农杆菌介导的高效玉米遗传转化体系的建立.pdfVIP

HEREDITAS (Beijing) 2009 11 , 31(11): 1158―1168 ISSN 0253-9772 研究报告 DOI: 10.3724/SP.J.1005.2009.01158 农杆菌介导的高效玉米遗传转化体系的建立 魏开发 , 363000 为了建立玉米高频再生及高效遗传转化体系, 对影响玉米胚性愈伤组织诱导的 11 个因素及影响胚性愈 伤分化的9 个因素用正交实验方法进行研究。结果显示, 基因型对胚性愈伤诱导有极显著影响。6-BA 、培养基、 AgNO3 、2,4-D 、ABA 对胚性愈伤诱导的影响达到显著水平。多重比较分析显示ABA 2 mg/L 每间隔 1 代添加 对胚性愈伤诱导率有显著影响。在影响分化的因素中, 基因型和6-BA 浓度表现出极强的主效应, NAA 、培养基、 KT 、2,4-D 对分化产生显著影响。Southern blotting 分析表明, 25 mg/L 潮霉素选择压下抗性愈伤率作为转化体 系优化指标是可靠的。在影响转化效率的因素中, acetosyringone (AS)使用浓度因基因型不同而表现出敏感度差 异, 共培养温度24~25 ℃、农杆菌浓度和浸泡时间0.7 OD×15 min, 以及pH 值5.5~6.2 是最高转化率的优选组合。 在整合后的玉米遗传转化体系中, 黄早4 和综31 自交系以抗性愈伤率为指标的GUS 基因稳定转化率分别达到 48.6%和46.2% 。 玉米; 正交实验; 胚性愈伤诱导率; 抗性愈伤率; 农杆菌介导; 遗传转化率 Establishment of high efficiency genetic transformation system of maize mediated by Agrobacterium tumefaciens WEI Kai-Fa Department of Biological Sciences and Biotechnology, Zhangzhou Normal University, Zhangzhou 363000, China Abstract: In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blottin