Genetic stability, environmental safety and field adaptation assessment of transgenic rice with chicken Newcastle disease HN protein

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In previous immunization experiments in chickens, immunization with two doses (0.5 µg, equivalent to 1/127 of a grain of rice) or a single dose (5 µg) provided complete protection. To advance the commercialization process of this product, in this study, we selected two transgenic rice strains (HN-1 and HN-2) and cultured them for three generations to evaluate their genetic stability, agronomic traits, and safety. Insertion site analysis showed that exogenous genes were stably integrated into nuclear chromosomes with no variants, as confirmed by PCR, qRT-PCR, and Western blotting. The transgenic strains exhibited germination rates, growth cycles, and 12 agronomic traits similar to those of the wild-type TP309, though HN-2 showed increased chalkiness. Pollen viability remained unchanged, and no transfer of the HN gene to weeds was detected. Field biodiversity analysis revealed no impact of the HN gene on pest and weed communities. These findings validate the transgenic rice’s genetic stability, agronomic adaptability, and environmental safety, providing critical data to support the acceleration of its commercialization as a plant-derived vaccine platform. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Plant sciences Intermediate tests Biosecurity field performance genetic stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Newcastle disease (ND), also known as Asian fowl plague or pseudo-fowl plague, is an acute, highly contagious viral disease of poultry caused by Newcastle disease virus (NDV) 1 . It is classified as a Category a animal disease by the World Organization for Animal Health (WOAH) and a Category I animal disease in China, reflecting its significant impact on poultry health and the economy worldwide. NDV possesses two major surface glycoproteins: the fusion (F) protein and the hemagglutinin-neuraminidase (HN) protein 2 – 4 . HN plays a dual role in receptor recognition and membrane fusion, and serves as a key immunogenic protein, making it an attractive target for vaccine development 5 – 7 . Currently, commercial ND vaccines mainly include inactivated vaccines, live attenuated vaccines and viral vector vaccines 8 – 14 . However, due to the genetic diversity and continuous evolution of NDV, especially the widespread prevalence of type VII strains, these traditional vaccines exhibit poor antigenic matching and the cross-protection rates are often less than 30% 15–19 . In addition, these vaccines require cold chain storage and transportation and involve multiple immunization procedures, which together increase costs and limit large-scale application 20 . Although ND vaccines have been used for decades, no subunit vaccines have been approved to date, This highlights the urgency of developing a new generation of vaccines that offfer higher safety, efficacy, affordability and genotype coverage 18 , 21 . Academician Zhang Gaiping's team recently published a research paper in the internationally renowned journal PNAS titled "A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice", which provides new insights into Newcastle disease vaccine development (The highest amount of the Osr2HN protein in transgenic rice seeds can reach 3.70 mg/g) 22, 23 . In the current experiments, this protein content reached a maximum of 1.2 mg/g. The study proposes a universal "head-to-tail" dimeric vaccine antigen model 24 . Using the hemagglutinin-neuraminidase (HN) protein, the receptor-binding protein of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, as an example, they successfully produced a highly effective recombinant antigen, Osr2HN, using a rice endosperm expression system. This study highlights the potential of rice-based vaccine production platforms. In recent years, plant bioreactors have made significant progress in several areas 25 – 27 . They are capable of producing a wide range of products; for instance, plant tissues can be processed for oral delivery of food proteins, thereby reducing downstream processing requirements 28 – 33 . Rice (Oryza sativa L.) is not only a staple crop for over half the world's population but also a well-established model organism for functional genomics research 34 . Its high productivity, broad adaptability, and established genetic engineering tools make it a strategic choice for molecular agriculture 35 . Compared to microbial or animal cell-based systems, rice endosperm bioreactors offer unique advantages, including genetic stability, scalability, low production costs, and the potential for oral vaccine delivery without cold chain reliance. Indeed, recent advances in transgenic rice technology have enabled high-level expression of various recombinant proteins, including vaccines, antibodies, and therapeutic peptides 35 – 38 . However, the commercialization of plant-derived vaccines faces significant challenges. The stability of transgenic strains must be verified across multiple generations, and comprehensive assessments of genetic stability, agronomic performance, and biosafety are essential. In particular, issues such as transgenic stability, environmental biosafety, gene flow, and ecological impacts must be addressed to meet regulatory standards and gain public acceptance. Building on previous success in expressing highly active NDV HN antigens in rice endosperm, this study aimed to systematically evaluate transgenic rice carrying HN vaccine antigens. Specifically, we focused on three key aspects: (i) multigenerational genetic stability, (ii) agronomic adaptability and phenotypic performance, and (iii) biosafety assessment, including survival competitiveness, gene spread potential, and impacts on ecological diversity. This study provides critical data to support the development and commercialization of rice-based NDV subunit vaccines and facilitates the establishment of an intermediate testing system for plant-derived vaccines. Materials and methods Materials In this study, two transgenic rice strains, HN-1 and HN-2, were successfully constructed using a rice endosperm reactor, and Taipei 309 (TP309) was used as a control variety. These strains were independently developed by our laboratory, and detailed information is available in our previous work 22 . EACKER straightedge (product no. 713319 3m) and electronic vernier calipers (model no. DL91150) were used as the measuring tools. Insertion site analysis Young leaves of rice seedlings from the T1, T2, and T3 generations were collected, sprayed with sterile water, blotted dried with absorbent paper, and placed into 50 mL Eppendorf tubes. The leaves were immediately frozen in liquid nitrogen for 10 minutes and then transported on dry ice to Wuhan Benagen Technology Co., Ltd. for insertion site analysis. DNA extraction from rice leaves and spikes Panicle tissues were randomly collected from transgenic rice strains (generations T1–T3) and TP309 rice plants at the anthesis stage. The collected panicle tissues and weeds were frozen with dry ice and then stored at -80 ℃ for subsequent DNA extraction. Weeds around the experimental fields were collected and preserved using the same method. For each generation, 6 samples were collected respectively for panicles of different transgenic rice varieties and for weeds from different areas around the corresponding paddy fields. DNA extraction was performed using the FastPure® Plant DNA Isolation Mini Kit (DC104-01, Vazyme), following the manufacturer’s instructions. The extracted DNA was stored at -20 ℃. DNA from surrounding weeds was extracted using the same procedure. PCR analysis was used to detect the inheritance of the target gene. Primers sequences specific to the HN gene are listed in Table 1 – 1 and the PCR reaction system is shown in Table 1 –2. The optimal reaction conditions for PCR were as follows: (1) pre-denaturation at 95 ℃ for 5 min; (2) 35 cycles of amplification at 90 ℃ for 30 s, 60 ℃ for 20 s, and 72 ℃for 30 s; (3) final extension at 72 ℃ for 5 min; (4) storage at 16 ℃. PCR products were analyzed by agarose gel electrophoresis. At least three replicate experiments were performed for each sample. DNA extraction was performed using the FastPure® Plant DNA Isolation Mini Kit (DC104-01, Vazyme), following the manufacturer’s instructions. The extracted DNA was stored at -20 ℃. DNA from surrounding weeds was extracted using the same procedure. PCR analysis was conducted to detect the inheritance of the target gene. Primer sequences specific to the HN gene are listed in Table 1 – 1 , and the PCR reaction system is shown in Table 1 –2. The optimal PCR reaction conditions were as follows: (1) pre-denaturation at 95 ℃ for 5 min; (2) 35 amplification cycles of 90 ℃ for 30 s, 60 ℃ for 20 s, and 72 ℃ for 30 s; (3) final extension at 72 ℃ for 5 min; (4) hold at 16 ℃. PCR products were analyzed via agarose gel electrophoresis. Acquisition and qPCR detection of cDNA in rice leaves Transgenic plants of the T1-T3 generation and TP309 plants were selected at the panicle flowering stage. Rice leaves were transported from Xinjiang to Henan on dry ice and stored at -80 ℃. Total RNA was extracted using the FastPure® Universal Plant Total RNA Isolation Kit (Cat: RC411-01, Vazyme). cDNA was synthesized via reverse transcription using the HiScript® II Q RT SuperMix for qPCR (+ gDNA wiper) (Cat: R223-01, Vazyme), following the manufacturer’s instructions. The cDNA extracted from leaves of T1-T3 generation transgenic rice and TP309 rice served as the template and was amplified by qPCR using primer pairs specific to the target genes and HygR genes. The primers used are listed in Table 1 – 1 , and the qPCR reaction system is shown in Table 1 –3. The relative expression levels of the exogenou gene and HygR gene in leaves were determined using the 2 −ΔΔCt method. At least three replicate experiments were performed for each sample. Western blot To determine the expression of the HN protein in mature rice seeds at the protein level, T1-T3 generation transgenic seeds and TP309 seeds, which were harvested simultaneously and stored under identical conditions, were used. Seeds from different strains were ground into a powder and then mixed with extraction buffer (25 mM PB, 20 mM Nacl pH 5.7) at a ratio of 1:5 (w/v, g/mL). The mixture was stirred for 1.5 h, followed by centrifugation at 9,000 rpm for 30 min at 4 ℃. The resulting supernatant was collected for Western blot analysis. Ar 10-180kDa Prestained Protein Marker were purchased from Henan Xianyan Biotech Co., Ltd. (Cat. No. ArP01201) and 180 kDa Prestained Protein Marker were purchased from Vazyme (MP102-01)..The primary antibody was chicken polyantibody were maintained in the laboratory. and the secondary antibody was HRP, Goat Anti-Chicken IgG purchased from Abbkine (Cat. No. A21080). Test strips for detecting antigen content in rice Antigenic titers were determined in the HN-1 and HN-2 transgenic rice strains across three consecutive generations. Immunochromatographic test strips developed in the laboratory were used to detect the HN antigen in harvested rice seeds. Rice extracts were prepared by mixing the ground seeds with extraction buffer (25 mM PB, 20 mM Nacl pH 5.7) at a mass-to-volume ratio of 1:5 (w/v), followed by stirring at room temperature for 2 h. The mixture was then centrifuged at 10,000 rpm and 4 ℃ to collect the supernatant, which was referred to as the 2^0 dilution. The extracts were subsequently serially diluted and used in antigen detection assays. Evaluation of germination and seedling emergence rates The materials used to evaluate germination and seedling emergence included T1-T3 generation transgenic strains and TP309 seeds, which were harvested simultaneously and stored under identical conditions. For each strain, 100 seeds were placed in 50 mL conical flasks and soaked in water at 25 ℃ in the dark for 36 h. The water was replaced every 12 h to maintain clean strains. After soaking, the seeds were transferred to 9-cm glass Petri dishes lined with