3.4. Variation in extranuclear genes: The genetic material of cytoplasmic organelles may also undergo changes under in vitro conditions. Mostly changes in mitochondrial DNA are observed and these changes in mt-DNA at molecular level can be very well observed while performing its RFLP restriction pattern. Hartman et al (1989) demonstrated significant changes in mt-DNA of the wheat plants obtained by the regeneration, the extent of change was determined by the length of the tissue culture period. The non-embryogenic cultures of wheat suffered about 8kb fragment loss of mt-DNA found in embryogenic cells.
4. Analysis of Somaclonal variants
Most useful somaclones are those which carry almost all of the good parental characters as well as incorporate within it certain desirable characters which were lacking in its parents. It becomes extremely important to select variants as early as possible, with minimal exposure of cells to tissue culture environment. With prolonged culture gross abnormalities may appear. The variants are generally assessed at the phenotypic level, and in over 50% cases it is based on R0 plants. However, this approach of screening R0 plants would the screening of only homozygous or dominant traits. The recessive mutations in heterozygous regenerants can be recognized only in the segregating R1 and R2 progenies. It is, therefore, important that the variants should be assessed in the sexual progenies of the in vitro regenerated plants so that their heritability is established.
The degree of variation of a plant can be determined by estimating the standard deviation for a particular quantitative trait. It is usually determined as the percentage of plants showing aberrations for one or more defined characteristics, such as plant height, time of flowering, fertility, flower and fruit color. The effect of environment on the phenotype of plant can also be detected using biochemical characterization mostly involving protein electrophoresis. These above mentioned methods can be very well used for the assessment of phenotypic variations but the variation or change at genome level cannot be monitored. In order to detect the variation at DNA level, use of certain molecular markers is encouraged. RFLP appears to be a better technique as it helps in identifying slight changes and also in studying plants grown in different environments.