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Plant and Cell Physiology Advance Access published online on January 4, 2008
Plant and Cell Physiology, doi:10.1093/pcp/pcm181
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics

Yoichi Ogawa1,{dagger}, Tomoko Dansako1,{dagger}, Kentaro Yano1, Nozomu Sakurai1, Hideyuki Suzuki1, Koh Aoki1, Masaaki Noji2, Kazuki Saito1,3 and Daisuke Shibata1,*

1Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
2Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
3Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan

*Corresponding author: Dr. Daisuke Shibata, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan, Tel: +81-438-52-3947, Fax: +81-438-52-3948, E-mail: shibata{at}kazusa.or.jp


  Abstract

We established a large scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal condition for Agrobacterium-mediated transformation of 96 independent binary vector constructs was established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

Keywords: Agrobacterium-mediated transformation - Arabidopsis thaliana - functional genomics - Gateway cloning system - high-throughput - suspension-cultured cells

{dagger} These authors contributed equally to this work.

(Received October 24, 2007; Accepted December 26, 2007)
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