Stable transformation of suspension-cultured Glycyrrhiza inflata Batalin cells with Agrobacterium tumefaciens

Abstract

A protocol for the effi cient genetic transformation of licorice (Glycyrrhiza infl ata Bata-lin) cells in suspension culture using Agrobacterium tumefaciens-mediated T-DNA delivery is described. G. infl ata cells in suspension culture were infected with A. tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1303, which contains the β-glucuronidase (GUS) reporter gene and a hygromycin resistance gene (hpt II), respectively, under the transcriptional control of the CaMV35S promoter. Optimal transformation effi ciency 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 μM acetosyringone. The transgenic cell lines have been maintained in suspension subculture for 5 months. PCR and Southern blot analyses confi rmed the stable integration of transgenes into the G. infl ata genome. The introduced genes had no discernable effect on cell growth or accumulation of total licorice fl avonoids in the transgenic cell lines. This study provides the basis for the development of transgenic G. infl ata cells. © 2012 Verlag der Zeitschrift für Naturforschung, Tübingen.