Establishment Of Efficient Plant Regeneration And Mutagenesis System In Brassica Napus

Ultraviolet (UV) irradiation, mutagenic agents ethylmethane sulphonate (EMS) and sodium azide (NaN₃) were applied to isolated microspores and microspore-derived embryos of Brassica napus in vitro. In the UV light irradiation experiment, the isolated microspores and microspore-derived embryos were exposed for 10, 30, 60 and 120 s. Embryo yield showed a sharp decrease with increasing UV exposure and only 10 embryos were developed following an exposure of 120 s of genotype M9. Embryo germination was also decreased when isolated microspores and microspore-derived embryos were treated by UV irradiation. The mutagenic treatments, NaN3 (1, 10, 100 μM) and EMS (0.001, 0.01, 0.1 %) were applied to the isolated microspores and embryos at early cotyledonary stage for various time intervals (1, 5, 15 h). With increasing of the mutagen concentrations and prolonging of the exposure time, embryo yield reduced gradually. Many fertile microspore-derived embryos died after the applying of the two chemical mutagens. Embryo germination was also decreased with the treatment of these two mutagens. Surprisingly, in this study the application of 1 and 10 μM NaN3 had a strong promotive effect on the rate of embryo germination and plant regeneration, with the significant increase over the control.UV irradiation, EMS and NaN3 were applied to cotyledons of B. napus in vitro. Cotyledons were exposured in the UV radiation for different time intervals (30, 60, 120, 180, 240 s), and the results showed that UV treatments strongly promoted the plant regeneration. When EMS was applied to cotyledons, the rate of induced calli and regenerated plants decreased with increasing of the mutagen concentrations and prolonging of the treatment time. When the treatment (1% EMS for 25 h) was applied to cotyledons, the rate of regenerated plants reduced to 0.00%. NaN3 significantly increased the callus induction along with the increasing of the mutagen concentrations. When the concentration of NaN3 increased to 1000 μM, the rate of induced calli reached the maximum.