*4.10. Analysis of Reactive Oxygen Species*

Histochemical staining of hydrogen peroxide (H2O2) and superoxide anion radicals (O2 .-) was performed using 3, 3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT), respectively [76]. Leaf tissue was immersed in NBT (1 mg/mL) solution for 2 h or in DAB (0.5 mg/mL) solution for 12 h in the dark. The stained leaves were decolorized in boiling ethanol (90%, *v*/*v*) for 2 h. H2O2 was quantitated according to Velikova et al. [77] and calculated using a standard curve of H2O2 reagent. The quantification of O2 .- was determined as described by Nahar et al. [78] and calculated using a standard curve of a NaNO2 reagent.
