DOUBLING WORLD

AGRICULTURAL PRODUCTION
BY ACHIEVING TWO CROPS
SIMULTANEOUSLY
WITH ONE SEED
CONCEPT

(https://mrsmatheson.wordpress.com/2012/11/30/higher-genetic-engineering-and-somatic-fusion/)

ABSTRACT
Todays Scenario is more or less like this.
1. Agricultural lands are used for industrial or mining or
for land filling of Solid Wastes or for dwelling thus
causing diminishing agricultural lands available.
2. Labour is becoming scarce and expensive
discouraging farmers to grow.
3. Wild Animals, Rats and untimely rains, hurricanes,
cyclones etc are damaging agricultural fields and thus
reducing total available yields.
4. Pests and insects are becoming more and more
pesticide resistant and reducing the yields.
5. Seasonal fluctuations in market prices sometimes
lead to heavy losses to the farmers leading to suicides
in many developing and under-delveloped countries
where agriculture is the main occupation.

6. Younger generations are attracted to more

remunerative occupations.
7. Over generations inherited lands got divided and
became smaller with lesser cultivable land after
volumes of the boundaries are increased.
In this context a need arises to solve all these problems.
A better proposition is to enable a farmer to use one
seed which can yield one crop above the ground and
simultaneously another crop under the ground like
POMATO which was designed to produce Tomatoes
above the ground and Potatoes under the ground;
thereby farmer will be having a chance to have a better
price atleast to one of them.
In this article efforts are made
1. to compile the available technologies
2. to better understand the protocols involved in
simpler terms
3. to use improvised versions of the methodologies to
augment sustained world food production in the
coming Years.
Years.

MISSION:
I am looking for associates who can involve with us in technology,
production and marketing on contract basis.

PROLOGUE

Food production must double by 2050 to meet the demand of the

worlds growing population and innovative strategies are needed to
help combat hunger, which already affects more than 1 billion people in
the world, several experts today told the Second Committee (Economic
and Financial) during a panel discussion on New cooperation for global
food security.
The 2008 food price crisis, the result of decades of insufficient
investment in agriculture and food security, swelled the ranks of the
poor and undernourished to 1 billion people, and, according to the Food
and Agriculture Organization (FAO), an extra 100 million people could
go hungry this year as a consequence of the combined negative effects

of the food price crisis, the financial and economic crisis, and climate
change.
Rajul Pandya-Lorch, Chief of Staff and Head of the 2020 Vision for Food,
Agriculture, and the Environment Initiative, International Food Policy
and Research Institute (IFPRI), said that, despite efforts to improve
food security by the international community, the number of people
going hungry has remained relatively constant over the last half
century, which forced the question: What do we need to do
differently?
On an inquiry about the tension between supporting smallholder
farmers and the need for substantial, larger-scale agricultural
investment to improve food security, he said it was an issue of
maximum efficiency, and that one way to encourage that could be
through support of cooperatives. Our main job now is to show that,
with the available resources, we can get good results during the next
few years, he said.
(http://www.un.org/press/en/2009/gaef3242.doc.htm)

HISTORY
The concept of grafting related potatoes and tomatoes so that
both are produced on the same plant was originally developed in
1977 at the Max Planck Institute for Developmental
Biology in Tbingen, Germany, and although healthy, the plant
produced neither potatoes nor tomatoes.[2] The Max Planck
Institute for Plant Breeding Research in Kln produced a plant
with fruit in 1994.[2]
As with all grafts, this plant will not occur in nature and cannot be
grown from seed, because the two parts of the plant remain
genetically separate, and only rely on each other for nourishment
and growth. Like most standard types of plant grafting, a small
incision is made in the stem of both plants and they are strapped
together. Once the cuts have healed and the plants are joined,
the leafy top of the potato plant can be cut away and the roots of
the tomato can be removed, leaving the leaves of the tomato
plant to nourish the roots of the potato plant.[3] The rootstock
(potato) acts as a stable and healthy root system and the scions
(tomato) are chosen for their fruit, flowers or leaves. The
tomatoes should be ready to harvest after about 12 weeks during
the summer months, the potatoes should be ready after the
tomato leaves begin to die back, normally in early autumn.
[4]
Grafting in this way can be used to produce many different
related crops from the same plant, for example the growing
popularity of 'Fruit salad' trees, which is a single tree that
produces multiple types of citrus fruits, or a tree with a variety of
fruits with stones (peach, plum etc.).

((http://en.wikipedia.org/wiki/Pomato)
In 1972, Carlson and his associates produced the first inter-specific
somatic hybrid between Nicotiana glauca and N. langsdorffii. In 1978,
Melchers and his co-workers developed the first inter-genetic somatic
hybrids between Solanum tuberosum (potato) and Lycopersicon
esculentum (tomato). The hybrids are known as Pomatoes or Topatoes
(http://www.biotechnology4u.com/plant_biotechnology_applications_cell_tissue_c
ulture.html)
Grafted pomato plants have been launched in the United Kingdom in
September 2013 by horticultural mail order company Thompson &
Morgan, who sold pre-grafted plants branded as the "TomTato".
The Incredible Ediblenursery in New Zealand announced a "DoubleUP
Potato Tom" in the same month
Grafting is a difficult process because the tomato and the potato stems
have to be the same thickness, and Thompson & Morgan trialled the
hybrid for several years before selling it. Production and grafting of
tomtatos begins in a specialist laboratory in the Netherlands, before
being shipped back to the UK and grown in greenhouses until they are
ready to be sold.

(http://en.wikipedia.org/wiki/Pomato)
In Florida, most orange trees have lemon roots. In California, nearly all
lemon trees are grown on orange roots. This sort of thing is not unique
with citrus. With the stone fruits, there is a certain latitude. Plums can
be grown on cherry trees and apricots on peach trees, but a one-to-one
relationship like that is only the beginning with citrus. A single citrus
tree can be turned into a carnival, with lemons, limes, grapefruit,
tangerines, kumquats and oranges all ripening on its branches at the
same timeMost of the trees on the Ridge [a mountain range in Florida
renowned for its orange tree orchards] are growing on Rough Lemon
As a rootstock, it forages with exceptional vigor and, in comparison
with others, puts more fruit on the tree.
Although grafting woody plants, like fruit trees, is an ancient
horticultural technique, grafting soft-stemmed vegetables is a much
more recent agricultural practice. Perhaps nurseries will soon start
selling mixed vegetable shrubs alongside fruit salad trees. Brassica
oleracea seems like a particularly good candidate for such an
experiment. This one species includes cabbage, broccoli, cauliflower,
Brussels sprouts and kale. Yes, all these plants are cultivars of the
exact same speciestheir appearances and characteristics have been
altered through artificial selection over the generations, in the same
way people have created so many different dog breeds. A
broccauliflower sprouts plant sounds particularly delicious. Maybe its
time for a family reunion.
(http://blogs.scientificamerican.com/brainwaves/2012/09/10/thescience-of-pomato-plants-and-fruit-salad-trees/)
Previously NONGWOO BIO developed a new CMS line (NWB-CMS) for
radish and the NWB-CMS line indeed provided lots of sub-lines of
Radish. And the Biotechnology Institute has developed the new
Cruciferae plants by fusing the NWB-CMS cells of Radish to the ordinary
cells of Chinese Cabbage and Cabbage.

(http://www.nongwoobio.com/nongwoo/html/tech_02.html)

SCIENCE

The seed is the mature, fertilized ovule. After fertilization, the haploid
cells of the embryo sac disintegrate. The maternally derived diploid
cells of the ovule develop into the hard, water-resistant outer covering
of the seed, called the testa, or seed coat. The diploid zygote develops
into the embryo, and the triploid endosperm cells multiply and provide
nutrition. The testa usually shows a scar called the hilum where the
ovule was originally attached to the funicle. In some seeds a ridge
along the testa called the raphe shows where the funicle originally was
pressed against the ovule. The micropyle of the ovule usually survives
as a small pore in the seed coat that allows passage of water during
germination of the seed.
In some species, the funicle develops into a larger structure on the
seed called an aril, which is often brightly colored, juicy, and contains
sugars that are consumed by animals that may also disperse the seed
(as in nutmeg, arrowroot, oxalis, and castor bean). This is distinct from
the fruit, which forms from the ovary itself.
The embryo consists of the cotyledon(s) , epicotyl, and hypocotyl. The
cotyledons resemble small leaves, and are usually the first
photosynthetic organs of the plant. The portion of the embryo above
the cotyledons is the epicotyl, and the portion below is the hypocotyl.
The epicotyl is an apical meristem that produces the shoot of the
growing plant and the first true leaves after germination. The hypocotyl
develops into the root. Often the tip of the hypocotyl, the radicle, is the
first indication of germination as it breaks out of the seed. Flowering
plants are classified as monocotyledons or dicotyledons (most are now
called eudicots ) based on the number of cotyledons produced in the
embryo. Common monocotyledons include grasses, sedges, lilies, irises,
and orchids; common dicotyledons include sunflowers, roses, legumes,
snapdragons, and all nonconiferous trees.
The endosperm may be consumed by the embryo, as in many legumes,
which use the cotyledons as a food source during germination. In other
species the endosperm persists until germination, when it is used as a
food reserve. In grains such as corn and wheat, the outer layer of the
endosperm consists of thick-walled cells called aleurone, which are high
in protein.

