Stable Transformation of Solanum lycopersicum L. With OmpK Antigen Using Hygromycin as Selective Agent

Abstract

Vibriosis, caused by Vibrio anguillarum, is a lethal bacterial infection that results in hemorrhagic septicemia and the death of various fish species in aquaculture as well as fish farms. It accounts for about 50% of the fish species' death and has been associated with significant economic losses in the aquaculture sector globally. Hence, there is a dire need to control vibriosis. Due to the drawbacks of using antibiotics and other drugs to treat vibriosis, vaccination is an effective method. OmpK from Vibrio anguillarum can be used as a potential vaccine candidate. Plants can be used as the expression system for the production of subunit vaccines as they have many advantages over other expression systems. The present research work aimed to optimize hygromycin concentration for the selection and to develop an efficient stable Agrobacterium-mediated transformation protocol for Solanum lycopersicum L. cv. Rio Grande, and to express OmpK antigen. The conditions for seed sterilization, germination, and tomato explants regeneration were tested. Promising results were obtained when seeds were germinated on half MS media after being sterilized with 0.1% mercuric chloride. Zeatin at a concentration of 2mg/L produced good regeneration efficiency. Solanum lycopersicum L. explants were treated with different concentrations of antibiotic hygromycin, and 25mg/L was found as the optimal concentration for the selection of transformed nodal explants. Successful transformation with OmpK antigen was carried out, in which 10 minutes of the infection time and 2 days of co-cultivation time showed the highest transformation efficiency of 60%. PCR was used to confirm the successful transformation using gene-specific primers. Transgene expression in transgenic tomato explants was analyzed by quantitative real-time PCR (qRT-PCR) in comparison to the β-actin gene. Further, protein expression was confirmed through Dot blot, Western blot, and ELISA. Taken together, the successful stable transformation of Solanum lycopersicum L. with OmpK may facilitate the development of a cost-effective subunit vaccine against vibriosis in fish species. Keywords: Solanum lycopersicum L., OmpK, Vibriosis, Aquaculture, Agrobacterium-mediated transformation, Hygromycin, qRT-PCR, Western blotting, ELISA