Establishment Of Soybean Transformation System Mediated By Agrobacterium

Soybean (Glycine max (L.) Merr) is an important source of a plant protein and edible oils and fats. Researches related to the soybean regeneration and genetic transformation have been intensively performed and improved. While the low efficiency and the complicated manipulation of the soybean genetic transformation procedure limits the improvement of soybean traits.The introduction of heterogonous gene into soybean depends on highly efficient receptor system, which includes regeneration system and appropriate transformation method. In this study, we import the GUS gene into soybean cultivars Heinong37and Heinong44using Agrobacterium strain EHA101and EHA105which contains binary vectors pCambia1305.2and pZY102. In addition, we optimized the transformation through monitoring every step of the transformation using GUS reporter gene expression. Based on PCR, Southern blot and GUS histochemical staining assay, the To transgenic seedlings were detected. Our study indicates that, the transformation’efficiency of the Agrobacterium-mediated cotyledonary node method is stable and closed to1%. Totally there are total three PCR-positive transgenic soybean seedlings are obtained using Agrobacterium-mediated embryonic tip transformation system with the system’s transformation efficiency about0.5%-1.0%.The factors including the seeds sterilization methods, the choice of Agrobacterium and soybean genotype, as well as the appropriate selection, which influence the transformation were optimized in this study. The results are as following:soybean cultivars Heinong44and Heinong37were chosen to be used for transformation explants sources, since the good performance during the transformation process; the optimal sterilization time of chlorine fumigation for soybean seed sterilization is16hours; the concentrations of glufosinate and hygromycin used for selection at the shoot induction step were4-8mg/L and5-10mg/L respectively; the appropriate growth status of agrobacterium strains such as EHA105and EHA101is at OD650=0.6-1.0for efficient transformation.