改良大豆子叶节再生体系的研究

Improvement of Cotyledon Node Regeneration System in Soybean(Glycine max)

采用大豆种子萌发5—6d后的子叶节作外植体，对农杆菌介导的大豆遗传转化系统及其影响因素进行r研究。结果表明，将子叶节与农杆菌共培养60h后放入含卡那霉素50mg／L的诱芽培养基上，待苗长至4-5cm后放入生根培养基中进行生根，最后移栽到培养土中，效果较好。对大豆遗传转化再生的主要因素，如大豆基因型、诱导出芽所需激素浓度、卡那霉素筛选压力等条件做了一系列的研究，建立了一个比较好的遗传转化系统，其转化率达到2．8％。

Transgenic crops such as soybean and cotton have been widely planted nowadays in China. Many efforts have been made to improve the quality of soybean, which is one of the main crops in China. Although transgenic legume has been put into the spotlight ever since 1984 when the first transgenic soybean was successfully obtained, setting up an effective transformation and regeneration system of soybean is still a challenge to investigators. Cotyledon nodes from soybean seeds germinating for 5-6 d were used in Agrobacterium tumefaciens-mediated transformation system as explants to study the factors affecting the transformation rate. The explants were first infected with Agrobacterium tumefaciens strain LBA440d containing pBI121 by 60 hours＇ co-cultivation. After washed with sterile water, the explants were then put onto shoot-inducing medium containing kanamycin for selection. Then, the induced seedlings in 4-5 centimeters long were transferred onto root-inducing medium. Finally, they were transplanted into soil when the roots had formed, and identified by PCR and Southern blot. The transformation rate was affected by three factors in this process. Hormone concentration was regarded as the most important factor in the regeneration system, so IBA as the phytohormone and 6-BA as the mitogen, with different concentrations were used to induce buds and roots formation. A relatively optimal system with 1.7 mg/L 6-BA and 0.5 mg/L IBA was acquired in which the budding percentage reached 81%. Genotype of the soybean was regarded as the second important factor in the transformation system. Four different kinds of soybean cultivars were used to test the influences of genotypes on transformation rate. Among them, the transformation percentage of Xiaoli Dou was up to 29%, nearly one fold higher than Heinong 10. Agrobacterium tumefaciens strain LBA440d could infect plants and transfer its gene into the plants. The process took place in the co-cultivation period. So the co-cultivation period was also important in the transformation system. 60 h co-cultivation with weak light was appropriate in our experiment, and faint mark of colonies could be seen after the co-cultivation. Transgenic plants were more resistant to kanamycin than untransgenic ones that could be killed on the medium with kanamycin. But high levels of kanamycin would also do harm to the transgenic ones. So, right concentration, about 50 mg/L in our test, was required to differentiate the transformed and untransformed plants. In conclusion, main causes that have obvious effects on the transformation and regeneration rates are the genotype of soybean,the hormone concentration for inducing buds and the kanamycin sensitivity on inducing and livability of the buds. A relatively good genetic transformation and regeneration system has been established, with which the transformation rate can reach to 2.8%.