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Title: Effect of temperature on green spore longevity for the ferns Equisetum ramosissimum and Osmunda regalis

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

Submitted to: CryoLetters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2010
Publication Date: 4/30/2011
Citation: Ballesteros, D., Estrelles, E., Walters, C.T., Ibars, A.M. 2011. Effect of temperature on green spore longevity for the ferns Equisetum ramosissimum and Osmunda regalis. CryoLetters. 32:89-98.

Interpretive Summary: Problem: Green spores produced by some fern species are extremely short lived under ambient conditions and die within a few months. Only a few reports exist about this storage physiology and necessary storage conditions to prolong life span. The objective of this work was to measure the effects of temperature and dehydration on viability and vigour of green spores of two fern species during long-term storage. Accomplishment: We analyzed final germination, time to 50% germination (T50) and % normal gametophyte development of green spores for a better understanding of spore aging and temperature effects. Spores were tolerant to desiccation and survive drying to < 5% RH. This feature allows us to expose the spores to subzero temperatures without risk of freezing damage from water crystallization. Despite protection from water freezing, these spores do not retain viability in the freezer, perhaps because triacylglycerols crystallize. Viability and growth potential of green spores were maintained by storage at -80oC or in liquid nitrogen. Impact: We have provided the first quantitative assessment of spore viability and health and studied changes in these parameters through time under different storage conditions. Contrary to the paradigm about the recalcitrant behavior of green spores, we have showed that green spores exhibit intermediate storage physiology or a new, previously uncharacterized storage behaviour.

Technical Abstract: Some fern species produce chlorophyllic or green spores. Green spores lose viability quickly compared to nongreen spores, and so need specialized treatment for long term conservation in germplasm banks. Dry storage at different temperatures (25 ºC, 4 ºC, -25 ºC, -80 ºC and -196 ºC) was studied in green spores of Osmunda regalis and Equisetum ramosissimum. Final (i.e., maximum) germination percentage, time to 50% of maximum germination (T50) and normal growth of the gametophyte to the bidimensional stage were assayed during 24 months of storage. Spores stored at 25 ºC died within 1 month. Spores stored at 4 ºC maintained high viability for about 3 months, and then aging was evident by a decrease of final germination percentage, an increase in T50, and abnormal development of the gametophyte. Germination of spores stored at -25 ºC was highly variable and depended on experimental conditions. Spores cryopreserved at -80 ºC and -196 ºC maintained high viability, rapid germination and normal growth throughout the study period, with no significant differences (P < 0.05) from freshly harvested spores. Green spores showed only a mild sensitivity to extreme desiccation (RH < 3%) that was indicated by an increase of T50 and abnormal growth. We conclude that cryopreservation of green spores is a feasible method to preserve viability and ensure normal gametophyte development for several years.