In order to check the amount of reactive oxygen species (ROS) produced in response to HS in transgenic Arabidopsis and WT plants, staining with nitro blue tetrazolium (NBT) was done (Agarwal and Khurana, 2018 (link)). For this, 2-week-old seedlings of Arabidopsis WT and overexpression transgenics were subjected to HS (42°C for 2 h) after which overnight staining of the plants was done by incubating them in NBT (2 mM NBT powder, 20 mM phosphate buffer). The seedlings were washed with water on the next day and subjected to removal of chlorophyll by dipping them in bleaching solution (ethanol, acetic acid, and glycerol in a ratio of 3:1:1). The plants were then visualized under a bright field light microscope (Leica), and pictures were taken for the comparison of ROS in transgenics and the WT Arabidopsis plants after heat stress treatment.
For rice, 1-month-old plants were taken, and a similar protocol was followed for the comparison of ROS in rice WT and overexpression transgenic lines. NBT staining was done after giving them heat stress.
For rice, 1-month-old plants were taken, and a similar protocol was followed for the comparison of ROS in rice WT and overexpression transgenic lines. NBT staining was done after giving them heat stress.