Abstract
Plants are susceptible to infection by a broad range of fungal pathogens. A range of proteins have been evaluated that can enhance tolerance to these pathogens by heterologous expression in transgenic carrot tissues. The protocols for carrot transformation with Arabidopsis NPR1 (Non-Expressor of Pathogenesis-Related Proteins 1) are described in this chapter, using the herbicide resistance gene bar, which encodes phosphinothricin acetyltransferase, as a selectable marker. In this protocol, petiole segments (0.5-1.0 cm long) from aseptically grown carrot seedlings are exposed to Agrobacterium tumefaciens strain LBA4404 for 10-30 min and cocultivated for 2-3 days. Herbicide selection is then imposed for 8-12 weeks on a series of different tissue culture media until embryogenic calli are produced. The transfer of the embryogenic calli to hormone-free medium results in embryo development which eventually gives rise to transgenic plantlets. Embryogenic calli can also be propagated in suspension cultures. This protocol has yielded transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern-positive independent events out of 100.
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