Hydrogen Peroxide (H2O2) and Lipid Peroxidation (MDA) Content Determination

For the determination of the H₂O₂ content in the leaves of different pumpkin cultivars, the fresh leaf sample (0.5 g) was homogenized in 5 ml of precooled HClO₄ (1.0 M) using the pre-chilled mortar and pestle and then centrifuged at 10,000 g for 10 min. The content of H₂O₂ was determined and calculated according to the method described by Willekens et al. (1997) and expressed as μmol g^–1 FW.

The level of lipid peroxidation was determined by quantifying the MDA accumulation in the leaves of different pumpkin cultivars according to the method described by Hodges et al. (1999) and Tian et al. (2015) with some modifications. Briefly, the fresh leaf sample (0.5 g) was homogenized in 2.5 ml of 0.1% trichloroacetic acid and then centrifuged at 10,000 g for 15 min. The absorbance of the supernatant was recorded at a 532-nm wavelength. The content of MDA was expressed as nmol g^–1 FW.