Epigenetic plasticity and regulatory remodeling of metabolism and cell wall dynamics drive genotype-specific embryogenesis in wheat
Microspore embryogenesis is a process in which immature male gametophytes are induced to form embryo-like structures that can regenerate into doubled haploid (DH) plants following chromosome doubling. By producing complete homozygosity in a single generation, DH technology accelerates cultivar development and is particularly valuable for breeding resilient crops under increasing biotic and abiotic stresses. However, bread wheat remains largely recalcitrant to microspore embryogenesis, with strong genotype dependence limiting its broad application in breeding programs. To investigate the molecular basis of this variation, we performed a comparative transcriptomic time-course in two spring wheat cultivars with contrasting embryogenic responses: Nanda (highly responsive) and Sadash (recalcitrant). Dynamic gene expression reprogramming was observed during embryogenesis, with Nanda showing distinct enrichment of biological processes linked to epigenetic regulation, including nucleosome assembly, chromatin remodeling, and chromatin organization, while the integration of stress perception, hormonal signaling, cytoskeletal and cell wall remodeling, and targeted metabolic shifts collectively establishes the cellular environment necessary for successful microspore embryogenesis. Differentially expressed gene (DEG) hotspots suggested possible structural variation underlying the divergent responses between genotypes. Machine learning highlighted potential biomarkers, notably a histone deacetylase gene TRAESCS1D02G454400 located within a DEG-enriched region. Subgenome-specific analysis further revealed pronounced suppression of B-subgenome homoeologs in Sadash, 65% of which overlapped with DEGs from the genotype comparison. These findings highlight the central role of epigenetic regulation, homoeolog expression bias, and genotype-specific genomic variation in determining embryogenic efficiency. These insights also provide new molecular targets and biomarkers to overcome recalcitrance and enhance the utility of microspore embryogenesis in wheat DH breeding pipelines. Overall design: RNAseq profiling of microspore cultures from three main stages of androgenesis – microspores at Day 0, proembryos at Day 7, and embryo-like structures at Day 21, using Nanda and Sadash, two spring wheat genotypes with contrasting embryogenic responses.