Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.Abbreviations EDTA ethylenediamine tetraacetic acid - HEPES 2-4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid - HPLC high performance liquid chromatography - MES morpholinoethane sulfonic acid - NADP⁺ ß-nicotinamide adenine dinucleotide phosphate - PEG polyethylene glycol - RA rosmarinic acid - Tris Tris(hydroxymethyl)aminomethane