Anatomy of the reproductive organs in angiosperms.

(http://www.biologyreference.com/Re-Se/Reproduction-inPlants.html#ixzz3T29y3X8n)

SEARCH OF LITERATURE
APPLICATIONS OF SOMATIC HYBRIDIZATION
a) Creation of hybrids with disease resistance - Many disease resistance
genes (e.g. tobacco mosaic virus, potato virus X, club rot disease) could be
successfully transferred from one species to another. E.g resistance has been
introduced in tomato against diseases such as TMV, spotted wilt virus and insect
pests.
b) Environmental tolerance - using somatic hybridization the genes conferring
tolerance for cold, frost and salt were introduced in e.g. in tomato.
c) Cytoplasmic male sterility - using cybridization method, it was possible to
transfer cytoplasmic male sterility.
d) Quality characters - somatic hybrids with selective characteristics have
been developed e.g. the production of high nicotine content.

CHROMOSOME NUMBER IN SOMATIC HYBRIDS

The chromosome number in the somatic hybrids is generally more than the total
number of both of the parental protoplasts. If the chromosome number in the
hybrid is the sum of the chromosomes of the two parental protoplasts, the hybrid
is said to be symmetric hybrid. Asymmetric hybrids have abnormal or wide
variations in the chromosome number than the exact total of two species.
(http://www.biotechnology4u.com/plant_biotechnology_applications_cell_tissue_c
ulture.html)

TRADITIONAL METHODS

Grafting or graftage[1] is a horticultural technique whereby tissues from

oneplant are inserted into those of another so that the two sets of
vascular tissues may join together. This vascular joining is
called inosculation. The technique is most commonly used
in asexual propagation of commercially grown plants for
the horticultural and agricultural trades.
In most cases, one plant is selected for its roots and this is called
the stock orrootstock. The other plant is selected for
its stems, leaves, flowers, or fruits and is called the scion or cion.[1] The
scion contains the desired genes to be duplicated in future production
by the stock/scion plant.
In stem grafting, a common grafting method, a shoot of a selected,
desired plantcultivar is grafted onto the stock of another type. In
another common form called bud grafting, a dormant side bud is
grafted onto the stem of another stock plant, and when it
has inosculated successfully, it is encouraged to grow by pruning off
the stem of the stock plant just above the newly grafted bud.
For successful grafting to take place, the vascular cambium tissues of
the stock and scion plants must be placed in contact with each other.
Both tissues must be kept alive until the graft has "taken", usually a
period of a few weeks. Successful grafting only requires that a vascular
connection take place between the grafted tissues. Joints formed by
grafting are not as strong as naturally formed joints, so a physical weak
point often still occurs at the graft because only the newly formed
tissues inosculate with each other. The existing structural tissue (or
wood) of the stock plant does not fuse.
(http://en.wikipedia.org/wiki/Grafting)

NOVEL ATERNATIVE- SOMATIC FUSION

Sexual hybridization since time immemorial has been used as a method

for crop improvement but it has its own limitations as it can only be
used within members of same species or closely related wild species.
Thus, this limits the use of sexual hybridization as a means of
producing better varieties. Development of viable cell hybrids by
somatic hybridization, therefore, has been considered as an alternative
approach for the production of superior hybrids overcoming the species
barrier. The technique can facilitate breeding and gene transfer,
bypassing problems associated with conventional sexual crossing such
as, interspecific, intergeneric incompatibility. This technique of hybrid
production via protoplast fusion allows combining somatic cells (whole
or partial) from different cultivars, species or genera resulting in novel
genetic combinations including symmetric somatic hybrids, asymmetric
somatic hybrids or somatic cybrids.
The most common target using somatic hybridization is the gene of
symmetric hybrids that contain the complete nuclear genomes along

with cytoplasmic organelles of both parents. This is unlike sexual

reproduction in which organelle genomes are generally contributed by
the maternal parent. On the other hand, somatic cybridization is the
process of combining the nuclear genome of one parent with the
mitochondrial and/or chloroplast genome of a second parent. Cybrids
can be produced by donor-recipient method or by cytoplast-protoplast
fusion. Incomplete asymmetric somatic hybridization also provides
opportunities for transfer of fragments of the nuclear genome,
including one or more intact chromosomes from one parent (donor) into
the intact genome of a second parent (recipient).
These methods provide genetic manipulation of plants overcoming
hurdle of sexual incompatibility, thereby, serving as a method of
bringing together beneficial traits from taxonomically distinct species
which cannot be achieved by sexual crosses. Several parameters such
as, source tissue, culture medium and environmental factors influence
ability of a protoplast derived hybrid cells to develop into a fertile
plant. The general steps involved in somatic hybridization and
cybridization methods are elaborated in Figure 13.1 and 13.2 .
Steps involved in somatic hybridization
The somatic fusion process occurs in four steps:
The removal of the cell wall of one cell of each type of plant
using cellulase enzyme to produce a somatic cell called a protoplast
1. The cells are then fused using electric shock (electrofusion) or
chemical treatment to join the cells and fuse together the nuclei.
The resulting fused nucleus is called heterokaryon.
2. The somatic hybrid cell then has its cell wall induced to form
using hormones
3. The cells are then grown into calluses which then are further
grown to plantlets and finally to a full plant, known as a somatic
hybrid.
Different from the procedure for seed plants describe above, fusion
of moss protoplasts can be initiated without electric shock but by the
use of polyethylene glycol (PEG). Further, moss protoplasts do not
need phytohormonesfor regeneration, and they do not form a callus.
[3]
Instead, regenerating moss protoplasts behave
like germinatingmoss spores.[4] Of further note sodium nitrate and
calcium ion at high pH can be used, although results are variable
depending on the organism.
(Torrence, James (2008). "Higher Biology" (2nd ed.). Hodder Gibson; Mahesh.
Plant Molecular biotechnology. 2009. Book.)

(https://www.google.co.in/url?
sa=t&rct=j&q=&esrc=s&source=web&cd=10&cad=rja&uact=8&ved=0CFUQFjA
J&url=http%3A%2F%2Fwww.scirp.org%2Fjournal%2FPaperDownload.aspx
%3FpaperID%3D35851&ei=t5PxVI-GI4yjugSi4LoBg&usg=AFQjCNEOVeQ2SGm5UNLSRVqlQBxp_Tkecw&bvm=bv.87269000,d.
c2E)

Schematic view of symmetric protoplast fusion producing somatic

hybrids.

Schematic view of asymmetric protoplast fusion using donor-recipient

method resulting into creation of alloplasmic somatic hybrid or cybrids.
2. Protoplast fusion
Protoplast fusion could be spontaneous during isolation of protoplast or
it can be induced by mechanical, chemical and physical means. During
spontaneous process, the adjacent protoplasts fuse together as a result
of enzymatic degradation of cell walls forming homokaryons or
homokaryocytes, each with two to several nuclei. The occurrence of

multinucleate fusion bodies is more frequent when the protoplasts are

prepared from actively dividing callus cells or suspension cultures.
Since the somatic hybridization or cybridization require fusion of
protoplasts of different origin, the spontaneous fusion has no value. To
achieve induced fusion, a suitable chemical agent (fusogen) like,
NaNO3, high Ca2+, polyethylene glycol (PEG), or electric stimulus is
needed.
i. Fusion by means of NaNO 3: It was first demonstrated by Kuster in
1909 that the hypotonic solution of NaNO 3 induces fusion of isolated
protoplast forming heterokaryon (hybrid). This method was fully
described by Evans and Cocking (1975), however this method has a
limitation of generating few no of hybrids, especially when highly
vacuolated mesophyll protoplasts are involved.

ii. High pH and Ca++ treatment: This technique lead to the development
of intra- and interspecific hybrids. It was demonstrated by Keller and
Melcher in 1973. The isolated protoplasts from two plant species are
incubated in 0.4 M mannitol solution containing high Ca ++(50 mM
CaCl2.2H2O) with highly alkaline pH of 10.5 at 37C for about 30 min.
Aggregation of protoplasts takes place at once and fusion occurs within
10 min.

iii. Polyethylene glycol treatment: Polyethylene glycol (PEG) is the most

popularly known fusogen due to ability of forming high frequency,
binucleate heterokaryons with low cytotoxicity. With PEG the
aggregation occurred mostly between two to three protoplasts unlike
Ca++ induced fusion which involves large clump formation. The freshly
isolated protoplasts from two selected parents are mixed in appropriate
proportions and treated with 15-45% PEG (1500-6000MW) solution for
15-30 min followed by gradual washing of the protoplasts to remove
PEG. Protoplast fusion occurs during washing. The washing medium
may be alkaline (pH 9-10) and contain a high Ca ++ ion concentration (50
mM). This combined approach of PEG and Ca++ is much more efficient
than the either of the treatment alone. PEG is negatively charged and
may bind to cation like Ca++, which in turn, may bind to the negatively
charged molecules present in plasma lemma, they can also bind to
cationic molecules of plasma membrane. During the washing process,
PEG molecules may pull out the plasma lemma components bound to
them. This would disturb plamalemma organization and may lead to the
fusion of protoplasts located close to each other (Figure 13.3). The
technique is nonselective thus, induce fusion between any two or more
protoplasts.

