Optimization of conditions for Agrobacterium-mediated transformation of 189Brassica rapa with a bacterial isochorismate synthase gene

S. Sanimah, J.T. Sundakar, H. Linthorst and R. Verpoorte

Abstract

The aim of the study was to transform Brassica rapa plants with a bacterial isochorismate synthase (entC) gene in order to obtain transgenic plants with elevated levels of salicylic acid (SA) to increase pathogen resistance. Transgenic plants of B. rapa ssp. oleifera carrying the entC gene which encodes for Escherichia coli isochorismate synthase were developed via Agrobacterium tumefaciens-mediated transformation. This method involved the use of hypocotyl explants co-cultivated with disarmed strain LBA 4404 harbouring the binary vector pCAMBIA 1301 carrying the gene for entC, hygromycin phosphotranferase and sGFP in the T-DNA region and driven by CaMV (Cauliflower Mosaic Virus) 35S promoter. Prior to this, several important parameters including type of agrobacterial strains, concentration of acetosyringone, preconditioning days, pH and light effect during co-cultivation were evaluated for their influence on the transformation efficiency and subsequently optimized. The presence of the transgene in the genome of the greenhouse-grown transgenic plants was verified by PCR (Polymerase Chain Reaction) whereas the expression of the entC gene in mRNA (messenger ribonucleic acid) was revealed by RT-PCR (reverse transcription-PCR). The result indicated that the exogenous gene was successfully integrated into the genome and expressed in transgenic plants of B. rapa ssp. oleifera.

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