Risk Evaluation and Molecular Characterisation of <scp> <i>AtNPR1</i> </scp> Transgenic Citrus Lines Tolerant to Citrus Greening Disease

Citrus greening disease, or Huanglongbing (HLB), has caused devastating losses to citrus production in Florida, with yields declining by over 90% since 2005. Despite extensive efforts, no sustainable solution has been widely effective. Here, transgenic 'Hamlin' sweet orange lines engineered to constitutively express the Arabidopsis NPR1 (AtNPR1) gene, a key regulator of systemic acquired resistance, are evaluated for health and environmental risks. These citrus lines exhibit strong HLB tolerance, with reduced disease symptoms, sustained fruit production, and no apparent negative phenotypic abnormalities. Comprehensive risk assessment reveals minimal exposure, health, or environmental risk. The AtNPR1 protein is: (1) barely detectable in fruit, (2) rapidly degraded in simulated gastrointestinal fluids, and (3) not similar to known allergens or toxins. Whole-genome sequencing identified the T-DNA insertion sites as heterozygous in either chromosome 1 or 6, with no disruptions in known fruit-producing genes. PCR markers were developed for rapid line identification. The selected lines are currently in a small field trial under high HLB pressure and continue to exhibit low visual HLB symptoms and positive horticultural traits. These findings support the initial requirements for regulatory approval of these transgenic citrus varieties, offering a promising strategy for sustainable citrus production.