影响基因枪法转化小麦幼胚的几个因素的研究

Studies on Some Factors Affecting Transformation of Wheat Immature Embryos by Biolistic Bombardment

近几年,利用基因枪直接转入外源DNA,已经成为禾谷类基因转化的主要方法。但在小麦中存在着DNA导入频率低且组织受轰击后再生能力降低等问题。通过实验发现,轰击前14天将小麦幼胚接种于附加0.5mg/L脱落酸(ABA)的诱导培养基上,可显著提高小麦盾片愈伤组织的再生能力。并且,在轰击前6h至轰击后18h,将愈伤组织保持在附加0.5mol/L甘露醇的培养基中,其报告基因的表达将比对照呈几倍的提高。另外,合适的轰击用金粉与DNA配比是转化的关键因素之一。轰击培养14天左右的愈伤组织,每枪用250μg金粉附着0.5μg质粒DNA较宜。

In recent years, the biolistic method, in which the alien DNA was directly introduced into cells via accelerated particles, has become a major way for cereal transformation. However, for wheat the transformation frequency is still low and the regeneration ability usually decreases after bombardment due to the particle wounding.It is found in our experiments that when wheat immature embryos were cultured 2 weeks before bombardment on ABA (0.5mg/L) containing medium the regeneration ability of scutellar calli could be improved significantly. Furthermore, we also found that from 6h before bombardment to 18h after it, keeping the target tissues on mannitol (0.5mol/ L) containing medium may increase several-fold in the expression of a report gene (GUS). Besides, optimization of the dosages of gold powder and DNA per bombardment is a key factor for the successful transformation of wheat. In our case, using twoweek cultured calli as target tissues, 250μg gold powder and 0.5μg DNA gave the best result.