Emerging factors that influence efficiency of T-DNA gene transfer into Phalaenopsis violacea orchid via Agrobacterium tumefaciens–mediated transformation system

Abstract

An early step in the Agrobacterium-mediated transformation of Phalaenopsis violacea orchid was investigated in the plant-bacterium interaction. Directed movement in response to chemical attractants is the crucial importance to Agrobacterium tumefaciens strains. Chemotaxis of Agrobacterium tumefaciens strains (EHA 101 and 105) towards wounded orchid tissues has been studied by using swarm agar plates. The results obtained indicate a minor role of chemotaxis in determining host specificity and suggest that it could not be responsible for the absence of tumorigenesis in Phalaenopsis violacea orchid under natural conditions. Various aspects of transformation were examined using transient gusA gene expression in efforts to improve the efficiency of producing transformants. Hypervirulent Agrobacterium tumefaciens strains, EHA 101 and 105, harboring the pCAMBIA 1304 plasmid containing gusA gene as a reporter marker, were used for transformation study.  The effects of different concentrations of L-cysteine, silver nitrate, and various temperatures (⁰C) during co-cultivation in half-strength MS medium supplemented with 5% of banana Mas extract on transformation efficiency were evaluated. Strain EHA 105 proved significantly better than EHA 101 based on higher percentage of gusA gene expression and even spread of GUS staining on PLBs. In addition, 200mg.L^-1 L-cysteine, 60µM silver nitrate, and under the temperature at 24⁰C gave the highest percentage of transient GUS expression. The results from transient transformation of PLBs suggested that Agrobacterium-mediated transfer of T-DNA is highly efficient. Therefore, by combining the best treatments, an efficient and reproducible of Agrobacterium-mediated transformation procedure could be continued for the production of transgenic Phalaenopsis violacea orchid.