Factors affecting Agrobacterium-mediated genetic transformation of marine microalga,Nannochloropsis sp.

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 β-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 um 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 hpt-specific primers on colonies resistant to 18 mg l1 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. © universiti malaysia terengganu publisher.