The Research On Optimization Of Soybean Embryonic Tips Regeneration And Genetic Transformation Of Resistance Gene GmPK

Drought and soil salinization is a serious global problem. In China, the drought area, saline, and secondary salinization of cultivated land increases every year, restricting the production of crops in China. Therefore, saline control, exploitation and stress resistance of new varieties has become a research hotspot in recent years. Soybean is the world's major grain and oil crops, the main human source of vegetable protein, but by the environmental impact, the soybean's output and quality is restricted. Agrobacterium-mediated method with the resistance gene into soybean, cultivation of new stress resistance varieties of soybean, is the focus of genetic transformation study. In this study, eight high-quality soybean varieties in Hebei Province as the test material are initially identified its salt sensitivity by morphology(jidou 7,jidou 17,wuxing 1,wuxing 2, wuxing 3,nf 58, jihuang 13, zhonghuang 13). Using four soybean varieties (wuxing 1, wuxing 2, wuxing 3, nf 58) and high protein varieties Jidou 12 as materials, the soybean embryonic tip regeneration system are optimizated, efficiency is improved significantly comparing with the somatic embryos system. On this basis, the wuxing 2 embryo tip regeneration system as the receptor system, genetic transformation of resistance gene GmPK (soybean protein kinase gene of SnRK2 family) is undertaken using agrobacterium-mediated method. The results show that:1,Under NaCl concentration of 35 mmol/L, the height and length of root of jidou 17, wuxing 2 and wuxing 3 were inhibited, and the difference is significant compared with the control. Under NaCl concentration of 70 mmol/L, height, length of root and length of hypocotyl of 8 genotypes tested were significantly inhibited.2,Different genotypes as well as the concentration of hormone in media impact on embryonic tip regeneration. In the five varieties, performance of Jidou 12 regeneration system is the best. Tip explants germination rate and the average amount of multiple shoots are higher on the induction medium supplemented with 0.5 mg/L 6-BA or with 0.2 mg/L IBA. Stem elongation medium supplemented with 0.5 mg / L GA3 was better than that with 0.2 mg/L 6-BA and IBA. The best effect of rooting was from one using 1/2 MS basic medium with addition of 20 g/L sugar and 0.5 mg/L IBA, rooting rate is 85.4%.3,The wuxing 2 as experimental material, genetic transformation of GmPK is undertaken using Agrobacterium-mediated method. Through the auxiliary ultrasonic treatment and addition of 2 mg/L of AgNO3 and 2 mg/L of Pro in co-culture period, PCR-positive rate of embryo tip conversion is significantly improved (the infection rate of embryo tip conversion); survival of transplanted regeneration is confirmed by PCR and PCR-Southern, it proves that gene GmPK has been integrated into the genome of soybean, conversion rate was 0.6%.