The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties
The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties
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DOI：
10.1007/s00299-007-0475-8
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发表时间：
2008-03-01
影响因子：
6.2
通讯作者：
Huang, Jian-Qiu
中科院分区：
文献类型：
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作者：
Liu, Sheng-Jun;Wei, Zhi-Ming;Huang, Jian-Qiu
本研究建立了一种高效的农杆菌介导的大豆Glycine max （L.）基因转化体系。基于对影响工厂改造效率的几个因素的研究。用添加0.02% （v/v）表面活性剂（Silwet L-77）的农杆菌接种大豆子叶节，可提高转化效率。在侵染期间施用0.02%的Silwet l -77，在共培养期间施用600 mg l（-1）的l -半胱氨酸，比单独施用这两种因素更显著地提高了转化效率。采用改良的选择方案（在不含潮霉素的诱导培养基上培养7天，在含3mg l（-1）潮霉素的诱导培养基上培养10天，再添加5mg l（-1）潮霉素的诱导培养基上培养10天，在含8mg l（-1）潮霉素的诱导培养基上培养10天）选育出了更高效的转基因再生苗。利用优化后的体系，从我国5个重要大豆品种中获得145株形态正常、可育的独立转基因植株。转化效率从3.8%到11.7%不等。通过分子和遗传分析证实了转基因的稳定整合、表达和遗传。对T-1植株进行了分析，并验证了转基因以孟德尔方式传递给T-1代。优化后的转化体系可用于农杆菌介导的大豆基因高效转化。
In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and L-cysteine (600 mg l(-1)) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22 degrees C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l(-1) hygromycin for 10 days, 5 mg l(-1) hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l(-1) hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T-1 plants were analyzed and transmission of transgenes to the T-1 generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.