卡那霉素筛选转基因棉花技术研究
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摘要
为了提高带有npt-Ⅱ标记基因的转基因棉花后代的筛选效率,通过利用不同浓度的卡那霉素溶液进行浸种,同时比较浸种时间的长短对棉花子叶片伤害程度,并用PCR分子检测相互验证。结果表明:用浓度1 000 mg/L的硫酸卡那霉素溶液浸种3 d,每5 mL溶液浸泡1粒棉花种子,播种出苗以后,拔除子叶上有白化斑的棉花苗,剩余的子叶上没有出现白化斑的棉花苗即为转基因阳性植株,经PCR检测筛选的准确率达100%。利用卡那霉素溶液浸种的方法筛选带有npt-Ⅱ标记基因的转基因棉花后代材料的技术流程经济有效、准确可行。
To improve the screening efficiency of transgenic cotton, based on the certain volume of kanamycin solution applied for cotton seed soaking, the different concentration of the solution, the soaking time and the degree of seed leave damages were measured.And combined with the comparison results with molecular detection, technical methods and test standards for resistance screening of cotton transgenic progeny were validated. The results showed that before sowing, every cotton seeds were soaked with 5 mL solution with1000 mg/L kanamycin sulfate for 3-4 d. After seedling emergence, the positive transgenic plants were seedlings with no white spots on cotyledons, and the screening accuracy was 100% validated by PCR detection. In conclusion, the method of screening transgenic cotton progeny labeled with npt-II marker by soaking seeds with kanamycin solution is accurate and feasible.
To improve the screening efficiency of transgenic cotton, based on the certain volume of kanamycin solution applied for cotton seed soaking, the different concentration of the solution, the soaking time and the degree of seed leave damages were measured.And combined with the comparison results with molecular detection, technical methods and test standards for resistance screening of cotton transgenic progeny were validated. The results showed that before sowing, every cotton seeds were soaked with 5 mL solution with1000 mg/L kanamycin sulfate for 3-4 d. After seedling emergence, the positive transgenic plants were seedlings with no white spots on cotyledons, and the screening accuracy was 100% validated by PCR detection. In conclusion, the method of screening transgenic cotton progeny labeled with npt-II marker by soaking seeds with kanamycin solution is accurate and feasible.
引文
[1]孙敬,唐灿明,袁小玲,等.利用卡那霉素间接鉴定法进行大规模的棉花转基因育种技术[J].棉花学报,2000,12(5):270-276.
[2]祝建波,王爱英,吴刚,等.转基因棉花的田间筛选技术研究[J].棉花学报,2000,12(5):230-233.
[3]包立生,杨晓红,郑寨生,等.应用卡那霉素快速鉴定苗床期抗虫杂交棉[J].湖北农业科学,2002(6):56-58.
[4]崔淑芳,李俊兰,葛朝红,等.卡那霉素浸种筛选转基因棉花的初步研究[J].华北农学报,2003,18(2):118.
[2]祝建波,王爱英,吴刚,等.转基因棉花的田间筛选技术研究[J].棉花学报,2000,12(5):230-233.
[3]包立生,杨晓红,郑寨生,等.应用卡那霉素快速鉴定苗床期抗虫杂交棉[J].湖北农业科学,2002(6):56-58.
[4]崔淑芳,李俊兰,葛朝红,等.卡那霉素浸种筛选转基因棉花的初步研究[J].华北农学报,2003,18(2):118.