High efficiency of agroinfiltration approach in Phalaenopsis amabilis (L.) Blume (moth orchid) with transient expression of green fluorescent protein reporter gene

Agroinfiltration has become a crucial technique in molecular biology, enabling the transfer of functional genes into target plants for rapid gene expression analysis. This study investigated the use of agroinfiltration for transient expression of the green fluorescent protein (GFP) reporter gene in Phalaenopsis amabilis (L.) Blume transformant leaves. The aim was to establish an efficient and rapid genetic transformation method for generating transformant plant candidates. Agroinfiltration was performed on the leaves using a suspension containing Agrobacteriumtumefaciens strain GV3101, which harbored a T-DNA construct in the pRI101AN vector carrying the 35S::GFP and neomycin phosphotransferase II (NPTII) genes. Plant development post-infiltration was monitored microscopically with a fluorescence microscope and confirmed molecularly using PCR (polymerase chain reaction) with specific primers for GFP (736 bp), NPTII (550 bp) and ACTIN (114 bp). The results showed that green fluorescence, indicating GFP expression, was detected under a blue laser (450 - 480 nm) within 24 hr post-infiltration on both the adaxial andabaxial leaf surfaces, particularly along the midvein (primary veins) and leaf margins. Anatomical analysis of transverse and longitudinal free-hand sections further confirmed the rapid occurrence within the epidermis, mesophyll and vascular tissues. The accuracy of the data was supported and confirmed by the positive results of all three P. amabilis transformant plants that were analyzed by PCR and it was proven that all positive transformant plants carried the GFP, NPTII and ACTIN genes as positive controls. These results indicate agroinfiltration and GFP approach offering efficient, rapid and valuable prospects for accelerating genetic engineering applications in plant research.