SEMINAR ON

Somatic Hybridization
 SOMATIC HYBRIDIZATION
Introduction
Definition
Somatic Hybridization process

Isolation of protoplast
Fussion of protoplast
Selection of hybrid cells
Regeneration of hybrid plants

Types of Somatic Hybridization:-

Symmetric Somatic Hybridization

Asymmetric Somatic Hybridization
Application
Limitations
Reference
INTRODUCTION :-

 Somatic hybridization or para sexual hybridization is a

process In which hybrids are obtained through the fusion off
protoplast.
Belonging to two distinct plant species somatic hybrid are
specifically implicated in the improvement of vegetatively
propagates crop plant such as a Potato, Cassava, Banana, etc.
Grater success in the production of Somatic hybrid involved
effective fusion of protoplast.
DEFINITION :-
When the two different cells of protoplast fussed
with each others & made a new species or (Hybrid cell) then
it is called protoplast fusion or Somatic Hybridization.

SOMATIC HYBRIDIZATION PROCESS :-

This process is completed in following steps :-

Isolation of protoplast
Fusion of protoplast
Selection of hybrid cells
Regeneration of hybrid plants
 ISOLATION OF PROTOPLAST :-

The plant cell is surrounded by cellulogenous cell wall and

connected with adjacent call by middle lamella.
Separation of plant cell and removal of cell wall either
experimentally or mechanically or enzymatic process.
Result in this procedure of breaked, naked plant cell
(with out cell walled cell ) is called protoplast.
This word is given by MANSTEEN in 1880.
The protoplast can be isolated from leaves and callus.
 Basically two methods are used for isolation of protoplast.

i) Mechanical method or non enzymatic method.

ii) Enzymatic Method

i) Mechanical method or non enzymatic method

This method is crude and oldest method for protoplast isolation

it is only an historical method.
It involves chopping and plasmolysis of tissue.
This method is time consuming and laborious.
The number of protoplasts through this method is not too much.
In 1892 KLERCKER first used this method
II. ENZYMATIC METHOD :-

This method is effective in protoplast isolation of many

plant part but in mesophill of leaves it is very useful &
effective.
The following enzyme are used in disslovation of middle
lamella and cell wall eg. Pectinase, cellulase, hemicellulase,
macerozyme etc.
The most widely used commercial enzyme are onozuka –R –10
macerozyme –R –10, pectolyse –Y –23 etc.

Advantage of this method :-

•Large quantity of protoplast isolated.
Cell are not injured as mechanical method.
• Osmotic shrinkage is minimum and the deleterious effects of
excessive plasmolysis are minimized.
• Cooking in 1960 was the first reported the isolation of
protoplasts from the fungus Myrothecium verrucosa.
PROTOPLAST FUSION :-
Protoplast fusion is a physiochemical phenomena.

Broadly speaking, protoplast fusion can be classified in two

steps, such as :-
(A) Spontaneous Fusion
(B) Induced Fusion
Protoplast Fussion

Spontaneous Induced Fussion

Fussion

Mechanical Electro Fussion

Chemo Fussion
Fussion
 SPONTANEOUS FUSION :-

In somatic hybridization spontaneous fusion is of little

significance.

The protoplast once they are freely isolated, do not fuse

spontaneously with each other.

An exception is the protoplast from microsporocytes of some

plants of lily family where the freely isolated protoplast fuse
spontaneously.

This type of spontaneous fusion has been used to produce

interygeneric fusion, e.g. the spontaneous fusion of
microsporocyte protoplast of Lolium lengiflorum and
Trilium kamtschaticum.
INDUCED FUSION :-

This technique has the possibility and ability to combine

different genotype beyond the limits imposed by sexual
process.

(A) – MECHANICAL FUSION:-

In this process the isolated protoplasts are brought into

intimate physical contact mechanically under microscope
using micromanipulator and perfusion micropipette.In this
way the protoplasts are retained and compressed by the
flow of liquid.
By this technique occasional fusion of protoplast has been
observed.
(B) CHEMOFUSION :-
Chemofusion are the must commonly used protoplast fusion
inducing agents.
It agents as known as chemically fusogens.
e.g.
Sodium Nitrate (NaNo3).
Potesium Nitrate (Kno3).
Polyvinyl alchohl and PEG.
A- In 1979, Kao and Michayluk from Canada discovered
an ther fusion inducing chemical PEG.
B- High concentration of Ca2+ & High pH.
• In 1973 KELLER and MELCHER from Germany,
developed this method.
• Take Tobacco protoplast at high temperture (370C) .
• Taken in centrifuge tube and centrifuge from 3 minutes.
Basic protocol for achieving protoplast fusions through the use of chemical
fusogens
 CaCl2

Conditions - H2O
Mannitol
pH – 10.5
• If these conditions are given to protoplast mixture.
• than result is protoplast fusion.

