Evaluation of reagents and methods for genome editing in potato (Solanum tuberosum L.)

Genome editing using sequence-specific nucleases (SSNs) is rapidly becoming a standard tool for genetic engineering in crop species. The implementation of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) for inducing double-strand breaks enables targeting of virtually any sequence for genetic modification. Targeted mutagenesis via nonhomologous end-joining (NHEJ) and gene targeting via homologous recombination (HR) have been demonstrated in a number of plant species but reports have been limited in vegetatively propagated crops, such as potato (Solanum tuberosum Group Tuberosum L.).;The aim of this dissertation was to develop reagents and methods for genome editing in potato. This was accomplished by demonstrating TALEN and CRISPR/Cas reagents targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene were successful in inducing targeted mutations in reporter and endogenous gene targets. Targeted mutations using CRISPR/Cas were capable of both clonal and germline transmission, making CRISPR/Cas the preferred reagent for this application. TALEN and CRISPR/Cas reagents were also used in combination with a geminivirus expression vector for gene targeting experiments to incorporate point mutations within the ALS1 locus. Transformed events modified by both TALEN and CRISPR/Cas reagents in the geminivirus expression vector carried gene targeting modifications that supported reduced herbicide susceptibility phenotypes. Gene targeting modification detection and reduced herbicide susceptibility phenotypes were enhanced by regenerating lines under high selection. The evaluated reagents and methods in this dissertation provide a frame work for genome editing in potato and other vegetatively propagated crops and have important implications for basic research and agriculture.