Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris

The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient beta-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24A degrees C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 mu M acetosyringone, Agrobacterium density of 1.0 units (OD600) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L-1) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.