Agrobacterium-mediated transient transformation of Flaveria bidentis leaves: a novel method to examine the evolution of C4 photosynthesis

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Abstract The genus Flaveria has been studied extensively as a model for the evolution of C4 photosynthesis. Thus far, molecular analyses in this genus have been limited due to a dearth of genomic information and the lack of a rapid and efficient transformation protocol. Since their development, Agrobacterium-mediated transient transformation protocols have been instrumental in understanding many biological processes in a range of plant species. However, this technique has not been applied to the genus Flaveria. Here, an efficient protocol for the Agrobacterium-mediated transient transformation of the leaves of the C4 species Flaveria bidentis is presented. This technique has the distinct advantages of rapid turnaround, the ability to co-transform with multiple constructs, and the capacity to assay coding and non-coding regions of Flaveria genomes in a homologous context. To illustrate the utility of this protocol, the quantitative transcriptional regulation of phosphoenolpyruvate carboxylase, the primary carboxylase of C4 plants, was investigated. A 24 bp region in the ppcA1 proximal promoter was found to elicit high levels of reporter gene expression. The Agrobacterium-mediated transient transformation of F. bidentis leaves will accelerate the understanding of the biology and evolution of C4 photosynthesis in the genus Flaveria as well as in other C4 lineages.

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