PROJECT REPORT

STUDIES ON EMBRYO RESCUE IN GRAPE

SUBMITTED BY
AJAY KUMAR
Roll No.: 6199002

SUBMITTED TO
DEPARTMENT OF BIOTECHNOLOGY
MEERUT COLLEGE, MEERUT
CCS MEERUT UNIVERSITY, MEERUT

CONTENTS
S. No.

Title

INTRODUCTION

REVIEWS OF LITERATURE

MATERIALS AND METHODS

EXPERIMENTAL RESULTS

DISCUSSION

CONCLUSION

REFERENCES

Page No.

I. INTRODUCTION
In the case of plants, the embryo is a multicellular structure endowed with potentiality to form a
new plant. It represents the beginning of a new sporophytic generation. The embryo may arise from a
specialized cell, i.e., zygote or from a somatic cell. The former is known as a zygotic embryo whereas
the latter is termed a somatic, adventive or asexual embryo. An embryo is essentially a bipolar
structure with root and shoots primordial structures.
Normally the embryos, miniature structures though they may be, develop into whole organisms, as
small as low lying grasses and as giant as Sequoia species. This vast potentiality of an embryo makes it
a very special structure. Although under normal and congenial conditions, young embryos attain
maturity and unfold their potential, there are certain situations, in which their subsequent development
is arrested at an early stage. This becomes a point of consideration when the embryos are of desirable
genotypes. In such cases, the embryos (or proembryos as the case may be) can be rescued from being
aborted by culturing them under suitable conditions.
Somatic embryogenesis in grapes and its potential application to biotechnology are well
recognized (Stamp and Meredith 1988; Mullins 1990; Gray 1992; Gray and Meredith 1992). These
researchers have emphasized the importance of inserting genes for viticulturally-significant traits into
genomes of economically important cultivars without changing their other characteristics. Others have
also demonstrated the application of gene insertion into embryos of many plants (McGranahan et al.
1988; Mullins et al. 1990; Scorza et al. 1990; Mante et al. 1991; Christou et al. 1991; Finer and
McMullen 1991; Machado et al. 1992). An additional strategy in grape would be to incorporate
beneficial genes into somatic embryos of zygotic origin, which could be used to enhance gerrnplasm
used in a breeding program. The success of this technology is dependent upon efficient in vitro plant
regeneration. Established transgenic plants could then be used as parents to transmit genes by
traditional or non-traditional breeding methods. This concept could be applied to germplasm used for
both scion and rootstock development.
In ovulo embryo rescue, used at the USDA's grape breeding program in Fresno, CA, now permits
the use of stenospermic seedless grapes as females (Emershad and Ramming 1982; Ramming and
Emershad 1982; Cain et al. 1983; Emershad and Ramming 1984; Emershad et al. 1989). Pollination
and fertilization occur in Stenospermic grapes with only partial seed development. Immature zygotic
embryos abort or become arrested in evelopment. Prior to abortion, immature seed traces are cultured
in vitro allowing the immature zygotic embryos to enlarge and develop into plants. This technology
has accelerated the development of seedless table and raisin grape germplasm (Vitis vinifera L.) by
significantly increasing the percentage of seedless progeny from 10-35% (Weinberger and Harmon
1964; Loomis and Weinberger 1979) to 40- 80% and by reducing the number of breeding generations
by half (Ramming 1990). Embryo rescue eliminates the need for seeded females as a bridge between
the genetic combinations of two seedless parents. This reduces the number of generations needed to
hybridize two seedless grapes from two to one. The zygotic origin of in-ovulo cultured embryos has
been established. Durham et al. (1989) and Chaparro et al. (1989) were able to show, by isozyme
analysis, the zygotic origin of progeny from Florida hybrid bunch grapes and Vitis vinifera x
Muscadinia rotundifolia crosses, respectively. Ramming et al. (1991), in addition to using isozyme
patterns, verified the zygotic origin of hybrids from 'Thompson Seedless' in field evaluations by using
phenotypic markers such as fruit colour, flavour and seed size.
A secondary benefit of in-ovulo embryo rescue in grape is the formation of somatic embryos from
immature zygotic embryos which could be used in the germplasm enhancement strategy proposed
above. In this report, we describe somatic embryogenesis and plant regeneration from immature
zygotic embryos of stenospermocarpic seedless grapes.

