Selection Of EPSPS Transgenic Cotton And Effects Of Glyphosate On Its Fertility

Glyphosate is a broad-spectrum herbicide. However, it causes severe damage to cotton (Gossypium hirsutum L.) development and reduces cotton yield. It is important to breed Glyphosate-resistant transgenic cotton (GR cotton) and to study the effect of glyphosate on GR cotton development, especially the sterility. It could provide information for the commercial production of GR cotton. In addition, if glyphosate be used as a chemical hybridizing agent, that will have an important effort on the seed production of hybrid cotton production and breeding of GR cotton.In this study, the EPSPS transgenic cotton seeds were used as material. After the primary selection by glyphosate and the test strip, two transgenic cotton cultivars were obtained. Semi-quantitative, quantitative RT-PCR as well as ELISA methods were used for detecting and testing the expression of EPSPs. The resistance of glyphosate was tested under field and laboratory conditions. Meanwhile, we studied the effect of glyphosate on fertility of GR cotton and the effectiveness of glyphosate as a chemical hybridizing agent.The results were as follows:1. The EPSPS transgenic cotton seeds were used as material. After the selection by PCR and the test strip, two transgenic cotton cultivars S06-1 and S06-2 were obtained. Genetic studies suggested that the EPSPS gene in GR cotton was a single-dominant gene, and they were none-allelic.2. Using semi-quantitative and the quantitative RT-PCR, the expression of EPSPS gene was tested and quantified in reproductive and vegetative tissues. It was shown that the EPSPS gene was differently expressed beween the two tissues. Its level in reproductive tissues was significantly less than that in vegetative tissues of S06-land S06-2.3. The expression of EPSP enzyme was quantified in reproductive and vegetative tissues using ELISA.The content of EPSPS enzyme is 0.18mg/g in stigma,0.17mg/g in anther and 0.24mg/g in leaf. Analysis of the EPSP enzyme content in various tissues of GR cotton revealed that the content of EPSPS enzyme in stigma and anther were significantly less than that in leaf as the observation on the mRNA level.4. The phytotoxicity of GR cotton caused by glyphosate showed that leaves appeared dry spots, and the edge of leaves was dry, even the plant dead. The appropriate concentration of glyphosate for identifying GR cotton in the seedling stage was 3g/L-6g/L. In addtion, the concentration of glyphosate used in commercial production of GR cotton should not exceed 9g/L. Otherwise, it would severely damage the cotton plant.5. The fertility of cotton induced by glyphosate was relative to plant timings and concentration of glyphosate application.The application of glyphosate in budding stage may result in male-sterile flowers, such as an increased distance between the anthers and the receptive stigma which caused reduced pollen deposition on the stigma surface, the anthers failed to open, undeveloped and non-viable pollen grains, and the pollen grains seemed to collapse. The concentration of glyphosate which caused a complete loss of pollen viability was 6g/L. Cross-pollination for plants made fructify normally.6. The effectiveness of glyphosate as a chemical hybridizing agent of GR cotton was studied. In this study, we used the transgenic lines S06-1, S06-2 as the female parent and glandless cotton 3055 as the male parent.The results showed that the purity of hybrid gradually increased with Glyphosate concentration raisied, even can reach 96.9%. However, the high concentration of glyphosate has negative impact on the yield and quality of cotton. Considering these factors, the best dosage of glyphosate as a chemical hybridizing agent was 6g/L and applied at budding stage. It is better to applied 3 times, 1time every 10 days.7. After analysis of the hybrid F1 of the crosses S06-1, S06-2 with the normal unland cotton cultivars H109, we concluded that there is no significant effect on the growth of hybrids by used glyphosate.