Spore Potential for Germination, Sporophyte Formation and Growth Young of Sporophytes of Four Fern from Species the Atlantic Forest (Brazil) ROSANE HiENDLMEYER and AUREA MaRIA RaNDI Laboratorio de Fisiologia Vegetal, Departamento de Botanica, Universidade Federal de Santa Catarina, 88040-900, Florianopolis. Santa Catarina, Brazil The world fern flora contains 9,000 to 12,000 species; about 3,250 are found American in the continent (Tryon and Tryon, Windisch, and 1982; 2002) 1,200 to 1,300 species are found Sehnem in Brazil (Prado, 1997). 493 (1977) listed species in the South of Brazil, the majority native to the Atlantic Forest. According "Conservation to International" the Atlantic Forest the third is 8% diversity "hotspot" in the world and only of remains original area its preserved. few In the last decades, several ferns with ornamental medicinal or proprieties have been indiscriminately exploited and them several of are considered be endangered to Examples some species. include tree ferns belonging the Cyatheaceae and to Dicksoniaceae ("xaxins" "samambaia- or which ?us"), have been exploited commercial for purposes, especially in the South of Brazil (Windisch, 2002). Dicksonia sellowiana Hook. (Dicksoniaceae) an endangered is tree fern in Brazil due to the extensive harvesting in its IBAMA, habitat (Sehnem, 1978, 1997) and Rumohra adiantiformis (Forst.) Ching., (Dryopteridaceae) Sphaeropteris gardneri (Hook.) R.M. Tryon, Cyathea atrovirens (Langsd. & Domin, Fisch.) Alsophila some setosa Kaulf, species of Adiantum, Asplenium, Blechnum, Selaginella, Marsilea, Adiantopsis and Lycopodium are extracted ornamental arrangements & for (Milton Moll 1988; Windisch, 2002). According Pence ex to (2000), the situ preservation of spores, particularly of rare species from threatened habitats, can be an important supplement to the maintenance of pteridophyte The species in the wild. conditions under which spores are stored have a notable impact on their viability. Generally, avoid to AMERICAN FERN JOURNAL: VOLUME NUMBER 100 4 (2010) deterioration, they are stored in dry, ambient or low temperatures, although in some cases this has resulted in loss of viability (Beri and Bir, 1993; Camloh, 1999). The aim was of the present study to verify the potential of germination of spores stored under refrigeration, the percentage of sporophyte formation after in vitro germination, and the relative growth rate (RGR) of young sporophytes room cultivated in a growth of four terrestrial species native from the Atlantic Blechnum Forest: brasiliense Desv. (Blechnaceae) an herbaceous species, Domin Cyathea corcovadensis (Raddi) and Cyathea delgadii Sternb. (Cyathea- ceae) both tree ferns and Saccoloma inaequale (Kze.) Mett. (Dennstaedtiaceaej an herbaceous fern. These species, with exception of Saccoloma inaequale, are routinely used in Brazil as ornamental plants and are exploited indiscrimi- nately from Saccoloma inaequale which their habitats. a rare species, also is has potential as ornamental plant. Such information will be useful for management and conservation programs in the future. Materials and Methods Blechnum brasiliense and Cyathea corcovadensis sporophylls were harvest- ed from several plants on January 12'^ of 2003; sporophylls of Cyathea delgadii were harvested on April 25**" 2002 and Saccoloma inaequale on March of of 29'*' of 2003. The collections were carried out in Natural City Park Sao Francisco de Assis, situated in Blumenau, Santa Catarina State, South Brazil, at 26 55'15" S and 49 05'30" W. The study site a 22.29 ha natural Atlantic is forest fragment. C Sporophylls were air-dried in an oven 30 three days on paper at for filter in order to induce dehiscence. The spores were removed and separated from debris by filtering through lens paper, and stored in glass jars under ± Spores Blechnum and Cyathea refrigeration at 7 1 C. of brasiliense corcovadensis were kept under refrigeration for eight months before the germination spores of Cyathea delgadii and Saccoloma inaequale were test; under months kept refrigeration for six before the germination Spores test. 10% were surface sterilized using a (v/v) solution of commercial bleach (2% of active chlorine) 20 min. before through paper and for filtering sterile filter washing several times in sterile distilled water. Spores of each species were mL sown in four conical flasks containing 20 of Mohr's mineral solution as mg modified by Dyer supplemented with Benomyl 0.01%. About 10 (1979) of spores were inoculated each which was plugged with two in flask, layers of (7X7 autoclaved transparent commercial polypropylene film cm) fixed with a rubber band. All tests were done in a completely randomized design. All procedures were carried out in a laminar hood. Flasks were kept in a growth chamber (25 ± 2 C) m"^ under a 16-hour photoperiod