II. REVIEW OF LITERATURE

1. Historical background
Probably the earliest record of embryo culture dates back to the work of Charles Bonnet in the
18th century, who excised embryos of Phaseolus and Fagopyrum and planted them in nutrient
solutions for rearing embryos were used around 1890 by Brown & Morris. More systematic steps in
embryo culture, however, started from the beginning of this century when Hannig (1904) cultured
embryos of Cruciferae excised from a few taxa such as Raphanus spp. and Chochlearia damica in a
medium containing mineral salt and sugar under aseptic conditions and succeeded in obtaining plants.
This study opened up a field for basic investigations in embryology. The main emphasis was laid on
understanding physiological and developmental aspects and effects of growth substances. Brown
(1906) studied the relative efficiency of various organic nitrogen compounds on growth and
development of excised barley embryos cultured on a medium containing mineral salts and sugar.
These studies gave a fillip for further investigations, mainly on the role of different embryonal parts,
for their subsequent development (Dubard and Urbain, 1913; Buckner and Kastle, 1917; Andronescu,
1919). Based on the information thus gathered, Knudson (1922) succeeded in rearing orchid embryos
to whole plants on a nutrient agar medium containing sugar in the absence of symbiotic fungus. It was
observed that in the absence of sugar, the embryos failed to develop beyond the protocorm stage.
Identification of this specific role of sugar led to the postulation that symbiotic fungi degraded starch
into sugars. The success of today's commercial propagation of orchids can rightly be ascribed to this
study which incidentally forms an excellent example of direct application of very basic research.
Dieterich (1924) concluded from his study that embryos grown in vitro germinate readily without
a rest period and that nutritional requirement of mature and immature embryos, for the formation of
plants, are different in such a way that the latter require richer medium. He noted that immature
embryos less than one third of their mature size did not form normal plants whereas less than 0.5 mm
failed to develop at all in the medium which was otherwise adequate to support the growth of mature
embryos of several plant families, viz. Cruciferae, Poaceae and Leguminosae .
Raghavan (1976, 1980) states in his reviews on embryo culture that there are heterotrophic and
autotrophic stages in embryo development. The embryo in the heterotrophic stage of development is
smaller (termed as proembryo) than in the autotrophic stage and usually requires presence of growth
regulators to allow for its proper development (Raghavan & Srivastava, 1982). In the autotrophic
stage, development of an embryo does not depend on exogenous sources of growth regulators. The
embryos usually have to be rescued from being aborted when they show inherent nutritional deficiency
or if they are the result of distant hybridization. In such situations the embryos usually abort without
germination but can, in most cases, be rescued following a proper in vitro procedure.
Laibach (1925) emphasised the significance of rescuing embryos of interspecific hybrids. The
embryos usually have to be rescued otherwise they fail due to embryo abortion and/or endosperm
degeneration. These observations have been subsequently well confirmed by several workers (Skirm,
1942; Asano & Myodo, 1977; Asano, 1978; Van Tuyl et al., 1986, 1987, 1989; Quazi, 1988). Tukey's
(1933) experiments on cherry embryo culture were a milestone in embryo culture of fruit crops. His
medium and procedure are widely accepted and successfully applied to other crops such as cucurbits
(Whitaker & Davis, 1962). Blake (1939) was the first to use Tukey's procedure in the breeding of
peaches.

2. Technique
The plant embryos are rescued from getting aborted by excising them from the ovaries or ovules. In
some cases, however, it is not technically possible to take embryos out of the ovules. To rescue these
embryos, whole ovules or even ovaries containing them are cultured. Such a contingency is
exemplified by embryo rescue in tobacco (Reed & Collins, 1978; Nikova & Zagorska, 1990); cotton
(Stewart, 1981), Brassica spp. (Bajaj et al., 1986; Agnihotri et al., 1988, 1990a,1990b), Brassica x
Raphanus (Takeshita et al., 1980), ovary slice culture in Lilium (Hayashi et al., 1986; Knoh et al.,
1988), seedless table grapes (Cain et al., 1983; Bouquet & Davis, 1989) , ovary culture in the presence
of anthers in culture medium in Lilium (Van Tuyl et al., 1990a,1990b) and ovule culture in Helianthus
(Espinasse et al., 1991) and Vitis (Fernandez et al., 1991).
Mathias et al. (1990) have presented a new technique that circumvents very early embryo abortion
after interspecific crosses between Cuphea paucipetala ssp. greenwoodii x C. laminuligera.
Embryonic sectors of young fertilized ovules are cultivated in vitro on a solid/liquid double layer
culture medium containing activated charcoal in the solid phase. Hybrid plants are obtained after
transfer of the developing embryos to solid media. This technique may also be applicable to other taxa.
Recently, ovule perforation and vermiculite support systems have been found to be more effective than
media formulations in promoting in vitro growth of immature embryos (< 10 mm long) of early
ripening peach cultivars (Pinto et al., 1993, 1994). Usually the following methods have been used to
rescue the embryos which otherwise may abort:
a) In vivo transplantation
Sting! (1907) observed that embryos of several Poaceae species could be grown successfully
when transplanted on each other's endosperm. Pope (1944) has shown that by means of his vivipary
technique quite young (1 week old) Hordeum embryos can be induced to germinate in the ear and
develop into normal plants only 15 days after pollination. Thus an immature endosperm can provide
sufficient nutrients and induce immediate embryo germination. This technique, however, did not find
much application.
b) In vivo/ in vitro culture
The in vivo technique was later modified and hybrid embryos were reared using an in vivo/in vitro
transplantation technique. This technique was pioneered by Kruse (1973, 1974a, 1974b). He succeeded
in obtaining hybrid plants from several distant crosses such as Hordeum x Triticum, Hordeum x Secale
and Hordeum x Agropyron. The hybrid embryos were grown on barley endosperm under in vitro
conditions. The endosperms were obtained from 14-18 day-old barley caryopses by squeezing out the
endosperms and discarding the attached embryos. These endosperms were cultured in the medium and
hybrid embryos planted on to them in place of the original embryos. In general the hybrid embryos
were excised 9-12 days after pollination. If, however, very young embryos are planted on mature
endosperms, up to 10 embryos could be placed on a single endosperm. Kruse (1974b) showed that
placing more than one embryo was better for the recovery of hybrid plants than transplanting
individual embryos. The medium used was simple and its composition did not appear to be critical for
the success of obtaining hybrid plants. This technique enables rescue of very young embryos which
may not grow on artificial media. Following this technique De Lautour et al. (1978), Williams (1978)
and Williams et al. (1982) succeeded in obtaining hybrid plants in Trifolium spp. and Asano (1980) in
Lilium.
c) In vitro culture

