Why Seeds and Spores Die in the Freezer and Sometimes Faster That Anticipated

Chris Walters, Research Leader of the Plant Germplasm Preservation Research team at USDA-ARS National Laboratory for Genetic Resources Preservation

Knowing how long storedgermplasmsurvives is critical for effective banking of genetic resources. Longevity is inherently difficult to predict because there are so many factors controlling how cells respond to storage conditions. Uncertainty increases forgermplasmcollections of natural populations, especially rare species that might have additional issues with the reproductive biology or with assessments ofviabilityor aging. Storage conditions invariably involve manipulation of temperature and moisture, and this presentation will describe some of the basics of why this leads to long-term preservation of somegermplasmand what we think is going wrong when the desired longevity is not achieved. Preserving germplasm involves slowing down the rate that ‘clocks tick,’ and this means that we need to slow down the rate that molecules move. The most effective way to do this is by having molecules impede their own movement by pushing them together tightly and forming a solid (like a traffic jam). This process begins during development when cells accumulate dry matter to replace water, allowing molecules to come into close proximity naturally without deforming stresses. Cells from orthodox seeds shrink a little and solidify during maturation drying, but major mechanical stresses are easily avoided. Once in the solid, the rules for molecular movement are mostly dominated by how tightly the molecules are packed (determined by properties of the molecules and concentration of water) and by how much energy they have (determined by temperature). Given a particular molecular configuration in solidified cytoplasm, the effect of lowering temperature on mobility is predictable, as is the kinetics of reactions, such as aging, that are regulated by mobility. Lowering temperature slows down aging reactions in the same way in diverse seeds and spores; thus, reducing storage temperature from 25 to -18oC will usually increase longevity about 30 fold (if moisture is optimized). The good news is thatgermplasmthat survives 4 years at 25oC will survive about 120 years in the freezer. The bad news is thatgermplasmthat survives only 40 days at 25oC won’t survive much longer than 3 years in the freezer. Freezer temperatures appear to be a nexus for how molecules move in biological systems. Below -18oC, aging reactions appear to be driven by molecules vibrating, which has a low temperature dependency. Thus, a large temperature decrease gives only moderate benefits. Currently, we estimate a 3 to 5 fold increase in longevity by storinggermplasmcryogenically rather than in the freezer. Further complexity in structure and mobility of solidifiedgermplasmis introduced by the presence of oil droplets in the cytoplasm. We have linked lipid crystallization with faster aging in the freezer and explain this as the condensed structure of solidified lipids causing greater pore space, hence increased mobility, in aqueous domains of the cytoplasm. Collectively our work provides a theoretical framework to explain why lowering temperature and moisture affect longevity and to predict how longgermplasmstored at -18C will survive.