SOMATIC HYBRIDIZATION&

CYBRIDS

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.

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

Procedure

Protoplast Fusion(Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion 2. Induced Fusion

Intraspecific Intergeneric ElectrofusionMechanicalFusion

Chemofusion

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.

Identification and Selection of somatic hybrid cells

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

• Pigmentation

• Cytoplasmic markers

• Fluorochromes like FITC (fluoroscein isothiocyanate) andRITC (Rhodamine isothiocyanate) are used for labelling ofhybrid cells

• Presence of chloroplast

• Nuclear staining

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

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

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

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

May be complete in two to several days

Although protoplast in culture generally start regenerating a cell wall within afew hours after isolation.

Protoplast lose their characteristic spherical shape once the wall formation iscomplete.

Regeneration of cell wall can be demonstrated using Calcalfluor White STfluoresecent Stain or Tinapol solution

Cell Wall Regeneration

• 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.

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.

Advantages of somatic hybridization

Production of novel interspecific and intergenic hybrid

Pomato (Hybrid of potato and tomato)

Production of fertile diploids and polypoids from sexuallysterile 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 whichcannot be propagated by vegetative means

Studies on the fate of plasma genes

Production of unique hybrids of nucleus and cytoplasm

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.

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

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.

Cybridization

Possibility of injection of nucleus of A to B.

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.

Usesi) 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.

Usesi) Chloroplasts replacement in Brassica confers resistance to herbicides such as

Atrazine.ii) CMS

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.

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