New data about pteridophytic spore conservation in germplasm banks

D. BALLESTEROS; A. M. IBARS & E. ESTRELLES ICBiBE. Jard Botnic, Universitat de Valncia. Quart, 80. 46008 Valncia. Spain. daniel.ballesteros@uv.es 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 25C, 4C, -25C, -80C and 196C 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 hasnt 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 its 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; GMEZ-CAMPO, 2001, ESTRELLES et al, 2003). Nevertheless, in the Pteridophyte conservation there arent 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 viabilitys 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

long-term storage of fern spores in germplasm banks, analyzing habitual storage methods and others less common. In this line, as a new approach respect to the remainder works in this field, we have analyzed so much the storage effects on the germination and the early development, as the effects on the late development of the gametophyte and its capacity to complete its vital cycle. Another objective is to know which are the causes of the loss of the viability of spores. Material and methods The work has been carried out with ten ferns species presents in the Mediterranean area; the species and populations collected were selected by their ecology. We have species of xeric habitats (Notholaena marantae, Ceterach officinarum), species of swamp soil habitats (Thelypteris palustris, Athyrium filix-femina), two tropical species (Cyrtomium falcatum, Pteris vittata), and one that, in Iberian Peninsula, inhabits high mountains (Polystichum lonchitis). The other species are typical of different under-woods presents in the Mediterranean area (Asplenium onopteris, Dryopteris filix-mas, Cystopteris fragilis, Polystichum aculeatum). We worked with almost 20 individuals by specie to obtain a significant sample of the variability in the natural population. To collect the spores the fertile fronds were pressed for a week until the spores were released. The spores that need to be dried, were introduced in hermetic jars with silica gel during one week at 4C, spores remain there until they were sowed. To carry out the different experiments spores were kept in darkness at 25C (laboratory temperature), 4C and 25C (habitual temperatures in germplasm banks) and 80C. Spores were kept at -196C (liquid nitrogen) too. After 6 months and a fast defrost, spores were sown on mineral agar medium (DYER, 1979) in seven petri plates of 5.5 mm diameter. All dishes were incubated for 120 days in a culture chamber at 20C with a 12 hours light photoperiod. For sugar estimation, alcoholic extraction was done with 50 mg of spores from different samples of Cyrtomium falcatum, stored at laboratory temperature or at 4C between 2001 and 2004. For the estimation of total soluble sugars we followed a colorimetric method with antrona-sulfuric acid reagent like describes MCCREADI et al (1950). It was periodically noted spore germination percentage, to observe the start of the germination and the development. The spores final viability was verified analyzing the germination percentage at 30 days, counting 100 spores by plate. It was analyzed also at 30 days the gametophyte percentage that reached laminar phase, taken like laminar phase the start of the 2D growth (transition phase). We opted for this date because in the controls all the gametophytes have arrived at this phase at 30 days. At 60, 90 or 120 days we noted how many replicas of the three storage temperatures had gametophytes in sexual phase. For the statistical processing, data were processed with arcsine transformation and they were analyzed with a one-way ANOVA or a t-test. To identify homogeneous groups in the average we utilize the test of Tukey. All the statistical tests were carried out with the program SPSS version 11.5. Results and discussion Germination and laminar gametophytes percentage mean and typical error are represented in table 1 for different treatments. In Polystichum lonchitis is observed that after 6 months at 4C, temperature and spores drying havent got effects in spore viability and gametophyte late development. We can observe furthermore that all the freezing treatments produce a significant spore viability loss except for the dry spores; moreover a smaller laminar gametophytes percentage at 30 days can be seen. Although this percentage of laminar gametophytes recovers above the 90 percent at 60 days and in all plates appear sexual gametophytes. This recovery is often due to the gametophytes damaged capacity to develop new gametophytes from apical cells (ATKINSON & STOKEY, 1964), despite in the most aggressive processing a high proportion of dead or abnormal gametophytes are observed. Repeated defrost are negative for spore viability and gametophyte development, even when spores are dry. In Ceterach officinarum the effects of different treatments

on germination dont exist. Differences in development are not reliable because there is a contamination excess in all the plates.

