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Unformatted text preview: SOMATIC HYBRIDIZATION Rubina Begum, MS. PhD1; Jayanti Tokas, MS, PhD1; Shalini Jain, MS, PhD2 and Hariom Yadav, MS, PhD2 1 Department of Biotechnology, JMIT, Radaur 2 NIDDK, National Institutes of Health, Bethesda, MD 20892, USA Corresponding: email@example.com 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 Isolation of Protoplast (Separartion 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, coleoptiles, 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 isolation process mainly due to physical contact Intraspecific produce homokaryones Intergeneric have no importance Induced Fusion Chemofusion- fusion induced by chemicals Types of fusogens PEG NaNo3 Ca 2+ ions Polyvinyl alcohal 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 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, acetocarmine or aceto-orcein stain Hybrid Selection (Several markers are used ) Genetic complementation Phytotoxins Specific amino acid Auxin autotrophy Antibiotics Auxotrophic and metabolic mutants Chromosomal analysis Herbicides 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 intergenic hybrid interspecific and 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 ...
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- Fall '11