SOMATIC HYBRIDIZATIONSOMATIC HYBRIDIZATION

Rubina Begum, MS. PhD1; Jayanti Tokas, MS, PhD1; Shalini Jain, MS, PhD2 and

Hariom Yadav, MS, PhD2

1Department of Biotechnology, JMIT, Radaur2NIDDK, National Institutes of Health, Bethesda, MD 20892, USA

Corresponding: yadavhariom@gmail.com

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

somatic hybridizationsomatic hybridization

Somatic hybridization technique

1. isolation of protoplast1. isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells4. Culture of the hybrid cells

5. Regeneration of hybrid plants 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

Collection of protoplasm

Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knifeRelease of protoplasm

1. Mechanical Method

Used for vacuolated cells like onion bulb Used for vacuolated cells like onion bulb scale, radish and beet root tissuesscale, radish and beet root tissues

Low yield of protoplastLow yield of protoplast Laborious and tedious processLaborious and tedious process Low protoplast viabilityLow protoplast viability

Enzymatic Method

Leaf sterlization, removal of epidermis

Plasmolysed cells

Plasmolysed cells

Pectinase +cellulase Pectinase

Protoplasm releasedRelease of

isolated cellscellulase

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 FusionProtoplast Fusion(Fusion of protoplasts of two different genomes)(Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion 2. Induced Fusion

Intraspecific Intergeneric ElectrofusionMechanical

FusionChemofusion

Spontaneous Fusion

Protoplast fuse spontaneously during Protoplast fuse spontaneously during isolation process mainly due to physical isolation process mainly due to physical contactcontact

• Intraspecific produce homokaryonesIntraspecific produce homokaryones• Intergeneric have no importanceIntergeneric have no importance

Induced Fusion

Chemofusion- fusion induced by chemicalsChemofusion- fusion induced by chemicals

• Types of fusogensTypes of fusogens• PEGPEG

• NaNoNaNo33

• Ca Ca 2+2+ ions ions• Polyvinyl alcohalPolyvinyl alcohal

Induced Fusion

Mechanical Fusion- Physical fusion of Mechanical Fusion- Physical fusion of protoplasts under microscope by using protoplasts under microscope by using micromanipulator and perfusion micromanipulator and perfusion micropipettemicropipette

Electrofusion- Fusion induced by electrical Electrofusion- Fusion induced by electrical stimulationstimulation

• Pearl chain of protoplasts is formed by low strength Pearl chain of protoplasts is formed by low strength electric field (10kv melectric field (10kv m-1-1) )

• Fusion of protoplasts of pearl chain is induced by the Fusion of protoplasts of pearl chain is induced by the application of high strength electric field (100kv mapplication of high strength electric field (100kv m-1-1) ) for few microsecofor few microseco

Identification and Selection of Identification and Selection of somatic hybrid cellssomatic hybrid cells

Hybrid identification- Based on difference Hybrid identification- Based on difference between the parental cells and hybrid cell between the parental cells and hybrid cell with respect to with respect to

• Pigmentation Pigmentation • Cytoplasmic markers Cytoplasmic markers

• Fluorochromes like FITC (fluoroscein isothiocyanate) Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC (Rhodamine isothiocyanate) are used for and RITC (Rhodamine isothiocyanate) are used for labelling of hybrid cellslabelling of hybrid cells

• Presence of chloroplastPresence of chloroplast• Nuclear stainingNuclear staining

• Heterokaryon is stained by carbol-fuschin, aceto-Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein staincarmine or aceto-orcein stain

Hybrid SelectionHybrid Selection

(Several markers are used )(Several markers are used )

• Genetic complementationGenetic complementation• Phytotoxins Phytotoxins • Specific amino acidSpecific amino acid• Auxin autotrophyAuxin autotrophy• AntibioticsAntibiotics• Auxotrophic and metabolic mutantsAuxotrophic and metabolic mutants• Chromosomal analysisChromosomal analysis• HerbicidesHerbicides

Culture of the hybrid cellsCulture of the hybrid cells

Hybrid cells are cultured on Hybrid cells are cultured on suitable medium provided with suitable medium provided with

the appropriate culture the appropriate culture conditions.conditions.

Regeneration of hybrid Regeneration of hybrid plantsplants

Plants are induced to regenerate from Plants are induced to regenerate from hybrid calli hybrid calli

These hybrid plants must be at least These hybrid plants must be at least partially fertile, in addition to having partially fertile, in addition to having some useful property, to be of any use some useful property, to be of any use in breeding schemes. in breeding schemes.

Advantages of somatic Advantages of somatic hybridizationhybridization

Production of novel interspecific and Production of novel interspecific and intergenic hybridintergenic hybrid Pomato (Hybrid of potato and tomato)Pomato (Hybrid of potato and tomato)

Production of fertile diploids and polypoids Production of fertile diploids and polypoids from sexually sterile haploids, triploids and from sexually sterile haploids, triploids and aneuploidsaneuploids

Transfer gene for disease resistance, Transfer gene for disease resistance, abiotic stress resistance, herbicide abiotic stress resistance, herbicide resistance and many other quality resistance and many other quality characterscharacters

Advantages of somatic Advantages of somatic hybridizationhybridization

Production of heterozygous lines in the Production of heterozygous lines in the single species which cannot be single species which cannot be propagated by vegetative meanspropagated by vegetative means

Studies on the fate of plasma genesStudies on the fate of plasma genes Production of unique hybrids of nucleus Production of unique hybrids of nucleus

and cytoplasmand cytoplasm

Limitations of Somatic Limitations of Somatic hybridizationhybridization

Poor regeneration of hybrid plantsPoor regeneration of hybrid plants Non-viability of fused productsNon-viability of fused products Not successful in all plants. Not successful in all plants. Production of unfavorable hybridsProduction of unfavorable hybrids Lack of an efficient method for Lack of an efficient method for

selection of hybridsselection of hybrids No confirmation of expression of No confirmation of expression of

particular trait in somatic hybridsparticular trait in somatic hybrids