and photon flux density of 30 jimol s~^ provided by cool white fluorescent tubes. The irradiance was analyzed by a PAR LICOR quantameter 250. of sensor (400 to 700 nm). The percentage of abnormal spores and germination were scored daily or every two days. One HIENDLMEYER & RANDI: SPOROPHYTE GERMINATION AND GROWTH from each was slide flask prepared, with 100 spores counted on each slide. Germinated and abnormal were spores recorded microscopy in optical (40X). Spores were considered to be abnormal when they were empty when totally or they presented few a storage grains between empty The mean spaces. germination time was calculated each for replication per treatment according the equation: to where tj is the time in days starting from day 0 and the number of spores rij is completing germination on day (Labouriau, Santana and 1983; Ranal, 2004). month one After of cultivation mineral young in solution, filamentous gametophytes were transferred to trays containing sterilized typic hapludult with soil (3 parts] addition of thermophilic organic compost The part). (1 thermophilic compost was obtained from decomposition and of vegetable fruit wastes at the University of Santa Catarina. The substrate with (soil thermophilic compost) was CIDASC analysis carried out in (Companhia Integrada de Desenvolvimento Agricola de Santa The Catarina). substrate pH N presented a low and high levels of P, K, Ca and (Table The were trays 1). covered with transparent film to avoid excessive water evaporation and plant dehydration. Substrate was sterilization carried out in high power a microwave oven for 20 minutes to avoid contamination with other ferns from the spore bank. To analyze soil the percentage of sporophyte originated from mature gametophytes and to obtain plants that were used growth later in 200 mature analysis, cordate gametophytes were transferred to four trays (50 gametophytes in each tray) containing the same substrate. Gametophytes were separated from each other by cm. During 1.0 the gametophytes were test, watered with water The sterile distilled three times a week. presence of was sporophytes recorded once a week. Plants were kept growth room in a as described above. When the sporophytes of the four species were big enough be manipulated to cm they were [ca 3.0 tall), individually transplanted to small pots containing AMERICAN VOLUME NUMBER FERN JOURNAL: 100 4 same the substrate used gametophyte development and were kept for in plastic trays covered with transparent film, in a completely randomized design (Time 1= Ti). For the growth analysis of Blechnum brasiliense, Cyathea delgadii and we Saccoloma inaequale, 20 sporophytes and growth utilized the analysis for we of Cyathea corcovadensis utilized 15 sporophytes. After 60 days of 2= individual transplantation {Time sporophytes were count T2), collected to number the of fronds and to measure the longest frond's length. Table 2 shows the period of growth spore inoculation time and time The after at 1 2. relative growth rate (RGR] was estimated according Bernabe to et (1999), as (Log^o al. - -10 L2 Logio T2 -Ti where Log the base logarithm, the La) / is Li is initial when leaf length the sporophytes were individually transplanted to the pots in time and 1, L2 is the leaf length at time 2 (after 60 days of transplantation to the individual pots). Data were analyzed by Windows Windows Excel Minitab for (Microsoft), for and Statgraphics software. Means and standard deviation were calculated. The Kolmogorov-Smirnov test for goodness of for normality and the Bartlett's fit test for the homogeneity of variance (0.05) were applied before the analysis. The parametric pairwise comparison among number test the of fronds of the four species, were done by the Duncan (5%) Muhiple Range The non- Test. Dunn parametric Kruskal-Wallis followed by was employed the test test to RGR analyze the (relative growth rate) because these data did not present residual normality or did not present homogeneity of variance (Santana and Ranal 2004; Zar, 1996). The Student "t" test was applied to compare the number of fronds and the longest frond's length between Tl and T2 each for species, Results The mean germination time for the four species studied in this work ranged from 9.9 ± 0.1 days for Blechnum brasiliense to 17.8 ± 0.1 days for Sacolloma The ± inaequale spores (Table percentage of germination varied from 32.0 3). ± 2.5% Cyathea corcovadensis 5.0% Cyathea for to 77.0 for delgadii (Table 3 and Fig. la-ld) and the percentage of abnormal spores varied from 5.6 ± HIENDLMEYER & RANDI: SPOROPHYTE GERMINATION AND GROWTH 0.7% for Sacolloma inaequale 29.0 ± 0.7% Blechnum to for brasiliense (Table 3). All species produced sporophytes during the period of analysis, but the percentage of mature gametophytes that produced sporophytes and the time for sporophyte