Stable Transformation of Suspension-Cultured Glycyrrhiza inflata Batalin Cells with Agrobacterium tumefaciens

: A protocol for the efficient genetic transformation of licorice (Glycyrrhiza inflata Batalin) cells in suspension culture using Agrobacterium tumefaciens-mediated T-DNA delivery is described. G. inflata cells in suspension culture were infected with A. tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1303, which contains the beta-glucuronidase (GUS) reporter gene and a hygromycin resistance gene (hpt II), respectively, under the transcriptional control of the CaMV35S promoter. Optimal transformation efficiency was achieved with an A. tumefaciens suspension having an OD600 of 0.4 and a period of 24 h of co-cultivation with 3-day-old cells in a medium supplemented with 200 microM acetosyringone. The transgenic cell lines have been maintained in suspension subculture for 5 months. PCR and Southern blot analyses confirmed the stable integration of transgenes into the G. inflata genome. The introduced genes had no discernable effect on cell growth or accumulation of total licorice flavonoids in the transgenic cell lines. This study provides the basis for the development of transgenic G. inflata cells.