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

Yoichi Ogawa | Zendy; Tomoko Dansako | Zendy; Kentaro Yano | Zendy; Nozomu Sakurai | Zendy; Hideyuki Suzuki | Zendy; Koh Aoki | Zendy; Masaaki Noji | Zendy; Kazuki Saito | Zendy; Daisuke Shibata | Zendy

Open Access

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

Author(s) -

Yoichi Ogawa,

Tomoko Dansako,

Kentaro Yano,

Nozomu Sakurai,

Hideyuki Suzuki,

Koh Aoki,

Masaaki Noji,

Kazuki Saito,

Daisuke Shibata

Publication year - 2008

Publication title -

plant and cell physiology

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 1.975

H-Index - 152

eISSN - 1471-9053

pISSN - 0032-0781

DOI - 10.1093/pcp/pcm181

Subject(s) - agrobacterium , transformation (genetics) , arabidopsis , arabidopsis thaliana , complementary dna , cloning (programming) , biology , functional genomics , transgene , computational biology , expression vector , genetics , genomics , genome , gene , computer science , recombinant dna , mutant , programming language

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.

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