ELECTRO FUSION :-

ZIMMER MANN and SCHEURICH (1981) improved

method for the large scale fusion of plant protoplast.

This method may increase fusion frequency reproducibity.

REGENERATION OF HYBRID PLANTS:-

The use of somatic hybridization in plant breeding depend

on two events
1 Regeneration of somatic hybrid plant.
2 Fertility of somatic hybrid plant.

Many regeneration are achieved by the somatic hybrid but

some somatic hybrid not gives regeneration this
characteristic is called somatic incompatibility.

The reason for somatic incompatibility are not

clearly understood.
 The somatic hybrid are of the following two types :-
A – Symmetric Somatic
Hybridization
B – Asymmetric Somatic Hybridization
SYMMETRICAL SOMATIC HYBRIDS :-

Some somatic hybrid plants retain the full or nearly full somatic
complements of the two parental species these are called
Symmetrical hybrids.

These somatic hybrids can now be used in breeding programmes for

limited gene/chromosome introgression from the species
contributing the haploid protoplast.
ASYMMETRICAL SOMATIC HYBRIDS :-

In this type of hybrid have only one parent character

(dominent )& receive character not high light.
e.g. Dacaus carota + Nicotiana toabaco .

Normally heterokaryon protoplast are selected for somatic

hybridization because :-
Color of both cells are different
Both protoplast cells are complementary of each other.
Different qualities mitochondria chromosomes number
Enzyme are protoplast in heterokaryon
Some important selected somatic hybrids selection of hybrid cells.
a) Interspecific hybridization]
b) Intraspecific hybridization
INTERSPECIFIC HYBRIDIZATION:-
1. Brassica juncea +B. spinescens
2. B.napus +B.carinata
3. B.napus +B.juncea
4. Citrus siensis +C.limon
5. C.sinensis +C.paradisi
6. C.sinensis +C.unshiu
7. Helianthus annuus
+H.gigateus
8. Oryza sativa +O.brachyantha
9. O.sativa +O.eichingeria
INTRASPECIFIC HYBRIDIZATION:-
1) Brassica campestris + Barbarea vulgaris
2) B.juncea + Diplotaxis muralis
3) B.napus + Arabidopsis thaliana
4) B. napus + B. tournefortii a
5) B. napus + Thalaspi perfoliatum a
6) C. aurantifolia + Swinglea glutinosa
7) C. sinensis + Murraya paniculata a
8) Oryza sativa + Echinochola oryzicola
9) Solanum tuberosum + Lycopersicon pimpinellifolium
APPLICATION OF SOMATIC HYBRIDIZATION :-

There are many important applications of somatic

hybridization
Production of novel interspecific & intergeneric cross between
plants that are difficult or impossible by other source.

1 Production of somatic hybrid and cybride.

2 Asymmetric hybrids production.
3 Direct DNA and macro molecule uptake.
4 Protoplast of sexual sterile plants can be fused with fertile
diploid & polyploid.
5 Production of Heterzygous.
6 Somatic cell fusion is useful in the study of cytoplasmic gene
& there activities
LIMITATION OF SOMATIC HYBRIDIZATION :-

It was one suggested that somatic hybrids would be of

great value in crop improvement.

In certain wide crosses elimination of chromosomes from

the hybrid cell is another limitation of somatic
hybridization. In protoplast fusion experiments the
percentage of fusion product is very low.
Lastly for hybrid identification selection and
isolation at the culture level.

There is no standardized method which is

applicable for all materials.
Regeneration capacity of hybrid cell is poor.
Lack of suitable selection.
 REFERENCE

T.A brown
Book – Biotechnology & plant Generic
Resources. 2005

S. S Bojwani and M.K Razdan

Book – Plant tissue culture 2005

B. D Singh
Book – Biotechnology 2007