Selection And Mechanisms Of Glyphosate Tolerant Mutant R0198

Weed is one of the major hazards that seriously affect cotton cultivation. Although manual weed control is a primitive method in the rural areas of China, it requires a lot of agricultural labor and can easly result in soil erosion. With the increase in labor cost, traditional weed control has become less suitable for modern agriculture. Glyphosate is a non-selective, broad-spectrum herbicide that is very effective against both annual and perennial weeds. It is widely applied to crop planting. However, glyphosate kills weeds and also injures crops. It is important to breed glyphosate-tolerant crops. There are mass glyphosate production and application in China every year. Glyphosate-tolerant cotton is a highly potential commercial crop in China. Breeding our own patents of glyphosate-tolerant cotton is essential to agricultural development in China. A glyphosate-tolerant cotton mutant (R1098) was obtained by the method of somatic cell radiation technology and stepwise selection. Tolerance identification and inheritance were systematically studied, as well as the mechanisms conferring glyphosate tolerance and its application in hybrid cotton. The main results of these researches are as follows:1. The selection of glyphosate-tolerant cotton mutantStepwise selection approach with mutation breeding technology was adopted to obtain glyphosate-tolerant upland cotton mutant from the embryogenic calli of Coker 312 (Gossypium hirsutum L.). The calli were mutagenized with y radiation emitted from 60Co. The mutant calli were transferred to selection medium and multi-step selection pressure process was carried out until the calli could proliferate in the presence of 20 mM glyphosate. Finally, a stable glyphosate-tolerant cotton germplasm (R1098) was obtained.2. Studies on tolerance characterization and inheritence of R1098Based on glyphosate tolerance test, R1098 could tolerate 1.48 kg ae ha-1 of glyphosate (the recommended applied dose in the field). The glyphosate tolerance of R1098 was stable, with the lint cotton yield equivalent to Sumian 12 and with better fiber characterization. With the resistance or tolerance to blight and Verticillium wilt, R1098 was considered an excellent cotton germplasm. Genetic tests demonstrated that glyphosate tolerance trait was controlled by one dominant gene, without the effect of cytoplasm.3. Studies on the physiological mechanisms conferring glyphosate toleranceBased on a whole-plant dose-response bioassay, R1098 was 71 times more tolerant to glyphosate than its genetic background which is a susceptible cotton germplasm (Coker 312) as measured by the IC50-Measurement of shikimic acid accumulation in response to glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a rapid and accurate assay to quantify glyphosate-induced damage in sensitive plants. Coker 312 accumulated 13.1 times more shikimate in leaves at 5 days after glyphosate treatment (1.50 kg ae ha-1) than R1098. Glyphosate treatment also resulted in a significantly increased activity of the enzyme glutathione S-transferase (GSTs, EC 2.5.1.18) in glyphosate-tolerant (GT) and glyphosate-susceptible (GS) cultivars; however, glyphosate increased the GSTs activity to a lesser extent in GT than in GS cultivars, implying that glyphosate induces much oxidative stress in GS cotton than in GT cotton.4. Studies on the molecular mechanisms that confer glyphosate tolerance.Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase, EPSPS-1 and EPSPS-2, were isolated from R1098. The two cDNAs were 97.7%identical within the common protein-coding region and the predicted sequences of the mature proteins had more than 83%identity with EPSPS proteins from other known higher plants. In comparison to the glyphosate-susceptible cotton Coker 312, sequence analysis of the EPSPS-1 gene indicated that R1098 has an alanine insertion at nucleotide position 1216 resulting in frameshift. It allows the original sequence of EPSPS-1 to be translated into a complete protein sequence. According to real-time PCR, glyphosate treatment also caused a significant increase in the EPSPS mRNA level of R1098. From these analyses, we conclude that glyphosate tolerance is conferred by two different mechanisms, mutation of the EPSPS gene and stable inducible EPSPS mRNA by glyphosate.5. The value of R1098 in controlling for the seed purity of hybrid cottonThe tolerance of R1098 was controlled by one dominant gene. So F1 of R1098 remains glyphosate tolerance. It can be used to identify the purity of hybrid cotton and removal fake hybrid cotton seeds automatically. An excellent hybrid cotton cultivar with glyphosate tolerance trait, ZHEZA14, was obtained be testing and screening widely for the crosses of R1098 with other upland cotton cultivars. Six different resource hybrid seeds of ZHERA14, which come from He Nan, An Hui and Zhe Jiang provinces, were tested for the seed purity, using the method of seed germination and seedling identification with glyphosate. The field experiment for the different purity rate of the hybrid seeds showed that there was a significant correlation between the seed purity and the lint cotton yield, the glyphosate treatment could increased the seed' purification and its yield, as well as the fiber quality, especially for the staple length, fiber uniformity and fiber strength. The results also showed that the seed purity rate for the upland cotton hybrid should be higher than 90%, otherwise the lint cotton yield and fiber quality will be greatly affected.