Plant Cell Rep (2006) 25: 1166–1173 DOI 10.1007/s00299-006-0172-z GENETIC TRANSFORMATION AND HYBRIDIZATION A. Richter · A. de Kathen · G. de Lorenzo · K. Briviba · R. Hain · G. Ramsay · H.-J. Jacobsen · H. Kiesecker Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera) Received: 23 February 2006 / Revised: 12 April 2006 / Accepted: 2 May 2006 / Published online: 27 June 2006 C  Springer-Verlag 2006 Abstract The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were trans- formed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes cod- ing for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S pro- moter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated. Keywords Agrobacterium . Expression stability . Pea . PGIP . Resveratrol Abbreviations FW: Fresh weight . PCR: Polymerase chain reaction . RT-PCR: Reverse transcriptase PCR Communicated by H. L ¨ orz A. Richter · H.-J. Jacobsen Department of Molecular Genetics, University of Hannover, Herrenh¨ auserstr 2, 30419 Hannover, Germany A. de Kathen de Kathen & Pickardt BioTechConsult GbR, Pestalozzi Strasse 35, 10627 Berlin, Germany G. de Lorenzo Dip. Biologia Vegetale Universit` a “La Sapienza”, Piazza A. Moro 5, 00185 Roma, Italy K. Briviba Institute for Nutritional Physiology, Federal Research Center for Nutrition, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany R. Hain Bayer CropScience GmBH, Global Biology Herbicides Biochemistry, 65926 Frankfurt-am-Main, Germany G. Ramsay Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK H. Kiesecker () German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany e-mail: hki@dsmz.de Tel.: +49-531-2616-148 Fax: +49-531-2616-418 Introduction Puonti-Kaerlas et al. (1990, 1992) first reported success- ful pea transformation and in the following years robust Agrobacterium-mediated gene transfer protocols were de- veloped (Schroeder et al. 1993; Bean et al. 1997; Polow- ick et al. 2000, Nadolska-Orczyk and Orczyk 2000; Grant et al. 1995, 2003). The average transformation efficiencies of about 2% clearly indicate that applied genetic engineer- ing for this important legume is practical in modern plant breeding. This work aimed to produce transgenic peas, car- rying antifungal genes encoding the stilbene synthase from grapevine and a PGIP from raspberry, to permit the stacking of genes for partial resistance by crossing. Phytopathogenic fungi penetrate the plant cell after the secretion of cell wall-degrading enzymes such as endo- and exo-polygalacturonases (PGs), which are considered to be important for pathogenesis (De Lorenzo et al. 2001). Inhibitors of these cell-wall degrading fungal enzymes are a part of the plant’s defense to limit fungal develop- ment and colonisation. Polygalacturonase-inhibiting pro-