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Isolation of protoplasts and vacuoles from cell suspension cultures ofColeus blumeiBenth.

✍ Scribed by Elisabeth Häusler; Maike Petersen; August Wilhelm Alfermann

Publisher
Springer
Year
1993
Tongue
English
Weight
326 KB
Volume
12
Category
Article
ISSN
0721-7714

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✦ Synopsis

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.

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