Submitted to: Cryobiology
Publication Type: Abstract Only
Publication Acceptance Date: 7/29/2002
Publication Date: 7/29/2002
Citation: Walters, C., l. Wheeler, and P. Stanwood. 2002. Viability losses in cryopreserved germplasm: A Pilot Study Using Lettuce Seeds. 39th Meeting of the Society for Cryobiology, July 28-31, 2002, Breckenridge, Colorado. p. 78. Interpretive Summary: This presentation reports the first quantitative study of viability of germplasm maintained in liquid nitrogen storage. The study uses 258 accessions of lettuce seeds and shows that storage in liquid nitrogen vapor certainly slows aging reactions. However, liquid nitrogen storage does not stop aging reactions, as is commonly assumed. After 18 years of storage, decreases in the percent germination of lettuce seeds could be measured. The results challenge current views on the mechanisms of aging reactions and how they are limited by low temperature storage.
Technical Abstract: It is generally assumed that germplasm stored at liquid nitrogen (LN) temperatures will remain alive indefinitely. The results of this study using 258 accessions of lettuce seeds lead us to question that assumption and to reconsider ideas of the underlying mechanisms that control aging of preserved cells. Lettuce seeds with initially high germination rates (>90%) were stored for 3 to 18 years under conventional storage conditions (6% water content and 5C or -18C) and then maintained at -18C or placed in LN vapor (-120 to -150C) for an additional 17-18 years. There was no evidence of damage after initial exposure to LN. During storage in LN, viability of seeds declined at an average rate of 1.4 % per year (std. dev. = 0.4 % per year) compared to an aging rate of 4 % per year (std. dev. = 1.6 % per year) for counterparts stored at -18C, resulting in an average reduction of viability of 24 and 68% respectively. Though the trend towards decreasing viability with storage time was consistent, there was a large difference in aging rate among samples. Seeds stored for longer times at 5C aged faster in both -18C and LN, even though initial viability was high when they were switched to the lower storage temperature. In LN, mobility within the aqueous and triglyceride matrices of cells is restricted according to differential scanning calorimetry scans showing Tg's > +25C for seeds containing 6% water and lipid crystallization between -20 and -80C. However, molecular mobility was not completely restricted as evidenced by a trend towards increasing dehydration of seeds stored in LN. Though low water contents and temperatures interact to increase cellular viscosity, the free energy that drives deteriorative reactions must be sufficiently great to allow aging to proliferate even at extremely low temperatures.