农杆菌介导大豆子叶节遗传转化体系的优化研究

Optimization of Genetic Transformation System from Soybean Cotyledon Mediated by Agrobacterium

以大豆品种"中豆32、Peking、早熟18、绥农14"子叶节为受体材料,用农杆菌介导法转入与抗逆相关的小麦Na+/H+逆向转运蛋白基因(TaNHX2),探索外植体大小、培养基主要成分、培养时间等因素对外植体分化的影响,旨在优化遗传转化条件,提高大豆转基因的遗传转化效率。研究结果表明,以健康外植体获得率、抗性丛生芽获得率和抗性芽伸长比率为指标,筛选并建立的优化转化系统为:大豆萌发和不定芽诱导时分别加入0.5mgL-16-BA和1.0mgL-16-BA,浸染时间为30min、共培养时间为3d;外植体大小为2/3子叶,kan抗性筛选浓度第一阶段和第二阶段分别是60和50mgL-1;利用上述方法,已获得中豆32转基因再生植株,经PCR分子检测,证明目的基因TaNHX2已导入并整合到大豆基因组中,转化率为3.78%。

Transgenic soybeans have been widely planted aboard, which leading the development of the transgenic crop in the world. Establishment of an efficient genetic transformation technology system will facilitate physiological and molecular biology studies as well as the improvement of cultivars in China. The objective of this study was to optimize the conditions of the cotyledon node system of Agrobacterium- mediated soybean transformation so that the transformation plants can be obtained. Four soybean genotypes of Zhongdou 32 ,Peking, Zaoshu 18 and Suinong 14 was used to introduce the wheat transcription factor Na^＋/H^＋ antiporter into soybean genome by Agrobacterium-mediated transformation. The proportions of healthy explants, resistance tufty shoots and elongated shoots used as criteria for selection of proper conditions. The results indicated that the optimal system included in 0.5 mg L^-1 and 1.0 mg L^-1 of 6-BA for germination medium and shoot induction ,2/3 cotyledon infected with 30 min for infection ,3 days for co-culture, selection with 60 and 50 mg L^-1 Kan during the first and second shoot initiation stages. Based on the optimized condition positive transformed plants containing TaNHX2 was obtained,which tested by specific PCR,indicating that Na^＋/H^＋ antiporter gene was into the genome of soybean. The results indicated that genotypes mainly affected rates of transformation and regeneration. The rate of genetic transformation in cultivar Zhongdou 32 was 3.78%.