SOMATIC HYBRIDIZATIONSOMATIC HYBRIDIZATION

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

Fusion

Chemofusion

Spontaneous Fusion

ProtoplastProtoplast fusefuse spontaneouslyspontaneously duringduring

isolationisolation processprocess mainlymainly duedue toto physicalphysical

contactcontact

•• IntraspecificIntraspecific produceproduce homokaryoneshomokaryones

•• IntergenericIntergeneric havehave nono importanceimportance

Induced Fusion

ChemofusionChemofusion-- fusionfusion inducedinduced byby chemicalschemicals

•• Types of fusogensTypes of fusogens•• PEGPEG

•• NaNoNaNo33

•• Ca Ca 2+2+ ionsions

•• Polyvinyl alcohalPolyvinyl alcohal

Induced Fusion

MechanicalMechanical FusionFusion-- PhysicalPhysical fusionfusion ofofprotoplastsprotoplasts underunder microscopemicroscope byby usingusingmicromanipulatormicromanipulator andand perfusionperfusionmicropipettemicropipette

ElectrofusionElectrofusion-- FusionFusion inducedinduced byby electricalelectricalstimulationstimulation

•• PearlPearl chainchain ofof protoplastsprotoplasts isis formedformed byby lowlow strengthstrengthelectricelectric fieldfield ((1010kvkv mm--11))

•• FusionFusion ofof protoplastsprotoplasts ofof pearlpearl chainchain isis inducedinduced byby thetheapplicationapplication ofof highhigh strengthstrength electricelectric fieldfield ((100100kvkv mm--11))forfor fewfew microsecomicroseco

Identification and Selection of Identification and Selection of

somatic hybrid cellssomatic hybrid cells

HybridHybrid identificationidentification-- BasedBased onon differencedifferencebetweenbetween thethe parentalparental cellscells andand hybridhybrid cellcellwithwith respectrespect toto

•• PigmentationPigmentation

•• CytoplasmicCytoplasmic markersmarkers

•• FluorochromesFluorochromes likelike FITCFITC (fluoroscein(fluoroscein isothiocyanate)isothiocyanate)andand RITCRITC (Rhodamine(Rhodamine isothiocyanate)isothiocyanate) areare usedused forforlabellinglabelling ofof hybridhybrid cellscells

•• PresencePresence ofof chloroplastchloroplast

•• NuclearNuclear stainingstaining

•• HeterokaryonHeterokaryon isis stainedstained byby carbolcarbol--fuschin,fuschin, acetoaceto--carminecarmine oror acetoaceto--orceinorcein stainstain

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

PlantsPlants areare inducedinduced toto regenerateregenerate fromfrom

hybridhybrid callicalli

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

ProductionProduction ofof novelnovel interspecificinterspecific andandintergenicintergenic hybridhybrid PomatoPomato (Hybrid(Hybrid ofof potatopotato andand tomato)tomato)

ProductionProduction ofof fertilefertile diploidsdiploids andand polypoidspolypoidsfromfrom sexuallysexually sterilesterile haploids,haploids, triploidstriploids andandaneuploidsaneuploids

TransferTransfer genegene forfor diseasedisease resistance,resistance,abioticabiotic stressstress resistance,resistance, herbicideherbicideresistanceresistance andand manymany otherother qualityqualitycharacterscharacters

Advantages of somatic Advantages of somatic

hybridizationhybridization

ProductionProduction ofof heterozygousheterozygous lineslines inin thethe

singlesingle speciesspecies whichwhich cannotcannot bebe

propagatedpropagated byby vegetativevegetative meansmeans

StudiesStudies onon thethe fatefate ofof plasmaplasma genesgenes

ProductionProduction ofof uniqueunique hybridshybrids ofof nucleusnucleus

andand cytoplasmcytoplasm

Limitations of Somatic Limitations of Somatic

hybridizationhybridization

PoorPoor regenerationregeneration ofof hybridhybrid plantsplants

NonNon--viabilityviability ofof fusedfused productsproducts

NotNot successfulsuccessful inin allall plantsplants..

ProductionProduction ofof unfavorableunfavorable hybridshybrids

LackLack ofof anan efficientefficient methodmethod forforselectionselection ofof hybridshybrids

NoNo confirmationconfirmation ofof expressionexpression ofofparticularparticular traittrait inin somaticsomatic hybridshybrids