The powerful capabilities of flow cytometry, including quantification of fluorescence and cell sorting, have been broadly applied in cell biology, molecular biology, and microbiology in various fluid and unicellular systems involving animals and microbes [20,21]. In plant systems, technologies using FCA have also been usefully applied to the comparative promoter analysis and the sorting protoplasts expressing fluorescent proteins [4,5,7]. Recently, technology using FCA has been developed to enable high throughput analysis of more than 108 samples per day [22]. In this study, we developed a useful and powerful strategy of FCA using protoplasts by using efficient translation systems and localization of sGFP into chloroplasts. Chloroplasts have been reported as an excellent reservoir for many kinds of ectopically expressed proteins, in which many kinds of foreign proteins could be kept stable [11]; in rice, 20–30 chloroplasts per a rice mesophyll cell have been reported to exist [23]. Similar numbers of chloroplasts were also shown to be gathered beneath the cell membrane in one protoplast (see Figure 1B).