RAPID EXTRACTION AND ANALYSIS OF NONSTRUCTURAL CARBOHYDRATES IN PLANT-TISSUES

Considerable time is necessary to determine the nonstructural carbohydrates (i.e., glucose, fructose, sucrose, starch) in plant tissues and some methods used for this purpose lack specificity. Two steps in such assays typically occupy much of the assay time: sample drying and homogenization/centrifugation. A laboratory method was therefore developed to carry out such assays without either of these steps. The new method involved separation of the ethanol soluble carbohydrates from disks or slices of plant tissue with hot aqueous ethanol and the in situ conversion of starch to glucose by enzymes. The amount of ethanol-soluble sugars and the glucose released from the tissue during the starch digestion were then determined in a microplate assay using an enzyme-coupled colorimetric reaction which was highly spe cific for glucose. The two starch-degrading enzymes (alpha-amylase and amyloglucosidase) and short digestion times (1.5 h) utilized in this method were selected to quantitatively release glucose from the starch in these samples without digesting significant amounts of beta-glucans. Results obtained with this technique were found to compare favorably with those obtained by homogenization of cotton (Gossypium hirsutum L.) leaf tissue. Besides cotton, this method has been successfully used to analyze soluble carbohydrates from a variety of other plant species. By this method it is possible to analyze plant samples two to five times faster than methods which employ sample drying and homogenization.