Trifolium pratense : friable calli, cell culture protocol and isoflavones content in wild plants, in vitro and cell cultures analyzed by UPLC

Abstract

Trifolium pratense L., Fabaceae, is a rich source of isoflavones and has become the focus of several stud-ies related to its phytoestrogenic activity. The aim of this study was to establish germination and cellcultures protocol for T. pratense and quantify isoflavones content in cell cultures, in vitro cultured andwild plants harvested in two different seasons. Murashige Skoog medium supplemented with naph-thalene acetic acid and kinetin was able to produce the highest formation of friable calli. Calli cultureswere analyzed qualitatively after 60 days of culture, and in vitro plants after 30, 45 and 60 days of cul-tivation. The chemical analysis was performed by ultra performance liquid chromatography, using thelinearity curves of daidzein, genistein, formononetin and biochanin A as standards. The concentrationsof isoflavones detected in wild plants were different in the two harvest periods and contrasted in con-tent when compared to the in vitro plants. Cell cultures exhibited diverse profiles and concentrationof isoflavones, none of which presented the isoflavonoid biochanin A. Pectinase was used to promotereduction of clumps and ended up altering the characteristics of secondary metabolites production insome cultures. Formononetin showed higher concentration in wild red clover samples (15.407 mg g−1),and in the in vitro grown plants the highest concentration was daidzein (17.591 mg g−1) at 60 days. Themethods used for this research were effective, and the red clover plants of the analyzed variety can becultivated in vitro aiming the commercial productivity by having contents greater than or equal to thewild plants in the periods studied, even without the use of elicitors during the cultivation.