TheDMACA-HCl Protocol and the Threshold Proanthocyanidin Content for Bloat Safety in Forage Legumes

A protocol using HCl-acidified 4-dimethylaminocinnamaldehyde (DMACA) was developed for screening proanthocyanidins (PA) in plants. Individual PA-containing cells could be observed following staining of decolourised leaves with DMACA. The quantitative assay using DMACA-HCl to detect soluble PA and butanol–HCl to detect insoluble PA could detect PA content lower than 0·4 mg g−1 dry matter (DM). The DMACA–HCl protocol is recommended for PA detection in low-PA plants because of its higher sensitivity than vanillin–acid protocols. Twenty-two forage legume species, most of which had been reported free of leaf PA but bloat-safe, were re-examined using the DMACA–HCl protocol. PA-positive cells were observed in leaves of 15 species. Measurable amounts of PA were detected in leaves of seven species, with the PA content ranging from 0·6 to 11 mg g−1 DM. White clover (Trifolium repensL), red clover (T pratenseL) and lucerne (Medicago sativaL) were also found to have PA-positive leaf trichomes. By analysis of these data and the literature, the threshold PA content for bloat safety in forage legumes was estimated to be 1–5 mg PA g−1 DM.