DEVELOPMENT OF A MESOPHYLL PROTOPLAST-BASED SYSTEM FOR GENE EDITING OF PAPAYA

Abstract

Papaya (Carica papaya L.) is an economically important food crop grown in tropical and subtropical regions. The global gross agricultural production value for papaya reached $6.2 billion in 2020. Papaya and its byproducts are used in commercial production, cosmetics, and pharmaceutical industries, offering many health benefits. While papaya is important economically, it faces threats from microbial pathogens. Genetic engineering and traditional breeding methods have been used to produce papaya with improved resistance to some pathogens. However, few innate disease-resistance genes are present in the papaya genome, and novel gene introgression from closely related species has reported F1 sterility with low viability and vigor. CRISPR-Cas9 gene editing technology has enabled gene editing in papaya using the Agrobacterium-mediated transformation method. When introduced to the market, papaya developed using this transgenic method will face increased regulatory constraints. A non-transgenic DNA-free alternative is available using CRISPR-Cas9 ribonucleoprotein complexes. A high-yield protoplast isolation method, producing highly viable, durable protoplasts, was developed during this study. Papaya protoplasts were transfected with fluorescent reporter constructs using our papaya-optimized PEG-mediated transfection method. Transfected cells were shown to express and shuttle proteins to expected locations and organelles viably. A system for DNA-free gene editing in papaya was developed using PEG-mediated transfection to deliver CRISPR-Cas9 ribonucleoprotein complexes into papaya protoplasts. The albino phenotypic marker gene phytoene desaturase and the susceptibility gene MLO homolog mildew resistance locus O-like protein 6 were targeted during this study.