ETD

• Ascorbic Acid
• CRISPR/Cas9
• Genome Editing
• Lactuca sativa
• VIGE

The discovery of the CRISPR/Cas system has transformed plant biotechnology by enabling precise and efficient genome editing. Lactuca sativa L., one of the most widely consumed leafy vegetables, requires rapid genetic improvement to enhance nutritional value and tolerance to biotic and abiotic stresses, beyond what traditional breeding and transgenic approaches can efficiently provide.
In the first part of this thesis, Virus-Induced Genome Editing (VIGE) was tested for the first time in lettuce using Lettuce mosaic virus (LMV) and Tomato spotted wilt virus (TSWV) as CRISPR/Cas delivery vectors. The LMV-based system showed high infectivity and successful editing in Nicotiana benthamiana but failed to induce detectable edits in lettuce, likely due to vector or host-specific limitations. In contrast, the TSWV-based vector, despite lower infection efficiency, enabled targeted genome editing in L. sativa, demonstrating the feasibility of virus-mediated CRISPR delivery in this species.
The second part of the thesis focused on the functional characterisation of the L. sativa monodehydroascorbate reductase gene family using CRISPR/Cas9 knockout mutants. Single-gene knockouts did not significantly affect ascorbic acid levels, suggesting functional redundancy or compensatory mechanisms. Transcriptomic analyses revealed altered expression of key transcription factors and highlighted the involvement of MDHAR genes in complex regulatory networks.