Protoplast fusion/Somatic fusion:

 Protoplast fusion is a physical method of fusion of somatic cells from different plant to form
hybrid.
 Mixing of protoplasts of two different genomes and can be achieved by either spontaneous
or induced fusion methods.

Methods of Protoplast fusion:

1. Spontaneous fusion:

 Cell fusion is a process integral to plant development.

 The most prominent process is egg fertilization.
 The breakdown of cell wall during protoplast isolation led people to believe that there
would be spontaneous fusion leading to the formation of homokaryons because
multinucleate cells were detected as soon as enzymatic protoplast isolation techniques
were applied.
 The argument that cell wall degradation would permit dilation of plasmodesmata, fusion
and complete mixing of cells was supported by electron micrographic studies.
 Spontaneous fusion of protoplasts is observed when protoplasts are isolated from callus
cultures.
 However, spontaneous fusion products do not regenerate in to whole plants except for
undergoing a few divisions.
 Later studies revealed that isolated protoplasts are usually characterized by smooth
surfaces and fusion has to be induced by one of a variety of treatments.

2. Induced fusion methods:

 Spontaneous fusion is of no value as fusion of protoplasts of different origins is required in

somatic hybridization.
 To achieve this, a suitable agent (fusogen) is added to fuse the plant protoplasts of
different origins.
 The different fusogens employed are: NaNO3, artificial sea water, lysozyme, high pH/Ca+
+, polyethylene glycol, antibodies, concavalin A, polyvinyl alcohol, electrofusion dextran
and dextran sulphate, fatty acids and esters.
 Some of the methods that have been employed are explained below.

i. Treatment with sodium nitrate:

 Power et al. first reported induced fusion by NaNO3 (1970).

 Isolated protoplasts are suspended in a mixture of 5.5% sodium nitrate in a 10% sucrose
solution.
 The solution containing the protoplasts is incubated in a water bath at 35°C for 5 min and
then centrifuged for 5 min at 200x g.
 Following centrifugation, most of the supernatant is decanted and the protoplast pellet is
transferred to a water bath at 30°C for 30 min.
 During this period, most of the protoplasts undergo cell fusion.
  The remaining aggregation mixture is gently decanted and replaced with the culture
medium containing 0.1% NaNO3.
 The protoplasts are left undisturbed for sometimes after which they are washed twice with
the culture medium and plated.
 This technique results in low frequency of heterokaryon formation, especially when
mesophyll protoplasts are involved.

ii. Calcium ions at high pH:

 The effect of high pH and calcium ions on the fusion of tobacco protoplasts was first
studied by Keller and Melchers (1973) .
 In their method, isolated protoplasts are centrifuged for 3 min at 50x g in a fusion-inducing
solution of 0.5 M mannitol containing 0.05 M CaCl2·2H2O at a pH of 10.5.
 The centrifuge tubes containing the protoplasts are then incubated in a water bath at 37°C
for 40–50 min.
 After this treatment, 20–50% of the protoplasts were involved in fusion.

iii. Polyethylene glycol (PEG) method:

 Kao and Michayluk (1974) and Wallin et al. (1974) developed PEG method of fusion of
protoplasts.
 This is one of the most successful techniques for fusing protoplasts.
 The protoplasts are suspended in a solution containing high molecular weight PEG, which
improves agglutination and fusion of protoplasts in several species.
 When adequate amount of protoplasts are available, 1 ml of the protoplasts suspended in
a culture medium are mixed with 1 ml of 28–56% PEG (1500 –6000 MW) solution.
 The tube is then shaken for 5 sec and allowed to settle for 10 min.
 To remove PEG, the protoplasts are then washed several times by the addition of
protoplast culture medium.
 The protoplast preparation is again suspended in the culture medium.
 The PEG method is popular for protoplast fusion as it yields in reproducible high-frequency
heterokaryon formation, low cytotoxicity to most cell types and the formation of binucleate
heterokaryons.
 PEG-induced fusion is non- specific and is thus applicable for interspecific, intergeneric or
interkingdom fusions.
 Both the molecular weight and the concentration of PEG are critical in inducing successful
fusions.
 PEG less than 100 molecular weight is not able to produce tight adhesions while that
ranging up to 6000 molecular weight can be more effective per mole in inducing fusions.
 At higher molecular weight PEG produces too viscous a solution which cannot be handled
properly.
 Treatment with PEG in the presence of/or by high pH/Ca++ is reported to be most
effective in enhancing the fusion frequency and survivability of protoplasts.

