QUANTITATIVE EVALUATION OF BIOACTIVE COMPOUNDS IN ALFALFA (MEDICAGO L.) AND THEIR CALLUS CULTURES IN VITRO

Abstract

Alfalfa ( Medicago sativa L. (Fabaceae)) is an important crop which accumulates vitamins A, D, E, and K. In addition to the nutritional components (proteins and carbohydrates) which are important in the use of alfalfa and other plants as animal feed or food supplements, the plants produce a variety of secondary metabolites. Alfalfa contains numerous secondary metabolites, but they are not yet fully characterized. The best studied secondary metabolite groups in alfalfa are carotenoids and saponins. Another important group of secondary metabolites in alfalfa are phenolic compounds. Phenolic compounds in plants include flavonoids. Flavonoids are usually found in plants as glycosides, i. e. provided with sugar substituents such as galactose, rhamnose or glucose, or glycoside malonates, and they are found in an almost bewildering diversity of forms. Biotechnological research has revealed additional benefits for the production of pharmaceuticals from alfalfa. The aim of the present research was to identify and select alfalfa ( Medicago L.) and callus cultures in vitro varieties which accumulate the highest amount of bioactive compounds. The main objects: -             to prepare nutrient media and to germinate alfalfa in vitro ; -             to form alfalfa callus cultures in vitro ; -             to determine proteins, ascorbic acid, chlorophyll a and b , carotenoids in alfalfa seedlings and callus cultures in vitro ; -             to determine the content of flavonoids, total phenolic compounds  in alfalfa seedlings and callus cultures in vitro ; -             to determine the antioxidant activity of alfalfa seedlings and callus cultures in vitro . The highest content of chlorophyll a was found in alfalfa Zydrune plants grown in a Murashige and Skoog (MS) medium – 76.7 ± 0.06 mg/100 g. The highest amounts of bioactive compounds were noted in alfalfa grown in an MS medium in vitro : protein in the cultivar Antane (32 ± 0.07 %), ascorbic acid  in the cultivar Birutė (70.4 ± 0.01 mg/100 g), flavonoids in the cultivar Malvina (1.5 ± 0.01 mg/g); and phenolic compounds in  the cultivar Antane (0.07 ± 0.01 mg/100 mg). The highest antioxidant activity against the stable DPPH radical (22.3 ± 0.01 %) was found in the cultivar Zydrune , and in callus culture from roots (26.36 ± 0.03 %) in the cultivar Malvina plants grown in the MS with 2,4-dichlorophenoxyacetic acid (2,4-D) (2 mg/l) and kinetin (0.25 mg/l) medium. It was detemined that the highest content of bioactive compounds were accumulated in alfalfa in vitro in the cultivars Antane and Zydrune and in callus cultures from roots in Malvina . Also, afalfa seedlings in vitro contained more bioactive compounds in comparison with callus cultures. DOI: http://dx.doi.org/10.5755/j01.ct.65.1.8717