Studies On The Establishment Of High Efficient Shoot Regeneration System And The Transformation Mediated By Agrobacterium In Tuber Mustard (Brassica Juncea Var.Tumida)

In this study, four cultivars of tuber mustard (Brassica juncea Czern. et Coss. var. tumida), 'Zhetong 1', 'Zhetong 3', 'Zhetong 4' and 'Zhongdu-zhacai', were used as plant materials to study the effects of key factors on the regeneration and the transformation mediated by Agro-bacterium. In the result, a high efficient regeneration system and a primary Agrobacterium-mediated transformation system were established.Sessile cotyledon, cotyledon, petiole and hypocotyls of 'Zhetong 1' were used as explants to study the effects of plant growth regulators on the differentiation of different explants of tuber mustard. The results showed that cotyledon and hypocotyls had the highest regeneration frequency in MS medium with BAP 2 mg.L-1 + NAA 0.2 mg.L-1. In which cotyledon had a high regeneration frequency of 100%, and hypocotyls had a high regeneration frequency of 60.7%. Sessile cotyledon showed the highest regeneration frequency in MS medium with BAP 2 mg.L-1 + NAA 0.4 mg.L-1; and the medium most suitable to petiole was MS + BAP 2 mg.L-1 + 2, 4-D 0.2 mg.L-1. Our results also displayed that the regeneration frequency of different types of explants was significantly different in MS medium with BAP + 2, 4-D. Furthermore, both the effects of seedling age and the effects of the types of explants on shoot regeneration from different explants of tuber mustard were significant in our study. In addition, the sessile cotyledon explants from 4 days seedlings and cotyledon and hypocotyls explants from 5 ~ 6 days seedlings showed higher regeneration frequency than the explants from other seedling ages. However, although these mediums were suitable to most of cultivars, the regeneration frequencys are different among genotypes.The effects of AgNO3 on the regeneration of different explants were also studied, and the results showed that the regeneration frequencys in mediums which added AgNOs werehigher than those without AgNO3. But the optimal AgNO3 concentration for different type of explants was different. And the optimal AgNO3 concentration for cotyledon, petiole and hypocotyls is 6 mg.L-1, but the medium with 4 mg.L-1 AgNO3 is most suitable to the regeneration of cotyledon explants. In addition, we developed the effects of agar concentration on the regeneration of different explants, and the results indicated that the medium with 0.8% agar had the highest regeneration frequency.When the shoots grown up, we observed the MS medium with NAA 0.2 mg.L-1 + 3% sugar + AgNO3 5 mg.L-1 + 0.8% agar or 1/2 MS with NAA 0.2 mg.L-1 + 3%sugar + AgNO3 5 mg.L-1+ 0.8% agar was the best for root induction.To eliminate Agro-bacterium and select transformats, the sensitivity of spectinomycin were tested, and the results showed that the suitable concentration of spectinomycin to hypocotyls was and 5 mg.L-1 respectively. Among carbemcillin, ampicillin and cefotaxime used for elimination of Agro-bacterium, ampicillin with 500 mg.L-1 should be preferable based on their effects on shoot regeneration.From our study we could see that hypocotyl was the best explant for transformation. And the shoot frequency with highest resistance was observed when explants were pre-cultivated 4 days and immersed in bacterium suspension with OD600 value about 0.5 for 10 ~ 15minutes followed by co-cultivated for about 3 ~ 4 days.At last, to detect the transgene in transgenic shoots, the specific premiers of CaMV35S was used in PCR reaction, and the result of PCR reaction showed that the transgenes had been integrated into the genome of tuber mustard.