Efficient production of doubled haploid plants by immediate colchicine treatment of isolated microspores in winter Brassica napus

The effect of colchicine on embryogenesis induction and chromosome doubling during microspore culture was evaluated in two F-1 hybrids of winter oilseed rape (Brassica napus L.). Colchicine treatment (50 and 500 mg/L) of isolated microspores during the first 15 h in culture stimulated embryogenesis and produced large amounts of healthy-looking embryos. These normal embryos germinated well at 24 degreesC after being transferred to solid regeneration medium and an initial period of low temperature (2 degreesC) for 10 days, and could directly and rapidly regenerate vigorous plants. A high doubling efficiency of 84-88% was obtained from 500 mg/L colchicine treatment for 15 h with low frequency of polyploid and chimeric plants. A colchicine treatment duration of 6 h was less effective on embryogenesis and doubling efficiency. The present experiment also showed that changing of induction medium 15 h after microspore isolation produced higher spontaneous doubling efficiency, as compared with medium change 6 h after isolation.