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Department of Genetics and Plant Breeding
Faculty of Agriculture
Sam Higginbottom Institute Agriculture, Technology and Sciences
(Formerly Allahabad Agriculture Institute)
Deemed-to-be-University
Allahabad
2012-13
Mail Id- mail2devhingra@gmail.com
Dev Hingra
HIGHLIGHTS
 Somatic hybridization technique
 Isolation of protoplast
 Mechanical method
 Enzymatic method
 Protoplast fusionProtoplast fusion
• Spontaneous fusion
• Induced fusion
Identification and Selection of somatic hybrid cells
 Hybrid selection
 Culture of the hybrid cells
 Regeneration of hybrid plant
 Advantage of somatic hybridization
 Limation of somatic hybridization
Defination
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 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 plants5. 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 knife
Release 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
Plasmolysed
cells
Pectinase +cellulase 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 FusionProtoplast Fusion
(Fusion of protoplasts of two different genomes)(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion 2. Induced Fusion
Intraspecific Intergeneric Electrofusion
Mechanical
Fusion
Chemofusion
Spontaneous Fusion
Protoplast fuse spontaneously during isolation process
mainly due to physical contact
• Intraspecific produce homokaryons
• 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, aceto-carmine 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
Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other quality
characters
Production of novel interspecific and intergenic
hybrid
Production of fertile diploids and polypoids from
sexually sterile haploids, triploids and aneuploids
 Pomato (Hybrid of potato and tomato)
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
Referance:-
 Singh, B.D. 2011. Biotechnology, 2nd
addition , Kalyani Pubilishers, New Delhi.
 Bhojwani, S.S. and Razdan, M.K. 1998. Plant Tissue Culture: Theroy and
Practice, 2nd
ed. Elsevier, Amsterdam.
 Singh, B.D. 2011. Plant Breeding : Principles and Methods, Kalyani Pubilishers,
New Delhi.
Somatic hybridization

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Somatic hybridization

  • 1. Department of Genetics and Plant Breeding Faculty of Agriculture Sam Higginbottom Institute Agriculture, Technology and Sciences (Formerly Allahabad Agriculture Institute) Deemed-to-be-University Allahabad 2012-13 Mail Id- mail2devhingra@gmail.com Dev Hingra
  • 2. HIGHLIGHTS  Somatic hybridization technique  Isolation of protoplast  Mechanical method  Enzymatic method  Protoplast fusionProtoplast fusion • Spontaneous fusion • Induced fusion Identification and Selection of somatic hybrid cells  Hybrid selection  Culture of the hybrid cells  Regeneration of hybrid plant  Advantage of somatic hybridization  Limation of somatic hybridization
  • 3. Defination Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization
  • 4. 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 plants5. Regeneration of hybrid plants
  • 5. Isolation of Protoplast (Separartion of protoplasts from plant tissue)) 1. Mechanical Method 2. Enzymatic Method
  • 6. 1. Mechanical Method Plant Tissue Collection of protoplasm Cells Plasmolysis Microscope Observation of cells Cutting cell wall with knife Release of protoplasm
  • 7. 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
  • 8. Enzymatic Method Leaf sterlization, removal of epidermis Plasmolysed cells Plasmolysed cells Pectinase +cellulase Pectinase Protoplasm released Release of isolated cells cellulase Protoplasm released Isolated Protoplasm
  • 9. 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
  • 10. 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 Electrofusion Mechanical Fusion Chemofusion
  • 11. Spontaneous Fusion Protoplast fuse spontaneously during isolation process mainly due to physical contact • Intraspecific produce homokaryons • Intergeneric have no importance
  • 12. Induced Fusion  Chemofusion- fusion induced by chemicals • Types of fusogens • PEG • NaNo3 • Ca 2+ ions • Polyvinyl alcohal
  • 13. 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
  • 14. 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
  • 15. Hybrid Selection (Several markers are used ) • Genetic complementation • Phytotoxins • Specific amino acid • Auxin autotrophy • Antibiotics • Auxotrophic and metabolic mutants • Chromosomal analysis • Herbicides
  • 16. Culture of the hybrid cells Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions.
  • 17. 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.
  • 18. Advantages of somatic hybridization Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters Production of novel interspecific and intergenic hybrid Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids  Pomato (Hybrid of potato and tomato)
  • 19. 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
  • 20. 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
  • 21. Referance:-  Singh, B.D. 2011. Biotechnology, 2nd addition , Kalyani Pubilishers, New Delhi.  Bhojwani, S.S. and Razdan, M.K. 1998. Plant Tissue Culture: Theroy and Practice, 2nd ed. Elsevier, Amsterdam.  Singh, B.D. 2011. Plant Breeding : Principles and Methods, Kalyani Pubilishers, New Delhi.