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D. BALLESTEROS; A. M. IBARS & E. ESTRELLES ICBiBE. Jardí Botànic, Universitat de València. Quart, 80. 46008 València. Spain. email@example.com Summary Pteridophyte spore conservation requires deep studies in order to explain the causes of viability loss, as well as to establish the optimal storage conditions. Germplasm Bank of the Botanical Garden of Valencia University is working to approach it. We report the effect of six months of storage at 25ºC, 4ºC, -25ºC, -80ºC and –196ºC on several pteridophytes species, over the viability of the spores and the further development of the gametophyte. Moreover we reported the response to desiccation and the repeated freezing and defrost effect on the stored samples. We can observe that successive freezing and defrost cycles decrease spore viability and have a negative influence over the subsequent development. Ultrafreezing hasn’t negative effects on germination and gametophyte development. We also study the relation of a loss of solutes with viability decreasing in the spores on Cyrtomium falcatum (Lf.) C. Presl. The amount of soluble sugar present in spores of different age and conservation conditions were analyzed. Introduction Pteridophytes are associated to ecosystems that are particularly sensitive to degradation, some of which are considered natural habitats of high priority by the present legislation. Some of these taxons are included in common interest listings species and require a strict protection. Pteridophytes are very sensitive to the environmental changes, so it’s essential the inclusion of its spores in germplasm banks. Germplasm banks play a very important role in the long-term ex situ conservation of the plant species. The conservation of seeds in germplasm banks is a reality vouched by numerous works (ELLIS et al, 1985; DICKIE et al, 1990; GÓMEZ-CAMPO, 2001, ESTRELLES et al, 2003). Nevertheless, in the Pteridophyte conservation there aren’t conclusive studies that assure a long-term conservation methodology. Fern spores have a relative rapid viability loss under environmental conditions (Lloyd & Klekowsky, 1970). Different works indicate possible causes of this rapid viability loss. For no green spores, is cited biochemistry and metabolic factors,as well as deficiencies of respiratory substrata, the lack of membranes integrity, the inactivation of enzymes and growth substances (BERI & BIR, 1993), or genetic factors as the chromosomic mutations (PAGE et al, 1992). In green spores has been indicated that the loss of viability is due to the high respiratory rate of these (LLOYD & KLEKOWSKY, 1970) or to the loss of photosynthetic activity recovery capacity after desiccation (LEBKUECHER, 1997). The scarce works that exist in Pteridophytes conservation have been centred in analyzing which are the optimum conditions to preserve through the years the viability of the spores. It has been tested with diverse habitual temperatures in germplasm banks, with both humid and dry conservation methods, even has been tested with techniques of cryopreservation (Jones & Hook, 1970, WINDHAM ET AL, 1986; LINDSAY et al, 1992; AGRAWAL et al, 1993; SIMABUKURO et al, 1998; PENCE, 2000; CONSTANTINO et al, 2000; QUINTANILLA et al, 2002). But, in general, only exist a few long-term and quantitative data that determine the best form and more lasting to conserve spores. And the causes of the viability’s loss have not remained clear because there is a little experimentation around this theme. In spore conservation PAGE et al (1992) indicated in their work the need of maintains spores viability and their capacity of growth and genetic integrity. In our entire project we want to know which are the optimum conditions for the
and one that. All dishes were incubated for 120 days in a culture chamber at 20ºC with a 12 hours light photoperiod. For the statistical processing. two tropical species (Cyrtomium falcatum. Results and discussion Germination and laminar gametophytes percentage mean and typical error are represented in table 1 for different treatments. In Ceterach officinarum the effects of different treatments . as a new approach respect to the remainder works in this field. For the estimation of total soluble sugars we followed a colorimetric method with antrona-sulfuric acid reagent like describes MCCREADI et al (1950). Pteris vittata). temperature and spores drying haven’t got effects in spore viability and gametophyte late development. All the statistical tests were carried out with the program SPSS version 11. Repeated defrost are negative for spore viability and gametophyte development. stored at laboratory temperature or at 4ºC between 2001 and 2004. Material and methods The work has been carried out with ten fern’s species presents in the Mediterranean area. Spores were kept at -196ºC (liquid nitrogen) too. to observe the start of the germination and the development. We opted for this date because in the controls all the gametophytes have arrived at this phase at 30 days.long-term storage of fern spores in germplasm banks. we have analyzed so much the storage effects on the germination and the early development. We have species of xeric habitats (Notholaena marantae.5 mm diameter. counting 100 spores by plate. At 60. Dryopteris filix-mas. The spore’s final viability was verified analyzing the germination percentage at 30 days. The other species are typical of different under-woods presents in the Mediterranean area (Asplenium onopteris. In this line. 