Genetic Engineering and Synthetic Biology approaches applied to the development of novel resistant or tolerant sweet orange (Citrus sinensis) varieties against Huanglongbing (HLB)

• Grant number: 22/10505-1
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: September 01, 2022
• End date: August 31, 2024
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Alessandra Alves de Souza
• Grantee: Guilherme Souza Prado
• Host Institution: Instituto Agronômico (IAC). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). Campinas , SP, Brazil
• Associated research grant: 20/07045-3 - Biotechnological and genomic strategies for quality, productivity and sustainable management of citrus, coffee and sugar cane in the State of São Paulo, AP.NPOP

Abstract

Brazil is the second largest citrus producer worldwide and leads the world ranking of sweet orange (Citrus sinensis (L.) Obseck) production. Due to their production method and ancestry, the genetic basis of citrus species is very narrow, which turns them susceptible to losses in productivity related to abiotic and biotic stresses. Among biotic factors, we can highlight bacterial diseases, such as citrus canker, citrus variegated chlorosis and Huanglongbing (HLB), from which HLB, caused by Candidatus Liberibacter spp. (CLs) bacteria, is considered the worst and most devastating disease in citrus growing all over the world, leading to huge losses for growers and for citrus industry. Currently, there are no comercial varieties of C. sinensis that are resistant to HLB, which makes imperative and urgent the development of plants with resistance phenotype. However, due to the reproductive cycle and multiplication mode of C. sinensis, classic techniques of genetic breeding become difficult or even unfeasible in a technical point of view, which suggests modern biotechnology techniques as suitable alternatives for the generation of these novel varieties. Among them, genetic transformation through transgenesis and genome editing through CRISPR technology are some possible ways to explore genes previously reported - but not yet used - for obtaining resistance and/or tolerance in C. sinensis. Thus, in this project I will use three modern biotechnology approaches with this aim: (1) genome editing through a plasmid CRISPR/Cas system; (2) genome editing through DNA-free RNP CRISPR/Cas system; (3) genetic transformation through transgenesis. Genome engineering approaches will be based on the knockout of SEOc gene and/or key genes of the pathogen in the context of cell proliferation and infectivity. In this way, embryogenic calli will be transformed by Agrobacterium tumefaciens harboring expression vectors for the plasmid CRISPR/Cas system, whereas RNP system will be used for protoplasts transfection by using lipofectamine associated to PEG. Finally, transgenesis approach will be based on genetic transformation of epicotyl through explants dip and cocultivation with A. tumefaciens. Hence, it is expected that regenerated plants derived from these three approaches give rise to unprecedented varieties and with some degree of contrast compared to the phenotype observed in plants which are susceptible to CLs. Therefore, it is expected that these varieties are resistant or at least tolerant against HLB, without threatening biosafety of the biotechnological products, allowing future approval to market levels, thus providing a robust solution to HLB problem and concerns in the context of worldwide citrus production. (AU)