SOMATIC HYBRIDIZATION

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

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

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.

 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.

History
Hanstein introduced the term Protoplast. The isolation of protoplasts from was first achieved through 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)

Isolation of Protoplast (Separation of protoplasts from plant tissue)

1. Mechanical Method

2. Enzymatic Method

1. Mechanical Method
Plant Tissue
Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knife

Release of protoplasm

Collection of protoplasm

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

Enzymatic Method
Leaf sterlization, removal of epidermis

Plasmolysed cells
Pectinase +cellulase

Plasmolysed cells
Pectinase

Protoplasm released

Release of isolated cells

cellulase

Protoplasm released

Isolated Protoplasm

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

Protoplast Fusion (Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion

2. Induced Fusion

Intraspecific

Intergeneric

Chemofusion

Mechanical Fusion

Electrofusion

Spontaneous Fusion
Protoplast fuse spontaneously during process mainly due to physical contact. Intraspecific produce homokaryones Intergeneric have no importance isolation

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.

 Protoplast fusion technique made it possible to produce a preliminary genetic map of 8 linkage groups for C. acremonium. Genes which enhance the production of antibiotics have been identified and allied to specific linkage groups. The other examples are : -Absidia glauca, Candida maltosa, Aspergillus niger, Fusarium graminearum, Penicillium verruculosum, T. reesei, etc.

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 may be produced. Some of the examples where interspecific hybrids were produced through protoplast fusion are: - S. cerevisiae x S. fermentali, - S. cerevisiae x S. lipolytica, - P. chrysogenum x P. notatum,

Induced Fusion
Chemofusion- fusion induced by chemicals

Types of fusogens
PEG NaNo3 Ca 2+ ions Polyvinyl alcohol

Induced Fusion
Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette. Electrofusion- Fusion induced by electrical stimulation 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 microsecond.

Protoplast fusion and somatic hybrids

the fusion process electrofusion protoplasts are aligned in a special chamber, electric current is applied, opening channels in cell membrane PEG fusion protoplasts are coated with PEG, then incubated together; where cell membranes fuse, channels begin to form after fusion, "fusion products" begin to "round up"

Protoplast viability
The most frequently used staining methods for assessing protoplast viability are: - Fluorescein diacetate (FDA) staining - Phenosafranine staining - Calcofluor white (CFW) staining

Fluorescein diacetate (FDA) staining

FDA, a dye that accumulates inside the plasma membrane of viable protoplasts. Viable intact protoplasts fluoresce Yellow green within 5 min. FDA is dissolved in CH3COCH3 & used at a concentration of 0.01%.

Phenosafranine staining
It is specific for dead protoplasts that turn Red in staining procedure. Viable cells remain unstained by Phenosafranine

Calcofluor white (CFW) staining

CFW binds to the -lined glycosides in the newly synthesized cell wall which is observed as a ring of fluorescence around the plasma membrane.

Protoplast density
Protoplasts have both maximum as wellas minimum plating densities for growth. Published procedures suggest that protoplasts should be cultured at a density of 5x103 to 106 cells/ml with an optimum of about 5x104 protoplasts/ml.

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

Culture of the hybrid cells

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

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.

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.

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

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

Application of Somatic hybridization

protoplast fusion to create somatic hybrids "wide crosses" where even embryo culture won't work Citopsis gilletiana (wild) x Citrus sinensis citrus sexually incompatible spp. wild relative has disease/nematode resistance somatic hybrid used as a rootstock

 protoplast fusion to create somatic hybrids Solanum somatic hybrids S. tuberosum dihaploids fused with wild diploid S. chacoense resulting somatic hybrid (4n) is backcrossed to S. tuberosum cultivars (also 4n) overcomes sterility due to ploidy differences between somatic and sexual hybrids

Protoplast culture and regeneration

From the protoplast solution of known density (about 105 protoplast/ml) about 1 ml suspension is poured on sterile and cooled down nutrient medium in Petri dishes. The plates are incubated at 25C in a dim white light.

Protoplast culture and regeneration

The protoplasts regenerate a cell wall, undergo cell division and form callus. The callus can also be subcultured. Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo develops into seedlings and finally mature plants.