狗尾草胚性愈伤高效农杆菌转化体系的建立

The Establishment of High Efficient Agrobacterium Transformation System of Setaria viridis

狗尾草是研究C4光合作用的优秀模型,具有热带植物的典型特征,也可作为研究热带作物的重要模型,高效的转基因技术是模式植物广泛服务于科研的基础,目前狗尾草作为模式植物的研究在国外越来越热门,但在国内还没有被关注。但作为模式植物狗尾草现有的转化技术还不完善,转化周期有待缩短,转化效率需要进一步提高。本研究以狗尾草ME34品系不同阶段胚性愈伤为外植体进行农杆菌转化体系研究,通过实验大大提高了狗尾草的转化效率,缩短了转化周期,为狗尾草转基因研究提供了新的技术方法,新方法主要包括以下技术环节:高效胚性愈伤诱导技术,受体胚性愈伤的选择,适宜转化的农杆菌株系及培养条件,浸染和共培养条件与方法,抗性愈伤筛选条件,抗性愈伤成苗条件等。

Setaria viridis is a wild-type species of Setaria italica and is an excellent model for studying the photosynthesis of C4. It is also a model for studying monocotyledonous plants, abiotic stress tolerance and energy plants. At present, the research of S. viridis is becoming more and more popular abroad, but the understanding of this model is still at the initial stage in domestic. In this study, the high efficient embryogenic callus induction technique was established, and the Agrobacterium transformation system of embryogenic callus was studied by using ME34 different stages of embryogenic calli. Which provided a new high efficient Agrobacterium transformation system for Setaria viridis： Light culture of seeds at early stage promoted embryogenic callus induction; Embryogenic calli with less than 3 subcultures was chosen for transformation; AGL 1 strain was used to transform and cultured at 20℃; The macro-elements of inoculation and cocultivation medium was halved and pH adjusted to 5.2; AGL 1 was activated 2-3 hours before inoculation; Inoculation with 30-60 minutes; Co-cultured with 5-7 days; Embryogenic calli selection with 40-50 mg/L hygromycin for at least month; In the late period of calli selection, light culture promoted regeneration; At the regeneration and rooting stage, the selection was 20 mg/L hygromycin; Seedlings with root hair were the positive plants.