Somatic hybridization involves the fusion of isolated plant protoplasts to generate hybrid cells and plants combining the genetic material of both parental species. It allows for the production of novel interspecific and intergeneric hybrids not achievable through sexual hybridization. The key steps are isolating protoplasts using enzymatic methods, fusing the protoplasts using techniques like PEG or electrofusion, selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Somatic hybridization has various applications in plant breeding and crop improvement.

1. SOMATIC HYBRIDIZATION AND
IT’S SIGNIFICANCE

2. INTRODUCTION
Somatic Hybridization:
The technique of hybrid production
through the fusion of isolated somatic
protoplasts under in vitro conditions
and subsequent development of their
product (heterokaryon) to a hybrid
plant.
Somatic Hybrids:
Hybrids obtained by somatic
hybridization.
In somatic hybridization the nucleus
and cytoplasm of both parents are fused
in the hybrid cell.

3. Types of Somatic Hybridization:
Based on taxonomic relationship of species involved in the
hybridization, somatic hybrids are of three types, viz.:
i. Interspecific Hybrids:
ii. Intergeneric Hybrids:
iii. Intertribal Hybrids:
Based on combination of chromosomes and cytoplasm from
parental species, somatic hybrids are of three types, viz.:
(i) Symmetrical hybrids,
(ii) Asymmetrical hybrids, and
(iii) Cybrids.

4. The term protoplast- Hanstein(1880).
The first isolation of protoplasts was
achieved by Klercker (1892) - mechanical
method.
In 1960, Cocking used an enzymatic method
for the removal of cell wall.
Somatic hybrids was firstly introduced by
Carlson in 1972 in Nicotiana guauca.
Literature......
.
Hanstein
Klercke
r

5. Somatic hybridization
Involves 4 major steps:
1.Isolation of Protoplasts.
2.Culture of Protoplasts.
3.Fusion of Protoplasts and
4.Subsequent regeneration of
hybrid cells into whole
plants.

7.  Sexual hybridization: involves fusion
of the nuclear genes of both the
parents.
 Somatic hybridization: involves
even cytoplasm from both
the parental species in hybrid
obtained by protoplast fusion.
 Cybridization: Hybrids
containing nuclear genome
of one parent but cytoplasm from
both the parents.

9. MECHANICAL METHOD:
A small piece of epidermis from a
plant is selected.
The cells are subjected to
plasmolysis. This causes
protoplasts to shrink away from the
cell walls.
The tissue is dissected to release
the protoplasts.

10. ENZYMATIC METHOD:
1.Sequential Method (Two Step):
The tissue is first treated with pectinase (macerozyme) to separate cells by
degrading middle lamella.
These free cells are then exposed to cellulose to release protoplasts.
Pectinase breaks up the cell aggregates into individual cells while cellulose
removes the cell wall proper.
2. Simultaneous Method (One Step):
This is the preferred method for protoplast isolation. It involves the
simultaneous use of both the enzymes — macerozyme and cellulose.

11. Protocol...
 The cells and tissue are incubated in the enzyme mixture for few to several
hours; naked protoplasts devoid of cell wall are gradually released in the
enzyme mixture.
 Protoplast have been isolated from virtually all plant parts, but leaf
mesophyll is the most proffered tissue, for this purpose.
 In general, fully expanded leaves are surface sterilized, their lower epidemies
is peeled off with a pair of forceps and the peeled areas are cut into small
pieces with the enzymes mixture; vacuum infiltration may be used to
facilitate the entry of enzymes into the tissues.
 After the period of incubation, protoplast are washed with a suitable
washing medium in order to remove the enzymes and the debris.
 The protoplasts may be cultured on a suitable medium in a variety of ways:
(1) Bergmann`s plating technique (in agar medium), (2) in a thin layer of
liquid medium, and (3) in small micro drops of 50-100 ul.
 Protoplast readily regenerate cell wall (within 2-4days)and undergo mitosis
to form macroscopic colonies, which can be induced to regenerate whole
plants.

12. PROTOPLAST CULTURE
Nutritional Media:
 In general, the nutritional requirements of protoplasts are similar to those of
cultured plant cells (callus and suspension cultures).

 MS and B5 media with suitable modifications are used.
 The medium should be devoid of ammonium, and the quantities of iron and zinc
should be less.
The concentration of calcium should be 2-4 times higher than used for cell cultures.
This is needed for membrane stability.
 High auxin/kinetin ratio is suitable to induce cell divisions while high kinetin/auxin
ratio is required for regeneration.
 Glucose is the preferred carbon source by protoplasts although a combination of
sugars (glucose and sucrose) can be used.
 The vitamins used for protoplast cultures are the same as used in standard tissue
culture media.

14.  As the isolated protoplasts are devoid of cell wall, in vitro
fusion becomes relatively easy. There are no barriers of
inability for the protoplast fusion.
 Somatic cell fusion appears to be the only means through
which two different parental genomes can be recombined
among plants that cannot reproduce sexually.
 Protoplast fusion involves the mixing of protoplast of two
different genomes. This can achieved by spontaneous,
mechanical, or induced fusion methods.
A. Fusion of protoplasts

15.  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 protoplast may fuse to
form homokaryons.
 These fused cells may
sometimes contain high number
of nuclei(2-40).
 Spontaneously fused protoplast,
however, cannot regenerate into
whole plants, except undergoing
a few cell divisions.

16.  Mechanical fusion
 The protoplast can be fused by
pushing them together
mechanically. Protoplast of lilium
and trillium can be fused in enzyme
solution by gentle trapping in a
depression slide.
 Mechanical fusion may damage
protoplasts by causing injuries.

