Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum L., and California poppy, Eschscholzia californica Cham., root cultures

An efficient protocol for the establishment of transgenic opium poppy (Papa ver somniferum L.) and California poppy (Eschscholzia californica Cham,) ro ot cultures using Agrobacterium rhizogenes is reported. Five strains of A. rhizogenes were tested for their ability to produce hairy roots on wounded opium poppy seedlings and California poppy embryogenic calli. Three of the strains induced hairy root formation on both species, whereas two others ei ther caused the growth of tumorigenic calli or produced no response. To cha racterize the putative transgenic roots further, explant tissues were co-cu ltivated with the most effective A. rhizogenes strain (R1000) carrying the pB1121 binary vector. Except for the cc-cultivation medium, all formulation s included 50 mg I-1 paromomycin to select for transformants and 200 mg I-1 timentin to eliminate the Agrobacterium, Four weeks after infection, parom omycin-resistant roots appeared on 92-98% of explants maintained on hormone -free medium, Isolated hairy roots were propagated in liquid medium contain ing 1.0 mg I-1 indole-3-acetic acid to promote rapid growth. Detection of t he neomycin phosphotransferase gene, high levels of beta-glucuronidase (GUS ) transcripts and enzyme activity, and GUS histochemical localization confi rmed the integrative transformation of root cultures. Transgenic roots grew faster than wild-type roots, and California poppy roots grew more rapidly than those of opium poppy. With the exception of a less compact arrangement of epidermal cells and more root hairs, transformed roots of both species displayed anatomical features and benzylisoquinoline alkaloid profiles that were virtually identical to those of wild-type roots. Transgenic root cult ures of opium poppy and California poppy are a simple, reliable and well-de fined model system to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis, and to evaluate the genetic engi neering potential of these important medicinal plants.