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

Abstract

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 β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) 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.

A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

“…Among the various methods established, agitation with glass beads needs protoplast generation [3]–[5], silicon carbon whiskers based-method has been reported to be toxic to human [5], [6], electroporation [5]–[7], [9] and biolistic microparticle bombardment [5]–[7] needs expensive instrumentation and protoplast generation, and Agrobacterium tumefaciens -mediated gene transfer is inefficient [5]–[8], [10]. Hence, in spite of such efforts, genetic engineering in eukaryotic microalgae is not well-established, compared with bacterial transformation [2], [5], [7], [11]–[13].…”

Vir gene inducers in Dunaliella salina ; an insight in to the Agrobacterium -mediated genetic transformation of microalgae

“…This method has several advantages over other direct transformation methods which include stability, inexpensive and simple procedure, integration of large DNA fragments with little rearrangements in the host genome and wide host range [2,3]. Recent studies involving genetic manipulation among microalgae through the Agrobacterium-mediated transformation has been demonstrated in different species such as Chlamydomonas reinhardtii [4,5], Haematococcus pluvialis [6], Schizochytrium [7], Chlorella vulgaris [8], Nannochloropsis [2], Scenedesmus almeriensis [9], and Dunaliella [10].…”

Agrobacterium-Mediated Transformation of Three Freshwater Microalgal Strains

“…Agrobacterium-mediated genetic transformation was successful in a variety of freshwater algal strains such as Chlamydomonas reinhardtii (Kumar et al, 2004), Haematococcus pluvialis (Kathiresan and Sarada, 2009), Chlorella vulgaris (Cha et al, 2012) and marine microalgal strains Parachlorella kessleri (Rathod et al, 2013), Schizochytrium (Cheng et al, 2012), Nanno chloropsis sp. (Cha et al, 2011) and Dunaliella bardawil (Anila et al, 2011).…”