Establishment Of Transformation System And Cloning Of The Vacuole Membrane Na～+/H～+ Antipoter Gene Of Black Locust Clone

Black locust (Robinia pseudoacacia ) is a nitrogen-fixing leguminous tree valued for its agroforestry qualities (ornamental, timber, animal feed, and honey production). Moreover, its fast growth rate has drawn attention to the species as one of the leading candidates for woody biomass plantations. The limited salt tolerance of black locust limits its planting area, so it is necessary to improve its tolerance using biotechnological programs. It is known that the vacuole membrane Na+/H+ antiporter gene which comparts Na+ into vacuole has shown a fine future in improving the salt tolerance of plants using biotechnological programs. The study has established a high-efficient transgenetic system of black locust, using Agro bacteria mediated method, 35S::MsNHX1 vector was transformed into leaf explants. At the same time, This study has cloned Na+/H+ antiporter gene from black locust and does some analysis on its role .In this study, the new stems of shoot, leaf of black locust clone 85043 and cotyledon, hypocotyls and embryo were used as explants to induce calii and buds producing in this study. 33.3% of all the stems explants had produced buds and each produced 2-3 .40% of all the cotyledon explants had produces calli and only 50% calli produced 1-2 buds. Using hypocotyls as explants, a great lots of calli appeared after seven days and average buds was five, the most could produce 7-8 buds each explant. In leaf culture, two steps method was used, average calli could produce 12 buds(>1.0 cm), this regeneration system is good and can use for transformation system of black locust.The effects of thidiazuron(TDZ) on adventitious bud formation from leaves of Robinia pseudoacacia were tested in combination with naphthalene acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid(2,4-D).The study showed that the combination of 2 mg/L TDZ with 0.1 mg/L NAA resulted in the highest frequency of average of 15 buds production. Higher concentrations of TDZ stimulated more adventitious bud production, but following the increase of TDZ, excessive callus formed, shoot growth and elongation was cessation...