Establishment Of Efficient Regeneration System Using Leaf Explant And Exploring Of Genetic Transformation Method In Ramie

Ramie [Boehmeria nivea (L.)] is one of the most important perennial fiber crops in China, hi vitro tissue culture of ramie can be used as an important method of rapid propagation, regeneration and genetic transformation of ramie. In this study, based on the leaf-blades of Zhongzhu No.1 and Xiangzhu No.3 as the explant, the different type of disinfectant and different duration were researched, The most appropriate type of disinfectant were screened out. The effect of the different size of leaf, different auxins (2,4-D, IAA, NAA, IBA) on callus formation and different solubility cytokinins (TDZ) concentration was further studied in ramie leaf callus induction. A high efficient regeneration system was selected for the two varieties. The pollen tube pass method to transgenic technology by spraying agrobacterium were explored. The agrobacterium tumefaciens (LB4404) infection solution carried PRI-47348 gene were directly sprayed on female inflorescences Before 3-4 days pre-flowering. Then the emale inflorescences were naturally pollened and the seed were harvested.The main results of this study are as follows:(1) To compare the effect of different disinfectant, it was found that the 1.67% effective chlorine content of disinfectant A was the best effect when it treated the explants for 10 minutes.(2) The callus inductivity frequency was different in different leaf sizes.The highest rate callus (90%-100%) could be inducted by0.90-1.10cm×1.80-2.00cm size leaf.(3) Callus induction stage in culture medium were supplemented with different auxins (2,4-D, IAA, IBA, NAA). The results showed that the callus induction rate was the highest.reach 95%-100% in adding 2,4-D medium using Xiangzhu 3 and Zhongzhu 1 blade.(4) Xiangzhu 3 and Zhongzhu 1 leaves were inoculated in the medium with different concentrations of cytokinins (TDZ) and auxin (2,4-D). It was found that the highest rate induced callus were 100%,91.6% when it were inoculated in MS+0.5 mg/LTDZ+0.5 mg/L 2.4-D(5)The callus sprouting of the optimal hormone combination screening test. It was found that the highest rate Sprouting rate wsa 48% when it were inoculated in MS+0.5mg/LTDZ+0.01mg/L2.4-D(6) The optimum rooting medium was MS+0.05mg/LNAA.(7) Different ramie varieties have different adaptability to antibiotic, from strong to weak in order, Xiangzhu 3> Wanzi Ramie> Huang Xiaoye>Yuanqing 5>polyploid 1> Zhongzhu 1(8) The transgenic seeds by pollen tube pass method were obtained and were screened in adding kanamycine MS medium, the results found no transgenic plants were grew. Therefore, the application of pollen tube pass method of transgenic ramie needs deeply explore.