Studies On Transformation Of Fast-growing Black Locust With AhDREB1

Black locust (Robinia pseudoacacia L.) is a multipurpose tree species in China with many excellent characters such as lumber, entironmental restoration and biomass production. It is significantly important for using saline licks effectively by improving the salt resistance of black locust in order to enlarge its planting region.The aim of this experiment is to transformate salt-resistant gene -AhDREB1-into fast-growing black locust silviculture and get high salt -resistant varieties. Then improve the application of fast-growing black locust in the large salt region in large scale.The research results of plant enduring salt gene engineering and black locust gene engineering provide stability foundation for this study,and made this study is possibal to success.In this work, high frequency regeneration and transformation systems were established base on the study of the factors affecting black locust stem regeneration and gene transfer efficiency. The AhDREB1 gene was introduced into black locust through the improved programs which was built in this study . The main research work and results are as follows:1. The effects of 6-BA,KT,NAA,IBA,sucrose and seedling age on stem regeneration, segment proliferation and rooting ability were studied through the orthogonal designs of L₉(3⁴), and the high frequency regeneration system of black locust was established. The results showed that the optimal medium compositions for adventitious buds regeneration from stem was MS + 6-BA 1.5 mg·L^-1 + NAA 0.75 mg·L^-1 + KT 1.5 mg·L^-1; the optimal medium compositions for segment proliferation was MS + 6-BA 0.10 mg·L^-1 + NAA 0.02 mg·L^-1; and the optimal medium compositions for rooting was 3/4MS + IBA 0.2 mg·L^-1 + NAA 0.2 mg·L^-1 + sucrose 20 g·L^-1.2. There was obvious difference among various seedling ages for the regeneration ability of stem.When sub-cultivateing for 20 days, the regeneration ratio is high and the number of adventitious buds is the most.3. The effect of Kanamycim (Km) in resistanting the stem regeneration and adventitious buds proliferation were studied. The concentration gradient experiment showed that Km could restrain the growing of stem regeneration and adventitious buds proliferation when its concentration reaches 30 mg·L^-1. So, the concentration of 30 mg·L^-1 was chosen as the choosing press in the course of genetic transformation.4 The bacteriostasises effect of Cefotaxime (Cef) was studied. The concentration gradient experiment showed that the Cef could restrain the growing of agrobacter when its concentration reaches 150 mg·L^-1, and there is no obvious effect in stem regeneration and adventitious buds proliferation.5. The important facts influencing the transformation efficiency were studied through the orthogonal designs of L₉(3⁴). An effective system for genetic transformation in black locust was developed as following: the stems were pre-culture 2 d, immersed in the bacterium solution (OD₆₀₀=0.7) which was added 10 μmol·L^-1 for 21 min, co-culture for 2 d, and the transformation efficiency would be increased if the selection pressures were changed from low to high.6. The resistance plants were identified by PCR. The results indicated that the AhDREB1 gene had been inserted into the genome of black locust .And two transgenic plants were gotten.