FCA showed the lowest sGFP fluorescence at 2 μg DNA and the highest at 10 μg DNA, however, the difference in fluorescence range was not very large among quantities ranging from 5–40 μg (Figure 5A). Similarly, the protein accumulations were not much different among 5–40 μg (Figure 5B). Comprehensively, an optimal amount of transfected DNAs resulted in 10 μg DNAs for FCA. Our results demonstrate that the use of only 5–10 μg DNA was enough for FCA using rice protoplasts and that large amounts of plasmid DNAs (>40 μg) are not necessary for FCA of our protoplast system. In this study, the smallest quantitiy (5 μg DNA) of the optimized DNA quantity (5–10 μg DNA) was used for various analyses.