Trypan Blue Staining

Cell death was visualized in rosette leaf tissue after staining with lactophenol-Trypan Blue (Keogh et al., 1980). Briefly, leaves were stained by incubation in 10 mL of ethanol-lactophenol solution (2:1 volumes of ethanol and phenol:glycerol:lactic acid:water [1:1:1:1]) containing 0.05% (w/v) Trypan Blue. After boiling for 10 min and cooling to room temperature for 30 min, the leaves were destained in 30 mL of chloral hydrate destaining solution (2.5 g mL⁻¹ water) for 2 d with shaking and replacing the destaining solution twice. Leaves were stored in 50% (v/v) glycerol, scanned at 600 dots per inch resolution (AtrixScan 3200XL; Microtek), and examined with a Zeiss microscope (Axio Observer A1; 100×). The cell viability was quantified by measurement of the staining intensity using ImageJ (version 1.51j8).