moistened sterile filter paper and incubated at 37 ℃ until radicle emergence. Once more than 80% of the seeds exhibited radicle protrusion through the seed coat, they were transferred to a 25 ℃ incubator and moistened regularly to promote germination. Germination was assessed every 12 h and recorded when radicles and shoots were visible. When the shoot reached half the length of the grain, the seeds were transplanted into moist nutrient soil in the culture room at 28 ℃ with a 14 h light/10 h dark photoperiod. Seedling emergence data were collected and recorded. Throughout the experiment, the seeds were kept consistently moist. Germination data and seedling emergence data were both recorded consecutively three times, and the experimental results were expressed as percentages. Developmental cycle assessment The developmental cycle assessment was executed utilizing T1-T3 generation transgenic rice transformants and TP309 control seeds, which were concurrently harvested and stored under uniform conditions. Seeds were subjected to sterilization and germination in growth medium. Post-germination, seedlings were transferred to growth chamber and subsequently transplanted into soil. Following 20 days, seedlings were replanting to outdoor environment and maintained under adequate irrigation conditions. The developmental cycle was monitored, with both vegetative and reproductive growth stages; the spikelet initiation stage delineates the transition between these two developmental phases. Evaluation of comprehensive agronomic traits A comprehensive evaluation of agronomic traits related to growth patterns was conducted on transgenic rice strains of the T1-T3 generations at the full maturity stage—which is defined as the period when over 90% of the glumes turn yellow and the basal seeds harden and become resistant to breakage. At this stage, rice spikes from each strain were collected into the corresponding numbered seed bags. After sun-drying for 3 days under outdoor conditions, agronomic traits related to spikes and grains were measured. The recorded agronomic traits related to growth pattern and their specific definitions were as follows: plant height, defined as the distance from the base of the plant to the tip of the second tallest leaf; effective tillers, referring to the number of tillers bearing spikes with more than five mature seeds, counted from the base upward; flag leaf length, measured from the base to the tip of the flag leaf; flag leaf width, indicating the maximum width of the flag leaf; and single-plant mass, which refers to the mass of the entire plant (with roots) after cleaning and blotting dry with a paper towel to remove surface moisture. The relevant agronomic traits recorded for rice spikes and their specific definitions were as follows: effective spike number, defined as the number of spikes with more than five mature grains per plant; spike length, the distance from the neck node to the tip of an effective spike; effective spike mass, the mass of the effective spike on a single plant; grain number per spike, the total number of grains in an effective spike; fruiting rate, the percentage of filled grains in an effective spike; grain density, the number of grains per centimeter of spike length; and thousand-grain mass, the mass of 1,000 filled grains. The recorded seed quality traits and their specific definitions were as follows: brown rice percentage, the ratio of brown rice mass (after hull removal) to the total grain mass (before hull removal); grain length, the average length of 10 grains; grain width, the average width of 10 grains; grain thickness, the average thickness of the rice grains; and chalkiness, the proportion of the white, opaque portion in the rice grain, calculated based on the chalkiness rate under fluorescent light and the average area of the chalky portion. Scanning electron microscopy observation of rice seeds Three seeds were randomly selected from each of the different T3-generation transgenic rice varieties. Intact grains were gently fractured using tweezers to expose a flat cross-section as uniformly as possible. The cross-sections were mounted on sample stubs with the exposed surface facing upward and then coated with platinum using an ion sputter coater (Cressington 108 Auto). Morphology and particle size of starch granules were examined using an environmental scanning electron microscope (FEI, Model Q45). Multiple observation areas were randomly selected and imaged. Identification of pollen viability using the iodine-potassium iodide staining method At the early anthesis stage of transgenic rice strains from the T1-T3 generations, mature anthers were collected from the upper, middle, and lower parts of panicles. Three samples were randomly collected from each variety of transgenic rice plants. These anthers were left at room temperature for 0h, 3h, and 6h, respectively, then transferred onto microscope slides. Each anther was gently crushed with forceps, and 1–2 drops of 1% K-KI solution were added using a Barton’s dropper to fully release the pollen grains. A coverslip was placed over the sample and gently pressed with forceps, followed by a 2–3 min staining. A 10x microscope objective was used to observe randomly selected fields to examine anther morphology and record the proportion of mature anthers. The results were used to assess differences in pollen viability between transgenic strains and the recipient variety. Field biodiversity evaluation Field insect diversity survey: When the T3 generation rice cultivated outdoors reached the milky stage, three consecutive rain-free days were selected for sampling. Sticky traps were placed around each rice strain plot, with three random sampling points per strain and two sticky traps at each point. After three days, the sticky traps were collected, and the trapped insects were identified and counted to provide a preliminary assessment of pest presence during the reproductive growth stage. Field plant diversity survey: For the rice cultivated outdoors, plant diversity was surveyed at the tillering stage (immediately after transplanting) and at maturity. At each stage, 3–5 plots per strain were randomly selected. All plants within a 0.25 m² area (50 cm × 50 cm) surrounding each plot were collected, and their species diversity and biomass were recorded to preliminarily assess changes in plant diversity before and after planting of the transgenic materials. Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). All experiments were conducted under a single-variable design, and P values were calculated using ordinary one-way analysis of variance (ANOVA) with α set to 0.05. All experiments were set up with at least three biological replicates. All data were verified using Levene's Test for Homogeneity of Variances, and met the assumption of equal variances (except for the flag leaf width of T3 and the plant weight of T1). For data that met the assumption of homogeneity of variances, the LSD Test and Duncan's Test were performed; for data that did not meet this assumption, Tamhane's Test was conducted. All graphs were generated using GraphPad Prism version 8.0. Results Analysis of the Insertion Sites of Exogenous Genes in Transgenic Rice Analysis of exogenous gene insertion sites was performed on the T1-T3 generations of the HN-1 transgenic rice strain, and the results showed: The reference genome size for gene sequencing was 385.71M. The comparison rates of the three samples ranged from 96.22% to 99.78%, coverage of the reference genome ranged from 98.31% to 98.38%, and the average sequencing depth ranged from 25.491X to 39.316X. The insertion sequences in all three samples were integrated into the nuclear chromosomes of the cells (Figs. 1A-1C). A comparison of different reads indicated that all three samples exhibited chromosomal translocations relative to the parental genome. Specifically, the regions from 32,680,503 to 32,681,743 bp on chromosome 1 (Chr1) were translocated to Chr3. This translocation allowed for the identification of the upper and lower boundaries of the insertion site on Chr1 and Chr3, respectively. The upper boundary of the insertion on Chr1 was 32,681,743 bp, whereas the lower boundary of the insertion on Chr3 was 31,802,579 bp (Figs. 1D-1F). The insertion intervals at the upper and lower borders within the genes were as follows: the upper boundary at Chr1:32,681,743 was located within the gene AGIS_Os01g048210 (Chr1:32, 681, 631–32, 683,642), and the lower boundary at Chr3:31,802,579 was found in the gene AGIS_Os03g046780 (Chr3:31,793,752–31,802,369) as well as in the gene AGIS_Os03g046790 (Chr3:31,803,572–31,804,793). Based on the available sequencing data, the three samples were analyzed using deep variant comparisons of the on-target reads for the upper exogenous sequences, and no variants were identified (Figs. 1G and 1H). Analysis of the genetic stability of exogenous genes at the DNA, RNA and protein levels Polymerase chain reaction (PCR) was used to detect the presence of exogenous genes HN-1 and HN-2 in TP309 and T1–T3 generation transgenic strains. DNA extracted from panicles (Fig. 2A, 2C) and leaf tissues (Fig. 2B, 2D) of transgenic rice plants from the T1–T3 generations of each strain served as templates. The exogenous HN gene was stably expressed throughout the T1-T3 generations. The relative expression levels of the exogenous genes HN-1 and HN-2 in the TP309 and T1-T3 generation transformant strains were assessed by quantitative reverse transcription polymerase chain reaction, using complementary DNA derived from the leaf tissues of each strain obtained through reverse transcription as a template. HygR served as an endogenous gene and the expression levels of exogenous genes in the rice spike tissues of TP309 were used as a basestrain reference. All exogenous insertion genes were expressed in the leaf tissues of HN-1, HN-2 at levels ranging from approximately 8- to 400-fold (Figs. 2E, 2F and 2G), demonstrating that the exogenous HN gene can persist across different strains of multi-generation transgenic rice. The chicken Newcastle disease virus HN protein was consistently expressed across all three generations of the transgenic strain, as confirmed by western blotting (Figs. 2H and 2I). In addition, the antigen titers of different generations of HN were evaluated using a test paper, revealing a relatively stable titer, which was maintained at 2^9 (Fig. 2J). Insertion site analysis, DNA, mRNA and protein levels confirmed that the HN protein of Newcastle Disease Virus (NDV) was stably present in the transgenic strains. Evaluation of germination rate, emergence rate and development period In the rice seed germination rate determination experiment, the germination rates of HN-1 at 12, 24, 36, and 48 hours were 30%, 70%, 86%, and 96%, respectively. The germination rates of HN-2 at 12, 24, 36, and 48 hours were 44%, 96%, 97%, and 97%, respectively. In the seedling emergence rate determination experiment, the germination rates of HN-1 at 8, 16, 24, and 32 hours were 30%, 70%, 86%, and 96%, respectively, while HN-2 had germination rates of 44%, 96%, 97%, and 97% at 8, 16, 24, and 32 hours, respectively. The results indicated that the germination (Fig. 3A) and seedling emergence rates (Figs. 3B and 3C) of HN-1, HN-2, and TP309 were comparable, with no statistically significant differences observed. Three successive generations of transgenic rice strains HN-1 and HN-2, along with wild-type rice, were cultivated outdoors to investigate differences in growth and development between the transgenic strains and wild-type plants and to monitor their fertility cycles. The total growth cycles of TP309 and the transgenic strains were relatively stable, fluctuating between 150 and 180 days (Fig. 3D). Comprehensive evaluation of agronomic traits The number of spikes, the number of grains per spike, and the grain quality of the rice directly determine the overall yield. Yield-related factors during rice development can be categorized as growth morphology, spike status, and grain quality. This study observed agronomic traits associated