Journal Article

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

Yoichi Ogawa, Tomoko Dansako, Kentaro Yano, Nozomu Sakurai, Hideyuki Suzuki, Koh Aoki, Masaaki Noji, Kazuki Saito and Daisuke Shibata

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 49, issue 2, pages 242-250
Published in print February 2008 | ISSN: 0032-0781
Published online February 2008 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcm181
Efficient and High-Throughput Vector Construction and Agrobacterium-Mediated Transformation of Arabidopsis thaliana Suspension-Cultured Cells for Functional Genomics

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  • Biochemistry
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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

Journal Article.  6154 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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