germplasm

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

Dr. Oliver Ryder, Barbara Durrant, Marlys L. Houck, Marisa Korody, Cynthia Steiner, Institute for Conservation Research, San Diego Zoo Global

Cryobanking of viable tissue culture cells at the San Diego Zoo, named the “Frozen Zoo® by its founder, Kurt Benirschke, has contributed knowledge advancing conservation science and species recovery from its beginnings in 1975. This diverse collection of cryopreserved diploid fibroblast cell cultures contributes to the legacy his endeavor. Banked gametes and cryopreserved reproductive tissues expanded the possibility of conservation applications. Identification of chromosomal errors affecting fertility of mammalian species as diverse as tigers, dik-diks, and gorillas has been enabled by its collections. Key studies delimiting species boundaries, evolutionary relationships, phylogeny and systematics of mammals and birds have utilized samples from the Frozen Zoo and its extended network of samples. Major contributions to the advancement of comparative vertebrate genomics have been facilitated by the collections of the Frozen Zoo. A crucial contributor to the 200 Mammals project, Genome10K, and the Vertebrate genome project, intact cells and high molecular weight DNA extracts from the Frozen Zoo have facilitated whole genome sequencing efforts and, notably, high quality de novo genome assemblies. Cryobanking of viable early passage diploid fibroblasts has allowed investigation of somatic cell nuclear transfer cloning technology to be pursued in the conservation context. Live offspring of two endangered species of wild cattle, the gaur, and the Javan banteng have been produced with cells banked for decades in the Frozen Zoo. Fibroblast cells from the Frozen Zoo were used for the first reported cellular reprogramming of endangered species to produce induced pluripotent stem cells (iPSC), including an African primate, the drill, and the critically endangered northern white rhinoceros were successfully reprogrammed. These studies have been extended and recently, using nonintegrating methods, iPSC have from two southern white and eight northern white rhinoceroses have been produced and characterized, an effort crucial to preventing its otherwise-certain extinction.

Date Recorded: 
Monday, July 22, 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