Optimization of Agrobacterium-Mediated Genetic Transformation of Soybean Using Glufosinate as a Selective Agent.

Abstract

Soybean [Glycine max (L.) Merr] is one of the most important oil crops. Genetic transformation techniques can provide new tools for soybean improvement. The production of transgenic soybean has been limited. This research integrated a modified soybean regeneration system into a transformation protocol to optimize the production of transgenic soybean. Soybean hypocotyls and cotyledonary nodes were cultured on Gamborg B5 medium containing thidiazuron (TDZ). TDZ at 0.16 muM was found superior to other concentrations for inducing multiple shoot formation. Explants isolated from seedlings germinated on medium containing 6-benzyl-aminopurine (BAP) greater than 7 muM produced more multiple shoots than when on lower concentrations of BAP. Elongation of shoots was achieved on Gamborg B5 medium containing 0.36 muM BAP for hypocotyl-derived, or containing 0.58 mg/l gibberellin acid (GA3) and 0.67 mg/l indole-3-butyric acid (IBA) for cotyledonary node-derived multiple shoots. Plant recovery was achieved on medium consisting of Gamborg B5 with 0.58 mg/l GA3, 0.67 mg/l IBA, 2.0% sucrose, and 7 g/l phytagar. Transgenic soybean plants were obtained with Agrobacterium-mediated transformation using glufosinate as a selective agent. Nodes with 1/3 cotyledons inoculated with Agrobacterium KYRT1 and subjected to vacuum infiltration during inoculation and 4 mg/l glufosinate in the selection medium, produced more glufosinate-resistant multiple shoots than other treatments. The optimal duration of vacuum infiltration was 10 minutes at 508 mm Hg. A glufosinate concentration of 1 to 1.2 mg/l was necessary to select transgenic shoots. Recovered plants were screened with a 0.3 ml/l solution of Liberty herbicide. PCR and southern hybridization analysis confirmed transformation. Progeny tests using herbicide leaf painting assay, PCR, and RT-PCR analysis, indicated that the transgene was transmitted to and expressed in the next generation. The effects of additional copies of virE and virG genes in Agrobacterium and activation of vir genes with acetosyringone on plant transformation were also studied. A significant enhancement was observed when additional copies of virE and virG genes were included in the Agrobacterium for transformation of Arabidopsis but not for soybean. Activation of vir genes with acetosyringone (100muM) increased the glufosinate-resistant multiple shoot formation rate in soybean, and reduced the rate in Arabidopsis.