Assessing Superoxide Accumulation in Plant Leaves

For analysis of ROS accumulation, the O₂⁻ in the B03S leaves was stained with NBT (Yuanye, Shanghai, China) according to the methods of Kumar [64 (link),65 (link)]. First, the leaves were cut into 10 cm long samples and immersed in phosphate buffer (pH 7.5) consisting of 2 mM NBT, 42 mM Na₂HPO₄ and 8 mM NaH₂PO₄, followed by vacuum infiltration at room temperature for 12 h. The NBT and O₂⁻ combined, forming visible blue stains in the leaves. Then, the leaves were decolorized with 100% ethanol for 1 h. Finally, the decolorized leaves were soaked in glycerol and imaged with a digital camera (EOS RP, Canon, Japan) under visible light.