iv. Electrofusion:

 Protoplasts are placed in to a small culture cell containing electrodes, and a potential
difference is applied due to which protoplasts line up between the electrodes.
 If now an extremely short wave electric shock is applied, protoplasts can be induced to
fuse.
  In this fusion method, two-step procedure is followed beginning with application of an
alternating current (AC) of low intensity to protoplast suspension.
 Dielectrophoretic collectors adjusted to 1.5 V and 1 MHz and an electrical conductivity of
the suspension medium less than 10–5 sec/cm generate an electrophoresis effect that
make the cells attach to each other along the field lines.
 The second step of injection of an electric direct current (DC) field pulse of high intensity
(750– 1000 V/cm) for a short duration of 20–50 μsec leads to breakdown of membranes in
contact areas of adjacent cells resulting in fusion and consequent membrane
reorganization.
 This electrofusion technique has been found to be simple, quick and efficient. Cells after
electrofusion do not show cytotoxic response.
 However, this method did not receive much acceptance because specialized equipment is
required.

Mechanism of Protoplast fusion

Protoplast fusion consists of three main phases:

Phase I: Agglutination or adhesion:

 Two or more protoplasts are brought into close proximity.

 The adhesion can be induced by a variety of treatments, e.g. concanavalin A, PEG, high
pH and high Ca++ ions.

Phase II: Plasma membrane fusion at localized sites:

 Membranes of protoplasts are stuck together by fusogen get fused at the point of adhesion
resulting in the formation of cytoplasmic bridges between the protoplasts.
 Plant protoplasts carry a negative charge from –10 to –30 mV.
 Due to common charge, the plasma membranes of two agglutinated protoplasts do not
come close enough to fuse.
 Fusion requires that membranes must be first brought close together at a distance of 10Å
or less.
 The high pH–high Ca++ ions treatment has shown to neutralize the normal surface charge
so that agglutinated protoplasts can come in intimate contact.
 High temperature promotes membrane fusion due to perturbance of lipid molecules in
plasma membrane and fusion occurs due to intermingling of lipid molecules in membranes
of agglutinated protoplasts.
 PEG agglutinates to form clumps of protoplasts.
 Tight adhesion may occur over a large or small localized area.
 Localized fusion of closely attached plasma membranes occurs in the regions of tight
adhesion and results in the formation of cytoplasmic bridges.
 It has been further suggested that PEG, which is slightly negative in polarity can form
hydrogen bonds with water, protein, carbohydrates, etc. which are positive in polarity.
 When the PEG molecule chain is large enough it plays role as a molecular bridge between
the surface of adjacent protoplasts and adhesion occurs.

Phase III: Formation of heterokaryon:

  Rounding off of the fused protoplasts due to the expansion of cytoplasmic bridges forming
spherical heterokaryon or homokaryon.

SOMATIC HYBRIDIZATION.
The somatic hybridization involves three aspects. The three aspects are: (A) Fusion of
Protoplasts (B) Selection of Hybrid Cells and (C) Identification of Hybrid Plants.
The conventional method to improve the characteristics of cultivated plants, for years, has
been sexual hybridization. The major limitation of sexual hybridization is that it can be
performed within a plant species or very closely related species. This restricts the
improvements that can be done in plants.
The species barriers for plant improvement encountered in sexual hybridization can be
overcome by somatic cell fusion that can form a viable hybrids. Somatic hybridization
broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its
subsequent development to form a hybrid plant.

