In this article, we will discuss about the isolation and selection techniques of somaclonal variation.
Isolation and selection of somaclonal variation is an important task. Since several changes are involved in producing somaclonal variation in different plant species, it is very difficult to sort out the somaclonal variants using a single selection system. A number of selection systems are now being used to select the variants.
A brief outline of commonly used techniques for detecting variations is given:
Analysis of Phenotypic Characters:
Phenotypic variations may arise among the regenerates during culture. Such variant characters Eire observed thoroughly. A detailed report on observed characters is to be prepared for each variant plants at the active level. After that, variants are transferred from culture flask to the field.
In the field, such variants plants are observed during their successive growth and development. Such qualitative and quantitative phenotypic characters viz., plant height, maturity date, leaf size, flowering date, yield, seed fertility, waxiness in different plant parts, flower morphology etc. are used as a parameter to sort out the variants.
Variants are also compared thoroughly with parental plants in all possible qualitative and quantitative phenotypic characters. Several consecutive seed generations of the variants are analysed to peruse whether the variant characters persist or not among the progeny (Fig 14.1).
Cytological Study of the Variants:
The traditional methods of acetocarmine and Feulgen-stained squashes of meristematic tissue (root tip, leaf tip cells) of the variants permit the study of the number and gross morphology of chromosomes. So any change in chromosome number or gross structures of chromosomes can be detected by this method. To have a better assessment of minor structural changes of chromosomes, banding techniques can be used.
DNA Content of the Variants:
DNA content of the Feulgen stained inter-phase nuclei can be measured by cytophotometer. An uniformly diploid state of cells always maintains its fixed amount of DNA. Any numerical changes of chromosome will show either higher or lower values of DNA content. So the measurement of DNA content can be used as a parameter for rapid screening of variants.
Gel Electrophoresis of Proteins or Enzymes:
A somaclone could be variant for a number of biochemical characters. Among them gel electrophoresis of the proteins or enzyme extract from the homogenized plant parts is a reliable parameter for detecting the variants. Any alternation in electrophoretic pattern of proteins or enzymes indicates that the variants have lost or gained some specific proteins or enzyme fractions.
The relative concentration of particular proteins or enzymes may alter relative to the parental proteins or enzymes. Assay of other biochemical products like pigments, alkaloids, amino acids etc. using the sophisticated instruments have also revealed the extent of variations among the regenerates.
Selection for Disease Resistance:
Sometimes, disease resistance character may appear among the somaclonal variants where the parent is highly susceptible to a particular disease. The pathogen or its toxin can be used as a selection agent during culture.
If the in vitro selection is not feasible on cell, tissue or protoplast culture level, screening at the seedling level is frequently possible. Behnke (1979) regenerated potato plants from callus selected for resistance to the toxin culture filtrate of Phytopthora infestans. Field resistance of some of the sugarcane variants has also been established.
Selection for Herbicide Resistance:
Selection for herbicide resistant phenotypes among the somaclonal variants has also been made. The herbicide is generally added to the cell culture system and the regenerated plantlets showing the tolerance to herbicide are selected. Chaleff (1980) has reported the selection of several Nicotiana tabacum mutants with increased tolerance to picloram.
Selection for Environmental Stress Tolerance:
Salt, water-logging and drought, low and high temperatures and mineral toxicity and deficiency are frequently cited as environmental stresses. Many attempts have been made to isolate stress-tolerant phenotypes in tissue culture. The persistence of stress-tolerant trait in regenerated plants has also been demonstrated.
Selection of high sodium chloride tolerant cell lines in tobacco and the regeneration of plants have been reported by Nabors et al. (1975). Regenerated plants showed salt tolerance through two successive sexual generations. But this may be due to physiological adaptation rather than genetic modification. Few attempts have been made to select for water-logging and drought resistance in cell culture. Handa et al. (1983) has reported somaclonal variation for resistance to polyethylene glycol in tomato cells.
Temperature stress is an unattractive in vitro selection agent. Breideubach and Waring (1977) studied the chilling response in tomato suspension culture and cell lines with enhanced chilling tolerance have been demonstrated by Dix and Street (1976) in tobacco. Wu and Wallner (1983) studied the heat response in suspension culture of pear cells in an effort to provide a biochemical basis of in vitro selection for tolerance to high temperature.
Mineral toxicities and deficiencies are frequently problems in strongly acidic and strongly alkaline soils. Aluminium toxicity at low pH is one of the best known examples. Meredith (1978) reported the stable aluminium resistant variant cell lines in tomato tissue culture. Sorghum plants have also been regenerated after selection against aluminium (Smith et al., 1983).