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Annals of Botany 86: 63-71, 2000
© 2000 Annals of Botany Company

Transformation and Characterization of Transgenic Plants of Solanum dulcamara L.—Incidence of Transgene Silencing

I. S. Curtis+, J. B. Power, P. Hedden, A. Phillips, K. C. Lowe, D. A. Ward and M. R. Davey§

Plant Science Division, School of Biological Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK IACR–Long Ashton, Department of Agricultural Sciences, University of Bristol, Long Ashton, BS41 9AF, UK

Received: 6 September 1999 ; Returned for revision: 16 October 1999 . Accepted: 16 March 2000

A transformation system is described for Solanum dulcamara using the supervirulentAgrobacterium tumefaciens strain 1065, carrying both the ß-glucuronidase (gus) and neomycin phosphotransferase II (npt II) genes adjacent to the right and left T-DNA borders, respectively. Leaf explants were more efficient for the production of transformed plants compared to stem explants on medium containing 50 mg l-1of kanamycin sulphate. A 1:10 (v:v) dilution of an overnight culture ofAgrobacterium gave optimal transformation in terms of transgenic plant regeneration. From a total of 174 kanamycin-resistant plants selected by their antibiotic resistance, 16 failed to exhibit GUS activity. Southern analysis revealed that these GUS-negative transformants originated from three independently transformed cell lines. Restriction enzyme analyses showed that the GUS-negative plants had both the gus and npt II genes integrated into their genome (one plant had a single copy of each gene; the other two plants had multiple copies), with major rearrangement of the gus gene occurring in plants with several copies of the transgene. GUS-negative plants showed leaf malformations, delayed flowering and a reduction in flower, fruit and seed production compared to GUS-positive and non-transformed (control) plants. Although gene silencing of the gus gene occurred, albeit at a low frequency (9.2%), the transformation system described generates large numbers of phenotypically normal, stably transformed plants. Copyright 2000 Annals of Botany Company

Agrobacterium -mediated transformation, gene silencing, Solanum dulcamara L. (Bittersweet, Woody Nightshade), T-DNA truncation, transgene expression


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