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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 direct transfer of exogenous DNA using gene gun has become the main method of cereal gene transformation. However, in wheat, there is a problem of low frequency of DNA introduction and reduction of regenerative ability of tissues after bombardment. It was found through experiments that wheat immature embryos were inoculated on the induction medium supplemented with 0.5 mg / L abscisic acid (ABA) 14 days prior to bombardment, which significantly enhanced the regenerative capacity of wheat scutellum callus. Moreover, the callus was maintained in medium supplemented with 0.5 mol / L mannitol 6 h before bombardment and 18 h after bombardment, and its reporter gene expression was several times higher than that of the control. In addition, the appropriate bombardment of gold powder and DNA is one of the key factors in transformation. Shells cultured for about 14 days were bombarded with 0.5 μg plasmid DNA per gun with 250 μg gold powder.