影响农杆菌介导玉米优良自交系遗传转化的因素

FACTORS INFLUENCING AGROBACTERIUM-MEDIATED TRANSFORMATION OF MAIZE ELITE INBRED LINES

以我国玉米骨干自交系9046,齐319,414,Mo17的幼胚为材料,在已经建立的农杆菌介导的玉米幼胚转化体系的基础上,研究了影响农杆菌介导玉米优良自交系遗传转化的因素,建立了优化的玉米优良自交系的遗传转化体系。研究结果表明,1.0-2.0mm的玉米幼胚是最适宜的转化受体;在感染液和共培养基中都加入乙酰丁香酮(200μmol/L)和抗坏血酸(50mg/L),能显著提高农杆菌对玉米的侵染能力;而感染前将幼胚高渗透压预处理未能提高转化率;延迟筛选有利于提高抗性愈伤组织的存活率。应用优化后的转化体系,获得了这4个玉米优良自交系的转基因植株,PCR阳性植株率为1.71%-4.09%。转化植株叶片总DNA的PCR和Southern杂交分析表明,T-DNA上的外源基因已经整合进了玉米基因组,并且在大多数转基因植株(71.4%)中为单位点插入。这一体系的建立,为进一步将有用基因导入玉米优良自交系奠定了基础。

Using the maize elite inbred lines 9046,Qi319,414,Mo17 as target genotypes,a highly efficient transformation system was developed based on the study of factors influencing the Agrobacterium-mediated maize transformation.The results showed that the immature embryos of 1.0-2.0 mm in length were optimal transformation explants.Inclusion of acetosyringone(200μmol/L) and ascorbatic acid(50mg/L) in both infection medium and co-cultivation medium led to a signif- icantly increase in the transformation efficieney.However,high osmotic treatment on the explants before inoculation didn't improve transformation efficiency.Delaying selection was beneficial to the survival of resistant calli.Using the optimized transformation procedure,42 PCR-positive transgenie plants were obtained from the 4 elite inbred lines and the frequency of PCR-positive plant ranged from 1.71%-4.09%.The integration of the transgenes into the maize nuclear genome was confirmed by PCR analysis using bar-and gus-specific primers and by Southern blot using gus-specific probe.Most of transgenie plants(71.4%) had one copy of T-DNA insert.The estab- lishment of Ihe transformation system in maize provides an efficient way tor transferring useful foreign genes to maize plants.