Total Anthocyanin:

Total anthocyanin will be determined by the method of Hodges and Nozzolillo (1996). 0.5 g of leaf material will be ground in pestle and mortar using 5ml of phosphate buffer (K2HPO4+KH2PO4, pH 7.8). Ice will be used to keep the pestle mortar cool. Centrifugation will be done at 10,000 rpm for 10 minutes while maintaining the temperature at 4C. The supernatant will be stored in a cool place. The supernatant will be directly taken in a cuvette and the absorbance will be measured at 600nm using a spectrophotometer.  Phosphate buffer will be used as blank to calibrate the spectrophotometer.

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