关键因子对棉花利用农杆菌介导法导入外源基因的影响

Influence of Some Factors on Transgenic Cotton by Agrobacteriummediated Gene Transfer

用 ５～ ６ｄ的棉花无菌苗下胚轴切段与农杆菌共培，将 Ｂｔ基因和 ｔｆｄＡ基因导入棉花。菌株质 植中 ＧＵＳ基因为标记基因， ＮＰＴ Ⅱ基因为选择基因。在含有 ０． ｍｇ／Ｌ ２， ４－Ｄ和 ０． １ｍｇ／Ｌ ＫＴ的 ＭＳ 培养基上共培 ４８ ｈ，转移到添加头孢霉素 ５００ ｍｇ／Ｌ、卡那霉素 ５０ ｍｇ／Ｌ培养基中诱导和筛选抗性愈 伤组织。７０～８０ｄ时，进行ＧＵＳ检测，选择ＧＵＳ阳性愈伤组织，经体细胞胚胎发生途径形成转基因再 生植株。

Hypocotyl segments from 5 - 6 day old seedlings of Grossypium hirsutum were cocultured with Agrbacterium tumefaciens in order to introduce Bt and tfdA genes into cotton crop. GUS gene is as marker gene and NPT Ⅱ gene is as selecting gene. The cultures were cocultured in MS medium contained 0. 1 mg/L 2, 4-D and 0. 1 mg/L KT for 48h, then transfered into the medium supplemented with 500 mg/L Cefotaxime and 50 mg/L Kanamycin to induce and select resistant calli. After 70-80 days, GUS gene was inspected and the positive calli were selected and cultured on embryogenesis medium until globular embryos developing and germinated plantlets forming.