含NPTⅡ标记基因的转基因抗虫棉室内快速鉴定方法

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4～7 d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.

Three Methods for Rapid Selection of Transgenic Cotton Plants in Laboratory with Kanamycin as Indirect Marker

Three methods were developed to rapidly identify Bt-transgenic cotton plants with kanamycin-resistant gene as indirect selective marker. The first method was to culture uncoated seeds on the kanamycin containing medium (kan-medium), the difference between transgenic and non-transgenic plants was revealed after seeds germinated on the medium. According to the color of the cotyledons, kan-resistant plants were discovered at optimal concentration of 0.75 g·L-1. The second method was to paint kanamycin solution (kan-solution) on the cotyledons directly with optimal concentration at 4.0 g·L-1, by which after 4-7 days, plants with cotyledon normal green were considered as kan-resistant plants. The third one was that a pore was dug on cotyledons and dropped kan-solution in . The best concentration of the kan-solution was 2.0 g·L-1. All the plants could be rapidly screened out in 4-7 days after kanamycin treatment by the three methods. The efficacy of above methods for transgenic plant indirect identification were tested using PCR analysis with Bt-gene specific primers, giving a examination ratio of more than 90%. Moreover, all the seeds decorticated could be more easily identified with kanamycin treatments.