Highly efficient transformation of the model zygnematophycean alga Closterium peracerosum-strigosum-littorale complex by square-pulse electroporation.

The zygnematophycean algae occupy an important phylogenetic position as the closest living relatives of land plants. Reverse genetics is quite useful for dissecting the functions of genes. However, this strategy requires genetic transformation, and there are only a few reports of successful transformation in zygnematophycean algae. Here, we established a simple and highly efficient transformation technique for the unicellular zygnematophycean alga Closterium peracerosum-strigosum-littorale complex using a square electric pulse-generating electroporator without the need for cell-wall removal. Using this method, the transformation efficiency increased more than 100-fold compared to our previous study using particle bombardment. We also succeeded in performing CRISPR/Cas9-based gene knockout using this new method. Our method requires only small amounts of labor, time, and incubator space. Moreover, our technique could also be utilized to transform other charophycean algae with available genome information by optimizing the electric pulse conditions.