Anthocyanin content was determined according to Gould et al. (2000) with a slight modification. Fresh leaf material (0.2 g, the third leaf from the top) was incubated in the presence of a mixture of chloridric acid/water/methanol (1/3/16) in the dark at 4 °C during 72 h. The absorbance was measured at 653 and 530 nm. Anthocyanin content was determined according to the following equation:
Anthocyanin (μg ml−1) = A(530) − 0.24 × A(653)