Genetic transformation via somatic embryogenesis to establish herbicide-resistant opium poppy

Abstract

A reliable genetic transformation protocol via somatic embryogenesis has been developed for the production of fertile, herbicide-resistant opium poppy plants. Transformation was mediated by Agrobacterium tumefaciens using the pCAMBIA3301 vector, which harbors the phosphinothricin acetyltransferase (pat) gene driven by a tandem repeat of the cauliflower mosaic virus (CaMV) 35S promoter and the beta-glucuronidase (gus) structural gene driven by a single copy of the CaMV 35S promoter between left- and right-border sequences. Co-cultivation of explants and A. tumefaciens was performed in the presence of 50 microM ATP and 50 microM MgCl(2). Root explants pre-cultured on callus induction medium were used for transformation. Herbicide-resistant, proliferating callus was obtained from explants on a medium containing both 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Globular embryogenic callus, induced by removal of the BA from the medium, was placed on a hormone-free medium to form somatic embryos, which were converted to plantlets under specific culture conditions. Plantlets with roots were transferred to soil, allowed to mature and set seed. Both pat and gus gene transcripts, and PAT and GUS enzyme activities were detected in the transgenic lines tested. Histochemical localization of GUS activity in T(1) opium poppy plants revealed transgene expression in most tissues of all plant organs. The protocol required 8-12 months to establish transgenic T(1) seed stocks and was developed using a commercial opium poppy cultivar that produces high levels of pharmaceutical alkaloids.