The in vitro technique is most widely used as a powerful tool to rescue embryos even if they are
immature or lack endosperms. The success of raising plants from weak and immature embryos largely
depends upon their stage of maturity and composition of the medium. So far as the stage of their
development is concerned, even very immature embryos have been successfully rescued, using
complex media. The growing embryo is a dynamic system that shows changing demands during its
growth and development. The smaller the embryo, the more complex is the requisite medium.
White (1932) cultured young heart shaped embryos of Protulaca oleracea in a medium containing
mineral salts, glucose and fibrin digest and induced growth in them to near maturity with root
primordia and cotyledons. However, heart shaped embryos of 200-300 um could not be cultured even
on complex media. Maheshwari and Baldev (1962) cultivated young embryos of Cuscuta reflexa on a
medium containing IAA and casein hydrolysate. The embryos proliferated to form several embryos
and on transfer to fresh medium, each embryo formed normal shoots.
3. Culture requirements
a) Culture media
Besides mineral salts which are essential components, several growth promoting substances are
needed in the culture media. Some of them are as follows:
(i) Sugars: From the literature, it appears that sucrose is the most commonly used sugar to serve as a
carbon source and osmotic stabilizer. In the majority of the cases, sucrose has given better results than
other sugars. However, in a few comparative studies, other sugars have also given good results. For
example, Datura stramonium embryos grew well on 4 per cent sucrose, whereas those of D.
metaloides grew on sucrose as well as on glucose, mannose or glycerol. In general, higher
concentration of sucrose is used to culture immature embryos. This concentration is more important for
the osmolarity of the medium than for providing nutrition; as is evident from the observations of
Raghavan & Torrey(1964a) that Capsella embryos which required 12 per cent sucrose (Rijven, 1952),
could also grow equally well on a medium containing 2 per cent sucrose plus a few growth regulators.
Sucrose concentration used for embryo culture shows an enormously wide range, from 0.5% to as high
as 18%. Considering the high price of purified sucrose, lower concentration along with other growth
promoting substances should be preferred, or comparatively less pure brands of sucrose should be
tried. Immature embryos are known to require higher osmotic strength of the medium compared to the
relatively mature ones. This fact seems to have been substantiated by the work of several investigators
(Tukey, 1934, 1938; Lammerts, 1942; Kent & Brink, 1947; Ziebur, 1951; Ziebur & Brink, 1951). This
is expected as the amorphous liquid endosperm in which young embryos are nourished has high
molarity (Ryczkowski, 1960, 1961, 1969; Mauney, 1961, Smith, 1973). High osmotic concentration of
the medium prevents precocious germination of young embryos and supports normal embryonic
growth. This effect can be produced by sucrose (Norstog, 1956), casein hydrolysate (Ziebur & Brink,
1951), mannitol or glycerol (Rietsema et al., 1953), etc. Tukey (1933) found that 2 percent glucose in
the medium supported an accumulation of chlorophyll and enlargement of the cotyledons at a very
early developmental stage of the embryos. On the other hand, this concentration of glucose inhibited
growth of more developed embryos. However, 0.5 percent glucose was ideal for embryos of various
developmental stages. It is to be noted that as the culture period of young embryos advances, their
osmotic requirements decrease (Pecket & Selim, 1965; Niederwieser et al., 1990).
(ii) Natural adjuvants: In vivo/in vitro culture of embryos has demonstrated that endosperm factors
are supportive of embryo growth. It was shown long ago that mature embryos of Datura could be
grown on a simple medium supplemented with glucose, whereas torpedo and heart shaped embryos
survived on the medium fortified with glycine, thiamine, pyridoxine, ascorbic acid, adenine, nicotinic