1964). After 6 months and a fast defrost. Cystopteris fragilis. the species and populations collected were selected by their ecology. species of swamp soil habitats (Thelypteris palustris. data were processed with arcsine transformation and they were analyzed with a one-way ANOVA or a t-test. Ceterach officinarum). spores were sown on mineral agar medium (DYER. 1979) in seven petri plates of 5. Polystichum aculeatum). moreover a smaller laminar gametophytes percentage at 30 days can be seen. were introduced in hermetic jars with silica gel during one week at 4ºC. In Polystichum lonchitis is observed that after 6 months at 4ºC. To collect the spores the fertile fronds were pressed for a week until the spores were released. analyzing habitual storage methods and others less common. despite in the most aggressive processing a high proportion of dead or abnormal gametophytes are observed. It was analyzed also at 30 days the gametophyte percentage that reached laminar phase.5. We worked with almost 20 individuals by specie to obtain a significant sample of the variability in the natural population. as the effects on the late development of the gametophyte and its capacity to complete its vital cycle. It was periodically noted spore germination percentage. Another objective is to know which are the causes of the loss of the viability of spores. even when spores are dry. The spores that need to be dried. This recovery is often due to the gametophytes damaged capacity to develop new gametophytes from apical cells (ATKINSON & STOKEY. inhabits high mountains (Polystichum lonchitis). spores remain there until they were sowed. For sugar estimation. 90 or 120 days we noted how many replicas of the three storage temperatures had gametophytes in sexual phase. Athyrium filix-femina). We can observe furthermore that all the freezing treatments produce a significant spore viability loss except for the dry spores. 4ºC and –25ºC (habitual temperatures in germplasm banks) and –80ºC. To carry out the different experiments spores were kept in darkness at 25ºC (laboratory temperature). in Iberian Peninsula. Although this percentage of laminar gametophytes recovers above the 90 percent at 60 days and in all plates appear sexual gametophytes. To identify homogeneous groups in the average we utilize the test of Tukey. taken like laminar phase the start of the 2D growth (transition phase). alcoholic extraction was done with 50 mg of spores from different samples of Cyrtomium falcatum.
and gametophytes develop sexual structures in practically all the plates. Emphasized on green colour.7±0.6±0.0±0.1±0. p-value<0.1±1. which maintains the same germination percentage after a 6-months storage as those spores either stored at -80ºC or in liquid nitrogen.6 92.8 74.9±2. And emphasized on yellow and red the treatments where loss exists.9±0. Dry -25ºC.6 4ºC.7±1.7±2.9 95.05). Respect to the differences in the viability loss percentage.2 79.9±3.6 94.1 76. Differences in development are not reliable because there is a contamination excess in all the plates. We don’t include Ceterach officinarum in this point because the plates have appeared very contaminated. Storage Polystichum lonchitis Germination (%) 81.4±1. except for Ceterach officinarum.9 97.8 85.5 95. neither the taxonomic group where different species belong.7±2.8 97. Dry 4ºC.0±0.3±2.9 96.9 90. we haven’t observed any relation with the ecology of the species.6 87.6 94. the ploidy level. the laesura type of the spores. Non dry -25ºC.3±9.4±7.4±9.9 98.5 60.9 98.8 95.4±0. These germination rates of spores after remaining ultrafreezing don’t differ of the controls taken after the collecting of the plant material.3 Ceterach officinarum Laminar Laminar phase Laminar phase Germination phase 30 30 days (%) 60 days (%) (%) days (%) 97.on germination don’t exist. Non dry -25ºC.7±0.9 66.6±0.8 85.4±0.3±3.3 85. We can observe that in all species and processing more than the 90% of the germinated spores arrive to the bidimensional gametophyte stadium after 30 days of cultivation.1±0.2 57. Defrost monthly -25ºC. the treatments in which the viability or development have no decrease respect to the control.0 88. Non dry Defrost monthly Table 1: Colours indicate tukey homogeneous groups.4±5.7±0. . Dry.9±0.4 56. If we observe the graphic 1 we can see that a significant viability loss has been verified in all the species used for the study when they were kept at 25ºC (ANOVA.3±3.8 73.9 95.7 65.7±3.5 95.0±0.7±1.
vittata Graphic 1: Bars shows means and twice the typical error of the germination percentage in the three storage temperatures (♦ 25ºC. We can observe in graphic 2 that total sugars amount is also independent of the spore’s viability in the samples.70 0. officinarum D. fragilis P. filix-femina C. There is a significant soluble sugar loss in spores along the time.60 0. ♦ -196ºC). marantae P. filix-mas T. A significant soluble sugar loss in stored spores isn’t synonymous with a significant viability loss or spores death.90 Germination proportion 0.00 * 0.10 * A. This loss is independent of the storage method. .1.50 0. p-value<0. Stars indicate groups significantly different (ANOVA.40 * * 0. lonchitis C. Other factors. aculeatum A. moreover soluble substances loss.20 0.30 0.80 * * * * * 0. onopteris N. should be implicated in viability loss and death of spores (BERI & BIR. palustris P.05). Sugar estimation data suggest that the total sugars amount is in relation to the stored time but not with the studied conservation method. ♦ -80ºC. 1993).
