Introduction:
Livestock plays a vital role in Pakistan’s agricultural sector, yet recurrent outbreaks of foot-and-mouth disease (FMD), particularly caused by FMD virus (FMDV) serotype O, result in significant economic losses. Existing inactivated and live attenuated vaccines have failed to provide sufficient protection against these outbreaks. This study aimed to generate a cost-effective, biosafe plant-based vaccine targeting FMDV serotype O antigens.

Methods:
A synthetic gene construct encoding the P1–2A polyprotein and 3C^pro protease of FMDV serotype O was engineered. To enhance immunogenicity, the cholera toxin B subunit (CTB) was genetically fused at the N-terminus of P1–2A via a flexible glycine–serine linker. The bar gene, under the CaMV 35S promoter, served as a selectable marker. The construct was introduced into young tobacco (Nicotiana tabacum) leaves (4–6 weeks old) via biolistic transformation, and transgenic lines were selected on medium containing 1.0 mg/L phosphinothricin (PPT). Wild-type (non-transformed) plants and vector-only transformants were maintained as negative controls.

Results:
Multiple putative transgenic plants were regenerated and confirmed by PCR using P1–2A, 3C^pro, and bar-specific primers. These lines were acclimatized and progressed to seed set. Segregation analysis and germination assays on medium with up to 3.0 mg/L PPT confirmed stable inheritance of the transgene in the T1 generation. Surviving seedlings consistently tested PCR-positive, validating successful transmission and stable integration of the vaccine construct. Controls (wild-type and vector-only) confirmed that the observed PCR amplifications were construct-specific.

Conclusion:
This work demonstrates the generation of genetically stable tobacco plants expressing key immunogenic proteins of FMDV serotype O. The results provide a foundation for subsequent immunological evaluation and highlight the potential of plant-based expression systems in developing cost-effective veterinary vaccines in Pakistan.