Restriction lift date: 2023-05-06
Agrobacterium mediated CRISPR Cas9 genetic engineering of Solanum tuberosum
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Full Text E-thesis
Date
2020
Authors
Barrett, Dylan
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Publisher
University College Cork
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Abstract
Global populations are continuing to rise and so too does the strain on the food production and agriculture industry. We are constantly in pursuit of new sustainable ways to feed the ever-increasing population. Genetic engineering of crop plants may prove to be a crucial tool in providing healthy sustainable food sources. The main aim of the project was to create an INDEL in The Rhamnose:beta-solanine/beta-chaconine rhamnosyltransferase (SGT3) gene region in Solanum tuberosum using Agrobacterium mediated CRISPR Cas9 genetic engineering. Sterol glycoside transferase (SGT) genes are involved in the biosynthetic pathway of glycoalkaloids in potatoes. This is a food safety issue which can result in reduced yields. In addition, the goal was to investigate the role of pre-transformation treatments on transformation efficiency. To that end, the effects of different wavelengths of light produced by light-emitting diodes on potatoes were examined in order to design a pre-transformation treatment. Red light and combinations of red and blue light were shown to have a significant positive impact on plant growth rate. Eight bacterial isolates from the soil rhizosphere were successfully isolated, identified and the volatile organic compounds produced by these bacterial isolates were measured in order to design a pre-transformation treatment. Six of the isolates were shown to produce known growth promoters through gas-chromatography mass-spectrometry and four of the isolates had a significant positive impact on plant growth rate in co-cultivation experiments. The role of heat shock treatment in transformation efficiency was also investigated. LED treatment with red-blue light (ratio of 3:1) and heat shock treatments were shown to increase Agrobacterium transformation efficiency in gus histochemical staining experiments. Finally, this project aimed to design guide RNA to target the SGT3 gene and transform Agrobacterium with the CRISPR vectors in order to successfully create a knockout in Solanum tuberosum. Three guide RNAs to target the SGT3 DNA were successfully designed and validated using an in vitro cleavage of SGT3 DNA by CRISPR/Cas9 ribonucleoprotein complex, however, it is unclear whether an in vivo knockout has been created due to the activity of the Agrobacterium-mediated CRISPR Cas9 transformation.
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Keywords
CRISPR , Agrobacterium , Potato , LED , Rhizobacteria , Transformation , SGT3 , Genetics , Bio-fortification , Plant genetics
Citation
Dylan, B. 2020. Agrobacterium mediated CRISPR Cas9 genetic engineering of Solanum tuberosum. MRes Thesis, University College Cork.