Tools for microalgal biotechnology: development of an optimized transformation method for an industrially promising microalga-Tetraselmis chuii

Tetraselmis chuii is a biotechnologically promising green microalga that is commonly used in aquaculture. Development of reliable and easy genetic transformation procedures is essential for the improvement of this microalga. Here, we report the development of a suitable method for the stable genetic transformation of T. chuii via Agrobacterium tumefaciens. Following a statistical experimental design, seven factors were tested for their effect on the transformation efficiency. The effects of temperature, pH, and the interaction between acetosyringone concentration and co-culturing period were statistically significant. These main factors were subsequently optimized to the following values: 27 A degrees C co-culturing temperature, pH 5.0, and 150 mu M acetosyringone concentration. Transformation was verified by PCR on different target genes present in the T-DNA as well as by the determination of GUS activity from the reporter gene. Genetic stability of the transformed clones was examined by culturing under non-selective conditions in phleomycin-free medium for 6 months. Transformed clones were stable as indicated by the maintenance of the resistance to phleomycin (Phl(R)). In summary, an easy and reliable genetic transformation method for T. chuii was developed. The use of A. tumefaciens provides a useful method for the genetic engineering of this biotechnologically promising microalga.