Study On Transgenic Black Locust(Robinia Pseudoacacia L.) With OsDREBL Gene

Black locust (Robiniapseudoacacia L.) is a multipurpose legume tree species in our country and it exhibits many excellent characteristics such as lumber, entironmental restoration and biomass production. Frost is an important antiblastic factor to black locust. Black locust is one of the important silviculture harbingers in loess hill of our country, but it usually shows abnormal growth in frost conditions. Genetic manipulation has made it possible to reach our breeding aim. This study was to transformate cold-resistant gene into black locust silviculture and get high cold-resistant varieties. Then improve the application of black locust in nothern region in large scale.The establishment of effective regeneration system is the foundation for Agrobacterium-mediated genetic transformation. Base on MS medium, NAA and BA were selected to be the best plant growth regulators for the regeneration of the compound leaf. Via the study for the regeneration of the folioles and rachis of the compound leaf, rachis is selected for the genetic transformation. The differentiation rate of rachis can reach the level as high as more than 80%, which were cultures on MS medium containing BA2mg/L, KTlmg/L, and NAA0.5mg/L. The compound leaves were placed on MS medium with BA1.0 mg/L and 2, 4-D1.0 mg/L for callus induction, and then the callus can regenerate adventitious buds on MS mediun supplemented with 1.5mg/L BA and 0.5mg/L. The effects of different explants types of adventitious shoots were studied, indicating that rachis had the highest level of adventitious buds differentiation.The Kanamycin and Ceftomine sensitivity of adventitious shoot and adventitious root were tested. The concentration of Kanamycin was 30mg/L for adventitious buds differentiation, and subculture selection was up to 50mg/L. Cef 200mg/L was the suitable bacteriostatic agent. Through optimizing transformation system, the results suggested: in vitro-germinated plantlet shoots (20~35d) were suitable for inoculation; selection of theinoculated rachis was delayed 7~9d; dark culture in former cycle was adopted, and when the transformed plants with Km resistance were observed, they were subculture transfered to light conditions, in order to allow the normal growth of the transformed plants. The results of molecular biological assays with PCR preliminarily indicated that the OsDREBL gene had been integrated into the genome of black locust, and two transgentic plants were obtained.