Somatic Embryogenesis-Based Tetraploid Induction Enhances Biomass and Freezing Tolerance in Liriodendron Hybrid

Polyploidization is a key mechanism driving plant evolution, environmental adaptation, and trait improvement. This study investigated the effects of artificial chromosome doubling on growth and freezing tolerance in a Liriodendron hybrid (T × T genotype). Tetraploids were efficiently induced during the liquid suspension stage of somatic embryogenesis using oryzalin, with a maximum induction rate of 33.33%. Compared with diploids, 3-month-old tetraploids exhibited compact growth, characterized by increased stem diameter and markedly enlarged leaf area. Tetraploids also showed larger but less dense stomata, enlarged leaf cells, and denser chloroplast organization. Under freezing stress, tetraploids displayed enhanced tolerance accompanied by coordinated transcriptional reprogramming. Transcriptome analysis revealed significant enrichment of defense-related pathways, including plant hormone signaling (JA, SA, and auxin) and MAPK signaling, together with ploidy-specific calmodulin expression, suggesting Ca²⁺-mediated regulation of hormone responses. Tetraploids preferentially upregulated genes involved in phenylpropanoid metabolism and lignin biosynthesis, promoting structural defense and redox homeostasis, whereas diploids showed stronger induction of flavonoid biosynthesis genes associated with rapid antioxidant protection. These transcriptional patterns were supported by physiological measurements, with tetraploids accumulating higher lignin content and enzyme activities, while diploids accumulated more flavonoids. Overall, tetraploids and diploids adopt distinct freezing adaptation strategies. Polyploidization drives coordinated regulation of hormone signaling and secondary metabolism, enabling improved biomass allocation and freezing tolerance. This study establishes an efficient tetraploid induction system for Liriodendron hybrid and provides mechanistic insights into polyploid-enhanced stress adaptation in woody plants.