An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation

At 4 to 5 weeks post-germination, use a sharp 4-millimeter biopsy punch to remove one leaf disk per Arabidopsis plant, avoiding the mid-vein and being careful to limit wounding. Collect the disks into individual wells of an unused 96-well luminometer plate containing 100 microliters of double-distilled water per well at axial side up to prevent desiccation.

If assessing multiple elicitors, remove a second leaf disk from the same leaf for each elicitor treatment, and cover the plate with the lid to allow the leaf disks to recover overnight at room temperature. The next morning, set the microplate reader parameters to a 1,000-millisecond integration time in 2-minute intervals over a 40 to 60-minute period to capture the dynamic oxidative burst.

Next, use a multichannel pipette to replace the water in each well with 100 microliters of freshly prepared reaction solution, including a control no-elicitor reaction for each genotype to assess the basal reaction oxygen species levels in the absence of elicitation. Then, immediately, measure the light emission for all of the wavelengths in the visible spectrum on the microplate reader.