Transformation and characterization of transgenic plants of Solanum dulcamara L. - Incidence of transgene silencing

A transformation system is described for Solanum dulcamara using the superv irulent Agrobacterium tumefaciens strain 1065, carrying both the beta-glucu ronidase (gus) and neomycin phosphotransferase II (nptII) genes adjacent to the right and left T-DNA borders, respectively. Leaf explants were more ef ficient 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) diluti on of an overnight culture of Agrobacterium gave optimal transformation in terms of transgenic plant regeneration. From a total of 174 kanamycin-resis tant plants selected by their antibiotic resistance, 16 failed to exhibit G US activity. Southern analysis revealed that these GUS-negative transforman ts originated from three independently transformed cell lines. Restriction enzyme analyses showed that the GUS-negative plants had both the gus and np tII 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. G US-negative plants showed leaf malformations, delayed flowering and a reduc tion in flower, fruit and seed production compared to GUS-positive and non- transformed (control) plants. Although gene silencing of the gus gene occur red, albeit at a low frequency (9.2 %), the transformation system described generates large numbers of phenotypically normal, stably transformed plant s. (C) 2000 Annals of Botany Company.