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Applied and Environmental Microbiology, April 2009, p. 1845-1851, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01856-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Construction of Disarmed Ti Plasmids Transferable between Escherichia coli and Agrobacterium Species{triangledown} ,{dagger}

Kazuya Kiyokawa, Shinji Yamamoto, Kei Sakuma, Katsuyuki Tanaka, Kazuki Moriguchi, and Katsunori Suzuki*

Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526, Japan

Received 11 August 2008/ Accepted 22 January 2009

Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1{lambda}pir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

* Corresponding author. Mailing address: Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi Hiroshima 739-8526, Japan. Phone: 81-82-424-7455. Fax: 81-82-424-0734. E-mail: ksuzuki{at}hiroshima-u.ac.jp

{triangledown} Published ahead of print on 30 January 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, April 2009, p. 1845-1851, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01856-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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