acid, succinic acid and pantothenic acid. Still smaller embryos failed to germinate even on this
complex medium, but could be accomplished with the use of non-autoclaved coconut water (Van
Overbeek et al., 1942). Since then, coconut water has been used extensively in embryo culture,
although not always with favourable effects. For example, Uttaman (1949a, 1949b) observed growth
inhibition by coconut water in cultured maize embryos. The reverse was true for barley embryos
(Norstog, 1956). Like coconut water, which is liquid endosperm, most of the adjuvants in embryo
culture media are of endosperm origin. Although chemical composition of the endosperms of different
plant species varies, usually they contain myo-inositol, sorbitol, auxins, cytokinins, nitrogenous
compounds, etc . (Pollards et al., 1961; Nakajima, 1962). Besides coconut water, casein hydrolysate
(Ziebur & Brink, 1951), and malt extract (Blakeslee & Satina, 1944) have also been extensively used
in the embryo culture media. Cold sterilized malt extract is known to contain zeatin and zeatin riboside
fractions (Van Staden & Drewes, 1975). Casein hydrolysate supplies amino acids, while yeast extract
has been found to be beneficial due to its richness in vitamins.
(iii) Growth regulators: Although growth regulators, such as GA 3, auxins, cytokinins, etc. are
extensively used, their effects have been found to be quite inconsistent and at times, contradictory.
Since the effects of these substances are not nutritional, it is likely that osmotic concentration and the
action of these substances are linked in some way with cell permeability and uptake of ions. In general,
low concentrations of auxins have favoured normal growth whereas their higher concentrations either
proved inhibitory or favoured unorganized callus growth from the cultured embryos (Raghavan &
Srivastava, 1982). Effects of GA3 are more or less consistent in the enlargement of the embryos (Iyer
et al., 1959), whereas, cytokinins have usually resulted in growth inhibition (Raghavan & Torrey,
1964b; Kano, 1965). Pinfield & Stobart (1972), on the other hand, obtained stimulation of growth by
kinetin in cultured embryos of Acer. The effect of these substances was more pronounced at lower
temperature (8 to 10 C). Under similar conditions with 2 mg/l GA3 the growth of cotyledons and
shoots was stimulated. Zdrujkovskaja-Richter (1982) achieved in 2 weeks a tenfold enlargement of
hybrid embryos of early ripening cherries cultured at the heart shaped stage on MS (Murashige &
Skoog, 1962) medium containing 400 mg/1 casein hydrolysate, 200 mg/l yeast extract and 2 mg/I GA3
or 0.1 mg/1 kinetin.
b) Effect of temperature, pH and light
Although there are some reports on the effect of these environmental factors, it is not easy to
postulate a particular set of conditions for the embryos of a particular taxon. However, it is interesting
to note that by and large, embryos reflect the growth requirements of their parental plants. In general
embryos excised from plants of winter season crops, require lower temperature as compared to the
embryos excised from the plants of summer season crops. For example, optimum temperature for the
embryos of potato is 20C whereas for those of cotton it is 32C (Narayanaswami & Norstog, 1964).
The normal range of media pH, i.e. 5 to 6 is well tolerated by the embryos of several plant species.
Light during the first few days of culture is to be avoided as it may induce precocious germination.
However, high light intensity is reported to suppress such precocious germination in some cases
(Rijven, 1952).
4. Applications of embryo rescue
It would be pertinent to make a distinction, albeit arbitrary, between embryo culture and embryo
rescue. The former is a broader term and covers the basic research as well as applied aspects, such as
breaking seed dormancy, testing seed viability, shortening breeding cycle, etc. On the other hand, the
term embryo rescue is restricted to only those cases where the embryos, if not rescued are endangered
and would not form seedlings. This situation is very frequently encountered when the embryos are

