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Licorice (Glycyrrhiza uralensis) is a medicinal plant that contains glycyrrhizin (GL), which has various pharmacological activities. Because licorice is a legume, it can establish a symbiotic relationship with nitrogen-fixing rhizobial bacteria. However, the effect of this symbiosis on GL production is unknown. Rhizobia were isolated from root nodules of Glycyrrhiza glabra, and a rhizobium that can form root nodules in G. uralensis was selected. Whole-genome analysis revealed a single circular chromosome of 6.7 Mbp. This rhizobium was classified as Mesorhizobium by phylogenetic analysis and was designated Mesorhizobium sp. J8. When G. uralensis plants grown from cuttings were inoculated with J8, root nodules formed. Shoot biomass and SPAD values of inoculated plants were significantly higher than those of uninoculated controls, and the GL content of the roots was 3.2 times that of controls. Because uninoculated plants from cuttings showed slight nodule formation, we grew plants from seeds in plant boxes filled with sterilized vermiculite, inoculated half of the seedlings with J8, and grew them with or without 100 µM KNO3. The SPAD values of inoculated plants were significantly higher than those of uninoculated plants. Furthermore, the expression level of the CYP88D6 gene, which is a marker of GL synthesis, was 2.5 times higher than in inoculated plants. These results indicate that rhizobial symbiosis promotes both biomass and GL production in G. uralensis.

Dried roots and stolons of licorice plants (Glycyrrhiza uralensis) are among the most important drugs (Glycyrrhizae radix) in traditional oriental medicine; they are also important commercial products used worldwide in sweetening and flavoring. Here, we describe the establishment of an in vitro stolon culture system for G. uralensis. Stolon formation was induced in single node stems (with axillary buds) grown in Murashige and Skoog (MS) liquid culture medium, supplemented with 0.01 μM α-naphthaleneacetic acid (NAA). Stolons were cultured at 26°C in the dark on a rotary shaker, gyrating at 100 rpm. The same NAA concentration produced the highest rates of stolon proliferation (6.58-fold in 4 weeks). A 6% sucrose concentration also enhanced stolon proliferation (6.34-fold in 4 weeks). GC/MS analysis confirmed the accumulation of small amounts of glycyrrhizin (14 μg g−1 dry weight) in cultured stolons. Interestingly, betulinic acid and oleanoic acid production in vitro were higher than in field-grown stolons. Adventitious root and shoot regeneration from cultured stolons were readily achieved under illuminated conditions in MS medium containing 0.01 μM of NAA and 0.2% gerlite. Regenerated plants produced glycyrrhizin (7,600 μg g−1 dry weight) in their roots. Our in vitro stolon culture system is suitable for studying glycyrrhizin biosynthesis and for rapid propagation of elite licorice clones.

Glycyrrhiza uralensis (Leguminosae, Fabaceae) is the most important medicinal plant used as a natural sweetener, in Chinese herbal medicines, and as a tobacco flavoring agent. Here, we report the generation of 56,857 expressed sequence tags (ESTs) derived from two full-length cDNA libraries produced from rhizomes (stolons) of two strains of G. uralensis, together with differential expression data obtained by MegasortTM analysis. The MegasortTM analysis was performed using two plant organs, namely, thickened roots and a rhizome-like organ, which differed in their glycyrrhizin content. After clustering of the 5′-end of ESTs, 5,542 contigs and 4,932 singletons were generated. A total of 10,474 nonredundant sequences were annotated on the basis of the TAIR database, of which 7,905 (75.5%) sequences exhibited homology to the sequences of registered genes. The gene functions of these 7,905 sequences were classified into Biological Process and Molecular Function on the basis of Gene Ontology (GO).