Trypan Blue Staining

Trypan blue staining was performed as described previously with minor modifications (Kim et al., 2009). Briefly, the third and fourth rosette leaves were soaked in trypan blue staining solution (10 g phenol, 10 ml glycerol, 10 ml lactic acid, 10 ml ddH₂O₂, and 0.02 g trypan blue) and stained in a boiling water bath for 3–5 min. Three leaves were completely submerged in trypan blue staining solution. After leaving overnight at room temperature, the leaves were carefully clamped into the decolorizing solution (2.5 g/ml chloral hydrate).