Agrobacterium-mediated genetic transformation of California poppy, Eschscholzia californica Cham., via somatic embryogenesis.

Abstract

An efficient Agrobacterium-mediated protocol for the stable genetic transformation of Eschscholzia californica Cham. (California poppy) via somatic embryogenesis is reported. Excised cotyledons were co-cultivated with A. tumefaciens strain GV3101 carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l-1 paromomycin as the selective agent and 200 mg l-1 timentin to eliminate the Agrobacterium. Four to five weeks after infection, paromomycin-resistant calli grew on 80% of explants in the presence of 2.0 mg l-1 1-naphthaleneacetic acid (NAA) and 0.1 mg l-1 6-benzylaminopurine (BAP). Calli were cultured on somatic embryogenesis induction medium containing 1.0 mg l-1 NAA and 0.5 mg l-1 BAP, and somatic embryos were visible on 30% of the paromomycin-resistant calli within 3-4 weeks. Three to four weeks after the somatic embryos were transferred to phytohormone-free plant regeneration medium, 32% converted to paromomycin-resistant plants. Detection of the neomycin phosphotransferase gene and high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all plant tissues confirmed the integrative transformation of the regenerated plants. The normal alkaloid profile of California poppy was unaffected by the transformation process; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of the benzophenanthridine alkaloid pathway.