Analysis of somaclonal variation through tissue culture and chromosomal localization of rDNA sites by fluorescent in situ hybridization in wild Allium tuberosum and a regenerated variant

Abstract

The effects of basal media and growth regulators on callus initiation and shoot regeneration have been investigated in wild Allium tuberosum (2n = 4x = 32). Callus initiation was greatest from flower bud explants cultured on MS medium supplemented with 2,4-D and BA at 1 mg l⁻¹ each. Maximum number of shoots was obtained from callus grown on MS medium supplemented with NAA and BA at 0.2 and 2 mg l⁻¹, respectively. The chromosome analysis of regenerants derived from callus revealed variation in ploidy, such as 2n = 28, 29, 30, 31, 33 as well as normal tetraploid. During the culture period for two generations, one aneuploid regenerant with 2n = 30 (named At30) showed better viability and growth than tetraploid plants and other aneuploid variants. In a karyotypic analysis of At30, the chromosomal positions of 5S and 18S-5.8S-26S rDNA were physically mapped by fluorescent in situ hybridization and compared to chromosomes of wild type A. tuberosum. Both wild type A. tuberosum and At30 exhibited two sets of 5S rDNA sites, one on the proximal position of the short arm of chromosome 3, and the other on the intercalary region on the long arm of chromosome 6. There was one 18S-5.8S-26S rDNA site in the secondary constriction including flanking short chromosomal segments of satellite and terminal regions on the short arm of chromosome 8 in wild type A. tuberosum. However, At30 showed only three labelled chromosome 8 indicating that this was one of the lost chromosomes of At30.