Rapid Analysis of Nonstructural Carbohydrate Components in Grass Forage Using Microplate Enzymatic Assays

Measurements of nonstructural carbohydrates (NSC) in plant tissues are important to estimate plant organ resources available for plant growth and stress tolerance or for feed value to grazing animals. A popular commercially available assay kit used to detect glucose with a lightsensitive dye reaction was recently discontinued and replaced by a test-tube-scale glucose kit (GAHK-20) that assays glucose through enzymatic coupled reactions and the formation of reduced nicotinamide adenine dinucleotide. The objective of this study was to develop a microplate assay method that uses the GAHK-20 to quantify forage NSC composition. A laboratory microplate enzymatic method was developed for the new glucose kit and evaluated for rapidly assaying NSC components, including glucose, fructose, sucrose, fructan, and starch in 11 species of cool-season perennial grasses. By standard addition, dilution, and temporal tests of enzyme reactions, we found that this microplate enzymatic assay is a rapid and reliable method to quantify NSC composition in grass forage samples. The microplate method allows analysis of many samples per day and considerably improved time and reagent use effi ciencies, especially for a large number of samples. In addition to forage, this method should be suitable for measuring NSC concentrations in fresh or dry tissues of a variety of other plant samples. D. Zhao, USDA-ARS, Sugarcane Field Station, 12990 U.S. Highway 441 N., Canal Point, FL 33438; C.T. MacKown, P.J. Starks, and B.K. Kindiger, USDA-ARS, Grazinglands Research Lab., 7207 W. Cheyenne St., El Reno, OK 73036. Received 20 Sept. 2009. *Corresponding author ([email protected]). Abbreviations: DI, deionized; EtOH, ethanol; EU, enzyme unit; G6P, glucose-6-phosphate; HEPES, 4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid; HK, hexokinase; HOAc, acetic acid; NADH, reduced nicotinamide adenine dinucleotide; NSC, nonstructural carbohydrates; PGI, phosphoglucose isomerase. Published in Crop Sci. 50:1537–1545 (2010). doi: 10.2135/cropsci2009.09.0521 Published online 20 Apr. 2010. © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.