ii.  Growth pattern and regeneration mode

In vitro growth may occur from meristem cultures, which may form callus (undifferentiated mass of cells) or direct shoot formation. Callus is further differentiated into organized structures by organogenesis or somatic embryogenesis. The departure from organized growth is a key element in somaclonal variation. In general, longer the duration of callus and cell suspension in culture phase, the greater the chances of generating somaclonal variation. These somaclonal variation can also occur in embryogenic cultures, if they are kept for a long time in cultures, depending upon the plant species.  

iii.  Length of culture and the subculture frequency

The age of callus and cell suspension in culture has a marked effect on the frequency of somaclonal variation which increased with the increase in the duration of in vitro cultures. The reduction or even total loss of regeneration ability is a general phenomenon observed in many long-term callus or cell culture lines. The level of DNA polymorphism has been found to be increased with length of time in culture. Although the chloroplast genome is generally considered to be more highly conserved and stable than nuclear and mitochondrial genomes, the prolonged culture resulted even in the deletion of parts of the chloroplast genome. These deletions were associated with changes in plastid morphology. The age affects the frequency of mutation which is primarily due to sequential accumulation of mutations over time than an increased mutation rate in old cultures. The frequent subcultures of callus and cell suspension favors stability in ploidy and chromosomal constitution compared to extended subculture intervals.

iv.  Physical factors

Chemical composition of the media to a great extent influences cytological behavior of cultured cells. Besides, the physical conditions, such as temperature and nature of media (liquid or semi-solid) also influences the rate of mutation.

v.  Ploidy and genotype

Genotype of the parent plant is a major determinant of variability in cultures. It influences somaclonal variation irrespective of regeneration mode. Somaclonal variation resulting from changes in chromosome number and rearrangements is easier to recover in regenerants of polyploids than diploids and haploids, since polyploidy can tolerate more gross genomic alterations as compared with diploids and haploids, particularly when such changes are deleterious. On the other hand, gene mutations or point mutations could be better expressed in haploids and diploids. Generally, ploidy levels lower than the usual ploidy level of the respective species are proved to be more or less unstable. Sacristan (1971) compared the cytological changes in long-term cultures of haploid and diploid strains and described that diploidization in haploid tissues was more common than the occurrence of tetraploids in diploid lines.