Transgenic rice lines show stable vaccine antigen production over three generations

Transgenic rice lines engineered to produce a vaccine antigen against Newcastle disease virus have held their genetic and biochemical stability across three successive generations under confined field conditions, according to a study published in the journal Animal Diseases. The work, carried out by researchers including Lei Zhang, Hongyan Chu and senior author Gaiping Zhang, evaluated two independent rice lines, designated HN-1 and HN-2, that express a dimeric form of the viral hemagglutinin-neuraminidase (HN) protein in their endosperm, the starchy tissue that makes up the bulk of the grain.

Newcastle disease is an acute, highly contagious viral illness of poultry with substantial economic consequences worldwide. The World Organization for Animal Health lists it as a notifiable disease, and China classifies it as a Category I animal disease, reflecting the threat it poses to poultry health and trade. The virus carries two key surface glycoproteins, one of which, the HN protein, plays a central role in receptor recognition and viral entry. Because genotype VII strains of the virus are now widespread, HN has become a preferred antigen target for vaccines designed to match the circulating viruses.

Existing commercial Newcastle disease vaccines include inactivated, live attenuated and viral vector formulations. The study’s authors note that, given the genetic diversity and ongoing evolution of the virus, these traditional vaccines often deliver cross-protection rates below 30 percent against genotype VII strains. They also require cold chain storage and repeated immunizations, which raise costs and complicate large-scale deployment, particularly in resource-limited settings. No commercial plant-derived subunit veterinary vaccine against Newcastle disease has yet been launched, which the researchers cite as part of the motivation for developing alternative low-cost, thermostable production platforms.

Rice endosperm has attracted attention as a bioreactor for veterinary vaccine antigens because of its low production cost, high biomass yield, well-established transformation system and the potential to store antigen at room temperature without a cold chain. Seed-derived antigens could in principle be mixed directly into poultry feed for oral immunization, reducing injection stress and labor on large farms. A previous proof-of-concept study by Ma and colleagues constructed a head-to-tail dimeric HN antigen and showed that a rice-expressed dimeric HN protein could provide full clinical protection against viral challenge in chickens. That earlier work, however, did not address multigenerational genetic stability, agronomic performance for large-scale planting, or field environmental biosafety, the three preconditions the new study set out to examine.

To assess genetic stability, the team performed whole-genome sequencing on plants from the T1, T2 and T3 generations of both lines. Sequencing reads mapped to the rice reference genome at rates between 96.22 and 99.78 percent, with reference coverage of 98.31 to 98.38 percent and average sequencing depths ranging from 25.5-fold to 39.3-fold. Insertion site analysis revealed a consistent interchromosomal translocation present in all three generations: a segment of chromosome 1 spanning positions 32,680,503 to 32,681,743 base pairs had been translocated to chromosome 3, with the upper boundary falling within the intron of a gene on chromosome 1 and the lower boundary in an intergenic region on chromosome 3. Deep variant analysis of on-target reads found no sequence variations in the coding region of the HN gene in any generation.

Expression analysis reinforced the stability picture. Polymerase chain reaction confirmed the presence of the HN gene in genomic DNA from the spikes and leaves of transgenic plants across all three generations, with no corresponding band in the wild-type TP309 control, the japonica cultivar Taipei 309 in which the lines were generated. Quantitative reverse-transcription PCR detected HN transcripts in leaf tissue of both lines in every generation, at levels roughly 8-fold to 400-fold above wild-type controls, though the authors attribute this wide range to individual plant variation and positional effects of the transgene insertion rather than any generation-dependent trend. Western blotting showed HN protein accumulation in mature seeds of the T1 through T3 plants, and a semiquantitative immunochromatographic strip test indicated that the relative antigen titer in seeds remained constant at 1:29 across all three generations. The authors emphasize that their multigenerational stability conclusion rests on seed proteins, not leaf transcripts, since leaf tissue is not the target tissue for the endosperm-specific promoter driving expression.

