Bee war et a!., 1987 described a method for quantification of the level of somatic
embryogenesis by dispersing in liquid by agitation and plated in a thin layer of medium containing 0.6%
low melting point agarose. This method is useful for identifying highly embryogenic callus lines among
phenotypically similar lines. Effect of various factors influencing somatic embryogenesis can also be
determined. In addition, better quantification in frequency of induction from explant, the frequency of
occurring embryogenic line, criteria used for selection, rate of successful movement of somatic embryos
through their development including germination, plantlet growth, transplanting and success in the field are
required.

4.4 Media requirements:

Somatic embryos have been induced and grown on a wide range of media from the relatively
simple to more concentrated formulations of Murashige and Skoog (MS), 1962, Gamborg et a!., (1968),
Schenk and Hildebrandt (1972), woody plant medium (Lloyd and Me Cown 1980) and DKW medium
(qriver and Kuniyuki, I 984 ). The Murashige ands Skoog medium is most commonly used for the induction
and proliferation of somatic embryos in arid zone plants. The strong mineral solution is beneficial for the
growth of embryos in plant species while weak salt solution appears to preserve the potentiality of
embryogenic suspension (Moures and Lutz, 1980; Ammirato, 1983). In most of arid zone plants two or
more distinctly different media may be required for the induction of embryogenic cells and subsequent
media for further development, maturation and germination of somatic embryos.

The nutritional requirements for somatic embryogenesis are yet not well understood. They are
neither specific nor exclusive since various media compositions produce similar results. The most common
factors responsible for the somatic embryogenesis are discussed. High inorganic nitrogen (ammonium
nitrate), a key element of Murashige and Skoog medium to fulfil the needs of nitrate requirement for the
growth of somatic embryos in many of arid zone plants species. However, the benefits of reduced nitrogen,
in addition to the nitrate for both embryo initiation and maturation is well established. For example
embryogenesis, the optimal concentration and form of the nitrogen supplies appears to be dependent on the
auxin concentration. The presence of reduced nitrogen appears to stimulate embryogenesis. The source of
reduced nitrogen is added in form of complex addenda such as casein hydrolysate (CH), coconut water
(CW), and malt extract (ME). A single amino acid L-glutamine or L-alanine or the presence of ammonium
ion is used for the somatic embryogenesis (Halperin, 1966; Ammirato and Steward, 1971; Raj Bhansali et
al., 1991). Among all the amino acids added singly, L-glutamine most commonly promoted Punica
granatum and Phoenix dactylifera somatic embryos (Raj Bhansali et al., 1988; Raj Bhansali, 1990). The
importance of nitrogen as well as reduced nitrogen sources has been studied in wild carrot cultures. In wild
carrot cultures the addition of 10 mM NH.tCI to an embryogenic medium already containing KN03 (12-40
mM) produced near optimal numbers of embryos. Glutamine, glutamic acid, urea, and alanine,
respectively, were found to partially replace NH.tCI as supplement to KN03 (Wetherell, 1978). The various
 159

nitrogen sources are not specific for the induction of embryogenesis, although at low concentrations organic
forms are much more effective than inorganic nitrogen compounds.

The potassium ion has also been shown to be essential for somatic embryogenesis (Reinert et al.,
1967). Another salt of MS and other media important to somatic embryogenesis is the presence of chelated
iron, often in the form of iron EDTA. In embryogenic cultures, iron EDTA has been found superior to other
iron chelates and to non-chelated iron salts (Heberle-Bros, 1980). An absence, the embryos fail to develop
from globular to heart shaped stage in Zi:zyphus species. Kononowicz and Janik (1984) showed that the kind
of carbohydrate influences the rate of embryogenesis in cotyledonary explant tissues. In citrus tissues, callus
is easily converted into somatic embryos in glycerol (200 mM) or galactose containing medium (Tulecke,
1987).

4.5 Growth regulators:

The most commonly used media (either in its original or modified forms) including growth
regulators, do well for inducing somatic embryogenesis, maturation and regeneration into complete plantlets
for a wide spectrum of arid zone plant species. The most important growth regulators involved in the
induction of somatic embryogenesis are the exogenously supplied auxins in the medium. Induction requires
the presence of auxin. However, subsequent development of embryogenic cells is severely restricted by the
presence of auxin, and cultures are then transferred to a medium having very low or lacking in auxins after
the induction stage. The inductive medium is often a simple basal medium lacking growth regulators and
provides for the expression of embryogenic potential taxa such as family Rutaceae, citrus species. These
plants have pre-determined embryogenic potential cells but 2,4-dichlorophenoxyacetic acid and benzyl
aminopurine are essential for some embryogenic systems of pomegranate, date palm, citrus, rose wood, ber,
Acacia nilotica, A. catechu and neem plants (Raj Bhansali, 1998; Rout et al., 1995; Garget al., 1996). In
general the role of cytokinins in embryogenesis is somewhat obscure because of conflicting results
(Ammirato, 1983; Tulecke, 1987; Vasil, 1985). Although kinetin, benzylaminopurine, iso-pentenylamino
purine, zeatin, thidiazuron (TDZ) and adenine sulphate stimulate somatic embryogenesis in many arid zone
plant species. (Tisserat, 1984; Raj Bhansali et al., 1988; Garget al., 1996; Murthy and Saxena, 1998)

Among auxins, 2,4-dichlorophenoxyacetic acid has proven extremely useful for the induction of
so,matic embryogenesis through indirect pathway. The auxin requirements for the primary (induction of
somatic embryos) and secondary media (development of embryos) may be the same or low concentration or
without auxin may be used in same medium. For most of embryogenic cultures of arid zone taxa, a change
in auxin type or concentration is necessary preludes to the development and maturation of somatic embryos.

Effective concentration of growth regulators range from 0.2-5 mg/1 2,4-dichlorophenoxyacetic

acid, naphtaleneacetic acid, napthoxyacetic acid or indoleacetic acid as auxins and 0.1-5 mgn
benzylaminopurine, iso-pentenylamino purine, kinetin as cytokinins. High levels of 2,4-
dichlorophenoxyacetic acid are necessary for date palm e.g., 100 mg/1 2,4-dichlorophenoxyacetic acid
(Reynold and Murashige, 1979). The optimal concentration of auxin for somatic embryogenesis for each
genotype, explant tissue is different and specific. The requirement for a cytokinin may also be specific in
some cases such as zeatin, TDZ but not benzylaminopurine or kinetin for the induction and development of
somatic embryos in date palm, neem and ber genotypes (Tisserat, 1984; Murthy and Saxena, 1998; Raj
Bhansali, 1999). Cytokinins are required for secondary embryogenesis and plantlet development from
somatic embryogenesis and for low sensitivity cultures (Raj Bhansali et al., 1998). Unfortunately, for each
plant species, auxin and cytokinin requirements and their levels must be determined empirically.

There are several reports where combination of auxin and cytokinin were important in initiating
and maintaining growth and in fostering somatic embryogenesis development. Beside these, equally
important is sequence of media (growth regulators) for various stages of somatic embryogenesis (Evans et
a!., 1981; Ammirato, 1983; Tulecke, 1987). On several occassion, specific stage has specific growth
hormone requirement for embryo development, maturation and germination into complete normal plantlets
(Raj Bhansali and Kaul, 1991). An elaborate sequence of media may be necessary in non - embyogenic
cultures.
160

4.6 Activated charcoal:

The addition of activated charcoal to the medium has influnced somatic embryogenesis
development in date palm, pomegranate and her. Charcoal reduces the salt and growth hormone
concentrations of the medium. It substantially lowers the levels of phenylacetic and p-OH benzoic acids,
which are inhibitory somatic embryogenesis. (Fridborg et a!., 1978). It also absorbs 5-
hydroxymethylfurfural, an inhibitor formed by the sucrose degradation during autoclaving as well as
amount of auxins and cytokinins (Weatherhead eta!., 1978). Thus charcoal may absorb various kinds of
inhibitors that would prevent growth as well as the continuous proliferation of friable callus cells. It has also
been reported that charcoal will absorb certain iron chelates, FeEGTA and FeEDDHA, preventing the
transition from globular and heart shaped embryos (Heberle-Bors, 1980).

4.7 Carbohydrates:
Sucrose as a carbon source appears to be the most potent carbohydrates for somatic embryogenesis
in almost all of arid zone plants, although many other mono and disaccharide can be successfully employed
(Verma and Dougall, 1977). High level of glucose (6-10%) produced optimal somatic embryo development
in one carrot line (Homes, 1967). Cotyledons for somatic embryos grown on glucose media, which
contained more tryglycerides than those grown on sucrose media, which contained more phosphor-and
glycolipids. Ben-Hayyim and Neumann (1983) working with citrus showed the greater conversion of
nucellar callus to somatic embryos in glycerol containing media (280 mM) than on sucrose (70 mM). On
glycerol media most of the callus converted to green somatic embryos. Galactose stimulated 6-12 fold
increase in somatic embryo development in citrus ovular callus cultures (Kochba et a!., 1978). Change in
carbon source may prove beneficial in the growth of habituated, non-embryogenic callus for 1 or 2 passages
without sucrose followed by transfer to media with sucrose stimulated somatic embryogenesis (Kochba and
Button, 1974). By adding inositol while simultaneously lowering the sucrose concentration (and
maintaining the culture in darkinetiness), somatic embryos matured free of extraneous proliferation and
without germinating precociously (Steward et al., 1975).

4.8 Environmental conditions:

Certain physical and environmental conditions may be crucial for the induction and development
of,somatic embryogenesis in some arid zone taxa (Raj Bhansali, 1994). The variables include the rate of gas
exchange in culture vessel (ethylene accumulation or dissipation), the volume of media per explant, dilution
and conditioning of media, light/dark regimes and their quality and quantity, the frequency of transfers, the
selection of explant tissues (nodular, colour or other criteria), temperature and characteristics of the
explants (Reinert, 1958; Tisserat and Murashige, 1977; Kochba eta!., 1978). The other conditions such as
effect of cold storage, media sequences and gradients are also important in somatic embryogenesis
(Tulecke, 1987).

There has been direct relationship between the density of embryogenic cells in the suspension or in
callus tissues and degree of embryo maturation. Several workers have reported the response of density is
critical for development, maturation and germination of somatic embryos. The effect of low density creates
the problem of synchronization of populations by sieving and centrifugation severely reduced the
population density (Huber eta!., 1978). Populations of somatic embryos typically show a wide range of
sizes and stages of development. At the time of transfer from the maintenance medium to the development
medium, there is a range of pro-embrygenic cell clusters including few to many cells. Somatic
embryogenesis is to some extent repetiti:ve so that new embryogenic center may arise, either from cell
clusters or maturing embryos. The most common method for attaining some degree of synchrony
(uniformity), at least in terms of populations may be using abscisic acid (ABA). For controlling somatic
embryogenesis ABA has proven effective in Carum carvi (Ammirato, 1974) and Pennisetum americanum
(Vasil and Vasil, 1981).

Changes in genetic stability of the ploidy level have been noted in many cultures leading to mixed
populations of polyploids and aneuploids. The plants regenerated from such cultures often show a range of
chromosome complements. The embryogenic capacity of cultures decreased and disappeared in progressive
sub-culturing. The maintenance of chromosomal and genetic integrity is essential for clonal multiplication
 161

via somatic embryogenesis Reduction of frequent sub-culturing can effectively minimise the extent of
chromosomal changes in cell culture. On the other hand somaclonal variation that has been arisen
spontaneously appears to have tremendous potential for producing novel and useful varieties (Larkin and
Scowcroft, 1981).

S. UNIFORMITY
Somatic sedlins are clonal material where as plant derived from zygotic embryos usually bring new
gene combinations into offspring (Larkin and Scowcroft, 1981; Finer, 1994). In some cases, somatic
explants need to be verified for their clonal trueness by detailed, morphological, progeny tests, isoenzyme
analysis, cytological studies, DNA base sequencing and by using other molecular techniques (Withers and
Alderson, 1986; Vasil, 1991; Phillips et al., 1995). Generation of variability is a common phenomenon in
plant cell, tissue and organ cultures (Bayliss, 1980).

Tissue culture derived Santalum plants showed a high incidence of variants (up to 90 %) and
some of them showed exceptional vigour under the field conditions (Rao et al., 1994). Very often at least
some of the variability can be observed in the regenerated plants. Vasil ( 1985) has reported remarkable
degree of morphological uniformity and the absence of polyploidy, aneuploids and other albinos in plants
regenerated from embryogenic tissue cultures of graminaceous species. In a study of the explants, callus
tissues and regenerated plants of P. americanum, it has demonstrated that the initial inflorescence explant
themselves contained some polyploid and aneuploid cells (Vasil, 1985). During callus formation and its
maintenance in cultures, an increase in frequency of polyploidy and aneuploid was noticed. It is thus clear
that cytological variant cells initially present in the explants divided faster than normal diploid cells in vitro.
More than 75 percent of the cells in the 6-month-old culture remain diploid, when tested through
cytophotometric and flowcytometric analysis. This indicated that there was a strong selection in favour of
normal cells during the somatic embryogenesis.

The generation and/ or the presence of variability in vitro can be a serious problem during clonal
propagation of date palm and pomegranate plants when absolute fidelity of genotype is implied as well as
required. Nevertheless, this problem has largely been ignored in the current enthusiasm for significant
application of such variability in plant improvement programmes.

It is vitually an axim of cytogenetics that meristematic or germ line tissues should be genetically
stable and that the daughter cells of mitosis should contain identical chromosome complements. The degree
of cellular and tissue organization has an important influence on the regularity of mitosis. Thus the
polarized cellular environment of meristematic cells ensures normal mitosis. Therefore, a very rare if any
cytological change takes place in embryogenic, organ or meristem cultures. The embryogenic cells have low
threshold for variability resulting in the selective development of somatic embryos from cytological normal
cells (Vasil, 1985; Withers and Alderson, 1986)..

Somatic embryos are similar to zygotic embryos having organized shoot and root meristems. These
meristems are not only maintained their morphogenetic competence, but also their genomic integrity
throughout the life of the plant. The high degree of uniformity is seen even in the long-term embryogenic
cultures and meristematic cells (Phillips et al., 1995). This indicates that embryogenic cells are similar to
meristematic cells in general structure and cytoplasm organization (Raj Bhansali, 1990). Thus it can be
considered that embryogenic cells are der.ived from active totipotent me!jstematic cells.

6. CONCLUSION AND FUTURE PROSPECTUS

Somatic embryos have been proved as potentially stable, true-to-type source of plant multiplication
system for most plants including arid zone plant species. However, variation in regenerants derived from
somatic embryos does occur, due to mutation, mitotic crossing over, transposable genes and variation in
explant etc (Durieu and Barber,l982; Meins, 1983). Somatic embryos of date palm, citrus and pomegranate
are potentially propagation materials of selected elite plant or derived plants from unusual genomes such as
162

from endosperm or ovular tissues. Early selection at single cell level or protoplast stage followed by
somatic embryogenesis is another useful tool for genetic engineering purposes in citrus, Eucalyptus
carru:~dulensis and other related species of arid zone woody plants. Somatic embryogenesis has number nf
advantages over the organogenesis in tissue cultures. One of the most distinct benefit is that somatic
embryos are bipolar structures having both root and shoot meristems and capable of regeneration of
complete plant that too in large number. The organogenesis, root and shoot meristems are often mutually
exclusive, and a sequence of various media changes is necessary to regenerate an entire plant. Somatic
embryogenesis process developed for arid zone woody species, can reduce the expenses in term of material,
time and labour, could offer an attractive alternative for plant regeneration in addition to production of
easily separable embryos per vessel. Embryos can be moved easily to tissue culture system for final
regeneration into complete plantlets. By this way large number of somatic embryos of fruit and forestry
woody plants of arid zone can be microprpagated through somatic embryogenesis.

Embryos are natural organs of perennation and if dormancy can be induced they could be used as
artificial seeds either by coating or encapsulation (Redenbaugh et a!., 1991). These can be handled like
normal seeds and stored, shipped, planted and so forth. But, the serious problems facing in the production
of synchronous or particular embrygenic stage population of uniform somatic embryos, inducing,
maintaining and breaking dormancy at choice and nourishing the young seedling besides the protection
from microbial rotting during storage and germination in field soil or potting mixture are some areas, which
need more studies. Somatic seeds may prove useful for long-term storage, such as storage of germplasm
banks (Gupta et al., 1991; Tay et al., 1993). Induced dormancy, artificial seeds, cold storage, dry storage or
cryogenic preservation may play big role in this endeavor.

One of the major goals of future work is the continuation of efforts in fostering somatic
embryogenesis in recalcitrant arid zone tree species. There is also variation in response of somatic
embryogenesis induction from variety to variety, from clone to clone and from experiment to experiment,
even in the species where somatic embryogenesis had been worked out. More basic research is needed to
understand better factors controlling and mechanisms of induction and regeneration of somatic embryos in
forestry and horticultural arid zone plants. The fundamental question of growth, differentiation and
development of somatic embryos and their endogenous and exogenous factors responsible for transition of
embryonic growth of hardy and intractable arid zone tree species should be answered by continuous
investigation in this area. Undoubtedly, somatic embryogenesis has been shown to be stable, long-term, and
highly efficient source for rapid and truly clonal propagation system of plants. Plants are regenerated from
somatic embryos that develop either directly or indirectly from single cells. These embryogenic cells
provide the excellent source of morphogenetically competent suspension cultured cells and even protoplasts
(Oghawara et al., 1989; Tusa et a!., 1990). It is to be used in future to employ somatic embryogenic
pathways in application of plant biotechnology towards genetic modification and improvement of arid and
semi-arid zone plants by using genetic engineering and molecular biological techniques. The technology
could lead to large-scale cloning of plants potentially has broad application in agriculture and forestry for
ultimate use to mankind. Further, application bioreactor technology for tissue culture products may give a
new dimension in production of somatic embryos for bulk quantities in short time by using bioreacters
(Bapat et al., 1990). The possibility of producing large number of plants of single genotype, evokes vision
of applications to plant breeding programmme (Gupta et al., 1991; Raj Bhansali, 1994; Jain et al., 1999).
Somatic embryogenesis technology in arid zone plants thus could be employed as highly mechanised
culture system, which able to produce efficiently large number of uniform propagules. The high frequency
cloning of desert plant would be a viable concept in near future to green arid lands.

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 5. Status of Somatic Embryogenesis in Indian Forest Trees

P. S. Rao, P. Suprassana, T.R. Ganapathi and V. A. Bapat

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre,
Trombay, Mumbai 400 085, India.

Chapter contents

1.0 Introduction 2.0 Case Studies

2.1 Sandalwood 2.2 Neem
2.3 Eucalyptus 2.4 Bamboo
2.5 Poplar 2.6 Teak
2.7 Dalbergia 2.8 Soapnut
2.9. Semul 2.10 Arjun
2.11 Brown oak 3.0. Conclusions
4.0. References

1. Introduction

India's forest area is about 64.20 M ha which constitutes approximately 19.52 percent of
the total geographic area of the country and has a great diversity of natural ecosystems.
The forest range covers from evergreen tropical rainforests in southern part extending to
western hills, Aravali/Satpura range and the north eastern states to alpine scrub in
Himalayas. The forest is divided into : a) reserve forests (54%), primarily for conservation,
b) protected forests (30%) for villagers to exercise certain rights; and c) unclassified
forests (16%) to which people have free access. Ninety percent of the forest areas are under
the State control (ITTO, Newsletter 1998). Population growth, urbanization and
agricultural development has put an unprecedented pressure on India's forest cover in the
past half century. Forests have also suffered severe degradation due to the enormous
demand for fuel wood, excessive grazing by cattle and ever increasing demands from
industries for paper, timber, medicines and other products ( Khoshoo 1989 ).

The new policy of the Goverment of India included an effort to increase tree cover thro}lgh
afforestation and social forestry initiatives. In addition, a large scale programme of the
waste land afforestation has been launched to develop new reserves for forest products. To
overcome the problems in traditional method of propagation of genetically superior
planting stocks and realizing the importance of biotechnology for speeding up genetic
characterization and mass propagation of elite clones, Goverment of India has set up two
Pilot Plant Facilities at National Chemical Laboratory, Pune and Tata Energy Research
Institute at New Delhi to generate identified clones of desirable plant species. Eucalyptus,
Poplars, Bamboo and Anogeissus are some of the tree plants which, have been produced
through tissue culture and around 4 million plants have been planted in an area of
approximately 3500 ha for field demonstration ( Annual Report Department of
Biotechnology, Govt of India. Report 1998- 1999 ) The field data has indicated better
performance of tissue culture raised plants vis a vis the control. A preliminary cost benefit
analysis has shown about 5 times higher returns in case of tissue culture raised plants.
169
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 169-191.
© 2000 Kluwer Academic Publishers.
170

The major route for tree multiplication has been via shoot organogenesis through the
culture of axillary bud from known mature trees. Standard protocols have also been
developed in various universities/research centers on other forest trees as well viz.
Wrightia arborea, Boswel/ia serrata, Tectona grandis, Azardirectha indica, Calligonum
polygonoides, Maytenus emarginata, Sa/vadora o/eoides, Prosopis cineraria and
Tecomel/a undula (Annual Report Deptartment of Biotechnology, Govt. of India, 1998 -
99). Although considerable success has been obtained in these tree species, progress is
very slow in several other species due to lack of rejuvenation in mature tissues, inadequate
supply of genetically superior planting material, secretion of phenolics in the medium,
poor germination of seeds, less survival of cuttings, problems in establishing clean cultures,
long duration of life cycle and insufficient information on genetic make up of several
valuable trees. Consequently routine regeneration of plants from tissue cultures has still
not been possible for many forest trees. Although the selection of elite plant material from
mature trees and culture of axillary buds/shoot tips is the standard protocol adopted in
many laboratories, tissues from seedlings have also been employed in some cases because
of lack of response from mature tissues.

Micropropagation of forest trees through somatic embryogenesis has been reported in

many cases albeit at low frequencies. This presents a challenge since high frequency
somatic embryogenesis with good quality of somatic embryos and an efficient rate of
embryo to plant conversion are a prerequisite for using this technology for large scale
multiplication of forest trees (Ahuja 1997). Since somatic embryos undergo through a
callus development phase, the relative genetic stability of somatic embryos and somatic
plants is necessary to be investigated at the morphogenetic and molecular level before
introducing into forestry programmes. (Ahuja 1997).

According to Park eta/ (1998), the most promising application of somatic embryogenesis
is in the high value clonal forestry in which a valuable genotype or tree varieties are cloned
for deployment on productive sites and such genotypes and tree varieties may have faster
growth with enhanced resistance to insects and diseases developed through combinations
of conventional tree breeding and genetic engineering. Once embryogenic system is
established, somatic embryos could be encapsulated and prepared as synthetic seeds which
would form source for novel delivery of tissue culture plants. In recent years, embryogenic
tissues have also been widely employed in transformation studies to transfer useful genes
into forest trees (Tzfira et a/.1998). Somatic embryos have also been identified as a better
source for clonal germplasm preservation.

Several studies have been conducted on the maturation and desiccation of somatic embryos
for high frequency conversion of somatic embryos to plants ( Bapat and Rao 1997, Pedroso
and Pais 1995, Mordhoret et al., 1997). Source of nitrogen, high level of sucrose, low level
of oxygen, chilling of cultures, incorporation of abscisic acid, and growth of cultures on
less nutrient medium are some of the key steps employed for maturation and desiccation of
somatic embryos (Bapat and Rao, 1997, Capuana and Derbergh 1997).

Somatic embryogenesis in several forest trees has been reported from various laboratories
in India. However, in very few cases, successful conversion of somatic embryos into plants
has been demonstrated beyond the laboratory scale. The reported data in these plant species
 171
sheds some light on several aspects of somatic embryogenesis and might eventually become
useful in setting up protocol development towards large scale multiplication. The present
article provides a research update on somatic embryogenesis in some Indian forest trees.

2. Case studies

2. 1 Sandalwood ( Santalum album L.)

Sandalwood is the moSt precious tree of Indian forest and is often referred as "jewel of
forest". Although, this tree is widely grown in several states of India, trees in the
southern part of the country are well known for their scented wood and essential oil.
Indian exports earn substantial foreign exchange in sandalwood trade. The wood and
roots are extracted for oil, which is widely used in perfumery, cosmetics, soaps,
incense sticks, talcum powder and in medicines. The wood is also in great demand for
furniture, carving idols, boxes, pillars and other related products. Sandalwood tree
produces prolific seeds and conventionally new trees are raised only through seeds.
Sandalwood tree is affected by spike disease causing serious damage to existing plant
population. Therefore, there is an urgent need for alternative method for propagation of
elite clones. Multiplication of elite clones through in vitro culture is desirable and might
provide an answer necessary for producing more planting material.

Somatic embryogenesis in sandalwood described way back in 1965 ( Rangaswamy and

Rao 1963, Rao, 1965, Rao and Rangaswamy 1971) was one of the first reports in forest
trees. Since then, extensive work has been carried out on this tree species and todate
sandalwood has become a versatile plant material for tissue culture manipulations and
offers as an excellent embryogenic system. Hypocotyl, endosperm, stem segments and
protoplasts are the main sources for induction of callus (Rao and Bapat 1995).
Rangaswamy and Rao (1963) reported for the first time the establishment of tissue
cultures from the endosperm tissue of Santalum album. Subsequently Lakshmi Sita et a/
(1980) and Rao and Raghav Ram (1983) reported callus induction and somatic
embryogenesis in endosperm of sandalwood. The induction of callus from stem segments
was achieved on MS (1962) medium supplemented with 2,4-D (1-3 mg/1 )and KIN
(0.2 mg/1) and upon transfer of callus to MS + IAA( 0.5mgll ) + BA (0 .. 5mgll ), callus
underwent differentiation and produced numerous globular embryos (Fig. 1 ; Bapat and
Rao, 1979, Rao and Bapat 1992). Actively growing embryogenic cultures through
regular subcultures at periodic intervals of 4 weeks on MS medium produced abundant
somatic embryos (Bapat and Rao 1979 ). For conversion of embryos to plants, it was
often essential to transfer the embryos to White's medium containing IAA (lmg/1) +
IBA ( 0.5mgll) + GA3 ( 0.5mgll). Although conversion frequency was not high,
plants that were regenerated could be grown to maturity and field planted. The
transplanted plants showed vigorous growth and yielded flowers and friiits. Several
experiments on the effects of ABA, high temperature, sucrose and drying of callus tissue
on maturation and desiccation of embryos have been conducted for increasing the
conversion frequency of embryos to plants (Rao and Bapat 1995).

Somatic embryos of sandalwood were successfully encapsulated in sodium alginate (3%)

and such encapsulated embryos were then cultured on the MS nutrient medium. Embryos
germinated within 8 weeks and subsequently produced shoots and roots establishing
172

contact with the medium. Well developed plantlets were obtained at the end of 8 weeks.
On MS + IAA (O.Smg/1 ) + BA ( O.Smg/1 ) encapsulated embryos started producing
secondary embryos instead of germination. and plants have been regenerated from these
embryos upon isolation from beads ( Bapat and Rao 1988). Because of the soft nature of
alginate gel, deformity of the beads was sometimes observed, and hence silica gel (hard
nature) was used along with alginate to make composite gel to combine the properties of
both the gelling agents. Composite gels were prepared by mixing 50% silica gel with 4
% alginate in 1 : 2 proportion. Similar to embryos in alginate beads, embryos in
composite gel also germinated and formed plants (Rao and Bapat, 1992).

The undifferentiated callus on MS+ 2,4-D (lmg/1) yielded good cell suspensions and
consisted predominately of small cell aggregates. The cell suspensions could be
routinely subcultqred every 4-5 days. Such cell suspensions formed a good source for
obtaining embryos. Cells after plating on MS + IAA (0.5mgll) + BA ( 0.5 mg/1 ) formed
within 10 days club and heart shaped embryos. In some cultures, embryos became
grossly enlarged, accumulated chlorophyll and anthocyanin and produced secondary
embryos. The embryos showed all the stages from filamentous proembryos to
dicotyledonous structures. Mature embryos developed into plantlets ( Rao and Bapat,
1992.).

The established cell suspensions in flasks were used as the inoculum for the two types of
bioreactors to scale up the embryogenic cell cultures. Firstly, a seven titre stirrer glass
tank bioreactor was used for the conversion of non embryogenic cells into embryogenic
cells. Secondly, a bell jar bioreactor of one litre capacity was used for the development of
preglobular embryos to mature embryos. Conversion of non - embryogenic cells to
proglobular embryos occurred in 7 1 capacity bioreactor in MS + IAA ( 0.5mgll) +
BAP (0.5mgll) after 3 weeks. In one litre capacity bioreactor mature embryos with well
developed cotyledons became visible after 4 weeks in medium consisted of MS + IAA (
O.Smg/1) + IBA (0.5mgll) + GA3 (0.5mg/l) + sucrose 5%. Normal plantlets were
obtained from these embryos ( Bapat eta/., 1990 ).

Protoplasts were successfully isolated from callus and cell suspensions, and these
underwent divisions and developed into microcolonies, which subsequently de-
differentiated into somatic embryos and plants (Rao and Ozias-Akins 1985, Bapat eta/.,
1985). High yields of protoplasts (8.73 x 10) were obtained with a mixture of cellulase
(1%), macerozyme (0.5%) and 0.5 M sorbitol or mannitol. Among the various media
tested V-47 medium was suitable for protoplasts from cell suspensions whereas MS was
suitable for protoplasts from callus. Sustained and repeated divisions were obtained from
protoplasts isolated from both sources resulting into cell colonies. Somatic embryos were
regenerated from protoplasts on MS + IAA ( 5.71 mM) + IBA (2.46mM) + GA3 (
2.89mM) ( Rao and Ozias Akins, 1985, Bapat eta/. 1985). Recently introduction and
expression of a marker gene through Agrobacterium vector has been reported in
Sandalwood ( Veena and Rao, 1998). Lakshmi Sita and Bhattacharya (1997) studied
molecular approaches for obtaining disease resistance in Sandalwood. Gene cloning,
sequencing and transformation with marker gene have been investigated ( Lakshmi Sita
and Bhattacharya, 1997).
 173
Using synchronous embryogenesis system. Shankar Rao et al (1996) observed that early
events in embryogenesis fall into four distinct stages viz., fresh callus (stage 0),
preglobular masses (stage 1), globular embryos (stage 2) and bipolar embryo (stage 3).
Stage 0 to stage 1 was characterized with stages specific appearance of two ploypeptides
of 15 and 30 kDa molecular weight. Activities of protein kinase, glycosidase and
xylanase increased markedly with progressing embryogenesis. The authors suggested
that in addition being controlled at the stage specific gene expression, sandalwood
embryogenesis is also regulated at the level of controls on cell wall flexibility and post
translational changes in the pool of existing proteins.

Veena Sri and Sankara Rao (1998) reported for the first time a system for the
introduction and expression of foreign genes. The torpedo and cotyledon stage embryos
.were infected with Agrobacterium tumefaciens harboring fl-glucorinidase (uidA) and
neomycin phosphotransferase II (nptll) genes on the binary vector. In total 20% of the
inoculated embryos, callused and developed secondary embryos. The transformation was
confirmed by growth in the presence of kanamycin, gus, nptll assays, PCR. The
integration of the marker gene into sandalwood plant genome was evidenced through
DNA dot blot hybridization.

The results on sandalwood highlight the development of an efficient method for the
regeneration of plants from somatic embryos derived from various explant sources.
Retention of regenerative capacity of cultures after several culture passages provides a
source for continuous harvest of somatic embryos and plants. Results on somatic
embryogenesis are being followed by several lines of research from cell and protoplast
culture to bioreactor production of embryos and genetic transformation. The transfer of
laboratory scale production to pilot plant production to regenerate thousands of plants
for planting at various sites is the next step towards mass propagation. The interaction of
the forest department and a well established tissue culture laboratory is necessary to
achieve this goal. Resistance to diseases and other stress conditions can be achieved by
in vitro selection using cell and protoplast cultures. The versatile nature of the
embryogenic system for tissue culture manipulations need to be tapped to understand
basic mechanisms involved in embryogenesis and to employ for genetic manipulation.

2.2 Neem (Azadirachta indica L.)

This well known forest tree of India is an evergreen, tall and rapidly growing tree,
which is propagated by seeds. This plant has received world wide attention due to its
biological activity against a wide range of insects and nematodes and also has other
applications in agriculture, pesticides, fertilizers, health, medicine and as a therapeutic
agent (Koul et al., 1990, Thengane et al., 1995). Many investigations have been carried
out on the isolation, identification and screening to find out the active principal
(Schutterer, 1995) and the most important ingredients of the neem seeds are the
Azadireachtins (Rembold 1990, Jacobson 1986, Khanna 1992).

Due to its immense importance, this tree species has been extensively investigated.
Thengane et al., (1995) reviewed the in vitro approaches for neem micropropagation and
allied research on neem. All parts of the neem plant have been cultured and successful
plant regeneration has been achieved. Leaves, stem, cotyledons, inflorescences, and
174

embryos were cultured to establish the callus and differentiation of shoot or root
meristem via callus was obtained on MS medium containing 2,4-D, IAA, NAA, IBA,
BA, Kinetin and 2iP, individually or in various combinations (Thengane eta/., 1995).
Induction of adventitious shoots followed by rooting was achieved from immature
embryos, shoot tips and axillary buds (Joarder eta/., 1993).

Somatic embryogenesis in neem was reported by Muralidharan and Mascarenhas (1989)

and Shrikhande eta/ ( 1993 ) in which cotyledons from unripe fruits were used for
callus induction on MS basal medium supplemented with 1000 mg/1. CH, 0.5 mg!l IAA,
1 mg!l BA, 5% sucrose and 0.4% agar. The cultures were initially were maintained in
dark. Embryo growth was promoted upon transfer of embryogenic callus to MS medium
with IAA (0.5 mg!l) + BA (2 mg!l) + 5% sucrose and 0.4% agar. Complete plants were
obtained from somatic embryos on Y2 strength MS liquid medium with 2% sucrose.
After 2 months of transfer to green house, 90% survival of the plants was observed
(Thengane et a/.,1995). Direct somatic embryo induction was also achieved without an
intervening callus phase on 2,4-D + BA medium. Precise information on callus
maintenance medium, embryo development and germination was recorded ..

Because of its several uses, neem has been widely recommended for rural development
projects which require quality supply of desirable clones in large numbers. There is a
greater need to identify elite trees and clone them through various propagation methods.
The main drawback in neem propagation is the rapid loss of seed viability (Chaney arid
Knudson 1988) and seeds need to be sown immediately after collection. The other most
important challenge is to standardize an alternative method for large scale cultivation of
somatic embryo derived plants at selected sites under the specific environmental
conditions. This needs continuous monitoring of their performance in the field. It is also
possible to expose the cells to physical and chemical mutagens and to select the plants
with novel genetic variations. In addition, the demonstrated feasibility of somatic
embryogenesis in neem could form the basis of genetic engineering programs by
subjecting the embryogenic cells to various methods for gene transfer.

2.3 Eucalyptus (Eucalyptus sps )

Eucalyptus is widely grown in India and is well known for its use as fuel wood, timber
and as a raw material for pulp and paper industry, honey, tannins and for extracting
essential oils. Eucalyptus is an evergreen tree and can grow in a wide variety of agro-
climatic conditions. In India about 170 sps I varieties have been identified and cultivated
under social clonal forestry schemes throughout the country. Multiplication through
cutting is the most preferred method to retain elite traits. To achieve high multiplication
rates, in vitro micropropagation method has been adopted in several species of
Eucalyptus and explants from both juvenile and mature trees have been employed. One
of the most critical factors in attempting to propagate genetically superior Eucalyptus
trees is the successful disinfection of mature tissues. The ability of Eucalyptus to coppice
and sprout, thus producing juvenile material which is less exposed to microorganism
reduces the difficulty of sterilization (Jones and Van Staden 1997). An increase in
biomass production of plants ( upto 49.5%) over seedling control was recorded in tissue
culture derived 34 month old Eucalyptus plantations (Khuspe eta/., 1987).
 175

Lakshmi Sita (1993) has demonstrated somatic embryogenesis in Eucalyptus citridora

and E. grandis and reported induction of globular embryos in nodular callus on media
combinations containing MS+ 1mgll GA; MS+ 1mg/l GA+ 0.3mgll BA; MS+ 1mgll
GA+ 0.3mgll IAA and MS + 2-Smg/1 BA+ 1mgll NAA. Embryogenic callus was
maintained on B5 medium (Gamborg eta/., 1968) supplemented with CH 500mgll +
glutamine 500mgll and sucrose 3% in darkness. Embryos could be multiplied by
repetitive embryogenesis to form clusters of embryos (Lakshimi Sita 1993). Direct
induction of somatic embryos, long term embryogenesis and plantlet regeneration have
also been reported by Muralidharan and Mascarenhas (1995) in E. citriodora. Seeds
cultured without seed coat produced embryogenic cultures on MS or B5 medium
supplemented with various concentrations (0.5 - 10 mg/1 ) of 2,4-D and NAA.
Repetitive embryogenesis resulting in the production of numerous somatic embryos was
achieved upon transfer to fresh medium fortified with NAA ( 3mgll ). Various
parameters such as effect of basal medium, auxins, organic nitrogen compounds,
abscisic acid and long time storage were standardized for maintaining embryogenic
status of the callus and suspension cultures yielding somatic embryos were established.
Germination of embryos was obtained on an auxin free medium consisting of B5
minerals and vitamins and 2gll sucrose. Incorporation of coconut milk (10%) in the
basal medium enhanced germination rate to 58%. Plantlets survived in vitro and
continued to grow in soil- sand mixture at 70-80% humidity. No abnormalities were
observed in the morphology and growth rate as compared to the controlled seedlings
(Muralidharan and Mascarenhas 1995).

With the knowledge gained so far in this plant, it is possible to multiply desirable
clones through the process of somatic embryogenesis. However, a lot needs to be done to
standardize the method in producing a large number of plants at an affordable price for
afforestation purposes. Selection of proper mother tissue from superior trees is
necessary for obtaining consistent and good yield of embryogenic cultures. A precise
information on maturation and desiccation of somatic embryos is needed for high
frequency conversion of somatic embryos to plants. Somatic embryos are useful for
genetic manipulation, selection and production of elite clones and for cryogenic storage
for future use (Muralidharan and Mascaernhas, 1995).

2.4 Bamboo ( Bambusa, Dendrocalamus )

Bamboo is a very common plant of grass family in the Indian forests and occupy an area
of 10.03 million ha, roughly 12.8% of the total forest area in the country. In India, there
are 125 indigenous as well as exotic species of Bamboo belonging to 23 genera notable
among them being Bambusa aurudinacea and Dendroca/amus strictus. The plant is
widely used as a raw material for shelter, wood, paper providing food and even
medicines in subtropical and tropical countries and has been identified as a potential
agricultural crop. This plant is now an integral part of daily life of rural communities
for making toys, baskets, hats, wall papers and wall hangings. The conventional methods
of vegetative propagation ofbamboo are by planting ofrhizomes, offsets, layering, nodal
cuttings and macro-cuttings (Raste and Bhojwani, 1998). Propagation through seed is
unreliable because of long flowering cycle (30-40 years), poor seed set, loss of seed
viability and heterozygosity.
176
In vitro studies on bamboo have been conducted with excised embryos for callus
induction and maintenance, morphogenesis and plant regeneration and culture of
protoplasts and for developing protocols for micropropagation of commercially important
species (Chang and Ho, 1997). Somatic embryogenesis has been reported in several
species (Rao and Rao 1988, Rao eta/., 1990, Yeh and Chang 1987, Woods eta/., 1992)
of Bamboo viz., Bambusa, Dendrocalamus, Sinoca/amus and Otatea. Zygotic embryo is
the best source for the induction of somatic embryos, however, in some cases, rhizomes,
nodes and leaf sheaths of juvenile plants also produce embryogenic cultures. Embryonal
axis of B. arundinacea produced somatic embryos on MS + 2,4-D + BA (Mehta et at.,
1982). Rao and Rao (1990) reported somatic embryogenesis from seed cultures of
Dendrocalamus strictus on B5 medium supplemented with 2, 4- D at 10 and 3x10. M.
Encapsulation of somatic embryos of D. calamus and plantlet regeneration was reported
by Mukunthakumar and Mathur ( 1992 ). The alginate matrix (6% Na alginate, IOOmM
CaC)z 2H20) containing MS + 1mgll NAA + 0.5 mg/1 Kin was used for encapsulation of
embryos. A germination frequency of 96% and 45% was achieved in vitro and soil
respectively. Conversion of somatic embryos to plants was achieved on a medium
supplemented with NAA (lmg/1) and Kin (0.5mgll). Mature embryos could be
germinated on filter paper bridges on a B5 liquid medium containing sucrose 2% +
IBA+NAA (Rao and Rao 1988). The germinating embryos produced long roots with a
few laterals.

Nadgauda eta/., (1990) and Rao and Rao (1988) succeeded in the induction of in vitro
flowering and more importantly, seed production in D. calamus and B.arundina.
Multiple vegetative shoots and flowers emerged from the nodes of explants derived from
in vitro grown young seedlings. The highest frequency of flowering (47%) was obtained
when cultured explants were transferred from BAP (22.2 mM ) containing medium to a
hormone free medium after 8 weeks of initial culture. Rout and Das (1994) observed
flowering B. vulgaris, D. giganteus and D. strictus on half strength MS medium
supplemented with 0.25mgll IBA, 0.5mg/l adenine sulphate and 0.5 mg/1 GA3 • Bag et
a/., (1999) reported callus and somatic embryogenesis from cultured sprouted buds
excised from mature plants of Dendrocalamus hamiltonii. Efficient embryogenesis was
recorded on MS + 7.5mM 2,4-D and 5.0mM BAP. Plantlet production continued even
after two years of culture without the loss of embryogenic potential. Well rooted plantlets
were successfully acclimatized and 70% of the plants survived in vivo.

Saxena and Dhawan (1999) developed a complete protocol for large scale propagation of
D. strictus through somatic embryogenesis from seeds cultured on MS medium
supplemented with 2,4-D ( 3 x 10·5 M). Upon transfer toMS containing NAA ( 3 x 10·6
M), Kin (5 x 10'6 M) and soluble PVP (250mgll), the dark green embryos developed
into healthy plantlets. Unrooted shoots ,if any, obtained on the multiplication media
were rooted on MS major salts reduced to half strength supplemented with NAA (3 x 10·
~ and IBA ( 2.5 x 10·6 M). Rooted plants were successfully transferred to soil with
80% survival and 100, 000 plants have been produced. The high conversion of embryos
to plants in Bamboo in this study has been attributed to the use of gerlite instead of agar
in the medium which resulted in a germination frequency of 90% in 85% of the
genotypes. At all the stages of regeneration of somatic embryos, a strong genotypic
control influenced the frequency of somatic embryo germination which holds the key to
success for commercial micropropagation of Bamboo. The factors influencing
 177
acclimatization process especially seasonal effects which were not earlier studied (
Saxena and Dhawan 1999).

Natural regeneration of Bamboo is either by seeds or by sprouts from the rhizome. After
the gregarious flowering and seeding, seeds are collected, sown and seedlings are reared
in nurseries. The other common method is by planting rhizome cuttings or by offsets just
before the rainy season. However, the supply of rhizome and offset material per clump is
very limited and quite inadequate for raising large scale commercial plantations of
desirable clones. The report by Saxena and Dhawan (1999) is promising in this
direction. However, research should also be aimed at screening for totipotent cells from
mature proven clones. The availability of embryogenic cell cultures for in vitro selection
techniques will permit the evaluation of bamboo plants tolerant to abiotic and biotic
stress conditions. The results on in vitro flowering will pave way for the investigations
on in vitro breeding for better characters. Research is needed to be concentrated on
employing somatic embryogenesis in modern-techniques of genetic engineering.

2.5 Poplar (Populus sps. )

Poplar in their natural range occur widely above 28 N latitude in northern India and
there are six Poplar species indigenous to India growing along water courses in the
higher hills, in valleys and also on hill sides. Poplar possesses high timber value for
industrial applications in packaging, pulpwood, sports goods, fodder and fuelwood.

Somatic embryogenesis has been reported in several poplar species (P. alba x P.
grandidentata, P. ciliata, P. deltoides, P. euramericana and P. nigra X P. maximowiczii
) and a variety of non embryogenic explants have been employed including leaf sections,
internodes and shoot tips. (Michler 1995). Regeneration of somatic embryos directly on
leaf veins or on hypocotyl and cotyledon explants has also been achieved. Callus ,cell
suspensions from nodal cultures have been used to raise indirect somatic embryo
initiation. Studies conducted on somatic embryo formation have revealed that non
chlorophyllus, densely cytoplasmic and nodular embryogenic callus type produces
somatic embryos. Induction of somatic embryos has been achieved on MS+2,4-D+BAP
containing 3 % sucrose. Exogenous application of ABA was not found to play a
necessary development role with embryo maturation, which was attributed to the high
water content in Poplar seeds. Further growth of somatic embryos was possible with
minimum cultural manipulations, where for example a pulse treatment of IAA or NAA
was all that was necessary to stimulate radicle elongation. ( Cheema, 1989).

It is always stressed that to initiate forest tree tissue cultures, mature elite trees having
promising characters have to be selected since seedling or any other juvenile tissue can
not be of much use. This is due to the heterogenous nature of seeds because of cross
pollination which do not guarantee production of good quality plants. In this regard, it is
noteworthy that, in Poplar it is feasible to raise the cultures from mature trees as
reported here. This encourages to tackle the micropropagation through somatic
embryogenesis from known plus trees. The technique would be helpful in the selection of
the clones to ameliorate research efforts necessary for large scale plantations. It also
offers to screen clones to a variety of stress conditions with a planting of tolerant and
adaptive clones to a given forest site.
178

2.6 Teak ( Tectona grandis L.)

Teak is a major source to obtain a high quality timber and combines attributes such as
strength, durability and resistance to termites. Under favorable conditions Tectona
grows into a large tree with a tall, clean and cylindrical bole (Mascarenhas eta/., 1993).
This is a common tree in Indian forests but grows well in a fairly moist warm and
tropical climate. Due to its immense economic value, several commercial organizations
have launched massive teak plantation schemes throughout India. Natural regeneration
is mainly through seeds and is widely employed in several nurseries. Other methods like
grafting, rooted stumps, and budding are also practiced for propagation.

Micropropagation protocol for teak has been well established by National Chemical
Laboratory, Pune. It includes selection of buds from genotypes, proliferation of shoots
followed by induction and elongation of roots, acclimatization and field planting. Field
evaluation studies have also been conducted (Mascarenhas eta/., 1993). In comparison
to organogenesis, studies on embryogenesis has lagged behind. However, National
Chemical Laboratory Group group reported globular embryos in teak by using different
explants such as apical meristem, in vitro leaves, stem, shoot tips and axillary buds.
Kushalkar and Sharon (1996) reported somatic embryos, both from callus derived from
apical buds on MS+ BAP (0.1mgll) + NAA (0.01mgll) and directly from axillary buds
on filter paper bridges on 1,-1 MS + BA ( 0.1mgll) + 2iP ( 1.5mgll).

Potential application of somatic embryos for a large scale production of plants via
somatic embryogenesis in teak is still far from reality, since few reports are available and
not much research on somatic embryogenesis has been conducted. It is necessary in
plants like teak to select proper genotypes for a large scale production of plants which
have the potential for producing significant gains in the field test. The protocol has to be
based on the extensive data applicable to plants collected from various locations and
preferably originated from a mature plus tree. Studies need to be focussed to obtain a
high rate of somatic embryo induction, maturation and germination. The results
presented show promise for small scale production of a few hundred plants necessary to
set up a field trial to evaluate the growth in comparison with tissue culture raised plants
through axillary bud culture. Depending on the data generated, further experimention
will be required to enhance embryogenic frequency in cell cultures and to use such
cultures for scale up studies. The other benefits offer by somatic embryogenic system can
be in applying a number of genetic manipulation techniques for improvement.

2. 7 Dalbergia (Dalbergia sps )

Dalbergia is an important leguminous timber yielding tree and is common in most of

the states of India. Dalbergia lanceolaria, D. latifolia and D. sissoo are the main
species and shoot multiplication has been achieved in all three species. Dalbergia
latifolia and Dalbergia sissoos are highly drought resistant suitable to arid land
reclamation programmes. Attempts have been made to clonally propagate this plant by
using seedling explants as well as tissues from mature plants (Mohan et a/ 1997).
Somatic embryogenesis was obtained in D. sissoo in callus cultures derived from 45
days old semi-mature zygotic embryo on MS + 2,4-D +Kin. ( Das et al., 1997). Somatic
 179
embryos proliferated rapidly by secondary embryos on transfer to Y:z strength MS
medium with Kin+ 2,4-D .. The light green embryos germinated on Y:z strength MS +
0.5mgll ABA. Scanning electron microscopic studies were also conducted by the same
group ( Das eta/., 1997 ).

At present, the efficiency of plant production from Dalbergia embryogenic cultures has
been reported only from a one laboratory and efforts are to be directed to augment the
methods to establish the cultures from mature trees possessing all promising features.
This is an important timber yielding tree in India and any success in micropropagation
by somatic embryogenesis is a much needed requirement. The reported work has given a
preliminary indication of the amenability of this plant for tissue culture manipulations.
Once a reliable protocol is set, the embryogenic cultures can also be handled for
advanced techniques of genetic engineering.

2.8 Soap nut (Sapindus trifo/iatus L.)

Sapindus trifoliatus is an economically important tree of tropical forest and is a major

source of saponins for industrial use. Fruits of the tree are used in excessive salvation,
epilepsy, and chlorosis. Soap nuts are used as detergent in textile and emulsifier in
insecticides. Kernels contain oil which is widely used in soap manufacturing and
exhausted cake as a filter and fertilizer. Wood is used for charcoal making.

Somatic embryogenesis has been induced from young leaf explants. ( Unnikrishnan
1992).The explants produced callus on MS + 2,4-D ( 2mgll) +Kin (0.5mgll) and upon
transfer of tissue to MS + BA + Kin development of proembryos to heart type embryos
took place followed by further growth into plantlets on MS + 4% sucrose. These authors
further indicated that somatic embryos of soap nut can tolerate concentration of NaCl
upto 100 mM without affecting the growth.

Soapnut is the another example besides Sandalwood and Poplar in which tissue (leaf)
from mature tree yields embryogenic cultures. The plant is of immense importance
commercially in several industries and rapid propagation through somatic embryogenesis
offers an viable alternative for plantation of superior clones. Success of regenerating
somatic embryos on an NaCl medium paves the way to test these clones on a saline land.
This, however, requires intense research work. The establishment of reliable and
repeatable protocol is a prerequisite for isolation of such mutants. The reported work
although is from a single laboratory it is worth pursuing to assess the utility of somatic
embryos in other studies for plant productivity and improvement.

2.9 Semul ( Bombax ceiba L. )

Semul is an important forest tree of tropical forest and belongs to family Bombacaceae.
It is commonly known as "red silk cotton tree" and is used for rearing silkworms. This
soft wood tree is also used in match industry. Axillary buds collected from mature trees
produced multiple shoots on MS + BA (0.5mgll) + NAA ( 0.1 mg/1) in 4-6 days. In vitro
multiple shoots could be rooted on medium containing MS + IBA (0.5mgll) + BA (0.1
mg/1).
180
Somatic embryogenesis in semul was achieved from immature zygotic embryos as
explant (Fig. 2 ; Bansal, personal communication). The 1-3 mm long immature zygotic
embryos within the ovules bearing two white cotyledons formed the explants. Both
direct and indirect somatic embryogenesis was observed. BAP triggered somatic
embryogenesis at higher frequencies and somatic embryogenesis was inversely related to
concentration of BA. A maximum of 31 embryos per explant were observed. In some
cases direct embryogenesis was observed within 6 days of inoculation from the root pole
of in vitro raised somatic embryos. It was reported by these workers that prolonged
supply of the low concentration of BA (0.44 ~ was essential for maturation, growth
and conversion of somatic embryos into plantlets (Fig.2). The somatic embryo
conversion was prolific and efficient plantlet formation was achieved in about 90% of
cultures. Somatic embryos not separated from the callus took to an aberrant course and
germinated into plants with anomalous roots or shoots or both. Even after prolonged
subculture of three years somatic embryos retained their regenerative capacity (Bansal,
personal communication).

This important tree is highly susceptible to insect and fungal attacks and prior to
pollination flower falls giving unisexual male flowers causing depletion in number of
desirable trees. The success of somatic embryos directly and indirectly has laid down the
frame for rnicropropagation to produce the abundant planting material. There is no loss
of regenerating capacity even after three years of culture ( personal communication. The
high conversion somatic embryos to plants has also been achieved. This plant promises
to offer as a test candidate to incorporate rnicropropagation method in social forestry
programme.

2. 10 Arjun ( Terminalia arjuna L.)

Termina/ia arjuna , is one of the important timber yielding Indian tree and is a ho~t to
tropical tasar silkworm. The bark and other plant parts have medicinal uses and are
source of tanin (Nishi Kumari et a/ 1998). There is an urgent need of propagating elite
plus trees, because of tremendous variation in the populations. Leaves collected from
mature trees were used for the induction of callus on MS medium supplemented with
2,4-D (5mg/l ) +kinetin (O.Olmg/1) + 3% sucrose and somatic embryos differentiated
upon transfer to MS basal medium (Nishi Kumari et a/ 1998). Somatic embryos
developed into complete plants on MS medium with 3% sucrose and 0.8% agar. Plantlets
grown on this medium showed 75% survival. During hardening, covering of plantlet
with a poythene bag or beaker for at least 3 weeks was essential and 25 such hardened
plants transferred to pots. Such in vitro raised plants showed luxuriant growth under
field conditions (Nishi kumari eta/., 1998).

Terminalia is yet another forest tree in which it has become feasible to employ mature
tissue to raise cultures ensuring retention of genetic fidelity in progenies. The
performance of plants in the field has already shown promise that this method can be
employed on a larger scale. It is also necessary to monitor the performance of plants at
regular intervals to evaluate the physiological and genetic changes. This plant can also
be exploited for basic molecular studies on somatic embryogenesis as well as for genetic
manipulation aimed at tree improvement.
 181
2.11 Brown oak (Quercus semecarpifo/ia L.)
The genus Quercus is represented by about 23 species in the Indian Himalayan region
and used for fodder, fuel and to some extent timber and planted at several places to
prevent soil erosion and for conservation of water. The conventional mode of
propagation is through seeds is limited and a big gap exists between mature trees and
seedlings. Extensive work on direct and indirect somatic embryogenesis has been carried
out at G. B. Pant Institute of Himalayan Environment of development, Almora, North
India (Bisht et al., 1998). Somatic embryogenesis was induced directly from cotyledons
of mature seeds of Quercus semecarpifo/ia L. on MS +rnA + 2,4-D or GA3. Ca}lus
also differentiated into somatic embryos on WPM medium (Lloyd and McCown 1980)
supplemented with 2,4-D. Embryos on subculture regenerated into secondary embryos.
The germinated somatic embryos had well developed shoots, leaves and a tap root
system. Removal of tap root and planting of shoots on WPM medium supplemented with
rnA helped for better survival of tissue culture plants in the field. In Quercus
/eucotrichophora L. and Quercus glauca, in vitro multiplication through shoot
regeneration was reported but induction of somatic embryogenesis was not achieved.
High frequency shoot multiplication was obtained on MS + BA (22mM) followed by
rooting on Y2 strength WPM+ 0.4- 24.6 mM rnA (Purohit eta/., 1999).

Somatic embryogenesis has been reported from juvenile and adult tissues of various
species oak species (Chalupa 1995). The major problem in oak is the low conversion of
somatic embryos to plants resulting in a low recovery of plants. At present only one
laboratory in India has reported work on Oak. The preliminary results indicate that
more research work needs to be done on selected clones. Studies are also needed on the
strategies for better conversion of embryos to plants.

4. Conclusions

There has been a steady increase in research activities in India on micropropagation of

woody tree species, primarily involving induction of multiple shoots and regeneration of
plants (Shekhawat et a/., 1998). Investigations have also focused on somatic
embryogenesis in forest tree species, underscoring the need to develop cost effective and
high volume propagation. The list of plants that can be successfully produced by somatic
embryos is now long enough to assume that embryo formation in cell culture in vitro is a
general process in numerous plant species. Somatic embryogenesis offers a better
system preferred over organogenesis in many ways : (1) uniformity in the regenerated
plants since the origin of somatic embryos is unicelluar, (2) prolific rate of production of
embryos which could be exploited for preparation of synthetic seeds ( Rao eta/ 1998)
and (3) somatic embryos are readily amenable to automation and mechanization and can
be scaled up using bioreactors (Gupta eta/ 1993, Vasil 1994). Thus somatic embryos
have the potential to produce large number of individual clones for afforestation purposes
and cloning of trees through somatic embryogenesis is considered by the industry as an
important new technology (Tamis-Roger 1998).

Biotechnological and molecular tools are now being introduced into forestry research and
improvement progranunes not only to evolve superior trees through plant genetic
engineering techniques but also to confirm the genetic fidelity of micropropagated
182

plants through RFLP/RAPD assays (Tzfira eta/ 1998, Report on Plant Tissue Culture
Technologies for large scale production and commercialisation, Dept of Biotechnology,
Govt of India 1999). Additionally, somatic embryogenic systems interface well with
genetic transformation techniques for the production of transgenic plants (Rutter et a/.,
1998) improved for accelerated growth, modified root structure, abiotic and biotic
tolerance, wood quality and productivity.

Genetic identity of individuals, hybrids, inbred lines and clones can be verified using
molecular markers. Both RFLP and PCR based markers have been used in Poplars (Vijay
Rani et al 1995), Acacia hybrids (Wickneswari and Lee 1993) and willows (Chang et al
1995). In Eucalyptus, molecular techniques have been employed in the discrimination
and verification of genotypes (Keil et al 1994). In addition, these techniques have also
been applied for tropical hard woods in the phylogenetic relationships. Chloroplast and
mitochondrial DNA based markers have also been successfully employed in clarifying
phylogenetic relationships (Zakri et al 1996). Marker assisted selection offers the scope
for an early selection of better wood quality at the young seedling stage. RFLPs were the
first molecular markers to be used for forest tree populations (Neale et al 1992). Success
has also been achieved in quantitative trait loci (QTL) mapping resistant to white pine
blister in sugar pine and wood density and wood volume in radiata pine (Devey 1994).
Concerted efforts are required to generate populations for species to employ molecular
marker techniques in tree improvement programmes.

Despite the noteworthy success in the induction of somatic embryogenesis in important

Indian forest trees, more research is warranted in understanding and exploitation of
somatic embryogenesis for scaling up aimed at mass propagation. Synchronization of
embryogenesis, production of high quality embryos, maturation of somatic embryos and
the efficient conversion of embryos into plants, remain the challenges for the forest
biotechnologists. The recurrent embryogeny which is a major influencing factor for
successful demonstration is not reported extensively in forest plants except in some
conifers. Other optimum requirements have so far not been determined conclusively for
various stages of initiation, proliferation, development, rooting and establishment in the
soil. Ongoing research in different research laboratories in India may form a base for the
pre commercial programme supporting a project concentrated on a cost effective method
for the large scale production of plants from somatic embryos. Cryopresrvation.
automation, and techniques of molecular biology are steps have to followed as
complimentary strategies for the delivery of desired plants for plantations in the forest.

Successful implementation of biotechnological tools in the genetic improvement of forest

trees and in the afforestation programs requires concerted and coordinated efforts from
the research scientists and the governmental agencies. Commercialization of the cloning
process through efficient and reproducible somatic embryogenesis and cost effective
methods will usher in an era of more productive forests. However, somatic
embryogenesis poses certain key bottlenecks for large scale plantations due to many
technological difficulties involved in the process. Desirable traits in woody plants are
expressed after 15 - 20 years of planting and it is not possible to earlier capture of the
genetic gains. An investment locked for such a long time does not make a profitable
venture for a private company. Hence, application of micropropagation protocol has
remained restricted to government forest agencies in India. Scientific manpower,
 183
adequate research facilities, technical information system and funding are key elements
need to be added for effective and viable and realistic productive output of somatic
embryogenesis.

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 187

TABLE 1. Somatic embryogenesis in Indian forest trees

Plant Explant Response*

Sandalwood Endosperm Callus,

Santalum album Embryos, Plantlets
Hypocotyl, stem, Embryos, Plantlets
Protoplasts

Neem Cotyedons Embryos, Plantets

Azadirachta indica

Eucalyptus
E. citridora & Embryos
E.grandis
E.citridora Seeds Embryos, Plantlets

Bamboo
Dendrocalamus strictus Seeds Embryos
Dendrocalamus strictus
Seeds Embryos, Plantlets
Bambusa arundinacia

Embryonal axis Embryos

Poplar
Populus spp. Embryos,Plantlets

Teak
Tectonis grandis Apical ~mds Embryos

Dalbergia
Dalbergia sissoo Semi-mature zygotic Embryos,Plantlets
embryos

Soapnut
Sapindus tri{oliatus Young leaves Embryos,Plantlets

Semul
Bombax ceiba Immature zygotic embryos Embryos,Plantlets

Arjun
Terminalia arjuna Leaves Embryos,Plantlets

Brown oak
Quircus semicarpi{olia Cotyledons Embryos,Plantlets

For details of references, see the text.

188

• Bhabha Atomic Research Centre (BARC), Nuclear Agriculture &

Biotechnology Division, Trombay, Mumbai 400 085.
• Indian Institute of Science (DSc), Department of Microbiology & Cell
Biology, Bangalore, 560 012.
:::.·
• National Chemical Laboratory, Pune 411 008.
• Department of Botany, University of Delhi, Delhi 110 007.
• Tata Energy Research Institute, New Delhi 110 003.

• National Chemical Laboratory, Pune 411 008.

..:(
• G. B. Pant Institute of Himalayan Environment & Development, Kosi
Katannal,

.:·.
RD. University, Department of Biological Sciences, Jabalpur 482 001.

Banaras Hindu University, Department of Botany, Varanasi

 189

Fig. 1. Somatic embryogenesis and plant regeneration in sandalwood.

A Somatic embryos.
B. Germination of somatic embryos.
190

Fig. 2. Somatic emryogenesis in Bombax ceiba L.

(Photo courtesy of Dr.Y.K. Bansal, RD. University, Jabalpur,India)

A & B. Initiation and development of somatic embryos from immature

zygotic embryos.
C. Plantlet development from somatic embryo.
 191

c
Somatic embryogenesis in neem (Photocourtesy of Dr. S.R. Thengane, NCL,
Pune, India)

A Embryogenic mass derived from cotyledons of unripe green fruits.

B. Germinating embryo mass
C. Somatic embryo derived plantlet.
 6. SOMATIC EMBRYOGENESIS RESEARCH ON FRUIT TREES
IN INDIA

Rajani Nadgauda, Gaurav Mathur and Shilpa Gogte

Tissue Culture Pilot Plant
National Chemical Laboratory
Dr. Homi Bhabha Road
Pune - 411 008, India

Chapter contents

1.1 Introduction
1.1.1 Background
1.1.2 Beginning
1.2 Somatic embryogenesis in fruit trees
1.2.1 Export potential of fruits
1.2.2 A brief survey of published reports
Citrus species
The palm family
Pomegranate
Banana
Guava
Mango - the emperor offruits
Other fruits
1.3 Cashewnut (Anacardium occidentale L.)- case study
1.3.1 Introduction
1.3.2 Explant
Source
Surface sterilization
Culture conditions
1. 3. 3 Initiation of embryogenic callus from nucellus
Initiation
Maintenance
Induction of somatic embryos
1.3 .4 Development and maturation of somatic embryos
1.3. 5 Problems and possibilities
1.4 Conclusions and prospects
1.5 References
193

S.M. Jain, P.K. Gupta and R.J. Newton (eds.). Somatic Embryogenesis in Woody Plants, Volume 6, 193-213.
© 2000 Kluwer Academic Publishers.
194
1.1. Introduction

The progress in the field of somatic embryogenesis of woody perennials

has been quite noteworthy in the past decade (Jain et al. 1995, 1999).
Although most of the economically important woody species still prove to
be recalcitrant, specially when propagated using mature tissues.
Nevertheless, limited success in propagation of a number of commercially
important angiosperm and gymnosperm tree species has been achieved
(Tzfira et a!. 1998). These encouraging results have given a tremendous
boost to the research in this field, especially the fruit trees. This chapter
will familiarize the readers with the work done over the years and the
ongoing research in India on somatic embryogenesis of fruit trees.

1.1.1. BACKGROUND

India's vast land area spreading over a number of climatic zones has been
responsible for it's rich and diverse flora. It features among the list of
twelve Mega-diversity' countries of the world, and represents almost all
eco-climatic zones (Rawat 1998). Various tropical, subtropical and
temperate fruit tree species grow on the different soil types, placing the
country in a unique position of harboring this tremendous bio-diversity.
This natural and 'exotic' wealth has always attracted merchants and
explorers from all over the world, thus encouraging trade and
strengthening relations with other nations for over twenty centuries. But
this last century has witnessed a rapid depletion of these natural resources
due to the ever-increasing population, placing the complete ecological
system under tremendous pressure. Forests too have not been spared by
this ruthless depredation (Chaturvedi 1998).
The Indian horticultural produce has played a major role in its
foreign earnings and has an attractive market worldwide. Further
improvement in quality and post-harvest techniques can go a long way in
substantial increase of this ever-growing export industry. India is the
second largest producer of fruits and vegetables in the world today (Ghosh
1999). Government agencies and forest departments in India have brought
forth various programs and projects for promoting the propagation of
fruits with export potential. Department of Biotechnology (DBT),
Government of India, has identified some priority crops based on their
economic importance for the country and multi-institutional network
 195
programs are being conducted. Some of the crops identified are mango,
cashew, grapes, forest trees, foliage plants and others (Mascarenhas 1998).
DBT has also been instrumental in supporting a large-scale co-ordinated
project on mango somatic embryogenesis taking into consideration its
market potential in the domestic circuit and for export (DBT Annual
Report 1997-1998).
Application of tissue culture techniques as a supplement to
vegetative propagation methods is relatively recent, and this technology is
yet to be efficiently exploited. With the advances in the field of
biotechnology in the past few decades, refinement of molecular techniques
and their application to horticulture industry has opened up a whole new
dimension for future investigations in the area of crop improvement. The
role of propagation through tissue culture thus becomes very crucial.
Selection of elite fruit tree varieties and their propagation through tissue
culture has proven to be beneficial in supplementing the demand for
nursery grown high quality seedlings for plantations. This can also be seen
clearly in the examples of date palm (Phoenix dactylifera L.) where the
majority of planting material is obtained through somatic embryogenesis
(Litz and Gray 1995).

1.1.2. BEGINNING

As compared to the micropropagation of tree species through axillary bud

proliferation, propagation via somatic embryogenesis is still in its infancy in
India. Although the earliest reports of somatic embryogenesis in woody
species date back to mid 1960s (Konar and Oberai 1965, Rao 1965), its
progress and application has comparatively been very gradual. There are a
number of reported cases of somatic embryogenesis of woody species in
the literature, but only a few have successfully been able to reach the field
(Rangaswamy 1986).
It would be a mere repetition if the advantages of somatic
embryogenesis to woody plants are elaborated here, as this topic has been
extensively covered in the previous volumes of this series. A few words on
the benefits of somatic embryogenesis to some economically important
fruit tree species, in the Indian context, would be useful. The major
advantage would be in the conservation of the depleting flora and the other
in rapid large-scale clonal propagation of elite varieties suitable for a
particular region. Apart from being useful in basic studies on embryo
development, somatic embryos can also be utilized for genetic
196

improvement through transformation, cryopreservation of endangered as

well as elite varieties and production of synthetic seeds.
In this chapter we would introduce the readers to the major work
carried out in India for the micropropagation, exclusively by somatic
embryogenesis, in various fruit trees with special reference to cashewnut.
The purpose of this exercise is to present an overall scenario highlighting
only the reported work, incurring problems and it's future significance in
the country's progress. We have tried not to include elaborate case studies
in this review, but have very briefly incorporated the work carried out on
somatic embryogenesis of cashewnut in our laboratory.

1.2. Somatic Embryogenesis in Fruit Trees

Among the various plantation crops, fruit trees form a distinct and a very
important group because fruits hold a major share in the economy and
export earnings of the total agricultural produce in India. Tremendous
progress has been achieved in the improvement of the final marketable
product of the fruit trees. Starting from selection, breeding, propagation,
cultivation and harvest, to, processing, packaging, transport and
marketing, the progress is commendable.
Tissue culture has cut down the time involved in breeding new
varieties and hastened large-scale propagation in fruit trees, effectively
complementing the conventional propagation methods (Alston and Tobutt
1989). Genetic engineering also promises better quality, flavor, shelf life
and resistance to various common diseases and pathogens (Gavinlertvatna
1992, Hammat 1992). Considering the above benefits, somatic
embryogenesis can be a very useful and a potential tool for both,
propagation and transformation studies for improvement of existing fruit
tree varieties.

1.2.1 EXPORT POTENTIAL OF FRUITS

Indian economy is largely based on agriculture, with horticultural crops

contributing to 25 percent of the total agricultural export in India and
fruits forming its major bulk (Ghosh 1999). As mentioned earlier, the
advantage of having different agro-climatic zones in India can suitably be
exploited by cultivating temperate as well as tropical fruits. Considering
the export potential, it is appropriate to identify the fruit trees based on
 197
their demand, improve upon their quality by genetic engineering, and
increase the production by large-scale clonal propagation, subsequently
boosting the export earnings.
The export of horticultural produce from India is yet to make a
mark in the international market. Except for cashewnut, where India stands
as the largest producer and exporter in the world, despite the acute
shortage of raw nuts for processing, which has lead to the import of raw
nuts to meet the demand of the processing industry. This clearly shows the
need to increase the domestic production of improved varieties (Nayar
1999).
This also holds true for the other major fruit crops in India viz.,
mango, banana, grapes, citrus, guava, papaya, pomegranate, pineapple and
sapota. Along with higher yield and variety improvement, crop
management, post-harvest processing, packaging and marketing are
equally important areas that need improvement to increase the export
earnings. As these other aspects are beyond the scope of this chapter, we
will be focussing on the possibilities of alternate methods for increasing the
yield and variety improvement.

1.2.2 A BRIEF SURVEY OF PUBLISHED REPORTS

This survey includes the published reports only of the work carried out on
somatic embryogenesis of fruit trees in India. If any reference has been
overlooked, it is completely inadvertent.

Citrus
Since the earliest reports of culturing developing ovules and seeds, before
and after fertilization (Maheshwari and Rangaswamy 1958, Rangaswamy
1958 a, b), ovules have been frequently used for initiating cultures in
various Citrus species. The initial studies by Mitra and Chaturvedi (1972)
reported embryoid formation and complete plants from unpollinated
ovaries of C. aurantifolia and C. sinensis, where ovary wall tissue was
responsible for this response. Later studies by Bhansali and Arya (1978 a,
b) showed indirect embryoid formation from stem segments in MS
(Murashige and Skoog 1962) basal medium supplemenled with
benzyladenine (BA). Somatic embryos were also reported to form directly
on cultured embryos and ovules (of both zygotic and nucellar origin),
which were dissected from seeds (Sabharwal1963).
198

Chaturvedi and Mitra (1974, 1975) also reported embryoid

formation in C. grandis from stem callus on MS basal medium
supplemented with zeatin and naphthalene acetic acid (NAA). Apart from
ovary and stem, embryoid and complete plantlet formation was also
obtained by using anthers of C. aurantifolia (Chaturvedi and Sharma
1985). Gill and co-authors (1991, 1994) have reported embryoid and
plantlet formation from immature embryos and different seedling explants
of C. reticuklta on MS basal medium supplemented with NAA and
Kinetin. Another widely used basal medium for inducing somatic
embryogenesis in Citrus is Murashige and Tucker (MT) medium
(Murashige and Tucker 1969). Moore (1986) used MT medium (50gll
sucrose) with ·0.5-1.0 gil malt extract for this purpose. Later, Grosser and
co-workers (1988) placed developed embryos (without roots) on MT
medium supplemented with 5 mg/1 BA and produced multiple shoots that
later rooted.
Kitto and Young (1981) had described the benefits of
micropropagation of non-nucellar vegetative tissue to Citrus, supported by
the following reasons. Sufficient amount of seeds are obtained only after
several years from newly introduced rootstocks, and some desirable
rootstock clones may be seedless, or may not produce nucellar seedlings.

The Palm family

Coconut palm forms a very important tropical fruit crop throughout the
world and known for its multifarious uses. Apart from obtaining high
yielding hybrid varieties, the primary concern of the farmers is disease-
resistance. Breeding of the locally available elite varieties can be hastened
by supplementing the planting material by clonal propagation, which will
be helpful in supplying uniform parental lines for hybrid production.
Success in clonal propagation in other palms (date and oil palms) has
encouraged the tissue culture of coconut, which had proven to be difficult
to regenerate.
Disease tolerance and improvement in cultivation and processing
practices is the present need for Indian farmers, where, there has been a
striking increase in the productivity in 1996-97 (6863 nuts/ hectare), since
1983-84 (4982 nuts/hectare) (Markose 1999). Since coconut is propagated
entirely by seed, clonal propagation method to obtain improved breeding
stocks is needed urgently. Prakash Kumar and co-authors (1985) reported
direct somatic embryogenesis (embryoid formation) from vascular tissue of
leaf explants on medium supplemented with NAA and IAA in presence of
 199
specific Mg/K ratio. Coconut somatic embryogenesis is usually indirect,
and has to pass through the callus phase. Gupta and co-workers (1984)
also reported embryoid formation from the young leaf explants of in vitro
germinated zygotic embryos of coconut on Y3 basal medium containing
226 mM of 2,4-dichlorophenoxy acetic acid (2,4-D). Cytokinins (BA and
kinetin) were added with auxins, namely NAA, IAA (indole acetic acid)
and 2,4-D (2,4-dichlorophenoxyacetic acid), singly and in combination, by
Bhalla-Sarin and co-authors (1986) for callogenesis, using zygotic
embryos of coconut as explants.
Phoenix dactylifera L. (date palm), another economically
important palm of the dry, subtropical regions, faces similar problems for
good quality planting stock and disease resistance. Sharma et al. (1984),
used axillary bud and shoot tip explants to initiate embryogenic callus
cultures on MS basal medium supplemented with activated charcoal (3
g/1), 2,4-D (100 mg/1), BA (5 mg/1) and thiamine-HCl (1mgll). A higher
phosphate concentration (170 - 200 mg/1) and substitution of 2iP (2-
isopentenyl adenine) by BA was recommended for good quality friable
callus production. Plantlet regeneration was achieved by placing the
embryogenic callus on hormone-free medium. Dass and co-workers (1989)
reported regeneration of plantlets from embryogenic callus obtained from
shoot tips by a three-step protocol (see Table 1). A pre-treatment of
explants with anti-oxidants and additional supplements in the MS basal
medium like calcium pantothenate, glutamine, biotin, adenine, polyvinyl
pyrollidone (PVP), were found to be helpful in producing viable and
normal plantlets.

Pomegranate
Jaidka and Mehra (1986) using seedling explants reported somatic
embryogenesis in pomegranate. Different explants taken from seedling
germinated in vitro were inoculated on MS basal medium with NAA (4
mg/1), Kinetin (2 mg/1) and coconut water (5% v/v), where, embryogenic
callus was formed. This callus was then shifted toMS medium with NAA
(2mg/l) and BA (2 mg/1), where it gave rise to somatic embryos, and
further, plantlets. Later, somatic embryos of Punica granatum L. were
obtained on modified MS basal medium supplemented with 2,4-D (5 mg/1),
BA (5 mg/1), kinetin (2 mg/1) and 500 mg/1 casein hydrolysate (CH) using
immature zygotic embryos as explants (Rajbhansali 1990). Maturation and
conversion of somatic embryos was achieved in a four-step protocol where
the concentration of 2,4-D was gradually lowered and the somatic
200
emblings germinated finally on hormone-free medium. Nataraja and
Neelambika (1996) have also reported somatic embryogenesis and
complete plantlet regeneration in pomegranate from petal cultures (Table
1).

Banana
Banana is the only plant presently being successfully produced by tissue
culture for the domestic market. Tissue culture plants have been tested in
several laboratories and found to give higher yields (Mascarenhas 1998).
The total area under banana cultivation in India is estimated at 433019
hectares with a production of 13095087 MT for the year 1995-1996 (A.K.
Misra, Ministry of Agriculture, Govt. ofindia, personal communication).
Though, presently the large-scale clonal propagation of banana is
not done by somatic embryos, there certainly is scope in improvement of
variety using this powerful technique. Recently, a report of establishment
of embryogenic cultures using young male flowers in five cultivars of
banana has been published (Ganapathi et al. 1999). Plantlet regeneration
with the emergence of plumule and roots was achieved within 6 to 8 weeks
and normal development of plant was observed under greenhouse
conditions. Most of the edible banana varieties are triploid and propagated
vegetatively by suckers. Also, sterility and polyploidy impedes breeding for
superior varieties. In their system, Ganapathi and co-workers have used
young male flowers as explants for induction of somatic embryos (Table
1).

Guava
In guava (Psidium guajava), recurrent somatic embryogenesis has been
reported (Akhtar and Jaiswal 1995) from immature zygotic embryos. The
development of somatic embryos was found to be asynchronous.
Developmental stage of the zygotic embryo and concentration of auxin
(2,4-D) were found to be important for the induction of recurrent
embryogenesis. The 8-week old torpedo stage zygotic embryo and 2,4-D
at concentration of 1 mg/1 gave the optimum embryogenetic response.

Mango - the emperor offruits

Two very important fruit crops of India, viz., mango and cashewnut, are
members of the same family Anacardiaceae. Both contribute to a very
large amount of export earning as compared to other fruit crops. The
popularity of these fruit trees has already led to initiation of a number of
 201
research programs in different parts of the country. Collaborative research
projects, giving priority to improvement of quality, disease resistance and
clonal propagation, have started showing promising results. Though, as
compared to mango, the research on cashew is lagging far behind.
Jana and co-workers (1994) reported a method for rapid
production of somatic embryos from nucellar tissue of three
monoembryonic Indian mango varieties, viz. Alphonso, Mundan and
Baneshan. Response of this direct somatic embryogenesis (without
intervening callus phase) varied among the three varieties tested. Alphonso
showed highest percentage of maturation and conversion, with 55.6%
embryos showing normal development. Somatic embryos up to the stage
of germination could be produced within 10-11 weeks showing a vast
potential for large-scale multiplication.
Somatic embryogenesis has also been reported from other
monoembryonic varieties of mango, namely, Amrapali, Chausa, Langra
and Mallika (Hussain Ara et al. 1995). Embryogenic callus was induced
from nucellar tissue in modified MS medium containing 2,4-D. Globular
somatic embryos were obtained on the same medium. Medium with B5
major, MS minor salts and 400 mg/1 glutamine were best for development
and maturation of somatic embryos.

Other fruits
There are also few reports of plantlet regeneration vta somatic
embryogenesis in other important fruit trees like apple (Mehra and
Sachdeva 1984), pear (Mehra and Jaidka 1985) and sapota (Sachdeva and
Mehra 1986). Mehra and Sachdeva (1984) raised callus cultures from all
parts of in vitro grown seedling tissue of apple, including the inflorescence.
Nitsch basal medium with NAA (2 mg/1) and BA (2 mg/1) was found to be
most suitable for the purpose. Coconut water (CW) was found to be
inessential. Mehra and Jaidka (1985) also successfully induced
embryogenesis from different seedling explants of Pyrus conmmunis var.
sativa, with Nitsch basal medium supplemented with NAA (2 mg/1) and
BA (2 mg/1). Sachdeva and Mehra (1986) obtained callus from root,
hypocotyl, stem and shoot tips of in vitro germinated seedling, on Nitsch's
medium supplemented with coconut water (15%), NAA (4 mg/1) and
kinetin (2 mg/1). Callus was also obtained from leaf, cotyledon, embryo
and endosperm explants on Nitsch's medium supplemented with coconut
water (15%), 2,4-D (4 mg/1) and kinetin (2 mg/1). The calluses from these
two media, when transferred to medium with BA (2-4 mg/1) instead of
202
kinetin, became nodular and differentiated early stages of embryoids in
great numbers. Development of embryos through globoid and heart shaped
stages was observed. The embryogenic calli were transferred to White's
basal medium containing NAA (2mg/l) and BA (2mg/l), where the
embryoids readily formed roots, albino shoots and plantlets.
In papaya (Carica papaya L. var. Honey Dew), protocol for
somatic embryogenesis is being worked out (Bhattacharya et al. 1999).
Somatic embryos were formed from immature zygotic embryos on
modified MS medium with 2,4-D (1-15 mg/1). Sixty percent of all the
zygotic embryos inoculated on lower levels of 2,4-D produced globular
somatic embryos on apical domes, cotyledons and root meristems of the
explants within 4-5 weeks of incubation. For maturation and germination
of the embryos, MS medium devoid of any growth regulators
supplemented with 3% sucrose was found to be suitable.

1.3. Cashewnut (Anacardium occidentale L.) - case study

1.3.1 INTRODUCTION

Cashewnut (Anacardium occidentale L. ), a native of Brazil, has

now completely acclimatized to the coastal regions ofthe Indian peninsula.
This important tropical fruit crop is valued for its delicious and nutritive
kernels. The oil from the shell (CNSL-cashewnut shell liquid) finds use in
the paint and varnish industry, brake linings, resins and laminations
(Kamath 1956). During 1997-98, 76,323 tonnes of cashew kernels and
4181 tonnes of CNSL were exported from India, fetching the country a
foreign exchange equivalent to USD 374 million. (Nayar 1999).
To improve the quality and yield, breeding programs have been
successfully i~plemented leading to commercial release of high-yielding
elite varieties (Bhaskara Rao et al. 1998). Although the propagation of
these chosen varieties is presently being carried out by conventional
grafting methods, it is inadequate in meeting the existing demand for
planting material (Nambiar et al. 1990). This existing propagation method
can be effectively supplemented by clonal propagation employing in vitro
techniques.
Regeneration protocols using organogenesis have been reported
for cashew (D'Silva and D'Souza 1992, Das et al. 1996, Hegde et al.
1991, Philip 1984, Thimmapaiah and Samuel 1999). In attempts to devise
a complete regeneration pathway using somatic embryogenesis, limited
 203
success has been achieved using immature embryos (Jha 1988), immature
cotyledon (Hegde et al. 1994) and seedling tissue (Lakshmi Sita 1989).
Recently, Ananthakrishnan and co-workers (1999) have reported somatic
embryogenesis from nucellus using a two-phase (solid and liquid) culture
method.
We have used nucellus explant (Gogte and Nadgauda 1999) from
immature fruits for the induction of somatic embryos, as described in
section 1.3.2. (this chapter).

1.3.2. EXPLANT

Source
Immature nuts of A. occidentale L. var Vengurla-1 were obtained from
Konkan Krishi. Vidyapeeth, Dapoli, Maharashtra. Collections of immature
nuts were made from open pollinated field-grown trees through February-
March from 2nd till 1Oth week post- fertilization.

Surface sterilization
Immature nuts were washed thoroughly under running tap water for 10-15
minutes, followed by laboratory detergent wash (1% Labolene®,
Qualigens, India) for 20 minutes. Nuts were then surface sterilized with
0.1% HgCh for 20 minutes and finally, rinsed 3-4 times with sterile
distilled water in laminar airflow.

Culture conditions
The nuts were dissected under sterile conditions in laminar airflow, the
ovules were removed and cut in halves. The ovule halves containing the
nucellus were placed on semisolid MS (Murashige and Skoog 1962) basal
medium supplemented with 3% sucrose, 0.5% activated charcoal and
different plant growth regulators. The ovule explants were placed in
disposable petri-plates. The pH of medium was adjusted to 6.0 prior to
addition of 0.5% agar. [The addition of activated charcoal to medium
results in increase in the pH after autoclaving (George E. F. 1993). A
higher pH was found to be more suitable for growth of nucellar tissue]. All
cultures were incubated at 25 ± 2°C in the dark.
204
1.3.3 INITITIATION OF EMBRYOGENIC CALLUS FROM
NUCELLUS

Initiation
In the preliminary experiments where different growth regulators were
tested for callus initiation, the number of nucellar explants showing
proliferating calluses was substantially more on media having both 2,4-
dicholorophenoxy acetic acid (2,4-D) and gibberellic acid (GA3). Medium
with 2,4-D (5J.!M) + GA3 (20J.!M) had the highest number of explants
(57.57%) with proliferating calluses. Though the number of explants with
proliferating qllluses was lower (46.18%), the callus initiated on medium
with 2,4-D (5~-tM) + GA3 (15~-tM) + BA (51-tM) gave rise to somatic
embryos on further subculture. This medium was therefore routinely used
as the initiation medium in our further experiments (Gogte and Nadgauda
1999, communicated). A yellowish callus (Fig. 1a) was obtained on the
initiation medium after 3 weeks. It was observed that nuts collected at 3-4
weeks post-fertilization showed the best response with respect to callus
induction.

Maintenance
This yellowish callus was maintained on the medium with 2,4-D (10~-tM) +
GA3 (15~-tM) and supplemented with 10% (v/v) coconut water (CW) and
0.05% casein hydrolysate (CH). The cultures turned black completely on
the maintenance medium within two weeks. Blackening occurred despite
addition of activated charcoal and transfer to fresh medium after every 4
weeks. Along with blackening, growth of the callus slowed down. The
completely blackened primary callus gave rise to a white granular mass
after 9 weeks of maintenance (Fig. 1b).

Induction of somatic embryos

The white granular mass was transferred to the induction medium. The
medium contained 2,4-D (SJlM) + GA3 (30~-tM), 10% (v/v) coconut water
and 0.05% casein hydrolysate. After 7-8 weeks, globular somatic embryos
appeared from the surface of the white granular mass. These somatic
embryos were then transferred to MS basal medium devoid of any growth
regulators and supplemented with 3% sucrose and 0.5% activated
charcoal. Further development of the globular somatic embryos through
heart (Fig. 1c), torpedo and cotyledonary stage occurred on this medium.
 205
Figure 1d shows a clump of somatic embryos obtained from the nucellar
tissue. The total period taken from initiation of callus to the development
of globular somatic embryos was 19-21 weeks.

1.3.4 DEVELOPMENT AND MATURATION OF SOMATIC

EMBRYOS

Few somatic embryos showed normal development from globular to heart,

torpedo, and early cotyledonary stages. Developmental abnormalities
including, fused embryos absence of cotyledon formation, fused
cotyledons, multiple cotyledons and asymmetric development of
"cotyledons were also observed. Secondary somatic embryos arose from
the root pole tip of some primary somatic embryos. These secondary
embryos mostly were in clusters and got easily detached from the primary
somatic embryo after the torpedo stage.
Effect of ABA ( abscisic acid), PEG (polyethylene glycol), maltose,
sucrose, gellan gum and agar was tested for maturation of somatic
embryos. Formation of roots from somatic embryos was observed on
medium containing 6% maltose. Embryos reverted to callus phase in
presence of ABA and PEG (unpublished data).

1.3.4 PROBLEMS AND POSSffiiLITIES

The sequential events during formation of a fully mature zygotic embryo

are very complex. The development of cotyledons and accumulation of
storage products plays a very important role in physiological maturation of
the embryo in vivo, particularly in species with large seeds. Lack of
complete understanding of these processes underlying maturation,
therefore, poses difficulties in replicating normal development of somatic
embryos in vitro (Wang and Janick 1984).
This fact needs to be considered in cashewnut (cotyledons form the
essential bulk of the mature zygotic embryo) in designing future
experiments for the maturation and conversion of somatic embryos.
Once a complete protocol for somatic embryogenesis is
established, it can be further exploited for:
(i) Mass propagation of elite varieties.
(ii) Cryopreservation of the embryogenic calluses and its use m
protoplast isolation and fusion.
206
(iii) Genetic manipulations for better quality kernels, disease resistance
and higher yields.
(iv) Somaclonal variants can be isolated and screened for desirable
traits.

1.4. Conclusions and Prospects

Although, work on somatic embryogenesis has been initiated and

regeneration protocols are available for a number of fruit tree species in
India, a convincing protocol leading to large scale field trails is still
lacking. There are still a number of obstacles such as abnormal somatic
embryos, low frequency of conversion and low survival rates of somatic
emblings in the field in obtaining normal true-to-type clones using somatic
embryogenesis. These must be overcome before anyone delves into
developing commercially competitive large-scale propagation protocol and
variety improvement programs for fruit trees. Combined efforts of various
groups working within India and collaborations with international groups
would definitely hasten the process of understanding, leading to
elimination of problems and tapping the full potential of this very
promising technology.
It can thus be concluded that, the overall status of somatic
embryogenesis research for fruit trees in India is in its developing stage and
is progressing in the right direction. The number of success stories, though
handful, are surely convincing enough to be able to reflect the tremendous
potential and usefulness of the technique to the horticulture industry in the
near future.

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 Table 1: Somatic embryogenesis in fruit trees - Indian scenario
S.No. Species Explant Medium Reference
Embryogenesis Maturation/Conversion
I. Achras sapata SG I. Nitsch+ NAA (4) +KIN (2) + CW {IS%) for R, White's medium + NAA(2mg/L) + BA(2mg/L) Sachdeva and Mehra, 1986
ST, SH, HY (callus)
2. Nitsch+ 2,40 (4) +KIN (2) + CW (IS%) for LE,
CO, EM (callus)
3. Nitsch+ BA (2-4) (embryoids)
2. Anacardium IZ MS +2,40{4)+ NAA (4)+ KIN (2) + PVP (250) Jha, 1988
Occidentale L.
A. occidentale L. NU I. MS+2,4-0 (5) + GAJ {15) + BA (5) Gogte and Nadgauda, 1999
2. MS + 2,4-0 (10)+ GA, {15) + CH (0.05%) + (communicated)
CW{IO%)
3. MS + 2,4- 0 (5)+ GAJ (30)
A. occidentale L. NU I. MS + 2,4-D (6.78) Ananthakrishnan et a/. ( 1999)
2. MS + 2,4-D (4.52) liquid
3. Citrus aurentifolia Stem MS+BA Bhansali and Arya, 1978a
segment

C. aurentifolia Anther MS + BA (0.5 mg/L) + IAA(Img/L) MS medium (without auxins) Chaturvcdi and Sharma, 1985

C. aurentifolia FL MS-A + KIN (0.5mgll) + IAA (0.25mgll) MS-A + IAA (0.5mgll) + GA3 ( IOmgll) + Mitra and Chaturvedi, 1972
and C. sinensis KIN (0.25-5mgll)

C. grandis Stem callus MS +Zea+NAA Chaturvedi and Mitra. 1975

C. reticula/a IZ, seedling MS +NAA(IOmg/L)+ Kin (0.5-1 mg/L) Gilleta/.,1991,1994
explants (L,
ECO,COT,
R)
4. Cocos nucifera LE Y3 + 2,4-0 (226) Gupla et a/., 1984
C. nucifera IZ I. 85 + IAA-asp (2mgll) + IAA-ala (2mgll) BS + NAA (0.5mgll) + BA (2mgll) + Bhalla-Sarin eta/., 1986
2. BS + IAA-asp (2mgll) + KIN (2mgll) PVP (I gil)
5. Malusspp. SG,FL White's + NAA (4) + KIN (2) + GA, (I)+ CW (15%) Nitsch + NAA (2) + BA (2) Mehra and Sachdeva, 1984

N
_.
_.
 Table 1 (continued) N
.......
N
S.No. Species Explant Medium Reference
Embryogenesis Maturation/Conversion
6. Mangifera indica var NU MS + 2,4·0 (22.62 )+ GAJ (14.43) I. MS +ABA (3.78) + CW (20"/o) + Jana el a/., 1994
Alphonso CH(0.1g/L)
2. MS + BA (22.2)
7. Musaspp. MaleFL MS + BA (0.22) + IAA (1.14) MS (112) +ME (0.5g/L) + AC (0.1%) Ganapathi el al., 1999
8. Phoenix dactyli/era Linn. R,LE, MS (modified)+ 2,40 (100) + BA (5) + AC (0.3%) Sharma el al., 1984
cv. Khadravi SH,BU
P. dactyl!fera SHTips I. MMS-1+NAA(1)+2,4-0 (20.5)+BA(0.1)+ Oass el a/., 1989
PVP (2g/l)
2. MMS-2 + NOA (3) + NAA (5) +IAA (I)+ KIN
(0.1) + BA (5) + 2,4·0 (0.1) + PVP (2g/l)
3. MMS-3 + KIN (2) + BA (5) + NAA (0.1-1 00)

9. Prunus persica co I. MS (modified)+ 7,40 (5) + BA (2) + MS+BA(2) Rajbhansali, 1990

KIN (2) + CH (500)
2. MS+AC
10 Psidium guajava MS +Sue (5%) + 2,4-0(0.1-2 mg/L) MS+BA(2) Akhtar and Jaiswal, 1995.

II. Punica granatum L. P,CO MS+IAA(1)+IBA(I) MS (112) +Sue (4%) Natarajael a/., 1996

P. granatum L P,CO I. MS (modified) + 2,4-D (5) + BA (2) + MS+BA(2) Rajbhansali, 1990

KIN (2) + CH (500)
2. MS+AC
P. granatum L. SG I. MS + NAA(4) + Kin(2) + CW(5% v/v) Jaidka and Mehra, 1986
2. MS + NAA(2) + BA(2)

12. Pyrus communis var. saliva SG,FL White's + NAA (4) +KIN (2) Nitsch+ NAA (2) + BA (2) Mehra and Jaidka, 1985
L_ _ _ _ _ +GA(l)+CWt1S%)
-

• All concentrations occurring in the various media fonnulations are in j1M, unless specified.

2,4-D- 2,4 dichlorophenoxyacetic acid, ABA- Abscisic acid, AC- activated charcoal, 85- Gamborg el a/. (1968), BA- benzy1adenine, BU- bud, CO- cotyledon, CW-
coconut water, EM- zygotic embryo, ECO- epicotyl, FL- flower, GA3- Gibberellic acid, HY- hypocotyl, lAA- Indole acetic acid, JBA- Indole butyric acid, JZ- immature
zygotic embryo, KIN- Kinetin, LE-leaf, ME- malt extract, MS- Murashige and Skoog (1962), MMS- Dassel a/. (1989), NAA-naphthalenc acetic acid, NOA-naphthoxy
acetic acid, NU- nucellus, PVP- po1yviny1pyrollidone, RT- root, SG- seedling, SH- shoot, Sue- sucrose, Y3- Eeuwens (1978), Zea ·zeatin
 213

Figure 1
7. Application of Somatic Embryogenesis for the improvement of Tropical Fruit
Trees

1NasimAkhtar and 2Shri. M Jain

1IIT-BREF Biotek, Indian Institute o[Technology, KHARAGPUR- 721 302 (WB), India. Phone: (091) 03222-
77028, 77890; Fax: (091) 03222- 77890; E-mail: E"or! Bookmark not thfrnetl 2Plant Breeding and
Genetics Section, FAOIJAEA Joint Division, International Atomic Energy Agency (1AEA), Wagramer Strasse
5, PO Box 100, A-1400, Vienna, Austria; Phone: 43 1 2600 21623; FAX: 43 1 26007; E-mail:
SMJain@iaea.org

Contents:

1. Introduction.
2. Somatic Embryogenesis, Scale-up Micropropagation and Mass Production in
Bioreactors.
3. Somatic Embryogenesis and Synthetic Seed Technology.
4. Somatic Embryogenesis and Genetic Transformation.
5. Somatic Embryogenesis and Protoplast Technology.
6. Somatic Embryogenesis and CryopreseJVation.
7. Somatic Embryogenesis and Selection of Somaclonal Variants.
8. Future Prospects and Concluding Remarks.
9. Acknowledgement.
10. References.

1. Introduction:

The improvement of tropical fruit trees through conventional breeding techniques has
been limited due to inherent problems such as long life cycle with extended juvenile
period, floral morphology, existing hybridization barrier, sterility, apomixis and long
term inbreeding depression. Sufficient ammount of work have been done on
regeneration of tropical fruit trees through somatic embryogenesis particularly in
banana, citrus, mango, papaya etc. (see Akhtar et a/. in this volume). Somatic
embryogenesis has several distinct advantages over the conventional micropropagation
(Litz and Gray, 1992). Application of somatic embryogenesis in fruit tree improvement
has been limited because of poor germination of somatic embryos and low frequency of
recovery of somatic seedlings. However, as a system for large-scale production in
bioreactors through repetitive induction, somatic embryogenesis has attracted the
attention of fruit breeders and pomologists for the improvement of fruit trees. The
215
S.M. Jain. P.K. Gupta and R.J. Newton (eds.). Somatic Embryogenesis in Woody Plants. Volume 6. 215-247.
© 2000 Kluwer Academic Publishers.
216
desirable traits for fruit tree improvement include delayed ripening, pest and disease
resistance, drought and cold tolerance, fruit quality, rootability of mature tissues,
reduced juvenility, regular bearing habit and alteration of tree forms and architecture
e.g. dwarfing and semidwarfing capacity. Strategies that have been developed for
improving existing fruit tree cultivars have been based upon the assumption that it is
possible to target cells having embryogenic competence and to subtly alter the genotype
to affect one or more specific horticultural traits (Litz eta/., 1997). The technique that
heve been utilized involved recurrent selection for recovering useful somaclonal
variation (somatic mutation) that occur in embryogenic cultures and genetic
transformation of embryogenic cells or protoplasts for a specific character. The recent
threat to the wild cultivars of some fruit species e.g. mango and its Mangifera spp.
relatives has been noted by Mukheijee (1985). The maintenance of in situ and ex situ
germplasm repositories requires a major long-term commitment by national and
international agencies. Ideally, germplasm would be stored more efficiently as
embryogenic calli, somatic embryos or as artificial seed. This would further
complemented by the regeneration of cryopreserved embryogenic calli, somatic
embryos or the artificial seeds at large-scale in bioreactors. In recent years, some
studies have been initiated in order to improve fruit trees through the use of somatic
embryogenesis (Bajaj, 1995e; Merkle eta/., 1990).

2. Somatic Embryogenesis, Scale-up Micropropagation and Mass Production in

Bioreactors:

Conventional micropropagation is a time consuming and labour intensive process.

Somatic embryogenesis has powerful advantages for mass multiplication in
comparision to both conventional clonal propagation (e.g. root cutting, grafting etc.)
and other regeneration systems (e.g. organogenesis). Micropropagation through
somatic embryogenesis offers not only a means for mass multiplication for biomass
energy production, but also provides the basis for the conservation of important elite
and rare plants that are threatened with extinction. The morphological and
physiological similarities of somatic to zygotic embryos indicate that they are almost
complete propagules with embryonic root, shoot and leaves (atleast cotyledons) and
most importantly the 'program' to make a complete plant. The high volume
multiplication of embryonic propagules is the most commercially attractive application
of in vitro somatic embryogenesis (Denchev eta/., 1992).
A theoretical model for the large scale production of somatic embryos in vitro
and their handling by various methods is presented by Sharp eta/. (1984). During the
last decade, there was increased interest in problems affecting scale-up operation
(Bajaj, 1991c). Some advances have been made towards bioreactor culture of somatic
embryos for mass propagation (Nishimura et a/., 1993) and towards environmental
control and automation with use of computer and robotic in plant tissue culture
(Aitken, et a/., 1995). The application of bioreactor technology, which was originally
developed for microbial fermentation using a stirred-tank, had apparently not met its
 217
potential to produce hundreds and thousands of clonal embryos capable of growing into
plants. The first report of large scale embryogenic cultures described attempts to grow
carrot cells in a 20 litre carboy which resulted in the formation of a few embryos
(Back-Husemann and Reinert, 1970). To increase the efficiency of production, various
improvement has been made in the biorector technology, with the possibility of using
computer, robots, and various other mechanized systems are being developed (Aitken,
et a/., 1995; Bajaj, 1991c; Smith, 1995). The use of bioreactor technology for mass
mutiplication of somatic embryos in tropical fruit trees has not yet been developed. In
oil palm (E/aeis guineensis Jacq.), scale-up of in vitro clonal propagation systems
through somatic embryogenesis has been described only recently by Rival eta/. (1997).
Various forms and modifications of culture vessels heve been designed and
used for biorectoer purpose. The culture vessels used to establish a scale-up system for
somatic embryogenesis in carrot were a commercially available spinner flask, an
specially designed airlift culture vessel and a screen column culture vessel with a
working volume of0.5, 1.2 and 2.0 L, respectively (Teng eta/., 1994). A batch culture
system in a stirred 10 L. culture vessel was used for somatic embryo production in
carrot (Ducos eta/., 1993). Somatic embryogenesis was carried out in similar types of
airlift vessels and magnetically stirred, hanging stirrer bars, and culture vessel of 1.8 L
in Capsicum annum (Mavituna and Buyukalaca, 1996) and Santalum album (Surajit et
a/., 1998). A hellical ribbon impellor bioreactor was used for embryogenic cultures of
transformed Eschscholtzia california cell lines EC12 (Archambault et a/., 1994).
Moorhouse eta/. (1996) designed a new culture vessel, a novel aeration and agitation
system to enhance gaseous exchange and reduce the shear stress on submerged cell
suspension cultures. Attree eta/. (1994) successfully produced somatic embryos from
suspension in a culture vessel on a flat adsorbent pad above the surface of a liquid
culture medium. Recently, an algorithm system of image analysis for automated
machine vision of maturing somatic embryos, automatic sorting, propagule counting,
bioreactor-feedback and production control for somatic embryogenesis has been
developed (Grantd' Esnon et al., 1989, Smith., 1995).
The effect of medium pH on carrot (Daucus carota) somatic embryos was
examined in a controlled 3 L culture vessels with MS medium and 11. 7 J.iM adenine
(Jay et a/., 1992). Maximum somatic embryo production occurred at pH 4.3 in
hormone-free condition, but embryos were unable to germinate. In contrast, there was
a three-fold decrease in production at pH 5.8, but embryos were normal and converted
into plantlets (Jay eta/., 1994). Teng eta/. (1994), studied the cell density and noted a
decrease in dissolved oxygen concentration to 33% during the initial phase which
increased to 80% during maturation and germination of carrot somatic embryos.
Embryos grown in culture vessels germinated with short cotyledons and long roots
whereas, flask-cultured embryos germinated with long cotyledons and short roots.
Recently, the effect of initial cell density, pH and dissolved oxygen on production of
carrot somatic embryos were investigated using a cell suspension culture induced from
hypocotyl segments in 0.2% gellan gum modified MS medium with 0.5 mg/12,4-D and
0.3% sucrose (Shigeta eta/., 1996). The best embryo production (140 torpedo-shaped
embryo/ml medium) was achieved with a medium at 5.2 pH, and one m1 packed cell
218
volume/L density after 2 weeks in culture. The dissolved oxygen played an important
part in the early phase of embryo development. At least 80% dissolved oxygen
concentration of saturated air was required for the development of globular and heart-
shaped embryos during the initial first week of culture, whereas the subsequent
development of torpedo shaped embryos required low concentration of dissolved
oxygen. The production level of 50,000 embryos/L per day was achieved with an
inoculum density of 0.1 % volume of tissues/volume of medium using a batch culture
in a stirred 10 lit. culture vessel system in carrot (Ducos et a/., 1993). Regular
changing of medium increased the viability of embryos. A filtering unit coupled with a
culture vessel yielded about 95% single embryos free of undifferentiated tissues.
Similarly, somatic embryos of sweet potato (Ipomoea batatas Lam. cv. 'White
Star') were produced in airlift culture vessels. The age of the embryogenic callus
influenced the number and morphology of the somatic embryo population in semisolid
and liquid medium. Maximum number of mature somatic embryos (35 embryos/lOg
inoculum) was obtained from 6 week old callus at 30 °C. Globular embryos formed and
continued developing in liquid suspension with 20mM NH.N03 and 30mM KCl, and
produced viable mature cotyledonary embryos by day 14 (Bieniek eta/., 1995). They
further noticed limited embryo production due to changes in gaseous composition of
culture vessel. The ethylene production enhanced to 6.4 ppm (day 16), maximum C02
(21.2%) on day 16 and oxygen depleted to a minimum of 8.1% by day 14. In bell
pepper (Capsicum annum var. Ace), proliferation was carried out with 1-1.2 g of
embryogenic callus in 50 ml MS medium (Murashige and Skoog, 1962) with 1.0 mg/1
2,4-D in conical flasks followed by re-suspension for two weeks in large culture
vessels. After embryo formation, half strength MS medium, with 3% sucrose and 0.5
mg/1 cis-trans abscisic acid, was used for embryo maturation. Under the optimum
conditions, they reported 98% germination with 57 embryos/mi. In transformed
Eschscholtzia california cell lines EC12, the best production of somatic embryos
(44/ml or 757/g dry wt. per day) was achieved at 60 rpm with dissolved oxygen at 20%
by surface oxy,genation alone (0.05 VVM kLa 1.4/hr) using hellical ribbon impellor
bioreactor (Archambault eta/., 1994). They further noticed that production of somatic
embryos was limited by availability of phosphate, magnesium, nitrogen salts and
carbohydrates. The negative effects on somatic embryo production were attributed to
the shear experienced by the embryogenic cells and/or embryo forming aggregates.
In sitka spruce [Picea stichensis (Bong.) Carr.], the culture vessel was
inoculated with a suspension culture. The profusion capacity of the culture vessel was
used to replace the initial proliferation medium with maturation medium in order to
induce the development of somatic embryos in submerged cell culture (Moorhouse et
al., 1996). They monitored size ratio of the somatic embryos heads and observed
similar growth during initial elongation to that of shaken flask cultures. In white
spruce, Attree eta/. (1994) recovered over 63% of cotyledonary stage somatic embryos
from suspension in a culture vessel on a flat adsorbent pad above the surface of a liquid
culture medium containing 20-50 jJM abscisic acid and 7.5% PEG. These embryos
survived well at 8% moisture content following treatments for 4 week at 63% relative
humidity. Following imbibition, embryos converted to plantlets at 92% frequency
 219
compared to 80% for embryos grown in petri-dishes. Tautorus et al. (1992; 1994)
worked out the growth pattern and studied the nutrient utilization during. bioreactor
culture and maturation of somatic embryos in black spruce and interior spruce using an
airlift culture vessel containing LP (von Arnold and Eriksson, 1981) medium with
60mM sucrose. A maximum of 7.1 gil dry wt. and 2,892 somatic embryos/ml in black
spruce and 5.9 gil dry wt. and 2,698 somatic embryos/ml of interior spruce were
obtained after 15 and 21-30 days, respectively. They further noted variation in
metabolic properties of the two species. Interior spruce metabolize fructose while, black
spruce metabolize glucose (Tautorus et a/., 1994). Very recently, Timmis (1998)
discussed some of the problems asssociated with conifer somatic embryogenesis and its
bioreactor production for the utilization of trees and their products in the forest
industry.
Hence, the success of bioreactor technology with other species, may greatly
facilitate the acceptance of somatic embryogenesis as the process of mass
multiplication on a commercial scale for fruit trees in near future.

3. Somatic Embryogenesis and Synthetic Seed Technology:

The successful demonstration of regeneration through somatic embryogenesis in some

tropical fruit trees (Akhtar eta/. in this volume), opened the scope for the utilization of
somatic embryos to direct sowing and an easy delivery system to seed beds as artificial
seeds. The regeneration of many tropical fruit tree species through somatic embryos
and the subsequent acclimatization of plants and transplantation to the field are the
major bottlenecks for the commercial acceptance of the technology. Somatic embryos
produced in vitro lack a seed coat and tend to either germinate immediately (if mature)
under favourable conditions, or become quiescent if dehydrated (see Akhtar et a/. in
this volume; Litz and Gray, 1992). In contrast, zygotic embryos inside the seed are
protected by seed coat and show a high degree of tolerance to desiccation (Senaratna et
al., 1990, 1995; Teng and Hor, 1977). Therefore, a system is required to induce
somatic embryos to behave like zygotic embryos, and thus, can be handled with
minimum of damage and be delivered to seed bed. Synthetic or the artificial seed is a
generic term assigned to somatic embryos and other similar clonal propagules
encapsulated with different coating gels to facilitate storage, delivery and
transportation. The concept was first put forward by Murashige (1978). Synthetic seed
technology, using somatic embryos, assumes great significance in crops where i) seeds
are not produced, ii) seeds are produced in small quantity, iii) zygotic seeds are short
lived, and iv) vegetative propagation is a problem and germplasm can not be
conserved. The major thrust of synthetic seed technology is on the direct sowing of
somatic embryos to the field. Several handling systems have been proposed to facilitate
the delivery of somatic embryos from in vitro condition to the field or the greenhouse
(Redenbaugh, 1993). These delivery systems include:
i. Encapsulation of somatic embryos in an alginate gel capsule (Gray et a/., 1995;
Jeon eta/., 1986; Redenbaugh, 1990; Redenbaugh eta/., 1984, 1986).
220
ii. Gel seeding of somatic embryos with fluid drilling equipments (Baker, 1985;
Cantliffe et a/., 1987; Drew, 1979; Kitto et a/., 1991, 1995; Schultheis et a/.,
1986a, b, 1990).
iii. Desiccation of somatic embryos encapsulated into water soluble resins (Janick et
a/., 1989, 1993; Kitto and Janick, 1985a, b).
iv. Desiccation and then planting dried somatic embryos with conventional drill (Gray,
1989; Gray eta/., 1987).
There are many factors, which contribute to the successful use of synthetic
seed methodology. The foremost factor is the establishment of somatic embryogenesis
and high frequency conversion of somatic embryos into plants. The production of large
number of somatic embryos in bioreactors and mechanized germination of
encapsulated somatic embryos under in vitro condition complete the protocol of
synthetic seed preparation on a commercial scale. Successful culturing of embryogenic
protoplasts in alginate beads (Neidz, 1993) has opened a new aspect of synthetic seed
technology. The ability to add nutrients, biofertilizer, antibiotics or other additives to
the matrix, and the easy handling, sorting, shipping and planting are major attractions
to employ synthetic seed technology as a unit of delivery.

3.1. ENCAPSULATION OF SOMATIC EMBRYOS:

The most widely used method for producing synthetic seeds is the hydrated
encapsulation of somatic embryos with sodium alginate (Bajaj, 1995c; Jeon et a/.,
1986; Redenbaugh et al., 1993). The gel matrix can be manipulated with nutrients,
plant growth regulators and cabohydrates to enhance the artificial endosperm for better
growth and survival of embryos (Gray, 1990; Gray and Purohit, 1991; Redenbaugh and
Walker, 1990). A number of plant species are reported to regenerate plants via
synthetic seed with very low frequency. In alfalfa, about 60% plant formation under in
vitro condition and 20% when grown in the greenhouse was reported from gel coated
synthetic seeds (Fujii et al., 1987; Senaratna et al., 1990), a slightly better recovery
frequency was 65% with celery somatic embryos (Janick et al., 1993). Cho (1990)
produced carrot synthetic seeds using alginate encapsulation followed by dehydration
to 80% water loss, and subsequent germination frequency was 90%. A frequency of
95% recovery on polyester fiber support in liquid MS medium is reported by Shigeta et
al. (1990). The cotyledonary stage somatic embryos (3-5mm long) from nucellar calli
of Mangifera indica cv. 'Amrapali' encapsulated in 2% alginate (Fig. 1A), were
germinated with 73.61% frequency on 0.6% agar gelled medium with B5
macronutrients, MS micronutrients, 3% sucrose and 1.0 mg/1 GA3, however the
treatment of these somatic embryos with abscisic acid or its inclusion in the gel coating
inhibited the germination frequency (Ara, 1998). In guava, elongated and short
torpedo somatic embryos (8 week old), encapsulated in 2% alginate gel (Fig. 1B),
converted into 92.63 and 69.64% plantlets, respectively, on full strength MS basal
medium with 3% sucrose as compared to a 98.33 and 83.05% recovery from non-
encapsulated somatic embryos (Akhtar, 1997; Akhtar and Jaiswal, 1994). In
Asparagus cooperi, only 45% of naked lffid 34.3% of encapsulated somatic embryos
 221
were converted into plantlets on full strength MS medium (Ghosh and Sen, 1994).
Similarly, Lakshamana Rao and Singh (1991) reported about 91% and 49.7%
conversion of non-encapsulated and encapsulated somatic embryos of Solanum
melongena. In Santalum album, Bapat and Rao (1988, 1992) recovered only a small
frequency of plantlets from artificial seeds. Somatic embryos in other species like
Terminalia arjuna (Fig. lC), Sterculia alata and Dendrocalamus strictus, produced
with 2% alginate in our laboratory, showed high frequency of plantlet recovery.
Although encapsulated somatic embryos have successfully been converted into
plantlets in several species, there are problems with the use of alginate capsule. Water
soluble nutrients have been reported to rapidly leach out of the capsule (Redenbaugh et
a/., 1987). Respiration has been shown to be reduced in encapsulated seeds due to poor
gas exchange (Redenbaugh eta/., 1993; Redenbaugh and Walker, 1990). Several othe~
hydrated gels have been proposed to over come some of these problems (Redenbaugh et
a/.,_1993). For tomato seeds, a molding system proposed where sodium alginate (1.5%)
222

Ficure 1: Synthetic seeds of tropical fruit and tree species. A. Synthetic seeds of mango encapsulating mature
cotyledonary stage somatic embryos (bar= I mm). B. A germinating synthetic seed of guava encapsulating 8-
week old elongated torpedo stage somatic embryos (bar= I mm). C. Germinating synthetic seeds of Terminalia
arjuna (bar=8.78 mm).
 223
was mixed in flat bottom microliter wells and injected with calcium chloride (50mM)
to complex the alginate. An automated molding process was also designed for large
scale encapsulation of seeds and somatic embryos (Redenbaugh eta/., 1993). Another
method for large scale production of synthetic seeds was described using a dual ramp
vibratory bowl and nozzle assembly which mixed embryos with sodium alginate just
prior to deliver into a calcium chloride bath (Redenbaugh eta/., 1991). However, the
principal limitation of commercialization of synthetic seeds is the quality and vigor of
somatic embryos. Very recently, a method for the encapsulation of high quality papaya
(Carica papaya L.) somatic embryos, produced in highly productive liquid system, was
developed (Castillo et a/., 1998). Callus was induced from embryos on semisolid
medium with 6% sucrose. Embryogenic calli were transferred to liquid medium and
under light conditions. Further, proliferation was achieved with 0.5 j.1M ABA. Three
encapsulation methods were investigated to test the effects of sodium alginate
concentration, presence or absence of medium in gel matrix, and duration of CaC12
exposure. Highest frequency of germination was obtained using 2.5% sodium alginate,
half-strength MS medium in the gel matrix and submerging for 10 min in 50j.IM
CaC}z.

3.2. FLUID DRILLING:

The concept of fluid drilling, i.e. sowing of zygotic seeds in a protective flowable gel
was first conceived by Currah eta/. (1974), while Drew (1979) was the first to suggest
the idea of fluid drilling of somatic embryos. Fluid drilling is a complex process (Kitto
et a/., 1995) and somatic embryos must be fully developed or be developmentally
mature and capable of continued growth after fluid drilling (Gray and Purohit, 1991).
The nutrient formulation and carbohydrate source of the gel amendment in sweet
potato (Ipomea batata L.) influenced normal development of somatic embryos
(Schultheis and Cantliffe, 1992). Kitto eta/. (1991, 1995) carried out extensive studies
on fluid drilling of carrot somatic embryos. The composition and volume of the fluid
drilling gel, the role of light and chilling and the percent embryo conversion were
determined. Sucrose as carbohydrate source and the fungicides Chitosan and Truban
appeared to be beneficial for plantlet recovery (Kitto eta/., 1995).
224
3.3. DEHYDRATION OF SOMATIC EMBRYOS AND ENCAPSULATION INTO
wATER SOLUBLE RESINS:

The concept of using desiccated somatic embryos as synthetic seeds was first reported
by Jones (1974). However, Kitto and Janick (1985a, b) studied the use of several
materials on carrot somatic embryos, of which polyethylene oxide homopolymers
(Polyox) showed the most desirable characteristics of artificial coating to produce
synthetic seeds. Citrus seeds and somatic embryos have been used for screening various
compounds, and the survival of somatic embryos decreased rapidly during drying of
polyox coated seeds (Janick et a/., 1993). Hardening treatments with 12% sucrose,
chilling at 4 °C, high inoculum density and/or 1 J.I.M ABA prior to desiccation
increased the embryo survival compared to non-treated controls (Kitto and Janick,
1985b). Recently, ABA treated somatic embryos of carrot were successfully dried in an
alginate encapsulation and about 68% of germination of synthetic seeds by dehydration
up to 92% is reported after storage for 10 days (Liu et a/., 1992). Although, these
studies indicate the feasibility of desiccated embryos for synthetic seeds. However,
more extensive work is required on conversion of desiccated somatic embryos to be
produced as synthetic seeds.

3.4. DESICCATION AND PLANTING OF DRIED SOMATIC EMBRYOS:

Desiccation of somatic embryos prior to encapsulation with a coating material was

previously proposed by Gray (1987a, b, c) and Gray eta/. (1987). Somatic embryos of
orchardgrass became quiescent when dried from 75 to 13% water (Gray eta/., 1987).
Developmental stage of somatic embryos before desiccation was critical factor for
germination and plantlet formation. A number of other factors also influence the
maturation and germination of dehydrated somatic embryos (see section on maturation
and germination of somatic embryos in Akhtar eta/. in this volume). Hence, there are
many challenging problems that must be overcome in relation to somatic embryo
development, maturation, storage and conversion to seedlings under stressful condition
to be delivered as synthetic seeds. Despite the many problems that remain, the promise
of commercial propagation through the synthetic seed technology has potential for
future research.

4. Somatic Embryogenesis and Genetic Transformation:

Genetic transformation is a process whereby, a desirable foreign DNA is introduced

into plant cell through the use of some vector (Bajaj, 1989a, b, 1993a, b, 1994; Ellis,
1995). The plant transformation technology is grouped into three main categories: 1)
Use of biological vectors (viruses and Agrobacterium mediated transformation); 2)
 225
Direct DNA transfer (chemical, electrical, microlaser induced permeability of
protoplast or cells), and 3) Non-biological systems (microprojectile, microinjection or
liposome fusion) (Dandekar, 1992; Ellis, 1995; Oliveira et al. 1996). Although some
efforts have been made in woody plant transformation, the recovery of transformed
plants has been limited mainly due to the reliance on the efficient regeneration
protocols for fruit trees (Martin, 1987; Oliveira eta/. 1996; Schuerman and Dandekar,
1993). Several problems in regeneration of woody plants have recently been resolved
by authors working with apple, grapevine, papaya and cherry (Cabrera eta/., 1996;
Emershad and Ramming, 1994; Monmarson et al., 1995; Norelli and Aldwinckle,
1993; Yepes and Aldwinckle, 1994). For fruit trees the genetic transformation
protocols are either not available or are suitable only .for juvenile materials. Zygotic
embryos are being used for DNA uptake and raised into complete plants (Christou,
1992; Fitch eta/., 1990; Sato et al., 1993). Since, zygotic embryos are the results of
sexual fusion, different developmental stages differ in their competence to genetic
transformation, as well as in embryogenic induction. On the other hand, large number
of developmentally uniform somatic embryos are available for the use in place of
zygotic embryos. Furthermore, a single cell on the explant surface is the target of DNA
introduction, expression of transgenes and regeneration of non-chimeric plants through
somatic embryogenesis in comparison to organogenic differentiation.
Embryogenic cultures and embryos have responded favourably to
Agrobacterium (Cabrera eta/., 1996; Cheng et al., 1996; Dandekar, 1992; Fitch eta/.,
1993; Mathews eta/., 1992, 1993), microinjection (Neuhaus eta/., 1987) and particle
gun bombardment (Christou, 1992; Fitch eta/., 1990, 1992; Kobayashi and Uchimiya,
1989; Wilde et a/., 1992; Ye et a/., 1994, 1997), direct DNA transfer via
electroporation (Hidaka and Omura, 1993). Transformation was reported, for
Theobroma sp., Feijoa and Mangifera sp. (Table 1) but transformed plants could not
be obtained. On the other hand, there have been reports on regeneration of transgenic
plants from well developed somatic embryos or the embryogenic cell cultures (Table
1). The particle gun bombardment has proved efficient for transformation of Citrus,
papaya, and peach (Table 1). However, Agrobacterium vectors have been more
frequently used in fruit trees achieve a high frequency of stable transformation
compared to biolistics (Ellis, 1995; Oliveira et a/. 1996; Mehlenbacher, 1995).
Transgenic plants have also been recovered after direct DNA transfer to protoplasts of
Citrus and kiwifruit (Table 1). The direct DNA transfer through electroporation of
cells or protoplasts has actually been suggested as a way of improving plant
regeneration of fruit trees by Ochatt and Patat-Ochatt (1995). Several factors shown to
affect the Agrobacterium-media:ted transformation in Citrus (Gutierrez et a/., 1997)
and other species (Dandekar, 1992; Ellis 1995; Schuerman and Dandekar, 1993).
Genes such as those encoding proteins with insecticidal or bactericidal
properties from Bacillus thuringiensis have been introduced in walnut (Dandekar et
al., 1994, 1998). A gene encoding herbicide resistance has been introduced in
tamarillo (Atkinson and Gardner, 1993). Papaya and Citrus have been transformed
with the capsid protein gene from papaya ring spot virus (PRV) (Fitch eta/., 1993) and
Citrus tristeza virus (CTV) (Gutierrez et a/., 1997) respectively. Fungal and viral
226
disease resistance have also been reported in transgenic plants regenerated through
somatic embryogenesis from single cell transformed with Agrobacterium in Musa spp.
(Vuylsteke eta/., 1998). In papaya, Gonsalves eta/. (1998) have reported some 207
kanamycin resistant embryogenic clusters from nine bombarded plates over a period of
7 month. Some 83 of these clusters produces transgenic plants of which 25 were
resistant to the homologous PRSV isolates from Hawaii and some of these were also
resistant to PRSV isolates outside Hawaii. Very recently the same group of worker
have developed a protocol for efficient transformation and regeneration of Carica
papaya L. (Cai et a/., 1999). Cruz-Hernandez et a/. (1997) have genetically
transformed embryogenic mango cultures with an engineered disarmed strains of
Agrobaterium tumifaciens (LBA 4404) containing each of the following genes in the
antisense configuration in binary vector pBI121: ACC oxidase from A. thaliana and
ACC synthase and alternate oxidase from mango. Among other genes that have been
integrated and expressed in fruit trees are the ro/ genes to improve rooting ability in
kiwifruit (Rugini eta/., 1991) (Table 1). With the success in regeneration of transgenic
plants in other crop species with some useful genes (Bajaj, 1989a, b, 1993a, b, 1994;
Oliveira et a/., 1996), the use of somatic embryogenesis to transform fruit trees for
other important traits like delayed ripening, tree architecture (dwarfing and
semidwafing character), regular bearing habit, insects, pests and disease resistance
could create research opportunities for the future.

5. Somatic Embryogenesis and Protoplast Technology:

Some aspects of basic biotechnology such as genetic transformation, cell wall

biosynthesis, macromolecule uptake, organelle isolation, membrane physiology and
somatic cell genetics, rely on protoplast isolation and plant regeneration. Protoplast
fusion and somatic hybridization technology facilitates gene flow between species
through bypassing the reproductive isolation barrier. Since the early work of Cocking
(1960), protoplasts have been isolated routinely through enzymatic digestion of plant
tissues. Protoplast-to-plant regeneration protocols have been developed for large
number of crops (Bajaj, 1989a, b, 1993a, b, 1994; Ochatt and Power, 1990).
The woody species, particularly fruit trees were considered difficult to
regenerate from protoplasts. In tropical fruit trees, protoplast-to-plant regeneration was
first accomplished with Citrus species (Vardi eta/., 1975). Historically, examples of
protoplast to plant regeneration in Citrus were limited to genotypes where friable
embryogenic callus was available (Ashi eta/., 1985; Grosser and Gmitter, 1990a, b, c;
Kobayashi eta/., 1983; Vardi eta/., 1982). Ovule derived nucellar callus of Citrus was
the most appropriate source for protoplast isolation (Vardi and Galun, 1988). Isolation
of viable protoplasts from leaf tissue (Grosser and Chandler, 1987) and fruit juice
vesicles (Echeverria, 1987) was possible, but conversion to plants was not reported.
Citrus plant regeneration directly from leaf protoplasts has been reported for several
genotypes (Ohgawara eta/., 1989, 1991; Tusa eta/., 1990). Culturing of embryogenic
protoplasts in alginate beads was also reported for 'Halmin' sweet orange (Neidz,
1993). Fusion of seedling leaf protoplasts of sour orange (Citrus aurantinum L.) with
 227
embryogenic callus of cleopatra mandarin (C. reticulata Blanco) has been reported to
produce 394 hybrid plants (Grosser, 1994). Protoplasts from embryogenic calli of
Citrus reticulata and Citropsis gabunensis were isolated and fused using electric
current (Ling and Iwamasa, 1994). Treated protoplasts were cultured on MT
(Murashige and Tucker, 1969) medium with growth regulators and 0.6% Bacto-Difco
agar. Protoplast-derived calli were proliferated on MT medium containing 1.0 mg/1
zeatin and 0.9% agar. A total of31lines of somatic hybrid were obtained by screening
on the basis of chromosome count and isozyme analysis. Tetraploid somatic hybrids
regenerated from the calli via somatic embryogenesis, exhibited intermediate
characteristics to the parental plants (Ling and Iwamasa, 1994). Somatic hybrid plants
were also regenerated following protoplasts production from of two sexually
compatible genotypes, Citrus sinensis with Poncirus trifoliata (Grosser eta/., 1988a, b;
Ohgawara, eta/., 1985), Citrus sinensis and Citrus unshui (Kobayashi eta/., 1988),
and C. sinensis and C. paradisi (Ohgawara eta/., 1989). In addition, somatic hybrid
between sexually incompatible species C. sinensis and Severinia disticha (Grosser et
a/., 1988b) and between C. sinensis and Citropsis gilleliana (Grosser and Gmitter,
1990a, b, c) were reported. The first successful production of a cybrid plant using a
donar-recipient system was reported between Citrus and Microcitrus genotype (Vardi
et a/., 1987, 1989, 1990). Grosser eta/. (1990) hybridized Citrus reticu/ata and an
incompatible species Citropsis gil/e/iana. Somatic hybrids were confirmed by
chromosome counts and analysis of banding profiles for one or more isozyme systems
(Grosser eta/., 1988a, b), or through EcoR-1 restriction analysis of rDNA (Ohgawara
eta/., 1985). Recently, putative Citrus cybrid plants were produced, by PEG-induced
somatic cells hybridization of nucellus-derived embryogenic protoplasts with nucellus
seedling leaf-derived protoplasts (Grosser et a/., 1996). The cybrid plants were
regenerated through somatic embryogenesis. Isozyme and RFLP analysis confirmed the
eybrids using a nuclear genome-specific DNA probe. Jumin and Nito (1996) have
reported regeneration of plantlets via somatic embryogenesis from protoplasts of six
plant species related to Citrus. The regenerated plants were acclimatized successfully
and transplanted in the greenhouse condition from all the six species viz. Fortune/Ja
polyandra, Ata/antia bilocularis, Triphasia trifolia, Hesperethusa crenulata,
Glycosmis petaphylla and Murraya koenigii. Earlier (Jumin and Nito, 1995), reported
the in vitro flowering of plants regenerated via somatic embryogenesis from protoplasts
of orange jasmine (Murraya paniculata).
Recently, Ara (1999) and Ara eta/. (1998) isolated 81% viable protoplasts
from proembryogenic masses (nucellar origin) derived from monoembryonic cultivar
'Amrapali' of Mangifera indica. These protoplast showed 89% division frequency in
liquid culture medium with 1.0 mg/1 2,4-D and 0.18 M sucrose. They also found an
inhibitory effect of mannitol on protoplast division. The viable protoplasts developed
into microcalli in liquid suspension culture, and proliferated on medium supplemented
with 6% sucrose, 0.1% phytagel and growth regulators, auxin or KIN or auxin+KIN.
These calli produced somatic embryos when transferred to 0.8% agar gelled medium
with 6% sucrose. Mature somatic embryos, incubated in liquid medium supplemented
228
with 3% sucrose and 1.0 mg/1 GA3 germinated (40-60%) and formed plantlets (20-
40%).
The isolation and culture of papaya protoplasts has been attempted in order to
facilitate wide interspecific crosses and induce somaclonal variants. Since plants have
been regenerated from papaya cotyledons (Litz et a/., 1983) studies have been
conducted on isolation of cotyledon protoplasts (Litz and Conover, 1979; Liu and
Yang, 1983). Similar work with papaya and PRY-resistant C. candamarcensis (C.
pubescens) led to actual fusion of protoplasts from these species (Jordan et a/., 1986).
Rapidly proliferating and highly regenerable suspension cultures of somatic embryos of
inter-specific hybrid of Carica papaya x C. cau/ijlora, were used for protoplast
isolation (Chen and Chen, 1992). Protoplasts were first cultured in liquid medium for 2
weeks and then plated on 1% agarose solidified medium. About 1.4% of protoplasts
developed directly into somatic embryos. Protoplast-derived somatic embryos
proliferated rapidly through direct somatic embryogenesis on modified MS medium
supplemented with 1.0 mg/1 ABA and converted into plantlets upon transfer to MS
medium with growth regulators. The plants derived from these protoplast were also
successfully transplanted to soil.
In Musa, the most effective protoplast isolation was achieved from sliced,
folded leaves of aseptically grown plants (Chen and Ku, 1985). Bakry (1984) obtained
protoplasts from callus tissues initiated from inflorescence, whereas Krikorian et a/.
(1988) recovered protoplasts from cell suspension cultures that originated from
immature seeds ofM ornata and diploidM acuminata 'Long Tavoy', respectively. The
protoplasts released from shoots maintained a high degree of viability (80%) after 21
days of culture (Chen and Ku, 1985). Recently, long term embryogenic suspension
cultures were used for protoplasts isolation in Musa spp. (Panis et a/., 1993). They
described five methods for protoplast culture. About 50% plant regeneration was
reported from microcalli via somatic embryogenesis by these authors when high
inoculum density or nurse cell culture techniques were used. Recently, Matsumoto et
a/. (1998) have isolated protoplasts from embryogenic calli and cell suspension of
Brazilian desert banana (Musa spp. AAB group) and cultured as embeded in solid
medium blocks floated on liquid medium containing rice cell suspension nurse culture.
Best results were onbtained at protoplast density 1xl0..6 g/ml were cultivated on a
membrane, lsopore filter with nurse cells in the dark and transferred on new nurse cell
after 10-14 days of incubation. Plantlets were regenerated on medium containing 2J.IM
BA and IAA (Matsumoto eta/., 1998).
In litchi (Litchi chinensis cv. Xiaofanzhi) a protoplast yield of 10-15 x10·6 g
fresh weight was reported from cell suspension produced from friable embryogenic
calli of young embryo origin (Yu eta/., 1996). They recoovered about 92-97% viable
protoplasts. Embeding protoplasts in alginate beads and coculturing this with nurse
cell promoted continuous division, small callus formation and direct formation of
somatic embryos. They found 100% frequency of embryo differentiation from
protoplasts derived callus on semi-solid medium. Somatic embryos suffered
hyperhydricity, however, some embryos after culture on different medium for 2-5
months initiated roots and buds and eventually plantlets.
 229
In avocado (Persea americana Mill.), Witjaksono and Litz (1997) have
reported a protoplast yield of 3 to 33 millionlg of suspension culture. They found that
plating density, nitrogen sources, medium osmolarity and dilution affected plating
efficiency and growth rate of protoplast-derived microcalli and somatic embryos in
agarose disks and in liquid medium. The plating efficiency of 25% (liquid) and 40%
(agarose) were obtained with 0.08 to 0.1 million protoplasts/ml, 0.4M osmolarity and
glutamine instead of ~N03 • Further dilution of proembryos after 2-3 weeks of
plating gave globular and early heart stage embryos after 1 month. Transfer of heart
stage somatic embryos ·to hormone-free semisolid medium facilitated maturation and
germination. However, only =:;;J% plantlet recovery was noted (Witjaksono et a/.,
1998).
In Actinidia spp., petiole segments were used as a primary explant source for
isolating viable protoplasts at a high yield, about 90% (Pais et al., 1987). Plant
regeneration from protoplasts of leave and leaf-derived calli has been repoted by Mii
and Ohashi (1988) and Tsai (1988). Plant regeneration from protoplasts of long term
callus culture has also been reported inActinidia sp. (Oliveira and Pais, 1991; Oliveira
eta/., 1994).
Hence, it is clear from these discussion that considerable advances has been
made in the protoplasts derived plant regeneration through somatic embryogenesis
(Bajaj, 1989a, b, 1993a, b, 1994). These development further paved the way for the
subsequent exploitation of the many recent advances in molecular biology for the
improvement of tropical fruit trees e.g. production of transgenic fruit trees with the
application of somatic embryogenesis.