with these three factors in T1, T2, and T3 rice generations from strains HN-1, HN-2, and TP309. In the T1 generation, the ear length of the HN-1 strain was slightly shorter than that of TP309, whereas the differences in the other traits were not significant. The seed-setting percentage of HN-2 was lower than that of TP309 with no significant differences in the remaining traits. When comparing the two strains, HN-1 exhibited a significantly higher seed-setting percentage and grain density per centimeter than HN-2, although the other traits did not show significant differences (Fig. 4A). In the T2 generation, the differences in the 12 agronomic traits between HN-1 and TP309 were not significant. However, the differences between HN-2 and TP309 were significant, particularly regarding the reduction in ear length and mass of the effective panicle. When comparing the two strains, the spike length and effective spike mass of HN-2 were lower than those of HN-1, whereas the differences in the other traits were not significant (Fig. 4B). In the T3 generation, the effective panicle mass of the HN-1 strain was significantly lower than that of the TP309 strain, with no significant differences in the remaining traits (Fig. 4C). Conversely, the mass of the effective panicle and thousand-grain mass of HN-2 were significantly higher than those of TP309, with no differences in the other traits. When comparing the two strains, the thousand-grain mass of HN-2 was greater than that of HN-1, although the differences in other traits were not significant. In addition, we measured the grain length, grain width, grain thickness, brown rice mass, chalkiness, and other qualities of the seeds harvested from the T1, T2, and T3 generations. The grain length (Figs. 5A and 5D), grain width (Fig. 5B and 5D), grain thickness (Fig. 5C), and brown rice yield of the transgenic rice strains HN-1 and HN-2 did not differ significantly from those of TP309 (Fig. 5E). However, the chalkiness rate and degree increased significantly compared with TP309, particularly in the HN-2 rice strain, whereas no significant changes were observed in the HN-1 rice strain (Fig. 5E). This finding suggests that the increase in the chalky white rate and degree in the HN-2 rice strain, attributed to insufficient grouting material, may be influenced by various environmental factors, such as climatic conditions and cultivation methods during the grouting period, or maybe an inherent characteristic of this transgenic strain. The data indicated that the growth and development of the rice strains HN-1 and HN-2 were comparable to those of TP309 plants, demonstrating that the agronomic traits of these strains remain robust. Scanning electron microscopy further revealed changes in the internal structure of transgenic rice, showing an increase in branched-chain starch and a decrease in straight-chain starch (Fig. 6). Consequently, the visible increase in chalkiness in the HN-2 rice strain makes it easier to break. Because we primarily extracted the target proteins from milled grains, this strain shows great potential for applications. Evaluation of survival competitiveness Rice typically produces seeds through self-pollination, and the viability of rice pollen is closely linked to its reproductive capacity, which directly influences the competitive survival of rice plants against surrounding vegetation. To evaluate the competitive survival ability of transgenic rice strains and TP309 in the field, mature anthers were collected from plants of the T1–T3 generations of transgenic rice at the early flowering stage, and these anthers were subjected to iodine-potassium iodide (I-KI) staining.The pollen status of the transgenic rice and TP309 was observed at various time intervals (t0, t3, and t6). Across the T1 (Figs. 7A and 7B), T2 (Figs. 7C and 7D), and T3 (Figs. 7E and 7F) generations, Statistical analysis revealed no significant changes in the normal pollen rate of the transgenic strains or TP309 over time, nor were there any significant differences in the proportion of normal fertile pollen between the transgenic strains HN-1, HN-2, and TP309. The results of this experiment indicated that the pollen longevity of transgenic rice was stable and highly viable when compared to TP309, demonstrating no significant difference in pollen viability between transgenic rice and TP309 plants. Consequently, both species exhibited similar capabilities in competing for survival against other species in the field. Evaluation of gene flow capacity and field biodiversity To assess the potential of exogenous gene transfer from transgenic rice strains to surrounding plants, leaves of various weeds in paddy fields of T1-T3 generations of transgenic rice grown outdoors were collected and analyzed. Specific primers were used to detect the presence of the Newcastle disease virus HN gene in non-rice plants (Fig. 8A, 8B).To evaluate whether transgenic rice would affect the populations and diversity of surrounding organisms in the field, an insect diversity experiment was conducted using the milky rice, which exhibited more severe insect pest issues. Sticky traps were placed in wild-type rice plots and T3 generation blocks of each transgenic rice strain (each block measuring 50 cm × 50 cm). After three days of placement, the number of Lepidopteran insects and arthropods on the sticky traps was counted. The results indicated that the number of lepidopterans around the rice-planting area was significantly higher than that of arthropods. However, there was no significant difference in the species and number of insects between the TP309 block and transgenic strains HN-1 and HN-2 (Fig. 8C). The plant diversity experiment randomly sampled blocks of TP309 and transgenic strains. During the harvesting periods, the species and masss of all other plants in the blocks were recorded. Compared with the TP309 control, the weed species and quality surrounding the transgenic strains HN-1 and HN-2 did not show any significant effects (Fig. 8D). Thus, it can be tentatively concluded that the impact of transgenic rice on the biodiversity of the surrounding environment is relatively small. Discussion Newcastle disease (ND) has been defined by the World Organisation for Animal Health as infection of poultry with virulent strains of Newcastle disease virus (NDV). Lesions affecting the neurological, gastrointestinal, respiratory, and reproductive systems are most often observed. The control of ND must include strict biosecurity that prevents virulent NDV from contacting poultry, and also proper administration of efficacious vaccines 1 , 9 . When administered correctly to healthy birds, ND vaccines formulated with NDV of low virulence or viral-vectored vaccines that express the NDV fusion protein are able to prevent clinical disease and mortality in chickens upon infection with virulent NDV. Live and inactivated vaccines have been widely used since the 1950’s 18 . Recombinant and antigenically matched vaccines have been adopted recently in some countries, and many other vaccine approaches have been only evaluated experimentally. Despite decades of research and development towards formulation of an optimal ND vaccine, improvements are still needed. Impediments to prevent outbreaks include uneven vaccine application when using mass administration techniques in larger commercial settings, the difficulties associated with vaccinating free-roaming, multi-age birds of village flocks, and difficulties maintaining the cold chain to preserve the thermo-labile antigens in the vaccines. Incomplete or improper immunization often results in the disease and death of poultry after infection with virulent NDV. Another cause of decreased vaccine efficacy is the existence of antibodies (including maternal) in birds, which can neutralize the vaccine and thereby reduce the effectiveness of ND vaccines. Based on this, there is an urgent need to develop an efficient Newcastle disease vaccine that is easy to mass-produce, easy to store and transport, and low in price 18 , 39 . Based on the results of previous animal experiments, in order to achieve the commercial production of this genetically modified rice (HN-1, HN-2), intermediate trials were conducted at the designated site (Transgenic Rice Experimental Base in Xinjiang, China) to evaluate the genetic stability, agronomic traits, and biosafety of the transgenic rice across three consecutive generations. In this study, we used a rice endosperm expression system previously established in our laboratory to advance the commercialization of the Newcastle disease vaccine. We verified the stability of exogenous gene integration, expression, and functional trait expression across three successive planting generations. Additionally, compared with TP309, the transgenic rice strains exhibited minimal differences in several physiological traits, including seed germination, developmental cycle, plant growth morphology, spike position, and seed quality. Importantly, the gene insertion did not affect these functional traits. The gene transfer test further demonstrated that the Newcastle disease virus HN gene in transgenic rice did not disperse to neighboring plants in the surrounding area, and preliminary surveys on the species and population sizes of insects and arthropods around the T3 generation transgenic rice plants preliminarily indicated that the two transgenic rice strains (HN-1 and HN-2) exerted no adverse effects on the local ecosystem. This underscores the advantages of this expression system, which is not only promising for vaccine applications but also serves as a significant reference for producing other medicinal proteins. These findings provide critical validation for rice-based molecular farming as a promising platform for vaccine development. Compared with traditional Newcastle disease vaccines—including inactivated, live attenuated, and viral vector-based formulations 40 – 42 —our rice-derived HN antigen offers several key advantages. First, it directly addresses the challenge of antigenic mismatch, as plant-based systems can be rapidly engineered to express antigens corresponding to prevalent NDV genotypes, such as genotype VII, which currently accounts for most field outbreaks. Second, plant-derived vaccines offer low-cost production without the requirement for specialized fermentation infrastructure or cold-chain logistics, greatly enhancing accessibility in low-resource settings. Third, rice endosperm bioreactors provide the possibility of oral vaccine delivery, which could simplify immunization programs by reducing the need for multiple injections and trained personnel 27 , 43 . Our findings align with and extend previous work on molecular farming 24 . For example, the “head-to-tail” dimer antigen design reported in PNAS demonstrated that rice-expressed HN proteins could induce potent immune responses and achieve complete protection in chickens at remarkably low doses 22 . The present study complements this by providing essential data on genetic stability and biosafety, thereby addressing critical prerequisites for commercialization. Taken together, these advances highlight the potential of transgenic rice to serve as both a cost-effective production platform and a novel oral delivery vehicle for NDV subunit vaccines. Despite these promising results, several challenges remain before rice-based NDV vaccines can reach practical application. First, although immunogenicity has been demonstrated in controlled experiments, larger-scale and longer-term studies are needed to confirm consistent efficacy under field conditions. Second, the scalability of downstream processing, including antigen extraction and dosage standardization, requires further optimization to meet industrial production standards. Third, public acceptance and regulatory approval of genetically modified crops used for pharmaceutical purposes remain major hurdles. Addressing biosafety concerns, particularly those related to gene flow and food chain contamination, will be crucial for the successful adoption of such vaccines. Finally, it remains necessary to