Transgene constructs and their expression in the Kaleidocell line. (A) The fusion genes were inserted downstream of the 35S cauliflower mosaic virus promoter (35SP). Expression cassettes of Gal4-CFP and CoxIV-YFP were inserted in tandem in the transgene region [between the right (RB) and left borders (LB)]. Expression cassettes of Cam53BD-GFP and RecA-RFP were inserted in the transgene region. Unique sites digested by Bam HI in the transgenes and the predicted sizes (kbp: kilo base pairs) between the Bam HI sites and to the RB were shown on the top of the constructs. The size of the 35SP is also shown on the bottom of the construct. A transgenic plant expressing Gal4-CFP and CoxIV-YFP was crossed with a transgenic plant expressing Cam53BD-GFP. The resulting plant was further crossed with a transgenic plant expressing RecA-RFP. The final transgenic plant expressing all four transgenes was designated as the Kaleidocell line. Gal4: Saccharomyces cerevisiae nuclear protein, CoxIV: Saccharomyces cerevisiae cytochrome oxidase IV, Cam53BD: petunia calmodulin CaM53 binding domain, RecA: Arabidopsis DNA repair protein. (B) Southern blotting analysis of the Bam HI digested genomic DNA of homozygous Kaleidocell. Lane M: 1 kb DNA Ladder (NEB), Lane BamHI: Bam HI digested genomic DNA. The sizes of the DNA ladder are shown on the left. The DNA probe is shown on the right bottom. (C) Fluorescence microscope images of a protoplast from the Kaleidocell line. The protoplast was observed with four different fluorescence filter sets: cyan, green, yellow, or red. Captured images were merged to generate a single image of the protoplast. Scale bars = 10 μm.