PhD student Swedish University of Agricultural Sciences Alnarp, Skane Lan, Sweden
Body of Abstract: Tomato is a crucial crop for agriculture and has high commercial value. However, most studies on the application of CRISPR/Cas9 in cultivated tomato have relied on Agrobacterium tumefaciens-mediated transformation, which poses the risk of foreign DNA integration. An alternative approach is to use Ribonucleoprotein (RNP) based CRISPR/Cas9, which allows for the production of DNA-free plants that may be considered non-GMO. However, shoot regeneration remains a significant challenge for this technology, with only a few old reports demonstrating successful shoot regeneration from unedited protoplasts and one recent report showing success in wild tomato from edited protoplasts.
In this study, we developed a transgene-free breeding method for cultivated tomato using RNP-based CRISPR/Cas9. This involved optimizing protoplast isolation and overcoming the challenge of shoot regeneration from transfected protoplasts. Our results demonstrate successful mutant plant generation targeting the SELF PRUNING (SP) and SELF PRUNING 5G (SP5G) genes simultaneously, with a high mutation rate of up to 60% in at least one allele of either SP or SP5G genes. These findings provide valuable insights for future studies on CRISPR/Cas9-mediated genome editing in cultivated tomato and its potential application in sustainable agriculture.