Loquat (Eriobotrya japonica Lindl.), a subtropical evergreen species of the Rosaceae family, faces industry constraints in industrial development due to its sensitivity to freezing temperatures and low photosynthetic efficiency. Polyploid loquats, particularly triploids, exhibit enhanced stress resistance, vigorous growth, and seedless fruit production. In this study, triploid F1 progeny (B431 × GZ23) was obtained through hybridization between diploid (GZ23) and tetraploid (B431) parents. Under −3°C stress, the triploid lines exhibited significantly improved freezing tolerance and photosynthetic performance, as evidenced by chlorophyll fluorescence parameters and ultrastructural integrity. Lipidomics profiling across all B431 × GZ23 lines revealed that phosphatidylcholine (PC), particularly the abundant unsaturated PC species 18:2/18:2, played a key role in these adaptive advantages. Compared to the parental lines, EjFAD2 expression was specifically upregulated in triploid loquats under freezing stress. Consistent lipidomic and gene expression patterns across three B431 × GZ23 lines ruled out line-specific mutations. Heterologous expression of EjFAD2 in Arabidopsis increased freezing tolerance. Co-expression analysis identified EjMYBS3 as a regulator that binds to the EjFAD2 promoter, and its overexpression in transgenic Arabidopsis enhanced freezing tolerance. Transient expression of EjFAD2 and EjMYBS3 increased the content of PC 18:2/18:2 in loquat, which contributed to the maintenance of photosystem activity under freezing stress, thereby enhancing the freezing tolerance of loquat. Collectively, these findings provide preliminary insights into the molecular mechanisms underlying cold resistance in polyploid loquats and highlight the regulatory role of EjFAD2 and EjMYBS3 in freezing stress response.

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