Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus

The effect of colchicine on induction of embryogenesis and chromosome doubling during microspore culture was evaluated in two F-1 hybrids of spring oilseed rape (Brassica napus L.). Immediate colchicine treatment of isolated microspores with the concentrations 50 and 500 mg/L for 15 h 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 83-91% was obtained from 500 mg/L colchicine treatment for 15 h with low frequency of polyploid and chimeric plants. The present experiment showed that a treatment duration of 30 h revealed less positive effects on embryogenesis and doubling efficiency, especially at higher colchicine concentration (1000 mg/L). Poor embryogenesis and embryo germination were observed from ordinary microspore culture without change of induction medium and colchicine treatment, and several subcultures were required for induction of secondary embryogenesis and plant regeneration.