inherently weak or they are the result of distant crosses. In some cases, endangered embryos may have
to be rescued by culturing whole ovules or even ovaries when the embryos are not amenable for
handling. Inspite of this, most frequently these two terms are used almost interchangeably.
a) Inherently weak embryos
In certain cases, seeds show very poor germination due to nutritional deficiencies and/or physiological
reasons. For example, in many fruit trees the early ripening types produce seeds which do not
germinate at all. In early ripening varieties of stone fruits and grapes, normal seed germination is
extremely low. Tukey (1933) observed that some varieties of sweet cherry ripen in less than 50 days of
anthesis but their seeds contain under-developed embryos which fail to germinate. However, when the
embryos were excised and cultured, normal plants could be obtained. Since then embryo culture has
been practised as a general method in horticultural crops including avocado (Skene & Barlass, 1983;
Torres et al., 1986), peach, nectarine and plum (Ramming, 1985), grapes (Gray et al., 1987; Tsolova,
1990), Pelargonium (Scemama & Raquin, 1990) and Ornithogalum (Niederwieser et al., 1990).
Perhaps the most interesting application of this technique involves hybridization between seedless
grape varieties (Ramming, 1990). In order to achieve a hybrid between seedless varieties with certain
desired characteristics using conventional breeding, a seeded variety must be used as the female
parent. Poor germination of seeds from early ripening grapes is probably due to poor embryo
development. Embryos have been rescued from early ripening varieties of peach (Tukey, 1934) and
Vitis (Ramming & Emershad, 1984), thus allowing their use as females. Many seedless grape varieties
are characterized by cessation of embryo development. Every grape breeder's dream has been to
interbreed stenospermocarpic seedless grapes. This step holds promise of eliminating the intermediate
seeded female and the second generation of intermating or back crossing. In addition, to saving one
generation approximately five years, land, labour, fertilizer and water required for raising one
generation are also saved. Fruit breeders have succeeded in rescuing embryos and growing them into
plants from stenospermocarpic seedless grapes (Emershad & Ramming, 1984; Spiegel-Roy et al.,
1985; Emershad et al., 1989; Gribaudo et al., 1993). Similarly, in Musa balbisiana, which is a relative
of commercial banana, seeds do not germinate in nature although plants can be obtained by embryo
culture (Cox et al., 1960). Embryo rescue in these cases offers a potential to increase the genetic
variability in otherwise vegetatively propagated plant species. In orchids, seeds are shed when their
embryos are only at the globular stage. Most of the prized varieties are difficult to propagate as their
seeds lack storage tissue and are just naked embryos without endosperms. Knudson (1922) succeeded
in germinating orchid embryos into plants on a simple medium. Thereafter, orchid propagation,
utilizing embryo culture became a commercial proposition. In fact this was one of the primary
biotechnological applications of in vitro culture and was later perfected by Morel (1960). Similarly,
weak embryos of other plants can also be rescued by culturing to obtain plants either directly or via
callus cultures (Pental & Grunckel, 1980).
b) Distant hybridization
The application of embryo culture technique was used by Laibach (1925) to recover hybrid plants from
a cross between Linum perenne x L. austriacum. Although the capsules from the crossed buds
developed normally, the seeds were shrunken and much lighter than the normal ones. These hybrid
seeds failed to germinate in soil. Excised embryos from these seeds germinated normally on cotton
soaked in sucrose solution. This successful rescue of embryos paved the way for a series of
applications of this technique to recover interspecific and intergeneric hybrids in a large number of
genera including Solanum (Jorgensen, 1928), Brassica (Nishi et al., 1959; Gossypium (Altman et al.,
1987), Populus (Thakur & Khosla, 1991; Raquin et al., 1993), and Lotus (O'Donoughue & Grant,
1988). Fruit breeders succeeded in obtaining distant hybrids in several horticultural species through

embryo culture. An increase in yield of Vitis vinifera x V. rotundifolia (Goldy et al., 1989) and seedless
progeny (Goldy et al., 1988; Ramming et al., 1991) has been achieved with the aid of embryo rescue.
Embryo rescue has been successfully used to produce hybrids involving intergeneric and interspecific
crosses in cereals. The important examples of inter-generic hybrids obtained via embryo rescue are
barley x rye (Fedak, 1986), wheat x barley (Koba et al., 1991), wheat x rye (Oettler, 1984; Kaltiskes &
Gustafson, 1986), oat x maize (Rines & Dahleen, 1990), and wheat x maize (Laurie et al., 1990).
Vernalization of isolated embryos has also been shown to reduce generation time by 40 days in wheat
(Sharma & Gill, 1982) and this saving can prove to be vital in breeding programmes.

III. MATERIALS AND METHODS

1. Materials
a) Plant material

The grapes embryos were collected for in vitro culture and growth studies were:
S.No.

Cross

No. of ovules

Code

Pearl of Csaba x Perlette

70

33

Pearl of Csaba x Hyb (BA x BE) x

BS

58

46

Pearl of Csaba x Pusa Urvashi

112

11

Pearl of Csaba x Pusa Navrang

138

24

Pearl of Csaba x Hyb. 70-56

60

48

Pearl of Csaba x Beauty Seadless

40

19

Perlette x Beauty Seedless

60

52

b) Chemicals
All the chemicals used for the preparation of basal media were procured from Hi-Media Laboratories
Pvt. Limited, Mumbai, India and vitamins and plant growth regulators from Sigma Chemicals Co., St.
Louis, USA.
c) Culture medium
Murashige and Skoogs medium (1962) is used in present project. The composition of this medium is
presented in Table 1.
Table 1: Composition of Murashige & Skoog (1962) medium.
S. No.
Chemical (1X)
Quantity (mg/l)
A.

MACRO

1.
2.
3.

NH4NO3
KNO3
MgSO4.7H2O

1,650
1,900
370

4.
B.

KH2PO4
CALCIUM

170

1.

CaCl2.2H2O

440

C.

MICRO- I

1.

H3 BO3

6.20

2.

MnSO4.4H2O

22.30

3.
4.
5.

