Isolation of Somaclonal Variants
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. Selection without Selection Pressure:
Unorganised callus and cells, grown in cultures for various periods on a medium that contains no selective agents, are induced to differentiate whole plants. The regenerated plants are ultimately transferred to the field and screened for variation. Somaclonal variants of various crops like sugarcane, potato, tomato, granium, ceneals and grasses and lucern have been isolated for various desirable traits.
B. Selection with Selection Pressure:
In this method variant cell lines are screened from cultures by their ability to survive in the presence of a substance in medium that may be toxic or under condition of environmental stress. E.g. Amino acid analogue and amino acid resistance, disease resistance, herbicide resistance environmental stress tolerance, auxotrophic lines, antibiotic resistance etc.
Different methods of selection and screening of Somaclonal variants are described below:
1. Analysis of Phenotypic Characters:
Phenotypic variations may arise among the regenerates during culture. Such variant characters are observed thoroughly. The variants are transferred from culture flask to the field. In field, such variants plants are observed during their successive growth and development. such qualitative and quantitative 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 variants. Variants are also compared thoroughly with parental plants in all possible quantitative and qualitative phenotypic characters. Several consecutive seed generations of variants are analysed to pursue whether the variants character persist or not among the progeny.
2. Cytological Study of the Variant:
The traditional methods of acetocarmine and feulgen-stained squashes of meristematic tissues of the variants permit the study of the number and gross morphology of chromosomes. So any change in chromosomes number or gross structure of chromosomes can be detected by this method. To have a better assessment of minor structural changes of chromosomes, banding technique can be used.
3. DNA Content of the Variant:
DNA content of the feulgen stained interphase nuclei can be measured by cytophotometer. An uniformly diploid state of cells always maintains its fixed amount of DNA. Any material changes of chromosomes will show either higher or lower vales of DNA content. So the measurement of DNA content can be used as parameter for rapid screening of variants.
4. 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 enzymes extract from the homonized plant is a reliable parameter for detecting the variants. Any alteration in electrophoretic pattern of protein or enxymes indicates that the variants have lost or gained some specific proteins or enzymes fractions. Assay of other biochemical products like pigments, alkaloids, aminoacids etc, using the sophisticated instruments have also revealed the extent of variation among the regruents.
5. Selection for Disease Resistance:
Sometimes, disease resistance character may appear among Somaclonal variants where the parent is highly susceptible to 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 seedling level is frequently possible. Behnke (1979) regenerated potato plants from callus selected for resistance to the toxin filtrate of Phytopthora infestans. Field resistance of some of the sugarcane variants has also been established.
6. Selection for Herbicide Resistance:
The growth of weeds in a population of agricultural important crop is generally controlled by herbicides. As a herbicides have a short residual life , they are applied repeatedly on the crops. Some crops become susceptible to a herbicide due to its repeated application on them. The herbicide is generally added to the cell culture system and the regenerated plantlets showing the tolerance to herbicide are selected. The examples of herbicide resistant plants regenerated from cell cultures include Nicotiana tabacum, bentazone, chlorosulphon, isopropyl N-carbamate, phenmedifarm, picloram and paraquat and Medicago saliva. Herbicide tolerance can also be introduced into cells by somatic hybridization or through genen transfer technology.
7. 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. Selection of high sodium chloride tolerant cell lines in tobacco and regeneration of plants have been reported by Nabors et.al. Regenerated plants showed salt tolerance through two successive generations. Few attempts have been made to select for water –logging and drought resistance in cell cultures. Handa et.al. ( 1983) has repoted somaclonal variation for resistance to polyethylene glycol ( PEG) in tomato cells.
Similarly, attempts were made to isolate variants to chilling stress in tomato, heat , tolerance in pear, aluminium toxicity and sorghum.
8. Auxotrophic Lines:
Auxotophs are continuously used for DNA transformation, somatic hybridization and study of metabolic processes. The use of haploid cell cultures and the application of effective mutagenic treatment have made feasible the recovery of large number of auxotrophs.
9. Antibiotic Resistance:
Cell lines resistance to the antibiotics streptomycin, linomycin, kanamycin, chlororamphenicol and cyclohexamide have been developed from various plant species.