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Title: Effects of temperature and desiccation on ex situ conservation of nongreen fern spores

item Ballesteros, Daniel
item ESTRELLES, ELENA - University Of Valencia
item Walters, Christina
item IBARS, ANNA - University Of Valencia

Submitted to: American Journal of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2012
Publication Date: 4/1/2012
Citation: Ballesteros, D., Estrelles, E., Walters, C.T., Ibars, A. 2012. Effects of temperature and desiccation on ex situ conservation of nongreen fern spores. American Journal of Botany. 99(4): 721-729.

Interpretive Summary: Problem: Fern spore storage physiology may be analogous to seed and pollen storage physiology and provides a single cell system to study anomalies of germplasm response to temperature and moisture. We are interested in why some germplasm stored in the freezer ages faster than expected. This study focuses on non-green spores, which are believed to survive longer than green spores. Accomplishment: We developed viability assays that allow us to quantify aging rates in ferns. We showed interactions between longevity and storage conditions for spores from 10 species of ferns that suggest spores have similar responses to moisture as seeds. We show that aging of ferns stored in the freezer is relatively fast, and this provides a good model to study the basis of freezer damage. We show that this type of damage is avoided during storage at -80oC or liquid nitrogen. Impact: We have developed a good model system to study freezer damage to germplasm. We have provided management procedures to preserve fern germplasm using cryopreservation. This will aid in ex situ conservation of many plants in danger of extinction.

Technical Abstract: Conservation of the genetic diversity of ferns is limited by the paucity of ex situ spore banks. Conflicting reports of fern spore response to low temperature and moisture impedes establishment of fern spore banks. There is little information available to evaluate longevity of fern spores under different conditions and to assess the risks, relative to the benefits, of cryogenic storage compared to freezer storage -- the standard condition used for seed germplasm. Comparing spore responses to different storage conditions requires tools to quantitatively assess changes in spore viability and growth over time. Here, we quantify spore aging through decreases in percent germination, increases in the time required for 50% of spores to germinate (T50), and decreases in the percent of normal development in germinated spores. Aging rates are compared among spores from 10 diverse fern species over a three year storage period at a range of temperatures including -196C (liquid nitrogen). Increase in T50 sometimes preceded loss of % germination and may be an early indicator of aging for some species. Generally aging rate decreased with lower storage temperature and moisture, except at -25C where aging was anomalously fast, especially if spores underwent repeated freeze/thaw cycles or were not dried appropriately. Anomalous response to -25C storage may be analogous to the syndrome of damage experienced by seeds categorized as “intermediate.” Storage in liquid nitrogen circumvents the damage and appears amenable to all species tested. Germination and subsequent gametophyte development of spores stored at -80C or in liquid nitrogen did not change over the 3 year study period for all species examined. Cryo-conservation appears to be an effective tool to extend fern spore longevity that is broadly applicable across diverse species.