Studies On Agrobacterium-mediated Transformation Of Embryogenic Cell Suspension And Plant Regeneration In Hevea Brasiliensis

The establishment of embryogenic cell suspension culture system and its regeneration system of Hevea brasiliensis can provide sufficient and fine materials for studying genetic transformation. In this study, embryogenic suspension cells of rubber tree clone Reyan 8-79 were used as recipients. The factors influencing genetic transformation such as bacterial concentrations, the way of co-culture, the pH value of co-culture medium, acetosyringone concentration supplemented in the co-culture medium, co-culture temperature and co-culture time were studied. The conditions of Agrobacterium-mediated transformation into embryogenic suspension cell of rubber tree were optimized. Concentration gradient screening of kanamycin was used for obtaining resistant embryogenic callus lines. Embryoids and regeneration plants were obtained through resistant embryogenic callus differentiation. GUS staining, PCR identification, putative transgenic plantlets PCR production sequencing analysis and Southern blotting results showed that the foreign genes have been successfully integrated into the genomic DNA of the rubber tree. The purpose of this study is to provide favourable conditions for improving the traits of rubber tree with purpose and provide new ways and conditions for creating new and good germplasm resources and provide powerful tools for gene function studies. The main results are the following:1. The influencing factors of Agrobacterium-mediated transformation of embryogenic cell suspensions of rubber tree:the frequency of transient expression of GUS gene (uidA) and the survival rate of suspension cells were used as indexes. The effects of bacterial concentrations, the way of co-culture, the pH value of co-culture medium, acetosyringone concentrations supplemented in the co-culture media, co-culture temperature and co-culture time on Agrobacterium-mediated transformation into embryogenic cell suspensions of rubber tree were studied. The optimal conditions for Agrobacterium-mediated transformation into embryogenic cell suspensions of rubber tree were determined as the following:the bacterial suspension OD600 was about 0.3 to 0.5, the pH value of co-culture medium was around 5.2, 100～150μM acetosyringone supplemented in co-culture media, the co-culture temperature was about 20 to 22℃, about 4～5 d co-cultured on solid co-culture medium.2. Resistant embryogenic calli. embryoids and regenerated plants obtained:In study of Agrobacterium-mediated genetic transformation of embryogenic cell suspensions, ticarcillin with concentration of 300 mg/L was used to inhibit Agrobacterium after co-culture, and kanamycin concentration gradient from low to high (50,75,100 mg/L) was used to select resistant embryogenic callus of rubber tree. Total of 10 lines of resistant embryogenic callus, 2177 putative transgenic embryoids and 165 putative transgenic plantlets were obtained.3. Molecular identification:GUS staining, PCR identification, sequence analysis and Southern blot analysis were used to identify the resistant embryogenic calli. embryoids and regenerated plants. The results showed that npt-Ⅱas foreign gene has been successfully integrated into the genomic DNA of the transgenic plantlets of rubber tree.4. In this study, embryogenic suspension cells of rubber tree were successfully transformed via Agrobacterium-mediated method. The Agrobacterium-mediated genetic transformation method with embryogenic suspension cells of rubber tree as recipients was preliminarily established.