Enhanced production and secretion of rutin and GABA in immobilized cells of mulberry tree (Morus bombycis K.)

Cell immobilization has been proposed as a useful technique for mass production and efficient purification of secondary metabolites. In this study, we compared the bio-productivity of ligand-free and Ca-alginate-immobilized mulberry cells for rutin and gamma-amino butyric acid (GABA). In the leaves of Subong mulberry plants (M. bombycis K.) grown in a greenhouse, GABA accumulated as the leaves aged; a more than a 20-fold increase of GABA was observed in leaves undergoing senescence than in younger leaves. In contrast, more rutin was detected in mature leaves than in young leaves and those undergoing senescence. The production of total proteins in ligand-free leaf callus cells dramatically increased until 6 days after incubation in liquid suspension media (from 6.5 mg/g callus at day 0-14.5 mg/g callus), and by day 15 dropped to levels similar to those seen in the 0-day control. In contrast, immobilized cells showed a slight increase and then an insignificant decrease in protein content during the 15-day incubation period. Interestingly, immobilized mulberry cells more efficiently produced and secreted rutin and GABA into the suspension media than ligand-free cells. KN, a cytokinin, enhanced this production while 2,4-dichlorophenoxyacetic acid(2,4-D), an auxin, alleviated the effect of KN. As a result, incubation of the immobilized Subong cells in a full-strength Murashige and Skoog (MS) liquid medium containing 1 mg/l of 2,4-D and 0.1 mg/l KN, among the hormone combinations in the medium we tested, produced the highest amounts of rutin (8.2 mu g/g callus cells) and GABA (305 mu g/g callus cells) and secreted the largest amounts into the suspension media.