Trypan Blue Staining for Leaf Cell Death

Lactophenol trypan blue (TB) was used to detect the dying cells [48 (link)]. Leaves of Fol 1322 infected were taken for cell death staining. Then, 10 mL of 85% lactic acid, 10 mL of phenol, 10 mL of distilled water, and 10 mg of trypan blue were used for preparing a stock solution of trypan blue (HiMedia). Diluting the stock at 1:1 with 95% ethanol yielded a workable solution. The leaves were incubated in a working solution for 1 h, then boiled for 1 min, cooled, and stored at room temperature overnight [48 (link)]. To remove chlorophyll content, the leaves were dipped in 95% ethanol and boiled for 8 min [49 (link)] and infected lesions on the leaves were examined and photographed [48 (link)]. The cell death/mm² were marked and quantified using Image J software [42 (link)]. Each experiment was repeated three times.