3. Mechanisms leading to genetic variations
3.1. Changes in ploidy level: The most frequently observed chromosomal abnormality in the cultured cells and the regenerated plants is the occurrence of the change in ploidy. Such ploidy changes are (i) euploidy, which is the increase in the chromosome number in simple multiples of the basic chromosome number (2n, 3n, 4n, 5n, etc.), and (ii) aneuploidy where the chromosome constitution is not simple multiples of the basic chromosome number.
Formation of a restitution nucleus due to the failure of spindle formation and chromosome lagging at anaphase and the fusion of spindles during synchronous divisions in multinucleate cells are common sources of the occurrence of euploid cells of even series (4,8,16 and so on) in tissue culture. While the odd euploid series in tissue culture arise through nuclear fusion or genome segregation during polyploidy mitosis. The haploid cells found in diploid calli may due to somatic pairing and reduction.
There is strong selection system exist in tissue culture which plays critical role in establishing dominant karyotype or model chromosome number. In mixed cultures of diploid and tetraploid cell lines of carrot (both lines show identical growth rates in monoculture), the frequency of tetraploid cells gradually increased, and the cultures attained a tetraploid mode. The regeneration process itself acts as a screen to eliminate a portion of varying karyotypes, generally, a strong selection exist in favour of diploids or at least euploids than aneuploids during plant regeneration from callus and suspension cultures.
3.2. Changes in chromosome structure: Structural changes in chromosome usually refer to the loss or gain of chromosomal segments which generally results into an altered karyotype but the chromosome number remains the same. In Haplopappus gracilis occurrence of accentric fragments, deleted chromosomes, dicentric chromosomes was frequently observed (Singh and Harvey, 1975a). Structural changes in chromosomes originate from breakage during the various stages of the cell cycle. The dicentric chromosomes can bring about continuing variation by initiating a breakage fusion bridge (BFB) cycle. The culture conditions may cause delay in DNA synthesis in the heterochromatin region of the chromosomes until mitosis, resulting in the formation of non-replicated heterochromatin bridges and breakage at anaphase.
3.3. Gene mutations and amplification: Several somalones due to dominant or recessive single or multiple gene mutations have been described by many workers. Recessive mutations may not express in the R0 generation of somaclones but can be detected in their self progeny. Many distinct gene mutations in several tomato plants regenerated from leaf callus have been well characterized and mapped to specific loci on the chromosomes.
It was suggested by Brown in 1981 that if a gene cannot modulate its expression, it is likely to undergo the gene amplification but if the right selection agent is available. Goldsborough et al. (1990) selected Nicotiana tabacum cell lines resistant to normally lethal concentrations of glyphosate and found that the level of enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), which is inhibited by increase level of glyphosate, was elevated. Selected cells maintained high levels of mRNA for EPSPS even in the absence of the herbicide which is due to the amplification of at least two genes encoding the enzyme. This implies that selection resulted in stable genetic modification.