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.
Over the years, microalgae have been identified to be a potential source of commercially important products such as pigments, polysaccharides, polyunsaturated fatty acids and in particular, biofuels. Current demands for sustainable fuel sources and bioproducts has led to an extensive search for promising strains of microalgae for large scale cultivation. Prospective strains identified for these purposes were among others, mainly from the genera Hematococcus, Dunaliella, Botryococcus, Chlorella, Scenedesmus and Nannochloropsis. Recently, microalgae from the Selenastraceae emerged as potential candidates for biodiesel production. Strains from the Selenastraceae such as Monoraphidium sp. FXY-10, M. contortum SAG 47.80, Ankistrodesmus sp. SP2-15 and M. minutum were high biomass and lipid producers when cultivated under optimal conditions. A number of Selenastraceae strains were also reported to be suitable for cultivation in wastewater. This review highlights recent reports on potential strains from the Selenastraceae for biodiesel production and contrasts their biomass productivity, lipid productivity as well as fatty acid profile. Cultivation strategies employed to enhance their biomass and lipid productivity as well as to reduce feedstock cost are also discussed in this paper.
In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150 mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120 s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains ( NS16, sp. NS6, sp. DPBC1 and sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae ( sp. NPA14 and sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9 ng/mg for NS16, 42.2-247.0 ng/mg for sp. NS6, 70.2-110.9 ng/mg for sp. DPBC1 and 142.8-164.8 ng/mg for sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from NS16 and sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.
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