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

Plant & Cell Physiology
Yoichi OgawaDaisuke Shibata

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.

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Citations

Jun 9, 2009·Plant & Cell Physiology·Takashi KuromoriMinami Matsui
Mar 23, 2012·Plant & Cell Physiology·Yoichi OgawaDaisuke Shibata
Jan 15, 2013·Journal of Experimental Botany·Wojciech StrzalkaKatsunori Tanaka
Jun 14, 2013·Scientific Reports·Takashi NakatsukaMasahiro Nishihara
Jan 14, 2009·The New Phytologist·Sakiko OkumotoWolf B Frommer
Oct 22, 2009·Natural Product Reports·Keiko Yonekura-Sakakibara, Kazuki Saito
Feb 26, 2010·The Plant Journal : for Cell and Molecular Biology·Satoshi KitamuraIssay Narumi
May 5, 2009·Journal of Plant Research·Motoaki Seki, Kazuo Shinozaki
Jan 1, 2017·Plant Biotechnology·Taichi KoshibaToshiaki Umezawa
Aug 9, 2019·Molecular Biology Reports·Natalya V PermyakovaElena V Deineko

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