Somatic hybridization and cybrids,plant tissue culture

1. SOMATIC HYBRIDIZATION
&
CYBRIDS

2. SOMATIC HYBRIDIZATION
Development of hybrid plants through the fusion of somatic protoplasts
of two different plant species/varieties is called somatic hybridization.
This is a non conventional genetic procedure involving fusion between
isolated protoplast under in vitro condition and subsequent development
of their product (heterokaryon) to a hybrid plant.

3. 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

4. Procedure

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

6. Identification and Selection of somatic hybrid cells
1.Selection of somatic hybrids by culturing them on such a medium on which
only somatic hybrids can grow.(Smith et al.1976).
2.Complimentary selection of somatic hybrids on specific culture
medium.(Melscher & Labib 1974).
3.Mechanical isolation by visual means and knowledge of identification of
somatic hybrids.
4.Morphology of the plant after regeneration.

7. 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

8. Hybrid Selection
• Genetic complementation
• Phytotoxins (soybean resistant to Hm T toxin whereas Zea mays sensitive to it)
• Specific amino acid (Conavalin present in soybean but not in sweet clover, alfalafa)
• Auxin autotrophy (N. glauca and N. langsdorffi)
• Antibiotics
• Auxotrophic and metabolic mutants
• Chromosomal analysis
• Herbicides

9. Culture of the hybrid cells
Hybrid cells are cultured on suitable medium
provided with the appropriate culture conditions.
• Iscove's MDM with 25 mM Hepes.
• DMEM/F-12 with 15 mM HEPES.
• Alpha MEM with Nucleosides
• RPMI 1640 Medium

10. 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.
Caipira sweet orange + Rangpur lime

11. Cell Wall Regeneration
 May be complete in two to several days
 Although protoplast in culture generally start regenerating a cell wall within a
few hours after isolation.
 Protoplast lose their characteristic spherical shape once the wall formation is
complete.
 Regeneration of cell wall can be demonstrated using Calcalfluor White ST
fluoresecent Stain or Tinapol solution

12. • Symmetric hybrids: These contain the somatic chromosome
of both the parental species. These are very significant as they
show all the properties exhibited by parent species.
Asymmetric hybrids: These are those hybrids which preserve
the genetic material of one parent organism. The
chromosome content of other parent species is lost.

13. Advantages of somatic hybridization
• 1. Symmetric hybrids can be produced between species, which cannot be
hybridized sexually. These hybrids can be readily used in breeding programmes for
transfer of useful genes to crops or may be useful as new species.
2. Hybrids can be produced even between such strains, which are completely
sterile, e.g., monoploids.
3. Cytoplasm transfers can be affected in one year, while backcrossing may take 15-
16 years. Even where backcrossing is not applicable, cytoplasm transfers can be
made using this approach.
4. Mitochondria of one species can be combined with chloroplasts of another
species. This may be very important in some cases, and is not achievable by sexual
means even between easily crossable species.
5. Recombinant organellar genomes, especially of mitochondria, are generated in
somatic hybrids and cybrids. Some of these recombinant genomes may possess
useful features.

14. 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

15. 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

16. Disadvantages of Somatic hybridization
• 1. Techniques for protoplast isolation, culture and fusion are not available
for many important crop species like many cereals and pulses.
2. In many cases, chromosome elimination occurs from somatic hybrids
leading to asymmetric hybrids. Such hybrids may be useful, but there is no
control on chromosome elimination.
3. Many somatic hybrids show genetic instability, which may be an
inherent feature of some species combinations.
4. Many somatic hybrids either do not regenerate or give rise to sterile
regenerates. Such hybrids are useless for crop improvement. All
interfamily somatic hybrids are genetically are unstable and/or
morphologically abnormal, while intergeneric and intertribal hybrids are
genetically stable but produce abnormal and/or sterile plants or only
teratomata.

17. Disadvantages 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

18. CYBRID
• Plants or cells witch containing nucleus of one species but cytoplasm from
both the parental species .
• Cytoplasmic hybrids.
• Involves fusion of two protoplasts (donor and recipient) in which donor
nuclear genome is eliminated while its plastome and/or chondriome are
merged with that of the recipient.
• For production of a cybrid cell uses two parental cells but should be one
complete cell (cytoplasm with nucleus ) and other should be without
nucleus or in active nucleus
i) Suppression of nuclear genome during fusion.(uses gamma rays and X-rays )
ii) Selection of the fusion products that have only the extranuclear genomes of
a donor.

19. Cybridization
Possibility of injection of nucleus of A to B.

20. Aims of cybridization
• To obtain biological information on intra- and intergeneric transfer of
organelles.
• To combine cytoplasm gene of one species to anther species witch have
nucleus with cytoplasm.
• This is also a way for those species witch don’t perform to sexual
reproduction with each other, so this way provided to facilitate to make a
desire species with combination of both species.
Uses
i) Interaction of nuclear genome and plastome and/or chondriome of another
species.
ii) Phylogenetic limit of compatibility between nucleus and plastome and/or
chondriome.
• To replace organelles such as chloroplast and mitochondria with that of
another genotype.
Uses
i) Chloroplasts replacement in Brassica confers resistance to herbicides such as
Atrazine.
ii) CMS

21. Benefits
• Transfer of plasma genes of one species to nuclear
background of another species.
• Production of wide range of genetic variations, sexually
incompatible combinations.
• Useful for sterile plants.
• Mitochondrial gene also combined with chloroplast gens of
another species.
• Plants like medicinal plant, crop plants, sterile pants, or many
other valuable species also included in this.

22. Thank you