6. Somatic Embryogenesis and Cryopresen-ation:

With the potential use of somatic embryogenesis in mass propagation (Bajaj, 1991c),
genetic transformation (Bajaj, 1989a, b, 1993a, b, 1994; Ellis, 1995), artificial seeds
(Redenbaugh, 1993; Bajaj, 1995a, b) and large-scale production using bioreactors
(Bajaj, 199lc; Denchev et a/., 1992), there is need to develop methods for their
storage, so that they can be used when desired (Bajaj, 1995c). Since, somatic
embryogenesis has been induced in a number of fruit tree species (Akhtar et al. in this
volume), cryopreservation would enable the conservation of that germplasm too.
Cryopreservation of encapsulated somatic embryos is a method for long-term
conservation and exchange of germplasm (Bajaj, 1995c, d). The first demonstration of
successful regeneration of cryopreserved Daucus carota cell cultures is reported by
Latta (1971). Since then, considerable improvements have been made over the
conventional method offreeze storage (Kartha and Engelmann, 1994; Bajaj, 1995c, d).
These developments include: (i) Vitrification, (ii) Simple freeze method, (iii) Gel
coating, (iv) Encapsulation and dehydration and (v) Cryoselection. High viability of
somatic embryos and regeneration of normal plants after freeze storage in liquid
nitrogen has been reported in a number of species (Bajaj, 1995c, d; Engelmann, 1991;
Kartha and Engelmann, 1994).
230
Several factors have been found to affect the viability and subsequent
regeneration of cryopreserved cultures. Sakai eta/. (1990) have studied the effects of
vitrification and cryoprotectants on the nucellar tissue of naval orange (Citrus sinensis
Osb.). Highest survival (91.2%) was observed with 2M glycerol followed in decreasing
order by 3M glycerol, 2M propylene glycerol, 2M DMSO, 3M DMSO, 2M ethylene
glycol, 3M ethylene glycol and 3M propylene glycol along with 0.4M sucrose dissolved
in MT (Murashige and Tucker, 1969), basal medium (Sakai eta/., 1991). Revived cells
resumed growth within 3 days and developed into plantlets via somatic embryogenesis.
Recently, nucellus derived embryogenic callus cultures of Salustiana sweet orange (C.
sinensis) were subjected to cryopreservation assay. Cryopreservation with 10% (v/v)
DMSO, frezzing by slow cooling and thawing by fast warming was suitable to recover
variable growing cultures and whole plants through somatic embryogenesis.
Evaluation of liquid phase R1 and solid phase R2 cooling rate, using a programmable
freezing unit indicate the 100% of embryogenic cultures survived when frozen using a
range of cooling rates (R1 not above 0.5 °C/min. and R2 not above 1 °C/rnin.) and
thawed by fast warming. Storage upto 2 years in liquid nitrigen did not affect the
growth of cryopreserved cultures and the recovery of whole plants (Perez, eta/., 1997).
Embryogenic suspension cultures of plantain (Musa balbisiana) in 7.5% DMSO, were
preserved in liquid . nitrogen, and plants were regenerated through somatic
embryogenesis on MS medium with 1 ~ BA and 100 mgll inositol (Banerjee eta/.,
1987; Panis eta/., 1991). Somatic embryos in oil palm were dehydrated prior to the
start of the cryopreservation process, and about 13-53% survival rate of embryogenic
clumps after freezing in liquid nitrogen has been reported (Dumet et a/., 1993).
Further, somatic embryos from leaf-derived calli of oil palm dehydrated to 60-80%
moisture content prior to immersion in liquid nitrogen, is reported by Engelmann et a/.
(1985). They were also successful in recovering somatic embryos and shoots from
cryopreserved cultures in medium containing 0.5-1.0 mM 2,4-D. Regeneration from
cryopreserved tissues has also been reported in cacao (Pence, 1991) and datepalm
(Tisserat et al., 1981). Slow freezing of somatic embryos followed by immersion into
liquid nitrogen is reported in sweet orange (Martin and Duran, 1988). The DMSO
treated cultures produced plantlets at a faster rate and survival rate was 90% compared
to 17% for untreated embryos.
7. Somatic Embryogenesis and Selection of Somaclonal variants:

Genetic and phenotypic variation produced during the cell and tissue culture cycle are
referred to as somaclonal variation (Bajaj, 1990; Hammerschlag, 1992; Jain et a/.,
1997; Larkins and Scowcroft, 1981). Genetic variation in plants or cell lines
regenerated in vitro were observed in early experiments with tobacco (Lutz, 1966) and
sugarcane (Heinz and Mee, 1971). Most of the observed variability was due to
physiological and developmental factors and was transient or epigenetic (not heritable)
(Evans and Sharp, 1986). Genetic variability among cell lines regenerated through
organogenesis has sometimes been attributed to the chromosomal abnormalities
(Bayliss, 1980), and therefore, may have limited potentials for plant improvement.
Although less frequent, variation among plants raised through somatic embryogenesis
 231
has also been reported and successfully utilised to generate plants with disease
resistance and other stresses (Duncan, 1997; Hammerschlag, 1992; Hitomi et a/.,
1998; Mandahar, 1997; Morrison eta/., 1988; Semal and Lepoivre, 1990). Somaclonal
variation were observed among plants regenerated through somatic embryogenesis
induced by NAA and 2,4-D in Solanum melongens L. (Hitomi et a/., 1998). They
found that the frequency of somaclonal variation in leaf shape, plant height, fruit shape
and pollen fertility in NAA experiment were higher than those observed in 2,4-D
experiment. They also observed that variation in leaf shape and fruit shape were
inherited while those in flower number were not. Shchukin et a/. (1996), frequent
somaclonal variation in regenerated plants in the presence of dicamba in the medium
compared to control in banana.
Among fruit crops, somaclonal variants have been recovered from a number
of species. Litz eta/., (1995), compared an isozyme extracted from leaves and roots of
a nucellar seedling of polyembryonic mango cultivar 'Philippine' with the same
isozyme extracted from leaves and roots of plants derived from somatic embryos of
same cultivar. They found that there was considerable variation (~ 20%) in the
banding pattern of esterases and alcohol dehydrogenases of somatic embryo-derived
plants, whereas, the nucellar seedling tissue showed much lesser variation (~ 2%).
They concluded that although genetic variation occurs in nucellar seedlings, variation
is much greater in plants regenerated from somatic embryos (Litz, 1997). In Citrus,
polyploidy has been induced in the embryogenic cultures through the use of colchicine
and plant were regenerated through somatic embryogenesis (Gmitter et a/., 1991).
Several techniques have been used to identify somaclonal variation, with RAPD
analysis being more frequently used in recent years (Rival et a/., 1998a). Genomic
methylation which thought to play important role in the induction of somaclonal
variation is being studied in oil palm. Preliminary result obtained by complementary
approaches showing epigenetic nature of somaclonal variation is discussed by Rival et
a/. (1998b). Chromosomal count of root-tip of plants raised via somatic embryogenesis
in grapevine (Vitis vinifera cv, Podark Magarcha) showed 6 (i.e. 2.5%) tetraploid
among 242 plants analysed (Kuksovo eta/., 1997). They found that gamma irradiation
used for inducing mutation, increased the frequency of tetraploid plants and
embryogenic calli (7.6%) along with some aneuploids. Kunitake et a/. (1998) have
reported that chromosome variation in embryogenic calli derived plant increased with
increasing duration of subculture particularly when low ploidy level of the plant such
as haploid and diploid were used as explant. Approximately 80% of haploid derived
plants showed morphological variations such as dwarfness and abnormal
morphological characteristics in Asparagus officina/is L.
In norway spruce (Lelu eta/., 1990), a variation in chromosome number was
observed for two mature somatic embryos. Heinze and Schmidt (1995) monitored the
genetic fidelity vs. somaclonal variation in norway spruce somatic embryogenesis by
RAPD analysis. Fourre eta/. (1997) observed 3 cases of variants with 1 trisomic and 2
chimeras in acclimated somatic plants, while RAPD analysis failed to identify any
somaclonal variation. In Lotus cornicu/atus L., 72 plants regenerated from leaf-derived
calli were evaluated for several morphological and agronomical traits (Damiani et a/.,
232
1990). They observed a high frequency of somaclonal variation and found that most of
the in-vitro induced variation as recessive and detected only after sexual propagation.
They concluded that tissue culture did not increase variation for any traits over that in
the initial donor plants.
Several excellent reviews pertaining to biotechnology of perennial fruit
species have been published in recent years (DeWald and Moore, 1987;
Hammerschlag, 1992; Karp and Bright, 1985), however, only in peach (Prunus persica
L. Batsch.) somaclonal variation has been observed in any details (Hammerschlag,
1990). There has been reports on the selection of resistance to diseases through the use
of somaclonal variation and in vitro induced variability in other plants (Duncan, 1997;
Mandahar, 1997). These include resistance to 'Panama' disease banana caused by
Fusarium oxysporum (Hwang, 1990; Hwang and Ko, 1987), strawberry with resistance
to Fusarium oxysporum (Toyoda eta!., 1991), peach with resistance to Pseudomonas
syringae and Xanthomonas compestris (Hammerschlag and Ognianov, 1990) and
Citrus with resistance to mal seccotoxin (Node! and Spiegel-Roy, 1987) and Phoma
tracheiphia (Gentile et a/., 1993). Recently, Jayasankar and Litz (1998) selected
embryogenic mango cell cultures for resistance to Colletotrichum gloeosporioides. In
addition, Jayasankar et a/. (1998), used RAPD markers to analyse genomic DNA
isolated from embryogenic nucellar cultures of two mango cultivar 'Himndi' and
'Carabao' that were selected for resistance to the culture filtrate of Colletotrichum
gloeospoioides. They found that in vitro selection cause changes in RAPD banding
pattern in selected embryogenic cultures with respect to unchallenged control cultures
and stock plants. The absence of differences between the unchallenged control of either
of cultivar and DNA from the leaves of parent trees confirmed that the changes were
not due to the prolonged maintenance in liquid medium. Some regulation systems have
also been developed that might be employed for selecting papaya somaclonal variants
with resistance to fungal diseases using callus and/or suspension culture (Manshardt,
1992). Selection at cellular level, for many other specific horticultural and agronornical
traits has been thoroughly reviewed only recently (Bajaj, 1990; Jain eta/., 1997; Karp,
1995).

8. Future Prospects and Concluding Remarks:

In the preceeding overview, we have presented an outline of some applications of

somatic embryogenesis for plant improvement, particularly tropical and subtropical
fruit species. Besides the limitation imposed by the genotype, the explants and the
cultural environments, somatic embryogenesis has been reported in large number of
fruit trees (see Akhtar et a/. in this volume). Clonal propagation of fruit trees by
somatic embryogenesis offers the benefit of producing large number of clonal
propagules in bioreactors in comparatively little time and enabling commercial
exploitation of somatic embryogenesis in fruits tree species. Non-synchronous
production of somatic embryos, poor germination rate and low frequency of recovery of
somatic seedlings has limited the application of somatic embryogenesis for fruit tree
 233
improvement. Further attempts should be made to synchronize production of somatic
embryos and to improve the bioreactor processes. The encapsulation of somatic
embryos and production of synthetic seeds have great potentials in crops where seeds
are not produced and vegetative propagation is impossible. Further, synthetic seed
technology provides an easy means of handling large number of propagules during
their transport, and can greatly facilitate automated sowing under green-house
conditions. Somatic embryos produced in vitro can not tolerate desiccation and drying
and therefore can not be used directly as artificial seeds. The potential use of fruit tree
somatic embryo as artificial seeds, in terms of long term storage and handling would
be greatly facilitated if desiccation tolerance could be induced. Hence, studies for
inducing desiccation tolerance in somatic embryos should be the focus of future
research. Cryopreservation of embryogenic calli, somatic embryos and synthetic seeds
and regeneration of plants has the potential for long term conservation and exchange
of germplasm. Propagation of improved clonal materials of fruit tree will require many
years of testing before they can be accepted by the industry and the public as reliable,
cost effective and free of adverse effects. Identification of stable and useful somaclonal
variation and development of an early testing methods for desirable traits in somatic
somatic embryo derived fruits tree species need to be investigated further. The ability
to regenerate plants from protoplasts via somatic embryogenesis facilitates direct gene
transfer, somatic hybridization, cybridization and polyploidization studies. Priorities
for genetic improvement will have to be established for individual species based on
industrial need, public demand and availability of suitable plantation sites. A more
serious effort needs to be made to elucidate the theoretical and practical problems
associated with somatic embryogenesis so that the technology can become
commercially competitive.

9. Acknowledgements:

The financial support to the Laboratory of Morphogenesis, Centre of Advanced Study

in Botany, Banaras Hindu University, Varanasi, provided by various agencies like
University Grant Conunision, Council of Scientific and Industrial Research, Central
Silk Board, Department of Biotechnology, Department of Science and Technology,
Government of India, is gratefully acknowledged.

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seed production-I: Encapsulation of somatic embryos by alginic acid, Korean J. Plant Tiss. Cult.; 13: 119-
128.
Jones, L H. (1974) Long term survival ofembryoids of carrot (Daucus carota L.), Plant Sci. Lett.; 2: 221-224.
Jordan, M., Ciudad, G., Rojas, M. L. and Valverde, F. (1986) Isolation, culture and fusion of Carica
candamarcensis and C. papaya protoplasts, Gartenbauwissenschaften; 51: 175-178.
Jumin, H. B. and Nito, N. (1995) Embryogenic protoplast culture of orange jasmine (Murraya paniculata) and
their regeneration into plants flowering in vitro, Plant Cell Tiss. Org. Cult.; 41: 277-279.
Jumin, H. B. and Nito, N. (1996) Plant regeneration via somatic embryogenesis from protoplast of six plant
species related to Citrus, Plant Cell Rep.; 15: 332-336.
Kaneyoshi, J., Kobayashi, S., Nakamura, Y., Shigemoto, N. and Doi, Y. (1994) A simple and efficient gene
transfer system of trifoliate orange (Poncirus trifoliata Raf.), Plant Cell Rep.; 13: 541-545.
Karp, A (1995). Somaclonal variation as a tool for crop improvement, Euphytica; 85: 295-302.
Karp. A and Bright, S. W. (1985) On the cause and origin of somaclonal variation, Oxford Survey of Plant
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Kartha, K. K. and Engelmann, F. (1994) Cryopreservation and germplasm storage, NRCS publication no. 32487,
in I. K. Vasil and T. A Thorpe (eds) Plant Cell and Tissue Culture, Kluwer Academic Publisher, The
Netherland. pp. 195-230.
Kitto, S. and Janick, J. (1985a) Production of synthetic seeds by encapsulating asexual embryos of carrot, J.
Amer. Soc. Hort. Sci.; 110: 277-282.
Kitto, S. and Janick, J. (1985b) Hardening treatments increase survival of synthetically-coated asexual embryos
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Kitto, S. L., Pill, W. G. and Molloy, D. M. (1991) Fluid drilling as a delivery system for somatic embryo-derived
plantlets of carrot (Daucus carota L.), Sci. Hort.; 47: 209-220.
Kitto, S. L., Pill, W. G. and Molloy, D. M. (1995) Fluid drilling as a delivery system for somatic embryo derived
plantlets, in Y. P. S. Bajaj (ed.) Biotechnology in Agriculture and Forestry, Vol. 30: Somatic
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Osb.) protoplasts by direct DNA transfer, Japanese J. Genet.; 64: 91-99.
Kobayashi, S., Ohgawara, T., Ohgawara, E., Oiyama, I. and Ishii, S. (1988) A somatic hybrid plant obtained by
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Tiss. Org. Cult.; 14: 63-69.
Kobayashi, S., Uchimiya, H., and Ikeda, I. (1983) Plant regeneration from 'Trovita' orange protoplasts, Japan J.
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Krastanova, S., Perrin, M., Barbier, P., Demangeat, G., Cornuet, P., Bordonnet, N., Otten, L., Pinck, L. and
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Krikorian, A D., Cronauver-Mitra, S. S. and Fitter-Corbin, M.S. F. (1988) Protoplast culture of perennials, Sci,
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Kuksovo, V. B., Piven, N. M. and Gleba, Y. Y. (1997), Somaclonal variation and in vitro induced mutagenesis in
grapevine, Plant Cell Tiss. Org. Cult.; 49: 17-27.
Kunitake, H., Nakashima, T., Mori, K. and Tanake, M. (1998) Somaclonal chromosomal effect of genotype,
ploidy and culture duration in Asparagus officinalisL.,Euphytica; 102:309-316.
Laimer da Camara Machado, M., da maara Machado, A, Hanzer, V., Weiss, H., Regner, F. Stein Kellner, H.,
Mattanovich, D., Plail, R., Knapp, E. Kalthoff, B. and Katnger, H. (1992) Regeneration of transgenic
plants of Prunus armericana containing the coat protein gene of plum pox virus, Plant Cell Rep.; 11: 25-
29.
Lakshmana Rao, P. V. and Singh, B. (1991) Plantlet regeneration from encapsulated somatic embryos of hybrid
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Larkin, P. J. and Scowcroft, W. R. C. (1981) Somaclonal variations a novel sources of variability from cell
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 Tab1e 1: Genetic Transfor.mation in Tropica1 Fruit Trees usinq Somatic Embryogenesis.
Plant Species Explant and Tnnsfonnation Result and Analyses References
Re2enention System Methods
Actinidia deliciosa In vitro grown stem Agrobacterium Crown gall induction and hairy root development. Atkinson et al., 1990
segment GUS analysis.
Actinidia deliciosa Leaf segment Agrobacterium Transfonnation frequency, Transient GUS Janssen and Gardner, 1993
expression, Shoot resistance to kan. PCR and
Southern.
Actinidia deliciosa Protoplasts of petiole Direct transfer using PEG Factors affecting transient expression oftransgenes Oliveira and Pais, 1991
derived calli analyzed by TLC and GC-MS.
Actinidia deliciosa Protoplasts derived calli Direct DNA uptake by Microcolony resistance to kan. Shoot regeneration, Oliveira et al., 1994
of petiole stem and root Electroporation, NPT II expression, Dot Blot.
segment Agrobacterium
Actinidia deliciosa leaf discs Agrobacterium Shoot resistance to kan. Increased rooting ability, Rugini et al., 1991
NPT II activity, Dot Blot, Southern analysis.
Actinidia deliciosa Hypocotyl slice, stem Agrobacterium Shoot resistance to kan. GUS and NPT II expression Uematsu et aL, 1991
segment (Dot Blot)
Ananas comosus Organogenesis and Agrobacterium Transformed embryogenic calli and somatic embryos Firozabady et al., 1997
Embryogenesis resistance to gentamycin .Transformation confirmed
by PCR and Southern analysis.
Carica papaya Leaf explant, Agrobacterium GUS expression. Studied sensitivity of transgenic Cabrera et al., 1996
Embryogenic calli plants to auxins. Expression of rol genes
Carica papaya Zygotic and somatic Microprojectile Transgenic plant and somatic embryos resistance to Cai et al., 1999
embryos bombardment kan., coat protein gene expression of PRSV
Carica papaya Embryogenic Tissue Agrobacterlum Transgenic somatic embryos resistance kan and Cheng et al., 1996
carbenicillin.
Carica papaya Hypocoty~ zygotic and Microprojectile Transformed somatic embryos and leafY shoots Fitch et al., 1990
somatic embryos bombardment resistance to kan. GUS and NPT II expression
Carica papaya Immature zygotic Particle gun Transgenic papaya plants confirmed by GUS and Fitch et al., 1992
embryos bombardment NPT II. PCR and Southern analysis. ELISA for
PRV-CP. Increased resistance to PRV.
Carica papaya Hypocotyl segment, Agrobacterium Shoot with GUS, NPT II and PRV-CP expression. Fitch et al., 1993
embryogenic calli, PCR analysis. Southern Blot hybridization and
somatic embryos ELISA for NPT II and PRV-CP expression
Carica papaya Zygotic and somatic Microprojectile Transgenic plant and somatic embryos resistance to Gonsalves et al., 1998
embryos bombardment kan., coat protein gene expression ofPRSV
Carica papaya Zygotic and somatic Microprojectile Transgenic somatic embryos resistance kan. Mahon et al., 1996 N
~
Vl

Carica papaya
Carica papaya
Carya illinoensis
Carya illinoensis
Citrns aurantium L.
Citrus reticulata
Citrus sinensis
Citrns sinensis
Citrns sinensis x Poncirns Internodal segments of 5
trifolius day old seedlings
Citrns sinensis x Poncirns
trifolius; Citrns paradisi x
Poncirns trifolius and
Citrns aurantifolia
Citrns sp.
Cyphomandra betaceae
Feijoa sellowiana
Mangifera indica
Musaspp.
Passiflora edulis
embryos bombardment Transformation confirmed by Southern N
.j::>..
hybridization. 0'\
Embryos Agrobacterium NOS expression Pang and Sanford, 1988
In vitro grown petiole Agrobacterium Somatic embryo resistance to kan and cabenicillin. Yangetal., 1996
segment, somatic embryos
Embryogenic culture of Agrobacterium Octopine expression Martin, 1987
immature zygotic embryo
Embryogenic culture Agrobacterium Resistance to kan. Transgenic plants recovered by McGranahan et al., 1993
repetitive embryogenesis. GUS expression. Southern
analysis.
Internodal segments Particle gun Resistance to kan. GUS expression Gutierrez et al., 1997
bombardment
Embryogenic callus Direct DNA transfer by Transient expression and colony formation GUS Hidaka and Omura, 1993
electroporation expression
Embryogenic cell Agrobacterium Embryoid resistance to kan or hygromycin and Hidaka et al., 1990
suspension regeneration of transgenic plants. Southern
hybridization
Internodal segment of Agrobacterium Transformation confirmed by GUS expression and Pena et al., 1995a, b
seedlings Southern analysis. Grafting ofmocroshoots or
seedling rootstocks
Agrobacterium Grafting of transformed shoots on rootstocks. Kaneyoshi et al., 1994
Transformation confirmed by PCR. Southern and
Nothern analysis.
Internodal segments of in Agrobacterium Resistance to kan. Low transformation frequency. Moore eta/., 1992
vitro grown seedlings PCR analysis. GUS positive shoots. Southern
analysis.
Protoplast Direct DNA uptake Resistance to kan. Kobayashi and Uchimiya, 1989
Growing leaves Agrobacterium Resistance to kan. and clorsulfuran. Inheritance of Atkinson and Gardner, 1993
transgenes confirmed by GUS, NPT II and Southern
analysis.
In vitro grown stem Agrobacterium Induction of calli. GUS expression in gall tissue. Atkinson et al., 1990
Somatic proembryos Agrobacterium Somatic embryo resistance to kan. GUS and NPT II Cruz-Hernandez et al., 1997;
expression, Southern analysis. Mathews et al., 1992, 1993; Litz
et al., 1990
Single cell, callus Agrobacterium, biolistic Fungal and viral disease resistance Vuylsteke et al., 1998
Leaf and stem explants Agrobacterium Transgenic plants resistance to kan. Nopalie Manderson et al., 1994
expression. PCR analysis. DNA Dot Blot
Prunus armeniaca Cotyledon of immature Agrobacterium GUS assay in regenerated transgenic plants Laimer da Camara Machado et al.,
embryo 1992
Prunus avium Internodal segment Agrobacterium Shoot resistance to kan, NOS expression and avoid Brasileiro et al., 1991
difficult regeneration procedure
Prunus domestica Hypocotyl slice from Agrobacterium Transgenic plants resistance to kan. NPT II and GUS Mante et al., 1991
seeds expression. Southern analysis.
Prunus domestica Hypocotyl slice from Agrobacterium Confirmation ofPRV-CP integration by Southern, Scorza et al., 1994
seeds Nothern and Western Blots. Transgenic clones
analyzed by PCR and GUS expression
Prunus domestica Hypocotyl slice from Agrobacterium Resistance to kan in transgenic plants. GUS assay. Scorza et aL, 1995a
seeds PCR., Southern, PRV-CP expression. Studies of
heterologous expression
Prunus persica Leaf segment of in vitro Agrobacterium GUS and NPT II expression. Southern Blotting. Archilleti et aL, 1995
grown stem
Prunus persica Leaf segment, Agrobacterium Transformation of embryogenic calli. Resistance to Scorza et al., 1990
embryogenic callus, kan., NPT II expression, Southern Blotting
immature embryos
Prunus persica Embryo derived call of Particle gun Transformation efficiency; GUS assay, PCR analysis Y e et al., 1994
seedling origin bombardment
Prunus subhirtella Embryogenic callus Agrobacterium Embryogenic callus selected by repetitive da Camara Machado et al., 1995
embryogenesis with kan. GUS assay. Nothern Blot

GFLV = Grapevine fanleaf nepovirus ELISA= Enzyme linked immuno sensitivity assay
GUS = P- glucuronidase GCMS = Gas chromatography- mass specrtoscopy
NOS = Nopaline synthase PCR = Polymerase Chain Reaction
CP = Coat Protein PRV = Papaya ringspot virus
TLC = Thin layer chromatography Kan. = Kanamycin
NPT-II = Neomycinphosphotransferase -II

N
.j::>.
-J
8. Somatic embryogenesis in oil palm

Alain RIVAL
CIRAD-CP/ORSTOM GeneTrop Lab. P.O. Box# 5045. F-34032 Montpellier Cedex OJ, France.
Email: rival(iiJ,orstom.(r

Chapter contents:

l. Introduction

2. Breeding

3. Somatic embryogenesis

4. Scaling-up to pilot stage

5. Agronomic performance

6. Bottlenecks for commercial propagation

7. Research programmes in progress

8. Future directions for research

9. Conclusions

I 0. Acknowledgements

11. References

249
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 249-290.
© 2000 Kluwer Academic Publishers.
250
1. INTRODUCTION

1.1 Botany of oil palm

The first description and subsequent botanical classification of the African

oil palm (Elaeis guineensis Jacq.) dates back to the end of the XVI1h century
(Figure 1).
Jacquin definitively named it in 1763 following his works carried out on the
Martinique island in the French West Indies. The generic name Elaeis
originates from the ancient Greek word elaion, which means oil ; the species
name guineensis refers to the geographic origin of this palm, in the Gulf of
Guinea (West Africa). Oil palm has been domesticated by man since ancient
times, even though its breeding was only initiated at the beginning of this
present century (Hartley, 1988; Jacquemard, 1995). The adaptation of oil palm
to new environments will continue and produce diversification puts new
demands on domestication (Gerritsma and Wessel, 1997)
The Elaeis genus is made up of three species: E. oleifera, E. odora and E.
guineensis; the latter being the only one exploited agronomically for its oil
production and planted in industrial plantations.
Oil palm belongs to the family of the Aracaceae in the order of the
Spadiciflores, sub-order of Palmales. The size of oil palm genome, as recently
estimated by flow cytometric analysis (Rival eta!., 1997a) is 3.4.10 9 bp with a
2n=32 caryotype.
At the adult age, this arborescent monocotyledon shows the typical aspect
of palms with a crown consisting of 40-50 opened palmate leaves (Figure 2).
Growth is ensured by a single terminal meristem which produces an average
of two leaves per month. Vertical growth rates may vary from 30 to 75 em per
year, depending on the genetic origin of the palm. The root system is composed
of a large number of fasciculate adventive roots which secure the anchorage of
the plant. Oil palm growths preferentially in rich and temporarily damp alluvial
soils (Hartley, 1988). It can reach 30m in height, with a crown of leaf of 10-16
m in diameter, and so requires a large soil area to properly ensure correct
growth. The standard plantation density in industrial conditions is 143 palms
per ha.
The oil palm is a temporal dioecious species (Cruden, 1988) which displays
alternate male and female flowering cycles throughout the life of the plant. The
50 em long inflorescence consists of a peduncule and a spadix enclosed in two
spathes. It emerges from base of the petiole after the leaf expansion is
completed. The male inflorescence has finger-like spineless spikes with 400 to
 251
1500 flowers composed of 6 perianth pieces and an androecium with 6
stamens. The female inflorescence is formed of shorter spikes bearin~ 10 to 30
trifloral groups with two non functional, aborted male flowers and one female
flower comprising a tricarpellary ovary and a trilobed stigma (Beirnaert, 1935).
Fruit bunches are harvested five to six months after flowering (Figure 3).
They weigh 10 to 90 kg depending on the age of the palm and its genetic
origin. The number of bunches produced per year depends on the ratio of
female and male inflorescences produced, the rate of leaf production and of
fruit abortion.
The fruit is a sessile drupe which is black to orange in colour depending on
the ripening stage, 2 to 5 em long and weighing 10 to 30g. The mesocarp
contains 30 to 60% palm oil. The endocarp (or shell) surrounds the seed (or
kernel) which contains kernel oil.

1.2 Geographical distribution

Oil palm is mainly cultivated in industrial plantations between 7°S and 7°N,
at an altitude lower than 400 m, in areas with a high rainfall (500 to 3000 mm),
well distributed over the year (Surre and Ziller, 1963). Spontaneous or
subspontaneous plantations grow in Africa, between 10-11 °N (Guinea) and
8-l0°S (Angola) (Hartley,1988) which is now considered to be the zone of
origin ofthe species (Zeven, 1965).

1.3 Economic importance

Oil palm is the second source after soybean of edible vegetable oil (Figure
4). Industrial exploitation of oil palm started at the beginning of the 201h
century, firstly in South East Asia (Malaysia, Indonesia), then in Africa on the
edges of the Gulf of Guinea and then in Brazil in the 1920's. Since the 60's, its
cultivation area has been extended to South America, mainly in the Amazon
basin (Equator, Colombia, Peru) and to the Pacific coast (Costa Rica). During
this period, intensive planting programmes were developed in South East Asia
and to a lesser extent in West Africa. World palm oil production has undergone
a tenfold increase since 1948 and it was over 17 million tonnes per year in 1997
(Table 1). Oil production is still growing due to a continuous increase in the
area planted. Malaysia and Indonesia together account for 80% of world
production, with an overall increase of 7% in 1996 and 8.5% in 1997, while
production remained static in Latin America, particularly in Colombia. The
consumption of oleaginous products continues to rise (Table 2), due to
demographic pressure, the increasing domestic use of oil palm in producing
countries and to the demand from highly populated countries such as India and
China, which are at present relatively small consumers of oleaginous products.
252
The price of palm oil, after a continuous growth (from 300 to 650 USD per ton
of CPO - Crude Palm Oil) during the 1990-95 period, stabilised at an average
price of 550USD/Ton during the 1996-97 period.

2. BREEDING

Due to the increasing demand for palm oil, the lack of plantable land and
the foreseeable increases in cultivation costs, it is necessary to make available
to the planters planting material with high genetic potential. Nowadays,
planting material consists solely of tenera hybrids (fruits with shell of
intermediate thickness), originating from crosses between dura (thick shell)
and pisifera (thin shell) types, the thickness character being controlled by a
monofactorial gene (Beirnaert and Vanderweyen, 1941 ). The breeding schemes
developed by seed companies aim to produce dura x pisifera hybrids with a
high productivity of oil with a high proportion of unsaturated fatty acids, a low
growth rate and, in particular cases, resistance/tolerance to diseases such as
vascular wilt caused by Fusarium oxysporum elaeidis in Africa (Hardon eta!.,
1976). Since each selection cycle lasts for around 10 years, genetic
improvement is very slow, even if much progress has been achieved over the
last 50 years (Jacquemard et a!., 1997). A very high heterogeneity is still
observed among hybrids, some palms producing 60 % more oil than the
average of the progeny of a given cross (Noiret, 1981). When these
characteristics are cumulated with the low planting density (generally 143
palms per hectare) and the necessity of establishing seed orchards for the
production of commercial material, it can be seen that oil palm improvement is
labour intensive, time consum·ing and therefore expensive. The constraints
which exist in oil palm breeding make desirable the development of a
vegetative propagation technique which would allow:
• the exploitation of the variability existing among the tenera hybrids by
cloning elite trees;
• an increase of the production of high quality seeds by cloning the best male
parents (pisifera), since pollen production can be a limiting factor (Hartley,
1988; Krikorian, 1989);
• the exploitation of the interspecific hybrids E. guineensis x E. oleifera, a
limited number of which are fertile, but which can show a good tolerance
to pests and diseases, notably in South America (Meunier, 1975).
• the production of biclonal seeds from somatic embryogenesis-derived
parents (Hartley, 1988).
 253
3. SOMATIC EMBRYOGENESIS

3.1 Current status of oil palm micropropagation

The biological characteristics of the oil palm do not allow its vegetative
propagation by conventional horticultural means, in spite of work carried out
on the reversion of floral buds or the study of viviparous palms (Chevalier,
1910; Henry, 1948; Davis, 1980). The in vitro culture of apices was
experimented with young palms (Staritsky, 1970; Abdullah, 1990) but they did
not produce satisfactory results. Moreover, the excision of the apex leads to the_
death of the mother tree. Therefore, the only possible way to clonally propagate
elite oil palms is by means of somatic embryogenesis. Cloning of oil palm
(Elaeis guineensis Jacq) is performed by inducing somatic embryogenesis on
calli derived from various origins, using tissue culture protocols which have
been recently reviewed in detail by Duval et al. (1995a).
For more than 25 years, two major groups have carried out research
programmes: in the UK and in Malaysia, the Unilever Plantations and
Harrisson and Crossfields Plantations group (Smith and Jones, 1970; Corley et
al., 1977) and in France and Cote d'lvoire, the ORSTOM/CIRAD (formerly
IRHO) group (Rabechault et al., 1970; Noiret, 1981). These programmes were
initiated by the organisations which develop their own breeding strategies
aimed at multiplying the elite germplasm available in their plantations for
commercial use. The results obtained leading to potentially important
commercial applications, only limited information was published concerning
the techniques set up, as underlined by various authors (Krikorian, 1989; Blake,
1990). During the eighties, several teams from producing countries started their
own research programmes. Naturally, due to the dynamism of oil palm industry
in Malaysia, several local teams developed large scale research programmes,
notably at PORIM (Paranjothy and Othman, 1982). Wooi (1990) estimated that
in Malaysia, around 10 commercial laboratories were carrying out research on
in vitro propagation of the oil palm through somatic embryogenesis. Most of
them are still conducting commercial or semi-commercial activities in oil palm
clonal micropropagation, sometimes with a diversification of their
micropropagation activities towards more remunerative planting material, such
as banana.
254
3.2 Recent improvements of current protocols

The performance of oil palm micropropagation by somatic embryogenesis

suggests that cloning is possible for most of the planting material currently
available, including E. guineensis x E. oleifera interspecific hybrids (Duval et al.,
1997).
Since 1981, 460 palms have been sampled for tissue culture at the La Me
CNRA Micropropagation Laboratory in Cote d'lvoire (Konan et al., 1996).
Sampling included pisifera palms, back-crosses and in vitro regenerants. All
the palms sampled in La Me using our standard procedure have produced calli
on foliar explants (Figure 5). Callus production may vary according to the
genetic origin of the mother palm. Palms of Deli x LaMe origin produced calli
on 31% of explants on an average, whereas this percentage reached only 7 and
9% respectively in palms of Deli x Nifor and Deli x Yangambi origin. Within a
given cross, callogenesis rates are highly variable (C.V. may vary from 20% to
175% depending on the genetic origin). Embryogenesis on nodular callus
(Figure 6) was obtained with an overall success rate of 87% (Table 3). This
phenomenon occurs at a very slow rate (taking up to 2 years) and remains
difficult to control. Further proliferation of somatic embryos cultures is ensured
by secondary adventive embryogenesis, giving rise to polyembryonic cultures
which are able to ensure mass propagation of oil palm clones. It consists of a
group of proembryonic formations composed of meristematic cells and of
somatic embryos at various developmental stages (Figure 7). On hormone-free
medium, these polyembryonic cultures produce new adventive somatic
embryos and simultaneously, the most advanced embryos develop into shoots.
When they have reached a sufficient size (5 to 7 em) the shoots are separated
manually and rooted on a medium containing NAA (Rival et a!., 1997b).
Success rates during the late phases of the micropropagation process were as
follows: 83% during acclimatisation, 79% during prenursery and 78% during
nursery. Thus the overall success rates were 62% from prenursery to nursery
and 51% from the acclimatisation to the nursery (end). To date, the total
production of the La Me Laboratory since its opening in 1981 has reached
750,000 plantlets, originating from 216 different clones. The overall area
planted with clonal material (genetic trials+ commercial plots) in Cote d'Ivoire
has now reached 800 ha (thus ca 110,000 palms).

With success rates of over 90%, the first stages of the procedure are now fully
mastered, although extremely slow: it takes at least two years to regenerate a
significant number of plants. However, the frequency at which proliferating
embryos are obtained in vitro, enabling mass production, is just 40%. This stage is
currently the major stumbling block for large-scale ramet production to multiply
all selected ortets. Wong eta!. (1996) recently reported 53% proliferating embryo
 255
lines, which is much the same as the values obtained by the French ORSTOM-
CIRAD group and its partners. The phenomenon of somatic embryo proliferation
is certainly worth studying in detail in order to increase the success rate for this
stage. It corresponds to the secondary embryogenesis described on other species
such as woody plants (Merkle, 1995).

Recent papers on the description of somatic embryogenesis in oil palm or

on the description of new regeneration protocols for oil palm remain very
scarce. As we will describe further, the impact of problems in commercial
development during the 80's has been severe and it has certainly provoked a
reduction of the research effort in this domain.
In the present paper, we will focus mainly on recent research work
developed in the aim of improving tissue culture protocols which have already
proven to be efficient for oil palm, using modem approaches in the fields of
plant physiology and molecular biology.

3.2.1 Embryogenic cell suspension cultures

Progress has recently been made in plant production through somatic

embryogenesis, particularly by developing systems based on the artificial seed
concept (Redenbaugh, 1993), in which the somatic embryogenesis process is
used to produce individual embryos with relatively synchronous development.
Embryo development can be halted at a given stage, either by following the
natural procedure that occurs during zygotic embryo quiescence or by using
artificial methods, such as low temperature storage. The somatic embryos,
which are often encapsulated with antifungal/nutritive coating are then used as
seeds, either in vitro or sown directly in the field (Fujii et al., 1992).
Embryogenic oil palm cell suspensions have been initiated by several authors
(Teixeira et al., 1990, de Touchet et al., 1990) thus demonstrating the
feasibility of this technique for a rather "recalcitrant" model.
Research work has been implemented in our group (de Touchet et al.,
1990, 1991; Duval et al., 1995 a&b) in order to develop new methods of large
scale micropropagation for oil palm (see Bajaj, 1991 for a review).
In oil palm, embryogenic suspensions are established from friable, nodular
calli, which are isolated from nodular compact calli (Duval et al., 1995 a&b ).
The embryogenic suspension was prepared by dissociating the initial callus in a
stirred liquid medium, then by selecting the meristematic nodules that multiply
in the presence of 2,4-D during subsequent subcultures. Between 10 and 12
mg.r 1 of biomass, which represents more than 105 aggregates, were inoculated
into a medium containing 100 mg.r 1 of 2,4-D and 2 mg.r 1 of activated
charcoal. A series of histological observations revealed the considerable
256
homogeneity of the tissues and how the suspension proliferates. The
suspension growth rate was about 2 to 3 per one month culture cycle.
Embryogenesis expression was then triggered by the transfer of cell aggregates
to a growth-regulators free liquid medium for 4 weeks. The cell aggregates then
differentiated into proembryos consisting of meristematic cells. This stage ,
which amounts to weaning in auxin, is essential to stop proliferation and to
induce the expression of totipotency. At the end of this stage, the suspension
was filtered (1 mm mesh) so as to keep only the smallest aggregates, which are
the only ones capable of developing into an individualised plantlets. Embryo
maturation was then achieved by spreading the filtrate onto a solid medium, at
a rate of 0.05ml of PCV (Packed Cell Volume) per Petri dish, leading to the
differentiation of ca 300 embryos per dish under these conditions. The early
phases of this step was characterised by the formation of an epidermis, giving a
shiny white appearance to the embryos, followed by the differentiation of both
cauline and root poles. In many cases, only root development could be
observed. Modifications of the auxin:cytokin ratio through the adjunction of
SflM BAP(Benzyl Aminopurine) in the maturation medium led to a significant
increase in the number of individualised shoots , with a concomitant halt in root
development. The production scheme currently used in our team involves 4
distinct stages, as shown in Figure 8.
To date, embryogenic suspensions have been successfully isolated for
more than 20 clones. The average concentration was ca 10 5 cell clusters per
litre with a multiplication factor reaching 4x per month. These characteristics
allow mass propagation. Son dahl ( 1991) reported on the successful culture of
oil palm cell suspensions in bioreactors. For oil palm, Sondhal (Pers. Com.)
estimated the price of an encapsulated embryo produced by bioreactor
technology in the USA at ca 0.20 USD, to which has to be added he cost of in
vitro culture for germination , rooting and acclimatisation. The direct transfer in
greenhouse of coffee (Coffea arabica) somatic embryos produced by temporary
immersion has been recently reported (Barry-Etienne et al, 1998). Such
improvements, aimed at improving the quality of somaplants while reducing
the production costs, have a direct impact on the future economic viability of
the industrial production of elite plant material.
In the case of oil palm, it is clear that research efforts on the
implementation of production strategies based on the large scale use of
embryogenic cell suspensions has been severely hampered by the critical
problem of somaclonal variation. Field trials are under way for the assessment
of clonal fidelity in plantlets originating from cell suspension cultures.
 257
3.2.2. Maturation of somatic embryos

Storage proteins
Somatic seedlings are generally less vigorous than plantlets resulting from
zygotic embryo germination. This relative weakness may be the consequence
of incomplete maturation of somatic embryos (Crouch, 1982). Storage proteins
might be appropriate markers for the assessment of the maturation of somatic
embryos (Redenbaugh et al., 1986) and thus their ability to withstand
desiccation. In oil palm, our research work is aimed at understanding patterns
of storage protein accumulation in somatic embryo~ in order to: i) improve the
vigour of the in vitro regenerated plants produced through embryogenic
suspensions and ii) carry out medium term conservation of embryos at room
temperature as encapsulated artificial seeds (McKersie et al., 1995) or
alternatively by using cryopreservation (Engelmann, 1991; Dumet et al.,
1993a,b).

Oligosaccharides
Oligosaccharides are generally involved in the desiccation tolerance of
embryos (Leprince et al., 1993). These compounds have been demonstrated to
play a central role in the creation of a vitreous state and in the protection of the
cellular structure (raffinose and stachyose) against the crystallisation of solutes.
The [sucrose/(raffinose +stachyose)] ratio thus may be considered to a reliable
indicator of the capacity of embryos to withstand desiccation. In oil palm
zygotic embryos, this ratio was found to decrease dramatically from 68 down to
14 between the 3rd and the 4th month after fertilization and to drop to 5.2 at the
6th month (Aberlenc-Bertossi et al., 1995; Chabrillange et al., 1996). In oil
palm somatic embryos, resistance to desiccation is improved when embryos are
treated with abscisic acid (ABA) and sucrose at the end of the maturation phase
(Aberlenc-Bertossi, pers. comm.).

Cytokinins
Embryogenic suspension cultures of oil palm allow mass propagation of
somatic embryos; however regeneration rates are low. Histological
observations have revealed that shoot development might be limited by the
absence of a shoot meristem. The addition of ~-benzyladenine during
development was found to induce shoot apex differentiation and thus increased
germination rates, by up to 70 %. However, multiple shoot formation was a
consequence of a longer period of cytokinin supply during the development of
the embryo. In contrast, a short period of culture on medium with 6-
benzyladenine at the beginning of embryo development was found to result in
single shoot production (Aberlenc-Bertossi et al., 1998).
258
3.3 Cryopreservation

Research for the development of a cryopreservation protocol for oil palm

somatic embryos started in 1984, when laboratories producing clonal oil palms
on a large scale were faced with management difficulties associated with the
increasing number of clones produced. A cryopreservation protocol was set up
(Engelmann et al., 1985) and experimented successfully on a large scale.
However, the development of this technique for routine use was considerably
slowed by the low and erratic production of finger shaped embryos which were
at that time the only structures likely to withstand freezing (Engelmann, 1991 ).
Dramatic improvements were made to the former process by adding an embryo
dehydration step using silica gel, following a pre-treatment on high sucrose
medium, before freezing in liquid nitrogen (Dumet eta!., 1993a). The 7 day
pre-growth period previously used was completed by an additional dehydration
period carried out either by placing the embryos in the air current of the
laminar flow cabinet or in an air-tight box containing silica gel (Table 4). This
approach allowed the freezing of standard embryos, thus suppressing the
limitation imposed by the low production of finger shaped embryos; moreover,
proliferation recovery after thawing was generally higher and more rapid,
reaching up to 100% in some cases. This process was successfully applied to
39 different oil palm clonal lines (Dumet et al., 1993b). The effect of various
sugars and polyols on the tolerance to desiccation and freezing of oil palm
embryonic cultures was subsequently investigated (Dumet et al., 1994). When
embryos were cryopreserved after a desiccation period, survival was optimal
with fructose, galactose, sucrose and glucose, intermediate with maltose and
lower with other compounds. The recovery of proliferation was best with
embryos conditioned with sucrose.
Routine application of cryopreservation to oil palm somatic embryos is now
performed in several production units using this improved process.

4. SCALING UP TO PILOT STAGE

The transfer of technology from the research laboratory to the pilot

production unit scale was initiated in 1982. A production laboratory designed
for the annual production of 250,000 clonal plantlets was set up in Cote
d'Ivoire (West Africa). In 1985-1986, two laboratories based on the same
model were opened in Indonesia, with two different partners, one from the
public sector (IOPRI - Indonesian Oil Palm Research Institute) and one from
the private sector (SOCFINDO - Socfin Indonesia). At the same time, one
laboratory was established in Malaysia, in collaboration with a national
 259
development agency (FELDA - Federal Land Development Authority). A
subsidiary of CIRAD for the commercial development of the process
(TROPICLONE S.A.) was created in 1987 and a production laboratory was
opened in France (Rival et al., 1997d).

5. AGRONOMIC PERFORMANCE

The performances of 41 clones produced in the La Me laboratory (Cote

d'Ivoire) and Socfindo laboratory (Indonesia) using the same process have
been recorded in 10 different trials planted in Cote d'Ivoire and Indonesia
(Duval et al., 1997). In the trials as a whole, 31 clones produced substantially
more than the hybrid LM2T x DA 1OD used as a standard, with yield increases
of between I 0 and 54%. To date, the results collected confirm the possibility of
yield increases using selected clones (Table 5).

6. BOTTLENECKS FOR COMMERCIAL

PROPAGATION

The scaling up of the tissue culture process at various locations with

different partners has revealed almost everywhere the same difficulties.
Problems have generally occurred in two major fields: production costs and
the genetic fidelity of clonal vitroplants.

6.1 Production costs

Tissue culture of palms is generally time-consuming and the biological

events involved in each step of the process progress very slowly, as has already
been reported for coconut (Verdeil and Buffard-Morel, 1995). The total time
necessary between the sampling of the mother palm and the hardening of the
first batch of plantlets is 18 months on average (Duval eta!., 1995a). The latter
has a negative impact on the production cost, as overheads for the propagation
facilities remain very high. Because of the very high production costs, the
selling price of clonal plantlets could not be lowered to less them 5 times the
price of selected seeds (2 to 3 USD). The process as a whole is very labour
intensive, as several steps require very skilful manual operations (selection of
calli and competent embryogenic structures, separation of shootlets before in
vitro rooting, etc ... ). Furthermore, the regeneration protocols currently being
260
developed are unsuitable for large-scale production, i.e. 10 4-10 5 units per clone
per year. At present, only a few clones can reach an annual production of
around 10 4 plants. Finally, it appears that for a given clone, the culture and
production management approaches used determine its availability and when it
can be supplied. Thus, customer requirements have to fit in with the vagaries of
production, which is not acceptable from a commercial point of view (Rival et
a!., 1997d).

6.2 Somaclonal variation

The occurrence of vegetative abnormalities (stunted plantlets, goffered

leaves) in oil palm clonal offspring remains rare. The culling rates in pre-
nursery and nursery are measured at comparable ranges between vitroplants
and seed-derived plants.
When a limited number (less than a few hundreds per clonal line) of clonal
plantlets were produced by several teams at the research laboratory scale at the
end of the 70's, no severe problem of somaclonal variation could be observed.
After scaling-up of the protocols in pilot production units, evaluation in the
field has revealed the occurrence of a small percentage (ca 5%) of variant
palms which show an abnormal flower development, whatever was the
procedure followed to obtain somatic embryogenesis (Duval eta!, 1995a). This
character, originally referred to as "mantled" by Corley et a!. (1986) has been
characterised as a feminisation of the male parts in flowers of both sexes
(Figure 9). The alteration results in an abnormal floral development, by
modifying two of the inner whorls. This leads to abnormal morphogenesis of
the fruit, which bears supernumerary carpels around the drupe; hence the term
«mantled». The severity ofthe phenomenon varies and may have only a slight
influence on oil yields if it does not prevent fruit set (as it is the case with
« slightly mantled » variants), or lead to abortion of flowers, thus to complete
sterility of the palm(« severely mantled»).
Genetic fidelity was monitored in the field on 29 415 ramets from 127
clones. An abnormal phenotype, termed mantled has been reported to occur on
9, 7% of regenerated palms but this trait has a significant impact on the yield for
only 6% of the palms observed in average (Table 6).
The early detection of« mantled-type » somaclonal variants is thus today of
critical concern in oil palm clonal micropropagation.
 261
7. RESEARCH PROGRAMMES IN PROGRESS

Difficulties faced during the implementation of the scaling up of somatic

embryogenesis-based processes for oil palm micropropagation have
necessitated the launching of new research programmes which are currently
underway.

7.1 Maturation of somatic embryos

Using the zygotic embryo as a reference, storage proteins have been

analysed as potential biochemical markers of maturation. This study involved
firstly the characterisation of storage proteins in the zygotic embryo. Secondly,
a comparison of the accumulation of these proteins and their corresponding
mRNAs during zygotic and somatic embryo development has been carried out.
Thirdly, experiments have been performed aimed at identifying factors
favouring their biosynthesis. Somatic embryos originating from embryogenic
suspensions (de Touchet eta/., 1991) were used throughout this study, as this
technique enables the rapid production of large amounts of homogeneous and
synchronous material.
Storage proteins accumulated during oil palm embryo development were
extracted, purified and characterised (Morcillo eta/., 1997). Only water- and low-
salt-soluble proteins, with respective sedimentation coefficients of 2S and 7S,
were detected in mature embryos. After purification by gel filtration, the various
protein classes identified were characterised by electrophoresis and amino acid
composition analysis. The 2S proteins comprise polypeptides of 22 kD and 19 kD,
which are acidic (pi<6) and basic (pi>9) respectively. The 7S proteins
predominate and are heterogeneous oligomers (Mr of 156 and 201 kD),
comprising a polypeptide triplet of Mr between 45 and 65 kD with no disulphide
bonds. Their amino acid composition is broadly similar to those of the 7S proteins
of other monocotyledon embryos, but differs from those of the legume 7S vicilins.
Histological examinations and electrophoresis showed that the 2S and 7S proteins
appeared at the 3rd month after fertilization, and no qualitative changes were
detected up to the 6th month of embryo development.
The accumulation of 7S globulins was studied in embryos during seed
development and in single somatic embryos cultured in vitro from embryogenic
suspensions (Morcillo et a/., 1998a). Antibodies raised against these proteins
were used for their detection by Western-blot and quantification with
E.L.I.S.A .. (Enzyme-Linked Immunosorbent Assay). In zygotic embryos, the
7S globulins were found to be deposited mainly between the 14th and the 17th
post-anthesis week, corresponding to the end of the embryo growth. This
262
represents 10% of dry weight and 50% of soluble proteins. The amount of
soluble proteins and 7S globulins in somatic embryos increased rapidly during
the early stage of development, but were almost 80 times lower than in zygotic
embryos. In somatic embryos, 7S globulins represented 0.3% of dry weight and
4% of soluble proteins. After 22 days of development, the protein content
declined slowly, suggesting a lack of embryo maturation and an early
germination.
In order to improve embryonic maturation and the vigour of regenerated
plantlets, the effects of modifying in vitro culture conditions were investigated
with respect to the accumulation of the major oil palm storage proteins, the 7S
globulins (Morcillo et a!., 1998b). In this study, the effect of arginine and
glutamine on globulin accumulation was studied using somatic embryos of two
different genotypes by means of E.L.I.S.A. Arginine and glutamine were both
found to enhance protein accumulation but in different ways which were best
illustrated by measurements of soluble proteins per embryo and 7S globulin
content per dry weight. Arginine increased soluble proteins by 46% irrespective
of the clone, and glutamine by 19% and 63% depending on the clone. The
clone which accumulated the least protein in the presence of glutamine was that
which contained the most protein initially. Only arginine favoured an increase
in 7S globulin content on a per dry weight basis, irrespective of the clone
considered (+ 26% ).
This study allows further investigations of specific storage proteins as
potential markers for the vigour of regenerated plantlets, opening the way for
the use of strategies based on the "artificial seeds" concept for oil palm.
These "artificial seeds" will be made of embryos produced at a high rate from
embryogenic suspensions, then properly matured (thus enriched in storage
proteins). Single somatic embryos will be encapsulated and stored (whether at
room temperature or at 0-4 oq before delivery in due time, according to the
planting season, to tissue culture labs/nurseries situated close to the fields, in
which further development of embryos is achieved, in vitro or not.
A strategy based on the sequential cryopreservation of embryogenic lines
should be followed, in order to lower the risk of loosing the embryogenic
capacities of suspensions throughout multiplication. It is necessary to plan the
simultaneous production of a limited number of embryos from a large number
(10-20) of clonal lines in the aim of maintaining the widest possible genetic basis
for clonal production.

7.2 Physiology ofvitroplants

7.2.1 Physiology of in vitro rooting

Following induction by auxin treatment, the in vitro rooting performance of

oil palm shoots varies considerably. A recent study (Rival et al., 1997b) has
 263
described a preliminary evaluation of gaiacol-peroxidase activity as a marker of
in vitro rooting. It was carried out on 17 different clones obtained through the
standard micropropagation procedure; no rooting occurred in the absence of
exogenous auxins. Changes in peroxidase activity were found to be similar to
those previously described for other species, peaking between 10 and 14 days
under optimum initiation conditions (see review Gaspar et al., 1992). The peak
was found to shift in time when the rooting success rate was low. Peroxidase
activity measurements, carried out prior to induction on 24 production batches
belonging to 13 different clonal lines, revealed a significantly greater
heterogeneity in batches with a low success rate (>50%). Thus it was found
that root initiation was applied to material with a highly variable physiological
status. This study has enabled major improvements in the standard rooting
protocol, which initially involved two separate induction/expression phases. A
single rooting step is now used, with an auxin treatment applied over a much
longer time (8 weeks) using lower NAA (Naphthalene Acetic Acid)
concentrations (0.5-1.0 mg.l" 1). This quite long induction/expression phase
probably acts as a buffer stage, which is able to stabilise the physiological
status of shoots (peroxidase activity, level of endogenous auxins) before the
inductive treatment by auxin may act.
Studies on changes in peroxidase activity during the in vitro rooting of oil
palm clonal plantlets have led to the implementation of an improved rooting
protocol, which has been successfully developed on a large scale in production
units.
Improved protocols involving procedures closer to the physiological events
recorded will be tested: initiation over 10 to 15 days, followed by an expression
phase on a medium containing peroxidase inhibitors such as polyphenols
(rutin) (Moncousin and Gaspar, 1983), or substances known to promote
adventitious rooting, such as polyamines (Hausman et a!., 1995) and group D
vitamins (Buchala and Schmidt, 1979).

7.2.2 In vitro photosynthesis and acclimatisation

Several studies have been conducted in oil palm in order to reduce

acclimatisation losses (Rival et al., 1994, 1996, 1997c,d, 1998b). This problem
has a very severe impact on production costs, because it occurs at the ultimate
stage of the tissue culture process.
The in vitro photosynthetic parameters of somatic seedlings have been
measured throughout the ORSTOM-CIRAD somatic embryogenesis-based
process, with the aim of characterising the physiological status of the in vitro
regenerated plants and thus optimising success rates during acclimatisation.
Various photosynthetic parameters (photochemical activities, C02 exchange
and carboxylase enzymatic activities) were studied during four characteristic
264
stages of in vitro micropropagation: proliferating somatic embryos, shoots (1st
and 2nd caulogenesis cycles) and rooted plantlets (Rival et al., 1997c).
In vivo chlorophyll fluorescence measurements indicated that the maximum
photochemical activity of photosystem II (PSII) was very low in proliferating
embryos and strongly increased in later development stages, finally reaching an
activity very close to that measured in acclimatised plants. The quantum yield
of photosynthetic electron transport followed the same trend except that a
marked depression of electron transport activity was observed in the rooted
plantlets. C02 exchange measurements showed that absolute levels of in vitro
photosynthesis were low, but measurable.
Photosynthetic activity was also investigated by focusing on the primary
steps of carbon metabolism in plants. The activities of two of the primary
enzymes of C02 fixation, namely PEPC (Phosphoenol pyruvate carboxylase,
EC 4.1.1.31) and RubisCO (Ribulose 1,5-bisphosphate carboxylase, EC
4.1.1.39) were measured throughout the micropropagation process. The
PEPC:RubisCO ratio progressively decreased, due to a substantial depletion of
PEPC activity. Specific RubisCO activity did not show any significant
alteration, except a transient increase during the first caulogenesis stage on
gelified medium (Table 7). Quantitation of RubisCO was carried out via rocket
immunoelectrophoresis (Figure 10), using a polyclonal antibody raised against
RubisCO from green tobacco leaves. The relative amount of RubisCO
increased during somatic embryo development (from 3.2% in proliferating
embryos to 38.8% in 2nd-cycle-shootlets), then it decreased during the rooting
treatment (26.4%). The impact of sucrose enriched media (60 g.l" 1) on
photosynthetic activity is most probably involved in the decrease observed
during the rooting phase.
In order to characterise the physiological phenomena which occur during
the acclimatisation of in vitro-grown oil palms, a comparison of the growth and
carboxylase activities of in vitro propagated plants and seedlings was carried
out over a 100 day period (Rival et al., 1998b). Growth parameters (total FW,
relative foliar FW and the number of expanded leaves) and biochemical
characteristics (total soluble protein and chlorophyll content, specific PEPC,
RubisCO activities and relative RubisCO content) were studied. Oil palm in
vitro propagated plants were found to undergo an original pattern of
acclimatisation, as their PEPC/RubisCO ratio was not affected during
transplanting to the greenhouse environment and remained at the same level
(ca. 0.05) as was measured in in vitro growing leaves. At about D60 after
sowing (or ex vitro transplanting) the main physiological characteristics
(chlorophyll and soluble protein contents, and PEPC/RubisCO ratio) were
similar in both seedlings and in vitro propagated plants, but growth
characteristics were markedly different. Rocket immuno-electrophoresis (Rival
et a!., 1996) revealed that relative RubisCO amounts (Table 8) were in a
-1
comparable range (ca. 230 mg.g prot· ) in leaves from in vitro grown and
 265
already acclimatised in vitro-propagated plants and were found to be lower than
-I
in greenhouse-cultivated adult oil palms (350 mg_g prot- )-
All the studied photosynthetic parameters (carboxylase activities,
photochemical activity, C02 exchanges) indicate that, in oil palm,
photosynthetic activity could be measured as early as during the first
caulogenesis step, showing a noticeable increase during the second step_
Subsequently, photosynthesis decreased during root growth_
With respect to Grout's classification (1988), it can be assumed that the oil
palm belongs to the class of plants in which in vitro-grown leaves can
contribute to autotrophy and then play an active part in acclimatisation.
It is therefore highly probable that acclimatisation losses could be due
principally to poor or incomplete rooting and/or to a poor management of the
environment of vitroplants (especially concerning RH) during this very critical
step.

7.3 Genetic fidelity

The early detection of « mantled-type » somaclones is today of great

concern in oil palm clonal micropropagation. The availability of a reliable
marker will be the key for :i) sorting and eliminating abnormal lines as far
upstream as possible in the process and ii) improving tissue culture protocols in
order to lower the risk of "mantled" abnormality down to a level which could
be accepted by end users (Rival eta!., 1998a).

7.3.1 Biochemical markers

Several potential biochemical markers of the « mantled » abnormality in

oil palm have been investigated in our group. Polypeptide patterns (Marmey et
al., 1991) and endogenous cytokinins (Maldiney et al., 1986; Besse et al.,
1992) have been studied. Nevertheless, it has been very difficult to assess the
validity of such markers on a large number of samples, because of the lack of
reproducibility (in the case of proteins) or the high cost (in the case of
endogenous cytokinins) of such estimations.

7.3.2 Ploidy level

Flow cytometric analysis performed on two different crosses of dura x

pisifera oil palm has given an accurate estimation of nuclear DNA content
(Rival et al., 1997a). The genome size of Elaeis guineensis was found to be 2C
= 3,76 ± 0.09 pg and, therefore, ca. 3.4xl0 9 bp. Embryogenic calli and plants
266
showed the same ploidy level (Figure 11), but the measured qDNA values
differed significantly. No variation was observed in the ploidy level among 3
different types of calli originating from foliar explants, namely nodular
compact calli, fast-growing calli and friable calli. Since fast-growing calli
(FGC), already identified as a source of« mantled » phenotype variants, did not
show any difference in their ploidy level, these results are consistent with the
hypothesis of an epigenetic origin for this type of somaclonal variant.
Our results confirm those obtained by Jones et al. (1982), who clearly
demonstrated the presence of only diploid cells in oil palm plantlets
regenerated from tissue culture. The latter authors demonstrated that deviations
from cytological normality in calli and suspension cultures occurred, but it was
supposed that these abnormal cells did not contribute to embryogenesis. In our
experiment, flow cytometry did not revealed such abnormalities in callus
cultures. Kubalakova et al. ( 1996) reported on the occurrence of polyploid and
mixoploid cells during the regeneration of Cucumis sativus from embryogenic
calli. Conversely, Libiakova et al. (1995) noted that both long-term callus
cultures and regenerated shoots of hybrid Abies remained at the diploid level
during tissue culture. Konan et al. (1994) obtained the same type of results
when analysing the ploidy level of somatic-embryo derived plantlets of
Manihot esculenta by flow cytometry.

7.3.3 Random Amplified Polymorphic DNA (RAPD) analysis

RAPD analysis of leaf genomic DNA was efficient for distinguishing oil
palm clonal lines, but failed to reveal any polymorphism in genomic DNA
which could be associated with either "mantled" somaclonal variants or with
the overall tissue culture process used to regenerate oil palms (Rival et al,
1997e).
RAPD analysis efficiently differentiated oil palm clonal lines originating
from different mother palms. This result will be useful for further studies such
as clonal identification, the monitoring of progenies in seed production
programmes and in genetic mapping, thus complementing the data obtained by
Shah et al. ( 1994). For all of the primers studied and all clonal lines, no
significant RAPD pattern changes were evident either when comparing the
mother palm with its clonal offspring or when comparing normal and variant
phenotypes (Figure 12). RAPD analysis has thus failed to reveal any
polymorphism associated with the "mantled" somaclonal variants, even though
they are clearly identifiable in the field through their phenotypic characteristics.
Our results indicate that gross genomic structural change is rare or absent in the
tissue culture protocol under investigation. The sensitivity of RAPD for the
identification of somaclonal variants has been discussed by several authors
(Taylor et al., 1995; Wallner et al., 1996; Munthali et al., 1996). By means of
RAPD analysis, Valles et al. ( 1993) did not detect any variants in Lolium and
 267
Festuca regenerants. Thakur et al (1999) also observed no somaclonal variation
in Quercus serrata somatic seedlings. In a study on clonal fidelity using RAPD
in spruce, Heinze and Schmidt (1995) concluded that a low frequency of
genetic instability was present in the population of somatic embryo-derived
plantlets. Conversely, Brown et al. (1991) assessed the RAPD approach as
being very efficient for the study of DNA polymorphism in Triticum tauschii,
avoiding the need for more complex and expensive methods. Moreover,
Damasco et al. ( 1996) reported the identification of a RAPD marker specific of
the dwarf off-type from micropropagation of two cultivars of Cavendish
banana (Musa spp. AAA) , ·following the screening of 66 random decamer
primers. Recently, Godwin et al. (1997) showed that rice somaclonal families
differed significantly from the parental material using RAPD, indicating that
genomic alterations occurred in all families regardless of phenotype. Fourre et
al (1997) did not find any difference in Picea abies somatic seedlings using
RAPD, although cytological analysis detected chromosomal variations such as
mixoploidy or trisomy.
In our experimental conditions involving 259 primers, the proportion of the
oil palm genome covered by the amplification products scored for
polymorphism roughly averaged 0.04%. The probability of detecting
polymorphisms resulting from macroevents (deletions, insertions, substitutions)
at the DNA level within a PCR amplified region is thus very low.

7.3.4. DNA methylation

The role of DNA methylation in the regulation of gene expressiOn

(Finnegan et al., 1993, 1998) and its implication in somaclonal variation
(Brown, 1989; Karp, 1991; Phillips et al., 1994) has been extensively
investigated.
Significant changes in methylation level of genomic DNA during
dedifferentiation (Durante et al., 1982) and during somatic embryogenesis (Lo
Schiavo et al., 1989) have been reported for higher plants. It is, therefore, likely
that changes in DNA methylation levels and/or patterns could be involved in
the determination of somaclonal variation in oil palm, as has been
demonstrated for maize (Kaeppler and Phillips, 1994).
Long-term observation in clonal field trials have shown that the« mantled»
phenotype is unstable (Duval et al., 1997). The fact that almost 50% of sterile
palms revert to a normal phenotype after seven year's flowering backs up the
hypothesis of an epigenetic mechanism that affects genome expression during
the juvenile phase ofthe palm.
Levels of global DNA methylation were estimated in oil palm after
enzymatic hydrolysis of genomic DNA to nucleosides and HPLC
quantification (Figure 13) of 5-methyl deoxycytidine (5mdC) according to
268
Palmgren et al (1990) and Gehrke et al (1984 ). Global genomic levels of DNA
methylation [(5mdC) I (5mdC+dC)] have been investigated in regenerated oil
palms, with the aim of comparing mother palm/regenerants and normal/variant
regenerants within the same clonal line. Global levels of genomic DNA
methylation in oil palm were found to range 20 to 25%, in agreement with
levels already observed in several other higher plants (Klaas & Amasino,
1989). Recently, levels of DNA methylation measured have been found to
discriminate the "mantled" variants at the adult age (Rival et al., unpublished
results). In some clonal lines, analyses of the global methylation rates of leaf
genomic DNA have revealed a substantial demethylation in severely "mantled"
palms (20.6 %versus 22.2 %). The phenotypic fidelity of regenerants depends
on the type of embryogenic callus lines used for micropropagation (Duval et al,
1995a). Clonal· oil palm plantlets originating from nodular compact calli (NCC)
have been shown to exhibit the « mantled » variant phenotype at an average 5%
level, whereas this rate attained 100% in plantlets derived from fast growing
calli (FGC). Estimation of global methylation rates in genomic DNA from calli
of the two different types has revealed a significant hypomethylation in FGC
(23.2% vs 18.7%) when compared with NCC from the same clonal line.
Analyses of global methylation rates are underway in our group for the
monitoring of genetic fidelity throughout the in vitro culture process, by
estimating the role of the various growth regulators involved in the tissue
culture process on the methylation patterns of genomic DNA (LoSchiavo et al.,
1989).
It has been recently shown that Arabidopsis seedlings with reduced DNA
methylation displayed a range of abnormalities including altered leaf size and
shape, reduced fertility and homeotic transformation of floral organs (see
review Finnegan et al, 1998). Evidence for a direct relationship in oil palm
between hypomethylation of genomic DNA and the determinism of the
"mantled" somaclonal variant phenotype is still yet to be obtained.

8. FUTURE DIRECTIONS FOR RESEARCH

8.1 Embryogenic suspensions

The aim of our research in this field is to use oil palm artificial seeds to
improve the management, distribution and conservation of the clonal material
produced from embryogenic suspensions. Our results as a whole show that, at
the end of the in vitro development, the oil palm somatic embryos do not
display the characteristics of zygotic mature embryos. Thus, further research is
needed in order to obtain somatic embryos capable of withstanding desiccation
 269
and medium term conservation as artificial seeds.
The immature zygotic embryos are considered as useful model for making
studies of the acquisition of tolerance to desiccation. Various osmotica, growth
regulators (ABA) and slow desiccation in monitored conditions of hygrometry
are currently being tested in order to improve the tolerance of embryos to
desiccation. Studies on the impact of these treatments on parameters such as
oligosaccharide concentrations and storage proteins patterns are underway in
our group.

8.2 Molecular markers for the « mantled » somaclonal

variant phenotype

In order to investigate patterns of DNA methylation in oil palm during in

vitro micropropagation in relation with the somaclonal variation, we are now
using RFLP in conjunction with oil palm eDNA probes and isoschizomeric
restriction .enzyme pairs (Kovarik eta!, 1994) showing differential sensitivity
to the methylation of dC residues (e.g. Mspi/Hpall).
Furthermore, in the case of the « mantled » somaclonal variant, it is
conceivable that only a very small fraction of the genome might be affected by
methylation changes. Klaas and Amasino (1989) have demonstrated that active
chromatin in plants show reduced methylation rates. It will be certainly worth
analysing differences in methylation rates in this active chromatin fraction in
normal and variant regenerants, since the "mantled" somaclonal variant is
phenotypically detectable and must thus affect genes which are expressed.
In-depth studies are in progress in our group, in order to characterise
genomic DNA polymorphism that could be related to the "mantled"
somaclonal variant phenotype using the Amplified Fragments Length
Polymorphism (AFLP) approach (Vos eta!., 1996). AFLP analysis, which is a
very powerful tool for the analysis of DNA polymorphism, has recently proven
successful in generating molecular markers in oil palm (Singh eta!., 1998).
We are now developing a novel approach based on the analysis of
differential genome expression in normal/variant plant material. This approach
is centred on techniques available to study differences in the abundance of
specific mRNA species between populations. We are currently using the PCR-
based Differential Display method (Liang and Pardee, 1992) in order to
characterise gene expression in calli and embryoids producing' normal and
abnormal plants, in the hope of identifying an early marker of the "mantled"
phenotype.
270
8.3 Genetic engineering

We are now at only the very beginning of research on genetic engineering

in oil palm. Several projects are under way, with the aim of introducing foreign
genes into oil palm in order, for example, to modify oil yield and quality or
resistance to pests and diseases. Preliminary work has recently been published
by Chowdhury et al. (1997) on the evaluation of promoters for use in the
genetic transformation of oil palm using microprojectile particle bombardment.
The transient expression of reporter genes has already been obtained. To our
knowledge, the production of transgenic oil palms has not yet been achieved.
Nevertheless, recent progresses in the genetic transformation of
monocotyledonous plants such as rice (Christou, 1997; Hiei et al., 1997) and
the development of molecular genetics for oil palm (Mayes et al., 1997) will
certainly lead to major breakthroughs in this very,promising area.

9. CONCLUSIONS

Once the quality control of regenerants can be achieved at a sufficient le

using molecular markers, oil palm clonal micropropagation through somatic e
certainly evolve to a larger commercial scale, by means of powerful and lo
techniques such as the use of embryogenic cell suspensions. These techniqu
lead to automation of somatic embryos production in bioreactors, using com
analysis. Genetic stability of somatic seedling is also very important. E
somaclonal variations in cell lines could be beneficial and therefore molecul
will be needed to identify genetic variation throughout the production process.
A coherent network for Research and Development is now established, linki
in basic research (Universities and Research Institutes) on one hand, and key
palm sector in producing countries (private companies, development agen
agricultural research institutes) on the other hand.
A very close relationship between Biotechnology and Plant Breeding prog
essential in order to: i) efficiently select the elite material to be propagated, a
propagated material in the producing areas according to statistically designed fi
Results presented in this article illustrate, through the example of oil palm mi
somatic embryogenesis, both the importance of the pilot scale step in the scali
the ways in which the need to perfect this step can stimulate important rese
involving a fundamental approach with modern research tools.
 271
10. ACKNOWLEDGEMENTS

Research work described in this chapter was conducted under a joint

research programme between ORSTOM (lnstitut Fran9ais de Recherche
Scientifique pour le Developpement en Cooperation) and CIRAD-CP (Centre
de Cooperation Internationale en Recherche Agronomique pour le
Developpement- Departement Cultures Perennes).
Research on somaclonal variation was partially funded with the aid of a
grant from the Palm Oil Research Institute of Malaysia (PORIM) through a
Collaborative Research Project (n° CRB-96-001).
Many thanks are due to all the colleagues involved in the Oil Palm Project:
Fabienne Morcillo, Caroline Hartmann and Andre Rode at IBP (Institut de
Biotechnologie des Plantes) in University ofParis-Orsay; Frederique Aberlenc-
Bertossi, Frederique Richaud, Jean-Luc Verdeil, James Tregear, Thierry Beule
and Yves Duval at GeneTrop in Montpellier.
Research would not have been possible without the excellent collaboration
of our colleagues working in producing countries : CNRA at La Me in Cote
d'Ivoire, IRAB at Pobe in Benin, SOCFINDO and IOPRI in Indonesia, and
FELDA and PORIM in Malaysia.
Special thanks are due to Dr James Tregear for his English corrections.

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Scaling-up in vitro clonal propagation through somatic embryogenesis: the case of oil palm
(Elaeis guineensis Jacq). Plant Tissue Culture and Biotechnology, 3(2), 74-83.
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 277
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278
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 279
Table 1 : Palm oil production statistics ( 103 Tons). Source : Oil World.

1995 1996 1997

World 15 000 16 073 17 450

Malaysia 7811 8 386 9 108

Indonesia 4 040 4 450 4 990
Nigeria 630 620 620
Colombia 388 411 440

Table 2: Palm oil consumption statistics (10 3 Tons). Source: Oil World.

1995 1996 1997

World 14 627 16 042 17 220

Indonesia 2 115 2 503 2 730

E.U. 1 698 1 803 1 915
China 1 305 1211 1 661
India 768 1 222 1 290
Malaysia 1 098 1 236 1192
Pakistan 1 177 1148 1118
Nigeria 698 732 752

Table 3 : Performance of the ORSTOM/CIRAD process for oil palm

micropropagation in CNRA La Me laboratory, Cote d'Ivoire .

Genetic Origin Age Nb of palm Nbof Average

sampled embryogenic cultivation
clones period
(months)
Deli x LaMe Young 85 67 15
Adult 317 229 17
Deli x Yangambi Adult 37 33 20
Deli x La Me x Sitibi Adult 18 17 20
Deli x Nifor Adult 8 7 28
(Deli x La Me) x (Deli x Yangambi) Young 13 3 9
LaMexLaMe Adult 9 6 19
Back cross E. g. x (E.g x E. o.) Young 9 6 15
280
Table 4 : Water content of oil palm standard somatic embryos and
survival of unfrozen control (-LN) and cryopreserved (+LN) embryos from 7
differents clonal lines.
Pre-growth consisted of a 7 day culture on a medium containing 0. 75 M
sucrose followed by a 16 hours ofdehydration with silica gel.

Clonal line n°
1 2 3 4 5 6 7
Water content (%) 75±2 78±1 78±2 73±1 81±2 75±2 75±2
after pregrowth

Water content(%) 23±2 32±3 36±4 19±2 35±2 20±5 28±1

after pregrowth + dehydration

Survival 15/30 20/30 4/30 21/30 15/30 6/30 12/30

(-LN) = control (50%) (66%) (13%) (70%) (30%) (20%) (40%)

Survival 5/30 4/30 5/30 16/30 13/30 11/30 4/30

(+LN) (16%) (13%) (16%) (53%) (46%) (37%) (13%)
 281
Table 5 : Performance of clones produced at CNRA La Me as compared
with control cross (LM2T x DAlOD) in various trials planted in Indonesia
(AK-GP and BB-CL) and the Cote d'lvoire (LM-GP).

Clone Trial FFB Oil production % of standard

{Kg.ealm-1 ~f12 {t.ha-l.yr-12 cross
LMC 007 LM-GP 71 153.95 5.0 125
LMC 020 LM-GP 76 145.53 3.8 131
LMC 024 LM-GP 64 115.88 3.5 125
LM-GP 70 114.89 3.7 128
LM-GP 71 171.44 5.2 130
LMC 025 LM-GP 64 121.32 4.0 143
LM-GP 70 122.67 4.1 141
LMC 030 LM-GP 76 121.58 3.2 110
LMC 036 LM-GP 70 113.48 3.3 114
LMC 036 LM-GP 71 •158.4 4.6 115
LMC 043 AK-GP24 183 6.1 115
BB-CL 4 199.6 6.5 127
LMC044 BB-CL4 167.6 6.2 122
LM-GP 64 139.47 4.3 154
LM-GP 64 140.13 4.3 154
LMC047 LM-GP 71 168.96 4.8 120
LMC 051 AK-GP24 196 6.3 119
BB-CL4 174.7 5.7 112
LM-GP 70 113.96 3.7 128
LM-GP 71 163.13 4.5 113
LM-GP 76 113.39 3.3 114
LMC 056 LM-GP 64 119.31 3.5 125
LM-GP 71 158.72 4.8 120
LMC 065 LM-GP 76 116.07 3.3 114
LMC 073 AK-GP 24 223 6.9 130
LMC 074 AK-GP 24 211 6.1 115
BB-CL4 188.6 5.7 112
LMC 079 LM-GP 76 118.6 3.4 117
LMC090 AK-GP 24 187 6.2 117
 282
Table 6: Percentage occurrence of the "mantled" abnormality in somatic
oil palm seedlings at CNRA, La Me, Cote d'Ivoire.

Number Flowering Normal Slightly Severely

of clones palms abnormal abnormal
127 29,415 26,571 1,092 1,752
Rate 100% 90.3% 3.7% 6.0%

Table 7 : Carboxylase activities (RubisCO and PEPC) during oil palm

somatic embryo development.
Each value is the mean of at least 3 different measurements in triplicate, except
(*) one measurement in triplicate.
a-b-e-d== homogeneous groups (95% Tukey HSD intervals)

Culture RuBisCO PEPC PEPC/RuBisco

stage activity activity ratio
(f.!moles.h-l.mg-l TSP) (f.!moles.h- 1.mg-ITSP)
a a a
Proliferating 3.37 ± 1.01 5.21 ± 0.75 1.42 ± 0.41
embryos
b b b
Shoots 6.38 ± 0.48 1.83 ± 0.40 0.28 ± 0.05
(1st subculture)
a c b
Shoots 2.54 ± 0.28 0.18 ± 0.07 0.07 ± 0.02
(2nd subculture)
a c b
Plantlets 3.34 ± 0.89 0.18±0.14 0.06 ± 0.04
Acclimatized plant* 5.01 0.023 0.005
 283
Table 8 : Comparison of RubisCO relative amounts and specific activities
in oil palm seedlings and in vitro-propagated plants at various culture stages.
The data are the means of four replicate samples. Means followed by the
same letter are not significantly different as determined by the Newman (1939)
and Keuls' test (1952).

Plant material RubisCO specific RubisCO

activity relative amount
-I -I -I
(~mol COfh mg rot) (mg g prot)

in vitro shootlet 11.6a 252

acclimatised in vitro propagated 11.4a 231 A
plant (100 days)
adult greenhouse grown vitroplant 14.5b 354B
(300 days)
F(2,9l 11.3 31.9
p-level 0.0035 <0.0001
284

Figure 1 : Drawings of an oil palm, a fruit and a nut by N.J. Jacquin (1763) . In Hartley (1988)

Figure 2: Adult oil palm in IOPRI Estate at Marihat, North Sumatra (Indonesia)
 285

Figure 3 : Harvesting oil palm bunches in North Sumatra (Marihat Research Station , IOPRI)

Peanut Olive
3%
7%
Cotton Soya bean
seed 33%
6%

Rapeseed
17%
Palm oil
3% 26%

Figure 4 : Distribution of world production in edible vegetable oils (USDA-FSA 1996/97)

286

Figure 5 : In vitro callogenesis on an oil palm leaf explant.

Figure 6 :Somatic embryo (SE) developing on an oil palm embryogenic callus culture (nodular
compact callus : NCC) originating from leaf explant.
 287

Figure 7 : Oil palm polyembryonic cultures during in vitro proliferation.

 N
PROUFERATION PRETREATMENT MATURATION DEVELOPMENT 00
00
Liquid medium Liquid medium Solid medium Solid medium

Proliferation of Expression of somatic Differentiation Shoot and root

meristematic and embryogenesis of somatic embryos development
embryogenic clumps

1 month 1 month 1.5 month

Subculture f t~ 1
1 month
washing sieving subculture
&
plating
Dehydration
Encapsulation
Conservation
Figure 8: Procedure for in vitro regeneration of oil palm from embryogenic suspensions
 289

Severely Slightly
« mantled » «mantled » Normal

Figure 9 :Cross sections of normal, slightly mantled and severely mantled oil palm fruits originating
from somaclones.

116 SIS 514 •512 511 1\4 112 1J 214 ·212 211 3\4 312 311 414 412 ~1

Figure 10: Quantitation ofRubisCO protein sample in oil palm by rocket immunoelectrophoresis
using polyclonal antibodies raised against RubisCO from green tobacco leaves.

~ S!I6 to SII: Purified spinach RubisCO (Sigma R-8000) with respectively 0.07; 0.14, 0.27, 0.54,
1.07 f.lg total soluble proteins (I'SP) per well,
~ ~ to I II: Proliferating somatic embryos with respectively I. 02, 2. 05, 4.I 0 f.lg TSP per well
)- 214 to 211: In vitro grown shootlets (I" cycle) with respectively 0.49, 0.98, 1.96 pg TSP per well
~ .Y. to 311: In vitro grown shoot/ets (2tri cycle) with respectively 0.45, 0.90, I.80 pg TSP per well
)- 4/4 to 4/1: In vitro rooted plantlets with respectively 0.44, 0.88, 1. 75 pg TSP per well
290

Q~c~. . . .MU-..u~~._~~~----,~~
Proplclum lodido F I -
(Channel unit$)

Figure 11 : Histogram of fluorescence intensities in oil palm nuclei isolated from in vitro nodular
compact calli (NCC) and stained with propidium iodide.

Figure 12 : Gel electrophoresis of RAPD fragments obtained with somatic embryogenesis-derived

adult oil palms from two different clonal lines: LMC 51 and LMC 63 : [N], normal; (AN], abnormal
«mantled» variants. Primer OPJ 024 (Operon Technologies).

-
CdC

-
., SmdC G
... dG
.
T
CQ
N -
u =
:
=
..
<(
i
I
.
"!

- = ....
..
ll- ..
0

dA
-
.!r
.
y_}.
• . 'y _'w 1
I I
10.00 20.00

Figure 13 : HPLC chromatogram of nucleosides obtained after hydrolysis (nuclease PI/Alkaline

phosphatase) of genomic DNA extracted from an oil palm embryogenic callus line- type
FGC.
9. APPLIED AND BASIC STUDIES ON SOMATIC EMBRYOGENESIS IN
HAZELNUT ( Corylus avellana L )

Somatic embryogenesis in hazelnut

Rodriguez, R• (1).; Berros, B (1).; Centeno, M Luz (2); Rovira, M (3).; Rodriguez,
A.(l); Radojevic, L (4)

(1) Laboratorio de Fisiologia Vegetal. Dpto. BOS. Universidad de Oviedo (Spain). (2)
Departamento de Biologia Vegetal. Universidad de Santiago de Compostela. E.P.S.
Lugo (Spain). (3) IRTA. Centre de Mas Bove, Tarragona ( Spain). (4) Institute for
Biological Research, Belgrade.
* Corresponding author. E-mail: rrodri@sci.cpd.uniovi.es. C/ Catednitico Rodrigo
Uria sin. Oviedo, E-33071-Espafia.

Chapter contents

1. Introduction
2. In vitro multipliction
3. Physiological studies related with In vitro and In vivo multiplication
4. Somatic embryogenesis
5. Biochemical and molecular studies
6. Cryopreservation
7. Conclusions and future prospects

291

S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 291-359.
© 2000 Kluwer Academic Publishers.
292

1. Introduction

The common names of hazelnut or filbert are the species belonging to the genus
Corylus, tribe Coryleae, family Betulacea. They are classified in the order Fagales,
subclass Hamamelidae, class Magnoliopsida (Cronquist, 1981). The number of species,
that are widely distributed throughout temperate regions of the northern hemisphere,
varies considerably based on taxonomy. The most widely recognised species include
five shrubby species: C. ave/lana L., C. americana Marshall, C. cornuta Marshall, C.
heterophy/la Fischer, and C. siebo/diana Blume; and four tree species, C. colurna L.,
C. jacquemontii Decaisne, C. chinensis Franchet, and C. ferox Wallich (Thompson et
al., 1996). These plant species are cultivated for nuts, timber, and as ornamentals. The
European hazelnut, C. ave !lana, is by far the most economically important species, due
to the edible and organolectic characteristics of its fruits. The interest in other species,
from the genetic improvement point of view, lays on their potential uses e.g. cold
hardiness (C. americana, C. cornuta, C. heterophylla and C. sieboldiana) and may
produce nonsuckering rootstocks (C. colurna, C. jacquemontii and C. chinensis)
(Romisondo, 1976; Bergougnoux et al., 1978; Mehlenbacher, 1991; Thompson et al.,
1996). The cytological investigations indicated that all Corylus species studied so far
are diploid with 2n=22, except for a few aberrant forms (Danielsson, 1946; Salesses,
1973; Botta et al., 1986).
C. avellana is very rich in genetic diversity. Both genetic diversity and the
geographical distribution of cultivars and wild seedlings with similar traits, were used
by numerous taxonomists to subdivide the European hazel into distinct species: C.
maxima Mill. and C. pontica Koch. However, lack of any barriers to hybridization
among these "species" and the continuous distribution of morphological traits in their
progeny, indicates that these classifications are unreliable. Therefore, these two
species, C. maxima and C. pontica, should be included in one large polymorphic
species C. ave/lana (Koval., 1976; Mehlenbacher, 1991; Rovira, 1997).
 293
1.1. Botany of the genus Corylus

Plants of C. ave/lana (Fig.l.l) are multi-stemmed shrubs, 3-10m tall, occasionally

growth as high as 15m. Shoots are glandular pubescent. Leaves are 5-lOcm long,
rounded broadovated, abruptly acuminate, slightly lobed, and doubly serrate.

Fig. 1.1. Morphology of vegetative and generative organs ofCorylus ave/anaL.

a - a 1• Branch and inflorescence with male flowers; b. vertical section of ovary; c - c 1 leaves det.ail; d. female
flowers with stamens; e - e 1 immature and mature seeds, respectively (drawing J. Radojevic) (Radojevic,
1977).

Nuts (Fig. 1.2) are borne in clusters of 1 to 12, each enclosed in a husk which may vary
in length. The husk may be flared out, tubular, or constricted belong the nut
(Mehlenbacher, 1991). Up to third year, it has only the vertical root and later on
294
horizontal root hairs appear. The bark of the stem is smooth, ash-gray or reddish with
noticeable lenticels.

Fig. 1.2. Nuts ar"' hom in clusters.

The buds are very large (3mm in length) and covered with light brown lash-shape
shells. The leaves are long (6 - 16cm) and wide (5 - 9cm) dark-green on the front side
and light-green on the back side. The flowers are bisexual. The male flowers have 8
stamens. Two to four flowers are usually assembled in shape of aments. One ament has
four million pollen grains. They are formed in autumn and in spring the flowers open
themselves and pollinate female flowers, and arc further grouped in two inflorescences
from which red thread-like stigmas stick out. Two stigmas belong to one style. The
flowering is in February and March. The fruits appear in groups of 1 to 4, surrounded
by leaf-like green shells that later become brown and leathery. The fruit has one ovary
which produces only one seed and it has oval shape. Hazel tree multiplicates by seeds
and, vegetative by sprouts.

1.2. Geographic distribution and main producing areas

 295
Cary/us avellana L. is distributed in the forests and underbrushes of the temperate
zone of the northern hemisphere (Fig. 1.3).

15u_--~ __ _ L_ _~~~~~~--~~~

15 0 15 30 45 60 75 90
Fig. 1.3. General range of distribution of C 01y/us ave/lana L (drawing J. Radojevic) (Radojevic. 1977).

It often appears in association with Querco-Carpinetum and Fagetum mantanum and

usually forms dense underscrubs. It grows in lowlands and up to an altitude of 1800m.
The European hazelnut, Cary/us ave/lana L., was one of the first trees to
move northward as temperatures moderated and glaciers receded after the last glacial
period (17,000-18,000 B.C.) in the British Isles, northern Europe, and Scandinavia.
Hazelnut became the dominant vegetation during the Boreal period.
296
This enormous "hazel maximum" has been found repeated in the British Isles and
several other parts of Europe. Since this period, C. ave/lana is scattered in the west by
the Atlantic cost of Europe and North Africa, from about 32° N latitude in the Atlas
Mountains of Morocco to 68° N in coastal regions of Norway, and bounded in the east
by the Ural Mountains in the Soviet Union. The southern boundary of its distribution
extends from Morocco, Algeria and Spain in the west, through Italy, Yugoslavia,
Greece, and Turkey, to northern Iran and Transcaucasia. From Turkey it ranges south
into the mountains of Lebanon and Syria. The northern boundary includes the British
Isles, Scandinavia, and northern regions of the former Soviet Union to about 60° N
latitude (Thompson et al., 1996). Although the geographical distribution of Corylus
ave/lana L. includes a wide range of climates, the world's major production areas are
limited to only a few areas near large bodies of water. All are characterised by mild,
humid winters and cool summers (Mehlenbacher, 1991).
The world's main hazelnut producing countries are Turkey with around
425,000t inshell, Italy lOO,OOOt, USA 24,000t and Spain 22,0001. Hazelnut is also
being produced in Greece, the former Soviet Union, Iran, Romania and France, but
these countries do not have a major input in the world hazelnut trade. Since 1990,
world's production seems to have stabilised between 500-600.000t inshell, meanwhile
consumption rounds 450-500.000t inshell. It seems that in coming years Turkish
production could become higher, although cultivation expansion has been restricted,
while other productions could remain stable (Tous and Romero, 1997a).

1.3. Importance of the hazelnut

Hazelnut is mainly known as a fruit tree, as its fruit has a pleasant taste and it is highly
nutritive. The fruits contain 55-72% fat (they produce high-quality edible oils but these
easily tum rancid); 3-11% assimilated carbohydrates; 10-22% protein; 5-7% diary
fiber; 5-6% water and 2-3% minerals. Energy value is 600 caVlOOg of kernel. They
 297
also contain vitamins B 1, B2 , and C; and are an excellent source of vitamin E (250-
550ppm) (Tous and Romero, 1997a).
Hazelnuts have multiple uses and are sold in two different markets -the in-
shell market and the kernel market. Cultivars suited for one use are quite different
from those suited for the other. The in-shell market accounts for 5 to 10% of the world
hazelnut crop (Mehlenbacher, 1991). The remaining 90 to 95% of the crop is cracked
and kernels are sold to bakers, candy makers and other processors.

1.4. Cultivation and orchard management

Hazelnuts are cultivated in very diverse situations due to the enormous agroecologica/..
technological and socioeconomical differences of the producing countries. The
traditional training system in hazelnut orchards (Fig. 1.4) in Turkey, Italy and Spain,
has been a multistem bush, according to the natural tendency of bushing growth of this
species.

Fig. 1.4. Hazel orchard in Tarragona (Spain).

However, the training system used in the new orchards of the United States, France,
Italy and Spain is in vase with only one stem (Tous et al., 1994a). Tree spacing is
298
highly variable (from 800 to 400 treeslha), as it depends on the fertility of the soil,
rainfall, variety vigour and mechanisation requirements. General cultural practices to
improve the orchard management efficiency, are: pruning, sucker control, soil
maintenance, fertilising, irrigation, pest control and harvesting.
Tons et al. (1994a) reported three pruning systems used in hazelnut in
different areas of cultivation: training young trees, pruning mature trees and
rejuvenation pruning in aged orchards. Sucker removal is done, traditionally, in
winter, but also several herbicides are used during spring and summer (Germain, 1973;
Baronet al., 1985; Tous et al., 1994b). During the first plantation years, periodical and
shallow mechanical labours have to be given, tending to eliminate weed competition
and favouring the ventilation and soil moisture. In mature orchards, soil management
varies according to areas, climatic conditions and harvest type (Tous et al., 1994a).
Fertilization practice presents a considerable criterion diversity, it seems that the most
recommended quantities offertilizer is the equilibrium (N:P:K 1:0.4:0.9). Hazelnut is
a species without excessive capacity for water consumption but highly sensible to water
deficits during the whole vegetative cycle (March-November) (Girona et al., 1992).
Numerous pests and diseases can become a danger for hazelnut when their
attacks exceed certain limits, most of them can be controlled by cultural or chemical
means. Among pests, "hazel weevil",[Curcu/io nucum (L.)] is the worst enemy of
hazelnuts in Europe and Asia Minor attacks the tender nut and produce a typical hole,
destroying afterwards the kernel and causing severe damage. The "bud mite"
(Phytoptus avel/anae Nal.), present in all hazelnut producing countries, causes the
swelling of the female inflorescence and vegetative buds, followed by their biting.
Other pest diseases are caused by Zeuzera pyrina, Archips rosana, Myzocallis corili,
Corylobium avellanae and Nezera viridula (Bergougnoux et al., 1978; Barrios et al.,
1997; AliNiazee, 1998).
Hazelnut has some serious diseases problems. "Antracnose" is caused by
Criptosporiosis cory/i (Barrios et al., 1997), is a serious problem throughout Europe
and Asia; the fungus attacks buds, stems and catkins of hazelnut trees, causing their
desiccation. The caused agent of "Eastern filbert blight", is ascomycete Anisogramma
 299
anomala (Peck) E. Muller, originally observed on the American hazelnut in the
eastern United States, and currently it is a problem in Oregon State. "Canker disease"
caused by Cytospora cory/icola Sacc. and destroys the older branches, mainly in Spain
and Italy. "Sphaceloma" is another serious disease of hazelnut which is caused by
Sphaceloma coryli and affects different plant parts: leaves, young shoots and nuts.
Several other fungal diseases are known to affect hazelnut: Botrytis cineria Pers.,
described in France (Bergougn9ux et al., 1978); "root rots" caused by Armillaria
me/lea (Vahl.) Pat. and Rossillinia necatrix (Hart.) Berl.; "oidium" in the reverse of
the leaves caused by Phyllactinia guttata (Mehlenbacher, 1991; Barrios et al., 1997).
"Bacterial blight" occurs due to Xanthomonas campestris pv. Corylina attach
(Miller et al., 1949), and is a serious disease in nurseries and young orchards in
Oregon, eastern Europe, France and Italy (Miller et al., 1949; Bergougnoux et al.,
1978; Viggianni, 1994 ). In layer beds, bacterial blight affects stems and lateral
herbaceous shoots, symptoms include necrosis and drying-out; this disease can girdle
and fill young trees (up to 5-year-old age) (Mehlenbacher, 1991).
The most significant virus diseases is "hazelnut mosaic", which is caused by
the apple mosaic virus (ApMV). ApMV has been found in a large number of cultivars
from Spain, southern Italy and Turkey. The symptoms of the diseases are: chlorotic
ringspots, line patterns and flecking on older foliage (Man!naud and Germain, 1975;
Ragozzino, 1980; Aramburu and Rovira, 1998). The virus can be readily detected in
young foliage by ELISA test (Postman and Cameron, 1987).
Harvesting takes place from the end of August until middle of October.
Hazelnuts are harvested on the ground, picking them up in one or two operations. At
present, harvest is quite mechanised, except in Turkey where the fruit is recollected by
hand from the tree (Tous et al., 1994a). In the remaining producing countries, there
are three generalised harvest systems: harvesting by metallic broomer or blower
followed by mechanical cleaning, used in mountain areas of Tarragona and Italy;
harvesting by sucking machines, is the mechanical system in Spain and Italy and
harvesting by sweepers and pickup machines, system diffused in big orchards of
Oregon in the USA and France.
300
1.5. Conventional Propagation Practices and Improvement

In fruit and nut tree culture, vegetative or asexual propagation is done to conserve and
multiply the cultivar with a desirable trait. Corylus ave/lana L. reproduces naturally by
suckering, producing several suckers every year, which develop from the roots in the
vicinity of the trunk. Cultivars differ markedly in suckering, (Vidal-Barraquer and
Tasias, 1976; Bergougnoux et al., 1978; Thompson et al., 1978; Tous and Romero,
1997b). In Turkey, Italy and Spain, traditionally, rooted suckers have been used to
replace the old~r stems in the plants and to plant new orchards. This propagation
system is the simplest and cheapest, although it has some problems: roots hardly
develop, which is detrimental to the development of the plantation, and the varietal
authenticity and phytosanitary control are difficult to guarantee. In France and USA
countries, where hazelnut cultivation is only 25-30 years old, layering is a very
common method of propagation in the nurseries (Bergougnoux et al., 1978, Solar et
al., 1994; Aleta et al., 1997). Materials obtained by layering from a controlled mother
plant guarantee varietal authenticity and allow phytosanitary control, but on the other
hand, this propagation method needs an orchard destined only to grow mother plants
of different cultivars.
Hazelnut propagation by cuttings would allow to get a high number of plants
at low prices. Several researchers have been working on hard and soft cuttings and
varietal differences in rhyzogenesis have been observed (Paglietta, 1965; Lagerstedt,
1970; Roversi, 1972; Pistomik et al., 1982; Rodriguez, 1983; Ponchia and Howard,
1988; Solar et al., 1994; Aleta et al., 1997; Soylu and Etiirk, 1997). The results are
encouraging, However are yet to be available to the nursery growers for
commercialization. Several kinds of budding and grafting have been assessed at the
research level with a different success (Fregoni and Zioni, 1968; Paglietta, 1966;
Lagerstedt, 1976 and 1981; Me et al., 1981; Mena and Rovira, 1983; Achim and
Pamia, 1997). On the other hand, whip grafting system on bank applicating localised
 301
head, "hot-callusing pipe", (Lagerstedt, 1981) is a very useful method for producing
grafted hazelnut (Tous et al., 1997). But, while selected rootstocks are not available at
the market yet this method does not show enough interest for nurseries in front of
traditional propagation.

2. In vitro multiplication

Micropropagation of filbert is an attractive alternative for the amplification and

multiplication of selected cultivars and/or selected elite trees due to high in vitro
potential of this species and the relatively high amount of work performed in the last
ten years. Recent advances in plant production by in vitro tissue culture have indicated
that commercialization of plant production could be developed at reasonable costs in a
short period (Fig. 2.1 A-I).
The fact that certain genotypes or physiological ages can present some
problems for propagation. Thereby, the extent of knowledge currently available would
ensure that the limited research would overcome these problems.
On the other hand, filbert in vitro techniques are useful tools for studying (as
mentioned in this chapter) the molecular basis of reinvigoration, the physiology,
pathology and genetics of this species. Adaptation of these findings to other woody
species would always be possible.

2.1. Methods of Sterilisation of the Explants

Surface and, even more, endogenous contamination, problems are faced normally
while initiating cultures as well as also during in vitro multiplication, Additionally,
sterilisation requirements change according to age, phytosanitary treatments and
seasons, and therefore, it is nearly impossible to generalise any sterilisation procedure.
302

J.
"J

'fir-
!":.

'
",.
$,;l.
...
' . ' ~~ :,· .
..-·-:-· _j

,([ J ·~·
~1\\
,,
/

~'-
, ....... '
;' ~~.& " .
4~

·-·
•••

~ .i;4
~~L-
...
Fig. 2. L Different aspects of hazel micropropagation: A) Seedlings, B) Field-grown branches of mature
selected tree, C) Forced outgrowth and daails of primary explants, D) In vitro cuhure establishment and
initiation, E) Multiple shoot-bud formation on a double phase cuhure system, F) Responses of isolated shoot
tips in vitro, G) Rooted plantlets showing a well fonned root system, H) Microplants growing ex vitro.
 303
However, all the reported treatments are very similar, and may only differ in treatment
time and the concentration of the conventional steriling agents used.
Concerning the use of embryogenic material (Fig. 2.1 A), most of the reports
indicate that applicatioh of 5% (w/v) calcium hypoclorite for 20 min, and subsequently
dissection of embryos without any risk of further contamination. Some of these reports
concentrate on callus induction from immature embryos; induction of somatic
embryogenesis from embryos and cotyledons at different developmental stages
(Radojevic et al., 1975; Berros eta!.; 1994)
As we reported earlier (Perez et al., 1983a ; Rodriguez et a!., 1989) surface
sterilisation of cotyledons and whole seeds can be done by submersion in 85% ethanol
for 5 min, washing in sterile deionized water for 10 min, and with sodium hypochlorite
for 20 min. Asepsis is completed by rinsing with sterile water several times, sometimes
tensioactive agent (Tween-20 - Polyxyethylene sorbitan monolaurate, Sigma- or
commercial detergent) is helpful to allow a better contact with tissues for recovery
sterile and healthy explants derived from seedlings (Fig.2.1 A) (Perez eta!., 1983b; Al
Kai et al.,1984; Anderson, 1984).
Conversely, it is very difficult to establish a satisfactory method with juvenile
and adult plant material (Fig. 2.1 B), even when the selected trees were phytosanitarily
treated. There are different methods based on the application of 100 mg/1 quinoleine
sulphate for 4 h (Messeguer and Mele, 1983, 1985), or on the effect of mercury
chloride, whose toxic effect can be partially eliminated by addition of 2.5g/1 calcium
chloride (AI Kai eta!., 1984). In our laboratory after the standard procedure previously
cited (Perez eta!., 1987; Rodriguez eta!., 1989), better results were obtained by using
field-grown branches forced to outgrow after cold storage for three months (Diaz-Sala
eta!., 1990a). In this case (Fig. 2.1 C), field branches were first pre-sterilised for 2 min
in 0.8% (w/v) ferrous sulphate and 0.4% wide range fungicide (Captan) solution.
Outgrowth of axillary buds was stimulated by immersing the basal ends of the branches
in distilled water under controlled conditions for 15-20 days. These primary explants
were pre-sterilised in 80% (v/v) ethanol for 5 min and surface sterilised with 2.5%
(w/v) sodium hypoclorite plus o: l% tween-20 for 20 min. Afterwards, they were rinsed
304
several times with sterile deionized water and kept in a 2 g/1 DIECA
(Diethyldithicarbamic acid, sodium salt, Aldrich) solution for l hour to avoid
oxidation. All the treated material was subjected to a conditioning treatment in the
b2:>al culture medium without plant growth regulators. This step enables the selection
of vigorously growing tissues and allows elimination of the contaminated and damaged
material (Fig. 2.1 C).
Performance of severe pruning on selected trees greatly increases the recovery
of healthy explants and also the adaptation to in vitro conditions (Rey et al., 1994d).
Contamination is not seen at the onset of culture establishment; some internal
contaminants become t:vident at later subcultures and are difficult to eliminate. This
internal bacterial contaminants in filbert tissue culture were eliminated by
combinations of two or more antibiotics. Combinations of Streptomycin with Timetin
were effective in eliminating persistent bacterial contamination in hazelnut plants
(Kiaoling and Reed, 1995; Reed et al.,1998). Phytotoxicity varies with antibiotic type
and plant genotype.

2.2. Media comfJOsition for Meristems, Shoot-Buds and Stem Segments Culture

After reviewing the literature, we can conclude that the most frequent basal media
used for filbert culture are the MS medium (Murashige and Skoog, 1962) and the K(h)
medium (Cheng, 1975) as it was earlier reported (Rodriguez et al., 1989). But several
other media formulations are also used for hazelnut such as Radojevic et al. (1975), AI
Kai et al. (1984), Anderson (1984), Mena et al. (1985), and also slightly modified MS
medium (Diaz-Sala et al., 1990a). Generally, sucrose has exclusively been used as a
carbon source (Anderson, 1984; Messeguer and Mele, 1985; Perez et al., 1987; Diaz-
Sala et al., 1990b) except in a report of Kiaoling and Reed (1993) indicating use of
glucose. Some special mineral nutrient media developed for other woody plants have
been successful in hazelnut (Driver and Kuniyuki, 1984; Tulecke and McGranaham,
1985). In conclusion, no exhaustive study on the effectiveness of different individual
 305
medium components on filbert species has been brought to our attention. In any case
from the practical point of view, and for all types of hazelnut explants independently
of age (Fig. 2.1 A-G) we recommend two different formulations to get high
propagation yields: a) DKW media (Driver and Kuniyuki, 1984) supplemented with 6-
benzylaminopurine (BA) (1.5 - 3 mg/1) and indole-3-butyric acid (IBA) (0.01 mg/1)
plus 3% (w/v) glucose as it was reported by Kiaoling and Reed (1993) for mature trees
of hazelnut cvs. Nonpareil and Tonda Gentil Romana, or b) according to our
experience (Diaz-Sala et al., 1990a,b; 1994), MS2 media (MS salts with half-strength
nitrates but double strength calcium chloride and magnesium sulphate, plus 100 mg/1
myo-inositol, 0.5 mg/1 nicotinic acid, 0.5 mg/1 pyridoxine HCl, 2 mg/1 glycine, 0.4
mg/1 thiamine and 2 mg/1 ascorbic acid and 30g/l sucrose) plus BA (5 mg/1), indole-3-
acetic acid (IAA) (0.001 mg/1) and gibberellic acid (GA 3) (0.1 mg/1). On this procedure
after in vitro establishment, BA was reduced (1 mg/1) to allow a better performing
cycloclonal chain (Fig.2.1 D-F). This method was successfully used on mature
materials of cvs. Gironella and Tonda Gentille Delle Langhe. The double-phase culture
system (Viseur, 1987) improved the results (Fig. 2.1 F), when a constant amount (5-
10 ml) of liquid media was added after placing the primary explants in a vertical
(single bud) or horizontal (nodal shoot segments) orientation. It is important to
underline that the nature of the iron supply seems to be very important as FeEDT A
gave poor results with chlorotic shoots, whereas sequestrene 138 Fe (Ciba-Geigy),
yielded better results (Table 2.1).
Meristems (Fig. 2.1 G) with two leaf primordia (3-4 mm long) were
successfully developed into shoot-buds on a medium containing MS mineral solution,
2% (w/v) sucrose, 0.8% (w/v) agar, supplemented with (in mg/1) myo-inositol 100.0,
thiamine 2.0, nicotinic acid 5.0, adenine sulphate 2.0, panthotenic acid 10.0, plus 2,4-
dichlorophenoxyacetic acid (2,4-D) 0.001, BA 1.0, GA 3 1.0 and IBA 0.1; after one
month of development, 2,4-D was omitted from the media.
Table 1. Seed and organ culture for micropropagation and somatic embryogenesis w
0
0\
Period and
Age Explant Medium PGR(mg/1) Culture conditions Success Reference
passages
.........................................................................................................................................................................................................................................................................................
Immature Embryos MS (mod.) Kn I, 2,4-D I Agar0.8-I%
42 weeks per Callus and Radojevic et al.
12 h light
year embryoids (1975)
25 ± I "C
Mature Cotyledons 1/2 K(h) IBA 10, Kn I Agar 0. 7% dark-
20 days Callus and root Perez and Sanchez
ness or 16 h light
I formation Tames (1981)
25 ± 4 "C
Agar0.7%
Mature Seeds 112 K(h) BAP0-10 Multiple shoot bud Rodriguez and
16 h light 30 days
formation Fernandez (1982)
25 ± I "C
Shoot section
Agar0.8%
Adult including a z 0.3 -I 45 days Messenguer and
MS (mod.) 16 h light Growth
I year vegetative IAA0.3 I Meh! (1983)
25 ± I "C
bud
Agar0.7% 15 days
Asexual embryoids Plantlet
K(h) BAP0.5 18 h light I Perez et al. (1983 a)
in cotyledonary stage regeneration
25±4"C
Agar0.7%
20-day-old Cotyledonary K(h) IBA0.1-1 20 days
18 h light Embryogenesis Perez et al. (1983 a)
seedlings nodes BAPO.I-1 3
25 ± 4 "C
10 years Apical buds Standardi GA3 1, Agar0.7%
30 days
2,4-D 0.01 16 h light Bud growth Standardi (1983)
I
Knl 25 ± I "C
MS +Zuc-
cherelli Agar0.7%
Seedlings Shoot segments 16 h light Root
vitamins IBAO.I AI Kai et al. (1984)
formation
Sequestre-ne 25 ± 1 "C
138 Fe
MS + Zuc-
cherelli NAA0.01 Agar0.7%
2 months Axillary
vitamins GA3 0.1 16hlight 5 weeks Multiplication AI Kai et al. (1984)
seedlings buds
Sequestre-ne BAP5 25 ± I "C
138 Fe
 0.5 X Agar0.6%
35 days Plantlet
Seedlings Shoot segments strength IBA0.5 16 h light Anderson (1984)
1 regeneration
Ander-son's 20 "C
Agar0.6% Lateral buds
Young
Lateral buds Ander-son's 2ip 1,BAP2 16 h light 30 days breaking and shoot Anderson ( 1984)
seedlings
20 "C growth
MS or 1/2 Agar0.8% Shoot bud Messenguer and
Seedlings M !lA IBA 1-3 g/1 30 days
MS development Melt\ (1985)
IAA0.01
GA12+BAP, Agar0.8% Different rates of
Young 112MS Messenguer and
Shoot segments 2ip,Kn,Z 16 h light - shoot muhiplication
seedlings Mele (1985)
several 25 ± l "C and·development
concentrations
Adult 112 MS
Shoot segments
trees
Seedlings 112 K(h) IBA 10 Liquid 5days Perez et a!. ( 1985 a)
Plantlet
Juvenile ~ No regulators Solid 15 days regeneration
Shoot segments
Agar0.7% Muhiple shoot bud
20-day-old
seedlings
-
and
cotyledonary
nodes
112 K(h) BAP 16 h light
25 ± 4 "C
20 days formation and
elongation
Perez et al. (1985 b)

Seedlings IBA1.5g/l
Shoot 1-2 30 days Plantlet
juvenile 112 K(h) 10 s pretreat- Solid Perez et al. (1985 c)
segments 1 regeneration
adult ment
Agar0.7% Muhiple shoot bud
Unstratified Seedlings BAP,Z,Ad, Rodriguez et al.
K(h)andMS 16 h light 35 days formation and
seeds Kn (1985)
25 ± 1 "C cotyledonary leaves
BAP 2 em shoot Agar0.7% Plantlet
IBA 1 gil Rodriguez et al.
treated segments 112 MSor 16 h light regeneration and
IBA 10 s 10 days - - - · : _ . _ .... ~&'
(1984)
and untreated apical, K(h) 25 ± 1 "C
pretreatment
seedlings media, basal 25±1
Agar0.7%
Isolated
1/2 K(h) BAP 16 h light 20 days rormauon ana Yerez er a1. ~ IYlS 1J
embryos
25 ± 4 "C elongation

Vol
0
-....}
 Vol
Agar0.7% Bud growth 0
Adult Axillary 00
l/2 K(h) BAP 16 h light 20 days and basal Perez et al. ( 1987)
trees buds
25 ± 4 "C proliferation
Selected
BAP5+IAA Agar0.7% 40 days
mature trees Single buds and Establishment of Diaz-Sala et al.
ModifiedMS 0.01+ GA3 0.1 16 h light
cv.Tonda nodal segments cycloclonal chain 1990b
Double phase 25 ± 4 "C
Gentille
Selected Agar0.7%
10 s 40 days
mature trees Isolated Diaz-Sala et al.
ModifiedMS immersion 16 h light Rooting
cv.Tonda microshoots 1990b
IBA1 m!Yml 25 ± 4 "C
Gentille
Isolated
Seedlings, ModifiedMS BAP+IAA+ Agar0.7%
meristems 40 days Shoot-bud Fernandez et at.
juvenile, solid and GA3 16 h light
Rinsed DIECA development 1990
Mature liquid various 25 ± 4 "C
2!YI
BAP5+IBA
Isolated 0.05+K9 40days
Developing Tulecke Agar0.7%
embryos and Somatic
fiuits. 16 h light
cotyledonar BAP5+IBA embryogenesis and
cv.Gironell ModifiedMS Berros et al. 1995
portions 0.5/ BAP 0.5+ 25 ± 4 "C plantlet regeneration
IBA5 25+25 days
40days
Isolated buds BAPhigh Agar0.7% Increase of
Mature sequential Diaz-Sala et al.
and nodal ModifiedMS followed by 16 h light proliferation and
cv.Gironella subcultures 1994
segments reduction 25 ± 4 "C rooting
IBA1 m!Yml
Agar0.7%
Isolated plus Put, Spd, 40 days Promotion of
Mature ModifiedMS 16 h light Rey et al.1994 a
microshoots Spm rooting
25 ± 4 "C
various
BAP5+IBA
0.05+K9
Various stages of
Developing Isolated 40 days
Tulecke somatic
fiuits embryos and BAP5+1BA Agar0.7%
embryogenesis Berros et al. 1995
cv.Gironell cotyledonar 0.5/ BAP 0.5+ 16 h light
ModifiedMS induction and
andCasina portions IBA5 25 ± 4 "C
Plantlet
25+25 days
regeneration
2,4-D various
 309
2.3. Shoot Production and Elongation

Shoot multiplication was attempted almost with all possible type of explants. The
simplest way to obtain shoot proliferation is to directly sow seeds on a suitable basal
media containing cytokinins (Rodriguez, 1982; Rodriguez and Fernandez, 1982;
Rodriguez et al., 1989). The use of 6-Benzylaminopurine (BA) (8 mg/1) effectively
stimulated multiple shoot bud induction (three to five shoots/seed) and development in
90% cultured seeds. From our point of view, BA is highly effective shoot proliferator
and seed germinator inductor, its combination with an appropriate amount of kinetin
(K) or zeatin (Z) can increase proliferation on whole seeds.
When cotyledonary nodal segments were used as primary explants, initiation
and elongation of shoot buds were obtained after 15 days of culture in half-strength
K(h) medium containing BA (5 mg/1), followed by a 20-day culture period on the same
medium with reduced BA concentration (0.1 or 0.5 mg/1) (Perez et al., 1984b). When
seedlings used as source explants, Anderson, (1984) described BA and N6 -
isopentenyladenine (iP) combination whose crucial for high multiplication rate. Al Kai
et al. (1984) reported good multiplication rates using naphthalenacetic acid (NAA),
gibberelic acid (GA3) and BA at 0.01, 0.1, 5 mg/1, respectively. While working with
shoot segments taken from 1 year-ola branches, Messeguer and Mele (1985) reported
successful shoot outgrowth and bud development on MS medium supplemented with Z
and indole-3-acetic acid (IAA) (3 mg/1), by regular subculture for 45 days. We had
obtained (Rodriguez et al., 1989; Diaz-Sala et al., 1990b; Diaz-Sala et al., 1994)
reliable results, proliferation rate of 3.8, using apical buds (0.5 em) and/or nodal
segments (1.5 em) excised from: A) field-grown branches, B) newly developed shoots
from the forced outgrowth of axillary buds on "A" branches and C) newly developed
shoots from the forced outgrowth of axillary buds on "A" branches subjected to cold
storage. Results indicate that 3-month cold storage greatly enhanced morphogenic
capacity and reduced contamination and oxidation of tissues. Consequently, a
310
multiplication protocol could easily be established by culturing the tissues on a
modified MS medium containing BA (5rnll), IAA (0.01 mg/1) and GA3 (0.1 mg/1).
Both proliferation and elongation rates were significantly higher when double-phase
culture system (Viseur, 1987) was used on either the initiation or proliferation phases.
To our knowledge, the effect of carbon source on filbert shoot multiplication rates has
only been studied by Kiaoling and Reed (1993). Glucose was most effective on
sustaining shoot production, in the presence of BA (1.5 mg/1) and indol-3-butiric acid
(IBA) (0.01 mg/1). It seems that, in general terms, high yield of shoot production in
vitro is linked to repeated subculture in the presence of high cytokinin concentrations,
which seems to stabilise the response, allowing attainment of a high proliferation
plateau.
The improvement in shoot production from adult tissues was determined by in
vitro subcultures and intensive pruning. Intensive pruning increases proliferation in
initial explants and facilitates the establishment and maintenance of culture lines.

2.4. Rooting and Plantlet Establishment.

Hazelnut microshoots do not present real problems for rooting. In our laboratory (Diaz-
Sala et al., 1990b) rooting is normally accomplished by 10 seconds immersion in IBA
solution (0.1-1 mg/ml) followed by a 20-day culture on half-strength solid honnone-
free medium (MS2) under 16h photoperiod. Over 80% juvenile microshoots formed
roots and between 60-70% from adult plant material (Fig. 2.1 H,I). The rooting ability
of adult plant material varies with the degree of in vivo or in vitro reinvigorization, as
it was proved during the study of temporary modification of adult hazelnut
proliferation capacity by sequential subculture intensive pruning as a pre-treatments for
in vitro reinvigoration (Diaz-Sala et al., 1994). The same procedure, but including ex
vitro manifestation, was successfully applied to mature tissues of cv. Gironell
achieving almost the same rooting response. The root systems developed after this last
procedure were more complex and functional.
 311
A strong positive effect of exogenous polyamines on rooting of microshoots of
adult hazel cv. Gironell was observed (Rey et al., 1994a). The effect of polyamines on
both, root induction solution and rooting medium was assessed to study the effect on
the successive rooting phases. Polyamines improved rooting of IBA-treated
microshoots in a synergistic fashion, perhaps by favouring a better induction of roots,
with an acceleration of the response (only half time required for rooting as compared to
the control). When applied without IBA, polyamines had only a limited positive effect
on rooting, although longer exposure times and/or higher concentrations could
increase their effect. Possible rapid uptake and traslocation of polyamines in the ll:ylem
was discussed. In these experiments IBA was applied only in the immersion solution,
whereas the polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were
added both in the immersion solution and in the rooting medium. Put gave the best
results inducing 100% rooting. All polyamines tested at 1 mM concentration promoted
not only the percentage of rooting but also the number of roots per microshoost. The
results offer a new approach to enhance rooting ability of species that are normally
difficult to root.
The efficiency of Agrobacterium rhizogenes strains was also tested for rooting
on the same cultivars, higher 65% root induction occurs. Optimal results were obtained
by the application of both Agrobacterium and IBA, not only 90% rooting response was
obtained but also excellent root system was generated. However, the problem with
system is that sometimes the initiation locus of roots is unpredictable, and sometimes
interferes with plantlet survival. This undesirable effect varies according to the
topophisis; apical segments show the greatest polarised response, where all the roots
emerge on the basal end of shoot cuttings. Conversely, on the medium and basal
segments, roots frequently appear anywhere along the explant.
Regenerated plantlets grow well and can be maintained for long periods on
basal medium without IBA. Coordinated growth of caulinar and radicular systems is
maintained during this culture period so that continuous proliferation and plantlet
regeneration can be induced once again. This is achieved by excising 2cm long
312
microcuttings from the in vitro plants and following the same procedure already
described. Results sum up to 5 x in 5 weeks .
Microplants obtained through any of the mentioned treatments are normally
adapted to soil (90%) without practicably any losses (Fig. 2.1 1). The best procedure for
acclimation is a gradual transfer to sterilised ground or sterile peat-perlite mixture
under conventional conditions of temperature and humidity.

3. Physiological Studies related with In vitro and In vivo Reinvigorization

During several years, along with the definition of micropropagation, protocols for
hazel explants taken from trees of different ages, the study of the molecular traits
related with tree aging, were followed according to the scheme presented (Fig. 3.1).
The main results obtained are briefly presented on the next sections.

3.1. Polyamines as Developmental Indicators

Variations in endogenous polyamines levels were determined in leaves and buds of

mature hazel trees (Rey et al., 1994b). Results indicated a specific correlation between
high Spd and Spm levels, and rapid shoot growth and leaf expansion. Conversely, low
Spd and Spm, along with increasing Put levels, may be associated with the imposition
of shoot-bud dormancy. In previous studies on the morphogenic ability of hazel, and
taking into account that these explants can be easily established in vitro during
dormancy imposition, we observed that high levels of Put and increasing Put to total
polyamine ratios, could be related with high morphogenic capacity. Genotype
differences affecting the morphogenic potential could account for the observed
differences in the polyamine levels of the cultivars.
TISSUES CULTURE ADULT TREES SUCCESSIVE INTENSIVE PRUNING
EX VITRO REJUVENATION'!

MORPHOLOGICAL A.B.C.D.E.f
I I~ I A.BCG I~
FEATURING
[I] (A)
ONCE
-' PRUNNED
....
[[] MICROPROPAGATION ~ ----- -----
(8)
.:~ - --
ROOTING PHASES
)
(C)
~
=*l / l
EMBRYOGENESIS PHASES .... --.
(D) STIMULATED I
I I
0 MATERIAL TWICE
I ~
PRUNl'<'ED
I A,B.E.G I ,~ "'
ANALYTICAL TECNIQUES ~
PROTEINS ~
mill. (E)
~
DNA
m METHYLATION ~\If~ THREE TIMES
PRUNNED
L2J (F) CYCLOCLONAL
l ~ SEQUENTIAL
:¥
"fc,
SUBCULTIVES r:---~1' J
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Fig. 3.1. Schematic representation of hazel research at Oviedo University showing the relationship between reinvigorization, in vitro W
morphogenesis induction, and molecular traits.
314
The data obtained could also be correlated with seasonal changes of other endogenous
growth regulators in hazel as it has been already described (Rodriguez, 1983 ).

3.2. Polyamines as Indicators of Phase-Change and Reinvigorization

We have studied the endogenous polyamine concentrations in leaves from both

juvenile and mature hazel shoots, as well as in leaves from shoots obtained by both
forced outgrowth and micropropagation of adult tissues (Rey et al., 1994c). In order to
determine the observed in vitro reinvigoration was associated with polyamine
metabolism, we tested the effect of serial subcultures on polyamine concentration.
Polyamines, mostly free Put, were higher in juvenile tissues. Adult tissues
micropropagated for 14 subcultures, had polyamine concentrations characteristic of
juvenile tissues. However, with additional subcultures, total polyamine concentrations
decreased. The Put to Spd plus Spm ratio was higher in juvenile and micropropagated
tissues than in adult tissues, but decreased in micropropagated tissues after 20
subcultures. This ratio may reflect a balance between vegetative growth and
reproductive processes. Thus, an analysis of polyamine concentrations may provide a
simple assay for determining the juvenility of plant tissues and, hence, their suitability
for micropropagation.
The effect of in vivo reinvigorization was also analysed respect to the
endogenous poliamine content (Rey et al., l994d) in leaves and buds of adult and
repeatedly several pruned hazelnut trees. Polyamine content in leaves from shoots
obtained by forced outgrowth of branches taken from adult and pruned trees was also
determined. Variations of poliamine levels relation to pruning treatments were
observed in all the tissues analysed. Free poliamines increased in response to pruning
treatment, mostly due to an increase in free Put. Free Spd and Spm seemed to decrease
 315
with pruning intensity, whereas bound poliamines did not seems to correlate with any
treatments. Significantly, in all the tissues the Put to Spd plus Spm ratio increased in
the free fraction. The results indicate that the poliamine metabolism could play an
important role as physiological marker for juvenility and rejuvenation in relation to
cloning of woody plants.
The effect of repeated severe pruning on endogenous free polyamines content
in leaves and buds of adult hazel cv. Negretta was also studied (Rey et al., 1994d, c).
Polyamine levels varied in relation to pruning intensity, indicating that a
reinvigoration phenomenon was taking place. The Put to Spd plus Spm ratio increased
significantly in relation to pruning, supporting the view that the metabolic dynamics of
polyamines, more than individual concentrations of any one of them, may participate
in the control of plant ontogeny and the subsequent morphogenic potential. The
differences found in the polyamine between cv. Negret and other hazel cultivars such
as Gironell, can be related to their different morphogenic potential. This may indicate
that genotype differences affect both polyamine biosynthesis and metabolism as well as
morphogenic expression, taking into account the genetic complexity of these processes.

3.3. Age De(Jendcnt PolyiJCl>tide and DNA Methylation Patterns

Leaves from adult hazel trees maintained by sequential in vitro subcultures were
analyzed (Diaz-Sala et al., 1995). Qualitative and quantitative variations were found in
the in vitro tissues as compared to both adult and juvenile forms. The comparison
between different tree sources may conclude that in vitro hazel trees conditions show
specific biochemical and molecular patterns. The specificity of the induced changes
could be a prerequisite related to the high morphogenic potential of adult plants when
they are subjected to sequential in vitro subcultures.
Although the SDS-PAGE protein profile of leaves from different tree sources
was relatively similar, some specific changes were observed, two polypeptides, bands 9
and 20 were not detected in the in vitro line as compared to the adult and juvenile
316
fonns. In addition, a relative decrease of Rubisco levels was clearly detected (bands 4
and 18). This decrease was confinned by immunoblotting using antibodies raised
against the 55kDa.
The DNA fragment distribution profile obtained with Hpall was similar
between juvenile and forced outgrowth adult trees; however, the DNA obtained from in
vitro adult leaves showed a different distribution profile, thus reflecting a different
pattern of DNA methylation at CCGG sites. Similar results were obtained for
methylation at the GG sites when DNA isolated from forced outgrowth and in vitro
adult leaves was digested with EcoRII.

4. Somatic Embryogenesis

This morphogenic pathway was developed in fil'"ert following different alternatives

depending on the explant used. Somatic embryo!;enesis was induced on immature
embryonic axes or cotyledons using several growth regulators combinations. Cotyledon
nodes which gave rise to 60% embryogenic induction, and embryonic axes with 45%
induction represent the main two alternatives for plantlet fonnation. Possible
differences may be due to genetic variability, depending on the cultivars used.

