Development of Haploids and Double Haploids in Oil Seed Crop through Anther Culture

Anther Culture or microspore culture is a very important and useful tool in plant breeding for efficient production of haploids and subsequent doubled haploid plants in many species, limited research has been conducted with Brassica rapa as it is more recalcitrant in cell and tissue culture than other Brassicas. In order to access the effect of genotype on microspore embryogenesis in B rapa, two high yielding brown Sarsoon cultivars (KS-101 and KOS-1) were studied for their response to haploid plant regeneration through Anther culture. Flower buds of 2.2 mm to 2.7 mm size representing late uninucleate stage of anther microspores were subjected to low temperature treatment 40C for 7days and anthers from these buds were cultured on MS medium having 13 % sucrose. Anthers were then sub cultured in MS media supplemented with various concentrations of auxins and cytokinins. The best callus development and proliferation was achieved in MS media supplemented with 2, 4-D 1mg/l and 0.5 mg/l NAA. Sucrose concentration, cold pre treatment and incubation time influenced embryogenesis. For regeneration of haploid plantlets anther derived calli were transferred to MS full strength medium supplemented with kn92.0mg/l) fallowed by the use of BA (2.0mg/l) and incubated at 22 + 20C in light and gave maximum regeneration of 75 %. The regeneration shoots (at three leaf stage with length of 5cm) were subculture in rooting media and maximum rooting (30.07 %) was achieved when MS medium was supplemented with IBA (0.4mg/l). Root tip mitosis chromosome counts revealed percentage of haploid frequency of KOS-1 (41.85) followed by KS-101 (38.84 %). After 5 days of rooting root samples were taken for confirmation of haploid nature of the plant, Haploid frequency was greater than 65 %.