SOMATIC HYBRIDIZATION

SOMAT MA IC HYBRIDIZAT A ION Rubina Begum, MS. PhD1; Jayanti Tokas, MS, PhD1; Shalini Jain, MS, PhD2 and Hariom Yadav, MS, PhD2 1Department of Biotechnology, JMIT, Radaur 2NIDDK, National Institutes of Health, Bethesda, MD 20892, USA Corresponding: yadavhariom@gmail.com

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

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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

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

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

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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

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

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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

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

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

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

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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 microseco

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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

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

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

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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.

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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

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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

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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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