Storage

Polystichum lonchitis Germination (%) 81.92.3 85.41.2 79.71.5 60.32.7 65.71.4 56.72.3

Ceterach officinarum Laminar Laminar phase Laminar phase Germination phase 30 30 days (%) 60 days (%) (%) days (%) 97.00.9 98.70.6 94.40.9 66.33.6 87.70.8 74.33.9 97.00.9 98.70.6 94.40.9 90.11.8 97.10.5 95.00.8 95.90.9 95.60.5 95.90.9 96.60.6 92.93.9 95.10.8 85.72.8 73.47.8 85.45.0 88.73.1 76.49.2 57.39.6

4C, Dry 4C; Non dry -25C; Dry -25C; Non dry -25C; Dry; Defrost monthly -25C; Non dry Defrost monthly

Table 1: Colours indicate tukey homogeneous groups. Emphasized on green colour, the treatments in which the viability or development have no decrease respect to the control. And emphasized on yellow and red the treatments where loss exists. 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 25C (ANOVA, p-value<0.05), except for Ceterach officinarum, which maintains the same germination percentage after a 6-months storage as those spores either stored at -80C or in liquid nitrogen. These germination rates of spores after remaining ultrafreezing dont differ of the controls taken after the collecting of the plant material. Respect to the differences in the viability loss percentage, we havent observed any relation with the ecology of the species, the laesura type of the spores, the ploidy level, neither the taxonomic group where different species belong. 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, and gametophytes develop sexual structures in practically all the plates. We dont include Ceterach officinarum in this point because the plates have appeared very contaminated.

1.00

*
0.90

Germination proportion

0.80

* * * * *

0.70

0.60

0.50

0.40

0.30

0.20

0.10

*
A. onopteris N. marantae P. aculeatum A. filix-femina C. officinarum D. filix-mas T. palustris P. lonchitis C. fragilis P. vittata

Graphic 1: Bars shows means and twice the typical error of the germination percentage in the three storage temperatures ( 25C, -80C, -196C). Stars indicate groups significantly different (ANOVA, p-value<0.05). Sugar estimation data suggest that the total sugars amount is in relation to the stored time but not with the studied conservation method. We can observe in graphic 2 that total sugars amount is also independent of the spores viability in the samples. There is a significant soluble sugar loss in spores along the time. This loss is independent of the storage method. A significant soluble sugar loss in stored spores isnt synonymous with a significant viability loss or spores death. Other factors, moreover soluble substances loss, should be implicated in viability loss and death of spores (BERI & BIR, 1993).

(mg. sugar / mg. spores)

(% germination) Graphic 2: We can observe for the different samples, the average and twice the standard error of the total sugar amount by weight that spores had according to the germination percentage. The colours of the bars indicate the different Tukey homogeneous groups. The colours of stars indicate the significantly homogeneous groups for the different germination percentage. Our results after six months of work contribute to the first quantitative data related to the ultrafreezing effects in no green spores. We have found that ultrafreezing doesnt affect spores viability after 6 months, neither gametophyte development. Gametophyte remains its capacity to complete the vital cycle. The preservation of biological material in cryogenic temperatures (cryopreservation) manages to stop completely the biological reactions, that permits the materials indefinite storage. References AGRAWAL, D.C.; S.S. PAWAR & MASCARENHAS, A.F. -1993-. Cryopreservation of spores of Cyathea spinulosa Wall. ex. Hook. f. - An endangered tree fern. Journal of Plant Physiol.ogy 142: 124126. ATKINSON, L.R. & A.G. STOKEY -1964-. Comparative morphology of the gametophyte of homosporous ferns. Phytomorphology 14(1): 51-70. BERI, A. & S.S. BIR -1993-. Germination of stored spores of Pteris vittata L. American. Fern Journal. 83(3): 73-78. CONSTANTINO S.; L.M. SANTAMARIA & E. HODSON -2000-. Storage and in vitro germination of tree fern spores. Botanic Gardens Micropropagation News, 2(4): 58-60. DICKIE, J.B.; R.H. ELLIS, H.L. KRAAK, K. RYDER & P.B. TOMPSETT -1990- Temperature and seed storage longevity. Annals of Botany 65, 197-204.

