Transformation and characterization of transgenic plants of Solanum dulcamara L.--incidence of transgene silencing
2000
Curtis, I.S. | Power, J.B. | Hedden, P. | Phillips, A. | Lowe, K.C. | Ward, D.A. | Davey, M.R.
A transformation system is described for Solanum dulcamara using the supervirulent Agrobacterium tumefaciens strain 1065, carrying both the β-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 −1 of kanamycin sulphate. A 1:10 (v:v) dilution of an overnight culture of Agrobacterium 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.
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