compare the performance of rice-based vaccines with other plant-based systems (e.g., maize, tobacco, or tomato) to determine the most suitable platform for different antigen targets and delivery strategies. Conclusion In conclusion, this study provides relatively comprehensive evidence supporting the genetic stability, agronomic feasibility, and biosafety of transgenic rice expressing NDV HN protein. Together with prior immunogenicity studies, these results establish a solid foundation for the development of rice-based NDV subunit vaccines. Integrating genetic engineering advances with rigorous field evaluation and regulatory frameworks will be essential to accelerate the translation of molecular farming technologies into commercial poultry vaccines. Beyond Newcastle disease, the strategies demonstrated here may also inform the broader development of plant-derived vaccines and therapeutics for both animal and human health. Declarations Funding This work was supported by Development of rapid detection and diagnostic technology for Giardia, Coccidioidomycosis and Anaplasmosis (2023YFD1801203), key scientific and technological projects of Henan Province (222102110210) and Major Science and Technology Projects in Henan Province (241110310200). Author Contribution "Conceptualization, G.Z., X.W. and W.G.; methodology, H.C., Z.H., L.Z., F.L. and Y.H.; software, H.C. and L.H.;validation, L.Z.,H.C., Z.H., Y.H., Y.L., S.B., W.C., J.Z. and Y.Z.; formal analysis, L.Z., H.C., and Z.H.; investigation, S.P., S.K.and H.C.; resources, G.Z., X.W.; data curation, H.C., Z.H., L.Z. and Y.H.; writing—original draft preparation, L.Z. and H.C.; writing—review and editing, L.Z. and H.C.; visualization, L.Z. and H.C.; supervision, X.W., and W.G.; project administration, X.W.; funding acquisition, G.Z. and X.W.." Acknowledgements We thank the reviewers and editors for their thoughtful comments and suggestions regarding the manuscript. We thank Editage for providing assistance with language editing.The authors would like to express their sincere gratitude to the staff and plantation managers of the Changji Comprehensive Experimental Base of the Chinese Academy of Agricultural Sciences for their assistance. In addition, the data for insertion site analysis of exogenous genes in transgenic rice (Fig. 1 in the article) were provided by Wuhan Benan Gene Technology Co. We would like to express our gratitude to this company. Data Availability The datasets used or analysed during the current study available from the corresponding author on reasonable request. References Ul-Rahman, A. et al. Zoonotic potential of Newcastle disease virus: Old and novel perspectives related to public health. Rev. Med. Virol. 32 , e2246 (2022). Costa-Hurtado, M. et al. Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication, disease, and mortality in chickens. Vet. Res. 46 , 97 (2015). Chen, L. et al. Phylodynamic analyses of class I Newcastle disease virus isolated in China. Transbound. Emerg. Dis. 68 , 1294–1304 (2021). Berihulay, H. et al. Exploring the genetic basis of Newcastle disease virus in chickens: a comprehensive review. Front. 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A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics. Vaccine 27 , 904–910 (2009). Miller, P. J. et al. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge. Vaccine 25 , 7238–7246 (2007). Zhu, Q. et al. Molecular farming using transgenic rice endosperm. Trends Biotechnol. 40 , 1248–1260 (2022). Tables Tables 1.1 to 1.3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table.docx Fignotes.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 25 Nov, 2025 Reviews received at journal 12 Nov, 2025 Reviews received at journal 29 Oct, 2025 Reviewers agreed at journal 20 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 15 Oct, 2025 Submission checks completed at journal 15 Oct, 2025 First submitted to journal 15 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":133671,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of shoot leaf insertion sites in rice seedlings of T1, T2 and T3 generations. Figures (\u003cstrong\u003eA\u003c/strong\u003e), (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eC\u003c/strong\u003e) Average sequencing depth as well as coverage of each window within the genome corresponding to the T1, T2 and T3 generation. Figure (D) Comparison plot of reads supporting exogenous sequence insertion of T1. Figure(E) Comparison plot of reads supporting exogenous sequence insertion of T2. Figure(F) Comparison plot of reads supporting exogenous sequence insertion of T3. Figure (\u003cstrong\u003eG\u003c/strong\u003e) Frequency distribution of structural variants across samples.\u003c/p\u003e\n\u003cp\u003eNote: INS: insertion, DEL: deletion, INV: inversion, DUP: duplication, TRA: interchromosomal translocation. Figure (\u003cstrong\u003eH\u003c/strong\u003e) Distribution of each type of structural variation across the genome.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/e65d11f28ba81ee4b0be33e6.jpg"},{"id":94664009,"identity":"797859dc-e384-47ec-b9f6-aaba334c40d5","added_by":"auto","created_at":"2025-10-29 12:16:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":381955,"visible":true,"origin":"","legend":"\u003cp\u003eStable expression of HN antigen in different generations of rice. (\u003cstrong\u003eA\u003c/strong\u003e) and (\u003cstrong\u003eC\u003c/strong\u003e) PCR (amplicon size: 1138 bp) was used to detect HN genes in rice leaves at the flowering stage and in rice spikes in different generations from HN-1; (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eD\u003c/strong\u003e) PCR (amplicon size: 1138 bp) was used to detect HN genes in rice leaves at the flowering stage and in rice spikes in different generations from HN-2; T11-T16: The T1 generation of the transgenic strain was randomly selected from six, T21-26: The T2 generation of the transgenic strain was randomly selected from six, T31-T36: The T3 generation of the transgenic strain was randomly selected from six, TP indicates TP309, + indicates positive plasmid (The labelling in the figures is based on this method); (\u003cstrong\u003eE\u003c/strong\u003e), (\u003cstrong\u003eF\u003c/strong\u003e) and (\u003cstrong\u003eG\u003c/strong\u003e) show the mRNA levels of the HN gene (amplicon size: 264 bp) detected in leaves of rice plants at the flowering stage for generations T1-T3, respectively.; HN-1-1 to HN-1-3 shown in the figure represent three randomly taken HN-1 plants; HN-2-1 to HN-2-3 represent three randomly taken HN-2 plants; (\u003cstrong\u003eH\u003c/strong\u003e) and (\u003cstrong\u003eI\u003c/strong\u003e) Western blot detection of HN gene expression in rice seeds of different generations from HN-1 and HN-2; In the figure, T11-T13 represent randomly selected plants of three T1 generations; T21-T23 represent randomly selected plants of three T2 generations; T31-T33 represent randomly selected plants of three T3 generations. (\u003cstrong\u003eJ\u003c/strong\u003e) Test strips were used to detect the titre of HN antigen in rice seeds of different generations (mass: volume = 1:5).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/d753353db1f4c38734c1dfc1.jpg"},{"id":94673165,"identity":"c2e7f6af-7cb8-425c-aedd-1f0be8e5eced","added_by":"auto","created_at":"2025-10-29 13:41:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":207411,"visible":true,"origin":"","legend":"\u003cp\u003eGermination and seedling percentage assessment of HN-1, HN-2 transgenic rice: (\u003cstrong\u003eA\u003c/strong\u003e) Germination of transgenic rice. (\u003cstrong\u003eB\u003c/strong\u003e) seedling percentage of transgenic rice. (\u003cstrong\u003eC\u003c/strong\u003e) Visualisation of germination of transgenic rice. (\u003cstrong\u003eD\u003c/strong\u003e) Development and growth cycle of transgenic rice and TP309.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/90dac0afb4c2d5293735de1b.jpg"},{"id":94664011,"identity":"a679c147-6ba1-4c7e-9fa3-8a852d9f4e19","added_by":"auto","created_at":"2025-10-29 12:16:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":438649,"visible":true,"origin":"","legend":"\u003cp\u003eCombined agronomic traits of T1-T3 generation transformant strains and TP309. (\u003cstrong\u003eA\u003c/strong\u003e), (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eC\u003c/strong\u003e) corresponded to 12 comprehensive agronomic traits recorded in T1-T3 generations, including: a, The height per plant ; b, The effective tiller number; c, length of flag leaf ; d, width of flag leaf ; e, The weight per plant; f, The effective spikes number ; g, Ear length; h, weight of the effective paicle ; i, grains number of per panicle; j, percentage of the seed setting; k, grain density per cm; and l, Thousands grain weight; The data were statistically significantly different from those of the above data by One-way ANOVA, where * is p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, and **** p\u0026lt;0.0001. Both HN-1 and HN-2 were assessed with five randomly selected plants.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/76a03c6c6b756955f69deea2.jpg"},{"id":94673271,"identity":"a3ad6769-33b0-4fc4-88e3-f7a80666562b","added_by":"auto","created_at":"2025-10-29 13:41:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109659,"visible":true,"origin":"","legend":"\u003cp\u003eGrain phenotypes of T1-T3 generation transformant strains and TP309. (\u003cstrong\u003eA\u003c/strong\u003e), (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eC\u003c/strong\u003e) Comparison of rice grain morphology data for each strain in the T1, T2 and T3 generation, from left to right, grain length, grain width and grain thickness; (\u003cstrong\u003eD\u003c/strong\u003e) Rice grain phenotypes (brown rice on top, fine rice on bottom); (\u003cstrong\u003eE\u003c/strong\u003e) Comparison of rice grain quality among the strains, from left to right, in order of brown rice percentage, chalkiness and chalkiness. The data were statistically significantly different from those of the above data by One-way ANOVA, where * is p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, and **** p\u0026lt;0.0001. Both HN-1 and HN-2 were assessed with five randomly selected plants.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/b6214c1bb1dbf057bf6506f5.jpg"},{"id":94673170,"identity":"061224f5-e787-4a1f-97ad-109000017485","added_by":"auto","created_at":"2025-10-29 13:41:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":289427,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope detection of TP309, Osr2HN-1, Osr2HN-2 respectively rice of T3 grain chalkiness. Three replicates for each strain.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/8d4627cdc78a0cd8c7f0c410.jpg"},{"id":94664020,"identity":"b88fc5f2-7cbd-447e-b72e-90e4ae0e6727","added_by":"auto","created_at":"2025-10-29 12:16:46","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":500389,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of pollen viability between transformant strains of T1-T3 generations and TP309. (\u003cstrong\u003eA\u003c/strong\u003e) and (\u003cstrong\u003eB\u003c/strong\u003e) corresponds to the comparison and statistics of pollen viability of T1 generation transformant strain and TP309, respectively; (\u003cstrong\u003eC\u003c/strong\u003e) and (\u003cstrong\u003eD\u003c/strong\u003e) corresponds to the comparison and statistics of pollen viability of T2 generation transformant strain and TP309, respectively; (E) and (F) corresponds to the comparison and statistics of pollen viability of T3 generation transformant strain and TP309,respectively. Each plant should have at least three replicates.