In Vitro Mutation Inducted By EMS In Stevia Rebaudiana Bertoni, Screening And Identification Of Salt-tolerant Mutant

Stevia (Stevia rebaudiana Bertoni) has become the focus in food and medicine field, as a kind of ideal sweetener and functional health care products. As a salt sensitive plant, selecting and breeding a new Stevia species of salt resistance and adaptability is very urgent and important with the salinization aggravation. The combination of chemical mutagenesis and tissue culture is an effective method to obtain Stevia salt-tolerant mutant. Using bud stem section with axillary of Stevia (’Wantian NO.1’-B1) as the material, the objective of this study were to confirm the salt tolerance threshold (critical NaCl concentration) of Stevia plantlet, to determine the suitable ethyl methyl sulfonic acid (EMS) concentration and treatment time, to select the salt-tolerant mutant by 1.0% NaCl, to identify the salt resistance of Stevia through physiology, biochemistry and SRAP molecular markers, and to test the salt mutant by salt stress in pot culture condition. The main results are as follows:1. The growth of Stevia plantlets became weak gradually and inhibited, including the survival rate and rooting rate reduced significantly with NaCl concentration increased from 0,0.2%,0.4%,0.2%,0.8%,1.0% to 1.2%. Stevia leaves became small and etiolated, and the survival rate was 3.13% and the rooting rate was zero of Stevia plantlets when the NaCl concentration was 1.0%. All the Stevia plantlets were dead under 1.2% NaCl concentration. Based on the principle of real salt tolerant variants, we preliminarily determined the salt-tolerant critical NaCl concentration was 1.0% of Stevia plantlets ’Wantian NO. l’-B1.2. Stevia plantlets were soaked with different concentration EMS solution (0,0.2%, 0.4%,0.6%,0.8%,1.0% and 1.2%, respectively) and shake 0h,2h,4h,6h, h,10h and 12 h respectively, then were inoculated in MS medium. Results indicated that the effects of low EMS concentration and short processing time on plant height, rooting rate and survival rate were lesser; with the increased of EMS concentration and the extension of processing time, Stevia plantlets grew gradually weakened, plant height became smaller, terminal bud and axillary bud grew slower, the rooting rate and survival rate reduced rapidly. When the EMS concentration was more than 1.0% and the processing time was longer than 8 hours or the EMS concentration was greater than 0.8% and the processing time was longer than 10 hours, survival rates of Stevia were below 50%. We concluded that the appropriate concentration and time range were 0.8-1.0% and 8-10 hours of Stevia plantlets ’Wantian NO.1’-Bl.3. The stem sections of Stevia plantlet were treated with 1.0% EMS for 8 hours and then were inoculated in MS medium with 1.0% NaCl, to select the salt resistance of variant plants. The survival rate was 13.67% after cultured 45 days, which was significantly higher than the control of 3.33%. We selected 41 salt-tolerant variant plantlets.4. We measured the content of chlorophyll, proline, malondialdehyde, soluble sugar and soluble protein, the relative conductivity, and the activities of POD, SOD and CAT of salt-tolerant mutant after cultured for 5d, 10d,15d,20d and 25d respectively. Results showed that the contents of chlorophyll, proline, soluble sugar and soluble protein were significantly higher than that of the control, the relative conductivity and MDA content significantly lower than the control, the activities of CAT, POD and SOD significantly higher than that of control in most time. These present results indicated that the salt resistance of Stevia plantlets that mutated by EMS were enhanced.5. DNA was extracted from the 41 salt-tolerant variant plants that were selected by 1.0% NaCl repeatedly, and the identification was conducted by SRAP molecular marker. The results showed that only 4 mutants displayed changes in the DNA level, the main changes were the lack or excess of several amplification bands, and salt resistance of the other mutants only expressed in the physiological and biochemical levels.6. Four mutants, control and those that were not mutanted in DNA level but mutanted in physiological and biochemical level were transplanted to pot to test their salt resistance.We found that salt-tolerant mutants were growing well than the control and those plants that were mutanted in physiological and biochemical level, the main variations were plant strong growth, fat leaves, and thick green leaves, etc.