Plant protoplasts are of immense utility in somatic plant cell genetic manipulations and
improvement of crops. Thus, protoplasts provide a novel opportunity to create cells with new
genetic constitution. And protoplast fusion is a wonderful approach to overcome sexual
incompatibility between different species of plants. More details on the applications of
somatic hybridization are given later.
Somatic hubridization involves the following aspects:
A. Fusion of protoplasts
B. Selection of hybrid cells
C. Identification of hybrid plants.

A. Fusion of Protoplasts:
As the isolated protoplasts are devoid of cell walls, there in vitro fusion becomes relatively
easy. There are no barriers of incompatibility (at interspecific, inter-generic or even at inter-
kingdom levels) for the protoplast fusion. Protoplast fusion that involves mixing of
protoplasts of two different genomes can be achieved by spontaneous, mechanical, or induced
fusion methods.
Spontaneous fusion:
Cell fusion is a natural process as is observed in case of egg fertilization. During the course of
enzymatic degradation of cell walls, some of the adjoining protoplasts may fuse to form
homokaryocytes (homokaryons). These fused cells may sometimes contain high number of
nuclei (2-40).

This is mainly because of expansion and subsequent coalescence of plasmodermal

connections between cells. The frequency of homokaryon formation was found to be high in
protoplasts isolated from dividing cultured cells. Spontaneously fused protoplasts, however,
cannot regenerate into whole plants, except undergoing a few cell divisions.
Mechanical fusion:
The protoplasts can be pushed together mechanically to fuse. Protoplasts of Lilium and
Trillium in enzyme solutions can be fused by gentle trapping in a depression slide.
Mechanical fusion may damage protoplasts by causing injuries.
Induced fusion:
Freshly isolated protoplasts can be fused by induction. There are several fusion-inducing
agents which are collectively referred to as fusogens e.g. NaN03, high pH/Ca2+,
polyethylene glycol, polyvinyl alcohol, lysozyme, concavalin A, dextran, dextran sulfate,
fatty acids and esters, electro fusion. Some of the fusogens and their use in induced fusion are
described. A diagrammatic representation of protoplast fusion is depicted in Fig. 44.4.
Protoplast Fusion
Treatment with sodium nitrate:
The isolated protoplasts are exposed to a mixture of 5.5% NaNO3 in 10% sucrose solution.
Incubation is carried out for 5 minutes at 35°C, followed by centrifugation (200 x g for 5
min). The protoplast pellet is kept in a water bath at 30°C for about 30 minutes, during which
period protoplast fusion occurs. NaNO3 treatment results in a low frequency of heterokaryon
formation, particularly when mesophyll protoplasts are fused.
High pH and high Ca2+ ion treatment:
This method was first used for the fusion of tobacco protoplasts, and is now in use for other
plants also. The method consists of incubating protoplasts in a solution of 0.4 M mannitol
containing 0.05 M CaCI2 at pH 10.5 (glycine-NaOH buffer) and temperature 3 7°C for 30-40
minutes. The protoplasts form aggregates, and fusion usually occurs within 10 minutes. By
this method, 20-50% of the protoplasts are involved in fusion.
Polyethylene glycol (PEG) treatment:
This has become the method of choice, due to its high success rate, for the fusion of
protoplasts from many plant species. The isolated protoplasts in culture medium (1 ml) are
mixed with equal volume (1 ml) of 28-56% PEG (mol. wt. 1500-6000 Daltons) in a tube.
PEG enhances fusion of protoplasts in several species. This tube is shaken and then allowed
to settle. The settled protoplasts are washed several times with culture medium.

