title: An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii.
abstract: Algal-based recombinant protein production has gained immense interest in recent years. The development of algal expression system was earlier hindered due to the lack of efficient and cost-effective transformation techniques capable of heterologous gene integration and expression. The recent development of Agrobacterium-mediated genetic transformation method is expected to be the ideal solution for these problems. We have developed an efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Pre-treatment of Agrobacterium in TAP induction medium (pH 5.2) containing 100 μM acetosyringone and 1 mM glycine betaine and infection of Chlamydomonas with the induced Agrobacterium greatly improved transformation frequency. This protocol was found to double the number of transgenic events on selection media compared to that of previous reports. PCR was used successfully to amplify fragments of the hpt and GUS genes from transformed cells, while Southern blot confirmed the integration of GUS gene into the genome of C. reinhardtii. RT-PCR, Northern blot and GUS histochemical analyses confirm GUS gene expression in the transgenic cell lines of Chlamydomonas. This protocol provides a quick, efficient, economical and high-frequency transformation method for microalgae.
authors.affiliation: School of Biosciences, Mahatma Gandhi University, Kottayam, 686560, Kerala, India, pratheeshpt@yahoo.com.
authors.forename: P T
authors.initials: PT
authors.lastname: Pratheesh
authors.forename: M
authors.initials: M
authors.lastname: Vineetha
authors.forename: G Muraleedhara
authors.initials: GM
authors.lastname: Kurup
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D005966:Glucuronidase