In FCA, the fluorescence intensity was normalized by the average value of K-sGFP and showed an eight-fold higher intensity in KR-sGFP as compared to sGFP and K-sGFP (Figure 2A). The transfection ratio was also higher in KR-sGFP (65.7%) than in sGFP (16.8%) and K-sGFP (14.7%), resulting in a 4.5-fold increase in KR-sGFP than sGFP and K-sGFP (see Figure 2B). As shown in Figure 2C, the large difference in the population distribution was observed on the plot data of FCA. The threshold value (103) was determined and the fluorescence below it was regarded as the background signals using mock control. In the case of K-sGFP, a large population (83.3%) was distributed below the threshold value, while the large portion of KR-sGFP (65.7%) was distributed on the overall intervals with higher fluorescence intensity than the threshold value. These results suggest that the localization of sGFP in chloroplasts largely enhances the intensity of fluorescence on FCA (>8-fold) and this causes an increase in the population with higher fluorescence than the threshold level of background signals and, consequently, increases transfection ratio, from 14.7% to 65.7%. In other words, it can be supposed that the population of K-sGFP under the threshold value might contain the transfected protoplasts with lower fluorescence intensity than the background signals. Additionally, the results of FCA between sGFP and K-sGFP are shown to be not considerably different. Thus, it can be supposed that a Kz sequence might not be a much more effective factor for FCA, even if it is helpful to increase the expression level.