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通过试验研究,提出了3种室内快速筛选和鉴定含NPTⅡ标记基因的转基因抗虫棉的方法。一是待检测棉子去种皮,在含卡那霉素培养基培养,根据幼苗子叶颜色和棉苗状态来辨别是否是转基因材料,卡那霉素最适浓度为0.75 g.L-1;二是去皮种子培养在无卡那霉素的萌苗培养基上,在棉苗子叶上涂抹卡那霉素溶液进行鉴定,最适浓度为4.0 g.L-1;三是在待测棉苗子叶上打孔、滴加一定浓度的卡那霉素,根据打孔并滴加卡那霉素部位的叶片颜色变化,鉴别并统计转基因植株,其最适浓度为2.0 g.L-1。3种方法均可在卡那霉素处理后4-7d内快速鉴定出转基因植株。同时,对转基因阳性植株进行PCR鉴定,结果表明3种卡那霉素方法鉴定的准确率均在90%以上。
Through the experimental research, three methods of rapid screening and identification of transgenic cotton with NPTⅡmarker were proposed. First, cotton seed to be tested to seed coat, cultured in kanamycin medium, seedling leaf color and seedling state to identify whether the transgenic material, kanamycin optimum concentration of 0.75 gL-1; the second is Peeled seed culture in kanamycin-free seedling culture medium, the cotton seedling leaf smear kanamycin solution identification, the optimum concentration of 4.0 gL-1; Hole, add a certain concentration of kanamycin, according to drilling and dropping kanamycin site leaf color change, identification and statistics of transgenic plants, the optimum concentration of 2.0 gL-1.3 kinds of methods are available at Transgenic plants were quickly identified within 4-7 days after kanamycin treatment. At the same time, the positive transgenic plants were identified by PCR. The results showed that the accuracy of three kanamycin methods were all over 90%.