Exploring Transcriptional Regulatory Network During Direct De Novo Shoot Regeneration in Poplar

Regeneration of transgenic cells into plants is well known to be an obstacle to genetic engineering, and woody plants are commonly among the most recalcitrant species. To elucidate the molecular mechanisms of regeneration in woody plants, we conducted transcriptional profiling during direct de novo shoot regeneration in Populus tomentosa cv. BJHR01, which supports efficient regeneration. We sampled RNA from leaf explants, callus-like tissues, and shoot-forming tissues during in vitro culture. We identified 27 765 expressed genes, 70% of which (19 386 genes) were differentially expressed. Gene Ontology (GO) and weighted gene co-expression network analyses revealed overrepresentation of GO terms related to DNA replication, nuclear division, cell cycle, transcription initiation, and auxin response during callus-like tissue initiation and development. Genes associated with chromatin remodelling, epigenetic modification, and cytokinin and stress hormone signalling were also differentially regulated, highlighting the role of transcriptional regulation and cell division in early regeneration. During shoot initiation, cytokinin and auxin signalling genes were dynamically regulated. One hub gene, PtoARF3.1, a key regulator of auxin signalling, significantly enhanced regeneration efficiency when overexpressed. This study provides insights into the molecular mechanisms of direct de novo shoot regeneration in Populus and paves the way for identifying potential regeneration enhancers.