Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger AAB)

摘要: Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg settled cell volume embryogenic suspension cells, depending upon the Agrobacterium strain used, gave rise to hundreds morphologically normal, transgenic plants groups. Development a highly efficient for recalcitrant species like banana, especially Cavendish group (AAA) cultivars, required identification optimisation factors affecting T-DNA delivery subsequent plant regeneration. We used male-flower-derived suspensions (Cavendish Lady Finger) strains AGL1 LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) gusA (β-glucuronidase) or nptII (neomycin modified gfp (green fluorescent protein) gene T-DNA, investigate optimise tissue culture variables. Factors evaluated included pre-induction Agrobacterium, conditions media inoculation co-cultivation, presence acetosyringone Pluronic F68 co-cultivation media. One factor that led significant enhancement frequency was introduction centrifugation step during co-cultivation. Post liquid-media wash recovery helped avoid overgrowth on filters supporting cells. Marker-gene expression molecular analysis demonstrated transgenes integrated stably into genome. T-DNA:banana DNA boundary sequences were amplified sequenced order study integration profile.