National Laboratory for Genetic Resource Preservation (USDA-ARS)

Jose Faria, Lisa Hill, Christina Walters, Tree Seed Laboratory, Federal University of Lavras, Brazil, USDA-ARS, National Center for Genetic Resources Preservation, United States

Quercus imbricaria is included in the red oak group (Lobatae) and is broadly distributed in the Midwestern US. The embryonic axes are about 1 mg dry mass and have 0.68 g H2O/g dw in the acorn. Their small size and considerable desiccation tolerance made them ideal to compare various approaches for cryopreservation. Drying over a stream of nitrogen gas for 120 min reduced axis water content to 0.15 g/g, with 55% survival following liquid nitrogen (LN) exposure. To test whether addition of cryoprotectants could improve survival, axes were bathed in Plant Vitrification Solution #2 (PVS2) for 5-60 min and tested for viability before and after LN exposure. Axes submitted to PVS2 treatments and exposed or not to LN attained survival of 100% (or close to) after two weeks of tissue culture. Differential scanning calorimetry (DSC) was used to infer water freezing and melting patterns. In axes dried without PVS2, water melted at about 178 J/g H2O and melting transitions were not observed in axes dried to less than 0.34 g/g when standard methods of 10oC/min scanning rates were used. Using similar DSC methods, the water melting signal was no longer apparent in axes soaked in PVS2 for 45 minutes. To evaluate water melting behavior during fast cooling and warming, axes were plunged into LN and scanned at 300oC/min, revealing broad endothermic events between -120 to -40oC in axes that received no cryoprotectant, which we interpreted as crystal growth during warming. The enthalpy for melting transitions was reduced to about 70 J/g H2O, but there was no effect on the water content at which the melting signal was not detected. From our collective results, we suggest that PVS2 alters the rate of lethal ice crystal formation during rapid cooling and warming allowing for greater survival of axes exposed to LN.

Date Recorded: 
Thursday, July 25, 2019

Gayle Volk, USDA ARS National Laboratory for Genetic Resources Preservation, United States

The USDA-ARS National Plant Germplasm System has over 30,000 clonally maintained accessions within its field, screenhouse, greenhouse, and tissue culture collections. These fruit, nut, tuber, and bulb crop collections are usually not duplicated at secondary locations and are vulnerable to bioticabiotic, and climatic threats. Only about 15% of the clonally maintained accessions are currently secured in long-term storage at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado. The labor required to cryopreserve the clonal collections at NLGRP exceeds that which is available, even when reliable, robust cryopreservation methods are available. We have sought to prioritize collection materials for cryopreservation and to identify methods that improve the efficiency of the shoot-tip cryopreservation procedure. In particular, we have used field-, screenhouse-, and growth-chamber harvested plant tissue as source material for shoot tip cryopreservation, rather than relying on in vitro grown cultures. This strategy has been particularly effective for garlic, citrus, and grape cryopreservation efforts. In addition, incorporation of antioxidants and shoot tip micrografting methods have made cryopreservation protocols widely applicable to diverse genetic resources for each crop.

Contributing Author(s): 
Date Recorded: 
Tuesday, July 23, 2019

Lisa Hill - National Laboratory for Genetic Resources Preservation (USDA-ARS)

Drying seeds to appropriate relative humidity is the first step to long term seed storage. NLGRP and CPC recommend drying seeds to 25-35% relative humidity at room temperature. One way to acheive specific relative humidities is to use saturated salt solutions. Here, Lisa shows the steps of creating a saturated salt solution from Magnesium Chloride Hexahydrate (Cl2H2MgO6) or Potassium Acetate (CH3CO2K). She also demonstrates the equipment needed to create dessication chambers of various sizes.

Contributing Author(s): 
Date Recorded: 
Friday, December 13, 2019

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.

Contributing Author(s): 
Date Recorded: 
Thursday, May 3, 2018