4.1. Explants and surface sterilisation methods

The best results were obtained with immature zygotic embryos (Fig. 4.1), but
embryogenesis induction was attempted with a great range of plant materials
including vegetative and reproductive tissues such as: a) Embryogenic phase:
endospenn, immature and mature embryos, cotyledon and carpellary portions; b)
Juvenile phase: cotyledon nodes, proximal cotyledon portions, shoot tips, and intact
seedlings; and c) Mature phase: leaves and isolated shoot tips from in vitro
proliferating mature tissues.
 317

Fig. 4.1. Morphogenic responses and plant formation in embryo cuhures of C. avellana L: A) hazel tree, B)
Fruit sterilisation, C) Immature embryo isolation, D, E) Embryo cuhure on MS medium, 2,4-D and K, F)
Callus formation and multiplication on the same medium, G) Formation of embryogenic callus (EC) and
somatic embryos (SE) on MS medium without hormones, H) Formation of adventitious buds on MS medium
with GA3, I, J) Multiplication of SE on MS without 2,4-D, K) Hazel plantlet on MS medium with G~

(drawing J. Radojevic) (Radojevic, 1977).

318
When embryos were the primary inoculum, independent of the maturation
degree, high tissue recovery was obtained after sterilisation of seeds in 5% (w/v)
calcium hypochlorite and subsequent dissection of embryos. For other types of
explants, any of the sterilization methods previously described (see section 2.1) could
be used.

4.2. Culture initiation media

For explants derived from embryogenic and juvenile phase two approaches, direct and
indirect induction can be used.

4.2.1. DIRECT SOMATIC EMBRYOGENESIS INDUCTION

Two different basal salt formulations can be used to induced direct somatic
embryogenesis, modified MS (Murashige and Skoog, 1962) medium or T medium
(Tulecke and McGranahan, 1985), supplemented with: 0.6 mM m-inositol, 0.04 mM
nicotinic acid, 0.03 mM pyridoxine hydrochloride, 0.04 mM glycine, and 0.01 Mm
thiamine hydrochloride. Two different growth regulators treatments could be
successfully used Kinetin (K) (9 j.LM) plus BA (5 j.LM) plus IBA (0.05j.LM) for 40 to
60 days, or IBA (5 j.LM) plus BA (0.5 J.lM} for 25 days, followed by IBA (0.5 j.LM) plus
BA (5 j.LM) during other 25 days.

4.2.2. INDIRECT EMBRYOGENESIS

Indirect embryogenesis is obtained using the same basal media supplemented with 2,4-
Dichlorophenoxiacetic acid (2,4-D) (1.0-4.5 JJ.M) in combination with K (0.1-1.0 j.LM)
and/or BA (O.lj.LM). Variable results can also be obtained in presence of several
concentrations of BA plus K or BA plus IBA after longer periods of culture.
 319
It is important to emphasise that the most suitable auxin is IBA. Although
2,4-D may be used, this phytohormone at a high concentration appears to inhibit
subsequent developmental steps at the globular and heart stages (Radojevic et al.,
1975). Among the cytokinins tested, BA was the more effective in embryo induction
but adenine (Ad 0.7-7 !J.M) could also induce the desired response but at a lower
extent. In general, the evolution of embryogenesis (Fig. 4.2 A-H ) can be determined
by the time of culture in presence of 2,4-D, which mainly affects the polarity (Fig. 4.2
C-E). Also, induction culture time affects the embling yield.

4.3. Maintainance of Somatic Embryogenesis

Further proliferation of somatic embryos normally occurs from the newly formed
tissues, which under go similar embryogenic pattern of differentiation (Fig. 4.3 A-E).
First, proliferation is easily maintained on the same induction media (Fig. 4.3 A-D),
and embryogenesis decline on the fresh media without growth regulators.
Unfortunately, callus formation is progressively induced and interacts with embryo
proliferation. Furthermore, since somatic embryogenesis in hazel is not a synchronous
response, different stages of embryo organisation, embryo germination, shoot
development, and root manifestation are visible at a given time.
In order to avoid drastic reduction in the number of embligs produced,
cotyledonary somatic embryos or isolated cotyledonary leaves derived from
proliferating clusters may be used. The induction phase may be renewed by
transferring to the fresh media (Fig. 4.3 D). The renewal of induction phase takes place
in short periods, so that after 5 to 10 days rosettes of cotyledonary leaves and globular
embryos (Fig. 4.3 A-D) appear in almost 60% of the subcultured tissues. This type of
transfers can be performed every 40 days.
320

G H -
Fig. 4.2. Various aspects of somatic embryogenesis: A) Embryogenesis on immature complete seed, B)
Embryogenic cells, C) Globular stage of somatic embryo, D 1, 2, 3) Heart, tubular and cotyledonar stages, E)
Mature somatic embryos, F, G) Embryogenic cluster showing precocious germination and plantlet formation ,
H) Plantlet formation.
 321

Fig. 4.3. Reiterative or secondary embryogenesis: A, B, C) Embryogenesis multiplication, D) Embryogenesis

on an isolated cotyledonary leaf, E) Plantlet formation with inhibition of the root system.
322
4.4. Embryo germination and plantlet formation

The recovery of total embryogenic ability of the explants is difficult mainly due to
anastomosis and nonsynchronous development (Fig. 4.4 A-G). The limiting factors
which affect the percentage of true to type emblings and plantlets are: type of initial
explants, growth regulator concentrations and culture time.
Both shoots and plantlets can be spontaneously developed from embryogenic
tissues on the same proliferating media, and regenerate normal plantlets (Fig. 4.5 left
and right). Transfer of embryogenic clusters to growth regulator-free medium or to low
growth regulator media allows plantlet formation from 40% clusters. Furthermore, due
to the culture conditions and the high embryogenic potential, precocious germination
of somatic embryos may occur and regenerate plantlets.
Both shoots and plantlets, originating from somatic embryos, are easily
multiplied by micropropagation program. The presence of reduced BA (2,5 J..LM) in MS
medium is needed to maintain a cycloclonal proliferation line. Rooting of the tissues is
readily accomplished by base dipping in a high IBA concentration for few seconds (see
2.5 section).

4.5. Maturation of somatic embryos

Somatic embryos are difficult to avoid secondary embryogenesis competition with

embling formation, probably due to the lack of maturation on these newly induced
organs. Treatments such as gibberelic acid (GA3), abscisic acid (ABA) and low
temperature were applied to both isolated or clustered embryos. The addition of GA3 ( 1
mg/1) facilitates regeneration of well developed plantlets, however, frequently
secondary embryogenesis occur along the newly obtained plantlets.
 323

Fig. 4.4. Plant regeneration: A) Embryogenic callus (EC) on MS medium with 2,4-D and K, B- D) EC with
somatic embryos (SE) at different &tages ofthe development on MS medium with 2,4-D and K, E) Secondary
SE (sSE) developing on the cotyledons on MS medium with GA3 and K, f) Adventitious buds (AB) formation
on the same medium, G) Hazel rooted plantlets on MS medium containing GA3 and K. (Radojevic, 1977).
324

~ (~~~
"7~"
Fig. 4.5. Nonual and twin plantlets of hazel obtained by somatic embryogenesis (left) and adventitious buds
via organogenesis (right) (drawing J. Radojevic) (Radojevic, 1977).

4.5.1. ABSCISIC ACID EFFECTS

Clumps of somatic embryos formed by a variable number of units (from 2 to 12

embryos) were used to favour embryo maturation. The inoculates were initially
cultured on MS medium supplemented with ABA (0,4 ~M) under continuos darkness,
yielding developed somatic embryos from 75 to 90%. However, culture of somatic
embryo clumps formed by 6-8 embryos helped to attain the development higher
number of cotyledonary leaves by using clumps after different culture periods (20, 40
and 60 days) we established that any type of cotyledonar embryo can develop in the
presence of ABA (0.4 ~), which simultaneously inhibits repetitive embryogenesis.
 325
Maximal cotyledonary development was achieved in the presence of ABA (0.4
j.!M) and only high ABA concentration (4 j.!M) inhibited precocious root development.
Secondary embryogenesis was only inhibited in the somatic embryo clumps treated
with high ABA concentrations. In any case, independent of ABA concentration used,
the percentages of germination and development of somatic embryos were about 40%.

4.5.2. EFFECTS OF TEMPERA,TURE

Low temperature (4°C) maintained during 60 days enhanced the number of germinated
somatic embryos, as well as of developed shoot-tips (85.7%). However, the percentage
of juvenile-looking shoots was very low (16%).

4.5.3. OTHERS

Sucrose, mannitol and polyethyleneglycol (PEG) (0.2 M) inhibited induction of

repetitive embryogenesis, and allowed development of cotyledonary leaves and
precocious gemination. These treatments resulted in 12.5% explants forming juvenile-
looking emblings. However, both mannitol and PEG induced partial necrosis in the
embryo cultures whereas this negative effect was not observed with sucrose treatment.
These treatments resulted in high germination rate but the number of juvenile-looking
plantlets was still rather reduced. Therefore, treatment with high ABA concentrations
followed by desiccation (with sucrose) could be compulsory to enhance percentages of
desired development.
The regenerated plantlets were transferred to peat: perlite (1: 1) and after 30
days in the growth room, further elongated shoots and newly formed leaves appeared.
Subsequent adaptation to greenhouse conditions was 100%.
The main histologycal differences between immature and mature somatic embryos
were:
a) Distinctive cell differentiation in cortical and medullar parenchyma of mature
embryos. No tissue diversification in immature embryos.
326
b) Primacy vascularization in immature embryos. Tracheas are present in mature
embryos.
c) Shoot-tip differentiation and development preceeding root-tip differentiation, in
both embryo types.
These characteristics confirm that there are different capabilities of plantlet
fonnation and development derived either from precocious or true to type germination
of somatic embryos.

4.6. MorJ)ho-histological studies

4.6.1. ANATOMICAL STUDY OF SOMATIC EMBRYOGENESIS

The cross sections through hazel embryogenic callus (EC) containing embryogenic
nodules (EN) show (Fig. 4.6 A) that below a layer of large, partly disrupted superficial
cells, there is a compact continuous layer of long and small, ovoid meristematic cells,
rich in organelles and cytoplasm. Towards the centre of the EN-s cells, become larger
and vacuolated; the small cells divided by many periclinal divisions and form
spherical outgrowths. At the surface of the EN, small ovoid meristematic cells divide
by· anticlinal division and form many somatic embryos at different stages of
development (Fig. 4.6 B-D).
After the transfer of EC, differentiation progressively takes place and three
month later, masses of vacuolated cells with small nuclei which surround somatic
proembryo-like structures can be seen (Fig. 4.7 A). Also, the embryogenic single cells
were frequent in these calli, and easily discernible, due to a thick and highly stained
cell wall, big nuclei and dense cytoplasm with numerous starch granules. The two (Fig.
4.7 A, arrow), three and early globular somatic embryos (GSE) (Fig. 4.7 B-C) are
similar to the corresponding stages of zygotic embryogenesis.
 327

, t00Mm ,
)

Fig. 4.6. Anatomy of C. ave/lana somatic embryogenesis. (Fix: Camoy's. stain: Delafielde hemato:--yline:
gret.>Jl titter): A) S"'ction !rom one embryogenic nodul"' (EN) of the embryog"'nic callus (a) continuous tissue
of small cytoplasmic cells (b). meristematic centres (me) and spherical outgrmnhs (d) in the surface of EN.
Scale bar~ lOO ~un, B-D) Somatic embryos in globular (GSE). torpedo (TSE) and on cotyledonary ;,tages
(SECo) of development. Scale bar ~ 50: 100 and 100 ~un . respecti vely. (Radojevic. 1975).
328

Fig. 4.7. Differentiation and development of somatic embryos in embryogenic callus (EC) of C. ave/lana
cuhivated on MS medium without 2,4-D. (Fix: Camoy's, stain: Delafielde hematoxyline; green fiher): A)
Numerous proembryo-like strucrures (Pe) in the EC (x 560), B) Three celled embryo (x 1920), D)
Muhicellular embryos (x 560). (Radojevic, 1977; Radojevic a al., 1983).

4.6.2. SOMATIC EMBRYOGENESIS THROUGH CALLUS FORMATION

Indirect responses are mainly obtained when embryonic axes or cotyledon portions are
cultured on 2,4-D containing medium. Macromorphological characteristics appear to
be represented mainly by callus proliferation without external manifestation of somatic
embryogenesis.
Calli are composed of large cells with wavy walls fonning vast and
disorganised masses. Internally, two type of organisation may be observed:
embryogenic structures and vascular and meristematic organisations.
From zygotic embryos, globular stages with a spherical appearance (Fig. 4.5,
4.6 and 4.7) and heart-shape formations are easily distinguished. Globular formations
are composed of small cells with a dense cytoplasm. The heart-shaped structures show
a bipolarity, not only through differential staining but also through the organisation,
which seems to be characteristic of more developed embryogenic stages (Fig.4.5 and
4.6).
 329
When cotyledon portions are cultured, vascular strands are induced.
Meristematic centres consist of small cells that may occasionally coexist with
prevascular elements located in the central zone, which later on develop into the
radicular primordium. This result is confirmed by the manifestation of external roots
when 2,4-D is used.

4.6.3. DIRECT SOMATIC EMBRYOGENESIS

Macromorphologically, all the responses induced on media deprived of 2,4-D

correspond to the formation of roots and somatic embryos without callus manifestation.
Induction occurs at the most superficial cell layers of the explant, originating from the
parenchyma cells, which give rise to continuous bands of embryogenic cells. However,
it cannot be excluded that these sources could be either the origin of embryos or
cotyledonary leaves. However, the constitution of diads, tetrads, and cellular groups
with densely stained cellular walls, may show that a high percentage of the somatic
embryos constituted is unicellularly originated.
The histology of embryos at the cotyledonary stage shows as a characteristic of
this phase, the differentiation of the pole surrounded by scarce vascular and
cotyledonary leaves. Histological sections of the cotyledonary leaves show that
differentiation gives rise to a mesophyll without a clear pallisade layer nor spongy
parenchyma. A completely independent ovoid bundle is located longitudinally and
transversally in the future embryonic axis, in which the radicular pole is not yet
differentiated.
The development and the complete singulation of the embryos can occur
simultaneously or subsequent to the differentiation of the radicular pole. At this point,
cells of the vascular system presenting helicoidal enlargements, typical of this stage,
can be observed. The parenchymatous cells of the somatic embryo present a superior
diameter when compare with less developed cotyledonary stages. The dominant
longitudinal growth can be explained by the location of meristematic cells, exclusively
at the caulinar and radicular meristems
330
Although most of the embryos give rise to a cluster, it has been shown that the
different four typical structures can coexist, giving a certain non-synchronous character
to this type of induction.

4.6.4. REITERATIVE EMBRYOGENESIS

According to the macroscopic organisation of the induction of embryogenesis in the

absence of callus, abundant bands and superficial formations would mean that the
embryos originated from parenchymatic cells. The anastomosis and the small number
of rooting stems from the embryonic cluster can be explained by cotyledonary stages,
usually characterised by the absence of differentiation from the caulinar meristem, and
ovoid vascularization composed of cells with more or less embryogenic characteristics
from which new immature, vascular systems develop. Occasionally, structures
corresponding to first precocious germination have been observed. These are
characterised by an abnormal development of the pole, corresponding to the hypocotyl.
In this area, a spherical structure with its own vascular bundle develops. There is no
connection with the vascular tissue of the initial embryo, and a typical ovoid structure
of the somatic embryos does not appear.

4.6.5. HISTOCHEMICAL CHARACTERISATION

Histochemical characterisation of embryogenic callus for sugars, lipids and proteins

was carried out (Radojevic, 1977). The large cells located at the callus surface as well
as deeper long cells, show very positive PAS reaction (Fig. 4.8 A, C). Conversely,
meristematic cells do not react with the same intensity, even when certain groups of
meristematic cells contain high number of different sizes grains, which are PAS
positive (Fig. 4.8 B) amylase test has showed that these structure are starch grains.
Other structures and starch grains which are PAS positive, become PAS negative after
the acetilation (Reaction C). Saponification (Reaction D) turns these structures back to
 331

Fig. 4.8. Histochemical detection of carbohydrates in embryogenic callus (EC) and somatic embryos (SE) of
C. avellana. (Fix: Bouin; stain: PAS-Groat hematoxyline; green fiher): A) Carbohydrates in EC. Note the

strong PAS reaction in the superficial layer of an embryogenic nodule (El'<') (x 310), B) Abundance of the
starch grains in the meristematic cells ofEC (x 480), C) Cells of globular somatic embryos (GSE) with poor
starch contains (x 490). (Radojevic et al. , 1979).

PAS positive. Cell wall and starch grains of globular somatic embryos (GSE) are PAS
positive (Fig. 4.8 C).
The localisation of total lipids in the EC was determined by black Sudan B
staining. Superficial layers contain more lipids than the rest (Fig. 4.9 A, arrow). These
cells and meristematic layers are full of lipid granules of different sizes (Fig. 4.9 A,
asterisc) in contrast with the cells from the middle of the callus. In GSE all cells
contain moderate quantities of lipid granules, which are irregularly distributed. With
332

Fig. 4.9. Lipid distribution on embryogenic callus (EC) and somatic embryos (SE) of C. avellana. (Fix;
Fonuol-calcium; stain: soudan black; orange filter): A) Numerous lipid granules (black stain) in the
superficial layer (arrow) of an embryogenic11odule (EN). Note the voluminous lipid granules (asterisk) in the
meristematic cells ofEC (x 490), B-C) Heart-shaped somatic embryos (HSE). In superficial layers of SE lipid
contains is reduced (x 200; x 490), D) Torpedo-;tage ofSE with high contains of lipid granules at superficial
layer (x 125). (Radojevic et al., 1979).
 333
the differentiation of GSE to heart-shaped stages (HSE), the distribution of lipids
follows the same pattern than in callus, e.g. most lipids are in cells at the surface of
the SE (Fig. 4.9 B-C). Lipid granules in the centre of SE, later at the developmental
stages have smaller size (Fig. 4.9 D). Nill blue colour permits to visualise simple
lipids, which stain pink and acid lipids stain pink-violet. The presence of acidic lipids
is very rare, but they can be found in large cells at the surface of callus, and only in the
latest developmental stages of somatic embryos.
The detection of proteins with terminal NH2 was accomplished by the
.alloxane Schiff reaction; the higher reaction was noticed in larger cells, EC
superficial cells, and also in the meristematic cells. The layers of ovoid meristematic
and parenchymatic cells from the centre of the callus do not react to this cytochemical
reaction. By the tetrazo reaction of Danielli, the presence of numerous amino acids
among which are tyrosine, tryptophane, histidine and arginine in EC is detected.
Positive results of this reaction are recognised by the presence of a violet colour in the
tissue. Some large cells at the surface of the callus, which react positively (Fig. 4.10 A,
B), often contain starch grains which are negative to the tetrazoreaction (Fig. 4.10 B,
arrow). To determine the individual nature of the detected amino acid we used Morel's
and Sisley's specific reaction. A colour change from rose to purple-rose indicates the
presence of tyrosine, mostly found in large cells at the surface of the callus and in the
long meristematic cells of the EC (Fig. 4.10 C). For the detection of reductive groups,
among which are SH-groups, in the callus tissue we used the reaction with ferric-
ferrycianide which gave similar results as DDD reaction. The cells showing positive
response to this reaction become blue. Blue bromophenol and picric acid stained
proteins in the cell nucleus of the callus. The nuclei in large, long cells have big
affinity for this stain. Cell's nucleoli from the meristematic layer appeared stained by
this reaction. Nucleoli are very small in SE at early stages of development and become
voluminous at the later stages of differentiation.
Among the applied reactions for monochromic staining of nucleic acids in the
EC and isolated SE, only the reaction with gallocyanine gave positive results.
334

Fig. 4.1 0. Presence of proteins in embryogenic callus (EC) of C. ave/lana. (Fix: Bouin; stain: truazoreaction
ofDanielli's and DDD reaction; green fiher: A) High protein cootents on superficial and meristematic cells (x
125), B) Detail of starch grains (arrow) surrounding the nucleus of superficial cell layers (x 490), C) Detail of
hazel EC. Note that SH-groups are more abundant in long meristematics cells (DDD reaction) than in ovoid
cells (x 310). (Radojevic a al., 1979).

Gallocyanine is connected with phosphate groups of nucleic acids yielding a blue

colour compound found mostly in nucleoli and a part of the nucleus close to the
nuclear membrane of EC long cells. A lower reaction was shown by the ovoid cells
from meristematic layer. Cells from the centre of the embryogenic nodule (EN) reacted
negatively (Fig. 4.11 A-B). Early stages of SE had very big affinity for the gallocyanine
dye (Fig. 4. 11 C) in contrast with the embryos at later stages of development, where
only cells from certain layers showed positive staining (Fig. 4. 11 D).

4.6.6. ULTRASTRUCTURAL STUDY OF SOMATIC EMBRYOGENESIS

The cells of somatic embryos and embryogenic nodules (EN) contain lots of cytoplasm
and small vacuoles. The cytoplasm is densely enriched with numerous ribosome,
mitochondria, plastids and endoplasmic reticulum (ER). The nucleus with prominent
 335

Fig. 4.11. Nucleic acids in embryogenic callus (EC) and somatic embryos (SE) of C. avellana. (Fix: Bonin;
stain: gallocyanine; orange fiher): A) Nucleic acid-rich meristematic cells (x 125). B) Detail. Note the
presence of voluminous nucleolus in meristemic cells, x 490. C) Nucleic acid-rich cells in early stage SE
(arrow) (x 125). D) SE with cotyledons (SECo). Superficial layers and meristem regions of SECo are more
basophilic than central parts (x 80). (Radojevic et al., 1979).
336
nucleolus occupies the central part. Plastids contain one or more starch grains (Fig.
4.12 A) (Radojevic et al., 1975). In the meristematic cells of EN there is a very specific
organisation of endoplasmatic reticulum (Vujicic et al., 1983), the rough endoplasmic
reticulum (RER) was found in the form of individual cisterns (Fig. 4.12 A and B), or
aggregated into massive parallel stacked cisterns (Fig. 4.13). Very frequently,
concentric parallel cisternae of endoplasmic reticulum (CER) were present in EC (Fig.
4.12 B). The ribosome, which bound to these aggregated cisterns, were associated into
tetrameral units, forming a pattern of four ribosome in a square which looked like
crystallised ribosomes (Fig. 4.13) (Vujicic et al., 1976; Radojevic, 1977). This special
disposition was observed in the cells of somatic embryos and plantlets. Early stages of
GSE and torpedo somatic embryos (TSE) were also characterised by the presence of
reticulum (RER) cisterns with orderly bound stacked ribosome (Vujicic et al., 1983),
but some of the cisterns became dilated in torpedo stages. The distance between two
neighbouring cisterns was 50J.Lm and the tetramers persisted in TSE. To confirm
whether CER and RER are structures specific of embryogenic callus and somatic
embryos, we studied the ultrastructure of zygotic embryos isolated from mature hazel
seeds. They contained a dense cytoplasm with numerous organelles, and RER. The
cisterns of RER were organised into massive flat, or circular aggregates, but without
regular ribosome alignment (Vujicic et al., 1983). It was concluded that the formation
of ribosomal tetramers is an inherent feature of Corylus somatic embryos and might
represent a pool of functionally viable ribosome (Morimoto et al., 1972), which are not
programmed for immediate protein synthesis, but will be used during somatic
embryogenesis (Radojevic et al., 1975; Vujicic et al., 1983).
 337

Fig. 4.12. Ultrastructure of embryogenic callus of C. Avellana. (Fix: 3% ghnaraldehyde, 2% osmium

trtrox.ide): A) Meristematic cells with nucleus (N), prominent nucleolus (Nu), numerous mitod10ndria (M),
plastids (P) and endoplasmic reticulum (ER) cisternae (x 12.000), Scale bar=! fUll, B) Concentric whorl of
parallel cisternaes of endoplasmic reticulum (CER). Note ribosomes in tetramers association (arrow) (x
44.000). Scale bar= I IUD. (Radojevic et al., 1975).
338

Fig. 4.13. RER aggregates with regular pattern of four ribosomes in a square (arrow), Fix: 3% glutaraldehyde,
2% osmium tetroxide (x I 10.000), scale bar=0.5 f.Ull· Reproduced from The Journal ofCell Biology, 1976, 69,
686 - 692, by C<lpyright permission of The Rockefeller University Press.

5. Biochemical and molecular studies

5.1. Endogenous l>lant growth regulators and somatic embryogenic ca1>acity

The foremost factors which affect somatic embryo formation, are: genotype, age and origin
of the initial tissues (Wann, 1988), which in turn are related to their endogenous hormonal
status. Furthermore, the importance of the culture medium, particularly the exogenous
application of plant growth regulators (PGRs) is much discussed (Terzi and LoSchiavo,
1990). Thus, the embryogenic potential of explants is closely associated with their contents
of both, natural and exogenously applied PGRs. Unfortunately, little is known about this
topic mainly due to the complexity and sophistication of the teclm.iques required for PGRs
analyses. Only a few research teams have found links between the embryogenic capacity of
plant tissues and a specific endogenous honnonal content (Rajaserakan, l987a,b; Wenck et
al., 1988; Besse et al., 1992; lvanova et al., 1994) but, in most cases, these results were
obtained in herbaceous plants and not in woody species.
 339
5.1.1. ANALYTICAL METHODS

The use of an analytical method combining inununoaffinity columns for cytokinin

purification, high performance liquid chromatographic (HPLC) separation and,
quantification by inununoassay (Fernfmdez et al., 1995; Centeno et al., 1996), allowed the
simultaneous evaluation of IAA, ABA and a nun1ber of cytokinins (dihydrozeatin, (diH)Z,
(diH)Z riboside, [9R](diH)Z, iP, N'-isopentenyladenosine, [9R]iP, Z, and Z riboside, [9R]Z).
Sample size is relatively small (100 mg dry weight) and, therefore, is rather homogeneous
an1ong the Cary/us avellana tissues analysed, which presented different embryogenic
potential under the same culture conditions.
To determine the endogenous polyamine content (Putrescine, Put; Spennidine,
Spd; and Spennine, Spm), the plant material was e:\.iracted, hydrolysed and dansylated as
described by Rey et al. (1994b). Afterwards, the dansylated derivatives were separated by
thin layer chromatography (TLC), using fluorimetric detection for quantitative analysis.

5.1.2. RELATIONSHIP BETWEEN PGRs AND EMBRYOGENIC CAPA CITY

Somatic embryogenesis in hazelnut can be successfully induced from inunatnre tissues,

taken from embryonic and juvenile phases, in presence of different PGRs. So, several
embryogenic models have been suggested for tllis species: Radojevic et al. (1975), Perez et
al. (1983a), Fernfmdez et al. (1990), Gonzalez et al. (1991), Berros et al. (1992), Berros et
al. (1994), Berros et al. (1995), Centeno et al., (1997). For basic research, our embryogenesis
model from hazel tissues (Berros et al., 1995; Centeno et al., 1997) offers a great advantage
because it is direct, onlitting the callus stage, wllich enables the establishment of a direct
relationship between the endogenous PGRs balance and t11e embryogenic response of t11e
explants (Ivanova et al., 1994).
We have analysed PGRs in two hazelnut e:\.1Jlants. The first one is cotyledonar
tissues from zygotic embryos differing in their genotype (cv. Casina and cv. Negretta) and in
developmental stage (collected in August and in September when fruits are inunature and
 340
mature respectively). The second one, is leaves differing in their differentiation stage
(immature leaves with cotyledonar appearance, and juvenile leaves well differentiated, both
from in vitro plantlets witl1 a zygotic origin) and in origin (zygotic and somatic embryos, in
this last case the leaves can1e from somatic embryos in a cotyledonar state). The induction
medimn of somatic embryogenesis was the same for all six kinds of explants, the earlier
described T medimn. When we use t11e cotyledons as initial explant, we found an inverse
correlation between their embryogenic response and the degree of maturity of hazelnut fruits
(Fig. 5.1). In the san1e manner, the response of leaf e>..'Plants in T medimn decreased witl1
the differentiation stage of tissues (Fig. 5.1 ). The results of PGRs analyses in these hazelnut
e>..'Plants showed that their embryogenic potential was associated with specific balances of
endogenous phytohormones.

Cotyledons from zygotic embryos

genotype (cv.) Cas ina Ca.<; ina Negldta

collection dme August Septembrer September

Maurity degree ofeyglltic embryos

iP-type!Ztype
7.47 3.23 0.36
cytokinin raio

Leaf tiss ues

origin omatic Zygotic Zygotic

stage Cotyledonar - - - - - - + Inmmure Juvenile
cotyledonar oppearance well dili!rentiared

Di5nntillim degree oflel'itissueG

IANABA ratio 14. 98 9.95 2.26

Fig. 5. L Plant growth regulators in cotyledons and leaves of Corylus ave Ilana. The figure relates the
embryogenic potential with the best hormonal marker, fowtd for each kind of explant.
 341
The endogenous ABA content in cotyledons did not show differences associated
with the genetic origin or developmental stage of fruits. So, it did not seem to play an
important role in the embryogenic response of these explants (Centeno et al., 1997).
Nevertheless, in leaf tissues we found high ABA content, characteristic of juvenile leaves
respect to immature and cotyladonary ones, which could be related with the absence of
response in the well differentiated tissues.
There is a controversy over the role of ABA in inducing embryogenic response of
cultured tissues. The reduction of endogenous ABA levels in cultured leaves of Pennisetum
purpureum (Rajasekaran et al., 1987a) inhibited their capacity to produce somatic embryos,
while promoting the acquisition and conservation of an embryogenic state in calli of Hevea
brasiliensis (Etienne et al., 1993). In C. avellana, the mean values of ABA levels found in
all tissues showing some embryogenic response in T medium, were between 0.61 and
0.67runol g·1 dry weight, whereas it was 1.54 runol g·1 dry weight in juvenile leaves. Tllis
opens a question: the lack of response in these last explants was due to its initial ABA levels
acting as inllibitors or to the advanced degree of tissue differentiation? Probably, both
characteristic are linked and both inllibited the embryo formation in well differentiated
leaves. In cotyledons of cv. Negretta the lack of response was related with othe! honnonal
factor, as we will further describe, since ABA levels (0.75 nmol g·1 dry weight) were more
similar to those from responsive tissues than from juveniles leaves.
With respect to the role of auxin in somatic embryo fonnation, many tissues
comprise cells which can be induced in vitro to an embryoge1lic state by the use of amdns in
the culture medium (Krikorian, 1995). Therefore, atLxins are considered phytonnones
promoting the embryogenic process, but this asseveration is not true in all cases as shown by
the endogenous levels found in several cultured cells and callus. In tllis sense, Sasaki et al.
(1994) found an IAA level fifteen times higher in embryogenic cells of carrot than in non-
embryogenic ones, meanwhile tllis relation was the reverse in callus cultures of the same
species (Michal.czuk et al., 1992). On the other hand, Besse et al. (1992) did not observe
great differences in the IAA content of callus lines of Elaeis guineensis differing in their
embryogenic response.
342
In cotyledons of C. ave/lana the IAA levels, although they were not associated with
the maturity degree of fruits from cv. Casina collected in August or September, they were
higher in explants from this cultivar than in cotyledons from cv. Negretta (Centeno et al.,
1997), which did not show any embryogenic response in T medium. The direct relationship
between IAA endogenous content and the embryogenic potential of tissues was clearer in
leaf explants: IAA levels decreased markedly with the differentiation degree and its increase
paralleled the embryogenic response (unpublished data). So, the endogenous levels of auxins
act as an hormonal factor promoting the fonnation of smnatic embryos in hazel tissues.
Moreover, IAA content in responding explants seems to be enough for the initiation of the
embryogenic process since presence of tllis hormone-type in the induction medimn is not
indispensable.
It is possible that the endogenous IANABA ratio in explants, more tllilll their IAA
and/or ABA content, has a main role in the induction and expression of somatic
embryogenesis in C. ave/lana tissues, as it was pointed out in leaves of Medicago falcata by
Ivanova et al. (1994). The authors established a positive correlation between auxin-
favourable values and the embryogenic capacity of the explants. Certainly, in cotyledonar
leaves of hazel somatic embryos this ratio (14.98) was almost double tlmn in immature
leaves (9.95) and seven-times higher than in well differentiated leaves (2.26). In cotyledons,
the IANABA ratio found in the embryogenic genotype (0.23 and 0.24) was nearly two
times higher than in the non-embryogenic one (0.14) (Centeno et a!., 1997) despite of the
small differences in ABA and IAA individually regarded. So, this parameter could be use as
a physiological index of the embryogenic capacity of explants in CoryJus sp., mainly for leaf
tissues.
The cytokinin analyses in C. ave/lana cotyledons showed cllilllges in the
distribution of the total cytokinin content between iF-type and Z+DHZ-type cytokinins along
seed maturation. So, the youngest cotyledons of cv. Casina exhibited the highest iP and
[9R]iP levels and the lowest Z, (diH)Z and (diH)[9R]Z contents; in contrast, cotyledons of
the most mature fruits of hazel, those corresponding to cv. Negretta, had the lowest iP and
[9R]iP amounts and the highest Z, [9R]Z and, mainly, (diH)(9R]Z levels (Centeno et al.,
1997). In the generally accepted pathway of cytokinin biosynthesis (Letl1alll and Palni, 1983;
 343
Kaminek, 1992; McGaw, 1995), iP has been proposed as a precursor of Z that, in its turn,
leads to (diH)Z through reduction of the N'-side chain; the cytokinin bases are easily
interconverted with their 9-ribosides. Therefore, it seems that a biochemical differentiation
of this metabolic pathway runs parallel to cell differentiation and tissue maturation in hazel
embryos. This hypothesis is further sustained by the finding of an enzymatic activity
converting Z to (diH)Z in isolated cotyledons of Cicer arietinum (Munoz et al., 1990) and its
partial purification from Phaseo/us embryos (Martinet al., 1989).
The progressive enhancement of (diH)Z-type cytokinins, respect to the others, in
hazelnut cotyledons could represent a mechanism to maintain the total cytokinin content,
which was very similar in the three cotyledonary systems studied. It is possible that
cytokinin oxidase activity is enhanced during seed maturation, as it occurs in developing
maize kernels (Dietrich et al., 1995), being (diH)Z and [9R](diH)Z resistant to the oxidation
by tllis enzyme (Chatfield and Armstrong, 1986).
The cytokinin results allow us to directly relate tl1e embryogenic potential of C.
ave/lana cotyledons with their endogenous balance iP-type/Z+(diH)Z-type cytokinins, being
this ratio 7.47 in the youngest and most responsive explants, 3.23 in cotyledons whit a
middle capacity to form somatic embryos and 0.36 in non responsive and most mature
explants. The content of iP and its 9-riboside is a better index of the embryogenic potential
than t11e IANABA ratio in this kind of hazel tissues.
The role of iP-type cytokinins in the embryogenic response of cotyledons may be as
biosynthetic precursors, to supply the great cytokinin amount required for the stimulation of
cell division previous to somatic embryogenesis. This hypothesis is supported by the results
obtained by Murthy et al. (1995) on the endogenous PGRs content of peanut seedlings
during somatic embryogenesis induced by in vitro treatment with thidiazuron. The authors
observed a decrease in the pool of available iP, but not of the other PGRs and a suppression
of the 10 days cyclic fluctuation of iP in the cotyledons of treated seedlings respect to the
control, indicating an increase of the rate of iP utilisation to form cytokinins along the
embryogenic response.
Respect to tl1e cytokinins levels found in leaf explants, they were double in
cotyledonary leaves from somatic embryos (6.29 runol g·1 dry weigth) than in those from
344
microshoots with a zygotic origin (immature or juvenile). So, the cytokinin content in leaf
explants seems to be directly related with their embryogenic potential, since cotyledonary
leaves showed the highest capacity to form somatic embryos. Nevertheless, immature and
juvenile tissues have a very similar amount of cytokinins although, it was expectable a lower
content in the well differentiated and non-embryogenic leaves. The endogenous levels of
cytokinins in hazel leaves may be associated with the previous exposition time of tissues to
BA and kinetin present in T medium, more than with the differentiation degree of explants.
Two findings support this idea: 1°- it is known the capacity of exogenous cytokinins to
increase the levels of natural cytokinins in cultured tissues and, 2°- the number of previous
subcultures in T medium to obtain leaves with somatic origin was 5 while it was one to
develop immature or juvenile leaves from zygotic embryos. The results obtained in
cotiledonacy leaves were in agreement with the idea that a great cytokinin amount is
necessary to initiate the embryogenic process, but tllis is in opposition with the report of
Wenck et al. (1988). The authors showed, in leaves of two Dactylus glomerata genotypes
differing in their embryogenic capacity, that a lligh endogenous content of Z, [9R]Z, (diH)Z
and [9R](diH)Z inllibited the response in non-embryogenic lines. If the relationsllip between
the hormonal content, or balance in C ave/lana tissues and their embryogenic potential
changes in function of explant type, it is not surprising a strong influence of the species. The
pattern found for hazel tissues is not necessary generalizable for other species.
Cotyledon and leaf explants of hazel showed one more difference: t11e iP-
type/Z+(diH)Z-type cytokinins were not pointing out t11e embryogenic potential of leaf
tissues. So, the capacity to form somatic embryos in T medium of the explants from C
ave/lana was marked mainly by the iP-type/Z+(dH)Z-type ratio for cotyledonar tissues and,
by the IAA/ABA ratio for the leaf explants. Therefore, choosing the adequate marker
depends both on the type of tissue and on the previous culture conditions, in vivo or in vitro.
Polyamines have been directly and indirectly related with cell division and tl1e
growth and potential morphogenic capacity of plant tissues (Bagni et al., 1982; Bagni and
Mengoli, 1992). Although the correlation between polyamine levels and somatic
embryogenesis has been demonstrated (Feirer et al., 1985), tl1e reported data on t11e
embryogenic process must be carefully interpreted (Slocmn and Galston, 1985). When the
 345
endogenous polyamine levels were detennined during the Corylus fruit maturation phases
previously described (Berros, et al. 1994), it was found that the content of these compounds
decreased parallel to fruit maturation. Similar results were described in other species for the
same embtyonary phases (Lin et al., 1984; Hamana et al., 1992). Moreover, the levels of
both, polyamines and enzymes related to their biosynthesis, were higher during the first
stages of fruit development (Slocum and Galston, 1985; Kushad et al., 1988; Townadje and
Richardson, 1988). However, it might had not been the absolute polyamine levels that define
the specific embtyogenesis induction abilities (Bagni and Mengoli, 1992). In this respect,
the results obtained for the different hazel fruit developmental stages showed that an active
polyamine metabolism occurs, and it was possible to relate the changes in single polyamines
to biochemical events occurring during specific phases of the embtyonic development and
growth (Lin et al., 1984). Put and Spd-were the most abundant polyamines in the free and
conjugated fractions, while Put showed the highest levels in the insoluble conjugated
fractions.
Developing embtyos of this species showed the highest polyamine levels,
overcoming those described in juvenile and adult plants (Rey et al., 1994b,c). Moreover, the
embtyonary tissues were characterised by having Spd as the most abundant polyamine,
while the greatest levels ofPut and Spm were found in juvenile and adult tissues.
In C. ave/lana cv. Casina the highest embtyogenic induction occurs when the
Put/Spd and Spd/Spm ratios are lowest and highest respectively (Berros, 1996). Similar
results were pointed out in soja by Lin et al. (1984); during the first embtyonary phases, the
Put level was superior to that of Spd, while in ulterior phases Spd was the most abundant
polyamine. Keeping in mind that it is not possible to induce an embtyogenic response
during the precotyledonary phases of hazel zygotic embtyo development and, moreover, that
it exists a critical period of response induction that coincides with the higher levels of
spermidine, it is possible to suggest that the somatic embtyogenesis induction from zygotic
embtyos may be conditioned by high levels of polyamines and specifically by the Put/Spd
ratio (Ranch et al., 1985; Mengoli and Bagni, 1992; Berros et al., 1994).
With respect to the polyamine levels detected in three types of leaves at different
developmental stages, derived from one single hazel clone and showing different somatic
346
embryogenic ability, we found that the most immature tissues yield both higher somatic
embryogenic induction and spermidine level (Berros and Rodriguez, 1994).

5.2. Specific polypeptide in relation with embryogenic competence

Bidimensional electrophoresis was used in the peptide content analysis of juvenile, non-
embryogenic, and of cotyledon, embryogenic leaves, of C. ave IIana. The protein pattern of
cotyledonary leaves characteristically presented abundance of high molecular weight
polypeptides (21-62 kDa. ), while that of juvenile leaves was richer in low size polypeptides
(14.4-31 kDa.). Furthermore, a 21 kDa. polypeptide, exclusive of colitedonary-embriogenic
leaves, was found which may be taken as an indicator of the embryogenic development
capacity of there leaves.
In conclusion, the results showed specific polypeptide patterns for both kinds of
leaves. This may indicate that, although it is not possible to ascribe a unique protein pattern
to the morphogenic-embryogenic competence of the analysed tissues, due to the fact that
they constitute anatomically and physiologically distinct organs, it would be feasible to
establish a relation between the polypeptide pattern of cotyledoruuy leaves and their
embryogenic potential.
As a final consequence of the findings indicated above, it has been postulated that
the high size polypeptides would be involved in the embryogenic response of C. ave/lana
(Berros, 1996). Even when in some species the embryogenic potentiality has been related to
low molecular weight polypeptides, in the largest part of the results published till now (Choi
and Sung, 1984; Chen and Luthe, 1987; Stirn and Jacobsen, 1987; Hahne et al, 1988;
McGee et al, 1989; Yuffa et al, 1994), the polypeptide characteristics of cotyledonary leaves
are included between those of high molecular weight (21-70 kDa.). Moreover, taking into
account that a similar developmental model was described for zygotic and somatic embryos
(Wann, 1988), it may be possible that specific sets of proteins would determine this model in
different taxonomic groups through induction of the expression of specific genes which, in
turn, would determine the cell division pattern observed (Meinke, 1991).
 347
To get further insights on the relation between the syntl1esis of proteins and the
embryogenic development, inhibitors of Spd syntl1esis, such as metllylglioxal-bis-
guanilhidrazone (MG) and ciclohexilanline were applied to the tissues. It was found tl1at the
absence ofSpd resulted in tl1e generation of polypeptides witl1 sizes between lO and 48 kDa.
However, in spite of reports in which tlle relation between the use of MG and the
inhibition of an organogenic patll\vay is established (Feirer et a!., 1985; Minocha et al,
1991 ), no study has been published tllat allows the correlation between embryogenic
induction in presence of the inhibitor and tl1e syntl1esis of proteins. The application of MG
on a tissue exerts multiple effects on tlle activity of different enzymes, whether involved in
the polyamine metabolism or not (Feirer et a!., 1985; SlOCUlll, 1991), precluding the
conclusion tllat its effect on the embryogenic induction is a direct consequence of tl1e
alteration in Put and Spd levels.

s.J. Isoenzymes and embryogenic activity

To detennine whetl1er tl1ere exist possible enzyinatic markers of tl1e embryogenic potential
of hazel tissues, tl1e differential expression oftl1e following systems was analysed: Aconitase,
phosphoglucomutase, alcohol-dehidrogenase, acid phosphatase, peroxidase, esterase,
superoxide-dismutase and catalase. The comparison was perfonned witll 12 clones of
cotyledonary-embryogenic and juvenile non-embryogenic leaves, all having tl1e same
genotype, tllat were maintained under tl1e same culture conditions. Most enzymatic systems
showed quantitative differences between botl1 leaf types, being specially relevant the
isoenzyme variations of aconitase, alcohol-dehidrogemse, acid phosphatase, peroxidase and
superoxide-dismutase.
Usually, high isoenzyme activity correlated witll preconditioned tissues and witll
stages of active development and growtl1 (Bredemeijer and Burg, 1986; Kay and Basile,
1987). This higher metabolic activity of immature tissues was related to the existence of
organogenic potential (Cornu, 1988; Sotak et al., 1991). Besides, in Triticum aestivum a
lower number of isoenzymatic markers was associated witl1 111ature tissues (BAat et al.,
348
1992).
In nwnerous species the isoperoxidase patterns have being correlated to organ
initiation and, specifically, to the induction of somatic embryogenesis (Fransz et al., 1989;
Coppens and Dewitte, 1990; McDouglall et al., 1992). In all cases, the differences shown by
tissues with different embryogenic potentials was seen at the level of cationic markers. The
cationic peroxidases have being related to the metabolism of auxins (Grarnbow and Langen-
Schwich, 1983), they may determine the level of auxins in the tissues which, in tum, may
condition their embryogenic potential. Furthermore, the peroxidases have been related with
the induction of somatic embryogenesis due to their effect on the plasticity control which
may affect cell extension.
The zymograrns of hydrolytic isozymes such as esterases, peroxidases and acid
phosphatases have being related to both the organogenic and embryogenic potentials
(Everett et al., 1985; Fransz et al., 1989; Coppens and Dewitte, 1990; Toshinori and
Futsuhara, 1992, Martinelli et al., 1993). However, there is not a common enzymatic-
developmental pattern for the different species analysed. It may be possible that the
establishment of homologies is hampered by the diversity of the systems used to develop the
enzyme activities, together with the multiplicity of culture conditions used. On the other
hand, evolutive mechanisms such as gene duplication, intron insertion in structural genes or
even the existence of multigene families lower the possibility that the same isoenzyme is
harlx>ured by different species (Davies, 1980). Given these conditions, it may be supposed
that gene expression modulators that influence embryogenesis and organogenesis do so
under a species specific base.
Taking into consideration all the variables indicated above, it appears that the
isoenzymatic systems that best characterise the embryogenic potential of hazel cotyledonary
leaves correspond to alcohol deshydrogenases, peroxidases and superoxido-dismutases
 349
6. Cryopreservation

Cryoconservation may be carried out by quick-freezing and storage in the liquid

nitrogen, or by slow-freezing methods. The later involves slow freezing to - 4°C by
gradual lowering of temperature (1 °C/min), followed by immersion and storage in
liquid nitrogen. After storage in liquid nitrogen for the desired period, the plant
material can be thawed and tested for viability (vital staining: fluorescein diacetate,
Evans blue, toluidine blue) and regeneration potential (tested in vitro culture).
Hazelnut seeds (Corylus ave/lana) is a recalcitrant species, and can be store
up to six months in most cold conditions. The storage difficulties of these seeds seem
to lay on their high fat content (Corylus sp. seeds as "desiccation-tolerant liquid
nitrogen-sensitive"). Gonzales and Perez (1994) reported that the embryogenic axes of
cv. "Morell" and "Butler" of hazelnut were cryopreserved in liquid nitrogen at -196°C,
with or without previous desiccation at different moisture contents. The highest
recovery rate was recorded after freezing at 11% moisture content in cv. "Butler" (50%
recovery). There was no loss in germination of desiccated seeds down to 5% moisture
(fresh weight basis), although desiccated whole seeds did not survive liquid nitrogen
exposure and the embryonic axes excised, after cryoexposure of whole seeds survived,
were grown in vitro conditions (Normah et al., 1994).
Stratification of stored hazelnut seeds improved shoot production from both
control and cryopreserved isolated embryonic axes (Reed et al., 1994). Axes from
freshly harvested nuts grown in vitro had 90% to 100% viability with 60% to 80%
shoot production but shoot production decreased to about 30% for both control and
cryopreserved axes from seeds stored for one month. Axes from stratified-stored seed
were dried to 8% moisture under laminar flow and cryopreserved with resulting 85%
viability and 70% shoot growth rates, while only 30% of unstratified axes produced
shoots.
350
7. Conclusions and future prospects

Hazelnut micropropagation through simultaneous shoot-bud formation is ready to be

applied, this alternative allows realising-up to industrial level for both mature and
juvenile tissues. But mature tissue micropropagation is feasible at this level only when
the reinvigorization by severe pruning is applied. The repetition of severe pruning
proportionally increases the multiplication rate and simplifies the micropropagation
protocols.
Direct somatic embryogenesis from immature embryos is a technique fully
developed, being possible to apply it at any genetic programme. The use of mature
explants greatly reduces the yield of emblings. Therefore, further research must be
performed mainly at the three following points:
- synchronisation of responses
- maturation of somatic embryos
- level of embling production
Indirect morphogenesis including both caulogenesis and somatic
embryogenesis have been explored at lower extent. No publications exist on indirect
caulogenesis, but reviewing the indirect embryogenesis protocols and keeping in mind
the good knowledge about physiological bases of morphogenesis and development in
hazelnut, it is clear that in not such a long time these alternatives could be exploited at
industrial level if agricultural demands would support the effect.
 351
Acknowledgements

The authors wish to sincerely thank Profs. C. Perez, A. Gonzalez, C. Diaz-Sala, M.

Rey and M. Albuerne for their consolidation of the "hazel-line" at our laboratory.
Their work and human qualities found our current results
The financial support of DGCYT, FICYT and UE.F AIR is kindly acknowledged.

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Tous, J. and Romero, A (1997a) Situaci6 mundial de Ia producci6 i el comer9 de l'avellana, in J. Santos, J.
Santacana, J. Plana, J. F. Cil, and F. J. Vargas (eds.), El conreu de l'Avellaner. Generalitat de
Catalunya (DARP), pp. 9-24.
Tous, J. and Romero, A. (1997b) Eliminaci6 de rebrots, in in J. Santos, J. Santacana, J. Plana, J. F. Cil, and F. J.

Vargas (eds.), El conreu de I 'Avellaner. Generalitat de Catalunya (DARP). pp. 65-67.

Tous, J., Romero, and Lloveras, J. (1994b) Control quimico de rebrotes en avellano, Invest. Agr.: Prod. Prot.

veg. 9,101-197.
Tous, J., Romero, A, Plana, J., Rovira, M. and Vargas, F.J. (1997) Perfonnance of'Negret' hazelnut cultivars on

several rootstocks, Acta Hart. 445,433-439.

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walnut, Juglans regia L., Plant Sci. 40, 57-63.
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10. SOMATIC EMBRYOGENESIS IN PISTACHIO (PISTACIA VERA L.)

A. Onay1 and C. E. Jetfree2

1Department ofBiology, Faculty of Science, University ofDicle, Diyarbakir, Turkey
2 University ofEdinburgh, Institute ofCell and Molecular Biology, Daniel Rutherford
Building, King's Buildings, Edinburgh EH9 3JH, UK

CONTENTS

1. INTRODUCTION
1.1. ORIGIN AND HISTORY
1.2. BOTANY
1.3. IMPORTANCE OF THE SPECIES
1.4. CLONAL IMPROVEMENT
2. SOMATIC EMBRYOGENESIS IN PISTACHIO
2.1. IMMATIJRE FRUIT EXPLANTS
Materials and methods
Culture initiation
Culture maintenance and proliferation
Maturation of somatic embryos
Germination of somatic embryos and embling development
Transfer ofemblings to soil
Growth ofemblings in soil substrate in the greenhouse
Encapsulation of somatic embryos and embryogenic mass
2.2. EMBRYOGENESIS FROM CULTIJRED LEAF EXPLANTS OF PISTACHIO
Culture initiation
Culture maintenance
Embryo development, germination and embling growth
3. THE CHROMOSOMAL BASIS OF SOMACLONAL VARIATION IN
PISTACHIO

4. CONCLUSIONS AND FUTURE PERSPECTIVE

5. REFERENCES

361
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 361-390.
© 2000 Kluwer Academic Publishers.
362
SOMATIC EMBRYOGENESIS IN PISTACHIO (PISTACIA VERA L.)

A. Onay 1 and C. E. Jeffree 2

1Department of Biology, Faculty ofScience, University of Dic/e, Diyarbakir, Turkey
2 University of Edinburgh, Institute ofCe/1 and Molecular Biology, Daniel Rutherford
Building, King's Buildings, Edinburgh EH9 3JH, UK

1. Introduction
Pistacia vera L. (Pistachio) is a dioecious tree species widely cultivated in Iran, the
Mediterranean regions of Europe, North Africa, the Middle East, China and California
for its edible nuts. Currently, interest in Pistachio regeneration is very high in Iran,
Turkey and California, as it is generally recognised that Pistachio nuts are easily
converted into liquid cash. In common with many other commercially important fruit
and nut tree species, Pistachio does not reproduce true-to-type when propagated from
seed. Therefore, in current and traditional horticultural practice, Pistachio nut trees with
desirable properties are usually propagated by budding or grafting onto rootstocks or
from rooted cuttings. Difficulties in rooting of cuttings favour the use of grafting and
budding methods, but incompatibility between rootstock and scion, frequently
necessitates inter-grafting. In addition, being labour-intensive, grafting is slow and
expensive, limiting the number of propagated plants which can be produced. The
expansion of Pistachio plantations has been limited by using the traditional methods,
and alternative methods are required for the clonal propagation of large numbers of elite
Pistachio nut varieties with proven desirable characteristics. Intensive clonal
propagation of elite Pistachio varieties through plant tissue, organ and cell techniques
could benefit the Pistachio industry just as it has already done with species such as
Carica candamarcensis and Phoenix dactilifera (Jordan et al. 1983; Reynolds 1982).
This review summarizes the current status of somatic embryogenesis in Pistachio, and
describes tissue culture studies carried out on one Pistachio species (P. vera L.) at the
University of Edinburgh.

1.1. Origin and History

The origin of the Pistachio was not known until the beginning of the present century.
The Queen of Sheba during her visit to Assyria commandeered the limited crop of nuts
for the exclusive use of herself and her guests (Whitehouse 1957). The nuts of Jacob
were Pistachio nuts, called "Gatoum" or "batam" by the Arabs (Moldenke & Alma
1952). The latter name may properly apply to Pistacia khinjuk rather than to Pistachio.
According to Dioskurides, the Latin word Pistachio is derived from "pissa" = resin and
 363
"aklomai" =to heal. That is to say, it is a plant with wholesome resin. Davatchi (1958)
sees its origin in the Persian name "peste". In Turkey, one of the centres of origin of
Pistachio (P. vera), the tree is called "Tree of Gold" and the Pistachio nut is known as
"King of Fruits" and "Fruit of Kings" (Ayfer 1990). Currently, the Pistachio nuts are
known as "Antep fistigi" by the Turks because they are mainly planted in Antep
province of South-east Turkey.
Botanists such as Linnaeus, Candolle, Boissier and Engler did not know of the
existence of the natural Pistachio savannahs. Syria and Mesopotamia were considered as
the natural habitat of Pistachio (Bailey 1947). According to Vavilov (1951) the origins
of Pistachio are:
(1) central Asia, including north-east India, Afghanistan, Tajikistan and Uzbekistan,
(2) the near-east which covers Asia Minor, Caucasus, Iran and the mountain region of
Turkmenistan.
The tree was introduced into Europe at the beginning of the Christian era
(Moldenke & Alma 1952). The cultivated varieties of Pistachio were introduced into
Syria from Turkey and into Italy towards the year 800, subsequently into Spain from
Italy and France, and were much later introduced into the USA in 1853-1854 (Lemaistre
1959). The first commercial plantations were mainly in Iran, Turkey, California, China
and Afghanistan, and other countries adjacent to wild Pistachio nut stands. Today it is
grown as an orchard tree mainly in Iran, the United States, Turkey, Afghanistan, Greece
and Syria.

1.2. Botany
Pistachio (Pistacia vera L.) belongs to the family Anacardiaceae (Ozbek & Ayfer 1958;
Crane 1984; Woodroof 1979) a virtually cosmopolitan family in the Sapindales I
Rutales comprising 77 genera and more than 600 species (Wannan & Quinn 1991). The
Anacardiaceae, comprises such widely-known and commercially-important trees and
shrubs as cashew (Anacardium occidentale), mango (Mangifera spp.), Rhus
toxicodendron (poison ivy), R. radicans, and R. coriaria (Chandler 1951; Whitehouse &
Stone 1941; Zohary 1952; Davis 1966; Joley 1969; Crane 1984; Ayfer 1963). The most
widely accepted classification divides the family into five tribes: Anacardieae, which
includes Anacardium, Rhoideae, which includes Pistacia, Cotinus and Rhus,
Semecarpeae, Spondiadeae, and Dobineae (Wannan & Quinn 1991). In a monographic
study of the genus Pistacia, Zohary (1952) recognised eleven species, of which six are
native to Turkey (Davis 1966); other authors recognize as many as 15 species
(Whitehouse 1957; Joley 1979). P. vera is the only species of Pistacia with edible nuts,
for which it is widely cultivated (Joley 1979).
The ability of Pistacia species to grow in diverse climates and various soil is an
advantage, and the rootstocks are adaptable to a wide range of soil types (Ioley 1979;
Woodroof 1979). The Pistachio tree is very xerophilous and gypsocalciphilous
(Woodroof 1979; Ayfer 1990). In Turkey it generally grows in the reddish-brown rocky
soils of the hills on sandy soils rich in potassium and magnesium and with very thin A
horizons (Tekin et al. 1990). Pistachio requires continental climatic conditions, with
364
cold winters and wann summers. One of the most important reasons for low yields of
Pistachio nuts in Turkey is that most cultivars cannot receive adequate chilling in those
areas with wann winters (Kaska et al. 1990). Although chilling must be sufficient to
break bud donnancy, long, hot and dry summers are essential for maturation of the nuts.
According to Ayfer (1963, 1990), growing areas suitable to meet the average chilling
requirements of this species (around 800-1000 hr below 7°C) have an average
temperature around 7.0-7.4°C for the three winter months December, January and
February, together with daily mean temperatures above 30°C for 98-110 summer days
per annum.
Pistacia vera is dioecious and wind pollinated, fruit bud differentiation occurring
during the calendar year prior to blossoming (Ayfer 1963). Shoot extension begins at
the end of March and tenninates between the end of April and the middle of May.
Generally, 1 or ;2 vegetative axillary buds are located distally on the new growth. They
are considerably smaller than the inflorescence buds, and may give rise to lateral
branches the following year, or they may remain donnant. Inflorescence buds begin
expansion at the end of the following March, and anthesis occurs generally in the later
part of May and for about three weeks their growth and differentiation is rapid (Ayfer
1963). Thus Pistachio bears its fruit laterally on wood produced in the previous season
(Crane 1984). Like other fruit trees, Pistachio is alternate-bearing, producing little or no
crop after a heavy cropping year.(Ayfer 1963; Woodroof 1979; Crane & Iwakiri 1981).
A strong apical dominance has also been reported to characterise developmental
patterns of vegetative and floral Pistachio organs (Crane & Iwakiri 1985).
Leaves of Pistacia are alternate, deciduous or evergreen, usually pinnate, more
rarely trifoliate or simple. Both staminate and pistillate inflorescences are panicles that
may consist of several hundred individual flowers. Both types of flowers are apetalous,
and the pollinating agent is wind. Staminate and pistillate cultivars having similar
flowering times must be provided to ensure adequate pollination of the latter. The fruit
is a semidry drupe. Its maturity is manifested by a change in the appearance of the
epicarp (skin) from translucent to opaque, and a softening and loosening of the epicarp
and mesocarp (hull) from the endocarp (shell) which encloses the embryo (kernel)
(Crane & Iwakiri 1981 ). The endocarp has a thin red-violet fleck and the seeds range in
colour from light to dark-green. Endocarp dehiscence is first noted along the ventral
suture in late July, about the time ultimate kernel size is attained and progress along
both sutures until physiological maturity about the middle of September. Physiological
maturity is indicated by easy separation of the epicarp (hull) from the shell, the
equivalent of flesh separation from the pit in a free-stone peach.

1.3. Importance of the species

In addition to being a valuable nut tree, P. vera L. has other practical and
commercial uses. The leaves, galls and bark are an important source of tannins for the
leather industry. Pistachio produces resins which are obtained by tapping, including a
mastic, and its volatile oils are distilled to make pistachio turpentine. The heavy,
narrow-ringed and colourful wood of P. vera is easy to work with and is also a raw
material for the production of high calorie charcoal. Besides these commercial uses of
P. vera, this and other species of Pistacia are used for ornamental planting or as a
 365
source of shade, as wind breaks and for erosion control. They are also used as stocks on
which to graft the commercial cultivars of P. vera.
Pistachio is consumed as a luxury table nut in its countries of origin and elsewhere
in the world. Its delicious flavour and attractive colour make it popular with most
people at first acquaintance, so as production and availability increase demand also
increases. It is widely used in the pastry, confectionery and ice-cream industries for its
pleasing flavour and green colour. Pistachio nuts are very nutritious with high oil (58%)
and protein (19%) contents, and most cultivars are relatively low in sugar (7%) (Bloch
& Brekke 1960). The outer green husk of the Pistachio nuts, P. vera L. contains a
mixture of phenolic acids (Yalpani & Tyman 1983). Other compositional studies have
been reported on the fatty acids, and amino acids of Pistachio kernels (Garcia et a!.
1992). Pistachio nuts for market are graded by quality classes, with size and colour the
most important attributes. In general smaller nuts are greener in colour (Ayfer 1990),
'and consequently, although the taste, aroma and texture of the kernels of large-sized
cultivars are inferior, large nuts with green kernels are very desirable and expensive and
are more acceptable for the trade.
World commercial production of pistachio table nuts increased four-fold from
about 56,000 tons in 1976 to 213,000 tons in 1992 (Monastra 1995). Output from Iran,
Syria and Turkey increased four, six and eighteen-fold respectively, and production in
the United States rose steadily from negligible in 1976 to 66,000 tons in 1992, making it
the second world producer after Iran. The average annual yield per tree, over 12-15
years of production, ranged from 56.5 kg (fresh weight) for Kerman to 11 kg for Red
Aleppo cultivars. (Joley 1979). Irrigation in pistachio orchards may significantly
increase fruit yield. Irrigation at 20-day intervals produced 57.1 kg/tree during the
normal yielding years, whereas without irrigation only 34.5 kg/tree were produced
during the same period. During the low yielding years, the yield from the non-irrigated
treatment was 17.3 kg/tree, whereas the irrigation treatment increased fruit yield to 40.3
kg/tree, yet irrigation treatments had no effect on fruit quality (Kanber eta!. 1990).

1.4. Clonal Improvement

With growing demand on world markets, the future appears optimistic for the Pistachio
nut. Therefore, there is an urgent need for large numbers of improved, fast growing
trees to establish new Pistachio orchards. However, the Pistachio is the most difficult of
all of the nut trees to propagate (Joley 1979), and the lack of adequate methods of
vegetative propagation is one of the outstanding problems in the expansion of Pistachio
plantations. Neither the traditional methods for Pistachio propagation and improvement
(which are costly and time-consuming), or the recent in vitro methods developed since
1982 (which are inefficient and, to date, unable to facilitate mass clonal propagation of
Pistachio trees of improved quality, quantity and yield) seem adequate to meet this
current and future demand.
In current and traditional horticultural practice, the propagation of P. vera can only be
achieved from seed via seedlings, or by grafting. Grafting and budding methods must be
employed for clonal propagation since difficulties in rooting of mature explants prevent
the use of cuttings and layering. Despite significant development of Pistachio
366
propagation, the expansion of Pistachio plantations is still limited by inadequate
supplies of nursery material from which to develop uniform, high-productivity
populations, and by the absence of any constructive breeding programme for the
improvement of scion material for such traditional approaches (Barghchi & Alderson
1983). As with so many fruit and nut tree species, propagation of Pistachio from seed is
unsatisfactory. Because the species is an outbreeder, the seed progeny are genetically-
variable. Seed-progeny also include undesirable male trees, which cannot be identified
until flowering age, thus wasting orchard space.
It is therefore essential to develop reliable methods for the improvement and mass
clonal propagation of "elite" commercial Pistachio varieties. However, at present, there
is no fully integrated approach for micropropagation of mature, proven Pistacia
varieties covering all steps from explanting and propagation of clones in vitro to the
establishment of the new plants in the soil. Micropropagation of temperate nut trees is
not, of course, a new process, and has been recognised and exploited by gardeners,
horticulturalists, and agriculturalists for some time (Hansman & De Novoa 1986).
Studies on the micropropagation of Pistacia spp. were started by Alderson & Barghchi
(1982). Several researchers have successfully used organogenesis to clone elite mature
Pistachio trees but have failed to regenerate them using embryos from mature tissues
(Barghchi 1982, Bustamante-Garda 1984, Abousalim 1990, Yang & Liidders 1993,
Gonzales & Frutos 1990, Parfitt & Almehdi 1994, Onay 1996). Ahmad et a/. (1994)
obtained nodular cell suspension cultures of P. vera and may have obtained
embryogenesis from seed materials in liquid cultures, but at that time no regeneration of
whole emblings from somatic embryos had been obtained from any Pistacia species.
The first somatic embryos of Pistachio (P. vera L.) to be successfully germinated to
form plantlets were obtained from zygotic tissues (immature fruits) cultured in vitro
(Onay eta!. 1995, 1996; Taskin eta!. 1996) and from juvenile leaf explants of Pistachio
(Onay 1996). Somatic embryogenesis from mature explants remains elusive.
Nevertheless, somatic embryogenesis offers the promise of a solution to the
problems of clonally propagating elite Pistachio varieties. However, to improve
Pistachio nut production by somatic embryogenesis, essential prerequisites will be to
overcome the reluctance of mature tissues from proven varieties to generate somatic
embryos, to optimize the storage of embryos as artificial seeds, and to develop an
efficient embling regeneration procedure. Recently, protocols were presented by us as
the first stage of a study of somatic embryogenesis in Pistachio (Onay eta!. 1995, 1996;
Onay 1996), and this work is outlined below.

2. Somatic Embryogenesis in Pistachio

2.1. Immature Fruit Explants

Materials and methods

Source ofplant material. Immature fruits were collected between mid-June and early-
August 1993, or between mid-July and mid-September 1994 from a single Pistachio nut
tree variety growing on the Ceylan-pinari state production farm in the Urfa province of
South-east Turkey. Fruits were transported to Edinburgh, by aircraft. The fruits were
 367
stored in paper bags at 4°C prior to extraction of the kernels.
Surface sterilisation. Immature kernels, from which the outer pericarp and shells had
been removed, were pre-sterilised by immersion in absolute ethanol for 2 min. followed
by a rinse with sterile distilled water. These pre-sterilised kernels were then exposed to
10% (w/v) hydrogen peroxide solution for 10 min followed by a 20% (v/v) sodium
hypochlorite solution (10-14% available chlorine) for 20 min. The testas were then
removed and the kernels washed three times with sterile distilled water before being
placed in contact with the culture medium.
Initiation of EMS. The extracted kernels were surface sterilised as described above. The
kernels were cultured on two different basal media: Woody plant Medium (WPM)
containing 500 mg r 1 casein hydrolysate and 50 mg r 1 !-ascorbic acid, and Murashige
and Skoog (MS) medium containing 500 mg r 1 casein hydrolysate and 50 mg r 1 !-
ascorbic acid. The above media were also supplemented with various combinations and
concentrations of the auxins NAA and 2,4-D and cytokinins BAP and TDZ at
concentrations of 1, 2, and 4 mg r 1 for NAA and 2,4-D, and I, 2, 4 and 8 mg r 1 for BAP
and TDZ. Combinations of 2,4-D and NAA at I to 4 mg r 1 with BAP at I mg r\ and
agar-solidified with 0.7% Difco Bacto-agar and treatments with BAP and TDZ were
also tested in both MS and WP liquid media. Erlenmeyer flasks of 250 ml capacity
containing 50 ml of liquid MS medium without agar. Four or five kernels were cultured
in each flask in 50 ml MS medium with Gamborg's vitamins. The flasks were then
sealed with a double layer of aluminium foil and placed on an orbital shaker at 98 rpm
at a light intensity of 25 f.!mol. m- 2 sec- 1 photon flux density and a temperature of 25°C.
Unless otherwise stated the media were supplemented with 3% w/v sucrose and
adjusted to pH 5.7 before autoclaving. They were subcultured every 10 or 12 days. The
presence of EMS was determined by morphological observations.
Maintenance and proliferation of EMS. An EMS was initiated by culture of immature
fruits for 30 days in liquid MS medium with 500 mg r 1 casein hydrolysate, 50 mg r 1 !-
ascorbic acid, 1.0 mg r 1 BAP, and 3% w/v sucrose. This EMS was then subcultured
regularly every 10-12 days on an embryogenesis expression medium having the same
composition with or without 1 mg r 1 BAP. Subcultured explants were maintained in
continuous light at 25°C, in 250 ml culture tubes sealed with aluminium foil. The
mother EMSs thus obtained were transferred to a tissue proliferation medium that
consisted of solidified MS medium supplemented with 500 mg r 1 casein hydrolysate
and 50 mg r 1 !-ascorbic acid, either with 1.0 mg r 1 BAP or without growth regulators.
The effects of the carbon sources sucrose, glucose, fructose, lactose, ribose, xylose,
mannitol, sorbitol and glycerol on maintenance and proliferation were tested at
concentrations of2, 4, 6, 8 10 and 12% w/v. The cultures were incubated in continuous
light (20 f.!mol m- 2 s- 1) at 25 °C.
Maturation of somatic embryos. Maturation experiments were carried out both in liquid
and on agar-solidified media. The maturation experiments were carried out with only
one embryogenic line. All media were also supplemented with 500 mg r 1 casein
hydrolysate, 50 mg r 1 !-ascorbic acid and 0.7% agar if used, and the pH medium was
adjusted to 5.7 before autoclaving. The cultures were placed under cool-white
fluorescent light (25 f.!mol m- 2 s' 1, continuous photoperiod) at 25°C.
368
Effects ofABA, BA and sucrose in agarified medium on the maturation of SEs. Ten days
after subculture on the proliferation medium, pieces of actively-growing EMS were
transferred onto agar-solidified MS medium with combinations of BAP at 0.5, 1.0, 2.0,
4.0 and 8.0 mg r 1 and ABAP at 0.25 mg r 1 together with sucrose at 2, 4, 6 and 8% w/v
and incubated at 25 °C. The combinations of ABA at 0.25, 0.5, 1.0 and 2.0 mg r 1
together with sucrose at 2, 4, 6, or 8% w/v were also tested. The embryogenic potential
of the EMS is defined as the number of somatic embryos produced in 4 weeks per 250
mg fresh weight of EMS. After 4, 5 and 6 weeks of culture the SEs were counted by
eye.
Effects ofABA, BAP and sucrose in liquid medium on the maturation of SEs. The aim of
these experiments was to investigate the positive effect of ABA, BAP and sucrose on
the development of mature SEs in the liquid MS medium. Ten days after subculture on
the proliferation medium, pieces of actively-growing EMS were transferred into liquid
MS medium with combinations of ABA and BAP at 0, 0.5, 1.0 and 2.0 mg r 1 together
with sucrose at 2, 4, 6; or 8% w/v, and the cultures were incubated under continuous
light (25 Jlmol m·2 s· 1) at 25°C. Cultures of EMS were placed in liquid MS medium
using 250 ml Erlenmeyer flasks continuously rotated at 98 rpm in continuous light at
25°C. Each experiment was performed twice. After 2 or 3 weeks of culture, the SEs
were counted by eye.
Germination and embling development. Germination and embling development studies
were carried out in full-strength MS medium without PGRs, supplemented with 4% w/v
sucrose, 500 mg r 1 casein hydrolysate, 50 mg r 1 !-ascorbic acid and 0.7% agar.
Transfer of plantlets to soil. In vitro germinated embryos were washed overnight in
running water before being potted up in a sterile I: I mixture of peat and perlite or peat
and grit. Plantlets were covered with a Pyrex beaker to maintain 90±5% relative
humidity for 4-5 weeks before transfer into glasshouse conditions (25°C day; 20°C
night; 18-h daylength).
Statistical analysis. A randomised complete block design was used for the initiation
experiments. The Chi square (X2) test was used to test the association effects of the
model. In the maturation experiment, each treatment used two blocks, 5 replicates (Petri
dishes) per biock and 5 explants (ca. 50 mg) per replicate. A General Linear Model was
performed and the t-test adjusted for the different number of observations used in
pairwise comparison of treatments. An original statistical technique involving logistic
regression analysis based on GENSTAT (5), has been applied for the analysis of
somatic embryos' germination and embling development data. The developed statistical
models are reported in elsewhere (Onay eta!. 1997; Onay eta!. 1999).

Culture initiation
There was evidence of a significant influence of the date of collection of kernels on the
frequencies of induced EMSs when kernels were cultured on liquid MS media. The
frequencies of EMSs obtained from fruits collected on July 15th, August 7th and
September lOth were 20%, 10% and 12.5%, respectively (P < 0.05, Table 1).
Initiation of EMSs was observed only in the kernels cultured on HAP-supplemented
 369
liquid MS medium in early-August 1993, and mid-July and mid-September 1994
harvested fruits. EMSs were not induced by liquid WPM medium nor on solid medium.
Therefore, these results will not count in the analysis. The morphological observations
recorded for the rest of the tested treatments for the initiation ofEMSs will be followed
hereafter for all tested dates. After 4 weeks, both the EMSs and the callus or callusing
tissues could be distinguished on the different treatments. The EMSs developed only
from liquid MS medium supplemented with BAP (1-4 mg 1" 1). This response appears to
be similar to that of mimosa (Albiziajulibrissin Durazz) (Bums & Wetzstein 1998).
Table 1: Effects of fruit collection dates on initiation of embryogenic mass (EMS) in liquid MS medium from
kernel cultures of Pistachio in liquid medium*.
Collection data Media No. of fruits cultured %of kernels induced
Cultured with EMS embryogenic mass (EMS)
June 15th 1993 MS 44 0 0.0
August 7th 1993 MS 40 4 10
July 15th 1994 MS 40 8 20
September I Oth 1994 MS 40 5 12.5

x' test P<0.05

.. .. . .
*Data show only the cytok1mn BAP treatments. lmt1at10n was best m med1a contwmng 2 mg r BAP.
Among several PGRs and their combinations tested, only the BAP treatments in liquid
MS medium initiated friable embryogenic masses (Fig.1a), depending on the
concentration of BAP in the induction medium. Similarly, somatic embryogenesis was
observed with explants taken from four types of Aesculus tissue using the cytokinin
BAP (Bergman et a/. 1996). The first stages of the development of EMS visible on the
external part of the explants were never observed before 4 weeks in culture. The
explants cultured at the highest concentration ofBAP (8 mg 1" 1) did not induce EMS and
formed degenerate black tissue. Two embryogenic lines were established from the fruits
cultured on August 7, and the more friable one was chosen for further studies.

Culture maintenance and proliferation

When shifted to reduced cytokinin medium or a medium devoid of any growth
regulator, EMSs proliferated into larger, globular "solid" masses. The mother EMSs
thus obtained were transferred to tissue proliferation medium consisting of solidified
MS medium supplemented with 500 mg 1" 1 casein hydrolysate and 50 mg 1' 1 1-ascorbic
acid, either with 1 mg 1" 1 BAP or without growth regulators. Attempts to establish a fine
cell suspension from clusters of EMSs failed. However, rapidly growing clusters of
embryogenic masses were obtained. In a series of experiments, attempts were made to
optimise growth in liquid cultures.
The basal salts mixture (MS, WP, SHand G-5) and nine carbohydrates with or without
1 mg 1' 1 BAP were all examined. Statistical analysis of the results showed that the dry
matter content and fresh weight were significantly influenced by the composition of the
basic media (Onay 1996). Froni the obtained results, it would appear that MS is superior
to the others in terms of its ability to support dry matter content, and is followed closely
370
by WP and G-5 media. SH medium supports dry matter to the same extent but less than
WP and G-5 media. In terms of the fresh weight of EMS, MS medium was again
superior to the others. Visual observation of the cultures showed that those in MS
medium were the most friable, followed by WP, SHand G-5 media respectively.

Figure 1: Somatic embryogenesis in cultured immature kernels of Pistachio (P. vera L.) Fig.la: Clusters
of embryogenic masses (EMSs), bar= 13 mm. Fig. lb: Clusters of maturing SEs after 2 week maturation on
MS medium containing 0.5 mgt·' BAP (10 days after on MS germination medium containing 4% sucrose),
bar= 6 mm. Fig. lc: Germinating SEs (after 2 week maturation on liquid MS medium in the absence of PGR)
after 40 days of culture in germination medium containing 4% sucrose, bar= 9 mm. Fig. ld: Well developed
emblings 40 days after on MS germination medium (matured in I mg 1· 1 ABA, bar= 10 mm.
Fig. le: Emblings undergoing acclimatisation in a soil and grit mixture in a growth room, bar= 45 mm.
Fig. 1 f: Emblings originated from somatic embryos six months after transplanting, bar= 83 mm.

MS medium was thus chosen as the standard medium for future experimentation
 371
because it was found better to support friable proliferation as well as high dry matter.
Similarly, embryogenic callus and somatic embryos were induced using a Murashige
and Shoog's semi-solid basal medium (Bonneau et al. 1994; Das et al. 1995).
Suspension cultures were started from EMSs (0.5 g wet weight) using Erlenmeyer
flasks of 250 ml capacity containing 50 ml of liquid MS medium without agar. The
flasks were then sealed with a double layer of aluminium foil and placed on an orbital
shaker at 98 rpm at a light intensity of 25 J.tmol. m·2 sec·' photon flux density and a
temperature of 25°C. They were subcultured every 10 days. Since the embryonic mass
cultures consist of a highly vacuolated suspensor and densely cytoplasmic embryonic
cells, the dry matter of these cultures is strictly correlated with the portion of embryonic
cells (Schuller and Reuter 1993). Growth was determined by taking wet and dry weight
measurement rather than by cell count data, because the cells are in very tight clusters,
and the clusters are not completely digested with enzyme peroxidase and speed shaking.
·For fresh weight (FW) measurements the embryonic tissue was weighed in a plastic
Petri dish, and was left at room temperature for at least a couple of hours in order to
remove the liquid medium. The total dry weight of the embryonic masses was obtained
after drying to constant weight on a pre-weighed paper case at 80°C. The dry EMSs
were cooled in a desiccator before weighing. If there was no decrease in weight for any
of the treatments, it was assumed that they were oven dry. All flasks were sampled after
10 days of culture, and the fresh weight and dry matter were determined as explained
above. Each growth measurement represents the mean of 5 flasks per treatment. Each
experiment was done at least twice. All the embryonic mass suspension experiments
described in the subsequent figures were done with the same embryonic line
(originating from a single fruit).
In order to study the influence of various carbohydrate sources on embryogenic
mass proliferation, 5-times subcultured embryogenic mass was used in experiments
with different carbohydrate concentrations on a MS medium modified by the addition of
2, 4, 6, 8, 10 and 12% of sucrose, glucose, lactose, fructose, ribose and xylose with or
without I mg r' BAP. Only four tested sugars (sucrose, glucose, fructose and lactose) in
various concentrations in the liquid MS medium had any pronounced effect on the dry
matter and the fresh weight (Onay 1996). Among the carbohydrates tested as sources of
carbon, sucrose gave the best results. EMS transferred directly to a maintenance
medium containing glucose, fructose, lactose, xylose, ribose, sorbitol, mannitol or
glycol as alternative carbon sources lost some of their friability (Onay 1996). The
effects of sucrose concentrations on dry matter as % of the fresh weight are shown in
Figure 2. The statistical analysis of data showed that there were very highly significant
differences in the dry matter content ofEMSs between the tested treatments (P < 0.001).
Cultures grown on a growth regulator-free medium attained approximately 10% higher
dry matter than cultures grown on a BAP treated medium.
There was a steady increase in the dry matter content of the embryogenic mass on both
BAP media and growth regulator-free media, and with an increase in the concentrations
of sucrose up to concentration 10% of sucrose and a slight decrease thereafter (Fig. 2).
In both treatments with BAP and without BAP there was a strong positive correlation
between dry weight and sucrose concentration (r = 0.98 and r = 0.96, respectively).
372
35 . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - .
c:::::J hormone-free
30 ~with BAP

25

20

t5

10

10 12 14
Sucrose concentration(%)

Fig. 2: Relationship between sucrose concentration (%) and dry matter of EMS (%) on media supplemented
with 1 mg 1"1 BAP and on growth regulator-free media. Different lowercase letters above any two columns
indicate that these two means are statistically different at p = 0.05 according to the Student t-test. The data for
the mean of two experiments with 5 replicates. Embryonic tissues had been subcultured 5 times before the
experiments performed.

cs::sJ hormone-free
= w i t h BAP

...

6 8 H 12 14
Sucrose concentrations(%)

Fig. 3: Relationship between sucrose concentration (%) and fresh weight of EMS (g) on media supplemented
with 1 mg 1'1 BAP and on growth regulator-free media. Different lowercase letters above any two columns
indicate that these two means are statistically different at p = 0.05 according to the Student t-test. The data for
the mean of two experiments with 5 replicates. Embryonic tissues had been subcultured 5 times before the
experiments performed. Vertical bars represent the standard error of the mean.

However, there was a negative correlation (Fig. 3) between fresh weight and sucrose
concentration (r = -92 and r = -0.91, respectively). The effects of sucrose concentrations
on fresh weight are shown in Fig. 3. With increasing sucrose the fresh weight
production declined. In all the other treatments tested, the ratio of fmal to initial fresh
weight of the embryonic tissue ranged from 3.5 to 8.9 on MS medium supplemented
10% and 4% sucrose, respectively. New embryonic tissue proliferated during the mid-
level sucrose treatments was slightly friable with a deep green texture. The embryonic
tissues of all treatments proliferated with a fme texture and immature somatic embryos
were just visible after 10 days of growth. The results shown in Figs. 2 and 3 indicate
that EMS showed grew best on medium with a sucrose content of 6% to I 0% of dry
matter, equivalent to a sucrose content of between 2% and 6% of fresh weight.
 373
Maturation of somatic embryos
Several media and growth regulators in agar-solidified or liquid MS medium (auxins,
cytokinins and ABA) together with sucrose were tested to promote the development of
high quality somatic embryos (Onay 1996). The embryogenic potential of the EMS was
defined as the number of SEs produced per mg fresh weight of the EMS. The formation
of high quality SEs was found to be dependent upon the presence or absence of growth
regulators in the maturation treatment and on the origin of EMS.
Ten days after subculture on the proliferation medium, pieces of actively growing
EMS were transferred onto agar-solidified MS, WPM, SH and G-5 media. SEs were
produced on all tested media (MS, SH, WPM and G-5). Regardless of medium, all
Pistachio SEs appeared to arise from proliferated embryogenic masses. However, The
MS medium gave the highest number of mature SEs after four weeks of culture, but
there was no significant difference between the responses on MS and SH media (Onay
1996). Sucrose, in the presence of BAP in terms of the number of SEs obtained, was
superior to other tested carbohydrates (glucose, fructose, mannitol and PEG) but not as
efficient as glucose in the absence of BAP. Therefore, sucrose was chosen to mature
somatic embryos.
Effects of ABA and sucrose in agar-solidified MS medium on the maturation of SEs.
Analysis of variance showed significant effects of sucrose and ABA concentrations on
the number of mature SEs, and more importantly, revealed an interaction between ABA
concentration and sucrose concentration (Table 2).
The highest quantities of SE were observed on media with 0.5 to 1 mg r' ABA and
4%, 8% sucrose (24.00 and 27.23 per 250 mg fresh weight respectively); when high
concentrations of ABA (2 mg 1" 1) were combined with 2%, 4% and 6% sucrose, the
number of SEs was reduced. In addition, this reduction was also seen when a low
concentration of ABA (0.25 mg 1" 1) was combined with different concentrations of
sucrose because there were only minor differences in the number of SEs produced at
2%, 4%, 6% and 8% sucrose combined with 0.25 mg r' ABA. The mid concentrations
of ABA (0.5 and 1 mg 1" 1) used on solid media and the higher sucrose concentrations
were most favourable for a high frequency of matured SEs, whereas at 0.5 mg r' ABA
an increase in sucrose concentration from :2% to 8% reduced the number of SEs (Table
2).
Effects of BAP and sucrose in agarified MS medium on the maturation of SEs has
been reported by Onay et al. (1995). Of the treatments tested only sucrose and BAP had
significant effects on the numbers of mature somatic embryos when somatic embryos
matured on agar-solidified MS medium. Increasing the BAP concentration from 0.5 to
32 mg 1" 1 resulted in fewer mature somatic embryos (Onay et al. 1995). Sucrose
concentrations above 6%w/v were not beneficial. At the lowest BAP concentrations
sucrose stimulated embryo production, but above 2 mg r' BAP, further increases in the
sucrose concentration above 4%w/v had an inhibitory effect, so that the highest
concentrations of BAP and sucrose produced the lowest yields of somatic embryos
(Onay et al. 1995).
374
Table 2: Number of mature SEs per 250 mg fresh weight of pistachio embryogenic mass obtained on solid
media with different ABA and sucrose concentrations after 4 weeks of culture.

Sucrose(%, w/v)
ABAmgl· 2% 4% 6% 8%
0.25 11.40 a 11.80 a 11.60a 15.10ab
0.50 20.00 b 24.00 be 19.60 b I 0.40 a
1.00 11.60 a 16.10 ab 14.40 a 27.23 c
2.00 9.86 a 15.60 a 14.20 a 16.80 ab
Mature SEs wlfh cotyledons were counted by eye. Means followed by the same letter are not srgnificantly
different at p = 0. 05 based on the Student t-test. After four weeks culture, the best maturation was obtained in
a medium containing 0.5 ABA and 4% wlv sucrose.

Effects ofABA, BAP and sucrose in liquid medium on the maturation of SEs. Pieces of
actively-growing EMS were also tested in liquid MS medium with combinations of
ABA and BAP at 0, 0.5, 1.0 and 2.0 mg r' together with sucrose at 2, 4, 6, or 8% w/v.
Table 3 shows the mean numbers of mature SEs on the liquid MS media supplemented
with different BAP and sucrose concentrations.
Table 3: Number of mature SEs per 250 mg fresh weight of Pistachio embryogenic mass obtained on media
with different BAP and sucrose concentrations after 2 weeks in liquid medium.

BAPmgl· 2% 4% 6% 8%

0.0 35.6 ± 2.29 c 39.2 ± 2.47 be 49.8±3.20a 33.3 ± 2.23 cd

0.5 50.1 ± 3.40 a 48.4 ± 2.98 a 42.8 ± 1.97 b 40.5 ± 2.30 b

1.0 34.5 ± 1.91 c 40.1 ± 2.28 b 36.7 ± 1.88 c 41.1 ± 2.76 b
2.0 38.5 ± 2.36 c 35.9 ± 2.35 c 32.8 ± 1.97 d 29.7 ± 2.12 d
Mature SEs wrth cotyledons were counted by eye. Means followed by the same letter are not srgnificant/y
different at p = 0.05 based on the Student t-test. Media with 0.5 mg /" 1 BAP and 2% sucrose induced the most
embryos (50.1 per 250 mgfresh weight).

The number of SEs on 0.5 mg r' BAP and the control treatments (without the
growth regulators) with sucrose concentrations 2% and 6% was significantly higher (P
< 0.05) than on the remaining treatments (Table 3), respectively. Some of the matured
somatic embryos were overgrown by callus, and others hypocotyls showed
malformations such as multiple cotyledons or trumpet-like cotyledons. Figure lb shows
clusters of fully mature somatic embryos before transferring onto germination medium
containing 0.5 mg r' BAP. There was also significant difference in the mean number of
mature SEs between the treatments tested when ABA as used to mature SEs in liquid
medium (Table 4). Increasing the ABA concentration from 0.5 to 2.0 mg r' resulted in
less mature SEs. ABA concentration above 0.5 mg r' were not beneficial. Sucrose
concentrations 2% and 4% were the most beneficial treatments. Further increases in the
sucrose concentration above 4% had an inhibiting effect, so that the highest
concentrations of ABA and sucrose produced the lowest yields of SEs.
 375
Table 4: Numbers of mature SEs per 250 fresh weight of pistachio embryogenic mass obtained on media with
different ABA and sucrose concentrations after 2 weeks in liquid medium.

Sucrose(%, w/v)
ABAmgl· 2% 4% 6% 8%
0.0 35.8 ± 2.12 c 35.8 ±2.24 c 46.3 ± 3.69 ab 34.5 ± 1.99 c
0.5 46.5 ± 2.90 ab 50.7 ±2.72 a 41.7 ± 2.10 b 34.6 ±2.08 c
1.0 22.6 ± 1.96 d 18.1 ± 1.41 e 19.4 ± 1.50 de 16.7 ± 1.05 e
2.0 16.7± 1.73 e 23.6 ± 1.00 d 17.6±2.13 e 23.5 ± 4.19 d
Mature SEs with cotyledons were counted by eye. Means followed by the same letter are not significantly
different at p = 0.05 based on the Student t-test. Media with 0.5 mg 1" 1 ABA and 4% sucrose induced the most
embryos (50. 7per 250 mgjresh weight).

Cultures maintained on agar-solidified medium produced somatic embryos as

much as on liquid medium. These results suggest that maturation may be achieved in
liquid or agar-solidified media but maturation on agar-solidified was longer than in
liquid medium. Media with 0.5 mg r~ ABA and 4% sucrose induced the most embryos
(50.7 per 250 mg fresh weight) (Table 4).

Germination and embling development of somatic embryos

Germination and embling development studies were carried out using plant growth
regulator-free MS medium supplemented with 500 mg r 1 casein hydrolysate, 50 mg r 1
1-ascorbic acid, 4% sucrose and 0.7% agar. Somatic embryos were considered
germinated when radicle emergence was observed. Germinated SEs developed into
emblings on the germination medium. As soon as a germinating SE elongated its
epicotyl, it was considered to be a embling. In this study, ABA or BAP were added only
during maturation and EMSs were obtained from liquid medium without growth
regulators. Neither germination nor embling development medium contained BAP or
ABA. Further development of Euonymus europaeus (Bonneau et al. 1994) somatic
embryos into plantlets was also achieved on a medium devoid of growth regulators.
Effects of solidified MS medium. To study the effects of maturation treatments on
germination and embling development, fully mature somatic embryos were transferred
onto the surface of the germination medium after 4, 5 and 6 weeks.
Ten days after subculture on the proliferation medium, pieces of actively growing
EMS were transferred onto MS medium solidified with 0.7% w/v agar and containing
either ABA and sucrose or a combination of ABA and BAP with sucrose, at the
following concentrations: ABA at 0.25, 0.5, 1.0 and 2.0 mg r 1 together with sucrose at
2, 4, 6, or 8% w/v. BAP at 0.5, 1.0, 2.0, 4.0 or 8 mg r 1 with sucrose at 2, 4, 6 or 8% w/v
and ABA at 0.5 mg r 1• Five or six somatic embryos were isolated and placed on 30 ml
agar-solidified MS medium in 90 mm Petri dishes. Forty somatic embryos were used
with each combination of the three treatments (BAP/ABA, sucrose and maturation
period); and each experiment was performed twice. The number of somatic embryos
germinated (out of 40) was counted after 10, 20, 30 and 40 days of culture. The
developed statistical models and results are reported in elsewhere (Onay et al. 1999).
376
Table 5 shows the combination of the maturation period, sucrose concentration and
BAP or ABA concentration which optimises each of these quadratic fits within the
ranges of treatment values used in the experiments. The corresponding fitted
probabilities are also shown, but since they are based on an estimated model they are
likely to be slightly optimistic. Except for the germination experiment using BAP, the
estimated optimum treatment combinations include the shortest maturation period used,
4 weeks.
Table 5. Estimated combinations of treatments for maximising the probabilities of germination at 40 days of
culture and for embling development at 28 days of culture. The values in brackets are those constrained to lie
within the experimental ranges: lower values would possibly increase the estimated probabilities.
Experiment Treatments
Estimated Maturation Sucrose BAP/ABA
probability periods (weeks) cone. (% w/v) cone. (mg r')
Germination (BAP) 0.65 5.5 3.68 2.41
Germination (ABA) 0.74 (4.0) 3.65 0.27
Embling development (BAP) 0.22 (4.0) 3.62 0.50
Embling development (ABA) 0.17 (4.0) 4.55 0.06

These results suggest that higher proportions may have been achievable with
shorter maturation periods or lower concentrations. The estimated optimum sucrose
concentrations are all within the range used in the experiment, but since no consistent
effect of sucrose was found between 2 and 6% w/v these are no grounds for selecting a
particular value in this interval.
The study allows tentative conclusions to be drawn about the best combination of
treatments of BAP and ABA to be used for germination and embling development. It is
clear that the longest durations of culture (40 days for germination and 28 days for
embling development) are the best of those used in the study. Also the optimum
combinations of BAP and ABA in the germination and embling development
experiments were 2.41 mg 1" 1 and 0.27 mg r\ and 0.5 mg 1" 1 and 0.065 mg 1" 1
respectively (Table 5).
Effects of liquid MS medium. The effects of ABA, BAP and sucrose treatments during
maturation on the subsequent germination and embling development of SEs has been
reported by Onay et a!. (1995). To determine the effects of ABA, BAP and sucrose
treatments during maturation on the subsequent germination and embling development
of SEs, clusters of immature SEs were transferred from the liquid onto the surface of
0.7% agar-solidified MS medium supplemented with 4% sucrose 500 mg 1" 1 casein
hydrolysate and 50 mg 1" 1 !-ascorbic acid for germination. Thirty SEs were used in each
treatment, and each experiment was repeated once. SEs were considered to have
germinated as soon as radicle elongation was observed. The numbers of germinated SEs
was counted after I 0, 20, 30 and 40 days of culture.
Most of the tested maturation treatments with or without ABA and BAP promoted
germination of SEs. There was no inhibition of SEs in media without growth substances
 377
but the maturation media applied especially 2 mg r 1 ABA inhibited germination of
some mature SEs and produced callusing tissues. The majority of the SEs showed
elongated shoots and radicles within 40 days (Fig. lc). SEs matured for 3 weeks in the
shaking flasks were dark green in colour. Single radicle elongation was observed in the
majority of the developing emblings but shoot elongation was usually poor in most of
the treatments tested (Fig. ld). Some developing emblings matured in the higher
concentrations of BAP or ABA were overgrown by recallusing, and radicles showed
malformations.
As a results of germination and embling development studies, it was concluded
that the resulting fitted probabilities of germination were as high as 0.65 with BAP and
0.74 with ABA and the highest estimated probabilities of embling development were
0.22 with BAP and 0.17 when the agarified medium used for maturation (Table 5).
However, when the liquid medium used for maturation the resulting fitted probabilities
of germination were as high as 0.56 with BAP and 0.47 with ABA and the highest
estimated probabilities of embling development were 0.52 with BAP and 0.24 with
ABA (Onay et al. 1997).
These results suggest that maturation may be achieved in liquid or agar-solidified
media but maturation on agar-solidified was longer than in liquid medium but
germination and plantlet development was highest when cultures had been grown in
agarified medium. In treatments with ABA the SEs did not germinate precociously,
whereas in treatments with ABA, generally the development of SEs was reduced and
additional time was required for the maturation of embryos.

Transfer of emblings to soil

Emblings developed in vitro from SEs were washed overnight under running water and
potted in a mixture of peat and perlite (1:1, v/v) or peat and grit (1:1, v/v). Emblings
were covered with a Pyrex beaker (Fig. 1e) to maintain 90±5% high relative humidity
for 4-5. Emblings were weaned in two stages first at 90±5% RH for 2 weeks and then at
70±5% RH for 2 weeks before transfer into greenhouse conditions (25°C day; 20°C
night; 18-h daylength; 65-85% RH).

Growth of emblings in soil substrate in the greenhouse

Strikingly uniform growth was observed for the regenerated emblings. Survival of
emblings improved when they remained under high humidity for several weeks before
transfer to greenhouse conditions. A two-week period of weaning by progressive
reduction of humidities from 90% to 65% RH was beneficial in order to obtain higher
embling survival rates. Under the acclimatisation conditions, the survival of emblings
was much higher in the peat-grit mixture than in the peat-perlite mixture. In the peat and
grit, emblings tended to resume shoot growth very quickly. Fungal contamination of the
substrate was occasionally observed.
Emblings with at least two pairs of new leaves reached 5-7 em in the mixture of
peat and grit in 4 weeks and there were at least two pairs of new leaves on each plant.
We observed a variation in the number of emblings that could be transplanted
378
depending on the maturation treatments. The vigorous emblings were those that
embryos matured on solidified MS media with BAP and ABA (70% and 60%,
respectively). The lowest number of emblings transferred (20%) was from the 3 week
maturation in liquid medium with 1 and 2 mg r 1 ABA, whereas they were 35 and 20%.
The entire process, from maturation to obtaining emblings in the greenhouse,
required 10 to 12 weeks (4 weeks of acclimatisation). Under the method studied in this
part, We have established in soil over 200 somatic emblings of Pistachio. Developing
emblings regenerated via embryogenesis showed a similar pattern of growth in the
greenhouse. In the greenhouse the majority of the emblings became dormant and did not
set new terminal buds. Acclimatised emblings resumed growth after transfer to a soil
and grit mixture, and developed to maturity (Fig. If). Only 20% (40 out of200) of them
managed to survive after 3 months in the greenhouse because environmental conditions
were not conclusive to good growth. Pistachio trees need dry atmosphere and high light.
Watering requirements are not fully understood. Pistachio trees prefer a deep root run.
Therefore, the emblings produced were frequently repotted. Plants do not like to be
disturbed. They were also prone to infestations of whitefly and red spider. The materials
propagated have not been planted onto a typical Pistachio regeneration site. Therefore,
the growth characteristics of the regenerated emblings may not be thoroughly obtained.

Encapsulation of somatic embryos and embryogenic mass

The use of somatic embryogenesis for the cloning of superior genotypes generates a
need for long term preservation during field evaluation of clones, since the culture value
of EMS or cell lines is unknown, owing to their juvenile origin. Encapsulation is a
practical procedure for short-term storage of embryogenic Pistachio tissue, and may be
applicable to the preservation of desirable elite genotypes. This research is in its infancy
but it has been shown that mature somatic embryos of P. vera can be encapsulated in
calcium alginate gel and still retain a useful level of germination capacity in vitro (Onay
et al. 1996). Although the conversion frequency of complete emblings from synthetic
seeds of Pistachio was low (14%), the results obtained were positive, demonstrating that
the method is feasible, and suggesting that it may be possible to optimise these
techniques to improve the rate of production of complete emblings after storage of
somatic embryos for 60 days at 4°C in dark. The results reported also demonstrate that
encapsulation of Pistachio EMS in beads of calcium alginate gel is an effective
procedure for short to medium term storage of embryogenic cell lines. While there is
some loss of dry matter content and proliferative capacity during storage, stored
encapsulated tissue rapidly recovers its original dry matter content and proliferative
vigour following return to normal culture conditions. It remains to be established
whether the storage period can be extended further than the 60-day period investigated
here, to facilitate longer-term tissue storage. Both of these approaches are applicable to
the preservation of desirable elite Pistachio genotypes and to the management of culture
stocks during production. The potential for long term storage of embryogenic cultures
of some fruit trees (coffee, pear, apple, and peach) after storage in liquid nitrogen has
been demonstrated by others (Dereuddre et al. 1994). Pinus caribaea plants from cryo-
stored and non-cryo-stored somatic embryos appeared similar when established in a
greenhouse (Laine et al. I 992). Conceivably, therefore, cryo-storage may also prove to
 379
be a suitable approach to long-term storage of Pistachio tissues.

2.2. Embryogenesis from cultured leaf explants of the Pistachio, Pistacia vera L.

Culture initiation
Leaf material from freshly-germinated leaf explants, regenerated seedling explants and
mature Pistachio (50-year-old) was the source of explants. The cultures were carried out
by incubating two pairs of fully expanded leaves from shoot cultures into 250 ml
conical flasks of 50 ml liquid MS medium containing BAP or TDZ. Mature leaf
explants from 50-year-old P. vera. were harvested and surface sterilised in 20% NaOCl
for 20 minutes, and rinsed three times in distilled water. Leaf explants of 5 mm 2 were
cut while submerged in, and cultured in, 50 ml liquid MS medium in 250 ml flasks
sealed with a double layer of aluminium foil. Alternatively, mature regenerated leaf
explants from 50-year-old P. vera were also used as a source of material. The plant
growth media for callus initiation consisted of liquid MS medium, and 0.5, 1, 2 and 4
mg r 1 BAP or TDZ. All media containing growth regulators were autoclaved for 16
min. The cultures were kept on a gyratory shaker at 98 rpm at 25°C under continuous
light (25 11mol m2 s· 1). The growth of callus from leaf explants was estimated by visual
observation. Callus formed on all leaf explants developed into a mass of friable deep
green-yellow with intensive red pigmentation up to 3 weeks of culture on MS medium
containing TDZ (Fig. 4a). The intensity of callus proliferation was greatest in medium
with 1 mg r 1 TDZ alone.

Culture maintenance
Attempts to maintain callus failed when the primary friable callus was transferred to a
reduced level of TDZ, a medium with the original concentration of TDZ. Four weeks
after the callus was transferred to liquid or agar-solidified MS medium supplemented
with the cytokinin TDZ, parts of the calli grew, and newly proliferated fresh callus
clumps appeared. Transfer of callus to a liquid MS medium devoid of growth substance
was moderately favourable in order to maintain callus. Transfer of callus to an agar-
solidified MS medium supplemented with 1 or 2 mg r 1 BAP was most favourable in
order to maintain calli. During the 4 weeks of subculture the cultures grew more and
more rapidly and proliferated new callus clumps with a change of the original colour.
Direct transfer of callus from liquid MS medium containing 2 mg r 1 to an agarified MS
medium having 1 mg r 1 TDZ or 1 mg r 1 BAP resulted in actively proliferating calli
within 4 weeks of culture. Transfer of callus from liquid MS medium to an agar-
solidified WPM, SH or G-5 media having 1 mg r 1 TDZ or 1 mg r 1 BAP also resulted in
the complete inhibition of callus within 3 days of culture. Transfer of callus from liquid
MS medium containing 2 mg r 1 TDZ and sucrose to an agarified medium having 1 mg
r 1 TDZ or 1 mg r 1 BAP + fructose, lactose and fructose resulted in the partial inhibition
of callus within 3 days of culture, but glucose was as beneficial as sucrose. Callus that
was initiated on any leaf explant did not become embryogenic after subculturing onto
agar-solidified MS medium supplemented with BAP or TDZ. Embryogenic callus
initiation was dependent not only on the age of explants but also on the individual leaf
used. There was a significant difference in the frequencies of embryogenic callus
380
obtained between the tested treatments (Table 6, P < 0.05).
Table 6: Effect ofTDZ on induction of embryogenic callus from young leaves from seed, young leaves
regenerated from l-year-old greenhouse grown seedlings and from leaf explants of 50-year-old plant.
Origin of explant No. of explants No. of explants with % of explants
cultured embryogenic callus developing embryogenic
callus
Leaves from seed 140 10 7
Leaves from seedling 80 3 4
Leaves from mature trees 80 0 0

X2 (2 df) P<0.05

Embryogenic callus formed on only I 0 leaf explants out of I40 seed-originated

leaves, 3 leaf explants out of 80 seedling leaves, and none out of 160 leaves from 50-
year-old trees. Embryogenic calli were initiated on liquid MS medium containing TDZ
(0.5-4 mg r') while maintained on agar solidified MS medium supplemented with or
without BAP and TDZ (1 or 2 mg r'), while agarified hormone-free MS medium was
used to mature SEs. Figure 4b shows differentiation of green nodular structures upon
r'
the transfer of calli from 1-4 mg TDZ to medium containing I mg BAP. Yellowish r'
nodular structures started to appear after the second subculture onto 1 mg r' BAP
containing medium. TDZ was less effective for the induction of embryogenic
competency than BAP despite being more effective for stimulating callus initiation.

Embryo development, germination and embling growth

After 3 weeks of culture all the calli which developed, were transferred to agar-
solidified MS medium supplemented with 1 mg r' BAP or TDZ and kept on that
medium for four weeks, and subcultured regularly every 4 weeks. Observations were
recorded for per gram embryogenic callus at the end of 120, 150, 180 and I90 days for
SE induction (Table 7). At the end of that period (approximately IOO days from the start
of culture) calli showing nodular structures started to produce SEs on MS medium with
I mg r' BAP (Fig. 4c). SEs were matured in the presence of BAP or TDZ but the
response varied in terms of the period of induction and emergence of SEs. SEs were
first observed within 4 months originating on the surface of the callus grown in the
presence of I mg 1" 1 BAP. Calli originating from mature material did not differentiate
somatic embryos. SEs were first seen emerging within the first four months in the
presence of BAP (1 mg r'), but few were observed within five months with TDZ. In
fact, the induction of somatic embryogenesis was significantly delayed when I mg r'
TDZ was used for embryo development. We reasoned that theSEs may be arrested at
the globular stage of development on media that contained TDZ. The frequency of
developing embryos in the BAP supplemented medium was higher than the TDZ
supplemented medium. Well-developed SEs (Fig. 4d), were removed from the calli and
transferred to a medium containing MS medium without growth regulators with
Gamborg's vitamins and sucrose 4% to allow further development. Few SEs were
germinated on medium containing cytokinins (BAP or TDZ) though most of the SEs
 381
formed well-differentiated single roots (Fig. 4e). The frequency of shoot elongation
remained poor in the majority of SEs. When the germinating embryos were cultured on
a fresh medium root elongation stopped and at least a couple of tiny leaves formed
within 2 weeks.
Table 7: Number of SEs obtained from leaf culture initiated on TDZ concentrations (1-4 mg 1"1) and
subsequently transferred to 1 mg 1"1 BAP containing MS medium and then transferred toMS medium with
BAPorTDZ.
Total number of somatic embryos
120 days ISO days 180 days 190 days
BAP TDZ BAP TDZ BAP TDZ BAP TDZ

Leaves from seed 5 0 13 3 21 8 25 11

Leaves from seedling 3 0 8 5 19 9 19 9
Leaves from mature trees 0 0 0 0 0 0 0 0
*Each value presents the sum of the results of one experiment (After 90 days of culture I gram
nodular or embryogenic callus per treatment were traniferred to MS medium supplemented
I mg- 1 BAP and kept on that medium for 100 days).

Shoot development was increased by using 1 mg r 1 BAP, however, at this time the
developing emblings started to produce calli from the roots and eventually the whole
developing embling recallused. Less than 10% of SEs which were induced in the BAP
supplemented media could be regenerated to emblings. However, none of the TDZ
supplemented plates did suppress the induction of shoots. In short, in this study it
should be noted that BAP and TDZ were found to be most effective for inducing
somatic embryogenesis in juvenile leaf explants of Pistachio. They were then
transplanted to 10 em diameter plastic pots filled with peat-perlite; the emblings were
maintained at 95% humidity under a glass cover in a growth room for several weeks.
Successful acclimatised emblings were planted in a greenhouse (Fig_ 4f)_
Regeneration of plants in vitro was accomplished by somatic embryogenesis only
using juvenile leaf explants. However, the compatibility of the embryo production from
EMSs of leaf explants was achieved less frequently than other regeneration materials of
Pistachio. The effects of conditions of incubation on the production of embryogenic
calli were examined. Calli were not formed even at the cut edges of the many explants
that were in contact with agar-solidified MS medium in places where the edges of the
leaves had curled up and away from the medium during 4 weeks of culture. Even fewer
calli were produced 8 weeks after culture. These results for culture initiation are not in
agreement with those obtained for Coffea canephora by Hatanaka et al. (1991) who
initiated embryogenic callus from cultured leaf discs placed on a agar-solidified MS
medium. When the leaf explants from the different age groups were cultured under the
same conditions, little callusing of tissues was obtained on agar-solidified MS medium
but all leaf explants of Pistachio produced callusing tissues in liquid MS medium
containing TDZ or BAP after 3 weeks of culture. The effects of plant growth regulators
on embryogenesis were examined in leaf cultures of Pistachio. Of all the cytokinins
tested, TDZ was found to be most effective for callus initiation and growth resulting in
382
some red pigmentation, and deep green in colour. Callus production using the cytokinin
TDZ has been reviewed by Huetteman & Preece ( 1993 ). The transfer of calli induced in
liquid MS medium with TDZ (0.5, 1.0, 2 and 4 mg 1" 1) from liquid to a agar-solidified
MS medium with or without BAP (1 or 2 mg 1" 1) was most favourable in order to
maintain callus. During the 4 weeks of subculture the cultures grew more and more
rapidly and proliferated new callus clumps with changes in the original colour.
Embryogenic callus initiation was dependent not only on the age of explants but also
the individual leaf used, and the type of growth substance and their concentrations.
Embryogenic callus formed on 7%, 4% and 0% of seed originated leaves, seedling
leaves and regenerated leaves from 50-year-old trees, respectively.
As shown in Table 6, the number of juvenile leaf explants producing embryogenic
callus varied significantly in terms of the leaf explants used. Not all calli that were
initiated on any leaf explant became embryogenic after subculturing onto agar-solidified
MS medium supplemented with growth substances. Embryogenic callus was friable and
light to deep green. There was a significant difference in the frequencies of
embryogenic callus obtained between the tested treatments (Table 7, P < 0.05). TDZ
was less effective for the induction of embryogenic competency than BAP despite it
being effective for stimulating callus initiation.
Cytokinin (BAP) induced embryogenesis in immature fruit cultures of Pistachio is
reported in the earlier part of this study. In the presence of TDZ, SEs never germinated
normally even after prolonged subculture; instead they began to recallus. We reasoned
that by producing excessive pigments, it may arrest developing SEs. Perhaps TDZ was
inhibiting shoot elongation and SE development although it was the most effective
agent for callus initiation. SEs were produced in the presence of TDZ at lower
concentrations in the range of 0.5-2 mg 1" 1• In contrast with our results, regenerated SEs
were capable of developing into plants in the presence of TDZ at the concentrations of
1.1-17 mg 1" 1 for Nicotiana tabacum (Gill and Saxena 1993). In these experiments, the
number of emblings (emblings) obtained through embryogenesis remained very low.
We have attempted to enhance the percentage of embling formation by increasing the
osmolarity of the medium during experiment studies (results not shown), since some
work has indicated that desiccation may be an important part of the maturation process
(Xu et al. 1990) but these experiments did not improve the number of developing
embryos. Nevertheless, embryogenic compatibility of leaf explants was not explored in
these studies. Overall, it should be noted that TDZ may be the most potent cytokinin for
callus initiation from leaf explants of Pistachio and the cytokinin BAP and the age of
the explants played a decisive role in the ability to produce embryogenic callus.
There are few reports of somatic embryogenesis using TDZ. Thidiazuron has been
used in the different concentrations to stimulate somatic embryogenesis from
cotyledons of eastern black walnut (Neuman eta/. 1993), Rubus (Fiola eta/. 1990),
grape (Vitis vinifera L.) (Matsuta & Hirabayashi 1989) and leaf segments of Prunus
persica (L.) (Declerck & Karban 1996). On the basis of the induction of embryogenesis
in Pistachio leaf explants by TDZ or BAP alone it may be logically concluded that
cytokinin regulated embryogenesis may involve modulation of endogenous cytokinin.
However, the question still remains whether mature leaf explants of Pistachio have
embryogenic potential.
 383

l' igure 4: Somatic embryogenesis and embling regeneration from juvenile leaf ex plants of Pistachio, P.
vera L.: Fig. 4a: Induction of greyish-reddish callus in liquid MS medium containing I mg 1" 1 TDZ 3 weeks
after incubation, bar = 13 mm. Fig. 4b: Differentiation of green nodular structures upon transfer of callus
from 1-4 mg 1· 1 TDZ to medium containing I mg 1" 1 BAP, note the embryogenic centre with yellowish colour
(arrow), bar= 5.8 mm. Fig. 4c: Proliferation of the embryos on embryogenic callus, bar= 10 mm. Fig. 4d:
Isolated SEs, bar = 3 mm. Fig. 4e: Genninated SEs 3 weeks after transfer to germination medium, bar = 7
mm. Fig. 4f: Transplanted emblings originated from somatic embryos three n.;;,,ths after transplanting, bar =
23mm.

3. The chromosomal bases of somaclonal variation in Pistachio

Somaclonal variation may be defmed as genetic variation observed among progeny of
384
plants regenerated from somatic cells cultured in vitro. Somaclonal variation has been
observed among woody plant regenerants (Skirvin et al. 1993). Variation in height,
number of branches, leaf traits and chromosome number may be the indication of
somaclonal variation.
To date, few chromosome reports have been made for this species (P. vera)
(Zohary 1952, Whitehouse 1957, Maggs 1973 and Barghchi & Alderson 1989). In view
of the high interspecific fertility, the available chromosome counts (2n = 30) of P. vera
are somewhat surprising (Scaramuzzi 1957; Rogers 1957; Zohary 1962; Maggs 1973).
However, Barghchi & Alderson (1989) have reported different chromosome numbers
(2n= 32). More to the point, none of these authors provided any morphological evidence
in their publications. As far as we are aware, no methods have been carried out in
Pistachio to study the identification of chromosome number, nor are there reports of sex
chromosomes in this species. The chromosomes of the somatic embryos were counted
after preparation with a squash method (Onay 1996).
To confirm the origin and nature of the micropropagated emblings, the
chromosome numbers were determined. Root tips were pre-treated overnight with I%
a-bromonaphthalene, acid hydrolysed and stained for a few minutes in 2% acetic
orcein. The chromosomes were counted in several regenerated emblings. The
chromosome number of somatic embryos was counted in more than 100 metaphase
plates and confirmed that somatic embryos of Pistachio derived from EMS of immature
fruits have a complement of 2n = 30 (Fig. 5). Mixploidy was never encountered,
although some differences in chromosome morphology could be seen within a
complement. In other words, all emblings regenerated via embryogenesis had the same
diploid chromosome number. Somaclonal variation has been detected in somatic
embryogenesis of both woody angiosperms and gymnosperms (Devemo 1995).
However, somaclonal variation may not always be detected during somatic
embryogenic culture or during regeneration of plants. For example, abnormalities
appeared after several years of growth in the field in oil palms (Corley et al. 1986). For
this reason, plants regenerated from somatic embryogenic cultures should be monitored.
Somaclonal variation, however, can also be advantageous. Selection for somaclonal
variants expressing important traits such as disease resistance, coupled with early, rapid
screening of regenerated plants, may be a technique that can be used for improvement
of woody plants (Devemo 1995).
From these preliminary results it may be concluded that neither mixploidy nor
aneuploidy were observed in either somatic embryos or regenerated emblings in
Pistachio. However, the sex of the regenerated emblings is still questionable because
those embryogenic lines produced could be a genetic male or female line. The
resolution of chromosome morphology was insufficient to conclude whether
recognisable sex chromosomes were present. If it is possible to determine sex by
chromosome states, the cloned emblings from juvenile tissues may be used in order to
study clonal effects of in vitro sex developed emblings. In addition it could be easier to
determine the sex chromosomes, once the haploid chromosome number is obtained
from anthers and pollen. Studies have started to examine dioecy in P. vera and have
initiated molecular and genetic factors involved in sex expression in the species
(Hormaza & Polito 1996). However, that determination will be eventually possible by
 385
improving the developing techniques. Recently, a complete karyotype analysis on I 0
Pistacia species was done by Lin Guo Yang and all of the pistachio species have either
28 or 30 chromosomes (pers.·comm., Dan Parfitt 1999).

Fig. 5: A diploid metaphase plate obtained from a somatic embryo produced through EMS cultures of
immature fruits of Pistachio, bar= 12 J.lm.

4. Conclusions and Future Perspective

Primary explants which produce somatic embryos in Pistachios, are restricted to
juvenile tissues such as immature fruits and juvenile leaf explants. Explants from
mature trees, when cultured, have not demonstrated embryogenic potential. Whether
embryogenic potential exists in meristematic tissue types in the mature tree, such as
vegetative and reproductive buds and apical buds remains to be determined.
Figure 6, summarises the possible routes of in vitro origins of somatic embryos in
Pistachio, both from the primary explants and from embryogenic callus. Once
embryogenic cultures are established from the primary explants, the multiplication of
somatic embryos might follow any one of the pathways described (Fig. 6).
Although initiation, maturation and germination of somatic embryos is being
controlled, high frequency regeneration, synchronous development of somatic embryos
and transplantation into the field is not very successful. Refinements in protocols are
necessary to get good quality embryos closer to zygotic embryos to facilitate storage,
germination and encapsulation of these embryos. The embryogenic potential of an
explant was observed to be not only on the age of explants but also on type of explants
used. At present, expression of the embryogenic potential is limited to juvenile tissues.
386
We have documented the morphological characteristics of possible routes of somatic
embyogeny on initiation, maintenance and development media in P. vera L. To extend
the expression of embryogenic potential to the explants from mature trees, it is
necessary to identify cell or tissue types which are physiologically identical to these
juvenile tissues.
Origin of Somatic Embryos
Pistacia vera L.

Primary explant
" ~ Primary explant
Immature fruits Juvenile leaf
~ ~
Liquid medium + BAP (1-4 mg n Liquid medium + TDZ (0.5-4 mg r 1)

~ ~
Nodular structures Nodular/globular structures+ BAP
~ ~
Multicellular embryogenic callus Embryogenic callus
~ ~
Liquid medium Solid medium
(low or no BAP) supplemented. with BAP
(suspension cultures) ~
~
Cleavage of pre-existing somatic Proliferation of embryogenic masses
embryos( On liquid or solid media) (Solid medium)
~
Somatic embryos

Figure 6: Possible routes of somatic embryogeny on initiation, maintenance and development media in
pistachio (P. vera L.).

Finally, the progress in Pistachio somatic embryogenesis described here shows

promise as a model system for mass clonal propagation of Pistachio trees. Although
somatic embryogenesis is a common feature of in vitro morphogenesis in many species
of higher plants this appears to be the first review on somatic embryogenesis for P. vera
L. Consequently, the establishment of a sound protocol for the production of somatic
embryos of P. vera L. is the first step towards a methodology for the production of
clonal stocks. Efforts are now under way to initiate EMS from mature explant materials
using maternal tissues.

Acknowledgements. The research was funded by a grant from University of Dicle, Faculty of
Science and Art, Diyarbakir, Turkey, and performed under Scottish Office Agricultural and
Fisheries Department licence. Grateful thanks are extended to Mr John Findlay of the Science
Faculty Electron Microscope Facility for photography of the tissue cultures.
 387
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 11. SOMATIC EMBRYOGENESIS IN PECAN
(CARYA ILLINOINENSIS)

Hazel Y. Wetzstein, Benjamin S. Jeyaretnam1 , Wagner A. Vendrame2 , and Adriana

P.M. Rodriguez 3 •

Department of Horticulture, University of Georgia, Athens, GA, 30602, U.S.A.

Present addresses: 1 Complex Carbohydrate Center, University of Georgia, Athens, GA 30602, U.S.A., 2
School of Forest Resources, University of Georgia, Athens, GA 30602, U.S.A., 3 Biologia Celular,
CENA/USP, Av. Centenario 303, C. Postal96, 13400-970, Piracicaba, SP, Brazil.

Chapter contents

1. Introduction
1.1. Importance of Caya
1.2. Crop improvement strategies and application of biotechnology
2. Induction of embryogenic cultures
2 .1. Explant and media components
2.2. Effects of auxin type during induction
3. Repetitive embryogenesis
4. Embryo conversion
5. Embryo development and maturation
5 .1. Zygotic embryogenesis
5.2. Comparison of somatic and zygotic embryo development
6. Growth in the field
6.1. Phenotypic analysis
6.2. Molecular analysis
7. Conclusions
8. References

1. Introduction

1.1. IMPORTANCE OF CARY A

Pecan, Carya illinoinensis (Wangenh.) K. Koch, is the most economically important nut
tree crop indigenous to North America. It is a member of the Juglandaceae family
which includes hickories and walnuts. Pecan is native to the Mississippi River Valley
391
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 391-414.
© 2000 Kluwer Academic Publishers.
392
and tributaries, and extends from the north central and eastern United States into
northern Mexico (Peterson, 1990; Harlow et al., 1991). Commercial production has
extended considerably beyond its native range from Ontario, Canada, south to Oaxaca,
Mexico, and from Virginia and the Carolinas west to California (Grauk.e and Thompson,
1996). Some commercial production occurs in Israel, South Africa, Australia, and
Brazil. However, the United States is the principal grower of pecans and produced 335
million pounds in 1997 (Agricultural Statistics 1999, USDA). The state of Georgia
alone produced about 30% of the U.S. crop. In addition to producing a prized edible
nut, pecan can be a valuable landscape tree forming an upright, cylindrical-to-rounded
crown. Pecan is a source of wood products for flooring, veneer and furniture, and
serves as a habitat and food source for wildlife. The oil extracted from the kernels is
edible and can be used to produce drugs and essential oils.

1.2. CROP IMPROVEMENT STRATEGIES AND APPLICATIONS OF

BIOTECHNOLOGY

Somatic embryogenesis has been shown to be a plant tissue culture technique with
potential applications for mass clonal propagation, genetic transformation, and use in
studies of embryo development. The integration of this technology into pecan crop
improvement programs could be very beneficial.
Propagation in pecan is primarily by budding or grafting of improved cultivars onto
seedling rootstocks. At present, there is no means for the commercial production of
clonal rootstocks in pecan. Thus, this crop has not been able to utilize rootstock
improvement strategies adopted for use in other tree crops. Somatic embryogenesis is
a promising means for clonal rootstock production. Potential uses of clonal rootstocks
in pecan include the introduction of desirable characteristics such as dwarfing for size
control, enhanced nutrient uptake, control of alternate bearing, salinity tolerance,
nematode resistance, and growth uniformity.
Disease and pests can be serious and comprise a major part of the production costs
in pecan. Common insect pests include aphids, casebearer, bud moth, bark beetle, and
borers. Pecan is also susceptible to a number fungal diseases of which pecan scab is the
most serious. The application of gene transformation methods to pecan would be
advantageous. Potential areas for improvement in pecan include disease and insect
resistance, improved oil quality, high yield, regular bearing, precociousness, tree size
control, and early maturity.
For pecan, reproducible methods exist for the initiation of embryogenic cultures and
the regeneration of plants (Wetzstein et al., 1996). For the most part, methods can be
applied to a wide range of genotypes. A current limitation is that success has been
obtained only when immature zygotic embryos are used as the explant. Refinements and
further developments of protocols will be described in this contribution. In addition, we
will report fmdings on pecan embryo development and maturation, as well as recent
results on field tests of regenerated trees.
 393
2. Induction of Embryogenic Cultures

2.1. EXPLANT AND MEDIA COMPONENTS

Successful reports of somatic embryogenesis in pecan have used zygotic embryo tissues
as starting material for induction of embryogenic cultures. Since earlier reports, several
aspects of the system have been tested and optimized. The first report on pecan somatic
embryogenesis (Merkle et al., 1987) presented low embryogenic frequency and
suggested that the developmental stage of explants may be limiting the induction of
somatic embryos. Embryogenic response was obtained in WPM containing casein
hydrolysate, 2,4-D, and BAP (defined as P2, Table 1). The use of cytokinins and
glutamine in the conditioning medium (P 1, Table 1) was not effective.
Following this work, the same P2 medium (now defined as WPM2) was tested
against a medium with high cytokinin:auxin ratio (WPM5, Table 1) in two cultivars,
Stuart and Desirable (Wetzstein et al., 1988). Somatic embryos were observed in up to
40% of cultures in both cultivars and culture media. Embryogenic response varied with
the developmental stage of the zygotic embryo explant. The best stage for embryogenic
response was approximately 15 weeks after pollination, characterized by rapid cotyledon
expansion and presence of liquid and gelatinous endosperm, and shell hardening to 2/3

TABLE 1. Composition of culture media used for conditioning/induction of embryogenic cultures of pecan

Reference Denomination Medium components

Merkle et al., 1987 P1- WPM, 30g/l sucrose, 0.8% agar, 250mg/l glutamine, 2mg/l
conditioning kinetin, 1mg/l BAP, 0.01mg/l IBA
Merkle et al., 1987 P2=WPM2 WPM, 30g/l sucrose, 0.8% phytagar, 1g/l casein hydrolysate,
Wetzstein et al., 1988 conditioning 2mg/l 2,4-D, 0.25mg/IBA
Wetzstein et al., 1988 WPM5 WPM, 30g/l sucrose, 0.8% phytagar, 1g/l casein hydrolysate,
0.5mg/12,4-D, 1mg/IBAP, 1mg/l 2iP
Wetzstein eta!., 1989 Modified WPM minus glycine, with 30g/l sucrose, 1g/l casein
WPM hydrolysate, 4g/l phytagar, 2mgll 2,4-D, 0.25mg/l BAP
Corle-Olivares et al., Induction BDS basal nutrient medium (Dunstan & Short, 1977).
1990 medium 0.51mm ascorbic acid, and one of the growth regulator
combinations: 1) 0.04,uM picloram + 4.4,uM BAP; 2)
0.41,uM picloram + 2.2,uM BAP; 3) 0.05,uM IBA + 4.4,uM
BAP + 9.3,uM kinetin; 4) 0.05,uM 2,4-D + 14.8,uM
adenine, 30days.
Wetzstein et al., 1990 Induction WPM minus glycine, with 30g/l sucrose, 1gll casein
medium hydrolysate, 4g/l phytagar, 0.25mgll BAP, NAA (10.7,uM,
32.2,uM or 54,uM) or 2,4-D (4.5,uM or 9,uM)
Yates & Reilly, 1990 Conditioning Tulecke & McGranaham (1985) basal medium with 0.7%
medium agar and one of the growth regulator formulations (,uM), for
4 to 6 weeks: 1) 0.5 IBA + 4.4 BAP + 9.3 Kin; 2) 5.0 IBA
+ 52.8 BAP; 3) 52.8 BAP; 4) 5.0 IBA.
Rodriguez & Induction WPM minus glycine, with 30g/l sucrose, 1g/l casein
Wetzstein, 1994 medium hydrolysate, 3g/l gelgro, 1.2 .uM BAP and either NAA (32
or 64 ,uM) or 2,4-D (9 or 27 ,uM).
394

of the total length (Wetzstein et al., 1989). Yates and Reilly (1990) confirmed the
importance of the zygotic embryo developmental stage for somatic embryo induction for
several other cultivars under different culture conditions (Table 1). These authors
defined a globular stage zygotic embryo, surrounded by abundant liquid endosperm for
highest embryogenic frequency.
The time on conditioning medium (supplemented with 2,4-D and BA, Table 1) prior
to transfer to basal medium was evaluated by Wetzstein et al. (1989), who observed that
one week was sufficient for good embryogenic response. The percent of embryogenic
cultures reached 85% and 55% in 'Stuart' and , 'Desirable', respectively. Genotypic
differences in embryogenic response among cultivars were also observed by Yates and
Reilly (1990), with 'Stuart' and 'Desirable' being the most and least responsive,
respectively (Table 1). 'Cherokee', 'Elliott', 'Schley', and 'Wichita' had greater
embryogenesis in medium 1, while 'Stuart', 'Barton', 'Cape Fear', and 'Desirable' in
medium4.
Other combinations of auxin:cytokinin (Table 1 ) were used by Corte-Olivares et al.
(1990), who obtained low percent embryogenesis (1.0-3 .5%) from zygotic embryo axes
of two new cultivars, Western Schley and Wichita. Although somatic embryo
development was limited, embryogenic callus proliferated rapidly. Plantlets were
recovered by induction of organogenesis from the somatic embryo tissues on medium
with high BAP:picloram ratio.
Wetzstein et al. (1990) compared two auxins (NAA and 2,4-D) in the induction
medium (Table 1) using cultivars Stuart and Desirable. The results suggested that the
type and concentration of auxin influenced embryo form and percent embryogenesis.
This trend was later confirmed by Rodriguez and Wetzstein (1994), who evaluated callus
and embryo formation and morphology in relation to the presence of NAA or 2,4-D in
the induction medium (Table 1). The percent of cultures with callus and/or somatic
embryos was 100% for all treatments 12 weeks after induction, except for 12 mg/12,4-
D (95% of the cultures were embryogenic). Cultures induced in NAA responded earlier,
with 100% embryogenic cultures by 4 weeks after induction.

2.2. EFFECTS OF AUXIN TYPE DURING INDUCTION

Differences in embryo morphology

Although high rates of embryogenesis can be obtained using a number of auxin
induction media, qualitative differences in embryo morphology and function were
observed depending on the type of auxin used (Rodriguez and Wetzstein 1994). Embryo
quality was assessed by classifying the cultures according to embryo morphological
characteristics, mainly in relation to cotyledon and shoot apex morphology. Cultures
induced in NAA had a higher percentage of embryos with well-defmed shoot apices and
cotyledons (34-41 %), while those induced in 2,4-D had 69-84% of cultures with a
predominance of embryos with weak or no shoot apex.

