The restricted exposure of the plants to light (20 h) was used to reduce the autofluorescence background resulting from the presence of chlorophyll. The formation of RuBisCo and the biosynthesis of chlorophyll in rice leaves have been reported to be the greatest during greening of etiolated leaf tissues [33,34]. During this process, photosynthesis-related proteins are detected within 2 h of illumination with white light [35], the stromal structures of chloroplast are evident following illumination for 16 h [36], and chlorophyll formation begins subsequently [33,36]. In several preliminary experiments, we established that the optimum illumination time was 16–20 h, which resulted in chlorophyll formation but not to a level where autofluorescence was evident in the images using a fluorescein isothiocyanate (FITC) filter (data not shown). Consequently, 20 h of illumination was used for the FCA analysis of protoplasts in this study.