-Mediated Nuclear Transformation of a Biotechnologically Important Microalga-.

() is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of mediated by (). was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. -mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene () (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of with the gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when LBA4404 (A = 1.0) and (A = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer for producing high-value products and fundamental studies.