Histological differences in initiation and development of embryogenic cultures

Detailed histological and morphological studies were conducted to compare somatic
embryo induction with NAA versus 2,4-D (Rodriguez & Wetzstein, 1998). In pecan
 395
and other systems, a higher incidence of embryo abnormality occurs with induction by
2,4-D compared to other types of auxin.
Cotyledon tissues in zygotic embryos during explanting were characterized by
compactly arranged mesophyll cells, a single epidermal layer and vascular strands (Fig.
lA). The parenchyma cells were usually isodiametric with a single large vacuole, a
condensed nucleus and cytoplasm either appressed to the cell membrane, or forming
cytoplasmic strands.
After 7 days in culture, a general enlargement of the explant was observed with both
auxin treatments. In general, the more superficial cell layers, closer to the epidermis,
exhibited mitotic activity with frequent periclinal divisions and occasional anticlinal and
oblique figures. However, subsequent tissue responses varied with the type of auxin
used. In NAA treatments, proembryogenic protrusions were observed after day 10 (Fig.
!B). In tissue induced with 2,4-D, cell division was more intense (Fig.lC). Extensive
mitotic areas produced large cellular proliferations as early as 7 days. The epidermal
surface in 2,4-D cultures frequently ruptured (Fig. 10), as a result of the intense mitotic
activity in internal cell layers. In contrast, explants induced with NAA has primarily
intact epidermal surfaces (Fig.lE,F). Somatic embryo formation occurred in all
treatments. However with 2,4-D, somatic embryos were present as early as 7 days.
Embryos formed on NAA were usually single with a defined shoot apex and continuous
epidermis (Fig. IE). In 2,4-D, diverse types of embryos (Figs. 2G,H) were observed
frequently with a discontinuous epidermis about the shoot apex area and asymmetric
cotyledon development. This could be an effect of the more intense proliferation of the
internal cells of the explant.
The intensity of cell divisions and the formation of large embryogenic protrusions
may influence the formation of abnormal embryo types. The higher incidence of
abnormalities in embJ;.yo morphology in 2,4-D may also be caused by the early
formation of embryos while still in medium containing auxin. Disruption or
malformation of the shoot apex may contribute to abnormal formation of cotyledons.
Liu et al. (1993) proposed that the shoot apex is the site of auxin synthesis and that
cotyledon formation is related to auxin polar transport. Choi eta!. (1997) also confirmed
the importance of polar auxin transport in normal cotyledon formation, by culturing
globular somatic embryos in medium with TIBA (2,3,5-tribenzoic acid), a polar auxin
transport inhibitor. Rodriguez and Wetzstein (1998) suggested that, in pecan extensive
cell proliferation and callus formation may cause malformation of the shoot apex and
subsequently poor cotyledon development.
In pecan, a wide range of auxin types and concentrations can initiate embryo
formation. However, embryo quality and form can vary with induction medium. This
stresses the importance of adjusting protocols to optimize not only embryo number, but
also quality.
396

F1g. I. Micrographs showing the initiation and development of pecan somatic embryogenic cultures. A)
Zygotic cotyledon cross section at time of explanting. Point=vascular traces B) Embryogenic protuberance
(day 10) induced on medium with NAA . C) Culture induced on medium with 2,4-D showing extensive
proliferation of embryogenic cells (day 7). Point= globular proembryogenic structures. D) Rupture of
epidermis and callus proliferation (ca) seen in cultures induced with 2,4-D. E) and F) somatic embryos
induced on medium with NAA. G) Somatic embryos induced on medium with 2,4-D. Tubular (arrow) and
fan-shaped (point) embryos are evident. H) Barrel-shaped somatic embryos with ruptured areas on the
shoot apex. Cotyledon are malformed. Bars : Figs. A,B,D,E,H= 100 I-' m; C, F=200 I-'m; G =400 I-'m.
(Reproduced with permission from Protoplasma).
 397
3. Repetitive Embryogenesis

Reports on pecan somatic embryogenesis are characterized by a repetltlve

proliferation of secondary embryogenesis after transfer to basal medium without growth
regulators. Some repetitive cultures that are maintained in our laboratory date back to
introductions made in 1986. Routinely, embryogenic cultures are maintained with
subculture at 4-8- week intervals to fresh modified WPM (Table 1). Subculturing
involves separating highly embryogenic clusters and transferring them to fresh medium.
Cultures are maintained in the dark, at 27 ±2°C. Repetitive embryos form individually
or in groups directly from somatic embryo tissues.
In the repetitive system, globular, heart shape and cotyledonary forms are observed.
Somatic embryos with different morphological characteristics, as reported by Rodriguez
.and Wetzstein (1994), continue to form in the repetitive system after years of
maintenance in basal medium: Class 1 embryos - somatic embryos resembling zygotic
embryos, with clear bipolarity and well-developed shoot apices and cotyledons (Fig.
2A); Class 2- somatic embryos with multiple or fused shoot apices, or fused embryos
(Fig. 2B); and Class 3- somatic embryos with poorly developed or absent shoot apices
(Fig. 2 C,D).
At any given time, class 1-type embryos can be harvested for plant regeneration.
These are individualised and cultured in fresh basal medium to enlarge. After they reach
8-10mm (width), conversion enhancement treatments are applied to facilitate
germination. If conversion enhancement treatments are not given to cotyledonary-stage
pecan embryos, a tendency for dedifferentiation is observed and new somatic embryos
or callus will form (Mathews and Wetzstein, 1993).

4. Embryo Conversion

Regeneration of plantlets from pecan somatic embryos was initially reported by

Wetzstein et al. (1989), who tested desiccation treatments (1 or 5 days) to promote
conversion of somatic embryos into plantlets. Root growth was greatly improved by the
longer desiccation treatment, with better lateral root branching in MS (Murashige &
Skoog, 1962) versus WPM medium. Somatic embryo conversion was obtained with
supplementation of calcium nitrate (0.5%) to avoid shoot necrosis, however, after
germination, continuous shoot growth was not observed.
Embryo conversion was further improved with additional treatments consisting of
a cold treatlnent (5 weeks at 5°C), followed by desiccation (5 days) (Wetzstein et al.,
1990). The combination of both treatments improved root emergence up to 71.9%,
compared to a maximum of 48.8% with desiccation only and 16.9% with cold treatment
alone. Shoot development after germination was still limiting high plant survival. Out-
planting was more effective when embryos had a strong root emergence.
Somatic embryos from eight cultivars germinated after approximately one year in
culture, with clear differences among cultivars (Yates and Reilly, 1990). 'Schley'
embryo germination rate was six times greater than from any of the other cultivars. The
authors suggest this behavior in culture might be related to the growth habit of this
398

Fig. 2. Pecan somatic embryos showing different morphological characteristics in repetitive embryogenic
cultures maintained for more than 4 ('Pabst ' ) or 10 ('Stuart' ) years. A) Well formed 'Stuart' somatic
embryos (class I), with well-developed cotyledons and shoot apices; B) 'Stuart' somatic embryos, the one
on the left has a well-formed cotyledons and prominent shoot apex (class I); the one on the right shows
fused cotyledons and a multiple, abnormal, malformed shoot apex (class 2); C) several 'Pabst' embryos with
different morphological aspects, mostly lacking a shoot apex (class 3); D) Tube-shaped 'Pabst' somatic
embryo with weak or no shoot apex (class 3). All bars=lOO,um. (NAP/MEPA , at ESALQ, Univ. Sao Paulo
is acknowledged for use of the electron microscopy facilities)

cultivar in the field which has a tendency to naturally sprout water shoots. For most of
the cultivars, shoot development was weaker than root development, except for 'Cape
Fear' .
Further studies by Mathews and Wetzstein (1993) focused on the strategies to
improve conversion of pecan somatic embryos and acclimatization of plantlets. Addition
of silver nitrate (5mg/l) to the germination medium (WPM) and application of BAP
( 10011M) directly on the shoot apex of somatic embryos following conversion-
enhancement treatments gave a dramatic increase in plant regeneration . On average,
conversion rates improved to 20% of the embryos, with a maximum of 71% in cultivar
Mahan. The role of silver nitrate may be related to inhibition of ethylene biosynthesis.
 399

Fig. 3. Schematic illustrating the treatments applied to pecan somatic embryos to promote embryo
conversion.

The application of BAP directly in the shoot apex avoids the detrimental effects that this
cytokinin may have in rooting. The whole procedure of conversion and hardening was
successful, with 70-80% of the regenerated plants reaching the hardening stage and 99%
of the hardened plants being transferred to the greenhouse, totaling more than 200
plants. A diagram illustrating embryo conversion and plant regeneration is shown in
Figure 3.
Observations that different morphological types of embryos are formed in culture
led to a study to determine their ability to germinate and convert into plantlets
(Rodriguez & Wetzstein, 1994). Three classes of somatic embryos induced in medium
containing NAA or 2,4-D were collected and cultured in basal medium to grow (see
description of embryo classes in repetitive embryogenesis section) . The embryos were
then given the conversion enhancement treatments defined by Mathews & Wetzstein
( 1993). Irrespective of the type of auxin used in the induction medium, somatic
embryos morphologically resembling the zygotic ones germinated better than those with
abnormal shoot apex formation or with weak or poorly defined apices.
Since NAA induced a higber percentage of well-formed embryos (Rodriguez &
Wetzstein, 1994), we have concluded that NAA is a better auxin for induction of pecan
400
somatic embryos than 2,4-D. Histological studies (Rodriguez & Wetzstein, 1998)
showed a less aggressive response of the explant tissue to NAA, when compared to
2,4-D, which can in part explain the higher percent of abnormalities. To what extent
the effect of 2,4-D is carried out into further subcultures has not been determined.

5. Embryo development and maturation

5.1. ZYGOTIC EMBRYOGENESIS

A clear understanding of pecan zygotic developmental stages, and the

morphological, physiological and biochemical changes that are associated with each of
these stages has been useful to compare and evaluate somatic embryo development in
culture. Comparisons of developmental timing, physiological maturity, and molecular
and biochemical changes have provided strategies that modify culture protocols to
obtain higher quality embryos. Pecan embryo development can be divided into a)
histodifferentiation, b) cotyledon, b) maturation, and c) post-abscission stages based on
morphological, physiological and biochemical properties and attributes (Jeyaretnam et
al., 1999) (Fig. 4 ). Because several unique changes occur during cotyledon expansion
in pecan, we considered it as a distinct development stage.
The embryo is microscopic during histodifferentiation and is characterized by
globular and heart-shaped forms. The histodifferentiation stage continues until about
97 days post pollination (DPP). This is followed by the cotyledon stage which spans
from about 97 to 125 days DPP. During the cotyledon stage, the cotyledons enlarged
to the full length of the ovule. In addition, marked changes are observed with the
transition of nuclear liquid endosperm to cellular endosperm, also referred to as the 'gel'
stage. The endosperm plays a major role in embryo development by providing nutrients
and developmental cues. Numerous soluble proteins and few or no insoluble (storage)
proteins are expressed during this stage based on SDS-PAGE analysis (Fig. 7). During
this period, both shuck and shell are pale green color.
The maturation stage follows the cotyledon stage and is characterized by an
exponential increase in embryo weight and a concomitant decline in water content.
Storage deposition takes place which is manifested by the lateral expansion and fllling of
the cotyledons. Also many insoluble proteins are expressed within a narrow period of
development. The severing of vascular connections to maternal tissues marks the end of
the maturation stage and the beginning of the post-abscission stage. During the post-
abscission stage, water content declines (from 28 to 15%) and a number of insoluble
proteins disappeared.

5.2. COMPARISON OF SOMATIC AND ZYGOTIC EMBRYO DEVELOPMENT

Zygotic embryos, which develop within the maternal tissue, are regulated and
programmed in response to changes in hormone and moisture regimes. In contrast,
somatic embryos develop in an artificial (in vitro) environment, which is very different
 401

./ Abscission
V

......
Embryo microscopic
Histodifferentiation r Mat
Jf PA Des D
-Globular
- - -&- •:
Heart Cot

Liquid ~ndosperm

Gelatinous Endosperm

11111111111111111111111111 Cotyledon Elonglljtion

l
Cotyledon Filling II II lj II II IIIII! II II II II II I
' ' I
~ Sheli harden~ng
. ' I

~ Green ~~ Greenish White ~k-- White ~~ Bro~n ~

SheiiMarking~l
I
Shuck DehisceF-.-.·,.-.-.;:;;•ww~

k Soluble proteins ~~+- ~~~~~~~i~~~teins t~ Stprage Proteins

: IIIII : ; liJo'

70 80 90 100 110 120 130 140 150 160 170 180

Days Post Pollination

Fig, 4. Time scale of events during pecan fruit development. Cot=cotyledon stage, Mat=maturation stage,
PA=post abscission, Des=desiccation, D=dehiscence. (Reproduced with permission of the Journal of
Horticultural Science and Biotechnology).

from what exists within the developing fruit. The culture medium exerts fairly constant
environmental influences. The different environmental conditions provided during in
vivo vs. in vitro embryogenesis may translate into divergent developmental programs that
affect embryo function and competence. Comparisons of somatic and zygotic embryos
reveal several morphological, biochemical, and physiological differences. The quantity
and quality (types and composition) of the lipids, proteins, and carbohydrates found in
the mature embryo play a major role in the germination and growth of the seedlings.
Different storage materials will require different cascades of enzymes (proteins) for
efficient germination and successful early seedling growth.

Anatomy
Profound cytological differences were observed between somatic and zygotic embryos.
Cotyledonary cells in zygotic embryos had extensive lipid deposits (Fig. 5A). Protein
bodies and occasional starch grains were evident. In contrast, somatic embryos collected
from repetitive cultures had few or little lipid deposits (Fig. 5B). Cells were highly
vacuolate. Subsequent biochemical analyses concurred with these observations.
402

,. .
Q

Fig. 5. Light micrographs of pecan cotyledons from a A) mature zygotic embryo, and B) somatic embryo.
Point=lipid, arrow=protein body

Storage lipids
Lipids make-up a large portion of pecan zygotic embryo weight, accounting for 68% of
the total weight (Rudloph, et a!., 1992). More than 98% are storage lipids, of which
triglycerides are the major component. Triglycerides are neutral lipids and are formed
from fatty acids and the trihydroxy alcohol, glycerol. In addition to trig1ycerides, very
low amounts of mono- and di-glycerides are present at varying levels in pecan embryos
(Kanamangala et a!., 1999). During germination, these ester bonds are cleaved due to
hydrolysis and free fatty acids are released. The fatty acids that are esterified to glycerol
show great variation in chain length and degree of saturation (number of double bonds).
The relative amount of fatty acids found in zygotic embryo occurs in the order of oleic
acid (C18: 1) > linoleic acid (C18:2) > stearic acid (C18:0) > palmitic acid (C16:0).
In contrast, somatic embryos have a much lower triglyceride content (both on a per
embryo and per unit weight basis) than zygotic embryos (Table 2). Treatments identified
to enhance conversion, i.e., enlargement and cold plus desiccation, increased triglyceride
content by 3and 5 fold, respectively, compared to small embryos. However, triglyceride
levels remained well below that found in zygotic embryos. Similarly, conifer somatic
embryos had a low level of storage reserves that affected the latter stage of somatic
embryo development and germination (Feirer eta!., 1989). In pecan, no qualitative
differences in the classes of fatty acids are observed between somatic and zygotic
embryos. However, quantitative differences were apparent (Fig. 6). In small and
enlarged somatic embryo, the abundant fatty acids are linoleic acid (C18:2) followed by
oleic acid (18: 1) and palmitic acid (16:0). This indicates that somatic embryos contain
relatively greater levels of unsaturated fatty acids. When the enlarged somatic embryos
were subjected to conversion treatment of 8 weeks cold and 5 days of desiccation, a
quantitative change of the component fatty acids was noticed. Treatments resulted in a
higher content of oleic acid relative to linoleic acid, a condition which mimics mature
 403

SmallS. E.
~18:1 (40%)
18:2 (46%)~

Internal Standard ~

_A~ 16:0 (12 %) \..._C 18:0 (2 %)

Enlarged S. E. ~18:1 (36%)

18:2(49%)~

A~- 16:0A(14 %) A
~18:0(2%)
l..J\
S. E. after Cold +
Desiccation treatment ~18:1 (49%)

18:2 (35 %)~

A~ 16:0 (12 %) 1\.

)
v \...../\~18:0(4%)
Z.E.
~18:1 (66%)

18,2 (24 %)+---~

1 ~18:0(2%)

I
30 35

Retention Time (min)

Fig. 6. GLC-MS analysis of the methy ester derivatives of fatty acids (of
triglycerides) present in somatic embryos (S. E.), S. E. subjected to conversion
enhancement treatment and zygotic embryos (Z. E.)
404

TABLE 2. Triglyceride content of fresh somatic embryos given various conversion-enhancement treatments.

Treatment Ave. Embryo Wt. Triglyceride

(mg) mgtembryo ~tglmg FW

Small embryos 31 0.8 24 a

Enlarged embryos 164 2.3 14 b
Cold + 5 d desic. 188 1.7 9c
Zygotic embryo· 1924.0 481
4000
Means with same letters are not statistically different (LSD, a =0.05). d desic: days desiccation; Wt: weight;
FW, Fresh weight; *Values were not included in statistical analysis.

zygotic embryos (Fig. 6). This observation points to the fact that, there are biochemical
changes taking place during conversion enhancement treatments that promote more
zygotic-like development which may improve conversion competence.

Proteins
In developing embryos several temporally expressed specific proteins (maturation-
specific, embryogenesis-abundant proteins and germination/growth-associated proteins)
have been identified in different species. We evaluated changes in protein profiles during
pecan embryo development by SDS-PAGE analysis. Soluble proteins were extracted in
physiological buffer and insoluble proteins were extracted with detergent. During pecan
zygotic embryo development insoluble proteins (i.e., storage and structural proteins)
were expressed after the onset of the 'maturation stage' (Fig. 7). Several (more than 11)
insoluble proteins were concurrently expressed within a narrow window of pecan zygotic
embryo development corresponding to the phase of rapid dry matter accumulation. This
indicates a possible storage function. In contrast, many soluble proteins were first
expressed during early cotyledon stage and continued to be present in maturing embryos.
However, several of these soluble proteins disappeared during the post-abscission stage,
potentially those that are germination-related.
Comparisons of protein profiles of somatic and zygotic embryos show that somatic
embryos lack prominent insoluble protein bands found in zygotic embryos (Fig. 7).
When enlarged somatic embryos were subjected to conversion enhancement treatments
of cold and desiccation, the total protein content per unit embryo weight increased to a
level similar to zygotic embryos (Table 3). The protein content on a per unit weight and
per embryo basis increased after 8 weeks cold (4 oq and subsequent 5 days desiccation.
However, the total protein levels in somatic embryos remained well below that found in
zygotic embryos.
 405

kD
116.2
66.2
45.0
31.0

21.5
14.4

116.2
66.2
45.0

31.0

21.5
14.4

M 1 2 3 4 5 6 7 8 M 9 10
a. ro
.... ....ro "'0
(].)
X ~
w >-
....0 -;::
~
I
-roro.....Ol
-
"0
ro E c

u
u w ~ (/)UJ

Zygoti c SE

Fig. 7. PAGE-analysis of pecan proteins extracted from zygotic embryos (ZE) (Lanes 1-8) and somatic
embryos (SE) (Lanes 9, 10). A) Soluble proteins. B) Insoluble proteins.
406

TABLE 3. Protein content of fresh somatic embryos given various conversion-enhancement treatments.

Treatment Emb. Wt. (mg) Protein

mg/embryo p.gl mg FW

Small embryos 31 3.0 95 a

Enlarged embryos 164 2.5 15 c
8 weeks Cold 205 15.0 73 b
Cold + 5d desic. 188 18.4 %a
Zygotic embryo* 4000 68.0 92

Means with the same letters are statistically different. (T test LSD, a=0.05). 5d desic. 5days desiccation;
* Values not included in statistical analysis.

6. Growth in the Field

Tissue culture-induced variation or somaclonal variation is a major concern in the

production of true-to-type regenerants. Several factors have been reported to affect the
nature and extent of somaclonal variation, such as length of time in culture, explant type,
frequency of subcultures and culture conditions (De Klerk, 1990). Before somatic
embryogenesis can be exploited for clonal propagation, the genetic fidelity of regenerated
plants must be evaluated and their field performance assessed.
A major difficulty in evaluating tissue culture regenerated plants is the need to
develop a method where material can be easily and rapidly screened to reveal any genetic
differences. Additionally, if changes occur, it must be determined whether they are
heritable (true somaclonal variation) or temporary (epigenetic or physiological). Some
common methods for evaluation of somaclonal variation are phenotypic analysis,
chromosome counting and structure analysis, electrophoresis patterns of proteins and
isozymes, and DNA restriction fragments (also called molecular markers) (De Klerk
1990). Despite the long period of time necessary for field evaluations, phenotypical
analysis is still largely applied to evaluate variation, based on the percentage of plants
showing mutations for one or more defined characteristics.
We have conducted field studies to assess if pecan trees regenerated using our
embryogenic culture protocol maintain clonal fidelity. Two approaches were used:
phenotypic analysis and molecular analysis. The trees used in this work were regenerated
from two different culture lines (designated A and B). Here, a culture line is defined as
all tissues derived from one original zygotic embryo explant. Trees were transferred into
the field in July to September 1994. Depending upon the characteristics assessed,
evaluations were made 3 to 4 years after field planting (Fig. 8). Phenotypic evaluations
were made of 7 trees from one line and 15 trees from another line. For molecular
analysis, 10 and 18 trees were evaluated from the same two lines used for phenotypic
studies.
 407

Fig. 8. Pecan tree regenerated from somatic embryogenesis .

6.1. PHENOTYPIC ANALYSIS

Table 4lists some of the phenotypic characteristics evaluated. Factors assessed included
general growth measurements, leaf morphological characteristics, and pest susceptibility .
Trees derived from embryogenic cultures exhibited vigorous growth. Comparisons
between trees regenerated from the two lines for tree height, total shoot growth, trunk
caliper, and shoot length per trunk cross-sectional area showed no significant differences .
However , trees regenerated from the 2 culture lines differed in branching habit. The
number of shoots per 1-yr-old branch was 1.7 times greater in trees from line A versus
line B. Aspects of tree form such as branch angle , amount of branching, shoot growth,
and the degree of apical dominance are under genetic control (Kramer and Kozlowski
1979). The extensive number of shoots observed in line A corresponds to characteristics
for 'Mahan' , the maternal parent. 'Mahan' has been characterized as having a multiple
branching habit (Sparks, 1992).
Trees generally exhibited foliage that appeared healthy and free of nutrient
deficiencies . Levels of elements were within normal concentrations for pecan leaves as
measured by plasma emission spectroscopy, and did not differ significantly between lines
(data not shown). In contrast, leaf morphological differences occurred between trees
from the two lines. Trees from line A had significantly lower specific leaf weights and
408

TABLE 4. Mean values in phenotypic evaluations for growth, leaf morphological characteristics and ratings
for the presence of scab (Cladosporium caryigenum) lesions and southern pecan leaf phylloxera (Russellae
stuetze{) galls in trees derived from two somatic embryogenic culture lines.'

Culture Line
Phenotypic Characteristics Significance w
A' B'
Tree height (em) 203 211 NS
Total shoot growth (em) 261 203 NS
Trunk caliper (em) 2.7 2.4 NS
Shoot length per x-sec. area (em/em') 53.1 62.5 NS
No. shoots per 1-yr-old branch 6.4 3.7
Specific leaf weight (mg/cm2) 7.7 9.6 *
Leaf length-to-width ratio (em/em) 1.7 1.5 *
Scab rating v 1.5 3.7 **
Phyl!oxera rating " 23 ·I 5 NS
' Trunk and shoot values were measured at the end of the 1997 growing season. Data for expanding buds were
collected in Spring 1998, and for leaf characteristics, scab and phylloxera ratings in Summer 1998; 'Mean of
7 trees; 'Mean of 15 trees; w Means compared by Tukey's test; v 1 = no scab lesions, 5 = severe scab in over
75% of leaflets;" 1 =no insect galls, 5 =galls in over 75% of leaflets; Ns,•.•• Nonsignificant or significant at
P~0.05 and P~O.lO, respectively.

significantly higher leaf length-to-width ratios compared to trees from line B (Table 4).
Most leaves were pinnate and compound, characteristic of mature pecan leaves. However
within a single tree, leaf morphology exhibited successive changes in maturation. Leaves
in the basal portion of trees had simple, juvenile characteristics. Such changes in phase
are commonly observed in seedling pecan trees and would be anticipated in trees derived
from somatic embryos.
Significant differences in susceptibility to scab were observed between trees from
the different culture lines (Table 4). Trees from line A exhibited little or no scab lesions
on leaves. In contrast, leaves from line B had frequent lesions that severely distorted leaf
morphology. The extremely low occurrence of scab lesions in line A is of interest.
Longer term evaluations will be needed to verify the extent of scab resistance in this line.
Significant differences in phylloxera galls were not observed between tree lines.
No marked differences, visible abnormalities or conspicuous branching patterns were
observed in trees within a line. Trees regenerated within a culture line exhibited some
variation in tree height, i.e., about 1. 6-fold in line A and 1. 7-fold in line B. Ritchie and
Long (1986) evaluated the field growth of tissue-cultured Douglas fir trees that varied in
tree size and plagiotropic growth in early evaluations. However, trees that exhibited
large differences in size when young, became more similar in height 5 years after
establishment. This suggests that early tree differences may not be due to inherent
variation but rather to tree establishment. Additional field observations are needed in
pecan to determine if tree size will become more uniform.
 409

TABLE 5. Comparison of morphological characteristics for trees regenerated from culture lines A and B.

Trees from Culture Line A Trees from Culture Line B

Tree
No. Specific leaf Leaf length-to-width Specific leaf weight Leaf length-to-width
weight ratio (em/em) (mg/cm2) ratio (em/em)
(mg/cm2)

9.9 a 2 2.0 a 10.7 a 1.8a

2 7.7 b 1.8 ab 10.4 ab 1.7 ab
3 7.6 b 1.7 ab 10.3 abc 1.7 ab
4 7.5 b 1.7 ab 10.3 abc 1.7 ab
5 7.4 b 1.6 ab 10.1 abc 1.6 ab
6 7.2 b 1.6 ab 10.0 abc 1.6 ab
7 6.8 b 1.4 b 9.9 abc 1.5 ab
8 9.8 abc 1.5 ab
9 9.6 abc 1.5 ab
10 9.4 abc 1.5 ab
11 9.2 abc 1.5 ab
12 9.1 abc 1.5 ab
13 9.0 abc 1.4 b
14 8.8 be 1.4 b

2 Comparisons by Tukey's test (Ps0.05), independently for each culture line.

Leaves in trees within a line were consistent and characteristic for the species. No
variegation or distortion was observed, unlike that seen in white spruce trees regenerated
from somatic embryos that were partially achlorophyllous (Isabel et al., 1996). In trees
from line A, one tree showed significant differences in specific leaf weight (Table 5). For
leaf length-to-width ratio, trees were generally similar to each other, although tree 1
differed significantly from tree 7. Likewise, in trees from line B, a range in values was
obtained for both specific leaf weight and leaf length-to-width ratio (Table 5). A few
trees showed significant differences from each other. However, overall all trees were
similar within the population observed.

6.2. MOLECULAR ANALYSIS

Although a number of studies have compared the field performance of tissue-cultured

versus conventionally-propagated fruit crops, fewer studies have attempted to address the
extent of genetic diversity in somatic embryogenic systems. A novel PCR-based assay
for plant DNA fingerprinting, AFLP (Amplified Fragment Length Polymorphism), was
used for molecular analysis of pecan regenerates. It is reportedly more efficient than
410

0.3 0.475 0.650 0.825 1.0

1A
SA
10A
2A
3A
8A
8A
9A GROUP1
188
268
228
248
4A
198
7A
21 a
158
118
168
258
128 GROUP2
138
148
178
238
208
278
288

Fig. 9. Phenogram for pecan trees regenerated from two somatic embryogenic lines. Trees 1-10 were
regenerated from line A; trees 11-28 were regenerated from line B. Similarity indexes are shown at the top
bar.

RFLP and RAPD (Faye et al. 1996), reliable and reproducible (Cato and Richardson
1996), requires small amounts of DNA, and can detect a great number of polymorphisms
with few primers (Hill et al. 1996, Majer et al. 1996). With recent fluorescence
techniques, the method is highly automated and eliminates the use of isotopes for
detection.
Recently, the applicability of AFLP analysis was evaluated for assessment of genetic
variability in pecan somatic cultures (Vendrame et al., 1999). We have now expanded
this work to assess field-grown trees. Phenotypic evaluations combined with an AFLP
strategy could be useful in assessing the genetic integrity of somatic embryogenesis-
derived materials. Once a diagnosis of stability has been obtained for a targeted species,
somatic embryogenesis could proceed with higher confidence at an industrial scale as part
of an improvement program as well as for studies involving genetic transformation.
DNA isolation, and AFLP™ procedures followed those previously described (Perkin
Elmer Applied Biosystems, 1997; Vendrame et al., 1999). A total of 361 polymorphic
fragments were identified using three primer pairs. The number was similar to that
obtained by Hill et al. (1996), who identified 320 polymorphic AFLP loci inLactuca spp.
with three pairs of primers. Other techniques such as RAPD and RFLP are more limited
in terms of the number of markers obtained. In another study with pecan, 20 primer
combinations produced 120 RAPD markers (H. Y. Wetzstein, unpublished data). Trees
regenerated from lines A vs. B generally could be distinguished by frequent
polymorphisms detected between the lines. As shown in the phenogram generated from
similarity data (Figure 9), clustering analysis resulted in two major groups: Group 1,
 411

Pecan Somatic Embryogenesis

Tree Immatu re Fn..11t Expla.nr Embrro lnduct•on Embryo Oevelopmem

(SEM ) ( SEM )

Plantlet> on Sool Convers1on ro Plandets

Fig 10. Scheme depicting pecan somatic embryogenesis culture initiation, and somatic embryo and plantlet
production.

composed primarily of trees derived from line A; and Group 2 comprised of trees
primarily derived from line B. However, in addition to the expected between-line
polymorphisms, some differences were detected within lines such as four trees from line
B (trees 18, 22, 24, and 26) which clustered with most of the line A trees in Group 1.
In addition, one tree from line A (tree 7) grouped within the majority of line B trees
(Group 2) . All trees exhibited some polymorphisms (which were repeatable in separate
AFLP analyses) regardless of whether they originated from the same or different lines.
However, trees regenerated from the same culture line generally showed higher similarity
values than those originated from a different culture line (Figure 9).
Somatic embryogenic cultures produced trees that appeared to be phenotypically
stable and thus far have maintained characteristics of the line. No marked phenotypic
variants have been observed after four years growth in the field . In contrast, AFLP
analysis showed polymorphisms L.1at apparently were not reflected in phenotypic
variations. The usefulness of AFLP to evaluate somatic embryo-derived plants for clonal
fidelity will be dependent on how closely polymorphic loci can be correlated with
economically important phenotypic characteristics.
412

7. Conclusions

In pecan, somatic embryogenic cultures can be routinely induced in a wide range of

genotypes and remain highly repetitive for long periods of time. We found that somatic
embryos differ markedly from zygotic embryos in anatomy and biochemistry. However,
conversion-enhancing treatments appear to modify somatic embryo development resulting
in embryos that more closely mimic zygotic forms. The importance of considering
embryo quality when developing culture protocols is illustrated by our observations that
embryo normalcy can be markedly affected by conditions during culture induction. A
diagram depicting pecan somatic embryogenesis is shown in Fig. 10. Regenerated plants
can be successfully transferred to the field and generally appear to be phenotypically
stable and maintain characteristics of the line. A limitation to current protocols is that
immature zygotic embryos are the only explant found to be responsive to date.
Investigations to develop methods for producing cultures from mature tissue would allow
somatic embryogenesis to be used for mass clonal propagation of improved genotypes.
Nonetheless, an application of current methodologies is in the production of clonal
rootstocks, which are not possible using conventional propagation methods. Efforts are
under way to identify promising genotypes for this application. In addition, somatic
embryogenesis can also be used in genetic transformation for crop improvement.

8. References

Cato, S. and Richardson T.E. (1996) Development and evaluation of AFLPs in Pinus
radiata. Plant Genome IV Program, 69. International Plant Genome Conference,
January 14-18, 1996. San Diego, Ca., USA.
Choi, Y.E., Kim, H.S., Soh, W.Y. and Yang, D.C. (1997) Developmental and
structural aspects of somatic embryos formed on medium containing 2,3,5-tribenzoic
acid Plant Cell Reports, 16 (11), 738-744.
Corte-Olivares, J., Phillips, G.C., Butler-Nance S.A. (1990) Somatic embryogenesis
from pecan zygotic embryo explants. HortScience 25,983.
De Klerk, G.-J. 1990. How to measure somaclonal variation. Acta Bot. (Nether!) 39,
129-144.
Faye, C., A. Murigneux, A. Bonjean, P. Lacaze, and Peleman, J. (1996) AFLP™
mapping in maize:distribution and stability of the bands. Plant Genome IV, 73.
International Plant Genome Conference, January 14-18, 1996. San Diego, Ca., USA
Feirer, R.P., Conkey, J.H. and Verhagen, S.A. (1989) Triglycerides in embryogenic
conifer calli: a comparison with zygotic embryos. Plant cell. Rep.8, 207-209.
Grauke, L.J. and Thonpson, T.E. (1996) Pecans and Hickories, in J. Janick and J.N.
Moore (eds.), Fruit Breeding, Vol III, Nuts, Wiley & Sons, New York.
Harlow, W.H., Harrar, E.S., Hardin, J.H., and White, F.R. (1991) Textbook on
dendrology covering the important forest trees of the United States and Canada, 7th
edn., McGraw-Hill, New York, pp. 269-271.
 413
Hill, M., H. Witsenboer, M. Zabeau, P. Vos, R. Kesseli, and Michelmore, R. (1996)
PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in
Lactuca spp. Theor. Appl. Genet. 93, 1202-1210.
Isabel, N., R. Boivin, C. Levasseur, P-M. Charest, J. Bousquet, and Tremblay, F.M.
(1996) Occurrence of somaclonal variation among somatic embryo-derived white
spruces (Picea glauca, Pinaceae). Amer. J. Bot. 83, 1121-1130.
Jeyaretnam, B., Levi, A., Phatak, S.C., and Wetzstein, H.Y. (1999) Changes in growth,
water content and protein reflect embryo development in pecan (Carya illinoinensis),
J. Hort. Sci. And Biotech., 74 (3) 315-320.
Kanamangala, R.V., Maness, N.O., Smith, M.W., Bursewitz, G.H., Knight, S. and
Chinta, B. (1999) Reduced lipid pecan: Chemical alteration and implications for
quality mainteneance during storage, J. Amer. Soc. Hort. Sci. 124 (4):389-398.
Kramer, P.J. and Kozlowski. (1979). Physiology ofwoody plants. Academic Press,
New York.
Liu, C-M, Xu Z-H, and Chua N-H (1993) Auxin polar transport is essential for the
establishment of bilateral symmetry during early plant embryogenesis. Plant Cell, 5,
621-630.
Majer, D., R. Mithen, B.G. Lewis, P. Vos, and Oliver, R.P. (1996) The use of AFLP
fingerprinting for the detection of genetic variation in fungi, Mycol. Res., 100, 1107-
1111.
Mathews, H., and Wetzstein, H.Y (1993) A revised protocol for efficient regeneration
of somatic embryos and acclimatization of plantlets in pecan, Carya illinoinensis. Plant
Sci 91, 103-108.
Merkle S.A., Wetzstein, H.Y. and Sommer, H.E. (1987) Somatic embryogenesis in
tissue cultures of pecan. HortScience 22, 128-130.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays
with tobacco tissue cultures. Physiologia Plantarum 15, 476-497
Perkin Elmer Applied Biosystems (1997) AFLP ™ plant mapping protocol. PIN
4303146 Rev. A., The Perkin Elmer Corp., Foster City, Calif.
Peterson, J.K. (1990) Silvics of North America, vol 2, Hardwoods. USDA For. Serv.
Agric. Handbook 654:205-210.
Ritchie, G.A. and Long, A.J. (1986). Field performance ofrnicropropagated Douglas
Fir. New Zeal. J. For. Sci. 16, 343-356.
Rodriguez A. P.M. and Wetzstein H. Y. ( 1994) The effect of auxin type and concentration
on pecan ( Carya illinoinensis) somatic embryo morphology and subsequent conversion
into plants, Plant Cell Reports, 13, 607-611.
Rodriguez A.P.M. and Wetzstein H.Y. (1998) A morphological and histological
comparison of the initiation and development of pecan (Carya illinoinensis) somatic
embryogenic cultures induced with naphthaleneacetic acid or 2,4-
dichlorophenoxyacetic acid, Protoplasma, 204, 71-83.
Rudolph, C.J., Odell, G.V., Hinrichs, H.A., Hopper, D.A. and Kays, S.J. (1992)
Genetic, environmental and maturity effects on pecan kernel lipid fatty acid,
tocopherol and protein composition, J. Of Food Quality 15, 263-278.
Sparks, D. (1992). Pecan cultivars: the orchard's foundation. Pecan Production
Innovations, Watkinsville, 446p.
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Vendrame, W.A., Kochert, G. and Wetzstein H.Y. (1999) AFLP analysis of variation
in pecan somatic embryos. Plant cell reports 18, 853-857.
Wetzstein, H.Y., Merkle, S.A. Ault, J.R. and Sommer, H.E. (1988) Somatic
embryogenesis in pecan (Carya illinoensis). SAAS Bul Biochem Biotech, 1, 64-67.
Wetzstein, H.Y., Ault, J.R. and Merkle, S.A. (1989) Further characterization of
somatic embryogenesis and plantlet regeneration in pecan (Carya illinoensis). Plant
Science, 64, 193-201.
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embryogenesis and plantlet regeneration in pecan, Carya illinoensis. Acta
Horticulturae, 280, 69-73.
Wetzstein H.Y., Rodriguez A.P.M., Burns J.A. and Magner H.N. (1996) Carya
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Vol. 35, Trees IV, Springer-Verlag, Berlin, pp 50-75.
Yates I.E and Reilly C.C. (1990) Somatic embryogenesis and plant development in eight
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12 . Somatic embryogenesis in Iongan [Dimocarpus Iongan Lour.]

Lai Zhongxiong, Chen Chunling , Zeng Lihui, and Chen Zhenguang

Lab of Horticulture. Biotech., Department of Horticulture, P 0 Box 94, Fujian Agricultural
University, Fuzhou 350002, P R China

Chapter Contents

I. Introduction
l.l Botany/Breeding
1.2 Geographic distribution
1.3 Major problems in diseases & pests
1.4 Economic importance
I.5 Conventional propagation methods
2. In vitro multiplication
2.I Organogenesis
2.2 Problems with organogenesis
3 Somatic embryogenesis
3 .I Culture initiation
3 .1.1 Explants
3 .I.2 Sterilization protocol
3 .2 Induction of somatic embryogenesis
3 .2.I Initiation of embryogenesis of embryogenic calli from explants
3.2.2 Somatic embryogenesis
3.3 Maintenance of embryogenic cultures
3 .3 .1 Maintenance of embryogenic calli
3.3 .2 Maintenance of embryogenic cell suspension
3.3 .3 Maintenance of other embryogenic cultures
3.3 .4 Synchronization of somatic embryogenesis
3.3.5 Paths for somatic embryo formation
3.4 Development and maturation of somatic embryos
3 .4 .1 Sucrose concentration
3.4.2 Organic addenda and active carbon
3.4.3 Light
3.5 Germination
3 .5 .1 Comparisons of somatic embryo germination through different processes of maturation
3 .5 .2 Effect of maturation time on the germination of somatic embryos
3.5 .3 Effect of somatic embryo morphology on the germination of somatic embryos
3 .5. 4 Effect of the sizes of somatic embryos on their germination
3 .5 .5 Effect of cultivars on the germination of somatic embryos
3.6 Transgenics
3.7 Conclusion
4. Acknowledgement
5. References
415
SM. Jain. P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants. Volume 6, 415-431.
© 2000 Kluwer Academic Publishers.
416

1 Introduction

1.1 Botany/Breeding
Longan (Dimocarpus Iongan Lour.) belongs to Sapindaceae family, and is one of the important
tropicaVsubtropical woody fruit trees. The number of chromosomes is 2n=2x=30. Longan trees
are usually 6-l 0 meters tall, however, many grow in wilderness up to 40 meters with a trunk
diameter of up to one meter. Sometimes trees are with buttresses, branches terete occasionally
with warty lenticles, densely tomentose; and leaves are normally glabrous, sometimes hairy.
Fruit are drupaceous, 1-3 em in diameter, ellipsoid to globular, smooth to warty, sometimes
granular, yellow-brown. Seeds are globular, with shining black or brown testa, and the seed
are enveloped by a fleshy white arilloid (Tmdalll995, quoting from Verheij & Comell991).
There are several problems in Iongan breeding. Firstly, genetic basis of cultivated Iongan is
narrow. Among 20 species of Dimocarpus, only D. Iongan is cultivated as the fruit tree. There
are many genetic resources such as wild Iongan, D. Iongan var. Magnifo/ius, D. Iongan var.
Obtusus, longepetiolulatus, other Dimocarpus species and related Genus litchi, etc., and
among some of them may have various resistant genes or other beneficial characters which may
be helpful in Iongan breeding. However, these resources have not been utilized until now.
Secondly, resistant cultivars with steady high yield are very few and Iongan growers are eager
to get new cultivars. Thirdly, the effect of traditional breeding methods is limited for Iongan's
long juvenile phase, genetic heterozygosity and lack of knowledge of genetic background.
Therefore, new approaches to Iongan breeding should be developed as soon as possible.
Biotechnology has played an important role in genetic improvement and is considered as a
new effective means for any breeding programme. For example, genetic transformation,
protoplast fusion and embryo rescue can be used to solve the above breeding problems, and
thereby, the success of biotechnology is very much dependent on an effective plant regeneration
system. In Iongan, the majority of plant regeneration occurs via embryogenesis. Therefore, we
should establish a high-effective embryogenesis system in Iongan, which is the basic work for
Iongan biotechnology.

1.2 Geographic distribution

Longan, originated from south China, is mainly distributed in south Asia. The main producing
countries are China and Thailand. The biggest producing region is Fujian Province in China
with an estimated planting area of 150,000 ha. Other secondary producing countries are
Vietnam, India, Philippines, Japan, etc. Longan has also been introduced to some
tropical/subtropical areas in America (e.g. the United States), Oceanica (e.g. Australia), Africa
and south Europe since 19th century and planted as fruit or ornamental tree.

1.3 Major problems in diseases & pests

There are several major diseases harming Iongan such as Iongan witches' broom, sooty mold,
tree European canker, heart rot; downy mildew, etc., among which witches' broom is the most
harmful, and so far there have been no any effective measures to control it. As to the pests,
there are over 10 species of pests. Most of them are harmful to Iongan fruit and they are as
follows: Acrocercops cramerel/a Snell., Homona coffearia Meyrick, Adoxophyes cyrtosema
Meyrick, Tessaratoma papillosa Drury, etc., among which Acrocercops cramerella Snell. was
most harmful to Iongan fruit.

1.4 Economic importance

 417

Longan is an important and rare kind of tropicallsubtropical fruit trees. It has been cultivated
as the fruit tree for over 2000 years in China. Longan fruits are very delicious and nutritious,
and valuable in Traditional Chinese Medicine(TCM). Longan trees are evergreen and large in
size and often used as ornamental trees. The Iongan wood is very hard and an excellent material
for making furniture or carving. The Iongan seeds contain starch as high as 50% of the weight,
which can be prepared for drinks, active carbon and starch gum. In addition, the. Iongan bark is
rich in tannin, which can be used for dying.

1.5 Conventional propagation methods

There are two conventional methods to propagate Iongan. Firstly, by seeds, which is usually
used for stock propagation or producing ornamental plants, but still used for seedling
propagation in some areas. Secondly, by vegetative propagation including grafting, layering by
girdling, etc., among which grafting is the main method for current Iongan propagation.
However, propagation by Iongan seeds will result in variation because of genetic heterozygosity
and requires long-term vegetative growth for passing through the juvenile phase; and vegetative
propagation is too slow in producing seedlings. Moreover, Iongan is subject to witches' broom,
a virulent disease. Therefore, virus-free planilets and further propagation via tissue culture are
of great importance to Iongan production.

2 In vitro multiplication

2.1 Organogenesis

In vitro culture of Iongan began by Wei & Yang (1981), and plant regeneration from various
explants, including cotyledon, immature zygotic embryo, anther, leaf of bearing tree, stem
apex and stem axis, etc., has been reported (Lai et al 1997c). However, so far, most of the
Iongan plants were regenerated via embrogenesis. There were some reports on organogenesis
(Lai et al 1997c, 1998a). Zhou ( 1986) reported for the first time plant regeneration from stem
apex or axis of seedlings or adult trees via organogenesis, but the shoot proliferation
efficiency was very poor. After modifying the methods of obtaining plant materials and
sterilization of stem tip culture in 1990s, the survival rate and shoot proliferation efficiency
increased to a certain extent; and witches' broom virus-free tube plants were also obtained by
stem culture combined with heat treatment (Chen 1991, 1996). In addition, Zeng (1998)
observed transgenic hairy roots (inducing from immature somatic embryos co-cultured with
Agrobacterium rhizogenes Rl600) in Iongan produced adventitious buds when cultured on
MS (Murashige & Skoog 1962) medium containing 5 mg/1 kinetin, 20 g/1 sucrose and 7gll
agar.

2.2 Problems with organogenesis

Despite of several reports on Iongan organogenesis, shoot proliferation is still poor and the
technique can not be used ·for large-scale Iongan production. It should be indicated tlllit the
culture of explants excised from adult trees via organogensis is very difficult. Usually,
adventitious buds could be induced but they die on the subsequent subculture; sometimes few
adventitious buds proliferated by subculture, but rooting was difficult .

3 Somatic embryogenesis

Since 1980s, there have been several studies on Iongan somatic embryogenesis, and plant
418

regeneration was achieved from cotyledon culture (Wei & Yang 1981), anther culture (Yang
& Wei 1984), embryo rescue (Yang & Chen 1987), leaf culture (Litz 1988). In all these
cases, embryogenic calli were first produced from explants and then plants were obtained via
somatic embryogenesis. However, there were several problems in earlier studies such as
instability of embryogenic calli, low percentage of somatic embryogenesis culture, low
numbers of formed somatic embryos, abnormalities and vitrification of somatic embryos,
difficulties in converting somatic embryos to plants. Recently, we induced somatic
embryogenesis from Iongan immature zygotic embryos or anthers, resulting in stable
embryogenic calli (Lai et al 1997a), and consistently high frequency of somatic
embryogenesis and plant regeneration (Lai et al 1997b, 1998b); and then we established
embryogenic cell suspension cultures with high embryogenic capacity (Lai et al 1995, Lai
1997); and next somatic embryogensis from single cells or protoplasts was induced from cell
suspension cultures (Lai & Chen 1996, Lai 1997). Until 1998, our laboratory also achieved
plant regeneration from transgenic hairy roots via somatic embryogenesis.

3.1 Culture initiation

3.1.1 EXPLANTS

So far, Iongan somatic embryogenesis has been reported from several explants, including
immature zygotic embryos(40 to 50 days after fertilization), cotyledons, anthers, leaves of
adult trees, transgenic hairy roots, embryogenic calli, cell suspension cultures, single cells,
protoplasts, etc.(Lai et al 1997c). However, selection of the proper explant is very important
to induce somatic embryogenesis in Iongan. Immature zygotic embryos (2.0-3.5 millimeters
in length) were optimal. The embryogenetic capacity of suspensions, single cells and
protoplasts was dependent on their resources.

3.1.2 STERILIZATION PROTOCOL

Young fruit or alabastrums were surface-sterilized for 10 minutes with 0.1 %. (WN) HgC)z,
and then immature zygotic embryos or anthers were taken out under sterile conditions (Wei &
Yang 1981, Yang & Wei 1984, Yang & Chen 1987, Lai 1997). Leaves were harvested and
soaked in a benomyl solution (250 mgll) for one hour. They were surface-sterilized for 10
minutes with 0.1% (WN) sodium hypochlorite to which 2-3 drops of Tween-20 were added
for each 100 ml of sterilant. The intact leaves were rinsed thoroughly with 3 changes of
sterile distilled water, This and all subsequent procedures were performed in a laminar flow
hood under sterile conditions. Leaflets were placed intact on sterile screening culture medium
in 9.0_1.5 em petri dishes so that the lower leaf surfaces were in contact with the medium.
The screening medium consisted of B5 (Gamborg et a! 1968) salts and organics,
supplemented with 400 mgll glutamine, 60 gil sucrose, 1.7 gil gelrite and 125 mgll benomyl.
After incubation of the leaf explants for 3-4 days in darkness at 25 •c, those explants that
were free of fungal contamination were transferred onto sterile plant growth medium in 9. 0,
1.5 em petri dishes (Litz 1988).

3.1.3 MEDIA

The basal media for Iongan in vitro culture were usually MS (Murashige & Skoog 1962) or
revised B 5(Gamborg et a1 1968, Litz 1988). In general, they were supplemented with some
plant growth regulators such as 2,4-0(2,4-dichlorophenoxyacetic acid),NAA(a-Naphthalene
acetic acid), KT(Kinetin), ABA (Abscisic acid), BA (6-benzylaminopurine), Ze(Zeatin), etc.
AgN0 3 was often used in the establishment and maintenance of embryogenic. cell lines. All
media were autoclaved at 1.1Kg/cm2 and 121 for 20 minutes.
 419

3.2 Induction of somatic embryogenesis

3.2.1 INITIATION OF EMBRYOGENIC CALLI FROM EXPLANTS

In most cases, Iongan somatic embryogenesis indirectly occurred from in-vitro cultures such
as embryogenic calli, cell suspension cultures, single cells, protoplasts isolated from cell
suspension cultures or calli, etc.; Sometimes it occurred directly from some organs or tissues
such as immature zygotic embryos, anthers, transgenic hairy roots, etc. Figure 2 shows
Iongan friable embryogenic callus.

Initiation ofembryogenic calli from immature zygotic embryos

Embryogenic calli have been initiatated from over 10 cultivars of Iongan such as "Honghezi",
"Dongbi", "Shieryuelongyan", ''Wulongling", "Jiuyuewu", "Youtangben", "Baihelongyan",
"Kohala"Koetc. (Lai 1997, Litz 1988,Yang & Chen 1987). The initiation of embryogenic
calli depended on the developmental stages of immature embryos, basal media, plant growth
regulators, carbon sources, AgN03, active carbon, light and genotypes, etc.

Effict of embryo developmental stages. The optimal developmental stage of immature zygotic
embryos was the cotyledonary embryos, length 2.0-3.5 millimeters, and percentage of initiation
was over 70%. The immature zygotic embryos less than 0.5 millimeters in length usually died
after culturing. The embryos over 5.0 millimeters in length which were subject to injury, often
died of browning when cultured on the medium without active carbon.

Effict of basal media. The initiation of Iongan embryogenic calli from immature zygdtic
embryos required high strength of inorganic salts. MS (Murashige & Skoog 1962) medium was
the optimal basal medium for producing calli. When culturing on the basal media with low
strengths of inorganic salts such as White or mMS(semi-strength ofMS macro-elements), the
callus induction decreased sharply.

Effect ofplant growth regulators. Embrogenic calli could be initiated on the medium without
plant growth regulators. However, the friable calli with strong embryogenic capacity formed
only on the media supplemented with high concentrations of 2,4-D (2.0-4.0 mg/1), NAA , BA,
kinetin, 3-indolelacetic acid(lBA) and Ze all suppressed the initiation of embryogenic calli but
promoted direct formation of somatic embryos. The calli produced on the media without plant
growth regulators were white, compact and hard and their embryogenetic capacity was very
poor. When cultured on the media containing low concentrations of 2,4-D (0.1-1.0 mg/1) or
combinations of cytokinins and high concentrations of 2,4-D, the immature zygotic embryos
formed the mixture of embryogenic calli and proembryos.

Effect of AgNOJ and active carbon. AgN03 could improve the quality of embryogenic calli,
especially when culturing on the medium with the combinations of 2,4-D and cytokinins. It
inhibited ethylene biosynthesis. Moreover, active carbon could reduce injury from browning,
but was harmful to the initiation of high-quality calli (friable calli with strong embryogenic
capacity).

Effict of carbon sources. Sucrose and fructose were both suitable for the initiation of
embryogenic calli from immature zygotic embryos and the optimal concentrations were 30 g/1
and 17 g/1, respectively. High concentrations of carbon sources increased the degree of
browning of explants and the hardness of calli.
420

Effict of light. Light increased the degree of browning in the culture of immature zygotic
embryos. When the light intensity increased to 0.055umol.m·2.s· 1, calli did not form and the
explants gradually turned brown and eventually died. Therefore, the initiation of embryogenic
calli from immature zygotic embryos should be conducted in darkness.

Effict of genotypes. The callus initiation was dependent on the genotypes of Iongan cultivars.
When culturing on the medium containing 2.0 mg/12,4-D, 7g!l agar and 30 g/1 or 20g/l sucrose
in the dark, "Honghezi" and "Jiuyuewu" directly formed the friable embryogenic calli; and
"Shieryuelongan", ''Wulongling", "Dongbi" and ''Youtangben" formed the embryogenic cultures
containing proembryos. The callus initiation rates were 73.3%, 66.7%, 63.3%, 33.3%, 40.0%,
36.4%, respectively. Nevertheless, the friable embryogenic calli could be screened from the
embryogenic cultures in the process of subcultures.

Initiation of embryogenic calli from anthers: Yang & Wei (1984) and Lai (1997) reported the
initiation of embryogenic calli by Iongan anther culture. The optimal stage of alabastrums for
anther culture was approximately 3 millimeters in diameter. At this stage, pollens were at the
uninucleate or middle or late stages of development. Yang & Wei (1984) considered that the
cold treatment (2-8 W) for 24 hours could improve the percentage of callus initiation within 30
to 40 days on the suitable MS basal medium supplemented with 2.0 mg/1 2,4-D, 1.0 mg/1
Kinetin, 5 g/1 active carbon, 6.5 g/1 agar, 50 g/1 sucrose (pH 5.8). However, Lai (1997)
reported that the cold treatment was not necessary and the suitable medium was MS basal
medium containing 2.0 mg/1 2,4-D, 7g!l agar and 50 g/1 sucrose, which was simpler than the
former one. Under the conditions, the friable embryogenic calli could be obtained from anther
culture.

Initiation of embryogenic calli from leaves: There was only one successful report concerning
leaf culture in Iongan. Litz (1988) reported that embryogenic callus was initiated from leaflets
of new flushes of mature 30-year-old Iongan (the cultivar "Kohala") trees. Callus initiation and
growth from whole leaflet explants was first observed 3-4 weeks after culturing. The initial
callus was white and friable, and formed from all sections of the leaves except for the veins and
midribs. No callus initiation was observed on the media containing 2,4-D alone, which was
different from callus derived from immature zygotic embryos; however, callus was initiated on
most medium formulations containing 2,4-D together with either BA or kinetin, but only in
some media callus was embryogenic. The basal medium was revised Bs, i.e., Bs (Gamborg et a1
1968) with 400 mg/1 glutamine, 60g/l sucrose, 200 mg/1 casein hydrolysate and 1.7 g/1 gelrite.
Embryogenic callus initiation was dependent on both auxin and cytokinin, and was clearly
dependent on the type of cytokinin. There was a strong interaction between 2,4-
dichlorophenoxyacetic acid and kinetin. Embryogenic callus was formed on most medium
formulations containing 0.25-3.0 mg/1 2,4-D together with all possible combinations of the
same concentrations of kinetin. The most effective range of plant growth regulator
concentrations was 0.5-2.0 mg/1 kinetin and 0.5-1.0 mg/1 2,4-D in all possible combinations.
Somatic embryos from embryogenic callus could be observed 75 days after the leaflets were
cultured. The entire callus was embryogenic on the media containing optimal concentrations of
2,4-D and kinetin.

Initiation of embryogenic calli from cotyledons: Wei & Yang(198l) for the first time reported
somatic embryogenesis from cotyledons in Iongan. They cut the cotyledons into sections and
cultured on the MS medium containing 2.0 mg/1 2,4-D, 1.0 mg/1 kinetin; 5g!l active carbon,
6.5-7.0 g/1 agar (pH 5.8) in the dark at 25-27•C.The formed calli were compact and hard, and
some of them were embryogenic.

3.2.2 SOMATIC EMBRYOGENESIS

 421

Somatic embryogenesis from embryogenic calli: Somatic embryogeneis occurred from all the
above embryogenic calli derived from different explants, but the embryogenic capacity was
different. Figure 4 shows somatic embryogenesis from Iongan embryogenic callus. The
embryogenic competency varied with the types of embryogenic calli, which were dependent on
the media and the culture conditions of callus initiation. In all the reports, embryogenic calli
could generally be divided into 3 types, i.e., Type A, Type B and Type C. Type A callus,
initiated on the media devoid of plant growth regulators or containing 2,4-
dichlorophenoxyacetic acid and active carbon, was white, compact and solid; Type C callus,
produced on the media with high concentrations of 2,4-0(2.0-4.0 mgll) was light, yellow,
vigorous, fine-grained and -friable and was usually called the friable embryogenic callus; Type
B callus, formed on the media containing low concentrations of 2,4-D(< 1.0 mgll) or
combinations of cytokinins and high toncentrations of2,4-D, was very similar to Type C, but it
contained proembryos. Type C could be selected during the subculturing of Type B calli. The
embryogenic calli initiated by Wei & Yang(1981), Yang & Wei(1984) and Yang & Chen (1987)
belonged to Type A; the embryogenic calli by Litz (1988) actually belonged to Type B ; and
Lai (1997) initiated all the 3 types of embryogenic calli. Somatic embryogenesis varied with the
types of embryogenic calli, cultivars, media, light, etc.

Effect of the types of embryogenic calli. The embryogenetic capacity of Type A callus was
poor, whereas Type B or C calli had high productive rates, a gram (fresh weight) of each callus
type could produce 8,000-11,000 somatic embryos under the optimal conditions. The
embryogenetic capacity of these embryogenic calli was in order as Type C, Type B, and Type
A calli. High embryogenic ability as observed in Iongan Type B and C calli, is rare in woody
plants. In general, cotyledonary somatic embryos directly formed from all types of calli after
the calli had been transferred onto the differentiation medium. Lai (1997) and Litz(l988) aiso
found that partial development of somatic embryos occurred on some calli, such as some kind
of Type B callus initiated on the media containing low concentrations of 2,4-D. However, the
somatic embryos were generally arrested at the globular developmental stage on the media
supplemented with higher concentrations of2,4-D.

Effect of Longan cultivars. The embryogenic capacity varied with different cultivars of Iongan
due to the differences of callus types initiated from different cultivars when culturing on the
same medium. However, the same types of calli produced from different cultivars showed the
similar embryogenic capacity.

Effect of media. Lai (1997) reported that the suitable medium for somatic embryogenesis
induction from Iongan embryogenic calli was MS medium supplemented with 20-30 gil
sucrose, 5% (VN) coconut water, 7 gil agar, 100mgll myo-inositol and the pH of the medium
was adjusted to 5.8 prior to autoclaving. For Type C callus, the low concentration of sucrose
(<3 gil) improved somatic embryo formation and decreased the number of abnormal somatic
embryos. When cultured on the media containing high concentration of sucrose (>50 gil), the
rate of somatic embryo formation decreased and the number of abnormal somatic embryos
increased remarkably. For Type B calli, culturing on the media containing high concentrations
of sucrose was also suitable, because they contained proembryos. Plant growth regulators were
not necessary, but they improved somatic embryogenesis in the presence of high sucrose
amount. Coconut water slightly increased the number of formed somatic embry9s and
facilitated somatic embryo growth. In addition, Litz (1988) reported that B 5 (Gamborg et al
1968) was the suitable basal medium; and Yang & Wei ( 1984) and Wei & Yang( 1981) indicated
that ER (Anderson 1975, 1978) was better than MS basal medium.

Effect of light. Longan somatic embryogenesis from embryogenic calli was susceptible to light.
422

Light greatly suppressed somatic embryogenesis, especially for Type C callus. Therefore,
induction of somatic embryogenesis in Iongan was performed in the darkness.

Somatic embryogenesis from embryogenic cell suspensions

Type C calli were friable and contained a large number of cell clusters (containing 5-10 cells),
which automatically dispersed when placed in the liquid medium. Figure 3 shows Iongan
embryogenic cell suspension. After transfer into liquid media of the same composition as for
callus formation and rotating at 100-120 rpm, well-scattered cell suspensions were rapidly
established within one or two weeks. The established cell suspensions retained a strong
embryogenic capacity. Each gram of suspended cultures (fresh weight) could form as many as
140,000 somatic embryos after they were transferred onto the solid differentiation medium
containing 20 g/1 sucrose, 5% (VN) coconut water, 7 g/1 agar, lOOmg/1 myo-inositol on MS
basal medium( Murashige & Skoog, 1962).

Somatic embryogenesis from single cells

Lai (1997) reported somatic embrogenesis from Iongan single cells. Single cells were obtained
from well-dispersed cell suspension cultures by sieving (40 microns), and then they were
transferred into liquid differentiation media for shallow-layer liquid culture. The formulations
of liquid differentiation media were similar to those of the solid differentiation media except for
agar. The culture densities ranged from 104 to 105 cells/ml. In many cases, single cells divided
first and then developed to form cell clusters with compact structure, from which somatic
embryos later formed. Some single cells formed linear 3-cell clusters aftertwo divisions. Such
linear 3-cell clusters have been regarded in other systems as initiation ·of embryogenic
cultures(Williams & Maheswaran 1986). Hence, we inferred that direct somatic embryogenesis
from single cells occurred under some circumstances.

Somatic embryogenesis from protop/asts

Longan embryogenic cell suspension cultures were highly suitable for isolation of protoplasts.
High yields (up to 108 protoplasts from each gram of fresh suspension cells) and high viability
(more than 95%) were achieved when they were inoculated into the enzyme mixture containing
l.Oo/o(WN) cellulase 'Onozuka R-10', l.Oo/o(WN) pectinase from Serva, 13% (WN) mannitol
for 14 hours. The viability of protoplasts was measured by the method of Evans blue
staining(Zhou & Yang 1989). When these protoplasts were embedded in calcium-alginate
beads, the frequency of visible rnicrocolony formation exceeded 5.0% in optimal conditions.
Usually protoplasts formed calli first and then somatic embryos formed from these calli; in few
cases, direct somatic embryogenesis was observed from protoplasts. After two or three
divisions on the third or fourth day, some regular linear or T -patterned 3 or 4-cell clusters were
observed under the microscope; early globular proembryos formed on the fifth to seventh day;
and globular embryos containing hundreds of cells occurred after 3 weeks or so. Cotyledonary
somatic embryos formed after the globular embryos were transferred into the liquid
differentiation medium( Lai & Chen 1996, Lai 1997).

Somatic embryogenesis from transgenic hairy roots

Our laboratory reported somatic embryogenesis from transgenic hairy roots in Iongan (Zeng
1998). Hairy roots were induced from small cotyledonary somatic embryos through
Agrobacterium rhizogenes Rl600-mediated transformation. When they were cultured under
the similar conditions of somatic embryogenesis from calli, some somatic embryos formed from
the surface of hairy roots. The somatic embryos had strong ability to produce secondary
 423

somatic embryos, which was possibly related to the changes of endogenous phytohormones in
transgenic hairy roots.

Secondary embryogenesis

Secondary somatic embryos produced from primary somatic embryos were observed in some
circumstances. Globular somatic embryos could proliferate directly when cultured on the media
containing low concentrations of 2,4-D, which was the most common path to form secondary
somatic embryos. In a few cases, secondary somatic embryos formed from cotyledonary
somatic embryos, and which were of poor quality. In fact, secondary embryogenesis should be
avoided from cotyledonary somatic embryos for improving the germination rate of somatic
embryos. Besides, secondary somatic embryos occurred from apical points or cotyledons of
transgenic somatic embryos framed from hairy roots. However, secondary somatic embryos
from apical points were usually abnormal.

Somatic embryogenesi~ from immature zygotic embryos, cotyledon, anthers, etc.

By culturing explants on the media (either MS or B5) devoid of 2,4-D or containing low
concentrations of 2,4-D, they usually formed somatic embryos directly. When the
concentrations of 2,4-D were below 0.1 mg/1, they often produced cotyledonary somatic
embryos directly. In the earlier reports, anthers, cotyledons, immature zygotic embryos all
formed somatic embryos directly. When the concentration of 2,4-D ranged from 0.1 to 1.0
mg/1, somatic embryo growth was generally arrested at the globular stage of development (Lai
1997, Litz 1988).

3.3 Maintenance of embryogenic cultures

3.3.1 MAINTENANCE OF EMBRYOGENIC CALLI

The maintenance of Iongan embryogenic calli was quite difficult. In many cases, partial
development of somatic embryos occurred in the subcultures of embryogenic calli on the media
with or without low concentrations of 2,4-D (Wei & Yang 1981, Yang & Wei 1984, Yang &
Chen 1987, Litz 1988). When they were continuously subcultured on the media containing high
concentrations of 2,4-D, the calli growth slowed down and turned browned. If the calli were
transferred onto the medium supplemented with AgN03 (5.0 mg/1) and high concentrations of
2,4-D, the callus growth was restored, but it slowed down 2 to 3 generations after subcultures.
However, the calli could be maintained over long periods (over 4 years) by alternately culturing
first on MS medium containing high concentrations of 2,4-D (1.0-2.0 mg/1) and then adding
5.0 mg/1 AgNOJ. Either Type C callus or Type B callus could be maintained by the methods.

3.3.2 MAINTENANCE OF EMBRYOGENIC CELL SUSPENSION

Longan embryogenic cell suspension cultures could be maintained according to the similar
method of maintenance of embryogenic calli (Lai et al 1995). In general, they were alternately
cultured in liquid MS medium amended with 2.0 mg/12,4-D, 1.0 mg/1 kinetin, 5.0 mg/1 AgN0 3
first and then with 100 mg/1 myo-inositol but without plant growth regulators. Moreover, the
embryogenic cell suspension could be maintained for over I year by taking turns on the solid
and liquid media. When Iongan embryogenic cell suspension cultures were transferred onto the
solid medium, they grew rapidly and formed friable embryogenic calli, which easily formed cell
suspension again. When cell suspension was not used, it could be maintained on the solid
culture medium. Whenever embryogenic cell suspension was needed, it could be established in
a short time. This method was economical and could also improve the quality of embryogenic
424

cell suspension.

3.3.3 MAINTENANCE OF OTIIEREMBRYOGENIC CULTURES

Except for embryogenic calli and cell suspension, other Iongan embryogenic cultures such as
the mixtures of cell clusters and proembryos, the cultures of globular embryos, etc., could also
be maintained (for over 4 years) by the similar methods as described earlier. The mixtures of
cell clusters and proembryos could be maintained according the method for maintaining the
embryogenic calli; and the cultures of globular embryos could be maintained on the MS
medium containing 2,4-D ranging from 0.5 to 1.0 mg/1.

3.3.4 SYNCHRONIZATION OF SOMATIC EMBRYOGENESIS

Synchronization of somatic embryogenesis at the globular and cotyledonary stages of somatic

embryo development from Iongan embryogenic calli was achieved with plant growth regulators,
nutrient control and modification of callus culture (Lai 1997).

Synchronization in the formation ofglobular somatic embryos

All embryogenic calli were transferred on the differentiation media for inducing somatic
embryos. After 10-15 days, proembryos or globular somatic embryos were visible. They were
transferred onto the media containing 1.0-2.0 mg/1 2,4-D, and as a result further development
was arrested at the globular developmental stage of somatic embryos. By this method, somatic
embryogenesis could be synchronized at the globular developmental stage. Alternative method
was to control nutrient in the medium. When excessive calli (> 0.9 gram of fresh weight
cultured in each 50 ml bottle) were inoculated to induce somatic embryogenesis, the
development of the induced somatic embryos stopped at the globular stage due to nutrient
deficiency, which did not any further develop (Lai 1997).

Synchronization of cotyledonary embryonic formation

When synchronized globular somatic embryos were transferred and spread evenly onto the MS
medium containing 20 gil sucrose, 400 mg/1 LH (lactoalburnin hydrolysate), 100 mg/1 myo-
inositol, 5% (VN) coconut water, 7 g/1 agar for approximately 20 days, all the globular
somatic embryos developed into cotyledonary somatic embryos which were about 2 millimeters
in length. An alternative method was via the liquid layer culture in the medium containing 20gll
sucrose, 400 mg/1 lactoalburnin hydrolysate and 100mgll myo-inositol with shaking at low
speed (30 to 50 rpm). The synchronized cotyledonary somatic embryos were small and they
should be transferred onto fresh media in time (generally within 2 to 3 weeks) for enlargement
and maturation (Lai 1997).

3.3.5 PATHS FOR SOMATIC EMBRYO FORMATION

There were at least 4 paths for somatic embryo formation in Iongan (Wei & Yang 1981, Yang &
Wei 1984, Yang & Chen 1987, Litz 1988, Lai 1997). The first path (Path 1) was somatic
embryogenesis from embryogenic cells, which was perhaps most common and also similar to
that of carrot (Williams & Maheswaran 1986); The second path (Path 2) was the multiplication
of globular somatic embryos, in which there were two ways for somatic embryo formation,
i.e., direct globular formation (Path 2-1) or somatic embryo formation from embryogenic cell
clusters which were produced by bursting of globular soamtic embryos (Path 2-2); the third
path (Path3) was somatic embryogenesis directly from the explants such as cotyledons, anthers,
immature zygotic embryos, etc.; and the final path (Path 4) was direct secondary
 425

embryogenesis (please see Figure 1).

Explants
(cotyledons, anthers, immature zygotic embryos,etc.)
Path 1 l I
Embryogenic cells (or calli) IPath 3
Path 2-1 ~ !
Globular somatic embryos-Globular somatic embryos-Embryogenic cells
lPath 4 ~ l 1Path2-2
Further developmental somatic embryos
~
Cotyledonary somatic embryos

Figure I Paths for somatic embryo formation in Iongan

3.4 Development and maturation of somatic embryos

All types of embryogenic calli or cultures could rapidly form directly cotyledonary somatic
embryos in Iongan after they were transferred onto the differentiation media containing low
concentrations of sucrose. However, these cotyledonary somatic embryos required a process of
special "maturation" culture before they could germinate; otherwise, they germinated at low
frequency or only some of them could regenerate into plantlets. The normal maturation of
somatic embryos was related to several main factors such as the concentration of sucro'se,
organic addenda, active carbon, light, etc.

3.4.1 SUCROSE CONCENTRATION

Either small white cotyledonary somatic embryos or small vitrified cotyledonary somatic
embryos matured normally and formed normal enlarged mature somatic embryos on the
maturation medium containing 50-60 gil sucrose in 3-month time. Small vitrified somatic
embryos should be transferred onto the maturation medium before their sizes were less than 0.5
centimeter long; Otherwise, the vitrification of somatic embryos was difficult to reverse. If the
maturation medium contained low concentrations (<30-40 gil) of sucrose, the somatic embryos
would mature abnormally and become more vitrified. After maturation culture, the somatic
embryos became white and hard, and 1-1.5 em in length.

3.4.2 ORGANIC ADDENDA AND ACTIVE CARBON

Coconut water (2.5-5.0%, VN) was beneficial to the enlargement of Iongan somatic embryos
during maturation. However, active carbon (> l.Ogll) affected the solidification of somatic
embryos and made them be sarcoid. Moreover, the higher concentrations of agar (8-1 0 gil)
were beneficial during maturation and increased the quality of somatic embryos.

3.4.3 LIGHI'

The effect of light on the maturation of somatic embryos varied with the sucrose concentration.
When the somatic embryos were cultured on the media containing high concentration of
sucrose (>50 gil), light (>0.05 umol.m-2.s-1) did not affect their maturation obviously; however,
when cultured on the media containing low concentrations of sucrose, light could decrease
426

somatic embryo death by vitrification, but usually resulted in precocious germination.

Therefore, the maturation of Iongan somatic embryos was generally conducted in the pressure
of 50 g/1 sucrose and weak light (<0.015 umol.m-2.s- 1).

3.5 Germination

Several factors such as maturation processes, maturation time, somatic embryo shapes and
sizes, cultivars, etc., affected germination of Iongan somatic embryos.

3.5.1 COMPARISONS OF SOMATIC EMBRYO GERMINATION THROUGH

DIFFERENT PROCESSES OF MATURATION

Longan germination of somatic embryos varied with different processes of maturation. The
white and hard somatic embryos matured in darkness on the medium containing 50g/l sucrose,
5%(VN) coconut water. They germinated and formed normal plantlets on MS medium
containing 0.25o/o(VN) coconut water, 20 g/1 sucrose, 7 g/1 agar, 100 mg/1 myo-inositol and
200 mg/llactoalbumin hydrolysate. The germination rate of normal somatic embryos in larger
sizes (> 1. 0 em long) could be up to 90%, which suggested that the better quality of the matured
somatic embryos and highly suitable germination medium for Iongan somatic embryos. When
the maturation media was devoid of coconut water, the germination rate and the number of
plantlets decreased remarkably. Similarly, maturation media containing active carbon or low
concentrations of sucrose was unsuitable for the somatic embryos to germinate into normal
plantlets and the regenerated plantlets were often vitrified.

3.5.2 EFFECT OF MATURATION TIME ON THE GERMINATION OF SOMATIC

EMBRYOS.

The maturation time greatly affected the germination of Iongan somatic embryos. The optimum
time for maturation culture was 75-90 days; and somatic embryos completely matured and
germinated normally. When the maturation time was less than 45 days, the somatic embryos
did not mature enough and usually only formed roots. It was difficult to regenerate buds. When
the maturation time was longer than 150 days, the bud germination rate increased, but rooting
was suppressed. These results suggested that the differentiation of root primordia should be
earlier than that of bud primordia. Over-maturation culture on the media containing high
concentration of sucrose should do harm to the root primordia. The results supported the
viewpoint that maturation and conversion of somatic embryos in most fruit trees required some
specific conditions (Tang & Wang 1999). For example, maturation and conversion of somatic
embryos required isolation from mother tissues (Deng & Corus 1992, Tulecke & Mcgranahan
1985) and low temperature (Deng & Corus 1992)in walnut, and subculture daily (Coutos et al
1992) and dehydration (Gray 1989) in grapevine, respectively. Figure 5 shows plant
regeneration from somatic embryos in Iongan.

3.5.3 EFFECT OF SOMATIC EMBRYO MORPHOLOGY ON THE GERMINATION OF

SOMATIC EMBRYOS

The somatic embryo morphology was related to the germination. Longan zygotic embryos had
2-3 cotyledons (the percentage of occurrence could amount to 5-30% in some cultivars) in the
nature, and we found that, in most cases, the Iongan somatic embryos with 2-3 cotyledons
could normally germinate. However, the somatic embryos with 1 or more than 3 cotyledons
germinated abnormally and the percentages of germination were below 20%. In addition, some
somatic embryos from anther culture were normal in morphology but germinated poorly, which
was possibly related to their complicated genetic origin; nevertheless, the generated plantlets
 427

from anther culture were nonnal in appearance.

3.5.4 EFFECT OF THE SIZES OF SOMATIC EMBRYOS ON THEIR GERMINATION

In general, the sizes of somatic embryos affected slightly the percentages of germination.
Except for too small somatic embryos(< 0.5centimeter in length), the differences of germination
percentages were not significant among various sizes of somatic embryos.

3.5.5 EFFECT OF CULTNARS ON THE GERMINATION OF SOMATIC EMBRYOS

The germination percentages of somatic embryos varied with different cultivars, however, it
could be enhanced by optimal matUration treatment. The regenerated plantlets appeared to be
morphologically nonnal and resembled Iongan seedlings. The leaves were typically compound
and lanceolate (Lai 1997, Litz 1988). They could survive after transfer into soil or other media
with a higher efficiency under proper conditions. Vermiculate was usually used for
transplanting medium. In south China, the highest survival rate was obtained between April
and May. However, no any data for field trials were reported until now.

3.6 Transgenics

Somatic embryos from transgenic hairy roots could germinate under the similar conditions of
plant regeneration from the non-transgenic somatic embryos. However, secondary
embryogenesis occurred frequently, which affected plant regeneration and might resulted from
the presence of abnonnal endogenous phytohormones due to the introduction of rooting genes
from Ri plasmid. Therefore, the transgenic plantlets were short and thin, and the frequency of
plant regeneration was far lower than that of the non-transgenic (Zeng 1998).

3.7 Conclusion

Our results demonstrated these key factors affecting somatic embryogenesis in Iongan: types of
in vitro cultures, concentration of sucrose, and light. Firstly, the friable calli and the cell
suspension cultures showed high embryogenic competency. Auxin 2,4-D was essential in
initiating embryogenic development, which was similar to that of some other fruit trees such as
mango(Dewald et al 1989), peach(Raj Bhansali et al 1990), chesenut(Gonzalez et al 1985),
Prunus avium(De March et al 1993).Secondly, sucrose greatly affected the number and quality
of somatic embryos, and these results were similar to the results in Codpnopsis /anceo/ata
(Choi 1995). Thirdly, light suppressed somatic embryogenesis, even resulted in the death of in
vitro cultures in Iongan. This is due to high polyphenols in Iongan tissues, which eventually
turned browning easily (Wei 1986). Light intensified browning and that led to suppressing of
somatic embryogenesis. So 1ongan somatic embryogenesis should be induced in the dark. Lai
( 1997) reported 100% frequency of somatic embryogenesis induction from calli, and the
number of formed somatic embryos was approximately 10,000 somatic embryos per gram
fresh weight. Such high somatic embryogenic ability was very rare in woody plants. Therefore,
we suggested Iongan somatic embryogenesis system to considere as a model system in woody
plants (Lai et al 1997c).
All types of embryogenic calli or cultures could rapidly form directly cotyledonary
somatic embryos in Iongan after subculture onto the differentiation media. They could
germinate at high frequency after passing through a process of special "maturation" culture,
which was of great importance for further applications to cell and genetic engineering,
mutagenesis, micropropagation, and production of artificial seeds in Iongan and also would be
beneficial in other tropicaVsubtropical woody trees. However, some aspects including the
mechanism of somatic embryogenesis, field trials, applications of somatic embryogenesis to
428

Iongan breeding and propagation, etc., need further immediate attention.

4. Ackowledgements Fianced by National Natural Sciences Foundation of China(No.

39870544) and Natural Sciences Foundation of FujianProvince of China (No. F99003,
C97030).

5. References

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and its applications in woody plants. Chinese higher education press, Beijing,pp 408-419.
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430

Figure 2. Longan friable embryogenic callus

Figure 3. Longan embryogenic cell suspension

 431

Figure 4. Somatic embryogenesis from Iongan embryogenic callus

Figure 5. Plant regeneration from somatic embryos in Iongan. A. Buds formation

from somatic embryos after maturation. B. Plant regeneration after rooting.
 13. SOMATIC EMBRYOGENESIS INDUCTION
IN TAMARILLO (Cyphomandra betacea)

M.L. Lopes, M.R. Ferreira, J.M. Carloto, G. S. Cruz and J.M. Canhoto
Departamento de Botfulica (Centro de Biologia Vegetal), Faculdade de Ci~ncias e
Tecnologia, Universidade de Coimbra, 3001-455 Coimbra, Portugal

Chapter Contents

1. Introduction
1.1. Diagnostic features
1.2. Distribution and economic importance
1.3. Previous in vitro culture studies
2. Methodology
2.1. Preparation of explants
2.2. Culture media
2.3. Cytological, histological and ultrastructural studies
2.4. Biochemical studies
3. Somatic embryogenesis induction
3.1. In zygotic embryos
3.2. In young leaves
4. Somatic embryo gennination
5. The role of auxin polar transport inhibitors on somatic embryo development
6. Somatic embryo origin
6.1. Zygotic embryos
6.2. Leaves
7. Genetic stability of the regenerated plants
7.1. Cytology
7.2. RAPD analysis
8. Embryogenic biochemical markers
9. Conclusions and future prospects
10. Acknowledgments
11. References

1. Introduction

1.1. DIAGNOSTIC FEATURES

Cyphomandra betacea (Cav.) .Sendtn. usually known as tamarillo or tree tomato, is a

solanaceous soft wood tree (Fig. 1A) grown for their edible fruits (Fig. 1B). The species,
433
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 433-455.
© 2000 Kluwer Academic Publishers.
434

which may reach 2 - 4 meter height (Slack, 1976), produces tomato-like red, orange or
yellow fruits according with the cultivars. The fruits of the red cultivar are the most popular
due to their more striking appearance and better flavour (Slack, 1976). They are generally 2
- 3 inches long and 2 inches in diameter possessing many seeds (Hooker, 1899). Also
included in the same genus are the species Casana (Cyphomandra casana), Mountain
Tomato (C. crassifolia) and Guava Tamarillo (C.fragans). The plant can be propagated by
seeds or cuttings (Fouque, 1973) or it may be grafted in Solanum mauritianum (Slack,
1976). In the first case, plants do not usually come true-to-type rendering difficult the
propagation of selected genotypes (Barghchi, 1998). Several authors (see section 1.3) have
also reported protocols for in vitro regeneration. The first pinkish flowers appear in spring,
while the mature fruits are collected from October to April (Guimarnes et at., 1996).
However, flower appearance and fruit maturity can be changed by pruning (Slack, 1976).
The chromosome number of the species is 2n=2x=24. Aneuploids and tetraploids
have also been reported (Standring et al., 1990). Fruits can be produced after self-or cross-
pollination (Dawes & Pringle, 1983) but the first type of pollination seems to be the most
common (Bobs, 1991). That could explain the little genetic variation occurring in natural
populations of this species (Barghchi, 1998).

1.2. DISTRIBUTION AND ECONOMIC IMPORTANCE

The plant seems to have its origin from the northern Andean region of Peru (Dawes &
Pringle, 1983; Hooker, 1899). Chile, Equator and Bolivia are other possible centers of
origin (http://www.crfg.org/pubs/ff/tamarillo.html). From there it has spread to other South
American countries namely Argentina, Brazil, Colombia and Venezuela
(http://www.crfg.org/pubs/ff/tamarillo.html). Nowadays tamarillo is grown in many
regions of the globe such as India, China, USA, Kenya, Australia, Southern Europe and
New Zealand (Guimarnes et al., 1996). In this last country, the plant is intensely cultured as
a crop and the fruits are exported (http://www.crfg.org/pubs/ff/tamarillo.html). In Portugal
the plant is usually grown as an outdoor ornamental although some attempts have been
made to explore this species commercially due to the high prices attained by its fruits in
market places (10 - 15 euros/Kg). However, spring and autumn frosts are important
limitations to a large-scale cultivation of tamarillos in our region. The fruits, after seed and
skin removal, can be eaten fresh or they may be used to make jam, sauces, and pickles
(Slack, 1976). In Jamaica and West Indies the plant is used in folk medicine against liver
infection. Fruit analysis (Cacciopo, 1984; McCane and Widdowson, 1992) has revealed a
low carbohydrate composition (4.7 g/100 g of fresh weight) as well as low energy values
(120 kJ/100g of fresh weight). Conversely, vitamin C and E, and citric acid are relatively
abundant.

1.3. PREVIOUS IN VITRO CULTURE STUDIES

Our laboratory (Guimarnes et al. 1988) reported for the first time success on somatic
embryogenesis induction in tamarillo by culture of mature zygotic embryos and hypocotyls
of seedlings. In a subsequent report (Guimarnes et al., 1996), the same group described the
regeneration of tamarillo plants by organogenesis and somatic embryogenesis from different
kinds of tamarillo explants (hypocotyls, cotyledons, roots and mature zygotic embryos) and
from protoplasts. Plant regeneration by organogenesis was also shown by Barghchi (1998).
 435

Binding et al. (1992) also developed a method for regenerating plants from mesophyll
protoplasts. Micropropagation of tamarillo plants through axillary bud explants have also
been carried out (Barghchi, 1986; 1998; Cohen & Elliot, 1979). Anther culture has been
tried by Barghchi (1998) and by our group. At our laboratory multicellular pollen grains (up
to 16 cells) were observed but no further development could be achieved. Barghchi (1998)
reported the induction of pollen calli on media supplemented with BA (benzyladenine) and
NAA (naphthaleneacetic acid) but no pollen plants could be regenerated. Transgenic
tamarillo plants were obtained by Atkinson & Gardner (1993) after co-culture of leaf pieces
and petiole segments with Agrobacterium tumefaciens, followed by shoot regeneration.
Further genetic transformation studies associated with efficient protocols of in vitro
regeneration may play a decisive role in developing new clones resistant to virus, pests and
diseases that, by conventional breeding, are difficult to obtain. Table 1 summarizes the
results so far achieved by in vitro culture of tamarillo.
ln the following sections, our recent results on somatic embryogenesis induction in
tamarillo are described.

2. Methodology

2.1. PREPARATION OF EXPLANTS

The methodology used for the induction of somatic embryogenesis in tamarillo has been
previously described (Guimaraes et al., 1988; 1996). In short, mature fruits of tamarillo
from trees growing at the Botanical Garden of the University of Coimbra or acquired at a
local supermarket (red cultivar) were used as a source of seeds. They were removed,
washed with sterile double distilled water and then immersed in a 7.5% calcium
hypochlorite solution for 15- 20 min, followed by three rinses with double distilled water.
Following this treatment the seeds were either immediately used for embryo isolation or
kept in a refrigerator at 4' C until they were used. Whole zygotic embryos were cultured in
test tubes containing the induction media (see section 2.2.). The cultures were maintained in
the dark at 25± 1' C. Maintenance of embryogenic calli was achieved by keeping the
cultures in the dark and by subculturing them monthly in the same conditions. Somatic
embryos germinated when embryogenic calli or the mature somatic embryos were
transferred to the same basal medium containing gibberellic acid (GA 3) under a 14h daily
illumination regime of 15-20 ,umol m- 2s- 1 photosynthetically active radiation provided by
cool fluorescent lamps. When young leaves were used for somatic embryo induction the
process was basically the same. The leaves were obtained from six-week-old seedlings
germinated in a MS (Murashige & Skoog, 1962) basal medium without growth regulators
or from micropropagated plantlets. After segmentation in 4 or 6 parts the leaf pieces were
inoculated on the induction media.

2.2. CULTURE MEDIA

Zygotic embryos were cultured on MS basal medium containing one of the following
auxins: NAA (5.0 mgll), 2,4-D (2,4- dichlorophenoxyacetic acid, 2.0 mg/1) and picloram
(5.0 mg/1). The media containing 2,4-D or picloram were also used for subculture of the
embryogenic calli. In some experiments, the embryogenic calli were subcultured in liquid
medium with the same composition, shaking at 80 rpm in the dark. When leaves were used
436

as explants, several concentrations of picloram (0.1 - 5.0 mg.'!) and sucrose (3 - 15%) were
tested. For somatic embryo germination, the embryogenic explants were cultured on MS
basal medium or MS supplemented with 0.1 mg/1 GA 3 and the sucrose concentration was
lowered to 2% (germination media). The role of auxin transport inhibitors on somatic
embryo development was evaluated by transferring the embryogenic calli to liquid
germination media containing different concentrations of the auxin polar transport inhibitors
2,3,5-triiodobenzoic acid (TIBA), 9-hydroxyfluorene-9-carboxylic acid (HFCA), trans-
cinnamic acid (TCA) and 2-p-chlorophenoxy-2-metil-propionic acid (CFA). In the case of
TIBA and HFCA 0.3 to 10 pM were used whereas CFA and TCA were tested in
concentrations ranging from 20 to 160 pM. All media had the pH adjusted to 5.6- 5.8 and
were solidified by adding 0.8% agar (bacto agar, Difco) before autoclaving at 121 'C for 20
mm.

2.3. CYTOLOGICAL, HISTOLOGICAL AND ULTRASTRUCTURAL STUDIES (SEM

ANDTEM)

For histological studies, small sections of explants (hypocotyls of zygotic embryos, leaves
or embryogenic calli) were fixed on the day cultures were initiated (day zero) and after
several periods of culture. Tissues were fixed in 2.5% glutaraldehyde (0.1 M phosphate
buffer, pH 7.0) for 90 - 120 min at room temperature. After three washes (10 min each)
with the same buffer, the samples were post-fixed in 1% osmium tetroxide prepared in
phosphate buffer for 60- 90 min at room temperature and then washed three times in the
same buffer. Tissues were dehydrated in an ethanol series (20- 100%) and the material
embedded in Spurr's resin (Spurr, 1969). Semi-thin sections 1- 2 pm were stained with
0.2% toluidine blue in acetate buffer for 30 min. Ultrathin sections (50- 70 nm) were cut on
a LKB ultramicrotome using a diamond knife and collected on uncoated copper grids. The
sections were stained first with uranyl acetate for 15 min followed by lead citrate treatment
for 10 min. The stained sections were observed under a Siemens Elmiskop-101 transmission
electron microscope at 80 kV. For SEM examinations, the dehydrated samples were critical
point dried with carbon dioxide as the transition fluid and coated with gold (250 nm).
Prepared specimens were mounted on aluminum stubs and examined in a JEOL JSM-T330
scanning electron microscope operating at 20 kV.
Chromosome counts of regenerated plantlets were taken from aceto-carmine
squashes of ethanol-acetic (3: 1) fixed root tips pretreated with colchicine 0.05% for 3h
according to the Feulgen method described by Singh (1993).

2.4. BIOCHEMICAL STUDIES

To compare the polypeptide profile between embryogenic and non-embryogenic calli the
following procedure was adopted: embryogenic calli were induced from leaf blade of
seedlings cultured in a medium containing 5 mg/1 picloram and 9% sucrose. Non-
embryogenic calli were induced in the same medium containing 3% sucrose. Callus (500-
1000 mg) was frozen in liquid nitrogen and stored at -80'C until use. Protein extraction was
done according to Pedroso et al. (1996). In short, calli were ground in liquid nitrogen in a
mortar and pestle, and then homogenized in 10% TCA (trichloroacetic acid) and 0.07% B-
mercaphtoethanol in cold acetone and incubated at -20'C for protein precipitation. This step
was repeated twice and the final pellet was ressuspended in SDS-PAGE sample buffer.
 437
Proteins were separated by SDS-PAGE using 15% polyacrilamide gels. Electrophoresis was
perfonned according to the method of Laemmli (1970). After electrophoresill, the gels were
stained with Coomassie Blue.
The DNA was extracted according to the protocol of Lodhi et at. (1994). PCR
(polymerase chain reaction) and RAPD (random amplified polymorphic DNA) analysis was
done with the method described by Yu & Pauls (1993). Electrophoresis of the amplification
products was carried out on a 1.5% agarose gel buffered with TBE (Tris + boric acid +
EDT A) and staining was perfonned with ethidium bromide.

3. Somatic Embryogenesis Induction

In the following sections we describe the process of somatic embryo fonnation in zygotic
embryos and leaves of tamarillo.

3.1. IN ZYGOTIC EMBRYOS

Mature or immature zygotic embryo explants are most commonly used for somatic
embryogenesis induction in woody plants (Kendurkar et at., 1995; Raemakers et al., 1999).
In tamarillo mature zygotic embryos also proved to be a good material for somatic
embryogenesis induction. Two types of somatic embryo differentiation can be induced from
zygotic embryos of tamarillo depending on the kind of auxin used: NAA or picloram or 2,4-
D. However, in any case, the somatic embryos or the embryogenic calli developed mainly
from the hypocotyledonary region of the zygotic embryos (see section 6.1). When NAA
was used, the hypocotyl of zygotic embryos fonned a small callus (Fig. 2A) from which
somatic embryos differentiated (Fig. 2B). These somatic embryos matured in the original
NAA containing medium after 4- 6 weeks of culture (Guimaraes et at., 1996). Since the
somatic embryos are induced and matured in the same culture medium, this process of
embryogenesis has been called "one step somatic embryogenesis" and has been reported in
several species including some woody plants (Barriga et at. 1998; Canhoto & Cruz 1996a,
Canhoto et at., 1999a; Pijut, 1999). When several concentrations of NAA were tested, the
best results (42.3% induction) were obtained with 2.0 mg/1 NAA, even though a wide range
of concentrations (0.1 - 10 mg/1) of this auxin were able to induce somatic embryogenesis
(Guimaraes et at., 1996).
In contrast with the results obtained with NAA, zygotic embryos cultured in the
presence of 2,4-D produce a slow-growing callus (Fig. 2C) after 4 weeks of culture. By the
8th week whitish clusters of embryogenic cells develop among the non-embryogenic cells
of the calli (Fig. 2D). The frequency of embryogenic calli also depends on the 2,4-D
concentration used, and the best results were obtained with 10 mg/12,4-D (Guimaraes et al.,
1996). Lower 2,4-D concentrations (less than 1.0 mg/1) were unable to induce embryogenic
calli. Subculture of the embryogenic calli on media devoid of auxins or containing 0.1 mg/1
GA 3 allowed the differentiation of the embryogenic masses in somatic embryos that
progress through the stages characteristics of embryo development. This kind of
embryogenesis, in which two different media are necessary to achieve full somatic embryo
differentiation, has been tenned as "two step somatic embryogenesis". The embryogenic
calli can be maintained in the induction media for extended periods of time (some lines
have been kept in culture since 1994) without loss of the embryogenic potential. Similar
results were obtained with picloram. Embryogenic callus fonnation and maintenance have
438

been reported in several woody plants (Canhoto et at., 1999b; Muralidharan et at., 1989;
Tennignoni et at., 1996; Tomar & Gupta, 1988; Watt et al., 1991). Embryogenic callus
fonnation offers a great potential for large-scale production (Merkle et al., 1995) and can be
used as a source of cell lines for assays of plant genetic transfonnation.

3.2. IN YOUNG LEA YES

Tamarillo leaf explants produced friable, yellowish non-embryogenic calli and nodular,
whitish embryogenic calli (Fig. 2E) in the presence of picloram. Small embryogenic areas
firstly appeared at the callus surface after 6 weeks of culture. When several concentrations
of picloram were tested it was shown that 0.1mg/l was unable to induce somatic
embryogenesis (Tab. 2). From all the concentrations tested 5.0 mg/1 picloram gave the best
(about 40%) results in induction rate (Tab. 2). The importance of proliferative somatic
embryogenesis either through the maintenance of embryogenic calli or by the proliferation
of secondary embryos has recently been discussed (Raemakers et al., 1999).
The role of different concentrations of sucrose in combination with 5.0 mg/1 picloram
on the induction of embryogenic callus was also tested. The results showed that 12% was
highly effective for embryogenic calli fonnation (Tab. 3). However, embryogenic calli grew
faster in the presence of 9% sucrose. Considering these results the medium containing 5.0
mg/l of picloram and 9% sucrose was used for the maintenance of embryogenic calli
derived from leaf segments. A positive effect of high sucrose concentrations on somatic
embryogenesis induction has been reported for other woody plants (Canhoto & Cruz, 1994;
Litz, 1984; Muralidharan et al., 1989). Several authors have pointed out that carbohydrates
may have a dual effect in plant tissue culture, acting as carbon source and as an osmotica
(Ammirato, 1989; Levi and Sink, 1990)
Leaves have been used by several authors to induce somatic embryogenesis in woody
plants (Cheema, 1989; Pedroso & Pais, 1993; SOndahl & Sharp, 1'fl7). Tamarillo leaves
could be an excellent explant alternative to zygotic embryos, since they are available at any
time of the year. Besides, if leaves from adult plants could be used, a protocol for clonal
propagation of elite trees could be developed (Kriebel, 1995). Attempts to regenerate
tamarillo plants by somatic embryogenesis in leaves from adult plants are being carried out.

4. Somatic Embryo Germination

When masses of embryogenic calli originated from leaves or zygotic embryos, were
separately transferred to an auxin-free medium containing 2% sucrose, the early phases of
somatic embryo development could be observed on the callus surface within 3 weeks. Later
on, fully developed embryos could be seen (Fig. 3A). Such embryos, subcultured on the
same medium for a further 4-5 week period developed in green normal plantlets (Fig. 3B).
Mature somatic embryos produced in the hypocotyl region of zygotic embryos also
germinated well in the same conditions. Experiments carried out to evaluate the role of
different growth regulators on the development of somatic embryos from embryogenic
masses have shown that 0.1 mg/1 GA 3 stimulated somatic embryo development and further
germination. In contrast, NAA inhibited root growth and kinetin stimulated the occurrence
of fused and accessory embryos (Guimariies et al., 1996).
 439

5. The Role of Auxin Polar Transport Inhibitors on Somatic Embryo Development

Auxin polar transport is an important mechanism in the control of several plant

developmental processes (Lomax et al., 1995), including zygotic embryo development (Liu
et al., 1993). The asymmetric distribution of membrane carriers for the uptake and efflux of
auxins at the cellular level is probably the main factor determining the direction of
movement and preferential accumulation of auxin (Raven, 1975; Rubery & Sheldrake,
1974). Studies of the effect of several auxin polar transport inhibitors on somatic embryo
development were carried out in embryogenic calli of tamarillo maintained in a MS medium
containing 5.0 mg/1 2,4-D. Portions of the embryogenic calli were transferred to an auxin-
free liquid medium containing several concentrations of TIBA, HFCA, TCA and CFA.
Somatic embryos developing in a MS basal medium were used as a control. Somatic
embryo development was evaluated by counting the number of embryos that reached at
least the torpedo stage after 30 days of culture. A qualitative evaluation of embryo
morphology was also performed. The results so far obtained showed that somatic embryo
development is strongly affected by the presence of TIBA and HFCA in the germination
medium (Fig. 4). TCA and CFA only impaired somatic embryo development at relatively
high concentrations (Fig. 5). Our results on auxin polar transport inhibitors concluded that
TIBA is the most powerful inhibitor of somatic embryo development, since 0.6 J4M TIBA
arrested all the embryos at the globular stage (Fig. 3C). Somatic embryos developing on
inhibitor containing media showed several morphological abnormalities such as fused
cotyledons (Fig. 3D) and fused hypocotyls. Liu et al. (1993) pointed out in Brassica juncea
that these anomalous embryos are phenocopies of the emb30 (gnom) mutants of
Arabidopsis displaying modifications in auxin polar transport (Mayer et al., 1993; Meinke,
1985). The transfer of arrested somatic embryos to an inhibitor free medium allowed them
to display the normal pattern of development. Schiavone & Cooke (1987) also reported the
inability of carrot somatic embryos to make the transition between the globular and the
heart stage in the presence of auxin transport inhibitors. These observations seem to indicate
auxin polar transport is a crucial mechanism for the establishment of polarity in somatic
embryos as well as in zygotic embryo development (Liu et al., 1993). Furthermore, some of
the morphological abnormalities found in somatic embryos of several species could be
related to some kind of anomaly affecting the transport of auxins.

6. Somatic Embryo Origin

Somatic embryo differentiation in the hypocotyls of zygotic embryos and in young leaves
was followed by histological and electron microscopic (SEM and TEM) studies.

6.1. ZYGOTIC EMBRYOS

At the time of culture (Fig. 6A) hypocotyl cells were rich in lipid (oil bodies) and protein
reserves (protein bodies). One week later, the first divisions were seen in epidermal and
subepidermal cells (Fig. 6B). When zygotic embryos were cultured on MS medium
(control) such divisions were not observed. By the lOth day of culture, the amount of lipids
and proteins was considerably reduced whereas epidermal and subepidermal cells keep on
dividing. The continuous proliferation of these cells gave rise to a meristematic layer of
cells just under the epidermis (Fig. 6C). This layer is easily distinguished from a more
440
internal zone of vacuolated cells (Fig. 6C). Until this stage, hypocotyledonary cells show a
similar behavior independently of the culture medium used (NAA or 2,4-D). Beyond this
stage, in the presence of NAA, somatic embryos (Fig. 6D) arose from the meristematic
layer in a process resembling somatic embryo differentiation in Feijoa setlowiana (Canhoto
& Cruz 1996b; Canhoto et at., 1996), Camellia japonica (Barciela & Vieitez, 1993) and
Hevea brasiliensis (Michaux-Ferriere et at., 1992). These embryos have probably a
multicellular origin. On other hand, cells of the meristematic layer gave rise to
proembryogenic masses (Fig. 6E) of meristematic-like cells in the presence of 2,4-D. From
the periphery of the embryogenic masses meristematic-like cells were continuously released
(Fig. 6F) giving origin to new embryogenic masses, thus explaining why embryogenic calli
can be maintained in culture for extended periods of time. Somatic embryos obtained from
these embryogenic masses have probably an unicellular origin and are a useful material for
tamarillo genetic transformation. Canhoto et at. (1999b) in Laurus nobilis, Jain et at. (1989)
in Pinus elliottii and Watt et at. (1991) in Eucalyptus grandis made similar observations.
Recently, a paper published by Raemakers et al. (1999) reviewed this kind of proliferative
somatic embryogenesis.

6.2. LEAVES

Transverse sections of leaves inoculated on a somatic embryogenesis induction medium

containing 2,4-D or piclorarn showed two distinct parenchyma: a one-layer thick palisade
parenchyma in the adaxial surface and a spongy parenchyma in the abaxial surface (Fig.
7A). One week later the only clear modification in leaf structure was the accumulation of
starch grains in both the parenchyma and epidermal cells (Fig. 7B). The first cell divisions
mainly occurred in the palisade parenchyma after 12-15 days of culture (Fig. 7C). Further
divisions spread throughout the mesophyll cells with the consequent formation of a callus
tissue (Fig. 7D). Later on, in this callus, some densely cytoplasmatic cells originated
globular proembryogenic masses (Fig. 7E) which, as described for hypocotyls, have the
potential to proliferate.

7. Genetic Stability of the Regenerated Plants

7.1. CYTOLOGY

Chromosomal aberrations are a common occurrence in plant cell and tissue culture (Singh,
1993). As suggested by Devemo (1995) and Jain (1999) in vitro derived plants need to be
screened for genetic stability to avoid potential losses. To evaluate the genetic stability of
tamarillo plantlets regenerated from long-term cultures of embryogenic calli, chromosome
numbers of root tips were counted. Two groups of plants were analyzed: one set of plants
was regenerated from a 6-month-old embryogenic callus (short-term callus) whereas the
other group was derived from a 4-year-old callus (long-term callus). Both calli were
maintained in the presence of 2.0 mg/1 2,4-D and subcultured monthly. The results showed
that all 18 plantlets of the short-term callus showed the diploid (Fig. 8A) set of
chromosomes (2n=2x=24). When plantlets from the long-term callus were analyzed it was
found that the normal diploid set of chromosomes and plants with an abnormal number of
chromosomes were obtained. These included tetraploids (2n=4x=48) and plants with 38, 40,
 441

42 and 44 (Fig. 8B) chromosomes. Furthennore, in long-tenn callus morphological

abnonnalities were often detected and the regeneration capacity was diminished. Abnonnal
somatic embryo formation has often been reported in various woody plants (Jha et al., 1992,
Perez et al., 1983, Canhoto & Cruz, 1994). The results so far obtained in tamarillo show
that a prolonged culture of the embryogenic calli in the presence of 2,4-D induce
chromosomal abnonnalities in the regenerated plants that can impair plant regeneration in
this species and may be the main responsible for abnonnal somatic embryo development
The data obtained also seem to indicate that chromosome aberrations occur during culture
and do not pre-exist in the cultured tissues. Similar results were obtained by Singh (1986) in
cultured immature embryos of barley. Modifications in the number of chromosomes of
somatic embryo derived plants have been signaled and were directly correlated to the
occurrence of somaclonal variation in some woody species (Lelu, 1987; Radojevic et al.,
1988).

7.2. RAPD ANALYSIS

Random amplified polymorphic DNA (RAPD) markers were also used to evaluate the
genetic stability of tamarillo plants regenerated from embryogenic calli. Two populations of
somatic seedlings were tested: one was obtained from a 4-year-old callus and another from
a short-tenn callus (six months in culture). A total of 32 primers (Operon Technologies)
were used to evaluate the genetic stability of 40 plantlets derived from the short-tenn callus.
From a total of 176 bands per plantlet, no variations in the pattern of bands could be found.
These results together with the chromosomal analysis of the regenerated plants seem to
indicate that plants derived from short-term callus are genetically homogenous. RAPD
analysis of the plantlets obtained from long-tenn callus showed polymorphisms in the
individuals 10 and 11 when the OPC 4 primer was used (Fig. 8C). However, further
experiments showed that these variations were not probably related to DNA modifications
of the regenerated plantlets since modified banding patterns were also observed when the
concentration of template DNA for the same plant changed. No somatic variation has been
found by RAPD analysis in somatic embryo plantlets of Picea abies (Heinze and Schmidt,
1995) and Quercus se"ata (Ishii et al., 1999; Thakur et al., 1999). However, as pointed out
by Jain & De Klerk (1998), the fidelity of somaclones is difficult to evaluate by RAPDs,
because this technique is not sufficiently sensitive. Furthennore, when polymorphisms are
detected they have to be confirmed by other methodologies (Jain & De Klerk, 1998), such
as amplified fragment length polymorphism (AFLP).

8. Embryogenic Biochemical Markers

Several authors (David eta/., 1995; De Vries et al., 1988; Hilbert et al., 1992; Pedroso et
al., 1996) have identified proteins related to the process of somatic embryogenesis
induction. Based on the protocols developed for the production of embryogenic calli of
tamarillo, the protein electrophoretic pattern of embryogenic and non-embryogenic calli
was compared to identify protein markers associated with these two types of calli. To obtain
embryogenic calli the cultures were induced on a medium containing picloram or 2,4-D plus
9% sucrose. To obtain non-embryogenic calli the level of sucrose was lowered to 3%
(Ferreira et al., 1998).
442
The SDS-PAGE analysis of the protein content of embryogenic and non-embryogenic
calli revealed some qualitative differences between them. A 26.5 kDa protein was
consistently found in non-embryogenic calli, whereas a labile 45 kDa protein appeared to be
specific to the embryogenic calli (Fig. 9). The 26.5 kDa protein was also found in non-
embryogenic calli derived from other plant sources, such as zygotic embryos and isolated
hypocotyls, cultured in the same conditions suggesting that this protein can be considered a
good marker for the non-embryogenic calli in tamarillo. To study the possible function of
this protein in the embryogenic process, a polyclonal antibody was generated and its
specificity was tested by Western blotting. The results showed that the antibody recognizes
specifically the 26.5 kDa protein in non-embryogenic calli and did not cross-react with any
of the proteins in the embryogenic calli (Ferreira, pers. com. in preparation).
Immunoblotting and SDS-PAGE analysis of the soluble and membrane fractions of the non-
embryogenic callus, showed that the 26.5 kDa protein is present in the soluble fraction
(Ferreira, pers. com. in preparation). Immunocytochemical studies to establish the
distribution of this protein within the cells are being carried out. Moreover, its eDNA is
being cloned from a non-embryogenic calli eDNA library to better characterize and to
determine its role on plant morphogenesis.

9. Conclusions and Future Prospects

Somatic embryos and plantlets of tamarillo have been produced from mature zygotic
embryos and young leaves. In both types of explants, embryogenic calli that can be
maintained in culture for several years have been established. The advantages of somatic
embryogenesis over other rnicropropagation techniques has been discussed (Jain, 1999). So
far, somatic embryogenesis in tamarillo has only been achieved from juvenile explants. The
same is true for most of the protocols developed in other woody plants. We have recently
initiated experiments to test the embryogenic potential of leaves from adult plants to
propagate selected cultivars. Another limitation to the success of somatic embryogenesis in
tamarillo is the genetic instability of the regenerated plants, particularly when they were
obtained from long-term calli. As we have shown, chromosomal abnormalities were
commonly found among these plants. The cryopreservation of short-term embryogenic calli,
where variations in chromosome number were not detected, can be a useful alternative to
the maintenance of embryogenic calli in culture.
The time-course of somatic embryo development was followed in hypocotyls and leaf
segments. The results so far obtained show that epidermal and subepidermal cells of the
hypocotyls and palisade parenchyma cells of the leaves are those involved on somatic
embryo differentiation or in embryogenic callus formation. However, these cells are not the
direct source of somatic embryos as has been found with other cell types (see Canhoto and
Cruz, 1996b). Instead, they first give rise to a layer of meristematic cells from which
somatic embryos further developed. At the moment we cannot explain why these cells
behave in this way and which factors are responsible by the induction of this type of
morphogenesis.
Further studies in the embryogenic system of tamarillo will be focused on
characterization of the embryogenic and non-embryogenic proteins found in the polypeptide
profiles and in the analysis of the factors controlling the transition of the embryogenic
cellular masses to somatic embryos.
 443
10. Acknowledgements

This work was supported by FCT (Fund~ao para a Ciencia e a Tecnologia). The technical
assistance of Mr. Jose Dias and Jorge Mascarenhas is gratefully acknowledged. We are
indebted to Prof. Jose Mesquita for allowing part of this work to be carried out in his
laboratory.

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Table I. Summary of in vitro culture studies in tamarillo.
Response
Somatic embryoS/plants
Shoot formation/plants
Shoot formation/plants
Transgenic plants resistant to
kanamycin
Shoot formation/plants
Shoot formation/plants
Somatic embryos/plants
Pollen calli
Shoot multiplicationlplants
Shoot formation/plants
V-KM(Binding&Nehls, 1978);B5(Gamborgefal., 1%8)
• concentrations in pM
.,..,.
0\
Explant Culture media(mg/1)/conditions References
--Zygotic embryos: hypocotyls MS +-2~4~D (2.0 anif.S.b) duimar~es et al.,-1988
Protoplasts from leaves •v -KM + BA (2.5) + NAA (5.0) + 2,4-D (0.5) Binding eta/., 1992
B5 + BA (2.5) + coconut milk
Leaves MSIB5 + BA ( 10) + NAA (0.1) Atkinson & Gardner, 1993
Leaves Coculture with Agrobacterium tumefaciens Atkinson & Gardner, 1993
Protoplasts from leaves V -KM + NAA (0.1) +zeatin (0.5) Guimaraes eta/., 19%
MS + BA (1.0); IBA (0.1)
Zygotic embryos, hypocotyls, cotyledons, MS + BA (0.1 -10) Guimaraes et al., 19%
roots MSIB5 + IBA (0.1) for rooting
Zygotic embryos MS. Various auxins (NAA, 2,4-D and Picloram) were tested. Guimariies eta/., 19%
The role of ABA, Kin and GA, on somatic embryo
development was evaluated
Anthers MS + BA (1.0) + NAA (0.3) Barghchi, 1998
Axillary and apical buds MS + BA (0.25 - 1.0) Barghchi, 1998; Cohen & Elliot, 1979
IBA (0.25- 1.0) for rooting
Leaves MS+ BA(4.0) Barghchi, 1998
IBA (0.25- 1.0) for rooting
 447
Table 2. Effect of different Picloram concentrations on somatic embryogenesis induction in leaf segments of tamarillo.
Leaf sewents were obtained from six-month old plants micropropaeated by sboot apex culture.

Number of inoculated Number of explants showing % of explants showing embryogenic

Picloram* (mg/1) explants embryogenic calli calli**

0 55 0 0

0.1 50 0 0

1.0 79 12 15.2

2.0 54 15 27.8

5.0 68 26 38,2

* on MS basal medium containing 9% sucrose.

• *Results taken after 12 weeks of culture.

Table 3. Effect of different sucrose concentrations on somatic en.bryogenesis induction in leaf segments of tamarillo. Leaf
segments were obtained from six-month old ptants micropropaaated by sboot apex culture.

Number of inoculated Number of explants showing % of explants showing embryogenic

Sucrose* (%) explants embryogenic calli calli**

3 44 5 11.4

6 50 13 26.0

9 49 22 44.9

12 48 34 70.8

15 48 13 27.1

* on MS basal medium containing 5.0 mgll Picloram.

* *Results taken after 12 weeks of culture.
448

Figure 1. Tree (A) and fruits (B) of tamarillo.

 449

ec
ec

Figure 2. Somatic embryogenesis induction in ·zygotic embryos and leaves of tamarillo. (A) Zygotic embryo after 2 weeks of
culture on a medium containing 5.0 mgll NAA (lOx). Note callus (c) formation in the hypocotyl. (B) Several somatic embryos
developing from a callus formed in the presence of NAA (5.0 mg/1) (!Ox). (C) Zygotic embryo after 4 weeks of culture on a
medium containing 2.0 mg/1 2,4-D (lOx). Note callus (c) formation in the hypocotyl and the absence of callus in the cotyledons
(ct). (D) Zygotic embryo derived callus showing embryogenic (ec) and non-embryogenic (ne) zones (22x). (E) Leaf derived
callus showing embryogenic (ec) and non-embryogenic (ne) zones in the presence of 5.0 mg/1picloram (30x).
450

Figure 3. Somatic embryo germination. (A) Germinating embryos (e) after 3 weeks on the germination medium
(2x). (B) Plantlets regenerated by somatic embryogenesis (lx). (C) Inhibition of somatic embryo development in the
presence of TIBA (30x). (D) Somatic embryo showing fused cotyledons (22x).
 451

I•
100
HFCA 13 TIBA
90
.....:::=
ell

80
CJ
tfD
,.s 70
=
t-="' 60
e'i!E"
Qj
so
CJ.s 40
;: ~

e-=
5l
30
20
~
10
0
0 0.3 0.6 1.2S 2.S s 10
Cone. (JIM)
Figure 4. Effect of HFCA and TIBA on somatic embryo development

100
90
I• TCA [:1 CFA

.a
ell
80
-5
~ Qj 70
,.s
~ ell

= "'
t>=
60
a'i!
~ E" 40
so
....CJ.S
.... Qj
30
e-=
= 20
"'
~ 10
0
0 20 40 60 80 160

Cone. (JIM)
Figure 5. Effect of CFA and TCA on somatic embryo development.
452

lb ........

Figure 6. Somatic embryo differentiation in hypocotyls of tamarillo. (A) Section of a hypocotyl cell packed with
protein (p) and lipid bodies (lb) (12000x.). (B) Transverse section of a hypocotyl showing (arrows) divisions in the
epidennal and subepidennal cells (640x). (C) Transverse section of a hypocotyl showing the differentiation in two
distinct zones: a peripheral layer of meristematic cells (me) and an internal zone of vacuolated (v) cells (130x.). (D)
SEM: aspect of a somatic embryo developing from the meristematic layer after 3 weeks of zygotic embryo culture
on a medium containing NAA (200x.). (E) Proembryogenic masses (pe) fanned on a medium containing 2,4-D
(200x). (F) Section of a proembryogenic mass showing some oells being liberated at the periphery (arrows) (250x).
 453

Figure 7. Fonnation of proembryogenic masses in leaves of tamarillo. (A) Transverse section of a leaf piece at the
time of inoculation showing the upper epidermis (ue), lower epidennis (I e), palisade parenchyma (p) and spongy
parenchyma (s) (210x). (B) Accumulation of starch grains (st) in a leaf piece cultured for one week in a medium
containing 2,4-D (BOx). (C) Divisions in the palisade parenchyma after 12 days of culture (boxed zone) (BOx).
(D) SEM aspect of a leaf derived callus (2.50x). (E) Proembryogenic masses (200x)
454

Figure 8. Chromosome counting and RAPD analysis of somatic embryo derived plantlets. (A) Root-tip cell showing tile
normal (2n=24) chromosome number (900x). (B) Root-tip cell showing an abnormal (44) number of chromosomes
(900x). (C) Agarose gel electrophoresis of amplified sequences from a RAPD reaction by using the primer OPC4. Lanes
I - 13 represent plants regenerated by somatic embryogenesis. M represents the 100-bp DNA marker. Note the
polymorphisms in the individuals 10 and II (arrows).
 455

s
kDa
67-
56-

42-

28-
23-

17-

11-

Figure 9. SDS-PAGE analysis of embryogenic and non-

embryogenic calli of tamarillo. S - standart, L - non-cultured leaf
(control), NE - non-embryogenic callus, E - embryogenic callus.
The black arrow indicates the 26.5 kDa protein and the white arrow
points to the 45 kDa protein.
14. SOMATIC EMBRYOGENESIS IN Araucaria angustifolia (BERT) 0. KTZE

Miguel P. Guerra 1, Vanildo Silveira, Andre L. W. dos Santos, Leandro V. Astarita,

Rubens 0. Nodari.
Laborat6rio de Fisiologia do Desenvolvimento e Genetica Vegetal, Departamento de
Fitotecnia, Centro de Ciencias Agrarias, Universidade Federal de Santa Catarina,
88040-900, Florian6polis, Santa Catarina, Brasil. E-mail (1): mpguerra@cca.ufsc.br.

Contents
I. Introduction 5. Culture maintenance
1.1. Importance of A. angustifolia 5.1. Solid medium
1.2. Botany and Ecology 5.2. Cell suspension
1.3. Geographical distribution 5.3. Characterization of embryogenic
2. Embryogeny in Araucariaceae cultures
3. Somatic embryogenesis in conifers 5.4. Biochemical aspects of embryogenic
4. Culture initiation cultures
4.1. Effect of explant 6. Culture maturation
4.2. Effect of the growth regulators 7. Conclusions and prospects
4.3. Effect of genotype 8. References

1. Introduction

1.1. IMPORTANCE OF A. angustifolia

Brazilian pine tree (Araucaria angustifolia), the only native conifer of

economic importance in Brazil (Figure 1A), representing the most exploited timber
source up to the 70's (Mattos, 1994; Shimizu and Oliveira, 1981 ). This tree can provide
seed, wood, fiber and resin. The seeds are of high nutritious value and are generally
consumed by humans and also by the wild fauna on the end of the fall and beginning of
the winter season. Young trees are typically used as Christmas trees in the region of
natural occurrence, and the wood of adult trees is used for furniture, structural timber,
and almost all other kinds of wood applications. The tonality of its wood varies from
yellow to brownish with a light and soft brilliant surface (Longhi, 1993). The wood of
A. angustifolia contains 58.3% cellulose and 28.5% lignin with long fibres, resulting in
the production of high quality paper. The resin serves as a base for the production of
varnish, acetone and other chemical products (Carvalho, 1994).
As the araucaria exploitation progressed, the natural reserves decreased, to the
point of elimination, due mainly to clear-cutting for timber export (Ondro et a/., 1995)
and for agriculture. Originally, the forests of araucaria covered an area of 182,295 km 2 •
457
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 457-478.
© 2000 Kluwer Academic Publishers.
458

Nowadays, only relicts of the natural vegetation are found, representing from 0.7%
(Longhi, 1993) to about 1% (Lima and Capobianco, 1997) of the original area. More
recently, logging became focused on the natural relicts in such a way that this species
was included on the official list of threatened Brazilian plants, in the category of
"vulnerable".
The development of technologies for conservation and genetic improvement of
A. angustifolia is required if we intend to establish reforestation programs with this
species. It has been demonstrated that, in good production sites, this species can reach
same biomass production as Pinus taeda or Pinus elliottii, the most common conifers
cultivated in Brazil (Roberto Pedro Born, Personal Communication).

1.2. BOTANY AND ECOLOGY

A. angustifolia is a heliophyte and pioneer plant. Its association with other

typical species forms the "araucaria forests" (Lorenzi, 1992). In the primary forest,
araucaria behaves as secondary initial the natural regeneration occurring in the margins
of the forests or in large glades (Klein, 1960; Carvalho, 1994).
Although, the species is dioecious, sometimes monoecious plants with unisex
flowers are found (Reitz et a/., 1978). The male flower size (1 0-13 em long) differs
from that of the other members of the Coniferales (Seward, 1906). The female cone
(Figure IB) is oval, with a wooden consistency and a diameter between 20 and 25 em
(Rizzini, 1976). There are usually 400-500 sporophylls on a cone. However, generally
only one sporophyll out of twenty is fertile and able to reach the stage of mature ovules
(Burlingame, 1914).
The reproductive cycle of the A. angustifolia, from the primordial carpel to the
seed, takes approximately four years. The female cone begins to develop in spring
(from August to October), followed by pollination in September of the next year.
Fertilization occurs only on the third year, between October and December. On the
fourth year, the cones reach maturity (from February to July), with subsequent shed of
seeds (Mattos, 1994).
This species has a high nutrient demand during the plant development, being
much more demanding in soil fertility than most of the other conifers (Handro, 1986).
This factor, along with the long life cycle and the long period of time between
pollination and seed formation (Haines et a/., 1984), imposes difficulties in the
establishment of breeding and large scale reforestation programmes with this species.

1.3. GEOGRAPHICAL DISTRIBUTION

The natural occurrence of A. angustifolia in Brazil is limited between the

latitudes 19° IS'S and 31 °30'S and the longitudes 41 °30'W and 54 °30'W (Seitz, 1986).
This species, restricted to altitudes above 500 m, originally covered a surface of 40%,
31%, and 25% of Parana, Santa Catarina, and Rio Grande do Sui, respectively, the three
most Southerly Brazilian states. There were also sparse patches in the states of Sllo
Paulo (3%), Minas Gerais and Rio de Janeiro (1%) and also in the east of the state of
 459

Missiones, Argentina.

__ t:L
Figura I. Sexual reproduction and somatic embryogenesis in Araucaria angustifolia. a) Mature tree (bar 2. 7
m). b) Female cone in the fall (bar 4.3 em). c) Isolated immature zygotic embryo double stained by
acetocarmine and Evan's blue (bar 115 11m). d) Embryogenic culture induction from immature zygotic
embry o on LP medium supplemented with auxin and cytokinin (bar 2 mm). e) Embryogenic culture
maintenance (bar 2 mm). f) Double stain with acetocarmine and Evan's blue on embryogenic culture on
early maturation treatment with ABA (bar 6.2 mm). g) Globular somatic embryos in embryogenic culture on
maturation treatment with ABA (bar 3.1 mm). h) Torpedo somatic embryos in embryogenic culture on
maturation treatment with ABA (bar 1.55 mm).

The deployment of natural reserves of A. angustifolia obliged the Brazilian

government to encourage reforestation programmes with exotic conifer species, mainly
Pinus elliottii and Pinus taeda. For A. angustifolia, the priority now is the conservation
of natural remnant populations and the establishment of a breeding programme for
460

competitive reforestation (Guerra eta/., 1999).

2. Embryogeny in Araucariaceae

The Araucariaceae family shows unique early embryogenic features (Kaur and
Bhatnagar, 1983) with a high degree of specialization (Buchholz, 1920). The free
nuclear stage is relatively protracted, with 32-64 nuclei being produced before cell
walls are formed (Gifford and Foster, 1989). The proembryo is central in the
archegonium (Haines and Prakash, 1980). While the upper peripheral proembryonal
cells form the suspensor, the lower cells originate the cap, and the central cells take part
in forming the embryo (Burlingame, 1915). In Araucariaceae, during the early
embryogeny, the lowermost cells do not contribute to the embryo formation, but are
associated with the cap organization (Haines and Prakash, 1980).
Dogra (1978) observed the absence of internal division in the primitive
gymnosperms. However, in Araucaria juss, the internal proembryo divisions give rise
to typical upper-suspensor-embryonal cell groups (U.S.E.). The E group includes the
conspicuous symmetrical cap, and the U cells are very ephemeral (Haines and Prakash,
1980). The secondary proembryo in Agathis robusta shows the U.S.E. pattern (Kaur
and Bhatnagar, 1983).
The archegonium produced by a single gametophyte in Araucariaceae varies
considerably in number, depending on the species (Gifford and Foster, 1989). In A.
angustifolia, the megagametophyte contains four archegonium cells, which once
fertilized can originate an equivalent number of cellular proembryonic complexes. This
phenomenon is denominated "simple polyembryony" or "polyzygotic polyembryony"
(Dogra, 1978). Due to competition, only one of these proembryos will develop into a
mature embryo (Gifford and Foster, 1989).
In conifers, there are two forms of polyembryony regarding the origin of the
embryos: simple, due to the fertilization of several eggs, and cleavage, due to the
splitting of the product of the individual zygotes (Buchholz, 1926). In Araucariaceae,
only simple polyembryony has been reported.

3. Somatic Embryogenesis in Conifers

Tissue culture techniques are valuable tools in tree-breeding programmes (Park

et al., 1998). Among them, somatic embryogenesis is a process analogous to zygotic
embryogenesis, where a single cell or a small group of somatic cells are the precursors
ofthe somatic embryos (Ammirato, 1983).
Somatic embryogenesis and plantlet regeneration in conifers was first reported
in Picea abies, using zygotic embryos as explants (Hakman et al., 1985; Chalupa,
1985), following by the induction of embryogenesis from megagametophytes in Larix
 461

decidua (Nagmani and Bonga, 1985). In woody plants, somatic embryogenesis has
been reported for 150 species and related hybrids (Dunstan et a/., 1995). The
applications of somatic embryogenesis include the provision of cell lines for genetic
transformation, the ex situ conservation of rare and endangered species or populations,
use in research to improve our understanding of conifer genetics, and the generation of
high-value clonal forestry, which is the most promising application (Park eta/., 1998).
High-value forestry is an intensive commercial forestry in which highly
valuable genotypes or tree "varieties" are cloned for deployment at productive sites.
The major advantages of clonal forestry are: (i) additional genetic gain achieved by
capturing non-additive genetic variation; (il) the speed in which such clones may be
introduced to meet changing breeding programmes or market goals; (iil) the ability to
introduce genetic variability into a clonal forestry; and (iv) the compensation for the
shortage of improved seeds from orchards (H6gberg et al., 1998; Park et al., 1998).
Somatic embryogenesis is a valuable tool in breeding programmes. The major
advantage is to obtain a large number of cloned plants in a short time (H6gberg et a/.,
1998). Both somatic embryogenesis and cryopreservation allow the development of
improved varieties by the regrowth of embryogenic cultures followed by the field
performance (Park et a/., 1998). The cryopreservation of embryogenic cultures
efficiently links the breeding programme to the mass clonal propagation strategies.
Thus, the inclusion of cryopreservation in the strategy of somatic embryogenesis allows
the use of superior clones in high-value forestry (H6gberg et a/., 1998). In addition,
cryopreservation of embryogenic cell masses allows the stable maintenance of
germplasm until the clonal progeny has been evaluated under field conditions (Park et
a/., 1998).
The most responsive tissue for plantlet regeneration in conifers is the
embryogenic cell masses obtained from culturing mature or immature zygotic embryos.
This tissue is the most adequate for studies on conifer transformation using the biolistic
bombardment approach, which enables the transference of foreign DNA to virtually any
cell types (Minocha and Minocha, 1999).
Previous studies on somatic embryogenesis for A. angustifolia were performed
by Guerra and Kemper (1992). Embryogenic cultures were obtained when immature
zygotic embryos were excised from young female cones and inoculated on LP basal
medium (von Arnold and Eriksson, 1981). The induction, establishment and
multiplication of embryogenic cultures and the effects of abscisic acid and
osmoregulators on the maturation of these cultures, were reported by Astarita and
Guerra (1998).

4. Culture Initiation

Several factors affect the initiation frequency of embryogenic cultures in

conifers. In A. angustifolia, these factors are: (i) choice of the explant, (ii) growth
regulators, and (iii) genotype of the mother plant. Currently, induction has been
462

obtained in satisfactory levels, corresponding to the rates related for species as

Pseudotsuga menziesii (20 to 25%), Larix decidua (53%) and Picea abies (23 to 39%)
(von Aderkas eta/., I987; Durzan and Gupta, I987; Harry and Thorpe, I99I; Mo and
von Arnold, I99I ).

4.I. EFFECT OF EXPLANT

Induction of embryogenic cultures (Figure ID) of A. angustifolia occurs in

high frequencies when zygotic immature embryos (Figure I C) are used as explants. The
inductive embryogenic ability of zygotic pre-cotyledonary embryos is restricted to a
short period from December to February, disappearing when cotyledon development
progresses (Astarita and Guerra, 1998). In Picea abies (von Arnold et a/., I996) and
Cryptomeria japonica (Ogita et a/., I999), the rate of embryogenic induction is also
correlated with the developmental stage of embryo. Megagametophytes from immature
seeds have also been tested as an explant source, resulting in induction frequencies of
2.2%. Usually in conifers, immature and mature zygotic embryos have been employed
to obtain embryogenic cultures, although in some species such as Larix decidua and
Pinus spp the same kind of cultures have been induced from megagametophytes
(Nagmani and Bonga, I985; Tautorus eta/., I991).

4.2. EFFECT OF GROWTH REGULATORS

In A. angustifolia, different concentrations of2,4-D (0-90 f.!M), NAA (O-I05 f.1

M), BA (0-II f.!M) and Kin (0-II f.!M) were tested in order to establish embryogenic
cultures (Figure ID). Astarita and Guerra (1998) obtained induction frequencies up to
68.7% (Table I) when immature zygotic embryos were inoculated in basal LP medium
(von Arnold and Eriksson, I98I) modified with NH4N0 3 (2.4 g.L- 1) and supplemented
with L-glutamine (450 mg.L- 1), nicotinic acid (0.5 mg.L- 1), pyridoxin-HCL (0.5 mg.L- 1),
thiamine-HCL (I mg.L- 1), sucrose 30 g.L-I, agar 0.7% (w/v), 500 mg.L- 1 of casein acid,
45 f.!M of 2,4-D, and 11 f.!M of both BA and Kin. However, a significant part of the
generated cell lines did not survive the gradual reduction of the growth regulator levels
in the maintenance phase.
Silveira et a/., (1999) demonstrated two options for the induction of
embryogenic cultures: (i) basal culture medium, free of growth regulators; and (ii) basal
culture medium supplemented with 2,4-D (2-1 0 f.!M), BA (0.5-4.0 f.!M), and Kin (0.5-4
f.!M). The data presented in the Table 2 reveal that the induction rates ranged from I6.7
to 43.7 %. This strategy represents an alternative for establishing embryogenic cultures
in culture medium free of 2,4-D. In Pinus sylvestris and Pinus pinaster (Lelu et a/.,
I999), the induction of embryogenic cultures was obtained in the absence of exogenous
growth regulators. The same authors showed that all stages of Pinus sylvestris somatic
embryogenesis occurred on the same culture medium which was free of growth
regulators.
The use of low levels of plant-growth regulators seems to be advantageous in
other conifers. In Pinus elliottii, low concentrations of growth regulators were optimal
 463

for embryogenic callus production (Jain et a/., 1989). The induction of embryogenic
masses in Pinus taeda was obtained in culture medium supplemented with low levels of
plant-growth regulators. The balance between auxin and cytokinin was more important
for the initiation of embryogenic cultures than the absolute concentrations (Li et a/.,
1998).

Table I. Induction rates of embryogenic cultures of A.

angustifo/ia from immature zygotic embryos inoculated on LP
basal medium supplemented with different levels of 2,4-D and
casein. BA and Kin were added at II J.!M each.
2,4-D Embryogenic cultures (%)'
Casein
Harvest dates
(J.!M) (mg.L·')
December January February
0 250 60aN 57.5 abA 48.3 abA
500 55 abA 52.5 bA 26.2 cB
22 250 39.1 bcB 68.7 aA 60.6 aA
500 40 bcAB 55 abA 36.6 bcB
45 250 27.5 cB 46.2 bA 51.6 ahA
500 60aA 52.1 bA 53.3 aA
90 250 50abA 58.2 aA
500 25 cA 53.2 aA
• Percentage of immature zygotic embryos induced to form
embryogenic cultures as average of four repetitions of 5 embryos
each.
b Means followed by the different small letters in the column or

capital letters in the line are significantly different at the 0.05

level according to LSD Test.

