Histochemical Detection of Superoxide and H2O2 Accumulation in Brassica juncea Seedlings

Plant cells continually produce reactive oxygen species (ROS) as a by-product of aerobic metabolism.Increased production of ROS occurs under unfavorable conditions imposed by various abiotic and biotic factors.Accumulation of ROS is damaging to various cellular components and macromolecules including plasma membrane, nucleic acids, and proteins and eventually leads to cell death.In this protocol, we describe the histochemical detection of hydrogen peroxide (H2O2) and superoxide (O2 -) anion, two of the most important ROS, in Brassica juncea seedlings by using 3, 3ʹ-Diaminobenzidine (DAB) and Nitrotetrazolium blue chloride (NBT) as the chromogenic substrate.DAB is oxidized by H2O2 in the presence of peroxidases and produces reddish brown precipitate.NBT reacts with O2 -to form a dark blue insoluble formazan compound.The protocol can be used in other plant species and for different plant tissues.