Abstract
<title>Abstract</title> <p> Background The establishment of adaxial-abaxial polarity in leaves is fundamental to their planar growth, regulated by intricate genetic networks. While ⁶⁰Co-γirradiation is a widely used physical mutagen for generating novel plant germplasm, the molecular links between radiation-induced morphological variations and endogenous developmental programs remain unclear. Results Using <italic>Phalaenopsis equestris</italic> as a model, we observed that 40Gy 60Co-γirradiation significantly induces heteromorphic leaf phenotypes. Morphological and cytological analyses revealed increased leaf length-to-width ratios and decreased surface area post-irradiation, characterized by disordered palisade and spongy tissue arrangement and a significant reduction in cell roundness. Transcriptomic analysis identified numerous differentially expressed genes enriched in cell wall metabolism and adaxial-abaxial polarity establishment pathways. Both the abaxial determinant <italic>PeKAN2</italic> and the adaxial determinant <italic>PeAS1</italic> were significantly downregulated post-irradiation. The synergistic downregulation of <italic>PeAS1</italic> and <italic>PeKAN2</italic> disrupted the dynamic balance between adaxial and abaxial determinants, driving abnormal anisotropic cell expansion and ultimately altering leaf polar growth. Virus-induced gene silencing of <italic>PeKAN2</italic> or <italic>PeAS1</italic> individually resulted in significant changes in Phalaenopsis leaf morphology and caused cascading dysregulation of the entire polarity gene network, including <italic>PeHD-ZIP</italic> , <italic>PeYABBY</italic> , and <italic>PeARF</italic> . Conclusions This study provides new insights into leaf polarity regulation in non-model plants and establishes a theoretical foundation and molecular targets for radiation mutagenesis breeding in orchids. </p>