Table 2. Induction rates of embryogenic cultures of A.

angustifo/ia from immature zygotic embryos inoculated on LP
basal medium supplemented with different growth regulators.
2,4-D BA Kin Explant Induction
J.!M J.!M J.!M Number Rate (%)
0 0 0 24 33.3 a'
0 4 4 24 16.7 b
2 0.5 0.5 24 43.7 a
5 2 2 24 35.4 a
10 4 4 24 43.7 a
• Means followed by the different small letters in the column or
capital letters in the line are significantly different at the 0.05
level according to Duncan Test.

4.3. EFFECT OF GENOTYPE

In A. angustifolia, the frequency of embryogenic culture initiation is dependent

on the genotype of the mother plant. Under the same conditions of culture medium,
differences of up to 29.16 % (Table 3) in the initiation frequency of embryogenic
cultures were observed (Silveira and Guerra, 1998).
Culture medium affects the genotype competence for somatic embryogenesis
(Jain et a/., 1989). According t~ von Arnold et a/. (1995), the quality of the seeds and
the genotype used may affect the induction rates of somatic embryogenesis. In Picea
464

g/auca, significant variances due to families and family x treatment interaction were
found (Hogberg eta/., 1998). In Abies alba x Abies cephalonica and Abies alba x Abies
numidica (Salajova et a/., 1996), and Picea abies (von Arnold et a/., 1996) the
frequency of embryogenic callus formation was dependent on the genotype of the
mother plant.

Table 3. Effect of different genotypes on the induction rates of

embryogenic cultures of A. angustifolia from immature zygotic
embryos inoculated in LP basal medium.
Induction Rate
Genotype Explant Number
(%)
Plant B 60 49.16 a•
Plant A 60 20b
• Means followed by the different small letters in the column or
capital letters in the line are significantly different at the 0.05
level according to Duncan Test.

5. Culture maintenance

The multiplication phase in A. angustifolia involves the transference of

embryogenic cultures to the same basal culture medium every 20 day interval (Figures
IE and IF), under constant level of growth regulators. In our laboratory, the repetitive
cell cycles were established by periodic transfers of the suspension cultures to either
solid and liquid medium.
The establishment of repetitive cycles of cell divisions may be considered the
starting point for the maintenance and scale-up of embryogenic cultures. The
regeneration potential of embryogenic cultures has been described by several authors
working either with agar-solidified medium or liquid suspension (Tremblay and
Tremblay, I99I; Dunstan eta/., 1993; Attree eta/., I994). The use of liquid cultures is
often considered as the most appropriate approach for mass propagation (Preil, 1991;
Tautorus eta/., 1994). In addition, embryogenic cultures in liquid medium facilitates
the appraisal of cellular organization (Durzan and Chalupa, 1976), analysis of
carbohydrate metabolism (Lulsdorf et a/., 1992), use of osmotic treatments to promote
embryo maturation (Roberts, 1991 ), release of extracellular proteins (Miies eta/., 1997),
and study of chemical composition and physical properties of the cell walls during
growth (Titel et a/., 1997). It is also a method for rapid clonal multiplication with less
labour when compared with multiplication on solid media.

5.1. SOLID MEDIUM

In our laboratory, several embryogenic cultures of A. angustifolia were

established either in LP basal medium supplemented with 2,4-D (7 f.!M), BA (2 f.!M)
and Kin (2 f.!M); LP medium supplemented with 2,4-D (5 f,!M), BA (2 f.!M) and Kin (2
f.!M), and LP medium free of growth regulators. These cultures were subcultured every
 465
20 days. When the subculture interval exceeded 30 days the cultures turned brown. The
same effect was noticed earlier in Picea rubens if the subculture intervals were longer
than 21 days (Harry and Thorpe, 1991 ). However, the cell browning affected neither the
growth rate in the maintenance phase nor its ability to produce embryos (Isabel and
Tremblay, 1995). However, the cell browning in A. angustifolia resulted in a reduction
of embryogenic culture proliferation and also a decrease in proembryo development.
After six months in culture, only 7.2 % of all induced cultures of A.
angustifolia were able to proliferate (Astarita and Guerra, 1998). Growth reduction or
cellular death following the induction phase is a usual feature in the somatic
embryogenesis of many conifers. In Picea abies, for example, between 5% and 15% of
the induced cultures were able to proliferate in the stabilization phase (von Arnold et
al., 1995). Similar responses were also observed for Picea glauca and P. engelmanii
(Mo and von Arnold, 1991), indicating that the main constraints in somatic
embryogenesis are not found in the induction phase but in the other stages.
Based on our experience, the growth of embryogenic cultures of A.
angustifolia increased the fresh weight from 0.1 to 0.2 fold when they were maintained
in solid medium for 21 days. These embryogenic cultures were kept for two years in
Petri dishes, preserving their ability to develop proembryos when submitted to
maturation treatment.

5.2. CELL SUSPENSION

Cell suspension system has been employed for the multiplication of

embryogenic cultures in many conifer species. A large number of somatic proembryos
can be obtained in cell suspensions maintained in bioreactores, rotating agitators or
conventional shakers, reducing the cost of clonal propagation (Tautorus eta!., 1991).
Cell suspension is very useful in the study of biochemical processes associated with
somatic embryogenesis (Roca and Mroginski, 1993).
Several parameters have been proposed to evaluate the growth in the cell
suspensions: sedimented cell volume, packed cell volume, mitotic index, and fresh and
dry weight (Lulsdorf et a!., 1992). The growth dynamic of cell suspension of A.
angustifolia was evaluated in our laboratory by the use of settled cell volume and
mitotic index techniques. The cell suspension cultures were initiated from embryogenic
cultures previously established in LP solid medium supplemented with 2,4-D (7 J..lM),
BA (2 J..lM) and Kin (2 J..lM). Graduated centrifuge tubes were adapted to nipple-flasks
for evaluation of the settled cell volume. Embryogenic cultures (1.5 g) was inoculated
in adapted nipple-flasks containing 100 ml of LP liquid medium containing or free of
growth regulators. The settled volume was evaluated every three days by inverting the
position of the flasks.
The evaluation of the mitotic index was achieved by collecting samples of
suspension cultures every five days. The protocol used is described in Table 4.
Cells were considered in dividing phase when the division stage was recorded
between the prophase and the telophase stages (Figures 4E and 4F). The value of the
mitotic index was obtained by scoring the percentage of mitotic cells in relation to the
466

total number of evaluated cells.

Table 4. Protocol for estimate mitotic index in cell suspensions of A. angustifolia

I. Take embryogenic cultures as initial material.
2. Fix the material using Camoy 3: I for 3 - 5 hours. In case the samples need to be
stored for further processing before the hydrolysis and staining, it is essential to
replace the Camoy fixing agent by 70% alcohol. Therefore, the samples can be stored
in the refrigerator for several months.
3. Wash with distilled water for 5 min.
4. Hydrolyse in HCI 5 Nat room temperature for 18 minutes and centrifuge at 7500 rpm
for 2 minutes.
5. Immediately after hydrolysis, add the feulgen reagent and incubate in the dark for 2
hours.
6. Prepare the slide by adding a drop of glacial acetic acid. Put the stained material onto
the slide and carefully place the cover glasses.
7. Observe under the microscope.

A significant growth was observed in the cell suspension cultures of A.

angustifolia in LP medium which was free of growth regulators, where its starting
volume increased 7.2 times in 99 days, and the sedimented cell volume at the end of the
growth phase was 58 mL. The growth dynamic of cell suspension cultures of A.
angustifolia in LP medium supplemented with 2,4-D (7 JlM), BA (2 JlM) and Kin (2 Jl
M) showed a 6.8-fold increase in its initial volume over a period of 54 days (Figure 2).

-+-RO --R7

70
3
.s 60
(
"
I
50
I
.,a
40
30 I
il 20 / I
"e
'6 10 / ____./
"'" 0 ~
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96
Days

Figure 2- Growth dynamic of embryogenic cultures of A.

angustifolia in LP culture medium (von Arnold and Eriksson,
1981) free of growth regulators (RO), and in LP culture medium
supplemented with 2,4-D (7 J.LM), BA (2 J.LM), and Kin (2 J.LM)
(R7).

The evaluation of the mitotic index was not possible in embryogenic cultures
which were free of growth regulators. These cultures accumulated storage material that
impaired visualization of the nucleus.
However, the mitotic indexes observed in cell suspension cultures maintained
in LP medium containing 2,4-D (7 JlM), BA (2 JlM) and Kin (2 JlM) were 12.1 %, 5.18
 467

%, and 2.13 % in the exponential, linear and stationary phases, respectively (Figure 3).
Cell divisions were not observed during the lag phase. These mitotic index values for
the embryogenic cultures may be considered high when compared with the exponential
phase of embryogenic cultures of P. abies (4.45 %) (Guerra, 1993).

Figure 3. Mitotic index (%) of suspension cultures of A.

angustifolia in LP culture medium supplemented with 2,4-D (7
j.!M), BA (2 j.!M), and Kin (2 j.!M).

Multiplication of embryogenic cultures of A. angustifolia in LP liquid medium

(free of growth regulators) resulted in the proliferation of embryogenic cells with the
competence to develop somatic proembryos. The cell proliferation in culture medium
which is free of growth regulators may be a prerequisite for the subsequent embryonic
development. It has been suggested that the presence of auxin in the culture medium
influences the formation of non-polar proembryos affecting the development of somatic
embryos in the maturation phase (Korlach and Zoglauer, 1995).

5.3 . CHARACTERIZATION OF EMBRYOGENIC CULTURES

Embryogenic cell lines of P. abies have been divided into two main groups
based on morphology and growth characteristics (von Arnold et al., 1995; Jalonen and
von Arnold, 1991 ). The first group of cell lines, named group A, consists of somatic
embryos with embryogenic regions of small and densely-packed cells, from which the
vacuolated suspensor cells extend. The second, named group B, consists of somatic
embryos comprised of only a few loosely-aggregated cells in their embryogenic region
(von Arnold et al., 1995).
The double-staining procedures with acetocarmine (2%) and Evan's blue
(0.1%) described by Gupta and Durzan (1987) have been used to analyze the
embryogenic cultures. The central cells in the embryogenic region become stained with
red (acetocarmine) and the suspensor cells become stained with Evan's blue. In Picea
abies (Jalonen and von Arnold, 1991) and Picea glauca (Dunstan et al. 1993),
observation has been made of development of somatic embryos as a result of
maturation when the proembryos resulting from the multiplication phase are of the
group A morphology alone.
468

Figure 4. Cell line characterization of embryogenic cultures of A. angustifolia. A) Somatic proembryos of

the group A (embryogenic culture) in culture medium without growth regulators (bar 115 J..Lm). B) Somatic
proembryo of the group B (non embryogenic culture) in culture medium with growth regulators (bar 57.5
J..Lm). C) Embryogenic cell stained with lugol for starch grain stain (bar 23 J..Lm). D) Embryogenic cell stained
with sudan III for lipid stain (bar 23 J..Lm). 4E - 4F) Mitotic cell figures stained with feulgen (bar 23 J..Lm).

The embryogenic cultures of A. angustifolia presented a morphology of both

groups A and B. These cultures, maintained in repetitive division cycles in liquid
medium supplemented with auxin and cytokinin, showed a morphology corresponding
to the group B (Figure 4B). However, the embryogenic cultures maintained in growth-
regulator-free culture medium presented a morphology of group A (Figure 4A). There
is evidence that auxin (2,4-D or IAA) levels above 2.5 fJ.M in the culture medium may
result in disturbances in the morphogenetic evolution of cell lines, affecting the
development (Korlach and Zoglauer, 1995). It is therefore reasonable to suggest that the
establishment and optimization of protocols for the large-scale multiplication of somatic
embryos derived from embryogenic cultures in growth-regulator-free culture medium
 469

may be the starting point for the progression and successful development of somatic
embryos in A. angustifolia.
In our laboratory, histochemical staining procedures using lugol and sudan III
(Johansen, 1940) have been performed in the cell lines of group A. Lugol stains starch
grains, and sudan III stains lipid bodies. In these cell lines, the presence of a high
amount of starch (Figure 4C) and lipid bodies (Figure 4D) was evident. According to
Tautorus eta/. (1991), the presence of these storage products is a prerequisite for the
maturation of somatic embryos. In conifers, during the development of the seed,
accumulation of proteins and lipids has been observed, ranging from 10 to 25% of the
fresh weight. The embryogenic cultures (group A) of Larix occidentalis were
additionally characterized by lipid and ribosome accumulation in the cytoplasm, and a
gradual increase in protein in the vacuoles (Bonga eta/., 1995).

5.4. BIOCHEMICAL ASPECTS OF EMBRYOGENIC CULTURES

In order to investigate some biochemic1l aspects, A. angustifolia cell

suspension cultures were established in basal LP medillm supplemented with 2,4-D (6.8
J..LM), Kin (2.3 J..LM), and BA (2.2 J..LM) (Astarita and Guerra, 1998). Total sugars
(Dub.ois et a/., 1956), glucose (Fales et a/., 1961) and fructose (Roe, 1934) were
assayed. Extracellular proteins were quantified by use of the Biureto colorimetric kit
(Bioclin, Brazil), using bovine serum albumin as the protein standard. Analyses were
carried out on samples taken from the cell suspension cultures every five days. The pH
was also measured. The experiments were performed with four repetitions per
treatment. The means of growth and cell-colony mass were calculated. Flasks
containing culture medium without cells were used as control for biochemical analysis.
The extracellular proteins released from the cell masses were measUred I 0 min
after inoculation under agitation. This initial value was subtracted from the
measurements of extracellular proteirts taken during the culture. This exclusion avoids
any interference from the mucilage extruded by the cell colonies.
The level of extracellular proteins in the culture medium ( 1.3 mg.L" 1) increased
slightly 10 min after inoculation (1.4 mg.L- 1). The quantity of extracellular proteins
continued to rise in the culture medium for five days after initiation of culture (Figure
5), corresponding to the transition from lag to exponential phase (Astarita and Guerra,
1998). Liquid culture medium without cells (control) showed low variation in the level
of extracellular proteins.
The increasing accumulation of extracellular proteins in the culture medium
suggested an intensive extrusion process, which could be due to biochemical
modifications or structural cell changes, resulting in cellular lysis. Secretion of proteins
from cell suspension cultures to the growth medium has been reported in several species
(Schimidt eta/., 1994). Analysis of carrot mutant has provided further evidence that the
medium conditioned with extracellular proteins can promote embryogenesis (Van
Engelen and De Vries, 1992). Cell lines of Picea abies secrete different extracellular
proteins in the culture medium which affect embryo morphology (Egertsdotter and von
Arnold, 1998).
470

r==e== Cell cultures ... • ... Control I

i 3
~
.§
... 2

J ........................................ ~
J 0 5 10 15
Days
20 25 30

Figure 5· Extracellular proteins released by suspension cultures

of A. angustifolia. Control treatment without cells. Vertical bars
denote one standard deviation from the mean.

Enhancement of membrane permeability and cell-wall porosity might also

contribute to the increase in protein release (Payne et al., 1991; Titel et a/., 1997). Cell
suspension of Chenopodium album shows changes in wall porosity (Titel eta/., 1997).
The same authors observed that the wall porosity was high at the beginning of the
intensive growth phase and declined in the transition to the stationary phase.
Transference of the embryogenic cultures from solid to liquid medium can also result in
cellular lysis. This may represent a shock reaction upon transfer to the new medium due
to sudden changes in osmolarity, ion composition, concentration and composition of
dissolved gas, pH of the medium, etc. (Sakano et a/., 1997). Cell disruption has been
associated with the lag and stationary phases (Payne et al., 1991 ).
Media cultures conditioned by cell cultures have been shown to contain a large
number of partially-soluble enzymes (Van Engelen and De Vries, 1992). Zygotic
embryos of Araucaria araucana can release numerous extracellular enzymes, which act
on the megagametophyte during the reserve mobilization (Cardemil and Reinero,
1982). Cultures of A. angustifolia have synthesized intercellular mucilage when
cultivated on semi-solid medium (Astarita and Guerra, 1998). Cell cultures of
Pseudotsuga have also produced and accumulated mucilage which contains
glycoproteins showing activity in the control of morphogenesis (Durzan, 1988).
Advanced stages of proteolysis in suspension cultures of Picea abies lead to the release
of mucilage into the medium which adheres to surface of cells in the embryonal group
(Durzan, 1996). The level of glycoproteins in the culture medium is correlated with cell
densities and inhibition of embryogenesis in Vitis vinifera-berlandieri and Citrus
cultures (Gavish eta/., 1991; Miles eta/., 1997).
Glucose concentration of A. angustifolia cultures peaked after fifteen days,
while fructose peaked after five days of culture (Figures 6 and 7). This suggests that
fructose was slowly consumed by the c"ells during the lag phase. After five days,
 471

fructose decreased quickly, corresponding to the start of linear growth. At a low

fructose level (day 20), glucose showed a decrease (Figure 7), indicating that fructose
was the main sugar metabolized by the cells. Biological growth processes are
influenced by the type of carbohydrate in the medium and by the species.

I____....___Cell cultures .. _•. __ Control I

20

...l
E
soil 15
§
-~ 10
c
g
__ ...•.... ,.•......•......•... ---·-·-···1
0
u
1A 5
0
<.J

"
(5
0+----+----r----r--_,----;---_,
0 5 10 15 20 25 30
Days

Figure 6- Changes in glucose levels in the suspension cultures of

A. angustifolia Control treatment: culture medium without cells.
Vertical bars denote one standard deviation from the mean.

Glucose has been utilized preferentially over fructose for many species, such as
Pinus el/iottii, Glycine max and Medicago sativa (Treat et a/., 1989; McDonald and
Jackman, 1989). However, cultures of Gingko biloba (Carrier et a/., 1990), Picea
mariana (Tremblay and Tremblay, 1991; Lulsdorf eta/., 1992) and cell lines of Picea
g/auca-engelmannii (Tautorus et al., 1994) show a preference for fructose in their
metabolisms. Previous studies have demonstrated that only sucrose and fructose support
A. angustifolia cells growth (Astarita and Guerra, 1998).
Several aspects can affect the uptake of sugars in tissues and cell cultures, such
as temperature, pH, nitrogen and specific activity of carriers in the plasmalemma (Dey
and Dixon, 1985). With A. angustifolia embryogenic cultures, the lowest medium pH
(pH 4.94) was observed after fifteen days in culture (Figure 8), corresponding to half
way through the linear growth phase. The pH of the culture medium began rising
immediately after this phase (Figure 8), reaching the highest value (pH 5.6) on the 30th
day. Control treatments without cells showed a slight pH decrease along the time of
culture. Acidification of culture medium is often observed in plant-cell cultures (Singha
et al., 1987; Cerana et a!., 1989; Sakano et a/., 1997) and is related to nitrogen
metabolism: the preferential assimilation of NH4+ over NH3- results in a metabolic
472

production and extrusion ofW to the medium (Raven, 1988).

!---Cell cultures ... • ... Control-!

L _________________ I

20

3e
oil
sc:::
15
0
·~
.l:l
c:::
Q)
g 10
0
uQ)

"'
B
....2
5
0 5 10 15 20 25 30
Days

Figure 7- Changes in fructose levels in suspension cultures of A.

angustifo/ia_ Control treatment: culture medium without cells_
Vertical bars denote one standard deviation from the mean.

! _ _ _ eencultures---::-_-..--:~ eontr:o(l
L____ ·----·-------- ·--

5.5 ·---- I. ......f. __ _

I --Ir·--...

4,5 +---+---+---+--+----t---1
0 5 10 15 20 25 30
Days

Figure 8- Changes in the pH of the culture medium of suspension

cultures of A. angustifolia. Control treatment: culture medium
without cells. Vertical bars denote one standard deviation from
the mean.
 473
Another aspect of medium acidification is the unbalanced uptake of ions. If
cells take up more cations than anions, the surplus positive charge is balanced by the
synthesis of organic acids and release W into the medium (Ltitge and Higinbotham,
1979).

6. Culture Maturation

In general, the maturation of zygotic embryos involves physiological processes

which ensure embryo dormancy, including the accumulation of abscisic acid (ABA),
growth inhibition and the maintenance of quiescence (Dodeman et a/., 1997). The
maturation of somatic embryos in vitro is dependent on the reduction in the growth of
the embryogenic culture and the accumulation of proteins, carbohydrates and lipids
(Tautorus et a/., 1991 ).
The maturation of conifer somatic embryos, starting from immature
proembryos, occurs in the presence of ABA and an osmotic agent (Attree and Fowke,
1993). ABA inhibits cleavage polyembryony, thus allowing embryo singulation, further
development, and maturation (Gupta eta/., 1993). The presence of an osmotic agent
allows the establishment of an environment with increased osmotic potential similar to
the level observed during the early stages of the zygotic embryo development.
Furthermore, ABA and water stress are involved reciprocally in the accumulation of
storage reserves in conifer somatic embryos (Attree and Fowke, 1993).
In our laboratory, the strategies used for maturation of somatic embryos of A.
angustifolia in a similar manner to those reported for most of the conifer systems, are
based on the use of different levels of ABA, carbohydrate sources, and osmotic agents.
Astarita and Guerra (1998) showed that maturation treatments with sucrose and fructose
supported cell growth but did not improve the formation of somatic embryos regardless
of addition of ABA (38 f.!M). When the culture medium was supplemented with
polyethylene glycol (PEG) 8000 (1 %) or ABA (7 and 19 11M), the development of
somatic proembryos was enhanced (Table 5). Experiments with different carbohydrate
sources - glucose, mannitol, sorbitol and inositol in isomolar concentrations (90 mM) -
resulted in necrosis or death of the colonies after two weeks in culture.
The culture medium containing ABA (50 J.!M) and PEG 4000 (1 %) resulted in
the development of stage 1 and 2 somatic embryos (Tautorus eta/., 1991) (Figures 1G
and 1H, respectively) after ninety days in culture. No further development to stage 3
was observed. These cultures showed cells with starch and lipid bodies, and
glycoproteins by histochemical stain with lugol, sudan III, and acetocarmine,
respectively (Silveira and Guerra, 1998).
Embryogenic cultures of A. angustifolia in the maintenance culture medium
show the presence of somatic proembryos. Our working hypothesis is that these
embryogenic cultures need pre-treatment in order to activate the genes associated with
the progression of embryonic development, before the cultures are prone to be
responsive to ABA and/or osmotic agents. Up to the present, the best results have
474
shown the enhanced formation of somatic embryos in stages 1 and 2 as the result of
treatment with PEG 4000 (3 %, 6 % and 9 %) in association with maltose (3 %, 6 % and
9%) and in response to treatment using bovine serum albumin (BSA) (lg.L- 1).
Certainly, it will be necessary to fmd the ideal proportion of these substances to the
maturation of A. angustifolia somatic embryos.

Table 5. Number of early somatic embryos of A. angustifolia

developed in LP basal culture medium supplemented with ABA
and PEG.
ABA PEG(w/v)
(J.1M) 1% 2% 5%
0 103 0.8 0
7 2.2 0 0.6
19 2.6 0.6 0
38 0.8 0 0
76 0.6 0 0

Maturation is currently the least efficient stage of conifer plantlet regeneration

from somatic embryos (Attree and Fowke, 1993). The maturation capacity varies
significantly among the cell lines. Up to the present, high frequencies of cell-line
maturation have only been observed in bipolar somatic embryos (Piiques et al., 1995).

7. Conclusions and prospects

A. angustifolia is the only native coniferous species of economic importance in

Brazil. The natural reserves of these species were almost totally exploited. The
development of technologies for germplasm conservation and genetic improvement is a
prerequisite for the ·establishment of reforestation programmes.
The application of somatic embryogenesis in conifer breeding programmes
allows the mass clonal propagation of elite genotypes for use in reforestation and
conservation of endangered natural populations.
This is the first report on somatic embryogenesis of A. angustifolia. The results
presented show that starting from young zygotic embryos, the induction of
embryogenic cultures of A. angustifolia may be obtained in LP basal culture medium
containing different levels of growth regulators (2,4-D, BA, and Kin). High levels of
growth regulators enhance the induction frequency. However, embryogenic cultures do
not survive under the gradual reduction of growth regulator concentrations. In A.
angustifolia, differently from other conifers, the induction and establishment of
embryogenic cultures may occur in basal culture medium which is free of growth
regulators. Currently, in the induction phases, two approaches are being employed: (i)
basal culture medium, free of growth regulators, and (ii) basal culture medium
containing low levels of growth regulators (2-10 JlM 2,4-D, and 0.5-4.0 JlM of each BA
and Kin).
Basically, the same culture media were employed to scale-up the embryogenic
 475

culture during the establishment or maintenance phase. The dynamic of cell suspension
and biochemical studies showed some relevant features of embryogepic culture
metabolism.
In the maturation phase ABA and PEG in the culture medium permitted the
progression development of torpedo somatic embryos. Experiments are in progress
using new embryogenic cultures, and osmotic agents. Methods for promoting the
maturation and production of somatic embryos are to be tested. Although much work
on somatic embryogenesis of A. angustifolia has been done in our laboratory, additional
work is needed to support the conservation and mass propagation of this endangered
conifer. The exploitation of somatic embryogenesis for large-scale plant multiplication
is very much dependent on the germination of somatic embryos. Our efforts are
focusing on these parameters.

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15. SOMATIC AND GAMATIC EMBRYOGENESIS IN QUERCUS SUBER L.

M.A. BUENO I , A. GOMEZ

' I
AND J.A. MANZANERA2
1INIA-CIFOR. Ctra. de La Corufia km 7. 28040 Madrid. Spain.
2E.T.S.I. Montes. Ciudad Universitaria s/n. 28040
Madrid. Spain.

1. Chapter contents

2. Introduction

2.1. QUERCUS SUBER L.: THE SPECIES.

2.2. GEOGRAPHICAL DISTRIBUTION
2.3. ECONOMIC IMPORTANCE
2.4. CONVENTIONAL MULTIPLICATION
2.5. VEGETATIVE PROPAGATION
2.6. MICROPROPAGATI ON

3. Somatic embryogenesis

3 .1. PLANT MATERIAL

3 .2. STERILISATION
3.3. CULTURE MEDIA
3 .4. INDUCTION MEDIA
3.5. SOMATIC EMBRYO DEVELOPMENT
3.6. GERMINATION

4. Gametic embryogenesis

4.1. PLANT MATERIAL

4.2. STERILISATION
4.3. INDUCTION MEDIA
4.4. GAMETIC EMBRYO DEVELOPMENT
4.5. GERMINATION

479
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 479-508.
© 2000 Kluwer Academic Publishers.
480

4.6. CHROMOSOME COUNTS

4.7. FLOW CYTOMETRY
4.8. BIOCHEMICAL MARKERS
Jsozymes
4.9. MOLECULAR MARKERS
RandomAmpli.fied Polymorphic Markers (RAPD)
Microsatellite markers
4.10. IDSTOLOGICAL STUDIES
Early microspore embryogenesis.
Microspore pro-embryos.
Responsive stages ofpollen development to embryogenesis.

5. Conclusions

6. Aknowledgments.

7. References

2. Introduction

2.1. QUERCUSSUBERL.: THE SPECIES.

Cork-oak (Quercus suber L., order Fagales, family Fagaceae) belongs to a group of
forest species of outstanding importance in the Boreal hemisphere. Oaks are the
dominant species in many ecosystems of the Holartic region.

The genus Quercus comprises more than 600 species, most of them trees, characterised
by their fruits (acorns). First fossils are recorded in the Cretacic, and oaks diversified
during the Coenozoic throughout the whole world. Presently, oaks appear in the
Northern Hemisphere only.

Cork-oak is a medium-sized tree (Fig. 1), up to 25m height, reaching sexual maturity at
10- to 12-year-old. Chromosome number is 24 and genome size is 1.9 pglnucleus
(Bueno et al. 1996). Cork-oak is highly heterozygous (Elena-Rossell6 and Cabrera,
1996). Natividade (1950) suggested that Quercus species are actually tetraploids
derived from species with 12 chromosomes, and their genetic similarities are bigger
than the mmphologic traits. This has also been demonstrated by Streiff et al. (1998)
between other oaks, such as Q. robur and Q. petraea.

The main feature of cork-oak is its corky periderm, which is of technical interest. The
periderm consists of three layers: phelem or cork, phelogen, and pheloderm. The outer
cork layer, plays a protective role due to the cell wall composition. This contains
suberin, 35% fatty acids, 20 to 30% lignin, and cellulose, polyterpens and tannins
 481
(Cooke, 1948). Cork is a dead tissue, with low density, highly insulating and
waterproof.

Figure 1. Recently debarked cork-oak tree from Oiceres, Spain (kindly provided by A.
Bachiller).

Cork-oak silviculture is based on high forests. The main product, cork is peeled from
the stem and branches every 9 to 12 years. Trees are pruned for low branching because
branch cork is usually of better quality than that from the stem. Fifty to 100 year-old
trees with a thick bark are commori.Iy used for this purpose.

Cork is peeled for the first time, when the tree reaches 20 em in diameter at breast
height, normally at about 25-years-old. Cork production decreases in 150 to 200- year-
old trees, however, may continue to produce until350-year-old.
482

2.2. GEOGRAPHICAL DISTRIBUTION

Cork-oak vegetates in the Atlantic and Western Mediterranean countries: Portugal,

Spain, France, Italy, Morocco, Algeria and Tunisia. The total estimated area is around
2,355,000 ha (Montoya, 1988), mainly in Portugal (34% of the total area). The area in
Spain is 487,000 ha (Montero, 1987).

Cork-oak has been introduced in Crimea, Georgia and the United States during the last
century with scarce success. Other introduction assays have been done in Japan, Greece,
Turkey, Chile, Argentina, Uruguay, Australia and New Zealand (Natividade, 1950).

2.3. ECONOMIC IMPORTANCE

Average cork yield amounts 80,000 tons/year in Spain, representing a 22.7% of the
world production, with an economic value in 1996 of almost 56 million Euros. This
production supports the activity of more than 300 enterprises, 12,000 employees and
240 million Euros (Madrigal et a/.1999).

2.4. CONVENTIONAL MULTIPLICATION

Cork-oak is usually propagated by acorns without special problems of germination.

Nevertheless, many forests have regeneration problems due to grazing. Furthermore,
acorn production is irregular, and dependent on climatic conditions. Good crops may
take place every three or four years.

Another feature of this species, in common with other oaks, is the periodicity of bud
growth. Apical bud growth is often stopped and substituted by an axillary bud, leading
to a tortuous shape.

During germination, the acorn produces a rapidly growing root to a considerable depth.
A three-year-old seedling of 9 em height may have a 60 em deep root.

2.5. VEGETATIVE PROPAGATION

There are no vegetative propagation methods applied to cork oak plant production.
Nurseries rely on seed propagation due to the good germination capacity of the acorns.
They are collected in autmnn and can be sowed directly, and start germinating in a
couple of weeks.

Research on vegetative propagation methods has been followed since a long time ago,
but with little success. The simplest method is mound layering. Stump sprouts root at a
56% rate (Natividade, 1950). Grafting is also possible, but a number of limitations
(training of skilled personnel, additional facilities, rootstock costs, incompatibility, care,
etc.) render this method impractical at a large-scale.
 483
Propagation by cuttings is usually the method of choice for practical purposes, but this
has not been so in cork oak. Komissarov (1946) reported up to 100% rooting using a
mixture of 1000 ppm naphthaleneacetic acid (NAA), 1000 ppm indole-3-butyric acid
(IBA) and 1000 ppm anthracene in talcum but this result has only been conflfllled for
juvenile material. When cuttings were taken from plants older than one year, the results
have been discouraging. Cuttings taken from etiolated stump sprouts form roots at a 5%
rate only. In young plants, a 1% IBA application in the greenhouse with substrate
heating at 28°C gave a 50% rooting (Roldao et al., 1992).

2.6. MICROPROPAGATION

Sterilisation methods
Young shoots are sterilised in a 0.1 to 0.25% NaOCl solution (174 gil active Cl) with a
few drops of Tween 20, by continuous shaking for 10 min, followed by three rinses in
sterile distilled water, 10 min each.

Also HgCh 0.2% with a few drops of Tween 20 for 2 min may be used for sterilisation.
This can be washed twice, the first one of 2.4 g/1 CaCh and the second one of 1 g/1
ascorbic acid for 10 min each. Calcium chloride removes mercury traces from the
explant surface and ascorbic acid has an anti-oxidant function (Boulay, 1984).

Medium composition for bud elongation and rooting

Different macronutrient formulations can be used for cork-oak bud culture. According
to previous works (Manzanera and Pardos, 1990), bud micropropagation of cork-oak
gave the best results in the following macronutrient formulations:

a) Heller (1953)
b) Gresshoff and Doy ( 1972)
c) Sommer et al. (1975)

In all cases, the micronutrients of Murashige and Skoog (MS, 1962) have been used,
with the following cofactors: ascorbic acid 1. 7 mg/1, nicotinic acid 1 mgll, m-inositol
100 mgll, Ca-panthotenate 1 mgll, pyridoxine 1 mgll, thiamine I mgll, sucrose 30 gil.
The medium was solidified with 6 g/1 agar, the pH adjusted to 5.6 ± 0.1with 0.5 M
NaOH or 0.1 M HCI, and autoclaved at 0.5 atmosphere (110 °C) for 20 min.

Similarly to other oaks (Manzanera et al. 1996), the best treatment for axillary bud
induction on stem segments is 6-benzyl aminopurine (BA) 0.1 to 1 mg/1 plus NAA 0.0 I
to 0.5 mg/1

Rooting
Dipping elongated shoots (ca. 2 em) for 30 s in IBA 1 g/1 solution gave up to 80%
rooting, depending on the clone. Good results were also obtained by culturing for 7 days
in medium with 1 mg/1 IBA, up to 70 % rooting, also depending on the clone. There
were no statistical differences between both methods.
484

In explants from adult trees, the best rooting treatment needed more auxin: 1 to 5 mg/1
IBA in the medium, for 7 days, and in some clones up to 14 days. Reduction in medium
concentration to one tenth (x 0.1) maintained excellent rooting mte and number of roots
per explant.

3. Somatic embryogenesis

3.1. PLANT MATERIAL

Somatic embryogenesis has been obtained in cork-oak from: whole immature zygotic
embryos (Bueno et al. 1992), nodal segments of seedlings (El Maataoui and Espagnac,
1987), and leaves of seedlings (Ferruindez-Guijarro et al. 1995).

Open pollinated acorns were taken during the period of fruit development (June to
September). In June, female flowers were pollinated and little ovules developed, and
only a few immature embryos were observed. Small heart-shaped embryos, enveloped
in a fluid endosperm, began to appear in July. By August embryos had filled the ovule
cavity, and the endosperm had a finn appearance. By September the embryos were
mature.

Nodal segments were obtained from 6- to 8-month-old seedlings. Explants were about 5
mm in length.

Apical leaves with petioles were removed from 4-month-old seedlings and used as
explants to induce somatic embryogenesis.

3.2. STERILISATION

Acorns are sterilised for 20 min with 2% NaOCl plus a few drops of Tween 20,
followed by three rinses in sterile distilled water for 10 min each. Complete embryos
were extracted from the ovule and cultured.

Nodal segments are sterilised in ethanol 70%, followed by 45 gil Ca(OClh for 25 min
and three rinses in distilled sterile water, 10 min each (El Maiitaoui and Espagnac,
1987).

Leave explants are washed for 15 min in 0,2% Benlate® (DuPont, 50% benomyl) and
2% Tween 20. This is followed by soaking in an agitated solution of 10% NaOCl (7%
active chlorine) plus a few drops of Tween 20 for 15 min, and three rinses in sterile
distilled water for 15 min each. Entire leaves were placed with their abaxial surface
touching the medium (Ferruindez-Guijarro et al., 1995).

3.3. CULTURE MEDIA

The basal culture medium for zygotic embryos comprises macronutrients of Sommer et
al. (1975) and micronutrients of MS, with the following additions: ascorbic acid (11.3
 485
IJ.M), nicotinic acid (8.1 IJ.M), glutamine (3.4 mM), calcium pantothenate (4.2 IJ.M),
pyridoxine-HCl (4.9 IJ.M) and thiamine-HCI (3 J.IM). Sucrose (88 mM) was used as
carbon source. The following growth regulators were used: BA and 2,4-
dichlorophenoxyacetic acid (2,4-D). The pH of the medium was adjusted to· 5.6 ± 0.1
with 0.5 M NaOH or 0.1 M HCl, and the medium was autoclaved at 0.7 atmospheres
(ll5°C) for 20 min, except for glutamine, which was filter-sterilised and added after
autoclaving.

For nodal segments, the.medium of choice isMS with 111 mM glucose, 8 g/1 gelose, 1
g/1 casein hydrolysate, 10 IJM IBA and 8.9 IJM BA. The pH is adjusted to 5.6 prior to
autoclave at 1l0°C for 15 min (El Maataoui and Espagnac, 1987).

The basal culture medium for leaves comprises macronutrients of Schenk and
Hildebrandt (1972) and micronutrients, Fe-EDTA, vitamins, inositol and sucrose ofMS
medium. The pH of the medium was adjusted to 5.7 ± 0.1 with 0.5 M NaOH or 0.1 M
HCl, and the medium was autoclaved at 50.66 kPa (ll4°C) for 20 min (Ferruindez-
Guijarro eta/., 1995).

3.4. INDUCTION MEDIA

The induction medium for zygotic embryos comprises of basal medium, solid (8 g/1
agar) and liquid supplemented with 2,4-D (2.3 IJ.M to 45 IJ.M). The treatments lasted 30
days, after which explants were transferred to growth regulator-free medium. In the
solid medium the higher concentrations of 2,4-D (22.6 IJ.M and 45 IJ.M) provided a
lower percentage of embryogenic tissue, but the differences were not statistically
significant.

Somatic embryos were formed either directly on the zygotic embryos or in the callus
during the second and third week after the beginning of the 2,4-D treatment. At day 30,
the explants were transferred to growth regulator-free medium for somatic embryo
development.

Explants collected during August gave the best results in liquid medium as well as in
agar-solidified medium, although embryogenic responses were also recorded in a few
case from June to September (Manzanera eta/. 1993).

For nodal segments, the induction medium is similar to the culture media. Two weeks
after culture initiation, the explants begin to form a white callus, mainly on both sides of
the nodal segment (El Maataoui and Espagnac, 1987).

The induction medium for leaf explants comprises of macronutrients from Gamborg
(1966) and the other components (micronutrients, Fe-EDTA, vitamins, inositol and
sucrose) from MS (agar 6 g r\ The following subculture sequence was used: first 30
days on medium with lOJ.iM BA plus 10 IJ.M NAA in darkness; second, 30 days nq
medium with 0.5 J.iM BA plus 0.5 IJ.M NAA under a 16 h photoperiod. After that
explants were subcultured on medium lacking plant growth regulators. After about 90
486

days from the beginning, embryonic structures were separated from small amounts of
soft white-yellowish callus and transferred to proliferation medium. Sometimes somatic
embryos developed directly from the swollen surface of the explant The percentage to
initial induction was low: 4% of the cultured leaves were embryogenic (Ferruindez-
Guijarro et al., 1995).

3.5. SOMATIC EMBRYO DEVELOPMENT

Somatic embryos, previously induced on immature zygotic embryos, were developed in

gibberellic acid (GA3) either alone or in combination with BA and NAA. Cultures were
kept in light (70 IJ.mol/m2/s, 16-h photoperiod). Instead of embryo development, de novo
embryogenesis was induced by BA (0.44 J.1M) plus NAA (0.05 J.!M). Maturation of
somatic embryo was stimulated by 3.78!-lM abscisic acid (ABA). Embryos changed
appearance from· translucent to opaque, cotyledons became white, while embryonic axes
acquired a yellowish pigmentation (Fig. 2.1). Microscopical studies (Fig. 2.2) revealed a
well developed histological structure of the somatic embryos, with a prominent apical
meristem between both cotyledons, a root meristem and the caliptra. Vascular bundles
are already visible in both the embryo axis and the cotyledons.

Calli from nodal segments are subcultured in the development medium with similar
composition as in previous steps. During proliferation, calli turn green and can be
maintained for one year. In a few cases (3%), five to seven weeks without subculturing,
small globular structures appear on the callus surface, easily detachable. Most of them
develop nodules, in which a root apex can be distinguished and cotyledon-like
structures differentiate at the opposite side. These primary embryos produce secondary
embryogenesis when subcultured on the same medium. At the globular stage,
secondary embryogenesis occurs all over the embryo surface, while in at the later stage,
i.e. cotyledonary secondary embryos appear mainly on the root apex (El Maataoui and
Espagnac, 1987).

The secondary embryogenesis from leaves simply by subculturing the somatic embryos
on media lacking plant growth regulators. Secondary embryogenesis have not been
observed on heart- or torpedo- stage embryos. Secondary embryos appeared primarily
on the root pole and secondarily on the main axis and cotyledons.

Isolated immature embryos of cork-oak had longer and more swollen cotyledons, and
the percentage of embryos giving secondary embryogenesis was significantly higher
when grown on SH than on 112G. In the latter case, approximately 60% of the embryos
remained arrested without any further growth. Alternating culture on proliferation and
growth retarding medium almost doubled the number of new embryos at a similar stage
of development at a given time (Fernandez-Guijarro eta/. 1995).
 487

Figure 2.1 . Cork-oak somatic embryo originated from zygotic embryo.

Figure 2. 2. Histological section of a somatic embryo showing differentiated tissues,

with a prominent apical meristem between both cotyledons, a root meristem and the
caliptra (bar: 1 mm).
488

3.6. GERMINATION

Germination of somatic embryos from zygotic origin is impeded almost completely

when an osmoticum (sorbitol 0,3 M to 0,7 M) is added to the medium. Better results are
obtained with treatments containing 3~ GA3 (12% germination), 4 llM ABA in the
dark (up to 22%), and with reduced concentrations (30 mM) of sucrose (24%
germination). Espermine (1 mM) was less satisfactory (up to 8% germination).

Cold storage treatment, i.e., ten weeks at 5°C or two weeks at 2°C, promotes radicle
and epicotyl growth especially on somatic embryos from basal medium of Sommer et
a/. (1975). Epicotyl dormancy is overcome by placing the somatic embryos on paper
bridges, in test tubes containing 10 ml medium of Sommer et al. (1975) supplemented
with 0.4 J.1M BA. Embryos genninated from a few days to three weeks after the end of
the cold treatment. Plantlets with normally developed shoots are transferred to soil, and
acclimated in the greenhouse.

In somatic embryos from nodal segments, subcultivation to growth regulator free

medium leads to the development of the root in a few cases but shoot development has
not been obtained whatever the medium assayed (El Maataoui and Espagnac, 1987).

Germination of somatic embryos developed from leaves was obtained in light (mixed
cool-white and grolux fluorescent lamps, 50 j.lmol/m%, 16-h photoperiod) after a cold
treatment at 3±1°C for 30 days. There was a statistically significant difference between
non-cold-treated embryos and those chilled for 30 days, not only in germination
response but also in reduced adventitious embryogenesis. The addition of BA to the
germination medium after chilling did not improve germination. The macronutrient
formulation had little influence on the germination of embryos that had matured in
darkness and had undergone cold treatment. However, SH macronutrients induced the
highest percentage of secondary embryogenesis while Y2 MS macronutrients gave the
lowest (Fernandez-Guijarro eta/. 1995).

Results on somatic embryogenesis from immature zygotic embryos of cork-oak are

similar to those obtained in other oaks, such as Quercus rubra (Gingas and Lineberger,
1989). In this species, the embryogenic period of immature zygotic embryos was
sharply limited to a period of 4 to 7 weeks after pollination, which takes place in June.
Chalupa (1990, 1995) also obtained embryogenesis in Q. robur embryos.

The role of ABA has been studied in the maturation of zygotic and somatic embryos. At
moderate concentrations, ABA permits normal growth and maturation, prevents the
initiation of new embryogenic centres and of aberrant forms, and precocious
germination of immature seeds (Walton, 1980; Zeevart and Creelman, 1988). ABA also
promotes accUmulation of storage substances, i.e. lipids, proteins and starch (Hakman
and von Arnold, 1988; Roberts et al. 1990; Roberts, 1991).
 489

Cold storage of somatic embryos matured in vitro at 5°C for 10 weeks was the best
treatment for breaking dormancy. This is similar to the treatment necessary for zygotic
embryos of other species (Manzanera eta/. 1993).

Although the frequency of somatic embryogenesis induction on leaves (4%) is lower

than on zygotic embryos, this result opens a new possibility to apply this technique to
selected phenotypes (Fernandez-Guijarro et a/.1995).

4. Gametic embryogenesis

4.1. PLANT MATERIAL

Branches bearing catkins were collected every week during the month of May, which is
flowering period of Quercus suber, from trees of different origins. The cut branches were
oc
preserved in darkness with moist cotton wrapped in aluminium foil at 4 for one week.

Anthers from the same catkins were squashed in acetocarmine [4% (w/v) carmine in 45%
(v/v) acetic acid] to determine the developmental stage of the microspores or pollen
grains. Pollen development appears to be highly asynchronous, as anthers contained all
developmental stages, from tetrads (Fig. 3) to late bicellular pollen grains, within the same
catkin, as previously observed in other Quercus species (Ostrolucka, 1983)

Figure 3. 1. Tetrads squashed in acetocarmine.

490

Figure 3. 2. Micrography of the anther cavity showing the tetrad stage.

4.2. STERILISATION

Catkins between 0.5 and 1 em in length were collected and sterilised by immersion in
96% ethanol for 30 s. Anthers were isolated under aseptic conditions and used to initiate
the cultures.

4.3. INDUCTION MEDIA

Anthers were plated in Petri dishes (12 em diameter, ca 100 anthers per plate), in a
medium containing macronutrients (Sommer et a/. 1975), MS rnicronutrients and
cofactors, 3% (w/v) sucrose and 1% (w/v) activated charcoal, at pH 5.6, and solidified
with 0.8% (w/v) agar. The medium was previously autoclaved at 1 atmosphere (121 °C)
for 20 min. For each series of experiments, plates with anthers isolated from the same
catkin were cultured in the dark for 2, 3, 5 or 7 days, at 25, 33 or 37°C, in all possible
combinations (1-4 plates per treatment), and then transferred to 25°C. Altogether, 2700
anthers were cultured under these different conditions, although about a 60% could not be
analysed due to fungal contamination.

In one of the plates, anthers pre-treated at 33°C for 5 days, gametic embryo formation
could be seen in 14 anthers (out of ca 100 anthers plated) after 20 days of culture at 25°C.
 491

In all cases, embryos grew from the interior of the anthers, breaking through the
degenerating anther walls. One of the anthers incubated at 33°C for 7 days also produced
an embryo culture, which apparently grew more slowly and could be seen only after 79
days of culture at 25°C. No visible embryos or calli were formed from any of the anthers
subjected to other pretreatments, and the anthers eventually degenerated and died (Bueno
eta/. 1997).

The initial translucent globular structures, observed growing from the anther, developed
later into heart-shaped and torpedo-shaped embryos, until formation of well-developed
cotyledons could be seen.

Figure 4. Anther culture showing embryo production at different stages, from globular
to cotyledonary.
492
4.4. GAMETIC EMBRYO DEVELOPMENT

After 20 days at 25°C in the darkness, formation of embryos was observed in some of the
anthers (Fig. 4). One month later, all the embryos obtained from the anther cultures were
independently transferred to individual plates containing induction medium without
activated charcoal and supplemented with 0.5 g r 1 glutamine (Gln), where the embryos
were clonally propagated.

Secondary embryogenesis was induced, and new embryos developed on the surface of the
primary embryos. The clones have been maintained for 6 months under these conditions,
with subculture into fresh plates once a month.

4.5. GERMINATION

For plant regeneration, the method established for somatic embryo cultures of Q. suber of
zygotic embryo origin was used (Bueno eta/. 1992, Manzanera eta/. 1993). Individual
cotyledonary embryos were vernalised for 10 weeks at 4°C, in the darkness, and then
cultured at 25°C with a photoperiod of 16 h light/8 h dark and a photon flux density of 100
J..lmol m-2 s-1 provided by Osram cool-white 18 W fluorescent lamps, until root formation
initiated. The embryos were finally transferred to tubes with sterile vermiculite and
Sommer's liquid medium (Sommer eta/. 1975) supplemented with 0.5 mg r 1 BA, where
shoot development and plantlet regeneration was obtained about one month later.

4.6. CHROMOSOME COUNTS

Embryogenic material formed in anther culture, or a previously established somatic

embryo clone (Bueno eta/. 1992), was fixed in glacial acetic acid: ethanol (1:3, v/v) after
pre-treatment with 2 mM hydroxyquinoline for 2-4 h. The samples were then washed in
distilled water, hydrolysed in 5 M HCl for 30 min at room temperature (22°C), washed
again and incubated for 1 to 2 h in Feulgen solution until staining of meristematic regions
was visible. Finally, each sample was squashed on a microscope slide in a drop of
acetocarmine. Chromosome preparations were examined with a Polyvar microscope under
inunersion oil (x 100).

The diploid chromosome number of Q. suber is 2n = 24. This was confirmed in several
metaphase plates of the standard, a somatic embryo of Quercus suber, showing the diploid
chromosome number (2n = 24, Fig. 5.1.). Figure 5.2 shows a metaphase plate of a clone,
obtained in anther culture, having the haploid chromosome number.
 493

Figure 5.1. Metaphase plate of a cork-oak somatic embryo, showing the diploid
chromosome number (2n = 24).

Fig. 5.2. Metaphase plate of a clone, obtained in anther culture, having the haploid
chromosome number (n = 12).
494

4.7. FLOW CYTOMETRY

Flow cytometry offers a sensitive, rapid and accurate method to estimate nuclear DNA
content (Galbraith et al. 1983) and the ploidy level in different cell populations. This
technique has been reviewed in detail by Galbraith (1989, 1990) for plant physiology
studies.

To investigate the ploidy level of the embryos by flow cytometry, small pieces of material
( 10 - 50 mg) were chopped with a razor blade in the presence of a lysis buffer. The
composition of lysis buffer is: 15 mM Tris, 2 mM Na2EDTA, 80 mM KCl, 20 mM
NaCl, 0.1 % (v/v) Triton X-100 and 0.1 % nonylpheno,.:y polyethoxy ethanol (NP40) in
deionised water at pH 7.5, sterilised through a 0.22 ~filter and stored at 4°C.

After filtration of the homogenate through a 30 ~ nylon mesh and centrifugation at

2500 rpm and 4°C for 20 min, the pellet was resuspended in 500 ~I of lysis buffer.
Finally, samples were stained at 20°C with a solution of 55 ~l propidium iodide (1
mg/ml) in 10% phosphate buffer saline (PBS) at pH 7.4 for 5 min and then analysed in a
Coulter Epics-xi flow cytometer. At least 10.000 nuclei were measured for each sample.
To avoid possible differences in the efficiency of DNA staining due to sample preparation,
the available material most closely related to these embryogenic cultures, i.e. a somatic
embryogenic callus culture originally induced from immature zygotic embryos of Q.
suber (Bueno et al. 1992), was used as standard. Chromosome counting confirmed the
diploid composition of the standard (Fig. 5.1). Nuclei isolated from the standard were
passed through the flow cytometer, and the Gl DNA peak (2C) was set at channel number
250 (Fig 6.1).

Nuclei prepared from one of the anther cultures showed a G I peak with lower DNA
content than the standard, appearing at about channel 125, and a very small G2 peak in
channel 250, suggesting that the sample contained only a very small fraction of actively
dividing cells (Fig. 6.2). This shows that the embryos formed in the anther were indeed
haploid, i.e. of pollen origin, which was also confirnied again by chromosome counting.
The rest of the samples were also measured, each of them first independently and then
together with the standard. Ninety eight cultures showed a Gl peak with half the DNA
content of the standard and were therefore haploid (91%), while diploid DNA amounts
were found for ten cultures (9%). Material from most of these cultures was measured
twice or three times after they had been propagated in vitro for 3, 6 or 10 months. The
same DNA patterns were obtained in each independent measurement, indicating that the
embryo cultures were stable with regard to ploidy level.

Flow cytometry has a good amount of applications to plant science. Among others,
nuclear DNA quantification. By this metlwd, cork-oak DNA content was reported for
the first time by Bueno et al. (1996), who 1nade an estimation of 1.9 pg per nucleus.
Therefore, cork-oak genome has ca. 1.7 x 10 9 bp. Nuclear DNA content also has been
measured in many other species, e.g., Quercus petraea (Greilhubert, 1988), Q.
canariensis (Bueno et al. 1996), Elaeis guineensis (Rival et al. 1997), etc.
 495

0 200 400 600

FL3
...
M
M 2

(!)
'<I'
ru

ru
(!)

0 200 400 600

FL3

Figure 6. Flow cytometry analysis of cork-oak embryo nuclei. l: The diploid standard
(the Gl peak was set at channel 250). 2: anther embryo showing the haploid DNA
content.
496

4.8. BIOCHEMICAL MARKERS

Jsozymes

During the process of embryogenesis on anthers, a variable number of embryos is

obtained. First embryos come forth 25 days after the induction treatment, but new
embryos can appear up to three months later. The question was raised, then, whether
those embryos were also directly induced on microspores or they were clonally
propagated from primary embryos as the result of a secondary embryogenesis process.
In the first case, a wider range of genetic variability would be available for breeding
purposes. This question could be elucidated using biochemical (isozymes) or molecular
(DNA) markers.

Some advantages of isozyme analysis are that the different alleles of a heterozygous
locus can be distinguished, it allows a non tissue-specific search of genotypes in
different structures, such as leaves and embryos, it is relatively simple and economic.
Enzyme gene marker analysis has proven to be efficient in identifying haploid origin of
anther embryogenesis in Fagus sylvatica (Miiller-Starck and Jorgensen, 1991). On the
other hand, isozyme analysis has some disadvantages, i.e., it is more tedious, only the
genome region coding for the analyzed isozymes is explored, sometimes they are not
expressed in some tissues, and small quantities of one gametic type may not be
traceable by enzyme gene markers but possibly by DNA markers.

Plant material. Embryo cultures from six different anthers from the same tree were
selected and 20 embryos from each anther were tested by isozymes (Bueno et al. 1999).

Jsozymes analysis. The following enzyme systems were tested: esterase, isocitrate
dehydrogenase, alcohol dehydrogenase, phosphoglucoisomerase, acid phosphatase and
shikimate dehydrogenase. Embryos for isozyme analysis were homogenized in 0.2 ml
of extraction buffer at 0°C.

Extracts for isozymes analysis were absorbed on filter paper wicks and inserted into
11.5% starch gels. Electrode buffers, gel buffers and electrical conditions for each
system are specified in table 1.

Studying the origin and segregation of isozyme loci in haploid embryos. A total of 6
enzyme systems was studied, comprising 11 loci. The parent tree was heterozygous for
shikimate dehydrogenase (SKDH) with two alleles of mobilities 0.297 and 0.266. This
means an individual degree of heterozygosity of 9.1 %.

After scoring the heterozygous locus in the parent tree, six anthers and 20 embryos
individually sampled from each anther were studied, all of which originated from the
same tree. Embryo samples carried only one of these two alleles, showing the haploid
origin (Bueno et a!. 1999). Haploid embryos segregated according to the Mendelian
ratio of gametic frequencies (1: 1) at p<O.OOO (x 2 test).
 497

TABLE 1. Buffer composition and electrical conditions for the isozyme analysis in
cork-oak.

Abbreviation Buffer system Electrode buffer Gel buffer electrical

conditions
TC Tris citrate pH 0.02MTris, 0.15 MTris 250 V (constant)
7.5 (Wendel & 0.02 M citric 0.05 M citric 200 rnA, 4h
Weeden, 1989) acid acid
M Morpho line pH 0.04 M citric 0.002 M citric 200 V (constant)
6.1 (Conkle et acid pH 6.1 acid pH 6.1 50-60 rnA, 5h
al. 1982)
L Lithium pH 8.3 0.2MLhB03 0.2 M 75 rnA
(Scandalios, pH 8.3 Tris/citrate (constant)
1969) 350-420 V, 5h
s Sodium pH 8.2- 0.06MNaOH, 0.067 M Tris, 250 V (constant)
8.7 (Conkle et 0.3 MH3B03 0.008 M citric 75-100 rnA, 4.5
al. 1982) oH8.2 acid, pH 8.7 h

Isozyme markers also have been used in other forest species to ascertain the haploid
origin of anther embryos. such as in Quercus petraea, Fagus sylvatica (Jorgensen,
1988) and Populus (Baldursson eta!. 1993)

4.9. MOLECULAR MARKERS

DNA markers are used for studies on genetic variability, genotype identification, etc.
Restriction fragment length polymorphism (RFLP; Bolstein et al. 1980) were previously
used as molecular markers for this purpose. Nevertheless RFLP is time consuming and
expensive (Milller-Starck and Jorgensen, 1991). The development of polymerase chain
reaction (PCR) for DNA amplification (Saiki et al. 1988) has been important for the
detection of DNA polymorphism.

Random Amplified Polymorphic Markers (RAPD)

One of the PCR-based applications, random amplified polymorphic DNA (RAPD;
Welsh and McClelland, 1990; Williams et al. 1990) uses single DNA primers of
arbitrary sequence. Amplification of genomic DNA occurs wherever the primers find
sufficient sequence identity at a favourable distance and in converging direction. RAPD
may be used to detect DNA variability at different levels, from single-base changes to
deletions and insertions.

RAPD markers are not only useful to detect polymorphism and produce DNA
fingerprints for the study of species and individual identity (Sanchez et a/. 1998), but
also to detect genetic variability in anther-derived embryos. This technique has several
advantages: it requires only a few nanograms of DNA, it is rapid, it is relatively
economical, and it explores a wider range of genome than isozymes. The main
498

disadvantage of the method is that they are dominant. Therefore, they do not detect
heterozygosity.

Plant material and DNA extraction. Embryo cultures from six different anthers from the
same tree were selected and 20 embryos from each anther were tested by RAPD
markers (Bueno et al. 1999). DNA from embryos was extracted as described by Doyle
& Doyle (1990).

PCR amplification. Concentration of DNA extracts was measured with a UV-

spcctrophotometer (GeneQuant II, Pharmacia).

Amplification of cork-oak embryo DNA RAPD markers was performed in a Perkin-

Elmer 9600 thermocycler, with a reaction mixture of 25 J..Ll final volume per tube. The
mixture was composed of 0.5 U EcoTaq DNA polymerase (Ecogen), 3 mM MgCh, 0.67
M Tris-HCl (pH 8.8), 0)66 M (NH4)2S04, 0.1% Tween-20, 0.1 mM of each dNTP, 0.2
mM primer and 20 ng sample DNA.

Six oligonucleotide primers from Operon® (OPC-05, OPN-15, OPN-16, OPN-18, OPS-
13 and OPS-18) were chosen according to previous studies on RAPD marker analysis
on cork-oak (Manzanera, personal communication; Gallego eta/. 1997).

Polymerase chain reactions were performed following this profile: first cycle 2 min at
94°C; 10 cycles 30 sat 94°C (slope 1.5°C/s), 1 min at 55°C (slope 1.5 min) and 4.5 min
at 72°C; 25 cycles 30 s at 94°C (slope l.5°C/s), 1 min at 45°C (slope 1.5 min) and 4.5
min at 72°C; a fmal cycle of 1 min at 72°C.

Amplification products were separated by electrophoresis in 1.5% agarose gel and

stained with ethidium bromide. A standard of 'A DNA/Pstl was charged in each gel.

Studying the origin and segregation of loci of haploid embryos from the same anther.
Five of the six RAPD primers tested gave a good amplification with embryo samples of
two different anthers. Polymorphic bands were found in four primers. One primer
showed identical band patterns for all embryos tested. For three primers, no. OPC-05,
OPN-15 and OPN-18, a polymorphic band was observed, while for the other primer
(OPN-16) there were two different polymorphic bands found in embryos of both
anthers.

RAPD banding patterns of these five polymorphic bands permitted individual

differentiation of the embryos tested (Fig. 7, Table 2). For two bands, segregation
occurred randomly in the case of one anther. In all other cases, the segregation clearly
occurred according to Mendelian inheritance.

The multiple origin of the anther embryo populations from different microspores has
been corroborated by RAPD markers, both because several genome patterns were
distinguished in each anther embryo population and because they segregate following
the gametic pattern (Bueno eta/. 1999).
 499

Figure 7. Amplification pattern of gametic embryos with primer OPC-05 . From left to
right, lanes 1 to 4 are embryos from the same anther. Lanes 5 to 8 are embryos induced
on other anther, and lanes 9 to 11 are embryos from a third anther. Lane 12 is the 'APstl.
Differences in banding pattern are visible among embryos from the same anther.

TABLE 2: Number of embryos induced in two anthers of the same tree, showing RAPD
polymorphism in five bands, amplified with four primers (OPC-05, OPN-15, OPN-16
and OPN-18). *: significant frequencies in the x.,2 test (p<0.05, 1 degree of freedom).

OPC-05 OPN-15 OPN-16A OPN-16B OPN-18

ANTHERB
No. of embryos with 13* 14 10* 4 11*
band
No. of embryos without 10* 9 12* 18 11*
band
Total No. Of embryos 23 23 22 22 22
analyzed
ANTHER A
No. of embryos with 11* 9* 8* 9* 9*
band
No. of embryos without 10* 11* 12* 11* 11*
band
Total No. Of embryos 21 20 20 22 20
anal zed
 503

Figure 8. 1: Histology of an anther after 20 days of culture in which embryogenesis has

been induced. In the pollen sack (PS) a large proliferative mass, a young embryo (E), is
emerging from the anther. Some small pro-embryos (arrows) of 2-4 cells are visible,
together with some microspores (M) which are not following the embryogenic pathway.
Several cell layers form the anther wall (A W). Fig 8.2. Microspore pro-embryo with a
few cells, detail of Fig. 8.1 at higher magnification. The microspore wall, the exine
(arrowheads) is still surrounding the pro-embryo. M, microspores; AW, anther wall.
Bars represent 50 llffi.
 501

Each 25 Ill amplification reaction contained: 20 ng of genomic DNA, 0.2 J1M of

fluorescently labelled forward primer and unlabelled reverse primer (Progenetic), 200
J.iM each dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 9), 2.5 mM MgCh and 0.5 U of
Taq-DNA polymerase (Ecogen). Fluorescent labelled PCR products were separated and
analysed on a semiautomated sequencer (ABI-Prism, Perkin-Elmer). Standards were
used for length determination of alleles.

TABLE 3. Microsatellite loci from Q. petraea used in Q. suber haploid embryo and leaf
analysis: Primer sequences and repeat units (Steinkellner eta!. 1997a).

Locus Repeat unit Forward primer Reverse primer

SsrQpZAG15 (AGb Cgatttgataatgacactatgg Catcgactcattgttaagcac
SsrQpZAG46 (AG) 13 Cccctattgaagtcctagccg Tctcccatgtaagtagctctg
SsrQpZAGllO (AG),s Ggaggcttccttcaacctact Gatctcttgtgtgctgtattt

Tree analysis. All loci were polymorphic in cork oak with an allele number per locus
ranging from 3 to 6, being 4.67 allele per locus on an average. The Diversity index (D)
and observed heterozygosity (h) range between 0.455 and 0.656 per locus, in the first
index, and between 0.475 and 0.685 in the second. Average values of these indices for
all three loci studied were 0.557 and 0.576, respectively. The discrimination power
between genotypes was 90.3%. Similar results were obtained by Steinkellner et a!.
(1997a) in Q. petraea by microsatellite analysis, while isozymes are less sensible for
heterozygosity detection in Q. petraea, Q. cerris, Q pubescens and Q. robur
populations, where average observed heterozygosity ranged between 0.04 and 0.07
(Samuel eta!. 1995). In American oaks (Q. rubra and Q. ellipsoidalis) a slightly higher
mean heterozygosity, 0.228, was obtained (Hokanson eta!. 1993). These results are in
agreement with Streiff eta!. (1998), who compared the spatial genetic structure of Q.
robur and Q. petraea with isozymes and microsatellites, finding that the average
heterozygosity for both species was 0.25 when assessed by isozymes and 0.81 when
assessed by microsatellites.

Microsatellite polymorphisms has provided a new approach to the genetic analysis of

cork-oak and a tree identification system due to the high discrimination power obtained
for genotypic differentiation. In our case, only three loci were necessary to identify 10
among 12 trees tested.

Embryo analysis. All alleles of the parent tree were also found in the haploid progeny,
but only one allele per locus was amplified in each embryo, corroborating the allelic
pattern of the parent tree. In one of the anthers, haploid embryos with different alleles of
locus SsrQpZAG15 were found. In another anther, haploid embryos with different
alleles of locus SsrQpZAG46 also were found. These results indicated their multiple
origin from different microspores or pollen grains of the same anther. In the few cases
where embryos of anther origin had a diploid genome, only one allele per locus was
found, confirming the hypothesis of a spontaneous duplication of the haploid genome.
502

Parental tree analysis by descendant embryo analysis. Allele segregation in the haploid
descendants can be used for the heterozygosity analysis of an individual so that direct
DNA analysis is not necessary for the parental tree. The high rate of polymorphisms
observed also permitted the identification of the parent tree by parental exclusion. The
parent tree of a embryo culture can be found out in the case of mistake in labelling
during manipulation.

The principle of parental exclusion could be applied in these embryo cultures and only
two loci were sufficient for parental identification with a better resolution than isozymes
inAesculus hippocastanum (Miiller-Starck and Jorgenesel\ 1991).

4.10. HISTOLOGICAL STUDIES

Early events in microspore embryogenesis.

Semithin sections, 1 J..lm thickness, provide interesting cellular and subcellular features
of early microspore embryogenesis in several species (Gonzruez-Melendi et al. 1996a,
b; Risuefio et at. 1998; Testillano et at. 1995). This approach has been extended to the
study of microspore embryogenesis in Quercus.

Before the first embryos appear on the surface of the responsive anthers (20-30 days after
plating), it is difficult to know what anther contains induced microspores, due to the
absence of differential features in the first weeks of culture. Interestingly, the
microscopical analysis of those anthers after 20-30 days in culture provides information
on different stages of microspore embryogenesis. Various induced microspores start
embryogenesis at different times in each individual anther.

Several embryogenic structures at different developmental stages are observed

together, inside the same anther, indicating the high level of asynchrony among
microspores following the sporophytic pathway. Structures with two, three or more cells
still surrounded by the pollen wall are observed inside the pollinic sack where, at the
same time, elongated multicellular structures, which have already broken the exine, and
much higher proliferative masses are present (Fig. 8.1). Two-celled structures display
cells of the same size and shape, with a cell wall dividing the original microspore in two
halves, therefore, symmetric divisions would take place in the beginning of microspore
embryogenesis. Subsequent divisions give rise to smaller cells still inside the exine,
with very similar morphological organization (Fig. 8.2).

Before the breakdown of the exine, multicellular structures or pro-embryos show small
polygonal cells with rounded and large nuclei, displaying one small nucleoli. The
cytoplasm is more dense than in other cells of the anther and shows very small vacuoles
or these may be absent. No starch accumulations appear at these early stages of
microspore embryogenesis. Cell walls separating these pro-embryo cells are straight
and thinner than the wall surrounding the whole structure. The young pro-embryos still
surrounded by the exine display a cellular organization similar to cycling or
meristematic cells (Fig. 8.2).
 503

Figure 8. 1: Histology of an anther after 20 days of culture in which embryogenesis has

been induced. In the pollen sack (PS) a large proliferative mass, a young embryo (E), is
emerging from the anther. Some small pro-embryos (arrows) of 2-4 cells are visible,
together with some microspores (M) which are not following the embryogenic pathway.
Several cell layers form the anther wall (A W). Fig 8.2. Microspore pro-embryo with a
few cells, detail of Fig. 8.1 at higher magnification. The microspore wall, the exine
(arrowheads) is still surrounding the pro-embryo. M, microspores; AW, anther wall.
Bars represent 50 llffi.
504

Microspore pro-embryos.
As embryogenesis proceeds the pollen wall, the exine breaks down and a high growth
of the inner multicellular structure together with changes in cellular organi:zation take
place. The subsequent proliferation process gives rise to rounded proliferative masses
clearly resembling globular-shaped embryos, which keep a small part of their volume
inside the pollinic sack and the rest is emerging out from the anther after the break of
the somatic cell layers surrounding it. Cellular morphology is different than that
observed in early stages. Cells are smaller than in early embryogenesis and show large
vacuoles occupying a high proportion of the cellular volume. Nuclei are elongated and
situated at the cell periphery. Different cellular types are forming these globular
microspore embryos (Fig. 8.1). Iodide-based staining, specific for starch demonstrates
that cells at the embryo periphery are rich in starch inclusions whereas almost no starch
is present in the inner cells (Risueii.o et al. 1999).

Responsive stages ofpollen development to embryogenesis.

Anther selection for in vitro induced microspore embryogenesis cannot be very accurate
in terms of developmental stages, since not much differences in size or morphology
appear in flowers containing microspores. The selected anthers are inside medium-sized
inflorescence buds of healthy appearance. Microscopical analysis revealed that the
anther population for culture mainly contains microspores at developmental stages
between tetrads and late vacuolate microspores, both stages being the most frequent
during microsporogenesis (Risueii.o eta/. 1999).

The subsequent studies of anthers in which embryogenesis has been induced revealed
that, together with the induced embryos, some vacuolate microspores were usually
present in the same pollinic sack. These vacuolate microspores could either be stopped
to develop further from the time of plating, or have developed in culture from younger
stages. The latter possibility is less feasible because if gametophytic development occur
in embryogenic cultures, many other different developmental stages would be found
after several weeks, which is not the case. Most probably, this fact seems to indicate that
one of the responsive developmental stages to embryogenesis in Quercus, as reported
for other species (Gonzalez-Melendi eta/. 1995, 1996b), is the vacuolate microspore.

5. Conclusions

Clonal propagation of cork-oak has been achieved through bud micropropagation as

well as through somatic embryogenesis from inunature zygotic embryos and leaves.

Haploid embryos have been induced in anther cultures by heat stress. Histological
studies have shown that embryogenesis has been induced in the vacuolate microspore.
The haploid genotype of the anther embryos has been corroborated by chromosome
counting and by flow cytometry.

The number of embryogenic microspores is high, producing many haploid genotypes,

as it has been confirmed by biochemical and DNA markers. In a few cases, spontaneous
genome duplication has been observed. The use of microsatellite mrukers concluded
 505

that these embryos are double haploids. The heterozygotic paternal tissue was discarded
as the possible origin for anther embryogenesis.

These results open new possibilities to the use of these propagation techniques for the
improvement of cork-oak, such as artificial seed production, the DNA duplication of
haploid embryos for the obtention of homozygotes, etc.

6. Aknowledgments.- This work was supported by project INIA SC-98-081,

Comunidad de Madrid 7B-0028-1998 and a Integrated Action HU-94-010 from the
Spanish-Austrian Mixed Commission for Scientific and Technical Collaboration. We
thank Mrs Dolores Salvador and Mrs Carmen Garda-Barriga for their technical
assistance.

7. References

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16. SOMATIC EMBRYOGENESIS IN Aspidospermapolyneuron Mull. Arg.

Luciana L. F. Ribas1, Miguel P. Guerra2 , Flavio Zanette3, Luiz Kulchetscki4

1-Plant Tissue Culture Laboratory, Department of Botany, UFPR, P.O. Box 19031,
Curitiba-PR, 81531-970, Brazil, e-mail: llfribas@bio.ufpr.br; 2-Plant Tissue Culture
Laboratory, Department of Plant Sciences, UFSC, Florianopolis-SC, Brazil, e-mail:
mpguerra@cca.ufsc.br; 3- Plant Tissue Culture Laboratory, UFPR, Curitiba-PR,
Brazil, e-mail: jlazan@agrarias.ufpr.br; 3- Faculty of Agronomy, University of Ponta
Grossa, Brazil, e-mail: luizkulc@uepg.br.

Contents
1 Introduction 3 Culture maintenance and repetitive
2 Culture initiation (secondary) embryogenesis
2.1 Explants 4 Somatic embryo maturation and
2.2 Culture media conversion
2.3 Effect of the growth regulators 5 Anatomical and cytochemical studies
Mature embryo response to 6 Conclusions and pt'ospects
combinations ofplant growth 7 References
regulators
Immature embryo response to
combinations of plant growth
re ulator:r

1. Introduction

Aspidosperma polyneuron, a representative of Apocynaceae family, is a species of

great economic importance for the forestry sector. It is under a serious risk of
extinction in Brazil and Venezuela (Figure 1A). Therefore, there is a great need of a
germplasm conservation program for this species. This species grows at the Semi-
decidous Stational Forest being listed as rare and endangered in the State of Parana,
Brazil, where it is a native tree (Hatschbach and Ziller, 1995).
A. polyneuron is also found at the Stational-Decidous Forest; less common at the
Mixed Ombrofilous Forest in southern Parana State; at the Dense Ombrofilous Forest,
in farther northwestern Mato Grosso State; and less frequent at the Pantanal (wetland)
region. Originally, in Brazil, this species was also distributed in southern Bahia State,
Goias, Espirito Santo, Minas Gerais, Rio de Janeiro and Sao Paulo, growing also in
Argentina, Colombia, Paraguay and Peru (Carvalho, 1994).
This species gives low yield and has preference for deep and fertile soil, avoiding
humid slopes and low regions (Inoue et al, 1984) being recommended for reposition of
the degraded riparian forest where there is no flood. According to Rizzini (1971) it has
an excellent wood quality, presenting high natural durability and mechanical
resistance. It does not decompose as long as it is not exposed to humid soil. In the past,
509
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 509-531.
© 2000 KhMer Academic Publishers.
510

its wood was very much used in the furniture industry, general carpentry, naval and
housing industry (Carvalho, 1994; Santos, 1987).
A. polyneuron poses serious problems in natural propagation due to seasonal seed
production, and large amount of seed production every four years that caused irregular
seed germination and difficult seed collection. Slow growth and hard to achieve root
cuttings are some of the main problems related to mass propagation of Brazilian native
trees (Carvalho, 1994). Thereby, seed propagation is the most preferred method of
propagation.
Seeds of superior genotypes are obtained from seed orchards. The implantation
and management of forestry seed orchards are time consuming and require a long-
cycle to obtain genetic improved seeds. The long-cycle of regeneration, the size of trees
and the seasonal seed production are major obstacles for most forestry improvement
programs (Grossnickles et al., 1996).
An alternative to increase forestry productivity is the establishment and
management of mono- and multi-clonal stands, employing vegetative propagation from
elite trees. Plant micropropagation offers advanced technical tools for mass
propagation of selected genotypes. The application of this technique depends on the
induction and in vitro control of morphogenesis in two preferential routes:
organogenesis and somatic embryogenesis. Somatic embryogenesis has potential to
mass propagate millions of forest tree plantlets, presenting higher multiplication rate
when compared with other in vitro techniques and conventional methods of
propagation (Handley, 1995). However, somatic embryogenesis is achieved less
frequently than other methods of regeneration, particularly in woody species (Chin-Yi
and Thorpe, 1987; Thorpe and Hasnain, 1988; Tautorus et at., 1991; Kulchetscki,
1993).
Somatic embryos can be obtained in liquid media, by using bioreactors to
generate a large number of elite genotypes on a regular basis or through the use of
encapsulation to obtain artificial or synthetic somatic seeds, which can be stored or
germinated later, as seeds (Ahuja, 1993; Merkle et al., 1995). In the forestry sector,
synthetic seed technology allows all year-round plant prooduction and ready access to
seed repositories through the use of somatic embryos, reducing significantly the risks
for some species which have asynchrony and limitations for natural seed production
(Gupta et al., 1991).
Somatic embryos can be used as a tool for germplasm storage and metabolite
production in vitro (Vicient and Martinez, 1998). Somatic embryogenesis still presents
limitations for some genotypes with difficulty in induction and development of somatic
embryos or in subsequent stages of maturation, conversion or the final goal to
obtaining viable germinated somatic plantlets (Vicient and Martinez, 1998).
 511

2. Culture initiation

2.1 EXPLANTS

Mature and immature fruits were collected from open pollinated trees at Sao
Paulo Forestry Institute headquarters from May until August at various stages of
maturation. Seeds from these fruits were treated with 1% sodium hypochlorite solution
containing 0.1% of Tween 20 for 20 min. These seeds were polyembryonic (2-6
embryos per seed).
A. polyneuron immature embryos induced direct (Figure 1C) and indirect
somatic embryogenesis, while mature embryos were able to induce the indirect pattern
(Figure 1B), which is in agreement with Sharp et at. (1980) and Williams and
Maheswaran (1986). Immature zygotic embryos are already embryogenic by nature,
just requiring the necessary conditions to induce somatic embryogenesis. These tissues,
apparently, require less media supplements to produce embryogenic response when
compared with other less juvenile explants. When the choice of explant is mature
embryos or seedlings, their respective epidermic tissues are still immature. In this case,
the origin of somatic embryogenesis induction has multicellular source coming from
meristematic cells located at immature epidermic tissqe. When mature or completely
differentiated tissues were employed as explants, somatic embryogenesis was just
possible via dedifferentiated cells through callus formation. Guerra and Handro (1998)
obtained identical patterns of somatic embryogenesis induction in Euterpe edulis.
Raemakers et at. (1995) made an observation that sometimes it is not possible to
predict or detect direct or indirect or both patterns.
In many systems where somatic embryogenesis was described as indirect,
callus formation or cellular embryogenic mass was present as somatic embryos at
juvenile phase (pro-embryogenic mass or pro-globular embryos) (Emons, 1994).
Merkle et at. (1990) supported that the most appropriated way to define indirect and
direct patterns of somatic embryogenesis is to relate unto the epigenetic state of explant
cells. Embryogenic somatic cells, generally, begin somatic embryogenic process more
easily than differentiated vegetative cells. Differentiated cells require epigenetic
changes that will promote the induction of an indirect route of embryogenesis. By this
way, the pattern of somatic embryogenesis is determined by the relation of the
epigenetic distance from the embryonary state that explant cells are to be found. Emons
(1994) and Yeung (1995) stated that one of most distinct features between direct and
indirect somatic embryogenesis depends on dedifferentiation time and the competence
acquisition. The former case involves cells that respond to experimental treatments and
become determined in a short time span, without callus proliferation. On the latter
case, a longer time is necessary for the acquisition of an embryogenic competent state,
and in many cases, this state is preceded by cellular proliferation. An average time for
the beginning of induction and expression events of somatic embryogenic process in A.
polyneuron was 4 to 12 weeks for immature embryos and 12 to 16 weeks for mature
embryos. During this time were visible and subcultured somatic embryos onto different
nutrient media once the globular state had been achieved.
512
A. potyneuron somatic embryo initiated mainly at the cotyledonary tissue located
at cotyledonary node and less frequent at the cotyledonary lamina, for the direct
(Figure lD) as well as the indirect pattern. Guerra and Handro (1998) studying
Euterpe edutis described that embryogenic initiation occurred on tissues located at
cotyledonary node. Emons (1994) and De Klerk et at. (1997) reported that only a very
limited number of competent cells respond to stimuli directed to embryogenesis, and
this is especially true during the first stages of regeneration. Competent cells reflect
embryogenic characteristics depending on culture environment, phytohormone
balance, osmotic conditions, amino acids and salt concentrations. Thorpe (1994)
observed that all cells don't respond to morphogenetic signals as a result of
regeneration blockade caused by genetic, epigenetic or physiologic sources.

2.2 CULTURE MEDIA

Following aseptic treatment, zygotic embryos at various stages of maturati<!m were

cultured in three different culture media: MS (Murashige and Skoog, 1962), WPM
(Lloyd and Me Cown, 1980) and LPm (Von Arnold and Eriksson, 1981). These culture
media were supplemented with 0.45% agar, 3% sucrose, 0.05% casein hydrolysate
(CH) and glutamine (Gln). The induction of somatic embryos occurred in these three
media, however, LPm gave higher response. Therefore that salt formulation was
selected as the best basal medium for further stages of somatic embryogenesis. Salt
formulation of LPm also gave good results for the induction and development of
somatic embryos in conifers, mainly with some Picea sps, as such: Picea gtauca and
Picea engetmannii (Roberts et at., 1990), Picea rubens (Harry and Thorpe, 1991),
Picea gtauca engetmannii (Carrier et at., 1997) and Araucaria angustifolia (Astarita
and Guerra, 1998).
WPM basal media supplemented with CH and Gln at 0.05% also improved
efficiency of producing somatic embryos in immature embryos. Immature embryos
cultured in MS basal medium supplemented with growth regulators also succeeded in
inducing somatic embryos, although less efficient when compared with other basal
media (LPm and WPM). High levels of nitrogen are also a requirement for induction
and differentiation of somatic embryos. Merkle et at. (1991) suggested that in order to
promote induction of somatic embryos, the basal media should be supplemented with 5
to 12,5 mM ammonium. For MS, LPm and WPM nutrient media, salt formulations of
ammonium nitrate are 20.6; 15.0 and 5.0 mM, respectively, and 0.05% organic
nitrogen was added as CH and Gln. It is probable that high levels of nitrogen added to
MS culture medium have inhibited the induction of somatic embryos of A. potyneuron.
The importance of nitrogen supply during somatic embryogenesis can be related to the
synthesis of nucleic acids and proteins, and storage of nutrient during somatic
embryogenesis process. An important reason for the supplementation of organic
nitrogen in the culture media is the fact that ammonium is the primary route to be
assimilated by this compound into organic compounds (Grey et at., 1987).
Addition of CH and Gin (0.05%) has stimulated formation and development of
somatic embryos of A. potyneuron. George (1996) noticed that addition of Gin (0.5-
 513

1.0mM) or CH (0.5-1.0 g. L-1) may increase the ratio of callus proliferation and further
development of somatic embryos because those substances when added to the culture
media will supply higher quantity of reduced organic nitrogen. Thorpe (1994) observed
that the addition of organic nitrogen is frequently employed during the induction of
embryogenic cultures and the subsequent development of somatic embryos. Similar
results were also obtained for Abies alba (Hristoforoglu et al., 1995) and Eucalyptus
citriodora (Muralidharan and Mascarenhas, 1995).

2.3 EFFECT OF THE GROWTH REGULATORS

Mature embryos were inoculated in MS, LPm or WPM basal media,

supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0; 0.45; 4.5; 11.3;
22.6j.JM), alone or in combination with kinetin (KIN) or 6-benzylaminopurine (BA) (0;
0.45; 2.25; 4.5 JlM). LPm culture media was also tested when supplemented with 2,4-
D (4.5 and 11.3 JlM) and thidiazuron (TDZ) (0.045 and 0.45 JlM). Immature embryos
at various stages of development, were plated after seed coat excision, onto LPm basal
medium supplemented with 2,5; 5 and 10 J.1M 2,4-D, alone or in combination with
0.5J.1M KIN, BA or TDZ, or testing also WPM and MS·-culture media plus 5 and lOJlM
2,4-D, combined with 0.5 J.1M BA, KIN or TDZ. Cultures were evaluated every four
weeks, by recording the observations: a) no response, b) embryo growth, c) callus
formation with several colors (white, yellow, brown), d) induction of embryogenic
cultures, and e) initiation of somatic embryos in several stages of development
(globular, heart-shape and torpedo). For each treatment tested, 30 embryos were plated
in test tube (25 x 150 mm). Explants were cultivated in the absence of light and plated
in primary culture media as long as the initiation of globular somatic embryos was
visible.

Matur{! embryo response to combinations of plant growth regulators

Relevant morphogenetic responses obtained through the use of mature embryos

are presented in table 1.
514
Table I. Morphogenic responses of zygotic mature embryos of A. polyneuron
cultured in different types and levels of growth regulators.

Growth Regulators (J,.LM) Morphogenic responses

0 and 0.45 2,4-D + 0.45 KIN, BA or TDZ - Germination of embryos.

4.5 and 11.3 2,4-D + 0.45 KIN, BA or TDZ
-Whitish, yellowish and brownish compact callus
formation;
- Embryogenic cellular mass;
-Globular, heart-shaped and torpedo somatic
embryos.

22.6 2,4-D + 0.45 KIN, BA or TDZ -Yellowish and brownish compact callus, presenting
low development;
- Increased rate production of phenolics.

The following salt formulations and growth regulator combinations induced an

indirect pattern of somatic embryogenesis A. polyneuron in mature embryo as explants:
LPm, supplemented with 4.5 ~ 2,4-D and 0.45 ~ TDZ or KIN and WPM, added to
11.3 ~ 2,4-D and 2.25 ~ BA or 2.25 ~ 2,4-D and 2.25 ~KIN.
Mature embryos plated for 12 to 18 weeks on semi-solid induction media
produced a yellowish to brownish callus in darkness. A whitish embryogenic culture
appeared at the surface of this callus, which were globular somatic embryos and some
of them were able to reach torpedo stage. Somatic embryo induction also occurred by
reducing major salt formulations, low levels of nitrogen, and growth regulator
concentrations (WPM, plus 2.25 ~ 2,4-D and 2.25 ~KIN).
Sharp et al. (1980) suggested that some effects of different sources of nitrogen at
somatic embryo induction could be better understood when they are acting in
synergism with other components of nutrient media, especially 2,4-D. Our results
demonstrated that the requirement of ammonium was directly correlated with 2,4-D
concentrations. In these cases, 2,4-D concentrations were 2.5 to 25 times higher as
compared to those that did not exhibit ammonium requirement. Apparently, the
ammonium requirement was associated with specific concentrations of 2,4-D in the
culture media. Preece (1995) reported that salt formulation optimization could reduce
the growth regulator requirement in the culture media.
Sharma and Kumar (1994), working with somatic embryogenesis in Thevetia
peruviana, species that belongs to the same family of A. polyneuron (Apocynaceae),
obtained similar results for growth regulator concentrations tested for A. polyneuron.
In other species, such as Quercus suber, 2.3 and 4.5 ~ 2,4-D was more efficient for
inducting somatic embryos (Manzanera et al., 1993), as well as with Tilia cordata
(Chalupa, 1990) and Ceratonia siliqua (Carimi et at., 1997). Rao and Sita (1996) also
presented similar results as obtained with A. polyneuron, that high levels of 2,4-D
(22.62 ~) inhibited the induction of somatic embryos of Dalbergia latifolia, and the
best growth regulator concentrations and combinations were 9 f.1M 2,4-D and 0.45 ~
KIN.
 515
Immature embryo response to combinations of plant growth regulators
Immature embryos inoculated on LPm culture media supplemented with 2,4-D
(2.5-10 J..IM), and when combined with KIN, BA or TDZ (0.5 J..IM) produced callus in
40 to 50% cultures. These callus formations appeared close to the cotyledonary node
and presented a proportional growth rate to the 2,4-D concentration. Growth regulator
combination of 5 J.1M 2,4-D and 0.5 J.1M KIN, BA or TDZ had also induced
embryogenic cellular mass formation.
Similar results were obtained when immature embryos were plated on WPM
culture media supplemented with the same growth regulator concentrations tested for
LPm culture medium. After a month of embryo cultivation on basal medium, cotyledon
expansion was noticed and continuing growth was visible at the hypocotyl-radicle axis.
This growth was proportional to the size of inoculated embryos, where about 20 to 30%
cultures also formed callus. The expression of embryogenic routes occurred also in
about 20% primary cultures inoculated on basal medium containing 2,4-D (5 aiJd 10
f.LM), combined with KIN, BA and TDZ (0.5 J.1M).
The process of globular somatic embryo induction and further development was
observed at cotyledonary node region or emerging at the surface of a whitish grainy
cellular mass and with less frequency happening onto cotyledonary tissue. After 12 to
14 weeks under culture induction an average of 3 to 6 somatic embryos were counted
and less frequently some somatic embryos at heart-shape stage were also visualized.
Immature embryos when inoculated onto MS culture medium were also able to induce
somatic embryos through direct route, although less frequently when compared to other
tested salt formulation (LPm and WPM).
In the first experimental evaluation done four weeks after explant culture,
growth and cotyledonary expansion occurred at the hypocotyl-radicle axis. In
approximately 20 to 30% of culture explant, callus formed from the cotyledonary node
and at the border of the hypocotyl-radicle axis. Embryos plated onto MS culture
medium, supplemented with 10 f.LM 2,4-D and 0.5 J.1M KIN or 5 J.1M 2,4D and 0.5 f.LM
TDZ were able to produce globular somatic embryos directly on cotyledonary node
region.
Further development onto advanced ontogenetic stages was prevented once those
somatic embryos stayed at the same induction medium. Some friable embryogenic
cellular mass or even compact callus was detected at the surface of some somatic
embryos. Carman (1990) and Normanly (1997) suggested that the time period of
exposition under 2,4-D determines the embryogenic competence. If time exposition is
too extended, culture cells will lose competence, being called non embryogenic cells.
Embryogenic competence is temporarily expressed and influenced also by
phytohormone regulation. Normanly (1997) noted that there is a trend to accept the
concept of endogenous indole-3-acetic acid (IAA) metabolism, which also influences
the setting of embryogenic competence. For the cells to acquire competence, high
endogenous IAA concentrations would be necessary and further ontogenetic
development would be implemented by decreasing IAA concentration.
One of the main objectives of this study was to observe induction time of somatic
embryos under culture media, although additional studies involving 2,4-D pulse
516

treatment would be necessary to get more information over acquisition of embryogenic

competence. Vasil (1982) hinted that embryogenic competence is apparently achieved
during initial stages in culture under high concentration of auxins. However, somatic
embryos are only formed if levels of 2,4-D are below optimal concentrations. Those
optimal concentrations are obtained through sequential subcultivation onto culture
media supplemented with low levels of 2,4-D or under long cultivation period in
primary culture medium. Continued exposition to 2,4-D seems to be antagonistic to
morphogenic organized development and these observations can be considered as of
general application to somatic embryogenesis.
Expression of somatic embryogenesis in A. polyneuron were achieved after
cultivating the explants for an extended induction period (12 weeks) onto culture
media supplemented with 2,4-D. Similar results were obtained for Eucalyptus
citriodora and Daucus carota (Gorst et al., 1987) where proembryos appeared after
transferring explants onto nutrient media with low levels or devoid of auxin content. In
some systems, auxin effect were present even after subcultivating the explant to
nutrient media without auxin (Parrot et al., 1991).
Low frequency somatic embryogenesis under primary culture were also described
for other species such as: Juglans regia (Tulecke and Me Granahan, 1985; Quercus sp.
(Gingas and Lineberger, 1989); Aesculus hippocastanum (Kiss et al., 1992) and
Castanea sativa x Castanea crenata (Vieitez, 1995), suggesting that primary somatic
embryos were inferior to secondary somatic embryogenesis. In Medicago sativa only 5
from 300 explanted zygotic embryos (1,7%) were able to produce embryos at priniary
embryogenesis, while every somatic embryo rescued was able to generate secondary
somatic embryos (Parrot and Bailey, 1993), as for Eucalyptus citriodora protocol
(Muralidharan and Mascarenhas, 1995) only 3,3% of explants produced somatic
embryos and 16% for Ceratonia siliqua (Carimi et al., 1997).
Induction of embryogenic competence in A. polyneuron was dependent on a
connection between explant physiological conditions, requiring culture setting and
growth regulator levels added to basal media. Induction and development of somatic
embryogenesis were successfully completed by using zygotic embryos cultivated in
culture media supplemented with 2,4-D (5 or 10 J.LM), combined with 0.5 J.1M KIN, BA
orTDZ.
Explants inoculated onto basal media supplemented with 2,4-D, in the presence
or absence of high levels of cytokinins (2.5 and 5.0 J.LM) were unable to promote
somatic embryogenesis. Contrary to these, George (1996) reported that addition of a
low concentration of cytokinins (0.1-1.0 J.1M BA or KIN) in nutrient media
supplemented with auxins has implemented the growth ratio of embryogenic callus for
many dicot species.
Embryos of A. polyneuron when inoculated onto culture media supplemented
with 5 J.1M 2,4-D and 0.5 J.1M TDZ induced somatic embryos formation and
embryogenic cellular masses, independently of the tested salt formulation and on the
explant (embryos) physiological conditions. There have been few reports related to the
use of TDZ to induce somatic embryogenesis, concentrations used were between 0.5-
10 IJ.M applied to cotyledonary explants, as in Fraxinus americana (Bates et at., 1992),
 517

Vitis vinifera (Matsuta and Hirabayashi, 1989), Arachis hypogaea (Murthy et al.,
1995). Merkle et al. (1987) and Neuman et al. (1993) reported successful results for
somatic embryo forniation of Juglans nigra employing WPM culture medium plus a
balanced concentration of 2,4-D and TDZ. Neuman et al. (1993) found that when a
combination of 4.5 ~ 2,4-D and 5.0 ~ TDZ was employed. somatic embryos were
induced in mature tissues of Juglans nigra.
A key factor to express morphogenesis in vitro is that competent cells are able to
respond to growth regulators. Some of the internal factors that control cell competence
are the endogenous levels of explant growth regulators which would act at the starting
of the in vitro cultivation together with the capacity of the cells of synthesizing growth
regulators or essential metabolites (George, 1993; Thorpe et al., 1991; Thorpe, 1988).
Since mass propagation through somatic embryogenesis is achieved involving
empirical manipulations of many factors especially phytohormone concentrations, the
knowledge about how growth regulators control plant morphogenesis is quite
superficial (Libbenga and Mennes, 1995). It is of general agreement that
phytohormooes are transported to tissues where responsive cells once activated play a
critical role over control of gene activity at transcription and translation levels of many
physiological processes. Those responsive cells are characterized ~y the presence of
protein-like receptors which recognize phytohormone signal and translate the
information within specific biochemical pathway (Potrykus, 1991). Those receptors are
localized in the cell membranes, cytoplasm and nucleus, and are, in many cases,
protein-like receptors (Van der Linde, 1990). Guerra et al. (1999) pointed out that
modus operandi of direct somatic embryogenesis would be the activation of receptors
located in the responsive cells containing regulators to specific growth regulator. Those
dedifferentiated cells would follow certain morphogenic routes generating embryonary
competent mother cells, which would, in turn, establish clonal population of
embryogenic cells. Indirect patterns of somatic embryogenesis would be a sort of callus
formation as a by-product of dedifferentiated cells containing cell-receptors.
Responsive cells from that callus would, through an induction process, acquire
embryogenic competence, then generating a clonal population of embryonic cells.
LoSchiavo et al. (1989) proposed a hypothesis to explain the mechanism of acting
auxins influencing cells and promoting somatic embryo formation. This hypothesis
suggests that auxins promote DNA hypermethylation on cell division, which is a
proembryo formation prerequisite. Contrasting to this requirement, embryo
development would need DNA hypomethylation, which process occurs in proembryos
when nutrient medium is depleted of auxin. The most important role of synthetic
auxins is to block developmental routes, possibly through DNA hypermethylation,
intensifying embryogenic cell formation from competent explant (Carman, 1990).
Hypermethylation process is not restricted only to 2,4-D since other auxins, as a-
naphthaleneacetic acid (NAA) or IAA were employed and have promoted high levels
of methylation (Lo Schiavo et al., 1989). Lambe et al. (1997) provided details about
methylation process, informing that up to 30% of cytosine residue are methylated into
5-methyl-cytosine. Auxin increases DNA methylation reversibly, while KIN tends to
block alterations in DNA methylation (De Klerk et al., 1997).
518

3. Culture maintenance and repetitive embryogenesis

The optimum conditions for each factor presenting some influence on the
formation of somatic embryos was determined experimentally and tested under
different conditions. This methodology was adopted for each critical stage in the
development of repetitive somatic embryogenesis. Embryogenic cellular mass was
obtained by using specific nutrient medium and source of explants as mentioned
previously to be the most responsive. The most responsive explants were mature and
immature embryos; which were plated onto LPm culture medium supplemented with
4.5 ~M 2,4-D and 0.45 ~ TDZ or KIN. Embryogenic cultures were recurrently
subcultivated and maintained after repetitive cycles. These embryogenic cultures
showed friable composition, of whitish tone and granular texture (Figure 1D).
Embryogenic cultures were cultivated in test tubes with LPm semi-solid basal
medium (0.45% agar), supplemented with sucrose 3%, 0.05% of Gln and CH plus
different combinations of growth regulator concentrations, like 2,4-D (0.5; 1.25 and
2.5 ~) plus 0.5 JJM KIN or NAA (0.5; 1.5 and 3.0 ~) plus 0.5 ~ KIN.
Embryogenic cultures were also cultivated in nutrient media devoid of growth
regulators and/or with 0.1% 'activated charcoal, and maintained in darkness, at
25±2°C.
From tested growth regulators in different combinations, 0.5 ~ 2,4-D or NAA,
in combination with 0.5 ~ KIN was considered the most efficient for growth yield
and culture maintenance, allowing the establishment of repetitive cycles of cellular
division and continuous subcultivation (15) up to three years. Globular somatic
embryos were observed arising from the surface of those cellular masses. Continuous
induction of proembryos and globular somatic embryo formation were achieved by
maintaining those cultures at basal media plus 2,4-D. Studies in other species like
Eucalyptus citriodora (Muralidbaran et al., 1989), Juglans nigra (Deng and Cornu,
1992), Camellia japonica (Barciela and Vieitez, 1993) and Camellia sinensis (Kato,
1996) indicated that recurrent or secondary somatic embryogenesis was effective to
maintain embryogenic capacity for over two years.
Parrot et al. (1991) suggested that after initiation of embryogenic cells,
uninterrupted presence of auxin could be harmful to normal development of somatic
embryos. When auxin concentration is sufficiently high, the somatic embryos don't
develop any further, instead produce a new cycle of somatic embi'yos. Raemakers et al.
(1995) suggested that this change in maturation process due to high concentrations of
auxin is also associated with loss of control of a cluster of organized somatic
embryogenic cells. Then some cells are excluded from this cluster and they start a new
cycle of somatic embryos (Williams and Mabeswaran, 1986).
Continuous cellular proliferation occurred when embryogenic cellular clusters
were transferred onto LPm nutrient medium containing 0.1% activated charcoal,
without growth regulators. Addition of activated charcoal in the culture medium could
benefit the embryogenic cells due to removal of auxin residue (Bucheim et al., 1989).
Self growth of embryogenic cells lineage could be indicative that cytokinin or auxin
concentration levels are adequate, or that cell sensitivity alterations to the growth
 519

regulators have occurred (Delbreil et al., 1994), and that event which occurs in vitro
culture is called habituation (Krikorian, 1995). Cell tissues that would require an
exogenous addition of auxin or cytokinin could, suddenly or gradually, lose that
exigency and become habituated. In many cases, but not as a rule, the process of
habituation could be reverted and then habituated cells would sustain their totipotency,
being able to produce roots, buds or somatic embryos. On the other hand, habituation
could be irreversible, inducing change at genetic level such as loss of totipotency, and
in that case, habituation would be responsible for mutation (Lambe et al., 1997).
Establishment and maintenance of embryogenic cultures for long periods of time
(two to three years) cultivated at basal media devoid of growth regulators has been
described for many species such as Juglans regia (Tulecke and McGranahan, 1985),
Daucus carota (Smith and Krikorian, 1990), Asparagus ofjicinallis (Delbreil et al.,
1994) and Carya illinoensis (Burns and Wetzstein, 1997). The most important aspect
for control of maintenance of cell lineage at repetitive cycles is related to a
considerable reduction of growth regulator levels.
Carman (1990) has indicated that reduced levels of 2,4-D have increased morpho-
physiologycal development of somatic embryos. In many somatic embryogenesis
protocols described so far for conifers, optimal conditions for growth regulator
concentrations were achieved between 2 to 5 J.LM for both auxins and cytokinins (Gupta
et al., 1993), although higher concentration of auxin, usually 2,4-D, has been
employed in most attempts to generate embryogenic tissue.
As for A. polyneuron very low concentration of growth regulators 0.5 J.LM 2,4-D or
NAA and 0.5 J.lM KIN has demonstrated to be effective for proliferation and
maintenance of embryogenic cultures. It is important to note that these tested growth
regulator concentrations for mature embryos of A. polyneuron at somatic
embryogenesis induction stage have stimulated mature embryo germination. However,
due to the fact that embryogenic cultures were maintained for a long period (20 to 24
weeks) at basal media supplemented with 2,4-D acting synergistically with other
growth regulators possessing cytokinin activity, it is probable that a residual effect of
those growth regulators has been a major fact in maintaining that cell lineage. For
basal media devoid of growth regulators, it was observed globular somatic embryos
formation at the surface of friable embryogenic mass cells, which embryos, by their
turn, have formed secondary globular somatic embryos at their surface, establishing
then repetitive cycles of secondary embryos formed on primary embryos. Merkle et al.
(1995) called this process of repetitive cycles as autoembryony, when it occured in the
absence of growth regulators.
However, normal development of globular somatic embryos of A. polyneuron
cultivated under basal media free of growth regulators has occurred slowly towards
more advanced ontogenetic stages but it happened at an even lower speed when
compared to those cultivated under basal media supplemented with (~- (2-
isopentenyl) adenine (2-iP) and NAA. Similar results were obtained by Linossier et al.
(1997), who have observed that basal media without growth regulators would provoke
a reduction on somatic embryo formation of Hevea brasiliensis at torpedo stage, and
this fact would be attributed to a lack of food reserve, like carbohydrates and lipid
520

content, which, nevertheless, would not cease autoembryony. Litz et al. (1995) have
noted that subcultivation onto semi-solid media at the absence of 2,4-D, is essential for
production of heart-shaped somatic embryos of Mangifera indica, although synchrony
was not observed and somatic embryos could be found at different developmental
stages.
This study has determined that proliferation of embryogenic cultures has occurred
at basal media free of growth regulators (Figure lB), with or without activated
charcoal, and with reduced combinations of synthetic auxins (0.5 J1M 2,4-D or NAA)
and KIN (0.5 J.LM), combined with 2-iP and NAA (Figure lE), abscisic acid (ABA)
(Figure 1D) and with polyethylene glycol (PEG), being subcultivated every eight
weeks. That has led to the conclusion that embryogenic cultures have maintained a
certain adequate level of endogenous growth regulators, which allowed cellular
proliferation and maintenance.
According to George (1993), once induced cells in vitro become determined to
follow a specific route of development, they retain that capacity for many cellular
divisions. This fact can be explained due to the capacity of embryos to regenerate
somatic embryos or cellular embryogenic masses. As for forest tree species, which have
a long life cycle, as is the case of A. polyneuron, somatic embryogenesis can be a useful
tool since embryogenic cellular lineage can be maintained and multiplied during very
long period until field test is conclusive.

4. Somatic embryo maturation and conversion

Proembryos and somatic embryos at various stages were transferred and

subcultivated onto maturation culture media. The LPm solid nutrient medium
containing 0.65% agar was supplemented with sucrose 3%, Gln (0.05%), CH (0.05%)
and growth regulators combinations at various levels like 2-iP (12.3; 24.6 J.LM) and
NAA (0; 0.25; 0.5 J.LM) or even levels of 2-iP plus indole-3-butyric acid (IBA) (0; 0.25;
0.5 J.LM); BA (1.0; 2.22; 4.44 J.1M) and NAA (0; 0.5 J.LM); KIN (11.6; 23.23 J.LM) and
NAA (0.5 J.LM). LPm basal medium was. tested without growth regulators
supplemented or not with activated charcoal (0.1% ). Cultures were maintained at
photon flux rate of 25 J.1 E s· 1 m·2, in a 16-h photoperiod at temperatures of 25±2°C.
Growth regulator concentrations at levels of 12.3 and 24.6 J.1M 2-iP in
combination with 0.5 J1M NAA have presented the best results so far, resulting in the
development of friable cellular masses showing somatic embryos in several
developmental stages (Figure lE). Somatic embryos at torpedo stage were selected and
transferred onto conversion basal media (Figure 1F). Gray et al. (1995) have indicated
that somatic embryos could be formed from cell proembryonic complexes that have a
tendency to develop asynchronously, with somatic embryos being cultured at various
developmental stages.
These somatic embryos asynchronously produced are subject to nutrient
depletion when basal medium is worn out; being restored to optimal nutrient level
conditions after subcultivating them onto new nutrient media. Under such variable and
unbalanced conditions, somatic embryos interrupt maturation and become
 521

unorganized, forming new cycle of embryogenic cells, which will contribute further to
asynchrony. Consequently, somatic embryos that reach maturity are able to develop
embryogenic tissue at different proportion, provoking, thus, precocious germination in
which only apical or root primordia tissues are formed, then preventing the occurrence
of further normal germination. Those situations were observed in somatic embryos of
A. polyneuron. Somatic embryos can also present anomalies like additional cotyledons
and underdeveloped apical meristems. These abnormal features happen due to culture
conditions and are not related to intrinsic factors of somatic embryos.
Combinations of two growth regulators (2-iP and NAA) were successful in
promoting friable embryogenic cellular mass formation during many subcultivations
and that feature was also observed when cultures were transferred onto basal media
supplemented with 0.1% activated charcoal but devoid of growth regulators. Similar
results to that were obtained when clusters of globular somatic embryos of Euterpe
edulis were able to achieve subsequent ontogenetic stages and repetitive
embryogenesis, when transferred to semi-solid culture supplemented with 12.3 f.1M 2iP
and 0.5 f.1M NAA.
Embryogenic cultures of Euterpe edulis cultured onto culture media supplied
with 24.6 f.1M 2-iP and 0.5 f.1M NAA allowed the growth of somatic embryos,
maintaining, at the same time, the embryogenic competence to original culture.
Besides, elongated and bipolar somatic embryos frequently originated new globular
somatic embryos in a continuous and repetitive pattern (Guerra and Handro, 1998).
Cytokinins were also important for the development of the somatic embryo
Thevetia peruviana (Apocynaceae). Embryogenic cells achievement frequency was
superior to 80% when cellular aggregates were inoculated into semi-solid culture
medium supplemented with 9.84 f.1M 2-iP and 0.45 f.1M 2,4-D (Sharma and Kumar,
1994). Merkle et al. (1995), have suggested that inclusion of cytokinins during
histodifferentiation stage could compensate for the deleterious effect at meristem
development promoted due to auxins present to the media. George (1996) has
described that when BA and KIN concentrations were raised to concentrations up to 5
and 10 f.1M respectively, they were able to stop the auxin effect, decreasing the growth
ratio of callus and initiating formation of somatic embryos.
After sequential subcultures of the cellular masses (around eight to nine) onto
nutrient media plus 2-iP and NAA, it was observed a reduction on expression and
development of somatic embryos of A. polyneuron but it did not interfere with the
proliferation rate of embryogenic cellular mass. This feature could be attributed mainly
to the presence and/or residual effect of 2,4-D in the culture media, making
ontogenetic development process of somatic embryos difficult, as reported by Attree
and Fowke (1993) and Zimmerman (1993). Lambe et al. (1997) implied that
habituated or hard to differentiate callus formation could occur due to the presence of
DNA hypermethylation that happens during in vitro formation process. Progressive
methylation of genes involved with cellular differentiation process at division and
multiplication stage and continuos elimination of cells capable of differentiation are
possible causes of progressive loss of totipotency in callus culture.
522

Induction of maturation for embryogenic cultures was also tested employing

solid (0.65% agar) LPm culture medium plus polyethylene glycol 8000 at 5 and 10mM
and abscisic acid at concentrations of 30, 60 or 120 f.!M. ABA added to the autoclaved
nutrient medium following filter sterilization procedures. Cultures were maintained at
dark conditions. LPm basal media complemented with high concentrations of ABA
(30-120 f.!M) was efficient in promoting maturation for embryogenic cellular lineage of
A. polyneuron where a high frequency of up 90 globular somatic embryos and
proembryos were detected by culture (Figure 1D). That concentration also allowed the
proliferation of embryogenic cultures when compared to culture media where
polyethylene glycol was present. Basal medium supplemented with 10 mM PEG also
induced formation of globular and heart-shaped somatic embryo at the surface of the
embryogenic cellular mass, however, it was less effective when compared with basal
media plus ABA.
Usually, high osmolarity requirement for somatic embryos is associated with
features observed in vivo, as for instance the osmotic potential verified in embryo sac
content (Raghavan, 1976). In the present work, ABA and PEG effect for maturation of
embryogenic cellular lineage was tested after many subcultivation procedures (12-14),
which results were not conclusive, being necessary additional tests to better understand
the question of lack of synchrony in somatic embryos at the developmental stage.
According to Zheng et at. (1996), the earlier the ABA addition to nutrient media, the
greater the intensification of the normal development of somatic embryos induced in
vitro, as well as their conversion into plantlets. Similar results were described by
Linossier et at. (1997) when studying the effect of concentration of PEG (140 g. L- 1)
and ABA (10- 5 M) on development of somatic embryos of Hevea brasiliensis. The
presence of osmotic substances has reduced secondary embryogenesis and improved
the conversion process of pro-embryogenic cellular mass into torpedo somatic embryos.
ABA applied alone at basal media has implemented globular somatic embryo
formation. Astarita and Guerra (1998) have also made similar observations of
increased number at proembryos formation in Araucaria angustifolia, when basal
media was supplemented with PEG 8000 .(1%) and ABA (7 and 19 f.!M).
Concentration of PEG 4000 at 5 and 10% associated with ABA triplicate frequency of
conifer somatic embryos at maturation stage, besides increasing food reserve and
survival capacity after desiccation treatment of somatic embryos (Attree and Fowke,
1993).
No further progression of somatic embryos of A. polyneuron at cotyledonary
stage cultivated at basal media with or without growth regulators was detected. As time
elapsed, it was not observed emergence of radicle, and these somatic embryos started to
become necrotic or to form new somatic embryos at the surface of hypocotyl and this
feature was confirmed through histological studies. Possible causes for somatic
embryos not being able to convert into plantlet could be the residual effect of 2,4-D,
due to its long time in culture at induction treatment or the somatic embryos became
necrotic once they were not able to accomplish morphophysiologic maturation
conditions to achieve plant status.
 523

Figure 2 shows a diagram of induction and development routes for

embryogenic cultures of A. polyneuron. A major challenge found at this point was
referred to a lack of an improved methodology to better evaluate somatic embryo
asynchrony at torpedo stage. Previous studies have suggested that additional· tests with
basal media supplemented with ABA and/or PEG could be employed for the first
stages of cellular mass to evaluate their effect for cellular lineage and somatic embryo
maturation. Nickle and Yeung (1993) have stated that ABA application affected
conversion rate of somatic embryos of Daucus carota. Somatic embryos at globular and
torpedo stage have demonstrated to be more efficient to maintain a cytoplasmic reserve
at the apical dome when compared to somatic embryos at late torpedo stage, in which
ABA has presented an inhibitory growth effect.
A general expectancy of ABA application is to reduce somatic embryo
abnormalities, which could be associated with apical meristem development. ABA
could also act as an inductor agent for meristem organized development or it could
interfere in a way that cells located at apical meristem can be canalized into meristem
cells. In Mangifera indica somatic embryos in the maturation phase showed necrotic
areas starting from cotyledons and hypocotyls and extending all over the somatic
embryos (Litz, 1985). This author asserted that this necrotic tissue could be avoided by
the addition of reductor agents or by altering the nutrient media composition. It was
observed, with frequency, secondary somatic embryos appearing at the hypocotyl
region and that feature was controlled by addition of low concentrations of cytokinin in
the culture media. During germination and conversion phases of Theobroma cacao,
many cells from embryo tissue presented degeneration and became oxidized causing
the death of somatic embryos so that no plantlet was obtained. For Alemanno et al.
(1997) this peculiarity for lack of conversion was attributed to deficiency of reserve
substances when compared to zygotic embryos. Here again the question of residual
effect of 2,4-D was considered as preventing maturation of somatic embryos, as cited
by Parrot et al., (1991) which residues could be removed from the tissues by addition of
activated charcoal to the nutrient media (Bucheim et al., 1989). Wetztein and Baker
(1993) have attributed the low conversion rate to the lack: of development of
meristematic areas.
Somatic embryos have plastic structures during their developmental stages, so
that a variety of embryonic shapes can be seen, and some of those types can interfere
with their capacity to turn into complete plant. Besides this characteristic, somatic
embryos can present normal morphological shape and be abnormal concerning cell and
tissue differentiation. It is of general agreement that alluded plasticity is a result of
changes in time and length of organizational events happening at culture environment
(Thorpe, 1988). In order to occur conversion it is important to get mature and totally
developed embryos. Somatic embryos should present normal external morphology and
internal characteristics similar to zygotic embryos. Those characteristics include
storage nutrients such as proteins, lipids and carbohydrates (Gupta et al., 1991).
Somatic embryo maturation and whitish appearance were essential conditions
for the germination of somatic embryos of Juglans regia (Deng and Cornu, 1992) and
Hevea brasiliensis (Cailloux et al., 1996). The translucent aspect of somatic embryos,
524

as observed with certain frequency in somatic embryos of A. potyneuron, could be a

symptom of absence of protein or starch, as described by Tulecke and Me Granahan,
(1985). Somatic embryos of Prunus avium were translucent and presented low
conversion rate into plantlets (Garin et at., 1997). Storage product accumulation is a
key factor to zygotic embryogenesis, so that the embryo can reach out to this reserve
during and after germination until the seedling achieves its autotrophic potential.
Feirer et at. (1989) have described that lack of food reserve, such as triglycerides,
affects the final stages of development and conversion of somatic embryos into
plantlets. These storage compounds can be employed as biochemical markers to
compare quality and somatic embryos fidelity, since storage accumulation is an
important phase for development of somatic embryo (Cailloux et at., 1996).
Although many somatic embryos turned to be normal plants, there is, still, a
large amount of variations in shapes, sizes, number of cotyledons, synchrony,
maturation rat~ and germination (Vasil, 1994). The major limitations for mass
propagation and field planting of somatic embryogenesis protocols for perennials are
related to low frequency and conversion rate, facts detected in this study too. For a
considerable number of species described so far, the conversion stage still represented
the major challenge to be surpassed in order to determine optimum protocols where
complete plants can be obtained, as for instance Abies nordmanniana (Norgaard and
Krogstrup, 1991), Castanea sativa x C. crenata (Vieitez, 1995), Quercus robur
(Chalupa, 1995) and Fraxinus americana (Preece and Bates, 1995). About 12% of
somatic embryos of Ceratonia siliqua were able to germinate and turn into complete
plant (Carimi et at., 1997) and for Simarouba gtauca only 20-25% of somatic embryos
has reached germination stage (Rout and Das, 1994). Similar results were obtained for
Vitis vinifera (Faure et at. 1998), whose somatic embryos were able to achieve torpedo
stage, although germination was precocious and frequency of viable plantlets was very
low. Wetzstein and Baker (1993) presented that low conversion rates for Arachis
hypogea was probably due to uncompleted development of meristematic areas. Those
different features associated with conversion of somatic embryos still pose the major
challenge for many species, including A. polyneuron, where additional investigation is
needed in order to understand some of the controlling agents for conversion.

5. Anatomical and cytochemical studies

Histological studies were performed to determine the target areas responding

to growth regulators, and to identify critical stages during the somatic embryogenic
process, through sequential examination of the histological events in a temporal frame.
Embryogenic cultures were selected and prepared for histological sections in order to
identify critical stages at various ontogenetic phases from friable cellular mass onto
somatic embryos. This selected culture was being cultivated under maturation medium
and presented asynchrony features of somatic embryos distributed all over its surface,
like globular, heart-shape and torpedo.
The samples were gently prepared and fixed using 2% glutaraldehyde and 4%
paraformaldehyde buffered with 0.1 M phosphate buffer, at pH 7.2 during 24 hours
 525

(Karnovsky, 1965). After absolute alcohol dehydration the specimens were transferred
into 100% tert-butyl step of the alcohol (TBA) series (10%-70% during 10 minutes
each following 70%, 96% and 100% during two hours in each concentration under
vacuum infiltration and room temperature). Parainfiltration was performed with 100%
ethanol and liquid resin (1:1) during 24 hours under room temperature. Liquid
embedding was performed with historesin glycol-methacrylate according to the kit
recommendations (kit JBA Polysciences/USA). Seriated sectioning 3-4 JlM thick was
obtained employing a circular autocut microtome. Slide preparations were stained for
30 min. According to recommendations proposed by Sakai (1973) (7.52 g Na2HP04,
9.6-g citric acid, 0.5-g toluidine blue, and 1000 ml distilled water). The cleaned slides
were then dried in a dust-free area and mounted with permouth. The specimen was
examined and photographed using a Zeiss MC80 optical microscope.
Figures 3A-3C present chronological events beginning with proembryos up to
developmental stages of somatic globular embryos. The presence of meristematic tissue
was also characterized by small (20-30J.1M), ,isodiametric shape, dense cytoplasm and
displaying a prominent and centrally located nucleus in most of which one, two or even
more dark stained nucleoli could be seen, and under active cellular division. It was also
observed that in the process of globular somatic embryo formation, various planes of
cellular division appeared, suggesting that typically embryogenic cells were involved in
this process.
Figure 3A shows some organized structures going from proembryos formation
up to globular somatic embryos framed from small cells (20-30Jlm), presenting,
isodiametric shape, dense cytoplasm, small vacuoles, displaying a prominent and
centrally located nucleus and dark stained nucleoli, under active cellular division.
Figure 3B shows a globular somatic embryo formation presenting many planes of
cellular division. This characteristic confirms its typically embryogenic cell origin,
agreeing with some features characterized for other species like Elaeis guineensis
(Schwendiman et al., 1988), Quercus suber (El Maataoui et al., 1990) and Zea mays
(Fransz and Schel, 1991).
DeJong et al. (1993), have indicated that periclinal and oblique planes of cell
division appear in the place of original anticlinal formation located at epidermis
providing then a precursor signal for the establishment of embryogenic cell induction
process. The effect of cytokinins added to culture media is not only to stimulate cell
division but also to promote changes in planes of cell division. Figure 3B displays a
typical somatic embryo, encircled by protoderm. West and Harada (1993) suggested
that the first indication towards protoderm formation was that cellular divisions
happened perpendicularly to the surface of somatic embryo.
526

Figure 1. A- Tree of Aspidosperma po(vneuron; B- fndirect somatic embryogenesis showing asynchrony of

somatic embryos (LPm, without growth regulators) (bar=3.l5 rnm); C- Globular somatic t!mbryos developed on
the cotyledonary tissue (bar=3.15 rnm); D- Globular somatic embryos formation at the surface of friable
embryogenic culture (LPm + 30 ~ABA) (bar=2.13 rnm); E- Maturation of somatic embryos (LPm + 12.3f,lM
2-iP + 0.5 f.lM NAA) (bar=I.47 rnm); F- Torpedo somatic embryo (LPm, without growth regulators) (bar=
2.29rnm).
 527

stabilization Multiplication Maintenance

Indirect somatic
Induction
embryogenesis

mature
embryo

LPm + LPm + LPm, without

2.25 .UM 2,4-D + 2·4-D or NAA (0. 5 1lM)+ growth regulators
LPm + 0.5 .UM KIN O.S !lM KIN
4.5 .UM 2,4-D +
0.5 .UM TDZ or KIN

Maturation Conversion
Direct somatic
embryogenesis Repetitive
embryogenesis

immature
embryo

LPm+
12.30 !lM 2-iP +
LPm, MS, WPM + 0.5 llM NAA or 30 !lM ABA
5.0-10.0 !lM 2.4-D + LPm, without
0.5/lM TDZ, KIN, BA growth regulators

Figure 2. Induction and development routes of embryogenic cultures of A. polyneuron.

528
In the present study it was not observed the presence of suspensor-like cells
located at basal region of somatic embryos. Embryogenic cells of A. polyneuron were
arranged as a compact cluster, presenting just small intercellulary spaces in between
cells. Others species such as Musa spp. (Lee et al., 1997) and Baraga officina/is
(Quinn et al., 1989) have presented similar features, like the absence of suspensor cells
or any other structures at longitudinal section of globular somatic embryos. Williams
and Maheswaran (1986) reported that when a suspensor-like structure is present to
somatic embryos, it is quite probable that their origin is unicellular. Based on that
indication one could assume that A. polyneuron somatic embryos would come from
multicellular source.
Dodeman et al. (1997) analyzing comparative histological studies done
between somatic and zygotic embryos detected two main differences between both
systems: lack of suspensor tissue and endosperm differentiation during the process of
somatic embryogenesis. El Maataoui et al. (1990) have noted that those characteristics
were more visible at initial stages of globular somatic embryos formation, features also
detected in this present work with A. polyneuron.
Figures 3C-3D depict somatic embryo development shown chronologically
through heart-shape, torpedo and cotyledonar stage. As suggested by Zimmerman
(1993) and Yeung (1995), globular stage has its start from small clusters of cells. An
oblong shape follows that stage, where cells suffered some modifications from
isodiametric to bilateral simetry, indicative of the ftrst steps of heart-shape stage.
Transition from globular to heart-shape stage is visualized through cotyledon
expansion or elongation, which appears as small protruding domes located at the
somatic embryo periphery, concurrently with hypocotyl elongation and radicle
formation. This frame continues to develop until it reaches the torpedo and plantlet
stage or somatic seedling formation. This plantlet formation stage can be identifted
through the presence of green cotyledons, elongated hypocotyl and already developed
radicle. As somatic embryos continue to develop towards torpedo and cotyledonary
stages, cells tend to become more vacuolized (Figure 3D). These results agree with
those described by Alemanno et al. (1996) who supported that during subsequent
somatic embryo ontogeny, cells did not present embryogenic characteristics. Canhoto
and Cruz (1996) noted the scarcity of data concerning somatic embryo ontogeny, ftrst
events leading to differentiation of embryogenic cells and the determination of which
tissues are responsible for somatic embryo induction.
This approach on induction of somatic embryogenesis from A. polyneuron
was characterized by a notable asynchrony, where proembryos and somatic embryos
organized into various ontogenetic stages at the same embryogenic cellular mass could
be seen. Similar observations were also noted for Quercus suber (El Maataoui et al.,
1990). Figure 3D shows slide preparation of a longitudinal sectioning of A. polyneuron
somatic embryo at cotyledonary stage where cotyledons can be visualized near the
shoot apical meristem (epicotyl) altogether with differentiation of tracheary elements of
protoxylem and the hypocotyl-root axis.
Somatic embryogenic cells at torpedo stage are characterized by being more
vacuolated. The exception to this pattern occurs at the axis border close to radicle
 529

formation region, where cells are to be small, isodiametric and present dense
cytoplasm. Figure 3D depicts the most superficial cell layer of somatic embryos named
protoderm, followed by many compact layers of cells which determine the so called
ground meristem and the central region where procambium differentiation takes place.
At this procambium region cells are to be more elongated and densely stained showing
the differentiation process of the first protoxylem tracheary elements delineated by a
thick spiraling structure. Faure (1989) has also described identical features in somatic
embryos vascular system of Vitis rupestris, supported by Nickle and Yeung (1993).
Those histological studies carried out in somatic embryos of A. polyneuron
have demonstrated, so far, that their inability to complete their morphogenetic process
towards mature somatic embryos was due to the absence or partial organization of an
apical meristem. In order to conclude the somatic embryogenesis it is necessary to have
primary meristems fully formed (Dodeman et al., 1997). Quinn et al. (1989),
analyzing histological sectioning of Borago officina/is, have concluded that the visible
presence of root apical meristem and cotyledons under development, proposed that
those tissues were indicative of somatic embryo-like structures. Despite the presence of
those structures, they could not be considered a bipolar structure because there was no
differentiation of the apical meristem. Alemanno et al. (1996) have also observed
similar features in isolated embryogenic structures of Theobroma cacao where
protoderm and procambium bundle were present, but apical meristem was not detected.
Those referred to structures that were not able to achieve further development and so
faded away, losing thus, their embryogenic characteristics. Faure (1989) described that
only 27% of somatic embryos presented meristem tissue at root and apical regions,
showing histological similarity when compared to zygotic embryos. The remruning
somatic embryos analyzed presented just root apical meristem.
This histological study has also shown the presence of callus-like cells that
were quite different from the embryogenic ones by their significantly larger size, size
heterogeneity, lack of organization and a high degree of cell vacuolation. Those cells
show the presence of a red-stained substance encircling callus cells. It is probable that
that substance is a remainder of cell culture debris close to unorganized callus cells.
In general, callus cells were bigger and were displayed as clusters of cells not
too much aggregated. Fransz and Schel (1991) mentioned that callus cells of Zea mays
exsudate a mucilaginous substance that would fill the intercellular spaces exiting
between cell aggregates, then facilitating the diffusion of cell nutrients and
metabolites. Considering the fact that somatic embryogenesis follows a pattern similar
to normal zygotic embryogenesis, it makes it a model to better investigate plant
development and morphogenesis (Kiyosue et al., 1993).
Proembryogenic cellular masses selected for cytochemical investigation, were
taken from multiplication and maintenance cellular lineage basal media (LPm,
supplemented with 0.5-1.25 J.LM 2,4-D and 0.5 J..LM KIN) or from globular somatic
embryos under maturation culture media (LPm plus 30 J.1M ABA) (Figure lD). To
characterize embryogenic cells, the specimens were double stained with Evan's blue
(0.1%) and acetocarmine (2%) as suggested by Durzan (1988). To determine starch
and lipid grains the specimens were stained with lugol and Sudam III respectively
530

(Johansen, 1940). Photograph recording was accomplished employing

stereomicroscope SZH 10, optical microscope BX-40 (B-max) and camera Olympus
PM-20.
Embryogenic cultures were unique and could be distinguished from
nonembryogenic cultures by its white mucilaginous appearance, being friable, with a
coarse texture and ability to become red. Those embryogenic cellular masses were
maintained for approximately three years, being subcultivated each eight weeks onto
maintenance culture media in order to select embryogenic lineage. Cytochemical
studies have confirmed the cells to be embryogenic due to its ability to stain red at
acetocarmine addition, which indicates presence of nucleoprotein (Figure 3E). Cells
that strongly react to acetocarmine and weakly to Evan's blue are embryogenic and
those that do otherwise are nonembryogenic or callus cells.
Globular somatic embryos developed at the surface of proembryogenic cellular
mass have reacted strongly at presence of acetocarmine, and many cells were visible as
a clump-like structure repetitively as a characteristic of somatic embryos at pro-
globular and globular stage, being those features in accordance to which was described
by Smith and Krikorian (1990) and Sharma and Kumar (1994).
Cytochemical analyses employing lugol detected the presence of a large
amount of stained black-bluish starch grains dispersed throughout the cell (Figure 3F).
Borders of cell clumps showed large amount of starch grains, where it was documented
an average of 15-18 starch grains per cell. This average agrees with studies done in
Daucus carota (Emons, 1994), where embryogenic cells presented an average of 5-25
starch grains in contrast with those of nonembryogenic cells that have only one or two
per cell. Barciela and Vieitez (1993) have also found starch grains accumulation at
superficial layers of globular somatic embryos, embryogenic and proembryogenic cells
of Camelliajaponica. Similar features where described for other species like: Quercus
suber (El Mafttaoui, 1990) and Vitis rupestris (Faure, 1989).
Considering the fact that starch grains are abundantly found in cells showing
potential in de novo process, some researchers have considered to employ this
characteristic as an embryogenic or morphogenic cell marker (Profumo et al., 1987;
Schwendiman et al., 1988 and Plata et al. (1991). Data presented by Stamp (1987) and
Plata et a{ (1991) suggested that starch should be promptly metabolized in
embryogenic tissues, supplying energy for localized mitotic and metabolic activities.
Figure 3G shows lipid content in cell aggregates, which stains orange when in
the presence of Sudam III. Lipid content was in less quantity when compared to starch
grains per cell. In many studies, reference has been made to nutrient storage as
potential markers to evaluate vigor and quality of somatic development, with emphasis
on accumulation of proteins and lipids (Merkle et al., 1995). Lipids storage has also
been considered a marker to predict if tissues cultivated in vitro have potential to
induce somatic embryo formation. Lipid content found in Elaeis guineensis have
initially presented small droplets that would further become aggregated to form one or
two bigger drops in each cell. Similar observations were described in cell aggregates of
A. polyneuron. Those lipid droplets were observed in Elaeis guineensis at their first
stages of somatic embryo formation (Schwendiman et al., 1988).
 531

Merkle et al. (1995), reviewing morphogenic aspects of somatic

embryogenesis have described that for many species most lipid storage happens at
maturation stage, which coincide with protein depletion. Authors have argued that
some differences in lipid and fatty acids could exist when both zygotic and somatic
embryos are compared, which would reflect the kind of maturation process employed.
Cailloux et al. (1996) noticed lipid accumulation at various stages of somatic embryo
development in Hevea brasiliensis. Quinn et al. (1989) described lipid accumulation at
cotyledonary phase in somatic embryos of Borago officina/is.
Data shown by Choi and Soh (1997) on somatic embryos of Apium
graveolens, supported results observed with A. polyneuron where the average size of
embryogenic cells was between 30 and 60 !JM, presenting large vacuoles, dense
cytoplasm and large quantity of starch grains, excluding isolated cells. Halperin (1995)
has supported that suspension and callus cells have various sizes of isolated cells
presenting different potential to develop. Some big and highly vacuolated cells rarely
divide, but when stimulated can be induced to divide and produce cell aggregate that
would turn into somatic embryos.

