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4. Verification and characterization of somatic hybrids
As no system is foolproof and they have their own advantages and disadvantages. Therefore, even after selecting the desired hybrids/cybrids following protoplast fusion, it is required to carry out one or more tests to compare the parent protoplast lines with the putative hybrids. Some of the techniques that can be tried are:
4.1. Morphology: Somatic hybrids in most of the cases show characters intermediate between the two parents such as, shape of leaves, pigmentation of corolla, plant height, root morphology and other vegetative and floral characters. The method is not much accurate as tissue culture conditions may also alter some morphological characters or the hybrid may show entirely new traits not shown by any of the parents.
4.2. Isozyme analysis: Multiple molecular forms of same enzyme which catalyses similar or identical reactions are known as isozymes. Electrophoresis is performed to study banding pattern as a check for hybridity. If the two parents exhibit different band patterns for a specific isozyme the putative hybrid can be easily verified. The isozymes commonly used for hybrid identification include, acid phosphatase, esterase, peroxidase.
4.3. Cytological analysis: Chromosome counting of the hybrid is an easier and reliable method to ensure hybridity as it also provides the information of ploidy level. Cytologically the chromosome count of the hybrid should be sum of number of chromosomes from both the parents. Besides number of chromosomes, the size and structure of chromosomes can also be monitored. However, the approach is not applicable to all species, particularly where fusion involves closely related species or where the chromosomes are very small. Moreover, sometimes the somaclonal variations may also give rise to different chromosome number.
4.4. Molecular analysis: Specific restriction pattern of nuclear, mitochondrial and chloroplast DNA characterizes the plastomes of hybrids and cybrids. Molecular markers such as RFLP, RAPD, ISSR can be employed to detect variation and similarity in banding pattern of fused protoplasts to verify hybrid and cybrid.