Background

Cyanidioschyzon merolae 10D is a unicellular red alga that inhabits sulfate acid hot springs (Matsuzaki et al., 2004). It has a simple cellular structure, composed of a minimum set of organelles: one nucleus, one mitochondrion, and one plastid in a cell. C. merolae is used as a model organism to investigate the basic architecture of eukaryotes. Thus, many molecular genetics techniques have been developed using this organism, including antisense suppression, transient expression, gene disruption, and stable insertion into the genome (Minoda et al., 2004; Ohnuma et al., 2008; Imamura et al., 2009; Imamura et al., 2010; Zienkiewicz et al., 2017a; Zienkiewicz et al., 2017b; Zienkiewicz et al., 2018). The plating method for the selection of transformants is important for transformation experiments. However, C. merolae shows considerably low plating efficiencies (less than 0.5%) on MA2 solid gellan gum plates. The direct spread of cells onto an MA2 solid gellan gum plate usually results in the death of the cells (Figure 1A). Hence, the plating method has been modified using the cornstarch embedding method which is commonly used for the cell-wall-less Chlamydomonas reinhardtii strain (Shimogawara et al., 1998). In the cornstarch embedding method, cells are grown on a starch bed prepared by spotting 20% slurry cornstarch on the MA2 solid gellan gum. Cells form colonies on the solid starch bed, but these are often diffused or blurred (Figure 1B).

To improve these points, we here devised a novel plating method using melted cornstarch (named in this protocol “top starch solution method”), similar to the top-agar plating method used in bacterial genetics. The 2% cornstarch in dH₂O was melted at 98 °C for 10 min. The melted cornstarch solution (= top starch solution) was mixed with an equal volume of the liquid MA2 medium containing C. merolae cells, and poured onto the surface of the MA2 solid gellan gum. Cells formed defined colonies using this plating method (Figure 1C). The plating efficiency of the top starch solution method was more than 2.0%, while that of the conventional method was less than 0.5%. In the conventional method, it was necessary to prepare many starch beds on solid plates prior to use, but the top starch solution method only requires the pouring of the melted starch-cell mixture onto the solid medium, making the plating procedure quick and easy. In this report, we describe the detailed procedure of the top starch solution method.


Figure 1. Effect of the top starch solution method on the colony formation in C. merolae. (A-C) In each approach, plates with the spotted cells were incubated for two weeks at 40 °C under continuous white light. A. Five hundred cells were directly spotted onto an MA2 solid gellan gum plate, which resulted in no visible colony formation. B. Solid starch beds were made using 15 μl of 20% slurry cornstarch in an MA2 liquid medium. Five hundred cells were spotted onto a solid cornstarch bed. Open triangles indicate diffused, blurred colonies. C. The 2% cornstarch in dH₂O was melted at 98 °C for 10 min to make the top starch solution. The top starch solution was mixed with an equal volume of cells in MA2 medium. Fifteen microliter aliquots containing 500 cells were spotted onto the MA2 solid gellan gum plate.