6. Conclusion and prospects

Although the induction and subsequent expression of an embryogenic route in

A. polyneuron has been asynchronic and with low frequency for the first generation
(induction of somatic embryos), repetitive somatic embryogenesis was able to set up a
lineage of embryogenic cells, with high proliferation ratios, being maintained in vitro
cultivation for more than three years up to this date.
Induction of somatic embryos has been achieved after mature and immature
embryos as explants cultivated in vitro under culture media supplemented with 2,4-D
(5 to 10 !JM), with combination of 0.5 !JM of KIN, BA or TDZ. Maturation of somatic
embryos has occurred in an asynchronic way when cultured on media supplemented
with 12.30 or 24.60 !JM of 2-iP and 0.5 !JM of NAA. Somatic embryos have mimicked
zygotic embryos developmental sequence until torpedo stage. However, it was not
possible to achieve full control for those factors related to conversion of somatic
embryos into plantlets or seedlings, showing, then, that technical problems of
engineering on the entire process of embryogenesis to seedling production and planting
need to be solved.
Some determinant factors related to cellular competence and determination in
somatic embryos and in embryogenic cell lineage were identified. Cytochemical and
histological analyses furnished the tools to determine embryogenic cells and somatic
embryos at various ontogenetic stages, and then to set the stage for this partial
embryogenic somatic embryo protocol.

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532

Figure 3. A-D Development stages of somatic embryos. Longitudinal section of: A- proembryo (bar= 50.74J1m);
B- globular (bar=85.7lJ1ffi); C- globular and heart stage (bar=l33.33J1m); D- cotyledonar stage (bar=333.33J1ffi);
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containing: F- stach grains stained with lugol (bar= 17.78 J1ffi) and G- small droplets of lipids stained with sudam
III (bar=37.78J1m).
 533

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 17. SOMATIC EMBRYOGENESIS IN A LEGUMINOUS TREE-
ACACIA SENEGAL (L) WILLD.

Saloni Shahana and Shrish C. Gupta

Department of Botany, University of Delhi, Delhi- 110007, India

Contents

I Introduction 4 Embryo Maturation and Embling

2 Culture Initiation Development
Induction of 5 Conclusions
Embryogenesis 6 Acknowledgements
7 References

Introduction

The development of somatic embryos was first observed in Daucus carota root
cultures by Reinert (1958, 1959) and Steward et al. (1958). Since then it has
been used as a model system to explore various facets of somatic embryogenesis
including the early events occurring in embryo induction (Sung and Okimoto,
1983; Zimmerman, 1993).
Somatic embryogenesis is a potentially efficient means of propagation. Somatic
embryos, being bipolar propagules. can develop into complete plantlets in a single
step, thus, circumventing the problern of root induction encountered in regeneration
studies, especially with hardwood perennials. Therefore, efforts have been made
to induce it in various taxa during the past four decades. The successful induction
and development of somatic embryos in legumes is very useful as it helps in
the production of transgenic legumes where regeneration of adventitious shoots
is problematic (Distabanjong and Geneve, 1997). Leguminous plants have been
difficult to regenerate in aseptic cultures (McHughen and Swartz, 1984 ). However.
during the last ten years, some reports have appeared demonstrating embryogenesis
in forage and grain legumes. but those on tree legumes are comparatively less.
Gharyal and Maheshwari ( 1981) published the first report on somatic embryogenesis
in juvenile tissues (hypocotyl) of Albizia lebbeck followed by Tomar and Gupta
(1988) in hypocotyl explants of A. richardiana. Only recently, it was induced
in the internodal and leaf petiole explants excised from field-grown (physiologically
mature) woody plants of Calliandra tweedii (Kumar and Gupta, 1993).
Acacias, belonging to the Fabaceae, are of immense value as fodder, for
afforestation and also for reclamation of wastelands. Most of its species yield
excellent firewood and some are rich sources of proteins, tannin and gum. They
can adapt to extremes of temperature as well as moisture stress and, therefore,
539
SJ!. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woodv Plants, Volume 6, 539-552.
£:: 2000 Kluwer Academic Publisher.>.
540

can grow in both arid and moist regions on a wide range of tropical soils. They
are also effective in checking soil erosion and help stabilisation of sand dunes
(Skolmen, 1986). Due to these attributes, they form an important component of
forest vegetation in India. There are about 800 species of Acacia (Willis, 1966)
of which somatic embryogenesis has been induced just in four so far (Table 1).

Table 1: In vitro somatic embryogenesis in Acacia species.

Species Explant Response Reference

A. auriculiformis Hypocotyl Embryos Wei eta/., 1996

A. catechu Cotyledon Emblings Rout eta/., 1995

A. koa Hypocotyl Embryo ids Skolmen, I 986

A. nilotica Immature Triploid plants (two) Garget a/., 1996

endosperm

Embryogenesis has been recently induced in another important species, Acacia

senegal (L.) Willd. (Shahana and Gupta, 1999). It is a small, deciduous tree with feathery
crown. A. senegal is native to Sudan and in India, occurs particularly in Haryana, Gujarat
as well as in the dry rocky hills of Rajasthan (the Aravallis). The leaves are bipinnate
with stipular spines. The flowers are fragrant and develop in axillary, long spikes. The
pods are straight, strap-shaped with 5 or 6 seeds each. The tree is extremely hardy and
resistant to drought. It is one of the main cash crops of the desert region in north-west
India.
A. senegal yields the true gum arabic of commerce, known as 'Senegal gum'. The
gum is produced in gum cysts developing in the inner bark and later on it is exuded. The
gum arabic is chiefly used as an emulsifier in confectionery, for preserving flavours of
soft drinks and spray-dried instant foods as well as in the manufacture of chewing gums.
In the pharmaceutical industry, it is used as a binding agent for the manufacture of cough
pastilles and other medical preparations or as a coating of pills. It is also used in the
manufacture of adhesives. It has several applications in the paint, ink and cosmetic in-
dustries too. Gum arabic being demulcent and emollient is used internally for intestinal
troubles and externally to cover int1ammed surfaces, such as burns, sores and nodular
leprosy.
The wood is used as fuel and for making wheels and agricultural implements. The
tough roots and stems of young trees are utilized for producing tool handles and weaver's
shuttles. Its leaves and flowers are used as cattle fodder. The roots cure dysentry and
nodular leprosy. The fibre obtained from the bark of flexible strands of the surface roots
is used for cordage, well-ropes and fishing nets. Though the tree occurs in Indian deserts,
still the gum arabic is imported to the extent of 5,000 tonnes annually. Also, as the
consumption of gum is rising. there is an urgent need to boost up the indigenous source
material, i.e. the trees of A. senegal.
 541

2 Culture Initiation

Green pods were collected between October and December from trees growing
on Kamla Nehru Ridge near the University of Delhi. The fruits were (i) washed
under running tap water for 15 min., (ii) treated with 5% Polysan (v/v, Polypharma,
Pvt. Ltd., Mumbai) solution for 30 min., and (iii) washed again under running
tap water for 10 min. Thereafter, (iv) the material was disinfected with 1%
sodium hypochlorite solution (Qualigens, Mumbai) for 15 min. and subsequently
(v) with 90% ethanol for I -2 min. Finally, (vi) it was thoroughly washed with
sterilized distilled water. Young seeds were excised from green pods under a
laminar flow cabinet. The embryonal axes were removed and each cotyledon
was cut longitudinally into two halves. The two parts of a cotyledon were inoculated
in one culture tube with their dorsal surface in contact with the semi-solid induction
medium. Superficial incisions were made on the ventral surface of cotyledons
at the time of inoculation.
To select the optimum medium, initially ex plants were cultured on the following
eight basal media: W (White, 1943), MS (Murashige and Skoog, 1962), LS
(Linsmaier and Skoog , 1965), B 5 (Gam borg eta/., 1968), MT (Murashige and
Tucker, I 969), N (Nitsch, I 969), SH (Schenk and Hildebrandt, I 972) and N 6
(Chu et al., I 975). As a maximum of 52% cultures organized an average of
9.1±I .54 embryos per explant on MS medium, it was chosen for further experimentation
(Table 2).

Table 2. Morphogenic response of cotyledonary ex plants excised from immature seeds of A. senegal
and cultured on different basal media for 70 d., under 16 h. photoperiod.

Medium Number of Explants forming Average number Explants

explants somatic embryos of embryos forming
(%) per explant callus(%)

MS 42 52'" 9.1 ± 1.54'" 52

LS 44 20b,, 10.8±5.16 61
MT 48 50' 6± 1.28 65
N 48 6' 9±3 2
N• 48 35'b 5.6± 1.48 88
SH 44 0" 0 43
B, 42 10' 6±4 36
w 48 O' 0 19

'Values in a column followed by the same superscript are not significantly different as determined
by Chi-square test at 5% Ieve I
"Mean± standard error

MS medium was supplemented with one of the cytokinins supplied by Sigma-

Aldrich Chemicals, like BA (benzyladenine, 0.44-13.2 J.LM),Z (zeatin, 0.46-13.8
11M), Kn (kinetin,0.46-I3.8 11M) or 2iP(2-isopentenyl adenine, 0.49-14.7 11M)
or auxins (Sigma-Aldrich Chemicals) such as NAA (a-naphthalene acetic acid,
542

0.54-16.2 J.1M) or 2,4-D (2,4-dichlorophenoxy acetic acid, 0.45-13.5 J.1M). As

a source of carbohydrate, 3% sucrose (Daurala, New Delhi) was added to the
medium and gelled with 0.8% agar (Qualigens, Mumbai). Cultures were maintained
at 25±2 °C and 55±5% relative humidity under white fluorescent light (450-
640 J.1 W cm- 2) emitted by 40 W Philips tubes.
For histological studies, tissues were fixed in formalin-acetic acid-ethyl alcohol
(F .A.A.; I: 1:18, v/v) for 24 h and dehydrated through ethanol-xylene series,
followed by infiltration with xylene, and embedding in paraffin wax. Serial sections
( 12-15 J.lm thick) were cut with a rotary microtome. The sections were placed
on slides, dewaxed and then double-stained with safranin and fast green. Finally,
they were dehydrated and mounted in canada balsam. Photomicrographs were
taken with a Leitz Orthoplan Universal Microscope.
Scanning electron microscopy of embryogenic ex plants was also carried out.
Cotyledonary explants with embryos at different stages of development were
fixed in 2.5% (v/v) glutaraldehyde in cacodylate buffer at pH 7.2, dehydrated
through a graded ethanol series and critical point dried . The specimens
were coated with gold and then examined under a scanning electron microscope
(Philips, 501B; SEM) at 10,000 kV.

3 Induction of Embryogenesis

Cotyledonary explants inoculated on MS medium alone or supplemented individually

with any one of the four cytokinins (mentioned earlier) differentiated somatic embryos
within four to five weeks of culture. The best response was elicited on zeatin fortified
MS medium. Cotyledonary explants cultured on MS medium alone or in combination
with different concentrations of zeatin, swelled and formed white or green friable
callus at places of incisions as well as on cut margins, within 15 d. of culture. Off-
white and green shining translucent calli developed at cut margins in some cultures, in
another 20 d.
Gradually, the amount of callus increased, covering half of explant's surface. After
50 d., some of the calli started turning brown, and in texture, they changed from
jelly-like to compact. Almost all the cultures developed callus at different levels of
zeatin. Somatic embryos via direct embryogenesis were induced within 30 d. of
inoculation. They differentiated at (i) places of incisions, (ii) cut margins (Fig. 1 A)
and (iii) general surface of explants. Margins of some of the explants became convoluted
due to irregular overgrowth and later on, increased in size, followed by induction of
somatic embryos as small globular or elongated structures. In some cases, embryos
were induced indirectly via translucent and compact callus which was green, light
brown or off-white. Of the different levels of zeatin tried, best response was elicited
on 9.2 J.1M (Fig. 1 B), wherein a maximum average of 23±2. 70 embryos developed
per explant in 87% cultures (Table 3). The maximum number of morphologically near
 543

Figure I. Somatic embryogenesis in cotyledonary explants excised from immature seeds of Acacia
senegal. (A) Green embryos developed all along the cut margin on 6.9 J.LM zeatin added to MS
medium, after 60 d. of culture (x 1.6), (B) Direct induction of green somatic embryos on the surface
and at cut margins on 9.2 J!M zeatin, after 60 d. of culture (x 3), (C) A shoot developed in between
two cotyledons.of a somatic embryo on 0.44 J!M BA + 0.49 J!M 2iP, after 28 d. of culture (x 4), (D)
A somatic embryo with leafy cotyledons and root at the radicular end on MS medium supplemented
with 0.68 J.LM glutamine, after 30 d. of culture (x 4.8).
544

Table 3. Somatic embryogenesis in A. senegal cotyledons excised from immature seeds and reared on MS
medium supplemented with zeatin for 70 d., under 16 h. photoperiod.

Zeatin Number of Explants forming Average number Explants

(J.LM) explants somatic embryos of embryos forming
(%) per explant callus(%)

0 40 35b' 3.6 ± 0.61" 85

0.46 48 49b.d 4.1 ± 0.83 94
2.3 44 70'-'·· 1.4 ± 1.57 100
4.6 48 73'·'·· 15.3 ± 2.19 100
6.9 47 5Jb.o 15.6 ± 3.09 91
9.2 46 87' 23 ± 2.70 100
ll.5 42 95' 16± 2.19 100
13.8 46 83'·' 19.1±1.86 100
'Values in a column followed by the same superscript are not significantly different as determined
by Chi-square test at 5% level
"Mean ± standard error

normal or normal embryos were also formed on 9.2 J.1M zeatin. Heart- and torpedo-
shaped as well as dicotyledonous embryos developed frequently. At other levels,
though normal embryos were observed but their average number per explant was low
and the frequency of responding cultures was also less. Some embryos had just a

60 z
o-------- 2i p

BA

1 1·5 2 2·5 3
CYTOKININ$ ( mg/1)

Figure 2. Percentage of normal and near normal embryos differentiated at different levels
of cytokinins in A. senegal cotyledonary explants, after 70 d. of culture.
 545

single cotyledon, a few were cylindrical and still others were enlarged structures with
expanded cotyledons. Some of them remained morphologically unorganized, i.e. without
any recognisable developmental stage. Though embryos were induced in appreciable
numbers on the other three cytokinins tried, the frequency of induction as well as the
number of normal embryos were relatively low (Fig. 2).
On 2,4-D supplemented medium, the somatic embryos mostly originated indirectly
through callus phase except a few that developed directly on the surface (Fig. 3).
Normal organization of embryos was not achieved on any concentration of 2,4-D
tried, as they were mostly green, swollen, pleuricotyledonary, funnel-shaped or
elongated and fused with each other. On NAA supplemented medium, the response
was better as normal, green heart- and torpedo-shaped as well as dicotyledonary
embryos org~ized on the surface of explants at 2.69 JlM level but the average number
of embryos per explant was very low as compared to those on the optimal regeneration
medium (MS + 9.2 JlM zeatin). The embryos were mostly abnormal in morphology on
other concentrations ofNAA tried.

~60
(2) 0----0 NAA
~50 (O)
0:: o------o 2, 4-D
::::>
~ 40
::::> (7)
u
1.!> 30 I
Z I

~0 20 \
------cr--------------0----
Bi 10 ------------o
I
6.-~-/(-g}- (O) (0)
~ (0) (0)
0 0·1 0·5 '1 1·5 2 2·5 3
AUXINS (mg/ I)
Figure 3. Effects of auxins on morphogenic response of A. senegal cotyledonary explants.
The percentages of normal embryos are indicated in parentheses.

A perusal of literature shows that mostly auxins have been used for the induction
of embryos, as in Cercis canadensis (Trigiano et a/., 1988), Arachis hypogaea
(Chengalrayan et a!., 1994) and Albizia julibrissin (Burns and Wetzstein, 1998).
But at times, a combination of auxin and cytokinin has proved beneficial as in Robinia
pseudoacacia (Merkle and Wiecko, 1989), A. nilotica (Garg et a!., 1996) and
Dalbergia sissoo (Das et al., 1997). Hatanaka eta/. (1991) found that only cytokinin
(2iP) is able to induce embryogenesis in Coffea canephora. During the present
546

investigations, zeatin has supported better differentiation of embryos in cotyledonary

explants while for hypocotyl of Albizia richardiana, 10-s MBA has been effective
(Tomar and Gupta, 1988) and in Calliandra tweedii, 0.5 J.lM NAA has proved
more efficient for leaf petioles while I J.1M 2iP for its internodal segments (Kumar and
Gupta, 1993). The other species of Acacia in which embryogenesis has been reported
in cotyledonary explants excised from green pods is A. catechu. In this taxon, embryos
developed from callus derived from immature cotyledons, cultured on Woody Plant
medium (Lloyd and McCown, 1981) fortified with 13.9 J.lM kinetin and 2. 7 J.1M NAA.
As compared to this, in our experimental system, i.e. A. senegal for the same explant
(cotyledon), the induction medium included only a single cytokinin, i.e. zeatin and
embryos differentiated directly in high frequency, whereas in A. catechu the mode of
differentiation was indirect.

Figure 4. Hist\)logy and scanning electron photomicrographs of embryogenic cotyledonary explants

of Acacia senegal. (A,B) Sections of the explant showing globular and heart-shaped somatic
embryos, respectively (x 400), (C) Several embryos developed at the margin and on the surface
of the explant (x 18.4), (D) A heart-shaped embryo (x 64).

Histological studies indicate that embryogenic cells are densely cytoplasmic with
prominent nuclei and the non-inductive cells possess scanty cytoplasm with large
vacuoles. Embryos gradually get isolated as they have no vascular connection with
 547

the mother explant, which has been considered as their characteristic feature (Haccius,
1978). In vertical sections, globular and heart-shaped embryos are often seen (Figs
4A, B). The scanning electron micrographs reveal a large number of embryos developed
on the surface of explants (Figs 4C, D).

4 Embryo Maturation and Embling Development

The major hurdle in the application of somatic embryogenesis as an effective means

of plant regeneration is lack of sufficient embryo maturation, resulting in a lower
frequency of plant development (Capuana and Debergh, 1997). The major event
taking place during this phase is the accumulation of storage proteins, carbohydrates
and lipids, and a decrease in water content associated with a gradual decline in the
metabolic activity (Gray, 1996).
During the present investigations, somatic embryos were isolated and transferred
from the differentiation medium to different maturation and development media. The

Table 4. The response of somatic embryos excised from cotyledonary explants of A. senegal, given
maturation treatment for 70 d. as well as desiccation pre-treatment for different durations, and cultured on
development medium for 30 d., under 16 h. photoperiod.
Media Number of Embryos Embryos Embryos
embryos forming forming forming
shoots(%) roots(%) both, roots
and shoots' (%)

163 0.6 0.6 o···

2 157 4 6 I'
3 191 3 3 0.5'
4 189 I I o•
5 170 5 2 0.6•
6 168 4 4 I'
7 200 2 2 0.5•
8 149 3 2 0.7•
9 164 9 10 I'
10 149 9 9 2'
II 180 3 4 I'
12 125 6 5 o•
13 212 I 3 0.5•
'14 189 7 7 I'

"(I) MS basal, (2) MS + 4.4 J.iM BA + 4.6 J.iM Kn, (3) MS + 4.4 J.iM BA + 9.2 J!M Kn, (4) MS + 4.4
J.iM BA + 18.4 11M Kn, (5) MS + 8.8 J.iM BA + 4.6 11M Kn, (6) MS + 8.8 11M BA + 9.2 11M Kn,
(7) MS + 9.211M BA + 18.4 11M Kn, (8) MS + 17.6 11M BA + 4.6 11M Kn, (9) MS + 17.6 11M BA
+ 9.2 11M Kn, (10) MS + 17.6 11M BA + 18.4 11M Kn, (II) MS + 4.54)!M TDZ, (12) MS + 9.08
J.iM TDZ, (13) MS +IS J.iM TDZ, and (14) MS + 4.4 J.iM BA + 4.6 J.iM Kn + 4.54 J.iM TDZ
.. Values in a column followed by the same superscript are not significantly different as determined
by Chi-square test at 5% level
548

maturation medium contained ABA, AC and PEG individually as well as in different

combinations. Subsequently, the desiccation treatment was given for different durations
(1, 24 and 72 h.). This was, in turn. followed by culture of somatic embryos on
development formulations, which included MS medium supplemented with BA, Kn
and TDZ at varying levels (Table 4). Different levels ofBA and combinations ofBA
and 2iP were also tried as conversion media. The other treatments included rearing
of embryos on medium fortified with different concentrations of glutamine, AC and
sucrose. MS medium, full- and half-strengths supplemented with 2% sucrose were
also tried. The embryos organized roots more frequently on these combinations (Fig.
1 D). Though, shoots did initiate but they were not sufficiently elongated, fully
developed and normal-looking (Fig. 1C). Leaflets in some of them were enlarged or
swollen. In a few cultures, a crown of leaflets developed at the elongated plumular
end. Some of the somatic embryos. which were difficult to separate from each other,
were transferred in small clusters to a~ove-mentioned development medium. They
supported the organization of additional somatic embryos. May be that standardisation
of the stage of embryo excision from the induction medium and their culture onto the
maturation and development media would help in the successful conversion of embryos
into emblings. Experiments in this direction are being carried out.
Several researchers have proposed the use of different ingredients in the culture
medium for maturation of somatic embryos. Chemicals which have proved effective
are abscisic acid (Fujii et al., 1989: Senaratna et al., 1989, 1990; Capuana and
Debergh, 1997), polyethylene glycol. an osmotically active compound (Capuana and
Debergh, 1997), activated charcoal (Buchheim et al., 1989; Ebert and Taylor, 1990),
an .increase in the osmoticum by adding high concentrations of sucrose in the medium
(Emons et al., 1993). as well as a few amino acids, particularly glutamine (Khlifi
and Tremblay, 1995).
Of the four investigations so far published on somatic embryogenesis in Acacia,
high frequency conversion of somatic embryos into plants has been reported only in
Acacia catechu (Rout et a/., 1995). In Acacia koa, embryoids developed from
shoot tip and hypocotyl explants, that too with great difficulty (Skolmen, 1986).
Whereas, only two triploid plants could be raised from endosperm explant of Acacia
nilotica (Garg et al.. 1996) and in A. auriculiformis, the globular embryos developed
roots but no shoots (Wei et al .. 1997). In contrast, in our experimental system, i.e.
A. senegal, somatic embryos form roots readily but the development of plumular
leaves was still a problem. Similar situation was also encountered earlier in the
development of somatic embryos of Dalbergia sissoo (Das et al., 1997).

5 Conclusions

During the past decade. concerted efforts have been made to understand the phenomenon
of somatic embryogenesis and success has been achieved to some extent. This is reflected
by the increasing number of species in which somatic embryogenesis is being induced
 549

continuously. But there are still some subtle problems involvmg embryo maturation which
have to be overcome if it has to become a viable method of propagation. In many species,
the embryos so differentiated are mostly aberrant in morphology, and thus difficult to
develop into emblings. Even if the embryos are normal, they do not form emblings,
resulting in very low conversion frequencies. This has been attributed to poor maturation
of somatic embryos (Lelu and Label, 1994 ). Although serious strides have been made
to unravel the molecular (Jung and Sung, 1984; Jung eta!., 1987) and biochemical
changes taking place during this phase (Sung and Okimoto, 1983), it still needs further
intensive investigations to unravel the control mechanism(s).

6 Acknowledgements

This chapter is based on the research which has been supported by U.S .I.F.
project No. FG-In-778 (IN-FS-1 02) sanctioned to S.C.G. by the U.S. Department
of Agriculture, and S.S. is grateful to the University Grants Commission for providing
the CAS fellowship.

7 References

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552

Abbreviations

ABA Abscisic acid

AC Activated charcoal
N6 Chu et al.'s basal medium
2,4-D 2,4-dichlorophenoxyacetic acid
d. days
F.A.A. Formalin-acetic acid-ethyl alcohol
Bs Gamborg et al.'s basal medium
h. hours
Kn Kinetin
fJM Micromolar
LS dnsmaier & •Skoog's basal medium
MS Murashige & Skoog's basal medium
MT Murashige & Tucker's basal medium
2iP N 6 -(2-isopentenyl) adenine
BA N 6-benzyladenine
NAA a-naphthalene acetic acid
N Nitsch's basal medium
PEG Polyethylene glycol
SH Schenk & Hildebrandt's basal medium
TDZ Thidiazuron
w White's basal medium
z Zeatin
 18. Somatic embryogenesis in cypress
(Cupressus sempervirens L.)
Maurizio Lambardi
CNR I National Research Council, Istituto sulla Propagazione delle Specie Legnose,
via Ponte di Fonnicola 76, 50018 Scandicci (Firenze), Italy.
E-mail: lambardi@ipsl.fi.cnr.it
Contents
1. futroduction 3.5. Proliferation and maintenance
2. Traditional and in vitro propagation of EST
methods 4. Embryo development and
3. fuduction of somatic embryogenesis maturation
3.1. Embryonal-suspensor tissue 5. Protoplast isolation and
purification
(EST) and non-embryogenic 6. Particle bombardment of EST
callus 6.1. DNA preparation and
3.2. Explant material microprojectile DNA-delivery
3.3. Culture conditions 6.2. Transient gene expression
3.4. Effect of genotype and 7. Conclusions
collection time 8. References

1. Introduction

The genus Cupressus, native to wann temperate climates of the Northern hemisphere,
can be found around the Mediterranean, in North America and in Asia. Twenty-five
taxa have been identified in the genus and described as species (Ducrey et a/., 1999), all
generically named "cypress". Here, we refer to the species Cupressus sempervirens L.,
also called "common", "Mediterranean" or "Italian" cypress, by far the most important
and widespread cypress in the Mediterranean basin. The species is native to northern
Persia, as well as Syria, Turkey, Cyprus and several Greek islands. However, during the
Roman Empire it was introduced into all the Mediterranean countries, where it can now
be considered naturalised. The cypress grows up to 30 m in height, it is monoecious, and
bears male and female strobili (cones) separately at the end of short branchlets.
Depending on the crown branch habit, the species is divided into two varieties, i.e.:
- C. sempervirens var. horizontalis, the most common in natural areas, characterized by
spreading branches and a broad conical crown;
- C. sempervirens var. pyramidalis (= var. fastigiata), the most popular for ornamental
use because of its erect branches, parallel to the trunk, which give the tree its typical
columnar shape, resembling a flame (Fig. 1).
In the Mediterranean area, the cypress is one of the few species with characteristics
of marked drought hardiness, and suitability for afforestation in difficult terrains such as
calcareous, clay or rocky soils. Due to its high natural durability and straightness,
cypress wood production is highly valued. As an ornamental tree, in Greece, Italy and
Spain it is typical of religious and archeological sites. In Italy, particularly in Tuscany, it
has from ancient times been closely associated with the traditional landscape, as well as
553
S.M Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 553-567.
© 2000 Kluwer Academic Publishers.
554

widely planted in parks, villas and boulevards (Pozzana, 1991). Finally, because of its
columnar and dense crown habit, it is used as wind-breaks for vegetable and fruit tree
crops, particularly in France, Spain and Portugal.
Since the seventies, a serious disease, named the "cypress canker" and caused by the
fungus Seiridium cardinale, started to spread over large Mediterranean areas (especially
in Italy, Greece and France) and led to extensive damage in forests, nurseries and
ornamental plantations. In Tuscany alone, hundreds of trees have died yearly, while
many others, even if still alive, have had the crown severely damaged. Soon, the disease
became a factor strongly limiting cypress planting. The losses were so devastating that a
large-scale breeding programme, with the main aim of selecting canker-tolerant clones,
was initiated in several Mediterranean countries, mainly Italy, Greece and France
(Raddi & Panconesi, 1981; Xenopoulos, 1990; Teissier du Cros et a/., 1991).
Unfortunately, long reproductive cycles of conifers makes the conventional breeding
techniques very time consuming. Hence, somatic embryogenesis can have important
implications in cypress improvement programmes, opening the door to the use of tissue
culture for faster propagation of disease-tolerant genotypes and for utilization in genetic
engineering of important silvicultural traits.

2. Traditional and in vitro propagation methods

Cypress is traditionally propagated by planting nursery-grown seedlings and by grafting.

In grafting, scions, collected from selected forms, are side-veneer or cleft grafted on
seedling rootstocks during the winter season. This is an extremely labour-intensive and
costly practice for the nursery, but it is widely utilized to propagate clones selected for
their columnar shape and planted for ornamental purposes. The species can also be
propagated by softwood cuttings, provided that the cuttings are collected in spring,
treated with talcum powder containing 1% indole-3-butyric acid (IBA), and maintained
under mist conditions. However, cutting propagation has never become· a common
practice, because of the poor rooting ability of some genotypes, and the strong seasonal
periodicity of rooting of stem cuttings (Capuana & Lambardi, 1995).
For these reasons, several attempts have been made in recent years to explore the
potentialities of tissue culture methodologies. The possibility of reproducing young and
adult plants by micropropagation has previously been reported (Capuana eta/., 1991;
Capuana & Giannini, 1997). However, the low efficiency of some phases (i.e., shoot
proliferation and rooting) prevented the procedure from becoming. a real alternative to
traditional propagation methods. A different approach used excised mature embryos as
initial explants, in order to induce the formation of adventitious buds and their
subsequent development into shoots, which can be maintained in continuous
proliferation (Lambardi eta/., 1995). Somatic embryogenesis from immature zygotic
embryos of C. sempervirens is reviewed in this chapter. Its applications in protoplast
isolation and in biolistic experiments are also described.

3. Induction of somatic embryogenesis

3.1. Embryonal-suspensor tissue (EST) and non-embryogenic callus

It is well known that morphological differences between embryogenic and non-

 555
embryogenic tissues provide the basis for selection of cultures with a high efficiency in
plant regeneration. The cypress is no exception in this respect, as EST and non-
embryogenic callus, originating from immature embryos, can easily be identified for
their characteristic traits. EST of cypress is white, translucent and mucilagil)ous, with a
high percentage of filamentous pro-embryos (Fig. 2A). Non-embryogenic callus is white
to yellow, never translucent or mucilaginous, and with low organogenic potential. While
the differentiating embryogenic tissue always arises from the basal portion of the
embryonic axis, presumably from suspensor cells, non-embryogenic callus can arise
from various parts of the explant. As a rule, once this type of callus starts to proliferate
on the embryo surface, no other type of tissue develops on the same explant. .
Proliferating EST of cypress consists primarily of clusters of somatic pro-embryos,
very similar to the late pro-em6ryo stage of zygotic embryos (Attree & Fowke, 1991).
The clusters are polarized structures, initially organised into an embryonic region
subtended by multiple, closed and short suspensors (Fig. 2B). By cleavage
polyembryogenesis, these structures continually initiate embryos which generally
develop simultaneously to the filamentous stage (Fig. 2C).

3.2. Explant material

C. sempervirens embryos reach full maturity in the autumn of the second year after
fertilisation. During this second year, the female cone turns brown and the embryo starts
to acquire firmness. At this point the seed coat can be removed and the immature
embryo safely excised from the megagametophyte. To obtain sterile isolated embryos, in
our procedure the seeds were mechanically removed from the cones and decontaminated
for one min with 70% ethanol plus 10 min in 0.1% HgCh, followed by multiple rinses
with sterile distilled water. The seeds were kept in the dark at 4°C for 5-7 days in a
small amount of sterile distilled water to soften the seed coat, after which the immature
embryos were dissected under a laminar-flow hood.
Only the cultivation of whole immature embryos proved to be effective in the
production of EST, while epi- or ipocotylar portions of embryo and isolated cotyledons,
as well as mature embryos, always originated non-embryogenic callus.

3.3. Culture conditions

The best medium for initiation of EST from C. sempervirens immature embryos was the
DCR formulation (Gupta & Durzan, 1985), supplemented with amino acids from AE
(von Arnold & Eriksson, 1981) plus 100 mg!L L-glutamine, 500 mg!L casein
hydrolysate, 200 mg!L myo-inositol, 30 giL sucrose, 10 J.lM 2,4-dichlorophenoxyacetic
acid (2,4-D) and 7 giL Bacto-agar (inductive medium). In these conditions, the first
small quantities of embryogenic tissue started to appear after 4-5 weeks of incubation at
23±1 ac in the dark.
In the above culture conditions, more than 3% of immature embryos collected from a
pool of clones initiated highly efficient lines ofEST (Table 1). Moreover, almost 50% of
the embryos that did not initiate embryogenic tissue were stimulated to produce non-
embryogenic callus. In conifers, the induction of embryogenic tissue is often promoted
or enhanced by the presence of both cytokinin and auxin in the medium. Here, the
addition of 2,4-dichlorophenoxyacetic acid (2,4-D) alone as growth regulator proved to
be critical for the induction and initial proliferation of EST, while the combination of
2,4-D with benzyladenine (BA), at a 5:1 ratio, was detrimental to the initiation of both
556
embryogenic and non-embryogenic tissue. Sallandrouze eta/. (1999) also reported the
initiation of a single C. sempervirens embryogenic line from an immature embryo
collected in mid-February and cultured on a hormone-free MS (Murashige & Skoog,
1962) medium, supplemented with 15 giL of both fructose and glucose, 4 giL charcoal,
10 mL/L coconut water and 7 giL Bacto-agar.

3.4. Effect of genotype and collection time

In cypress, the ability to initiate EST is strongly influenced by the genotype. In our
experiments, immature zygotic embryos were collected during the years 1991/1994 on
open-pollinated adult grafted trees, belonging to 24 different clones selected for
tolerance to the cypress canker disease (Lambardi eta/., 1994). The embryos were then
cultured separately in the embryogenic inductive medium. Only embryos from 5 of the
24 clones were able to initiate EST. Moreover, two clones (namely 'Clone 47' and
'Clone 267') gave the highest percentages of embryogenic lines (20% and 15%,
respectively) when embryos were collected at specific dates, i.e., April 26 for 'Clone 47'
and May 11 for 'Clone 267' (Table 2).
The developmental stage of the zygotic embryo has repeatedly been reported as
critical for the induction of embryogenic tissue in conifers, and the period during which
the embryo responds to inductive treatments can be as short as a few days (Becwar et a/.,
1990; David eta/., 1995) during its course of maturation. Cypress immature embryos
were collected weekly over 2 months, i.e., from late-April to late-June (1993). For each
of the 5 clones, while non-embryogenic calli originated on explants collected throughout
almost the entire sampling period, the appearance of EST was mainly restricted to
embryos collected over a 2-week period. The beginning of this "embryogenic window"
ranged from late-April ('Clone 47') to the end of May ('Clone 327'). However, this lack
of coincidence reflected only slight differences among the clones in the course of their
embryo maturation processes. In fact, EST always originated from embryos that were
morphologically at the same stage of maturity, i.e., an early cotyledonary stage,
characterized by the two cotyledons just differentiated and still tightly joined. In this
respect cypress is similar to Picea species, where early cotyledonary embryos (also
described as embryos with tiny cotyledons) appear the most responsive to EST induction
(von Arnold, 1987; Hakman & Fowke, 1986, 1987; Nagmani et a/., 1987; Attree &
Fowke; 1991). However, differently from Picea, where also mature zygotic embryos have
been successfully used for the establishment of embryogenic cultures (Jain eta/., 1988;
Tautorus et a/., 1990; Tremblay, 1990; Wilson & Thorpe, 1995), EST was never
observed from mature embryos of C. sempervirens which proved to be competent only for
non-embryogenic callus (unpublished data).

3.5. Proliferation and maintenance of EST

Following the production of EST from the radicle region of the zygotic embryo, the
embryogenic tissue was initially subcultured every 21 days onto fresh inductive medium
in the dark. However, in order to avoid the appearance of extensive browning and
necrosis, after the third subculture the EST was transferred onto a DCR medium with a
reduced auxin concentration. Either a combination of a-naphthaleneacetic acid (NAA)
and BA (5 ~ each), or the addition of 2,4-D alone (5 ~. proved to be equally
 557

effective in the avoidance of culture browning. Regularly subcultured every 1-2 weeks in
one of the above maintenance media, the embryogenic lines were .prolific (the culture
volume doubled approximately every two weeks), showed high concentrations of
filamentous somatic embryos (Fig. 3A), and could be maintained for about two years
without any sign of decline in embryogenic nature.
Embryogenic lines initiated on immature embryos from different C. sempervirens
clones often showed dissimilarities, in terms of morphological characteristics,
proliferation rate and somatic embryo production. In our experience, the best
embryogenic lines were repeatedly obtained from a specific clone, namely the 'Clone
162'.

4. Embryo development and maturation

:rhe development and maturation of cypress somatic embryos were obtained in the dark
on hormone-free OCR medium, supplemented with 0.5 giL activated charcoal
(maturation medium). The culture time on the maturation medium required by the
filamentous somatic embryos to move to the next stages of development was always
unpredictable. While some embryogenic lines started to show large areas of
asynchronically-developing somatic embryos after 3-4 weeks (Fig. 3B), ·other lines
required sev~ral months to actively enter the maturation stage. Finally, a few lines never
developed further and, in time, turned to a non-embryogenic state.
In conifers, the addition of abscisic acid (ABA) to the culture medium has been
repeatedly reported as essential to stimulate embryo maturation (e.g., Klimaszewska
1989; Roberts eta/., 1990; Attree & Fowke, 1991; Thompson & von Aderkas, 1992; von
Arnold et al., 1995). Embryogenic cultures of cypress never got significant benefits from
the inclusion of ABA in the maturation medium; on the contrary, at a concentration of
10 pM or higher, the EST either turned brown and necrotic, or it became extremely
mucilaginous and did not undergo further development. As already suggested for other
species (Pan & van Staden, 1998), it is hypothesizable that the stimulatory effect of
activated charcoal in cypress somatic embryogenesis may be due not only to a general
absorbtion from the medium of phenolic compounds and/or inhibitory substances, but
also absorption of ABA naturally released during the culture.
In the charcoal-containing medium, somatic embryos differentiated until the
cotyledonary stage, but the hypocotyl and radicle regions remained poorly developed
(Fig. 3C). In our experience, only cotyledonary somatic embryos which were isolated
and singly transferred on filter-paper bridges, soaked with liquid hormone-free DCR
medium, regenerated plantlets (Fig. 3D).
Sallandrouze eta/. (1999) obtained the maturation of cypress somatic embryos to the
cotyledonary stage only after the addition to the culture medium of bovine serum
albumin (BSA). When transferred onto a BSA-free medium, the cotyledonary embryos
generated whole plants. The authors hypothesize that BSA, in promoting somatic
embryos maturation, may act as an osmotic agent (similarly to sucrose and polyethylene
glycol) and/or play a metabolic role as an organic source of nitrogen.

5. Protoplast isolation and purification

Protoplasts have been obtained from 3-day-old embryogenic suspension cultures of C.

sempervirens, derived from proliferating EST. Cell wall digestion solution (a mixture of
558

1.67% Cellulase Onozuka, 0.67% Macerase, 0.067% Pectolyase, 8% D-sorbitol, 5 mM

CaCh. and 50 mM MES at pH 5.8) was added at a rate of 2mL per gram FW, and
digestion was allowed to proceed overnight with gentle shaking. The protoplasts were
separated from undigested cell masses and debris by sequential filtration through 100
J.llil and 62 J.llil nylon filters. They were then pelletted by centrifugation at 37 x g and,
after the removal of the cell wall digestion solution, washed three times with 8% D-
sorbitol. The protoplasts were purified using a two-phase Ficoll gradient, according to
Wilson et a/. (1989). In this procedure, the final centrifugation separated viable
protoplasts, by floating in a tight band, above a 20% Ficoll:8% sorbitol and below a 8%
sorbitol layer. Typical yields of C. sempervirens protoplasts from these cultures were 3.5
x 105 protoplast g·1 FW, with 40-50% viability as determined with the Evans' Blue test
(400 mg/L in 0.65 M mannitol). After plating on DCR proliferation medium (containing
20 giL glucose, 30 giL sucrose, and 10 giL D-sorbitol), the protoplasts regenerated cell
wall within 48 h and produced cell colonies, showing the characteristics of EST.
Filamentous pro-embryos occasionally originated from these structures, but they never
developed further.

6. Particle bombardment of EST

With conifers, substantial progress has been recently made in gene transfer technologies
for gene expression studies and for recovery of transgenic trees. Most recent work used
microprojectile-mediated DNA delivery to obtain transient gene expression in various
conifer tissues (Seguin et al., 1996). Success in recovering transgenic trees using this
technology with embryogenic tissue was obtained with white spruce (Picea glauca; Ellis
et al., 1993), black spruce (Picea mariana; Charest et al., 1996), and tamarack (Larix
laricina; Klimaszewska et al., 1997). One-year-old EST of cypress proved to be suitable
as targets for the delivery of chimeric marker genes through microprojectile
bombardment (Lambardi et al., 1998). The variables of the delivery method were
optimized using the ,P-glucuronidase (GUS) gene, and two other marker genes (NPTII,
neomycin phosphotransferase, and CAT, chloramphenicol acetyltransferase) were tested
for their effectiveness.

6.1. DNA preparation and microprojectile DNA-delivery

Three plasmid vectors were used and evaluated for their efficiency for EST
transformation, i.e., plasmid pBI426, containing a double 35S promoter with the alfalfa
mosaic virus enhancer (AMVE) (Datla et al., 1991), plasmid pRT99GUS, containing a
single 35S gene of the Cauliflower mosaic virus (Topfer eta/., 1988), and plasmid
pCGUOO, containing the sunflower ubiquitin promoter (Binet et a/., 1991). Plasmid
DNAs were coated onto gold particles (1.6 J.llil diameter) using the CaCh precipitation
method developed by Klein et a/. (1989). DNA delivery was carried out using the
Biolistic® particle delivery system PDS-1000/He (DuPont, Wilmington, Del.; described,
e.g., by Kikkert, 1993). Embryogenic tissue of cypress, collected during the proliferation
stage, was weighed under aseptic conditions and spread (200 mg per plate) on the
surface of filter papers (0 5.5 mm, medium porosity), placed in the centre of 9-cm
diameter Petri dishes containing a gelled (0.7% Bacto-agar) DCR medilll;fi. For each
bombardment, 1 Jlg of plasmid DNA was used. The plates were placed 12.5 em from the
 559
stopping net, under a vacuum of 800 mm of Hg.

6.2. Transient gene expression

After EST bombardment, the plates were placed at 25 oc in darkness for 48 h, after
which GUS gene expression was detected by both histological and :fluorometric assays in
accordance with Charest eta/. (1993). Both the NPT II, and the CAT ELISA assays
were performed according to the manufacturer's recommendation (5 Prime~ 3 Prime
INC., CO, USA).
Transient expression of the GUS gene was easily detected using the histochemical
assay, and the expression was based on the number of expression units (i.e., the number
of islands of cells showing blue coloration; Fig. 4A) per 200 mg of sample tissue
(Duchesne & Charest, 1991). Clusters of cylindrical and spherical cells were the most
frequent components of the observed blue spots. However, GUS gene expression was
occasionally detected also from the whole surface of somatic pro-embryos (Fig. 4B).
Both the number of days after the last subculture (at the time of bombardment), and
the plasmid vector used strongly influenced transient gene expression (Table 3). With
two of the three plasmids tested (pCGUBO and pBI426), EST of cypress yielded the
highest GUS gene expression when bombarded 15 days after subculture. An effect of the
subculture time on transient gene expression has been also reported for larch and black
spruce (Duchesne & Charest, 1991; Duchesne et a/., 1993). Comparing the three
vectors, differing for their promoter sequences, the relative levels of gene expression
detected histologically were: sunflower ubiquitin > 35S-35S-AMVE > 35S. Indeed, with
reference to the means of the three subculture periods, the number of expression units
after bombardment with pCGUBO was respectively 1.5 and 3 times that of pBI426 and
pRT99GUS plasmids.
The plasmid pCGUBO carried NPT II and CAT reporter genes that were also tested
for their level of transient gene expression following bombardment of 9-day· subcultured
EST. Both the enzymes were easily detectable (1652.5 ± 193.7 pg NPT II proteinlmg
total protein, and 1751 ± 146.4 pg CAT proteinlmg total protein), showing that the
genes were functional in the tested cypress tissue, in accordance with other conifer
species, e.g. larch (Charest et al., 1991) and spruce (Charest et al., 1996). The high level
of transient gene expression with the GUS gene of the pCGUBO plasmid makes it a good
candidate to test for stable transformation experiments using cypress embryogenic tissue.

7. Conclusions

The common or Mediterranean cypress is one of the most important conifer species for
ornarnental use in several Mediterranean countries. Many breeding programmes have
been developed by public organizations, in order to select genotypes of both superior
growth habit, and high tolerance to the cypress canker disease. Hence, the exploitation
of tissue culture systems can offer a valuable alternative to traditional propagation for
large-scale clonal reproduction of selected genotypes. To date, C. sempervirens is the
only species inside the genus Cupressus for which a procedure of somatic embryogenesis
has been described, and the tecnique, as well as in many other conifer species, seems to
be the most promising among the in vitro regeneration systems, taking into
consideration its superior multiplication potential. Moreover, embryogenic tissue is an
560

ideal material for use in genetic transformation technologies, which would be highly
advantageous in cypress, considering the time required for the production of new
genotypes by sexual crossing. In conifers, achievement of this goal is often hampered by
the non-availability of efficient tissue culture systems, and by the lack of information
about screenable markers and gene promoter combinations. Effective transient gene
expression has been obtained from EST of cypress following microprojectile DNA-
delivery, and the Cupressus genus can therefore be added to the other conifer genera
successfully used for this type of experiments. This result can be regarded as an
important step for Cupressus sempervirens towards gene expression studies and
transgenic tree selection.

Acknowledgements

The author thanks Lorena Sozzi and Daniele Menabeni for their excellent technical
assistance.

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 563
Table 1. Percentages of immature zygotic embryos of C. sempervirens initiating embryonal-suspensor tissue (EST),
non-embryogenic callus (NEC) or non-regenerating, after culture on inductive medium, containing 2,4-D alone or
in combination with BA Percentages recorded after 8 weeks of incubation at 23 ± 1•c in the dark

Growth regulators No. of cultured Embryos forming Embryos forming Non-regenerating

embryos EST(%) NEC (%) embryos(%)
10 ~tM2,4-D 937 3.2 47.1 49.7
15 ~tM2,4-D+3~tMBA 896 0.3 13.8 85.9

Table 2. Effect of collection date on the initiation of embryonal-suspensor tissue (EST) or non-embryogenic callus
(NEC) from zygotic embryos of 5 different C. sempervirens clones. The dates refer to the year 1993. Percentages
recorded after 8 weeks of culture on inductive medium, containing 10 ~tM 2,4-D, and incubation at 23 ± 1°C in the
1lark
o/o of immature embryos forming EST or NEC
Date of 'Cione47' 'Clone 162' 'Cione267' 'Cione302' 'Clone 327'
collection
EST NEC EST NEC EST NEC EST NEC EST NEC
April26 20.0 70.2 0 0 3.6 63.6 0 0 0 0
May 4 0 9.3 0 0 4.4 12.5 0 11.7 0 0
May11 2.6 9.0 0 10.4 15.0 9.8 0 10.8
May 18 2.6 9.8 2.8 26.7 1.6 23.3 0 9.3
May24 0 24.1 0 60.0 0 15.0 0 26.7 0 24.1
May31 0 61.1 l.S 78.5 0 9.5 1.2 52.4 0 25.0
June 8 0 62.5 0 61.7 0 36.7 0 36.7 1.4 52.8
June 16 0 51.4 0 64.3 0 58.3 0 12.5 2.8 51.4
June 23 0 48.6 0 57.4 0 63.3 0 0 1.4 68.0
0 70.2 0 70.2

Table 3. ~-glucoronidase gene activity by histological assay in EST of C. sempervirens after microprojectile DNA-
delivery, with reference to different plasrnids and subculture periods (Lambardi et aL, 1998)

Subculture PI as mid (means± standard errors)

(days) pBI426 pRT99GUS pCGUilO

9 166.6 ± 50.6 cd<•> 177.6 ± 30.8 cd 314.3 ± 58.9 be

15 335.2 ± 27.5 b 81.8 ± 16.4 d 491.8 ± 10.2 a
21 173.6 ± 16.1 cd 7l.S ± 8.5 d 193.2 ± 35.2 bed
X 225.2 A(ll 110.3 B 333.1 c

<•> Means followed by different small letters are significantly different at p~.05
(2) Overall means referring to each plasmid are significantly different when followed by different capital letters
564

Figure I. An age-old cypress with a typical pyramidalis crown.

 565

Figure 2. Induction and evolution of EST in C. sempervirens. (A) Mucilaginous and translucent embryogenic tissue
just initiated on inductive medium from an immature zygotic embryo. A cluster of somatic embryos at tbe
filamentous stage is visible. (B) A typical embryonal-suspensor mass, consisting of multiple meristematic heads
(arrows), formed by densely packed cells and subtented by vacuolated suspensor cells. (C) By cleavage, tbese
polarized structures originate multiple pro-embryos (arrows), each consisting of a meristematic head and an
elongating suspensor. Bars = 0.5 mm (Lambardi et al., 1994).
566

Figure 3. Development and maturation of somatic embryos. (A) An embryogenic line cultured on maintenance
medium, showing a high concentration of pro-embryos. Bar = I nun. (B) Many somatic embryos (arrows)
developing asynchronically from EST, during subculture on the charcoal-containing maturation medium. Bar = 5
nun. (C) A somatic embryo at an early cotyledonary stage, showing no sign of radicle development. Bar = I nun.
(D) Somatic embryo maturation after isolation on a filter-paper bridge, soaked with liquid hormone-free OCR
medium. Bar = 5 nun.
 567

-
Figure 4. Transient gene expression in cypress EST. (A) High GUS gene expression in embryogenic tissue spread
on a filter paper and bombarded with the pCGUW plasmid. Bar = 10 mm. (B) A single pro-embryo showing
extensive GUS gene expression. Bar= 0.1 mm (Lambardi eta/., 1998).
 19. SOMATIC EMBRYOGENESIS
IN RATTAN (Calamus spp.)

D.K.S. Gohi, 0. Monteuuis 1•2 and M-C Bon 1

Plant Biotechnology Laboratory, CIRAD-Fon::t/Innoprise Corporation,
1

P.O. Box 60793,91017 Tawau, Sabah, Malaysia;

2Current address: CIRAD-Foret, Campus de Baillarguet, B.P. 5035,

34032 Montpellier Cedex 1, France

Chapter Contents

1. Introduction
2. Propagation of rattans: a review
2.1. By seeds
2.2. By conventional vegetative techniques
2.3. In vitro propagation by axillary budding
2.4. In vitro propagation by somatic embryogenesis
3. Recent advances in somatic embryogenesis of rattans
3.1. Protocols
3 .1.1. Initial explants
3 .1.2. Culture conditions
3.2. Initiation and development of somatic embryos
3 .2.1.Morphological observations
3 .2.2.Histological observations
4. Discussion
5. Conclusions and prospects
6. Acknowledgments
7. References

1. Introduction

Rattans are climbing palms (Fig.1) belonging to the large subfamily Calamoideae
(Moore, 1973). World wide, the rattans in total may be represented by 600 species in 13
genera (Uhl and Dransfield, 1987). Of these, Calamus is the largest genus distributed
from tropical Africa, Indian subcontinent, south China and east through the malesian
region to Fiji, Vanuatu and eastern Australia. It consists of about 370-400 species all
sharing the presence of overlapping reflexed scales on the fruit. Their climbing growth
habit is associated with the presence of particular climbing organs: the cirrus which is
569
S.M Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 569-585.
© 2000 Kluwer Academic Publishers.
570
an extension of the leaf rachis, or the flagellum which is a sterile inflorescence
originating from the top of the leaf sheath obliquely opposite the petiole (Dransfield,
1997). Some rattan species are growing as single-stemmed while others may be
clustered, which is of silvicultural importance. Rattan stems, covered with leaf sheath
usually armed with spines, can grow to enormous length - up to I 00 meters for Calamus
manan- and their diameter varies from species to species. Most of the asian rattans are
dioecious, which means that there are separate male and female plants.

Figure I. Calamus subinermis growing in its natural environment in Sabah

(East Malaysia, north of Borneo).

As a non-wood forest product, the uses of rattans and especially Calamus

species are numerous and varied. Due to their aesthetic features, strength, flexibility and
uniformity, the canes are an attractive source of raw material for such uses. These range
from furniture making to various local applications such as the making of baskets, mats,
traps and bird cages, to the provision of food for local villagers through the consumption
of young shoots or the fruits (Dransfield, 1979; Basu, 1985; Umali-Garcia, 1985).
In Southeast Asia, the commercial trade of furniture has contributed
significantly to the local economies and export sales of producer countries. In Indonesia,
the Philippines, Thailand, and Malaysia, an annual figure of almost US$400 millions
within a span of I 0 years was reported (Kiew, 1989; Dransfield and Manokaran, 1993).
As a result of this striking rattan trade, the cane supply, mostly from the wild (90%),
 571

begins to show signs of depletion in this region. An awareness of the impending problem
with the dwindling supply of the raw material began to surface and spread among these
countries.
It was thus during the 1980s that producer countries banned the export of raw
rattan except as semi-processed or finished products. This was in order to stimulate the
development of rattan-based industries locally, as well as to protect the remaining wild
resources. A management system of both timber and non-timber forest species such as
rattans within gazetted forest areas was developed (Singh and Yuan, 1999). In particular,
state forestry departments were encouraged to replenish the rattan resource in logged-
over forests through large-scale cultivation or undertake silvicultural practices with a
view to establish rattan plantations on a commercial scale. Efforts to assess and
determine the status of natural rattan stands, particularly those with economic
importance, were made in order to come up with a sustainable management plan
(Appanah et al., 1999; Unchi, 1999). Conjointly, emphasis has been given to research
on rattan reproduction and cultivation to counteract the problem of depletion. Some
priority areas on the list are the development of suitable programs and technologies for
proper rattan management, improvement and particularly, the propagation of
economically important rattan species such as those with large diameter cane or with
faster growth or earlier maturity (Alloysius and Bon, 1996). Research on the latter aspect
is crucial as it generally takes more than 15 years for the large-caned species before most
rattan cane is mature enough to be harvested.
There are different possibilities of propagating rattans species, as described
thereinafter. However, this review is based primarily on the research work carried out
within the Plant Biotechnology Laboratory in Sabah, Malaysia, on the micropropagation
through axillary budding and more particularly, somatic embryogenesis ofthree large-
caned Calamus species namely: C. manan Becc., C. merrillii Becc., and C. subinermis
H. Wend! ex. Becc.

2. Propagation of rattans: a review

2.1. BY SEEDS

Conventionally, rattans are propagated through seeds. Rattans produce large quantities
of fruits during each growing season. For example, C. manan can produce as many as
5,000 fruits that can all mature at the same time. However, more and more immature
rattans from the wild are extracted before fruiting, which drastically affects the
production of seeds. Another impediment is that the viability of rattan seeds decreases
in a short span of time (Baja-Lapis, 1998). According to Pritchard and Davies (1999),
although the germination rate is likely to be a function of the quality of the seed lot, the
harvesting time and post-harvest treatments may also be important in germination
response. In most cases, the timing of fruit harvests and sowing of seeds do not coincide.
Mori eta!. ( 1980) have demonstrated that C. manan fruits stored in closed plastic bags
can maintain viability of up to only I month at room temperature and 3 months at
temperatures between I 0 and 14 a C. The moisture content of the fruits appears to be
572

crucial to the viability of the seeds, especially in the case of long-term storage. A study
made by Pritchard and Davies ( 1999) showed a direct correlation between moisture
content (about 40%) and cool temperature (16°C) for high germination rates (60 to
90% ), depending on species.
Alternatives to the use of seeds as a planting source are therefore crucial as a
counter-measure to this depletion. This is where the propagation of rattan by vegetative
means becomes important. Although the vegetative propagation for plant improvement
programs has been extensively developed for many species, there are only a few reports
for rattans (Yusoff and Manokaran, 1985; Umali-Garcia, 1985; Alloysius and Bon,
1996; Goh et al., 1997).

2.2. BY CONVENTIONAL VEGETATIVE TECHNIQUES

Rattans grow either as single-stemmed individuals or as clusters with the production of

numerous stems (Dransfield, 1979). Conventional vegetative propagation has employed
offsets from these clusters and also from rhizomes as planting material especially when
seeds are of limited supply. Their survival in the nursery after they had been separated
from the mother plant depends on factors such as the ease of extraction, the size of the
offsets, the number of roots present, the potting medium used, and the season of
collection (Umali-Garcia and Canlas-Mendoza, 1996). However, offsets can be difficult
to come by for extensive propagation. In addition, such shoots are bulky, expensive,
difficult to handle and transport from the natural forests to the sites of planting, and
generally show a low survival rate after transplanting in the field (Yusoff and
Manokaran, 1985). The use of offsets is particularly valuable when considering the
clonal propagation of superior genotypes particularly for the establishments of seed
orchards of economically important species. However, for single-stemmed species such
as C. manan, the alternative means of propagation is only by tissue culture (Goh et al.,
1997).

2.3. IN VITRO PROPAGATION BY AXILLARY BUDDING

In vitro multiplication of shoot apices through axillary budding is carried out by using
explants consisting of the collar region from either in vitro germinated seedlings or from
nursery plants (Umali-Garcia, 1985; Goh et al., 1997). Early works on the tissue culture
of rattan were on the multiplication of vegetative shoots within Calamus spp .. Patena et
al. (1984) reported on the multiplication of about 2,335 shoots from a single seed of C.
manilensis H. Wend. within thirteen months under the given in vitro conditions. Yusoff
( 1989) managed to induce the formation of multiple shoots from the collar region of 2-6
months old in vitro germinated seedlings of C. manan. Goh et al. ( 1997) demonstrated
the possibility of micropropagating through multiple shoot production, the three most
economically valuable rattan species i.e. C. manan, C. merrillii and C. subinermis, either
from in vitro germination, or from outdoor seedlings. However, the multiplication rates
were shown to vary a lot from one explant to another within the same species.

2.4. IN VITRO PROPAGATION BY SOMATIC EMBRYOGENESIS

 573

So far, the most widely used procedures for micropropagating palms in tissue culture
conditions have been somatic embryogenesis, or indirect organogenesis via callus
(Tisserat, 1984; Schwendiman et al., 1988; Paranjothy, 1993; Bhaskaran and Smith,
1995; Verdeil and Buffard-Morel, 1995). Clonal propagation via somatic embryogenesis
offers prospects for mass cloning of genotypes selected for outstanding traits (Haines,
1994; Jain et al., 1995). However, the prerequisites to commercial scale clonal
propagation of any species would be the genotypic responsiveness for high frequency
somatic embryogenesis, genetic conformity, normal development and behavior including
seed and fruit production (Paranjothy, 1993; Rival et al., 1997).
The possibility of using this technique to multiply selected individuals offers
a practical means particularly for species such as the single-stemmed C. manan. To
avoid the risks of losing superior genotypes associated with the use of the sole shoot
apex as the explant, research on the development of somatic embryogenesis protocols
for the efficient and reliable production of planting material has been keenly pursued in
the past decade.

3. Recent advances in somatic embryogenesis of rattans

Previous works in this area on rattan were reported only several years ago and involved
the regeneration of plantlets from callus. Umali-Garcia (1985) attempted to culture
eleven species of Calamus and two species of Daemonorops with several media
compositions. Callogenesis from the shoot primordia was achieved in all thirteen species
and regeneration in two species.
Plantlets were also successfully obtained from friable callus arising from
cultured zygotic embryos of C. manan on media containing either 2,4-di-
chlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) at varying
concentrations (Yusoff and Manokaran, 1985). In another study, Mulung (1992)
cultivated apical meristem shoot tissues on 2,4-D and regeneration of multiple shoot
initials from massive callus was possible. However, for these studies, somatic
embryogenesis has not been unequivocally demonstrated. The most recent report by Goh
et al. (1999) involving the application of histology for the analysis of somatic embryos
derived from roots of C. manan presented for the first time evidence of the process in
rattan.
This chapter will attempt to assess the strategies that need to be developed for
somatic embryogenesis of rattan plants based on results obtained for three large-caned
species of major economical importance, namely:
Calamus manan, single-stemmed species, found naturally in Sumatra, Southern
Kalimantan, Peninsular Malaysia and southern Thailand (Dransfield et al., 1994);
Calamus subinermis, multi-stemmed species, endemic to Sabah (Malaysia),
Palawan and part of Sulawesi (Indonesia);
Calamus merrillii Becc., multi-stemmed species, endemic to the Philippines.

3.1. PROTOCOLS
574

3 .1.1. Initial explants

Initial explants generally consisted of shoot apices, very young leaves and root tips from
in vitro germinated seedlings as well as zygotic embryos. Callogenesis could also arise
from new roots or very young leaves from plants growing in the nursery or field.
However, the use of in vitro- germinated seedlings as source explants avoided the critical
disinfection step required for materials collected from outside plants. As such, the
technique of somatic embryogenesis was tested first on explants from 3 to 4 months old
seedlings, with the ultimate objective to adapt the protocol to mature selected field
plants. Zygotic embryos were used as explants depending on the seasonal availability of
fresh fruits each year. Zygotic embryos were excised from mature seeds after scrubbing
the fleshy sarcotesta off the fruits with a hard brush. The seeds were then disinfected
with 10% commercial grade sodium hypochlorite for 10 min followed by three washings
in sterile water. Extraction of the fairly large embryos (2-4 mm), located beneath the
operculum, resulted in more than 95% contamination-free samples for C. manan (Goh,
1997). In contrast, the excision of the smaller zygotic embryos (less than 2 mm) from
C. subinermis was more difficult. Additionally, as the surface ofthe seeds is deeply
pitted, the level of contaminations from inoculation of only the tiny embryo remained
high at more than 70%.

3.1.2. Culture conditions

For the induction of callus, root tip fragments of about 1-2 em in length or zygotic
embryos or leaf explants (1 em square) were inoculated onto a basal Murashige and
Skoog (1962) macro- and micronutrients culture medium supplemented with I 00 mg ]· 1
myo-inositol, 500 mg ]· 1 casein hydrolysate, 2 mg ]· 1 glycine, 1 mg ]· 1 thiamine, 1 mg 1· 1
pyridoxine-HCL, 1 mg ]· 1 nicotinic acid, 30 g ]· 1 sucrose. The selected auxin, picloram,
was added to the basal medium at different concentrations according to the stages of
culture as indicated thereinafter. After pH adjustment to 5.6-5.8 with IN KOH and the
addition of7 g ]· 1 "high gel strength" Sigma agar, 12.5 ml of the medium were dispensed
into 21 x 150 mm glass test tubes prior to sterilization by autoclaving at 120°C and 95
kPa for 20 min. This constituted the primary culture medium for the induction of
embryogenic cells. For the further development of embryogenic cells, the maturation
medium constituted a lower concentration or complete absence of picloram. All the
cultures in glass tubes were maintained in total darkness at 26± 2 oc and relative
humidity (RH) of90-95% for the induction process, and under a 16 h photoperiod (50-
60 f.!mol m·'s·', "TLD 36W/84 Philips" fluorescent lamps) at 28±2°C, and RH 70% for
the germination of the somatic embryos.

3.2. INITIATION AND DEVELOPMENT OF SOMATIC EMBRYOS

3.2.1. Morphological observations

At the morphological level, the induction of calli was noted to vary according to the
types of explant and species studied (Table I). Upon the transfer of calli with potential
for further development to the maturation medium, the development of the somatic
embryos was similar regardless of the species.
 575

TABLE I. Proportions of calli obtained for different types of primary explants expose.d.to the same
experimental conditions for each of the three Calamus sp.

Type of primary explant

Species
Zygotic embryo Leaf portion Root tip

C. manan I 03/200=51. 5% 1150=2% 39/50=78%

C.subinermis 96/200=48% 0/50=0% 34/50=68%

C. merrillii 180/200=90% 42/50=84% 37/50=74%

Callus induction. In C. manan, root explants appeared to be the most responsive for the
induction of callogenesis (Fig. 2a and b) in contrast to zygotic embryos or leaves that
rapidly became oxidized even when placed in darkness. The calli that arose from about
50% of the zygotic embryos introduced in culture consisted of large compact masses of
haustorium-like tissues that failed to differentiate upon transfer to the maturation
medium. Eventually, the surface of these tissues became·fuzzy after which vitrification
or oxidation occurred. By contrast, when placed in the same initiation conditions, less
than 2% of leaf explants responded and were therefore disregarded for further studies.
The best scores were observed for root tips explants with.78% of them producing callus
3 to 6 months after inoculation. Primary calli occurred on apices of primary or secondary
roots or along the cut surface of the root as protuberances of the central cylindrical zone.
In some cases, the transfer of friable calli to the maturation medium consisting of a lower
picloram level resulted in a reversion to a soft non-friable callus stage. Conversely, calli
which were sub-cultured several times to the primary medium with the same
concentration of picloram (7.5 mg 1' 1) became progressively translucent. New
proliferation of yellowish calli was observed and upon transfer, progressively
differentiated under suitable growth conditions.
In C. subinermis (Table 1), the response of zygotic embryos to the induction
medium followed a similar morphogenetic pattern as in C. manan, resulting in whitish
callus masses in about 50% of the samples introduced. In the case ofleaf explants, none
of the introduced samples responded. For root explants, 68% of the samples were
responsive (Fig. 3a), however, the subsequent development of callus was much slower.
It took more than 6 months for the induction of callus and another 6 months for the
formation of nodular structures. Reversion of some callus masses was also noted upon
the transfer of the calli to media with lower auxin concentrations. Owing to the slow
response of most samples, phenolic oxidation ultimately limited further morphogenesis
Among the three rattan species, C. merrillii demom;trated the most prospects
for regeneration via the process of somatic embryogenesis (Table 1). Although
callogenesis was induced in about 75% of the introduced root explants, no embryogenic
structures evolved from the calli during the course of our study. The calli eventually
became translucent or brownish due to phenolic oxidation after subsequent transfers.
Zygotic embryos and young leaves (Fig. 3b) from in vitro seedlings used as explants
were equally and highly responsive at 90% and 84%, respectively, of the samples
introduced. Within 6 weeks after inoculation, callogenesis was observed in both types
576

Figure. 2. Morphological aspects of somatic embryogenesis in Calamus manan. (a) Primary and creamy
callus proliferating on an apex of a secondary root explant; bar=Smm. (b) Primary callus proliferating at
the cut parts and the apex of a primary root explant; bar=Smm. (c) Nodular callus after several months of
culture; bar=lcm. (d) Translucent and milky white globules on callus; bar=lcm;. (e) Embryo-like
structures at different stages of development; bar=3mm. (f) Somatic embryo-derived plantlet or embling;
bar=lcm. (g) Seedling; bar=Smm (From Goh et at., 1999. Copyright c 1999 by the Society for In Vitro
Biology -formerly the Tissue Culture Association. Reproduced with permission of the copyright owner).

of explants
In the early phase of the process, a mixture of white and friable calli was noted,
the friable calli tending to become yellowish in its later stage. Thereafter, it was
necessary to sub-culture the calli several times at 4 to 8 week intervals to either the
 577

Figure 3. Morphological and histological aspects of somatic embryogenesis in Calamus subinermis and
Calamus merrillii. (a). Primary friable callus proliferating from the apical zone of a secondary root explant
from a Calamus subinermis in vitro germinated seedling; bar=5 mm. (b). Pro-embryos (P) originated
from a callus formation (C) produced by a leaf explant (L) from a Calamus merrillii zygotic embryo;
bar=5 mm. (c). Section of a somatic pro-embryo of Calamus subinermis showing the shoot (sa) and the
root (ra) apices; bar=260 11m. (d). Section of a somatic pro-embryo of Calamus merrillii showing the shoot
meristem (sm) and the pro-cambium (pc); bar=26011m

primary medium or onto maturation medium with a lower concentration of picloram

ranging from 1 to 5 mg J·'.

Maturation and germination ofsomatic embryos. The subsequent development of calli

to somatic embryos was similar in all three species and as such, will be described
without specific reference to any species unless otherwise noted. When transferred onto
media containing lower auxin levels (1 to 5 mg J·'), a mixture of non-friable calli,
578

nodular and/or irregularly shaped globular structures was observed within the same
callus (Fig. 2c and d and Fig. 3b). Discrete structures with a dome-shaped appearance
usually glossy and translucent were preferably selected and considered as promising
somatic embryos (Fig. 2e). From these, however, only a low percentage of germination
was observed for C. manan and C. subinermis, at 10 and 5%, respectively. In contrast,
70% of the obtained somatic embryos germinated for C. merrillii. Germination was
indicated by the bipolar development with the appearance of the plumule and radicle
(Fig. 2t). The whole process from callogenesis to the formation of the first leaf and
elongation of the primary radicle was quite slow and took more than 12 months for the
three species (Goh et at., 1999). In a few cases, it was interesting to note on primary
somatic embryos the presence of secondary embryos; the formation and historical origin
of which remain unknown (Fig. 4).
Some batches of somatic embryos did not go through a normal developmental
pattern as described. Abnormalities such as the appearance of multiple shoot apices and
profuse rooting from compact aggregate of embryogenic structures were npted in about
5% of the samples in all three species. Attempts at elongating these shoot apices using
different growth regulators or culture conditions failed to produce any positive
morphological changes. Instead, some clusters developed into white compact masses
similar to those obtained for zygotic embryo explants of C. subinermis and C. manan.
The dissection of these tissues revealed the presence of greenish shoot-like structures
inside, indicating a form of reversion most likely due to some ontogenetical defects.

Figure 4. Occurrence of secondary or adventitious somatic embryogenesis (arrows); bar =5 mm.

 579

3.2.2. Histological observations

To better understand and ascertain the process of somatic embryogenesis, an histological
examination of the various structures obtained was undertaken applying the procedure
described in Goh eta!. (1999).
These histological observations showed that primary calli proliferated mainly
from the perivascular zone of root explant for C. manan and C. subinermis, and ofleaf
and zygotic embryos for C. merrillii (Aliotti, 1999; Gob eta!., 1999). In general, at the
initial stage, two types of calli could be distinguished visually and were selected for
histology: soft and whitish calli, and friable and yellowish calli. Soft whitish calli
consisted of highly differentiated cells that were. vacuolated and with few starch
reserves. Over time, in accordance with the morphological state, such calli comprising
of necrotic cells and cells with cytoplasm containing high levels of polyphenolic
compounds failed to respond and could not be maintained in culture. In contrast, friable
and yellowish type of calli appeared to arise from a mixture of undifferentiated cells and
actively dividing embryogenic cells with an enlarged nucleolus and a higher
nucleoplasmic ratio and few starch reserves. Active division of these cells giving rise
to embryonic zones, could be observed (Fig. 5). These progressively became embryonic
and could be easily located by the intense staining of the cytoplasm due to the presence
of abundant soluble proteins and very big nucleolus.
In C. subinermis and C. merrillii, embryonic cells were also often surrounded
by a polysaccharidic mucilage which originated from the modification of the median
lamella. This feature was generally indicative of healthy cells with prospects for further
development. The clusters of embryonic cells, while losing their starch reserves, when
transferred onto media with decreasing concentration of picloram, delimited into zones
that later evolved into proembryos.
Proembryos at the globular stage initially consisted of undifferentiated cells
with uniformly thin walls except in the peripheral region. This characteristic
progressively isolates these structures from surrounding degenerating cells. At this stage,
it was apparent that degeneration of proembryos would occur when culture conditions
were unsuitable. During the maturation of the proembryos, a protoderm was formed
through the establishment of a peripheral zone consisting of one and then several layers
of cells and vascular tissues appeared. These changes were associated with the formation
of bipolar embryos in which both shoot and radicle appeared at each end (Fig. 3c and d,
Fig. 5h), notwithstanding the fact that often shoot pole develops first. Unlike in zygotic
embryos that exhibited limited starch and protein reserves, no reserves at all were
detected in the embryonic body. This could account for the limited germination potential
of some somatic embryos.

4. Discussion

The process of somatic embryogenesis has been reported for numerous tropical plants
such as cocoa (Pence eta!., 1980), coffee (Berthouly and Michaux-Ferri<!re, 1996),
papaya (Fitch, 1993) and rubber (Michaux-Ferri<!re and Carron, 1989). The only palm
580

Figure 5. Histological aspects of the evolution of somatic embryogenesis in Calamus manan. (a) Callus
originating from perivascular cells of the root explant; bar=l60 11m. (b) Undifferentiated cells within a
callus; bar=20 11m. (c-e) First mitosis (c; bar=811m) giving rise to a zone of embryonic cells (arrows: d;
bar=80 11m; e; bar= 40 Fm). (f) Proembryos arising (arrows) from degenerating callus ; bar= 80 Fm. (2g)
Shoot (sa) and root apices (ra) of a zygotic embryo; bar=300 11m.(h) Histological aspect of a somatic
embryo. sa= shoot apex; bar=600 11m (From Goh eta/.. , 1999, Copyright c 1999 by the Society for In
Vitro Biology· formerly the Tissue Culture Association. Reproduced with permission of the copyright
owner).
 581

species most similar to rattans, particularly C. manan in respect to the single-stemmed

feature of the species, which have extensive studies on the propagation of plant materials
via this type of regeneration process is the African oil palm, Elaeis guineensis ( Ahee et
a!., 1981; Pannetier et a!., 1981; Jones, 1983; Paranjothy, 1984). The procedures
developed for the oil palm have been successful enough to be transferred from the
laboratory to the industrial production units, resulting in the mass production of high
quality planting materials (Hannower and Pannetier, 1982; Duval eta!., 1988; Wooi,
1995; Zamzuri eta!., 1998). These possibilities have prompted similar studies on the
three Calamus species lising primarily picloram as the most effective callus-inducing
auxin, similarly to date palm (Benbadis, personal communication). Additionally, the
microscopic analysis has been very useful for ascertaining the somatic embryogenesis
process while increasing our understanding at the histological and cellular level.
In general, the onset of embryogenesis in palms, with special mention for oil
palm, is recognized to be of multicellular origin (Schwendiman eta!., 1988). In rattans,
as established by our histological investigations (Aliotti, 1999; Goh eta!., 1999), the
process of embryogenesis appeared to be of a unicellular origin under the given culture
conditions unlike many palms but similarly to coconut, Cocos nucifera (Verdeil eta!.,
1994). The sequence of histological events- cell separation, wall thickness increase and
internal segmenting divisions leading to proembryos- in this specie·s were similarly
observed in rattans, strengthening the single cell origin hypothesis of the somatic
embryos obtained.
Our results showed an indirect somatic embryogenesis model, asynchronous,
and of moderate frequency. As for many palms (Tisserat, 1984; Guerra and Handro,
1991; Paranjothy, 1993; Bhaskaran and Smith, 1995; Verdeil and Buffard-Morel, 1995),
induction and expression were dependent strictly on the stage involved, the type and age
of explant, and the auxin concentration. Although these influencing factors may appear
in most plant species, they may not interact similarly or at the same level for the
successful development from callus to the plantlet stage (Thorpe, 1988). In addition,
callus proliferation in rattans might be dependent upon the genetic background of the
explant source as observed for the oil palm (Ointing and Fatmawati, 1995; Wooi, 1995).
In coffea species, differences in both genotypes and optimal culture conditions are
contributing influences on the percentages of high frequency calli (Sondhal and Sharp,
1979; Berthouly and Michaux-Ferriere, 1996). Thus, in the three Calamus species, visual
observations such as the reversion of the embryogenic stages to unresponsive compact
tissues, the presence of numerous irregularly-shaped structures or the production of
multiple shoots could be due to these factors. The defect in ontogenesis thereby led to
an overall inefficient production of somatic embryos.
For mass cloning of selected plants, embryogenesis is preferred over
organogenesis as this process bypasses the difficult step of rooting in the production
procedure for rattans. This is especially so when bearing in mind somatic embryos
develop both root and shoot on the same culture medium, although most of the. time the
shoot pole develops first. Rooting prior to their transfer to the nursery for ex-vitro
acclimatization is quite crucial to the final survival of the rattan plants. The tendency to
root is typical of most monocots in the presence of auxins. Indeed, at the morphological
level, profuse rooting could be observed for some clusters in C. merrillii after their
582

transfer.
Results from our study have demonstrated the process of somatic
embryogenesis in the three rattan species. There is up to now limited information at the
organogenesis level related to rattan zygotic embryos. However, our observations
revealed similar morphological and developmental patterns (Fig. 6). This has been
further confirmed by a histological analysis of the embryogenic tissues for each of the
three species.

Figure 6. In vitro development of a somatic embryo-derived plantlet (E)- also called somatic seedling or
embling -, and of a seedling (S) in Calamus subinermis

5. Conclusions and prospects

The protocol for plant regeneration of rattans through somatic embryogenesis has still
to be improved to fulfil the requirements where mass cloning is concerned, and
particularly when starting from mature selected plants. In this respect, the possibility of
using inflorescences as primary explants, as successfully experienced in date palm
(Bhaskaran and Smith, 1992) and coconut (Verdeil eta/., 1994) for instance, seems
particularly attractive for rattan species, especially for the single-stemmed ones. The
other option could be to use root tips, as practiced on oil palm (Jones, 1983), but all the
attempts tested so far on C. manan have failed, mainly due to unadapted disinfection
protocols. In addition, collection of root tips seems harder than for oil palm.
Another concern is whether the regeneration system is reproducible, i.e. can
the callus be indefinitely sub-cultured and retain its regeneration potential or whether the
regeneration is a single occurrence oflimited multiplication potential. Further assessment
of the culture conditions would be necessary and useful to ascertain this .
The next question is whether the plant originating from the callus is genetically
identical to the mother plant originally selected, i.e. is not affected by any somaclonal
variation problem. In oil palm, evaluation of clones in the field has shown the occurrence
 583
of about 5% of variants with abnormal floral development which then may have a slight
influence of fruit setting and thus, yields (Corley, 1993; Paranjothy, 1993; Rival eta!.,
1997).
Ultimately, an evaluation of the performance of the somatic embryogenesis-
derived plants in the field will be important to establish the possible existence of these
variations. This is currently underway (Fig. 7). If somaclonal variations do occur, it will
be important to assess the effect of such anomalies, positive or negative, on the further
development and behavior of the somatic embryogenesis-issued plants. Once the genetic
fidelity of somatic embryogenesis process is demonstrated, only then can the process for
propagating improved quality rattan plants be envisaged. However, based on the
information available so far, cost and time can be serious impediments to the utilization
of somatic embryogenesis for mass producing planting material for large-scaled clonal
industrial plantations of rattans. The returns may simply not warrant such an investment
compared to plantations from low cost nursery-produced seedlings. An interesting option
could be to apply somatic embryogenesis to a limited number of selected genotypes to
be utilized as seed producers within vegetative orchards. The cost of the somatic
embryogenesis-issued individuals will be diluted through the large number of seeds
produced every year. These may be biclonal orchards, taking advantage of the dioecy of
the rattan species. At this point in time however, more information is needed, especially
in the field of genetics, to justify this option and to figure out experimentally what can
be practically expected from such clonal orchards.
Lastly, somatic embryogenesis can be judiciously combined with
cryoconservation technology as successfully developed for oil palms (Engelmann et a!.,
1985; 1988, Ashmore, 1997) for the storage of endangered rattan genotypes and even
species. This could prevent them from total extinction as the result of wild harvesting
operations which are increasing dramatically.

Figure?. Calamus merrillii somatic seedlings ready to be planted.

584

Acknowledgments

This work was supported as part of the European Col111l1ission-funded project on the
"Conservation, Genetic Improvement and Silviculture of Rattans in South-east Asia"
project NE-TS3*-CT940285-. We would also like to express our appreciation to Dr.
Nicole M-Ferriere, CIRAD-Biotrop, Montpellier, France, for her helpful comments and
assistance in the histological analysis.

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 20. Somatic embryogenesis in jojoba (Simmondsia chinensis)

Veena Agrawal, Surya Prakash and Shrish C. Gupta

Department of Botany, University of Delhi, Delhi - 110007, India

Contents

Introduction 2 In vitro propagation

1.1 Properties of jojoba oil 2.1 Organogenesis
1.2 Uses 2.2 Somatic embryogenesis
1.2.1 Lubricants 2.2.1 Explant source and
1.2 .2 Factices and adhesives initiation of callus
1.2.3 Acids and alcohols 2.2.2 Differentiation of
1.2.4 Cosmetics somatic embryos
1.2.5 Pharmaceuticals 3 Conclusions
1.2.6 Feed 4 Acknowledgements
1.2.7 Other uses 5 References

1 Introduction

Simmondsia chinensis (Link) Schneider, commonly known as "jojoba" or "hohoba".

is a dioecious, evergreen perennial plant belonging to the monotypic
family Simmondsiaceae. It is a native of Sonoran desert of the south western
United States of America, north western Mexico and Baja California (Gentry,
1958; Benzioni, 1995). This species is distributed over about 256,000 square
km area from 25 to 31° North latitude and from 109 to 117° West longitude
(Gentry, 1958).
Jojoba is cultivated mainly for its seeds which store liquid wax whose
properties are similar to spermaceti (sperm whale oil) - a substitute for
some petro products and a valuable high-priced lubricant. Spermaceti is found
in the heads of sperm whales, an endangered marine mammal and the oil
can be extracted only after killing them. During the II World War, US trade
imposed a ban on killing of sperm whales due to the danger of their becoming
extinct. Their Director of Priorities, Office of the Production Management.
at the same time declared that no adequate substitutes were available for
spermaceti. Simultaneously, to fulfil its demand, scientists began to search
an alternative source. Amongst 15,000 plant oils tested and chemically
analyzed by U.S. Department of Agriculture, S. chinensis was the only one
587

S..'v!. Jain. P.K. Gupta and R.J. Newton {eds.). Somatic Embryogenesis in Woody Plants, Volume 6. 587-604.
© 2000 Kluwer Academic Publislrers.
588

whose seeds contained liquid wax similar to that of spermaceti (Anonymous,

1985). As early as in 1933, Greene and Foster discovered that its seeds
contained liquid wax. Since then, S. chinensis has occupied an important
place as an agronomically valuable crop for genetic manipulation and large
scale exploitation.
In India, it was introduced at the Central Arid Zone Research Institute
(CAZRI), Jodhpur, from Israel in 1965, but could not gather any momentum
for several years due to its slow growth rate. However, realizing its commercial
importance, as a source of liquid wax, added interest in this species
was revived all over the globe in 1978. Since then, over 70 collections
have been introduced in India from USA, Mexico, Tanzania, U.K., Israel
and USSR (Harsh et a/., 1987). Of these, EC 33198 is the most productive
clone.
Jojoba has a wide ecological amplitude and it grows from sea level up
to 1,220 m. It is capable of withstanding extremely high temperatures (ranging
from 35 to 48° C) and water stress (Al-Ani et a/., 1972). Jojoba can be
cultivated on well drained, coarse soil with silt and clay in lower zones
(Gentry, 1958).
Till date, growers have been mainly using seeds to raise orchards. However,
seedling based propagation is not advisable because (i) of poor seed germination,
and (ii) high ratio of male to female plants. Gentry (1958) reported as high
as 5: 1 ratio of male to female plants, if developed through seeds. On the
contrary, for plantation, 1:6 ratio of male to female has been recommended
(Laidig et al., 1984 ). In Indian environment, one male plant is sufficient
to pollinate five female plants (Harsh et al., 1987). The causes of disparity
in the sexual ratio in nature are not clearly understood but it is speculated
to be related to environmental factors and not the genetic (Gentry, 1958).
However, the chromosome number in male, female and hermaphrodite
plants is 2n = 52. Sex chromosomes have not yet been observed (Benzioni
and Forti, 1985).

1.1 Properties of jojoba oil

Greene and Foster ( 1933) and later Green et a/. (193 6) discovered that seeds
of jojoba contain 50-60% liquid wax by dry weight. However, composition
of wax varies over wide range of habitats (Miwa, 1971), even plants
growing at one and the same site may produce seeds with different amounts
of wax (Yermanos and Duncan, 1975). Raw oil extract of jojoba seeds comprises
97% linear wax esters. The rest contains tocopherols and free fatty alcohols
as well as fatty acids. It also has an amazing internal homogeneity as
more than 87% of the esters are present in combination with acids and alcohols
with carbon chains extending to 20-22 atoms with two double bonds, one
on each side of the ester bond (Sanchez et a/., 1992; Binman et a/., 1996).
 589

Each acid and alcohol that make up jojoba oil has a single double bond
(Mirov, 1952; Anonymous, 1985). In contrast to this, common vegetable
oils have fatty acids with carbon chain lengths of 16 to 18 atoms (Ayerza.
1993). The golden colour oil contains only traces of saturated wax, steroids.
tocopherols and hydrocarbons. It has promising physical properties
such as high viscosity index, high flash and fire points, high dielectric constant.
high stability and freezing point (Mirov, 1952), which can be used in various
industries. It does not become rancid due to the presence of natural antioxidants
such as alpha, gamma and delta tocopherols. It is also not damaged by repeated
heating to temperatures above 2 85° C or by heating to 3 7 0° C for four days
(Daugherty et a!., 1958; Miwa, 1971; Anonymous, 1985). Other physical
and chemical properties are summarised in Table 1.

1.2 Uses

Jojoba oil and its products have diverse uses as lubricants, factices and
adhesives; acids and alcohols, cosmetics, feeds, electric insulator, foam
coating agents, heating oil, plasticizers, fire retardants, transformer oil as
well as in pharmaceutical industries (Anonymous, 1985; Benzioni and Forti.
1985; Harsh et al., 1987).

1.2.1 Lubricants

Addition of sulfur or sulfur containing compounds enhances the lubrication

qualities. It is considered similar to or even much better compared to sperm
whale oil. In addition to sulfur, many molecules such as phosphorus, chlorine.
bromine, etc~ if added to it, form better lubricant additives. Sulfur chlorinating
jojoba oil particularly enhances wear properties, while sulfur brominating
oil improves the load carrying capacity (Wisniak and Benajahu, 1978).
Phosphonated and sulfurized jojoba wax derivatives possess good extraction
properties compared to the compounds commonly used in solvents for the
extraction of metal ions such as uranium and mercury (Binman eta!., 1996 ).

1.2.2 Factices and adhesives

By altering the conditions of sulfurization factices, rubber-like tacky masses

are obtained. These factices are suitable for the manufacturing process of
590

Table /. Physical and chemical properties of S. chinensis seed liquid wax (after Daugherty
et al., 1958).
Constituent /Parameter Level
Glycerol None
Oleic acid 0.66%
Palmitoleic acid 0.24%
Saturated acids 1.54%, 1.64%
Eicosanoic acid (C 20 ) 30.3%
Docosanoic acid (C 2) 14.2%
Eicosanol (C 20 ) 14.6%
Docosanol (C 22 ) 33.7%
Melting point 11.2-11.8° c
Solidifying point 6.7° c
Flash point JC.O.C.) 555° F
Fire point (C.O.C.) 640° F
Viscosity S.U. at 100° F/Sec. 127
Viscosity S.U. at 210° F/Sec. 48
Viscosity index (Dean and Davis) 173
Color number (A.S. T.M.) 2
Corrosion at 212° F, copper strip Nil
Pour point 10" c
Carbon residue 0.01%
Refractive index at 25° C 1.4648, 1.4650
Density at 25° C 0.8642, 0.8990 l
Specific gravity 25/25° C 0.8635, 0.8640
Saponification number 92.2, 95.0, 165.7
Acid number 0.23, 0.32, 0.57
Iodine number (Hanus) 81.7, 88.4
Acetyl number 6.8
Reichert Meiss1 number 0.70
Polenske number 0.31
Unsaponifiable matter 37.62%, 48.3%
Iodine number of unsaponifiables 77.2, 79.3-80.2
Acetyl number of unsaponifiables 171.8, 172
Soluble acids (as butyric) 2.43%
Insoluble acids 59.43%
Iodine number of total fatty acids 76. I
Acid number of total fatty acids 172
 591

linolium and printing ink. It has also been recommended as a valuable

material for paints, adhesives, varnishes, chewing-gums and flooring material
industries (Bhatia and Gulati, 1981; Anonymous, 1985).

1.2.3 Acids and alcohols

It can be a good source of monounsaturated alcohols and acids. These

can be used as intermediates in the preparation of disinfectants, detergents,
driers, emulsifiers, resins, plasticizers, protective coatings, fibres, and corrosion
inhibitors.

1.2.4 Cosmetics

The jojoba oil is mainly used in cosmetic industry due to its high aesthetic
quality and stability towards rancidity. More than 300 cosmetic products
with jojoba oil as ingredient have been developed in USA. Among them
shampoos, hair conditioners, hair sprays, facial oils, body oils, shaving creams,
hand lotions, make-up removers, lipsticks, lip glosses, vanishing creams
and skin fresheners are already being marketed (Anonymous, 1985).

1.2.5 Pharmaceuticals

It is also being used in the production of antibiotics like penicillin G and

cephalosporin. In comparison to sperm whale oil, the use of jojoba oil can
enhance the production of penicillin by more than 20 per cent (Yermanos,
197 4). Joj o ba oil is believed to have the potential of curing several skin
diseases. It prevents hydrolysis of vitamin A ester and has a strenuous inhibiting
effect on the growth of tubercle bacilli (Daugherty e t al., 19 58).

1.2.6 Feed

After the extraction of oil, the residue contains about 30 per cent proteins
as well as carbohydrates and fibres with balanced amount of essential amino
acids like lysine but poor content of methionine (Yermanos, 1974). Therefore,
it can be used as animal feed. However, due to the presence of four
toxic factors like, cyanomethylenecyclohexyl glucosides, collectively known
592

as 'simmondsin', its direct use as animal feed is prevented. Detoxification

and palatability improvement methods for this compound are in progress.
The preliminary research conducted on rats by Nestle Company, Switzerland,
have shown that it can be used as edible oil (Anonymous, 1985). Treatment
of jojoba. seed meal with selected strains of Lactobacillus acidophilus
lowers cyano levels by 90% in three weeks and improves palatability
(Verbiscar and Banigan, 1983).

1.2.7 Other uses

Jojoba oil can also be used as carrier for pesticides and plant hormones.
water-evaporation retardant', products .for sizing as well as water proofing,
formulations for softening leather, as an additive to some plastics, in the
treatment of waste water and recovery of rare metals (Anonymous, 1985).

2 In vitro propagation

2.1 Organogenesis

During the past few decades, in vitro technique has been successfully
used for propagating plants at least for a few taxa. It has been achieved
through either (i) inducing adventitious shoots which are then rooted or
(ii) somatic embryogenesis. The latter has a known potential for efficient
and rapid micropropagation (Ammirato, 1987; Roberts eta/., 1995). In addition.
it can assist in the application of recombinant DNA technology (Haissig
et a!., 1987; Rao and Bapat, 1995). Somatic embryos are formed by the
differentiation of dedifferentiated somatic cells and thus are theoretically
a clone of the donor plant (McKersie and Van Acker, I 994).
The first attempt on in vitro morphogenesis in S. chinensis was made by
Aragao in 1977. A little later, Rost and Hinchee ( 1980) published preliminary
report on the production of callus and regeneration via different explants
like shoot tip, leaf, stem and cotyledon on MS medium augmented with
3% sucrose along with various auxins and cytokinins. They reported that
all explants induced callus on medium containing auxins such as 2,4-D.
NAA and IAA as well as cytokinins namely IPA, kinetin and zeatin. In vitro
regeneration in S. chinesis has been attempted by several workers using
different explants like shoot tip (Aragao, 1977; Birnbaum, 1978; Mandani
et al .. 1978; Wochok and Sluis, 1979), polynodal segments (Arce and Jordan.
 593

1988), binodal and uninodal cuttings (Jacoboni and Standardi, 1987; Chaturvedi
and Sharma, 1989), epicotyl (Scaramuzzi and D' Ambrosio, 1988), leaf, stem
and cotyledon (Rost and Hinchee, 1980) as well as immature zygotic embryos
(Lee and Thomas, 1985; Wang and Janick, 1986). The results so far obtained
have been included in Table 2. In most of the investigations, juvenile explants
were used and in a few, the age of the donor plant has not been mentioned.
However, regeneration of juvenile explants could not be exploited for large
scale propagation primarily because the sex of the seedling-raised plants
cannot be determined and secundly, the ratio of male to female seedlings
is very high in nature (5:1, Harsh eta/., 1987).
Despite its immense economic value, only scant attention has been paid
to regeneration potential of mature explants. Chaturvedi and Sharma (1989)
developed plants through shoot proliferation in long term cultures of jojoba.
According to them, single node stem segments of both sexes were cultured
on modified Schenk and Hildebrandt s medium (1972) with kinetin or BAP
and IAA. Multiple shoots developed on 1 mg/1 each of BAP and IAA
augmented medium. The in vitro raised shoots induced roots on SH medium
with 7 mg/1 IBA + I mg/1 NAA + 1 mg/1 caffeic acid. In a subsequent
study, multiple shoots were induced in explants excised from field grown
female jojoba plants on a much simpler medium (MS + 20 JlM BA; Prakash
eta/., 1997). An year later, differential hormonal requirements for cloning
male and female plants were evaluated (Agrawal et a/., 1998). Explants
of male plants produced maximum shoots on MS medium supplemented with
10 JlM BA while those of female yielded best results on MS + 20 JlM BA.
Nearly 80% of the in vitro raised shoots induced roots if a pulse treatment
of filter sterilized 50 JlM IBA was given for 20 min. to female and 40
min. to male plant regenerants, prior to their transfer to semi-solid MS
medium supplemented with 10 11M IBA + 1 J.!M BA + 0.5% activated
charcoal.

2.2 Somatic embryogenesis

Somatic embryogenesis in S. chinensis has so far been achieved through

callus derived from immature zygotic embryos (Lee and Thomas, 1985; Wang
and Janick, 1986). Callus developed in 3-4 mm long embryos if transferred
to MS semi-solid medium containing 4.5 ~-tM 2,4-D. The callus organised
somatic embryos which could not develop into plantlets. For clonal propa-
gation of this value added crop, the zygotic embryo raised somatic embryos
may not be the right choice because this species is cross-pollinated and
each zygotic embryo is expected to possess its own genotype. On the contrary,
embryogenic callus raised from tissues, other than zygotic embryos, of selected
mature donar plants that too of known sexuality, should be ideal for mass
594

Table 2. In vitro morphogenic responses of S. chinensis Uojoba).

Explant Status Medium Mode of Result Reference

of regeneration
tissue

Shoot tip Juvenile MS+NAA+K Indirect Shoots and Aragao, 1977

organogenesis roots on
separate
explants
-do- -do- MS+IBA+BA -do- Shoot tip Mandani et a!.,
growth and 1978
root initiation
Leaf Mature (i) MS+NAA+ -do- Callus Rost and
IPA/K/Zea Hinchee, 1980
(ii) MS+NAA+ -do- Roots -do-
IPA/K
Stem -do- (i) MS+NAA+ -do- Callus -do-
IPA/Zea
(ii) MS+NAA+ -do- Roots -do-
25% cw
Cotyledon Juvenile MS+NAA+Zea -do- Roots -do-
orMS+IAA+IPA
Shoot tip -do- (i) MS+NAA+ Direct Multiple -do-
IPA/Zea organogenesis shoots
(ii) MS+NAA+ -do- -do- -do-
IPA+GA
(iii) MS 1/2 +IBA -do- Plantlets -do-
Immature -do- MS+BA+NAA Indirect Somatic Lee and Thomas,
embryo + 2,4-D embryogenesis embryos 1985
Immature -do- MS+2,4-D+BA -do- -do- Wang and
zygotic Janick, 1986
embryo
Node Four- MS+G~+NAA Direct Shoot Jacoboni and
year-old organogenesis proliferation Standardi, 1987
potted
plants
Node Mature (i) SH+BAP+ IAA -do- Multiple Chaturvedi and
shoots Sharma, 1989
(ii) SH+IBA+NAA -do- Plantlets -do-
+Caffeic acid
Node Juvenile MS+BA -do- Multiple Agrawal et a/.,
shoots 1996
Node Mature (i) MS+BA -do- -do- Prakash eta/.,
1997
(ii) MS+1BA+BA -do- Plantlets Agrawal et a/.,
+AC 1998, 1999
 595

production of desired plants. Lee eta!. (1984) and Lee (1988) have also
mentioned that improvement of jojoba plants using cellular and molecular
biology techniques is not well established mainly due to the difficulties
faced in regenerating plants from callus and protoplast cultures.
Recently, attempts have been made by us to differentiate somatic embryos
from the callus derived from leaf explants of physiologically mature female
plants.

2.2.1 Explant source and initiation of callus

The aseptic cultures were established by rearing nodal explants excised from
17 to 20-year-old female plrants, (\fter rigorous methods of sterilization.
The twigs, 8-12 inches long, were thoroughly washed under running tap
water for half an hour. After removing the leaves, the twigs were cut into
nodal explants. Initially 1-1.5 em long nodal explants bearing two opposite
leaves were treated with 0.5% (w/v) bavistin solution, keeping the vessels
on table top rotary shaker (Model T -112, Infors AG, Switzerland) at !50
r.p.m. for 15 min. and then washed thoroughly under running tap water for
15-20 min. with a view to remove even the traces of fungicide. These were
subsequently surface sterilized with 0.5% HgCI, (w/v) solution for 10 min.on
table top rotary shaker. Finally, the explants were washed four or five times
with sterilized distilled water under laminair flow cabinet and stored till
inoculation. At the time of inoculation, both the cut ends of nodal explants
were trimmed. By employong this multi-step method for sterilization, nearly
98% healthy nodal explants were obtained. The explants were cultured on
MS medium supplemented with 20 ).tM BA, containing 3% sucrose as carbon
source and gelled with 0.8% agar (Agrawal et a!., 1998). The pH of the
media was adjusted to 5.8 before autoclaving. 20 ml of molten medium
was poured into 25 x 150 mm borosil test tubes. The cultures were incubated
at 25±2 oc under continuous light of 450-460 j1W.cm· 2 intensity emitted by cool
and white fluorescent lights ( 40W Philips). Nearly 100% cultures differ-
entiated an average of 4.7 ± 2.0 shoots per explant on the aforesaid medium.
within two months. The in vitro raised shoots were subcultured on the same
but fresh medium at one month's interval for more than two years. Thus.
multiplied shoots were used as the source material for leaf explants (Fig.
lA).
The young leaves excised from in vitro raised shoots were cultured
on MS basal medium alone or along with I, 5, 10 and 20 11M 2,4-D. Basal
medium did not support any callus but all the explants reared on 5 and
I 0 11M 2,4-D containing medium induced it. At higher level of 2,4-D (20
11M), the percentage of explants forming 'callus declined to 75% (Table 3).
The callus was mainly induced at the cut ends, the margins as well as along
the midrib and on the surface of leaves, in direct contact with the medium.
596

Table 3. Induction of callus in leaf explants of female jojoba plants reared on MS +

2,4-D medium, after 30 d. of inoculation.

2,4-D Number Explants Ex plants Nature Amount

(!lM) of developing forming of of
ex plants callus (%) somatic callus callus*
embryos (%)

0 24 0''' 0

24 83b 0 greenish-white, +
shiny, friable

5 24 100• 0 ye !low ish-green, ++

friable

10 24 too· 0 yellowish-white, +++

watery, friable

20 24 75b 0 pale-yellow, ++
friable
* Relative growth of callus on visual basis: - = nil; + = poor; ++ = good; +++ = profuse
** Values in a column followed by the same superscript are not significantly different as determined
by Chi-square test at 5% level.

within 15-20 d. of inoculation. Of all its concentrations tried, 10 JJ.M 2,4-D

proved optimum for raising maximum amount of yellowish-white, friable
and watery callus in cultures. At 1 and 5 JJ.M 2,4-D, the growth of callus was
relatively less. Thus, 10 JJ.M 2,4-D was selected for raising callus. Initially,
the callus was whitish-green and semi-compact but subsequently turned pale-
yellow and friable after 30 d.

2.2.2 Differentiation of somatic embryos

Since the callus, developed on leaves incubated on 2,4-D containing medium,

did not organise any somatic embryo even after 30 d. of inoculation, the
callus pieces were subcultured on medium lacking 2,4-D but containing cytokinins
such as BA .or K alone at 1, 5 and 10 JJ.M concentrations. Soon after their
transfer to BA or kinetin containing media, the callus began to proliferate
further and turned pale yellow to greenish-brown within 15 d. In next 10-
15 d., these cultures started developing nodular structures, some of which
differentiated into embryos. It was easy to locate globular-, heart- and torpedo-
shaped as well as dicotyledonous embryos (Figs lB, C; 2A, B).
 597

Figure I. Induction of somatic embryogenesis in the leaf derived callus of in vivo grown
sexually mature Simmondsia chinensis female plants. (A) In vitro differentiated multiple
shoots on two nodal explants reared on MS medium supplemented with 20 f.IM BA
for 30 d. x 1.36, (B) Organisation of globular· to heart-shaped somatic embryos in callus
reared on MS + I f.IM BA for I 0·15 d. The callus was raised on MS + I 0 f.IM 2,4-D x
1.92, (C) Developmental stages (torpedo-shaped to early dicot., arrows) of somatic embryos
on medium mentioned in "B" after 15 d. of subculture x 2.4, (D) Somatic embryos showing
early and late. dicotyledonous stages on MS + l J.iM BA after 30 d. of culture x !.92 .
598

Figure 2. Scanning electron micrographs of S. chinensis leaf callus raised on MS +

10 ~tM 2 ,4-D which has differentiated somatic embryos on MS + I ~tM BA. The globul a r
(A) and dicot. (B) embryos developed in large numbers. Ax 648 ; B x 1352 .

At early stages of development, clusters of embryos, at different devel-

opmental stages, were observed all over explants (Figs 1B; 2A). Gradually ,
they passed through various stages like heart-, early torpedo- and torpedo-
shaped as well as early dicotyledonous, if the explants were retained on
the same medium for 30 d. (Figs I B, C; 28). At times, embryos either developed
only single or unequal cotyledons. A few of them fused to form multiple
cotyledons. Precocious growth of somatic embryos at the radicular end has
also been observed. Complete embryo with two prominent cotyledons developed
in cultures reared on 1 J.1M BA (Figs 1D; 28).
Of the two cytokinins tried , BA was more effective for differentiating
somatic embryos. Somehow, none of the cultures developed shoot buds even
at higher level (I 0 J.1M) of BA or K. 1 J.1M BA was optimum for inducing
 599

Table 4. Differentiation of somatic embryos on callus explants transferred after 30 d.

from 2,4-D containing medium to BA or K augmented MS medium.
Cytokinin Number Explants Average Nature of
(liM) of inducing number of callus
explants somatic somatic embryos
embryos (%) per culture

BA

0 21 14b" 3.33± 1.24'" yellowish-brown,

friable

21 70• 7.00±4.78• brownish-white

to green, friable

5 18 22b 2.75±0.82d whitish-yellow

to dark-green,
friable

10 20 15b 4.00± 1.41 b dark-green, friable

Kinetin

20 20b 3.25±1.21' yellowish-white

to green, friable
5 20 0' 0.00±0.00' yellowish-white,
friable
10 20 0' 0.00±0.00' whitish-yellow
to green, fri abIe
• Values in a column followed by the same superscript are not significantly different as determined
by Chi-square test at 5% level.

embryogenic cultures where 70% callus pieces differentiated an average of

7.00 ± 4.78 somatic embryos per culture. At higher levels (5 and 10 J.lM),
a significant decrease in the percentage of cultures producing somatic embryos
was recorded (Table 4).
On kinetin augmented medium, the percentage of cultures differentiating
somatic embryos was relatively poor. The maximum response was 20% at
1 !JM. With an increase in its concentration, the proliferation of callus enhanced
significantly but without organising any somatic embryo. Further research
on the synchronization of somatic embryos as well as their development
into plantlets is in progress.

3 Conclusions

Jojoba has emerged as one of the promising agronomically valuable crop

600

for its seeds which store liquid wax which is utilized mainly as high pressure
lubricant for heavy machineries in the world. In the past, in vitro regeneration
of jojoba has been tried by several workers employing different explants
but limited success has been achieved (Aragao, 1977; Mandani eta!., 1978;
Rost and Hinchee, 1980). Being (i) a dioecious crop, (ii) with poor seed
germination, and (iii) disproportionate ratio of male and female seedlings
(5: 1), its regeneration through seeds is not an economically viable propo-
sition. Consequently, in vitro clonal propagation of plants, of known sexu-
ality, has a great signific~nce and application. Protocol for development
of male and female plants' has already been worked out in our laboratory
(Agrawal et al., 1998). However, rooting of in vitro raised shoots and their
subsequent acclimatization to field, still need some more investigations.
An alternative method is the regeneration of emblings through somatic
embryos derived from expla,nts of k.nown sexuality. Earlier, Wang and Janick
(1986) induced somatic embryogenesis m jojoba callus derived from immature
zygotic embryos. It was not advantageous since jojoba is a cross-pollinated
species where each zygotic embryo represents its own genotype. At the
same time it is not possible to determine if the immature embryo raised
somatic embryos will finally yield male or female plants. Regeneration through
somatic embryos differentiated on explants of known sexuality, on the contrary,
should prove rewarding in raising emblings of desired sexes. Therefore,
somatic embryogenesis was induced in callus derived from leaf explants
of female plants. The somatic embryos, thus produced, shall hopefully carry
the same genotype. Though 100% explants developed callus on 5 or 10 1-1M
2,4-D, yet none of the explants differentiated somatic embryos on the same
medium. However, embryos developed only when 2,4-D was removed from
the medium and a cytokinin was adjuvanted. Somatic embryo-s formed in
clusters from leaf derived callus in the University of Delhi represent almost
all the developmental stages, but some of them had abnormal morphology
as has also been recorded earlier for some woody taxa (Williams and Maheswaran,
1986; Ammirato, 1987; Young eta!., 1987, Rout et al., 1991).
Wang and Janick (1986) have reported that globular structures, when enlarged.
retained their shape and did not form cotyledon-like structures inS. chinensis,
while we have documented frequent organisation of dicot. embryos. Arrested
development of early embryos, usually at globular stage, has been defined
as "incomplete embryogenesis" by Sharp eta!. (1980). The structure observed
by Lee et at. (1984) also resembled more to globular than to dicot. somatic
embryos, but they might be representing only one of the stages of the developmental
sequence.
Somatic embryogenesis in leaf explants of other plants has been reported
earlier, such as in Coffea arabica L. (Sondhal and Sharp, 1977); Leucosceptrzrm
corum (Pal et a/., 1985); Euphoria Iongan Stend. (Litz, 1988); Vitis spp.
(Stamp and Meredith, 1988); Camelliajaponica L. (Pedroso and Pais, 1993;
Kato. 1996) and Cichorium intybus L. (Mohamed- Yasseen and Splittstoesser.
 601

1995). In the present study, the somatic embryos/embryogen ic calli were

raised axenically on leaves of female jojoba plants. This protocol can be
useful in investigations concerning genetic engineering of jojoba plants.
In addition, leaftissues shall be quite suitable as a source of isolated protoplasts
for genetic transformation studies.

4 Acknowledgement s

S.C.G. and V.A. are grateful to the University Grants Commission for financing
the research project "In vitro clonal propagation of male and female jojoba.
elite plants", under which S.P. worked as a Project Fellow.

5 References

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604

Abbreviations

2,4-D - 2,4-dichlorophenoxyacetic acid

2iPIIPA - N 6 -(2-isopentenyl) adenine
AC - activated charcoal
BA/BAP - N 6 -benzyladenine
CW - coconut water
d. - days
GA/GA - gibberellic acid
IAA - indole-3-acetic acid
IBA - indole-3-butyric acid
K - kinetin
m - meter
MS - Murashige & Skoog's (1962) medium
NAA - a-naphthaleneacetic acid
PVP - polyvinylpyrrolidone
SH - Schenk & Hildebrandt's (1972) medium
Zea - zeatin
 21. SOMATIC EMBRYOGENESIS IN AEGLE MARMELOS (L) CORR.,
A MEDICINAL TREE

S.ARUMUGAM AND M.V.RAO

DEPARTMENT OF PLANT SCIENCE, BHARATIDDASAN UNIVERSITY
TIRUCHIRAPALLI-620 024, TAMIL NADU, INDIA.

CONTENTS

l.llltroduaion 4. Indirect somatic embryogenesis in solid medium

1.1. Botany and genetics 4.1. Initiation
1.2. Economic importance 4.2. Maturation
1.3. Geo8J3Pbical distribution 4.3. Germination
1.4. Diseases and pests 5. Indirect somatic embryogenesis in liquid medium
l.S. Conv<ntional practices fer propagation 5.1. Induction
1.6. In vitro propagsticn-orl!llllogenesis 5.2. Maintenance
1.7. Need to develop somatic embryogenesis 5.3. Maturation
2. loitiaticn of somatic embryogenesis 1
6. Synth<tic seed productioo
2.1. E>plant 6.1. Induction
2.2. Stcrilizaticn 6.2. Encapsulation
2.3. Culture medium and growth regulators 6.3. Storage
2.3.1. Direct somatic embryogenesis 6.4. Germination
2.3.2. Indirect somatic embryogenesis 7. Conclusion
2.3.3. Synthetic seed produaion Aclmowledgements
3. Direct somatic embryogenesis Ref..-ences
3.1. Induction
3.2. Maturation
3.3. Germination

l.Introduction

Traditional herbal medicine bas always played a key role in the heallh system of many countries. An
estimated three-quarter of prescnlied drugs are derived from plants, and rediscovered because of their
prior use in indigenous medicine. The natural resources for planl-based chemicals are limited but the
consumer preference for natural producls is greater.
In India, the use of. different pans of several medicinal plants to cure specific ailments bas
been in vague from ancient times. Nowadays, a group of medicinal plants bas industrlal value, which
bring an appreciable amount of foreign earnings to the country. There is a growing trend all over the
world to shift from synthetic to natural based products including medicinal plants. Several thousands of
medicinal plants are being destroyed due to human neglect and industrialization (Anonymous,
1993; Zenk, 1978). Therefore, there is a need to make intensive research for genetic improvement,'
conservation, and cultivation methods to expand arable land under medicinal and aromatic plants.

1.1. Botany and genetics

The bael tree (Aegle marmelos (L.) Corr.) is an important ayurvedic medicinal tree, belongs to the
family Rutacaeae. It is a moderate sized, slender aromatic tree, 6.0-7.5m in height and 90-120 em in
girth. Branches armed with straight, sharp, axillary, 2.5 em long spines, bark soft, corky, light grey,
exfoliating in irregu1ar flakes; leaves attenuate, trifoliate, occasionally five-foliate, leaflets ovate or
ovate-lanceolate, crenate, acuminate, lateral sessile, terminal long-petioled; flowers large,
greenishwhite, sweet scented, in short axillary panicles; fruits globose, grey or yellowish, rind woody;
seeds nunterous, oblong, compressed, embedded in sacs covered with orange - coloured sweet pulp.
The tree flowers during May-July. The fruits require a year for ripening. The bael tree may yield on
average 300-400 fruits (200-250 kg) per tree. There are twelve varieties have been identified sofar
in India. Some have a very strong nauseating odour, where as some are mild and sweet scented
(Chadha, 1985).
The bael tree is a diploid species with a somatic chromosome number 2n= 18 (Banerji and Pal,
1957; Ragbavan, 1957; Nanda, 1962; Sanjappa, 1979) and36 (Mehra and Khosla, 1973).
605
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 605-655.
© 2000 K/uwer Academic Publishers.
606

1.2. Economic importance

The bael tree is extensively planted near Hindu temples for its leaves and wood, which are used for
worship, and for its edible fruits which are valued in indigenous medicine. The root is an ingredient of
'dasamula' (ten roots), a medicine commonly used by the Ayurvedic practitioners. A detailed account
on chemical constituents in various plant parts of the tree and their commercial and /or therapeutic uses
are given in Table.l.

1.3. Geographical distribution

The bael tree is a native of Indo-Malayan region (Hooker, 1975), and is being cultivated in southeast
Asia, especially in India, Pakistan, Bangladesh, Srilanka, Bunna and Thailand (Hossain et a/., 1994a;
Islam eta/., 1996). It was also introduced into tropical countries of Western Africa and South America
for afforestation purposes (Arya, 1986; Zaman, 1988). It can withstand various types of soils, climatic
conditions and a pH range of 5 to I 0 (Chadha, 1985).

1.4. Diseases and pests

The bael tree is infected with various microorganisms such as- Xanthomonas bi/vae causes a shot hole
and fruit canker; Sarcinel/a fumosus and Cercospora aegle-marmeli cause leaf spot disease; Apergilius
nidulans produces green rot; Fusarium so/ani causes soft fruit rot. The young leaves and shoots are
eaten by caterpillars of Papilio demoleus and P.erithonius. The beetles of Amblirrhymus poricol/is,
Aspidiotus orienta/is, Lecanium colemanii, L. viride, Myllocerus discolor feed on the plant sap and
defoliate the trees. The larvae of Argyroploce illepida, Chaetodacus zonatus, Euzophera niveicoste/la
bore the fruit and feed on the fruit pulp (Chadha, 1985).

1.5. Conventional practices for propagation

Genemlly bael tree is propagated by seed, although it seldom produces a plant true to type. Seeds have
short viability and are prone to insect attack. Besides, seedlings from seed show slow growth and are
liable to diseases and pest in the initial period. Natural reproduction from seed, therefore, is not so
satisfactory.
Bael tree can also be propagated by root-cuttings and layers. Under natural conditions the
plant produces root suckers copiously. The plants are also raised by gootee or inarching from a
desirable tree.

1.6. In vitro propagation-Organogenesis

Conventional methods of propagation (both sexual and vegetative) are associated with problems, like
seasonal seed production (takes one year), pest problems, low percentage of seed germination etc.
These disadvantages can be overcome by in vitro multiplication. There are basically three morphogenic
processes involved in rapid multiplication in vitro (micropropagation): organogenesis, shoot
proliferation via rapid axillary branching, and somatic ebmryogenesis. Plant regeneration may be
accomplished by employing callus, organ, cell and protoplast culture. Micropapagation may be
accomplished by either organogenesis or somatic ebmryogenesis. Organogenesis involves
differentiation of micro shoots and roots at different time periods during plantlet development.
Organogenesis has been greatly influenced by the genotype, physiological state of the explant, age of
the explant, and the in vitro environment, both the light/temperature regimes and the constitution of the
medium, in particular hormone concentrations.
Extensive studies have been made by several workers on in vitro multiplication of forest tree
species either through micropropagation or organogenesis. Some examples on micropropagation and
organogenesis in angiosperms are Acacia senegal and Acacia tortilis (Philips and Nanda, 1998), Betula
sp. (Kurten eta/., 1990; Brand and Lineberger, 1992a.b; Jokinen and Tormala, 1991; Welander, 1993),
bamboo (Rao eta/., 1990; Arya, 1999}, Citrus sp. (Navarro eta/., 1985; Duran-Vila and Navarro 1989.
Oh eta/., 1991; Singh and Chaturvedi, 1999), E/aeis guineenis (Dublin eta/., 1991), Eucalyptus sp.
(Warrag eta/., 1991; Boxus eta/., 1991; Osser and Boxus 1992: Bell eta/., 1993; LeRoux and van
Staden 1991; Mullins eta/., 1996; Geetha eta/., 1999), Rosa hybrida and Rosa chinensis minima (Hsia
 607
and Karban, 1996), Coffea arabica (Dublin et a/., 1991; Neunsch Wander and.. Baumann, 1992),
Azadirachta indica (Muralidharan and Mascarenhas, 1989; Shrikhande et a/., 1993; Raguman and
Ramanujam ,1998; Nirmala Kumari eta/., 1993; Thengane eta/., 1995; Joshi and Thengane, 1993 ;
Kaura eta/., 1999; Thengane and Joshi, 1999; Sharma eta/., 1999), Hevea brasiliensis (Montoro eta/.,
1992; Michaux- Ferriere eta/., 1992; Dublin eta/., 1991; Blanc eta/., 1998), Phoenix dacty/ifera
(Tisserat, 1979; Sharma eta/., 1984; Bhaskaran and Smith, 1992; Letouze eta/., 1998), Pau/ownia sp.
(Sharma and Dhiman, 1998), Da/bergia sisso (Singh et a.,/1999), Prunus avium (Hammat and Grant,
1997), Quercus sp. (El Maataoui eta.,/ 1990; Feraud-Keller et a/ ., 1989; Rancillac and Klinguer, 1991;
Chalupa ,1993; Gebhardt eta/., 1993; Evers eta/ .,1993; Schwarz and Schlaibaum, 1993; Purohit et
a/., 1999), Juglans regia (Leslie and McGranahan, 1992; Preece eta/., 1989}, Santa/urn album (Bapat
and Rao, 1989; Rao and Bapat, 1992}, Juniperus exce/sa (Negussie , 1997), Saraca asoca (Harikrishnan
eta/ .,1998); and among gymnosperms, Picea g/auca and Picea mariana (Attree eta/., 199la,b),
Pinus /ambertiana, Psedostuga menziesii (Gupta and Durzan, 1985) and Agathis australis (Aitken-
Christie et a/ .,1992).
In bael tree, the first attempt on in vitro propagation studies has been made by Singh et a/
(1976) but they failed to regenerate plants from nucellar tissue. Arya eta/ (1981) regenerated plants
from cotyledon and hypocotyl explants through organogenesis, but the regeneration frequency was
very low in both explaiJtli. Further regeneration attemptli have been made from root segments (Bhati et
a/ .,1992), seedling leaves (Islam eta/ .,1993), stem (Varghese eta/ .,1993)., nucellus tissue (Hossain et
a/., 1994), cotyledon (Hossain et a/., 1994), hypocotyl (Hossain et a/., 1995), cotyledonary node
(Arumugam and Rao, 1996a), immature and mature embryo axes (Islam et a/., 1995), epicotyl
(Arumugam and Rao, 1996b), cotyledon and hypocotyl (Arumugam et a/., 1997), and immature leaflet
(Arumugam and Rao, 1998).

1. 7. Need to develop somatic embryogenesis

Somatic embryogenesis, the development embryos from somatic cells, is analogous to the development
of zygotic embryos, and results in the production of a complete somatic seedling (embling) with the
potential to grow into a whole plant It carries a low risk of genetic diversity. It potentially provides
many advantages: I) a large number of plantlets can be produced inexpensively, 2) both root and shoOt
meristem development occur in the process step, 3) quick and easy scale-up can be achieved via liquid
culture, 4) long term germplasm conservation via cryopreservation can be utilized, 5) manufactured
seed can be used for embling establishment, and 6) it also provides a means of genetic transfer and the
multiplication of genetically-transformed cells mass production of transgenetic trees.
There are two different patterns of the origin of somatic embryos from in vitro grown
explants: 1) direct production of somatic embryos from the explant cells called the pro-embryo-
determined cells (PEDC), and 2) indirect production of somatic embryos from unorganized callus
tissue mass called the induced embryogenic determined cells (IEDC). In the former, somatic embryos
are presumed to originate from explant cells that require only in vitro environment to be released from
some repressive condition composed by the organization of the explant By contrast, IEDC pattern not
only requires the release of previously differentiated state through mitotic cell divisions but also an
induction of the new pattern of cell divisions to form organized embryos (ShaJ:p eta/ .,1980, 1982).
Some examples of somatic embryogenesis in woody angiosperms are: Acacia ni/otica ( Garg
eta/ .,1996),Azadirachta indica (Shrikhande eta/ .,1993), bamboo (Mascarenhas eta/ .,1988; Zamora
eta/., 1988; Rao eta/ .,1991;Mukunthakumar and Mathur, 1992; Woods eta/., 1992; Bag eta/., 1999),
Citrus spp (Gmitter eta/ .,1990; Oh et-a/ 1991,1992; Gillet a/ 1991,1994; Kunitake eta/ .,1991),
Betula pendula (Nwtila and Kanppinen, 1992), Coffea arabica (Neuenschwander and Baumann,
1992), Da/bergia latifo/ia (Muralidhara Rao and Lakshmi Sita ,1996), E/aeis guineensis (Jones and
Hughes ,1989; Durand-Gasselin et a/ .,1989; Teixeira et a/ .,1993; De Touchet et a/ .,1991),
Eucalyptus sp (fermignoni et ·a/ .,1998), Fagus sy/vatica(Vieitez eta/ .,1992), Hevea brasiliensis
(Hadrami eta/ .,1991; Montoro eta/ .,1993; Blanc eta/ .,1998), Jug/ans nigra (Neuman eta/ .,1993),
Mangifera indica (Jaiswal ,1990; Sahijram ,1990; Jana eta/ .,1993; Litz eta/ .,1984; Hnssain eta/.,
1998; Jose eta/ .,1999), Phoenix dactylifera (Bhaskaran and Smith, 1992; Sudhersan eta/ .,1993,
Letouze eta/ .,1998), Populus sp (Cheema, 1989; Park and Son, 1988, Vmcour eta/., 1998), Rosa
hybrida (Salm eta/ .,1996), Quercus spp (Chalupa ,1990; Manzanera eta/ .,1993; Brezinova et a/
.,1998; Cvikrova eta/ .,1998), Santa/urn album (Bapat and Rao,l984; Rao and Ozias-Akins 1985;
608
Bapat eta/ .,1990; Bapat, 1993; Ravishankar and McComb ,1998), Sapindus trifo/iatus (Desai eta/
.,1986).
Among gymnosperms, they include Abies alba (Schuller eta/., 1989; Hristoforoglu eta/.,
1992), Abies nordmonniana (Norgaard and Krogstrup, 1991), liriodendron tu/ipifera (Merkle eta/.,
1990), Picea abies (Hak:man eta/., 1985; Hakman eta/., 1990; Gupta and Duizan. 1986a; Gupta eta/.,
1991; Jain eta/ .,1988; Hakman and von Arnold, 1985; Filonova eta/ .,1998; Vagner eta/ .,1998;
Gupta et aJ .,1998; Bozhkov and von Amold,1998), Picea g/auca (Attree eta/ .,1990b; A1tree et aJ
.,1990a; Dunstan et a/ .,1993; Tremblay, 1990; Lulsdorf et aJ .,1992), Picea mariana (Attree et al
.,1990a), Pinus elliottii (Jain et al .,1989), Pinus Jambertiana (Gupta and Duizan. 1986b), Pinus nigra
(Salajova and Salaj, 1992), Pinus taeda (Gupta and Duizan. 1987; Becwar et aJ .,1990; Gupta and
Pullman, 1990; Gupta et aJ .,1998), Pinus radiata (Smith et al., 1991), Psedatsuga menziesii (Gupta et
a/ .,1993; Gupta et aJ .,1998), Prunus 411ium (Garin et aJ .,1997), Pinus sylvestris (Hany and Thorpe
,1998), Picea sichensis( Ingram andMavituna,1998) and Pinus patula (Jones et aJ .,1998).
Islam et aL (1996) reported somatic embryogenesis and plant regeneration in bael tree. They
produced somatic piantlets from zygotic embryo axis of matured seed through direct somatic
embryogenesis.
Somatic embryos do not have seed coat that make them susceptJ.ble to microbial
contamination and desiccation resulting in their inabilit¥ to survive when they are sown directly in the
field Isolated somatic embryos could be encapsulated by a protective gel-like substance, so that
embryoids are able to survive and prevent them from desiccation even after planting into soil. Such
encapsulated embryos can be used as synthetic seeds, which are generally planted immediately after
encapsulation. Redenbaugh (1986,1993) defined synthetic seed as a somatic embryo inside a coating,
and as being directly analogous to a zygotic seed Synthetic seed is also called as somatic seed,
artificial seed, manufactured seed etc. Synthetic seed offer the potential of delivering somatic embryos
to the field or greenhouse utilizing standard agronomic, horticultural and forestry practices for seed
sowing and crop culture. The technology will be valuable at implementation but economical
implementation demands major improvements in somatic embryo quality and in manufactured seed
performance under normal conditions.
Kitto and Janick (1986) first developed desiccated synthetic seed. This seed consisted of
desiccating an embryo coated with Polyox®. Germination, albeit at low levels, was achieved by
placing the dried w3fer in tissue culture media then placing on media moistened filter paper for
germination.
Hydrated synthetic seed was developed by Redenbaugh (1986). This method involves
encapsulation of the embryo in a drop of sodium alginate followed by complexing with calcium ions to
form a calcium alginate gel Synthetic seed must mimic natural seed functions if they are to perform in
the agricultural field in a manner that will allow economical application of the technology.
Several examples of synseed production are: alfalfa (Fuji et al., 1992), bamboo
(Mukunthakumar and Mathur, 1992), carrot and celery (Sanada eta/., 1993), interior spruce and black
spruce (Lulsdorf eta/., l993),Eucal)plus citriodara (Mascarenhas eta/., 1989), Pistacia vera (Onay et
a/., 1996), sandalwood (Bapat andRao, 1988; Fernandes et al., 1992; Bapat, 1993), sugar pine, loblolly
pine and Norway spruce (Gupta and Dmzan, 1985, 1987; Durz.an and Gupta, 1988).
Critical reviews on woody plant species (Tulecke, 1987; Thorpe et al., 1991; Ahuja, 1993;
Jain et al., 1995), tropical and sub tropical fruit crops (Litz and Jaiswal, 1991), perennial fruit and nut
crops (Litz and Gray, 1992), temperate zone fruit and nut crops (Zimmerman, 1991), cover the status of
somatic embryogenesis and other modes of in vitro propagation methods for these crops.
In the present chapter, the phenomenon of somatic embryogenesis and synthetic seed
production of Aegle manne/os is reviewed in light of existing literature by exploring past and pesent
approaches to somatic embryo initiation, maintenance, maturation, germination and plant development

2. Initiation of somatic embryogenesis

2.1. Explant

Ripened fruits were scarified and washed thoroughly with running tap water for 45 min to remove
mucilaginous sheaths. Seeds were surface sterilized with ethanol (70% vlv) for one min, disinfected
with (0.1 % wlv) mercuric chloride (HgCI2) for 5 min, and rinsed 5 times with sterile distilled water.
The sterilized seeds were germinated in a 250ml Erlenmeyer conical flask, containing sterile moist
 609
cotton, by incubating in darkness for five days, and subsequently, cultures were shifted to light at 16-
hrs/day condition. In direct somatic embryogenesis, cotyledon and hypocotyl explants were excised
from in vitro raised seedlings, whereas, in indirect somatic embryogenesis, cotyledon, hypocotyl
immature leaflet and mature leaf were collected from in vitro raised seedlings (15-days old).

2.2. Sterilization

Explants collected from15-days old in vitro raised seedlings such as cotyledon, hypocotyl, immature
leaflet, and mature leaf were thoroughly washed in running tap water for 45 min. Thereafter, the
explants were washed with liquid soap solution for 3 minutes and then soaked in 70% (vlv) ethanol for
45 seconds and finally disinfected with 0.5% (w/v) HgC12 for 5 min and rinsed three times in sterile
distilled water.

