Methods of Hybridisation and Cybridisation
The method of production of somatic hybrid is called somatic hybridisation, and that of somatic cybrid as cybridisation.
Both are also referred to as parasexual phenomenon. Somatic hybrids and cybrids are not produced through sexual fertilisation but produced through fertilisation of somatic cells.
The methods of hybridisation and cybridisation are discussed below:
(a) Formation of Hybrids and Cybrids:
First the protoplasts of two different plants are isolated. Then these are allowed to fuse in the presence of fusogen chemical (i.e. fusion inducing chemicals e.g. 0.6 ml of polyethylene glycol), NaNO3, lysozyme, dextran, etc. PEG is the most widely used fusogen chemical.
Electrofusion [use of electric pulse (i.e. 5-12 amp DC pulse) to induce fusion] is also used to induce protoplast fusion. Generally, 250- 100% protoplasts are involved in fusion event.
But 50-100% heterokaryon formation has been reported. The nuclei of fused protoplast may fuse together or they remain separate cells containing two different nuclei are called heterokaryon.
When two nuclei of binucleate heterokaryon fuse, there develop somatic hybrids. In contrast, in binucleate heterokaryon, when one nucleus degenerates, the resulting fusion product is called cybrid (i.e. cytoplasmic hybrid or heteroplast).
(b) Selection of Somatic Hybrids and Cybrids:
After fusion protoplasts on culture media regenerate .cell walls and undergo mitosis. This results in mixed population of parental cells, homokaryotic fusion product and hybrids. Hybrid cells should be differentiated from the other cells.
There is various selection methods used for selection of fusion products. The selection methods are dependent on: (i) physical properties of fused cells, (ii) biological properties of fused cells, and (iii) biological properties of colonies formed from fused cells.
The somatic hybrids cannot be identified. Hence, biochemical markers are required for this purpose.
In 1972, for the first time P.S. Carlson and co-workers produced first interspecific somatic hybrids between Nicotiana glauca and N. langsdorfii.
In 1978, Melchers and co-workers developed first intergenic somatic hybrids between potato (Solatium tuberosum) and tomato (Lycopersicon esculentum). The somatic hybrid was called pomato or topato.
P.S. Carlson and co-workers (1978) used nutrition requirement of mesophyll chloroplasts. They found that protoplasts of somatic hybrid grew and formed callus, while parental types did not form calli.
1. Besides, drug sensitivity test and auxotrophic mutant-based selection are also applied.
2. In most of somatic hybridisation experiment, selection procedure includes fusion of chlorophyll-deficient (non-green) chloroplasts of one parent with the green protoplasts of the other parents. This help in visual selection of heterokaryon under microscope.
3. Biochemical method is used for selection of hybrids and cybrids by staining some isozymes (e.g. esterases and peroxidases) and fluorescent dyes (FACS). Radio-immunoassay techniques were employed in selection of high yielding lines of Catharanthus and Anchusa cells. It could be made more efficient by making automatic via using flow-cytometric sorting systems.
4. Fluorescent antibodies can also be used to detect the desired component present on the cell surface.