PEG treatment method is widely used protoplast fusion as it has several advantages:
i. It results in a reproducible high-frequency of heterokaryon formation.
ii. Low toxicity to cells.
iii. Reduced formation of bi-nucleate heterokaryons.
iv. PEG-induced fusion is non-specific and therefore can be used for a wide range of plants.
Electro-fusion:
In this method, electrical field is used for protoplast fusion. When the protoplasts are placed
in a culture vessel fitted with micro- electrodes and an electrical shock is applied, protoplasts
are induced to fuse. Electro-fusion technique is simple, quick and efficient and hence
preferred by many workers.
Further, the cells formed due to electro-fusion do not show cytotoxic responses as is the case
with the use of fusogens (including PEG). The major limitation of this method is the
requirement of specialized and costly equipment.
Mechanism of fusion:
The fusion of protoplasts involves three phases agglutination, plasma membrane fusion and
formation of heterokaryons.
1. Agglutination (adhesion):
When two protoplasts are in close contact with each other, adhesion occurs. Agglutination
can be induced by fusogens e.g. PEG, high pH and high Ca2+.
2. Plasma membrane fusion:
Protoplast membranes get fused at localized sites at the points of adhesion. This leads to the
formation of cytoplasmic bridges between protoplasts. The plasma membrane fusion can be
increased by high pH and high Ca2+, high temperature and PEC, as explained below.
(a) High pH and high Ca2+ ions neutralize the surface charges on the protoplasts. This allows
closer contact and membrane fusion between agglutinated protoplasts.
(b) High temperature helps in the intermingling of lipid molecules of agglutinated protoplast
membranes so that membrane fusion occurs.
(c) PEG causes rapid agglutination and formation of clumps of protoplasts. This results in the
formation of tight adhesions of membranes and consequently their fusion.
3. Formation of heterokaryons:
The fused protoplasts get rounded as a result of cytoplasmic bridges leading to the formation
of spherical homokaryon or heterokaryon.
B. Selection of Hybrid Cells:
About 20-25% of the protoplasts are actually involved in the fusion. After the fusion process,
the protoplast population consists of a heterogenous mixture of un-fused chloroplasts,
homokaryons and heterokaryons (Fig. 44.5). It is therefore necessary to select the hybrid cells
(heterokaryons). The commonly used methods employed for the selection of hybrid cells are
biochemical, visual and cytometric methods.
Fusion Products of Protoplasts
Biochemical methods:
The biochemical methods for selection of hybrid cells are based on the use of biochemical
compounds in the medium (selection medium). These compounds help to sort out the hybrid
and parental cells based on their differences in the expression of characters.
Drug sensitivity and auxotrophic mutant selection methods are described below:
1. Drug sensitivity:
This method is useful for the selection hybrids of two plant species, if one of them is sensitive
to a drug. Protoplasts of Petunia hybride (species A) can form macroscopic callus on MS
medium, but are sensitive to (inhibited by) actinomycin D. Petunia parodii protoplasts
(species B) form small colonies, but are resistant to actinomycin D.
When these two species are fused, the fused protoplasts derive both the characters —
formation of macroscopic colonies and resistance to actinomycin D on MS medium. This
helps in the selection of hybrids (Fig. 44.6). The parental protoplasts of both the species fail
to grow. Protoplasts of P. parodii form very small colonies while that of P. hybrida are
inhibited by actinomycin D.
Drug Sensitivity Method
Drug sensitivity technique was originally developed by Power et al (1976) for the selection of
hybrids of Petunia sp. A similar procedure is in use for the selection of other somatic hybrids
e.g., hybrids between Nicotiana Silvestre’s and Nicotiana knightiana.
2. Auxotrophic mutants:
Auxotroph’s are mutants that cannot grow on a minimal medium and therefore require
specific compounds to be added to the medium. Nitrate reductase deficient mutants of
tobacco (N. tabacum) are known. The parental protoplasts of such species cannot grow with
nitrate as the sole source of nitrogen while the hybrids can grow.
Two species of nitrate reductase deficiency— one due to lack of apoenzyme (nia-type
mutant) and the other due to lack of molybdenum cofactor (cnx- type mutant) are known. The
parental protoplasts cannot grow on nitrate medium while the hybrid protoplasts can grow
(Fig. 44.7).

