In vitro callus selection in Brassica species

Abstract

The study mainly concerns the potential of in vitro selection as an aid to plant breeders. Three species, of the genus Brassica, were used in the investigations as model crop plants. Callus from these species were selected for NaCl tolerance. The study has three main sections dealing with the needs of tissue culture in Brassica species, in vitro selection of callus for NaCl tolerance and estimation of somaclonal variation.in vitro culture. The study also describes investigations in the optimum growth regulator concentrations for continued callus cultures and subsequent plant regeneration, in these Brassica species. All the explant types i.e. leaf discs with vein, leaf discs without vein, petioles and hypocotyls, of all the species studied, showed successful callus induction and subsequent callus cultures, but with varying frequencies. Plant regeneration from callus was not successful in all the species and with all the explant sources. Curly kale (Brassicaoleracea L.) showed the most successful plant regeneration. Among the explant sources, hypocotyls demonstrated the most potential with respect to plant regeneration. Optimum concentrations of NAA and BAP were different for different species. In general, BAP and NAA in balance produced more callus; and as expected, BAP enhanced shoot formation while NAA promoted root formations.The second section deals with procedures and problems associated with the in vitro selection of different Brassica species for salt tolerance. In addition the study also deals with the maintenance of selected callus and stability of the selected calli, with respect to salt tolerance. Nevertheless, callus was selected successfully from all the Brassica species, studied. In general, selection was found effective giving more salt tolerant cell lines from selected callus in comparison with the unselected callus. However, the stability of the salt tolerance varied between the species. Only Brassica rapa L. callus showed stable salt tolerance even after sub- culturing on salt free medium after selection, for few generations. The other two species showed unstable salt tolerance. In these species the selected callus was not significantly more salt tolerant than unselected callus, when exposed to the salt free medium after selection.The third section deals with the an estimation of the variation found with regenerated plants and callus from different culture cycles and culture conditions. Somaclonal variation was detected and quantified using RAPD-PCR analysis. Regenerated plants and callus from different cultures, showed different frequencies of variation to the parent plants. In general, variation showed by different cultures ranged from 1 to 11%. No DNA polymorphism was observed specific to the NaCl tolerance. Effect of culture duration on somaclonal variation was also not detected by RAPDs analysis.The final discussion reviews in vitro selection of callus for salt tolerance. The wider applicability of in vitro selection for crop improvement along with limitations and future prospects are also reviewed in the context of somaclonal variation and its application.