Study On Agrobacterium-mediated Genetic Transformation System Of Soybean

Soybean( Glycine max) is the most important source of both edible vegetable oil and protein supplement for livestock. It also has a wide range of industrial uses. Now, A number of techniques are currently in use for genetic modification of plants. These include Agrobacterium-mediated transformation, polyethyleneglycol-mediated transformation, pollen tube pathway method, microinjection, protoplast and intact cell technology, and micro particle bombardment technology. Plant transformation mediated by Agrobacteriwn tumefaciens has become the most common method for the introduction of foreign DNA into plant cells. Agrobacterium tumefaciens also has been used extensively for genetic engineering in soybean , but to date, transformation frequency of soybean is still low relative to that obtained in other plants. A successful transformation of soybean using Agrobacterium relies on several factors including the frequency of regeneration in transgenic soybean cells, soybean genotype, Agrobacterium strain, bacterial infection and transformation competency of the target tissue.At an attempt to improve transformation efficiency, present experiment was conducted to compare the effection of several factors on transformation of cotyledonary nodes in soybean via Agrobacterium-mediation.In experiment, multiple shoots were induced on cotyledonary nodes of soybean via organogenesis. In the same media and concentration of phytohormone, different regeneration frequency of multiple shoots was found in different genotypes. Cultivars "JilinSO", "Jilin47" and "Meiyin T'have a higher regeneration frequency; high efficiency regeneration multiple shoots appeared when cotylednoary nodes were induced on the media contains 4mg/L 6-BA+0.2mg/L IBA and 2mg/L 6-BA+0.2mg/L IBA+lmg/L KT. Soybean cultivars "JinongDG3256", "Meiyin 1" and "Jinong D9513" are more sensitive to Agrobacterium strain EHA101 than other cultivars. Since the T-DNA transfer process is mediated by the cooperative action of proteins encoded by genes in the Ti plasmid virulence regions (Vir genes) and the bacterial chromosome. Vir genes inducer such as AS , AS-OH, etc. can enhance Vir gene expression and increase the transformation rate, experiment result indicated thatin2mg/L proline or 2mg/L AgNOs has the optimal effect on soybean transformation, they improve the transformation rate.The GUS gene has a higher expression rate when the soybean cotyledonary nodes were pre-cultivated 3 days before infected by Agrobacterium. It shows 3 days pre-cultivation time is suitable for soybean transformation.During different Agrobacterium growth phase, its infecting ability is different. When Agrobacterium grow reach log phase (a density of OD6oonm=0-7), it has strong infecting ability. We recommed infecting time for 26 mins is better in soybean transformation.A new and potentially more efficient method for transforming soybean has been developed to facilitate Agrobacterium infection, it called "sonication-assisted Agrobacterium-mediated transformation "(SAAT). SAAT can be used efficiently to transform soybean embryogenic suspension culture tissue, producing a much higher rate of transformation than with Agrobacterium infection alone. However, we did not see a higher tansformation rate form SAAT-treated soybean cotyledonary nodes.