DNA-free genome editing of Solanum tuberosum. A CRISPR/Cas9-mediated proteolistic approach for novel crop production

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Meehan, Dara Eoin
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University College Cork
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Climate change is an ever-growing concern for global food security and crop production. Further adding to these issues is population growth and legislative changes governing the availability of agrichemicals for pest control. Novel crop production methods, which include genetic modification, must be considered for addressing these global food security issues. This project focuses on using one such method, CRISPR/Cas9, to alter the genome of the potato crop, Solanum tuberosum. The importance of potato cannot be overstated, being the third-largest food crop globally, and historically tied to Ireland. The rapidly changing climate and demand for increased yields calls for improvement to the potato, which historically has proven difficult to enhance, due to its tetraploid nature. The project specifically targeted the SGT3 gene, (Rhamnose:beta-solanine/beta-chaconine rhamnosyltransferase), a key gene involved in the production of solanine. The solanine production pathway is crucial to glycoalkaloid synthesis. While low levels of glycoalkaloids contribute to the flavour of potato, higher levels are toxic to humans and it is estimated that 14-27% of the U.S. potato crop is rejected due to dangerous glycoalkaloid levels. By inducing a small, 20 base-pair, deletion in the SGT3 gene, it is anticipated that its function would be disrupted, thus causing glycoalkaloid production to be supressed at an early stage of the pathway. In order to conduct this transformation in a “DNA-free” manner, a modified version of the CRISPR-Cas9 system was used, in which synthetic Cas9 endonuclease and synthetic sgRNA molecules were combined to form a ribonucleoprotien (RNP) complex. This RNP was then delivered to the plant tissue via particle bombardment. Following this, some plants were used for DNA analysis while others were subjected to regeneration protocols. A deletion in the SGT3 region was not detected in vivo, but was successfully digested in vitro, and important steps have been taken to ensuring this method will be more successful in the future. In addition, studies were conducted to examine how two cultivars of potato, Golden Wonder and Maris Piper, grow when exposed to different wavelengths of light and longer photoperiods. The strongest growth response was observed when plants were grown under various combinations of blue and red wavelengths.
CRISPR , Genetics , Plant biology , Plant genetics , Biology
Meehan, D. E. 2020. DNA-free genome editing of Solanum tuberosum. A CRISPR/Cas9-mediated proteolistic approach for novel crop production. MRes Thesis, University College Cork.