Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 13, 2007
Publication Date: May 1, 2008
Citation: Belknap, W.R., Rockhold, D.R., Mc Cue, K.F. 2008. An improved plant transformation vector based on pbinplus. Biotechniques. 44:739-750. Interpretive Summary: Binary transformation vectors represent a primary tool for introducing foreign genes (transgenes) into dicotyledonous crop species. While a variety of these vectors have been constructed, all so far released for general use contain proprietary DNA sequences that limit utility in applied research programs. We have constructed a binary vector that utilizes no proprietary sequences, and is designed for facile alteration of the selection procedure for generation of transgenic plants. pBINPLUS/ARS based on the pBINPUS vector that has been improved by the introduction of of non-proprietary promoter and terminator sequences for transcription of the NptII selectable marker (Ubiquitin-3) gene of Solanum tuberosum) and introduction of rare (8 bp) restriction enzyme sites flanking both the NptII coding sequence (PmeI) and the entire selectable marker gene (FseI). As both experimental and intellectual property limitation affect the choice of selectable marker, a variety of these markers have been employed in binary vectors. The introduction of separate rare restriction sites bordering the selectable marker coding sequence and the entire marker gene allows modification of this domain in completed binary constructs. For example, a binary construct containing multiple (stacked) transgenes to confer a complex phenotype could be easily modified for applications requiring alternative selectable markers.
Technical Abstract: Binary plant transformation vectors are widely employed for introduction of transgenes into plants via Agrobacterium tumefaciens-mediated transformation. We report the construction of a binary plant vector pBINPLUS/ARS based on the pBINPUS vector. Improvements introduced into pBINPLUS/ARS include the use of non-proprietary promoter and terminator sequences for transcription of the NptII selectable marker (Ubiquitin-3) gene of Solanum tuberosum) and introduction of rare (8 bp) restriction enzyme sites flanking both the NptII coding sequence (PmeI) and the entire selectable marker gene (FseI). This vector offers all of the advantages of its predecessor pBINPLUS and its helper plasmid pUCAP, that employ the proprietary Nopaline Synthase promoter and terminator, while allowing for facile modification of selectable marker sequences in complex binary vector constructs. pBINPLUS/ARS has been used to introduce transgenes into potato and in other crop species, and is available to all researchers in academic, government and industrial laboratories for “proof of principle” and commercial applications.