Wheat Leaf DAB Staining Assay

In this study, ddH₂O, 1 μM elf18 (Sangon, Shanghai, China), 1 μM flg22 (Sangon, Shanghai, China), 100 μg/mL Chitosan (Sigma, Beijing, China), 100 μg/mL Chitin (Sigma, Beijing, China), and 100 μg/mL (GlcNAC)₆ (Qingdao BZ Oligo Biotech, Qingdao, China) were injected into wheat CN19 seedling leaves, and each treatment group had 3 biological replicates per sample. After 6 h of treatment, the treated wheat leaves were stained with 5 mM DAB dissolved in 10 mM 4-Morpholineethanesulfonic acid (MES) (pH 3.8) for 8 h in darkness. Then, the stained samples were decolorized in glycerol: acetic acid: ethanol (1:1:3, v/v/v) in a boiling water bath for 0.5–2 h and photographed, as described previously [68 (link)].