Developmental and Molecular Insights into AgNO3-Induced Partial Masculinization and Stamen Abortion in Siraitia grosvenorii C. Jeffrey ex A.M. Lu and Zhi Y. Zhang

This study focuses on Siraitia grosvenorii, an economically important crop endemic to Guangxi Province, China, with significant value in both the pharmaceutical and food industries. However, its dioecious reproductive system results in low natural pollination efficiency, necessitating reliance on costly artificial pollination in commercial production, which severely constrains large-scale cultivation and the breeding of improved varieties. Herein, we systematically investigated the molecular mechanisms of silver nitrate (AgNO3)-induced partial masculinization of female flowers, resulting in the formation of morphologically bisexual flowers with aborted stamens. Morphological observations revealed that untreated female flowers exhibited permanently arrested stamen primordia, whereas spraying treatments with 0–300 mg/L AgNO3 triggered the development of morphologically complete bisexual flowers. Nevertheless, AgNO3-induced stamens exhibited severely disrupted male reproductive development, as indicated by the absence of mature pollen grains at anthesis. Cytological evidence demonstrated that pollen abortion was initiated at the pollen mother cell (PMC) stage, accompanied by abnormal vacuolation and impaired nutrient metabolism in tapetal cells. The dominant abortive phenotype was that PMCs exhibited abnormal development and progressive degeneration during the meiotic developmental window. Methylation-sensitive amplification polymorphism (MSAP) assays further revealed markedly elevated CpG-type methylation levels in AgNO3-induced bisexual flowers at early developmental stages. Expression analysis revealed that SgWIP1 exhibited distinct temporal expression patterns among female, male, and AgNO3-induced bisexual flowers, with reduced expression during later stages of stamen development in induced bisexual flowers. Collectively, our findings confirm that AgNO3 only partially activates the stamen developmental cascade in S. grosvenorii. Extensive epigenetic reprogramming triggered by AgNO3, together with the inability to sustain the expression of core male-determining genes, is tightly linked to tapetal dysfunction and subsequent pollen sterility. This study aims to elucidate the unique mechanism by which AgNO3-induced partial masculinization in S. grosvenorii from multiple perspectives—including tissue and cellular structure, DNA methylation epigenetic modifications, and gene transcription regulation—using techniques such as morphoanatomy, histochemistry, epigenetics (MSAP), and gene expression analysis. The findings of this study will contribute to molecular breeding efforts focused on sex regulation and promote industrial development.