Studies On Several Important Culturing Factors In The Isolated Microspore Culture Technical System In Brassica Napus L.

The microspore culture system of Brassica napus was investigated using 18 materials growing under natural conditions as donors. Several important techniques in the culture system including improving the frequency of embryogenesis, the rates of embryoids directly developing into normal plantlets and the rates of doubled haploid plants were explored in the present study. The results showed as follow:1. Different genotypes of Brassica napus showed remarkable difference in ability of embryos yields. Of the 18 experimental materials, 13 genotypes produced embryoids. Among them, 3 lines (G330, G338, ZZ38) produced higher embryo yield. Under the same experimental condition, difference between genotypes resulting in variation of the embryo yield in Brassica napus, was one of most important reason.2. Addition of activated charcoal to NLN medium had an obvious effect on the yield of microspore embryoids and the development of embryoids shape. The results showed that the range of genotypes capable of culturing, the yield of embryoids and the percentage of cotyledonous embryoids substantially increased by culturing in the medium with activated charcoal. Therefore, it played a very important role in the microspore culture.3. The cool pre-treatment on flower buds had remarkable effect on the embryo yield. Different genotypes of Brassica napus need different time length of pretreatment. The embryo yield of the donor was increased by cool pre-treatment. On the other hand, when the flower buds were pretreated at 4℃ before the culturing of microspores at 32℃ for two days, the embryo yield was much higher than that of direct high-temperature culture(32℃ ). The results showed that the cool pre-treatment was essential for increasing the frequency of embryoid induction in microspore culture.4. The medium with relatively high concentration of agar, cool-treatment (4℃) and drying treatment of cotyledonous embryoids could significantly increase the frequency of the microspore embryoids to form plantlets directly. Furthermore, the results showed that the rate was related to genotype. And the cool-treatment at 10℃ for 10 days was found to be most favorable. Investigation on secondary embryos formation and embryoids forming the plantlets directly showed that the two had negativecorrelation. As results, the frequency of plantlets formed directly could be increased effectively by inhibiting secondary embryo formation. The study screened for effective techniques and ways to improve the rate of embryoids developing into the plantlets directly.5. Different methods of chromosome doubling to obtain doubled haploid plants from isolated microspores culture in Brassica napus were compared: colchicine treatment on microspore-derived plantlets and isolated microspores. The results showed that direct chromosome doubling of isolated microspores was more efficient and resulted in significantly higher frequency of whole doubled plants, resulting in a extreme reduction of chimeric plants with both diploid and haploid branches. In view of this, colchicine could be easier to take effect on the vigorously dividing microspore cells, and thus increased the frequencies of doubled haploid plants.6. Addition of colchicine to NLN medium in microspore culture could not only significantly increase the doubled haploid plants yield, but also stimulate the microspore division and embryogensis, leading to increase embryoids yield. In the two aspects, 10mg/L colchicine were found to be most favorable. Thus it was clear that using of colchicine solved two technical problems in isolated microspore culture system. And it may provide an effect ensurance for the further widespread application of microspore culture in the rapeseed breeding.7. In isolated microspore culture in Brassica napus, many genotypes have been induced into embryoids. And doubled haploid plants were obtained by doubling technique. It showed that the combination of the microspore culture techniques and breeding could accelerate the breeding process. The...