Creation Of Artificial Male Sterile Plant In Broccoli (Brassica Oleracea Var. Italica) And Chinese Kale (B. Oleracea Var. Alboglabra) By Anti-sense Gene CYP86MF Transformation

Brassica crops is a kind of crops that the most successful in utilizing of heterosis in China. Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops. A cytochrome P450 gene CYP86MF was obtained in floral bud of Chinese cabbage pak-choi (B. campestris ssp. chinensis (L.) Makino var. communis Tsen et Lee) between A/B line by mRNA differential display PCR technology (DD-PCR) and rapid amplification of cDNA ends (RACE) technology. Chinese cabbage-pak-choi and flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis (Bailey) Tsen et Lee) were inoculated with Agrobacterium tumefaciens strain LBA4404 containing pBI35S-AMF carrying antisense gene CYP86MF. More than 130 plantlets KanR seedlings were obtained. In order to find out the function of gene CYP86MF in other Brsssica crops, we study further in this paper. After the efficient shoot regeneration system of B. oleracea L. var. italica P. and B. oleracea var. alboglabra were established and using hypocotyl as explants inoculated with the antisense gene. More than 260 plantlets KanR seedlings were obtained after we constructed an efficient genetic transformation system. The stamen of transgenic B. oleracea var. italica. were browned and there are little pollen in anther, but the stamen of transgenic B. oleracea var. alboglabra were natural. The pollen of transgenic couldn't germinate normally. Therefore, the expressing characteristics of gene CYP86MF were analyzed by Northern hybridization in transgenic plant and non-transgenic plant, the morphology of microspore were observed in transgenic plant and non-transgenic plant, the difference protein between transgenic and un-transgenic plant were observed. The primary results obtained from the research provide some clues for the study on the biological function of gene CYP86MF and molecular mechanism of the male sterility. The main results are as follow:(1) the efficient plant regeneration of B. oleracea var. italica was established by orhtogonalty design, optimized the concentration of NAA, BA, AgNO3 and sucrose, the type of explant was selected. Results showed that the regeneration frequency reached 100 % in this medium with MS + NAA 0.02 mg L-1 + BA 4 mg-L-1 + 2 % sucrose + 0.8 % agar, the best explant is hypocotyls, and the livability of regeneration plantlet is 98%. This formula is fit for other cultivars in B. oleracea var. italica.(2) the efficient plant regeneration of B. oleracea var. alboglabra was established by orhtogonalty design, optimized the concentration of NAA, BA, AgNO3 and sucrose, the type of explant was selected. Results showed that the regeneration frequency reached 97.5%in this medium with MS + NAA 0.03mg L-1 + BA 2mg L-1 + 3 % sucrose + AgNO3 10.5 mg L-1 + 0.8 % agar, and the best explant is hypocotyls, and the livability of regeneration plantlet is 90%. This formula is fit for other cultivars in B. oleracea var. alboglabra.(3) the difference of explant on differentiation medium and un-differentiation medium were analysised by cDNA-AFLP and two-dimension electrophoresis. The results showed that there are 13 bands which distributed from 200 bp to 600 bp, and the difference protein distributed PI 5 ~ 7, MW 40 ~ 70 kD.(4) the efficient gene transformation system of B. oleracea var. italica and B. oleracea var. alboglabra were established by orhtogonalty rotation design, optimized the pre-culture time, co-culture time, infective time, the concentration of Kan and Amp. The procedure was as follow: pre-cultured 3 days {B. oleracea var. italica) or 2 days (B. oleracea var. alboglabra), A. tumefaciens strain LBA4404 infected 8 min, co-cultured days. Then the co-cultured explants were inoculated into the regeneration medium containing the 5 mg-L"1 Kan to obtain the KanR shoot, and the explants were exchanged to fresh medium per 3 weeks. When the KanR seedlings grew to 2 ~ 3 cm, cut them and inoculated into the root induced medium. Roots were induced about 20 days, then opened the plastic fi...