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Plant and Cell Physiology Advance Access originally published online on January 4, 2008
Plant and Cell Physiology 2008 49(2):242-250; 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 email: journals.permissions@oxfordjournals.org

Efficient and High-Throughput Vector Construction and Agrobacterium-Mediated Transformation of Arabidopsis thaliana Suspension-Cultured Cells for Functional Genomics

Yoichi Ogawa1,4, Tomoko Dansako1,4, 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: E-mail, shibata{at}kazusa.or.jp; Fax, +81-438–52-3948.


  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 conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were 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

Abbreviations: CaMV, cauliflower mosaic virus; GST, glutathione S-transferase; GUS, β-glucuronidase; Hm, hygromycin; Km, kanamycin; MEPM, meropenem; NAA, 1-naphthalene-acetic acid; RAFL cDNA, RIKEN Arabidopsis full-length cDNA; SATase, serine O-acetyltransferase.

4These authors contributed equally to this work.

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