Optimization of Tissue Culture and Regeneration Conditions for Spinacia oleracea and its Agrobacterium mediated Transformation with OmpK Antigen for Developing a Vaccine Against Fish Vibriosis

Abstract

Vibriosis, caused by Vibrio anguillarum, also called Listonella anguillarum, is a deadly haemorrhagic septicaemic disease that affects fish, bivalves, and crustaceans in marine, fresh and brackish water. Vibriosis is one of the most prevalent and harmful infections in marine aquaculture, causing considerable economic losses. To eradicate vibriosis, an innovative, secure, and more stable vaccine must be created. Edible plants can be used as bio-factories for the expression of vaccine antigens. Outer membrane protein K (OmpK) of Vibrio anguillarum can be used as vaccine antigen against Vibriosis. The present research work aimed to optimize the tissue-culture conditions for Spinacia oleracea and efficient transformation protocol for stable transformation as well as transient transformation of Spinacia oleracea with OmpK antigen. Parameters like seed sterilization and seed germination media were optimized before the transformation. Sterilization with 0.2% mercuric chloride and germination on full MS media for spinach seeds provided good results. For tissue culture, nodal explants of Spinacia oleracea showed the highest regeneration efficiency on full MS supplemented with BAP 5mg/l and IAA 0.5mg/l. For Spinacia oleracea, different concentrations of antibiotic hygromycin were used with the optimal concentration being 10mg/l for nodal explants. Successful transformation with OmpK antigen was carried out using the above mentioned conditions. Transformed explants were regenerated on selection media supplemented with antibiotic hygromycin. Further, explants with 8 minutes infection time and 3 days co-cultivation time showed 60% regeneration efficiency. The leaves of the spinach plant were infiltrated with Agrobacterium tumefaciens, harboring plasmid containing OmpK gene Positive results were shown on all four days of collected leaves. The transformation was confirmed through PCR by using gene-specific primers. Transgene expression in spinach plants were analyzed by quantitative real-time PCR (qRT-PCR) in comparison to the β-actin gene as a control. Further, protein expression was confirmed through Dot-blot analysis, Western blotting, and ELISA. Taken together, the successful expression of the OmpK antigen of Vibrio anguillarum in edible plants may facilitate the development of an economical, cost-effective, and subunit edible plant-derived vaccine against vibriosis. Keywords: Spinacia oleracea, OmpK antigen, Vibriosis, Edible vaccine, Agrobacterium-mediated transformation, qRT-PCR, Western blotting, ELISA