2.7. Callose Staining and Quantification

Arabidopsis leaves of wild-type, mutant, Atpdbg4 OE, and atpdbg4 RNAi lines, as described by a previous study [23], were cut into 6 × 4 mm leaf sections, cultured on 1% agar dishes for 2 days, and then soaked in 85% ethanol overnight until bleaching. The leaves were transferred into a mixture of 0.1% aniline blue double-distilled water and 1 m glycine with pH 9.5 and a volume ratio of 2:3 for 5 h. The fluorescence signal of aniline blue was observed and photographed under ultraviolet conditions. Fiji (Fiji is just ImageJ, https://imagej.net/Fiji/Downloads, 27 August 2022) [30] was used to perform pixel signal intensity quantification. One-way ANOVA was used for significance analysis.