The following points highlight the six main screening strategies that are to be enforced for the selection of ideal somatic hybrids. The strategies are: 1. Microscopic Visual Selection 2. Auxin Autonomy 3. Chlorophyll Complementation 4. Biochemical Selection 5. Verification by Molecular Screening 6. Chromosomal Analysis.
Strategy # 1. Microscopic Visual Selection:
It is based on the fusion between coloured and colourless protoplast. Microscopic observation of heterokaryons, formed due to complete integration of structural characters of both parental protoplasts and subsequent culture under non-selection conditions and the development of heterokaryons facilitates the selection of potential hybrid cell line.
Strategy # 2. Auxin Autonomy:
In this selection process, protoplasts are subjected to screening by its potential to grow into cell on the medium devoid of auxin. Fusion of protoplasts between the same genotype and unfused protoplast fails to grow in absence of auxin in the medium.
However, fusion between the two target genotype potentiates to grow on the medium in absence of auxin. Mixture of two genetic materials allows hybrid cell lines to become auxin autonomy. Selection of the hybrids based on these approaches has been successful in certain members of leguminaceae.
Strategy # 3. Chlorophyll Complementation:
This approach has been successfully implicated in the selection of somatic hybrids in light sensitive tobacco varieties. Development of green colour colony in culture medium ensures hybrids. This was accomplished by fusion between two homozygous recessive albino mutants of tobacco.
Strategy # 4. Biochemical Selection:
This is based on conferring resistance due to dominant character against certain chemicals like antibiotics, herbicides, etc. These are being considered as resistant markers in the selection of somatic hybrids. For example, protoplast obtained from each parent, and grown separately in the medium contains antibiotics or herbicides, each parental line exhibiting sensitivity.
However, protoplast fusion between two parental types when cultured in the medium containing these chemicals exhibit resistance. The sensitivity trait of each parent will be dominated by resistant trait and will grow on the medium containing antibiotics or herbicides.
Isoenzymes are multiple molecular forms of the same enzyme and execute the same function. Depending on the genotype, isoenzyme acts as specific blue print and exhibit specific banding pattern with respect to their complementation of each parental type. In biochemical analysis, electrophoretic banding of isoenzyme can be analyzed for the verification of hybridity.
Different nature of protoplasts (fused, unfused) are subjected for electrophoretic separation of isoenzyme bands on acrylamide gel. Somtic hybrids display characteristic banding pattern of both the parents. Comparative enzyme profiles of each parental line and hybrid can be seen. The isoenzymes, which have been extensively used in biochemical analyses, are esterases, peroxidases, amylases and alcohol dehydrogenases.
Strategy # 5. Verification by Molecular Screening:
Several molecular techniques like RAPD, RFLP and availability of microsatellites are employed in the screening as well as verification of specific somatic hybrids. Restriction digestion of DNA obtained from unfused and fused protoplast exhibit specific banding profiles and ensures confirmation of hybrids.
Restriction digestion of organelle DNA can boost up effective screening process of hybrid lines and verification of somatic hybrid plant in germplasm. Verification of somatic hybrid, Nicotiana glauca, was successfully carried out by assessing restriction fragments of nuclear DNA, which encodes ribosomal RNA. Recently, availability of specific primers for somatic hybrid has been utilized for hybrid identification through PCR technology.
Strategy # 6. Chromosomal Analysis:
Chromosome count can be adapted for the identification of somatic hybrid cell lines. Somatic hybrid contains sum of chromosomes in the protoplst of two parental types. Besides, variation in chromosome number is common in hybrids. Genetic variation due to structural alteration in chromosome might help in the identification of hybrids.
Polyploid conditions have been witnessed in the protoplast culture, which involves the production of inter specific and inter generic somatic hybrids. On several occasions, variation in the chromosome number is mainly due to multiple fusion of protoplast. In addition, unequal rate of DNA replication in the hybridoma cells results in asymmetric hybrids and consequently exhibit chromosomal variations.