2.3. Culture medium and growth regulators

2.3.1 Direct somatic embryogenesis

Sterilized explants (cotyledon and hypocotyl) were inoculated on Murashige and Skoog (1962) (MS)
basal medium for the induction of direct somatic embryogenesis. The induction medium was fortified
with different concentrations of indole-3-acetic acid (IAA), a.-naphthalene acetic acid (NAA) (0.01 -
0.8 mg/1) in combination of with 6-benzylamino purine (BA) (0.1 mg!l) or kinetin (KIN) (0.8 mg/1),
and adjusted to pH 5.8. Agar (0.8 %w/v), and sucrose (3% w/v) were added and autoclaved at 121°C
for 15 min.
The cultures were incubated at 25 ± 2°C under white fluorescent light at 80 !ill M 2 s-1 with
16h photoperiod At the end of 5 weeks, percentage of exp1ants with somatic embryogenesis was
recorded Explants were subcultured at 2-week interval onto the fresh medium of similar composition.
Each treatment had 7 replicates and the experiment was repeated thrice. Data were taken after 35 days
with two subcultures.
The explant cultures in the induction medium were given different photoperiod treatments
(dark/light treatment) (5/19, 10/14, 15/9 and 20/4 hrs/day) for 15 days to examine their effect on direct
somatic embryogenesis.
After 5 weeks, embryos were transferred to the maturation medium composed of half- strength
MS medium containing BA (0.1 - 2.0 mg!l), ascorbic acid (AA) (1.0 mg!l) and gibberellic acid (GA3)
(0.1 mg/1). After 21 days of maturation treatment, embryos were subcultured on the same fresh medium
for germination and conversion into plantlets. Total number of plantlets with well developed root and
shoot systems were counted
After removing the cotton plugs, plantlets were taken out carefully from culture tubes and
washed in the running tap water to remove adhered culture medium to the roots. Complete plantlets
with well-developed roots were undergone to the hardening process. Thereafter, the plantlets were
transferred to small plastic container containing a mixture of sand, garden soil, and vermiculite (1: 1:2)
and, subsequently, transferred to the field

2.3.2..Indirect somatic embryogenesis

Sliced sections of cotyledon, hypocotyi; immature leaflet and mature leaf (5 mm) collected from in
vitro raised 15-days old seedlings were placed onto MS solid medium containing B5 (Gamborg eta/
.,1968) vitamins with 0.5mg!l 2,4-dichlorophenoxyacetic acid (2,4-D). Callus (21- day- old) was
subcultured onto fresh medium with same growth regulator concentrations for further proliferation.
Cultures were incubated in dark for first one week and then transferred to light, (80 J1E M-2 s-1) with
16h photoperiod at 25 ± 2°C. Callus was subcultured thrice at 2- week interval.
Three -week- old white friable embryogenic calli were cultured on induction medium
containing various concentrations of 2A-D (0.0 1 - 0.8 mg/1), NAA (0.00 1 - 1.0 mg/1) either alone or in
combination with BA (0.1-1.0 mg/1) for further proliferation of somatic embryos. The influence of
amino acid like glutamine (1-20 mg/1) was investigated on somatic embryo production. At the end of
35 days culture period somatic embryo induction data was recorded
610

After initiation of somatic embryos, half-strength MS medium supplemented with BA (2.0

mgll) + AA (0.001-0.1 mgll), BA (0.2 mgll) +ABA (0.01-0.5 mgll), BA (2.0 mgll) + GA3 (0.5- 2.0
mgll) combinations were used for maturation of embryos. The data was taken at the end of 21 days.
Matured somatic embryos (cotyledonary stage) were transferred on the same medium the germination
data was taken after 21 days. Rooted plantlets were baidened and transferred to the field
Suspension cultures were initiated through liquid culture by transferring 500 mg 3-week old
callus (obtained from cotyledon, hypocotyl, immature leaflet and mature leaf grown on MS + B5
vitamins+ 0.5 mgll2,4-D to 25 ml of induction medium) to a 100 ml Erlemneyer flask containing MS
liquid medium (with out ag;tt). The growth regulators were added in the liquid medium with various
concentrations and combinations of 2,4-D (0.01 - 2.0 mgll), 2,4-D (0.1 mgll) + BA (0.01 - 2.0 mgll),
and 2,4-D (0.1 mgll) + NAA (0.1 - 1.0 mg/1.) and glutamine (10.0 mgll). The flasks were agitated on
rotary shaker with 100 rpm, in darkness at 25 ± 2°C, for first seven days and then transferred to 16-h
photoperiod (80~8-1 ). Embryogenic suspension cultures (1g fresh mass) were subcultured at every
2- week interval for two months. The somatic embryo induction data was recorded within 35 days.
After induction of somatic embryos, different developmental stages were recorded on different
days (5, 10, 15 and 20) of incubation. Samples of suspension cultures were taken at random and the
number of somatic embryos was counted under the stereomicroscope. Observations were taken from 7
different independent cuhures, and the frequency of somatic embryos and the total number of embryos
in each culture were calculated
Various concentrations of glutamine (1, 5, 10, 15 and 20 mgll) were tested for the indnction
and enhancement of somatic embryogenesis on a medium containing 2,4-D (0.5 mgll).
Torpedo shaped embryos were transferred toMS liquid medium containing AA (0.01 - 1.0
mgll) + BA (0.001 - 0.5 mgll), ABA (0.5 mgll) + BA (0.01 - 1.0 mgll), AA (0.5 mgll) +ABA (0.01 -
1.0 mgll) for the matmation of somatic embryos. Matmation .data was recorded within 21 days of
culture.
Well-matured embryos were cultured on MS solid medium containing BA (0.01 - 0.8 mgll),
BA (0 4 mgll) + GA3 (0.5- 4.0 mgll), andBA (0.4 mgll) + AA (0.001 - 1.0 mgll) + GA3 (2.0 mgll) for
germination and conversion into plantlets. Somatic embryo germination response was recorded within
21 days. Rooted plantlets were banlenedand transferred to the field

2.3.3. Synthetic seed production

Cotyledonary stage somatic embryos obtained from both solid and suspension cultures were dried on a
filter paper and dipped in sodium alginate gel (0.5 - 5 % w/v) for few seconds. They were dropped into
calcium chloride (CaCl2) (2.5 % wlv) solution in a 500-ml flask for encapsulation. The encapsulated
embryos were kept for 30 min on a shaker (80-rpm) in light (80 J.LEM2S-1). After incubation period,
CaCl2 solution was removed by decanting and capsules were washed with sterile distilled water.
Somatic embryo germination response was recorded after 21 days.
To test the effect of storage on gennination efficiency, the encapsulated emblyos were stored
at 4°C in the refrigerator for various days (1, 2, 3, 5, 10, 15, 20, 25 and 30) in the darkness.
The synthetic seed germination was determined by incubating them on half-strength MS
medium under both light and darlc conditions at 25 ± 2°C. Encapsulated embryos (50 per pelridish)
were grown on half- strength MS medium containing BA (0.1 - 1.0 mgll), KIN (0.1 - 1.0 mgll), NAA
(0.01- 0.5 mgll) or BA (0.5 mgll) +KIN (0.1- 1.0 mgll), BA (0.5 mgll) + NAA (0.1- 0.8 mgll), BA
(0.5 mgll) + GA3 (0.1 - 1.0 mgll) combinations for germination. The germination response was
recorded within 21 days.
Healthy "syn seeds" were placed on various substrates such as cotton, sterile soil and half-
strength MS solid medium containing BA (0.5 mgll) + GA3 (0.1 mgll) combination. Rooted plantlets
were taken out from the culture vessel and roots were washed in running tap water, and transferred to
plastic container containing a mixture of sand, g;lrden soil, and vermiculite (1:1:2) and covered with
polythene bags. Plantlets maintained for one week in the controlled environment (25±2°C) and
transferred to 35±2°C for 3 weeks for banlening and finally shifted to the field
The cultures were examined periodically and the morphological changes were recorded on the
basis of visual observations. The experimental design was random and factorial with growth regulators
 611

as independent variables. The data, pertaining to frequencies of somatic embryos induction, maturation
and germination, were subjected to Mean± SD and mean separation was done by using Duncan's New
Multiple Range Test (DMRn.

3. Direct somatic embryogenesis

All steps comprise of somatic embryo induction, maturation, germination, acclimatization and field
transfer have been affected by: explant type, media formulation, growtb regulators, and culture
conditions. All tbese factors were investigated for tbe induction of somatic embryogenesis in Aegle
mannelos.

3.1. Induction

Four sets of growtb regulators (IAA + KIN, IAA + BA, NAA + BA and NAA + KIN) were tried for
tbe induction of direct somatic embryogenesis. The results indicate tbat NAA + BA combination gave
tbe highest response and tbe highest number of somatic embryos. After 4 weeks of culture, white
granular calli were formed only on cotyledon and hypocotyl explants. The granular callus gave rise to
numerous somatic embryos on tbe subsequent sulx\Uturcs, but only nodules appeared on otber explants
(epicotyl and mature leaf).
In many instances, tbe epidermal cell layer of explants split up and organized structures
emerged from tbe subepidermal layers. The number of organized structures was higher on hypocotyl as
compared to cotyledon explants. These organized structures closely resembled globular and heart
shaped somatic embryos.
Nodular structures were seen in most of tbe growtb regulator combinations witb varied
response. Hereafter, tbese nodular structures are referred as embryogenic masses, since tbe
development of embryos was restricted to tbese protuberances. The frequency of embryogenic mass
formation varied witb tbe type and concentration of growtb regulators used.
MS basal solid medium fortified witb various concentrations of IAA (0.01 - 0.8 m!ifl)
combined witb a fixed concentration of BA (0.1 m!ifl) were examined for embryo induction efficiency.
Among various combinations, IAA (0.2 m!ifl) + BA (0.1 m!ifl) was found to be the best for higheSt
response of somatic embryo production in hypocotyls (38.6 %) and cotyledons (30.8 %) (Table 2). The
highest number of somatic embryos was formed on cotyledons (2.4 embryos/explant) and hypocotyls
(1.2 embryos/explant). Number of embryos varied witb growtb regulator concentrations and type of
explant (Table 2).
Various concentrations ofiAA (0.01- 0.8 mgll) witb optimal concentration of KIN (0.8 m!ifl)
produced somatic embryos witbout callus phase. The maximum percentage of response was observed
on medium supplemented witb IAA (0.4 mgll) +KIN (0.8 m!ifl) combination in hypocotyl (17.1 %)
and cotyledon (16.4 %). The maximum number of somatic embryos in hypocotyl and cotyledon
explants was 2.8 embryos/explant, and 2. 7 embryos/explant respectively (Table 2).
Fixed concentration ofBA (0.1 mgll) combined witb different concentrations ofNAA (0.01-
0.8 m!ifl) were tested for induction of somatic embryos. NAA (0.2 m!ifl) + BA (0.1 m!ifl) combination
induced tbe highest embryogenic response in hypocotyls (45.2% embryos/explant) and in cotyledons
(43.3 %). The highest number of somatic embryos in hypocotyls was 6.6 embryos/explant, whereas in
cotyledons 4.8 embryos/explant (Table 2 & Plate 1 (a, b)].
The frequency rate of somatic embryo production was highest in tbe presence of NAA (0.1
m!ifl) and KIN (0.8 m!ifl) combination in hypocotyls (39.5 %) and in cotyledons (35.2%). The highest
numbers of embryos were recovered in hypocotyl (4.2 embryos/explant), whereas cotyledons produced
only 3.9 embryos/explant Epicotyl and leaf explants failed to produce direct somatic embryos by any
used treatment (Table 2).
For tbe initiation of direct somatic embryos, tbe cultures were pretreated by keeping in
darkness. Among tbe different photoperiods tested for induction, 10/14 hrs (dark/light) treated (upto
one week) explants induced highest embryogenic response in hypocotyls (72 %) and cotyledons (67 %)
(Fig 1).
In tbe present study, MS medium having NAA, IAA, BA and KIN combination were found to
produce somatic embryos developed from adaxial side of cotyledons, and hypocotyls. Addition of AA,
GA3 and BA to tbe medium promoted tbe growth, development, maturation and conversion of somatic
embryos into plantlets. Cytokinin is known to enhance shoot morphogenesis. The present results
612

indicated that somatic embryos were produced directly only on cotyledon and hypocotyl explants.
However, other expiants developed granules and nodular like structures. Our results indicated that
direct induction of somatic embryogenesis and plant regeneration from cotyledon and hypocotyl
explants of Aeg/e mannelos.
Of the four growth regulators combinations tested for embryo initiation, NAA + BA gave the
best results. The highest frequency (45.2 % and 43.3%) and 6.6 and 4.8 somatic embryos were
obtained directly from hypocotyl and cotyledon explants respectively. on medium supplemented with
NAA (0.2 mgll) + BA (0.1 mgll).
Islam et al. (1996) reported the highest direct somatic embryogenesis initiation response
(18%), and highest number of somatic embryos (12) from zygotic embryos after 42 days in the
presence ofMS medium containing 2,4-D (1.0 iJM) and BA (1.0 iJM) inAegle monnelos. Further, they
observed that media supplemented with 2,4-D alone did not produce any somatic embryos. In
combination with BA (0.5-2.0!JM), 2,4-D at greater than l.OOJ.IM also showed inhibitory effects on the
induction of somatic embryos. After six weeks of culture initiation, somatic embryos were transferred
to half-strength MS medium with 2% sucrose and l.OJ.IM BA, radicals and apices of the somatic
embryos became active within 15 days, cotyledons turned datk green and shoots elongated. The
conversion percentage was very low, approximately 12%.
Mumlidbar Rao and Lakshmi .Sita (1996) •induced direct somatic embryogenesis from
immature zygotic embryos of Dalb(Jrgia latifolia. Direct somatic embryogenesis was expressed in the
presence of 2,4-D along with high sucrose concentration. Somatic embryos were matured on MS
medium along with 0.5-I.Omgll BA and further stated that there was no somatic embryogenesis
withoutBA
Joshi and Thengane (1993) induced direct somatic embryogenesis from cotyledonary explant
ofAzadirachta indica on 2,4-D and BA supplemented medium.
Somatic embryos appeared on the edge of cut end of explant Sequential development of
somatic embryos from globular to heart-shaped stage and finally cotyledonary stage was observed on
embryo induction medium. High frequency of somatic embryo induction was achieved with NAA +
BA than IAA in combination with BA These results showed that addition of cytokinin with auxin was
important for direct embryo induction Ahmed et al. (1996), and Dutta eta/. (1991) reported that tl}e
winged bean cultures grown for 28 days on MS basal medium supplemented with NAA + BA
combination enable to induce somatic embryo formation Stimulation of direct somatic embryos by
cytokinin has also been reported in Phaseolus sp (Malik and Saxena, 1992). On contrary, direct
embryo formation from Apium explants on a medium supplemented with cytokinin alone has also been
observed (Halperin and Jensen, 1961). Nataraja and Neelambika (1996) induced somatic embryos
from petal cultures of Punica granatum on MS medium amended with auxins and cytokinins.
Ravishankar and McComb (1998) achieved direct somatic embryogenesis from zygotic embryos of
sandalwood on MS medium containing BA A low percentage of embryos germinated on half-strength
MS medium supplemented with IAA For the induction of direct somatic embryogenesis, the most
often used explant was immature zygotic embryo in forest trees (Schuller eta/., 1989; Hristoforglu et
a/., 1992; NorgaardandKrogstrup, 1991; Hakmanetal., 1990; Guptaandl>wzan, 1986a; Gupta eta/.,
1991; Dunstan et al., 1993; GU)U and Pullman 1990; Jain et aJ 1989; Garin et a/1997; Merkle eta/.,
1990; Finer, 1994), andZea mays (Amstrong and Green, 1985; Vain et al., 1989). There are several
examples on direct somatic embryogenesis in dicot and monocot plants (Garget a/., 1996; Shrikhande
eta/., 1993; Rao eta/., 1991; Gill eta/., 1994; MuralidbarRao andLakshmi Sita, 1996).
Our results indicated that NAA + BA combination showed the highest direct somatic
embryogenesis both in cotyledon and hypocotyl explants, followed by NAA+KIN combination The
effect of KIN could be maintaining the embryo-forming potential in solid cultures of carrot for a long
period (Steward et al., 1988). In carrot, 0.1 mgll KIN at 0.1 mgll promoted somatic embryogenesis
(Halperin and Jensen. 1961).
Somatic embryos at globular stage were characteristically creamy-yellow in color on 50 %
explants in Aegle marme/os. Benjamin eta/. (1996) described that after four weeks most of Agave
explants were covered with globular somatic embryos in MS medium supplemented with 1.4 J.IM 2,4-
D.
3.1.2. Maturation

Induced embryos (torpedo stage) were transferred to the maturation medium containing BA (C I - 2.0
mgll) + AA (1.0 mgll) combinations for the maturation and germination of somatic embryos. BA (0.8
 613

mgll) + AA (1.0 mgll) combination induced high frequency response (60.5 %) of maturation within 21
days of culture (Table 3).
Different concentrations and combinations ofBA (0.1 - 1.0 mgll) + GA3 (0.1 mgll) were used
for embryo maturation. Among these combinations, the BA (0.8 mgll) and GA3 (0.1 mg/l) combination
showed the highest response (16. 7 %) of embryo maturation (Table 3).

3.1.3. Germination

The germination efficiency of cotyledonary stage of somatic embryos varied with different
combinations of BA, AA, and GA3. Embryo germination was characterized by simultaneous
production of root and shoot systems. The matured embryos (cotyledonary stage) when subcultured
onto the same medium for germination, the embryos turned green and developed by elongation of
hypocotyl revealing the folded cotyledon. Later on, the cotyledons unfolded and shoot region
developed further with the emergence of first leaves.
BA (0.8mgll)+AA (l.Omgll) combination induced the highest frequency (6.3%) of somatic
embryo germination. Among different concentrations ands combinations of BA (0.1 - 1.0 mgll) + GA3
(0.1 mgll) were used, BA (0.8 mgll) + GA3 (0.1 mgll) combination showed the high frequency (4.2%)
of somatic embryo germination [(Plate 1(c, d, e, & f and Table 3].
After germination, cotyledons turned dark green and shoots elongated, and root formation
occurred simultaneously [Plate 1 (g)]. The plantlets were transferred to plastic cups containing a
mixture of garden soil, sand and vermiculite (1:1:2) for acclimatization [Plate 1 (h)]. Somatic embryos
derived plantlets or somatic seedlings established well in soil.
Among the various concentrations and combinations, BA (0.8 mg/l) + AA (1.0 mg/l)
combination gave the highest frequency rate of somatic embryo maturation and germination in Aegle
marmelos. Plantlets could be ensued from the embryoids after subculturing on half-strength MS
medium containing 1.0 mgll each 1AA or IDA with BA to the same medium (Nataraja and Neelambika,
1996). However, Hartmann eta/. (1990) reduced nutrients in the culture medium for maturation and
germination of somatic embryos in few woody species. Our results showed that conversion rate of
somatic embryos into plantlets was dependent on the type and concentration of auxin used in the
somatic embryo induction medium. The best plant conversion rate was obtained with the
supplementation of BA + AA combination in the medium. In contrast, Rao and Bahadur (19~)
suggested that addition of AA with BA promoted the formation of more somatic embryos than BA
alone in 0/den/andia umbel/ata. Earlier report by Islam et a/. (1996) indicated very low plant
conversion rate in Aegle marme/os. Kamada and Harada (1979) hypothesized that the addition of
glutamine in the embryonic initiation medium enhance the differentiation of predetermined direct
embryogenic cells.

4. Indirect somatic embryogenesis on solid medium

The most important factors affecting the induction of embryogenic callus and plant regeneration are
explant type, media formulation, and growth regulators. All these factors were investigated in the
indirect somatic embryogenesis of Aegle marme/os. The effect of growth regulators and explant type
on somatic embryogenesis on solid medium is summarized in Table 4.

4.1. Induction

Callus induction from all explants was observed on the medium containing 2,4-D. Calli were highly
variable in colour and texture. The various colours of callus were green, yellowish, brown and
yellowish green. Some cultures were friable and had rough surface, while others were smooth surfaced
and hard Calli with yellowish green colour and friable were visually selected and subcultured
The callus derived from different explants viz., cotyledon, hypocotyl, mature leaf and
immature leaflet were investigated for their embryogenic potential on MS basal medium with 2,4-D,
NAA, BA and glutamine combinations. After one week of culture, calli became enlarged with
yellowish brown in colour. Embryogenic callus development was observed from all over the surface of
the explants within 14 days of culture. Globular shaped embryos were observed on the surface of the
calli after 28 days with two subcultures. Repeated subcultures on the same medium increased the
614

frequency of somatic embryos at the end of 15 days of each culture. It was observed that 2,4-D and
NAA with glutamine stimulated embryogenic calli, however, 2,4-D in combination with BA and
glutamine on MS solid medium stimulated the induction of embryogenic callus with highest response.
In our study, 2,4-D (0.2 mg/1) + BAP (0.2 mg/1) and glutamine (10.0 mg/1) combination was found to
be the most effective combination for embryogenic callus induction. An optimal2,4-D concentration
was determined to obtain highest response of calli to form somatic embryos.
Explant type also influenced the percentage of somatic embryogenesis. Hypocotyl explant
derived callus was ideal for somatic embryogenesis induction followed by cotyledon, immature leaflet
and mature leaf explant derived calli. The data showed that the embryogenic mass induction was high
in 2,4-D (0.2 mg/1) + BA (0.2 mg/1) and glutamine (10.0 mg/1) combination, which was highly
significant at 5 %level (Table 4). At lower concentrations of 2,4-D (0.01 mg/1), the initial hump
formation was noted at low frequency, Put it was associated with callusing. Similar results were also
noted in the presence of NAA. At higher concentrations (above optimal level) of 2,4-D, embryogenic
mass induction was low by the end of the sixth week and finally tissues turned brown. Of the two
auxins tested, 2,4-D was ideal for embryogenic callus induction. Various stages of embryos were
observed in the same induction medium and embryo development was synchronous.
The embryogenic effect was studied on callus by supplementing 2,4-D (0.01 - 0.8 mg/1) +
glutamine (10.0 mg/1) in the mediutn. 2,4-D (0.2 mg/1) + glutamine (10.0 mg/1) combination induced
the highest response in hypocotyl derived callus (42.0 %), whereas other explant derived calli
responded poorly . The highest number of somatic embryos (30.8 embryos/calli) also produced in
hypocotyl derived callus (Table 4).
When 2,4-D (0.2 mg/1), glutamine (10.0 mg/1) and different concentrations of BA (0.1 - 1.0
mg/1) were tested, 2,4-D (0.2 mg/1) + BA (0.2 mg/1) and glutamine (10.0 mg/1) combination produced
the highest frequency of somatic embryogenesis (49.3 %) with an average number of somatic embryos
(46.4 embryos/callus) from hypocotyl derived callus followed by other explants (cotyledon, immature
leaflet and mature leaf) rrable 4 & Plate 2 (a, b, c & d)].
Among the various concentrations ofNAA (0.001 - 1.0 mg/1) in combination with glutamine
(10.0 mg/1) tested, NAA (0.1 mg/1) and glutamine (10.0 mgll) combination induced high percentage of
embryogenesis in hypocotyl derived callus (18.9 %) with highest number of somatic embryos (10.9
embryos/callus) followed by cotyledon, immature leaflet, mature leaf derived calli (Table 4).
The synergistic effect of BA (0.1-1.0 mg/1), in addition to NAA (0.1 mg/1) and glutamine
(10.0 mg/1), was studied for their embryogenic potential NAA (0.1 mg/1) + BA (0.4 mgll) +glutamine
(10.0 mg/1) combination gave the highest response of response in hypocotyl derived callus (40.8 %)
with highest number of embryos (31.5 embryos/callus) (Table 4).
To find out the role of glutamine in somatic embryo induction, different concentrations of
glutamine (1, 5, 10 and 20 mg/1) were added in the culture medium having 2,4-D (0.1 mg/1). The
highest frequency of embryogenic callus formation on hypocotyl and cotyledon explants derived callus
recorded was 87% and 75 o/o, respectively at glutamine (10.0 mg/1) (Fig 2). For further development,
embryogenic mass was transferred to the fresh medium. During development, the single cells stopped
dividing and differentiated into large, curved and vacuolated cells. Some cells in clumps were small,
relatively round, dense and often green. These cells kept on dividing to form compact, globular
proembryo masses. The whole embryo became round and yellowish green. this was the early globular
stage of development of somatic embryo and it was equivalent to zygotic embryo, but without the
attached suspensor. About 85 % of the globular staged embryos developed into heart, torpedo and
cotyledonary stages after two subcultures at 2-week interval
Most of the embryos were morphologically normal with distinct stages like globular, heart,
torpedo and cotyledonary stage. Abnormal embryos varied in shape and structure having one, two, or
even more unequal cotyledons which sometimes fused into a cup shaped structure. The squash
prepcuation of callus revealed the presence of several stages, which simulated stages in normal
angiospenn embryogeny, globular, heart shape, torpedo and cotyledonary staged embryos.
Somatic embryogenesis was never observed without exogenous growth regulators. Of the two
auxins used, 2,4-D was more potent than NAA for somatic embryo induction and gave the highest
response in producing number of embryos per culture. Addition ofBA and glutamine slightly enhanced
the frequency of embryo production. Auxin is the most important factor for the regulation of induction
and development of embryogenesis and it has different effects on different phases of embryogenesis.
This indicated that auxin is essential for the induction of embryogenesis. Both BA and glutamine had a
promotory influence along with auxin on somatic embryogenesis.
 615

Little differences in the frequency of embryogenesis and the number of somatic embryos were
noticed between cotyledon and hypocotyl derived calli. Hypocotyl exhibited a high frequency of
embryogenesis and more number of somatic embryos than cotyledon explant. When somatic embryos
were transferred to the germination medium a wide range of developmental changes were seen. Some
somatic embryos formed callus, roots or shoots, and some failed to develop into plants. The embryos
developed with dicotyledonary leaves were able to grow into normal plantlets.
Cytokinin (BA) combined with auxins (2,4-D or NAA) yielded maximum embryo production
in the solid medium. By increasing BA concentration, there was a significant and positive effect on
somatic embryogenesis. Cytokinin treatment alone also increased the number of somatic embryos with
multiple cotyledons (Ammirato, 1977; Lee and Soh, 1993). Wilson eta/. (1996) reported that BA (2.0
j1M) increased both total number of embryos and a proportion of cotyledonary stage embryos.
Our data showed the importance of exogenous auxin in regulating the induction of somatic
embryogenesis in Aegle mannelos. Media containing 2,4-D, BA and glutamine produced the largest
number of somatic embryos, when compared with other combinations tested. 2,4-D, BA and glutamine
combination was marlcedly more active than NAA, BAP and glutamine combination. Of all evaluated
concentrations, 2,4-D (O.lmg/1}, BA (0.1 mg/1} and glutamine (IOmg/1} combination was the best
suitable for the induction of somatic embryogenesis. The combination of growth regulators, showed
better response than individual growth regulator$. In contrast, addition of cytokinin to the induction
medium containing 2,4-D did not enhance the average number of somatic embryos per explant (Eapen
and George, 1993).
The effectiveness and efficiency of the type of auxin used appears to be tissue specific on
somatic embryo production. Higher concentration of auxins (2,4-D or NAA) not only decreased the
mean number and frequency of somatic embryos but also delayed the embryogenesis. The decreased
response of embryogenesis in the presence of higher concentration of auxins might be due to ·altered
hormonal levels in the culture medium, which are essential for embryo induction. These results are in
agreement with the result reported by Ramadev Reddy and Reddy (1993) in Arachis hypogaea.
Saadet and Felda (19%) reported that NAA stimulated non-embryogenic callus and 2,4-D
stimulated embryogenic callus formation. Our results indicated that 2,4-D, BA, glutamine produced
large number of somatic embryos and favored subsequent conversion into plants. Both 2,4-D and
NAA were highly suitable to induce somatic embryogenesis through callus culture. Of the two auxilrl;,
2,4-D showed a superior response for embryo induction. The inclusion of BA with auxin produced
higher frequency of somatic embryos. Kao and Michayluk (1981) reported in alfalfa a combination of
2,4-D or NAA with cytokinin was essential for the induction of somatic embryos. In Populus deltoides,
MS + 2,4-D + BA produced large number of globular shaped embryos (Michler and Bauer, 199!).
Evans eta/. (1981) reported similar results. Auxin 2,4-D is an effective inducer of somatic embryos in
more than 50 %of plant taxa reported for successful somatic embryogenesis. Glutamine (10 mg/1) has
been shown to be a partial or complete substitute for inorganic ammonium in somatic embryo
production inAegle mannelos. Wetherell and Dougall (1976) in carrot, Stuart and Strickland (1984) in
Medicago sativa reported that glutamine with 2,4-D produced highest of somatic embryos. Organic
nitrogen sources (glutamine), other aminoacids, and casein hydrolysate have improved the proliferation
and development of somatic embryos as compared to inorganic nitrogen sources (Boulay et a/., 1988;
Finer eta/., 1989; Tremblay and Tremblay, 1991).
The addition of glutamine in the embryogenic initiation medium has been hypothesized to
enhance differentiation of predetermined direct embryogenic cells (Kamada and Harada, 1979). The
nucellar-derived embryoids in culture have exhibited better maturation and subsequent germination
upon culturing on whites medium supplemented with casein hydrolysate (400 mg/1) and KIN (2mgll)
(Rangaswamy, 1958,1961). Tulecke and McGranahan (1985) obtained somatic embryogenesis from
Juglans hindsii and Juglans regia grown on medium supplemented with L-glutamine. Merckle et al.
(1987) and Neuman et al. (1993) observed a positive effect casein hydrolysate inJuglans regia somatic
embryogenesis. Whereas, Preece and Bates (1995) stated that casein hydrolysate was not necessary for
somatic embryo development in whiteash.
In this study, it was observed that most of the somatic embryos were loosely attached to form
an aggregate of embryos that could easily be separated. Some of the somatic embryos arose from the
base of other embryos to form clusters indicating formation of secondary somatic embryos.
Maheshwaran and Williams (1986}, and Ammirato (1987) also obtained similar results. Also, some
reports showed that amino acids were effective on embryo development in alfalfa (Stuart and
Strickland, 1984 a, b; Redenbaugh et al., 1991 a; Shetty andMcKersie, 1993; Nichol et al., 1991).
616

4.2. Maturation

Both the maturation and germination processes occurred in the same medium with 3 subcultures at 7-
day interval (21 days).
The highest somatic embryo maturation response was (59.2 %) on half-strength MS medium
containing BA (0.2 mgll) and AA (0.01 mgll) combination By adding BA (0.2 mgll) and ABA (0.1
mgll) in the culture medium, the highest (65.5%) response was recorded. Maturation on half-strength
MS medium containing (ABA, BA) combination was statistically significant at 5 % level .BA (0.2
mg/1) and GA3 (1.0 mg/1) combination showed the highest response (24.6 %) of maturation (Table 5).
Cytokinin BA together with ABA was highly effective in high maturation rate of somatic
embryogenesis in the solid culture medium. ABA could be the limiting factor needed to regulate cell
division, cell size and differentiation and, thereby, reduce overall embryo size and attain normal
morphology (Michler and Bauer, 1991). About 65 % somatic embryos were matured and germinated
within 3 days of culture. Our findings are in agreement with the of several woody species apple and
cherry (James eta/., 1984), Rosa hybrida (Rout et al., 1991), Aescu/us hippocastanum (Redojevic, et
a/., 1988).

4.3. Germination

Germination of embryos was chamcterized by simultaneous production of root and shoot systems. In
the germination medium, embryos turned green and developed into plantlets by elongation of
hypocotyl, and shoot primordia developed further with the emergence of first leaves. BA (0.2 mg/1) and
ABA (0.1 mg/1) combination was the best for highest response (28.6%) to the germination of somatic
embryos. BA (0.2 mgll)+AA (O.Olmgll) combination showed high response of (18.3%) embryo
germination, and BA (0.2mgll)+GA3 (1.0mgll) combination showed 1Ul"lo of germination [Plate 3(a-e)
and Table 5)].
Stereomicroscope observations indicated that embryo became matured, firm, large and
smooth. The continuous light and full-strength MS medium were deleterious to embryos, which turned
brown and eventually died. The highest somatic maturation and conversion frequencies were obtained
in dark treated cultures of half- strength MS medium with a combination of AA, ABA, GA3 and BA
The somatic embryo derived plantlets or somatic seedlings were transferred to plastic cups
[Plate 3 (f)] containing a mixture of garden soil, sand, and vermiculite (1:1:2) and then finally
transferred to the field condition
During germination, somatic embryos developed root first and then emergence of shoot.
Ammirato (1988) reported that root develop first from spherical nodules and by further transfer of
nodules to the solid medium formed shoots. Redenbaugh eta/. (1991 a, b) reported the maturation in
alfalfa, carrot and celery somatic embryos and their conversion into plantlets.
ABA has successfully been used in a large number of somatic embryogenic studies. It
prevents precocious embryo germination, and it allows normal embryo maturation, generally, increase
the uniformity of embryos and reduce the development of abnormal embryos (Am.mirato, 1983).
Somatic embryos, developed due to exogenous application of ABA, closely resembled zygotic
embryos in both structure and behavior (Am.mirato, 1988). Islam eta/. (1996) reported that BA (1.0
J1M) alone in half- strength MS medium with 2% sucrose induced the highest number of mature
somatic embryo production and germination within 15 days of culture.

5. Indirect somatic embryogenesis in liquid medium

5.1./nduction

Callus suspension cultures were established from the callus derived from hypocotyl and cotyledon
segments in half-strength MS liquid medium. The yellowish, green, friable and fast growing calli
obtained on the solid medium, were transferred to the liquid medium having similar growth regulator
composition. Uquid medium contained different growth regulator combinations of 2,4-D, NAA, BA
and glutamine. The dispersal of these calli produced a suspension culture mainly composed of free
cells, small clusters of 10-15 cells and irregular aggregates. Majorities of the cells were aggregated
 617

randomly on the callus, however 3-week- old callus by transferring to liquid medium broke up into
single cells and small cell clumps. Table.6 summarizes the results concerning embryogenesis from both
cotyledon and hypocotyl explants derived calli. The results indicated that half-strength MS basal liquid
medium with growth regulators yielded highest amount of embryogenic cell suspension.
Prior to transfer of callus for the induction of somatic embryogenesis, the culture was
pretreated in complete darkness for 3 days to enhance the embryo initiation. Although cultures grew
slowly during pretreatment period in the liquid medium, packed cell density increased, and had also
most pronounced effect on the number of heart ·shaped embryos. The center core of the proembryo
from the suspension culture continue to grow in the liquid medium and outer enlarged vacuolated cells
become crescent-shaped and were lost from the surface.
500 mg of callus grown on solid medium was transferred to conical flask containing quantity
of liquid medium (half-strength MS with growth regulators) and kept on rotary shaker at lOOrpm in
complete darkness for 3 days. Different stages of embryos such as globular, heart, torpedo, and late
torpedo shaped embryos were observed from both cotyledon and hypocotyl derived callus within 35
days of culture [Plate 4 (a, b, c& d)]. After embryogenic callus induction, callus proliferation was done
in the liquid MS basal medium containing various growth regulators. Such embryo development was
observed in all combinations with varying frequencies. Embryos were very loosely hold on the surface
of calli and matured synchronously. Somatic embryogenesis did not occur on the basal medium
without any growth regulator supplementation.
The effect of 2,4-D with glutamine was studied for embryo induction from callus on half-
strength MS basal medium containing different concentrations of 2,4-D (0.01-2.0 mgll) with glutamine
(10.0 mgll). 2,4-D (0.1 mgll) and glutamine (10.0 mgll) combination produced the highest frequency of
somatic embryogenesis (41.1 %) and highest number of somatic embryos (36.8 embryos/culture) in
hypocotyl callus (Table 6).
Somatic embryos were induced from various concentrations ofBA (0.01-2.0 mgll) with 2,4-D
(0.1 mgll) and glutamine (10.0 mgll) combination. A combination of 2,4-D (0.1 mgll), BA (0.1 mgll)
and glutamine (10.0 mgll) induced the highest frequency of somatic embryogenesis in hypocotyl (70.2
%) and cotyledon (68.2 %) calli. The highest number of somatic embryos was noticed in hypocoytl
(59.2 entbryos/culture) and cotyledon (52.8 embryos/culture) explants (Table 6).
Optimum concentrations of 2,4-D (0.1 mgll) and glutamine (10.0 mgll) with variods
concentrations of NAA (0.1-1.0 mgll) combinations were used for embryo induction. Among them
2,4-D (0.1 mgt) + NAA (0.4 mgll) + glutamine (10.0 mgll) combination induced the maximum
percentage of somatic embryos in hypocotyl (38.1 %) and cotyledon derived callus (32.2 %) explant
The maximum number of somatic embryos was observed in hypocotyl (26.6 embryos/culture) and
cotyledon (25.7 embryos/culture) (Table 6).
Proembryos underwent further cell divisions to give rise to the globular embryos on a medium
containing 2,4-D, BA + glutamine. The changes from globular to torpedo shaped embryos were
brought about by increased mitotic cell divisions in the shoot apical region and mid central region of
embryos and further divisions in the cotyledon. The frequency of somatic embryogenesis was studied
in the cell suspensions after 28 days of culture. More than 75 % of the cells were converted into
different stages of somatic embryos.
Induction of somatic embryogenesis in suspension cultures, resulted in the formation of large
clumps of globular and latter staged embryos and the embryos underwent proliferation and
development in the same medium. The number of somatic embryos was dependent on 2,4-D, BA and
glutamine concentrations in the induction medium. Among the two combinations-2, 4-D + BA +
glutamine, and NAA + BA + glutamine, the former one gave the highest embryogenic response. Till
today, there is no report in somatic embryogenesis in Aegle marmelos via suspension culture. Both
2,4-D + glutamine together in the culture medium were highly effective in inducing somatic
embryogenesis from cotyledon and hypocotyl explants within 28 days of culture. Stuart and Strickland
(1984) reported that differentiation of suspension culture and an increase in embryoid formation was
attained in Medicago sativa by adding glutamine together with 2,4-D. 1n the present study, the rate of
somatic embryogenesis induction was the result of an interaction between explant type, auxin and
cytokinin concentration. Michler and Bauer ( 1991) also reported similar observation in Populus sps.
The auxin 2,4-D generally has been the preferred auxin used for the initiation of embryonal
suspensor mass (ESM) of most conifer species (Gupta eta/., 1991; Tautorus eta/., 1991). NAA has
also been used for ESM induction of Picea abies (Verhagen and Wann, 1989). However, no difference
has yet been reported in ESM proliferation or embryo development in conifers with NAA vs 2,4-D as
618

the sole auxin source. Birch cultures have an ability to form embryos even on media containing higher
concentration of auxin (2,4-D) (Kurten et a/., 1990). Joarder eta/. (1993) induced embryogenic callus
from nucellus in Azadirachta indica grown on Ms medium with 2mgll BA and 0.2 mg/1 NAA. They
further stated that callus induction was genotypic dependent. In Abies sp., ESM culture was initiated by
cytokinin (BA, KIN) alone, with auxin found to be inhibitory (Nogaard and Krogstrup, 1991). Basal
medium supplemented with cytokoinins such as BA (Bhansali and Arya, 1978 a, b), KIN (Hidaka,
1984; Hidaka and Omura, 1989; Kochba eta/., 1972; Pasqual eta/., 1988), zeatin (Chaturvedi and
Mitra, 1975; Hidaka and Kajiura, 1988) have resulted in embryoid formation.
A 5: I ratio of auxin 2,4-D to cytokinin BA stimulated embryogenic callus initiation (Park and
Son, 1988). The same auxin and cytokinin ratio stimulated in the greater number of globular somatic
embryos (Michler and Bauer, 1991). 2,4-D induced embryogenic callli from nucellar and ovular
explants of Citrus limon and C.vo/ckameriana (Saad, 1975) and immature ovules (Moore, 1985 a,b).
2,4-D (0.5 mg/1) and KIN (0.25 mg/1) induced embryoids in C.sinensis (Pasqua! eta/., 1988). Apart
from 2,4-D, NAA (Chaturvedi and Mitra, 1975; Nito and Imaasa, 1990), IAA (Kochba eta/., 1972)
have also been used from initiation cultures in Citrus. NAA is better than 2,4-D for establishing callus
of Citrus reticulata (Gill et a/., 1991). Embryogenesis in Citrus was induced even on the medium
lacking growth regulators (KobaYashi eta/., 1984; Sim et al 1988; Vardi eta/., 1986; Wu, 1990;
Yang, 1983).
Vieitez (1995) noted that both high salt medium such as MS and high hormone levels resulted
in compact calli and abnormal embryo development (thickening of cotyledons, over growth, fused
embryos), whereas media lower concentration such as WPM (woody plant medium) or half-strength
MS medium, supplemented with low concentrations (< 2.2 J!M) of a cytokinin (zeatin) plus an auxin at
lower concentrations (0.05-0.25J!M) resulted in the proliferation of embryogenic callus as masses of
yelllowish globular structures from which well-formed embryos formed.
Dameri eta/. (1986) induced somatic embryogenesis in Aescu/us hippocastanum grown on
MS medium with 2,4-D (0.1-1.0 mg/1). Whereas MS medium supplemented with NAA (2 mg/1) and
KIN (2 mg/1) reduced callogesnesis. It was assumed that a balance of 2,4-D and KIN was necessary for
the induction of somatic embryogenesis in horse chestnut. Radojevic (1988) demonstrated that
exogenously applied auxins were essential for somatic embryogenesis in horsechestnut.
Auxin and cytokinin ratio influenced the induction of somatic embryogenesis in cotyledonary
explants (Profumo eta/., 1976,1980,1990,199la), leaf segment (Dameri eta/., 1986) in horsechestnut.
Sondahl and Sharp (1977) obtained best yield of somatic embryogenesis in Coffea arabica leaf-derived
callus in KIN exceeds NAA.
Theo et a/. (1996) induced callus in Rosa hybrida from excised roots after incubation for 4
weeks in the dark on SH-medium (Schenk and Hidebrandt, 1972) containing 50J.!M 2,4-D.For callus
induction, calli were transferred to hormone-free SH medium and incubated for 8 weeks. The use of
gerlite instead of agar during callus induction stimulated somatic embryogenesis (upto 16%), whereas
the presence of BA inhibited subsequent regeneration. Shoot development was faster on solid callus
than on friable callus. Garg et a/., (19%) induced embryogenic callus in Acacia ni/otica on MS
medium with 2,4-D, BA, casein hydrolysate.

5.2. Maintenance

Suspension cultures were maintained for 2 months, with a 2-weeks interval subcultures on the same
induction medium. After 2 months of culture, the viability of embryogenic cell suspensions began to
decline.
Durzan and Gupta (1988) maintained ESM in lower concentrations of growth regulators (1-2
mg/12,4-D and 0.1 -0.5 mg/1 KIN and BA). ESM were also maintained in liquid medium using 250 ml
Erlenmeyer flasks, continuously rotated at 100-120 rpm in the dark, and sub cultured at approximately
7 days intervals. Krogstrup (1990) reported that culture density is critical and often determined the
quality of early stage embryos in suspension culture.
Dunstan eta/. (1993) observed changes in embryogenic potential of ESM cultures and their
growth has been reported after several months of weekly subcultures. Gupta and Pullman (1990)
reported the use of increased osmolality of the maintenance medium during early stage embryogensis
by increasing it froml20mmlkg to 180-25mmlkg depending upon the species and genotype. This was
done by adding myo-inositol, sorbitol, mannitol etc to the maintenance medium. Gupta and Pullman
(199lb) found that further increasing the osmolality of maintenance medium to 250-350mmlkg for
 619

long periods of time altered development by stimulating the multiplication of embryonal head cells and
inhibiting the growth of the suspensor system. Loss of embryogenic ability has been attributed to the
presence of 2,4-D in the medium (Halperin, 1966) and eventual loss of embryogenic diploid cells
(Fujimura and Komamine, 1975).
Cheema (1989) reported that embryogenic potential of cell lines was not maintained by cell
suspension after repeated subcultures but was maintained on solid agar based medium. Starrantino and
Caponnetto (1990) have maintained friable embryogenic calli derived from ovules of Citrus sinensis
for 2 yrs with good viability and embryogenic potential. Starrantino and Russo (1980) maintained
Citrus sinensis and C.limon embryogenic cultures for more than 3 yrs through periodic transferring
onto fresh medium. Mitra and Chaturvedi (1972) reported that embryo and ovary derived calli was
decreased and subsequently ceased with prolonged subculture. Speigel-Roy and Vardi (1984) have
mentioned that even 12-yr old Citrus calli have been found to possess the embryogenic competence.
Muralidharan and Mascarenhas (1995) maintained embryogenic cultures of Eucalyptus for more than 9
yrs without loss of any regenerability. Juretic and Jelaska (1991) reported the retention of embryogenic
potential for 15yrs in a callus line of pumpkin. Gromos et a/. (1989) maintained embryogenic callus on
MS medium with 51-!M 2,4-D and 051-!M BA in darkness at 25°C in Salix. Litz eta/. (1982) reported
somatic embryogenesis in mango cultivars on modified MS medium with 400mlifl glutamine, I OOmlifl
ascorbic acid, 60 lifl sucrose, 200/o coconut water and BA (4.4 and 8.8!-IM). Further it was mentioned
that efficiency of somatic embryogenesis could be greatly enhanced if the cultures were maintained in
liquid medium. Vieitez (1995) found that glutamine was necessary for the maintenance since in its
absence callus proliferation and embryo development ceased after 4-5 successive subcultures.

5.3. Maturation

Somatic embryos at late torpedo stage were transferred to 100 ml flasks containing 25 ml of half-
strength MS medium fortified with various growth regulator concentrations of ABA, AA, BA, GA3
and placed on a rotary shaker for the maturation of somatic embryos.
Various concentrations of AA (0.01-l.O mlifl) and BA (0.001-0.5 mlifl) combinations were
tested for the maturation of somatic embryos. Combination AA (0.5 mlifl) + BAP (0.1 mlifl) induc:eP
highest maturation response (23.3 %) with maximum number of matured embryos (16.5) [Plate 4 (e),
and Table 7].
Optimum concentration of AA (0.5 mlifl) added with ABA (0.01-1.0 mlifl) in order to
investigate the maturation efficiency. Among them, AA (0.5 mlifl) + ABA (0.5 mlifl) combination
responded very well for maturation of somatic embryos, as well as producing highest number of
somatic embryos per culture (Table 7).
ABA (0.5 mlifl) with various concentrations of BA (0.01-1.0 mlifl) was tested for the
maturation of somatic embryos. ABA (0.5 mlifl) + BA (0.1 mlifl) combination yielded the highest
response of embryo maturation (33. 7 %) as compared to other growth regulators tested (Table 7).
The cotyledonary stage of embryos had fused cotyledons, which did not open further. The
shoot meristems with primary leaves did not emerge in such cases.
Maturation of somatic embryos in suspension cultures appeared to be the major problem. Both
AA and ABA together stimulated the maturation of somatic embryos. The addition of ABA with BA in
the maturation medium led to the formation of uniform and viable mature embryos. In contrast,
Ammirato ( 1974) reported that abnormal morphology of somatic embryos was prevented by exogenous
of ABA In future, large-scale industrial applications of somatic embryogenesis require recovery of
large number ofplantlets directly from liquid cultures. As for Picea abies, Boulay eta/. (1988) reported
that ABA treatment was necessary for singulated somatic embryos and elongation of globular embryos.
Moreover, ABA increased the frequency of normal embryos in different species (Ammirato and Stryer,
1985; Attree and Fowke, 1991). Growth inhibitor such as ABA (0.04-4.0 !-1M) in the basal medium
induced somatic embryogenesis in Citrus (Spiegel-Roy and Vandi, 1984). ABA has been used for
cotyledonary embryo development in many plant species (Skriver and Mundy, 1990) including conifers
(Durzan and Gupta, 1987). It has been hypothesized that ABA inhibits cleavage of polyembryony and
allows embryo singulation and further development (Durzan and Gupta, 1987). The direct transfer of
cultures to ABA, with or without charcoal (Boulay et a/., 1988; von Arnold and Hakman, 1988).
However, ABA has not always been the most critical component for embryo development and
maturation. The osmotic potential of the media may also play an important role (Gupta and Pullman,
1990; Roberts, 1991).
620
Different concentrations of ABA (O.l-2.5mgll) have been used to improve embryo maturation
(Tautorus et a/., 1991). Roberts et a/. (1990b) observed precocious germination, accumulation of
storage proteins, and maturation of white spruce embryos with ABA (40-60J.LM) containing medium
(Roberts eta/., 1990b). Becwar eta/. (1988) used mA with ABA in the maturation medium to improve
the quality and germination of somatic embryos of Picea abies. Pullman and Gupta (1991) added ABA
with activated charcoal in their development and maturation medium. In some conifers, increasing the
osmolality of the development and maturation medium along with ABA was necessary to inhibit
precious germination (Cornu and Geoffrion, 1990; Gupta and Pullman, 199la; Attree eta/., 199lb).
ABA, high levels of protein and carbohydrates, and less water content are main factors to
influence the maturation and desiccation of somatic embryos (Kim and Janick, 1991; Janick eta/.,
1992; A1tree et al., 1991). This has been demonstrated in alfalfa (Mckersie eta/., 1989), grapes (Gray,
1989), white spruce (Attree et al., 1991) and soybean (Hammit and Davey, 1987). It appears that ABA
and sucrose in combination with other factors play a vital role in maturation and desiccation of somatic
embryos. ABA also "implicated in the regulation of storage proteins and in the synthesis of mRNAs
(Gomez eta/., 1988).
In Santa/um album, ABA and high sucrose imposed on somatic embryos influenced growth
and resulted in embryo maturation following dehydration. Encapsulation was not necessary for the
revival of germination from desiccated embryos (Rao and Bapat, 1995). Addition of ABA (7.6J.LM) to
the callus medium prevented callus browning to some extent and seemed to stimulated callus growth
(Gronoroos et a/., 1989). ABA may function as an osmoregulator causing cell water content to
decrease, and so lead to more normal development (Gray and Purohit, 1991).
The addition of ABA has proved effective for controlling repetitive embryogenesis
(Ammirato, 1974, 1977, 1983; Kamada and Harada, 1981; Vasil and Vasil, 1981), for improving
synchronization of development and maturation and for enhancing conversion of somatic embryos of
angiosperms (Ammirato, 1974,19n,l983; Vasil and Vasil, 1981; Lutz et a/., 1985; Higuchi and
Maeda, 1990) and conifers (Durzan and Gupta, 1987; Dunstan et a/., 1988,1991; Hakman and von
Arnold, 1988; Krogstrup eta/., 1988; von Arnold and Hakman, 1988; Thompson and von Adeikas
,1992) into plantlets.
In rosewood, ABA or mannitol did not favor somatic embryo germination, whereas BA at 0.5-
1.0 mg/1 was foond to be effective (Muralidhar Rao and Lakshmi Sita, 1996). Laurent et a/. (1997)
observed a positive effect of ABA and high COlicentration of polyethylene glycol on Hevea brasiliensis
somatic embryogenesis. Vagner et al. (1998) triggered somatic embryo development in Picea abies by
replacing of auxin and cytokinin in the medium by 10-40uM ABA Presence of PEG4000 in the
maturation medium was found beneficial for embryo development and maturation. However, complete
embryo development and maturation cannot be achieved by PEG alone in the absence of exogenous
ABA Use of 3. 75% PEG fastened embryo development, lowered the water content in mature embryos
and significantly increased the number of developing embryos and their germination ability. Gupta et
a/. (1998) achieved a good number of cotyledonary somatic embryos in Pseudotsuga menziesii, Pinus
taeda, and Picea abies with a combination of PEG (4000-8000), ABA and activated charcoal. Jones et
a/. (1998) increased somatic embryo production in Pinus patu/a by an inclusion of osmoticum
(PEG4000) at 5-100/o together with lOmg/1 ABA in the medium.

5.4. Germination

After maturation of somatic. embryos, they were transferred to half - strength MS solid medium
containing three combinations of growth regulators for germination.
For germination of somatic embryos, various concentrations of BA (0.0 1-0.8 mgll) alone were
tested. Among them, 0.4 mgll BA induced the highest percentage response of embryo germination
(21.3 %) and 6.6 embryos/culture germinated (fable 8).
The optimal concentration of BA (0.4 mgll) was tested with various concentrations of GA3
(0.5-4.0 mgll) for somatic embryo germination. Of these, BA (0.4 mgll) and GA3 (2.0 m!¥) together
stimulated embryo germination higher than BA alone (fable 8).
The synergistic effect of different concentrations of AA (0.001-1.0 mg/1) in combination with
the optimal concentrations of BA (0.4 mgll) + GA3 (2.0 mgll) was tested for improving somatic
embryo germination. Our results indicated that AA (0.01 mg/1), BA (0.4 mg/1), and GA3 (2.0 mgll)
combination was the best among growth regulator combinations tested and induced the highest
 621

germination of somatic embryos and an average number of germinated somatic embryos per culture
within 21 days of culture (fable 8).
Somatic embryos enlarged gradually and turned green and germinated with shoot and root
pole within 15 days [Plate 4 (f & g)]. The plantlet conversion was 90% in half-strength MS solid
medium [Plate 4 (h)].
Maximum number of globular stage embryos (24.6% embryos) on 5th day, heart shaped
embryos on lOth day (24.2%) and torpedo stage embryos (28.2%) on 20th day, were observed [Fig 3
and Table 9].
Suspension culture derived somatic embryos on half- strength MS medium containing BA +
GA3 induced highest response in both somatic embryogenesis and embryo germination. The response
of embryogenic tissue following transfer to a medium depended on the use of growth regulators.
The development of globular ethbryos was observed on the proliferation medium within 28
days of culture. The developmental stages such as heart, torpedo (early stages) and dicotyledonary
shaped embryos (latter stage) were obseiVed on embryo maturation medium after second subculture. It
clearly showed that both BA, AA and GA3 are very essential for simultaneous development of shoot
and root poles from the embryos. Ammirnto (1988) reported that exogenous auxin is needed for
development of somatic embryos. Marsolais et aJ (1991) described the presence of four classes of
somatic embryos in Geranium within 28 dayl; of cultures.
Of the different combinations used, ABA and BA combination was the best for embryo
conversion. The present investigation clearly indicates that the BA, AA and GA3, combination is
highly suitable for embryo germination.
Our results indicated that unbalanced levels of endogenous and exogenous growth regulators
might result in the abnormalities of somatic embryos. Genernlly, zygotic embryos of dicotyledonous
plants always have two distinct cotyledons lateral to the embryo axis, but in somatic embryos the
cotyledon shows great diversity. Soh (1996) reported similar observation.
The suspension culture was initially agitated for one week in complete darkness for the
induction of highest response. There were few reports on pretreatment, which was a prerequisite for
somatic embryogenesis (Nichol et al., 1991). Dark pretreatment may induce more embryogenic cells
or cause an alternation in cellular metabolism towards cell differentiation and cell proliferation
(Redenbaugh et al., 1991 b).
von Arnold and Hakman (1988) determined that germination of Picea abies cotyledonary
embryos is better in dark than in light The germination rate of selected cotyledonary embryos varies
between 35-90o/o(von Arnold and Hakman 1988, Becwar et al., 1988; Gupta et al., 1993). Germination
rate of conifer embryos is strongly related to the storage protein contents of the embryo (Roberts eta/.,
1990a). Germination can be influenced by a high relative humidity treatment (Webster eta/., 1990) that
avoid the rapid depletion of major storage proteins observed when somatic embryos of Picea sichensis
are directly transferred from maturation condition onto a growth regulator-free medium (Roberts et a/.,
1991). Poplar somatic embryos mature and germinate with minimal culture manipulations. Cheema
(1989) and Michler and Bauer (1991) reported the necessity for transfer of globular shaped embryos
from liquid to solid medium to promote somatic embryo maturation and germination. In addition, it
was necessary to provide pulse treatments of either lAA or NAA in the culture medium to stimulate
radical elongation (Michler, 1995). The same was found in Carya (Wetzstei et al., 1989), Robinia
(Merkle and Wiecko, 1989).
Germination, the process in which the embryo is awakened, is necessary for getting plantlets
and requires specific physical conditions. Similarly, the somatic embryos although developed
artificially need precise cultural conditions for initiation and development of shoots and roots leading to
complete plantlet formation.
Kochba et a/. (1972) reported that the lower KINIIAA ratio favored root formation and
inhibited stem elongation. The presence of GA3 in the germination medium stimulated rooting as well
as stem elon~tion. Nita and Iwamasa (1990), and Kochba eta/. (1974) stated that GA3 and adenine
sulphate also enhanced shoot as well as root formation. Chalupa (1987, 1990, and 1992) observed that
germination of Quercus robur somatic embryos and formation of plantlets occurred rarely on media
containing a high cytokinin concentration. Chalupa (1995) stated that desiccation-improved
germination and conversion of embryos into plantlets. Embryogenic tissues cultured on the same
medium without transfer for 3-4 months, produced more plantlets after transfer to fresh woody plant
medium (WPM), which compared with tissues subcultured to a fresh medium every month. Similarly,
622

embryogenic tissues with induced embryoids, cultured for 3 weeks on MS medium supplemented with
cytokinin and sorbitol (6%) and then transferred to WPM containing a low concentration of BA
(0.88j.IM), produced more plantlets than tissues cultured on a medium lacking sorbitol. Sucrose
enhances the initiation of embryo maturation. In embryogenic suspensions, ammonium and nitrate were
taken up only when the embryos were visibly germinating and ammonium was used faster than nitrate
Nuutila and Kauppinen 1992). Chalupa (1987a) observed the germination of birch somatic embryos
after transfer of embryogenic tissue with developing embryos on WPM containing 0.25uM mA or
lacking growth regulators. Kurten et a/. (1990) found that birch somatic embryos either fonned on N70
medium or later transferred onto it, developed and germinated nonnally. Dewald eta/. (1989b) found
that half B5major salts, MS minor elements, 40gll sucrose, and 400mgll glutamine are suitable for
embryo maturation. Addition of zeatin or BA to the medium inhibited horse chestnut somatic embryo
germination (Radojevic, 1995). Gray (1992) induced germination in 80-100% Vilis rotundifolia
somatic embryos by adding lj.IM BA toMS medium. Michler and Bauer (1991) stimulated somatic
embryo germination in Populus sp by addition of BA (0.05 mg/1) and IAA (5.0mgll) to the medium.
Chilling of the somatic embryos decreases endogenous ABA content and as a consequence the
donnancy breaks enabling embryos to germinate including somatic embryos of Coryllus ave/lana
(Radojevic, 1977) and Vilis vinifera (Rajasekaran and Millins, 1979), androgenic (Radojevic, 1991)
and somatic embryos of horse chestnut (Profumo eta/., 199la).

6. Synthetic seed production

The reports on synthetic seed production in Aegle marmelos are scanty. The main objective of this
investigation was to encapsulate of somatic embryos of Aegle marmelos for producing synthetic seeds.
Synthetic seed technology has a potential for clonal propagation from tissue culture to the field 'Syn
seeds' are encapsulated of somatic embryos that mimic the shape and function of zygotic seeds.
Sowing artificial seeds into soil eliminates special plant hardening or acclimation steps nonnally
associated with transferring tissue-cultured material to the greenhouse or field (Bapat and Rao, 1988).
The encapsulation of somatic embryos, conversion of encapsulated embryos into plantlets and factors
affecting the conversion frequency were described below.

6.1./nduction

Abundant somatic embryos were produced indirectly via callus on MS solidified medium
supplemented with 2,4-D (0.2 mgll) + BA (0.2 mgll)+ glutamine (10.0 mgll); and 2,4-D (0.1 mgll) +
BA (0.1 mgll) +glutamine (10.0 mgll) in the liquid medium.

6.2.Encapsulalion

Sodium alginate (0.5-5.0 %) with CaC12 (2.5 %) was tested for making of synthetic seeds. Among
them, 3.0 %sodium alginate was suitable for encapsulation. For maximum frequency of 'syn seed'
germination, 3.0% sodium alginate was most suitable. The highest of response was 70.4% and the
number of syn seeds' per culture (45.5) germinated on the culture medium containing suitable growth
regulators, thereafter, reduced the gennination rate in low and high concentrations of sodium alginate
within 25 days of culture [Table 10, Plate 5(a)]. This is in consistent with results of George and Eapen
(1995) encapsulation of somatic embryos of finger millet with 3.0 %sodium alginate+ 2.5% CaC1 2.
Redenbaugh eta/. (1986) discovered that hydrogels such as sodium alginate could be used to
produce single-embryoid artificial seeds. Similarly Fernandes et a/. (1992) reported that somatic
embryos were isolated and were placed on 3 %sodium alginate matrix dropped in into CaC1 2 (0.69 %).
Sanada eta/. (1993) reported increased conversion of carrot and celery embryos encapsulated with
microencapsulated sucrose. Microcapsules were 0.5mm in diameter and were coated with ethylene-
vinyl acetate copolymer wax. Friend ( 1993) suggested that low encapsulation efficiency could have
caused the poor results apparently due to rapid release of sugar, synthetic seed using sucrose
microencapsulated with either cellulose acetate butyrate or gelatin. Bapat and Rao ( 1988), Bapat ( 1993)
reported inclusion of nutrient salts in manufactured sandalwood seed which were beads of a composite
of alginate and silica gel. Silica gel should adsorb nutrients, acting to control their release to the
embryo. Lulsdorf eta/. (1993) added activated charcoal to alginate beads encapsulating interior spruce
 623

(Picea glauca engelmannii complex), and black spruce (Picea mariana). Such seed survived on one-
month cold storage at 4°C on nutrient media Mnknnthalmmar and Mathur (1992) obtained germination
of male bamboo with encapsulation in 6% calcium alginate with MS medium with or without 3%
sucrose. They coated seeds with paraffin oil to reduce microbial invasion and desiccation. Somatic
embryos of sugar pine (Pinus lamberciana), loblolly pine (Pinus taeda) and Norway spruce (Picea
abies) have been encapsulated in sodium-alginate gel (Gupta and Dwzan, 1986a, 1987). The best
results were obtained with 1.5-2.0% sodium alginate and O.lM calcium nitrate in half-strength MS
medium containing 1-% sucrose. Redenbaugh et a/. (1993) reviewed a method for making self-
breaking capsules as a modification of the alginate bead as a modification of the alginate bead
technology. Such capsules break when they are exposed to water after sowing. 0nay et a/. (1996)
stated that encapsulation is a practical procedure for short- term storage of embryogenic Pistacia vera
tissue and may be applicable to the prese1Vlltion of desirable elite genotypes. The somatic embryos
were encapsulated by using sodiuni alginate (3.0 %) and calcium chloride (2.5 %) suitable for bead
formation inAegle marmelos.

6.3. Storage

The effect of storage of artificial seeds was investigated ilr embryo germination. In the present study,
75% encapsulated embryos germinated when stored for 1-3 days in refrigerator at 4°C and, thereafter,
germination rate declined. Similar results were obtained on the storage of 'syn seeds' in sandalwood at
4°C for more than 14 days (Bapat and Rao, 1988). Fernandes eta/. (1992) recorded 17% germination
of encapsulated sandal wood embryo. Effect of storage on encapsulated embryos was studied. The
highest response was 88.2% and an average of 28.8 'syn seeds' germinated per culture in 2 days stored
syn seeds. After 2nd day, germination starts decreasing gradually. The effect of synthetic seed storage
on germination was statistically significant at 5 %level (Table 11).

6.4. Germination

Various substrates were tested for 'syn seed' germination on half-strength MS solid medium [Plate 5
(c)], cotton [Plate 5 (b)] and sterile soil [Plate 5 (d)] containing half-strength MS salts with growth
hormones such as NAA (0.1 mgll) and BA (0.5 mg/1). On half-strength MS solid medium, 60% 'syn
seed' germinated in 30 days of culture (Fig 4). Among the three substrates (MS solid medium, cotton
and soil) used, MS solid medium was effective in syn seed germination than other two substrates.
Growth regulators BA and NAA together were responsible for highest synseed germination response in
MS solid medium. The germination rate of encapsulated seeds was very low on sterilized soil
moistened with MS nutrients and cotton with growth regulators. The synthetic seeds retained their
viability upto 3 days and gradually decreased (Table 11).
Synseed germination was tested on half-strength MS medium fortified with several
combinations of BA, KIN, NAA and GA3 individually and in combinations. The highest germination
response (75.5%) was observed in BA (0.5mgll)+NAA (0.2mgll) with 23.4% germination and 16.2%
plantlet survival (Table 12).
The encapsulated embryos germinated within 15 days of culture. The alginate matrix ruptured,
green leaves emerged, and roots developed from encapsulated embryos [Plate 5(e)]. The plantlets from
encapsulated embryos were normal when compared to non-encapsulated embryo derived plant [Plate 5
(f)].
Mascarenhas eta/. (1989) observed better synseed germination of Eucalyptus dtriodora on
MS medium with2% sucrose rather than on the soil directly. Fuji et a/. (1992) demonstrated naked
alfalfa embryos placed in the field under Styrofoam cups had a 25% conversion to autotrophic plants.
Sowing artificial seeds into soil eliminates special plant hardening or acclimatization steps normally
associated with transferring tissue-cultured material (Fernandes eta/., 1992)
Plantlets derived from synseeds were normal when compared with non-coated somatic
seedlings. These results were in concurrence with Fuji eta/. (1992). George and Eapen (1995) reported
that 6 % mannitol in the medium was. highly effective in syn seed germination, survival of plants in MS
solid medium, and successful field planting of synthetic seeds. Fernandes eta/. (1992) reported that
MS solid medium containing IAA, IDA, and GA3 was the best for synthetic seed germination.
The use of encapsulated somatic embryos can reduce the cost of production when compared
with non-encapsulated embryos. Also two stages such as rooting of embryos and hardening of the
624

plantlet, could be totally eliminated by directly sowing the encapsulated embryos in the soil.
Furthermore, ttansportation and exchange of gennplasm by encapsulated embryos rather than seeds
would be easier and relatively inexpensive and safer (Ganapathi eta/., 1992; Lu et a/., 1990).

Conclusions

The bael tree, an important ayurvedic medicinal tree, is being used very extensively in indigenous
medicine.
Conventional methods of propagation are associated with problems, like seasonal seed
produc5tion, pest problems, low percentage of seed germination etc. In vitro culture methods can
overcome these problems and provide faster multiplication of selected genetically elite clones.
Aegle marmelos somatic embryogenic cultures were initiated from different type explants, viz.
cotyledon, hypocotyl, immature leaflet, and mature leaf from in vitro raised seedlings.
Direct somatic embryo induction, matmation, germination have been effected by explant type,,
media fonnulation, and culture conditions. NAA and BA combination was found to be more effective
in somatic embryo induction10/14hrs {daikllight) treated (upto one week) explants showed the
highresst embryogenic response. Both embryo maturation and germination was obtained on the same
medium i.e. half-strength MS medium supplemented with BA and AA
Indirect somatic embryogenesis on solid medium showed that 2,4-D, BA and glutamine
combination was the most suitable for embryo induction Cytokinin BA together with ABA was highly
effective in embryo maturation and germination on half-strength MS medium. A combination of 2,4-D,
BA and glutamine combination supplemented to MS medium showed more effective induction of
somatic embryogenesis in suspension cultures. Snspeusion cultures were maintained for 2months with
2-week interval subcultures on the same induction medium. ABA and BA combination yielded the
hil!hest respqnse of emb~o maturation 9n half~ MS medium. The synergistjc effect of BA, AA
ana GA3 on half-strength MS medium ytelded the highest somatic embryo germmation Among various
explants used, hypocotyl explant showed proved to be more suitable for somatic embryogenesis.
3% sodium alginate was the most suitable for synseed encapsulation Prolonged synseed
storage at 4°C gradually decreased germination process. The highest synseed germination was
observed on BA and NAA combination on half-strength MS medium.

Besides the ;tbove mentioned, the following problems may be considered for future research.
1. To initiate somatic embryogenesis from vegetative tissues of old trees and /or matured trees
to achieve a large-scale propagation of selected adult clones
2. To optimize medium for long tenn maintenance of cultures
3. To obtain high frequency production of morphologically normal somatic embryos with a
capacity to germinate
4. To improve the process of efficiency of maturation and conversion rates of somatic
embryos into emblings
5. To improve the synseed storage conditions to obtain more germination and
6. To improve the suitable conditions for hardening the emblings after transfer to soil.

Acknowledgements

Authors are thankful to Prof. Dr.K. V. Krishnamurthy, Head, Department of Plant Science, for
providing laboratory facilities. The senior author is grateful to the University Grants commission, New
Delhi for providing Junior and Senior Research Fellowships.
 625

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 639

Table I. Chemical constituents in various plant parts of Aegle marmelos

Plant Part Chemical properties I content Therapeutic Uses

Root Auraptene,marmin, Anti-amoebic, hypoglycaemic,

umbelliferone, lupeol intermittent fevers, palpitation
of heart.

Root Bark Intermittent fevers, fish poison

Leaf
Aegeline; 0-(3,3-dimethyl
allyl)-halfordinol; N-2-ethoxy- Cardiotonic, fever, opthalmic,
2-(4-methoxy phenyl)-ethyl antifungal, asthma.
cinnamide; N-2- methoxy 2-[4-
(3', 3'- dimethyl allyloxy -
phenyl}- ethyl cinnamide; N-2-
methoxy-2-(4-methoxy phenyl)-
ethyl cinnamide; aegelenine;
a-13-phellandrene; y- sitosterol.

Marmesin, y-fagarine, Dyeing and tanning.

Stem Bark umbelliferone, 13-sitosterol,

Astringent, digestive,
Fruits &--«- Phellandrene, allo- stomachache, chronic diarrhoea,
imperatorin, 13 - sitosterol, dysentry, tonic for heart and
water-soluble gum. brain, antiviral, hypoglycaemic.

Wood Furoquinoline, dictamine, Timber.

mannesin, 13 - sitosterol.

Source: Chadha, 1985; Chopra, 1956.

640

Table 2. Effect of growth regulators added in MS medium on direct

somatic embryogenesis from cotyledon and hypocotyl explants of
Aegle mar.melos data (Mean±SD) were collected after 35 days.

Explants response Number of somatic

Growth Percentage Mean ± SD) embryos/explant
regulators (Mean ± SD)
(mg/1)
c H c H

IAA BA
0.01 0.1 21.1±0. 97d 18.3±0.76f 1. 3±0. 45d 2. 3±1. ooc
0.1 0.1 26.4±1.34° 28. 7±1.22d 1. 6±0. 66d 1.8±0.74d
0.2 0.1 30. 8±1. 85b 38. 6±1.56b 2.4±0.83° 1.2±0. 67d
0.4 0.1 24, 4±1. 97° 29. 8±1. 95d 1. 4±0. 48d 1.2±0. 42d
0.8 0.1 19. 4±1. 35d 25.2±1.47° 1.4±0. 48d l.o±o.oo•
IAA KIN
0.01 0.8 O.O:t:O.O O.O:t:O.O O.O:t:O.O O.O:t:O.O
0.1 0.8 3. 3:t:O.llb 3.3±0.24i 1. a±o. o• 1. oM. oo•
0.2 0.8 7. 6±0.12 9 8.1±0.55h Lo±o.o• 1. 0±0. 40°
0.4 0.8 16. 4±1. 23° 17 .1±1. 31 f 2.8±0.45° 2.7±0.78"
0.8 0.8 15. 5:U. 59• 13.3±1.989 1.2±0.40d 1.3±0.45d
NAA BA
0.01 0.1 25. 7±1.21° 23.5±1.35° 1. 7±0. 71d 2.1±0.82c
0.1 0.1 32. 5±1. 65b 32. 8±1. 66° 2.2±0.78° 2.5±0.87°
0.2 0.1 43.3±1. 95• 45.2±1.97. 4.8±0.82. 6.6±0.98.
0.4 0.1 34. 8±1.22b 34 .1±1. 23° 3. IHO. 75b 4. 8±0. 7-3b
0.8 0.1 21. 8±1. 56d 19. 3±"6. 9e-£ 3.7±0.7eD 2 ;2±0. 77c
NAA KIN
0.01 0.8 24.2±1.16° 37. 5±1. 86b 2.1±0.83° 1. 7±0.12d
0.1 0.8 35.2±1.8~ 39-. 5±1..1~ 3. 9±0-. 9-4b 4 .2-±0. 6eP
0.2 0.8 21.2±0. 98d 18.2±0.87f 2.8±0. 74° 2.6±0.59c
0.4 0.8 16.2±0.95° 14.2±0. 75 9 1. 4±0.48d 2.0±0.50"
0.8 0.8 11.5±0. 70f 9.3±0.22h 0.0±0.0 0.0±0.0

Each value represents an average of 7 replicates mean

separation using Duncan's New Multiple Range Test, me~ within
column with different letters are ~igrrificant at the 5 % level.
C-Cotyledon ; H-Hypocotyl.
 641

Table 3. Effect of half-strength MS medium fortified with

BA, GA3 and AA on maturation and germination of somatic
embryos (direct) in Aegle marmelos data (Mean±SD) were
collected after 21 days,both for maturation and germination.

Growth Percentage of Percentage of embryo

regulators embryo maturation Germination
(mg/1) (Mean ± SD) (Mean ± SD)

BA AA
0.1 1.0 20. O±o. 55d 2.8±0.21e
0.5 1.0 35. 0_±0. 30° 3. 6±0.17d
0.8 1.0 60.5±0.35° 6.3±0.26~
1.0 1.0 50.5±0.72b 5.6±0.22b
2.0 1.0 34.2±0. 65c 2. 4±0.19e
BA GA3
0.1 0.1 14.2±0.12f 1. o±o. oof
0.5 0.1 14.5±0.90f 3.5±0.02d
0.8 0.1 16.7±0.85e 4 .2±0. osc
1.0 0.1 12.5±0.719 2. 3±0. 02e

Each value represents -the average of 7 replicates

mean separation using Duncan's New Multiple Range Test,
means within column with different letters are significant
at the 5 % level.
Table 4. Effect of MS medium fortified with 2,4-D, NAA and BA in combination with 10 mg/1 Glutamine on
indu~tion of indirect somatic embryos from different explant derived callus of Aegle marmelos data ~
(Mean±SD) were collected after 35 days.

Explants response Number of somatic

embryos/culture
Growth (Percentage Mean±SD) (Mean±SD)
regulators
(mg/1)

c H IML ML c H IML ML

2,4-D
0.01 18. 5±0. 9"9. 25 .1±1. 23°" 24.3±1.22° 12.0±0.84f 13. 4±1. 29• 16.0±2.48' 14.0±1.48" 4 .1±0. 83°
0.1 28. 2±1. 56° 37. 2±1. 97° 30.1±1. 76° 18.2±1.22" 22. 8±1. 24°0 23.8±2.16° 19.3±1.90° 6.1±0. 83°
0.2 31.0±1.99" 42. 0±2. 34b 35.6±1.98b 25.8±1.44' 26.0±1.00° 30.8±1.77° 21.4±2.05° 10.4±1.44°
0.4 29. 2±1. 56° 45. 3±2. 45b 28. 3±1. 65° 20.0±1.21° 22. 9±1. 44cd 25.9±1.57° 13.8±1.53d 8.4±1.49°
0.8 26. 0±1. 35" 29. 4±1.65" 29.8±1. 93° 12.2±0.68f 17 .1±1. 86° 19.6±1.68" 15.3±1.84" 4. 6±1. 01°
2,4-D BA
0.2 0.1 30. 6±1. 57° 22. 2±1. 22" 36. 3±2. 65b 12.2±0.87f 26. 0±1. 50° 25.8±1.16" 21.8±1.72cd 11. 1±1. 92•
0.2 0.2 48.2±1.89" 49. 3±1. 65" 42.4±1.85" 38.4±1.21" 45. 5±2. 45" 46.4±2.08" 38.4±1.8" 32. 2±1. 93"
0.2 0. 4 32. 3± 1. 67bc 32. 7±2.68° 32. 5±1. 40° 30. 4±1. 87b 30. 8±2. 03b 36.8±1.83b 24.6±2.33° 16.3±1. 79b
0.2 0.8 18.0±0.97• 44.1±2.34b 25. 9±1.27° 25. 8±1. 25° 26. 8±1. 60° 25.8±2.18" 19.6±2.57° 11. 4±1. 35°
0.2 1.0 15.8±0.68. 16.5±1.87f 13. 3±1. 23' 11. 6±1. 44f 11.3±1.48" 11.3±2.33. 12.1±1.01' 11. 3±1. 55°
NAA
0.001 4.8±0.21' 4. 9±0. 21 h 3.8±0.10. 4.2±0.12. 2 .1±0. 83h 2. 4±0. sol 1. 9±0. 70" 2.1±0. 70"
0.01 7.1±0.45j 7.4±0.35•• 8.0±0.22. 8.8±0.23. 3.8±0.97 .. 4.2±1.16i 2. 3±1. oo• 4.1±0.83d
0.1 15.2±0.50. 18. 9±0. 67"' 18. 4±0.17" 9.1±0.34. 10. O±l. oo•• 10. 9±3. 59. 4.5±0.80.. 5. 4±1. Old
0.5 10. 5±0. 87 1 11. 0±0. 65. 12.1±0.23f 8.1±0.23 9 8. 5±1. 36' 1. 6±1. 2a• 1. O±l. oo• 3. 4±0. 91 d
1.0 9.0±0. 75 1 10.5±0.52. 10. 4±0.so• 4.2±0.21. 5.2±0.87 9 4.8±0.87' 6. 0±0. 89 9 1.8±0.74'
NAA BA
0.1 0.1 22.2±1.21' 21. 4±0. 98" 21.4±0.78° 17.8±0.98.. 17. 3±1. 79° 10.6±1.56 9 11.1±0.13' 10.9±1.44°
0.1 0.2 29. 5±1. 47° 35. 0±1. 25°0 27 .1±1. 45° 22. 0±1.034cd 23.6±1.11° 19.5±1.11" 16.1±1.57~ 14.2±1.40.
0.1 0.4 35. o±l. as• 40. 8±1. 27bc 34. 0±1. 67b 25. 6±1. 22° 30. 9±1. 64b 31.5±1.91° 27.3±2.20b 14. 5±1. 91.
0.1 0.8 29.5±1.64d 38. 2±2 .10° 26 .1±1. 33d 19.3±0.98d 25.6±1. 49° 16.4±2.20£ 16.1±1.57~ 12.3±1. 79°
0.1 1.0 26. 8±1. 55" 30. 0±1. 21 d 18.5±0.86" 17. 0±0. 76" 19.9±2.21° 14.0±2.09f 8.3±1.41. 8. 6±2 .15°

Each value represents the average of 7 replicates mean separation using Duncan's New Multiple
Range Test, means within column with different letters are significant at the 5 % levele
C-Cotyledon ; H-Hypocotyl ; IML-Immature leaflet ; ML- Mature leaf.
 643

Table 5. Effect of half-strength MS medium fortified with

BA, AA, ABA and GA3 on maturation and germination of
somatic embryos (indirect) in Aegle mar.melos data (Mean±SD)
were collected after 21 days,both for maturation and germination.

Percentage of Percentage of
Growth embryo maturation ~mbryo germination
regulators (Mean±SD) i(Mean±SD)
(mg/1)

BA AA
0.2 0.001 33.6±0.52e 13.4±0.17d
0.2 0.01 59.2±0. 7lb 1B.3±0.18c
0.2 0.1 45.3±0.55c 17.8±0.08c
BA ABA
0.2 0.01 45. 7±0.21c 16.0±0.12c
0.2 0.1 65.5±0.15a 28. 6±0.10a
0.2 0.5 40.0±0.60d 22. 4±0. 07b
BA GA3
0.2 0.5 20.0±0.35q 10.5±0.14de
0.2 1.0 24.6±0.85f ll.l±O.lld
0.2 2.0 16.2±0. 7lh 8.4±0.75f

Each value represents the average of 7 replicates mean separation

using. Duncan's New Multiple Range Test, means within column with
different letters are significant at the 5 % level.
 644

Table 6. Effect of half-strength MS medium fortified with various

concentrations of 2,4-D, NAA, BA in combination with 10 mg/1 glutamine
on induction of somatic embryos in suspension cultures from hypocotyl
and cotyledon derived callus of Aegle mar.melos data (Mean±SD)were collected
after 35 days.

Frequency of embryogenesis Number of somatic embryos/culture

Growth (Percentage Mean±SD) (Mean ± SD)
regulators
(mg/1)
c H c H

2,4-D
0.01 37 .1±1. 34d 33.1±1.21" 21.0±2.00" 16.2±1.63!
0.1 40. 2±1. sse 41.1±1. 60cd 34. 0±1. 73ed 36. 8±1. 83e
0.5 3g. 8±1. aoed 3g. 7±1. 85d 31. 3±3. 42d 33, 5±1. 41c
1.0 3g,5±1.80ed 35.2±1.g6" 2g.2±2.16d 28 .2±1. 63d
2.0 28.g±1.55" 32. 6±1. 44" 22. 3±1. 86" 24.1±1.76de
2,4-D BA
0.1 0.01 2g,l±l,gg• 45, 3±1. g7e 21. 6±2. 44" 36, 0±1. 73e
0.1 0.1 68.2±2.75. 70.2±3.22. 52. 8±3. 82. 5g,2±1. n•
0.1 0.5 58.3±2.12b 61.1±2. gob 50.3±1. 7gb 4g,2±2.58b
0.1 1.0 42.2±2.11e 42. 6±1. gee 43.3±3.4ge 47.1±1.sgbJ
0.1 2.0 36.2±1.88d 41. 2±1. g7ed 2g,3±2.33d 33.6±2.44e
2,4-D NAA
0.1 0.1 14.3±g,87 9 17 .1±0. 66 9 1g .1±1. 53"f 16.3±1.65f
0.1 0.2 31. 1±1. 7 ode 22. 3±1. 22f 21. 7±2 .16" 24. 3±1. 65de
0.1 0.4 32. 2±1. g3d 38.1±1. g7d 25. 7±1. 3g" 26.6±1.57d
0.1 0.8 27. 8±1. 67" 30. 2±1. 83" 20. 5±1. so• 20.8±2.71"
0.1 1.0 22. 5±1. 57f 23. 6±1.18f 16.7±2.gsf 16. 3±1. 40f

Each value represents the average of 7 replicates mean separation using

Duncan's New Multiple Range Test, means within column with different
letters are significant at the 5 % level.
C-Cotyledon ; H-Hypocotyl.
 645

Table 7. Effect of half-strength MS medium fortified with

various concentrations of AA, ABA and BA on maturation
of somatic embryos derived from suspension cultures of
Aegle mar.melos data (Mean±SD) were collected after 21 days,
both for maturation and germination.

Growth Maturation response Number of matured

regulators (Percentage Mean±SD) embryos/culture
(mg/1) (Mean ± SD)

AA BA
0.01 0.001 18.2±0. 5be 13. 5±0.10e
0.1 0.01 18.2±0.61e 11. 3±0.19f
0.5 0.1 23. 3±0. ggd 16.5±0.34d
1.0 0.5 20. 3±0. 98e 13. 3±0. 43.
AA ABA
0.5 0.01 24.1±1.22d 16.3±0.10d
0.5 0.1 26. 3±1.29c 20.2±0.18c
0.5 0.5 28 .2±1.27c 24 .1±0.10b
0.5 1.0 21. 2±1. 34de 16.2±0.14d
ABA BA
0.5 0.01 30.1±1. 37" 22. 8±0. 18bc
0.5 0.1 33. 7±1. 20b 26.4±0.07"
0.5 0.5 27. 6±0. 90c 20.5±0.14c
0.5 1.0 22.3±1.62d 17. 6±0.17d

Each value represents the average of 7 replicates mean

separation using Duncan's New Multiple Range Test, means
within column with different letters are significant at
the 5 % level.
646

Table 8. Effect of half-strength MS medium fortified with var-

ious concentrations of BA, AA and GA3 on germination of soma-
tic embryos derived from suspension cultures of Aegle maxmelos
data (Mean±SD)were collected after 21 days.

Growth Percentage of embtyo Number of somatic embryos

regulators germination(Mean±SD) germinated/culture(Mean±SD)
(mg/1)

BA
0.01 10.5±0.50h 0.0±0.0
0.1 15. 7±0. 7r 0.0±0.0
0.2 18. 5±6,. 3 f. 2.7±0.15h
0.4 21.3±0.99e 6. 6±0 .18 9
0.8 25. 2±1. 35d 2.5±0.22h
BA GA3
0.4 0.5 25. 3±1.22d 11. 6±0 .14f
0.4 1.0 28.7±0.70° 16.5±0.10e
0.4 2.0 31.2±0.9lb 28.1±0.23b
0 .. 4 3.0 21. 2±0. 99e 21.8±0.lld
0.4 4.0 19 .1±0. 56ef 17.7±0.5le
BA AA GA3
0.4 0.001 2.0 34. 8±1. 55b 16.7±0.10e
0.4 0.01 2.0 40. 6±1. 99a 32. 3±0 .15a
0.4 0.1 2.0 39. 5±1. 51 a 23. 3±0. 08°
0.4 0.5 2.0 32. 5±1. 33b 21. 3±0. 07d
0.4 1.0 2.0 27.3±1.22° 19.9±0.12d

Each value represents the average of 7 replicates mean

separation using Duncan's New Multiple Range Test,_means
within column with different letters are significant at
the 5 % level.

Table 9. Frequency (number/culture) of various stages of

somatic embryos(Mean ± SD) on different days count in
Aegle maxmelos.

Stages of Number of days

embryo
5 10 15 20
Globular 24. 6±1. 01 14.8±0.97 5.4±0.48 2.2±0.4
Heart 9.2±1. 72 24.2±0.74 10. 8±1. 72 3.6±0.48
Torpedo 2.8±0.74 5.0±0.63 20. 6±1. 01 28.2±0.74
 647

Table 10. Effect of sodium alginate with CaC1 2 (2.5 %)

on germination of synthetic seeds in half-strength MS
medium containing BA (0.5 mg/1) + NAA (0.2 mg/1) in
Aegle mazmelos, after 21 days.

Sodium Germination response Number of 'synseeds'

alginate (Percentage Mean±SD) germinated/petridish
(%) (Mean ± SD)

0.5 so. 5±0.11" 5.3±0.15

1.0 62. 0±0.10° 16.5±0.29"
2.0 65. 7±0.2lb 33.8±0.12°
3.0 70. 4±0 .12a 45. 5±0. 50a
4.0 54. a±·o .laC! 41. 6±0. 55b
5.0 36.5±0.20f 23. 7±0. 7ld

Each value represents the average of 7 replicates

mean separation using Duncan's New Multiple Range
Test, means within column with different letters are
significant at the 5 % level.

Table 11. Effect of storage conditions on germination

of 'syn seeds' on half-strength MS medium containing BA
(0.5 mg/1) plus NAA (0.2 mg/1) in Aegle mazmelos.

Storage ·syn seeds' Number of syn seeds

conditions germination response germinated/culture
(days) (Percentage Mean±SD) (Mean ± SD)

0 80.5±0.716 28.7±0.17a
1 87.4±0.asa 28. 6±0. 54a
2 88.2±0.5la 28.8±0.20a
3 85.7±0.75a 28.6±0.31a
5 70.5±0.34° 27. 7±0. 1-7a
10 65.3±0. 45d 11.7±0.17b
15 62.0±0.92d 8. 0±0. 27°
20 55.8±0.48" 5.6±0.36d
25 35.1±0. 68f 1. 6±0 .12"
30 32.5±0.95f 0.0±0.00

Each value represents the average of 7 replicates mean

separation using Duncan's New Multiple Range Test, means
within column with different letters are significant at
the 5 % level.
648

Table 12. Effect of half-strength MS medium containing BA,

KIN,NAA and GA3 on germination of 'synseeds' of Aegle maxmelos,
after 21 days.

Growth Germination response Number of 'syn seeds' Survival of

regulators (% Mean±SD) germinated/petridish
(mg/1) (% Mean±SD) Plantlets

BA
0.1 18.8±0.099 4.6±1.01 9 2.2±0. 74f
0.5 21. 0±0. 07f 7, 4±1. Ole 3.6±0.80
1.0 19. 5±0 .10 9 4. 2±0. 97 9 2. 6±1. Olt
KIN
0.1 16},5±~ .. o8h 2. 5±0.15h 2. 2±0. 21 f
0.5 17 .5±0.169 4. 2±0 .12 9 2.6±0.0lf
1.0 16:8±0.12h 2.3±0.12h 2 .1±0.14 9
NAA
0.01 11. 7±0. o5i 1. 8±o. o5i 1. 0±0. 08h
0.1 16. 5±0.15h 2.3±0.05h 1. 7±0. 07 9
0.5 16. 7±0 .14h 2 .1±0.12hi 1. 6±0 .11 9
BA KIN
0.5 0.1 20.1±0.22f 5.7±0.08f 2.6±0.18f
0.5 0.5 35.4±0 .19d 8.7±0.18e 5.3±0.30d
0.5 1.0 30.7±0.24e 5.0±0.15f 3.8±0.07e
BA NAA
0 .. 5 0.1 57.5±0.52° 19.3±0.14b 8.5±0.28°
0.5 0.2 75.5±0.75a 23.4±0 .lOa 16.2±0.10a
0.5 0.4 67 .2±0. 61b 15.2±0.07° 10.5±0.23b
0.5 0.8 55.4±0.57° 12. 6±0.19d 10. 0±0. OBb
BA GA3
0.5 0.1 10. 7±0.09i 1. 7±0 .lSi 1.3±0.21 9
0.5 0.5 14.3±0.12h 3. 0±0 .15 9h 2.2±0.10f
0.5 1.0 12. 6±0. OBi 2.5±0.25h 1. 8±0 .10 9

Each value represents the average of 7 replicates mean separation

using Duncan's New Multiple Range Test, means within column with dif-
ferent letters are significant at the.5% level.
 649

Fig .1 Effect of Photoperiod on Direct Somatic Embryogenesis

"0
"0
.
[
.."'
0::

c
..
!'l
Q.
w "
0

"'

5/19 10/14 15/9 2014

Dark/Light (Hrs)

Fig .2 Effect of Glutamine on Somatic Embryogenesis

90

80 D Colyledon

70 \ II Hypocotyl 1

"c
~

"
0
....c..
0:: 50
c."' 40
Q.
.n 30
0
~
20

5 10 15 20
2,4·0 (0.1 mg/1) + GLN (mg/1)
650

30 Fig .3 Different Stages of Somatic Embryos

0 Globular Embryos
25

,.
\1
-c
s 0 Torpedo stage embryos

0"
~
"'wE
0
~

5 10 15 20
Number of Days

Fig A Selection of Substrate for 'Syn Seed' Germination

Soil
7%
 651

Plate 1. Direct somatic embryogenesis

a. Direct somatic embryo induction from cotyledon explant X20

b. Somatic embryo induction from hypocotyl explant
c. Matured somatic embryos and germination from cotyledonary explant
d. Matured somatic embryos and germination from hypocotyl explant X20
e. Plantlet from cotyledon derived somatic embryos XIO
f. Plantlet from hypocotyl derived somatic embryos
g. Plantlet showed bipolar (shoot and root) structure
h. Acclimatized plantlet
652

Plate 2. Indirect somatic embryogenesis -via callus cultures

a. Indirect somatic embryos from cotyledon derived explant XIO

b. Indirect somatic embryos from hypocotyl derived callus XIO
c. Indirect somatic embryos from immature leaflet derived callus XIO
d Indirect somatic embryos from mature leaf derived callus XIO
 653

Plate 3. Somatic embryo germination and hardening- via callus cultures

a Germinated embryos X30 d. Embryo derived plantlet showed taproot

b. Germinated embryos on callus e. Shoot elongation
c. Germinated embryos showed shoot tip and root tip f . Potted plant
654

Plate 4. Indirect somatic embryogenesis-via suspension cultures

a. Globular-stage embryo XlO e. Cotyledonary -stage embryo xlO

b. Heart-stage embryo X20 f. Bipolar structure showed shoot pole and root pole
c. Torpedo- stage embryo g. Embryo derived plantlet
d. Late stage of torpedo-stage embryo Xl 0 h. Potted plant
 655

Plate 5. Synseed -via callus culture derived embryos (Suspension culture)

a. Somatic embryos encapsulated by Sodium alginate d Germination of synthetic seeds on sterile soil
b. Germination of synseeds on cotton e. Development and germination of synthetic seeds
c. Germination of synseeds on MS solid medium f. Acclimatized plant
22. ENVIRONMENTAL AND BIOCHEMICAL FACTORS CONTROLLING
THE IN VITRO EMERGENCE OF SOMATIC EMBRYOS IN EUROPEAN
SPINDLE TREE (Euonymus europaeus L)

Laurent Mo Bonneau
Laboratoire de Physiologie et Biochimie du Developpement de Ia Plante Universite de 0

Bourgogne, Dijon, France

Present address: Universite de Bourgogne, Laboratoire de Phytobiologie Cellulaire, BP
47870, 21078 Dijon cedex, France

Chapter contents

1. Introduction

20 In vitro multiplication

2010 Micropropagation-Organogenesis

2020 Callus culure and regeneration of organs and plantlets

2030 Protoplast isolation and culture

3 0 Somatic embryogenesis

3 olo Culture initiation

3020 Effects of the sugar type and of the osmotic potential of the medium

3030 Role ofpolyamines and polyamine metabolism

3040 Somatic embryo regeneration using a wild strain ofAgrobacterium rhizogenes

40 Conclusions and the future prospects

50 References

657
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 657-6690
© 2000 Kluwer Academic Publishers.
658

ENVIRO~MENT AL AND BIOCHEMICAL FACTORS CONTROLLING THE

IN VITRO EMERGENCE OF SOMATIC EMBRYOS IN EUROPEAN SPINDLE
TREE (Euonymus europaeus L)

Laurent M. Bonneau
Laboratoire de Physiologic et Biochimie du Developpement de Ia Plante . Universite de
l3ourgogne, Dijon, France
Present address: Univcrsite de Bourgognc, Laboratoire de Phytobiologie Cellulaire, BP
4 7870, 21078 Dijon ccdcx.

1- Introduction

The European spindle tree (Euonymus europaeus L. C:elastraceae) is a shrubby tree

widely distributed in temperate regions of western European countries from the
northern part of France to St. Petersburg and the western part of Russia. The
Celastraceae comprises 55 genera, including at least 850 known species of trees and
shrubs which are found in tropical and temperate regions. Various secondary
metabolites are elaborated in the family including steroids, triterpenoids, sesquiterpene,
peptide ore alkaloids. The spindle tree is commonly found in quickset hedges and small
broad-leaved forests when the soil is deep, moist, clayey and chalky. The leaves are
relatively small (3-4 em long) simple and are finely dentate. The green bark of young
branches is characterised by three to four parallel and longitudinal dark fine straight
lines. 1t bears pink berries (capsules) each containing four orange-colored seeds (5-6
mm diameter). The fruits produce a toxic alkaloid ( evonymin) and are poisonous to
humans. In Burgundy, this shrubby tree occurs as a wild plant and is the only species of
this genera. Ecotypes have never been reported. The spindle tree is naturally propagated
by seeds.
The zygotic embryo of the spindle tree is deeply dormant when mature. The
break of dormancy naturally occur during winter under the effects of cold temperahtres.
The embryo docs no! stick to the endosperm and can be easily removed from the rest of
the seed. The isolated embryo is a favorable system for shtdying the embryonic
dormancy or the physiology of germination and dormancy breaking, and to perform
experiments on growth and differentiation .
The dormancy break can be achieved in laboratory conditions by two different
ways:
1. Fully imbibed mahtrc seeds of the spindle tree are exposed to low
temteratures under moist conditions (stratification). Layering the seeds in moist sand for
several weeks at 4 °C allows both a post-maturation phase and the break of dormancy of
the embryos (Monin. 1966 ).
2. A rapid and homogenous germination of isolated zygotic embryos can be
achieved when cultivated in vitro in the presence of gibberellic acid. Isolated dormant
embryos are cultivated on a 0.8'% agar solidified basal medium containing a half-
strengh concentration of Knop's medium major salts, the minor elements of the Heller's
medium, supplemented with 100 mg/ L of vitamin B I. B6, and nicotinic acid (NG basal
 659
medium), and 0.24 mM of a filter-sterilised solution of gibberellic acid (Monin, 1971,
Nicolaeva, 1977).

2- In vitro multiplication

The main objectives of our in vitro cultures have been to elucidate the biochemical and
molecular mechanisms involved in cell, tissue, and organ differentiation. Euonymus
europaeus has been used as a ligneous angiospermous model to investighate rapid
propagation of difficult-to-root trees.

2-1- Micropropagation-Organogenesis

.Sterile cultures were established with shoot tips or meristems from young seedlings or
plantlets. Zygotic embryos were used as a source of shoot tips or meristems because
seeds can be easily desinfected by pure calcium quinolate and the sterile embryos easily
excised and cultivated in vitro. Moreover, all tips or meristems can be taken at the same
developmental stage of the seedlings, conferring a great homogeneity to the cultures,
compared to shoot tips or meristems collected from field-grown plants. Attempts to
obtain a rapid and homogenous seed germination were unsuccessful due to the lack of
maturity of numerous seeds when collected in the countryside, and to a frequently
installed dormant state of the embryos. Vigorous plantlets were finally obtained from
seeds stratified for 3 months at 4 °C or, more rapidly, from isolated zygotic embryos
from non stratified seeds, and cultivated in vitro on a 0.7% agar solidified NG basal
medium supplemented with gibberellic acid (Thalil-Rada, 1986). Well formed and
vigorous shoots were rarely observed after 5 days of germination on a Murashige and
Skoog's medium (Murashige and Skoog, 1962) solidified by 0.7%, agar. The
development of well formed leafy stems successfully occurred without callus
production when the explants were cultivated on a basal MS medium supplemented
with low concentrations of the following growth regulators: indole-3-butyric acid (IBA)
2.5 I o-'' M, mixed with 6-benzyl amino purine (BA) 88 I O" M) and gibberellic acid
( GA,) 1.4 J0-9 M. The active development of axillary buds was then achieved on a MS
basal medium with a reduced concentration of Nitrogen. Root differentiation was
successfully obtained when the cuttings were left for 6 days on complete darkness on a
4-fold diluted MS basal medium supplemented with IBA 25 10-" M and 5 g/L of active
charcoal.

2-2-Callus culture and regeneration of organs and plantlets

Numerous type of calli, more or less friable and colored, root or shoot bearing, or botl1.
can be initiated from the cotyledons or the axis of the isolated zygotic embryo, or from
in vitro germinated seedlings (roots, hypocotyl, epicotyl or young leaves) by using
various concentrations of a-naphtaleneacetic acid (NAA) (4.5 to 4500 10-9 M), 2,4-
dichlorophenoxyacetic acid (2,4-D) (4.6 to 4600 10 9 M), indole-3-acetic acid (IAA) (5.7
to 5700 10-9 M) in combination with BA (4.4 to 4400 w-''M) or kinetin (4.6 to
4600 I o-''M) in a basal MS medium. Once initiated, callus can be kept for a long time
by monthly subcultures under long days conditions ( 16 hours of light with an irradiance
660
of IOOflE.m-".s- 1 and 8 hour of darkness. IAA or NAA were not found to have
significantly distinct effects on callus formation and growth or on organogenesis. On
the contrary, the induction of rhizogenesis was never observed in the presence of 2,4-D.
In the presence of low concentrations of kinetin with an auxin/cytokinin ratio = I 00,
root differentiation was highly stimulated. Shoot differentiation was stimulated by
adding BA rather than kinetin in the culture medium (the optimal auxin/cytokinin ratio
was 0.0 I). Once initiated and elongated, shoots can de cut and transferred to a root
inducing medium as described for meristem-derived shoots.

2-3-Protoplast isolation and culture

Viable protoplasts from young leaves collected from 5-6 days old plantlets cultivated in
vitro were obtained by using cellulase and macerozyme (0,3%1) for 12 hours after
incubation in a half-strengh major salts and minor salts of the basal MS medium
supplemented with mannitol (0.45M), CaCl",2Hp (13.6mM) and BA (22 ~tM). The
digestion was performed in complete darkness at 23°C and the purification was
achieved by centrifuging the cells in a discontinuous glucose gradient. Purified
protoplasts were cultivated in the basal MS medium supplemented with IAA (5_7f!M),
BA (2_2f!M) and CaCl", 2H"O (4rnM). Divisions were observed while the cell wall
regenerated, and callus formation occurred following the transfer of the microcolonies
after I week in culture from the basal medium to a 2,4-D (9f!M) and kinetin (1.15f!M)
supplemented medium in weak light conditions (light irradiance of 100f!E.m-".s- 1).

3- Somatic embryogenesis

Somatic embryogenesis in the spindle ·tree was first obtained by Bonneau et al. ( 1994)
from zygotic embryo explants. The duration of cold seed storage, the type and
concentration of growth regulators, the sugar type and the osmotic potential of the
culture medium are the major critical factors for somatic embryo induction and
emergence. Free and bound polyamines, as well as' key enzymes of their biosynthesis
were also found to be essential determining factors for this process.

3-1 Culture initiation

Explants were dissected and cultivated as described by Bonneau (Bonneau et al., 1994 ).
Briefly, isolated zygotic embryos were used as explant source. After they were
harvested, the seeds were weakly dried in the open air for 3 weeks, then stored at 4°C in
a cold chamber. When needed, seeds were surface sterilized with pure calcium
quinolate for 5 mn and zygotic embryos were dissected. The cotyledons were cut in
square pieces and put on a 0.7'% agar (Difco) solidified MS medium supplemented with
I mM mesoinositol, I mM glutamine and 1,6%1 (w/v) glucose. The pH was adjusted to
5. 7 before autoclaving. In a first set of experiments, 8 concentrations of NAA, IAA,
2,4-D or IBA, in combination with 4 concentrations of BA and kinetin were
investigated. TI1e effects of the auxin exposure time were also tested from 1 to 4 weeks
before transferring the explants or explant-derived calli on a medium devoid of growth
regulators or containing a low auxin concentration. Embryogenesis occured for a wide
range of hormone type and ratio but was never observed with 2,4-D or IBA, whatever
 661
their concentrations, the associated cytokinin or the duration of exposure to the auxin.
The emergence of somatic embryos was only observed when a cytokinin waii present in
the culture medium, even in a very lo.w concentration (0.04 !JM). The minimal auxin
concentrations tested were 0.54 !JM NAA or 0.57 !JM IAA. Both the emergence of
somatic embryos and the percentage of embryogenic calli increased with the increase in
IAA concentrations until 22.8!JM. Higher concentrations were observed to be
inhibitory. In the presence of NAA, neither the duration of the exposure time to this
growth regulator, nor its concentration did significantly modified the expression of
somatic embryogenesis.
The first emergence of globular stages was observed between week 6 and week
8 in culture. Embryogenic calli were formed around the cut sections of the initial
explant. They were all white or yellowish with a few previously differentiated roots.
Frorri week 6, callus growth was reduced. The emergence of somatic embryos was
continuous over a long period (at least 20 weeks on the same medium without
subculture). During this phase of continuous emergence of new somatic embryos, callus
growth ceased and root development of some somatic embryos can occur. The
embryogenic callus can be maintained over one year by subculturing every two month
on the same medium with a 10-fold diluted auxin (secondary medium) and new somatic
embryos form continuously. No new emergence of somatic embryos was observed
when· the auxin (or cytokinin) was ommited in the secondary medium. New secondary
somatic embryos were frequently initiated from the basal part of the embryo axis,
conesponding to epidermal cells of the collar. Clusters of somatic derived one from the
other can be observed around this area. The number of somatic embryos which were
initiated from each zygotic embryo-derived callus was 2 to 20. Somatic embryos were
well formed and resembled to their zygotic counterpmts, although an abnormal number
of cotyledons was sometimes observed (from one large cotyledon, which could
originate from two fused cotyledons, to 3 distinct cotyledons which were rarely
observed). Whatever the number of cotyledons, the epicotylar axis generate a shoot
with normal expanded leaves. Development of somatic embryos can take place on the
induction medium; 20'% of the embryos at the cotyledon<}ry stage are able to germinate
in these conditions, but the germination process often require a specific medium. Our
recent findings show that the radical de.velopment occurs more rapidly when the
cotyledonary embryos were placed for 3 days on a maturation-activating MS basal
mediun supplemented with abscissic acid (7.5!JM). Somatic embryos were then
transferred to a "germination" medium devoid of growth regulators and containing a
half-strength concentration of major salts of MS and 44.4 mM glucose. The average
conversion percentage was 15 and well rooted plantlets were then transferred to soil in
the greenhouse.
During cold storage, the embryogenic potential of zygotic explants is strongly
modified. Somatic embryogenesis from zygotic embryos increased with the duration of
the cold storage with a maximum at 7 to 11 months. At this time, somatic embryos were
formed from more than 25'% of the cultivated explants after 6 weeks in culture. It
appears that somatic embryogenesis is related to the dormancy break during the seed
cold storage .
Somatic embryogenesis was not dependant on light and occurs in complete
darkness.
662
3-2 Effects of the sugar type and of the osmotic potential of the culture
medium

Cotyledonary explants dissected from seeds stored at 4°C for 6 to 8 months were
cultured in the presence of IAA and kinetin, in a MS basal medium supplemented with
glucose (89nu\tl). The increase in sugar concentration (glucose or sucrose) set off two
types of responses (Biahoua & Bonneau, 1999). In the prese nce of increasing
concentration of glucose, the embryogenic response was inversely proportional to the
glucose concentration and both the number of somatic embryos per embryogenic callus
and the number of embryogenic derived calli decreased (Fig. I).

F
v
v
v
v =
v
F -
F
- -
[]
1-

a~lJ ~ ~
S 47 S87 G 89 S1JO 5175 G178 G<66 SJ50 GJ55 G710
su~ar concentration (mM)

Figure /. Effects of sugar concentration in the culture med1um (suc rose:S or

glucose:<.i) on the fi·equency of somat1c embryogenesis of spind le tree cotyledonary
explants (da ta are average of three rep licates of% ex plants)

Increasing sucrose concentrations in the culture medium led to an opposite response.

The frequency of somatic embryogenesis strongly increased from 4 7 to 350 mM,
reaching 75'% for 350 mM sucrose (700 mM hexose equivalent). The embryogenic
response observed with both glucose and fructose in equimolar ratio in the culture
medium led to the same type of response as with glucose alone .
In the presence of a constant sucrose concentration , increasing the osmotic
potential with mannitol s imilarly increased the expression of somatic embryogenesis
(Fig. 2).
 663

~
"'
'iii 60
Ql
c
Ql
Ol
0 50
<::'
n
E 40
_!,!
~ 30
E
0

0"' 20
>-
0 (1 ,2)
c 10
Ql
6
~
lL 0
-0,45 -0,57 -0, 79 -1,02 -1,3
Osmotic potential (Mpa)

Figure 2, Eft'ects of the osmot ic potentia l of the cu lture med ium (sucrose 47 mM
+ 44 to 329 mM mannitol) on the frequency of somatic embryogenesis (data in
parenthesi s represent the number or somat ic embryos per embryogenic callus) -
Average number of three re pli cates of 96 ex plants.

-0,45 -0,68 -0.92 -1 ,14 -1,42

Osrootic potential (Mpa)

Figure 3 Effec ts of the os moti c potent ia l of the culture medium (glucose 89

mM + 44 to 32'! mM mannitol) on the frequency or somati c embryogenes is
(data in parenthes is represent the number or somati c embryos per
embryogenic ca llus ). Average number of three replicates of% explants.

A slight stimulation of the number of calli becoming embryogenic was

observed from a minimum threshold osmotic potential. No similar effects were
observed with the presence of glucose (Fig_ 3 ).
664
In this case, the frequency of somatic embryogenesis was not modified by
increasing the osmotic potential, but the number of somatic embryos emerging was
significantly enhanced for elevated osmotic potentials ..

These findings suggest that ( 1.) the induction of an embryogenic development

program of the explants is closely related to the osmotic potential of the culture
medium; and (2.) the presence of sucrose as both a carbon source and an osmotic
potential factor is needed for embryogenesis improvement; and (3.) the emergence of
somatic embryos is partly dependant of a minimum level of the osmotic potential in the
culture medium.
Interestingly, the emergence of somatic embryos is highly stimulated by an
elevated osmotic potential applied on explants collected from two to four month-old
seeds conserved at 4 oc. Perhaps an osmotic shock allows zygotic embryo explants to
undergo an embryogenic differentiation program. Somatic embryos differentiated
following an osmotic shock do not require abscissic acid. These embryos rapidly
developed into well rooted plantlets with a very high conversion percentage (85'%),
suggesting that the osmotic potential is also involved in the maturation program of
newly formed somatic embryos.

3-3 Role of polyamines and of polyamine metabolism

Embryogenic and non embryogenic calli were obtained by using the following
concentrations of growth regulators : IAA (22.8f!M) plus kinetin (0.046 11M), or 2,4-D
(4.6 11M) plus BA (4.4 nM), in the basal medium supplemented with 89 mM glucose
(Bonneau et al., 1995). Tissue were extracted according to the method of Flores and
Galston (Flores & Galston, 1982). Analysis of free polyamines levels (putrescine,
spermidine and spermine) was performed by high performance liquid chromatography
of their dansyl derivatives according to Smith and Davies (Smith & Davies, 1985).
Briefly, tissues were extracted with cold HCL0 3 , then dansylation was achieved by
adding 500f1l of dansyl chloride in acetone together with 70 mg sodium carbonate to
200f1l extract. The mixing was incubated in darkness for 16 hours at room temperature.
Dansyl reagent was removed by adding 10 mg proline. Dansylated polyamines were
extracted by toluene, separated and quantified by HPLC, in combination with
fluorescent spectrophotometry with a reverse phase 11Bondapak™ C 18 (Waters
Associates) and a programmed gradient of solvents methanol/water changing from 65
to 85% in 23 mn at a flow rate of 1 ml.mn· 1• Eluates were detected by using a
fluorescent spectrophotometer with an exitation wavelength of 365 nm and an emission
wavelength of 510 nm.
Initial cotyledon explants have relatively low levels of putrescine, spermidine
and spermine. The main amine found in both cultures, embryogenic and non
embryogenic was putrescine which represents 80 to 90 % of the total free amine pool.
Early changes in putrescine levels were observed before the emergence of roots or
somatic embryos from the zygotic embryo explant-derived calli. In the explants
cultivated on the non embryogenic medium, a weak and transient increase in putrescine
levels was observed during the first week in culture. This transient increase was
followed by a second and continuous increase until week 7. The concentrations of
potrescine in the calli were multiplicated by 5 and reached 50f1mol.g DW- 1•
 665
In the embryogenic medium, putrescine levels increased from the beginning of
the culture . The accumulation of this diamine was observed during the first three weeks
in culture, then the levels of putrescine decreased slowly before the first emergence of
somatic embryos (Bonneau et al. , 1995).
The transitory and early accumulation of putrescine on the embryogenic
medium could be correlated to the induction of somatic embryogenesis. The effects of
various polyamine biosynthetic inhibitors on putrescine levels and differentiation were
tested (Fig. 4 ).

METH ION INE

ARGIN INE

SAM ORN ITHINE AGMATINE

a-DFMO

DSAM PUTRESCINE

>n cHA
V
aminopropyle

Spermidine synthase

SPERM IDINE

aminopropyle

Spermine synthase

MTA SPE RMINE

Figure 4. Main steps or the poss ible bi osynthetic path ways leading to free polyamines in higher
plants. Arrows in grey represe nt the site o r action of the enzyme inhibitors: e< -DFM A: n -
dill uoromethy larginine, e< -DFMO : n -dtll uoromet hylortllt hi ne ancl C HA: cyclohexy lam ine.
(S AM: S-adcnosy lmethioninc , DSA M: clecarbo xy lated SA M, MTA : meth yl thioadcnosin e, ADC:
arginine decarbo xy lase. O DC" ornithin e decarboxylase) .

The supply, from the beginning of the culture of 10-' M a-

ditluoromethylornithine ( DFMO) , a specific irreversible inhibitor of the ornithine
decarboxylase ( ODC) strongly stimulated root differentiation on explants cultivated on
the non embryogenic medium (Bonneau et al., 1995). On the embryogenic medium,
somatic embryogenesis was highly stimulated the presence of DFMO (Fig. 5.1 and 5.2).
666

~ 35
"'<>
·;;;
c: 30
<>
C>

~ 25
.c
E
Q)
20
.II
15
E
0
15
0"' 10
>.
'-'
c:
Q)

5- 5
~ L:::::::::::::7
u. 0
Contro l DFMA DFMO CHA

Figu re 5. /. Effects ol· the add ition ol· Irr'M po lya mine biosynthetic inhib itors on day
0 on the emerge nce or somatic em bryos ti·om spindle tree cotyledons-deri ved ca lli
culti vated on the embryogenic medium (DFMA : n -di tluoromethylargini ne, DFMO:
n -di tluoromethylomi th ine, C' HA: cyc lohexylamine)

100
"'~"' 80
~ "
.0=
~ 3
=
p,c: 60
u 11

.,r:-
0 0
-.c
o E
~ Q)
40
.0 -
E ~
20
:>

B
:z

~ c:::7
0
Control DFMA DFMO CHA

Figure 5.2. Effec ts of the addi tion of Io·'M polya mine biosynthetic inhi bitors on day
0 on the number or somatic embryos emerg ing lium spin dle tree cotyledons-derived
call i cu lt ivated on the embryogenic medi um (DFMA : r1 -di lluoromethylarg inine ,
DFMO: u -ditluoromcthy lornit hine, CHA : cyc lohexy lam ine)

Mo reover, the emergence of somatic embryos occurred o ne week earlier than

in the contro l cond itio ns wi thout this inhibitor (the first globular somati c emb ryo
became visible between day 32 and 35 in culture) . After a rapid dec rease in putresc ine
le ve ls betwee n wee k I and week 3 in the presence of DFMO (putresc ine concentration
was reduced by 75%), a transient increase in putresc ine levels was observed between
week 3 and 5, prior to the precocious emergence of somatic embryos (Bonneau et al. ,
1995 ). T hese sti mulatory effects of the spec ifi c inhibition of the O DC pathway o n root
 667
differentiation and somatic embryogenesis suggest that the alternative pathway for the
synthesis of putrescine via the decarboxylation of arginine catalysed by arginine
decarboxylase (ADC) is the determinqnt pathway to allow the expression of somatic
embryogenesis from cotyledons of the spindle tree. Complementary experiments were
achieved dealing with the supply of a-difluoromethylarginine (DFMA), the specific
inhibitor of ADC to the media from the beginning of the culture. With the presence of
DFMA, a total inhibition of somatic embryogenesis was observed (Fig. 5.1 and 5.2).
Root differentiation was also completely inhibited by this treatment. When the explants
were grown in the presence of cyclohexylamine (CHA), a competitive inhibitor of
spermidine synthase. root differentiation was inhibited while somatic embryogenesis
was highly enhanced in the embryogenic medium (more than 75%, compared to the
control). The number of embryogenic calli was increased 1.3 fold and the number of
somatic embryos per embryogenic callus were increased 3-fold (Fig. 5.1 and 5.2). In the
presence of CHA the emergence of somatic embryos did not occur earlier. Somatic
embryos became visible at the globular stage, exactly after the same duration of culture
than in the control conditions. In the non-embryogenic medium, CHA weakly
stimulated root differentiation but was without effect on somatic embryogenesis. These
results suggest that an increase in free polyamines (putrescine and spermidine),
synthetized through the ADC pathway is directly involved in the stimulation of somatic
embryogenesis in the spindle tree when the hormonal conditions are favourable. Future
experiments dealing with microscopy will answer the question whether polyamines
stimulate callus induction or the development of previously induced cell or cell clusters,
or both.

3-4 Somatic embryo regeneration using a wild strain of A~robacterium

rhizo~enes

A wild strain (A4) of an agropine Agrohacterium rhizogenes carrying 4 rol A, B, C and

D genes (open reading frames, ORFs, I 0, II, 12, and 15 pelonging to the TL-DNA, and
rol BTR gene of the TR-ONA, characterised by a strong homology with ORF II), was
used for experiments, aimed at achieving. stable transformation in E. europaeus and
exploring the effects of the genes carried by the plasmid Ri on somatic embryogenesis.
Four weeks old plantlcts and cotyledons dissected from zygotic embryos were
submitted to bacterial attack. The plantlets were obtained from the germination of
isolated zygotic embryos on a basal NG medium supplemented with gibberellic acid.
Cotyledons were obtained from isolated zygotic embryos and cultivated on a basal MS
medium without any growth regulators. The· plantlets were decapitated and a droplet of
the bacterial suspension was placed on the cut section. Cotyledons were wounded with
a razor blade to favor the bacterial entry. The cotyledons were soaked for several
minutes in an overnight culture of A. rhizogenes and then placed on the basal medium.
After 3 days, the explants were transferred to the basal medium containing cefotaxime
to kill the bacteria, then subsequently maintained on this medium for 60 days on long
days conditions at 22°C with a an irradiance of I OOJlE.n,-".s- 1• Adventitious roots were
initiated after 15 days in culture from the cut sections of the plantlets, and after 45 days
from wounded cotyledons. These roots were dissected, decontaminated one more time
with cefotaxime and cultivated on a MS basal medium with a 5-fold diluted
concentration of major salts. Root growth was very rapid. compared to control roots.
668
and were highly ramified. The transformed status of these roots was confirmed by their
ability to produce agropine. Agropine was detected from root extracts by paper
electrophoresis followed by a coloration with silver nitrate according to Dahl et
al.(1983). Root cuttings, cultivated on the embryogenic medium described previously
never differentiated any somatic embryo. The organogenetic ability of roots was tested
by cultivating them on various culture media (MS basal medium supplemented with an
auxin alone, IAA or 2,4-D, or in association with a cytokinin, kinetin or BA, or with a
cytokinin alone). Somatic embryo emergence was only observed when the roots were
cultivated with BAP as the sole source of growth regulator (2.2 11M). Somatic embryos
became visible at the globular stage after 8 weeks of culture. These somatic embryos
are well formed, compared to somatic embryos regenerated from zygotic embryo
explants, and were easily converted into plantlets following the transfer to a medium
devoid of growth regulators. The unique taproot of these plantlets, derived from the
rhizogenic pole of the somatic embryo axis, rapidly became highly ramified. An opine
test did confirm that the somatic embryos and derived plantlets produced agropine.
The morphological characteristics of these plantlets were not the same as those
usually described for other plants transformed by A rhizogenes. For example, the
reduction of apical dominance and the reduction of the internodes were never observed.
After eight weeks in culture, transformed plantlets were 2-fold higher than control
somatic embryo-derived plantlets and have developed 5 pairs of leaves instead of 3.
Moreover, the leaves were larger (the leaf area was multiplicated by 3), weakly
wrinkled and less green. The root system was very developed (the roots were longer ant
highly ramified). Transformed plantlets show an active growth which can be a
consequence of the important development of the root system. The micropropagation of
these plantlets by monthly microcutting can be easily performed. The plantlets
regenerated continued to show the same growth characteristics over the subcultures and
during at least 6 month after transfer to greenhouse conditions.

Conclusion and future prospects

Somatic embryos were induced from zygotic embryos-derived calli when tissues are
plated on a MS medium containing IAA and kinetin. Kinetin was always needed in the
culture medium for somatic embryo formation, even at a very low concentration.
Cotyledons were shown to be the most favorable source of explant to produce somatic
embryos. Other explants from mature seeds or from seedlings never produced
embryogenic calli. Somatic embryos are readily induced in 6 weeks. Abberant
phenotypes were never observed although embryos with 3 cotyledons were sometimes
initiated. The development of somatic embryos into plantlets can be achieved directly
on the induction medium; however, root development is more efficient after a 3 days
exposure to a maturation medium containing abscissic acid. The frequency of somatic
embryogenesis greatly depends on the cold conservation time of seeds after rippening.
The percentage of embryogenic calli was greatest after 7 to 11 months of cold storage at
4°C, suggesting that a relationship could exist between somatic embryo induction and
the break of dormancy of zygotic embryos.
In the spindle tree, the osmotic potential nf the culhtre medium and the type of sugar
play a crucial role for the somatic embryogenesis process. In this process, an elevated
level of sucrose (350 mM), providing an osmotic potential of -1.30 MPa was shown to
 669

be very favorable for somatic embryo formation, while glucose have an inhibitory
effect.
Compared to control conditions, somatic embryos were produced in shorter
time in the presence of a specific inhibitor of putrescine formation through the ornitine
decarboxylase pathway. In these conditions somatic embryos emergence was highly
stimulated, suggesting a determinant role of the arginine decarboxylase pathway in the
somatic embryo induction process.
A. rhizogenes strain A4 has been shown to be a very effective mean of
inducing adventitious root differentiation on both cotyledonary explants and plantlets of
the spindle tree. Moreover, it is and efficient system to regenerate somatic embryos and
derived plantlets with improved growth characteristics. Future experiments dealing with
the transformation of explants or plantlets with selected ORFs encoded by A. rhizogenes
should answer the question of the origin of the growth stimulation of plants regenerated·
after a bacterial treatment. Preliminary results show that genes from this T-DNA leads
to interferences with polyamine biosynthesis functions in plants. It can be hoped that
the molecular dissection of the Ri TL-DNA will provide precise tools for establishing
the importance of polyamines in the somatic embryogenesis process.
The somatic embryo formation process of the spindle tree supplies interesting data for a
better understanding and control of somatic embryogenesis and could find applications
for rapid propagation of other ligneous species.

References

Beranger-Novat N., Monin .1., .lassey Y. & .1. Martin-Tanguy, 1997. Polyamine catabolism in dormant
embryos of the spindle tree (Euonymus europaeus L.) and in dormancy break obtained after
treatment with gibberellic acid. Plant Growth reg. 21 (I), 65-70
Biahoua A. & L. Bonneau, 1999. Control of in vitro somatic embryogenesis of the Spindle tree (Euonymus
europaeus L ) by the sugar type and the osmotic potential of the culture medium. Plant Cell Rep. in
press
Bonneau L., Beranger-Novat N. & .1. Monin, 1994. Somatic embryogenesis and plant regeneration in a woody
species: the European spindle tree (Euonymus europaeus L.) Plan! Cell Rep. 13, 135-138
Bonneau L., l3eranger-Novat N., Monin .1. & .1. Martin-Tanguy, 1995. Stimulation of root and somatic embryo
production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis. Plant Growth Reg.
16, 5-10
Dahl G., Guyon P., Petit A. & .1. :Tempe, 1983. Silver nitrate positive opine crown gall tumours. Plant Sci.
Lett. 32, 193-203
Flores H.E. & A.W. Galston, 1982. Analysis of polyamines in higher plants by high performance liquid
chromatography. Plant Physiol. 69, 701-706
Monin .1., 1966. Modalite d'elimination de Ia dormance des graines d'Evonymus europaeus L au cours de leur
stratification. C. R. Soc. Bioi. 161,: 2262-2264
Monin .1., 1971. Action de ditferentes gibberellines sur !'elimination de Ia dormance des embryons
d'Evonymus europaeus L. C. R. Soc. Bioi. 165,237-240
Murashige T & F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue
cultures. Physiol. Plan/. 15,473-597
Nicolaeva M.G., 1977. Factors controlling the seed dormancy pattern. In The physiology and biochemistty of
seed donnanc;- and germinllfion, Kahn A.A. eds, North Holland Publishing Company, Amsterdam,
New-York, Oxford, 51-74
Smith M.A. & P..l. Davies, 1985. Separation and quantification of polyamines in plant tissue by high
performance liquid chromatography of their dansyl derivatives. Plant Physio/. 78, 89-91
a
Thalil-Rada N., 1986. Contibution !'etude de Ia dormance embryonnaire chez le Fusain d'Europe it !'aide de
a
cultures in vitro. Mise au point de Ia multiplication vegetative de cette espece partir de cultures de
meristemes. PhD thesis.
 23. SOMATIC EMBRYOGENESIS IN QUERCUS ACUT/SS/MA
Kim Yong-Wook
Dept. of Biotechnology, Forestry Research Institute, Suwon, Republic of Korea

I. Introduction 3-3. Immature seeds of five family

2. Somatic embryogenesis
2-1. Plant materials
2-2. Initiation of embryogenic cultures
2-3. Maturation of somatic embryos
2-4. Germination of somatic embryos
3. Initiation of embryogenic cultures
3-1. Mature seeds
3-2. Immature seeds
4. Maturation of somatic embryos
5. Germination
5-1. Somatic embryos induced from mature seeds
5-2. Somatic embryos induced from immature seeds
5-3. Somatic embryos induced from five family
6. Conclusion
7. References

1. Introduction

The genus Quercus including over 500 species is widely distributed throughout the temperate regions of the
Northern Hemisphere and, is crops of major importance to forest industries such as timber, tan bark, or cork.
Among them, Q. acutissima Carruth. is deciduous tree, growing in the temperate zone of eastern Asia
including Korea and Japan. This species, is one of the most valuable tree species used for timber, fuel, tool
handles, and bed logs for the cultivation of the Shiitake mushroom.
This species is mainly propagated by seed. However, seedling production from acorns is sometimes not
advisable because of the high heterozygosity due to wind pollination.
In addition, clonal propagation of this species by conventional methods of cuttings or grafts has not been
satisfactory because of low rate of rooting or of graft incompatibility (Moon eta/., 1989).
Previous work has revealed the possibility of asexual multiplication of juvenile oak trees via in vitro culture
of axillary buds (Yieitez et a/., 1994, Romano et al., 1995), however, the Quercus species have proved
difficult to culture in vitro. (Cai et a!., ·1987, McCown and McCown, 1987), possibly due to their tannin
production, acutissimin A and B together with various other tannins being isolated from the bark of Q.
acutissima (Nonaka el a!., 1984, lshimaru eta/., 1987a, b).
Somatic embryogenesis has been regarded as the in vitro system of choice with the potential for eventual
mass propagation of superior and genetically engineered forest tree genotypes in both coniferous and
hardwood species (Gupta et a/., 1991 ). Current trends in forest breeding strategies emphasize combined
breeding and cloning (Park and Bonga, 1993). Moreover, this method has the potential to be an important tool
for obtaining efficient true-to-type vegetative propagation (Bonga, 1991 ).
In Quercus, somatic embryogenesis has been reported for several species including Q. bicolor (Gingas,
671
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 671-685.
© 2000 Kluwer Academic Publishers.
672

1991 ), Q. robur (Chalupa, 1990, Cuenca eta/., 1999, Manzanera, 1992), Q. petraea (Jorgensen, 1988, 1993),
Q. rubra (Gingas and Lineberger, 1989, Rancillac eta/., 1996), Q. suber (Bueno eta!., 1992, Celestino et at.,
1998, El Maataoui eta/., 1990, Manzanera eta/., 1993, Fernandez-Guijarro eta/., 1995), and Q. acutissima
(Kim eta/., 1992, 1994, 1997). An alternative was to produce somatic embryos and plants using embryonic
axes (Sasaki el a/., 1988, 1993,), cotyledons (Ide et a/., 1992), mature (Kim et a/., 1992) and immature
zygotic embryos (Kim et a/., 1994). Subsequent studies were initiated to determine the frequency of somatic
embryo formation and germination as a function of genetic origin, i.e. seed family (Kim eta/., 1997).
This chapter provides some culture methods concerning the induction of somatic embryogenesis as the
materials of mature or immature seeds and following germination conditions in Q. acutissima.

2. Somatic embryogenesis

2-1. Plant materials

Open pollinated mature acorns of Q. acutissima purchased at the market, Chuncheon, Korea 1990.
Immature embryos were also collected weekly from a single pollinated tree located in Suwon, Korea,
during early August and early September, 5 to I0 week post-fertilization. Time of fertilization was defined as
the point at which one ovule continued to develop and the other five began to degenerate (Gingas and
Lineberger, 1989).
Immature embryos were isolated from open pollinated seeds of 5 families (Chungnam 11,14, 15,
Chungbook 23, and 29), growing at a clone bank located in Yongin, Kyeonggido, Korea. All immature
embryos used were half-sibs. The acorns were collected weekly from 6 to I0 week post fertilization.
After the pericarps were removed, acorns were surface-sterilized in 95 % ethanol for I min followed by
dipping them in 4 % sodium hypochlorite solution for 5 min. They were rinsed three times with sterile
distilled water. Mature or immature embryos were excised from the acorns, bisected transversally, and
cultured on nutrient medium.

2-2. Initiation of embryogenic cultures

Mature seeds
The mature embryos were cultured either on· MS (Murashige and Skoog, 1962) medium containing I,000
mg/L glutamine, 5 mM proline, 3 % (w/v) sucrose and 0.8% (w/v) Difco-Bacto agar, or on WPM (Lloyd and
McCown, 1981) medium with the same additives but lower sucrose concentration (2%, w/v). An auxin {2,4-
dichlorophenoxyacetic acid (2,4-D), a-naphthaleneacetic acid (NAA), indole-3-butyric acid (JBA), or indole-
3-acetic acid (IAA)} and a cytokinin {benzyladenine (BA), 6-furfurylaminopurine (kinetin), or 6-[4-hydroxy-
3-methyl-2-butenylamino]purine (zeatin)} in various concentrations and combinations were added to both
media (initiation medium) (see Table 1). L-glutamine was sterilized by filteration, then added to partially
cooled medium (45-50 t) after autoclaving (Kim eta/., 1992).

Immature seeds
As for immature embryos culture, the medium was composed ofMS salts and vitamines, I giLL-glutamine,
5 mM proline, 30 giL sucrose, and 0.5-10.0 mgiL IBA and 0 or 1.0 mgiL BA, solidified with 0.8% Difco-
Bacto agar (see Table 2) (Kim eta/., 1994).

Immature seeds offive families

The medium for the initiation of embryogenic cultures from five families immature seeds was composed of
full-strength MS salts and vitamines, I giLL-glutamine, 5 mM proline, 30 giL sucrose, and 1.0 mgiL IBA and
1.0 mg/L BA, solidified with 0.8% Difco-Bacto agar (Kim eta/., 1997).
 673

The pH of the medium was adjusted to 5.7. Initially, cultures were kept at 25± I "C under a 16/8 h
photoperiod (50 f1 Em' 2s' 1) for 4 weeks.

2-3. Maturation of embryogenic cultures

For further embryogenic cultures development, the cultures obtained from all initiation media were
subcultured to MS medium lacking plant growth regulators under light condition (50 f1 Em·2s' 1) for 4 weeks.
After that, the frequency rate of embryogenic cuitures initiation was recorded and the only cotyledonary
somatic embryos were selected from cultures, and used in somatic embryo germination.

2-4. Germination

Germination ofsomatic embryos induceedfrom mature seeds

Cotyledonary somatic embryos were cultured on WPM medium containing 0.1 mg/L BA in the darkness at
4 "C for I0 weeks, and then transferred to )1! MS medium without plant growth regulators under light (50
f1 Em·2s' 1 ) at 25 ± I "C for 8 weeks (see Table 3).
The somatic embryos were cultured on WPM medium with 0.1 mg/L gibberellic acid (GA1) and 0.1 mg/L
abscisic acid (ABA) in light (50 f1 Em·'s- 1) at 25 ±I "C for 8 weeks.
The somatic embryos were also cultured on WPM medium containing 0.6 M sorbitol in the darkness at 25
±I "C for 2 weeks and then transferred to WPM medium lacking growth regulators in light (50 f1 Em·'s- 1)
at 25 ± I "C. After 2 weeks, somatic embryos were transferred to MS medium at 25 ±I "C in light (50 f1
Em' 2s' 1) for 4 weeks.
For desiccation treatment, the somatic embryos were placed on empty petri dish, wrapped with parafilm, and
kept in the darkness at 25 ± I "C for 2 weeks. The desiccated somatic embryos were rehydrated on MS
medium at 25 ± I ·c in light (50 f1 Em·'s- 1) for 4 weeks.

When shoots and roots began to develop, all cultures were transferred on WPM medium containing 0.2
mg/L BA for further growth.

Somatic embryos induced from immature seeds

Four separate experiments were conducted.
In the first experiment, somatic embryos were chilled on MS medium having 0.5 mg!L BA in the darkness
at 4 ·c for 8 weeks and then transferred to WPM medium containing 0.2 mg/L BA in light (50 f1 Em·'s- 1)
at 25 ±I "C (see Table 4).
In the second experiment, somatic embryos were incubated on MS medium containing 0.5 mg/L BA and
0.6 M sorbitol in light (50 f1 Em·'s- 1) at 25 ±I "C for 2 weeks and transferred to WPM medium containing
0.2 mg/L.
For the third experiment, somatic embryos were cultured on MS medium with 0.1 mg/1 ABA and 0.1 mg/1
GA1 at 25 ± I "C for 4 weeks in light and then transferred to WPM medium containing 0.2 mg/L BA.
For the fourth experiment, somatic embryos were also cultured on WPM medium supplemented with 0.1
mg/1 BA at 25 ± I "C for 4 weeks in light, and transferred to WPM medium with 0.2 mg/L BA.

