Propagation in vitro and ex situ cultivation of Woodsia alpina (Bolton) S.F. Gray

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1 15 Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray 15 Propagation in vitro and ex situ cultivation of Woodsia alpina (Bolton) S.F. Gray KRYSTYNA KROMER 1, ANDRZEJ RAJ 2, LUDWIK O NIERZ 3, DOROTA POTURA A 1 1) Botanical Garden, University of Wroc³aw, Sienkiewicza 23, PL Wroc³aw, Poland; kromer@biol.uni.wroc.pl 2) Karkonoski National Park, Cha³ubiñskiego 23, PL Jelenia Góra, Poland 3) Department of Botany and Plant Ecology, Wroc³aw University of Environmental and Life Sciences, Pl. Grunwaldzki 24a, PL Wroc³aw, Poland; ludwik.zolnierz@up.wroc.pl 3) Institute of Natural Sciences, Wroc³aw University of Environmental and Life Sciences, Pl. Grunwaldzki 24a, PL Wroc³aw, Poland ABSTRACT: Woodsia alpina is a rare fern with circumpolar distribution. The species is protected or endangered in many countries. In Poland, it is known from the Sudetes Mts. (1 location) and the Tatra Mts. (3 locations). The reproduction of this plant by spores is difficult and thus there is no method of cultivation available. An in vitro tissue culture technique is promising in propagation of many recalcitrant and demanding species. This technique was used to determine the propagation method on purpose to maintain the Sudeten population of Woodsia alpina in botanical collections and in the supplementary habitat in the Karkonoski National Park. Spores collected in the glacial circus Ma³y Œnie ny Kocio³ were sown in vitro and germinated during 3 9 months. Prothalli cultured in vitro spread vegetatively in various manners. Gametangia were formed on prothalli in different developmental stages. On heart-shaped prothalli archegonia and occasionally antheridia were formed, whereas on filamentous ones only antheridia occurred. Sporophytes were hardly ever produced with single sporophytes present per thousands of gametophytes. At apical meristems of the primarily formed sporophytes, green globular bodies (GGB) proliferated. These bodies could further regenerate forming secondary sporophytes, which developed nearby the initial ones. Sporophytes at this stage of development often became brown and lost fronds, however many of them renewed their growth after the dormancy period. Plants, which were transferred in spring to a greenhouse and planted in a commercial mix for ferns or neutralized peat substratum, acclimated in 70%. The growth cycle of W. alpina cultivated at the altitude of 120 m a.s.l. in the Botanical Garden of the University of Wroc³aw was short and fronds were lost KROMER K., RAJ A., O NIERZ L., POTURA A D Propagation in vitro and ex situ cultivation of Woodsia alpina (Bolton) S.F. Gray. In: E. Szczêœniak, E. Gola (eds), Club mosses, horsetails and ferns in Poland resources and protection. Institute of Plant Biology, University of Wroc³aw, Wroc³aw, p

