FACTORS AFFECTING AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION OF MARINE MICROALGA, NANNOCHLOROPSIS SP.

Abstract

The optimisation condition of an Agrobacterium tumefaciens-mediated transformation method for the marine microalgae Nannochloropsis sp. (UMT-M3) is reported using a binary vector harbouring the green fluorescent protein and P-glucuronidase (gfp-gusA) as reporter genes and a hygromycin phosphotransferase (hpt) as a selection marker. For an efficient Agrobacterium mediated genetic transformation of marine microalgae Nannochloropsis sp., parameters, namely pre-culture duration, inoculum density, co-cultivation temperature, co-cultivation duration, pH of co-cultivation medium and the concentration of acetosyringone (AS) were evaluated in this study by monitoring P-glucuronidase (gusA) expression two days post-infection. Results showed that 5 days of pre-culture, 3 days of co-cultivation at 24°C, medium pH 5.5, bacterial density of OD600= 1.0, and 50 JiM AS are optimal in the transformation of Nannochloropsis sp. The use of combined optimised parameters resulted in an average transformation frequency of 24.5%. The transgenic nature of transformants was confirmed by polymerase chain reaction with the gfp-gusA and hptspeci:fic primers on colonies resistant to 18 mg L-1 hygromycin. The developed transformation method will enable manipulation of important biochemical pathways in Nannochloropsis sp. and facilitate the genetic improvement of this commercially-important microalga.