Sequential stages in protoplast fusion. (A) two separate protoplasts, (B)

agglutination of two protoplasts, (C and D) Membrane fusion at
localized site, and (E and F) development of spherical heterokaryon.
iv. Electrofusion: The chemical fusion of plant protoplast has many
disadvantages (1) The fusogen are toxic to some cell systems, (2) it
produces random, multiple cell aggregates, and (3) must be removed
before culture. Compare to this, electrofusion is rapid, simple,
synchronous and more easily controlled. Moreover, the somatic hybrids
produced by this method show much higher fertility than those
produced by PEG-induced fusion.

Zimmermann and Scheurich (1981) demonstrated that batches of

protoplasts could be fused by electric fields by devising a protocol
which is now widely used. This protocol involves a two-step process.
First, the protoplasts are introduced into a small fusion chamber
containing parallel wires or plates which serve as electrodes. Second, a
low-voltage and rapidly oscillating AC field is applied, which causes
protoplasts to become aligned into chains of cells between electrodes.
This creates complete cell-to-cell contact within a few minutes. Once
alignment is complete, the fusion is induced by application of a brief
spell of high-voltage DC pulses (o.125-1 kVcm -1). A high voltage DC
pulses induces a reversible breakdown of the plasma membrane at the
site of cell contact, leading to fusion and consequent membrane
reorganization. The entire process can be completed within 15 min.
3. Selection of fusion products
The somatic hybridization by electrofusion of protoplasts allow one-toone fusion of desired pairs of protoplasts and, therefore, it is easy to
know the fate of fusion products. However, protoplast suspension
recovered after chemical treatments (fusogen) consists of the following
cell types:

i. unfused protoplasts of the two species/strains

ii. products of fusion between two or more protoplasts of the same
species (homokaryons), and
iii. hybrid' protoplasts produced by fusion between one (or more)
protoplasts of each of the two species (heterokaryons)
The heterokaryons which are the potential source of future hybrids
constitute of a very small (0.5-10%) proportion of the mixture.
Therefore, an effective strategy has to be employed for their
identification and isolation. Various protocols have been proposed and
practiced for the effective selection of hybrids, including morphological
basis, complementation of biochemical and genetic traits of the fusing
partners, and manual or electronic sorting of heterokaryons/hybrid
cells.
3.1. Morpho-physiological basis: The whole mixture of the protoplasts
are cultured after fusion treatment and the resulting calli or
regenerants are screened for their hybrid characteristics. Occasionally
the hybrid calli outgrow the parental cell colonies and are identified by
their intermediate morphology, i.e. green with purple coloured cells.
However, the process is labour intensive and requires glasshouse
facilities. It is limited to certain combinations showing differences in
their regeneration potential under specific culture conditions.
3.2. Complementation: In this case complementation or genetic or
metabolic deficiencies of the two fusion partners are utilized to select
the hybrid component. When protoplasts of two parents, (one parent
bearing cytoplasmic albino trait and the other parent bearing green
trait) each parent carrying a non-allelic genetic or metabolic defect are
fused, it reconstitutes a viable hybrid cell, of wild type in which both
defects are mutually abolished by complementation, and the hybrid
cells are able to grow on minimal medium non-permissive to the growth
of the parental cells bearing green trait. Later, the calli of hybrid nature
could be easily distinguished from the parental type tissue (albino trait)
by their green color. The complementation selection can also be applied
to dominant characters, such as dominant resistance to antibiotics,
herbicides or amino acid analogues.
3.3. Isolation of heterokaryons or hybrid cells: The manual or electronic
isolation of heterokaryons or hybrid cells is the most reliable method.
Manual isolation requires that the two parental type protoplasts have
distinct morphological markers and are easily distinguishable. For
example, green vacuolated, mesophyll protoplasts from one parent and
richly cytoplasmic, non green protoplasts from cultured cells of another
parent. The dual fluorescence method also helps easy identification of
fusion products. In this case, the protoplast labeled green by treatment
with fluorescein diacetate (FDA, 1-20 mgl-1) are fused with protoplasts
emitting a red fluorescence, either from chlorophyll autofluorescence or
from exogenously applied rhodamine isothiocyanate (10-20 mgl -1). The
labeling can be achieved by adding the compound into the enzyme

mixture. This can be applied even for morphologically indistinguishable

protoplasts from two parents.

4. Verification and characterization of somatic hybrids

As no system is foolproof and they have their own advantages and
disadvantages.
Therefore,
even
after
selecting
the
desired
hybrids/cybrids following protoplast fusion, it is required to carry out
one or more tests to compare the parent protoplast lines with the
putative hybrids. Some of the techniques that can be tried are:

4.1. Morphology: Somatic hybrids in most of the cases show characters

intermediate between the two parents such as, shape of leaves,
pigmentation of corolla, plant height, root morphology and other
vegetative and floral characters. The method is not much accurate as
tissue culture conditions may also alter some morphological characters
or the hybrid may show entirely new traits not shown by any of the
parents.

4.2. Isozyme analysis: Multiple molecular forms of same enzyme which

catalyses similar or identical reactions are known as isozymes.
Electrophoresis is performed to study banding pattern as a check for
hybridity. If the two parents exhibit different band patterns for a
specific isozyme the putative hybrid can be easily verified. The
isozymes commonly used for hybrid identification include, acid
phosphatase, esterase, peroxidase.

4.3. Cytological analysis: Chromosome counting of the hybrid is an

easier and reliable method to ensure hybridity as it also provides the
information of ploidy level. Cytologically the chromosome count of the
hybrid should be sum of number of chromosomes from both the
parents. Besides number of chromosomes, the size and structure of
chromosomes can also be monitored. However, the approach is not
applicable to all species, particularly where fusion involves closely
related species or where the chromosomes are very small. Moreover,
sometimes the somaclonal variations may also give rise to different
chromosome number.

4.4. Molecular analysis: Specific restriction pattern of nuclear,

mitochondrial and chloroplast DNA characterizes the plastomes of
hybrids and cybrids. Molecular markers such as RFLP, RAPD, ISSR can
be employed to detect variation and similarity in banding pattern of
fused protoplasts to verify hybrid and cybrid.

5. Cybrids or cytoplasmic hybrids:

Sexual hybridization involves fusion of the nuclear genes of both the
parents but somatic hybrids involves even cytoplasm from both the
parental species in hybrid obtained by protoplast fusion. However, in
another case somatic hybrids containing nuclear genome of one parent
but cytoplasm from both the parents, are termed as cybrids. The
approach is time consuming and require several years of crossing
plants provides an opportunity to study interparental mitochondrial,
chloroplast fusion giving rise to plants with novel genomes.

5.1. Methods to produce cybrids: They are produced in variable

frequencies in normal protoplast fusion experiments due to one of the
following methods:
1. Fusion of normal protoplast with an enucleated protoplast. The
enucleated protoplast can be produced by high speed centrifugation
(20,000-40,000xg) for 60 min with 5-50% percoll.
2. Fusion between a normal protoplast and another protoplast with a
non-viable nucleus or suppressed nucleus.
3. Elimination of one of the nuclei after heterokaryons formation.
4. Selective elimination of chromosomes at a later stage.
5. Irradiating (with X-rays or gamma rays) the protoplasts of one
species prior to fusion in order to inactivate their nuclei.
6. By preparing enucleate protoplasts (cytoplasts) of one species and
fusing them with normal protoplasts of the other species.
Cybrids provide the following unique opportunities: (i) transfer of
plasmogenes of one species into the nuclear background of another
species in a single generation, and even in (ii) sexually incompatible
combinations, (iii) recovery of recombinants between the parental
mitochondrial or chloroplast DNAs (genomes), and (iv) production of a
wide variety of combinations of the parental and recombinant
chloroplasts with the parental or recombinant mitochondria.

6. Applications of somatic hybridization

1. Novel interspecific and intergeneric crosses which are difficult to
produce by conventional methods can be easily obtained.
2. Important characters, such as resistance to diseases, ability to
undergo abiotic stress and other quality characters, can be obtained in

hybrid plant by the fusion of protoplasts of plant bearing particular

character to the other plant which may be susceptible to diseases.
3. Protoplasts of sexually sterile haploid, triploid, aneuploid plants can
be fused to obtain fertile diploids and polyploids.
4. Studying cytoplasmic genes may be helpful to carry out plant
breeding.
5. Most of the agronomically important traits, such as cytoplasmic male
sterility,
antibiotic
resistance
and
herbicide
resistance,
are
cytoplasmically encoded, hence can be easily transferred to other plant.
6. Plants in juvenile stage can also be hybridized by means of somatic
hybridization.
7. Somatic hybridization can be used as a method for the production of
autotetraploids.

