Quantitative Trypan Blue Leaf Staining

trypan blue stain was used to detect the dead cells as described by Wang et al. (2011) (link) and Bartsch et al. (2006) (link) with slight modifications. To prepare trypan blue (HiMedia) stock solution, 10 mg of trypan blue was dissolved in 10 mL of distilled water along with 10 mL phenol and 10 mL of 85% lactic acid. Then, 95% ethanol at a ratio of 1:1 was used to dilute the stock solution to yield a workable solution. In the working solution, the leaves were dipped and incubated for 1 h; after that, they were boiled for 1 min, cooled, and stored at RT overnight (Bartsch et al., 2006 (link); Liu et al., 2016 (link)). The chlorophyll content was removed by dipping the leaves in 95% ethanol and boiling them for 8 min (Rao et al., 2020 (link)). Then, the leaves were photographed and examined under a 4×/0.25 numerical aperture objective under bright-field microscopy (Leica microsystem, Germany) (Bartsch et al., 2006 (link)). The cell death per square millimeter was marked and quantified using Image J software (Schneider et al., 2012 (link)). The level of significance (*P\0.05, **P\0.01, ***P\0.001) was calculated by pairwise Student’s t-test using the PRISM GraphPad 9.0 software. The experiment was repeated thrice (Supplementary Table S9).