formation were among Blechnum variable species. brasiliense presented the first sporophytes after 86 days of cuftivation and 91.5 ± 2.5% of gametophytes produced sporophytes 303 days The after (Fig. 2a). first sporophytes appeared Cyathea in corcovadensis 117 days after of cultivation and ± after 264 days of cultivation, 35.8 16.2% gametophytes produced of sporophytes Cyathea (Fig. 2b). delgadii presented the sporophytes first after 109 days and of cultivation after 249 days of cultivation, 79.2 ± 13.5% of gametophytes produced sporophytes The sporophytes (Fig. 2c). first of Saccoloma inaequale appeared 78 days and after of cuftivation after 295 days, ± 24.4% 48.4 of gametophytes produced sporophytes (Fig. 2d). The number produced of fronds ranged from Blechnum 4.5 to 7.0 for from brasiliense, 4.7 to 5.0 for Cyathea corcovadensis, from 5.8 to 8.5 for Cyathea delgadii and from Saccoloma 6.0 to 9.0 for inaequale 3a (Fig. to 3d). AMERICAN VOLUME NUMBER FERN JOURNAL: 100 4 (2010) The longest frond length ranged from 3.0 to 6.9 cm for Blechnum brasiliense, from 2.7 to 6.1cm for Cyathea corcovadensis, from 4.0 to 10cm for Cyathea and from 9.5cm Saccoloma The delgadii inaequale 4a 3.3 to for (Fig. to 4d). RGR of Saccoloma inaequale, an herbaceous fern, was statistically greater than RGR Cyathea was the of corcovadensis, a tree fern, but similar to the other species (Table The number of fronds produced two months was 4). after among statistically smaller for Cyathea corcovadensis but did not differ the other species (Table 4). Discussion common under method Preservation refrigeration a of spore storage. is According to Pence (2000), the ex situ preservation of spores can be an important supplement to the maintenance of pteridophyte species in the wild. -20 Pence (2000) stored spores of 33 species at 4 C, C, or in liquid nitrogen and observed that spores of three species did not lose viability after more than HIENDLMEYER & RANDI: SPOROPHYTE GERMINATION AND GROWTH months. 75 Eighty-two percent of Dicksonia sellowiana Hook, spores germinated more than under C after 2 years refrigeration at 10 (Filippini et ah, 1999). Previously sterilized spores of D. sellowiana remained viable after cryopreservation and in liquid nitrogen cryopreservation seems improve to the germination of the spores {Rogge et al, 2000). Aragon and Pangua analyzed (2004) the spore germination of four rupicolous Asplenium taxa of adiantum-nigrum [A. adiantum-nigrum, L. var. A. adiantum. nigrum L. var. silesiacum, A. septentrionale Hoffm subsp. (L.) and septentrionale A. ruta-muraria subsp. ruta-muraria) and L. after 6, 1, months 12 of storage in Eppendorf tubes on (dry storage) or agar plates (wet -20 C and storage) 20 at C, 5 C. In all cases, except for A. ruta-muraria, germination percentage was maintained wet and in dry storage, but in the dry method storage percent germination was Though higher. the authors did not carry out a thorough post-germination study, the plates used in the various experiments remained chambers C in cufture 20 months and at for 6 the gametophytes appeared to develop normally. They suggested that storage time might not affect the subsequent development of the prothalli. , AMERICAN FERN VOLUME NUMBER JOURNAL: 100 4 (2010) b 12 ^ 8 n n h T r " 0 , , . 270 337 397 3 Days of cultivation 12 12 1 8 8 n n 1 0 , 0 252 312 386 4A 6 Days of cultivation Cyathea corcovadensis Cyathea (b), i), red soil with the addition of organic In comparison to studies done by Simabukuro et (1993) the mean al. Blechnum germination time for spores of brasiliense and Cyathea delgadii were delayed However, Simabukuro in the present study. et (1993) al. inoculated fresh unsterilized spores 25 C under continuous white The at light. surface sterilization could delay or reduce spore germination but necessary is in order to obtain higher percentages of gametophytes. Indeed, Camloh (1993, 1999) reported that the best germination of Platycerium bifurcatum (Cav.) C. when Chr. occurred unsterilized spores were used, but contamination that always occurred 10 days Simabukuro after of culture. et (1998) indicated al. that before the germination of dry-stored spores, in order to avoid the incidence of fungal growth, there is the need to sterilize them. The differences mean observed in the present paper the germination time for of B. brasiliense and C. delgadii could be an effect of the time and location of collection, method spore germination and time under sterilization for of storage For Blechnum and Blechnum refrigeration. chilense (Kaulf.) Mett. cycadifo- Thompson lium (Colla) Sturm, the germination of spores in agar-gelled medium 6-10 started after days of spore soaking (Perez-Garcia et 1996). al., The Blechnum germination results of stored spores of brasiliense are similar who to the results of Simabukuro et