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Towards a high throughput system to produce and regenerate gene-edited events in Solanum lycopersicum

Abstract

Plant genetic transformation and gene expression studies enable engineering plants to meet the ever-increasing demands of a growing global population while reducing the environmental impact of agriculture. The development and application of more efficient methodologies, which are applicable to a diversity of species and cultivars in study are central to effective research strategies. Plant protoplasts provide a versatile platform for testing gene function and the efficacy of gene editing tools, such as CRISPR-Cas. Plant protoplast isolation and further plant regeneration were devised decades ago and more recently associated with new breeding technologies. However, plant protoplast protocols require optimization when working with different plant species and even cultivars. Optimizing the culture conditions for protoplast isolation and callus and plant regeneration, including the selection of appropriate growth factors, is crucial for success. The current proposal aims at the improvement of the regeneration of gene-edited plants from tomato protoplasts and to make it sufficiently efficient to allow high-throughput screening of gene-editing outcomes at the microcallus stage. This would constitute a big leap in the use of gene-editing for functional genomics and improvement of tomato, and applicable to gene expression studies in tobacco, the model plant used in the thematic project to which this proposal is associated. The groups involved in this proposal have had several research collaborations with positive results due to complementary expertise of the members, thus the possibility of interaction and intensification of collaboration will be fundamental for the improvement and use of more efficient methodologies. We also foresee that the involvement of researchers from both groups, doctorate students and staff will integrate knowledge and experimental expertise from both sides, resulting in academic training and joint publications, as well as the elaboration of a joint research proposal to be submitted to funding agencies. (AU)

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