Glycerol-Based Cryopreservation of Sinorhizobium/Ensifer Strains from Sweet Acacia (Vachellia farnesiana [L.] Wight & Arn.).

The genus Sinorhizobium (Ensifer) exhibits remarkable metabolic versatility and adaptive capacity, establishing effective nitrogen-fixing symbioses with diverse legume crops, including the pioneer shrub Vachellia farnesiana. This association is crucial for ecological restoration in arid and nutrient-deficient Mexican dryland ecosystems and for the remediation of contaminated sites. However, the prolonged preservation of specialized native strains through continuous serial subculturing risks genomic instability and the loss of extrachromosomal replicons harboring essential nod, nif, and heavy metal tolerance determinants. Here, we describe an optimized, reproducible, and effective glycerol-based cryopreservation protocol specifically adapted for Sinorhizobium strains associated with V. farnesiana. The protocol is based on the use of a glycerol solution for immediate ultra-low-temperature freezing at -80°C. Strain reactivation via a frozen stock-scraping technique minimized the physiological shock associated with freeze-thaw cycles. Post-preservation validation confirmed the typical Sinorhizobium morphology and, critically, demonstrated the retention of symbiotic competence through in vitro root nodulation assays with V. farnesiana seedlings. This optimized procedure ensures the viable and functionally intact preservation of microbial genetic resources for potential large-scale reforestation and restoration applications.