2.4. Anthocyanin contents measurement Anthocyanin contents were measured according to the method of Nakatsuka et al. (2008) with some modifications: 300 mg petal
tissues were ground in liquid nitrogen and the anthocyanins were extracted with 1 mL of 1% (v/v) HCl methanol solution for 24 h at 4◦C. After clearing the extractions by centrifugation at 13,000 × g for 30 min, the supernatant was analyzed with a Beckman DU-800 spectrophotometer (Beckman Instruments, Fullerton, CA). The absorbance of the extract at 530 nm was used as a measure of the anthocyanin contents; values were normalized to the fresh weight of each sample to indicate anthocyanin content. Anthocyanin content was determined on three replicates (each of one flower).