ZnSO4.7H2O
Na2 MoO4.2H2O
KI

8.60
0.25
0.83

D.

MICRO -II

1.
2.

CuSO4.5H2O
CoCl2.6H2O

0.025
0.025

E.
1.

EDTA IRON-SODIUM
FeSO4.7H2O

27.8

2.
F.
1.
2.
3.
G.
1.
H.
I.
J
K
d) Glassware

Na2 EDTA.2H2O
ORGANIC-I
Nicotinic acid

37.3

Thiamine-HCl
Pyridoxine-HCl
ORGANIC II
Glycine
Agar-agar
Sucrose
Myo-inositol
Activated charcoal

0.1
0.5

0.5

2.0
8,000
30,000
100
150

The glassware, i.e., beakers, glass bottles with polypropylene lids, conical flasks, pipettes, test tubes,
etc. were of borosilicate glass from Borosil/Corning and petri plates, beakers, measuring cylinders, etc.
were of polypropylene which are autoclavable.
2. Methods
a) Cleaning of glassware
The glassware was steam sterilized(autoclaved) and then cleaned with detergent (Teepol/ Tween 20 @
0.1%) and thoroughly washed with running tap water, then rinsed with double-distilled water and
dried in a hot-air oven.
b) Preparation of stock solution
The stock solutions of various chemicals were prepared in autoclaved double distilled water and
stored in sterile reagent bottles in a refrigerator after filtering the contents. Benzylaminopurine (BAP),
Gibberellic acid (GA3), kinetin and Indole-3-butyric acid (IBA) were dissolved first in 1N KOH
solution and then, the required amount of sterile double distilled water was added to make the required
concentration of the stock solution. Component chemicals of M.S. medium were also stocked e.g.
Macro, Micro-I, Micro-II, Organic-I, Organic-II, Iron (Fe-EDTA), calcium chloride or readymade
M.S. media could also be used.
c) Preparation of culture media
Double-distilled water was used for the preparation of medium. The required amounts of macro- and
micronutrients, organic salts, vitamins, growth regulators and sucrose were added to the double
distilled water kept in a vessel. The final volume was made in a graduated cylinder/beaker by adding
double distilled water. The pH of the solution was adjusted to 5.78- 5.80 using either 1N HCl or 1N
KOH. For solidification of the medium, agar powder (Tissue culture grade/Bacteriological; agar-agar
type) @ 0.8% w/v was added to the luke warm solution and then, boiled for proper dissolving and
melting of agar powder.
d) Autoclaving
The prepared medium was then poured immediately into the 1L conical flasks; after plugging
(cotton plug), the vessels were covered with aluminium foil and then loaded for autoclaving at 121 0C
for about 27-28 min. at 21 psi.

The glassware, forceps, scalpels, cotton flakes (in glass jars), blotting papers, glass plates, double
distilled water in air tight bottles wrapped/covered with aluminium foil were carefully autoclaved at
1210C for 45 min. in a vertical autoclave device at 21 psi and kept in UV hood till inoculation.
e) Laminar
Before using the laminar it should be given UV for about 20 min. and then its surface is to be
sterilized with absolute ethanol using autoclaved cotton flakes before keeping the glass wares in it for
any kind of work.
After autoclaving the media was kept in the laminar for about 40 min. in UV for cooling and
sterilization along with the glass wares which were also autoclaved empty covered with cotton plugs
and ultimately with aluminium foil; for about 45 min.
f) Pouring
As the temperature of the media reaches to about 40-45 C the media is poured into the respective
glass wares for solidification of agar. Antibiotics could also be added to the media while pouring in the
laminar with the help of syringe and micro filter.
The antibiotics used were injections of streptomycin/ gentamycin. Before mixing the antibiotic
into the media the syringes as well as micro filter were rinsed twice in the autoclaved water. Syringes
and micro filters are to be used only once and discarded after using them.
g) Explant collection
Different fruit varieties of grapes were collected from the field in the early morning and brought to the
lab for inoculation in different bags having name tags or codes of the varieties collected.
h) Surface sterilization of explant
The collected fruits were washed in a solution containing 3-4 drops of liquid detergent teepol/teen 20.
Thereafter, the detergent was completely drained out from the fruits by 3-4 washings with vigorous
shaking by hand. The mouth of the flask was covered with a piece of cheese cloth and then, kept in
running tap water for 30 min. To remove the microbial load and dust particles adhering to the surface
of the explant.
For surface sterilization of berries; the berries were treated with carbendazim (Bavistin) (0.1%) + 8HQ (200 ppm) + Ridomil (0.1%) for 1/2 h followed by mercuric chloride (0.1%) for 4 min.
The fruits were then dried by keeping them onto the blotting paper for 5 min., and then cultured on MS
medium supplemented with 0.5 mg/l BAP, 1 mg/l NAA and 2 mg/l GA3.
i) Inoculation
Before inoculation, the laminar air-flow chamber was fumigated carefully by putting small amount of
KMnO4 and few drops of formaldehyde solution (35%) in a petriplate and closing the door
immediately for at least 12 h. The working table of laminar airflow chamber was wiped thoroughly
with ethanol before use.
The material required (i.e. forceps, scalpel, Petri plates, test tube, etc.) for inoculation was steam
sterilized and then flame sterilized (forceps, scalpel) by making their tips red hot. The hands were
cleaned with 70% ethanol. Then, the fruits were crushed to remove embryos by chopping its distal part
and making an incision vertical to the first incision, 2 nd incision shouldnt be deep, as it could harm the

