| weed infestation constitutes a major hazard in cotton production resulting in about 14-16% loss in yield. Manual tending is a primitive and traditional method for weeds control. However, due to the accelerated urbanization process, the transfer of rural population and the increase in labor cost, manual tending can no longer meet the needs of modern agricultural production. Glyphosate is a non-selective herbicide. Because of its broad-spectrum effect, high efficiency, and low toxicity and residual effect, it occupies 30% of the total herbicide market and has ranked the first agrochemical for many years. Selection and breeding glyphosate-resistant cotton varieties is the prerequisite to achieve chemical weed controlling on the cotton field. It is very necessary to breed our own patent of glyphosate-tolerant GM cotton for cotton production in China. In this study, Glyphosate-resistant EPSPS-G6 gene, which was cloned by Zhejiang University, was introduced into upland cottons culitivars, using coker 201 and CCRI-49 as the receptors by Agrobacterium- mediated method and pollen tube pathway method, and glyphosate-resistant transgenic cotton germplasms were obtained, which provided the new germplasm for development of glyphosate-tolerant cotton cultivars with our own intellectual property rights. Meanwhile, comprehensive identification and evaluation of germplasms provided a scientific basis for breeding and commercial production of transgenic glyphosate-resistant cotton. The major results in this study are as follows: 1. Developing of transgenic glyphosate-resistant cotton germplasmsThe EPSPS-G6 gene driven by ubiquitin promoter from maize was introduced into the upland cotton through agrobacterium- mediated method using coker 201 hypocotyls as explants and 50 mg.L-1 kanamycin as screening agent. Two T0 transgenic plants were obtained and the T1 lines was planted from the self-crossed seeds. The EPSPS-G6 gene was confirmed by PCR in the T0 plants and their T1 lines. But the resistance to glyphosate of the transgenic plants by agrobacterium-mediated method was not satisfied, probably because the Ubi promoter derived from monocot maize was not effective enough in expression of EPSPS-G6 gene in dicot cotton.Another vector with the EPSPS-G6 gene driven by Camv 35S promoter was introduced into CCRI-49 by the pollen tube pathway, and 26 transformants with glyphosate-resistance were obtained. PCR, dot blot hybridization, Southern hybridization (single and double restriction enzymes digestion) showed successful integration of the foreign EPSPS-G6 gene into the cotton genome. Field resistance test in T1 plants showed that the 20 of 26 transformants were consistent with single-gene Mendelian inheritance.152 homozygous resistance lines were obtained derived from 26 transformants, respectively. 2. Field test of glyphosate-resistanceThe test materials were treated with 15 mM,20 mM,30 mM and 40 mM of glyphosate. Non-transgenic CCRI-49 and TM-1 is highly sensitive to glyphosate herbicide, and their plants were died even treated by 15 mM glyphosate. While the transgenic plants obtained from the pollen tube pathway expressed glyphosate resistance with different levels. All the transgenic cotton plant were not affected with the treatment of 20 mM glyphosate, while a light wilting occurred in two transformants (EHC09-045 and EHC09-029) when they were treated with 30 mM glyphosate. At the treatment of 40 mM glyphosate, the resistance to glyphosate were greatly difference among the transformants,11 transformants of them showed no reaction at all, which indicated to be high resistant ones.13 transformants initially wilting or light wilting but recovered eventually, which shown moderate resistant ones; and 2 transformants died which indicated to be low resistant.3. Genetic analysis of glyphosate-resistant trait in transgenic cottonReciprocal crosses of five selected transgenic cotton lines, namely EHC09-002, E HC09-009, EHC09-036, EHC09-038 and EHC09-045, with TM-1 and CCRI-49 were performed. The glyphosate resistant trait of the transgenic cotton in F1 showed that there was no cytoplasmic effect as the same results of the crosses used the transgenic cotton germplasm either as female parent or male parent. The separation and distribution of the glyphosate resistant trait in F2 showed that EHC09-002, EHC09-009 and EHC09-036 were consistent with 3:1 ratio, according toχ2 test and confirmed by southern blot. However, EHC09-038 and EHC09-045 were inconsistent with the ratio 3:1, probably due to gene insertion sites and the integration of the gene.4. Agronomic traits and fiber quality in transgenic glyphosate-resistant cottonThe overall performance of agronomic traits and fiber quality of 26 transformants obtained by pollen tube pathway was normal and some even better than the control. The agronomic traits of T1 showed that plant height, fruiting branch number and boll number were not significantly different from those of the control. However, the integration of exogenous gene into cotton had a great impact on lint percentage and boll weight. The results in T2 plants showed that the transgenic cotton had little impact on fiber uniformity and fiber length, but had major impact on micronaire value, fiber elongation, and fiber strength, It suggested that the breeding of the transgenic cotton should focus on not only goal-oriented characters but also non-target traits during the selection.5. The effects of commercial glyphosate on the growth of the transgenic cotton seedlingsThe effects of three commercial glyphosates, (two 10% glyphosate aqueous solution and one 95% glyphosate powder) on the seed germination and seedling growth of transgenic cotton with glyphosate resistance were studies, using the transgenic glyphosate-resistant cotton, EHC09-008 and EHC09-020, as the materials, and their non-transgenic genetic background cultivar CCRI-49 as control. The results showed that 10% glyphosate aqueous solution may contain some harmful substances and affect the growth and development of the transgenic glyphosate-resistant cotton seedlings. Therefore the kinds of glyphosate should be selected carefully when they were used in chemical weed controlling in the transgenic glyphosate-resistant cotton field.
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