Lignin staining

Lignified tissues were visualized using Weisner staining (Phloroglucinol-HCl, Sigma-Aldrich) (Clifford, 1974). This stain reacts with aldehyde groups in lignin, giving a characteristic deep reddish-purple coloration to the xylem in vascular bundles. In detail, fruits at the red stage were cut in slices and incubated in 1% phloroglucinol in a 70% (v/v) ethanol solution until they were totally cleared. Then the phloroglucinol solution was removed, and a few drops of 37% (v/v) HCl were added. The lignified tissues appeared with a pink-red coloration about 5 min later. Pictures were taken immediately since color faded in around 30 min.