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小麦芽生长点的基因枪转化技术研究

标题: 小麦芽生长点的基因枪转化技术研究
英文标题: RESEARCH ON SHOOT APICAL POINT TRANSFORMATION OF WHEAT GERMINATED SEEDS BY PARTICLE BOMBARDMENT
作者: 董福双,张艳敏,杨帆,梁新朝,刘桂如,王海波
英文作者: DONG FU-SHUANG,ZHANG YAN-MIN,YANG FAN,LIANG XIN-CHAO,LIU GUI-RU,WANG HAI-BO
出版时间: 2009-01-01
机构: 河北农业大学
关键词: 小麦,芽生长点,基因枪,转化
英文关键词: WHEAT,SHOOT APICAL POINT,PARTICLE BOMBARDMENT,TRANSFORMATION
刊名: 华北农学报
英文刊名: ACTA AGRICULTURAE BOREALI-SINICA
ISSN: 1000-7091
卷号: 005
基金: ^A转基因生物新品种培育重大专项^B2009ZX08010-011B^D1
页码: 1-6
分类号: S512.03
摘要: 为建立简易的不依赖组织培养和抗性筛选的小麦转基因技术,以小麦品种石4185为对象,以GUS和BADH/BAR为外源基因,围绕适于基因枪转化的种子芽生长点受体的制备、基因枪轰击参数的优化等开展了试验,建立了"用解剖刀去除种子根、用镊子把芽鞘掰去、用镊尖扫去包盖生长点的未展开叶、将带生长点的部分与胚乳分离"为特点的受体制备方法;确定1 100 PSI+9 CM为最佳轰击参数组合.对轰击后的小麦芽生长点(70个*3批)进行GUS基因瞬时表达检测,以受体数统计时,转化率为22.9%~35.7%;以生长点数统计时,转化率为5.7%~8.6%.用含BADH/BAR的PABH9质粒转化1 000个受体,发育成了802个T_0植株,对T_0植株所结种子长成的穗行喷200 MG/KG的PPT进行抗性筛选,获得了105个抗性株,究其来源为42个T_0株,由此计算抗性率为5.2%.用PCR检测T_1植株,6个呈阳性.初步建立了基因枪法转化小麦种子芽生长点的技术.
英文摘要: In order to set up a simple and easy method for genetic transformation of wheat, which do not depend on tissue culture and resistance selection, experiments of target-preparation of shoot apical point of buds for particle delivery and parameter-optimization of bombardment were conducted with a cultivar of Shi4185 as a recipient, and with gus, badh/bar as foreign genes.A suitable way was successfully founded to prepare shoot apical point targets from germinated seeds of wheat to bombardment, and the optimum parameters combination was determined as 1 100 Psi + 9 cm. Statistics of gus detection of transient expression with 70×3 batches indicated that 22.9%- 35.7%of targets and 5.7%-8.6%of shoot apical points were transformed. With the bombardment of pABH9 containing badh/bar, 802/1 000 targets devel-oped to normal plants. 105 resistant T_1 plants were obtained by spraying PPT of 200 ppm to the seedlings of seeds corre-sponding to 42 T_0 plants with a resistance rate of 5.2%. PCR analysis showed that 6 T_1 plants were positive. Preliminari-ly, the technique to transform shoot apical point of wheat buds by particle bombardment was established.