CRISPR-Cas9 Induced Knockout of BEL5 in Tetraploid Potato: Optimized Methodology via Repeated de novo Regeneration and Impact on Tuberization

CRISPR-Cas9 has emerged as a powerful tool for targeted genome editing in plants; however, its application in tetraploid potato (Solanum tuberosum ssp. tuberosum) remains challenging due to its vegetative propagation and complex highly heterozygous genome. Availability of whole-genome sequence data for the specific genotype is crucial to ensure complete knockout of all alleles of target genes while minimizing off-target mutations. In this study, using the tetraploid potato cultivar Desiree, we report, a complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, known as one of the key regulators driving tuber formation. We employed Agrobacterium-mediated transformation and demonstrated that repeated de novo regeneration could improve editing efficiency by promoting emergence of new mutations. BEL5 knockout plants exhibited a delayed onset of tuberization under inductive short-day conditions in hydroponics; however, their overall tuber yields were comparable to wild type plants. Based on our results, we propose a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato. Besides providing functional insight into the BEL5 role in potato, this study includes a methodological approach for efficient CRISPR-Cas9 gene editing in this vegetatively propagated polyploid crop, along with strategies for detecting mutations in genes that lack clear phenotypic manifestation.