Assessment of conditions affecting Agrobacterium rhizogenes-mediated transformation of soybean
Assessment of conditions affecting Agrobacterium rhizogenes-mediated transformation of soybean
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DOI：
10.1007/s11240-008-9458-x
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发表时间：
2009-01-01
影响因子：
3
通讯作者：
Han, Tianfu
中科院分区：
文献类型：
--
作者：
Cao, Dong;Hou, Wensheng;Han, Tianfu
根癌农杆菌介导的转化方法因其快速、简便的特点，已成为研究基因功能和根系生物学的有力工具。这种转化方法特别适用于那些植物，包括豆科植物，其使用根癌农杆菌的转化具有挑战性。虽然有一些关于A.发根农杆菌介导的豆科植物转化产生“复合”植物，影响A.发根农杆菌介导的大豆[Glycine max（L.）梅尔。]尚未得到充分调查。为了更好地理解A。在大豆的发根农杆菌介导的根转化中，我们评估了基因型、感染的植株年龄、细菌接种浓度、接种温度和其它因素对大豆转化的影响。结果表明，不同基因型大豆对A.发根大豆品种自贡冬豆对A.在10个供试基因型中，K599菌株的产根瘤菌能力最强。研究了苗龄对毛状根诱导的影响，发现1日龄苗是毛状根诱导的最佳苗龄。OD_（600）= 0.2 ~ OD_（600）= 1.2的菌液对毛状根的诱导无显著差异。在16 h光周期下，23 ℃/20 ℃和28 ℃/25 ℃两种温度条件下均可诱导毛状根，而33 ℃/30 ℃昼夜温度条件下均不能诱导毛状根。用此转化方法，在2周内几乎100%的复合植株形成毛状根，根据GUS组织化学分析，转化率达到94.2%。转基因整合也已通过Southern印迹分析证实。
Agrobacterium rhizogenes-mediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple methodology. This transformation method is particularly suitable for those plants, including legumes, whose transformation using Agrobacterium tumefaciens has been challenging. Although there are some reports on A. rhizogenes-mediated transformation of legumes to produce 'composite' plants, conditions influencing A. rhizogenes-mediated transformation of soybean [Glycine max (L.) Merr.] have not been yet fully investigated. To better understand A. rhizogenes-mediated root transformation in soybean, we have evaluated the impact of genotype, plant age for infection, bacterial inoculating concentration, inoculation temperature, and other factors on transformation of soybean. The results have shown that there are significant differences among soybean genotypes in their susceptibility to A. rhizogenes. Soybean cv. Zigongdongdou is the most susceptible to A. rhizogenes strain K599 among 10 genotypes tested. The effects of seedling age have been evaluated, and 1-day-old plantlets are found to be optimal for hairy root induction. There are no significant differences in hairy root induction for bacterial suspension from OD600 = 0.2 to OD600 = 1.2. Under 16 h photoperiod, hairy roots can be induced both at 23 degrees C/20 degrees C and 28 degrees C/25 degrees C, but not at 33 degrees C/30 degrees C as day/night temperature regimes. Using this transformation protocol, almost 100% of the composite plants formed hairy roots within 2 weeks, and based on GUS histochemical analysis, 94.2% transformation frequency is obtained. Transgene integration has been also confirmed by Southern blot analysis.