Multigenerational stability and environmental safety of transgenic rice expressing a dimeric HN protein as a plant bioreactor for poultry Newcastle disease vaccine antigen production

Newcastle disease (ND) remains among the most economically important viral diseases in poultry, and novel thermostable, low-cost and genotype VII-matched veterinary vaccine platforms are urgently needed to overcome the limitations of conventional vaccines, such as cold chain dependency, high application cost and insufficient cross-protection against prevailing genotype VII strains. Rice endosperm has emerged as an ideal plant bioreactor for veterinary vaccine antigen manufacturing owing to its low production cost, cold chain-independent antigen storage, high biomass yield and great potential for oral or feed-based poultry immunization. In this study, we systematically evaluated two independent transgenic rice lines (HN-1 and HN-2) expressing a dimeric hemagglutinin-neuraminidase (HN) antigen of Newcastle disease virus (NDV) across the T1–T3 generations under confined field conditions and aimed to screen elite plant bioreactor lines for large-scale production of an ND vaccine antigen. The exogenous HN gene exhibited stable genomic integration, with no sequence variations detected in its coding region, and maintained consistent transcriptional and protein expression levels over three successive generations. Agronomic assays demonstrated that the major agronomic and grain quality traits of the HN-1 line were highly comparable to those of wild-type TP309, whereas the grain chalkiness defects of HN-2 were clearly line specific. No detectable transgene flow to adjacent weeds was detected, and pollen viability, seed germination performance and nontarget arthropod diversity were not altered in the transgenic lines. Collectively, the HN-1 line possesses excellent genetic stability, agronomic adaptability and ecological safety and can serve as a promising elite plant bioreactor candidate for the industrial production of NDV HN vaccine antigens. This work provides genetic stability, agronomic performance, and preliminary biosafety data to support the further development and regulatory evaluation of rice-derived veterinary ND vaccines.