A new micro assay experimental protocol for measuring alpha-amylase activity in plant tissues was developed. The alpha-amylase micro assay utilizes a 96-well microplate using only micro liter volumes of the various reagents. The alpha-amylase activity was determined by measuring the blue color developed by the reaction of starch with the iodine-potassium iodide reagent using a microplate reader. The effect of plant hormones, gibberellic acid (GA), and abscisic acid (ABA) on the induction of alpha-amylase activity in barley half-seeds (embryo-containing or embryo-less) was investigated using the micro assay method. The alpha-amylase activity in the embryo-containing half-seeds was much greater than in the embryo-less half-seeds of barley. GA caused the induction of alpha-amylase activity in embryo-less half seeds, whereas ABA inhibited the GA-induced induction of alpha-amylase activity. This new method will be used by undergraduate research students for their independent research projects in an upper-level Plant Physiology and Plant Development research-enriched course. The new micro assay method is more convenient, rapid, space-saving, and produces less chemical waste than the traditional alpha-amylase assays.
