Preliminary Factors Influencing Transient Expression of Gusa in Dendrobium Savin White Protocorm-like Bodies (PLBs) Using Agrobacterium-mediated Transformation System

A critical step in the development of Agrobacterium tumefaciens-mediated transformation in orchid genus is the establishment of optimal conditions for T-DNA gene delivery into protocorm-like bodies from which the whole plantlets can be regenerated. As a first step in the development of a successful Agrobacterium tumefaciens-mediated transformation for Dendrobium savin white, factors influencing the successful T-DNA integration and expression (as measured by the transient GUS expression) were investigated. Various parameters were included such as different acetosyringone concentrations used during co-cultivation in Agrobacterium suspension, the length of the preculture period of PLBs prior to infection, co-cultivation period, different calcium concentrations, silver nitrate concentrations, different bacterial density (OD ) and duration 600nm of immersion periods. Results obtained were based on the percentage of GUS expression which was observed 3 days post-transformation. The results based from transient gusA gene expression of PLBs were suggested that the Agrobacterium-mediated transfer system of T-DNA is highly efficient. Therefore, the investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of Dendrobium savin white protocorm-like bodies tissue.