Cryopreservation of Rhizobium Strains Associated with Common Bean (Phaseolus vulgaris L.): Evaluation of Functional Stability and Nodulation Capacity.

The conservation of rhizobial diversity is essential to sustain the functionality of symbiotic associations that underpin biological nitrogen fixation in ecologically and agriculturally important legumes. In particular, Rhizobium strains associated with the common bean (Phaseolus vulgaris L.) represent a strategic genetic resource in both productive systems and natural ecosystems of Mexico. However, the loss of viability or symbiotic plasmids during laboratory maintenance may compromise their nodulation capacity and nitrogen-fixing ability. In this study, we describe a standardized glycerol-based cryopreservation protocol at -80°C for the long-term preservation of Rhizobium strains symbiotic with P. vulgaris, along with the evaluation of their functional stability after thawing. The method relies on the use of glycerol as a penetrating cryoprotectant, which minimizes cellular damage associated with ice crystal formation and osmotic stress, thereby preserving cell viability and genetic integrity. Furthermore, the capacity of preserved strains to form nitrogen-fixing nodules is assessed as an indicator of symbiotic stability. This protocol provides a reliable tool for the preservation of microbial genetic resources of high ecological and agronomic value, contributing to the safeguarding of rhizobial biodiversity and to its potential application in sustainable agriculture.