Quantitative determination of amylase activity in germinating cereal grains using agar plates and ImageJ software

The main energy source during cereal seed germination is soluble carbohydrates formed by the hydrolysis of stored starch. This process is carried out by a complex of hydrolytic enzymes, the most important of which are α- and β-amylases. Classical methods of amylase analysis are based on the extraction of enzymes from homogenised grains with a buffer and subsequent determination of their activity by incubating the enzyme extract with starch solution and determining one of the reaction products (reducing sugars) or residual starch by spectrophotometric methods. At the same time, there are also methods for detecting amylase activity by incubating grain halves on starch-containing agar plates, with cleavage fixed by staining the surface with solutions containing I2. However, these simple methods are only used for an approximate qualitative assessment of amylase activity. This work demonstrates the possibility of quantitatively determining the total amylase activity in germinating wheat and triticale grains by using ImageJ software to estimate the size of the non-iodine-stained halos formed on the plates because of the hydrolysis of starch by amylases. The analysis protocol and examples of the application of the method in the study of the effect on the amylase activity of wheat and triticale grains of priming with physiologically active substances that enhance the germination of grains (hydrogen sulphide donor NaHS and β-aminobutyric acid) are presented. The method is proposed to evaluate the effectiveness of physiologically active substances for priming seeds to increase their germination and for selecting seeds with potentially high germination energy. Keywords: amylase, analysis of activity, cereal seed germination, ImageJ, seed priming