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

item Zhao, Duli
item Mackown, Charles
item Starks, Patrick
item Kindiger, Bryan

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/20/2009
Publication Date: 4/20/2010
Citation: Zhao, D., Mackown, C.T., Starks, P.J., Kindiger, B.K. 2010. Rapid analysis of nonstructural carbohydrate components in grass forage using microplate enzymatic assays. Crop Science. 50:1537-1545.

Interpretive Summary: Determination of nonstructural carbohydrate (NSC) composition and concentrations is often necessary for estimating the resources that are available for plant growth and for evaluating forage energy value to grazing animals. A commercially available chemical reagent kit that was previously used by research laboratories was removed from the market and replaced with a biochemical reagent kit that has not been evaluated for forage NSC. We adapted the new kit to a microplate assay format and demonstrated the suitability for cool-season forage NSC assays of glucose, fructose, sucrose, starch, and fructan. The adapted microplate methods produce precise, accurate, and reliable assays. It is especially useful for a large number of samples. Our research provided detail information about reagent and chemical solution preparation and the optimal reaction conditions for each of the NSC components. This microplate assay method should be suitable for other researchers wishing to measure NSC concentrations in fresh or dry tissues of a variety of plant based foods, biofuel feedstocks, and other forages grasses.

Technical Abstract: 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 light sensitive dye reaction was recently discontinued and replaced by a test-tube scale kit that assays glucose through enzymatic coupled reactions and the formation of reduced nicotinamide adenine dinucleotide (NADH). A laboratory microplate enzymatic method was developed for the new glucose kit and evaluated for rapidly assaying NSC components, including glucose, fructose, sucrose, fructans and starch, in 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 efficiencies, 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.