摘要
利用农杆菌〔ＧＵＳ基因（β－葡萄糖苷酸酶基因）为标记基因，ＮＰＴⅡ为选择基因〕，用共培法对棉花下胚轴切段直接转化。在０．１ｍｇ／Ｌ２，４－Ｄ和０．１ｍｇ／ＬＫＴ的ＭＳ培养基上共培４８ｈ，转移到加头孢霉素５００ｍｇ／Ｌ和５０～１００ｍｇ／Ｌ卡那霉素的上述培养基中诱导和筛选抗性愈伤组织，７０～８０天计算愈伤组织的诱导频率，并用组织化学定位法检测ＧＵＳ基因的表达，统计转化频率。结果表明：平均出愈率为４０％左右，最高达６０％以上。愈伤组织转化率平均为３０％左右，最高达７０％。再生植株转化频率一般在３０％左右，最高达６０％。中国农科院生物技术中心和中国科学院遗传所对ＤＮＡ分子杂交检测结果，证实ＢＴ基因已整合到转化棉株的基因组中。
Hypocotyl segments from 5 6 day old seedling of Gossypium hirsutum were co cultured with Agrobacterium tumefacience,GUS gene as marker gene,NPT as selecting gene.After the cultures were co cultured in MS medium contained 0.1 mg/L 2,4 D and 0 1mg/L KT for 48h,then they were transferred into the medium described above suplemented with 500mg/l cefotaxime and 50 100mg/L kanamycin to induce and select resistant calli.After 70 80d the induction frequence of calli was calculated.The expression of Gus gene was tested with histochemistry location method and the transformation frequency was recorded.The experimental result indicated that the average rate of callus was 40% or so, and the highest was over 60%.The average rate of callus transformation was 30% or so,and the highest was 70%.The frequency of regenerated plant transformation was 30% or so,and the highest was over 60%.According to the result of DNA molecular hybridization,the presence of introduced gene in the genome of the transgenic plants was confirmed. It was also confirmed that BT gene had been integrated into genome of transgenic plants and was inherited into its progenies.
出处
《华北农学报》
CSCD
北大核心
1997年第2期82-85,共4页
Acta Agriculturae Boreali-Sinica
