Construction Of Tobacco Mutant Population And Screening System

The establishment of the tobacco mutant library will provide valuable resources for tobacco breeding, which in order to achieve important agronomic traits such as high-yield, high quality, high resistance, etc. And it lays a solid foundation to accelerate the cloning and characterization of important genes of tobacco, will accelerate the tobacco functional genomics research process. In this study, tobacco chemical mutant (EMS) and T-DNA activation tagging mutant library were constructed, and mutant loci were screening by SSR markers, TILLING technology and PCR-walking methods. The paper includes the following five aspects:(1) The chemical mutagen EMS was used to generate mutation in tobacco seeds, half-lethal concentration of EMS was determined in the different varieties of tobacco seeds. The appropriate EMS concentration was 0.35%-0.52%, and the lethal concentration was all 1.3%. Among the tobacco varieties, for zhongyan100, the semi-lethal concentration of EMS was 0.49%.(2) In order to generate more mutations, the concentration of 0.6% and 0.8% EMS were used in mutation of zhongyan100. In continuous cultivation of mutants of the M1, M2, M3, agronomic traits of them were observed in the field investigations. 1427 M2 generation seeds, 832 M3 generation seeds were obtained, and phenotype variations were found in 392 M3 plants. Mutant phenotypes presented in flower color, sterility, plant type, axillary bud and so on.(3) SSR markers were used to identify mutants which had phenotypes in plant height, sterility, flower color, leaf shape etc., the results show that among the 58 mutants, 40 appeared polymorphism compared with the control, indicating that EMS treatment resulted in differential fragments. SSR polymorphism reflects the difference between mutants on the molecular level, and provides molecular evidence for the linkage between agronomic traits and mutants.(4) Crude CEL ? were extracted from celery, and a hybrid double strands DNA of 632 bp length which contained the G→C mismatch was cut by this enzyme. The results showed that the crude enzymes extract precisely cut at the predicted mismatch site and produced right fragments on both agarose and polyacrylamide gel electrophoresis. Purification of this enzyme can greatly reduce research costs, and this technology can be used in TILLING technology platform creation. It has great significance for the large-scale functional genomics studies of tobacco.(5) The T1 generation of activation tagging T-DNA inserted mutant library of honghuadajinyuan were grew, and the field phenotypes were investigated. The results showed among all the 638 transgenic lines, 298 lines presented phenotypes. 464 Morphological variants of plant overground part were observed, accounting for 4.85% of all the plants. According to phenotypic differences, mutants can be divided into different type variants as axillary buds, stem-leaf intersection angle, florescence, fertility, plant height, leaf shape and other types of mutations. Some mutations traits often show recombination on phenotypes, such as complex mutations in both axillary buds and florescence, while the dwarf and sterile double mutations occur and so on.