Anthocyanin and chlorophyll measurements

Five-day-old seedlings grown under white light were used for anthocyanin measurement. Anthocyanin extraction and determination was according to Datta et al. (2007). Four-day-old seedlings grown under red light were used for chlorophyll determinations. The protocol for extracting chlorophyll was modified based on the previous report (Ni et al., 2009). Fresh leaves were frozen and ground into fine power in liquid nitrogen, and then 50 mg tissue powder was extracted with 1 mL of 80% acetone at 4°C and incubated for 15 min in the dark. The extract was then centrifuged at 4°C for 15 min, and the chlorophyll in supernatant was measured by spectrophotometer at the wavelengths of 645 and 663 nm, respectively. The chlorophyll concentration was calculated as follows: Concentration: a+b (mg/g) = [8.02 × A663 + 20.20 × A645] ×V/1000×W (Chlorophyll a+b), where V = volume of the extract (mL), and W = weight of fresh leaves (g).