Agrobacterium-mediated transformation of opium poppy. Papaver somniferum, via shoot organogenesis

An efficient Agrobacterium-mediated protocol has been developed for the sta ble genetic transformation of intact opium poppy, Papaver somniferum L., pl ants via shoot organogenesis. Excised cotyledons were cocultivated with the disarmed A. tumefaciens strain GV3101 carrying the pBI121 binary vector, a nd incubated on an optimized shoot induction medium consisting of B5 salts and vitamins, 30 g L-1 sucrose, 2 mg L-1 6-benzylaminopurine, 5 mgL(-1) AgN O3, and 3 g L-1 Gelrite. Except for the cocultivation medium, all formulati ons included 30 mg L-l paromomycin as the selective agent, and 200 mg L-1 t imentin to eliminate the Agrobacterium. Eight-week-old, paromomycin-resista nt shoots were transferred to an optimized root induction medium consisting of B5 salts and vitamins, 0.5 mg L-1 indole-3-acetic acid, 0.5 mg L-1 indo le-3-butyric acid, and either 5 mg L-1 AgNO3 or 40 mg L-1 putrescine. About 15 % of the regenerated shoots developed roots within eight weeks. Regener ated plants were transferred to soil, where they grew normally and set seed . Detection of the neomycin phosphotransferase gene, the high levels of bet a-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical l ocalization of GUS activity in all organs, confirmed the genetic transforma tion of the regenerated plants. The transformation process did not alter th e normal alkaloid content of opium poppy; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regula tion of benzylisoquinoline alkaloid biosynthesis.