Establishment of regenerative callus and cell suspension system of licorice (Glycyrrhiza glabra) for the production of the sweetener glycyrrhizinin vitro

Abstract

Regenerative callus and cell suspension of licorice,Glycyrrhiza glabra, is a prerequisite to the possible production of the non-nutritive sweetener “Glycyrrhizin” in cell suspension. The primary embryogenic callus (PEC), initiated from young leaflets of four-weeks-oldin vitro-grown plantlets regenerated by shoot culture, was developed on modified Gamborg’s B5 medium supplemented with 2,4-D, Kinetin and low level sucrose (B5DK), at concentrations of 1.0, 1.0 mg L−1 and 2% respectively. Embryogenic Cell suspensions (ECS) were developed in liquid B5DK medium under orbital shaking at 120 rpm with 6cm stroke for 4 weeks in the dark at 25 ± 2°C and subcultured at two weeks interval. Browning of the cell suspension media of L33, L58 and L112 limited the viability to a short time span of 19, 26 and 21 days, respectively. Adding 500 mg L-1 PVP eliminated browning, however, the cell suspension of L33, L58 and L112 were viable over significantly shorter time span of 9, 12 and 10 days, respectively. Subculturing at two weeks interval was found to improve viability time span to 28, 31 and 25 days and was followed in subsequent control of browning. Characteristics of the ECS including packed cell weight, % packed cell volume, dry cell weight (dcw), growth rate (u) and cell viability (cv) were assessed. Morphogenesis of ECS and regeneration of plantlets from PEC and Secondary embryogenic callus (SEC) was achieved on B5 medium lacking growth regulators. According to the TLC-tentative analysis using glycyrrhizin standard at Rf value 0.28, low level of glycyrrhizin was detected in the ethanol-extractable samples of L58, however, detected no traces of glycyrrhizin in cells of L33 and L112 and in the media of the three genotypes; confirming that glycyrrhizin can be produced in suspension cultures of licorice. This result and the occurrence of phenolic compounds in the cultures, especially in L58, reflect the ability of licorice cell suspension to produce secondary metabolites. In light of the growing knowledge of glycyrrhizin metabolism, the application of direct genetic manipulation to restore the productive capacity of cell cultures and production of glycyrrhizin in a large scale may prove valuable and viable possibility.