Obtaining double haploid plants by isolated microspore culture can speed up the breeding process and improve the selection efficiency, which is favored by breeders all over the world. Since Litcher and others first obtained embryoids from microspore culture of Brassica napus, microspore culture of Brassica vegetables such as Chinese cabbage, cabbage and mustard has also been successful. Li Guangtao et al. (1996) used isolated microspores of purple Chinese flowering cabbage to culture and regenerate plants, but the embryo formation rate was extremely low.
Gu Honghui et al. (2003) studied the flowering characteristics of early-maturing Chinese flowering cabbage. The isolated microspores were heat-shock cultured in NLN- 17 medium at 32℃ for 2 days, then changed to NLN- 10 medium and continued to be cultured at 24℃. Compared with direct treatment with NLN- 10 medium without changing the medium, the yield of embryoids was significantly improved, and the quality of embryoids could be improved by updating the medium. Direct treatment of isolated microspores with 0.8mg/L colchicine can obviously increase the yield of embryoids, while too high colchicine concentration is not conducive to embryo formation and embryoid germination. The chromosome ploidy of microspore regenerated plants of Chinese flowering cabbage was identified by flow cytometry (FCM). The results showed that the spontaneous diploid rate (70%) and tetraploid rate (8%) were higher, while other polyploids and mixed ploidys were relatively less.
Zhu Yunhua et al. (2003) conducted microspore culture on Chinese flowering cabbage. The results showed that the main factors affecting isolated microspore culture were as follows: (1) genotype difference of Chinese cabbage significantly affected isolated microspore culture; Before microspore separation, the inflorescence was treated at low temperature of 4℃ 12 days, which obviously improved the frequency of microspore separation. When the sucrose concentration reaches 13%, that is, the culture medium has a certain osmotic pressure, which will promote the induction and development of embryoids.
Wang Taotao et al. (2004) used five genotypes of purple flowering, China flowering, China flowering, China flowering, China flowering, China flowering, China flowering, China flowering, China flowering, China flowering, China flowering, China flowering, China flowering. The results showed that genotype 8902 had the highest embryo yield, reaching 42 embryos per dish, with a minimum of 0. Adding proper amount of activated carbon can increase embryo yield by nearly 3 times. At the same time, the factors of further regeneration of embryoids were also studied: the regeneration rate was the highest when 1.2% agar was added to the culture medium, reaching 50.1%; Treatment of 10d at 4℃ can increase the regeneration rate from 45% to 65%. With the extension of embryo age, the rate of regenerated plants decreased obviously, and the optimum embryo age was 20 ~ 24 days. However, medium B5 and MS had little effect on microspore regeneration rate.
For Chinese cabbage, whether it is anther culture or pollen culture, low-temperature pretreatment of inflorescence before inoculation (4℃ 1 ~ 2 days) and high-temperature pretreatment after inoculation (33℃ 1 ~ 2 days) can promote the formation of embryos and increase the frequency of induction. The induction rate of embryoid in pollen culture is higher than that in anther culture. The sucrose concentration in the medium and the addition of effective factors will affect the induction rate of embryoids in anther and pollen culture. The sucrose concentration in the two media was 13%, and the optimum AC addition was 0.5g/L. In anther culture, adding100 ~120 mg/l agno 3 _ 3 to the medium can promote the generation of embryos. Spraying 200 mg/L paclobutrazol or 40 mg/L methylprednisolone on plants, soaking seeds with 1.0 mg/L or 0.6 mg/L L L Met, and adding 0.5 mg/L paclobutrazol or 0.3 mg/L methylprednisolone to the culture medium can promote embryogenesis. The frequency of embryoid induction varies greatly among different genotypes (Zhu Yunhua, 2003).