For the measurement of lipid peroxidation in leaves, the thiobarbituric acid (TBA) test, which determines malonaldehyde (MDA) as an end product of lipid peroxidation, was used. Leaf material (500 mg) was homogenated in a potassium phosphate buffer (pH 7.8).
The homogenate was centrifuged at 12,000×g for 20 min,and 1 mL of the supernatant was incubated in boiling water for
30 min. The reaction was stopped by placing the reaction tubes with 1 mL of TBA solution containing the above reagents plus 0.65% TBA in an ice bath.
The samples were centrifuged at 1500×g for10 min, and the absorption of the supernatant was read at 532 nm.
The value for non-specific absorption at 600 nm was subtracted.The amount of MDA was calculated from the extinction coefficient 155 mM−1 cm−1.