| To aid transformation of anthurium, tissue culture and regeneration was achieved through either somatic embryogenesis or shoot regeneration using in vitro grown etiolated internodes, laminae, and root segments. Two gene transfer methods were used to transform anthurium tissues. Using bombardment of DNA-coated microprojectiles into anthurium calli and etiolated internodes, transient expression of {dollar}beta{dollar}-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII) was observed in these tissues. No transformed plants were recovered using this method. Antibacterial genes, including an insect attacin gene (Att), phage P22 gene P13, phage T4 lysozyme gene e, and a gene encoding an analog of insect cecropin B (Shiva-1) were driven by either double CaMV35S or potato wound inducible promoter, in the plant expression vector pBI121. Agrobacterium tumefaciens LBA4404 carrying either pCa2Att, pCa2P13, pCa2T4, pWIAtt or pWIShiva was used for cocultivation with internode or lamina explants of UH965 and UH1060. Following culture on selection media containing kanamycin and carbenicillin or cefotaxime, shoots regenerated from various calli pieces. Kanamycin-resistant plantlets were recovered from UH965 and UH1060 etiolated internode explants cocultivated with Agrobacterium with or without tobacco cell line 'Su' as nurse culture. GUS activity as determined by histochemical staining was absent in the kanamycin-resistant plants evaluated. Western blot analysis of total proteins from lamina calli formed de novo from kanamycin-resistant UH965 plants showed the presence of attacin protein. Polymerase chain reaction was used to amplify DNA fragments from the introduced genes. In six UH965 plants, Att and nptII genes were amplified in the expected sizes of 546 and 1054 bp, respectively. In UH1060 plants, P13 and nptII genes were amplified. A GUS gene fragment was amplified in one of the UH965 plants. No amplification of the above-mentioned genes was observed in DNA samples of untransformed control plants. Southern hybridizations using amplified sequences from Att, P13 and NPTII all showed positive hybridization. Kanamycin-resistant UH965 plants with Ca2Att, Ca2P13 or Ca2T4 were challenged with the blight pathogen Xanthomonas campestris pv. dieffenbachiae strain D150 in the petiole end. The result of two challenge experiments indicated that most transgenic UH965 plants were partially resistant and a few were resistant to the blight bacteria. |