Hybrid Selection Based on Auxotrophic Mutant

The selection of auxotrophic mutants is possible only if the hybrid cells can grow on a
minimal medium. Another limitation of the technique is the paucity of higher plant
auxotroph’s.
Visual methods:
Visual selection of hybrid cells, although tedious is very efficient. In some of the somatic
hybridization experiments, chloroplast deficient (albino or non-green) protoplasts of one
parent are fused with green protoplasts of another parent.
This facilitates the visual identification of haterokaryons under light microscope. The
heterokaryons are bigger and green in colour while the parental protoplasts are either small or
colourless. Further identification of these heterokaryons has to be carried out to develop the
specific hybrid plant.
There are two approaches in this direction — growth on selection medium, and mechanical
isolation.
1. Visual selection coupled with differential media growth:
There exist certain natural differences in the sensitivity of protoplasts to the nutrients of a
given medium. Thus, some media can selectively support the development of hybrids but not
the parental protoplasts. A diagrammatic representation of visual selection coupled with the
growth of heterokaryons on a selection medium is given in Fig. 44.8.
Visual Selection of Hybrids Coupled with Growth on Selection Medium
2. Mechanical isolation:
The visually identified heterokaryons under the microscope can be isolated by mechanical
means. This involves the use of a special pipette namely Drummond pipette. The so isolated
heterokaryons can be cloned to finally produce somatic hybrid plants. The major limitation of
this method is that each type of hybrid cell requires a special culture medium for its growth.
This can be overcome by employing micro drop culture of single cells using feeder layers .
Cytometric methods:
Some workers use flow cytometry and fluorescent-activated cell sorting techniques for the
analysis of plant protoplasts while their viability is maintained. The same techniques can also
be applied for sorting and selection of heterokaryons. The hybrid cells derived from such
selections have proved useful for the development of certain somatic hybrid plants.
C. Identification of Hybrid (Cells) Plants:
The development of hybrid cells followed by the generation of hybrid plants requires a clear
proof of genetic contribution from both the parental protoplasts. The hybridity must be
established only from euploid and not from aneuploid hybrids. Some of the commonly used
approaches for the identification of hybrid plants are briefly described.
Morphology of hybrid plants:
Morphological features of hybrid plants which usually are intermediate between two parents
can be identified. For this purpose, the vegetative and floral characters are considered. These
include leaf shape, leaf area, root morphology, flower shape, its structure, size and colour,
and seed capsule morphology.
The somatic hybrids such as pomatoes and topatoes which are the fused products of potato
and tomato show abnormal morphology, and thus can be identified. Although the genetic
basis of the morphological characters has not been clearly known, intermediate
morphological features suggest that the traits are under the control of multiple genes. It is
preferable to support hybrid morphological characters with evidence of genetic data.
Isoenzyme analysis of hybrid plants:
The multiple forms of an enzyme catalysing the same reaction are referred to as isoenzymes.
Electrophoretic patterns of isoenzymes have been widely used to verify hybridity. Somatic
hybrids possess specific isoenzymes (of certain enzymes) of one or the other parent or both
the parents simultaneously.
There are many enzymes possessing unique isoenzymes that can be used for the identification
of somatic hybrids e.g. amylase, esterase, aspartate aminotransferase, phosphodiesterase,
isoperoxidase, and hydrogenases (of alcohol, lactate, malate). If the enzyme is dimeric
(having two subunits), somatic hybrids usually contain an isoenzyme with an intermediate
mobility properties. The isoenzymes are often variable within the same plant. Therefore, it is
necessary to use the same enzyme from each plant (parents and somatic hybrids), from a
specific tissue with the same age.
Chromosomal constitution:
The number of chromosomes present in the hybrid cells can be directly counted. This
provides information on the ploidy state of the cells. The somatic hybrids are expected to
possess chromosomes that are equal to the total number of chromosomes originally present in
the parental protoplasts. Sometimes, the hybrids are found to contain more chromosomes than
the total of both the parents. The presence of chromosomal markers is greatly useful for the
genetic analysis of hybrid cells.
Molecular techniques:
Many recent developments in molecular biology have improved the understanding of genetic
constitution of somatic plant hybrids.
Some of them are listed below:
1. Differences in the restriction patterns of chloroplast and mitochondrial DNAs.
2. Molecular markers such as RFLP, AFLP, RAPD and microsatellites.
3. PCR technology.
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.
However, wide variations are reported which may be due to the following reasons:
1. Fusion of more than two protoplasts.
2. Irregularities in mitotic cell divisions.
3. In fusogen or electro-induced fusions, about one third of the fusions occur between more
than two protoplasts.
4. Differences in the status of protoplasts (actively dividing or quiescent) from the two
species of plants result in formation of asymmetric hybrids.
5. Asymmetric hybrids may be due to unequal replication of DNA in the fusing protoplasts.
6. Protoplast isolation and culture may also lead to somaclonal variations, and thus variations
in chromosome number.
A selected list of interspecific hybrids produced through protoplast fusion along with the
number of chromosomes in the hybrids is given in Table 44.3.
Interspecific Hybrids
Symmetric and asymmetric hybrids:
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. Symmetric hybrids between incompatible
species are usually sterile. This may be due to production of 3n hybrids by fusing 2n of one
species with n of another species.

