Study On Salt-tolerant Genetically Modified Alfalfa

Alfalfa(Medicago sativa L.) is one of the world's most widely distributed perennial legume forages with relatively salt-tolerance,which belongs to dicotyledonous(Dicotyledoneae) bean(Leguminosae) alfalfa(Medicago),but its salt resistance still need to be improved to adapt to actual cultivation in moderate or severe saline soil.Traditional breeding is slower and less precise because of the barriers between different species.However,transgenic technology is expected to solve the problem.The SOS(Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in plants.The important physiological function of SOS gene is to regulate ionic balance and enhance resistance of sodium in plants.SOS1 SOS2-SOS3 and SOS1-SOS2-SOS3 genes from Arabidopsis thaliana were transferred for the first time into alfalfa(Medicago sativa L.cv Zhongmu No.1) mediated by Agrobacterium tumefaciens with precultured leaves,cotyledons and embryogenic callus from hypocotyls,cotyledons and leaves as receptors and transgenic plants were obtained.In the mean time efficient regenerative and genetic transformation systems of alfalfa had been set up.PCR detection preliminary proved that SOS genes had been integrated into the genome of alfalfa and the tests of tube seedlings showed that NaCl resistance of transgenic plants,which could grow normally on the medium containing 0.6%NaCl, had been enhanced significantly compared with control plants.1.The best seed disinfection scheme:Full seeds of Zhongmu No.1 were first treated for 3 min by 75%alcohol,then handled for 30 min by HgCl₂,after those seeds were flushed for 6 times by sterile water,then back in sterile water,shook overnight on wave bed.The seeds were washed twice by sterile water,then were put onto MS(B5) medium in the next day.Seed contamination and germination rates were 0 and 100% respectively after 14 days.Plants germinated from those sterilized seeds grew well according to subsequent investigation.2.Induction,proliferation and differentiation of callus:seeds hypocotyls cotyledons and leaves of alfalfa Zhongmu No.1 could induce embryogenic callus and then regenerate into plants.Order from quick to slow of embryogenic callus induction was hypocotyls(two months),cotyledon and leaf(two and a half months) seed(four months).It was needed half a month that embryogenic callus regenerated into seedlings.The best solution obtained from different experiments is:(1)Hycopotys and seeds were cultivated on medium D4 for 20 days and then on medium D1 for the next 14 days,on MS(B5) 0 medium till differentiation;leaves and cotyledons were induced on XGMM medium,then on MS(B5) 0 medium.3.We have selected an appropriate medium MS(B5) D for seedlings to grow strong through the comparison of the three kinds of media.4.Experiments showed that the best PPT screening concentration was 6mg/L for callus and 2mg/L for plantlet.5.The influence factors of Agrobacterium-mediated transformation efficiency were investigated on genotypes,explant types,strains,infection time,the use of AS and explant pretreatment with 70%alcohol before infection.The conclusion was that: Zhongmu No.1 was the ideal genotype with higher transformation efficiency; embryogenic callus transformation efficiency is the highest of all the explant types, and the shortest time needed to regenerate plants,but lower regeneration rate was its defect;resistant callus rate of EHA105 strains is 4.46 times of LBA4404;the best infection time is 120min;With 70%alcohol pretreatment explant before infection and the use of AS can improve the resistance of callus rate.According to above results,we optimized the transformation system.6.The scheme of regeneration after selection is:the cocultured calluses were screened on MS6P/MS100H medium for 20 days(yellow-green calluses could be continued screening for 1-2 times to improve the positive rate),then were transferred to MS (B5) 0 medium to differentiate and regenerate whole plants.The scheme of regeneration before screening:the coincubated blades or cotyledons were induced on XGMM medium for 40 days,then transferred to MS(B5)0 medium till regenerated into seedlings.The seedlings then were filtered on MS2P medium for 2-3 times.7.GUS staining results of resistance callus with SOS2-SOS3 showed that transgenic callus appeared blue dots while control callus did not appeared GUS dyeing cites. This indicates that the gene has been expressed.8.Whole genome DNA was extracted from transgenic alfalfa plants which had been screened for 60-120 days.PCR detection was determined and analyzed,which showed that target positive bands were obtained.PCR positive rate of SOS1 was 31.5%;PCR positive rate of SOS2-SOS3 was 31.5%and PCR positive rate of SOS1-SOS2-SOS3 was 59%.The main reasons of high PCR positive rates were as following:(1) Genetic transformation system was optimized and employed.(2) Seedlings detected had been screened for 60-120 day,those non-transgenic seedlings had already been killed.9.The high concentrations of salinity will lead to oxidation stress.The PCR positive transgenic plants 4-3,4-5 and control plants were treated for 20 days by different concentrations(0,0.3%,0.6%and 0.9%) of NaCl and salt-tolerance physiological and biochemical indexes related were determined.The results showed that chlorophyll and proline content,activities of Super Oxide Dismutase(SOD) and Peroxidase(POD) were significantly higher than those of non-transgenenic plants, while electrolyte leakage(EL) and the content of Malonaldehyde(MDA) were significantly lower than those of control.These results above demonstrated that the salt tolerance of transgenic alfalfa was enhanced.