Limitations of somatic hybridization

1. Application of protoplast methodology requires efficient plant
regeneration system from isolated protoplasts. Protoplasts from two
species can be fused, however, production of somatic hybrids is not
easy.
2. Lack of a proper selection method for fused products (hybrids) poses
a problem.
3. The end product of somatic hybridization are often unbalanced
(sterile, misformed and unstable)
4. Somatic hybridization of two
amphidiploids which is unfavorable.

diploids

leads

to

formation

of

5. It is not sure for a character to completely express after somatic

hybridization.
6. The regeneration products of somatic hybridization are often
variable due to somaclonal variation, chromosome elimination,
organelle segregation.
7. All diverse intergeneric somatic hybrids are sterile and, therefore,
have limited chances of development of new varieties.
8. To transfer useful genes from wild species to cultivated crop, it is
necessary to achieve intergeneric recombination or chromosome
substitution between parental genomes.
(http://nptel.ac.in/courses/102103016/13)

A simple method for the isolation of plant protoplasts

2. Materials and methods 21 Source tissue Tissue for protoplast
isolation consisted of fully expanded leaves excised from greenhousegrown plants (Basella, Centella, Withania, Amaranthus, Catharanthus,
Phlox, Petunia) as well as from actively-growing shoot cultures (tomato
cultivars viz, Arkavikas, pusa Ruby and Punjab Kesari; chilli, Nicotiana
rustica, N. glutinosa and N tabacum cv. Thompson), cotyledons of
aseptically-germinated seedlings (sunflower and niger), placental tissue
of fruits (tomato) and friable embryogenic callus (sandalwood). Shoot
cultures were stabilized in vitro The culture conditions and media for
the shoot cultures of each species were as follows. Surface sterilized
seeds of N. tabacum cv Thompson, N. rustica and N. glutinosa were
germinated on half strength MS medium for explanting shoots
Thereafter, shoot cultures were maintained by routine subculture onto
the same medium viz., half strength MS medium. Leaves were obtained
for protoplast isolation 3-4 weeks after every subculture Tomato
axillary shoots were obtained from 34 weeks old greenhouse grown
plants and taken to culture in half strength MS medium. Surface
sterilized seeds of sunflower and niger were germinated on half
strength MS medium. Seeds were dark-incubated for germination and
were shifted to 14 h exposure to light of 200 Em-2 s- 1 intensity at 25
2C temperature. Rapidly-growing embryogenic sandalwood callus
was obtained from foully developed endosperm as tissue explant.
Callus was induced on MS medium incorporated with 2,4dichlorophenoxyacetic acid (2,4-D) (1 mg/1) and kinetin (05 mg/1) and
then transferred to MS medium supplemented with 1 mg/1 of 2,4-D for
eliciting embryogenic competence.
2.2 Explant preparation prior to enzyme exposure Leaves of field-grown
plants were thoroughly washed in running water for about 10 min,
cleaned gently with dilute Labolene of Glaxo India Ltd., and quickly
rinsed with tap water first and then with deionized water Sterilization
was carried out with 2% sodium hypochlorite for 5 min followed by
several rinses with sterile distilled water. Surface decontaminated
leaves of greenhouse plants as well as those excised from shoot
cultures were chopped into 2-3 mm strips in most cases. But leaves
with epidermal layers peeled off were also used except in Basella,
Centella, Amaranthus, tomato, and chilli where peeling was not very
efficient. Cotyledons of sunflower, niger, chilli seedlings and placental
tissue of tomato fruits were similarly chopped into segments for
enzymatic digestion The embryogenic callus of sandalwood was lightly
squashed with a spatula and dropped into protoplast isolation solution.
All tissues were weighed in sterilized petriplates for fresh weight
determination before they were subjected to enzyme digestion. 2.3
Protoplast isolation, purification and culture The protoplast isolation
solution consisted of digesting enzymes, the osmoticum, a protecting
chemical, CaCl2 (10 mM), all dissolved in distilled water. 2.3a Enzymes:
The digesting enzymes used viz, cellulase and pectinase (Celluclast
and Pectinex/SP249) are a gift from Novo Industri (Denmark)'s Regional
Liaison Office, 101, 'Shah Sultan', Cunningham Road, Bangalore. These
are liquids and are readily and completely miscible with water at all

concentrations which occur in normal usage. The optimal working

conditions with these enzymes viz., temperature and pH are ideally
suited for their use in protoplast isolation. 2.3b Osmotic conditions
(stabilization): Osmotic stabilization was manipulated by adding to the
protoplast isolation and culture solutions, mannitol as the sole
osmoticum or mannitol in combination with either sorbitol or
magnesium sulphate. For tobacco leaves, niger cotyledons and
sandalwood callus, mannitol alone was finally used as the osmoticum.
Balancing of isolation and culture media to protoplast isotonic
concentration was carried out for different species and explants
individually using a cryoscopic osmometer (Osmomat 030 Gonotec)
(tables 1 and 2). 23c Incubation for protoplast release: Protoplasts
were isolated by incubating the various explants in filter sterilized
isolation solution. Incubation was carried out in dark at temperatures of
25 1C initially. To determine the time required for complete release
of protoplasts at incubation temperatures 25 1C, aliquots were
microscopically examined at different time points for all the
species/explants studied (table 1). To identify factors that might
influence the efficiency in terms of protoplast release, rapidity and
viability, incubation of tobacco leaf tissue was carried out at different
temperatures, at pH ranging from 56 to 6 and with different osmotic
stabilizers used either singly or in combination. The quantity and the
ratios of enzymes required for protoplast release were also determined.
2.3d Protoplast purification: The suspension of digested tissue in the
isolation mixture was sieved through a nylon mesh of appropriate pore
size to remove the debris and the filtrate was centrifuged at 100 g for 5
min to further separate the protoplasts from fine debris that remained
floating. The pellet of protoplasts was resuspended in 5 ml of washing
medium (table 2) and the process was repeated three times. The
protoplasts in 2 ml of washing medium were then layered on 9 ml of
flotation medium (table 2) in 15 ml capacity glass screw cap vials. The
tubes were left undisturbed for 15 min and then centrifuged at 160 g
for 10 min. The floating protoplast fraction that formed a clear band at
the interphase of washing and flotation medium was recovered with a
Pasteur pipette and was mixed with equal volume of fresh wash
medium. Protoplasts at this stage were allowed to equilibrate in the
wash medium in dark for about 30 min before they were plated. The
density and the yield of protoplasts were estimated using a
hemocytometer and the plating density adjusted. The competence and
viability of protoplasts was determined by dye testing (the viability test
with fluorescein diacetate (FDA)). Protoplast isolation by this method
was compared with that obtained by a standard method (Sankara Rao
and Gunasekari 1991). Leaf explants of tobacco and tomato axenic
shoot cultures raised under identical conditions were digested by these
two methods and the digestion time, and the yield at different
temperatures were Determined. 23e Protoplast culture: Aliquots of
purified protoplasts were plated in 2 ml of modified 8E liquid culture
medium (Niedz et al 1985) supplemented with appropriate growth
regulators and taken into 60 15 mm plastic or glass petriplates (table
2). The osmolarity of the culture medium was adjusted for each species
separately by adding appropriate amounts of mannitol. The plating
density was adjusted to obtain 2 104 protoplasts per ml of medium.
The cultures were initially incubated in dark at 25 2C temperature

