The Research On Optimization Of The Agrobacterium-Mediated Genetic Transformation System Of Soybean Cotyledonary Node With Non Tissue-Culture And Transformation Of Gene Bar

As the important oil and economic crops, soybean [Glycine max (L.) Merrill] is the major source of edible oil and protein, occupying an important position in food industry and agricultural production. With the continuous development of genetic engineering, cultivating the new varieties of soybean by transgenic technology has been widely used. At present, the problems of stem elongation and rooting are still existing in the Agrobacterium-mediated genetic transformation system of soybean cotyledonary node. Establishing a genetic transformation system of soybean with non tissue-culture is an effective way to solve these problems. In this study, we used the high-quality soybean varieties which are popularized in Hebei province as the materials, by investigating the factors influencing the transformation efficiency of Agrobacterium, the genetic transformation system of soybean cotyledonary node with non tissue-culture was optimized according to the transient expression of GUS in the region of cotyledonary node. The gene Bar was transformed into soybean jidou 16 by using the optimized system. An effective screening method of the genetic transformation system of soybean cotyledonary node with non tissue-culture was established by comparing the screening efficiency of glufosinate injected and painted on leaves. The screening concentrations of eight high-quality soybean varieties in Hebei province were studied by glufosinate injected on leaves. The results showed that:1. By investigating the concentrations of sugar, surfactant, ways of infection, times of infection, concentrations of infection medium and soybean genotypes, we found that infection medium contains 3% sugar and none Silwet L-77, OD600=0.6, with the absorbent cotton as attached media, infect one time have the highest percentage of GUS positive, was 62.13%. Jidou 16 is the susceptible soybean genotype.2. The screening efficiency of glufosinate injected on leaves was 27.2%, while the screening efficiency of glufosinate painted on leaves was only 9.1%. Consequently, the screening method with glufosinate injected on leaves was confirmed to be the effective screening method of this genetic transformation system. According to the scorch and death of leaves and stem apexes, the screening concentrations of eight high-quality soybean varieties in Hebei province with glufosinate injected on leaves were preliminary confirmed respectively to be: 100 mg/L (jidou 15, wuxing 2); 150 mg/L (jidou 16, jidou 17, wuxing 1, zhonghuang 13); 200 mg/L (jidou 7, nf 58).3. The gene Bar was transformed into soybean jidou 16 which is a high-quality soybean varieties in Hebei province by using the optimized genetic transformation system of soybean cotyledonary node with non tissue-culture, the regeneration rate was 71.2%. We obtained ten positive plants in T0 generation after detected the transgenic plants which resistant to glufosinate by PCR, the transformation efficiency was 2.5%. Three positive plants of T1 generation were obtained by detection of PCR and RT-PCR, preliminary demonstrated that the target gene was integrated into the soybean genome.