The mechanism of optimized two-step osmo-protection in regulating the cell homeostasis of 'Light Yellow' Petunia × Calibrachoa callus.

Cryopreservation is essential for long-term plant germplasm preservation, yet the mechanisms underlying cellular responses during the critical dehydration phase remain poorly understood. In this study, callus tissues of 'Light Yellow' Petunia × Calibrachoa were used to compare the cytological, physiological, biochemical, and molecular effects of one-step (T1) and two-step (T2-1, T2-2) osmo-protection. The one-step method (T1) directly exposed tissues to MS medium with 2 M glycerol and 0.35 M sucrose for 30 min, while the two-step strategy involved 15 min in glycerol alone (T2-1) followed by 15 min in glycerol-sucrose (T2-2). The two-step method markedly improved cell viability, reaching 90.15 % after 30 min, compared with 12.32 % in T1. LF-NMR revealed that two-step treatment reduced total water content (71.35 %) and free water proportion (52.6 %), while increasing bound water (7.26 %), accompanied by upregulation of aquaporin genes (TIP, PIP), indicating enhanced water regulation. After 30 min, cell shrinkage reached 69.65 % in T1 but only 49.59 % in the two-step treatment, suggesting a more moderate dehydration rate. Transmission electron microscope (TEM) confirmed that two-step treatment could protect the integrity of cell membranes, vacuoles, and organelles, while T1 caused plasmolysis, membrane collapse, and mitochondrial damage. Biochemical assays showed reduced malondialdehyde (MDA) and conductivity in two-step treatments, alleviating lipid peroxidation and permeability change. Analysis of cell wall by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS) revealed that T2-2 treatments not only promoted accumulation of hemicellulose (54.52 mg/g) and pectin (303.82 μmol/g) for enhancing wall strength, but also facilitated the accumulation of osmoprotective sugars, such as glucose (171.42 μg/mL), fructose (25 μg/mL), and xylose (13.76 μg/mL). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results further elucidated alongside upregulation expression of sugar metabolism-related the genes (INV, HK). Antioxidant enzymes (CAT, POD) and wall remodeling genes (XTH, PMEI) were also regulated during two-step treatment, suggesting multiple pathways mediate osmotic stress responses. In conclusion, the two-step osmo-protection effectively mitigates dehydration stress, preserves viability and structural integrity, and activates molecular defense mechanisms, demonstrating clear advantages for cryopreservation of high-moisture, freeze-sensitive plant materials.