for two weeks and then shifted to alternating light (of intensity 200
m2 s 1) and dark regimes of 14 and 10 h respectively. Samples
were tested periodically for viability and wall regeneration Further
culturing of protoplasts to obtain cell colonies and plant regeneration
was followed for N. tabacum cv Thompson applying the procedure
described earlier by Sankara Rao and Gunasekari (1991). All media
were autoclaved for 15 min at 15 p.s.i. Growth regulators used were
filter sterilized. All operations were carried out under aseptic
conditions. 3. Results The results obtained from the various protoplast
isolation experiments performed are summarized in table 1. With the
method described now, tissues of species explanted were most readily
acted upon by the enzymes and microscopic examination carried out
following digestion showed that the protoplast release was complete.
All species investigated yielded protoplasts (figures 1, 2, 5 and 6).
These species include herbaceous dicotyledonous plants, woody
sandalwood and the mucilage-containing herbaceous vine, Basella. On
an average, the incubation time required for complete release of
protoplasts varied between 4 and 10 h depending on the species
examined. Protoplast release from the explants of N. tabacum,
Lycopersicon esculentum cultivars and Capsicum frutescens cultivars,
particularly, needed only 4-6 h. Digestion of these explants carried out
by another method which was adopted earlier by Sankara Rao and
Gunasekari (1991) required a longer time (8-12 h) for protoplast release
(table 1). The yield of protoplasts was also comparatively low. The Novo
enzymes used in the present method maintained protoplast releasing
ability at temperatures beyond 25C. Increase of temperature during
tissue incubation using these enzymes, in fact, aided tissue digestion
further. The yield of protoplasts also increased slightly in N. tabacum cv
Thompson (table 1). More than its beneficial effect on protoplast yield,
increased incubation temperature had reduced the time needed for
complete release of protoplasts (table 1). The optimal conditions
identified with the protocol were, an incubation temperature range
between 28-30C and a pH between 58 and 6. These were also
conditions observed to be ideal for protoplast preparation from the
plant species used in the experiments as the protoplast viability did not
suffer the least. Using the present protocol, the yield of protoplasts
derived from chopped explants as also from leaves with epidermal
layers peeled off were comparable (table 1). Filtration of incubation
mixture through nylon mesh reduced the amount of contaminating
debris but only incompletely. That isolated protoplasts were free of cell
walls was determined by microscopic observation. Virtually a pure
population of intact protoplasts was readily isolated by flotation
separation on sucrose. The yield data for each of the plant species used
is shown in table 1. Washes and sedimentation helped further
purification of the protoplast preparation. Protoplasts were most stable
if allowed to equilibrate in the wash medium for about 30 min before
plating. In aliquots of purified protoplasts tested for viability, a fairly
good number were found positive. Protoplasts plated in modified 8E
medium formed cell wall readily (figures 3, 4, 6 and 8). The growth
regulators and osmolarity of the culture medium were different for each
species/cultivar investigated
Protoplasts of niger, tobacco cultivars, C. frutescens cv Punjab Jwala
and L. esculentum cv Arkavikas divided within six to seven days of

culture In tobacco, numerous cell colonies became visible after six

weeks of culture
Plants were regenerated for N. tabacum cv Thompson from the cell
colonies (figures 11 and 12) as described earlier (Sankara Rao and
Gunasekari 1991).
Most protoplast isolation protocols generally aim at higher yields, rapid
recovery and regenerability of protoplasts so obtained. But all these
objectives are seldom accomplished with any one method as there has
been no demonstrated whole plant regeneration from protoplasts
obtained by rapid preparations (Moore et al 1988; Hedrich et al 1985;
Thirumala Devi et al 1992) and similarly, high-yielding protocols are
rarely less simple procedures. Using the method described, we have
overcome some of these problems. The protocol provides a simple and
an easy-to-handle procedure that ensured satisfactory yields and quick
recovery of viable protoplasts.
The method owes its efficiency to elevated digestion temperatures and
a set of new enzymes synergistically acting on tissues at these
temperatures. The Novo cellulase and pectinase used are stable at
higher temperatures. The rate of enzymatic digestion also increased
with increase in incubation temperature. The tissue incubation,
however, was carried out at temperatures at which protoplast viability
remained unaffected. The time required for protoplast release was less
than it was observed with methods utilizing other enzymes (Sankara
Rao and Gunasekari 1991), tested on the same tissue under identical
conditions. Thus, the extended periods required for enzymes to release
protoplasts are avoided by this method and the protoplasts are
protected from the deleterious effects of long-term exposure to
digestion environment. The protocol is simple in that the various
pretreatment steps such as preconditioning of donor plants, presoaking
of explanted tissue, shaking during incubation, etc., were totally
avoided. Generally different plant species require cell wall degrading
enzymes from different sources. In the method presented, the same set
of enzymes were found effective on many different and unrelated
species equally well. Consistently, higher yields were obtained in all
species investigated with an average of 6l06 protoplasts per gram
fresh weight of explanted tissue. Competence of these protoplasts for
regeneration was ascertained by their survival and division in culture.
Plant regeneration could not be worked out for all the plants
investigated as nutritional and other culture requirements are highly
specific for individual species and cultivars. Nevertheless, cell wall
regeneration and division were observed with protoplasts of Guizotia
abyssinica (Niger), N. glutinosa, C. frutescens cv Punjab Jwala, and L.
esculentum cv Arkavikas, while further development leading to whole
plant regeneration was demonstrated for protoplasts of N. tabacum cv
Thompson. The method is routinely being used in our laboratory and is
extended to isolate protoplasts from other plant species. The release of
compounds that inhibit enzymatic cell wall digestion and consequent
poor protoplast release from chopped explants (leaves etc.) has been
reported (Butt 1985). In our experiments with the digestive enzymes
used, chopped explants yielded as many protoplasts as leaves with
epidermal layers peeled off would do. The process of wall digestion by

these enzymes, obviously, is not impeded by inhibitory compounds

exuding from chopped explants. The possibility that the Novo enzymes
have a greater penetration ability through multiple layers of tightly
packed cells in the explants, could also be a contributing factor to the
overall efficiency of these enzymes. They are supplied in a liquid state
and are easily miscible with osmoticum mixture. The tissues are readily
attacked by these enzymes and therefore practices such as vacuuminfiltration or shaking to provide uniform exposure of all cells to the
enzyme were totally eliminated.
Taking advantage of the extraordinary lytic properties of the enzymes
under study, the exposure time can be optimized for a given species or
cultivar to alleviate problems of over- or under-digestion of the
protoplasts and higher yields of viable protoplasts can be obtained. We
find that the method is simple and relatively rapid It is advantageous
over other methods in the overall ease of the procedure and
reproducibility. The ultimate range of applicability of the method,
however, has yet to be fully determined but may be limited in some
plant species
(http://www.ias.ac.in/jarch/jbiosci/20/645-655.pdf)

Somatic Embryo

The process of formation of an embryo is called embryogenesis.

Embryogenesis starts from a single embryogenic cell, that can be
a zygote (the product of the fusion of an egg and a sperm during
fertilization), or an undifferentiated callus cell. Embryos developing
from zygotes are called zygotic embryos, while those derived
from somatic cellsare called somatic embryos. During the embryonic
development, the polar axis of the plant is established, domains that
set up the organization of the plant body are defined, and the primary
tissue and organ systems are delineated. Somatic embryogenesis is
another important way to regenerate new plants in plant tissue culture.
Embryo development occurs through an exceptionally organized
sequence of cell division, enlargement and differentiation. Zygotic and
somatic embryos share the same gross pattern of development. Both
types of embryos develop as passing through typical developmental
stages . Embryo development is bipolar, having a shoot pole and a
radicular pole at opposite ends. Embryos are not organs because they
are structurally independent from their parent body (i.e. they do not
have a vascular system connecting them with their parent plant body).

Progenitor cell undergoes

unequal division.

The physical, observable transition from a nonembryogenic cell to an

embryogenic cell in somatic embryogenesis appears to occur when
the progenitor cell undergoes an unequal division, resulting in a
larger vacuolate cell and a small, densely cytoplasmic (embryogenic)
cell . The embryogenic cell then either continues to divide irregularly to
form a proembryonal complex or divides in a highly organized manner
to form a somatic embryo.

Somatic
Embryo Chart

Somatic embryos usually do not mature

properly. Instead, due to environmental factors such as
constant contact with inducing medium, somatic embryos often deviate
from the normal developmental pattern by bypassing embryo
maturation producing callus, undergoing direct secondary
embryogenesis and/or germinating precociously. Somatic embryos
growing from proembryonal complexes tend to develop asynchronously
so that several stages are present in culture at any given time.
(http://passel.unl.edu/pages/informationmodule.php?
idinformationmodule=956783940&topicorder=4&maxto=10)

LIMITATIONS AND SOLUTIONS

a) Somatic hybridization does not always produce plants that give

fertile and visible seeds.
b) There is genetic instability associated with protoplast culture.
c) There are limitations in the selection methods of hybrids, as many of
them are not efficient.
d) Somatic hybridization between two diploids results in the formation
of an amphidiploid which is not favourable therefore haploid
protoplasts are recommended in somatic hybridization.
e) It is not certain that a specific character will get expressed in
somatic hybridization.
f) Regenerated plants obtained from somatic hybridization are often
variable due to somaclonal variations, chromosomal elimination,
organelle segregation etc.
g) Protoplast fusion between different species/genus is easy, but the
production of viable somatic hybrids is not always possible.
(http://www.biotechnology4u.com/plant_biotechnology_applications_cell
_tissue_culture.html)

(https://www.google.co.in/url?
sa=t&rct=j&q=&esrc=s&source=web&cd=10&cad=rja&uact=8&ved=0CFUQFjAJ&url=ht
tp%3A%2F%2Fwww.scirp.org%2Fjournal%2FPaperDownload.aspx%3FpaperID
%3D35851&ei=t5PxVI-GI4yjugSi4LoBg&usg=AFQjCNEOVeQ2SGm5UNLSRVqlQBxp_Tkecw&bvm=bv.87269000,d.c2E)

PLAYERS IN THE FIELD

Phytowelt (Head office: Klsumer Weg 33 D-41334 Nettetal P

+49.2162.77859 F +49.2162.89215 www.phytowelt.com) has established
optimal systems for many plant species, including even difficult ones
like grasses, cereals and legumes.
Phytowelt already applied the SH technology for many different
applications.

