Plant Science 180 (2011) 766–774 Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector Rajasekaran Sripriya, Manoharan Sangeetha, Chidambaram Parameswari, Balamani Veluthambi, Karuppannan Veluthambi ∗ Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Palkalai Nagar, Madurai 625 021, Tamil Nadu, India article info Article history: Received 6 November 2010 Received in revised form 17 February 2011 Accepted 22 February 2011 Available online 1 March 2011 Keywords: Co-transformation Marker elimination Osmotin Oryza sativa abstract A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19nptII was constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene (ap24) was cloned in pBin19nptII and the resultant plasmid pBin19nptII-ap24 was mobilized into the Agrobac- terium tumefaciens strain C58C1 Rif r harbouring the single-copy cointegrate vector pGV2260::pSSJ1. The T-DNA of the cointegrate vector harboured the hph (SMG) and gus genes. Transformation of Oryza sativa L. var. Pusa Basmati1 with Agrobacterium tumefaciens (pGV2260::pSSJ1, pBin19nptII-ap24) yielded 14 independent hyg + /GUS + transgenic plants. Southern blot analysis with hph and ap24 probes revealed that 12 out of the 14 transgenic plants were co-transformed and harboured hph, gus and ap24 genes. The new multi-copy binary vector yielded 86% co-transformation efficiency. SMG elimination by genetic separa- tion of the cointegrate T-DNA with the hph/gus genes and binary vector T-DNA with the ap24 gene was accomplished in four out of ten primary co-transformants that were forwarded to the T 1 generation. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Despite the high commercial adoption rate of genetically mod- ified crops, many concerns are raised about the biosafety of these crops [1]. The major concern is the persistence of the selectable marker genes encoding antibiotic and herbicide resistance. To alle- viate the risks of the selectable marker gene (SMG) in transgenic crops, many strategies have been designed for its elimination. These include the strategy of avoiding the usage of SMG [2,3], excision of the SMG by site-specific recombination [4,5], repositioning of the transgene or the marker gene by transposition [6,7], and co- transformation of two independent T-DNAs, one with the gene of interest (GOI) and the other with the SMG [8,9]. Co-transformation by Agrobacterium is a simple and clean tech- nique for SMG elimination and it does not leave behind residual DNA sequences such as recombination sites and invert repeats in the SMG-eliminated transgenic plants [10]. Efficient SMG elim- Abbreviations: ap24, osmotin gene with antifungal activity towards Phytoph- thora infestans; 2,4-D, 2,4-dichlorophenoxyacetic acid; GOI, gene of interest; gus, -glucuronidase gene; hph, hygromycin phosphotransferase gene; MS, Murashige and Skoog; nptII, neomycin phosphotransferaseII; SMG, selectable marker gene. ∗ Corresponding author. Tel.: +91 452 2458683; fax: +91 452 2459105. E-mail address: kveluthambi@rediffmail.com (K. Veluthambi). ination by co-transformation requires a high co-transformation efficiency and unlinked integration of an SMG and a GOI. Although particle bombardment yields a high frequency of co-transformation [11–13], it is of limited use to obtain SMG-free plants because it fre- quently results in linked integration of multiple copies of the SMG and GOI. Successful co-transformation has been reported using Agrobac- terium [8,14,15]. In order to achieve high co-transformation efficiency using Agrobacterium, ‘twin T-DNA’ binary vectors were constructed in which the same binary vector harboured an SMG and a GOI in two separate T-DNAs [9,16–18]. In a modified twin T-DNA strategy, one T-DNA carrying the nptII gene as the posi- tive SMG and codA as the negative conditional SMG was deployed. The second T-DNA carried the non-selected gus gene. SMG-free plants were obtained in the T 1 generation by negative selection on 5-flurocytosine-containing medium [19]. By applying a transient positive selection step followed by negative selection using codA, SMG-free potato was obtained at a frequency of 6.1% [20]. One disadvantage of the twin T-DNA approach is the high frequency of ‘linked co-delivery’ of T-DNA along with the adja- cent intervening non-T-DNA sequences [16,21]. High frequency of unlinked integration of the T-DNAs with the SMG and the GOI was achieved in the conventional co-transformation system in which the GOI and the SMG were placed on two separate plasmids in a single Agrobacterium strain [14,22–24]. By separating the SMG and 0168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2011.02.010