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

Abstract

Summary An efficient Agrobacterium -mediated protocol has been developed for the stable genetic transformation of intact opium poppy, Papaver somniferum L., plants via shoot organogenesis. Excised cotyledons were cocultivated with the disarmed A. tumefaciens strain GV3101 carrying the pBI121 binary vector, and 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 mg L −1 AgNO 3 , and 3 g L −1 Gelrite. Except for the cocultivation medium, all formulations included 30 mg L −1 paromomycin as the selective agent, and 200 mg L −1 timentin to eliminate the Agrobacterium . Eight-week-old, paromomycin-resistant 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 indole-3-butyric acid, and either 5 mg L −1 AgNO 3 or 40 mg L −1 putrescine. About 15 % of the regenerated shoots developed roots within eight weeks. Regenerated plants were transferred to soil, where they grew normally and set seed. Detection of the neomycin phosphotransferase gene, the high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all organs, confirmed the genetic transformation of the regenerated plants. The transformation process did not alter the normal alkaloid content of opium poppy; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis.