Mechanisms underlying the formation of seedlessness in Leigong No. 1 pummelo

【Objective】Seedlessness is a highly desirable horticultural trait in citrus and has emerged as a major breeding target due to consumer preference for seedless fruit. Multiple mechanisms can generate seedless citrus, including male sterility, female sterility, embryo abortion, and self-incompatibility. For pummelo cultivars, self-incompatibility is the predominant cause of seedlessness. Recent studies on several local pummelo varieties, such as Juxianghong pummelo, Dongshi Zaoyou pummelo, and Gongshui Baiyou pummelo, have demonstrated that their seedlessness primarily results from self-incompatibility. Citrus self-incompatibility has been demonstrated to be a gametophytic system mediated by S-RNase, whereby pollination between identical S-genotypes fails to produce normal seeds, whereas cross-pollination between different S-genotypes yields viable seeds. The combination of self-incompatibility and parthenocarpic ability enables stable production of seedless citrus fruit. Leigong No. 1 pummelo is an excellent local cultivar from Chenzhou, which is highly favored by consumers for its crisp flesh and distinctive flavor. However, this variety often presents a mixture of seedy and seedless fruit types in the market, which adversely affects its fresh consumption and industrial promotion. The under-lying cause of seedlessness in Leigong No. 1 pummelo remains unclear. Therefore, this study aims to investigate the mechanism of seedlessness in Leigong No. 1 pummelo, providing a theoretical basis for stable production of seedless fruits and for seedless breeding in citrus.【Methods】Mature flowers of Leigong No. 1 pummelo were dissected to examine floral organ morphology. Pollen viability was assessed using acetic carmine staining. Artificial emasculation was performed to evaluate the parthenocarpic ability of Leigong No. 1 pummelo. Self-pollination was conducted on Leigong No. 1 pummelo, followed by cross-pollination using it as the female parent and Cocktail grapefruit, Chunxiang tangelo, and Sanhong Miyou pummelo as male parents. At 5 days post-pollination, aniline blue staining was used to observe the pollen tubes from self- pollination and cross- pollination to determine whether fertilization was successful. The number of fruits and seeds was counted to calculate the fruit set rate and the number of seeds per fruit. Previously reported S-RNase-specific primers were used to identify the S-genotype of Leigong No. 1 pummelo, and semi-quantitative PCR was performed to analyze the expression pattern of S- RNase in this variety.【Results】Morphological examination of mature floral buds from Leigong No. 1 pummelo revealed normal floral structures, with both pistils and stamens well developed. Pollen viability assessed by acetic carmine staining was high across three consecutive years: 95.51% in 2022, 93.55% in 2023, and 96.50% in 2024, indicating male fertility. Artificial emasculation without pollination resulted in a fruit set rate of 14%, and all fruits were seedless, demonstrating that Leigong No. 1 pummelo exhibits a certain degree of parthenocarpy. Pollination experiments were performed to observe pollen tube growth 5 days after pollination. The results showed that, following selfpollination, pollen grains of Leigong No. 1 pummelo germinated abundantly on the stigma, but tube growth was arrested within the style, indicating self-incompatibility. In contrast, when Leigong No. 1 pummelo was used as the female parent and pollinated with Cocktail grapefruit, Chunxiang tangelo, or Sanhong Miyou pummelo, pollen tubes grew normally to the base of the style, demonstrating crosscompatibility and female fertility. Statistical analysis of fruit set and seed number in Leigong No. 1 pummelo showed that the self-pollination fruit set rate was 12.00%, with an average of 0.92 seeds per fruit. All 11 seeds obtained were shriveled and abnormal, and none were able to germinate into seedlings. In contrast, cross-pollination with Cocktail grapefruit, Chunxiang tangelo, or Sanhong Miyou pummelo resulted in fruit set rates of 8.00%, 43.94%, and 50.77%, respectively, with average seed numbers per fruit of 38.63, 45.72, and 75.52. The majority of these seeds were plump and viable, capable of developing into seedlings after germination and sowing. These results further confirm that seedlessness in Leigong No. 1 pummelo is primarily caused by self-incompatibility. Using published primers for citrus S-RNase genes, we identified the S-genotype of Leigong No. 1 pummelo as S3S5. Semi-quantitative PCR revealed that both S3- and S5-RNase alleles were weakly expressed in petals, filaments, and anthers, but exhibited strong, tissue-specific expression in pistils, including the ovary, stigma, and style, with both showing higher relative expression levels in the style than in the ovary and stigma.【Conclusion】The seedless fruit formation of the local cultivar Leigong No. 1 pummelo from Chenzhou is mainly attributed to the combined effects of self-incompatibility and parthenocarpy. In production practices, implementing isolation planting to prevent cross-pollination can achieve stable production of seedless fruits. Furthermore, the self-incompatibility in this cultivar is a gametophytic type mediated by S-RNase, with the S-genotype identified as S3S5. These results provide valuable reference data for the design of pollination combinations in future seedless citrus breeding programs, facilitating the development of improved citrus varieties with reliable seedless traits.