Agrobacterium-mediated Genetic Transformation of Phalaenopsis bellina Using GFP and GUS Reporter Genes

Genetic transformation protocols of Phalaenopsis bellina protocorm-like bodies (PLBs) were established using gfp (green fluorescent protein) and gus ((Beta)-glucuronidase) genes as the reporter system. Agrobacterium tumefaciens strain, LBA 4404 containing the binary vector, pCAMBIA 1304, with the hptII gene as the selectable marker and gfp and gus-intron genes as the reporter genes. Horizontally dissected PLBs were immersed in A. tumefaciens suspensions with 200uM acetosyringone (AS) for 45 minutes. This was followed by cocultivation until the growth of A. tumefaciens was observed surrounding the PLBs on the co-cultivation medium. GFP detection and GUS histochemical assay were carried out to investigate the transient expression of both GFP and GUS reporter genes. The selection of proliferating PLBs was carried out on 4 mg/L hygromycin and 100 mg/L cefotaxime. GFP could be used as the reporter system as it is an effective, rapid and non-destructive system to monitor the transformed tissues.