SOMATIC HYBRIDIZATION

PRESENTED BY :Neha AgrawalM.Sc. 3rd Sem Biotech

SUMMITED TO :Dr. GARIMA SHARMA

Development of hybrid plants through the fusion of somatic protoplasts of two different

plant species/varieties is called somatic hybridization.

Somatic hybridization technique1. 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

Isolation of Protoplast (Separartion of protoplasts from plant tissue)

1. Mechanical Method 2. Enzymatic Method

Mechanical MethodPlant Tissue

Collection of protoplasm

Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knife Release of protoplasm

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.

Enzymatic Method

Leaf sterlization, removal of epidermis

Plasmolysed cells

Plasmolysed cells

Pectinase +cellulasePectinase

Protoplasm released Release of isolated cells

cellulase

Protoplasm released

Isolated Protoplasm

Enzymatic MethodUsed 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 .

SOMATIC HYBRIDIZATIONThe 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.

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.

Protoplast fusionIt 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.

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.

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

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.

Poly ethylene glycol treatmentThis 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.

Electro fusion techniqueThis 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.

SELECTION OF HYBRID CELLThe 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.

Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions.

Culture of the hybrid cells

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.

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.

Advantages of somatic hybridizationProduction 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.

Limitations of Somatic hybridizationPoor 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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