Asymmetric hybrids have abnormal or wide variations in the chromosome number than the
exact total of two species. These hybrids are usually formatted with full somatic complement
of one parental species while all or nearly all of the chromosomes of other parental species
are lost during mitotic divisions. Asymmetric hybrids may be regarded as cybrids but for the
introgressed genes.

As given in Table 44.3, protoplast fusion between N. tabacum (2n = 48) and N. nesophila (2n
= 24) results in a symmetric hybrids, while asymmetric hybrids are formed when B. napus
and B. junea are fused.

Cybrids:
The cytoplasmic hybrids where the nucleus is derived from only one parent and the
cytoplasm is derived from both the parents are referred to as cybrids. The phenomenon of
formation of cybrids is regarded as cybridization. Normally, cybrids are produced when
protoplasts from two phytogenetically distinct species are fused. Genetically, cybrids are
hybrids only for cytoplasmic traits.

Hybrids and Somatic Incompatibility:

Many a times, production of full-pledged hybrids through fusion of protoplasts of distantly
related higher plant species is rather difficult due to instability of the two dissimilar genomes
in a common cytoplasm. This phenomenon is referred to as somatic incompatibility.

Hybrids formed despite somatic incompatibility may exhibit structural and developmental
abnormalities. Several generations may be required to eliminate the undesirable genes. Due to
this limitation in somatic hybridization, cybridization involving protoplast fusion for partial
genome transfer is gaining importance in recent years.

Methodology of Cybridization:
A diagrammatic representation of the formation of hybrids and cybrids is given in Fig. 44.9.

Hybridization and Cybridization

As the formation of heterokaryon occurs during hybridization, the nuclei can be stimulated to
segregate so that one protoplast contributes to the cytoplasm while the other contributes
nucleus alone (or both nucleus and cytoplasm). In this way cybridization can be achieved.
Some of the approaches of cybridization are given hereunder:

1. The protoplasts of cytoplasm donor species are irradiated with X-rays or Ƴ-rays. This
treatment renders the protoplasts inactive and non-dividing, but they are efficient donors of
cytoplasmic constituents when fused with recipient protoplasts.

2. Normal protoplasts can be directly fused with enucleated protoplasts. Enucleated

protoplasts can be isolated by high-speed centrifugation.

3. Protoplasts are inactivated by metabolic inhibitors such as iodoacetate. In practice,

iodoacetate treated protoplasts are fused with X-rays irradiated protoplasts for more efficient
formation of cybrids.

4. It is possible to suppress nuclear division in some protoplasts and fuse them with normal
protoplasts.

Genetic recombination in Asexual or Sterile Plants:

There are many plants that cannot reproduce sexually. Somatic hybridization is a novel
approach through which two parental genomes of a sexual or sterile plants can be brought
together. Thus, by fusing parental protoplasts, fertile diploids and polyploidy can be
produced.

Overcoming Barriers of Sexual Incompatibility:

Sexual crossing between two different species (interspecific) and two different genus (inter-
generic) is impossible by conventional breeding methods. Somatic hybridization overcomes
the sexual incompatibility barriers.

