Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics.
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.
Ogawa
Yoichi
Y
Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
Dansako
Tomoko
T
Yano
Kentaro
K
Sakurai
Nozomu
N
Suzuki
Hideyuki
H
Aoki
Koh
K
Noji
Masaaki
M
Saito
Kazuki
K
Shibata
Daisuke
D
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D018745:Genome, Plant; D023281:Genomics / Q000379:methods*; D030821:Plants, Genetically Modified; D012231:Rhizobium / Q000502:physiology*; D014170:Transformation, Genetic