匍匐型地被菊再生及遗传转化体系的建立

为构建有效的地被菊再生和遗传转化体系,本研究以匍匐型地被菊雨花落英叶片为外植体,研究不同苗龄和激素配比对叶盘再生的影响;通过农杆菌介导法将外源基因导入野生型植株中,研究预培养、延迟培养、抗生素浓度等遗传转化条件对转化效率的影响。结果表明,雨花落英叶盘转基因最适再生条件为,以苗龄为30 d的组培苗幼嫩叶片为外植体,设置再生培养基配方为MS+6-BA 1.5 mg·L^-1+NAA 0.8 mg·L^-1,此时再生率高达91.11%,平均芽数为5.90;最优遗传转化组合为预培养2 d,侵染5 min,共培养2 d,延迟培养5 d,之后进行选择培养。羧苄青霉素在延迟培养阶段最适抑菌浓度为400 mg·L^-1;潮霉素叶盘筛选的最适选择压为8~10 mg·L^-1,生根筛选最适浓度为11 mg·L^-1;培养后期加入6-KT可以有效提高抗性芽的再生率;调节6-BA浓度为1.0 mg·L^-1,蔗糖浓度为40 g·L^-1,可以将得到的玻璃化苗转化为正常苗。分子鉴定结果表明,15株抗性苗中有10株阳性苗,阳性苗概率为67%;与野生型植株相比,9个株系的CmERF12基因均受到了明显的抑制表达。本研究建立了地被菊雨花落英再生和遗传转化体系,为进一步进行基因功能研究和遗传改良奠定了一定的理论基础。

Establishment of Efficient Regeneration and Transformation System of Ground-Cover Chrysanthemum

In order to establish an efficient regeneration and transformation system, and provide the basis for gene function verification, leaf disk from ground-cover chrysanthemum Yuhualuoying was used as explant in this study, and the effects of the ratio of hormones and the age of the tissue culture seedlings on regeneration efficiency were studied. What’s more, we screened the concentration of hygromycin and carbenicillin, explored the influence of several conditions such as preculture and delayed culture on transformation efficiency. The results showed that the best regeneration system contains the medium MS+6-BA 1.5 mg·L^-1+NAA 0.8 mg·L^-1, with the seedling age was 30 days, with which the regeneration rate was up to 91.11%, and the average number of buds is as high as 5.90. The optimal genetic transformation was supposed to be: pre-cultured for 2 days, infected for 5 minutes, co-cultured for 2 days, postponed antibiotics selecting for 5 days and then followed by selective culture. During the whole process, the optimum concentration of carbenicillin in the postponed culture medium was 400 mg·L^-1. As for hygromycin, the optimal selection pressure for leaf disk was 8-10 mg·L^-1, while the best concentration for rooting screening was 11 mg·L^-1. What’s more, studies have shown that 6-KT can effectively increase the regeneration rate of resistant shoots. And vitrified seedlings can be transformed into normal seedlings successfully by changing 6-BA to 1.0 mg·L^-1 sucrose to 40 g·L^-1. Finally, we obtained transgenic 15 resistant lines among which there were 10 positive seedlings. The CmERF12 genes of 9 lines were significantly inhibited compared with WT. So the positive rate as 67%. In general, the efficient regeneration and transformation system of ground-cover chrysanthemum Yuhualuoying has been established, which may lay a foundation for further studies on gene functions and genetic improvements.