Desenvolvimento de plantas de tomate resistentes contra vírus, obtidas de acordo com a nova regulamentação europeia

Ensuring food security for a growing global population in a changing climate presents a great challenge for agriculture and in this scenario, emerging diseases pose a major threat to the production of many crops, such as tomato, one of the most consumed food crops worldwide. In terms of economic value, tomato constitutes over 70% of the value of fresh vegetables produced worldwide and in Portugal, it is the most competitive agro-industrial product, with productions above the European production average, over 1,7 million tons (FAO, 2017). One of such emerging diseases causing devastating yield losses (up to 70%) is caused by Tomato brown rugose fruit virus (ToBRFV). At present, control of this virus rely on preventive sanitary measures and on the very few ToBRFV-resistant varieties that are slowly arising greatly dependent on the labour-intensive, cumbersome and time-consuming traditional breeding techniques. Conventional breeding can only tackle this task to some extent; and producers are continuously confronted with the losses caused by emerging diseases that keep arising. The continuous resurgence and spread of viruses such as ToBRFV makes essential rapid, timely and efficient modes of control. Recent advances in genome editing, such as genetic engineering using CRISPR/Cas, have opened many opportunities to accelerate plant breeding and to bridge the gap between conventional breeding and the knowledge acquired at molecular level to improve traits. In this way, the main objective of this work plan is to produce ToBRFV tomato resistant plants so that they can be available for producers as rapid as possible using the knowledge and tools that we currently have available. For that, we will produce ToBRFV tomato resistant plants, through gene editing, more specifically through the use of a CRISPR/Cas system to knockout tomato susceptible genes, genes that are essential to virus proliferation. Different tomato susceptible genes will be edited. Moreover, plants will be edited using ribonucleoproteins to deliver the CRISPR system, these are completely free of DNA, so that the risk of DNA integration into the genome can be excluded. The following hypotheses will be tested: 1) there will be significant differences on viral accumulation in plants edited using sgRNAs from different susceptibility genes; 2) there will be significant differences on viral accumulation in plants edited using sgRNAs from different combinations of susceptibility genes; 3) there will be significant differences on viral accumulation in plants edited using sgRNAs from a different number of susceptibility genes. Ensuring DNA free plants will allow to produce plants that are beyond the reach of genetically modified organism legislation. In this way, ToBRFV-resistant tomato lines established as in this proposal could soon be cultivated globally without any restrictions.