Journal Article

<i>Agrobacterium rhizogenes‐</i>mediated transformation of opium poppy, <i>Papaver somniferum</i> L., and California poppy, <i>Eschscholzia californica</i> Cham., root cultures

Sang‐Un Park and Peter J. Facchini

in Journal of Experimental Botany

Volume 51, issue 347, pages 1005-1016
Published in print June 2000 | ISSN: 0022-0957
Published online June 2000 | e-ISSN: 1460-2431 | DOI: https://dx.doi.org/10.1093/jexbot/51.347.1005
Agrobacterium rhizogenes‐mediated transformation of opium poppy, Papaver somniferum L., and California poppy, Eschscholzia californica Cham., root cultures

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An efficient protocol for the establishment of transgenic opium poppy (Papaver somniferum L.) and California poppy (Eschscholzia californica Cham.) root 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 either caused the growth of tumorigenic calli or produced no response. To characterize the putative transgenic roots further, explant tissues were co‐cultivated with the most effective A. rhizogenes strain (R1000) carrying the pBI121 binary vector. Except for the co‐cultivation medium, all formulations included 50 mg l−1 paromomycin to select for transformants and 200 mg l−1 timentin to eliminate the Agrobacterium. Four weeks after infection, paromomycin‐resistant roots appeared on 92–98% of explants maintained on hormone‐free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l−1 indole‐3‐acetic acid to promote rapid growth. Detection of the neomycin phosphotransferase gene, high levels of β‐glucuronidase (GUS) transcripts and enzyme activity, and GUS histochemical localization confirmed 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 cultures of opium poppy and California poppy are a simple, reliable and well‐defined model system to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis, and to evaluate the genetic engineering potential of these important medicinal plants.

Keywords: Agrobacterium rhizogenes; benzylisoquinoline alkaloids; California poppy; Eschscholzia californica; hairy root cultures; opium poppy; Papaver somniferum.

Journal Article.  7168 words.  Illustrated.

Subjects: Plant Sciences and Forestry

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