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/ecd7c63758ba13edfda417c9.jpg"},{"id":94673460,"identity":"3a8c46e4-c556-42b5-bcaf-190634261531","added_by":"auto","created_at":"2025-10-29 13:41:24","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":319419,"visible":true,"origin":"","legend":"\u003cp\u003eSafety evaluation of rice strains transgenic for HN-1 and HN-2 genes. (\u003cstrong\u003eA\u003c/strong\u003e) and (\u003cstrong\u003eB\u003c/strong\u003e) PCR result of the spread of foreign gene to surrounding weeds from HN-1 and HN-2. T11-T16: The T1 generation of the transgenic strain was randomly selected from six, T21-26: The T2 generation of the transgenic strain was randomly selected from six, T31-T36: The T3 generation of the transgenic strain was randomly selected from six, TP indicates TP309, + indicates positive plasmid (The labelling in the figures is based on this method); (\u003cstrong\u003eC\u003c/strong\u003e) and (\u003cstrong\u003eD\u003c/strong\u003e) Outdoor field biodiversity analysis of transgenic rice and TP309.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/874a85abad0d1b59fdb80ef5.jpg"},{"id":94731077,"identity":"7265a4ca-91f4-4b23-ad35-75c581ecd830","added_by":"auto","created_at":"2025-10-30 07:07:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3380594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/f0570043-c6bb-43ee-84a1-50f6200bc67f.pdf"},{"id":94672697,"identity":"f559c011-fc31-4015-9abc-fac8829a1c5c","added_by":"auto","created_at":"2025-10-29 13:40:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13153,"visible":true,"origin":"","legend":"","description":"","filename":"Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/0dfa972db560efb4f7a25022.docx"},{"id":94672564,"identity":"8d368089-71c5-4d6a-a1a3-4ac6611c82a1","added_by":"auto","created_at":"2025-10-29 13:40:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1774783,"visible":true,"origin":"","legend":"","description":"","filename":"Fignotes.docx","url":"https://assets-eu.researchsquare.com/files/rs-7453085/v1/8f9c996183b87311834f54a2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic stability, environmental safety and field adaptation assessment of transgenic rice with chicken Newcastle disease HN protein","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNewcastle disease (ND), also known as Asian fowl plague or pseudo-fowl plague, is an acute, highly contagious viral disease of poultry caused by Newcastle disease virus (NDV)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is classified as a Category a animal disease by the World Organization for Animal Health (WOAH) and a Category I animal disease in China, reflecting its significant impact on poultry health and the economy worldwide. NDV possesses two major surface glycoproteins: the fusion (F) protein and the hemagglutinin-neuraminidase (HN) protein\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. HN plays a dual role in receptor recognition and membrane fusion, and serves as a key immunogenic protein, making it an attractive target for vaccine development\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCurrently, commercial ND vaccines mainly include inactivated vaccines, live attenuated vaccines and viral vector vaccines\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, due to the genetic diversity and continuous evolution of NDV, especially the widespread prevalence of type VII strains, these traditional vaccines exhibit poor antigenic matching and the cross-protection rates are often less than 30%\u003csup\u003e15\u0026ndash;19\u003c/sup\u003e. In addition, these vaccines require cold chain storage and transportation and involve multiple immunization procedures, which together increase costs and limit large-scale application\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Although ND vaccines have been used for decades, no subunit vaccines have been approved to date, This highlights the urgency of developing a new generation of vaccines that offfer higher safety, efficacy, affordability and genotype coverage\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAcademician Zhang Gaiping's team recently published a research paper in the internationally renowned journal PNAS titled \"A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice\", which provides new insights into Newcastle disease vaccine development (The highest amount of the Osr2HN protein in transgenic rice seeds can reach 3.70 mg/g)\u003csup\u003e22, 23\u003c/sup\u003e. In the current experiments, this protein content reached a maximum of 1.2 mg/g. The study proposes a universal \"head-to-tail\" dimeric vaccine antigen model\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Using the hemagglutinin-neuraminidase (HN) protein, the receptor-binding protein of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, as an example, they successfully produced a highly effective recombinant antigen, Osr2HN, using a rice endosperm expression system. This study highlights the potential of rice-based vaccine production platforms. In recent years, plant bioreactors have made significant progress in several areas\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. They are capable of producing a wide range of products; for instance, plant tissues can be processed for oral delivery of food proteins, thereby reducing downstream processing requirements\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Rice (Oryza sativa L.) is not only a staple crop for over half the world's population but also a well-established model organism for functional genomics research\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Its high productivity, broad adaptability, and established genetic engineering tools make it a strategic choice for molecular agriculture\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Compared to microbial or animal cell-based systems, rice endosperm bioreactors offer unique advantages, including genetic stability, scalability, low production costs, and the potential for oral vaccine delivery without cold chain reliance. Indeed, recent advances in transgenic rice technology have enabled high-level expression of various recombinant proteins, including vaccines, antibodies, and therapeutic peptides\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHowever, the commercialization of plant-derived vaccines faces significant challenges. The stability of transgenic strains must be verified across multiple generations, and comprehensive assessments of genetic stability, agronomic performance, and biosafety are essential. In particular, issues such as transgenic stability, environmental biosafety, gene flow, and ecological impacts must be addressed to meet regulatory standards and gain public acceptance.\u003c/p\u003e\u003cp\u003eBuilding on previous success in expressing highly active NDV HN antigens in rice endosperm, this study aimed to systematically evaluate transgenic rice carrying HN vaccine antigens. Specifically, we focused on three key aspects: (i) multigenerational genetic stability, (ii) agronomic adaptability and phenotypic performance, and (iii) biosafety assessment, including survival competitiveness, gene spread potential, and impacts on ecological diversity. This study provides critical data to support the development and commercialization of rice-based NDV subunit vaccines and facilitates the establishment of an intermediate testing system for plant-derived vaccines.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch3\u003eMaterials\u003c/h3\u003e\n\u003cp\u003eIn this study, two transgenic rice strains, HN-1 and HN-2, were successfully constructed using a rice endosperm reactor, and Taipei 309 (TP309) was used as a control variety. These strains were independently developed by our laboratory, and detailed information is available in our previous work\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. EACKER straightedge (product no. 713319 3m) and electronic vernier calipers (model no. DL91150) were used as the measuring tools.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eInsertion site analysis\u003c/h2\u003e\n \u003cp\u003eYoung leaves of rice seedlings from the T1, T2, and T3 generations were collected, sprayed with sterile water, blotted dried with absorbent paper, and placed into 50 mL Eppendorf tubes. The leaves were immediately frozen in liquid nitrogen for 10 minutes and then transported on dry ice to Wuhan Benagen Technology Co., Ltd. for insertion site analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDNA extraction from rice leaves and spikes\u003c/h3\u003e\n\u003cp\u003ePanicle tissues were randomly collected from transgenic rice strains (generations T1\u0026ndash;T3) and TP309 rice plants at the anthesis stage. The collected panicle tissues and weeds were frozen with dry ice and then stored at -80 ℃ for subsequent DNA extraction. Weeds around the experimental fields were collected and preserved using the same method. For each generation, 6 samples were collected \u003cstrong\u003erespectively\u003c/strong\u003e for panicles of different transgenic rice varieties and for weeds from different areas around the corresponding paddy fields. DNA extraction was performed using the FastPure\u0026reg; Plant DNA Isolation Mini Kit (DC104-01, Vazyme), following the manufacturer\u0026rsquo;s instructions. The extracted DNA was stored at -20 ℃. DNA from surrounding weeds was extracted using the same procedure. PCR analysis was used to detect the inheritance of the target gene. Primers sequences specific to the HN gene are listed in Table \u0026lt;link rid=\u0026quot;tb3\u0026quot;\u0026gt;\u003cspan class=\"InternalRef\"\u003e1\u0026lt;/link\u0026gt;\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and the PCR reaction system is shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;2. The optimal reaction conditions for PCR were as follows: (1) pre-denaturation at 95 ℃ for 5 min; (2) 35 cycles of amplification at 90 ℃ for 30 s, 60 ℃ for 20 s, and 72 ℃for 30 s; (3) final extension at 72 ℃ for 5 min; (4) storage at 16 ℃. PCR products were analyzed by agarose gel electrophoresis. At least three replicate experiments were performed for each sample.\u003c/p\u003e\n\u003cp\u003eDNA extraction was performed using the FastPure\u0026reg; Plant DNA Isolation Mini Kit (DC104-01, Vazyme), following the manufacturer\u0026rsquo;s instructions. The extracted DNA was stored at -20 ℃. DNA from surrounding weeds was extracted using the same procedure. PCR analysis was conducted to detect the inheritance of the target gene. Primer sequences specific to the HN gene are listed in Table\u0026nbsp;\u0026lt;link rid=\u0026quot;tb3\u0026quot;\u0026gt;\u003cspan class=\"InternalRef\"\u003e1\u0026lt;/link\u0026gt;\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, and the PCR reaction system is shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;2. The optimal PCR reaction conditions were as follows: (1) pre-denaturation at 95 ℃ for 5 min; (2) 35 amplification cycles of 90 ℃ for 30 s, 60 ℃ for 20 s, and 72 ℃ for 30 s; (3) final extension at 72 ℃ for 5 min; (4) hold at 16 ℃. PCR products were analyzed via agarose gel electrophoresis.\u003c/p\u003e\n\u003ch3\u003eAcquisition and qPCR detection of cDNA in rice leaves\u003c/h3\u003e\n\u003cp\u003eTransgenic plants of the T1-T3 generation and TP309 plants were selected at the panicle flowering stage. Rice leaves were transported from Xinjiang to Henan on dry ice and stored at -80 ℃. Total RNA was extracted using the FastPure\u0026reg; Universal Plant Total RNA Isolation Kit (Cat: RC411-01, Vazyme). cDNA was synthesized via reverse transcription using the HiScript\u0026reg; II Q RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (Cat: R223-01, Vazyme), following the manufacturer\u0026rsquo;s instructions. The cDNA extracted from leaves of T1-T3 generation transgenic rice and TP309 rice served as the template and was amplified by qPCR using primer pairs specific to the target genes and HygR genes. The primers used are listed in Table\u0026nbsp;\u0026lt;link rid=\u0026quot;tb3\u0026quot;\u0026gt;\u003cspan class=\"InternalRef\"\u003e1\u0026lt;/link\u0026gt;\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, and the qPCR reaction system is shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;3. The relative expression levels of the exogenou gene and HygR gene in leaves were determined using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. At least three replicate experiments were performed for each sample.