Integrated Multi-Omics Reveals Mechanism of Adventitious Buds Regeneration in In Vitro Cultures of Cinnamomum parthenoxylon

A pluripotent callus is central to genetic transformation in <i>Cinnamomum parthenoxylon</i>; however, the molecular and cellular mechanisms regulating callus formation and subsequent differentiation remain unelucidated, hindering progress in its genetic improvement. This study systematically investigated the dynamic changes during the in vitro regeneration of <i>C. parthenoxylon</i> through morphological observations, physiological assays, and transcriptomic analyses, while comparing differences in callus formation under varying induction conditions to elucidate the mechanism of its high-efficiency regeneration. The results showed that the formation of a pluripotent callus is a critical step in <i>C. parthenoxylon</i> regeneration, characterized by the presence of highly proliferative cell zones. Compared to an ordinary callus (P3C), a pluripotent callus (P3) exhibited higher activities of polyphenol oxidase (PPO) and indole-3-acetic acid oxidase (IAAO), as well as elevated levels of zeatin riboside (ZR) and abscisic acid (ABA). In contrast, P3 showed lower levels of soluble sugars, soluble proteins, malondialdehyde (MDA), indole-3-acetic acid (IAA), and gibberellins (GA), a reduced IAA/ZR ratio, and diminished peroxidase (POD) activity. Weighted gene co-expression network analysis (WGCNA) identified 27 hub transcription factors (TFs) strongly associated with IAA/ZR, primarily from the ERF, bHLH, MYB, WRKY, and C3H families. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed the significant enrichment of differentially expressed genes (DEGs) related to plant hormone signal transduction and cell wall metabolism during pluripotent callus acquisition. Further investigations revealed that five genes encoding a putative indole-3-acetic acid-amido synthetase GH3.1, protein TIFY 10A, a two-component response regulator ARR2-like isoform X2, and xyloglucan endotransglucosylase/hydrolase, likely promoting callus pluripotency by modulating plant hormone signaling and cell wall metabolism, thereby enhancing in vitro regeneration in <i>C. parthenoxylon</i>. In summary, this study provides critical insights into the molecular mechanisms of <i>C. parthenoxylon</i> regeneration and offers valuable germplasm resources for establishing an efficient and stable genetic transformation system via tissue culture.