Generation of the First Cisgenic Fire Blight Resistant Apple Line of the Cultivar 'Gala'

The fire blight susceptible apple cultivar ‘Gala’ was genetically engineered to carry the fire blight resistance gene FB_MR5 originating from the wild apple genotype Malus x robusta 5 (Mr5). A previously established method involving A. tumefaciens mediated transformation was used to develop marker free genetically modified plants matching the definition of cisgeny. Following regeneration of transgenic lines a heat induced recombinase-mediated excision was used to remove all foreign helper genes. Thirteen transgenic lines were produced and out of these a single marker free line could be regenerated so far. The regenerated line is considered to be cisgenic as in the final product only the cisgene FB_MR5 controlled by its native regulatory sequences and no apple exogenous genes (transgenes) were found. Currently the molecular characterization of this line (determination of the insertion site, the copy number and transcription level of the resistance gene) is in progress. Inoculation experiments with a Mr5 avirulent strain of Erwinia amylovora, the causal agent of fire blight, were performed using both scissors and syringe shoot inoculation methods. Significantly less disease symptoms were detected on shoots of the cisgenic line compared to untransformed Gala. To our knowledge this is the first report of a cisgenic apple with increased resistance against fire blight.