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eTo determine the expression of the HN protein in mature rice seeds at the protein level, T1-T3 generation transgenic seeds and TP309 seeds, which were harvested simultaneously and stored under identical conditions, were used. Seeds from different strains were ground into a powder and then mixed with extraction buffer (25 mM PB, 20 mM Nacl pH 5.7) at a ratio of 1:5 (w/v, g/mL). The mixture was stirred for 1.5 h, followed by centrifugation at 9,000 rpm for 30 min at 4 ℃. The resulting supernatant was collected for Western blot analysis. Ar 10-180kDa Prestained Protein Marker were purchased from Henan Xianyan Biotech Co., Ltd. (Cat. No. ArP01201) and 180 kDa Prestained Protein Marker were purchased from Vazyme (MP102-01)..The primary antibody was chicken polyantibody were maintained in the laboratory. and the secondary antibody was HRP, Goat Anti-Chicken IgG purchased from Abbkine (Cat. No. A21080).\u003c/p\u003e\n\u003ch3\u003eTest strips for detecting antigen content in rice\u003c/h3\u003e\n\u003cp\u003eAntigenic titers were determined in the HN-1 and HN-2 transgenic rice strains across three consecutive generations. Immunochromatographic test strips developed in the laboratory were used to detect the HN antigen in harvested rice seeds. Rice extracts were prepared by mixing the ground seeds with extraction buffer (25 mM PB, 20 mM Nacl pH 5.7) at a mass-to-volume ratio of 1:5 (w/v), followed by stirring at room temperature for 2 h. The mixture was then centrifuged at 10,000 rpm and 4 ℃ to collect the supernatant, which was referred to as the 2^0 dilution. The extracts were subsequently serially diluted and used in antigen detection assays.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eEvaluation of germination and seedling emergence rates\u003c/h2\u003e\n \u003cp\u003eThe materials used to evaluate germination and seedling emergence included T1-T3 generation transgenic strains and TP309 seeds, which were harvested simultaneously and stored under identical conditions. For each strain, 100 seeds were placed in 50 mL conical flasks and soaked in water at 25 ℃ in the dark for 36 h. The water was replaced every 12 h to maintain clean strains. After soaking, the seeds were transferred to 9-cm glass Petri dishes lined with moistened sterile filter paper and incubated at 37 ℃ until radicle emergence. Once more than 80% of the seeds exhibited radicle protrusion through the seed coat, they were transferred to a 25 ℃ incubator and moistened regularly to promote germination. Germination was assessed every 12 h and recorded when radicles and shoots were visible. When the shoot reached half the length of the grain, the seeds were transplanted into moist nutrient soil in the culture room at 28 ℃ with a 14 h light/10 h dark photoperiod. Seedling emergence data were collected and recorded. Throughout the experiment, the seeds were kept consistently moist. Germination data and seedling emergence data were both recorded consecutively three times, and the experimental results were expressed as percentages.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDevelopmental cycle assessment\u003c/h3\u003e\n\u003cp\u003eThe developmental cycle assessment was executed utilizing T1-T3 generation transgenic rice transformants and TP309 control seeds, which were concurrently harvested and stored under uniform conditions. Seeds were subjected to sterilization and germination in growth medium. Post-germination, seedlings were transferred to growth chamber and subsequently transplanted into soil. Following 20 days, seedlings were replanting to outdoor environment and maintained under adequate irrigation conditions. The developmental cycle was monitored, with both vegetative and reproductive growth stages; the spikelet initiation stage delineates the transition between these two developmental phases.\u003c/p\u003e\n\u003ch3\u003eEvaluation of comprehensive agronomic traits\u003c/h3\u003e\n\u003cp\u003eA comprehensive evaluation of agronomic traits related to growth patterns was conducted on transgenic rice strains of the T1-T3 generations at the full maturity stage\u0026mdash;which is defined as the period when over 90% of the glumes turn yellow and the basal seeds harden and become resistant to breakage. At this stage, rice spikes from each strain were collected into the corresponding numbered seed bags. After sun-drying for 3 days under outdoor conditions, agronomic traits related to spikes and grains were measured.\u003c/p\u003e\n\u003cp\u003eThe recorded agronomic traits related to growth pattern and their specific definitions were as follows: plant height, defined as the distance from the base of the plant to the tip of the second tallest leaf; effective tillers, referring to the number of tillers bearing spikes with more than five mature seeds, counted from the base upward; flag leaf length, measured from the base to the tip of the flag leaf; flag leaf width, indicating the maximum width of the flag leaf; and single-plant mass, which refers to the mass of the entire plant (with roots) after cleaning and blotting dry with a paper towel to remove surface moisture.\u003c/p\u003e\n\u003cp\u003eThe relevant agronomic traits recorded for rice spikes and their specific definitions were as follows: effective spike number, defined as the number of spikes with more than five mature grains per plant; spike length, the distance from the neck node to the tip of an effective spike; effective spike mass, the mass of the effective spike on a single plant; grain number per spike, the total number of grains in an effective spike; fruiting rate, the percentage of filled grains in an effective spike; grain density, the number of grains per centimeter of spike length; and thousand-grain mass, the mass of 1,000 filled grains.\u003c/p\u003e\n\u003cp\u003eThe recorded seed quality traits and their specific definitions were as follows: brown rice percentage, the ratio of brown rice mass (after hull removal) to the total grain mass (before hull removal); grain length, the average length of 10 grains; grain width, the average width of 10 grains; grain thickness, the average thickness of the rice grains; and chalkiness, the proportion of the white, opaque portion in the rice grain, calculated based on the chalkiness rate under fluorescent light and the average area of the chalky portion.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eScanning electron microscopy observation of rice seeds\u003c/h2\u003e\n \u003cp\u003eThree seeds were randomly selected from each of the different T3-generation transgenic rice varieties. Intact grains were gently fractured using tweezers to expose a flat cross-section as uniformly as possible. The cross-sections were mounted on sample stubs with the exposed surface facing upward and then coated with platinum using an ion sputter coater (Cressington 108 Auto). Morphology and particle size of starch granules were examined using an environmental scanning electron microscope (FEI, Model Q45). Multiple observation areas were randomly selected and imaged.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification of pollen viability using the iodine-potassium iodide staining method\u003c/h2\u003e\n \u003cp\u003eAt the early anthesis stage of transgenic rice strains from the T1-T3 generations, mature anthers were collected from the upper, middle, and lower parts of panicles. Three samples were randomly collected from each variety of transgenic rice plants. These anthers were left at room temperature for 0h, 3h, and 6h, respectively, then transferred onto microscope slides. Each anther was gently crushed with forceps, and 1\u0026ndash;2 drops of 1% K-KI solution were added using a Barton\u0026rsquo;s dropper to fully release the pollen grains. A coverslip was placed over the sample and gently pressed with forceps, followed by a 2\u0026ndash;3 min staining. A 10x microscope objective was used to observe randomly selected fields to examine anther morphology and record the proportion of mature anthers. The results were used to assess differences in pollen viability between transgenic strains and the recipient variety.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eField biodiversity evaluation\u003c/h2\u003e\n \u003cp\u003eField insect diversity survey: When the T3 generation rice cultivated outdoors reached the milky stage, three consecutive rain-free days were selected for sampling. Sticky traps were placed around each rice strain plot, with three random sampling points per strain and two sticky traps at each point. After three days, the sticky traps were collected, and the trapped insects were identified and counted to provide a preliminary assessment of pest presence during the reproductive growth stage.\u003c/p\u003e\n \u003cp\u003eField plant diversity survey: For the rice cultivated outdoors, plant diversity was surveyed at the tillering stage (immediately after transplanting) and at maturity. At each stage, 3\u0026ndash;5 plots per strain were randomly selected. All plants within a 0.25 m\u0026sup2; area (50 cm \u0026times; 50 cm) surrounding each plot were collected, and their species diversity and biomass were recorded to preliminarily assess changes in plant diversity before and after planting of the transgenic materials.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). All experiments were conducted under a single-variable design, and P values were calculated using ordinary one-way analysis of variance (ANOVA) with \u0026alpha; set to 0.05. All experiments were set up with at least three biological replicates. All data were verified using Levene\u0026apos;s Test for Homogeneity of Variances, and met the assumption of equal variances (except for the flag leaf width of T3 and the plant weight of T1). For data that met the assumption of homogeneity of variances, the LSD Test and Duncan\u0026apos;s Test were performed; for data that did not meet this assumption, Tamhane\u0026apos;s Test was conducted. All graphs were generated using GraphPad Prism version 8.0.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of the Insertion Sites of Exogenous Genes in Transgenic Rice\u003c/h2\u003e\u003cp\u003eAnalysis of exogenous gene insertion sites was performed on the T1-T3 generations of the HN-1 transgenic rice strain, and the results showed: The reference genome size for gene sequencing was 385.71M. The comparison rates of the three samples ranged from 96.22% to 99.78%, coverage of the reference genome ranged from 98.31% to 98.38%, and the average sequencing depth ranged from 25.491X to 39.316X. The insertion sequences in all three samples were integrated into the nuclear chromosomes of the cells (Figs.\u0026nbsp;1A-1C). A comparison of different reads indicated that all three samples exhibited chromosomal translocations relative to the parental genome. Specifically, the regions from 32,680,503 to 32,681,743 bp on chromosome 1 (Chr1) were translocated to Chr3. This translocation allowed for the identification of the upper and lower boundaries of the insertion site on Chr1 and Chr3, respectively. The upper boundary of the insertion on Chr1 was 32,681,743 bp, whereas the lower boundary of the insertion on Chr3 was 31,802,579 bp (Figs.\u0026nbsp;1D-1F). The insertion intervals at the upper and lower borders within the genes were as follows: the upper boundary at Chr1:32,681,743 was located within the gene AGIS_Os01g048210 (Chr1:32, 681, 631\u0026ndash;32, 683,642), and the lower boundary at Chr3:31,802,579 was found in the gene AGIS_Os03g046780 (Chr3:31,793,752\u0026ndash;31,802,369) as well as in the gene AGIS_Os03g046790 (Chr3:31,803,572\u0026ndash;31,804,793). Based on the available sequencing data, the three samples were analyzed using deep variant comparisons of the on-target reads for the upper exogenous sequences, and no variants were identified (Figs.\u0026nbsp;1G and 1H).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of the genetic stability of exogenous genes at the DNA, RNA and protein levels\u003c/h2\u003e\u003cp\u003ePolymerase chain reaction (PCR) was used to detect the presence of exogenous genes HN-1 and HN-2 in TP309 and T1\u0026ndash;T3 generation transgenic strains. DNA extracted from panicles (Fig.