| Abstract: | The CRISPR/Cas9 genome editing system has been tested, validated, and applied in plants since its introduction in 2012. For plant breeders, the CRISPR system⁰́₉s ability to provide targeted genetic modifications is an exceptionally valuable discovery as it provides an efficient and precise transformation method that can edit target genes down to a single base pair, improving upon older methods that have documented challenges with precision and accuracy. Despite these qualities, CRISPR has not been applied in many plant species outside model organisms and the more common staple crops. Cowpea (Vigna unguiculata) is a legume staple crop grown across Sub-Saharan Africa that has very limited genetic resources. Although it is considered an orphan crop, its superior traits, such as drought tolerance, nitrogen-fixing capabilities, and ability to grow in marginal soils, have brought it newfound consideration as an archetype of the climate resilient and sustainable crop currently of great interest in the agriculture industry. In this study, we have optimized multiple steps of the CRISPR/Cas9 genome editing pipeline in cowpea, including developing an improved protoplast isolation protocol for sgRNA validation and testing single-walled carbon nanotubes (CNTs) as a novel carrier for the CRISPR/Cas9 complex with phytoene desaturase (PDS) as the target gene. The cowpea protoplast isolation method developed in this document improves on previously available protocols by combining easy procedures, high yield, consistent results, and a reasonable experiment run time. Using this new protoplast protocol, we tested the efficacy of four single-guide RNA (sgRNA) sequences in vivo. The CNT study showed that CRISPR/Cas9 editing was achieved in CNT-infiltrated plants at an average efficiency of 6.7%, indicating possible plasmid DNA delivery; however, this result needs further validation. The electronic version of this dissertation is accessible from https://hdl.handle.net/1969.1/197086 |
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