Agronomic evaluation of twelve core traits related to growth, yield and grain quality showed a clear divergence between the two lines. The HN-1 line was highly comparable to wild-type TP309: in the T2 generation none of the twelve traits differed significantly, and in T1 and T3 only panicle length and effective panicle weight, respectively, showed small but significant differences. The HN-2 line, by contrast, displayed several significant phenotypic differences across generations, including a lower seed setting rate in T1, reduced spike length and effective panicle weight in T2, and increased effective panicle weight and thousand-grain weight in T3. Grain morphology parameters such as length, width, thickness and brown rice percentage did not differ between either transgenic line and the wild type, but grain chalkiness told a different story. HN-1 showed no significant changes in chalkiness rate or degree across generations, whereas HN-2 showed significant and consistent increases in both measures. Scanning electron microscopy revealed that starch granules in wild-type and HN-1 seeds were regular, polyhedral and tightly packed, while the chalky endosperm of HN-2 seeds contained irregular, large, loosely arranged granules. The authors attribute this line-specific chalkiness phenotype to the T-DNA insertion position effect disrupting endosperm starch structure in HN-2.

Reproductive and developmental characteristics were largely unaffected. Seed germination rates and seedling emergence rates exceeded 90 percent in all lines and generations, with no significant differences among HN-1, HN-2 and wild type. The total growth cycle from sowing to maturity ranged from 150 to 180 days without line-specific variation. Pollen viability, assessed by iodine-potassium iodide staining at zero, three and six hours after anther collection, showed no significant differences between transgenic and wild-type plants in any generation, with the proportion of normally fertile pollen remaining stable over time.

The preliminary environmental safety assessment produced cautiously reassuring results, with important caveats. PCR analysis of leaves from weed species growing adjacent to the transgenic plots detected no amplification of the HN transgene, indicating no detectable transgene flow under the single-time-point, limited conditions of the study. Arthropod monitoring with yellow sticky traps placed during the milky stage of grain development, when insect pressure is typically highest, captured significantly more lepidopterans than other arthropod orders across all plots, but species richness and abundance did not differ significantly among wild-type, HN-1 and HN-2 plots. The authors stress that this survey was a preliminary observational assessment with limited sampling scope and short monitoring duration, conducted at a single site in a single growing season in small-scale plots, and that it cannot substitute for the multilocation, multiyear trials required for formal biosafety approval of transgenic veterinary bioproducts.

The study also acknowledges several further limitations. Soil microbiota and below-ground nontarget organisms were not evaluated, and low-level long-term gene flow cannot be completely excluded. Antigen expression stability under extreme field stress such as drought and high temperature remains untested, as does long-term antigen stability under high-temperature, high-humidity storage. The oral immunogenicity and protective efficacy of the HN antigen produced by the HN-1 line still require independent verification in chickens, and the economic advantage of rice-based vaccines over traditional production platforms awaits quantitative technoeconomic analysis. Within these constraints, the authors conclude that the HN-1 line, with its stable transgene inheritance, wild-type-like agronomic performance and grain quality, and no observed ecological risks under confined field conditions, qualifies as an elite plant bioreactor candidate for industrial production of Newcastle disease virus HN vaccine antigens, and that their findings provide genetic, agronomic and preliminary biosafety data to support further development and regulatory evaluation of rice-derived veterinary Newcastle disease vaccines.

Subject of Research: Multigenerational stability and environmental safety of transgenic rice expressing a Newcastle disease virus HN vaccine antigen

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

Article References: Zhang, L., Chu, H., Lou, Y., Hao, Z., Hua, Y., Wang, N., Li, F., Han, L., Wang, X., Wang, A., & Zhang, G. (2026). Multigenerational stability and environmental safety of transgenic rice expressing a dimeric HN protein as a plant bioreactor for poultry Newcastle disease vaccine antigen production. Animal Diseases, 6(1), Article 56. https://doi.org/10.1186/s44149-026-00266-2

Image Credits: AI Generated

DOI: 10.1186/s44149-026-00266-2

Keywords: transgenic rice, Newcastle disease virus, HN protein, plant bioreactor, veterinary vaccine, genetic stability, grain chalkiness, transgene flow, agronomic traits, poultry immunization, biosafety, endosperm