Agrobacterium rhizogenes-mediated transformation of β-glucuronidase reporter gene in hairy roots of Angelica gigas Nakai

Korean Angelica (Angelica gigas) produces pyranocoumarins (decursin and decursinol angelate), which have neuroprotective, anticancer, and anti-androgen receptor-signaling activities. An efficient protocol for the establishment of transgenic Angelica gigas root cultures using Agrobacterium rhizogenes is reported. To further characterise the putative transgenic roots, explant tissues were co-cultivated with A. rhizogenes strain R1601 carrying the pK2GW7-GUS binary vector. Except for the co-cultivation medium, all formulations included 50 mg L-1 kanamycin to select for transformants and 250 mg L-1 cefotaxime to eliminate the Agrobacterium. After 6 to 8 weeks co-cultivation with Agrobacterium rhizogenes, kanamycin-resistant roots appeared on 50% of explants maintained on hormone-free medium. Isolated hairy roots were transferred in liquid medium containing half-strength Schenk and Hildebrandt (SH) salt and 30 mg L-1 sugar. Detection of the neomycin phosphotransferase gene, high levels of beta-glucuronidase (GUS) transcripts, and GUS histochemical localisation confirmed the integrative transformation. In the future, these protocols should facilitate the extraction and study of valuable metabolites, such as decursin and decursinol angelate from A. gigas hairy root cultures.