New technologies for transfection of RNP complex and obtention of commercial varieties of Citrus sinensis with DNA-free genetic editing

• Grant number: 23/09068-9
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: March 01, 2024
• End date: February 28, 2025
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Alessandra Alves de Souza
• Grantee: Dhiôvanna Corrêia Rocha
• Supervisor: Yiping Qi
• Host Institution: Instituto Agronômico (IAC). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). Campinas , SP, Brazil
• Institution abroad: University Of Maryland, United States
• Associated to the scholarship: 21/03466-7 - Editing Citrus sinensis genes to attenuate the accumulation of callose associated with HLB Huanglongbing (HLB) symptoms, BP.DR

Abstract

HLB, a major challenge in citrus farming, necessitates the development of disease-resistant citrus varieties. DNA-free gene editing utilizing the CRISPR/Cas system has revolutionized the production of genetically modified (GM) plants by enabling precise edits without the introduction of foreign DNA. This approach allows for the creation of natural mutants, eliminating the requirement for GM plant regulation during commercial viability assessment. Consequently, DNA-free editing systems have become the preferred strategy for obtaining improved plants. In citrus, the prevailing method for developing DNA-free edited plants involves transfecting ribonucleoproteins (RNPs) into plant protoplasts, which are cells lacking cell walls. However, the isolation, purification, and regeneration of protoplasts pose significant challenges and time constraints. Thus, new techniques are sought to enable editing of explants that are more manageable and exhibit faster regeneration. Recently, microneedle arrays (MNAs) have been successfully adapted for RNP delivery to the soybean apical meristem. Considering that insects frequently target plant tissues, this study aims to employ microneedle arrays that mimic insect mouthparts for RNP delivery into citrus embryogenic callus. This approach offers improved manipulability and regeneration potential compared to protoplasts, thus enhancing the editing process. (AU)