Study On Technology System Of Processing Tomato Mutations By Ethyl Methane Sulfonate(EMS)

Using processing tomato “JW9”as the material, this project aimed to study the effect of different concentrations of chemical mutagen EMS on processing tomato seed germination, chose appropriate mutagenesis concentration and mutagenesis time. a total of 13 processing to study seed germinating and seeding growth, seeds germination potential, germination rate and hypocotyl length, radicle length and number of lateral root, seed germination index and vigor index. And the use of PCR-SSCP molecular marker technology to build the mutant population of mutant population physiological and biochemical and molecular testing, in order to establish a system of tomato chemical mutagenesis techniques, and optimize PCR-SSCP molecular marker technology, access to a large number of mutants, and through targeted screening get low temperature tomato mutants. The results of the study are as follows:1、The effects of EMS treatment on germination energy of processing tomato: The appropriate concentration of EMS treatment processing tomato dry seed “JW9” was 4% EMS for 12 h. The appropriate concentration of EMS treatment processing tomato wet seed “JW9” was 3% for 12 h. The physical damage of seeds treated by EMS(M1) increased with the increase of EMS concentration, the absorption of EMS mutagen was more effective for wet seeds, and the inhibition was larger, the reaction was more intense.2、Optimization of PCR- SSCP molecular marker technology system:Single strand conformation ploymorphism(SSCP)technique is to be affected by many factors, such as DNA extraction method, PCR reaction process, electrophoretic factors(denaturalization temperature and time, and electrophoresis temperature, electrophoresis time, electrophoresis buffer and electrophoresis power), denaturant. The influences of these factors on SSCP technique were studied in processing tomato. The PCR-SSCP reaction system and reaction process which were high polymorphism detection rate, good repeatability, clear stripe were screened and established. The results showed when extracting tomato leaves DNA using the CTAB method, adding Nacl 5M/L and the first centrifugation speed and centrifugal extraction time was 8000r/min, 15 min can obtain high quality DNA in CTAB buffer, the amplification program was 94℃ pre-denature 5min, 94℃ denature 1min, 52℃annealing 30 s, 72℃extension of 1min, a total of 29 cycles, final extension at 72℃ 10 min, primer size was 100-300 bp, denaturalization temperature was 98℃, denaturalization time was 10 minutes, non-denaturing polyacrylamide gel concentration of 8%, electrophoresis time was 1.5-2.5h,no added glycerin, the bands of the single strand DNA were clear and easy to read in processing tomato.3、The analysis of EMS treatment on PCR-SSCP of processing tomato’s M2 generation mutant: the use of chemical mutagen EMS treatment processing tomato seeds(M0), for processing tomato phenotypic traits in M2 groups had a serious influence, EMS pharmaceutical treatment for the majority of the material height, stem diameter, fruit festival location, number of fruit per plant immature, unripe fruit weight per plant has a role in promoting the effective number of branch number for most materials, plant ripe fruit, ripe fruit weight per plant inhibited. EMS cause plant seedlings, leaves, flowers, fruits and other organs yellowing, roots, fruit malformation, affect the overall yield, less beneficial mutations. The use of PCR-SSCP molecular markers be screened to obtain 36 mutation material mutation frequency was 3.6%, and finally offspring(M3) of 36 mutant materials were identified, but the result is not obvious, the difference is less bands.4、The analysis of EMS treatment on M3 generation low temperature resistant mutants of processing tomato: the results showed that the use of chemical mutagen EMS treatment processing tomato seeds(M0), and their offspring(M3) for bud and seedling low temperature stress. Compared with the controls, for superoxide dismutase(SOD) and peroxidase(POD) activity of M2 generation plants produce inhibition in processing tomato. Mutant SOD and POD activity was significantly lower than the control, malondialdehyde(MDA) activity higher than the control. Cryogenic mutant library PCR-SSCP screening and identification, access to 378 mutant populations, no significant difference after identification bands.