Schematic of the method used to generate mutant Venus flytrap plants. Leaves were cut from a clonal wild-type Venus flytrap line in tissue culture and callus (red) generated. The callus was removed from the plant and bombarded with gold particles coated in plasmid DNA. Following bombardment, a mosaic transgenic plant was regenerated and identified by the presence of fluorescent mCitrine (mCit) protein expression under the 35Spromoter (dark green). Further splitting of the mosaic plant at the rhizome was used to isolate a transgenic line with ubiquitous mCit expression (red box). From this, plantlets for genotyping were generated either by (1) splitting at the rhizome or (2) through leaf dissection, callus generation, and plant regeneration, which in principle generates new plants from a smaller pool of cells more likely to be genetically uniform. Plantlets showing mosaicism for one or more mutations were positively selected for further rounds of splitting and regeneration until plants exhibiting evidence of non-mosaic, deleterious mutations on bothFLYC1(orFLYC2) alleles were found. These were clonally propagated in culture before moving to soil.
