An Assay for Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation

Cut the sheath from 10-day-old rice seedlings into 3-millimeter segments with a sharp razor blade or surgical blade for pretreatment one day before the ROS assay. Place 5 sheath segments in an individual well of a 96-well microtiter plate containing 100 microliters of double-distilled water for 10 to 12 hours.

Cut the leaf disks from four to six-week-old rice plants using a biopsy punch with a plunger. Always cut the leaf disks from the middle third of the second leaf of the main tiller to reduce data variation. Next place one leaf disk in an individual well of a 96-well microtiter plate containing 100 microliters of double-distilled water for 10 to 12 hours for pretreatment. Keep all the leaf disks floating and facing up in the wells of a microtiter plate for water pretreatment to avoid leaf-side associated variation.

Prepare elicitation solution by combining 9.4ml of 50 micromolar tris-HCl, 400 microliters of L-012 solution, 100 microliters of horseradish peroxidase, and 100 microliters of flg22. Add 200 microliters of elicitation solution to the wells.

Start the software, and click on the Experiments button to create a new protocol or use an existing protocol. Click Procedure in the pop-up to set up the plate and select the wells from the plate to be monitored. Click Start Kinetic, and set the runtime to 30 minutes or longer, depending on the experimental requirements. Select minimum interval to obtain the readings as frequently as possible, and for integration time, choose 1 second or longer, depending on the signal intensity.

Click on Validate followed by OK to confirm the settings. Then click on Detect a New Plate in the pop-up and wait for the software to prompt the load plate dialog box. Carefully remove the double-distilled water from the wells containing the pretreated tissues, avoiding any tissue damage or desiccation. Use a multichannel pipette to add 200 microliters of the elicitation solution to the wells containing the tissues. Place the plate to be tested on the carrier, and begin detection.