2 16 Krystyna-Kromer,-Andrzej-Raj,-Ludwik- o³nierz,-dorota-potura³a 16 in the middle of August. Next year not all rhizomes initiated the growth; only the biggest ones displayed this ability. Plants cultivated in the supplementary habitat in the Jagni¹tków nursery of the Karkonoski National Park at the altitude of 650 m a.s.l. performed perfectly well. ABSTRAKT: Woodsia alpina jest rzadk¹ paproci¹ o rozmieszczeniu wokó³biegunowym. Gatunek ten w wielu krajach uznawany jest za zagro ony i gin¹cy, w Polsce zaœ wystêpuje w Sudetach (1 stanowisko) i w Tatrach (3 stanowiska). Reprodukcja tej roœliny przez zarodniki jest trudna, a adna metoda uprawy nie jest obecnie dostêpna. Kultura in vitro jest obiecuj¹c¹ technik¹ w rozmna aniu wielu opornych i trudnych gatunków roœlin. Zosta³a ona zastosowana w opracowaniu metody rozmno enia i zabezpieczenia sudeckiej populacji Woodsia alpina w kolekcji ogrodu botanicznego i na stanowisku zastêpczym w Karkonoskim Parku Narodowym. Zarodniki zebrane z Ma³ego Œnie nego Kot³a wysiane in vitro kie³kowa³y podczas 3 9 miesiêcy. Uprawiane przedroœla rozrasta³y siê wegetatywnie kilkoma sposobami. Gametangia powstawa³y na przedroœlach znajduj¹cych siê w ró nych stadiach rozwoju. Na gametofitach sercowatych ró nicowa³y siê rodnie i plemnie, a na nitkowatych jedynie plemnie. Sporofity powstawa³y bardzo rzadko; na tysi¹ce gametofitów rozwija³y siê zaledwie pojedyncze sporofity. Na inicjalnie utworzonych sporofitach z merystemu wierzcho³kowego proliferowa³y zielone globularne cia³a (GGB). Cia³a te mog³y wytwarzaæ nastêpne sporofity, które rozwija³y siê w pobli u inicjalnego. Powsta³e sporofity w tym stadium rozwoju czêsto br¹zowia³y i traci³y liœcie, chocia wiele z nich odnawia³o wzrost po okresie spoczynku. Roœliny wysadzone wiosn¹ do szklarni w handlow¹ mieszankê ziemi dla paproci lub zneutralizowany substrat torfowy aklimatyzowa³y siê w 70%. Okres wzrostu W. alpina we Wroc³awiu, na wysokoœci 120 m n.p.m. by³ krótki, a roœliny zrzuca³y liœcie w po³owie sierpnia. Nastêpnego roku nie wszystkie k³¹cza inicjowa³y wzrost; zdolnoœæ tê posiada³y tylko te najwiêksze. Roœliny uprawiane na stanowisku zastêpczym w Gospodarstwie Szkó³karskim Karkonoskiego Parku Narodowego w Jagni¹tkowie, na wysokoœci 650 m n.p.m., ros³y i rozwija³y siê bardzo dobrze. KEY WORDS: Woodsia alpina, in vitro cultures, endangered ferns Introduction Plants are the foundation of all life on earth, without which we cannot survive. It is our responsibility to preserve them for the future generations. In 1997 the IUCN prepared a Red List of Threatened Plants with the goal to protect the worlds flora. Out of known species of vascular plants, which include ferns, fern allies, gymnosperms and flowering plants, the 12.5 percent is threatened with extinction at a global level. According to Zaj¹c and Zaj¹c (1995) the endangered species of rare vascular plants are represented in Poland by taxa with the limited area of distribution, taxa in isolated sites and those with a significant disjunction from their compact ranges. Also, high

3 17 Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray 17 threatened are endemic plants with a limited number of specimens in their populations. From the botanical point of view, particularly interesting are uncommon and rare species which significantly contribute to the local (Nilsson et al. 1988) and regional (Gentry 1986) species biodiversity. Such species are extremely endangered due to the small population size and restricted distribution. Detailed information on the biology of species and a knowledge of the factors essential for their growth make it possible to define conservation priorities for plant taxa. Pteridophytes are an ancient group of plants with a large number of relict and endemic species. As evolutionary old plants they represent a specific biology, which determines on their weakness and strength and their ability to colonize new habitats. The limitations are identified as the handicap of an independent gametophyte stage, a single initial cell in the growing-points of sporophytes, the slow plant growth rate, intolerance to fluctuating environmental conditions, poorly controlled evaporative potential, uncontrolled high reproductive commitment and dependence on water to breed (Page 2002). Woodsia alpina is a rare or endangered plant in several countries, including Canada, the USA, Great Britain, and Spain. This species is listed by the U.S. federal government and the state governments of Main and Michigan as threatened, and of New York and Vermont as endangered (Goff et al. 1982; USDA Natural Resources Conservation Service). Woodsia alpina an alpine cliff fern, alpine woodsia or northern woodsia is a species with a circumpolar distribution, which includes Europe, Asia, Greenland and North America, and the Arctic Islands. In Europe it occurs in the Pyrenees, the Apennines, the Alps, the Sudetes Mts. and the Carpathians. The alpine cliff fern is an arctic-alpine glacial relict. It grows in Poland in single location in the Sudetes Mts. and the Carpathians. In the Sudetes Mts. it is known only from one location in a glacial circus Ma³y Œnie ny Kocio³ (Karkonosze Mts.). The other reported locations in this mountain range, at which woodsia however became extinct, were in the southern part of the Karkonosze Mts. and Pradziad Massive in the Czech Republic. In the Tatra Mts. the plant is known from 3 locations (Fabiszewski, Piêkoœ-Mirkowa 2001; Piêkoœ-Mirkowa, Delimat 2002). The plant is a small, rock loving fern typically growing in dense, upright clumps. Its leaves are linear to narrowly oblong, reaching cm in length, singly pinnate; pinnae are ovate and obtuse, pinnately lobed, smooth above, hairy but not scaly. W. alpina grows in clumps that usually have several crowns (Brown 1964; It has a short erect rhizome, compact and stiff; fronds usually are shorter than 10 cm. It sporulates in summer and in early autumn.

