*2.4. Anthocyanin Determination*

A 1.33 cm2 disc cut from stipule, and the entire petiole were each used per leaf, and anthocyanin extraction was done according to Abdel-Aal and Hucl [27]. Samples were placed in 5 mL glass tubes with 3 mL of acidified ethanol (85:15, V/V) ratio using 95% ethanol and 1.428N HCl) at pH 1, tubes were capped and then incubated overnight at room temperature for extraction. Samples were vortex mixed and centrifuged at 5000 rpm for 5 min. The supernatant solution was measured with spectrophotometer and absorbance was read at 535 and 663 nm, wavelength peaks for absorbance of anthocyanin and chlorophyll a, respectively. Total anthocyanin concentration in μg cm−<sup>2</sup> was calculated according to Murray and Hackett [28] as:

$$\text{Anthocyama}(\text{\(\mu g ml}^{-1}\text{\)} = \text{A}\_{535} - 0.24(\text{A}\_{663}) \tag{5}$$