197-204. Annals of Botany 65. HODSON -2000-. Cryopreservation of spores of Cyathea spinulosa Wall. that permits the material’s indefinite storage.ogy 142: 124126.H.. Botanic Gardens Micropropagation News. Comparative morphology of the gametophyte of homosporous ferns.M. ATKINSON. ELLIS. S. f.. sugar / mg..S.B.B. L. The preservation of biological material in cryogenic temperatures (cryopreservation) manages to stop completely the biological reactions. Journal of Plant Physiol.F.An endangered tree fern. H. Gametophyte remains its capacity to complete the vital cycle. ex. . & A. References AGRAWAL. Fern Journal.(mg. . PAWAR & MASCARENHAS. K. SANTAMARIA & E. L. Germination of stored spores of Pteris vittata L. RYDER & P. DICKIE.Temperature and seed storage longevity. American. The colours of the bars indicate the different Tukey homogeneous groups. the average and twice the standard error of the total sugar amount by weight that spores had according to the germination percentage. neither gametophyte development. The colours of stars indicate the significantly homogeneous groups for the different germination percentage. -1993-. spores) (% germination) Graphic 2: We can observe for the different samples. A. STOKEY -1964-. KRAAK.G. R.R. BIR -1993-. Phytomorphology 14(1): 51-70. We have found that ultrafreezing doesn’t affect spores viability after 6 months. BERI. D. 83(3): 73-78. A.S. 2(4): 58-60. J. Our results after six months of work contribute to the first quantitative data related to the ultrafreezing effects in no green spores. TOMPSETT -1990.C.L. Storage and in vitro germination of tree fern spores. Hook. CONSTANTINO S. & S.
Jermy AC & Paul AM eds. Centro de Estudios Ramón Areces. D.E. -2001-. A. Probert RJ eds. DYER -1992-. J. -1979. Pritchard HW. J. This study was supported by Spanish Ministry of Science and Technology Project REN2002-03697.F. AMIGO. Intercept. Conservation of pteridophytes: the ex situ approach. IBARS -2003-.M. LEBKUECHER. R. Application to peas.W. WINDHAM. Growth and development in microculture of gametophytes from stored spores of Equisetum.. MANN -1992-.. Hook -1970-.Handbook of seed technology for genebanks. L.B. J.G.F. 76(3): 141-148.. BALLESTEROS & A.B. SIMABUKURO. 88(2): 72-80.A.A. LLOYD. Madrid JONES L. BOSCAIU. Biotropica 2: 129-137. Journal of Botany 54(4): 430-435. GÓMEZ-CAMPO. In: Ide JM. P. Roma. N.F. S. -2000-. Principles and methodology. Determination of starch and amylose in vegetables.Effect of storage method on spore viability in five globally threatened fern species. 859-868. Ed. GUGGOLZ. Fern Horiculture: past. International Board for Plant Genetic Resources. London: Academic Press. present and future perspectives. ROBERTS -1985. Rossello from ICBiBE (Institut Cavanilles de Biodiversitat i Biologia Evolutiva of Valencia University) for useful comments on an earlier version of this manuscript.The culture of fern gametophytes for experimental investigation. A. S. La práctica de la conservación de semillas a largo plazo. I. American. 285-294. ESTRELLES E. A.J.M. 269-278. J. C. Vol.C. & E. PAGE. LINDSAY. PANGUA & S. In: Ide JM. Threatened Valencian Flora: Initiatives for its Conservation in Smith RD. DYER & G. V. Royal Botanic Gardens. was in receipt of an FPU grant from the Spanish Ministry of Science and Technology. MCCREADY. UK. “Seed conservation: Turning into Practice”. 90(4): 119-126. FUENTES. Survival of chlorophyllous and non-chlorophyllous fern spores through exposure to liquid nitrogen. Kew. LINDSAY & D.G. American.S. ELLIS. Journal of Botany 84(6): 792-797. E.G. Dickie JB. HONG & E. N. Jermy AC & Paul AM eds. The effect of sterilization and storage conditions on the viability of the spores of Cyathea delgadii. The experimental biology of ferns. FELIPPE -1998-. D. American Fern Journal. American Fern Journal. . Fern horticulture: past... Andover.G. J. 255-266.F. WOLF & T. RANKER (1986) Factors affecting prolonged stored spore viability in herbarium collections of three species of Pellaea. During this study.D. Andover: Intercept.H. OWENS -1950-. EA. R. Anal Chem 22: 1156-1158. V. SILVEIRA & H. In: Dyer AF ed. Linington SH. Spore germination and viability in Pteridophyta: evolutionary significance of chlorophyllous spores. M. Annals of Botany.M. present and future perspectives.. PAJARON -2002. 90(4): 461-467.D. QUINTANILLA. C. A. Kew. WILLIAMS. American Fern Journal. DYER. -1997-. 253-305.M. T.. M. PENCE. Desiccation-time limits of photosynthetic recovery in Equisetum hyemale (Equisetaceae) spores. PRIETO. Acknowledgements The authors thank Dr. KLEKOWSKI JR -1970-.DYER. & P. R. In: Conservación de especies vegetales amenazadas en la región mediterranea occidental.N. Wet storage of fern spores: unconventional but far more effective!.
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