4.6. NBT Staining and ROS Measurement

O₂⁻ levels were detected as previously described with minor modifications [43]. The seedlings were placed in the NBT staining solution containing 1mg mL⁻¹ NBT in 50 mM sodium phosphate buffer (pH 7.5). The seedlings were then incubated for 6 h in the dark with gentle shaking. The staining solution was replaced with absolute ethanol in a boiling water bath for 30 min to remove chlorophyll. The seedlings were transferred to filter paper with 60% v/v glycerol until images were taken.

Briefly, swollen root peels were ground using a mortar and pestle in buffer (9 mL mg⁻¹ fresh weight) containing 50 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 12,000 rpm for 20 min. The supernatant was transferred into a new tube for measurement. The procedure of ROS detection used was Plant Reactive Oxygen Species (ROS) ELISA Kit (MBS281870) followed the manual as described.