The Research On Optimization Of Soybean Cotyledonary Node Regeneration And Genetic Transformation Of Resistance Gene AtNHX5

Soybean is one of the most important foods and oil crop in the world, but its production and quality is restricted by environment partly. Salinity is a major constraint of crop productivity because it reduces yield and limits expansion of agriculture onto previously uncultivated land in china. For this reason, the incubation of new antireversional variety is becoming an emphasis and a hot research as well for the past few years. So cultivating new antireversional variety of soybean by the Agrobacterium-mediated method is an efficient way to protect the environment and increase economic.In this study, three high-quality soybean varieties in Hebei Province were used as speciments. An efficient Agrobacterium-mediated gene transformation system based on the examinations of several factors affecting plant transformation efficiency was developed. Using the optimized system for the transformation, the wuxing2 cotyledonary node regeneration system as the receptor system, genetic transformation of the Arabidopsis thaliana salt-tolerant gene-AtNHX5(Arabidopsis Na+/H+ antiporter gene in Arabidopsis thaliana CPA1 family)is undertaken using agrobacterium-mediated method. Results are as follows:1. In this study, Soybean cotyledonary node of Hebei excellent cultivars were used as speciments. An efficient Agrobacterium-mediated gene transformation system based on the examinations of several factors affecting plant transformation efficiency was developed for soybean. The results showed that increased transgenic shoots were obtained when seeds germinated 6 days and low temperature pretreatment. The application of 20 mg/L anti-oxidant silver nitrate during co-cultivation can ease browning phenomenon. The optimized ultrasonic treatment for infected explants was 30 S. Using the system for the transformation of wuxing2, the explants were incubated in the buffer for histochemical assay of GUS activity. Finally, we obtained 18 transgenic plants which PCR-positive rate was 0.97%.2. In this study, used the optimized system, investigated AtNHX5 transformation by cotyledonary node method. The result showed that the AtNHX5 had already integrated to the genome of Wuxing2 soybean regeneration plants initially by PCR verification, and the T0 transformation efficiency was 0.23%. The expression of AtNHX5 was assessed by RT-PCR analysis.3. The progeny of control plants and T0 plants which PCR and RT-PCR analysis positive grown in the greenhouse were examined for herbicide resistance. Under Basta concentration of 100 mg/L, we found the positive transgenic plants can limit Basta within a small range, but the control became a large area of death, or even the whole leaf brown. Further evidence showed inheritance in T1 generation plants.4. The progeny of control plants and T0 plants which PCR and RT-PCR analysis positive grown 30 d in the greenhouse were examined for salt stress. Under NaCl concentration of 200 mmol/L for 10 d, we found the positive transgenic plant grew normally, but the control were significantly inhibited. Demonstrated salt-tolerance in T1 generation plants initially.