embryos inside the fruit. Now the fruit is pressed onto the 2 nd incision with the help of the flat scalpel
and the embryos are squeezed out of the fruit.
Now the embryos are collected and implanted into the conical flasks having solidified MS medium,
with the help of forceps.in each conical 10-15 embryos are implanted.
j) Incubation of cultures
The cultures were incubated in culture room and provided with a photoperiod of 16/8 h light/dark at
252C temperature maintained by automatic photoperiod and temperature control devices.
(i) Culture Establishment (Stage-I)
The surface sterilized fruits embryos were cultured on MS medium supplemented with BAP (0.5
mg/l), IBA (1.0 mg/l) and GA3 (2.0 mg/l) under aseptic conditions and kept for incubation in the
culture room.
(ii) Shoot Proliferation (Stage-II)
The surface sterilized embryos were cultured on MS medium supplemented with BAP (0.5 mg/l), IBA
(1 mg/l) and GA3 (2 mg/l) under aseptic conditions and kept for incubation in the culture room.
(iii) Shoot Elongation (Stage-III)
MS medium supplemented with IBA (1 mg/l) and kinetin (1 mg/l) was used to elongate the microshoots.
(iv) Rhizogenesis (Stage-IV)
For obtaining successful and quicker root initiation, auxin, i.e., IBA (1 mg/l) is added to the MS
medium. A dose of sucrose, i.e., 30 g/l is also supplied to the rooting medium.
(v) Sub-culturing (Stage-V)
For the multiplication of the plantlets they are sub-cultured, for that we could make 5-6 plants
from one. The sub-cultured plants are now supplemented with IBA (1 mg/l) and Kinetin (1 mg/l) under
aseptic conditions by transferring them into the freshly prepared medium and kept for incubation in the
culture room.
(vi) Acclimatization (Stage-VI)
Aseptically, the microplants are transferred to autoclaved glass bottles with polypropylene screw lid
containing agropeat supplemented with half-strength of MS liquid medium devoid of calcium, organic,
growth regulators and sucrose components. The pH of the medium is adjusted to 5.7-5.8. The cultures
are kept in culture rooms for one week. The relative humidity inside the glass bottle is gradually
reduced by unscrewing and finally, removing the lid.
(vii) Transfer of plantlets to field conditions
The hardened plantlets are transferred to the earthen pots (12 size) filled with FYM: sand:
garden soil (2: 1/2: 1/2) and supplemented with one full tea spoon of bone meal and 20 g neem cake
per pot and kept under field conditions.
Observations recorded

The observations recorded are as follows:

Culture Establishment
(i) Percent survival: It was worked out by calculating the number of grapes embryos that germinated,
out of the total number of grapes embryos inoculated.
(ii) Duration required for germination of grapes embryos (days): The number of days taken for
sprouting of grapes embryos was recorded by visually observing the cultures, and counting the days
from the date of inoculation to date on which germination starts.
(iii) Contamination-free cultures (%): It was worked out by counting the infected cultures with
bacteria or fungus or both out of the total number of grapes embryos inoculated.
(iv) General growth: The visual observations were recorded on the basis of comparative growth of the
cultures such as stunted or normal.
Shoot Proliferation
(i) Average number of multiple shoots per grapes embryo: The number of multiple shoots formed
per grapes embryo after 20 days of transfer to proliferation medium was recorded.
(ii) Culture appearance: It was recorded by visually observing the colour and growth of cultures.
(iii) Visual observations: The visual observations were recorded on colour and comparative growth of
the culture.
Rhizogenesis
(i) Percent rooting: The number of micro-shoots rooted out of total number of micro-shoots cultured
on rooting medium was counted and rooting percentage was worked out.
(ii) Duration required for root initiation (days): It was calculated by noting the day on which the
first root appeared on the microshoots after they were placed in the rooting medium.
Acclimatization
Per cent survival: Percentage of plantlets survived after transfer to ambient conditions was worked out
by counting the plantlets that survived out of the total plants transferred for hardening after 20 days.