al. (1993), worked with sporophytes of B. brasiliense collected in the Biological Reserve of Moji-Guagu localized in S. Paulo The State (southeast region of Brazil). data presented in this paper are in HIENDLMEYER & RANDI: SPOROPHYTE GERMINATION AND GROWTH b 10 10 -^ 6 n.n. u, ^ \'- 2 0 270 330 337 397 Days Days of cultivation of cultivation d 15 - 10 E (cm) n n n.n'. ? FL 5- - 0 386 446 252 312 Days of cultivation Days of cultivation Fig. 4. Longest frond length (FL] of Tl and T2 of Blechnum b), Cyathea delgadii (c) and Saccoloma inaetqiia e (d) cult vated in red soil'^with the addition of und 2 C) er a 16-h photoperiod and photon flux accordance with similar studies in Cyathea corcovadensis (Felippe et ah, and 1989) C. delgadii (Randi and Felippe, 1988 Simabukuro a, b). et al. (1998) -12 stored previously sterilized spores of delgadii 12 months C and C. for at 74% observed germination days from sowing. The germination after 7 of Table 4. Relative growth rate (RGR) and mean number fronds per month (NF) of Blechnum brasihense, Cvatbea corcovadensis, Cyathea delgadii, and Saccoloma inaequale cultivated in red compound soil with the addition of organic (3:1) in growth chamber (25 ± 2 C) under a 16-h photoperiod and photon flux density of 30 nmol m"^ s \ is the statistic for the Bartlett test; D„„„ H Kolmogorov-Smirnov the the is statistic for test, is the statistic for the Kruskal-Wallis test: sd is the standard deviation. Letters denote statistical differences. * Data did not show normality. AMERICAN FERN VOLUME NUMBER JOURNAL: 100 4 (2010) Saccoloma Sterilized spores of inaequale took 24 days. For spores of related Odontosoria (Dennstaedtiaceae), germination took six to 15 days to reach maximum percentage (Granados The abnormal et al, 2003). spores observed in this study seems to be related with the reduction in percentage of spore Blechnum germination and for brasiliense Cyathea delgadii, but seems be to it not the only reason for the low germination of Cyathea corcovadensis and Sacolloma inaequale. Large differences in percentages of germination are the rule in simultaneous experiments on different taxa. Spore age, storage conditions and culture conditions, have on all a great influence fern spore germination (Camloh and Gogala, Camloh, Camloh, 1992; 1993; 1999; Sheffield et 2001). Differences in al., the time for sporophyte formation and percentages of sporophyte formation were common also in studies on sporophj^e development (Sheffield et al, 2001). In this study, the first sporophytes of Blechnum brasiliense were observed after months 2.9 of cultivation (86 days). Meanwhile, the sporophytes were first observed months 4.5 after spore inoculation for B. chilense and after 7 months for B. cycadifolium (Perez-Garcfa The growth was et al, 1996). of B. brasiliense probably by facilitated the substratum, temperature and photoperiod employed throughout this work. This methodology can then be utilized for the cultivation of B. brasiliense. In fact, sporophytes of this species were transplanted to the garden of the Department of Botany and showed normal development. a However, the sporophytes of C. corcovadensis, C. delgadii and S. inaequale were not transplanted the garden because they showed to sensitivity to desiccation and require special cares during acclimatization, needing high humidity, shadow and mild temperatures during Blechnum this period. brasiliense appears to be a plant adapted to regimes of oscillating humidity and greater light being more intensfty, easily acclimatized. The gametophytes of Sacolloma inaequale that did not produce sporophytes A grew successively until the end of this study. similar observation was found by Sakamaki and Ino working with (1999) Thelypteris palustris Schott They (Thelypteridaceae). concluded that sporophytes appeared on most gametophytes that reached a critical size for sporophyte formation, but some gametophytes that did not produce sporophytes became very and large lived for a long time without producing sporophytes. Our data for frond production by young sporophytes of Blechnum and Cyathea brasiliense delgadii are similar for the data found for Acrostichum & daneaefolium Langsd. which were ± ± Fisch. (Pteridaceae) 1.0 0.03 to 1.3 month 0.04 fronds per (Mehltreter and Palacios-Rios, 2003; Mehltreter et al., 2003). However, for adult sporophytes of several Cyatheaceae, the number of fronds produced each year varied from 14 (Tanner, Ash, 3 to 1983; 1987; Arens, 2001). RGR RGR In this study, the of Sacolloma inaequale was greater than the of Cyathea corcovadensis. were Statistically significant differences not observed among RGR the other species. Data for the of some angiosperms from the Atlantic Forest, presented by Paulilo showed herbaceous et (1993), that al. RGR showed RGR species greater than tree species. Concerning for fern