摘要
Kasalath是目前用于水稻转基因研究的籼稻模式材料。运用组织培养和农杆菌介导的遗传转化技术,对水稻Kasalath愈伤组织诱导和遗传转化体系进行了优化。结果表明:以Kasalash的幼嫩种子或新鲜的成熟种子为外植体诱导愈伤组织的效果优于贮藏1年的种子;用0.5、1、1.5、2、2.5、3、10、20、30 mg/L的2,4–D诱导愈伤组织,以2.5 mg/L的2,4–D诱导效果最佳;用菌液OD600 nm为0.05、0.1、0.15、0.2、0.3、0.4的农杆菌Ag10和EHA105与愈伤组织共培养介导遗传转化,皆以OD600 nm为0.15的农杆菌溶液效果最好,且EHA105的介导效果比Ag10更佳,其抗性愈伤率可达82.0%;EHA105的介导(共培养)时间以3 d为宜;分化培养前对愈伤组织进行6～8 h快速干燥或48～72 h慢速干燥处理,可以明显提高愈伤组织的再生成苗率,并缩短分化时间;在对愈伤组织侵染处理前的预培养处理和对抗性愈伤进行分化培养前的预分化培养处理对Kasalath的遗传转化效率影响不大,可省略以缩短遗传转化时间。遵循以上优化条件,从愈伤组织诱导至遗传转化获得再生植株全过程的时间只需57～60 d(已报道的遗传转化全过程需70 d以上),遗传转化效率可达到已报道的最高值65%。
At present, Kasalath is a model Indica material for rice transgenic research. By using the technique of plant tissue culture and Agrobacterium-mediated genetic transformation, we optimized the condition for the callus induction and the performance of genetic transformation of Kasalath. The results showed that immature or newly harvested mature seeds as explants were more effective to generate callus than the seeds stored for 1 year. Among the induction media with 0.5, 1, 1.5, 2,2.5, 3, 10, 20 or 30 mg/L 2,4-D solutions, the medium with 2.5 mg/L 2,4-D had the best performance in callus induction; in callus infection by co-culturing with Agrobacterium Agl 0 or EHA105 solution with 00600 am being 0.05, 0.1, 0.15, 0.2, 0.3 or 0.4, the efficiency of transformation reached the optimum when 0060o am of the working solution of both Agl 0 and EHA105 was 0.15, but EHA105 was superior to Agl0, its hygromycin-resistant callus reached up to 82.0% and 3 d co-culture with EHA105 was perfect. When the resistant calli suffered rapid dehydration for 6-8 h or slow dehydration for 2-3 d, the regeneration frequency was evidently improved and the time needed for regeneration was shortened; while pre-culture before callus infection and pre-differentiation processing before hygromycin-resistant callus getting onto differentiation culturing had little effect on genetic transformation efficiency, so these procedures could be omitted. By the optimized procedures, the overall time from callus induction to regeneration of transformant plantlets was shortened to 57-60 d, and 65% of transformation efficiency was attained.
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第5期471-477,共7页
Journal of Hunan Agricultural University(Natural Sciences)
基金
国家自然科学基金项目(31071487
31270348)
湖南省生态学重点学科建设项目(0713)
关键词
籼稻
2
4-D
农杆菌介导
遗传转化
影响因素
Indica rice
2,4-D（2,4-dichlorophenoxyacetic acid）
Agrobacterium-mediated
genetic transformation
influence factors
作者简介
周艳（1987-），女，湖南益阳人，硕士研究生，主要从事种子生物学研究，315658395@qq．com；
通信作者，jxclc@hunnu．edu．cn
