This document provides an introduction to somatic hybridization, which is the fusion of somatic protoplasts from two different plant species or varieties to create hybrid plants. It discusses the history and development of the technique, including early work isolating protoplasts. The key steps in somatic hybridization are described: isolating protoplasts using either mechanical or enzymatic methods, fusing the protoplasts spontaneously or through induction methods, identifying and selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Advantages include creating novel hybrids and transferring desirable traits between species, while limitations include low regeneration rates and viability of fused products.

1. WELCOME

2. SOMATIC HYBRIDIZATION

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

4. History
 Hanstein introduced the term ‘Protoplast’.
 The isolation of protoplasts was first achieved by Klercker (1892) on
plasmolysed cells.
 Cooking (1960) for the first time isolated the protoplasts of plant tissues by
using cell wall degrading enzymes viz., cellulase, hemicellulase, pectinase,
and protease extracted from a saprophytic fungus Trichoderma viride &
Myrothecium verrucaria.
 First achievement in protoplast fusion by Power (1970)

5. Protoplast
 Protoplast also known as a naked plant cell refers to
all the components of plant cell excluding the cell
wall.
 Protoplast is the biologically active and most
significant material of cells.

6.  Plant cell wall acts as physical barrier and protects
cytoplasm from microbial invasion and environmental
stress.
 It consists of a complex mixture of cellulose,
hemicellulose, pectin, lignin, lipids, protein,
 For dissolution of different components of the cell wall it
is essential to have the respective enzymes.

7. 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

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

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

10. 1. 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

11. 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

12. Enzymatic Method
 Used for variety of tissues and organs including leaves,
petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot
apices, embryo microspores
 Mesophyll tissue –
1. most suitable source
2. High yield of protoplast
3. Easy to perform
4. More protoplast viability

13. Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion 2. Induced Fusion
Intraspecific Intergeneric Electrofusion
Mechanical
Fusion
Chemofusion

14. Spontaneous Fusion
 Protoplast fuse spontaneously during isolation process mainly due to
physical contact
• Intraspecific
• Interspecific

15. Intraspecific protoplast fusion
 Intraspecific protoplast fusion is the cross between the same
species in an individual which involves the isogenic strains or
the non-isogenic ones.
 The true value of protoplast fusion as a mean for establishing
parasexual crosses has been realized so far in a few fungi. For
example, in Cephalosporium acremonium.
 This technique offers the only way of carrying out crosses and
genetic analysis.

16. Interspecific protoplast fusion
 Interspecific protoplast fusion is the crosses between two different
species.
 Interspecific protoplast fusions are of much importance in the area
where new products are to be produced. Due to new genetic set up
many noval secondary metabolites such as, antibiotics, phenolics.,
 Some of the examples where interspecific hybrids were produced
through protoplast fusion are:
- S. cerevisiae x S. fermentali,

17. Induced Fusion
 Chemofusion- fusion induced by chemicals
• Types of fusogens
• PEG
• NaNo3
• Ca 2+ ions
• Polyvinyl alcohal

18. (i) NaNO3 Treatment:
 Isolated protoplasts are suspended in mixture of 5.5%
NaCO3 and 10% sucrose soln. and incubated in a water bath at
35°C for 5 min. and centrifuged for 5 min. at 200 x g, following
centrifugation the supernatant is decanted and the pellet is
incubated at 30°C for 30 min.
 During this period most of the protoplasts undergo cell fusion.
After sometimes these are washed with osmoticum and then
plated properly in culture medium.

19. (ii) Calcium Ions at High pH:
 Here the isolated protoplasts are incubated in a solution of
osmoticum containing 0.05 M CaCl2 at pH 10.5 (using 0.05 µ
glycine— NaOH buffer) and at temperature 37°C.
 Aggregation of protoplasts generally takes place at once and
fusion occurs within 10 mins.
 After this treatment, 20-50% of the protoplasts have been
found to be involved in fusion.

20. (iii) PEG Treatment:
 PEG has been found to be used as a fusogen in most of the successful
cases of protoplast fusion. 1 ml of protoplast suspension is equally
mixed with 1 ml of 28-56% PEG (1500-6000 MW) solution. The tube is
then allowed to settle for 10 min to 40 min at room temperature.
 Both the mol. wt. and the conc. of PEG used is critical in inducing
successful fusions. After PEG treatment the elution of fusogen is done
by using high pH/Ca++ containing solution which is most effective in
enhancing the fusion frequency and survivability of protoplasts

22. (iv) Electro-fusion:
 Protoplasts are placed into a small cell culture containing electrodes
and a potential difference is applied due to which protoplasts line up
between the electrodes. In this fusion method, two step procedures is
followed:
 Pearl chain of protoplasts is formed by low strength electric field (10kv
m-1)
 Fusion of protoplasts of pearl chain is induced by the application of
high strength electric field (100kv m-1) for few microsec
 Heterokaryons produced by this electro-fusion divide normally in
culture medium and have the capability of regenerating the plantlet

24. Electro-fusion instrument

26. Identification and Selection of somatic
hybrid cells
 Hybrid identification- Based on difference between the
parental cells and hybrid cell with respect to
• Pigmentation
• Cytoplasmic markers
• Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC (Rhodamine
isothiocyanate) are used for labelling of hybrid cells
• Presence of chloroplast
• Nuclear staining
• Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein
stain

27. Hybrid Selection
(Several markers are used )
• Genetic complementation
• Phytotoxins
• Specific amino acid
• Auxin autotrophy
• Antibiotics
• Auxotrophic and metabolic mutants
• Chromosomal analysis
• Herbicides

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

29. Regeneration of hybrid plants
 Plants are regenerated into whole plant.
 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.

30. Advantages of somatic hybridization
 Production of novel interspecific and intergeneric
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

31. Advantages of somatic hybridization
 Production of heterozygous lines in the single species
which cannot be propagated by vegetative means
 Studies on the fate of plasma genes
 Production of unique hybrids of nucleus and
cytoplasm

32. 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

33. Cybrids
 are cells or plants containing nucleus of one species but
cytoplasm from both the parental species.
 Cybridization – production of somatic cybrid. The process of
protoplast fusion resulting in the development of cybrid is
known as cybridization.

34. New Murcott + Dancy somatic hybrid Somatic cybrid plant of Lee hybrid
(Murcott x Clementine)
pomato
Night shade and tobaco

35. Refrence
 SINGH, B. D., (2011), biotechecnology, 2nd edition, kalyanyani
pub.
 SINGH, B. D., (2011), plant breeding: principles and methods,
kalyanyani pub.
 RAJ, A., (2016), Protoplast fusion and somatic hybridization |
biotechnology.
 http://slideshare,in

36. Thank you