Lettuce (Lactuca sativa) is a popular leafy vegetable with global production of ~28 million Mt, cultivated >
1 million hectares, with a market value of US4 billion in 2022. However, lettuce is highly susceptible to fungal pathogens that drastically reduce biomass and quality due to spoilage/rot. Therefore, in this study, we investigated the expression of chitinase genes via the lettuce chloroplast genome to enhance biomass and disease resistance. Site-specific integration of the expression cassette into chloroplast genomes was confirmed using two sets of PCR primers. Homoplasmy in transplastomic lines was confirmed in Southern blots by the absence of untransformed genomes. Maternal inheritance of transgenes was confirmed by the lack of segregation when seedlings were germinated in the selection medium. Chitinases expressed in chloroplasts are active in a broad range of pH (5-9) and temperatures (20-50 °C). Exochitinase expression significantly increased the number of leaves, root or shoot length and biomass throughout the growth cycle. Endochitinase expression reduced root/shoot biomass at early stages but recovered in older plants. Plant extracts expressing endochitinase/exochitinase showed activities as high as purified commercial enzymes. Antifungal activity in Candida albicans cultures inhibited growth up to 87%. A novel Carbotrace 680™ Optotracer binding to the ß-1,4 linkages of chitin, evaluated for the first time in plant systems, is highly sensitive to measure chitinase activity. To the best of our knowledge, this is the first report of chitinase expression via the chloroplast genomes of an edible plant, to confer desired agronomic traits or for biomedical applications.