4.8. Measurement of Total Anthocyanin Content

Total anthocyanin were extracted from the rosette leaves of 25-day-old Arabidopsis plants and apple calli cultured for 7 days. Anthocyanin extraction was performed using a methanol–HCl method [71]. Approximately 0.1 g of each sample was incubated in 5 mL of 0.1 % acidic methanol solution (CH₃OH:HCl:H₂O = 70:0.1:29.9, v/v/v) overnight in the dark at 4 °C. The absorbance of each extract was measured at 530 and 657 nm with a UV-1600 spectrophotometer (SHIMADZU, Kyoto, Japan). The total anthocyanin content was calculated using the following equation: Q_{Total Anthocyanin} = (A530 − 0.25 × A657) × FM⁻¹. There were three biological replicates for each sample.