Optimizing the AGROBEST transient transformation protocol for the expression of the KAT1 potassium channel in Arabidopsis thaliana

Transgenic plant research ­­­­­­­­investigates genetic underpinnings of basic plant biology and aims to increase agriculture productivity to meet the demand of feeding a growing world population. The genome sequencing... [ view full abstract ]

Transgenic plant research ­­­­­­­­investigates genetic underpinnings of basic plant biology and aims to increase agriculture productivity to meet the demand of feeding a growing world population. The genome sequencing initiatives of the late 20^th century revealed many uncharacterized genes in model plants. Cellular transformation of these model systems is a vital method of studying genes of interest in organisms that have been extensively characterized and allows researchers to examine the expression and regulation of a target protein in a highly controlled internal environment. Various transformation protocols have been published for a variety of plants, with the most popular method utilizing a phytopathogenic soil bacterium, Agrobacterium. Biotechnologists have harnessed this bacterium’s natural mechanisms of gene transfer by isolating plasmid DNA destined for integration into the plant cell genome and replacing specific genes with cloned genes of their choosing to view expression in various plants, including Arabidopsis thaliana and Nicotiana benthamiana. There is a demonstrated need for a more consistently successful transformation protocol resulting in high expression of transgenes in A. thaliana, as the sensitivity of this plant combined with the variable virulence of different Agrobacterium strains has proven to be a challenge in reaching high efficiency transformation. A recent publication of a transient transformation protocol in  Arabidopsis  using Agrobacterium tumifaciens, called AGROBEST has shown promising preliminary results. Systematic manipulation of specific steps of this protocol was attempted to optimize KAT1 potassium channel expression visualized by fusion to yellow fluorescent protein (YFP). Refining the incubation and infection conditions of Agrobacterium with Arabidopsis seedlings increased the amount of cellular transformation and target gene expression, evidenced by fluorescent microscopy visualization of the control YFP and the KAT1-YFP fusion protein. Transformation of the same fusion protein with two tandem copies of the 35S promoter from the Cauliflower Mosaic Virus did not increase expression in Arabidopsis. The study presented here helps to define an efficient transient transformation method in Arabidopsis that will expand the possible scope of investigation by allowing for transient expression of any selected candidate gene in the extensive mutant library available for Arabidopsis thaliana.