Somatic hybridization is a technique for developing hybrid plants by fusing somatic protoplasts from different plant species/varieties, involving steps like protoplast isolation, fusion, selection, and regeneration. Two primary methods for protoplast isolation are mechanical and enzymatic, with the latter providing higher yields and viability. The process enables the creation of novel hybrids, but it also faces limitations such as poor plant regeneration and viability of the fused products.

2. Development of hybrid plants
through the fusion of somatic
protoplasts of two different
plant species/varieties is called
somatic hybridization.

3. Somatic hybridization technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants

4. Isolation of Protoplast
(Separartion of protoplasts from plant tissue)
1. Mechanical Method 2. Enzymatic Method

5. Mechanical Method
Plant Tissue
Collection of protoplasm
Cells Plasmolysis
Microscope Observation of cells
Cutting cell wall with knife
Release of protoplasm

6. Mechanical Method
 Used for vacuolated cells like onion bulb scale, radish and beet
root tissues .
 Low yield of protoplast.
 Laborious and tedious process.
 Low protoplast viability.

7. Enzymatic Method
Leaf sterlization, removal of epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase
Pectinase
Protoplasm released Release of
isolated cells
cellulase
Protoplasm
released
Isolated
Protoplasm

8. Enzymatic Method
 Used for variety of tissues and organs including
leaves, petioles, fruits, roots, coleoptiles,
hypocotyls, stem, shoot apices, embryo
microspores.
 Mesophyll tissue - most suitable source .
 High yield of protoplast.
 Easy to perform.
 More protoplast viability .

9. SOMATIC HYBRIDIZATION
 The technique of hybrid production through the
fusion of isolated somatic protoplast under in vitro
conditions and subsequent development of their
product (heterokaryon) to a hybrid plant is known as
somatic hybridizaton.
 In somatic hybridization the nucleus and cytoplasm of
both parents are fused in the hybrid cell.

10.  Sometimes; nuclear genome of only one parent but
cytoplasmic genes (plastome) from both the parents
are present in the fused hybrid, which known as cybrid
or cytoplasmic hybrid.
 Somatic hybridization involves the following aspects:
1) Fusion of protoplasts .
2) Selection of hybrid cells .
3) Identification of hybrid plants.

11. Protoplast fusion
 It involves mixing of two different genomes and can be achieved
by spontaneous or induced fusion method.
 Spontaneous fusion : -
I. cell fusion is natural process as is observed in case of egg
fertilization.
II. During the course of enzymatic degradation of cell walls,
adjoining protoplasts may fuse to form homokaryocytes
(homocaryons).
III. These fused cells may sometimes contain high number of
nuclei (2-40).
IV. The frequency of homocaryon formation was found to be high
in protoplasts isolated from dividing cultured cells.

12. Induced fusion method :-
I. Isolated protoplasts can be fused by induction.
II. There are several fusion inducing agents which are
collectively referred to as fusogen.
III. example: sodium nitrate ,high gylcol, polyvinyl
alcohol, lysozyme, concavalina, electrofusion dextran
and dextran sulphate , fatty acid and esters.

13. TREATMENT WITH SODIUM NITRATE
 Induced fusion by NaNO3 was first reported by power
et al .(1970).
 Isolated protoplasts are exposed to a mixture of 5.5%
carried NaNO3 in 10% sucrose solution.
 Incubation is carried out for 5 minutes at 35 ⁰C .
 Centrifugation for 5 minutes at 200x g

14.  High pH and high sodium Calcium ion treatment :
The different protoplast in one solution are together
treated with condition like high calcium and high ph
so that they fuse together . In some cases such extreme
conditions has proved to be toxic to certain
protoplasts.

16. Poly ethylene glycol treatment
 This has proved to be one of the most effective
methods for protoplast fusion .
 The cells are treated with a concentration of around
30% poly ethylene glycol which binds to plasma
membrane .
 This is treated with calcium solution which being
cationic binds to PEG.
 During washing the PEG pulls out the plasma lemma
leading to fusion of protoplasts in close proximately.

17. Electro fusion technique
 This process involves passing low voltage electric
pulses in a solution of protoplast to be fused so that
they line of for fusion .
 High voltage electric current which leads alteration of
membrane the adjacent protoplast fuse.

18. SELECTION OF HYBRID CELL
 The protoplasts are actually involved in the fusion.
 After the fusion process, the protoplast population
consists of heterogenous mixture of unfused
chloroplast, homokaryons 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 method.

19.  Hybrid cells are cultured on suitable medium
provided with the appropriate culture conditions.
Culture of the hybrid cells

20. Regeneration of hybrid plants
 Plants are induced to regenerate from hybrid calli .
 These hybrid plants must be at least partially fertile, in
addition to having some useful property, to be of any
use in breeding schemes.

21. 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 regarded as
cybridization .
 Normally cybrids are produced when protoplast from
two pythogenetically distinct species are fused.
 Genetically are hybrids only for cytoplasmic traits.

23. 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.
 Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other
quality characters.

24. Limitations of Somatic
hybridization
 Poor regeneration of hybrid plants.
 Non-viability of fused products.
 Not successful in all plants.
 Production of unfavorable hybrids.
 Lack of an efficient method for selection of hybrids.
 No confirmation of expression of particular trait in
somatic hybrids.

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