Effect of Cryopreservation Techniques on the Proteomic Profile of Cryoderived Vanilla planifolia Plants Maintained Under Greenhouse Culture Conditions

Cryopreservation is an effective strategy for the long-term conservation of plant germplasm. However, the metabolic responses of cryoderived plants under ex vitro conditions have been little studied. This work evaluates the proteomic profile of Vanilla planifolia plants regenerated from shoot tips recovered after cryopreservation using the Droplet-vitrification (D-V), D-Cryoplate (D-Cp), and V-Cryoplate (V-Cp) methods. The plants were maintained under greenhouse conditions for four years, and protein expression was analyzed using nano-LC-MS/MS. A total of 3523 proteins were identified, and 1019 showed differential expression compared to non-cryopreserved control plants. Nine proteins were consistently upregulated across all cryopreservation methods, including chalcone-flavanone isomerase, Glucose-Methanol-Choline oxidoreductase, calreticulin, glutamine synthetase, 3-ketoacyl-CoA synthase, pectinesterase, BAHD acyltransferase, α/β-hydrolase, and peroxidases. These proteins are associated with stress protection, primary metabolism, cell wall remodeling, and lipid and nitrogen metabolism. Conversely, down-regulation was observed in EG45-type expansin and in proteins containing RNA recognition motifs involved in growth regulation. Functional enrichment analysis revealed enhanced photosynthetic capacity and structural reinforcement in cryoderived plants compared to non-cryopreserved control plants. Among the procedures evaluated, the D-Cp technique, followed by the D-V technique, proved to be the most effective, demonstrating that cryopreservation enabled stable metabolic reprogramming during the greenhouse development of V. planifolia plants.