Establishment Of High-efficiency Agrobacterium-mediated BADH Gene Transformation System Of Tetraploid Black Locust And Detection Of Transgenic Plants

Tetraploid black locust is a new variety, which is first introduced into our country from Korea in 1995. Compared to general black locust, Tetraploid black locust shows many features of own, such as speeding growth, dense branches, and broader leaf containing more protein. It can be used as pioneer species in ecological construction, because of its some resistance to drought and tolerance to saline-alkali, and also used as better fodder, which could be expanded to a wider range. Tetraploid black locust not only has high ecological value, also has vary high economic value at the same time. For the purpose of improving tolerance to saline-alkali and resistance to drought of tetraploid black locust by genetic engineering, the Agrobacterium-mediated transformation of BADH gene in tetraploid black locust has been researched.In this research, the BADH gene was transformed through Agrobacterium-mediated, and all kinds of factors that affect transformation have been studied and optimized. Then the high-efficiency Agrobacterium-mediated BADH Gene transformation system of tetraploid black locust has been established on the base of high frequency regeneration system that had been established, and many transgenic plants were obtained. The main results were as following:1 .The high-efficiency Agrobacterium-mediated BADH gene transformation system of tetraploid black locust has been established. In the Agrobacterium-mediated gene transformation, frequency of GUS+ positive explants, frequency of resistant callus, and other transformation indexes were used to optimize the parameters affecting transformation. Results showed the efficiency of transformation were varied greatly with the explant type and its physiological state, and the efficiency of BADH gene transferring into callus was higher remarkably than that of stems. At the same time, the transformation frequency was affected greatly by bacterial concentration and infection time. The highest transformation efficiency was obtained when the bacterial cell density was OD600 0.3-0.7 with 6-10 minutes of infection. The activation of Agrobacterium,acetosyringone, co-culture medium type and its pH, trauma ways, infection ways, and selection ways also affect transformation, and other optimum factors included 3-4 days of co-culture at 22-25, 1-2 days of pre-culture for callus.2.Many transgenic plants were obtained. In a series of replicate experiments, a great many kanamycin resistant lines was obtained. The result of PCR analysis indicated BADH genes were integrated into genomes of 221 out of 316 lines. It revealed that the transformation system was efficiency and stability, and certified BADH gene was transformed successfully.3.Many transgenic plants with better tolerance ability to saline-alkali were obtained. Through tissue culture, the transgenic tetraploid black locust was investigated with variant NaCl salt gradient. The results showed transgenic tetraploid black locust differentiated normally on the medium with 0.8% NaCl, while the control plant lost ability of diffrerntiation. Salt tolerance of the transgenic tetraploid black locust to NaCl was increased by 0.3%-0.4%.The transgenic tetraploid black locust grew normally on the medium with 0.5% NaCl, and growth was inhibited obviously on the medium with 0.6% NaCl, while the control plant kept from growing normally on medium with 0.3% NaCl, and growth was inhibited severely on the medium with 0.4% NaCl. The salt tolerance of the transgenic tetraploid black locust to NaCl was increased by 0.2%-0.3%. These results indicated that the transgenic tetraploid black locust had better tolerance ability than the control plant, and demonstrated the expression of BADH gene in tetraploid black locust.