用LBA4404/pCAMBIA系列转化水稻的最佳条件

Optimization of the Conditions for Rice Genetic Transformation Mediated by Agrobacterium Tumefaciens LBA4404/pCAMBIA Series

用带有ｇｕｓ报告基因的农杆菌ＬＢＡ４４０４／ｐＣＡＭＢＩＡ１３０１ 转化水稻品种鄂宜１０５ 成熟胚诱导的愈伤组织，以ｇｕｓ基因瞬间表达活性为指标测定转化频率，对于利用ｐＣＡＭＢＩＡ 表达载体系列进行水稻遗传转化的最佳条件进行了初步的研究．结果表明：最适菌液浓度值为Ｄ（６００）＝ １．０；最适共培养温度为２２～２８ ℃；共培养培养基的ｐＨ 值在５．２～６．２ 这一较宽的范围内均可．共培养时间以不超过３ ｄ 为宜；在共培养培养基中加入乙酰丁香酮对提高转化效率非常重要，而在菌液中加入乙酰丁香酮处理对提高转化效率作用不大．但不同品种，甚至不同转化受体之间存在一定的差别，其最佳转化条件应视具体情况在此基础上加以摸索．

Mature seed derived callus from EY105 was transformed by gus gene containing Agrobacterium tumefaciens LBA4404/pCAMBIA1301. By using the transient expression of gus gene as a criteria, the transformation frequency was detected, and the optimal conditions for rice genetic transformation with pCAMBIA vector series were determined. The optimal concentration D (600) of bacterium cell suspension was 1.0 to 1.6. The optimal co cultivation temperature was between 22 ℃ and 28 ℃, the pH of medium for co cultivation was between 5.2 and 6.2, the duration of co cultivation should be less than 3 d. To achieve high transformation frequencies, it was very important to add As into co cultivation medium but not necessary into the bacterium cell suspension. It should be noted that there might be some fine difference of transformation conditions when different explant materials were used, therefore the optimal genetic transformation conditions for different explant and different target genes should be further studied.