Exogenous progesterone and β-estradiol promote the proliferation of callus in Agapanthus praecox by modulating hormone signaling and antioxidant system

Abstract

Callus proliferation is essential for large-scale in vitro culture of Agapanthus praecox . However, long-term continuous subculture frequently leads to physiological decline of callus, accompanied by slowed proliferation rate, which restricts the efficiency of callus maintenance and large-scale culture systems. In this study, two-year continuously subcultured calli was used to explore how exogenous progesterone (Prog) and β-estradiol (EE) regulate callus proliferation through combined morphological observation, physiological measurement, RNA-seq and quantitative real-time polymerase chain reaction (qRT-PCR) validation. The results showed that both steroids significantly promoted callus proliferative capacity, with optimal effects at 1.0 mg·L –1 Prog and 0.5 mg·L –1 EE. Morphological and transcriptional data revealed divergent regulatory modes: Prog was predominantly associated with single-cell expansion, whereas EE was correlated with increased cell division frequency to facilitate overall callus growth. Both treatments elevated endogenous IAA and cytokinin (CTK) contents, modulated multi-hormone signaling networks, and upregulated genes involved in cell cycle progression, stem cell maintenance and cell wall remodeling. Meanwhile, Prog and EE activated multiple antioxidant enzymes, reduced the accumulation of H₂O₂ and MDA, and stabilized intracellular redox homeostasis. Transcriptome profiling coupled with weighted gene co-expression network analysis (WGCNA) identified six core candidate hub genes represented by CYP85A1 and ERF2 , which are associated with hormone signal transduction, stress adaptation, and cellular growth. In conclusion, this study indicates that the animal-derived steroid hormones Prog and EE may promote the proliferation of long-term subcultured callus by potentially modulating endogenous hormone homeostasis and antioxidant pathways through two separate cell growth regulatory routes. These results provide a preliminary theoretical reference for optimizing long-term subcultured callus growth systems with steroid regulators.