EFFICIENT PRODUCTION OF DOUBLED HAPLOID BRASSICA-NAPUS PLANTS BY COLCHICINE TREATMENT OF MICROSPORES

The effect of colchicine on isolated microspore cultures of Brassica n apus was evaluated in order to combine a positive effect of colchicine on the induction of embryogenesis with the possibility to induce chro mosome doubling at an early developmental stage, thus avoiding the pro duction of haploid or chimeric plants. Colchicine was added to the cul ture medium immediately after isolation of B. napus microspores. The c ultures were incubated from 6 to 72 h with various concentrations of c olchicine. Samples were taken from the regenerating embryoids after 6 weeks for ploidy determination by flow-cytometry. The highest diploidi zation rate was obtained after a 24 h treatment of microspores with 50 mg/l colchicine, leading to 80-90% diploid embroids. A concentration of 100 mg/l colchicine applied for the same duration resulted in a low er diploidization rate (76-80%). Treatment durations of 6 h were not l ong enough to induce a high rate of diploidization, whereas the applic ation of 10 mg/l for 72 h was also very effective. A sample of the pla nts regenerated from the colchicine treated microspores was transferre d to the greenhouse. The plants looked similar to normal diploid rapes eed plants and showed reasonable pod and seed set. Thus, an additional generation for seed increase in the greenhouse is rendered unnecessar y. The advantage of applying a minimum volume of colchicine under cont rolled in vitro conditions means a considerable saving of time and lab our in DH-breeding programs.