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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (18): 94-99.

所属专题： 生物技术； 棉花

• 农学 农业基础科学 • 上一篇    下一篇

以茎尖分生组织为受体用农杆菌介导法转化激发子基因pemG1棉花植株的获得

徐大伟 邱德文 檀根甲 李淼 曾洪梅   

  • 收稿日期:2011-02-22 修回日期:2011-04-08 出版日期:2011-07-25 发布日期:2011-07-25
  • 基金资助:

    国家转基因生物新品种培育重大专项

Agrobacterium-mediated Transformation of Shoot Apex of Cotton and Production of Transgenic Plants Carrying Elicitor Gene pemG1

  • Received:2011-02-22 Revised:2011-04-08 Online:2011-07-25 Published:2011-07-25

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摘要：

为了解决棉花常规遗传育种周期长，农杆菌介导的转化下胚轴再生难，转化率低，胚变异率高等难题。通过改善茎尖的遗传转化，利用农杆菌介导法转化棉花茎尖，试图缩短转化周期，获得转化苗。构建了稻瘟菌激发子基因pemG1的植物表达载体pCAMBIA2300-pemG1，分别以‘珂字312’、‘中棉24’棉花茎尖分生组织作为外植体，摸索无菌苗的最佳培养方案，确定最佳共培养温度、共培养时间及卡那霉素筛选浓度。经过共培养，茎尖在诱导芽培养基上诱导生芽，从不同浓度的BAP和NAA生长激素的组合中，最终确定0.1 mg/L的BAP和NAA最适合芽的诱导；经过两个月的继代培养，在含有GA3的茎增殖培养基上完成茎的增殖和延伸；切取幼芽并使其在含有0.1 mg/L的IBA生根培养基中生根；生根的抗性植株移栽到温室中成苗，获得了T0代转基因株系4株。进行了转pemG1阳性植株的初步验证。

关键词: 坡耕地, 坡耕地, 土壤侵蚀, 坡度, 降雨量, 耕作方式

Abstract:

In order to solve the long cycle of conventional breeding of cotton and the difficult regeneration, the low conversion rate and high rate of embryo variability in Agrobacterium-mediated transformation of hypocotyl of cotton. Agrobacterium-mediated transformation using cotton tip as explants, by improving the genetic transformation of the tip, try to shorten the conversion period and get into seedlings.The fungal elicitor gene pemG1 was cloned from Magnaporthe grisea and its plant expression vector pCAMBIA2300-pemG1 was constructed. The apical meristems of cotton cultivar ‘Coker312’ and ‘CCRI24’ were used as explants for agrobacterium-mediated transformation. The optimum co-culture temperature, incubation time and the concentration of kanamycin were determined. The 0.1 mg/L of BAP and NAA was selected as the best concentration for bud induction. After subculture for two months, the stem was transferred to the medium containing GA3 for proliferation and elongation, and then to the medium containing 0.1 mg/L of IBA for rooting. Finally, the resistant plants were transplanted in the greenhouse and 4 transgenic plants were acquired by preliminary molecular verification.