SOMATIC HYBRIDIZATION AND SCREENING TECHNIQUES OF SOMATIC HYBRID MATERIAL
Somatic Hybridization involves fusion of two closely related or distantly related plant protoplast at Interspecific, Intraspecific, Intergeneric and interfamily level for the regeneration of hybrid cell into a hybrid plant.
Somatic hybrids containing complete nuclear complements of both parents are generally rare but have recently been produced in various plant families.
The possible reason for this cytological phenomenon could be that the chromosomes are highly homologous and chromosomal rearrangement occurred during fusion and regeneration. However, most regenerated fusion products don’t have an additive genome. Different causes of elimination have been proposed by Wang et al
(1) A different cell cycle of the remote parents
(2) Smaller centromeres of eliminated chromosomes
(3) DNA methylation of genes involved in centromere function and
(4) The existence of secondary metabolites in the cytoplasm of fusion partners. Whole chromosome block elimination and fragment loss upon genomic rearrangements are other possible explanations.
Hybrid Analysis Methods:
During recent years, a wide array of screening methods has been applied for hybrid analysis during or after protoplast fusion.
They can be subdivided into 4 types.
The first type of tools was used to monitor or direct the fusion event and thus to optimize the entire process. This is done by:
Labelling of Heterokaryons with different fluorescing agents,
Magnetic cell sorting using antibiotic microbeads
Process monitoring using GFP transformed genotypes as fusion partners.
When using fluorescing agents to label parental protoplasts, however, cytotoxic side-effects could disable regeneration.
The second group comprised of cytogenetic tools. Information on ploidy level was obtained directly, through chromosome counting and/or indirectly, through flow cytometry. Genomic/Fluorescence in situ hybridization (GISH/FISH) for hybrid genome determination and meiotic analyses were also described.
The third type of tools was the most popular one, namely molecular markers. In a majority of the publications reporting on complete plant regeneration, the genomic constitution of the hybrids material was investigated with molecular tools. DNA markers were sometimes complemented with isozyme analysis, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or sequence analysis. The most frequently employed molecular markers were Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR), Amplification Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP) and Cleaved Amplified Polymorphic Sequence (CAPS). PCR-RFLP and CAPS analysis using mitochondrial or chloroplast universal primer pairs have proven to be efficient and reliable methods for characterizing the cytoplasmic genome.
Southern blotting for chloroplast DNA and mitochondrial DNA was employed to screen cytoplasm DNA, whereas Northern blotting was used once for chloroplast evaluation. Also, sequencing of common bands and searching for restriction endonuclease sites could be cheaper and more convenient than actual CAPS analysis (though after sequencing CAPS could be used to confirm the results). Sequence analysis yielded SNPs that were used for restriction mapping and CAPS marker development.
Somewhat less conventional techniques were occasionally used, such as
Inter-Retrotransposon Amplified Polymorphism (IRAP)/ Retrotransposon-microsatellite Amplified Polymorphism (REMAP)
Microsatellite anchored fragment polymorphism,
Semi-quantitative real-time PCR
Inter-simple Sequence Repeat (ISSR)
Internal Transcribed Spacer (ITS)
DNA methylation-sensitive amplification polymorphism (MSAP) technologies
Spectrometric methods like High-performance Liquid Chromatography (HPLC) (Han et al., 2009), constituted the fourth group of screening tools but were not used very often. Finally, tools were created to determine the practical value of the screening products, such as bio tests for abiotic and biotic tolerance or resistance, pollen fertility testing, fatty acid analysis or determination of diverse agronomical traits.
Applications of Somatic Hybridization:
Production of Novel interspecific and intergeneric hybrid
For example Pomato hybrid of potato and tomato
Production of fertile diploid and polyploidy from sexually sterile haploids, triploids and aneuploidy.
Transfer gene for disease resistance, biotic stress resistance, herbicide resistance and many other quality characters.
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.