4 18 Krystyna-Kromer,-Andrzej-Raj,-Ludwik- o³nierz,-dorota-potura³a 18 The species is included in the Polish Red Data List (Zarzycki, Szel¹g 2006) and according to the Polish Red Book is classified as critically endangered (Fabiszewski, Piêkoœ-Mirkowa 2001). Isozyme studies confirm the longstanding hypothesis that W. alpina is an allotetraploid derived from hybridization between W. glabella and W. ilvensis. A considerable disagreement exists concerning the chromosome number of W. alpina but 2n=160 seems to be the most likely, given the numbers reported for the two parental species. Additionally, hybrids between W. alpina and W. ilvensis have been reported from both Europe and North America. These morphologically intermediate triploids with malformed spores have been named W. gracillis (Lawson) Butters (Wagner 1987; Windham 1987). Woodsia alpina typically occurs in crevices, steep slopes and cliffs, ridges containing slate and calcareous rocks, especially limestone of ph 5.1 to 7.8. Occasionally it grows on slopes covered mostly by grained rocks. The plant colonizes substrates with a low organic content. The only location in the Ma³y Œnie ny Kocio³ glacial circus is unusual habitat for this plant, because there it grows on a basaltic body of sub-volcanic intrusion (Zago d on, Zago d on 2006). Basalts differ from other crystalloid rocks in respect of their acidity and belong to rocks richer in base, which explains why W. alpina can grow there. The reproduction of this plant by spores is difficult and there is no method of cultivation available. An in vitro tissue culture technique is promising in propagation of many demanding and resistant species. It was used to determine the propagation method on purpose to preserve the Sudeten population of Woodsia alpina in botanical collections and in the supplementary habitat in the Karkonoski National Park. The best method of the species protection is in situ conservation based on preserving of natural habitats. Particular species, such as Woodsia alpina, require ex situ conservation either by multiplication by conventional methods or by in vitro tissue culture or spore culture technique. Such multiplied plants may be protected and maintained in the botanical gardens or in chosen supplementary habitats. 1. Material and methods Several leaf blades of Woodsia alpina with sori were collected at the beginning of July ( ) and August ( ) from the only natural locality in Lower Silesia in the Ma³y Œnie ny Kocio³ glacial circus. Collected fragments of sporophylls were dried out between sheets of paper at room temperature. The obtained spores were packed in small packages of blotting-paper, and after superficial sterilization sown on culture media. Spores were sown at different period of time according to the collection date (13, 42, 76, 88, 119 days).

5 19 Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray 19 In sterilization procedure packages were immersed in 70% alcohol for 3 minutes and followed by shaking for 5 or 10 min in a solution of 3% sodium hypochlorite. In next year (2005), spores from four plants were sterilized by immersion in 70% alcohol for 30 s followed by treatment in 5 8% sodium hypochlorite for 5 12 min. Spores were sown on 1/2 or 1/4 of MS (Murashige, Skoog 1962) medium supplemented with vitamins, glycine (2.0 mg l -1 ), sucrose (30 g l -1 ) and agar (8 g l -1 ). The ph of media was adjusted to 6.8 with 1M KOH or HCl prior to autoclaving for 15 min at 121 o C. Flasks with spores were incubated in different temperatures and light regimes: 25 o C and 16-h light photoperiod at the irradiance of 14.2 ìmol m 2 s -1 ; 10 o C at the irradiance of 0.15 ìmol m 2 s -1. The influence of sucrose concentration on the growth of gametophytes and sporophytes was also analysed. Prothalli were subcultured in a three-month interval; newly formed sporophytes were separated and cultured on 1/2 MS medium. Older sporophytes of various sizes were planted in a greenhouse in a neutralized peat soil or a commercial mix for ferns (Krone, Floro-hum). Pots were covered with polyethylene foil, which was gradually removed. Five weeks later, the potted plants were transferred to a hotbed and the growth of ferns was observed in the following years. Two supplementary habitats were created, one in the Botanical Garden of the University of Wroc³aw and the second, bigger one, in the Jagni¹tków nursery of the Karkonoski National Park. 2. Results and discussion Sterilized spores of W. alpina coming from the first trial in 2003 and sown on 1/2 MS medium were infected in a low percent (13.6%). In the sowing experiment in 2004, 29% of contaminations were noted. To prevent infections it is necessary to separate carefully spores from debris before sterilization. The germination rate was very low, and only single prothalli emerged after 7 months in culture. In the second experiment, spores of four individual plants collected in the natural location were sown separately on medium. The bestgerminated spores came from the plant signed with the number 4, weaker from the plant numbered as the first, and spores of a specimen with the number 3 did not germinate at all. The differences in the ability to form prothalli are probably connected with the degree of the spore maturity. Spores of the specimen No. 4 were much darker brown than the others, which means that they were fully ripe, however only 6% of them germinated.