Tubers of different somatic hybrids in comparison to the fusion partners

(cultivar: upper lines, wild species: lower lines, hybrids: in-between).

In mint cultivars, which are sterile and can only be vegetatively

propagated, SH was applied for increasing the gene pool by new
combinations for quantitative (biomass) and qualitative traits
(metabolites).
In potato cultivars
new
pathogen
resistancegenes
were
transferred from wild species. Various resistant somatic hybrid
lines with high tuber yield were selected (Fig. 2).
In fodder grasses new traits from wild grasses were transferred
by SH. Biomass yield was increased significantly.
Phytowelt has developed the first intersectional hybrids
of poplars via protoplastfusion! The gene pools of the different
poplar species contain all traits needed for breeding cultivars that
are ideally adapted for short rotation coppice (SRC), yet
intersectional
crosses
are
extreme
difficult.
Phytowelt`s
technology enhances the breeding of optimal SRC poplar clones.
Polyploid poplar clones derived from self fusion experiments show
an increased biomass.

PHASES OF THE STUDIES

1. Somatic fusion of seeds of two different plant species.
2. Efforts to grow them in Hydroponics and Aeroponics
3. Efforts to instill components to improve factors like colour,
flavor, taste, appearance, size, ripening time, control of
vegetative and reproductive phases

IDENTIFICATION OF PLANTS TO BE MERGED

Criteria to be followed:
1. Both species may prefer similar climatic conditions.
2. Both species may have same season and/or crop time or cycle.
3. One species is to give crop above the ground and the second
one should give crop below the ground.
4. Coats of the Seeds of both the species either should have no
ANF and no Antibiotics.
5. One species should produce yield containing more
carbohydrates or sugars while the other one should yield other
nutrients like FAT so a comprehensive nutrition helps to achieve
better results in both the crops.
6. Sizes of the seed should be more accommodative for the
somatic fusion
7. Seed coats should be more accommodative for the somatic
fusion
PROBABLE COMBINATIONS THAT MAY BE CHOOSEN
1. Blackgram (Vigna
(Vigna Mungo)
Mungo) + Peanut (Arachis
(Arachis hypogaea)
hypogaea)
2. Soy (Glycine
(Glycine max ) + Onion (Allium
(Allium cepa)
cepa)
3. Pudina (Mentha
(Mentha Arvensis ) + Carrot (Daucus
(Daucus carota)
carota)
4. Palak (Spinacia
(Spinacia oleracea) + Muli (Raphanus
(Raphanus sativus)
5. Coriander (Coriandrum
(Coriandrum sativum ) + Carrot (Daucus
(Daucus carota)
carota)
6. Chana(Cicer
Chana(Cicer arietinum)
arietinum) + Carrot (Daucus
(Daucus carota)
carota)
7. Guar (Cyamopsis
(Cyamopsis tetragonoloba ) + Peanut (Arachis
(Arachis hypogaea)
hypogaea)

Protoplast Isolation:

PROTOCOLS

Isolation of protoplasts (Fig. 8.9) is readily achieved by treating cells/tissues with

a suitable mixture of cell wall degrading enzymes. Usually, a mixture of

pectinase or macerozyme (0.1-1.9%) and cellulose (1-2%) is appropriate for most

plant materials. Hemicellulase may be necessary for some tissues. Generally
crude commercial preparations of enzymes arc used.
The pH of enzyme solution is adjusted between 4.7 and 8.0 and the temperature
is kept at 25-30C. The osmotic concentration of enzyme mixture and of
subsequent media is elevated (usually, by adding 500-800 m mol l -1 sorbitol or
mannitol) to stabilize the protoplasts and to prevent them from bursting. Usually,
50-100 m mol l-1 CaCl2 is added to the osmoticum as it improves plasma
membrane stability. The cells and tissues are incubated in the enzyme mixture
for few to several (generally, 16-18) hours; naked protoplasts devoid of cell wall
are gradually released in the enzyme mixture.
Protoplasts have been isolated from virtually all plant parts, but leaf mesophyll is
the most preferred tissue, at least in case of dicots, for this purpose. In general,
fully expanded leaves are surface-sterilized, their lower epidermis is peeled off
with a pair of forceps and the peeled areas are cut into small (Ca. 1 cm 2) pieces
with a scalpel and suspended in the enzyme mixture.

When epidermis cannot be peeled, leaf may be cut into Ca. 1 mm 2 pieces and
treated with the enzyme mixture; vacuum infiltration may be used to facilitate
the entry of enzymes into the tissues. After the period of incubation, protoplasts
are washed with a suitable washing medium in order to remove the enzymes and
the debris.
The protoplasts may be cultured in a suitable medium in a variety of ways: (i)
Bergmanns plating technique (in agar medium), (ii) in a thin layer of liquid
medium or (iii) in small microdrops of 50-100 l. Protoplasts readily regenerate
cell wall (within 2-4 days) and undergo mitosis to form macroscopic colonies,
which can be induced to regenerate whole plants. The conditions for isolation
and culture of protoplasts and regeneration of complete plants have been
standardized for a large number of plant species, but cereals still present some
problems.

Generally, MS and B5 media, and their modifications are used for protoplast
culture. The media are supplemented with a suitable osmoticum and, almost
always, with an auxin and a cytokinin, their types and concentrations depending
mainly on the plant species.
After 7-10 days of culture, protoplasts regenerate cell wall, and the osmolarity of
medium is gradually reduced to that of normal medium. The macroscopic
colonies are transferred onto normal tissue culture media. Protoplasts are very
sensitive to light; therefore, they are cultured in diffuse light or dark for the first
4-7 days.
2. Protoplast Fusion:
The techniques for protoplast fusion are pretty well refined and highly effective
for almost all the systems. A number of strategies have been used to induce
fusion between protoplasts of different strains/species; of these the following
three (Fig. 8.10) have been relatively more successful.
Protoplasts of desired strains/species are mixed in almost equal proportion;
generally, they are mixed while still suspended in the enzyme mixture. The
protoplast mixture is then subjected to high pH (10.5) and high
Ca2+ concentration (50 m mol l-1) at 37C for about 30 min (high pH-high
Ca2+ treatment). This technique is quite suitable for some species, while for some
others it may be toxic.

Polyethylene glycol (PEG) induced protoplast fusion is the most commonly used
as it induces reproducible high frequency fusion accompanied with low toxicity to
most cell types. The protoplast mixture is treated with 28-50% PEG (MW 1,5006,000) for 15-30 min, followed by gradual washing of the protoplasts to remove
PEG; protoplast fusion occurs during the washing.

The washing medium may be alkaline (pH 9-10) and contain a high Ca 2+ ion
concentration (50 m mol l-1); this approach is a combination of PEG and high pHhigh Ca2+ treatments, and is usually more effective than either treatment alone.
PEG is negatively charged and may bind to cation like Ca 2+, which, in turn, may
bind to the negatively charged molecules present in plasma lemma; they can
also bind to cationic molecules of plasma membrane.
During the washing process, PEG molecules may pull out the plasma lemma
components bound to them. This would disturb plasma lemma organisation and
may lead to the fusion of protoplasts located close to each other (Fig. 8.11).

The above fusion techniques are nonselective in that they induce fusion between
any two or more protoplasts. A more selective and less drastic approach is the
electrofusion technique, which utilizes low voltage nonuniform alternating
electric current pulses to bring the protoplasts in close contact (Fig. 8.12). Fusion
of protoplasts is brought about by a short pulse of high voltage.
The duration of high voltage is a few microseconds, and the voltages ranges
from 500 to 1,000 V/cm. The high voltage creates transient disturbances in the
organisation of plasma lemma, which leads to the fusion of neighbouring
protoplasts. The entire operation is carried out manually in specially designed
equipment, called electroporator.

Electrofusion has been mostly used with the members of Solanaceae often with
very high rate (over 50%) of fusion. This approach induces general fusion among
protoplats and there is no control on the type of protoplasts entering fusion. In a
modification of electrofusion, protoplast pairs are individually transferred into
microfusion chambers with the help of a micromanipulator set up.
Thus each microfusion chamber has one pair of protoplasts, which are induced to
fuse by a single or multiple, negative DC-pulse of 800 to 1,800 V /cm for 50

microseconds after mutual dielectrophoresis (1 MHz; 65-80 V/cm). This

technique is called microelectrofusion; it leads to highly specific and nearly 100%
pairwise fusion. Many workers feel that this fusion technique is more desirable
than the others for a number of important reasons.

3. Selection of Hybrid Cells:

The protoplast suspension recovered after a treatment with a fusion inducing
agent (fusogen) consists of the following cell types: (i) unfused protoplasts of the
two species/strains, (ii) products of fusion between two or more protoplasts of
the same species (homokaryons), and (iii) hybrid protoplasts produced by
fusion between one (or more) protoplast(s) of each of the two species
(heterokaryons) (Fig. 8.13).

