Establishment Of Regeneration System And Investigation Of Factors Influencing Transformation System Of Alfalfa

As one of the most widely distributed grass,alfalfa (Medicago sativa. L) was regarded as "the king of grass". Because the fall dormancy of alfalfa is related with production performance and cold resistance, now it is regarded as the first index when choosing suitable variety in different areas. But the alfalfa fall dormancy mecha-nism is not clear, we are urgent to make breakthrough in the alfalfa fall dormancy research.In this study, we established the regeneration system of alfalfa firstly, then we tried to get the RNA interference plants by the investigation of the genetic transformation system conditions in order to study the function of photochrome A and photochrome B and the molecular regulation mechanism of alfalfa fall dormancy Main results are as follows1. Establishment of regeneration system:Different cultivars and different explants had different callus induction ability, the callus induction rates of vernal and WL-525HQ are both higher than WL-343HQ and WL-232HQ, either hypocotyl or cotyledons; For explants, the callus induction rate of hypocotyl was significiantly higher than cotyledon; The appropriate concentration of2,4-D is2mg/L, when concentration of2,4-D is high, it is easy to cause browning; As for different cytokinin, when the concentration of2,4-D is2mg/L,with the increasing concentrations of KT and6-BA, the explants callus induction rate is constant reducing. So we chose the most appropriate media for inducing callus which was MS supplemented with2,4-D2.0mg/L and KT0.25mg/L and sucrose30g/L and8g/L agar; The effective medium for differentiation culture was MS containing0.5mg/L KT and0.01mg/L NAA and30g/L sucrose and8g/L agar. Therefore,taking fall dormancy level and regeneration ability into consideration, we chose variety vernal as the best transformation receptor.2. Results from the exploration of the transformation system conditions were as follows:The target fragments of phyA and phyB in the Agrobacterium bacteria were not lost, we could use the Agrobacterium to infect the explants at ease;The best Agrobacterium infection time was lOmin; The right inhibitory concentration of carbenicillin inhibitory concentration was300mg/L; The suitable kanamycin selection pressure was50mg/L. Therefore the etablished transformation system were as follows:the pre-culture medium was MS supplemented with2,4-D2.0mg/L and sucrose30g/L and8g/L agar; co-culture medium was the same with pre-culture medium; selective medium is MS supplemented with2,4-D2.0mg/L and KT0.25mg/L and carb300mg/L and kan50mg/L and sucrose30g/L and agar8g/L; the differentiation medium was MS containing0.5mg/L KT and0.01mg/L NAA and carb300mg/L and kan50mg/L and30g/L sucrose and8g/L agar.