Enhancing Cryopreservation Efficiency in Populus davidiana × P. tremuloides Shoot Tips: Optimization of Vitrification Protocols and Mechanistic Insights into Flavonoid-Mediated Stress Adaptation

Cryopreservation is vital for conserving the elite germplasm of the hybrid poplar <i>Populus davidiana</i> × <i>P. tremuloides</i>, which is difficult to propagate conventionally. This study established optimized vitrification and encapsulation-vitrification protocols, achieving high regeneration rates of 85.91% and 79.70%, respectively, with confirmed genetic stability. The process induced oxidative stress, altering markers (MDA, H₂O₂) and antioxidant enzyme activities (SOD, POD, CAT). Integrated transcriptomic and metabolomic analysis of key steps-preculture/loading (DLA) and osmotic dehydration (DLB)-revealed extensive stress-responsive reprogramming. A central finding was the robust activation of the flavonoid biosynthesis pathway during DLB, marked by upregulation of key genes (PAL, CHS) and accumulation of flavonols (e.g., quercetin). This response was linked to hormone signaling and antioxidant systems, forming a coordinated defense network. Our multi-omics findings demonstrate that successful cryopreservation relies on an adaptive response where flavonoid biosynthesis plays a critical role in conferring oxidative stress tolerance, providing a theoretical basis for improving woody plant cryopreservation.