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Fig. 2 | BMC Plant Biology

Fig. 2

From: A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers

Fig. 2

Selection and isolation of BS-resistant transformant clones on a gellan gum-solidified medium supplemented with BS. (a) Schematic diagram of the MA2 gellan gum plate supplemented with 1 mg/mL BS. MA2 gellan gum supplemented with 1 mg/mL BS was solidified in one well of a 24-well plate. The medium was covered with a cornstarch bed to facilitate the colony formation of C. merolae. (b) Colonies of BS-resistant clones on MA2 gellan gum plate supplemented with 1.0, 1.25 and 1.5 mg/mL BS. After PEG-mediated transformation, cells were recovered in a drug-free MA2 liquid medium for 2 days in the light. Cells were spread on MA2 gellan gum plate supplemented with BS and incubated for 14 days in the light. (c) Colony PCR analyses of BS-resistant clones. The positions of the primers are indicated as arrows below the chromosomal structure of the WT and BSD-MITmS transformant. For detection of the BSD marker insertion either by off-target and on-target insertion (the primer set, nos. 15/16), the predicted size of the PCR product was 0.4 kb. In the PCR amplifying CMD184C-CMD186C loci (the primer set, nos. 11/12), the predicted size of the PCR product of on-target insertion of the BSD-MITmS construct was 6.3 kb and the size for off-target insertion or the WT chromosome was 3.3 kb. The exact positions and sequences of the primers are indicated in Supplementary Table S1. Full length unprocessed gel image of Fig. 2c is shown in Supplementary Fig. S1a.

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