The germination frequency of the somatic embryos was recorded after all somatic embryos were culturec
on WPM medium containing 0.2 mg!L BA for 4 weeks. The somatic' seedlings were subcultured on WPM
medium having 0.2 mg/L BA.

Somatic embryos inducedfromfivefamilies immature seeds

The germination of somatic embryos was investigated by using three different culture conditions.
Firstly, somatic embryos were cultured on WPM containing 0.1 mg/L BA, under cool white fluorescen
light (50 f1 Em·'s- 1) and ·a 16/8 h photoperiod at 25 ± I "C for 4 weeks. They were then transferred· to WPIV
674

medium containing 0.2 mg!L BA for 4 weeks under the same culture conditions as described before (see
Table 5).
Secondly, somatic embryos were cultured on WPM medium containing 0.1 mg!L BA in the darkness at
4 OC for 8 weeks, and then subcultured on WPM medium containing 0.2 mg!L BA in the light and a 16/8 h
photoperiod at 25 ± I OC for 4 weeks.
Thirdly, somatic embryos were cultured on WPM medium having 0.1 mg/L BA and 0.1 mg!L GA 3 for 4
weeks in light as described above and then tran'Sferred to WPM medium containing 0.1 mg!L BA for 4 weeks.
They were then cultured on WPM suppl~mented with 0.2 mg!L BA for 4 weeks.

All media used for germination were solidified with 0.4 % gelrite (Sigma). When somatic embryos began
to develop epicotyl and primary root, all these germinated plantlets were transferred to WPM medium
supplemented with 0.2 mg/L BA for further development.

Plant regeneration
After 8 weeks of culture under germination condition, plantlets with shoot and root were transferred to
trays containing a I: I: I mixture of perlite, peatmoss and vermiculite. They were watered once a day. After 6-
8 weeks the lid was gradually opened to reduce humidity, and removed completely when new shoot growth
started. The surviving plants with well developed shoots and roots were grown out of doors.

3. Initiation of embryogenic cultures

3-1. Mature seeds

The ability to produce callus and somatic embryos from mature embryos varied with the media and growth
regulators tested (Table 1). The best initiation of callus occurred from the explants cultured on WPM medium
supplemented with 0.5 mg!L BA, 0.5 mg!L kinetin and 2.0 mg/L 2,4-D (58.1 %) (Table 1). As these calli were
transferred to the initiation medium, further induction of somatic embryos were not observed after
subculturing. With regard to the response to media and growth regulators for induction frequency of callus. Q.
acutissima appeared to be different from that required for immature embryos of Q. rubra which need 1.0
mg/L BA and 1.0 mg!L GA 1 (Gingas and Lineberger, 1989).
Explants began to produce callus after 2 week initial culture and callus was formed from the side parts of
explants. Most of those calli were hard, shiny and yellow in color. The high frequency of callus occurred
generally in the presence of 2.0 mg/L 2,4-D. As the cultures were transferred to hormone-free induction
medium in light (50 fl. Em-'s-') after 8 weeks of initial dark culture, the callus turned into green-white colored
bristle textures within 2 weeks. After more 4 weeks culture on the initiation medium, somatic embryos were
formed from brown callus cultured on MS medium containing 0.2 mg/L BA and 1.0 mg!L IBA and WPM
medium containing 0.2 mg!L BA and 1.0 mg!L IBA (Table 1).
The somatic embryos first appeared as tiny globular and petal-like lobes or various developmental stages
structure. In some case, cotyledonary stage somatic embryos with fused cotyledons and root pole formed from
explant cultured on MS medium containing 0.2 mg!L BA and 1.0 mg/ L IBA. The somatic embryos were not
observed when other plant growth regulators (2,4-D, NAA or lAA) were added to medium except 1.0 mg/L
IBA. It seemed to be essential to use combination of 0.2 mg/L BA and 1.0 mg!L IBA for somatic
embryogenesis from mature embryos in Q. acutissima.
Generally, MS medium seemed to be more effective than WPM for induction of somatic embryos (Table I).
The effect of various plant growth regulators on somatic embryogenesis has been reported. Auxin, 2,4-D
improves the induction rate of embryogenic callus or somatic embryos (Gui eta/., 1991; Neuenschwander and
Baumann, 1992). At this study, however, 2,4-D apparently induced callus in Q. acutissima, but it had to be
removed to induce somatic embryos.
 675

Table I. The effect of media and plant growth regulators on induction of callus and somatic embryos from mature embryos of
. acutissima
Medium Plant growth regulators % of induced callus No. of induced
(m~L) Somatic emb!}:o
MS 0.2 BA+l.O 2,4-D 8.1 0
0.5 BA+O.S kin•+2.0 2,4-D 16.0 0
0.2 kin+ 1.0 2,4-D 0 0
1.0 kin+ 1.0 2,4-D 16.3 0
0.2 BA+0.5 NAA+ 1.0 2,4-D 7.5 0
1.0 BA+O.S NAA+2.0 2,4-D 21.2 25
0.2 BA+LO IBA 0 0
0.2 zeab+ 1.0 IAA 6.5 0
WPM 0.2 BA+l.O 2,4-D 25.9 0
0.5 BA+0.5 kin+2.0 2,4-D 58.1 0
0.2 kin+l.O 2,4-D 31.9 0
1.0 kin+ 1.0 2,4-D 24.6 0
0.2 BA+0.5 NAA+ 1.0 2,4-D 12.8 0
1.0 BA+O.S NAA+2.0 2,4-D 27.9 0
0.2 BA+l.O IBA 0 0
0.2 zea+ 1.0 1AA 0 0

' kin : kinetin

bzea : zeatin

3-2. Immature seeds

Q. acutissima seed maturation occurs over a two year period. Pollination occurs approximately from mid t'
late Apri I of the first year and fertilization takes place in late June of the following year.
Most im.mature embryos harvested from 5 to 10 weeks post-fertiliZation produced embryogenic culture
within 4 weeks (Table 2). The highest frequency (58%) of embryogenic cultures was obtained from culture'
immature embryos harvested 6 weeks post-fertilization. These results were similar to that found in Q. rubr,
(Gingas and Lineberger 1989). Initiation of embryogenic cultures was highest on modified MS mediun
containing 0.5 mg/L IBA (74 %) or 1.0 mg/L BA and 0.5 mg/L IBA (72 %) at 6 weeks post-fertilization. InC
robur, initiation of embryogenic cultures was not accomplished by 2,4-D but adding 1.0 mg/1 BA containin:
0.1 or 1.0 mg/IIBA in the medium (Chalupa, 1990). With older than 6 weeks post-fertilization embryos, th
higtest frequency of embryogenic cultures initiation occurred with the addition of 1.0 mg/L BA and 1.0 mg/1
IBA in the medium {Table 2). Addition of 1.0 mg/L BA was generally beneficial for inducing embryogeni
cultures throughout the period collecting of immature embryos.
The immature embryos cultured in I ight (50 f.!. Em·2s" 1) changed to white-yellow embryogenic tissu
within 2 weeks. The development of embryogenic cultures continued and the cultures were transferred ont
MS medium without plant growth regulators after initial culture of 4 weeks. Embryogenic cultures forme
tiny cylindrical somatic embryos within 2-3 weeks of culturing immature embryos.
Generally, a higher number of somatic embryos were obtained from explants cultured on modified MS
676

Table 2. Effect of collection date and plant growth regulators on somatic embryogenesis from
immature embryos of Q. acutissima

Time after fertilization (weeks)

5 6 7 8 9 10
Growth regulators % of cultures wtth somatic embryosb
(mg/L)'
BA IBA

0.0 0.5 14 74 43 31 28 10

0.0 1.0 23 46 37 28 21 6

0.0 2.0 42 53 42 26 30 26

0.0 10.0 31 40 33 33 26 18

1.0 0.5 62 72 45 46 20 3

1.0 1.0 28 53 71 69 41 17

1.0 2.0 68 56 53 60 19 3

1.0 10.0 50 67 42 50 18 10

Mean' 40 58 46 43 25 12

• Medium: MS+ 1,00 Om giL glutamine+ 5 mM proline, pH 5. 7

' The data were recorded after 4 weeks initial culture.
' Means based on three replicate experiments.

medium supplemented with 1.0 mg/L BA and 0.5-2.0 mg/L IBA. It is difficult to calculate exactly the
number of somatic embryos, since they were clustered on embryogenic masses.
In all the media supplemented with IBA a few primary roots were observed from the original immature
embryos.

3-3. Immature seeds of five families

Most immature embryos collected from acorns at the time of 6- I0 weeks post fertilization and cultured in
vitro produced somatic embryos visible to the naked eye within 4 weeks. The frequency of somatic
embryogenesis was influenced by both collection date and seed family of immature embryos in Q. acutissima.
While the best initiation rates obtained were 90.9% (6 weeks post fertilization) and 91.2 % (9 weeks post
fertilization) with the family Chungnam II, poorer results were achieved with the families Chungnam 14, 15,
and Chungbook 29 showing 0% when employing material collected 10 weeks post fertilization, respectively
(Figure /.). Significant embryogenesis difference (P=0.05) among families were detected in the seed
collection dates. With Duncan's multiple range test, the embryogenesis percentages of family Chungnam
 677

100
;e "' .c .c - Chungnam 11
~
"' IZZl Chungnam 14
tJl 80 "' E8:l Chungnam 15
'iii ISSl Jeonbook 23
(j)
c: g Jeonbook 29
(j)
Ol 60
g,
.....
..0
E
(j)
40
(J

~ 20
E
0 Ol
(/)

0
6 7 8 9
Post-fertilization (weeks)

Figure I. Effect of collection date and seed family on formation of somatic embryos in immature
embryos ofQ. acutissimll. The results are shown as percentage of zygotic embryos forming
somatic embryos in two replicate experiments.
Means with the same letter are not sigoificantly different as determined by Duncan's multiple
range test (P=0.05).

II were equivalent in both 6 and 9 weeks post fertilization. Gingas and Lineberger (1989) also showod that
the highest somatic embryo numbers were obtained from immature embryos cultured 4-7 weeks post
fertilization in Q. rubra. Similar results were obtained with immature embryos collected 6 weeks post
fertilization in this species (Kim et at., 1994). Therefore, a careful determination of the developmental stage
of zygotic embryos should always be made in order to get the highest rate of production.

4. Maturation of somatic embryos

The embryogenic cultures derived from mature, immature, and immature zygotic embryos of 5 families
have been cultured in light (50 f..l Em·2s-') for 4 weeks, simply by transferring the embryogenic cultures on
MS medium lacking plant growth regulators.
The induced embryogenic cultures (Figure /./) looked as petal-like lobes or bell shaped bodies when
observed under a stereo microscope. Somatic embryos were often loosely intermingled with each other. After
4 weeks of culture, they were transferred to MS medium without plant growth regulator. When embryogenic
cultures were cultured continuously on plant growth regulator-free MS medium, repetitive somatic
embryogenesis from original somatic embryos occurred frequently, thereby forming many new tiny somatic
embryos. After 3-4 weeks culture, the development of globular (Figure 1.2), heart (Figure 1.3) and torpedo
(Figure 1.4) shaped somatic embryos continued in subcultured embryogenic tissues and embryogenic cultures
was continued to increase in volume. Globular and torpedo shaped somatic embryos continued in
development, finally giving rise to cotyledonary somatic embryos (Figure 1.5).
678

Figure 2. /: Numerous somatic embryos produced from the embryogenic cultures. 2-5: Scanning electron micrographs of somatic
embryos of various developmental stages, g:globular, h:heart-shaped, t:torpedo-shaped, c:cotyledonary shaped. 6; Epicotyl (arrow)
arising from between cotyledons of a genninating somatic embryo
 679

Occurrence of various abnormal structures, such as fused somatic embryos or fused cotyledons, were also
observed.
Before transfer to the germination medium, only cotyledonary somatic embryos were separated manually
from matured embryogenic cutltures.

5. Germination

5-l. Somatic embryos induced from mature seeds

Germination of cotyledonary somatic embryos and formation of plantlets were rarely observed on the
maturation medium. So induced somatic embryos were transferred to each germination medium.
Epicotyl dormancy of somatic embryos was overcome by chilling (Bueno et a!., 1992; Rajasckaran and
Mullins, 1979; Tulecke and McGranahan, 1985). The somatic embryos were cultured on WPM medium
containing 0.1 mg/L BA in darkness at 4 'C for I 0 weeks and then transferred to half-strength MS medium
without growth regulators in light (50 J.1. Em·2s. 1) for 8 weeks. In our experiment, total2 plantlets (7.1 %) were
regenerated by chilling treatment (Table 3). However, there has been a report that plantlets do not germinate
after chilling in Q. rubra (Gingas and Lineberger, 1989).
After chilling for 10 weeks at4 'C, a few somatic embryos became dark brown.
The addition of 0.2 mg/L GA 3 found to stimulate in breaking the dormancy of embryos or to promote shoot
regeneration from somatic embryos (Sasaki et a/., 1988). Four plantlets (16.0%) were produced from GA,
treatment in light (50 JL Em·'s·') after 8 weeks. Therefore, WPM medium with 0.1 mg/L GA 3 and 0.1 mg/L
ABA was most effective in germination of somatic embryos. Gmitter and Moore ( 1986) reported that
gibberellic acid stimulates germination of Citrus somatic embryos, and differentiation of dicotyledonous
somatic embryos from callus of Q. lebani (Srivastava and Steinhauer, 1982).
After 8 weeks of culture on WPM medium supplemented with 0.1 mg/L GA 3 and 0.1 mg/L ABA, somatic
embryo started to form the epicotyl (Figure 2.6.) and a few globular somatic embryos were adventitiously
induced on the necrotic somatic embryo.
Use of osmoticum like sorbitol is well known to stimulate germination of somatic embryos to reproduce
plantlets (Finkelstein and Crouch, 1986: Gingas and Lineberger, 1989). However, such treatment was
ineffective in germinating somatic embryos in Q. acutissima. No germinated plantlets were recovered from
WPM medium containing 0.6 M sorbitol for 2 weeks (Table 3). In Q. rubra, however, 2 plantlets were
regenerated on the medium containing 0.6 M or 0.87 M sorbitol (Gingas and Lineberger, 1989). After somatic
embryos were incubated on osmoticum medium for 2 weeks, browning of embryos was occurred. No
secondary somatic embryos were formed on either hypocotyl or root.
As an alternative to desiccation via use of an osmoticum, somatic embryos were subjected to air-drying
(Gingas and Lineberger, 1989; Gray, 1987). In air drying-rehydration experiment, nine somatic embryos were
put in sterile empty petri-dish, sealed with paratilm, and placed at 25 ±I 'C for 2 weeks. Within one week,
however, all embryos became brown, and lost their viability. It is impossible that air-drying was treated
prematurally at an early somatic embryo stage. Embryos might be not hardy enough to tolerate desiccation at
the stage. By air drying-rehydration for 2 weeks, 3 plantlets were recovered from embryos in Q. rubra
(Gingas and uneberger. 1989). In peduncculated oak, somatic embryos germination was also stimulated after
desiccation with 6 % sorbitol (Chalupa, 1990), and in interior spruce, partial drying at high humidity
promoted germination of the somatic embryos up to 90% (Roberts eta/., 1990).
When germinated plantlets from somatic embryos were cultured on WPM medium supplemented with 0.1
mg/L BA. the secondary embryos were formed on the apical bud (Figure 3.1) or hypocotyl of original one.
A total of six plantlets were regenerated from somatic embryos that induced from immature seeds. The
plantlets were cultured on WPM medium containing 0.1 mg/L BA for. further growth. The regenerated
plant lets were transplanted to a mixture of perlite: peatmoss: vermiculite (I: I: I, v/v/v) and hardened in
culture room with occasional spraying of tap water.
The results showed that the use of immature embryos rather than mature embryos is more suitable for
somatic embryogenesis. It is also necessary to refine the techniques in order to achieve a high conversion
680

frequency of plantlets from somatic embryos.

Table 3. Effect of plant growth regulators and osmoticum on germination of somatic embryos induced from mature seeds in
Q. acutissima

Treatments Produced roots Produced shoots Produced plants

(%) (%) (%)

WPM + 0.1 mg/L BA (4 'C, I Oweeks,

darkness) -> 7f MS (8 weeks, light, 39.3 14.3 2 (7.1)
50 11 Em·'s· 1)

WPM+ O.lmg/L GA 1 + 0.1 mg/L ABA 52.0 16.0 4 (16.0)

(8 weeks, light, 50 11 Em·'s-1)

WPM+ 0.6M sorbitol (2 weeks, darkness)

36.8 10.5 0 (0)
-> WPM (2 weeks, light, 50 11 Em·'s· 1) ->
MS (4 weeks, light, 50 11 Em·'s' 1)

Air drying-rehydration 0 0 0 (0)

5-2. Somatic embryos induced from immature seeds

Most of somatic embryos for germination experiments were derived from embryogenic cultures cultured on
medium containing 8 different combinations of plant growth regulators. For the germination study,
cotyledonary somatic embryos were selected. After 4 weeks of culture in hormone-free MS medium, a few
somatic embryos developed into plant lets with epicotyls or primary roots and the majority of somatic embryos
did not show any further development.
In the germination study, the best result was obtained on WPM medium containing 0.1 mg/1 BA (38
plant lets) (Table 4), and shootand root formation were also highest on this medium. The addition of 0.1 mg/1
ABA having 0.1 mg/1 GA 1 to MS medium (15 plantlets) resulted in a higher germination frequency than did
the chilling treatment (2 plantlets) or high osmoticum treatment (10 plantlets). Both GA 3 and BA are known
to promote shoot elongation from somatic embryos in Q. acutissima (Sasaki et a/., 1988). Our results
corresponded closely to the results of Sasaki eta/. (1988).
Generally, oak seed dormancy must be broken by cold treatment at 4 'C for 30-70 days (Gingas and
Lineberger, 1989). Also, epicotyl dormancy of somatic embryos was broken by chilling in Quercus suber
(Bueno et a/., 1992), Vitis vinifera x Vitis rupestris (Rajasekaran and Mullins, 1979) and Juglans regia
(Tulecke and McGranahan, 1985). By contrast, chilling treatment was ineffective for the germination of
somatic embryos when compared with other treatments (Table 4).
Germination frequency could also be increase by using a high osmoticum (sorbitol) concentration in
Brassica napus (Finkelstein and Crouch, 1986) somatic embryos. This treatment however, was less effective
in germinating somatic embryo in our study.
All somatic embryos incubated on each germination medium were transferred to WPM medium containing
0.2 mg/L BA. When cultured continuously, both cotyledons elongated and primary roots formed within one
 681

Table 4. Effect of plant growth regulators and osmoticum on germination of somatic embryos induced from immature
seeds in Q. acutissima

Treatments Produced roots Produced shoots Produced plants

(%) (%)

MS + 0.5 mg/L BA' 7' 7 2

MS + 0.5 mg/L BA + 34 16 10
0.6 M sorbitol

MS + 0.1 mg/L ABA + 19 37 15

0.1 mg/LGA3

WPM+ 0.1 mg/L BA 60 70 38

' The somatic embryos were chilled on MS medium containing 0.5 mg/L BA in darkness at 4 "C for 8 weeks and
then transferred to WPM medium containing 0.2 mg/L BA and the results were recorded after 4 weeks.
'Values indicate means !rom 30 somatic embryos : means based on at least three replicate experiments

week (Figure 3.3). After 2 weeks, epicotyl development occurred (Figure 3.2). Repetitive secdndary
embryogenesis was frequently observed on the hypocotyls of germinated plantlets. In some instances, the
germinating plantlets formed adventitious buds on the epicotyls, leading to multiple shoot formation.
Generally, a long primary root without root hairs formed from the lower part of hypocotyls of germinated
plant lets.
After 8 weeks incubation on WPM medium having 0.2 mg/L BA, plantlets were transplanted to a mixed
soil and hardened off in the culture room at 25 ± I OC with occasional spraying of tap water for 8 weeks
(Figure 3. 4). A total of 210 plantlets including 65 plantlets as in Table 2 were produced and 178 plantlets
were transplanted into pots. To date, 156 plantlets (92.7 %) have survived.

5-3. Somatic embryos induced from five families

To study germination, we selected and used cotyledonary somatic embryos. In previous experiment,
germination of somatic embryos, specifically epicotyl and/or radicle formation was rarely observed on the
initiation medium (Kim et a/., 1992, 1994). So, somatic embryos were subjected to three germination
treatments as shown in Table 5.
The best epicotyl induction rate was achieved under germination treatment obtained with somatic embryos
induced from the seed family Chungnam II (44.0 %). Similar results were obtained with the seed family
Chungnam 15 (43.5 %) under treatment III. On the other side, chilling treatment was not effective for the
epicotyl formation in Chungnam II (0 %).
The addition of 0.2 mg/L BA (treatment I) or 0.1 mg/L BA plus 0.1 mg/L GA 3 (treatment III) to WPM
medium resulted in effective epicotyl formation in this family. Prior to the present work, we tested BA
concentrations. The somatic embryos were cultured on the medium containing 0.1 mg/L BA for 4 weeks.
They were then transferred to either the same medium (i.e. containing 0.1 mg/L) or plant growth regulators-
free medium. The results indicated that the concentration was not enough to elongate both hypocotyls and
682

;
Figure 3. I; Primary leaves from a germinating somatic embryo. 2; Secondary somatic embryos (arrows) developed between
cotyledons of the germinated plantlet. 3; Germinating plantlets with leaves and a primary root. I; Plants regenerated from
somatic embryogenesis, growing in soil.

radicles of somatic embryos. Therefore, in the present study, we changed the concentration to 0.2 mg!L.
And it was appropriate to add BA in all the germination media. Both GA 3 and BA demonstrated
germination of shoot elongation in Q. acutissima somatic embryos (Kim eta!., 1992).
Chilling treatment at 4 t was less effective than any other treatment in radicle formation regardless of
seed families (0-4.2 % and 0-26.1 %, respectively) (Table 5). There was wide variation in the rate of the
radicle induction from somatic embryos among seed families tested. The best results for the radicle induction
were obtained with the family Chungnam II (26.1 %) by treatment ill . However, the poorest results were
obtained with the families Chungnam 14 (0 %), Chungbook 23 (0-6.3 %), and 29 (0-4.0 %) in all treatments.
The best seed family for the formation of epicotyl (43.5 %) and radicle (26.1 %) was Chungnam II with
treatment Ill. Manzanera eta/. (1993) reported that the epicotyl dormancy of somatic embryos was broken by
chilling treatment in Q. suber.
 683

Table 5. Effect of plant growth regulators and chilling treaunent on germination of somatic embryos induced from 5 families
immature seeds in Q. aculissima

Frequency of Frequency of
Treatments Origin of No. of'plantlets
epicotyl formation radicle formation
somatic embryos produced
(%) (%)

['
CN lid 44.0
16.0 10
CNI4 4.0
0.0 0
CN 15 28.0
4.0 0
JB 23 4.0
0.0 0
JB29 20.0
0.0 0
IP CN II 0.0
0.0 0
CNI4 4.3
0.0 0
CN 15 16.7
4.2 2
JB 23 20.0
0.0 0
JB 29 12.0
4.0 2

ill' CN II 43.5
26.1 15
CNI4 10.0
0.0 0
CN 15 11.0
5.3 3
JB23 6.3
6.3 I
JB29 22.2
0.0 0

'WPM+O.I mg/L BA (4 weeks) ~ WPM+0.2 mg/L BA (4 weeks)

'WPM+O.l mg/L BA (4 t. 8 weeks) ~ WPM+0.2 mg/L BA(4 weeks)
'WPM+O.l mg/L BA (4 weeks) ~ WPM+O.l mg/L BA+O.l mg/L GA3
(4 weeks) ~ WPM+ 0.2 mg/L BA (4 weeks)
• CN : Chungnam. JB : Jeonbook

The germination frequency could also be increased by using a high concentration of osmoticum (sorbitol)
in Q. robur (Chalupa, 1990) and by desiccation via air-drying in Q. rubra (Gingas and Lineberger, 1989).
Previously, however, these treatments were shown ineffective for the germination of somatic embryos for this
species (Kim et a/., 1992, 1994). In Carya illinoensis, Mathews and Wetzstein (1993) reported that the
addition of silver nitrate (3 mg/L), an ethylene biosynthesis inhibitor, in the germination medium and
application of BA (I 00 Jl M) on shoot apices appreciably increased shoot regeneration frequency.
All somatic embryos cultured on each germination medium were transferred to WPM medium
supplemented with 0.2 mg!L BA. When cultured continuously in light (50 Jl Em·2s·' ), both cotyledons were
enlarged and the epicotyl and primary root were developed further. After 1-2 weeks, the epicotyl developed,
and new leaves were induced within 4 weeks. Repetitive secondary embryogenesis was observed on the
epicotyl of the germinating plantlet. After 8 weeks of culture on WPM medium containing 0.2 mg/L BA, the
plantlets were transplanted into soil mixture. Among 33 plantlets produced, 20 were transplanted into pots.
Twelve plantlets (35 %) survived and after hardening off, they were transferred outdoors and 8 plants
survived in the field.
684

6. Conclusion

Plants of sawtooth oak, Q. acutissima, have been successfully regenerated by somatic embryogenesis, using
mature or immature seeds as the donor material. Although micropropagation of oak species, via somatic
embryogenesis, allows for mass multiplication, when transferred to germination medium, the germination
percentage is still not satisfactory and much research relative to conversion and acclimatization to soil need to
be done. Further studies are required to establish i the culture system to be utilized for the commercial
production. In this regard, the establishfuent of ,embryogenic cell suspension, cryopreservation, and the
identification of cell line which produce easily the plants are important.

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Neuenschwander, B., and Baumann, T. W. (1992) A novel type of somatic embryogenesis in Coffea arabica,
Plant Cell Rep. 10,608-612.
Non aka, G., Ishimaru, k., and Nishioka, I. ( 1984) Tannin and related compounds XI II. Galloyhamameloses
from Castanea crenata L. and Sanguisorba officina/is L. Chern. Pharm. Bull. 32, 483-489.
Park, Y.S. and Bonga, J.M. (1993) Conifer micropagation: its function in tree improvement programs, in
M.R. Ahuja (ed.), Micropagation of Wooy Plants, Kluwer Academic Publisher, Dordrecht, pp. 457-470.
Rajasekaran. K., and Mullins, M.G. (1979) Embryos and plantlets from cultured anthers of hybrid grapevines,
1. Exp. Bot. 30, 399-407.
Rancillac, M., Klinguer, A., Klinguer, S., and Millet, B. (1996) Preliminary investigations on somatic
embryogenesis from leaf discs of red oak (Quercus rubra L.), Plant Growth Reg. 20,67-73.
Roberts, D.R., Sutton. B.C.S., and Flinner, B.S. ( 1990) Synchronous and high frequency germination of
interior spruce somatic embryos following partial drying at high relative humidity, Can. J. Bot. 68, 1086-
1090.
Romano. A., Horonha, C., and Martins-Loucao, M.A. (1995) Role of carbohydrates in micropropagation of
cork oak, Plant Cell. Tiss. Org. Cult. 40, 159-167.
Sasaki, Y., Shoyama, Y., Maruyama, T., and Oda, M. (1993) Effects ofTG-19, a new cytokinin, on the shoot
and secondary somatic embryo propagation of Quercus acutissima, J. 1pn. For. Soc. 75, 150-153.
Sasaki, Y., Shoyama, Y., Nishioka, 1., and Suzaki, T. ( 1988) Clonal propagation of Quercus acutissima
Carruth by somatic embryogenesis from embryogenic axes, J. Fac. Agr. Kyushu Univ. 33, 95-10 I.
Sasamoto. H., and Hosoi, Y. ( 1989) Somatic embryogenesis in suspension cultures of Quercus serrata Thunb,
1. 1pn. For. Soc. 71,20-22.
Tulecke, W., and McGranahan, G. ( 1985) Somatic embryogenesis and plant regeneration from cotyledons of
walnut. Juglans regia L., Plant Sci. 40. 57-63.
Srivastava, P.S .. and Steinhauer, A. ( 1982) In vitro culture of embryo segments of Quercus febanii
organogenesis and callus growth as differential response to experimental conditions, Z. Ptlanzenphysiol.
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Tiss. Org. Cult. 37. 287-295.
 24. CRYOSTORAGE OF CITRUS EMBRYOGENIC CULTURES

Rosa M. Perez 1
Departamento de Protecci6n Vegetal y Biotecnologfa, Instituto Valenciano de
Investigaciones Agrarias. Apartado Oficial. 46113 Moncada (Valencia) Spain
1Present adress: Instituto Biologfa Molecular y Celular de Plantas. UPV-CSIC.

Departamento de Biologfa del Desarrollo. Campus de Ia UPV. Camino de Vera s/n.

46022 Valencia (Spain).
Phone number: 34 9633877870
e-mail: roperez@ibmcp.upv.es

Contents
1. Introduction 4.2. Cryoprotectant Treatment
2. Production of Embryogenic Callus 4.3. Freezing Method
Cultures 4.3.1. Dehydrative Slow Cooling
3. Cryopreservation 4.3.2. Vitrification
3.1. General Aspects 4.4. Storage
3.2. The Use of Cryoprotectants in 4.5. Thawing
Cryopreservation 4.6. Viability Assays and Regrowth
3.3. Cryopreservation Techniques 5. Conclusion
4. Protocols for Cryopreservation of Acknowledgements
Embryogenic Citrus Cultures References
4.1. Pregrowth

1. Introduction

Genetic improvement of citrus species has been long hampered by the complexity of
their genetic systems. Their partial or complete sterility prevents the hybrydization of
some potentially useful clones, which is as a consequence of selection over time of
infertile cultivars because of the consumer's preference for seedless fruit. Adventive
somatic embryogenesis from nucellus cells is a widespread phenomenon in citrus and it
prevents zygotic embryo development, resulting in seedling populations that are
genetically identical to the maternal parent (Soost and Cameron, 1975). Nucellar
embryony is the greatest impediment to genetic recombination among Citrus species,
regardless of their infertility. The long juvenile period of most citrus seedlings is also a
great limitation to the evaluation and selection of desirable fruit characters.
Furthermore, the large size of mature plants requires large area for the evaluation. Most
687
S.M. Jain. P.K. Gupta and R.J. Newton (eds.). Somatic Embryogenesis in Woody Plants, Volume 6, 687-705.
© 2000 Kluwer Academic Publishers.
688
citrus species have a high degree of heterozygosity (Ollitrault and Faure, 1992; Herrero
et a/., 1996) which produces a high segregation of genetic characters in the progeny,
reducing the possibility of recovering genotypes with desired traits.
The development of new technologies based on protoplast culture and fusion to
obtain somatic hybrids is promising for improving rootstocks and cultivars (Ohgawara
et al., 1985, 1989; Grosser et al., 1988a,b; Vardi and Galun, 1989; Tusa eta!., 1990). In
citrus, the utilization of protoplast technologies and further recovery of whole plants
relies on the availability of embryogenic cultures from the desired genotypes. The
availability of embryogenic callus and cell cultures is also important to develop genetic
transformation strategies (Hidaka et al., 1990; Kobayashi et al., 1994; Pefia pers. com.),
for true-to-type mass propagation of rootstocks (Ollitrault and Michaux-Ferriere., 1992)
and for genetic resources conservation (Engelmann et al., 1994a; Duran-Vila, 1995,
1997; Perez, 1995; Perez et al., 1997).
The conservation of gcrmplasm from wild Citrus relatives, the primitives and
currently cultivated citrus species is crucial for its use in current or future breeding
programmes. Conventionally, citrus germplasm has been maintained under the field
conditions in botanical gardens or at research institutes, where it is subjected to
potential losses due to pests, diseases and climatic hazards. As an alternative, genotypes
of interest can be preserved in the greenhouses and screenhouses where they can be
more easily protected. The high cost of this conservation system limits the number of
accessions that could be preserved (Navarro, pers. comm.). Most citrus species cannot
be conserved with seed storage technologies due to their recalcitrant nature, this,
together with the problems associated with the traditional storage methods, make
necessary the development of alternative genetic resources conservation methods.
The collection of calli derived from elite cultivars and rootstocks is of vital
importance, since these embryogenic cultures could be a major source of totipotent cells
for genetic transformation and biotechnologies based on protoplast fusion. The
demonstration of embryogenic cultures are amenable to cryopreservation indicates their
potential for long-term storage of citrus genetic resources.

2. Production of Embryogenic Callus Cultures

Most Citrus species are polyembryonic, and the adventitious embryos arise in vivo from
nucellar tissue. Studies of in vitro somatic embryogenesis have involved the culture of
isolated nucellus in monoembryonic species (Rangan et al., 1969; Juarez et al., 1976)
and fertilized or unfertilized ovules in polyembryonic cultivars (Button and Bornman,
1971; Kochba et al., 1972; Navarro et al., 1979; Perez et al., 1998). The published
reports have indicated the embryogenic callus induction by culturing the undeveloped
seeds taken from ripe ti-uits 8-15 months after anthesis (Starrantino and Russo, 1980),
embryogenic cell lines obtained from pollen of sweet orange (Hidaka and Omura.,
1989) and plant regeneration from callus derived from styles of lemon (Carimi et al.,
1994; De Pasquale et al., 1994).
 689
The production of embryogenic callus from the nucellar tissues of in vitro cultured
ovules is greatly cultivar dependent (Galiana, 1995; Perez et al., 1998). The percentage
of ovules producing callus varies widely among cultivars, ranging from 60 % in
'Murcott' tangor (Perez et al., 1999 in press) to 0.01 %in C. Volkameriana (C. Ortega,
pers. comm.). Embryogenic cultures could not be produced from cultured ovules of
monoembryonic cultivars (Moore, 1985; Perez et al, 1999 in press).
The different embryogenic response between polyembryonic and monoembryonic
species is due to their different endogenous hormonal balances or unknown components
that prevent embryogenesis that are apparently present in chalaza! ends of
monoembryonic cultivars (Tisserat and Murashige, 1977; Gmitter and Moore, 1986).
For embryogenic callus induction , ovaries and immature fruits (2-8 weeks after
anthesis), are surface disinfected and cut open under aseptic conditions. The ovules are
excised and placed on the modified MS culture medium (Murashige and Skoog, 1962)
supplemented with malt extract (Bitters et al., 1970; Kochba and Spiegel-Roy, 1973;
Navarro and Juarez, 1977; Vardi and Spiegel-Roy, 1982). In some reports, other
additives such as adenine sulphate (Button and Bornman, 1971; Kobayashi et at., 1983,
1988), 2,4-dichlorophenoxyacetic acid (Kobayashi et al., 1983, 1988), 3-indol-N-
acetyl-alanin and 6-benzylaminopurine (Vardi et at., 1986) and kinetin (Hidaka and
Kajiura, 1988; Hidaka et al., 1990) have also been added in the culture medium.
The embryogenic callus could be recovered by subculturing the small colonies of
proliferating callus and globular structures produced from the nucellar tissues (Fig. 1).
Alternatively, callus is transferred to the liquid medium for making cell suspension
cultures. For maintenance of these callus cultures, different media have been reported.
Their composition is given in Table 1. Once the embryogenic cultures have' been
established, they must be regularly subcultured, especially cell suspension cultures at
every 2-week interval and callus subcultured at one month interval.

Figure I. Citrus embryogenic callus production. (A) Longi tudinal section of an inmature fruit. (o) ovules.
(B) Structures produced from cultured ovules: (e) somatic embryo at cotyledonar stage, (g) somatic embryo
at globular stage, (p) pseudobulbi, (u) unorganized cells (C) Embryogenic callus obtained by subculturing
the unorganised cells produced directly from the nucellar tissues in the cultured ovules. Bar= 5mm.
690
Table I. Culture media for Citrus embryogenic cultures maintenance.

Culture medium composition References

MT" + 5% sucrose+ 0.05% ME" Kochba and Spiegel-Roy, 1973
MT" + 5% sucrose+ 10 mgi 'BA Kobayashi et al., 1983
MT " (half concentration of nitrates) +0.05% ME"+ Grosser and Gmitter, 1990; Tusa eta/., 1990
1550 mgl'glutamine + 750 mgi 'KCI + 5% sucrose
MT" + 0.05 % ME"+ 50J..lM Kin Hidaka and Kajiura, 1988
MS inorganic salts +I 00 mgr' i-inositol, 0.2 mgr ' Galiana, 1995; Perez et al., 1998
thiamine HCI +I mgl" 'pyridoxine-HCI + I mgl" ' nicotinic
acid+ 4 mgl"'glycine + 5% sucrose+ 0.05 % ME"
" Murashige and Tucker ( 1969) Medium
" Malt extract
BA: Benzylaminopurine, Kin : kinetin

However, these cultures lose, with the time, their ability to produce somatic
embryos spontaneously: Different approaches taken to restore the embryogenic capacity
in these cultures have been, more or less, successfully employed. They include: a)
substitution of sucrose by lactose or maltose in the culture medium (Button, 1977;
Galiana, 1995; Perez et al., 1998); b) addition of growth-regulator inhibitors
(hidroxinitrobencil bromide, 7-aza-indol, 8-aza-guanine) (Kochba and Spiegel-Roy,
1977) and c) addition of abscisic acid, ethephon or 2-cholroethyl-trimethyl-amonium
cloride (Kochba et al., 1978) to the culture medium (Fig. 2).

FiKure 2. Embryogenic callus of sweet orange, (A) Callus devoid of embryos obtained after monthly
subculture to fresh medium, (B) Induction of embryogenesis in maltosa containing medium. Bar= Smm.

A high labor input is required for the maintenance of these cultures by periodical
subculturing. The risk of culture contamination increases with time as well as the risk of
losses due to technical failures in growth chambers. Somaclonal variation increases with
long-term subculturing and the cultures may no longer be suitable for further
manipulations (Harding, 1996).
Cryopreservation of embryogenic cultures obviates the above mentioned problems
associated with the maintenance of cultures in active growth phase and arises as an
 691
alternative method for the long-term conservation of Citrus genetic resources.
Cryopreserved embryogenic cultures are highly suitable for somatic cell fusion, genetic
transformation, germplasm bank and minimize somaclonal variation (Kobayashi et al.,
1994; Duran-Vila, 1997; Olivares, 1998).

3. Cryopreservation

3.1. General Aspects

Cryopreservation, also called "freeze preservation" or "cryogenic storage" involves

transfer of biological material for long-term storage in the liquid nitrogen (LN)
_(Meryman, 1966; Bajaj, 1979a,b; Ashwood-Smith and Farrant, 1980; Kartha, 1985;
Meryman and Williams, 1985; Towill, 1991). At this temperature (-196°C), almost all
metabolic activities of cells are at a standstill and the specimens can be preserved for an
extended period of time (Kartha, 1987). The only process that might contribute to the
deterioration of cells in storage involves free radical damage caused by ionizing
background radiation (Whittingham et al., 1977; Benson, 1990). However, physical and
chemical protection can alleviate this small risk (Withers, 1992).
Cryoconservation of plant tissue cultures can obviate several problems associated
with cultures maintained in vitro in an active growing state. With successive subcultures
there is an increased risk of growing cultures being contaminated with bacteria and
fungi (Petru et al., 1971; Bertaccini, 1992). The morphogenetic competence and genetic
stability of tissue cultures can change over time, which has undesirable consequences
for in vitro genetic resources management and preclude their use for biotechnological
applications (Harding, 1996). The most effective means of stabilising in vitro plant
cultures is via cryopreservation (Ashwood-Smith, 1985; Harding, 1991; Benson et al.,
1998). No phenotypic, genotypic or epigenetic changes have been reported as result of
cryopreservation process (Withers and Engelmann, 1998).
There are several reports indicating that cryopreserved cells maintain their
biosynthetic and morphogenic potential (Withers, 1985; Kartha, 1987; Kartha and
Engelmann, 1994). Electrophoretic profiles for aminoleucine peptidades and amylases
were comparable in plants regenerated from control and cryopreserved apices of
sugarcane (Paulet et al., 1994) and sweet orange (Citrus sinensis (L.) Osb.) somatic
embryos (Marin et al., 1993). With the later material, no modification was observed in
the pattern of total soluble proteins. The ploidy level was not modified by
cryopreservation in plants regenerated from oilseed rape somatic embryos (Uragami et
al., 1993) and in dihaploids of potato (Ward et al., 1993). Restriction Fragment Length
Polymorphism (RFLP) patterns of plants regenerated from cryopreserved potato shoot-
tips (Harding, 1991 ), embryogenic cell suspensions and apices of sugarcane (Paulet et
al., 1994; Chowdhury and Vasil, 1993) showed no differences when compared to the
unfrozen controls. Furthermore, Kobayashi et al. ( 1994) found that transgenic cell
suspensions of sweet orange maintained their transgenic nature, analysed by southern,
after one year of cryostorage.
692
3.2. The Use of Cryoprotectants in Cryopreservation

The nature and causes of cryodamage are closely related to the events which occur
during the unfrozen-frozen transitions. At a gross level, rupture of the cell organelles
can occur during intracellular freezing and by recrystallization during thawing (Sakai et
at., 1968; Withers, 1978). Local overdehydration may also occur in the cytoplasma as a
result of intracellular ice growth. Proximity of ice to macromolecules may cause
configurational changes (Withers and Davey, 1978). Solution effects, the consequences
of cytoplasmic dehydration and exposure of the plasmalemma to concentrated
extracytop1asmic solutes, may lead to increased electrolite concentrations, the
accumulation of enzyme reaction intermediates, the concentration of carbohydrates and
enzymes, pH changes, macromolecular cross-linking due to the proximity, and
disturbances in the structured water in proteins and lipoprotein membranes (Meryman,
1966, 1971; Mazur et al., 1972).
Most cryodamage is related to membrane structure and function, particularly to
the plasma membrane (Steponkus, 1984 ). In contrast, nucleic acids are very resistant to
freezing and thawing (Levitt, 1966). Membrane lesions observed in frozen and thawed
cells include physical rupture, quantitative and qualitative loss of lipoprotein, loss of
specific enzyme activity and loss of selective permeability (Meryman, 1971; Araki,
1977). Some lipid components convert from a liquid-crystalline to a gel phase during
cooling and may not reconstitute during warming (Quinn, 1985).
Plant germplasm can not survive exposure to the cryogenic temperatures unless it
is subjected to physical or chemical cryoprotection. Examples of the former are dried
citrus seeds (Munford and Grout 1979; Perez, 1995), dried mature orthodox seeds and
buds from cold hardy temperature woody species (Sakai and Nishiyama, 1978; Sakai,
1985; Stanwood, 1985).
Chemical cryoprotection may relieve damage in frozen materials. There is more
than one way of action of cryoprotectants, in the same way that there is more than one
mechanism of freezing damage. Among the proposed mechanisms of chemical
cryoprotection are: (a) a reduction in ice crystal growth rate, size and amount, (b)
colligative action in which the cryoprotectant lowers the effective concentration of
solutes in equilibrium with ice inside and outside the cell at any temperature, (c)
osmotic dehydration before freezing, (d) increased membrane permeability permitting
water transport out of the cell, and (e) stabilization of macromolecules and membranes
(Meryman, 1966; Leibo and Mazur, 1970; Withers, 1978; Finkle et al., 1985; Benson,
1990).
Cryoprotectant properties are shared by a miscellaneous group of chemicals which
have in common a high affinity for water. Among them are dimethylsulfoxide (DMSO),
glycerol, sugars, sugar-alcohols, high molecular weight additives such as
polyvinylpyrolidone, dextran,hydroxyethyl starch, etc. (Meryman, 1966; Kartha, 1987).
Generally a concentration of 5 to I 0% for DMSO or I 0 to 20% for glycerol is adequate
for most materials (Kartha, 1987). Most of these cryoprotectants exhibit varying
degrees of cytotoxicity at high concentrations (Bajaj et al., 1970; Bajaj, 1979a). In some
 693
cases, where the application of a single cryoprotectant does not result in high survival, a
mixture of these compounds has improved the survival rates after thawing (Finkle and
Ulrich, 1979; Withers, 1985).

3.3. Cryopreservation Techniques

Most reports of successful cryopreservation of higher plant in vitro systems involve

procedures based upon dehydrative slow cooling (Withers and King, 1980). The
freezing protocol consists in slow freezing down to a temperature of -30 ° to -40 °C,
followed by the immersion of the vials containing cells or callus in liquid nitrogen. Ice
crystallization occurs first in the external medium causing dehydration of the cells.
A few reports describe ultra rapid freezing by dropping the samples directly in
liquid nitrogen (Seibert, 1976; Grout and Henshaw, 1978; Bajaj, 1983). The
intracellular ice crystallises in microcrystals of a size which is harmless to the integrity
of the cell components (Mroginski et al, 1991 ).
A novel approach to cryopreservation IS based on the use of
encapsulation/dehydration techniques (Fabre and Dereudre, 1990). This method entails
the encapsulation of germplasm in an alginate matrix, forming a calcium alginate bead,
followed by two subsequent treatments which involved the osmotic dehydration of the
beads in highly concentrated sucrose solutions, followed by air desiccation in a laminar
flow bench. After these procedures, the germplasm is transferred to cryovials and
piunged directly into LN.
The basis of the cryoprotective mechanism of encapsulation/dehydration is largely
due to the inhibition of the nucleation in the alginate matrix and the preserved
germplasm. Water within the bead forms an amorphous glassy state, circumventing the
damaging effects of ice. This is now one of the main methods of choice for germplasm
systems which have previously prov~d very difficult to cryopreserve using traditional
cryoprotection strategies (Benson, 1994 ).
Recently, an alternative cryopreservation technique has been developed which is
based on vitrification phenomenon (Fahy et al., 1984 ). In vitrification- based
procedures, the tissue is embedded and infiltrated in so called vitrification solutions,
which become highly viscous at low temperatures and solidify into a metastable glass
without the formation of ice crystals. This is followed by rapid cooling. Vitrification
avoids the damages caused by ice formation inside the cells. However, this procedure
has problems related to the necessity of using very high concentration of
cryoprotectants in the vitrification solution, which are toxic to the cells and also cause
physical damages to the cells during devitrification processes.

4. Protocols for Cryopreservation of Embryogenic Citrus Cultures

There are several stages in a freeze-preservation protocol, which are: pre-growth,

cryoprotection, freezing, storage, thawing, viability assays and recovery. Different
cryopreservation procedures have successfully been assayed in embryogenic cell
694
cultures of a wide range of citrus species and cultivars (Table 2).

Table 2. List of citrus species and cultivars that have been succesfully cryopreserved using different freezing
methods.

Specie-cultivar Freezing method Reference

Navel sweet orange Slow-cooling Kobayashi et al., 1990
Sakai et a/.,1991 b
Perez et al., 1993, 1997
Navel sweet orange Vitrification Sakai et a/.,1990, 1991 a
Grapefruit Vitrification Sakai et al., 1991 a
Sudachi Vitrification Sakai et a/.,1991 a
Hybrid: Murcott tangor Vitrification Sakai eta/.,1991 a
Common mandarin Slow-cooling Aguilar etl al., 1993
Willow leaf mandarin Slow-cooling Engelmann et al., 1994 a,b
Chios mandarin Slow-cooling Engelmann et al., 1994 a,b
Cleopatra mandarin Slow-cooling Perez et al., 1993, 1997
Engelmann et al., 1994 a,b
Shamouti sweet orange Slow-cooling Engelmann eta/., 1994 a,b
Hamlin sweet orange Slow-cooling Engelmann eta/., 1994 a,b
Mexican lime Slow-cooling Engelmann et al., 1994 a,b
Perez et al., 1997
Salustiana sweet orange Slow-cooling Perez eta/., 1993, 1997
Pineapple sweet orange Slow-cooling Perez eta/., 1993, 1997
Succari sweet orange Slow-cooling Perez eta/., 1997
Sour orange Slow-cooling Perez eta/., 1997
Lac lemon Slow-cooling Perez et a/., 1997
Star Ruby grapefruit Slow-cooling Perez et al., 1997
White grapefruit Slow-cooling Perez eta/., 1997
Red Marsh grapefruit Slow-cooling Perez et al., 1997
Avana Api reno mandarin Slow-cooling Perez et al., 1999
Hybrid: Murcott tangor Slow-cooling Perez et al., 1999
Hybrid: Minneola tangelo Slow-cooling Perez eta/., 1999
Hybrid:Orlando tangelo Slow-cooling Perez et at. , 1999
Hybrid: Fairchild mandmin Slow-cooling Perez eta/., 1999
Hybrid: Kinnow mandarin Slow-cooling Perez et al., 1999
Hybrid: Nova mandarin Slow-cooling Perez eta/., 1999
Hybrid: Sunburst mandarin Slow-cooling Perez et al., 1999
Hybrid: Page mandarin Slow-cooling Perez et al., 1999
Kumquat Hong Kong Slow-cooling Perez, 1995
Kumquat Meiwa Slow-cooling Perez, 1995
Glycosmis pentaphylla Slow-cooling Perez, 1995
Microcitrus papuna Slow-cooling Perez, 1995

4.1. ?regrowth

The age of cells at the time of harvesting for cryopreservation can affect their survival.
This is linked to cell size and water content. Cells at the early lag phase and stationary
phase are large, highly vacuolated and have high percentage of water content. These
factors reduce survival potential because ·there is an increase in the requirements for
 695
cryoprotective dehydration whilst reducing the capacity of cells to dehydrate through a
reduction in the surface area of cells area to volume ratio, and increasing its
susceptibility to plasmolysis and deplasmolysis injury (Withers, 1980). Rapidly
growing cells, in the late lag and early/mid exponential phase, are small and have
relatively low water content. These cells are the best suited for cryoconservation.
For cryogenic assays, citrus cell suspensions have been sampled at the beginning
of their exponential growth phase, 6 days (Kobayashi et at., 1990; Sakai et at., 1991b)
or 8-10 days after the last transfer to fresh medium (Sakai et at., 1990; Sakai et at.,
1991a; Aguilar et al., 1993; Engelmann et at., 1994a,b) and 3-4 weeks after
subculturing in the case of callus (Engelmann et al.,.1994a,b ).

4.2. Cryoprotectant Treatment

The. application of cryoprotection is essential for the survival of embryogenic cultures

subjected to cryopreservation assays. There are two types of cryoprotectants used a)
permeating and b) non-permeating, with respect to the plasma membrane. The most
commonly used permeating additives are DMSO ·and glycerol. The non permeating
compounds include sugars, sugar-alcohols and high molecular weight additives, such as
polyethylene glycol (PEG). Most of them exhibit cytotoxicity at relatively high
concentrations, and therefore, mixtures of cryoprotectants at lower concentrations are
recommended.
A mixture of cryoprotectants involving DMSO and sucrose at different
concentrations has commonly been used for dehydrative slow cooling. A solution
containing 5% DMSO and 0.15M sucrose has been employed for the cryopreservation
of Citrus deticiosa Tan. cell suspensions. Higher than 15% DMSO concentration was
toxic to the cells and they could not grow again after thawing when sucrose
concentration was increased from 0.15M to 0.9M in the cryoprotective mixture (Aguilar
et al., 1993; Engelmann et at., 1994a,b). On the contrary, Kobayashi et al. (1990)
reported in Citrus sinensis, that the survival rate increased with increasing sucrose
content in the mixture with 5% DMSO, reaching the highest survival with 1.2M
sucrose, and rapidly decline in a 1.5M solution after thawing of embryogenic cell
suspensions. Other sugars or polyhydric alcohols (glucose, sorbitol, glycerol) together
with 5% DMSO gave lower rates of survival in C. sinensis cell cultures (Kobayashi et
at., 1990).
In callus, a mixture of 10-15% DMSO and 0.15M sucrose provided an optimal
cryoprotection in all species and cultivars assayed (Engelmann et at., 1994a,b; Perez,
1995; Perez et al., 1997) (Table 2). Cryoprotection may be achieved by exposing callus
to cryoprotective solution for 1 h at 0 °C (Kobayashi et at., 1990; Engelmann et al.,
1994a,b) or 30 min at 4 °C (Perez, 1995; Perez et al., 1997).
A mixture of 2M glycerol and 0.4M sucrose provided cryoprotection to cell
suspensions using the simplified freezing method proposed by Sakai et at. (1991b). In
this case, the cells were exposed to cryoprotective mixture for 10 min at 25 °C.
For vitrification procedures, the temperature and the duration of treatment of the
plant material with the vitrification solution are highly critical parameters due its high
696
toxicity. The cryoprotection treatment defined by Sakai et al. (1990) for the vitrification
of cell suspension, has a two-step application of the vitrification solution designated as
PVS2. This is composed of MT (Murashige and Tucker, 1969) medium containing
0.15M sucrose and 30% (w/v) glycerol, 15% .Cw/v) ethylene glycol and 15% (w/v)
DMSO. Firstly, the cells must be treated with 60% PVS2 at 25 °C for 5 min, then they
are exposed to chilled PVS2 for 3 min, and finally are plunged in LN. Dehydrating
samples at low temperatures (0 °C) reduces the damaging effects of high osmolarity
provided by the vitrification solution. It also reduces cell permeability, increasing the
length of time that cells must be exposed to osmotic stress. Brief exposure, sufficient
for dehydration at 25 °C minimizes toxicity. The vitrification protocol has been
improved and simplified to a single application, that consists of direct exposure of the
cells to PVS2 at 25 °C for 3 min.

4.3. Freezing Method

The following methods have been successfully employed for citrus cell suspensions and
callus cryopreservation.

4.3.1.Dehydrative Slow Cooling

Slow cooling permits the tlow of water from inside to the outside of cell, thereby,
promoting extracellular ice formation and preventing lethal intracellular freezing. The
slow cooling rate is done only at temperatures when freeze-dehydration proceeds at the
higher rates (Sakai and Yoshida, 1967).
Citrus cell suspension cultures and callus had high survival rates when prefrozen
slowly to -40 °C before being immersed in LN (Kobayashi et al., 1990; Aguilar et al.,
1993; Engelmann et at., 1994 a,b; Perez, 1995; Perez et al, 1997).
Most cryogenic assays with cell suspensions and callus were performed with
programmable freezers [Cryoembryo-HP, Hoxan, Japan (Kobayashi et al., 1990),
Minicool, L'air Liquide, France (Aguilar et al., 1993; Engelmann et al., 1994a,b; Perez,
1995; Perez et al., 1997)]. The programmable freezing unit controls the automatic
injection of LN in to the cooling chambers. The freezing program is defined by several
parameters: (a) starting temperature T, at which the chamber is held before introducing
the samples; (b) freezing point T, of the cryoprotectant solution, which must be
determined for each cryoprotectant solution; (c) liquid phase cooling rateR, between T,
and T,; (d) cooling power W needed to overcome the latent heat of fusion, which is
applied as soon as the temperature of the liquid phase sample falls below T,; (e) time
during which the cooling power W is applied; (t) temperature T 1 at which the cooling
rate can be changed; (g) solid-phase cooling rate R, between T, and T1; (h) cooling rate
R, between T, and the final temperature T4 • The freezing program used by Perez et al.
(1997) was defined as follows: T, = 4 °C, T, = -6 °C, T, = -40 °C and T4 = -150 °C.
The cooling power, set at W= 5 and applied for 40 s, provided a smooth cooling curve.
In preliminary experiments, performed with sweet orange 'Salustiana' calli, several
cooling rates R, and R, were assayed and the final cooling R1 was set at 20°C min·'.
 697
After reaching the final temperature provided by the freezer, the cryotubes were
immersed in LN. Values of R, ranging from 0.1 to 0.5 °C min·' and R2 ranging from 0.1
to 1°C min-' did not ·affect the survival of the cultures (Table 3). The cryop~eservation
program developed for 'Salustiana' sweet orange has been successfully employed for
other citrus species and cultivars (Table 3) (Perez, 1995; Perez et al., 1997).

Table 3. Effect of liquid-phase and solid-phase cooling rates on the survival and growth of callus cultures of
Salustiana sweet orange. Liguid-phase cooling rate R, from 4 °C to - 6 °C. Solid-phase cooling rate R,
from -6 °C to- 40 °C. Control cultures were subjected to cryoprotective treatments but were not frozen.

Cooling rates Percent viable (number of cultures)

R,-R, (°C min-') Control Frozen
0.1 -0.1 95 (19) 96 (57)
0.1 -0.2 100 (7) 100 (20)
0.1 -0.5 100 (5) 100 (24)
0.1 - 1.0 100 (9) 100 (26)
0.1-5.0 I 00 (20) 0 (19)
0.2-0.5 100 (9) 100(19)
0.2- 1.0 100 (3) 100(21)
0.5-0.5 100 (10) 100 (35)
0.5- 1.0 100 (7) 100 (18)
1.0- 0.5 100 (10) 0 (24)

Freezing procedures without using the sophisticated and costly freezing apparatus
have been also reported. Cooling rate of about 0.5 °C min-' down to -40 °C can be
consistently achieved with cooling baths using pure methanol as a coolant. The cooling
bath Heto CB 10 is highly reliable (Perez et al., 1997).
A simple freezing device consists of a plastic box containing 250 ml isopropylic
alcohol (Nalgene, USA) has successfuly been used for the cryopreservation of cell
suspensions. This thermocooler placed in a deep-freezer, thermostated at -80°C for±
65 min, prefreezes the cells to - 40°C. This cooling pattern allows sufficient cell
dehydration to achieve high survival rates after cryogenic assays (Engelmann et al.,
1994a).
Acording to Sakai et al. ( 1991 b), cells cryoprotected with 2M glycerol and 0.4M
sucrose were frozen spontaneously by placing cryotubes in a freezer at -30 °C for 20-
30 min prior to direct immersion into LN. The cooling rate was about 2°C min-'.

4.3.2. Vitrification
In vitrification-based procedures, cells are dehydrated prior to freezing by exposing
them to high concentrated cryoprotective solution. This is followed by rapid cooling,
and then transfer of samples to LN (Sakai et al., 1990, 1991 a).
698
4.4. Storage

It is essential to store frozen cultures at a sufficiently low temperature to prevent ice

recrystallization. When the cultures arc not stored at a low enough temperature, an
additional injury to the cells may cause.
To evaluate the effect of temperature and duration of storage, 'Salustiana' sweet
orange cultures were frozen by slow-dehydrative cooling, placed in LN and transferred
to different storage conditions: -196 °C (immersed in LN), -150, - 80 and -20 °C (in
electrical freezers). As shown in Table 4, storage up to 2 years at -196 °C did not affect
the survival rate of cryopreserved cultures. Storage at -80 and -20 °C impaired further
growth of cryopreserved cultures (Perez et al., 1997). Storage at -150 °C, in electrical
freezer, did not affect the survival of callus cultures of several citrus species and
cultivars (Perez eta!., data not published). The use of this commercial electrical freezer,
which maintains a temperature of- !50 °C, can simplify the storage since obviates the
permanent requeriment of LN. An additional advantage of this freezer is its low
maintenace cost.

Table 4. Survival and growth-of Salustiana sweet orange cultures atier storage. Survival was estimated as the
percentage of cultures that grew after thawing. At least 20 cultures were used in each treatment.

Storage Temperature

-20 °C -80 °C - 150 °C -196 °C

I day 0 60 100 100
I week () () 100 100
I month () () 100 100
3 months 0 0 100 100
I year 0 () 100 100
2 ears 0 0 100 100

4.5. Thawing

Ice not only can damage when formed during the freezing process, but can also damage
during thawing as a result of recrystallization phenomenon. lee melts and reforms a
thermodynamically favourable, larger and more damaging crystal size. This can be
mitigated by rapid thawing (Farrant et al., 1977; Meryman and Williams, 1985).
Cultures cryopreserved by dehydrative slow cooling were successfully recovered
when thawing was performed by fast warming, immersing the cryotubes in a water bath
at 37-40 °C (Kobayashi et al., 1990; Sakai et al., 1991 b; Aguilar et al., 1993;
Engelmann eta!., 1994a,b; Perez, 1995; Perez eta/., 1997). When samples were thawed
slowly at the room temperature until ice melted completely, cell growth recovery was
unsuccessful (Aguilar et al., 1993; Perez et al, 1997).
In vitrification-based protocols, samples must be thawed rapidly for preventing
devitrification, otherwise ice crystals would form and that would be detrimental to
 699
cellular integrity. Samples thawed rapidly, by immersing them in a water bath at 25 °C,
showed high survival rates whereas slowly thawed (air exposure at 0 °C or 25 °C)
vitrified cells had very low survival rates (Sakai et al., 1991 a).

4.6. Viability Assays and Regrowth

Rapid viability tests using vital stains such as fluorescein diacetate (FDA) have been
widely used to estimate the viability of cryopreserved cultures (Aguilar et al., 1993;
Engelmann et al., 1994a,b; Kobayashi et al., 1990; Nadel, 1989; Sakai et al.,
1990, I 991 b; Widholm, 1972). Nevertheless, FDA staining performed immediately after
thawing over-estimates cell survival (Aguilar et al., 1993 ; Perez et al., 1997). The use
of this viability test requires caution especial when the ultimate aim is to recover
.growing cultures and whole plants. When cell survival assessment test is conducted one
week after the thawing, the results would be more realistic. However, the growth
recovery and embryogenic potential must remain the most reliable and accurate test for
assessing viability of cryopreserved cells (Perez, 1995; Perez et al., 1997). All cultures
subjected to slow cooling and fast thawing (Table 3) resulted in growth patterns similar
to unfrozen controls and produced embryos. Embryos from cryopreserved cultures
produced plants that had similar morphology to the unfrozen controls (Fig. 3).

Fi~ure 3 . Cu ltures of Salustiana sweet orange subjected to freezing. storage and thawing. Bar= 5 mm .
(A) Unfrozen control. (B) Cu ltures frozen by dehydrative slow cooling, stored in LN overnight, and thawed
by fast warming, (C) Plants I year after transplanting to soil from cryopreserved cultures (left). unfrozen
contro l (centre) and subjected to cryoprotection (right).

Factors that affect post-thaw regrowth include removal of cryoprotectant, mode of

culture and culture medium composition (Withers, 1985). Conveiltionally, the
cryoprotectant solution is removed after thawing and the cells are washed with liquid
medium before transferring them to the solid medium for growth (Perez et al., 1997). A
modified approach widely used is to place the freshly thawed cells on a filter paper to
facilitate their transfer to the fresh medium and thereby to reduce the level of potentially
700
toxic cryoprotectants in the cells (Kobayashi et al., 1990; Sakai et al., 1990, 1991 a,b;
Aguilar et al., 1993; Engelmann et al., 1994a,b).
In vitrification-based procedures, the highly concentrated vitrification solution
must be removed progressively in order to minimize osmotic shock after warming. This
is performed by diluting the vitrification solution in liquid medium supplemented with
!.2M sucrose before transferring the explants to the standard medium (Sakai et al.,
1990, 1991a).
Results of citrus callus and cell suspensions cryopreservation indicate that, after an
initial retardation of growth, there is an apparent enhancement of somatic
embryogenesis (Engelmann eta!., 1994b), leading the recovery of whole plants.
Plants derived from frozen-thawed cells are morphologically uniform and do not
display differences as compared to unfrozen controls (Fig. 3). Genetic studies on plants
derived from protoplasts derived from frozen and unfrozen embryogenic cultures
demonstrate that no genetic change is caused in the callus freezing-thawing process
(Kobayashi eta!., 1994; Olivares, 1998).

5. Conclusion

Citrus embryogenic cultures can withstand a wide range of cryopreservation protocols,

ranging from conventional cryoprotection followed by slow dehydrative cooling
(Kobayashi et at., 1990; Aguilar et al., 1993; Perez et al., 1993, 1997; Engelmann et al.,
1994 a,b) to vitrification and fast cooling (Sakai et al., 1990, 199la). The survival rate
and proliferation recovery of embryogenic cultures were not modified after up to 2
years of storage at cryogenic temperatures, into a LN container or in a -150 °C
commercial freezer (Perez et a!., 1997). A project has been initiated to produce
embryogenic cell cultures from all the polyembryonic species and cultivars of the
germplasm bank maintained at the Instituto Valenciano de Investigaciones Agrarias
(Spain) greenhouses for the establishment of a germplasm bank of cryopreserved
cultures that may totally or partially replace some of the genotypes presently maintained
as living plants.
The maintenance of cryopreserved cultures of citrus calli and embryogenic cell
suspensions makes easier and safer to manage collections of embryogenic calli to be
used for varietal improvement via somatic cell fusion and genetic transformation.
Moreover, transformed or fused cell lines could be cryopreserved during the whole
regeneration and evaluation period. The selected genotypes could be recovered and
multiplied again though somatic embryogenesis.

Acknowledgements
Deep thanks are due to Dr. L. Navarro for the critical review of the manuscript. The
helpful comments and suggestions of Dr. N. Duran-Vila and Dr. L. Pefia are also
acknowledged. This work would not have been possible without the participation of Dr.
A. Galiana, C. Ortega, J. Juarez, J. M. Arregui in the research described in this chapter.
 701
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(Citrus sinensis Osb. var. brasilensis Tanaka) cooled to -196 °C. J. Plant Physiol. 137:465-470.
Sakai, A., Kobayashi, S. and I. Oiyama, 1991 b. Cryopreservation of nucellar cells of navel orange (Citrus
sinensis Osb.) by a simple freezing method. Plant Sci. 74: 243-248.
Sakai, A , Otsuka, K. and S. Yoshida, 1968. Mechanism of survival in plant cells at super low temperatures
by rapid cooling and rewarming. Cryobiology, 4. 165-173.
Seibert, M., 1976. Shoot initiation from carnation shoot apices frozen to -196°C. Science 191: 1178-1179.
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 705
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25. CRYOPRESERVATION OF EMBRYOGENIC CULTURES OF CONIFERS

Hely M Haggman, Tuija S. Aronen and Leena A. Ryynanen

The Finnish Forest Research Institute, Punkaharju Research Station,
Finlandiantie 18, FIN-58450 Punkaharju, Finland

1. Introduction
2. Cryopreservation protocols appropriate for the embryogenic cultures of coni-
fers
2.1 THE SLOW-COOLING METHOD
2.1.1 Material
2.1.2 Pre-treatment and cryoprotection
2.1. 3 Freezing, thawing and post-treatment
2.1.4 In vitro cultivation after cryopreservation
2.2 THE ALTERNATIVE CRYOPRESERVATION METHOD BASED ON
EN CAPSULATION-DEHYDRATION TECHNIQUE
3. Genetic fidelity
4. Applications and future prospects
5. Conclusions
6. References

1. Introduction

The cryopreservation methodology, currently used for plant material, originates from
the research done on animal cells and tissues. The epoch-making finding in cryobiology
was that made by Polge et al. (1949), who demonstrated the ability of fowl spermatozoa
to survive freezing at -79 °C in 10-20% glycerol for up to ten weeks. Other classical
studies on mammals are those of Lovelock and Bishop (1959) and Mazur (1963).
Lovelock and Bishop ( 1959) investigated the effectiveness of cryoprotectants in modu-
lating the rise in salt concentration during freezing, and Mazur (1963) focused on the
effect of cooling rate and the likelihood of intracellular freezing. Cryopreservation
studies on plant material started in the 1960's when Sakai (1960, 1965) investigated the
cryotolerance of twigs of woody plants. The woody plants investigated for cryopreser-
vation studies in the 1970's and 1980's were the in vivo and in vitro material of fruit
trees, such as Malus spp (Sakai & Nishiyama 1978, Katano et al. 1983, Kuo & Lineber-
ger 1985, Tyler & Stushnoff 1988) and Citrus (Marin & Duran-Vila 1988), other woody
perennials such as Rubus spp. (Reed 1988) and Vaccinium spp. (Reed 1989), Coffea
arabica (Bertrand-Desbrunais et al. 1988), Morus bombycis (Yakuwa & Oka 1988) and
707
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants. Volume 6. 707-728.
© 2000 Kluwer Academic Publishers.
708
the moncotyledons, including Elaesis guinensis (Grout et al.1983) and Phoenix dactylif-
era (Tisserat et al. 1981 ). The first reports on cryopreservation of conifers were pub-
lished in the late 1980's, the target species being Picea abies, Pinus taeda (Gupta et al.
1987) and Picea glauca (Kartha et al. 1988). During the 1990's the number of target
species, both deciduous woody plants (for review see Bajaj 1995, Ryynlinen 1999) and
conifers, has increased rapidly, partly, due to the increased emphasis on cryopreserva-
tion technology in gene conservation, biodiversity, and in maintaining juvenility.

Cryopreservation is based on the reduction and subsequent standstill of metabolic ac-

tivities, as well as cell division in the explants, achieved usually at the ultra-low tem-
o
perature of liquid nitrogen (-196 C). The cryopreservation protocols of in vitro plant
material can be categorised in the classical protocols and the presently developed alter-
native ones. The classical procedures are based on slow-cooling and the new ones on
fast-cooling i.e. vitrification or the encapsulation-dehydration technique. The cryopre-
servation protocol is usually selected and optimized separately for each species and
explant type. In several cases, the use of'cryoprotectants is important due to their colli-
gative or antifreezing effects, i.e. reduction of extracellular ice formation. The cryo-
protectants used for plant material are mainly the same as those used for animal cells:
those permeating the plasmalemma, e.g. dimethylsulfoxide (DMSO) and glycerol, and
the non-permeating cryoprotectants. The non-permeating additives, most of which act as
osmotic agents, are sugars (e.g. sucrose and glucose), sugar alcohols (e.g. sorbitol) and
high molecular weight compounds such as polyethylene glycol (PEG) and polyvinyl-
pyrrolidone (PVP) (Chen & Kartha 1987). Cryoprotectants can also be used as a mix-
ture, because it is often more efficient than only one component at the same total osmo-
larity (Ulrich et al. 1979). Cryoprotectants are not always needed in the encapsulation-
dehydration technique, nor in the cryopreservation of in vivo buds.

In deciduous woody species, winter buds and actively growing meristematic tissue such
as in vitro shoot tips or apical meristems and somatic embryos have usually been used
as explants (for review see Bajaj 1995, Sakai 1995, Ryynlinen 1999). In conifers, the
explant material has almost solely originated from embryogenic cultures. In deciduous
species micropropagation has usually been performed through organogenesis and winter
buds, in vitro shoot tips, and apical meristems are common explants. In conifers, on the
other hand, the rate for micropropagation is in most cases low and the methods devel-
oped are primarily appropriate for research purposes. Somatic embryogenesis was first
reported in the middle of the 1980's in Picea abies (Chalupa 1985, Hakman et al. 1985)
and, since then, extensive progress has been achieved in this technique and it has proved
to be applicable for several gymnosperms (Jain et al. 1995, Jain et al. 1999). However,
the reports on somatic seedlings in field trials or in practical forestry are still limited to a
few Picea species, Pseudotsuga menziesii and Pinus radiata. Embryogenic cultures of
conifers are mainly established using immature or mature embryos. This means that
cryopreservation is needed for maintaining the juvenility and regenerability of cultures,
and for storing the individual genotypes during progeny testing in the field. In addition
to the above-mentioned purposes, cryopreservation technology could also be used for
long-term gene conservation of somatic embryos initiated from selected mature elite
trees in breeding programmes. As a matter of fact, the use of tissues from adult gymno-
sperms for somatic embryogenesis has recently been successful in Picea abies and
 709
Pinus pinaster (Piiques & Harvengt 1998), indicating that this may be possible for other
conifers in the future.

2. Cryopreservation protocols appropriate for the embryogenic cultures of coni-

fers

The classical cryopreservation protocols, reviewed by Withers (1985), for in vitro plant
material include choice of material, pre-treatment, freezing, storage in liquid nitrogen,
thawing and post-treatment, and further in vitro cultivation. Of the classical protocols,
the slow-cooling method has generally been used to cryopreservate conifer embryogenic
cultures. The alternative cryopreservation protocols, vitrification and encapsulation-
dehydration combined with cryopreservation, have so far not been applied to gymno-
sperms. The vitrification procedure, which is based on pre-treatment with high concen-
tration of cryoprotectants followed by very fast freezing, was initially developed for
nucellar cells of Citrus sinensis (Sakai & Kobayashi 1990, Sakai et al. 1990), and later
on also for the meristems of several woody dicotyledons. In this context only the slow-
cooling method and the. cryopreservation of encapsulation-dehydrated cells and tissues
will be covered in detail. The application of encapsulation and dehydration techniques
combined with cryopreservation for conifers might be relevant to artificial seed tech-
nology or to avoid the use of cryoprotectants known to be hazardous at higher concen-
trations.

2.1 THE SLOW-COOLING METHOD

2.1.1 Material
The recommended explant material for cryopreservation purposes has usually been
young and meristematic tissue (Engelmann 1991 ), because of small cells with a dense
cytoplasm and low water content. In conifers, embryogenic cultures consist of small
embryonal head cells and long, highly vacuolated suspensor cells with high water con-
tent. During cryopreservation, the suspensor cells usually die and only the embryonal
head cells remain alive. This phenomenon has been observed in several gymnosperm
species such as Picea abies (Galerne & Dereuddre 1988, Gupta et al. 1987, von Arnold
et al. 1995, Find et al. 1998), Picea sitchensis (Kristensen et al. 1994, Find et al. 1998),
Pinus taeda (Gupta et al. 1987), Pinus caribaea (Laine et al. 1992) and Pinus sylvestris
(Haggman et al. 1998).

The importance of the physiological stage of the material has also been emphasized. In
angiosperm cell cultures, growth state and cell density of suspension cultures are im-
portant factors for cell survival. The highest cryotolerance has been achieved when the
material has either been in the late lag phase or undergoing the exponential growth stage
(Sugawara & Sakai 1974, Withers & Street 1977, Withers 1985, Reinhoud et al. 1995).
There are few corresponding reports on conifers. Find et al. (1998) found that maximun
crytolerance in both Picea abies and Picea sitchensis occurred when cultures were har-
vested at the stationary growth phase in terms of dry weight. They suggested that these
differences may reflect the different proliferation patterns of angiosperm and gymno-
sperm suspension cultures; angiosperm cultures consist of single cells and cell aggre-
710
gates (Withers & Street 1977), whereas coniferous cultures consist of more organized
structures. In Pinus caribaea (Laine et a!. 1992) and Pinus radiata (Hargreaves & Smith
1992), it has been emphasised that for optimal recovery of viable cells the embryogenic
cell suspensions have to be vigorous.

In general, high percentage of genetically different cell lines survive cryopreservation

by slow-cooling protocol (Nmgaard eta!. 1993a, 1993b, Cyr eta!. 1994, Bercetche &
Paques 1995, Hliggman et a!. 1998, Park et a!. 1998, Aronen et a!. 1999). In Picea
glauca engelmanni complex, cryotolerance was not related to family ranking in height
increment (Cyr eta!. 1994), and in Picea abies significant family differences were nei-
ther associated with frost hardiness nor with the growth characteristics of the progeny
(Nmgaard et a!. 1993b, von Arnold eta!. 1995), indicating no directional selection in
these traits during cryopreservation.

2. 1. 2 Pre-treatment and cryoprotection

In conifer cryopreservation, more attention has been paid to optimizing the preculture
phase prior to freezing. In the preculture phase, the non-permeating cryoprotective agent
with an osmoregulating function has been either sucrose or sorbitol (Table 1). However,
there are no comparative reports on these two agents within individual species. Other-
wise, preculture with the cryoprotective agent has usually been performed in darkness
or in dim light. The temperature has been about the room temperature (20°C - 25 oq at
which in vitro culture takes place (Table 1), or at the same temperature as the preceding
longer adaptative cultivation period (Hliggman et a!. 1998, Aronen et al. 1999).

The cryoprotective substances are added to the last preculture medium containing the
osmoregulating cryoprotective agent shortly before freezing. The cryoprotectants or
cryoprotectant mixtures are added either dropwise over a short period of time as de-
scribed by Kartha et al. (1988), or explants are incubated in a series of cryoprotectant
solutions of increasing concentration as described by Gupta eta!. (1987). In most ofthe
published reports, 5% (v/v) permeating DMSO (dimethylsulfoxide) has been used as a
cryoprotective agent for conifers (Table 1). A mixture of cryoprotectants has mainly
been used for the in vitro material of woody shrubs (Reed & Yu 1995) and deciduous
trees such as· Pyrus spp. (Reed 1990) and Betula pendula Roth (Ryynlinen 1996). In
conifers, the applicability of PGD (mixture of polyethylene glycol, glucose, and
DMSO) has been investigated in Picea abies and Pinus taeda (Gupta et a!. 1987), as
well as in Pinus sylvestris (Hliggman et a!. 1998) and Abies cephalonica hybrids
(Aronen eta!. 1999). In all the cases in which DMSO treatment has been replaced by
PGD treatment, the recovery and/or growth of the cultures improved. Already in 1979,
Ulrich and co-workers pointed out that a mixture of cryoprotectants is often more effi-
cient than a single component at the same total osmolarity.

2. 1. 3 Freezing, thawing and post-treatment

After the cryoprotection phase, the samples in cryotubes are usually allowed to stand at
ooc for a period ranging from 30 - 120 minutes. Cryopreservation of conifer cultures is
performed following the two-step slow-cooling method. The samples are frozen slowly
in a programmed freezer at a defmed cooling rate (0.16-0.5 oc min- 1depending on the
case) until a terminal temperature of about - 40°C has been reached. The cooling rate
 711
per se is a form of compromise: slow enough to allow cells to dehydrate in order to
avoid intracellular ice formation, and fast enough to reduce any damage due to the in-
creased solute concentrations. After reaching the terminal temperature, the cryotubes
containing the samples are immediately immersed in liquid nitrogen. There are, how-
ever, some modifications of this cooling system. In Pinus radiata, the precultured mate-
rial is placed directly in a - 30°C freezer for two hours and then immersed in liquid
nitrogen (Hargreaves & Smith 1994).
0
The most common way to thaw the samples is fast thawing in a +37 C water bath. After
thawing, the cryoprotective solutions are drained from the cryotubes and the samples
are washed with a growth regulator-free tissue culture medium containing an osmo-
regulating cryoprotective agent. After the washing step the samples are transferred onto
solid proliferation medium containing the same osmoregulating cryoprotective agent.
This post-treatment stage usually lasts for 24 or 48 hours. The samples are finally trans-
ferred onto the normal proliferation medium.

2. 1. 4 In vitro cultivation after cryopreservation

The beginning of in vitro cultivation after cryopreservation is generally characterized by
a lag phase lasting for a few days to several weeks depending on the species, genotype,
explants etc. The length of the lag phase after cryopreservation in Picea abies has varied
from a few days in suspension cultures (Galeme & Dereuddre 1988) to several weeks in
some cell lines grown in a semi-solid medium (N0rgaard et al. 1993b). Both in Abies
nordmanniana (N0rgaard et al. 1993a) and Pinus sy/vestris (Hiiggman et al. 1998) the
lag-phase varied from 2 to 4 weeks. In Pinus radiata, on the other hand, the use ofnurse
cultures helped to reduce the lag-phase, especially in cell lines that had been stored in
liquid nitrogen for a couple of years (Hargreaves & Grace 1998).

Reports on the effects of cryostorage on the regrowth of embryogenic cultures of coni-

fers are somewhat contradictory. The regrowth of cryopreserved cell lines is lower, at
the same level, or higher than that of control cultures. Cryopreservation of Abies nord-
manniana decreased the growth rate of embryogenic cultures (N0rgaard et al. 1993a).
The regrowth of frozen cultures of Picea abies was, in some cases, not at the same level
as the unfrozen cultures during the first months after thawing (von Arnold eta/. 1995).
After prolonged culture, however, the difference appeared to disappear. In Picea
mariana (Klimaszewska 1995) and in Picea sitchensis (Find et al. 1993) the regrowth
was comparable to the non-frozen, cryoprotectant-treated samples, whereas in Pinus
radiata (as reviewed by Smith et al. 1994) and in Pinus sylvestris (Hiiggman et al. 1998)
the growth rate of cryopreserved tissue subsequent to thawing was superior to that of
serially propagated tissue. Other factors, such as the cooling rate and terminal freezing
temperature, concentration of osmoregulating agent (Find et al. 1993), and genotype
(N0rgaard et al. 1993a, 1993b, Hiiggman et al. 1998, Aronen et al. 1999), have also
been reported to have an influence on the regrowth of embryogenic cultures.
 -....]
TABLE I. Cryopreservation protocols for embryogenic cultures of gymnosperms. .......
N

Species Precul ture/time/ Cryoprotectants Time in Recovery and regeneration Genetic fidelity Reference
temperature, light during freezing cryostorag tested at
molecular level

Abies cephalonica

8 cell lines 0.2 M sucrose/ PGD 7 days Average growth rate was improved. yes Aronen eta!. 1999
24 hand No maturation experiments
0.4 M sucrose/
24 hi
5°C, dark

A. nordmanniana

5 cell lines 0.2 M sorbitol/ 0.4 M sorbitol 2h Average growth rate was decreased. no Nergaard et al. 1993a
24 hand and5%DMSO No maturation experiments
0.4 M sorbitol/
24 hi
24°C, dark

Pseudotsuga 0.2 M sorbitol/ 0.4 M sorbitol nm Highly significant yield improvement no Gupta et al. 1995
menziensii 24 hand and5%DMSO
0.4 M sorbitol/
24 h, dark

Piceaabies

nd PGD !Omin Average growth rate was decreased. no Gupta et al. 1987
Mature somatic embryos
 0.5 M sucrose/ 0.5 M sucrose nm Average growth rate was improved. no Galeme et al. 1992
18 hi and 5 %DMSO Plants
25°C, dark

81 cell lines 0.4 M sorbitol/ 0.4 M sorbitol 2h Average growth rate was decreased. no Norgaard et al. 1993b
48 hi and 5% DMSO Mature somatic embryos
20°C, dark

0.2 M sorbitol/ 0.4 M sorbitol I week Maximum regrowth with 50% (v/v) no Find et al. 1998
24 hand and 5 %DMSO sedimented cell volyme.
0.4 M sorbitol/ Mature somatic embryos
24 h, dark

P. glauca

I cell line 0.2 M sorbitol/ 0.4 M sorbitol I h, Maximal growth rate was 95 % no Kartha et al. 1988
24 hand and 5 %DMSO I year of that of the control.
0.4 M sorbitol/ Plants
24 h/
20°C, low light
intensity

P.glauca
engelmanni

357 cell lines 0.2 M sorbitol/ 0.4 M sorbitol I day- Mature somatic embryos yes Cyr et al. 1994
24 hand and 5 %DMSO 1 year
0.4 M sorbitol/
24 hi
20°C, low light
intensity

.....:)
_.
.....,
P. mariana -.....1
........
+:>.
0.4 M sorbitol/ 0.4 M sorbitol 30 min Fast recovery after a short no Klimaszewska et at.
24 hi and 5 %DMSO lag phase. 1992
25°C, continuous Plants.
dim light Field trials

P. sitchensis

0.3 M sorbitol/ 0.4 M sorbitol I week Fast recovery without any no Find et at. 1993
24 hand and 5% DMSO lag phase. Kristensen et al. 1994
0.4 M sorbitol/ Plants
24 hi
24°C, dark

0.2 M sorbitol/ 0.4 M sorbitol I week Mature somatic embryos no Find et al. 1998
24 hand and 5 %DMSO
0.4 M sorbitol!
24 hi
24°C, dark

Larix x eurolepis

0.4 M sorbitol/ 0.4 M sorbitol 30min Fast recovery after a short lag phase. no Klimaszewska ·et al.
24 h/ and 5 %DMSO Plants. 1992
25°C, Field trials established
continuous
dim light

Pinus caribaea

0.4 M sucrose/ 0.4 M sucrose 30 min Embryogenic potential no Laine et at. 1992
24 h/ and 5% DMSO 4 months similar to controls.
20°C, LD Plants
 P. sylvestris

9 cell lines 0.2 M sucrose/ PGD 24 h Average growth rate was improved. yes Haggman et al. 1998
24 hand No maturation experiments
0.4 M sucrose/
24 hi
5°C, dark

P. taeda

nd PGD 10 min Average growth rate was decreased. no Gupta et al. 1987
Mature somatic embryos

P. pinaster

0.5 M sucrose/ 0.5 M sucrose nm Lag phase less than 3 days. no Bercetche & Paques
24 h, dark and 5 %DMSO 95% of the cell lines regenerated 1995

P. radiata

0.4 M sorbitol/ 0.4 M sucrose 6 months, Nurse tissues reduced lag-phase. no Hargreaves & Smith
18-24 h, dark and 10% DMSO 4 years Plants 1992, 1994
Hargreaves & Grace
1998

DMSO = dimethylsufoxide; LD =long daylight photoperiod; nd =not done; nm =not mentioned; PGD =mixture of polyethylene glycol, glucose, and DMSO

-J
......
Vl
716

The effect of cryostorage on the embryogenic capacity of conifers has, in most cases,
improved or facilitated embryo maturation and further development into plants (Table
1). In Picea mariana and Larix x eurolepis, post-thaw cultures gave rise to mature so-
matic embryos and plants with normal morphology (Klimaszewska et al. 1992). In
Picea glauca (Kartha et al. 1988), and in Picea abies (Bercetche et al. 1990), the em-
bryogenic potential was similar to that of the unfrozen control. In Pinus taeda (Gupta et
al. 1987), on the other hand, somatic embryo production decreased after cryopreserva-
tion. This might be due to the use of an earlier and more injurious cryopreservation
protocol (Gupta et al. 1995). Galeme et al. (1992) have suggested that the beneficial
effect of cryogenic storage on embryogenic capacity is related to the elimination of non-
embryogenic cells from the cultures (i.e. a selection process) or it may also be due to
increased synchrony of development from embryo head cells.

2.2 THE ALTERNATIVE CRYOPRESERVATION METHOD BASED ON ENCAP-

SULATION-DEHYDRATION TECHNIQUE

The encapsulation-dehydration technique combined with cryopreservation, as reviewed

by Villalobos & Engelmann (1995), was initially developed for the apices of several
temperate species. In woody species, this technique has been successfully applied to the
fruit trees Malus domestica (Niino & Sakai 1992), Morus bombysis (Niino & Sakai
1992) and Pyrus sp. (Niino & Sakai 1992, Scottez et al. 1992), as well as Eucalyptus
gunnii (Poissonier et al. 1992), Vilis vinifera (Plessis et al. 1993), Juglans nigra x regia
hybrid (De Boucaud et al. 1994), and Salix hybrid (Blakesley et al. 1996). Fabre and
Dereuddre (1990) described the methodology for Solanum shoot tips, which included
the following steps: culture of apices to recover from the dissection stress, encapsulation
in alginate in order to form alginate beads, preculture of the encapsulated explants in a
medium containing sucrose (0.1-0.75 M) for 24 to 72 h, dehydration of the encapsulated
apices by exposing them to filtered dried air for 4 h, and then rapid or two-step free~ing.
We now know, however, that the preculture treatment and duration of the procedure
especially have to be determined for each species separately. According to Villalobos &
Engelmann (1995), the advantages of the technique are high survival rates, rapid recov-
ery, the callus phase in regrowth is usually omitted, the regrowth and freezing condi-
tions and manipulation of the encapsulated apices are relatively simple, and a program-
mable freezing device is not necessary if rapid freezing is used.

The encapsulation-dehydration technique combined with cryopreservation has, so far,

not been applied to gymnosperms. Artificial seed technology has been developed to
integrate the vegetative propagation of conifers for commercial production of clonal
propagules for reforestation use. The prerequisite for the application of this technology
is inexpensive production of large numbers of high-quality somatic embryos (Attree &
Fowke 1993). Development of the somatic embryogenesis of several gymnosperm spe-
cies is currently underway (Jain et al. 1995), but there are only few reports on encapsu-
lated somatic embryos of conifers. Encapsulation of somatic embryos is successful in
Pinus taeda (Gupta & Durzan 1987) and Picea abies (Fourre et al. 1991), the encapsu-
lated embryos retaining a high germination capacity in vitro. In Picea glauca engel-
mannii complex and Picea mariana, plants were established in the soil from encapsu-
lated somatic embryos (Lulsdorf et al. 1993). The main problems with hydrated gels are
 717
poor gas exchange and possible rehydration of previously desiccated somatic embryos
during storage, leading to a limited storage life (Attree & Fowke 1993). This is the rea-
son why alternative encapsulation methods have been studied. In Pinus radiata, syn-
chronous development of high-quality, bullet-stage somatic embryos in batch culture
has been achieved in bioreactors and, based on studies on the mobilisation of reserves
during the germination of zygotic embryos, an artificial megagametophyte has been
developed (Smith et a!. 1994). Desiccated somatic embryos of Picea glauca have been
encapsulated in non-hydrated, low-melting point compounds, polyethylene glycols and
dextrans, which allow somatic embryos to be desiccated under controlled conditions and
0
regeneration into plantlets after one year of storage at - 20 C (Attree & Fowke 1993,
Attree et a!. 1995).

In conclusion, the results of encapsulation and dehydration of somatic embryos of coni-

fers are promising and, hypothetically, it might be possible to combine these techniques
with cryopreservation for prolonged storage purposes. Aronen et a!. (1999) pointed out,
as stated below, that the use of DMSO as a cryoprotectant may cause genetical aberra-
tions in embryogenic cultures. The possibility to avoid the use of deleterious cryopro-
tectants makes encapsulation-dehydration, combined with cryopreservation, even more
attractive. Time will tell whether this technique does in fact represent an advance com-
pared with the slow cooling method for embryogenic cultures of conifers in the future.

3. Genetic fidelity

The last stage of cryopreservation protocols should be the confirmation of genetic fidel-
ity. Monitoring the genetic fidelity of cryopreserved material is particularly important in
the breeding of long-living conifers, since the effects of occasional mutations or genome
rearrangements may not be readily observed in young plants, but expressed substan-
tially at a later stage in mature trees. Due to the ultra-low temperature of liquid nitrogen,
it has been postulated that biological material, i.e. ge.rmplasm, could be stored and
maintained for an indefinite period of time with guaranteed genetic stability (Ashwood-
Smith & Friedmann 1979, Kartha 1985). However, only a few studies that include ge-
netic fidelity testing have been carried out. In fact, even the reports concerning the pos-
sible effects oflong-term storage of woody plant material are few.

In most cases, true-to-type observations have been based on morphological observa-

tions. According to N0rgaard et a!. (l993b), the culture morphology of Picea abies
remained unaffected by cryopreservation. The same was also true in Pinus sylvestris
(Haggman et a!. 1998) and in Abies cephalonica hybrids (Aronen et a!. 1999). In Pseu-
dotsuga menziesii, Gupta et a!. (1995) found that cryostored and non-stored somatic
seedlings had similar morphology and growth rate when established in soil. Pinus
caribea and Larix x eurolepis plants from cryostored and non-cryostored somatic em-
bryos appeared similar when established in the greenhouse (Laine et al. 1992) and nurs-
ery (Klimaszewska eta!. 1992), respectively. Embryogenic clones of Picea glauca have
been cryostored for 3 and 4 years in liquid nitrogen and, on the basis of the morphologi-
cal characters of in vitro development and ex vitro growth, there was a high degree of
stability especially in growth traits (Park et al. 1998). In this case, however, in vitro and
718
ex vitro comparisons were made using cryopreserved material, and thereby it is impos-
sible to conclude the possible effects of cryopreservation on genetic fidelity.

Genetic fidelity of cryopreserved cultures at the molecular level has been studied in
only a few cases (Table 1). In Picea glauca engelmanni complex, a DNA fmgerprinting
probe used for clonal identification demonstrated no evidence of somaclonal variation
caused by cryopreservation (Cyr et al. 1994). No variation was either found in Pinus
sylvestris when cryopreserved cultures were compared with unfrozen ones using ran-
dom amplified polymorphic DNAs (RAPD markers) (Hiiggman et al. 1998). In Abies
cephalonica hybrids, RAPD analysis showed considerable genetic variation in DMSO-
treated non-frozen samples (Aronen et al. 1999). Molecular markers are, however, not
necessarily able to detect rare point mutations or the presence of extra copies of either
some genes or whole chromosomes, which may also cause changes in genetic composi-
tions (Weising et al. 1995, Jain 1996, Jain et al. 1998). On the other hand, molecular
markers can be used to detect genetic changes that are not readily expressed as mor-
phological or physiological variations of the phenotype. Thus, molecular markers
should preferably be used together with other approaches, such as morphological and
cytological observations (Fourre et al. 1997).

Possible genetic variations after cryopreservation may be caused by several factors,

including those attributable to the use of cryoprotectants or the plant material itself. In
Abies cephalonica material, 16.8 % of the RAPD profiles indicat€d intraclonal genetic
changes in DMSO-treated, non-frozen samples, while background variation was found
in 1. 7% of the control amplifications and none in the cryostored samples treated with
DMSO (Fig. 1) (Aronen et al. 1999). It has been suggested that a 2-10 % solution of
DMSO in is involved in generating genetic or epigenetic changes not only in micro-
organisms and animal cells, but also in higher plants (Finkle et al. 1985). The mutagenic
potential of DMSO is most probably derived from several factors. DMSO interferes
with enzyme systems and alters 0 2 uptake, interacts directly with chromatin and nucleic
acids, inhibits DNA synthesis, alters the secondary structure of DNA and RNA, causes
scissions in DNA and alterations in folded genomes, decreases the mitotic index, and
causes mitotic irregularities (Hervas & Gimenez-Martin 1973, Ihrke & Kronstad 1975,
Bose & Basu 1977, Friend & Freedman 1978). Plant cells immersed in liquid nitrogen
are metabolically inactive, and in embryogenic cultures of conifers usually only em-
bryonal head cells are able to survive cryostorage. Thus, although cryostorage does not
remove the mutagenic potential of DMSO, it probably eliminates a high proportion of
the cells bearing genetic changes. On the basis of results obtained with Abies cephalo-
nica material (Aronen et al. 1999) and Pinus sylvestris (Hiiggman et al. 1998), the use of
DMSO as the sole cryoprotectant for coniferous cultures should probably be reconsid-
ered in favour of the PDG mixtures that retain good growth potential of the cultures,
and also seem to be less mutagenic.

One additional factor contributing to the genetic changes or genome rearrangements

reported in Abies cephalonica could be the type of explant used (Aronen et al. 1999).
The cultures derived from open-pollinated Abies cephalonica were, probably, inter-
specific hybrids. According to Lester (1973), interspecific F 1 hybrids of forest trees
often contain incompatibilities or genetic imbalances involving chromosomes, genes,
 719
cytoplasm, plastids, as well as embryo-endosperm relationships. Thus hybrid material
can be assumed to be more susceptible to external mutagenic effects than the genomes
of pure species. In a comparable study with embryogenic cultures of Pinus sylvestris
with and without cryostorage, no evidence of genetic changes due to cryoprotectant
treatments could be found (Haggman eta!. 1998).

Fig. I. An example of RAPD profiles of Abies cephalonica embryogenic cell line 2 generated with OPA-06
arbitrary I O-rner primer (Operon Technologies, CA). The embryogenic cultures were treated with different
cryoprotectants DMSO (10 %dropwise added dimethylsulfoxide), PGD I (dropwise added mixture of poly-
ethylene glycol , glucose, and DMSO), and PGD II (mixture of polyethylene glycol , glucose, and DMSO
added as a series of solutions with increasing concentrations) both with and without cryopreservation. The
amplification results of untreated controls are also presented. Lanes I and 2 represent DMSO-treated and
cryostored samples; lanes 3 and 4, DMSO-treated but non-frozen samples showing variable banding pattern;
lanes 5 and 6, PGD !-treated and cryostored samples; lanes 7 and 8, PGD !-treated but non-frozen samples;
lanes 9 and I 0, untreated control samples; lanes II and 12, PGD 11-treated and cryostored samples; lanes 13
and 14, PGD 11-treated but non-frozen samples; lane 15, I 00 bp ladder (Aronen et al. 1999).

The origin of embryogenic cultures, i.e. initiation characteristics may lead to genetic
mosaics already before cryopreservation. In Pinus species, the initiation of somatic
embryogenesis most often takes place when immature zygotic embryos have been used
as explants. In Pinus seeds, multiple zygotic embryos may be formed due to simple and
cleavage polyembryony (Sarvas 1964, Singh 1978), indicating the possibility that mul-
tiple embryos proliferate to form embryogenic tissue, i.e. genetic mosaics, also in the
initiation of somatic embryogenesis. In Pinus taeda, it was possible to initiate embryo-
720

genic tissue from more than one zygotic embryo of a seed, resulting in individual cell
lines with different genomes (Becwar et al. 1991). Recognizing the variation in growth
and regrowth patterns of Pinus genotypes (Haggman et al. 1998), it is possible that
unpredictable selection may occur during the cryoprescrvation of genetic mosaics for
instance due to different cryotolerance of the cell lines. In Picea species the initiation of
cell lines that are genetic mosaics is avoided because the initiation of somatic embryo-
genesis is successful from mature embryos and cotyledons.

4. Applications and future prospects

Cryopreservation of the embryogenic cultures of trees has generally been considered to

prevent the loss of embryogenic potential and somaclonal variation caused by the long-
term maintenance of actively growing embryogenic cultures, and of storing a high num-
ber of genotypes until the results of progeny testing become available. The maintenance
of embryogenic cultures is also time consuming, and cryopreservation removes the
necessity for serial transfer of the cell lines. Also, the risks of contamination increase
along with prolonged culture time.

Embryogenic cultures of conifers may maintain their growth potential and somatic em-
bryo production for several years, as in Picea glauca (Attree et al. 1989). In Picea
abies, cell lines may remain stable for years (Boulay et al. 1988, Mo et al. 1989) or lose
their embryogenic potential after 6 months (Hogberg et al. 1998). In Pinus species both
the growth and embryogenic potential may vary over the course of time or they may be
lost after some months of subculturing, as in Pinus radiata (Smith et al. 1994), Pinus
caribaea (David et al. 1995) and in Pinus taeda (Becwar & Pullman 1995). Thus the
storage of embryogenic cell lines in liquid nitrogen provides fresh material and a way to
avoid cell lines decline due to prolonged culture. Therefore, in P. radiata (Smith et al.
1994) and in Picea abies (Hogberg et al. 1998) it is recommended that several samples
of each cell line (genotype) should be cryopreserved as early as possible or within 6
months of initiation in order to secure a high-quality inoculum. In Pseudotsuga menzie-
sii, cryostorage has improved the number of mature cotyledonary embryos per ml of
culture plated, the germination percentage of the embryos and the survival of germi-
nated somatic embryo plants significantly after 3 months in the soil (Gupta et al. 1995).

Tissue culture per se is known to be a mutagenic procedure and it has been assumed that
all forms of mutational events that can occur in nature will also take place in tissue-
cultured cells (Phillips et al. 1990). There are still only a few reports on the genetic
fidelity of embryogenic cultures of conifers. The flow cytometry analysis indicated
similarity in DNA content of long-term embryogenic cultures and of somatic seedlings
of Picea abies (Mo & von Arnold 1991). Chromosome counting has, on the other hand,
revealed trisomy or polyploidy in embryogenic cultures in several species (Lelu 1988,
Salajova & Salaj 1992, Nkongolo & Klimazewska 1995, O'Brian et al. 1996, Fourre et
al. 1997). Somaclonal variation in embryonic cultures of Larix has been detected using
molecular markers (De Verno et al. 1994). There are, however, also cases such as Picea
engelmannii complex showing no variation by isozyme (Eastman et al. 1991) and
RAPD markers (Isabel et al. 1993). The above mentioned examples indicate that so-
 721

maclonal variation indeed occurs in conifer embryogenic cultures. Therefore, cryopre-

servation technology is expected to provide a means of evading possible somaclonal
variation caused by the long-term maintenance of actively growing embryogenic cul-
tures.

To satisfy the increasing demand of wood the forest productivity will have to be in-
creased. Somatic embryogenesis has potential for rapidly multiplying high value geno-
types for reforestation (Gupta et al. 1993). For the integration within operational for-
estry (Carson 1986), the somatic embryogenesis technology must not only be able to
multiply desirable individuals on a large-scale, but also preserve superior clones without
genetic change or loss of regeneration ability while the selection program is being car-
ried out. In this respect, the successful cryopreservation of embryogenic cultures is
highly desirable. The potential for integrating somatic embryogenesis in conifer breed-
ing programmes for transferring the genetic gains, achieved by breeding, into practical
silviculture, has been demonstrated in Pseudotsuga menziesii (Gupta et al. 1993), Pinus
radiata (Smith et al. 1994), Picea glauca engelmannii complex (Grossnickle et al.
1996), Picea glauca (Park et al. 1998) and in Picea abies (Gupta et al. 1993, Hogberg et
al. 1998). The essential role of cryopreservation as a link between breeding and subse-
quent mass propagation after clonal selection has also been emphasised. In several
countries, large-scale field testing is also required prior to approval of commercial pro-
duction of clonal reforestration material. For fast-growing conifers such as Pinus taeda
and Pinus radiata, it has been estimated that progeny tests of 6-1 0 years' duration are
required to evaluate the form and yield potential of individual clones (Smith et al. 1994,
Becwar & Pullman 1995). In slow-growing conifers such as Pinus sylvestris, the time
needed for reliable field-testing is estimated to be between 10 and 20 years (Mikola
1985, Wilhelmsson & Andersson 1993, Hannrup 1998). At present, however, the expe-
rience and reports on the effects of prolonged storage in liquid nitrogen as well as the
genetic fidelity testing of cryopreserved material are still limited. In addition, some
practical questions such as what is the number of half-sib and full-sib cell lines to be
cryopreserved to provide a representative sample of the family for breeding or mass
propagation purposes need to be answered.

In future, it may also be possible to cryostore for long-term gene conservation of elite
adult conifers. The importance of the evaluation of gene resources and gene conserva-
tion has been emphasised during the past few years. Strategies for the management and
conservation of the genetic resources have been created for several conifer species
(Libby 1990, Wilson 1990, Dvorak I 990, Anon. 1992). In the conventional in situ and
ex situ gene conservation methods, the germplasm is exposed to environmental effects,
natural selection, mutations etc., and the conservation of individual genotypes is not
possible. The advantages of cryopreservation as a gene conservation method include the
conservation of individual genotypes, maintenance of the juvenility of stored material,
and minimum space requirements. In some cases, cryopreservation has been calculated
to be cost-effective, and less labour is needed than in in vitro conservation by tissue
culture. On the other hand, cryopreservation is appropriate only for those species for
which effective tissue culture methods are available. Long-term gene conservation of
specific coniferous genotypes might become possible because the production of em-
bryogenic tissue from nonembryogenic explants of 2, 6 and 26-year-old Picea abies
722
trees has been successful (Westcott 1994). Recently, somatic embryo plants have also
been produced from mature gymnosperms, Picea abies and Pinus pinaster (Paques &
Harvengt 1998), indicating that this could also be possible for other conifers in the fu-
ture.

5. Conclusions

A considerable progress has been made in the development and optimisation of cryopre-
servation protocols for embryogenic cultures of conifers as a result of advances
achieved in somatic embryogenesis during recent years. The slow-cooling method has
been widely applied to cryopreserve conifer embryogenic cultures. The optimized pro-
tocols have turned out to be universally appropriate for several genotypes. Using cryo-
preservation, it is generally possible to maintain the embryogenic potential of cell lines,
avoid somaclonal variation connected to long-term tissue culture, store large numbers of
genotypes during progeny testing, and in the future it might also be possible to conserve
mature elite individuals for prolonged periods for gene conservation purposes. At pres-
ent, however, the experience and reports on the effects of prolonged storage in liquid
nitrogen are still limited, as well as the genetic fidelity testing of cryopreserved mate-
rial. Furthermore, field testing of cryopreserved material has been performed in few
species. The results achieved so far on the cryopreservation of embryogenic cultures of
conifers have been encouraging, but further information is needed on the field perform-
ance of cryopreserved material.

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26. CRYOPRESERV ATON OF EMBRYOGENIC CALLI OF Hevea brasiliellsis

Florent Engelmann 1 and Herve Etienne 2

I: lPGRI (International Plant Genetic Resources Institute), Via delle Sette Chiese 142,
00145 Rome, Italy
2: CA TIE/CIRAD-CP (Centro Agronomico Tropical de Investigacion y
Ensefienza/Centre de cooperation internationale en recherche agronomique pour le
developpement-Departement cultures perennes), Apartado II, 7170 Turrialba, Costa
Rica

Chapter Contents

1. Introduction

2. Methodology

3. Results and Discussion

3.1. CLASSICAL FREEZING PROTOCOL

3.2. SIMPLIFIED FREEZING PROTOCOL

4. Conclusion and future prospects

Acknowledgements

Table 1
Table 2
Table 3
Table 4
Table 5
Figure 1
Figure 1

References

729
S.M. Jain, P.K. Gupta and R.J. Newton (eds.), Somatic Embryogenesis in Woody Plants, Volume 6, 729-746.
© 2000 Kluwer Academic Publishers.
730
1. Introduction

Various problems are encountered in the establishment and maintenance of embryogenic

cultures. The production of embryogenic material is often a difficult and lengthy
process, as observed notably with oilpalm and coconut (Duval et al. 1995; Verdeil and
Buffard-Morel 1995). The progressive decrease of embryogenic potential over time is a
phenomenon which is commonly observed with embryogenic cultures (Withers 1985).
Moreover, risks of somaclonal variation increase in line with the extension of the culture
period (Reisch 1984). Finally, it is difficult and costly to maintain large numbers of
proliferating cultures in large-scale production laboratories. Tools which would allow to
overcome these difficulties are urgently needed to enhance the applicability of somatic
embryogenesis processes for the mass propagation of elite material.

Hevea brasiliensis Mull. Arg. is an arborescent species from the Euphorbiaceae family
originating from the Amazon basin, which represents the almost exclusive source of
natural rubber. It is indeed by large the highest producer of the nine species of the Hevea
genus. The culture zones of Hevea are comprised between 20°N and 20°8. This tropical
tree can be up to 40 m high and 5 m in circumference in the wild but the trees
encountered in plantations are much smaller. Hevea is allogamous and monoecious.

Rubber growing is relatively recent. Its expansion, at the beginning of the 20th century, is
directly linked with the increasing needs of industry. Its development was based on the
use of non-selected seeds. However, using seedlings introduces a very high
heterogeneity (Compagnon 1986). Moreover, selected materials display only a transitory
and insufficient rhizogenic capacity (Musik and Cruzado 1958), thus excluding the
possibility of multiplication through cuttings. It is in fact by using graftings instead of
seedlings that rubber growing could be significantly improved. However, the current
classical propagation method used for rubber trees, i.e. grafting onto unselected
seedlings, maintains intraclonal heterogeneity according to vigor and productivity; that
is why a great improvement may be expected by using micropropagation in vitro.
Micropropagation techniques appear to be more interesting for propagation and varietal
improvement of trees than herbaceous species since the improvement of most tree
species through conventional methods is very slow because of their very long life cycles
(Haissig 1989). With rubber tree, two techniques have been developed simultaneously,
propagation through microcuttings and somatic embryogenesis. Due to the difficulties
encountered with rooting of microcuttings produced from selected clones, the emphasis
is now on the development of somatic embryogenesis.

It is well established that the production of somatic embryos in liquid medium can be
used for the inexpensive mass production of improved varieties with an increasing
number of species (Gupta et al. 1991; Ducos et al. 1993; Etienne-Barry et al. 1999)
including rubber tree (Etienne et al. 1997b). Moreover, somatic embryogenesis allows to
envisage the production of synthetic seeds (Le Deunff 1993), the long-term storage of
 731
material through cryopreservation (Engelmann 1997), and represents also the ideal tool
for genetic transformation through the establishment of systems with high regeneration
frequency or of embryogenic suspensions (Vasil eta/. 1992). Finally, the regeneration of
somatic embryos and whole plants from plant tissues, which is based on the expression
of cellular totipotency is an ideal model which is widely used today for studying the
morphogenic and regulatory events of plant ontogenesis (Zimmerman 1993).

For all the above reasons, somatic embryogenesis has therefore been the subject of many
studies since the pioneering work of Bouychou ( 1953). Wang et a/. ( 1980) were the first
to report the production of plantlets coming from somatic embryos obtained from
anthers. The reproducible production of somatic embryos with various cultivars of
rubber tree was described more recently by El Hadrami et a/. ( 1991) and Etienne et a/.
( 1991 ). Owing to improvements in the protocol leading to the production of friable
emqryogenic calli, long-term embryogenic cultures of several commercial genotypes
could be established and routinely maintained on semi-solid medium (Montoro et a/.
1993, 1994; Etienne eta/. 1997a). Conditions for the production of somatic embryos and
plantlet regeneration from long-term embryogenic cultures using a temporary immersion
bioreactor have been described recently (Etienne eta/. 1997b).

However, the possibilities of large scale production are hampered by various problems
encountered with the establishment and maintenance of embryogenic cultures. It is
currently impossible to produce embryogenic material from all commercial genotypes:
until now, embryogenic cultures have been established from only three genotypes (PB
280, PB 260, PR I 07) out of the eighteen introduced in vitro (Carron et a/. 1998). Four
main problems are observed with embryogenic cultures: i) the production of
embryogenic material from the internal seed coat is a very tedious process, both in terms
of manipulations and of time requirement; ii) the establishment of a proliferating friable
callus is a highly infrequent event; iii) within the same genotype, different strains can
have different embryogenic potentials (Carron et a/. 1998); and iv) the risk of losing a
strain either through aging, i.e. the progressive decrease of its embryogenic potential or
through contamination is always present.

Cryopreservation, i.e. the storage of biological material at ultra-low temperature, usually

that of liquid nitrogen ( -196°C) represents an efficient solution to solve these problems.
At this temperature, all cellular divisions and metabolic processes are stopped. The plant
material can thus be stored without alteration or modification for a theoretically
unlimited period of time. Moreover, cultures are stored in a small volume, protected
from contamination, requiring a very limited maintenance.

Most of the experimental systems employed in cryopreservation (cell suspensions, calli,

shoot tips, embryos) contain high amounts of cellular water and are thus extremely
sensitive to freezing injury since most of them are not inherently freezing-tolerant. Cells
have thus to be dehydrated artificially to protect them from the damages caused by the
crystallization of intracellular water into ice (Mazur 1984). The techniques employed
and the physical mechanisms upon which they are based are different in classical and
732
new cryopreservation techniques (Withers and Engelmann 1997). Classical techniques
involve freeze-induced dehydration, whereas new techniques are based on vitrification.
Vitrification can be defined as the transition of water directly from the liquid phase into
an amorphous phase or glass, whilst avoiding the formation of crystalline ice (Fahy eta/.
1984).

Classical cryopreservation techniques involve slow cooling down to a defined

prefreezing temperature, followed by rapid immersion in liquid nitrogen. With
temperature reduction during slow cooling, the cells and the external medium initially
supercool, followed by ice formation in the medium (Mazur 1969). The cell membrane
acts as a physical barrier and prevents the ice from seeding the cell interior and the cells
remain unfrozen but supercooled. As the temperature is further decreased, an increasing
amount of the extracellular solution is converted into ice, thus resulting in the
concentration of extracellular solutes. Since cells remain supercooled and their aqueous
vapour pressure exceeds that of the frozen external compartment, cells equilibrate by
loss of water to external ice. In optimal conditions, most or all intracellular freezable
water is removed, thus reducing or avoiding detrimental intracellular ice formation upon
subsequent immersion of the specimen in liquid nitrogen. Rewarming should be as rapid
as possible to avoid the phenomenon of recrystallization in which ice melts and reforms
at a thermodynamically favorable, larger and more damaging crystal size (Meryman and
Williams 1985).

New techniques are vitrification-based procedures. In such procedures, the freezable

intracellular water is removed prior to freezing by exposing the samples to highly
concentrated cryoprotective media and/or air desiccation. Dehydration is followed in
most cases by rapid cooling. As a result, the internal solutes vitrify and deleterious
intracellular ice formation is avoided. Glass transitions (i.e. changes in the structural
conformation of the glass) during cooling and rewarming have been recorded with
various materials using thermal analysis (Sakai et a!. 1990; Dereuddre et a/. 1991;
Tannoury et al. 1991; Niino eta!. 1992).

Cryopreservation protocols are now available for cell suspensions, calli, apices, zygotic
and somatic embryos of several hundreds of species of temperate and tropical origin
(Kartha and Engelmann 1994; Engelmann 1997). The development of these protocols
has been mainly performed in the framework of academic studies and, in most instances,
only one or a few genotypes were employed. However, during recent years, there has
been an increasing number of cases involving large scale experimentation of
cryopreservation techniques. As a result, there are now various examples of routine use
of cryopreservation, in genebanks for the long-term storage of apices of crops such as
potato and apple (Schafer-Menuhr et a/. I 997; Forsline et a/. I 999) and in laboratories
for the conservation of cell lines with special attributes (Withers I 985), as well as for
that of embryogenic lines in the framework of large-scale clonal propagation
programmes (Cyr 1999; Florin eta!. 1999).
 733
Cryopreservation involves a series of stresses which might lead to modifications in the
recovered cultures and regenerated plants. It is therefore necessary to verify that the
genetic stability of the cryopreserved material is not altered before using this technique
routinely for the long-term storage of germ plasm. There is now increasing evidence that,
provided that the cryopreservation technique applied ensures to the extent possible the
maintenance of the integrity of the frozen specimen, no modification at the phenotypic,
biochemical, chromosomal or molecular level of plants regenerated from cryopreserved
material, which could attribute to cryopreservation has been reported (Engelmann 1997).

In the case of embryogenic cejl suspensions and calli, the protocols developed are
mainly classical freezing protocols (Mazur I 984). However, new cryopreservation
techniques, which are based on the vitrification of internal solutes have also been
employed: the technique termed vitrification has been successfully applied to cell
suspensions and calli of various species (Sakai I 993; Engelmann 1997) and the
encapsulation-dehydration technique to cell suspensions of Catharanthus roseus,
Papaver somniferum and Polygonum aviculare (Bachiri eta!. I 995; Gazeau eta!. I 998;
Swann et a!. I 998). Most classical freezing procedures require the use of expensive
programmable freezing devices which allow to achieve precise freezing conditions.
However, sophisticated apparatus are not always necessary to obtain high survival.
Withers and King (I 980) cryopreserved various cell suspensions with an improvised and
simple apparatus that can offer reproducible but non-linear slow cooling. Other authors
successfully employed domestic or laboratory deep-freezers to perform the slow cooling
step (Maddox eta/. 1982/83; Sakai eta!. 1991; Nishizawa eta!. I 992; Engelmann eta!.
I 994; Martinez-Montero et al. I 998).

The cryopreservation protocols established for embryogenic material are usually

efficient: under optimal conditions, little or no differences are observed in the survival
rates of control and cryopreserved material (Engelmann I 992; Dussert et a/. I 992), and
higher regrowth rates of cryopreserved samples have been observed in some instances,
due to the selection of meristematic cells during the cryopreservation process (Bercetche
eta!. I 990).

Until now, very limited research has been conducted on cryopreservation of Hevea.
Normah et a/. (1986) demonstrated that embryonic axes could withstand
cryopreservation following partial desiccation. More recently, Veisseire eta/. (1993)
succeeded in freezing embryogenic cell suspensions of one commercial clone. Their
study focused mainly on the effect ofpregrowth and preculture conditions on survival.

The work reported here aimed at developing a classical freezing protocol for
embryogenic calli of one Hevea commercial clone. A simplified freezing protocol was
then established with the same clone and tested with a second commercial cloHe.
734
2. Methodology

The material used for cryopreservation studies consisted of a friable embryogenic callus
of the commercial clone PB 260, which had been maintained in culture for 2 years.
Long-term cultured embryogenic callus of a second commercial clone, PR I 07, was
utilized to evaluate the efficiency of the simplified cryopreservation protocol. The
friable calli were obtained from the internal seed coat of immature Hevea seeds
(Montoro et al. 1993, 1994). Their culture conditions and the protocol for the
regeneration of somatic embryos have recently been established by Etienne et al.
(1997a, b).

For cryopreservation experiments, fragments of embryogenic calli (± 300 mg fresh

weight) were sampled from cultures 12 days after the last transfer on proliferation
medium and placed into 2 ml sterile polypropylene cryotubes. They were precultured for
I h at ooc in I ml cryoprotective medium containing 0.25 to 1.25 M sucrose and 0 to
15% dimethylsulfoxide (DMSO). DMSO was added progressively over the first 30 min
of preculture until the final concentration was reached. The classical freezing protocol,
performed with a programmable freezer (Minicool LC 40, L'Air Liquide) comprised
first slow freezing at various cooling rates (0.2 to 2°C/min) down to various prefreezing
temperatures (-20 to -80°C), followed by direct immersion of the cryotubes in liquid
nitrogen (LN). For simplified freezing, the cryotubes were placed in a· simple freezing
device consisting of a plastic box filled with isopropanol (Nalgene©, "Mr Freeze"),
which was itself enclosed, or not, in a polystyrene box. The simple freezing device was
placed in a -80°C deep-freezer. Once the temperature of -40°C was reached (measured
with a thermocouple placed in a cryotube containing 1 ml cryoprotective solution),
cryotubes were immersed rapidly in liquid nitrogen (LN). Average cooling rates down to
-40°C were 0.2 and O.SOC/min with and without the polystyrene box, respectively. In
both protocols, crystallization in the cryoprotective medium was induced manually at a
temperature intermediate between the crystallization and the nucleation temperature of
the medium, by briefly pinching the cryotubes with forceps previously cooled in LN.
Thawing was performed by immersing the cryotubes in a water-bath maintained by
thermostat at 40°C until ice was completely melted.

For recovery, the contents of the cryotubes were poured on a filter paper placed in a
Petri dish containing solid proliferation medium with sucrose concentration equal to that
of the cryoprotective medium employed. After 1 h, the filter papers with callus
fragments were transferred onto new solid medium with an intermediate sucrose
concentration. After 24 h, the filter papers with callus fragments were transferred onto
standard proliferation medium in a Petri Dish for recovery. After 15 days, calli were
assessed for regrowth and transferred into test tubes on the same medium for further
growth. Viability was measured immediately after thawing by using staining with
fluorescein diacetate (FDA, Widhalm 1972), by calculating the mean percentage of
living cells on a total of 100 cell aggregates (20 cell aggregates chosen randomly on 5
 735
plates observed with a microscope) according to the method of Dussert et a!. ( 1991 ). All
viability percentages were expressed as percentage of the control value.

3. Results and Discussion

3.1. CLASSICAL FREEZING PROTOCOL

During experiments aiming at determining the optimal cryoprotective medium, the

viability and regrowth of preculture controls were generally high for all cryoprotectant
combinations tested (Table I). Embryogenic calli of Hevea tolerated exposure to
relatively higher concentrations of cryoprotectants than calli of some other species such
as grape, sugarcane and Citrus (Eksomtramage et a!. 1992; Dussert et a!. 1992;
Engelmann et at. 1994). Prefreezing down to -40°C induced a drastic drop in viability
and regrowth intensity. Regrowth was obtained only when DMSO was present in the
cryoprotectant .medium. This result underlines the higher efficiency of binary
cryoprotective solutions in comparison with single cryoprotectants and the importance
of incorporating DMSO in a cryoprotective solution (Withers 1985). This might be due
to the properties of DMSO which penetrates cells very rapidly and allows penetration of
the other cryoprotectants (Stanley and Herschler 1986), thus improving survival by
increasing the concentration of intracellular solutes. Optimal viability and regrowth were
obtained for different conditions: the highest viability percentages were noted with
mixtures of0.75 to 1.25M sucrose and 0 to 10% DMSO, whereas regrowth was optimal
with 0.25, 0.75 and l.OM sucrose and 10 to 15% DMSO. This illustrates the fact that
viability data give only gross indications of the success of an experiment and that the
selection of optimal conditions should be based on the regrowth data only, as observed
with other materials such as banana and grape embryogenic suspensions (Panis et a!.
1990; Dussert eta!. 1991 ). After cryopreservation, a further slight drop in viability was
generally noted. Viability rates higher than 20% were noted with 1 and 1.25M sucrose
with 0 to 10% DMSO. The highest viability rate (49%) was obtained with I M sucrose
and 10% DMSO. Good regrowth (scale= 2+) of cryopreserved calli was obtained with
0.5-0.75 M sucrose and 10% DMSO, and IM sucrose and 15% DMSO. A combination
of I M sucrose and I 0% DMSO was selected as standard preculture medium for further
experiments.

Viability and regrowth of prefrozen controls decreased progressively in line with

decreasing prefreezing temperatures for all cooling rates experimented (Table 2). After
cryopreservation, no or low viability only and no regrowth were obtained for a cooling
rate of 2°C/min. Survival was noted for prefreezing temperatures between -30 to -80°C
with cooling rates of 0.2, 0.5 and 1°C/min. However, regrowth was achieved for a
narrower range of prefreezing temperatures, from -30 to -4SOC with 0.2°C/min, -30 to -
60°C with 0.5°C/min, and -40 and -4SOC only with 1.0°C/min. The highest regrowth
intensity (scale = 2+) was obtained for calli frozen at 0.5°C/min to -40°C. These
conditions have also been successful for the cryopreservation of numerous embryogenic
736
cell suspensions and calli (Withers 1985; Kartha and Engelmann 1994). Hevea
embryogenic calli were amenable to cryopreservation using a relatively wide range of
freezing parameters, like other materials such as sugarcane calli or oilpalm embryonic
cultures (Engelmann and Dereuddre 1988; Eksomtramage et a!. 1992). whereas very
precise conditions are requested for other materials such as grape cell suspensions
( Dussert et a!. I 991 ).

The regrowth rates of control and cryopreserved calli during the 3'd subculture on
proliferation medium were comparable, as was the production of somatic embryos
(Table 3). A tendency to obtain higher numbers of somatic embryos from cryopreserved
calli than from controls was noted consistently during this experiment and confirmed
during other experiments (data not shown). The observation that larger numbers of
somatic embryos seemed to be regenerated from cryopreserved calli than from control
calli might be due to the fact that non-embryogenic cells are preferentially destroyed
during the freeze-thaw cycle, thus leading to a selection of embryogenic material.
Similar observations have been reported notably with Picea abies and grape
embryogenic calli (Bercetche et a!. 1990; Dussert eta!. 1992). These authors suggested
that cryopreservation could be used as a tool to "rejuvenate" embryogenic calli and
suspensions when their proliferation potential starts to decrease after extended culture
periods.

3.2. SIMPLIFIED FREEZING PROTOCOL

When comparing the results obtained with classical and simplified protocols, a
progressive decrease in viability in line with decreasing prefreezing temperatures was
noted under all experimental conditions (Table 4). Slightly higher viability percentages
were obtained with both simplified protocols in comparison with equivalent freezing
rates performed with a programmable freezer. After cryopreservation, an important drop
in viability occurred for a freezing rate of O.SOC/min with both protocols, whereas the
decrease in viability was limited for a freezing rate of 0.2°C/min. The highest viability
was obtained with both protocols for prefreezing temperatures of -40 and -45"C.
Regrowth was achieved under all conditions experimented, but was higher for a cooling
rate of 0.2°C/min with both protocols (data not shown). With materials for which a
comparison of classical and simplified freezing protocols has been performed,
conditions could be determined which allowed not to modify the standard cryoprotective
treatment and to achieve viability and regrowth levels comparable to those obtained
using a classical freezing protocol (Kobayashi and Sakai 1993; Engelmann eta!. 1994).

Good survival was obtained with both clones cryopreserved using the simplified
freezing protocol (Table 5). A difference was noted between both clones as regards the
number of calli regrowing after freezing, since the clone displaying higher survival (PB
260) had less calli regrowing after freezing. This might be due to differences in the
reactivity in vitro of these two clones, as observed notably with sugarcane embryogenic
calli (Eksomtramage eta!. 1992). Growth recovery of cryopreserved calli of both clones
was rapid since no differences in regrowth intensity in comparison with controls was
 737
noted from the 3'd subculture onwards. Somatic embryos were regenerated from
cryopreserved calli of both clones (data not shown) (Figs. I and 2).

4. Conclusion and future prospects

The present work allowed the establishment of two efficient cryopreservation protocols
for embryogenic calli of Hevea using either a classical or a simplified freezing process.
Under optimal conditions, survival was high, regrowth of cryopreserved cultures was
rapid since no difference in regrowth intensity was noted in comparison with controls
from the 3'd subculture on proliferation medium onwards. Somatic embryos could be
regenerated in equivalent numbers from cryopreserved and control cultures.

Additional experiments should be performed to test the cryopreservation protocols

developed with other clones of commercial importance and to follow the development of
regenerated somatic embryos further than the cotyledonary stage. Cryopreservation
protocols developed in this study will facilitate the management of embryogenic cultures
of commercial clones of Hevea in research and production laboratories.

Acknowledgements

The authors gratefully acknowledge the excellent technical assistance provided by

Nathalie Chabrillange (Institut de recherche pour !e developpement (IRD), Montpellier,
France) and Marc Lartaud (CIRAD-CP, Montpellier, France) during the
cryopreservation experiments.
738
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 743
Table 1: Effect of sucrose and Dl'viSO concentration in the preculture medium on the viability (in%) and
regrowth intensity (scaled- to H+) of control. pretrozen and cryopreserved embryogenic calli ofhevea clone
PB 260. Embryogenic calli were frozen at 0.5'C/min down to -40°C before immersion in liquid nitrogen.
Results are the average of three independent experiments. (Adapted tram Engelmann et a/. 1997. with
permission).

Sucrose (M)
DMSO 0.25 0.50 0.75 1.00 1.25
(%)
0 97 85 89 94 91
++ +++ +++ ++ +
Control 77 58 71 83 62
+++ + +++ +++ ++
10 72 42 63 84 67
++ +++ +++ +++ ++
15 2 7 54 69 68
++ ++ +++ +++ ++
0 0 3 II 32 30
+
Prcfrozen IS 37 52 43 69
+ + + +
10 5 4 21 24 19
++ + ++ ++ +
15 4 13 6
++ + ++ ++ +
0 0 2 9 21 23

Cryopreserved 17 17 41 24
+ + + +
10 2 19 49 24
+ ++ ++ ++ +
15 0 0 8 8 8
+ ++ +
744
Table 2: Effect of freezing rate and prcfreezing temperature on the viability (in % of unfrozen control) and
regrowth intensity (scaled- to+++) ofpretrozen and cryopreserved embryogenic calli othevea clone PB 260
after 15 days of recovery. Embryogenic calli were precultured with !.OM sucrose and 10% DMSO. Results are
the average of three independent experiments. (Adapted !rom Engelmann eta/. 1997, with permission).

Pretreezing temperature (0 C)
Freezing rate -20 -30 -35 -40 -45 -50 -60 -80
(°C/min)
Pre Frozen 0.2 65 46 65 57 47 27 25 20
++ +++ + ++ ++ ++ +
0.5 70 81 76 49 59 18 3 2
+++ +++ ++ ++ + +
1.0 67 81 68 69 39 37 9 22
+ +++ +++ + + + +
2.0 89 92 90 75 82 50 66 47
++ ++ ++ ++ ++ ++ + +
Cryopreserved 0.2· 0 41 63 59 41 25 18 6
+ + + +
0.5 0 II 45 40 47 38 17 13
+ ++ + + +
1.0 0 3 12 35 46 22 10
+ +
2.0 0 0 0 2 I 12 38
 745
Table 3: Regrowth rate during the 3'd subculture on proliferation medium and production of somatic embryos
(No. of cotyledonary embryos produced per g fresh weight) from control and cryopreserved embryogenic calli
of hevea clone PB 260. Embryogenic calli were precultured with 10% DMSO and IM sucrose, and frozen at
0.5°C/min down to -40°C before immersion in liquid nitrogen. Values presented are means ± standard error
from data from 6 replicate·s. (From Engelmann eta/. I997, with permission).

Regrowth rate Somatic embryos

Control 4.5 ± 1.1 202 ±57
Cryopreserved 3.87 ± 2.2 250 ± 60

Table 4: Effect of freezing rate, prefreezing temperature and freezing procedure on the viability of pre!rozen
and cryopreserved calli of hevea clone PB 260. Freezing was performed using a programmable freezer
(cooling rates of 0.2 and 0.5°C/min), a simple freezing device placed in a polystyrene box (N I: average
cooling rate 0.2 °C/min) and a simple freezing device (N2: average cooling rate 0.5 °C/min). Results are the
average of three independent experiments. (Adapted from Engelmann eta/. 1997, with permission).

Prct)'eezing temperature (0 C)
Freezing rate -35 -40 -45
(°C/min)
Pre frozen 0.2 65 6I 36
0.5 61 57 12
N I (±0.2) 80 68 58
N2 (±0.5) 82 66 28
Cryopreserved 0.2 42 51 55
0.5 40 36 I6
Nl (±0.2) 28 54 48
N2 (±0.5) 2 21 9

Table 5: Viability (in %) of prefrozcn (prctr.) and cryopreserved (cryo.) calli, percentage of control and
cryopreserved calli showing regrowth at the end of the first subculture on proliferation medium, and regrowth
intensity (scaled 0-3) of control and cryopreservcd calli during the first (S I) and third (S3) subculture on
proliferation medium of two commercial hevea clones cryopreserved using a simplified freezing protocol
(simple freezing device placed in a polystyrene box; average cooling rate of0.2°C/min down to -40°C). Results
arc the average of three independent experiments. (From Engelmann eta/. I997, with permission).

Viability(%) Regrowing Regrowth Regrowth

calli(%) Intensity (S I) intensity (53)
pretr. cryo. control cryo. control cryo. Control cryo.
PB260 50±14 40±6 100 40 3 I 3 3
PRI07 80±12 30±12 100 60-70 3 3 3
746

Figure /· Regrowth of embryogenic callus of Hevea clone PB 260 one month after cryopreservation using a
controlled freezing protocol.

Figure 2: Production of somatic embryos trom embryogenic callus of Hevea clone PB 260 at the end of the 3'd
subculture on proliferation medium.