Measuring Chitin-Induced ROS Burst in Rice

The method for measuring the chitin-induced ROS burst levels is described in [54 (link)]. Briefly, leaf discs were cut from 7-day-old rice seedlings and floated on water overnight, and then the water was removed. After that, the leaf discs were incubated with 100 µL reaction solution (20 µM luminal, Sigma Aldrich, Saint Louis, MO, USA, Cat. No. 123072 and 2.5 µg/mL peroxidase, Solarbio Science & Technology Co., Ltd. Beijing, China, Cat. No. P8020) containing 400 nM chitin (Sigma-Aldrich, Saint Louis, MO, USA, Cat. No. H1396) to immediately detect the ROS burst. The luminescence was measured by a Berthold Mithras luminometer (Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany) every 1~2 min for 55 min. Eight replicates were performed for each sample.