In somatic hybridization experiments, only the heterokaryotic or hybrid

protoplasts, particularly those resulting from fusion between one protoplast of
each of the two fusion partners, are of interest.
However, they form only a small proportion of the population (usually 0.5-10%).
Therefore, an effective strategy has to be employed for their identification and
isolation. This step is called the selection of hybrid cells, is the most critical, and
is still an active area of investigation.
A number of strategies have been used for the selection of hybrid protoplasts.
(1) Some visual markers, e.g., pigmentation, of the parental protoplasts may be
used for the identification of hybrid cells under a microscope; these are then
mechanically isolated and cultured.
For example, the protoplasts of one species may be green and vacuolated
(derived from mesophyll cells), while those of the other may be non-vacuolated
and non-green (obtained from cell cultures). Where such features are not
available, the protoplasts of two parental species may be separately labelled
with different fluorescent agents.

This would allow the microscopic identification of hybrid protoplasts from the
parental ones. This approach can even be adapted to automatic cell sorting
permitting the recovery of large numbers of heterokaryons in a very short time.
This approach is, however, time consuming, and requires considerable skill and
effort.

Several workers have attempted to devise systems, which specifically select for
hybrid cells. In simple words, (2) these systems exploit some properties (usually,

deficiencies) of the parental species, which are not expressed in the hybrid cells
due to complementation between their genetic systems.
These properties may be sensitivity to culture medium constituents,
antimetabolites, temperature, etc., inability to produce an essential biochemical
(auxotrophic mutants), chlorophyll or some other pigmentation, etc. These
properties may be naturally present in the parental species or may be artificially
induced through mutagenesis.

The following example should be enough to clearly bring out the essential
features of the complementation approach. Protoplasts of Petunia hybrida form
calli on the MS medium, while those of P. parodii produce only small colonies.

Further, actinomycin D (1 g ml-1) inhibits cell division of P. hybrida protoplasts,

but it has no effect on those of P. parodii. Thus protoplasts of both these Petunia
species fail to produce macroscopic colonies (calli) on MS medium supplemented
with 1 g ml-1 actinomycin D.
However, their hybrid cells (P. hybrida + P. parodii; it may be noted that somatic
hybrids are denoted by a + sign as against the sexual hybrids being designated
by a X symbol) divide normally on this medium to produce macroscopic colonies.

This selection strategy exploits those natural properties of the two parental
species, which show complementation in the hybrid cells and, at the same time,
permit their selection. These strategies are simple, highly effective and the least
demanding, but their application is drastically limited by the nonavailability of
suitable properties (both natural and induced) in most of the parental species of
interest to the experimenters.

A variation of the complementation approach uses selectable markers. In this

approach, (3) different selectable markers like antibiotic and herbicide resistance
are engineered into the two fusion partners. The hybrid cells, in such cases, are
resistant to both the concerned selection agents, while the parental cells will be
sensitive to one and the other selection agent.

A more general and widely applicable strategy, but demanding more work than
the previous approach, is (4) to culture the entire protoplast population without
applying any selection for the hybrid cells. All the types of protoplasts form calli;
the hybrid calli are later identified on the basis of callus morphology,
chromosome constitution, protein and enzyme banding patterns, etc.
In some cases, the identification may be delayed till plants are regenerated. In
such an approach, it will be desirable to culture the protoplasts in very low

densities since neighbouring colonies are likely to fuse at higher densities;

ideally, they should be cultured in microdrops, each drop containing but a single
cell. Many workers tend to favour this approach since it does not depend the
presence of appropriate but difficult to find markers in the parental species.
4. Regeneration of Hybrid Plants:
Once hybrid calli are obtained, plants are induced to regenerate from them since
this is a prerequisite for their exploitation in crop improvement. Further, the
hybrid plants must be at least partially fertile, in addition to having some useful
property, to be of any use in breeding schemes. The culture techniques have
been refined to a state where plant regeneration has been obtained in a number
of somatic hybrids (Table 8.5).

Table 8.5. A list of some distant somatic hybrid plants:

But even today, it has not been possible to recover hybrid plants and/or calli
from a number of somatic combinations; this phenomenon is called somatic
incompatibility. The reasons for somatic incompatibility are not clearly
understood. The somatic hybrids are of the following two types: (1) symmetric
and (2) asymmetric hybrids.
Symmetric Hybrids:
Some somatic hybrid plants retain the full or nearly full somatic complements of
the two parental species; these are called symmetric hybrids. Such hybrids
provide unique opportunities for synthesizing novel species, which may be of
theoretical and/or practical interest. Frequently, somatic hybrids between
distantly related sexually incompatible species are sterile, precluding their
incorporation into a breeding programme.

This may be circumvented by producing 3n somatic hybrids by fusing somatic

(2n) cells of one species with haploid (n) cells of the other species; such 3n

plants may be expected to be partially fertile. These somatic hybrids can now be
used in breeding programmes for limited gene/chromosome introgression from
the species contributing the haploid protoplast.

A possible approach to the improvement of apparently useless somatic hybrids,

e.g., nonflowering somatic hybrid Daucus carota + Aegopodium podagraria, is to
fuse protoplasts from the hybrid with those of one of the parental species. The
fusion of a somatic hybrid protoplast with that from one of its parents is called
somatic back hybridization.
When protoplasts from the above somatic hybrid were fused with carrot
protoplasts, the resulting somatic hybrid produced flowers. Such hybrids can now
be ordered into breeding programmes with the aim of gene/chromosome
introgression.
Asymmetric Hybrids:
Many somatic hybrids exhibit the full somatic complement of one parental
species, while all or nearly all chromosomes of the other parental species are lost
during the preceding mitotic divisions; such hybrids are referred to as
asymmetric hybrids.

The available evidence suggests that such hybrids are likely to show a limited
introgression of chromosome segments from the eliminated genome(s) due to
drastically enhanced chromosomal aberrations and/or mitotic crossing over in
vitro.

Asymmetric hybrids can be obtained even from those combinations, which

normally produce symmetric hybrids by the following approach: protoplasts of
one of the parental species are irradiated with a suitable dose of X-rays or
gamma-rays to induce extensive chromosome breakage.
In such cases, chromosome segment introgression may be markedly enhanced. It
may be pointed out that asymmetric hybrids are essentially cytoplasmic hybrids
or cybrids, except for the introgressed genes.
Fate of Plasma-genes:
In contrast to sexual hybrid cells, i.e., zygotes, which contain the cytoplasmic
genes (plasmon) from the female parent only, somatic hybrid cells, contain
cytoplasmic complements from both the parental species.

The cytoplasmic genes (generally studied in terms of chloroplast types or

chloroplast DNA, cp-DNA mitochondrial DNA, mtDNA) appear to be distributed
randomly during the mitotic cell divisions. As a result, some cells receive
chloroplasts of one parental species, some others of the other species and a
small proportion retains the chloroplasts of both the species.

This is reflected in the plants regenerated from these cells. The same applies to
mitochondria as well. In addition, the distribution of chloroplasts is independent
from that of mitochondria.
Therefore, a somatic hybrid plant may contain chloroplasts from one parental
species and mitochondria from the other fusion parent. There is considerable
evidence that the genomes of both chloroplasts and mitochondria, particularly
the latter, undergo recombination in the hybrid cells; this produces recombinant
organelles in the progeny.
5. Cybrids:
Cybrids or cytoplasmic hybrids are cells or plants containing nucleus of one
species but cytoplasm from both the parental species. They are produced in
variable frequencies in normal protoplast fusion experiments due to one of the
following: (i) fusion of a normal protoplast of one species with an enucleate
protoplast or a protoplast having an inactivated nucleus of the other species, (ii)
elimination of the nucleus of one species from a normal heterokaryon, or (iii)
gradual elimination of the chromosomes of one species from a hybrid cell during
the subsequent mitotic divisions. Cybrids may be produced in relatively high
frequency by (i ) irradiating (with X-rays or gamma-rays) the protoplasts of one
species prior to fusion in order to inactivate their nuclei, or (ii) by preparing
enucleate protoplasts (cytoplasts) of one species and fusing them with normal
protoplasts of the other species.
The objective of cybrid production is to combine the cytoplasmic genes of one
species with the nuclear and cytoplasmic genes of another species. But the
mitotic segregation of plasmagenes, as evidenced by the distribution of
chloroplasts, leads to the recovery of plants having plasmagenes of one or the
other species only; only a small proportion of the plants remain cybrid, which
would further segregate into the two parental types.