\u0026nbsp;2A, 2C) and leaf tissues (Fig.\u0026nbsp;2B, 2D) of transgenic rice plants from the T1\u0026ndash;T3 generations of each strain served as templates. The exogenous HN gene was stably expressed throughout the T1-T3 generations. The relative expression levels of the exogenous genes HN-1 and HN-2 in the TP309 and T1-T3 generation transformant strains were assessed by quantitative reverse transcription polymerase chain reaction, using complementary DNA derived from the leaf tissues of each strain obtained through reverse transcription as a template. HygR served as an endogenous gene and the expression levels of exogenous genes in the rice spike tissues of TP309 were used as a basestrain reference. All exogenous insertion genes were expressed in the leaf tissues of HN-1, HN-2 at levels ranging from approximately 8- to 400-fold (Figs.\u0026nbsp;2E, 2F and 2G), demonstrating that the exogenous HN gene can persist across different strains of multi-generation transgenic rice. The chicken Newcastle disease virus HN protein was consistently expressed across all three generations of the transgenic strain, as confirmed by western blotting (Figs.\u0026nbsp;2H and 2I). In addition, the antigen titers of different generations of HN were evaluated using a test paper, revealing a relatively stable titer, which was maintained at 2^9 (Fig.\u0026nbsp;2J). Insertion site analysis, DNA, mRNA and protein levels confirmed that the HN protein of Newcastle Disease Virus (NDV) was stably present in the transgenic strains.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of germination rate, emergence rate and development period\u003c/h2\u003e\u003cp\u003eIn the rice seed germination rate determination experiment, the germination rates of HN-1 at 12, 24, 36, and 48 hours were 30%, 70%, 86%, and 96%, respectively. The germination rates of HN-2 at 12, 24, 36, and 48 hours were 44%, 96%, 97%, and 97%, respectively. In the seedling emergence rate determination experiment, the germination rates of HN-1 at 8, 16, 24, and 32 hours were 30%, 70%, 86%, and 96%, respectively, while HN-2 had germination rates of 44%, 96%, 97%, and 97% at 8, 16, 24, and 32 hours, respectively. The results indicated that the germination (Fig.\u0026nbsp;3A) and seedling emergence rates (Figs.\u0026nbsp;3B and 3C) of HN-1, HN-2, and TP309 were comparable, with no statistically significant differences observed. Three successive generations of transgenic rice strains HN-1 and HN-2, along with wild-type rice, were cultivated outdoors to investigate differences in growth and development between the transgenic strains and wild-type plants and to monitor their fertility cycles. The total growth cycles of TP309 and the transgenic strains were relatively stable, fluctuating between 150 and 180 days (Fig.\u0026nbsp;3D).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eComprehensive evaluation of agronomic traits\u003c/h2\u003e\u003cp\u003eThe number of spikes, the number of grains per spike, and the grain quality of the rice directly determine the overall yield. Yield-related factors during rice development can be categorized as growth morphology, spike status, and grain quality. This study observed agronomic traits associated with these three factors in T1, T2, and T3 rice generations from strains HN-1, HN-2, and TP309. In the T1 generation, the ear length of the HN-1 strain was slightly shorter than that of TP309, whereas the differences in the other traits were not significant. The seed-setting percentage of HN-2 was lower than that of TP309 with no significant differences in the remaining traits. When comparing the two strains, HN-1 exhibited a significantly higher seed-setting percentage and grain density per centimeter than HN-2, although the other traits did not show significant differences (Fig.\u0026nbsp;4A). In the T2 generation, the differences in the 12 agronomic traits between HN-1 and TP309 were not significant. However, the differences between HN-2 and TP309 were significant, particularly regarding the reduction in ear length and mass of the effective panicle. When comparing the two strains, the spike length and effective spike mass of HN-2 were lower than those of HN-1, whereas the differences in the other traits were not significant (Fig.\u0026nbsp;4B). In the T3 generation, the effective panicle mass of the HN-1 strain was significantly lower than that of the TP309 strain, with no significant differences in the remaining traits (Fig.\u0026nbsp;4C). Conversely, the mass of the effective panicle and thousand-grain mass of HN-2 were significantly higher than those of TP309, with no differences in the other traits. When comparing the two strains, the thousand-grain mass of HN-2 was greater than that of HN-1, although the differences in other traits were not significant.\u003c/p\u003e\u003cp\u003eIn addition, we measured the grain length, grain width, grain thickness, brown rice mass, chalkiness, and other qualities of the seeds harvested from the T1, T2, and T3 generations. The grain length (Figs.\u0026nbsp;5A and 5D), grain width (Fig.\u0026nbsp;5B and 5D), grain thickness (Fig.\u0026nbsp;5C), and brown rice yield of the transgenic rice strains HN-1 and HN-2 did not differ significantly from those of TP309 (Fig.\u0026nbsp;5E). However, the chalkiness rate and degree increased significantly compared with TP309, particularly in the HN-2 rice strain, whereas no significant changes were observed in the HN-1 rice strain (Fig.\u0026nbsp;5E). This finding suggests that the increase in the chalky white rate and degree in the HN-2 rice strain, attributed to insufficient grouting material, may be influenced by various environmental factors, such as climatic conditions and cultivation methods during the grouting period, or maybe an inherent characteristic of this transgenic strain. The data indicated that the growth and development of the rice strains HN-1 and HN-2 were comparable to those of TP309 plants, demonstrating that the agronomic traits of these strains remain robust. Scanning electron microscopy further revealed changes in the internal structure of transgenic rice, showing an increase in branched-chain starch and a decrease in straight-chain starch (Fig.\u0026nbsp;6). Consequently, the visible increase in chalkiness in the HN-2 rice strain makes it easier to break. Because we primarily extracted the target proteins from milled grains, this strain shows great potential for applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of survival competitiveness\u003c/h2\u003e\u003cp\u003eRice typically produces seeds through self-pollination, and the viability of rice pollen is closely linked to its reproductive capacity, which directly influences the competitive survival of rice plants against surrounding vegetation. To evaluate the competitive survival ability of transgenic rice strains and TP309 in the field, mature anthers were collected from plants of the T1\u0026ndash;T3 generations of transgenic rice at the early flowering stage, and these anthers were subjected to iodine-potassium iodide (I-KI) staining.The pollen status of the transgenic rice and TP309 was observed at various time intervals (t0, t3, and t6). Across the T1 (Figs.\u0026nbsp;7A and 7B), T2 (Figs.\u0026nbsp;7C and 7D), and T3 (Figs.\u0026nbsp;7E and 7F) generations, Statistical analysis revealed no significant changes in the normal pollen rate of the transgenic strains or TP309 over time, nor were there any significant differences in the proportion of normal fertile pollen between the transgenic strains HN-1, HN-2, and TP309. The results of this experiment indicated that the pollen longevity of transgenic rice was stable and highly viable when compared to TP309, demonstrating no significant difference in pollen viability between transgenic rice and TP309 plants. Consequently, both species exhibited similar capabilities in competing for survival against other species in the field.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of gene flow capacity and field biodiversity\u003c/h2\u003e\u003cp\u003eTo assess the potential of exogenous gene transfer from transgenic rice strains to surrounding plants, leaves of various weeds in paddy fields of T1-T3 generations of transgenic rice grown outdoors were collected and analyzed. Specific primers were used to detect the presence of the Newcastle disease virus HN gene in non-rice plants (Fig.\u0026nbsp;8A, 8B).To evaluate whether transgenic rice would affect the populations and diversity of surrounding organisms in the field, an insect diversity experiment was conducted using the milky rice, which exhibited more severe insect pest issues. Sticky traps were placed in wild-type rice plots and T3 generation blocks of each transgenic rice strain (each block measuring 50 cm \u0026times; 50 cm). After three days of placement, the number of Lepidopteran insects and arthropods on the sticky traps was counted. The results indicated that the number of lepidopterans around the rice-planting area was significantly higher than that of arthropods. However, there was no significant difference in the species and number of insects between the TP309 block and transgenic strains HN-1 and HN-2 (Fig.\u0026nbsp;8C). The plant diversity experiment randomly sampled blocks of TP309 and transgenic strains. During the harvesting periods, the species and masss of all other plants in the blocks were recorded. Compared with the TP309 control, the weed species and quality surrounding the transgenic strains HN-1 and HN-2 did not show any significant effects (Fig.\u0026nbsp;8D). Thus, it can be tentatively concluded that the impact of transgenic rice on the biodiversity of the surrounding environment is relatively small.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNewcastle disease (ND) has been defined by the World Organisation for Animal Health as infection of poultry with virulent strains of Newcastle disease virus (NDV). Lesions affecting the neurological, gastrointestinal, respiratory, and reproductive systems are most often observed. The control of ND must include strict biosecurity that prevents virulent NDV from contacting poultry, and also proper administration of efficacious vaccines\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. When administered correctly to healthy birds, ND vaccines formulated with NDV of low virulence or viral-vectored vaccines that express the NDV fusion protein are able to prevent clinical disease and mortality in chickens upon infection with virulent NDV. Live and inactivated vaccines have been widely used since the 1950\u0026rsquo;s\u003csup\u003e18\u003c/sup\u003e. Recombinant and antigenically matched vaccines have been adopted recently in some countries, and many other vaccine approaches have been only evaluated experimentally. Despite decades of research and development towards formulation of an optimal ND vaccine, improvements are still needed. Impediments to prevent outbreaks include uneven vaccine application when using mass administration techniques in larger commercial settings, the difficulties associated with vaccinating free-roaming, multi-age birds of village flocks, and difficulties maintaining the cold chain to preserve the thermo-labile antigens in the vaccines. Incomplete or improper immunization often results in the disease and death of poultry after infection with virulent NDV. Another cause of decreased vaccine efficacy is the existence of antibodies (including maternal) in birds, which can neutralize the vaccine and thereby reduce the effectiveness of ND vaccines. Based on this, there is an urgent need to develop an efficient Newcastle disease vaccine that is easy to mass-produce, easy to store and transport, and low in price\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBased on the results of previous animal experiments, in order to achieve the commercial production of this genetically modified rice (HN-1, HN-2), intermediate trials were conducted at the designated site (Transgenic Rice Experimental Base in Xinjiang, China) to evaluate the genetic stability, agronomic traits, and biosafety of the transgenic rice across three consecutive generations. In this study, we used a rice endosperm expression system previously established in our laboratory to advance the commercialization of the Newcastle disease vaccine. We verified the stability of exogenous gene integration, expression, and functional trait expression across three successive planting generations. Additionally, compared with TP309, the transgenic rice strains exhibited minimal differences in several physiological traits, including seed germination, developmental cycle, plant growth morphology, spike position, and seed quality. Importantly, the gene insertion did not affect these functional traits. The gene transfer test further demonstrated that the Newcastle disease virus HN gene in transgenic rice did not disperse to neighboring plants in the surrounding area, and preliminary surveys on the species and population sizes of insects and arthropods around the T3 generation transgenic rice plants preliminarily indicated that the two transgenic rice strains (HN-1 and HN-2) exerted no adverse effects on the local ecosystem. This underscores the advantages of this expression system, which is not only promising for vaccine applications but also serves as a significant reference for producing other medicinal proteins.