6 20 Krystyna-Kromer,-Andrzej-Raj,-Ludwik- o³nierz,-dorota-potura³a 20 Fern spores usually persist in a viable, yet metabolically inactive state for a long period of time. The sequence of sowing 13, 42, 76, 88, 119 days after collecting did not answer our question of the effective time of germination, because of a generally very low level of spore germination. The ability to germinate persisted from 1.5 to 9 months for W. alpina spores cultured in vitro. The developmental block, resulting in the failure of perfectly viable spores to germinate, even when they are in conditions that promote germination, points to their dormancy. Spores of several ferns including those of Hymonophyllaceae (Atkinson 1960) and Grammitidaceae (Stokey, Atkinson 1958) hardly ever exhibit any dormancy, whereas others like the members of Schizaeaceae and Adiantaceae are definitely dormant. Apart from the primary requirement to hydrate the spores prior to germination, in many cases the mechanisms triggering their germination are not understood (Raghavan 1989). Performed experiments showed a very low germination rate of W. alpina spores in comparison to W. ilvensis sterilized by the same method (unpublished data). Also in natural conditions, the germination of these two species is different and much more difficult in case of W. alpina, what can significantly limit the spreading of this species populations. Germinating spores form two cells rhizoid and protonema initials. The spore germination in W. alpina is according to the Vittaria-type pattern (Brown 1964), and prothallial development is similar to Drynaria-type (Nayar, Kaur 1971). This means that germinating spores develop the primary rhizoid and protallus initials based on the fixed sequence of cell divisions and the direction of growth. According to literature mentioned above, germination starts from the unequal cell division, perpendicular to the spore polar axis. A primary rhizoid is formed from a smaller cell, whereas a prothallus initial from a bigger one at the opposite side. The second division of protonema initials is perpendicular to the first one. W. alpina spores cultured in vitro formed filaments. The meristematic activity of the apical cell of a filament gives rise to adult prothalli. The characteristic feature of prothallus formation in Woodsia alpina is the variability in the process of development. In the next step, a broad plate is formed, which consists of the meristem located at the bottom of the notch, and a thick, meridian midrib surrounded by broad, one-cell-thick wings on its either side. The adult prothallus, after 3 4 months of culture, has a cordate-thalloid shape with doubled curved wings and hairs on the surface (1 5 mm). The morphology of prothalli depends on the density of their growth and vary from cordate to more elongated structures in loose and compact groups on the medium, respectively (Fig. 1). Gametophytes spontaneously propagated in culture at least in two ways: from filaments and in the adult stage of growth. The filament has an unlimited ability to branch out. The cordate-like prothalli also regenerate the progeny

7 21 Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray 21 usually as outgrowths of one of the wings. In this way, gametophytes prolong their growth. In the older regions of adult prothalli, progressive degeneration takes place and this part forms additional numerous filamentous protonema and extra branches of the independent prothallus. The adult prothallus forms in the middle an elongated cushion, which bears the sexual organs, i.e. archegonia, and rhizoids on the ventral surface (Fig. 2). Antheridia in W. alpina are formed in the posterior half of the thallus (Fig. 3), while archegonia occupy marginal regions in the anterior region. The archegonia are visible only in the adult heart-shaped gametophytes, mainly on the midrib, whereas antheridia arise rather occasionally in adult thalli, mostly in filaments (Fig. 4). During 3 months of culture, the flasks were filled with gametophytes, which only rarely formed sporophytes (Fig. 5). In one flask, a limited number of sporophytes occured. In spite of a big number of prothalli, the amount of sporophytes born was extremely low. Usually only 1 3 sporophytes were noticed in one flask with thousands of gametophytes. In some cases, from one clump of prothalli several sporophytes arose. Prothalli after 2 months of culture comprised of elongated heart-shaped structures and filaments, which entwined and covered them. This type of expansion, where heart and filamentous prothalli were close together, created favorable conditions for fertilization. The examination of such clumps of W. alpina revealed that initially formed sporophytes decayed but from an apical meristem, green globular bodies (GGB) proliferated similarly to those described by Fernández and Revilla (2003). These bodies could further regenerate into new sporophytes in a vegetative way of propagation. The phenomenon of regeneration may explain why in one clump of prothalli the new sporophytes arise more numerously close to the initial, first formed sporophyte (Fig. 5). Sporophytes left on the same medium become brown and lost fronds after next 3 months of culture. They could renew their growth after separation from gametophytes. Rhizomes transferred to a fresh medium may renew gametophyte-like growth as a result of proliferation of growing centers (Fig. 6). The reason of this phenomenon is unknown. It can be a natural rest time of the plant or it can be forced by leaf necroses induced by unfavourable growth conditions and insufficient nutrition. Generally, it is almost impossible to separate two W. alpina generations, gametophytes and sporophytes. Probably, quick growing gametophytes cover the sporophytes and impede the uptake of nutrition. Fernández and Revilla (2003) stated that gametophytes of Dryopteris affinis sp. affinis cultured on MS media and supplemented with growth regulators for one month, produced sporophytes from the apical notch strictly in an apogamic way, since sexual reproduction was not possible due to a lack of archegonia. However, facultative apogamy is possible but not obligatory in this species. The developing sporophytes were of the same size as those found in natural