IV. EXPERIMENTAL FINDINGS

Experimental findings pertaining to the project entitled Studies on Embryo Rescue in Grape are
presented in this chapter.
Culture Establishment (Stage-I)

The embryos of the different varieties of grapes were subjected to various surface sterilization
treatments to establish the contamination free cultures. Data are presented in Table 2. Significant
differences were observed among the treatments tried.
Table 2 : Effect of various surface sterilization treatments on establishment of different varieties
of grapes cultured on MS medium supplemented with BAP (0.5 mg/l), IBA (1 mg/l) and GA3 (2
mg/l)
Sl. No. Treatment details
Surviva Germinatio Contamination Duration required
l
n
for
germination
(Days)
T-1
Mercuric
chloride
(HgCl2) (0.1%) for 3
min.
T-2
Carbendazim (0.2%) +
8-HQC (200 ppm) for
2 h followed by
mercuric
chloride
(0.1%) for 3 min.
(i) Survival percentage of grapes embryos:
The cultures infected with fungus and bacteria did not survive for long period or were unable to
germinate and died subsequently. On the other hand, those free from infection germinated and
considered as surviving cultures. Highest survival (93.48%) was recorded when the fruits were treated
with carbendazim (0.2%) + 8-HQ (200 ppm) + Ridomil (0.2%) for 1/ 2 h followed by dipping in
mercuric chloride (0.1%) for 4 min whereas, the grapes embryos exhibited more contamination rate
when treated with only mercuric chloride (0.1% for 3 min.) (Table 2).
(ii) Microbial contamination
The treatment with carbendazim (0.1%) + 8 HQ (200 ppm) + Ridomil (0.2%) for 1/ 2 h
followed by mercuric chloride (0.1%) for 4 min. reduced the microbial infection from ------------(Table 2).
(iii) Duration for germination
It is evident from table 2 that the grapes embryos cultured after treating with carbendazim
(0.1%) + 8 HQ (200 ppm) + Ridomil (0.2%) for 1/ 2 h followed by mercuric chloride (0.1%) for 4
min. (T-1) significantly reduced the duration required for germination (---------- days) as compared to
those treated with mercuric chloride (0.1%) for 3 min. (------- days).
2. Shoot Proliferation (Stage-II)
Sub-culturing of germinated grapes embryos on M.S. medium supplemented with various
concentrations of different growth regulators i.e. Kinetin and IBA indicated significant response in
respect of shoot proliferation and growth of cultures (Table 3).
Duration required for root initiation

Perusal of data presented in Table 5 reveals that earliest root initiation was recorded in the microshoots treated with 0.5 mg/l NAA (6.78 days) followed by 1.0 mg/l NAA (8.56 days) compared to
control days.

VI. SUMMARY
Somatic embryo formation occurred from immature zygotic embryos within ovules of
stenospermoearpic seedless grapes (Vitis vinifera L.), when cultured for two months on liquid
Emershad/Ramming medium. Somatic embryos continued to proliferate after excision and transfer to
Emershad/Ramming medium supplemented with 1M benzylaminopurine and 0.65% TC agar. Plant
development from somatic embryos was influenced by genotype, medium, phase (liquid, agar), stage
(torpedo, mature) and their interactions. Optimal plant development occurred on Woody Plant Medium
supplemented with 1.5 % sucrose + 1/M benzylaminopurine + 0.3% activated charcoal and 0.65%
TC agar.

VII.CONCLUSION
The survey of literature presented here focuses on the methods used to save embryos from
degeneration and to obtain viable plants. How many field trials have been made from the materials
thus raised, and how much plant material of horticultural and agricultural importance has actually been
generated through embryo rescue is difficult to assess. The embryo rescue technique is simple in use
and holds a vast potential in rescuing the desirable embryos which may otherwise degenerate. The
technique requires simple facilities, usually available in plant tissue culture laboratories. However,
under suboptimal conditions, there are some constraints which warrant consideration. For example, in
the case of annual plants, if embryo rescue is practised when the crop is nearing its end, the coming
season may not be congenial for the growth of the crop in question. Therefore, phytotrons may be
necessary if, the desired material raised through embryo/ovule culture is not to perish in the unfriendly
ensuing weather. On the other hand, it is easier to handle the plants of perennial species as the plants
can be kept in laboratories, transplanted at any time of the year and acclimatized till they become
sufficiently hardy to survive in nature or domestication. Consequently there has, therefore, been a
major emphasis on raising hybrid fruit and forest trees using this technique. There is an additional
advantage in these cases, as the elite material thus raised can be vegetatively multiplied by cuttings, or
through the shoot tip culture method. Taking into account the life span of the trees, these methods
would prove very economical in the long run.
A new dimension has been added to embryo culture by the production of somatic embryos in
tissue culture. Now it has become possible to obtain somatic embryos from cultured cells of a large
number of plant species, both annuals ( Bhojwani & Razdan, 1983), and perennials (Tisserat, 1979;
Sharma et al., 1984; Bhaskaran & Smith, 1992). Availability of vast literature on embryo culture bears
testimony to the fact that there is already a sizeable amount of basic information needed to realize the
gains of this area of biotechnology. It now should be a matter of fixing judicious priorities to achieve
genetic gains in economic plant species in near future.

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