Study On Agrobacterium Mediated XinJiang Cantaloupe Transformation Of NP-1 Gene

An efficient system of regeneration of XinJiang cantaloupe (Jia Shi and Huang Hou) was developed. Several regeneration protocals were analysed with particular emphasis on the regeneration efficiency. The explants excised from 3 days old seedlings cultured in MS medium supplemented with 6-BA1.0mg/L and IBA0.2mg/L,resulted in a high qualifed buds regeneration frequency (28% and 25.6% in 35days ). The shoot propagation condition is MS supplemented with 6-BA 1.0 mg/L and IBA 0.1mg/L,pH 5.8.The highest index of shoot propagation was 7.3.On the 1/2MS medium with 0.5 mg/L IBA, The high rooting initiation frequency was achieved.The cotyledons of XinJiang cantaloupes were transformed with strain of Agrobactrium tumefaciens, LBA4404, harboring expression vector pBIC-35s NP1.By selection of regenerated plantlets with Kanamycin resistance, the putative transgenic lines were believed to be obtained, In this research, the influence factors of cantaloupe transformation were discussed, the preferable protocol is 3 days cotyledon culture and resuspended bacterium, O.D600=0.6, with liquid MS before transformation. Inoculation and co-culture duration were 8~12min, 3d. And Km100mg/L, Amp500mg/L supplied to development media(MS+ BA1.0mg/L＋IBA 0.2 mg/L) is proper. During transformation, 0.2mg/L mannitol treatment is effective (efficiency increased 6.6%) but As is not so. After strict Kanamycin selection, 12 lines of putative transgenic cantaloupe were obtained. Molecular analysis of was carried to confirm the transgenic nature. The transformation of NP-1 gene was verified by PCR and RT-PCR analysis, respectively. And at the same time, the total protein was extracted to carry out the bacterium restraining experiment in vitro. The result confirmed that the transgenic cantaloupes were obtained.