摘要
报道了采用微粒轰击（Ｍｉｃｒｏｐｒｏｊｅｃｔｉｌｅ ｂｏｍ ｂａｒｄｍ ｅｎｔ） 幼胚将除草剂抗性基因导入小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ Ｌ．）的转化研究。实验共使用了１３ 个小麦品种， 从开花后１４～１８ ｄ 的籽粒中剥取幼胚， 植物表达质粒含有ＣａＭＶ ３５Ｓ启动子控制的除草剂溴苯腈抗性基因ｂｘｎ 以及筛选标记基因ＮＴＰⅡ。采用高压放电基因枪，用质粒ＤＮＡ 包被的钨粒轰击预培养３ ｄ 的幼胚。在含有卡那霉素类似物ｇｅｎｅｔｉｃｉｎ Ｇ４１８ｓｕｌｐｈａｔｅ 的ＭＳ培养基上， 经过多步骤筛选和分化， 从８００ 多个幼胚中获得了１６ 株转化苗。除草剂抗性鉴定和Ｓｏｕｔｈｅｒｎ 杂交分析证明， 其中４ 株为转基因植物，具有溴苯腈抗性， 并且自交可育。转化工作从分离幼胚到转化苗鉴定完毕， 最短时间为６ 个月， 因此，
Genetic manipulation of wheat (Triticum aestivum L.) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation method. The authors report a reproducible protocol for rapid production of transgenic wheat via microprojectile bombardment. The experiment was carried out by using the immature embryo excised from caryopsis 14 to 18 days postanthesis and the plant expression plasmid carrying a CaMV 35S controlled bxn gene, for resistance to herbicide bromoxynil and a selectable marker gene NPTⅡ. After bombarding the precultured immature embryos isolated from 13 wheat varieties with plasmid DNA coated tungsten particle, these embryos were transferred on MS medium containing 10 mg/L geneticin G418 sulphate to select and regenerate transformants step by step. As a result, 16 transformed plants were obtained from a total of 849 bombarded embryos. The characterization of these plants by inoculation with herbicide bromoxynil and Southern analysis with bxn gene as a probe showed that 4 of the self fertile transformed plants contained the target gene and presented herbicide resistance. In several independent transformation experiments, the fastest one took only 6 months from embryo excision to characterization of regenerated plants. Therefore, this procedure is a rapid and efficient technique for delivering foreign DNA into wheat.
关键词
小麦
遗传转化
转基因小麦
抗溴苯腈
Wheat
Genetic transformation
Microprojectile bombardment
Immature embryo
