Transgene‐Free, Gene‐Edited Cavendish Bananas ( <i>Musa acuminata</i> , <scp>AAA</scp> )

Global consumer acceptance of gene-edited food crops is increasing with new breeding technologies that can modify the genome without foreign DNA integration. Here, we report an Agrobacterium-based system for transgene-free, gene editing of the banana cultivar, Cavendish. The protocol uses a three-tiered approach whereby cells containing T-DNA are enriched by positive antibiotic selection over a 48-h period, CRISPR/Cas9-mediated gene editing occurs over a short transient window and negative selection of cells containing T-DNA is achieved by the addition of 5-FC, which is converted to cytotoxic 5-FU by the co-expressed CODA enzyme. Two key enzymes in the carotenoid biosynthesis pathway were targeted as visual markers of editing: phytoene desaturase (pds) and lycopene β-cyclase (LCYb). Disruptive edits of the LCYb gene were easily identifiable at the calli stage of the regeneration process with cells appearing pink due to lycopene accumulation. Eight of 32 plants for pds and 34 of 125 plants for LCYb contained edits and were likely free of integrated vector DNA as determined by targeted genome sequencing and T-DNA-specific PCR, respectively. Plants containing disruptive tri-allelic edits in either gene displayed an albino phenotype. A subset of potentially transgene-free, edited plants was verified by whole genome sequencing. The transient editing protocol has an estimated 17.6% to 21.9% efficiency in generating plants containing disruptive tri-allelic modifications that are free of novel DNA. The protocol overcomes the regulatory limitations associated with the release of gene-edited, vegetatively propagated crops and provides an effective means of creating new disease-resistant and agronomically superior Cavendish cultivars.