普通小麦基因枪转化高效受体系统的建立

Establishment of efficient acceptor system for gene transformation by micro-projectile bombardment in wheat

以小麦幼胚愈伤组织为转化受体,以植物表达载体质粒pROK2为外源DNA,以蔗糖和甘露醇为渗透剂,研究了愈伤组织生长状态、渗透剂种类及其处理时间和浓度对基因枪转化效率的影响。结果表明,在采用基因枪轰击法对小麦幼胚愈伤组织进行转化的体系中,受体愈伤的最佳发育阶段为胚性启动前期;高渗处理可以使基因枪转化频率得到明显提高,但处理时间过长对愈伤组织的增殖和分化又有明显的抑制作用,用蔗糖进行高渗处理对愈伤组织生长及再生的抑制作用较用甘露醇弱;用0.6 mol/L蔗糖和0.5 mol/L甘露醇分别在轰击前6 h和轰击后18 h进行高渗处理转化效果均最佳。文章建立了高效蔗糖高渗基因枪转化受体系统。

An efficient acceptor system for gene transformation was established in callus of wheat immature embryos by improving the condition of osmotic treatment before bombardment. In this experiment, the acceptors were from 3 wheat cultivars including Qianjinzao,Xiaoyan 22 and 9848. Particle bombardment was carried out by using pROK2 containing the Npt Ⅱ gene and HAL 1 gene. The factors affecting the Biolistic transformation efficiency like physiological state and time of osmotic treatment with different osmosis concentration were studied. The result showed that osmotic treatment could enhance the differentiation frequency of bombarded calli. The callus before embryo switching on was optional. Sucrose as osmosis had less inactive effect on the growth and regeneration of callus than mannitol before bombardment. The callus treated with 0.6 mol/L sucrose and 0.5 mol/L mannitol for 6 hours before bombardment or 18 hours after bombardment could significantly achieve the optional transformation efficency.