17. Mechanism of fusion
The fusion of protoplast involves three phases
1.Agglutination
2.Plasma membrane fusion and
3.Formation of heterokaryons.
1.Agglutination(adhesion):
when two protoplast are in close contact with each other, adhesion
occurs.Agglunation can be induced by fusogens eg.PEG,high pH and high Ca2+.
2. Plasma membrane fusion:
Protoplast membrane 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.
High pH and high Ca2+ions neutralized the surface charges on the protoplasts.
This allows closer contact and membrane fusion between agglutinated
protoplast.

18. Cont...
 High temperature helps in the intermingling of lipid molecules
of agglutinated protoplast membrane so that membrane
fusion occurs.
 PEG causes rapid agglutination and formation and formation
of clumps of protoplast. This results in the formation
membrane fusion occurs.
 3.Formation of heterokaryons
 The fused protoplast get rounded as a result of cytoplasmic
bridges leading to the formation of spherical homokaryon or
heterokaryon.

19.  Induced fusion
 Freshly isolated protoplasts can be fused by
induction.
 There are several fusion-inducing agents
which are collectively referred to as
fusogens eg. NaNO3,high
pH,ca2+,polyethylene glycol, polyvinyl
alcohol….
A) Sodium nitrate Treatment
– Transfer protoplasts to 0.25M NaNO3
and then centrifuge.
– Low rate of heterokaryons.
– Not used much today.
• This method was successfully utilized for
fusion of protoplasts from root tips of oat
and maize seedlings but is not
preferred due to low frequency of fusion,
particularly when highly vacuolated
mesophyll protoplasts are used.

20. Click to add Title
b) High pH/Ca++
Induced Fusion Treatment
– Solution of 0.4M mannitol containing 0.05 M
CaCl2 with pH at 10.5, 370C
– Aggregates within 10 minutes
– Successful method.
This method involves centrifugation of the
protoplasts in a fusion inducing solution
(0.05M CaCl2 2H2O in O.4M mannitol at pH
10) for 30 minutes , after which the tubes are
placed in a water bath (37°C) for 40-50 min.
• This leads to fusion of 20-50% of the
protoplasts.
• The method has been found superior to other
methods in some cases, but high pH may be
toxic in other cases.

21. Reproducible high production of heterokaryons
Add 0.6 ml of PEG solution to pellet of protoplasts in a tube.
Incubate at room temperature for 40 minutes with occasional rocking.
After fusion, add 0.5 - 1.0 ml culture medium and centrifuge.
Re-suspend protoplasts in culture medium
Disturbs the charge on the membrane
Protoplast fusion has been successfully
achieved in several crops, using
polyethylene glycol as a fusogen.
The technique gives high frequency of
fusion with reproducible results and
involves low cytotoxicity.
The technique can be used for fusion of
protoplasts from unrelated plant taxa
(e.g. soybean tobacco, soybean-maize
& soybean-barley), and also between animal and plant
cells.)
C) PEG (polyethylene glycol) - 6000-8000 MW

22. The protoplasm are introduced into a
small fusion chamber containing parallel
wires or plates which serve as
electrodes.
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.125kVcm-
1).
A high voltage DC pulse 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.
ELECTROFUSION

23.  B. Selection of hybrid cells:
 About 20-25% of the protoplast are actually involved in the
fusion. After the fusion process, the protoplast population
consists of a heterogenous mixture of un-fused
chloroplast,homokaryyons and heterokaryons.
 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.
 Biochemical methods.
 The biochemical method 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 difference in the expression of characters.
 There are two approaches in this direction – Drug sensitivity and
auxotrophic mutant selection method.


24. Visual method
• Visual selection of hybrid cells, although tedious is very efficient. In
some of the somatic hybridization experiments, chloroplast deficient
protoplast of one parent are fused with green protoplast of another
parent.
• This facilitates the visual identification of heterokaryons under a light
microscope. The heterokaryons are bigger and green in colour while
the parental protoplast 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.

25. 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 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.
Isoenzyme analysis of hybrid plants –The multiple forms of an enzyme catalysing the
same reaction are referred to as isoenzymes. Electrophoretic pattern of isoenzymes
have been widely used to verify hybrids.
Symmetric and asymmetric hybrids-
If the chromosome number in the hybrid is the sum of the chromosome of the two
parental protoplast, the hybrid is said to be symmetric.
Asymmetric hybrids have abnormal or wide variations in the chromosome number than
the exact total of two species.

28. CULTURE OF THE HYBRID CELLS
Hybrid cells are cultured on suitable
medium provided with the appropriate
culture conditions.

30. REGENERATION OF HYBRID PLANTS

31. SOMATIC HYBRIDIZATION IN
CITRUS/ORANGE

33. Solanaceae
⊷ The family Solanaceae contains the most commonly
used species for somatic hybridization.
⊷ The genera from this family that have been often used
for somatic hybridization include Nicotiana,Datura,
Petunia, Solanum, Lycopersicon, etc. Other families
include Umbellifereae.

34. ADVANTAGES OF SOMATIC HYBRIDIZATION
Production of novel interspecific and intergenic hybrid
Pomato (Hybrid of potato and tomato)
Production of fertile diploids and polypoids from sexually sterile haploids,
triploids and aneuploids.

37. conclution
Somatic hybridization is an important tool of plant breeding
and crop improvements through the production of inter-
specific and inter-generic hybrids. It is valuable for plants
which is asexual and sterile.

38.  Ignacimuthu S.J.,1997, Plant Biotechnology-somatic hybridization and cybridization,5th
edition, Oxford and IBH publishing Co.Pvt.Ltd., New Dehli. Pp: 224-247.
REFERENCES
 http://www.biologydiscussion.com/somatic-hybridization/somatic-hybridization-
aspects-applications-and-limitations/10686