ISSR Analysis Of Genetic Diversity In The Sterile Plantlets Of Pogostemon Cablin Mutagenized By Colchicine And EMS

Pogostemon cablin was one of the important traditional Chinese medicines. It was distributed in Philippines, Malaysia, India and so on, then into China. Because the temperature of China was lower than that in the origin, Pogostemon cablin what was cultivated in China rarely came into bloom. Cutting propagation was used in the agricultural industry after it was introduced to China. Pathogen was transferred by vegetative propagation in the long period of cultivation, so the varieties were degenerate and insect pests were aggravated finally. In order to keep the existence and development of this important Chinese traditional medicine, one of the measures to solve the above-mentioned problems was that the new technology and method were introduced to breed the new variety. In this study, shoots of Pogostemon cablin were mutagenized by colchicincs, EMS and ISSR analysis was used to reveal the relationship and genetic diversity between mutant materials and the original donor of Pogostemon cablin. The main results in the paper were as follows.1. Taking the young leaves of Pogostemon cablin as explants, a large number of shoots were obtained through the way of callus induction. The shoots were transferred onto the multiplication media with different concentrations of 6-BA and onto the rooting media with different concentrations of IBA. As a result, the optimum multiplication medium was "MS+0.1 mg/L-0.2 mg/L 6-BA" and the optimum rooting medium was "1/2 MS+0.25 mg/L-1.0 mg/L IBA". Taking the shoots of Pogostemon cablin cultured on the multiplication medium of MS+0.1 mg/L 6-BA as experiment materials, they were mutagenized by colchicine and EMS with different concentrations and different treating time. Finally, the half lethal doses of the shoots were:mutagenized by 0.2% Colchicine for 3 d, and by 1.2%EMS for 7.5 h, respectively.2. In order to establish the optimized ISSR-PCR experiment system of Pogostemon cablin, the single factor experiments were at first used to detect the suitable concentration ranges of the different influential factors, and then the orthogonal design was used to optimize ISSR-PCR amplification system of Pogostemon cablin in 5 factors (Mg2+, dNTPs, primer, Taq polymerase, DNA template) at 4 levels, respectively. Through comprehensive analysis, a optimized ISSR-PCR reaction system was established as follows.10×buffer 2.5μL,40 ng DNA template,2.5 mmol/LMgCl2,0.3μmol/Lprimers,1.5 U Taq polymerase,150μmol/LdNTPs were contained in 25μL reaction solution, and the optimized amplification program was that predenaturing at 94℃for 5min, then denaturing at 94℃for 45 s, primer annealing at 50.9-58.1℃for 45 s, extension at 72℃for 90 s, for 40 cycles, at last extension at 72℃for 7 min. The productions were stored at 4℃. Using the optimized reaction system and PCR amplification program, six temperature gradient were designed in order to screen the suitable primers and the optimal annealing temperature according to the theoretical annealing temperature of 60 primers. Finally, Twelve effective primers with abundant polymorphism, good reproducibility and clearly discernible bands, were selected out from the 60 test primers and the optimal annealing temperature were ascertained.3. More than 100 shoots of Pogostemon cablin were mutagenized respectively by 0.2 % colchicine for 3 d and 1.2% EMS for 7.5 h,71 mutant materials mutagenized by colchicine and 62 mutant materials mutagenized by EMS were obtained finally. Twelve effective ISSR primers selected with optimal polymorphism were used for the amplification on two different kinds of mutant materials and 1 original donor of Pogostemon cablin (CK), then the apparent shape of plants mutagenized by mutagen were observed. The results showed that mutant materials mutagenized by colchicine almost became dwarfed, thicker and more branches, but the genetic similarity (GS) was higher and the genetic distance was closer according to the analysis of genetic diversity via ISSR. The apparent shape of the mutant materials mutagenized by EMS had no variation except a few plant that had mutant on the shape and colour of leaves. According to the analysis of genetic diversity via ISSR, the results showed that the GS of mutant materials mutagenized by EMS was relatively lower and the genetic distance was faster in comparison with the mutant materials mutagenized by colchicines. The result showed that the materials mutagenized by EMS was highly variable in DNA molecular level. The author thinks that the possible reason was that EMS mostly led point mutations while colchicine mostly led chromosome double.