| Dioscorea zingiberensis C. H. Wight, containing high concentration of diosgenin, is one of the important raw materials that can be synthesized steroid hormone drugs. Since D. zingiberensis has been over-intensively harvested in the past years, few wild populations can be found and current production of this medicinal plant comes from cultivation. Since polyploid is reported to have the potential in accumulation of secondary metabolism products and higher tolerance to environmental stress, application of polyploid in Dioscorea breeding has been considered a hopeful way to improve rhizome quality and resistance to environmental stress. In this study, colchicine inducing method was applied on seeds and tissue cultured mirco-tubes of D. zingiberensis. The tetroploid were screened out by root tip chromosome counting and FLC (flow cytometer). The tolerance of these tetroploid plants to high temperature was evaluated through their physiological indices under controlled high temperature.The main results are as follows:1. Tetroploid induction(1) Seeds as material: The results indicated that the highest inducing rate (11.5 %) was achieved by treatment with 0.2 % colchicine for 12 h.(2) Micro-tubes as marterial: Tetroploid could be obtained from any combination of 0.10 %-0.20 % colchicines treating 24 h-48 h, but the most efficient inducing rate was achieved by treatment with 0.15 % colchicine for 12 h.Seed treatment is more convenient for manipulation and produces higher genetic diversity, which might be potential in further breeding; Material of tissue cultured micro-tube gives higher inducing efficiency and uniform genetic background, which might be favorable for a given cultivar to improve resistance.2. Tolerance comparison of diploid and tetraploid plants to high temperature stress.Tissue cultured diploid and tetraploid from the same genotype were transplanted into pots after acclimation. The survival plants were grown under 28℃(control) or 42℃for 5 d and then recovered at 28℃for 2 d. Leaves should be sampled every two days from the first day to 7th day to examine the physiological indices related to heat-resistance. The results are listed as following:(1) REL and MDA concentration: REL (P < 0.01) and MDA (P < 0.01) concentration in tetraploid were significantly lower at all treatments than in diploid. The result suggests that tetraploid plants have greater membrane stability under high temperature stress.(2) Reactive oxygen species (ROS): Superoxide radical ((V) production rate and H2O2 concentration were accumulated when the plants exposed to high temperature. The indices of ROS in tetraploid were lower than that in diploid, which implies that the same high temperature stress did less injury to the tetraploid.(3) Antioxidant enzymes(SOD,POD,CAT,APX and GR): Under high temperature stress, the activities of SOD and GR displayed similar trend, which climbed slightly within first 1 or 3 d and then declined gradually, whereas the activities of POD, CAT and APX were declining with the continuance of treatment time. Except POD, all the antioxidant enzymes exhibited higher activities in the tetraploid plants than in the diploid, and the differences are significance, which indicated that tetraploid plants have the ability to maintain high level of antioxidant enzyme activities to removal of oxygen substances.(4) Antioxidant (ASA and GSH): Under high temperature stress, AsA and GSH concentration decreased when plants exposed in high temperature stress, but the tetraploid plants maintained higher level of AsA (P < 0.01) and GSH (P < 0.01) than the diploid, suggesting the tetraploid have greater ability to scavenge ROS.In conclusion, comparing with diploid, the tetroploid has lower accumulation of reactive oxygen species (ROS), lower REL and MDA concentration, but higher productions of antioxidant and antioxidant enzyme activities, which indicated they has suffered lower injury and better protecting mechanism, allowing them to tolerate a certain extent of high temperature stress. |
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