Two examples are given hereunder:

1. Fusion between protoplasts of potato (Solanum tuberosum) and tomato (Lycopersicon

esculentum) has created pomato (Solanopersicon, a new genus).

2. Interspecific fusion of four different species of rice (Oryza brachyantha, O. elchngeri, O.

officinalis and O. perrieri) could be done to improve the crop.
A list of selected examples of somatic hybrids developed by interspecific protoplast fusion is
given in Table 44.4.

Somatic Hybrids

A Novel Approach for Gene Transfer:

Somatic hybridization has made it possible to transfer several desirable genetic characters
among the plants (Table 44.5).

Applications of Cybrids:
Cybridization is a wonderful technique wherein the desired cytoplasm can be transferred in a
single step. Cybrids are important for the transfer of cytoplasmic male sterility (CMS),
antibiotic and herbicide resistance in agriculturally useful plants.

Some of the genetic traits in certain plants are cytoplasmically controlled. This includes some
types of male sterility, resistance to certain antibiotics and herbicides. A selected list of
agronomic characters transferred through cybrids is given in Table 44.5 (along with somatic
hybrids).

Cybridization has been successfully used to transfer CMS in rice. Cybrids of Brassica
raphanus that contain nucleus of B. napus, chloroplasts of atrazinc resistant B. campestris and
male sterility from Raphanus sativas have been developed.

Applications of Somatic Hybridization:

Somatic hybridization has opened new possibilities for the in vitro genetic manipulation of
plants to improve the crops.

Some of the practical application are briefly given:

1. Disease resistance:

Several interspecific and inter-generic hybrids with disease resistance have been created.
Many disease resistance genes (e.g., tobacco mosaic virus, potato virus X, club rot disease)
could be successfully transferred from one species to another. For example, resistance has
been introduced in tomato against diseases such as TMV, spotted wilt virus and insect pests.

2. Environmental tolerance:

The genes responsible for the tolerance of cold, frost and salt could be successfully
introduced through somatic hybridization, e.g., introduction of cold tolerance gene in tomato.

3. Quality characters:

Somatic hybrids for the production of high nicotine content, and low erucic acid have been
developed (Table 44.5).

List of Genetic Traits

4. Cytoplasmic male sterility:

ADVERTISEMENTS:

A modification of hybridization in the form of cybridization has made it possible to transfer

cytoplasmic male sterility.

Other Application of Somatic Hybridization:

1. Somatic hybridization has helped to study the cytoplasmic genes and their functions. In
fact, the information is successfully used in plant breeding programmes.

2. Protoplast fusion will help in the combination of mitochondria and chloroplasts to result in
a unique nuclear-cytoplasmic genetic combination.

3. Somatic hybridization can be done in plants that are still in juvenile phase.

4. Protoplast transformation (with traits like nitrogen fixation by incorporating exogenous

DNA) followed by somatic hybridization will yield innovative plants.
Limitations of Somatic Hybridization:
Although somatic hybridization is a novel approach in plant biotechnology, there are several
problems and limitations.

The success of the technique largely depends on overcoming these limitations, some of which
are listed below:

1. Somatic, hybridization does not always produce plants that give fertile and visible seeds.

2. Regenerated plants obtained from somatic hybridization are often variable due to
somaclonal variations, chromosomal elimination, organelle segregation etc.

3. Protoplast culture is frequently associated with genetic instability.

4. Protoplast fusion between different species/genus is easy, but the production of viable
somatic hybrids is not possible in all instances.

5. Some of the somatic hybrids, particularly when produced by the fusion of taxonomically
different partners, are unbalanced and not viable.

6. There are limitations in the selection methods of hybrids, as many of them are not efficient.

7. There is no certainty as regards the expression of any specific character in somatic

hybridization.

8. Somatic hybridization between two diploids results in the formation of an amphidiploid

which is not favourable. For this reason, haploid protoplasts are recommended in somatic
hybridization.