The Research Of Biological And Biochemical Method To Detect The Reoundup Ready Soybean

The cultivation and promotion of t Genetically Modified soybean increased the efficiency of weeding observably,obtained great economic benefit。As researching deeply, the ecological security of Genetically Modified soybean.the flow of resistance genes and food security by alien genes in transgenic herbicide resistants crops has caused more and more controversies and inhibited its developing. So it has important significance to establish a perfect detect GM soybean method for China's soybean breeding research, environmental security and food safety.Currently the method to detect Roundup Ready soybeans mainly focused on molecular detection and field (greenhouse) whole plant identification method. As the former method,the majority of the research institutes can not carry out because of the precision of equipment, reagents ,high cost or high specificity and also only for target genes were detected, and can not verify whether the expression of target genes which although visually accurate; The latter method equired a long time, heavy workload, and vulnerability to seasonal effects even though intuitive and accurate.In this study,we constructed the biological and biochemical method to detect Roundup Ready soybeans combining with herbicide biometric technology for the current status of research. We discussed the detection index, the different culture conditions and temperature which is the method applicable and verified it by field testing of phenotypic and molecular mean.The main results are as follows:ⅠThe establish of biological method to detection Roundup Ready soybeans1. we took for soybean hypocotyl inhibition rate as an indicator to establish the biological method of detection Roundup Ready soybeans at 26℃, continuous cultured 72 h in darkness. In the method , the hypocotyl inhibition LD50 of GM soybean was1620.86-2269.33mg a. i./L while Non-GM soybean was 78.04-97.43 mg a. i./L and it has 20 times between the two soybeans. So we defined the discrimination dose as 100 mg a.i /L or 2000 mg a.i /L whlile GM soybean and Non-GM soybean can effectively distinguish in the two doses.2. we established the biochemical method of detection Roundup Ready soybeans for the biological method. In the concentration of 100 mg a.i /L, shikimic acid accumulation of Non-GM soybean was 1237.7 mg a.i /L and GM soybean was 175.10 mg a.i. /L. There was a significant difference between the two soybeans and can effectively identify GM soybean and Non-GM soybean.3.We detected the samples of Longkang630 and Suinong10-6047 based on the biological, biochemical method。The results showed that Longkang630 and Suinong10-6047 was GM soybean and EPSPS gene was transferred into soybean which obtained the resistannce. Field testing of phenotypic showed that Longkang630 and Suinong10-6047 was Roundup Ready soybean and the two samples was identified as positive by molecular mean. Three kinds of test results confirmed each other and proved biological and biochemical determination viablely creditable.Ⅱ.The determination of physiological and biochemical mechanism to resistance in the biological and biochemical method1. The shikimic acid is the main way of targe pathway with the stress of glyphosate. Because of a EPSPS gene into GM soybean,which can germination and growth normally and the shikimic acid accumulation was smaller and not affected thus to in a higher concentration of glyphosate in water culture conditions , while non-transgenic soybeans do not have this capability.2.The GSTS activity has significant difference between GM soybean and Non-GM soybean.with the stress of glyphosate,which shown that GSTS involved in the detoxification metabolism of soybean and the metabolizable ability of GSTs is the other important reason of herbicide resistance to glyphosate . Late activity was gradually reduced with the concentration increase and time extension and shown that the detoxification capacity is limited.3.Although the SOD, POD activity rised in the first in the process of glyphosate Stress between the GM soybean and Non-GM soybean in vivo, but both no significant difference between them and shown that this mechanism is not the main reason of their resistance. Maybe the free radicals and active oxygen and other harmful substances was induced by the herbicide stress and water stress ,which caused the SOD, POD activity increased,. It was an instinct of self-defense, played a synergistic resistance Role.Ⅲ.Application of biological, biochemical method in the HybridizationWe detected the the offspring 06-698,1568, HF1, HF9, Sui BC1F6006, in as J9331, H04-7-11 efficiently by the method,which shown that the resistance extent to glyphosate significantly higher due to EPSPS gene transfer into successful and increased 20-30 times than their than hybrid parents。So we identified them as Roundup Ready soybeans and the results are consistent with the molecule detection.