Technique Optimization Of Microspore Culture In Rapeseed (Brassica Napus L.)

Rapeseed microspore culture is a highly effective ways of haploid breeding, can effectively shorten crossbreeding cycle, but in practical applications, there are some problems, such as work efficiency is not high and regenerated plants must be subcultured for several times in the laboratory until the normal growing season. This research focuses on optimizing the microspore culture technology system, simplifing the microspore culture method, and establishing the technology system of long-term preservation of embryos in order to establish microspore culture system that is appropriate in large-scale application.The main findings are as follows:1 Studies on microspore culture technology:For the 64 donor materials used, the yield of embryo ranged from 0 to 36.94/bud; The cold pretreatment on buds was beneficial to the microspore embryogenesis, and with the extension of the pretreatment time, the yield of embryo increased first and then decreased; The yield of embryo in early flowering stage was significantly higher than the full flowering stage; Using 55 mg/L colchicine dealing with in vitro microspores directly, the doubling rate of different genotypes ranged from 48.94% to 86.18%; The cold pretreatment on embryos was beneficial to plant regeneration of embryos, but the factor of light in process of cold pretreatment didn’t have obvious effect.2 Simplification of microspore culture method:There is no significant effect on the yield of embryo when simplify the separation lotion formula and reduce the times of centrifugation.The percentage of microspore enlargement after one day’s heat shock culture is a reference index for embryogenesis.3 Established the long-term embryos preservation technology system:Advanced replacement with fresh liquid NLN-13 medium during the process of shaking culture before preservation at low-temperature circumstance could significantly improve the storage capacity of embryos. Liquid NLN-13 medium is more suitable for long-term preservation of embryos compared with solid B5 medium.The cotyledon-shaped embryo could be safely stored for more than four months in 4℃ NLN-13 liquid medium if we replaced the culture medium timely with fresh medium during the process of shaking culture.4 Preliminary studies on plant regeneration of embryos in soil:Soil sterilization and the cold pretreatment on embryos could increase the frequency of plant regeneration of embryos in soil, adding 1/2 B5 liquid medium could help embryos turn green and hypocotyls elongate vertically, but had little effect on plant regeneration of embryos in soil.