Simplification of a haemolytic micromethod for toxic saponin quantification in alfalfa

Abstract

A simplification of a haemolytic micromethod is presented. In the orginal method, alfalfa (<i>Medicago media</i> Pers.) leaf sap is spotted on the plate covered with a blood-gelatine suspension. In the presented method, the mashed alfalfa pulp is used instead of sap. Due to saponin diffusion and the reaction with erythrocytes, a haemolytic ring appears, which has a width proportional to the concentration of toxic saponins. It is shown that the width of haemolytic ring does not depend on the sample weight ranging from 20 to 100 mg. This allows for the omission of laborious sap pressing and sample quantification. Individual alfalfa plants with different saponin contents were tested using leaf sap and leaf pulp for analyses. Good agreement was found with sap and leaf pulp methods. The correlation obtained by both methods was high, <i>r</i> = 0.87. The modified method requires only a small amount of plant material and makes the analyses of large numbers of individual plants per day possible. The method is especially recommended for breeding purposes.