A batch processing protocol for construction of expression vector plasmids from a cDNA collection and Agrobacterium-mediated transformation of suspension-cultured cells of Arabidopsis

Abstract

In order to accelerate functional analysis of large transcription factor families, development of a simple and easy protocol to generate transgenic cells of Arabidopsis overexpressing the transgenes using collections of cDNAs for coding sequences (CDSs) of the family genes is promising. We conceived a batch processing protocol for construction of plant expression vector plasmids and Agrobacterium-mediated transformation of suspension-cultured cells using a Gateway system-compatible cDNA collection and demonstrated its feasibility using a cDNA collection of the DOF family. The present results suggested that the batch processing from the LR reaction to the transformation of Agrobacterium cells was properly performed. The specific overexpression of a single DOF transgene was observed in at least 6 of 8 lines, and a transgenic callus obviously overexpressing multiple DOF transgenes was not obtained. These results show the potential utility of the present protocol to gain a clue for detailed functional analyses of Arabidopsis genes. © 2009 The Japanese Society for Plant Cell and Molecular Biology.