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Plant Physiology 58:656-662 (1976)
© 1976 American Society of Plant Biologists

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Articles

Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L. 1

Roger A. Leigh2,3 and Daniel Branton

a The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138

Morphologically intact and osmotically active vacuoles were isolated from root storage tissue of the red beet Beta vulgaris L., and the factors influencing both yield and stability of the vacuoles were determined. Successful isolation depended upon slicing the tissue in an apparatus specifically designed to cut open plant cells without the use of high shear forces and to liberate cellular organelles into an undisturbed reservoir of osmoticum. The resulting brei was centrifuged at 2,000g for 10 min to yield a pellet which contained many vacuoles but which also contained tissue fragments, nuclei, mitochondria, and plastids. The vacuoles were further purified by accelerated flotation through a Metrizamide step gradient. Biochemical assays, light microscopy, and electron microscopy confirmed that there was only trace contamination of the final vacuole preparation by other organelles. Isolated vacuoles were intact and retained their in vivo coloration.

2 Present address: Botany School, Cambridge University, Downing Street, Cambridge CB 2 3EA United Kingdom.

3 Recipient of a Maria Moors Cabot Fellowship in Botanical Research.

1 This work was supported by funds from the Maria Moors Cabot Foundation for Botanical Research.




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Copyright © 1976 by the American Society of Plant Biologists