DYER, A.F. -1979- The culture of fern gametophytes for experimental investigation. In: Dyer AF ed. The experimental biology of ferns. London: Academic Press, 253-305. ELLIS, R.; T.D. HONG & E.H. ROBERTS -1985- Handbook of seed technology for genebanks. Vol. I. Principles and methodology. International Board for Plant Genetic Resources. Roma. ESTRELLES E.; N. FUENTES, J. PRIETO, M. BOSCAIU, D. BALLESTEROS & A.M. IBARS -2003-. Threatened Valencian Flora: Initiatives for its Conservation in Smith RD, Dickie JB, Linington SH, Pritchard HW, Probert RJ eds. Seed conservation: Turning into Practice. Kew. Royal Botanic Gardens, Kew, UK. 859-868. GMEZ-CAMPO, C. -2001-. La prctica de la conservacin de semillas a largo plazo. In: Conservacin de especies vegetales amenazadas en la regin mediterranea occidental. 255-266. Ed. Centro de Estudios Ramn Areces. Madrid JONES L.E. & P.W. Hook -1970-. Growth and development in microculture of gametophytes from stored spores of Equisetum. American. Journal of Botany 54(4): 430-435. LEBKUECHER, J.G. -1997-. Desiccation-time limits of photosynthetic recovery in Equisetum hyemale (Equisetaceae) spores. American. Journal of Botany 84(6): 792-797. LINDSAY, S.; N. WILLIAMS, A.F. DYER -1992-. Wet storage of fern spores: unconventional but far more effective!. In: Ide JM; Jermy AC & Paul AM eds. Fern Horiculture: past, present and future perspectives. Intercept, Andover. 285-294. LLOYD, R.M. & E.J. KLEKOWSKI JR -1970-. Spore germination and viability in Pteridophyta: evolutionary significance of chlorophyllous spores. Biotropica 2: 129-137. MCCREADY, R.M., J. GUGGOLZ, V. SILVEIRA & H.S. OWENS -1950-. Determination of starch and amylose in vegetables. Application to peas. Anal Chem 22: 1156-1158. PAGE, C.N., A.F. DYER, S. LINDSAY & D.G. MANN -1992-. Conservation of pteridophytes: the ex situ approach. In: Ide JM, Jermy AC & Paul AM eds. Fern horticulture: past, present and future perspectives. Andover: Intercept, 269-278. PENCE, V.C. -2000-. Survival of chlorophyllous and non-chlorophyllous fern spores through exposure to liquid nitrogen. American Fern Journal, 90(4): 119-126. QUINTANILLA, L.G.; J. AMIGO, E. PANGUA & S. PAJARON -2002- Effect of storage method on spore viability in five globally threatened fern species. Annals of Botany. 90(4): 461-467. SIMABUKURO, EA; A.F. DYER & G.M. FELIPPE -1998-. The effect of sterilization and storage conditions on the viability of the spores of Cyathea delgadii. American Fern Journal, 88(2): 72-80. WINDHAM, M.D.; P.G. WOLF & T.A. RANKER (1986) Factors affecting prolonged stored spore viability in herbarium collections of three species of Pellaea. American Fern Journal, 76(3): 141-148. Acknowledgements The authors thank Dr. J.A. Rossello from ICBiBE (Institut Cavanilles de Biodiversitat i Biologia Evolutiva of Valencia University) for useful comments on an earlier version of this manuscript. This study was supported by Spanish Ministry of Science and Technology Project REN2002-03697. During this study, D.B.B. was in receipt of an FPU grant from the Spanish Ministry of Science and Technology.