Improved Procedure for the Estimation of Nanogram Quantities of Indole-3-acetic Acid in Plant Extracts using the Indolo-α-pyrone Fluorescence Method

Abstract

The indolo-.alpha.-pyrone fluorescence method of determining IAA was improved by adding butylated hydroxytoluene (BHT), an antioxidant, to samples: addition of BHT increases the fluorescence intensities and decreases their variability so that amounts of IAA as small as 0.1-1 ng become measurable. Interfering compounds, 4-chloroindole-3-acetic acid and 5-hydroxyindole-3-acetic acid, can be separated from IAA by TLC using polyamide as the solid support, and benzene-ethyl acetate-acetic acid (70:25:5, vol/vol) as the developing solvent. Polyamide TLC is also superior in purifying IAA without significant loss or decomposition. A serious problem in extracting IAA from plant tissues is the considerable loss. Losses can result from decomposition of IAA when its solutions in organic solvents are evaporated to dryness, apparently due to some impurity in the solvents. Decomposition can be eliminated by simple treatments of the solvents, i.e., addition of BHT, washing with water, and passage through cotton wool. The latter is particularly effective in the case of ethyl acetate and ether. Loss of IAA by photodecomposition is also substantial even in dried samples. Ways to minimize the conversion of indol-3-pyruvic acid to IAA during extraction procedure are also described. A revised procedure of IAA extraction and determination is proposed. Applied to etiolated shoots of maize seedlings, it enables content to be estimated (18 ng/g fresh weight) with over 90% recovery in terms of [14C]IAA added at the beginning of the extraction.