| Barley is the main cereal crop,ranking as the forth crop in the world agriculture in terms of acrage and productivity,just behond wheat,rice and maize.Fusarium head blight(FHB) disease of barley is the main disease in barley growing aeras with warm and humid environment in world,and is the main disease in the middle and lower reaches of the Yangtze River and the southern china.FHB not only can cause yield loss,but also produces a variety of mycotoxins that can potentially pose serious threat on human health and animal production.One main objective is to breed barley cultivars with FHB resistance and high yield potentials in FHB epidemic regions of barley in China.In this study antimicrobial peptide genes were used for transformation of barley by using the Agribacterium-mediated transformation.Calli of immature embryos of barley were used as the explants with pmi gene as the selection marker in the vector.We obtained the transgenic plants using the mannose selection and proved the integration and expression of the foreign gene through identification by CPR,PCR and Southern blot analysis.The main results are followings:1.The barley genotype and the concentration of the mannose are the main factors affecting the transformation frequency.Different barley genotypes have different sensitivities to Agrobacterium tumefaciens and mannose.We transformed three barley cultivars and have obtained the transgenic plants only from the Zaoshu 3.In this case 228 plants were obtained and 71 plants were positive by PCR identification and the transformation frequency was 3.06%.2.We can improve the transformation frequency when use the vacuum and the sonication during the process of transformation.The time for sonication is 30 sec and the vacuum is 15 min at 80 kPa for 30 min.We compared the three methods and the results indicated that we can improve the transformation frequency under the conditions used for vacuum and sonication,producing a high trasnformation frequency when vacuum was used.3.We can improve the transformation frequency after high osmotic treatment of callus just before transformation.We used three different times,5h,12h and 26h for osmotic treatment.The results indicated that the highest transformation frequency was obtained after the treatment for 12h.4.The callus was transferred to the regeneration medium following the mannose selection of 20 g/L and 25 g/L in the induction.We used three concentrations for differentiation,15 g/L,20 g/L and 25 g/L in media.The results indicated that mannose can reduce the regenetation of the callus because less non-transgenic plants were produced following the increase of the concentration of mannose.We obtained the highest transgenic plants in the 20 g/L mannose.5.Chlorophenol red(CPR) assay is the fast and effective method for the identifcation of the expression of PMI.In this study we detected the transgenic plants using CPR under the optimzed conditions of 15 g/L mannose,15 g/L sucrose and 100 mg/ml CPR.From 228 plants selected,150 plants were positive,and thus the positive frequency is 65.7%.Then the transgenic plants were identified by PCR and the positive frequency is 31.1%.Some transgenic plants identified by PCR were further analyzed by Southern blots. |
