Early GFP gene assessments influencing Agrobacterium tumefaciens-mediated transformation system in Phalaenopsis violacea orchid

Abstract

An initial step in the genetic transformation of Phalaenopsis violacea orchid species was investigated in the plant-Agrobacterium interaction. Agrobacterium tumefaciens strains EHA 101 and 105, harboring the pCAMBIA 1304 plasmid contains gfp gene as the reporter gene marker, were used in this transformation study. The spectrophotometric GFP assay provides information on the amount of inoculated Agrobacterium tumefaciens that effectively bound to various orchid tissues. Different temperatures during co-cultivation period, the concentration of Lcysteine, calcium (CaCl2) and silver nitrate (AgNO3) in co-cultivation medium during cocultivation period were identified to be major and important factors in enhancing the increase percentage of transient gfp gene expression in PLBs. Agrobacterium tumefaciens EHA 105 was proved to be better bacterial strain in transforming the targeted PLBs than EHA 101, based on the notably higher transient expression of gfp gene in all the optimization parameters that were tested. Highest T-DNA delivery efficiencies were obtained when P. violacea PLBs were co-cultivated with Agrobacterium tumefaciens strain EHA 105 in half-strength MS medium supplemented with 5% of banana cultivar, Mas extract containing 200 mgL-1 L-cysteine, 60µM silver nitrate, without calcium in the medium during co-cultivation in the dark condition at 24°C. The results from the transient gfp gene expression of PLBs suggested that Agrobacterium-mediated transfer of T-DNA to the naturally recalcitrant P. violacea is feasible and is highly efficient. Hence, the use of the gfp marker gene during in vitro screening of the transgenic cells has enabled the visual selection of orchid transformed by Agrobacterium tumefaciens at higher frequency rates.