Genome editing for crop improvementchallenges and opportunities

Resumo

This doctoral thesis, entitled "Genome Editing for Crop Improvement", explores the potential of genome editing technology to improve crops, particularly those of high economic interest. Four scientific articles were published addressing different aspects of this topic. The first article reviewed genome editing tools, focusing on the advantages of CRISPR/Cas9. The second article emphasized the role of long ncRNAs in gene regulation in plants. After gathering all the information, the RNAi technique was used to analyze the GIP and NPP1 genes in Phytophthora cinnamomi, thus avoiding the risks of genome editing (the destruction associated with CRISPR). Ink disease is one of the most significant causes contributing to the decline of chestnut orchards. The reduction in Castanea sativa Mill yield can be attributed to two main species: Phytophthora cinnamomi and Phytophthora cambivora, the first being the main pathogen responsible for ink disease in Portugal. P. cinnamomi is a highly aggressive and widely distributed plant pathogen, capable of infecting nearly 1,000 host species. This oomycete causes substantial economic losses and is responsible for the decline of numerous plant species in Europe and worldwide. The remaining two articles addressed combating P. cinnamomi using RNAi to silence the NPP1 and GIP genes. Silencing these genes will either make it impossible or reduce the production of the proteins NPP1 and GIP, which are responsible for the onset of ink disease. To silence part of the coding region of the NPP1 and GIP genes, the integrative vector pTH210 was used. The integration of the cassette was confirmed by PCR and sequencing in hygromycin-resistant P. cinnamomi transformants. The transformants obtained with gene silencing were later used to infect Castanea sativa, allowing the effects of gene silencing on the plant's phenotype to be evaluated. The publications collectively demonstrate the potential of gene editing for crop improvement, emphasizing RNAi and CRISPR/Cas9, but also highlighting the importance of ncRNAs in gene regulation and offering strategies to combat pathogens. Combating this type of phytopathogen increases crop yields and, ultimately, benefits global food security and sustainability.