一种改进的水稻成熟胚愈伤组织高效基因转化系统

Improved High-efficiency System for Rice Transformation Using Mature Embryo-derived Calli

以成熟胚愈伤组织为材料的农杆菌介导水稻转化法虽已建立,但转化频率仍有待提高。本文以粳稻(Oryzasativa)品种(中花10号和中花11号)的成熟胚诱导的愈伤组织为受体材料,对组织培养体系及影响遗传转化的因素进行优化,建立了一套改进的农杆菌介导的水稻高效遗传转化系统。农杆菌菌株为EHA105,质粒载体是pUN1301/OsRAA1,其中含有标记基因GUS和筛选基因HPT。愈伤组织诱导培养基为NBD2(NB+2mg·L^(-1)2,4-D),继代培养基为NBD0.5,预分化与分化培养基为RE1(MS+1mg·L^(-1)6-BA+0.25mg·L^(-1)NAA+0.5mg·L^(-1)KT+0.2mg·L^(-1)ZT)和RE2(MS+1mg·L^(-1)6-BA+0.5mg·L^(-1)NAA+0.5mg·L^(-1)KT+0.2mg·L^(-1)ZT)。另外,还分析了影响T-DNA转移的多种因素,如外植体种类、愈伤组织预培养基和愈伤组织继代次数等。采用优化的转化程序,水稻愈伤组织转化率和植株转化率可达70%以上。

Rice has been transformed via Agrobacterium transfection with use of mature embryo-derived calli, but the transformation efficiency has remained relatively low in general. In this study, we optimized different factors affecting transformation and established a highly efficient transformatiom system mediated by Agrobacterium using mature embryo-derived calli from two japonica cultivars （Zhonghua 10, 11 ）. The transformation was performed with A. tumefaciens strain EHA105 harboring the plasmid pUN1301/OsRAA1, GUS used as the reporter gene and HPT as the selectable marker gene. First, we established a high-frequency regeneration system for rice, defined NBD2 （NB＋2 mg.L^-1 2,4-D） as callus induction medium, NBD0.5 as subculture medium, and RE1 （MS＋1 mg.L^-1 6-BA＋0.25 mg.L^-1 NAA＋0.5 mg.L^-1 KT＋0.2 mg.L^-1 ZT） and RE2 （MS＋1 mg.L^-1 6-BA＋0.5 mg.L^-1NAA＋0.5 mg.L^-1 KT＋0.2 mg.L^-1 ZT） as regeneration medium. Second, factors such as the selection of explants, the pre-culture medium and the subculture times were shows to affect the efficiency of T-DNA delivery. The transformation efficiency using the optimized method was increased to 70%, which was measured either by counting the number of hygromycin-resistant calli or the number of transgenic plants.