Molecular Breeding 3: 15–28, 1997. 15 c 1997 Kluwer Academic Publishers. Printed in Belgium. Expression of intron modified NPT II genes in monocotyledonous and dicotyledonous plant cells Christoph Maas 13 , Craig G. Simpson 2 , Peter Eckes 3 , Hedva Schickler 4 , John W.S. Brown 2 , Bernd Reiss 1 , Klaus Salchert 1 , Ilan Chet 4 , Jeff Schell 1 and Christoph Reichel 1 1 Max-Planck-Institut f¨ ur Z¨ uchtungsforschung, Abteilung Genetische Grundlagen der Pflanzenz¨ uchtung, Carl-von-Linn´ e-Weg 10, D-50829 Cologne, Germany; 2 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK; 3 Hoechst-Schering AgrEvo GmbH, Forschung Biochemie H 872 N, 65926 Frankfurt, Germany ( present address; author for correspondence); 4 Hebrew University Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel; Received 21 November 1995; accepted in revised form 23 July 1996 Key words: barley, intron, NPT II, reporter genes, selection, tobacco Abstract Intron sequences from monocotyledonous and dicotyledonous origin were used to abolish marker gene expression in prokaryotes (Escherichia coli and Agrobacterium tumefaciens) but permit expression in selected eukaryotic systems using the eukaryotic specific splicing mechanism. A 1014 bp maize Shrunken-1 (Sh 1) intron 1 flanked by exon1 and exon 2 sequences was cloned into the N-terminal of the NPT II-coding region. Transient gene expression analysis revealed that the modified neomycin phosphotransferase II (NPT II) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, is expressed in barley protoplasts, but poorly expressed in tobacco protoplasts. In dicotyledonous cells AU-rich sequences are known to be important for efficient splicing and therefore an attempt was made to improve expression of the NPT II gene, containing the Sh 1 intron 1, in tobacco by increasing the AU content from 57% to 69%. Reverse transcriptase PCR analysis of RNA from transiently expressed NPT II transcripts from tobacco protoplasts revealed that despite the increase in AU-content, NPT II was still poorly expressed. Cryptic splice sites were identified as one possible cause for missplicing of the Sh 1 intron 1 in dicots and poor levels of expression. Alternatively, cloning of the 198 bp intron 2 of the potato STLS 1 gene (81% AU) into the N-terminal part of the NPT II-coding region resulted in proper expression of NPT II in tobacco as well as in barley protoplasts and abolished marker gene expression in prokaryotes. The successful insertion of an intron into a selectable marker gene which completely abolishes gene expression in prokaryotes, without affecting expression of chimeric genes in monocotyledonous and dicotyledonous plant cells provides a suitable system to reduce the number of false-positives in transgenic plant production. Introduction The cauliflower mosaic virus (CaMV) 35S promoter system is widely used for the constitutive expression of a wide array of genes in plant cells. However, detailed analysis has revealed that the CaMV 35S promoter is not only active in higher eukaryotes, but also in prokaryotes such as Escherichia coli and Agrobac- terium tumefaciens [1, 38] and in lower eukaryotic organisms such as Saccharomyces cerevisiae [49, 50] or fungi such as Trichoderma harzianum, Fusarium solani or Cryphonectria parasitica [6, 17, 34]. There- fore, it is crucial for the production of transgenic plants to design plant vectors which prevent the detection of ‘false transformants’ resulting from promoter activity in endophytic prokaryotes or fungi which are quite common in plants. For example, Leifert et al. [22] isolated 198 endophytic bacterial strains from 9 plant species micropropagated in vitro under ‘aseptic’ condi- tions which were found to colonize leave tissues and/or the vascular system of plants [8, 13, 21]. Endophytes were also detected in mycorrhizal fungi such as Glomus