Cybrids provide the following unique opportunities: (i) transfer of plasmagenes of

one species into the nuclear background of another species in a single
generation, and even in (ii) sexually incompatible combinations, (iii) recovery of
recombinants between the parental mitochondrial or chloroplast DNAs
(genomes), and (iv) production of a wide variety of combinations of the parental
and recombinant chloroplasts with the parental or recombinant mitochondria.
When cybrids are produced by irradiating the protoplasts of one species prior to
fusion, they provide the additional opportunity for (v) the recovery of
chromosome segment introgressions from the lost genome in combination with
variations in the plasmon. The cybrid approach has been used for the transfer of

cytoplasmic male sterility from N. tabacum to N. sylvestris, from P. hybrida to P.

axillaris, etc. (vi) In addition, mitochondria from one parental species may be
combined with the chloroplasts of the other parental species.
(http://www.yourarticlelibrary.com/biotechnology/plant-tissues/somatichybridization-of-hybrid-plants-explained-with-diagram/33226/

RESULTS
DISCUSSION
IP

The UPOV convention is the basis for legislation on plant breeders

rights (PBR) in currently 59 single countries and the European Union.
The chapter explains - on the basis of the UPOV convention - who is
entitled to plant breeders rights and the scope of such rights including
its conditions, restrictions, and exemptions. Furthermore, the document
details keywords of the legislation to enable applicants for plant
breeders rights to understand the legal procedure of the granting
process. The grant of PBR is independent from regulations on the
production or commercialization of a given variety.

EXPLORING MARKETEERS
STATUTORY REGULATIONS TO BE TAKEN
CARE

When comparing somatic hybridization to transgenic approaches,

somatic hybridization enables broadening of the germplasm base,
allows the transfer of uncloned multiple genes and generates products
that are not subjected to the same legal regulations as transgenic lines
[J. W. Grosser and F. G. Gmitter, 2004 SIVB Congress Symposium
Proceedings of Thinking Outside the Cell: Applications of Somatic
Hybridization and Cybridization in Crop Improvement, with Citrus as a
Model, In Vitro Cellular & Developmental Biology Plant, Vol. 41, No. 3,
2005, pp. 220-225. doi:10.1079/IVP2004634
J. Grosser and F. Gmitter, Protoplast Fusion for Production of
Tetraploids and Triploids: Applications for Scion and Rootstock Breeding
in Citrus, Plant Cell, Tissue and Organ Culture, Vol. 104, No. 3, 2011,
pp. 343-357. doi:10.1007/s11240-010-9823-4

Also, it transfers both mono- and polygenic traits

[R. Thieme, U. Darsow, L. Rakosy-Tican, Z. Kang, T. Gavrilenko, O.
Antonova, U. Heimbach and T. Thieme, Use of Somatic Hybridization to
Transfer Resistance to Late Blight and Potato Virus Y (PVY) into
Cultivated Potato, Plant Breeding and Seed Science, Vol. 50, No. 1,
2004, pp. 113-118.].

Regulation of GMOs

The overseeing of testing, use and commercialisation of GMOs whether plants,

animals or microorganisms requires a special regulation system. This system
establishes the legal and institutional framework for the control of potential
negative effects of GMOs on the environment or human and animal health
(Bbeanu, 2003). In the US, transgenic plants are only introduced into the
environment or on the market following approval from the following
governmental agencies responsible for environmental, and human and animal
health protection:
1. US Department for Agriculture (USDA),
2. Environmental Protection Agency (EPA),
3. Food and Drug Administration (FDA).
In the US and Canada, transgenic plants are grown and used for human and
animal food, and separate storage and labelling are not mandatory.
Since 1990, in the European Union, special legislation has been drawn up,
enhanced and extended, with the purpose of providing environmental and
human health protection, and creating a common market in the field of
biotechnology. Thus: - EU Directive No. 219/1990 (amended by Directive No.
81/1998) regulates the contained use of genetically modified microorganisms
(for research and commercialisation), - EU Directive No. 220/1990, concerning
the deliberate release of genetically modified organisms into the environment,
was the main initiative taken by the EU, and was subsequently supplemented by
several Commission Decisions (623, 811, 812, 813/2003), - EU Directive No.
18/2001 regulates the deliberate release of genetically modified organisms into
the environment. This Directive repealed Directive No. 220/1990. Having come
into force on 17 October 2002, Directive 18/2001 both updates and consolidates
15 existing regulations. This Directive also deals with mandatory information to
the public, the long-term monitoring of effects, labelling and traceability, in all
stages of GMO introduction on the market. Two other acts have been adopted
and published in the Official Journal of the European Communities, with respect
to the Community system of GMO traceability, the labelling of genetically
modified food and fodder, and the continuous procedure of authorisation or
introduction of GMOs in the environment as food or fodder: - EC Regulation No.
1829/2003 (22 September 2003) on genetically modified food and fodder, and EC Regulation No. 1830/2003 (22 September 2003) on the traceability and
labelling of genetically modified organisms, and GMO-based food and fodder.
These regulations amended EU Directive No. 18/2001.

WEB REFERENCES
REFERENCES

1. Greene, David (27 September 2013). "TomTato Is The Latest

Wonderplant". NPR News. Retrieved 30 September 2013.
2. a b Renneberg, Reinhard (2008). Biotechnology for Beginners. Elsevier.
p. 210. ISBN 9780123735812.

3. "How to create a pomato plant". Retrieved 29 May 2013.

4. "Pomato Plants". The Guru (36). 2010. Retrieved 29 May 2013.
5. a b Jabr, Ferris. "The Science of Pomato Plants and Fruit Salad Trees".
Scientific America. Retrieved 29 May 2013.
6. "Kenyan farmers produce pomato plants to improve land use".
Retrieved 29 May 2013.
7. "Prison grows unique pomato to fight hunger".
www.businessdailyafrica.com. Retrieved 29 May 2013.
8. Jude Gillies. "Potato Tom opens fresh doors". Stuff.co.nz. Retrieved 201309-30.
9. Hall, John (27 September 2013). "The TomTato: Plant which produces
both potatoes and tomatoes launched in UK". The Independent.
Retrieved 30 September 2013.
10.Wilkes, David (25 September 2013). "The TomTato... or how you can
make ketchup AND chips from the same plant! |". Mail Online. Retrieved
30 September 2013.
11.Bellincampi D and Morpugo G 1987 Conditioning factor affecting growth
in plant cells in culture; Plant Sci. 51 83-91
12.Butt A D 1985 A general method for the high-yielding isolation of
mesophyll protoplasts from deciduous tree species; Plant Sci. 42 55-59
13.Cocking C 1960 A method for the isolation of plant protoplasts and
vacuoles; Nature (London) 187 927-929
14.d'Utra Vaz F B, Slamet I H, Khatum A, Cocking C and Power J 1992
Protoplast culture in high molecular oxygen atmosphere; Plant Cell Rep.
11 416-418
15.Hedrich R, Raschke and Stitt 1985 A role for Fructose 2,6Bisphosphate in regulating carbohydrate metabolism in guard cells;
Plant Physiol. 79 977-982
16.Kao and Michayluk 1975 Nutritional requirements for growth of
Vinca hajastana cells and protoplasts at a very low population density in
liquid media; Planta 126 105-110
17.Kyozuka J, Hayashi and Shinomoto 1987 High frequency plant
regeneration from rice protoplasts by novel nurse culture methods; Mol.
Gen.Genet. 206 408-413
18.Lee L, Schroll R E, Grimes D and Hodges 1990 Plant regeneration
from indica rice (Oryza sativa L.) protoplasts; Planta 178 325-333
Maheshwari S C, Gill R, Maheshwari and Gharyal 1986 The isolation
and culture of protoplasts; in Differentiation of protoplasts and
transformed plant cells (eds) J Reinert and Binding (New York: SpringerVerlag) pp 20-48
19.Mei-Lei C T, Reitveld , Van Marrewijk G A M and Kool A J 1987
Regeneration of leaf mesophyll protoplasts of tomato cultivars (L.
esculentum): factors important for efficient protoplast culture and plant
regeneration; Plant Cell Rep. 6 172-175
20.Moore D, Monson R K, Ku S and Edwards G 1988 Activities of
principal photosynthetic and photorespiratory enzymes in leaf mesophyll
and bundle sheath protoplasts from the C3-C4 intermediate Flavaria
ramosissima; Plant Cell Physiol. 29 999-1006
21.Niedz R P, Rutter S M, Handley L W and Sink C 1985 Plant regeneration
from leaf protoplasts of six tomato cultivars; Plant Sci. 39 199-204
22.Sankara Rao and Srikantha 1986 A simple method for the
preparation of mesophyll protoplasts; in Proceedings of the VI
International Congress of Plant Tissue and Cell Culture, Minnesota, p 37
23.Sankara Rao and Gunasekari 1991 Control of morphogenesis in
tobacco protoplast cultures: Organogenesis Vs. embryogenesis; Indian J.
Biochem. Biophys. 28 467-471
24.Shahin A 1985 Totipotency of tomato protoplasts; Theor. Appl. Genet.
69 235-240 Thirumala Devi M, Vani T, Malla Reddy and Raghavendra A

S 1992 Rapid isolation of mesophyll and guard cell protoplasts from pea
and maize leaves; Indian J. Exp. Biol. 30 424-428