Study On EPSPS Gene As The Selection Marker Of Soybean Genetic Transformation System

Glyphosate is Phosphonomethyl Imino Acetic Acid, which was found to be aherbicide in1971. It has been widely used for thirty more years since it was put intomarket. Now it is the biggest usage of herbicide. Glyphosate can inhibit5-enolppyruvylshikimate-3-phosphate synthetase(EPSPS)to block the synthesis ofaromatic amino acids then plants die as a result. With the no-tillage technique adoptedand the level of mechanic agriculture increased, weeding efficiency is required to behigher and higher. Thus, it's very important to breed herbicide-tolerant (for example,glyphosate-tolerant) varieties. For transgenic breeding, there are three ways to getglyphosate-tolerant crops: transferring modified or mutant EPSPS gene, transferringoverexpressed EPSPS, or transferring glyphosate-degrading genes.In the research of soybean transformation, how to select and use selection agentsdoes not only affect the efficiency of transformation but also the application ofresultant transgenic plants. Antibiotics such as kanamycin and hygromycin, andherbicides such as glufosinate are usually used as selection agents in soybeantransformation. Kanamycin is low cost and widely applied, but the tolerance ofdifferent receptor varieties varies significantly. Hygromycin has good selection effectsin soybean transformation, but in some reports, it can seriously inhibit the growth ofsome plant tissues. At the present, glufosinate is the most-used selection agent insoybean transformation. Glufosinate can inhibit glutamine synthetase resulting in theaccumulation of ammonium ion, and then plant cells die of ammonium poisoning. Asa high-efficiency, low-toxicity, and broad-spectrum herbicide, glyphosate has beenwidely used for thirty more years and its characteristics and mechanisms are wellunderstood, but it is hardly publicly reported as the selection agent of soybeantransformation.In this study, herbicide glyphosate was used as the selection agent, and EPSPS asthe target gene. The glyphosate tolerance was explored in three stages of soybeantransformation with cotyledonary nodes as explants, that is, the stages of induction,screening, and growth of shoot, in order to know the feasibility of glyphosate as aselection agent, provide the reference glyphosate concentrations and makesuggestions in the selection of selection agents in soybean transformation.Results showed that glyphosate can be used as a selection agent in soybean transformation with cotyledonary nodes as explants and the reference glyphosateconcentrations are140-160mg/L,240-260mg/L and5-10mg/L respectively in thestages of induction, screening, and growth of shoot. Based on it, EPSPS genetransformation was conducted and two transgenic events were gotten. Therefore, thisstudy does not only provide another option of selection agent in soybeantransformation, but also increase the application value of transgenic materials andprovide theory and suggestion during the selection of selection agents in soybeantransformation and glyphosate-tolerant breeding.