Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris.
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.
Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030, Kuala Terengganu, Terengganu, Malaysia. cha_ts@umt.edu.my
Thye San
TS
Cha
Willy
W
Yee
Ahmad
A
Aziz
D000098:Acetophenones / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics*; D048408:Chlorella vulgaris / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D005966:Glucuronidase / Q000032:analysis / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000235:genetics; D013696:Temperature; D014170:Transformation, Genetic
D000098:Acetophenones; D049452:Green Fluorescent Proteins; C051667:acetosyringone; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D005966:Glucuronidase