Seedless is a highly valued commercial characteristic in the citrus industry, both for fresh consumption and for processed markets. In this study, the ‘Succari Sweet Orange’ (WT) and its seedless mutant ‘Juxiangyuan’ (MT), which originated from a bud mutation, were selected to study the formation of a citrus seedless phenotype. Microscopic analysis of MT’s floral organs, including anther and ovary cross-sections, provides insights into its seedless phenotype compared to the original seeded cultivar. Additionally, pollen features, viability, and in vitro germination were examined to determine the cause of seedlessness. MT exhibited significant developmental deformities in both male and female gametes, with pollen grain analysis indicating a high rate of deformity (41.48%), low viability (9.59%), and minimal in vitro germination (9.56%). Hybridization experiments were conducted to assess male and female sterility and pollen incompatibility. Both WT and MT exhibited parthenocarpic development. Notably, MT fruit produced with an average of 3.51 seeds pollinated to WT, despite severe pollen abortion of MT. MT, however, produced seedless fruit through self-breeding or cross-breeding with other varieties, demonstrating stable female sterility. Despite reduced pollen quantity and viability in the seedless mutant ‘Juxiangyuan’, its seedlessness primarily stems from female sterility. This study contributed to a deeper understanding of seedless formation in ‘Juxiangyuan’ and provided valuable information relevant to its commercial cultivation.

Cold stress is a critical abiotic factor that severely limits plant growth and agricultural productivity in subtropical regions. Poncirus trifoliata exhibits exceptional cold hardiness and is widely used as a rootstock in Citrus. However, the key genes and mechanisms conferring this resilience remain largely unexplored. Here, we characterized PtCOR8, a cold-induced gene isolated from P. trifoliata. Phylogenetic and subcellular localization analyses confirmed that PtCOR8 encodes a plasma membrane-localized protein belonging to the WCOR413 family. Functional validation revealed that heterologous overexpression of PtCOR8 in tomato significantly enhanced cold tolerance, concomitant with reduced malondialdehyde (MDA) content, elevated peroxidase (POD) activity, and upregulation of cold-responsive genes (e.g., CIN8). Notably, expression profiling of COR8 in 16 citrus accessions under natural overwintering conditions indicated a strong positive correlation between its expression level and cold tolerance of different genotypes. Transgenic tomato plants with PtCOR8 driven by its native promoter also presented enhanced cold tolerance, confirming that the native promoter is sufficient to drive functional expression under cold stress in the tomato system. Through promoter deletion and β-glucuronidase (GUS) staining experiments, the ATCT motif was further identified as a cis-acting element capable of mediating cold-induced promoter activity. Our findings uncover a dual-layered mechanism in which the PtCOR8 protein alleviates membrane lipid peroxidation and oxidative damage, while its transcription level is precisely modulated by a novel promoter regulatory mechanism, thereby improving freezing tolerance. This study provides important genetic insights and a valuable gene resource for cold-resistant citrus breeding.