| Tetraploid black locust is a variety has been bred in South Korea, the leaves of which are 3 times larger than those of diploid varieties and contain 1.4 times more protein. And it has no prickles on its surface. It can be used not only as pioneer species in environmental beautifying and ecological restoration, also as fodder, bee forage, timber production, and so on. Tetraploid black locust not only has high ecological value, also has very high economic value at the same time. In this research project, the Agrobacterium tumefaciens mediated transformation of BADH gene of tetraploid black locust has been studied, for the purpose of improving resistance to soline-alkali and drought of tetraploid black locust; of playing more important role in developing of waste lands, ameliorating of soline-alkali soil, and greening and beautifying of surface-mined lands, mine waste dumps, slopes of roads and railroads where restoration of vegetative cover has proven difficult; of fully making benefits of its ability to fix atmospheric nitrogen in the soil. According to reference, this research is the first time to transfer practically productive aim-gene to tetraploid black locust, and it is also the first time to transfer BADH gene to forest-tree. This research provide a useful way in improving resistance to soline-alkali and drought of forest tree.In this study, the stem segments of new shoot with axillary buds of well-growth tetraploid black locust trees were used as explants. The effects of different basic mediums, different hormone kinds and their concentrations ratios, different sucrose concentrations on calli induction, buds differentiation and rooting in the process of establishment of high frequency regeneration system of tetraploid black locust were studied. On the base of high frequency regeneration system, the effects of various factors on transformation efficiency of BADH mediated by Agrobacterium tumefaciens were discussedin the light of GUS histochemical assays. The transformation procedure discussed in this research is reproducible and effective, allowing routine introduction of genes. The results of this research were listed as following:1 .The high frequency regeneration system of tetraploid black locust has been established. In the process of calli induction, MS medium supplemented with 6-BA l.Omg.L"1, NAAO.Smg.I/and sucrose 30g.L"!is the most effective medium, and the calli induction frequency is as high as 88.4%. MS medium supplemented with 6-BA 2.0mg.L-1, IBA O.2mg.L-1and sucrose 30g.L-1is the most suitable medium for differentiation of buds, the mean number of buds differentiated from a callus is up to 11.3; and 1/2MS medium supplemented with NAA l.Omg.L-1and sucrose SOg.L-1 is very efficient in the rooting process, the rooting frequency is higher than 959o. Before planting into outdoor soil, the tube-plants should be acclimated in green house for 15~20days firstly, the surviving rate could up to 93%.2. The high efficient transformation system of tetraploid black locust has been established. In the transformation of BADH gene mediated by Agrobacterium tumefaciens strain LBA4404, the selecting pressure is 50 mg.L-1 Kan. And the most suitable bacteriostatic agent is 400 mg.L"1 Cef. By using GUS histochemical assays the transformation system was optimized. Using calli as explants, the transformation frequency could be greatly improved. Using low pH medium MS to activate Agrobacterium, and when its concentration is about OD0.3~0.7, are the best conditions for inoculation. Acetosyringone(AS) could dramatically improve transformation frequency, its suitable concentration is 20 mg.L"1. Before inoculated with Agrobacterium, calli should be precultured 1 ?days, and the appropriate inoculation time is 4~8 minutes, and then cocultured for 3~4days. Then the cocultured calli were transferred to MS medium containing 400 mg.L"1 Cef. After about ISdays, the calli were transferred to the same medium with SOmg.L'1 kan to select Kan resistant shoots.
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