| Soybean [Glycine max (L.) Merrill] is one of the most important protein and oil crops worldwide. There is great interest to improve soybean crops by genetic transformation techniques. A rapid and efficient shoot organogenesis and regeneration system was established for soybeans using whole cotyledonary node explants. Based on this protocol, genetically transformed soybeans were produced by co-cultivating whole cotyledonary node explants with Agrobacterium tumefaciens carrying a binary vector (EHA105/pCAMBIA3300) coding for CryIA and pta genes.Whole cotyledonary node explants were obtained from aseptic seedlings cultured on MSB5 medium supplemented with N6-benzyladenine (BA) at the concentration of 0.4 mg L-1 for 5~7 d. Then the explants were transferred to various shoot induction media for 14 d. Shoots were elongated on the MSB5 medium to 3~4 cm. The fertile plants were collected after rooting on MSB5 medium containing indole-3-butyric acid (IBA) 0.5 mg L-1. Plantlets with well-developed roots were hardened and planted in greenhouse to maturity. The process of shoot development was also observed; BA or N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) were used to shoot regeneration and BA was more effective; The effects of the plant growth regulators BA, kinetin (KT), IBA as well as the explants'genotype on shoot induction were evaluated by using a standard orthogonal design, BA exhibiting the greatest benefit and the best combination for shoot regeneration was MSB5 medium supplemented with 3.0 mg L-1 BA, 0.2 mg L-1 IBA and 0.5 mg L-1 KT on Hefeng 25 genotype. Under these most favorable conditions, one explant could regenerate 30~35 shoots. Overall, most of the regenerated shoots elongated and rooted well. In comparison with traditional cotyledonary node and embryonic tip regeneration system, whole cotyledonary node regeneration system appeared superior in shoot regeneration frequency and number of shoot.In the present study, we first report a more efficient and successful regeneration system for soybean using whole cotyledonary nodes as explants. Using this system led to a significant increase in regeneration efficiency and could overcome genotype-associated problems by selecting the proper medium. The results showed that shoot regeneration frequency could reach upwards of 95% and the procedure enabled the production of a large number of regenerated plantlets in a relatively short time (60 days).An efficient transformation system was established for soybean using Agrobacterium-mediated transformation with three cultivars and some important factors that affected transformation efficiency were identified. The results showed that the effect of different Agrobacterium strains on transformation were different and EHA105 was proved to be a better transformer than GV3101 and LBA4404; Hefeng 48 showed a high frequency of transformation; Cef 300 mg L-1 + Cb 200 mg L-1 was used for inhibiting Agrobacterium growth and PPT 5.0 mg L-1 was used to selection of transgenic cells. Using this system, the efficiency of transgenic plant production ranged from 5.50% to 12.07%. PCR and Southern analyses confirmed the stable integration of the insect resistant genes in the primary transgenic plants. The results of T1 plants analysis showed the inheritance and stable integration of transgenes. Insect bioassays showed that the transgenic soybean plants exhibited aphid resistance activity.The results showed that the whole cotyledonary node regeneration system can be used for effective Agrobacterium-mediated transformation for soybean.
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