Studies On Transformation Of AVP1 Gene Into Lotus Corniculatus L. And Identification The Salt And Drought Tolerance Of The Transgenic Plant

Soil salinity and desertification has become the cosmopolitan problem all over the world. Lotus corniculatus L. is one of well-known leguminous forges around the world. But it can not grow under salinity and drought conditions because of its poor salt- and drought-tolerance. Therefore, breeding salt- and drought-tolerant Lotus corniculatus L.via biotechnology has an important significance for improving and making use of the large area of saline-alkali and desertified land on the basis of conventional breeding.Many researches have shown that overexpression of the AVP1 gene can enhance plant salt- and drought-tolerance. In this research, we chose Lotus corniculatus L. as experimental material, and obtained transgenic Lotus corniculatus L. overexpressing the AVP1 through Agrobacterium-mediated successfully. PCR was performed on the 8 resistant plants together with wild-type, and then the salt and drought tolerance of the transgenic plant were detected. The main results were as following:1. The regeneration system of Lotus corniculatus L. was established. Cotyledon was the optium transformation receptor, B5+0.5mg/L 6-BA was the optium culture medium for differentiation, 1/2MS+0.05mg/L NAA was propitious for rooting.2. The transformation system mediated by Agrobacterium tumefaciens was established. 300mg/L Carb could inhibit the reproduction of the Agrobacterium strain GV3101. The selection pressure of Kanamycin for somatic embryo induction was 50mg/L. The best pre-culture time, the bacterial concentration, the co-culture time, were 3-4d, 0.5, 3d, respectively.3. The resistant plants were detected by PCR. 8 resistant plants were all positive. This indicated AVP1 has been integrated into the genome of Lotus corniculatus L.4. When plants were exposed to 200mmol/L NaCl for 7 days, the transgenic plants overexpressing AVP1 gene were more salt-tolerant and can grow better, however, the wild-type plants were inhibited seriously, even died. After 5 days of drought stress, wild-type plants showed growth reduction and wilt, whereas the transgenic plants continued normal growth, it wilted after the 7 days of drought stress. All plants were rewatered, the transgenic plants survived and resumed normal growth, but wild-type plants died completely. This showed that the growth of the transgenic plants was better than that of the wild-type plants under stress condition.5. The shoot dry weigh of all the plants decreased gradually as the NaCl concentration increased, but the shoot dry weight of the wild-type plants showed a faster decline than that of the transgenic plants. At 200mmol/L NaCl, the shoot dry weight of the wild-type plants decreased 52%, whereas the shoot dry weight of the transgenic plants decreased only 33%.6. Compared with wild-type plants, the transgenic plants retained more water under salt stress and drought stress. After 7 days of 200mmol/L NaCl or drought stress, the leaves of the wild-type plants had a higher water loss, the decline of the relative water content in the wild-type is 36% and 38%, respectively, but only 18% and 24%, in transgenic plants, respectively.7. The MDA content and the relative membrane permeability of the transgenic plants and the wild-type plants increased when the NaCl concentration or the drought time increased, but the wild-type plant showed a sharp increase. At 200mmol/L NaCl or day 7 of drought stress, in transgenic plants, the content of MDA was 35% and 27% , respectively, lower than that in the wild-type plants; The relative membrane permeability was 28% and 27%, respectively, less than that of the wild-type plants.8. The overexpression of the AVP1 can protect the photosynthetic system of the transgenic Lotus corniculatus L. At 200mmol/L NaCl, there was 60% reduction of the Pn in wild-type, whereas only 40% in transgenic plants. After 7 days of drought stress, similarly, the decline of the Pn in wild-type plants was 68%, but only 47% in transgenic plants.9. Transgenic plants accumulated more Na⁺, K⁺, Ca²⁺ in their leaves and roots. At 200mmol/L NaCl, in transgenic plants, the amount of Na⁺, K⁺, Ca²⁺ in leaves was 30%, 41%, 81%, respectively, higher than that in wild-type plants, the amount of Na⁺, K⁺, Ca²⁺ in roots was 19%, 49%, 41%, respectively, higher than that in wild-type plants. After 7 days of drought stress, the amount of Na⁺, K⁺, Ca²⁺ in leaves was 74%, 55%, 88%, respectively, higher than that in wild-type plants, the amount of Na⁺, K⁺, Ca²⁺ in roots was 63%, 61%, 200% higher than that in wild-type plants.These results indicated that the overexpression of the AVP1 enhanced the salt- and drought-tolerance of Lotus comiculatus L.. This study pave a way for breeding transgenic Lotus comiculatus L. resistant to salt- and drought-stress.