Protoplast Extraction and Cell Size Analysis

Leaf protoplasts were extracted from 10 leaves of 4-week-old rosette of A. thaliana plants in WT and #236 background (n = 5 from each genotype) using the “Tape-Arabidopsis Sandwich method” described in Wu et�al., 2009 (link). For cell wall digestion, 1.25% of Cellulase Onozuka R-10 (Serva) and 0.3% of Macerozyme R-10 (Serva) were used. Protoplasts were washed with W5 solution [5 mM Glucose, 154 mM NaCl, 125 mM CaCl₂, 5mM KCl, 5 mM MES (Sigma-Aldrich), and imaged using an Olympus BX61]. Thirty-seven and forty-five pictures containing approximately 10,000 protoplasts were analyzed using ImageJ (https://imagej.nih.gov/ij/) to calculate cell size. The images were segmented using the "find edges" process and a color threshold. Touching cells were separated from the segmented binary images using the watershed process. Cell size was then measured using the analyze particles option.