Alfalfa contains proteins and other metabolites such as alkaloids, phenolic compounds, and triterpenoid saponins. Alfalfa saponins cause bitterness and decreasing in palatability for animals. The second role is attributed to the formation of protectants against pests, pathogenes and nematodes. This factor is considered as an antibiotic against biostresses. So, it is expected that alfalfa necessarily is a resistant plant. However, specific pests and pathogens have been found to damage alfalfa and reduce yield. In order to study variability and also biological activity of saponins, the behavior of Therioaphis maculata was evaluated after aphids fed on the seedlings of 4 alfalfa cultivars. The neonates were transferred on seedlings. Number of suvivals, nymphs, and the size of aphids were recorded. Crude saponins were extracted using methanol 80% after cutting the tissues following freezing in liquid nitrogen and making powder in a mortar. Crude saponins were then purified with butanol following ether precipitation. TLC was used to analyze pure saponin mixtures. Different spots were expressed in variable colors but in same pattern for cultivars on TLC plate. The measurment of Rf showed no difference in saponin patterns. The usage of spotted alfalfa aphid was highly also useful for evaluation. Statistical analysis showed that survivals and nymphs were nearly the same. Although no difference of aphid sizes as well as survivals and production of nymphs in each growth stage was found between aphids feeding on
different cultivars but aphid size in wide was significant when aphids were fed on cultivar Euver. However, saponins had no significant role in resistance to spotted alfalfa aphid. Further research is necessary to fmd out the factor increasing wide of aphid body.