\u003c/p\u003e\u003cp\u003eThese findings provide critical validation for rice-based molecular farming as a promising platform for vaccine development. Compared with traditional Newcastle disease vaccines\u0026mdash;including inactivated, live attenuated, and viral vector-based formulations\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e\u0026mdash;our rice-derived HN antigen offers several key advantages. First, it directly addresses the challenge of antigenic mismatch, as plant-based systems can be rapidly engineered to express antigens corresponding to prevalent NDV genotypes, such as genotype VII, which currently accounts for most field outbreaks. Second, plant-derived vaccines offer low-cost production without the requirement for specialized fermentation infrastructure or cold-chain logistics, greatly enhancing accessibility in low-resource settings. Third, rice endosperm bioreactors provide the possibility of oral vaccine delivery, which could simplify immunization programs by reducing the need for multiple injections and trained personnel\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur findings align with and extend previous work on molecular farming\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For example, the \u0026ldquo;head-to-tail\u0026rdquo; dimer antigen design reported in \u003cem\u003ePNAS\u003c/em\u003e demonstrated that rice-expressed HN proteins could induce potent immune responses and achieve complete protection in chickens at remarkably low doses\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The present study complements this by providing essential data on genetic stability and biosafety, thereby addressing critical prerequisites for commercialization. Taken together, these advances highlight the potential of transgenic rice to serve as both a cost-effective production platform and a novel oral delivery vehicle for NDV subunit vaccines. Despite these promising results, several challenges remain before rice-based NDV vaccines can reach practical application. First, although immunogenicity has been demonstrated in controlled experiments, larger-scale and longer-term studies are needed to confirm consistent efficacy under field conditions. Second, the scalability of downstream processing, including antigen extraction and dosage standardization, requires further optimization to meet industrial production standards. Third, public acceptance and regulatory approval of genetically modified crops used for pharmaceutical purposes remain major hurdles. Addressing biosafety concerns, particularly those related to gene flow and food chain contamination, will be crucial for the successful adoption of such vaccines. Finally, it remains necessary to compare the performance of rice-based vaccines with other plant-based systems (e.g., maize, tobacco, or tomato) to determine the most suitable platform for different antigen targets and delivery strategies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study provides relatively comprehensive evidence supporting the genetic stability, agronomic feasibility, and biosafety of transgenic rice expressing NDV HN protein. Together with prior immunogenicity studies, these results establish a solid foundation for the development of rice-based NDV subunit vaccines. Integrating genetic engineering advances with rigorous field evaluation and regulatory frameworks will be essential to accelerate the translation of molecular farming technologies into commercial poultry vaccines. Beyond Newcastle disease, the strategies demonstrated here may also inform the broader development of plant-derived vaccines and therapeutics for both animal and human health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Development of rapid detection and diagnostic technology for Giardia, Coccidioidomycosis and Anaplasmosis (2023YFD1801203), key scientific and technological projects of Henan Province (222102110210) and Major Science and Technology Projects in Henan Province (241110310200).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e\"Conceptualization, G.Z., X.W. and W.G.; methodology, H.C., Z.H., L.Z., F.L. and Y.H.; software, H.C. and L.H.;validation, L.Z.,H.C., Z.H., Y.H., Y.L., S.B., W.C., J.Z. and Y.Z.; formal analysis, L.Z., H.C., and Z.H.; investigation, S.P., S.K.and H.C.; resources, G.Z., X.W.; data curation, H.C., Z.H., L.Z. and Y.H.; writing\u0026mdash;original draft preparation, L.Z. and H.C.; writing\u0026mdash;review and editing, L.Z. and H.C.; visualization, L.Z. and H.C.; supervision, X.W., and W.G.; project administration, X.W.; funding acquisition, G.Z. and X.W..\"\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe thank the reviewers and editors for their thoughtful comments and suggestions regarding the manuscript. We thank Editage for providing assistance with language editing.The authors would like to express their sincere gratitude to the staff and plantation managers of the Changji Comprehensive Experimental Base of the Chinese Academy of Agricultural Sciences for their assistance. In addition, the data for insertion site analysis of exogenous genes in transgenic rice (Fig.\u0026nbsp;1 in the article) were provided by Wuhan Benan Gene Technology Co. We would like to express our gratitude to this company.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eUl-Rahman, A. et al. Zoonotic potential of Newcastle disease virus: Old and novel perspectives related to public health. \u003cem\u003eRev. Med. Virol.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, e2246 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCosta-Hurtado, M. et al. Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication, disease, and mortality in chickens. \u003cem\u003eVet. Res.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 97 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, L. et al. Phylodynamic analyses of class I Newcastle disease virus isolated in China. \u003cem\u003eTransbound. Emerg. Dis.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, 1294\u0026ndash;1304 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerihulay, H. et al. Exploring the genetic basis of Newcastle disease virus in chickens: a comprehensive review. \u003cem\u003eFront. 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Newcastle disease virus: is an updated attenuated vaccine needed? \u003cem\u003eAvian Pathol.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 467\u0026ndash;478 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu, Z. et al. Current situation and future direction of Newcastle disease vaccines. \u003cem\u003eVet. Res.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, 99 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHein, R. et al. Review of Poultry Recombinant Vector Vaccines. \u003cem\u003eAvian Dis.\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e, 438\u0026ndash;452 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGomes, C. W. C. et al. Newcastle disease vaccination in captive-bred wild birds. \u003cem\u003eTrop. Anim. Health Prod.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 1349\u0026ndash;1353 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuma, I. et al. 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Molecular farming using transgenic rice endosperm. \u003cem\u003eTrends Biotechnol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 1248\u0026ndash;1260 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1.1 to 1.3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intermediate tests, Biosecurity, field performance, genetic stability","lastPublishedDoi":"10.21203/rs.3.rs-7453085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7453085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBased on the previously verified head-to-tail dimer vaccine model, a dimer of the HN protein (defined as Osr2HN) expressed in rice endosperm was designed. In previous immunization experiments in chickens, immunization with two doses (0.5 \u0026micro;g, equivalent to 1/127 of a grain of rice) or a single dose (5 \u0026micro;g) provided complete protection. To advance the commercialization process of this product, in this study, we selected two transgenic rice strains (HN-1 and HN-2) and cultured them for three generations to evaluate their genetic stability, agronomic traits, and safety. Insertion site analysis showed that exogenous genes were stably integrated into nuclear chromosomes with no variants, as confirmed by PCR, qRT-PCR, and Western blotting. The transgenic strains exhibited germination rates, growth cycles, and 12 agronomic traits similar to those of the wild-type TP309, though HN-2 showed increased chalkiness. Pollen viability remained unchanged, and no transfer of the HN gene to weeds was detected. Field biodiversity analysis revealed no impact of the HN gene on pest and weed communities. These findings validate the transgenic rice\u0026rsquo;s genetic stability, agronomic adaptability, and environmental safety, providing critical data to support the acceleration of its commercialization as a plant-derived vaccine platform.\u003c/p\u003e","manuscriptTitle":"Genetic stability, environmental safety and field adaptation assessment of transgenic rice with chicken Newcastle disease HN protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 12:16:41","doi":"10.21203/rs.3.rs-7453085/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-25T09:56:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T13:27:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-29T16:57:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307715636039437277542057161986339290750","date":"2025-10-20T06:59:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235919764362978461353593191201702797356","date":"2025-10-15T12:55:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T06:12:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-15T04:13:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-15T04:07:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc67cd08-912d-4612-8edc-b6abf5908866","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":56882189,"name":"Biological sciences/Biological techniques"},{"id":56882190,"name":"Biological sciences/Biotechnology"},{"id":56882191,"name":"Biological sciences/Genetics"},{"id":56882192,"name":"Biological sciences/Molecular biology"},{"id":56882193,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-02-12T04:09:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 12:16:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7453085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7453085","identity":"rs-7453085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}