8 22 Krystyna-Kromer,-Andrzej-Raj,-Ludwik- o³nierz,-dorota-potura³a

9 23 Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray 23 environments, what may indicate their diploid character. During cultivation in vitro the growth regulators were excluded. It should in some way prevent the unwanted proliferation of asexual sporophytes. Apogamy is triggered in the callus and gametophytes growing on the medium containing 2,4-D, high doses of sucrose and ethylene (Raghavan 1989). To determine the sexual or asexual origin of sporophytes, the content of DNA in the prothalli and in sporophytes should be estimated. The life cycle of ferns comprising of two generations, haploid gametophytes and diploid sporophytes, requires creation of specific conditions in culture in vitro for the proper development of gametophytes and formation of an increased number of embryos. In spring, specimens of W. alpina propagated in vitro were planted in a greenhouse (Fig. 7) in a commercial mix for ferns or in a neutralized peat substratum. Transplanted micropropagules were covered with polyethylene foil or with inverted glass beakers inside the growth chamber. After developing new fronds, ferns were transferred to a hotbed initially covered with a shadowed glass and later with a green plastic net. Propagules survived in about 70% after the weaning process (Fig. 8). The species had fronds, which did not persist throughout the winter. The growth cycle of W. alpina was rather short in our altitude (the Botanical Garden of the University of Wroc³aw) and fronds became brown and lost in the middle of August. Next year not all the plants renewed the growth. Probably, specimens that did not form the rhizomes big enough during the previous season hardly ever renewed their growth in the following season (Fig. 9). According to literature, the species endures temperatures up to -40 o C in winter but requires a cold summer. At our altitude it may be too warm in summer, because the comparison of plants in the Botanical Garden (120 m a.s.l.) and at the supplementary locality at the Jagni¹tków nursery (650 m a.s.l.) showed the better state of Woodsia alpina plants in the second location. Fig. 1. Cordate prothallium of W. alpina with a marginal meristem at the notch (phot. K. Kromer) Ryc. 1. Sercowaty gametofit W. alpina z tkank¹ merystematyczn¹ we wciêciu (fot. K. Kromer) Fig. 2. Formed archegonia on gametophyte of W. alpina (phot. K. Kromer) Ryc. 2. Wykszta³cone rodnie na gametoficie W. alpina (fot. K. Kromer) Fig. 3. Antheridia on cordate prothallium of W. alpina (phot. K. Kromer) Ryc. 3. Plemnie na sercowatym przedroœlu W. alpina (fot. K. Kromer) Fig. 4. Antheridia on filaments of W. alpina (phot. K. Kromer) Ryc. 4. Plemnie na nitkowatych przedroœlach W. alpina (fot. K. Kromer)

10 24 Krystyna-Kromer,-Andrzej-Raj,-Ludwik- o³nierz,-dorota-potura³a 24 Fig. 5. In vitro culture of W. alpina gametophytes with formed sporophytes on 1/2 MS medium (phot. D. Potura³a) Ryc. 5. Kultura in vitro gametofitów W. alpina z powstaj¹cymi sporofitami na po ywce 1/2 MS (fot. D. Potura³a) Fig. 6. Sporophytes developing from resting rhizomes of W. alpina after 11 months in culture (phot. D. Potura³a) Ryc. 6. Sporofity rozwijaj¹ce siê ze spoczynkowych k³¹czy W. alpina po 11 miesi¹cach uprawy (fot. D. Potura³a)

11 25 Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray 25 Fig. 7. Sporophytes of W. alpina planted to flower-pots (phot. D. Potura³a) Ryc. 7. Sporofity W. alpina wysadzane do doniczek (fot. D. Potura³a) 3. Conclusion To our knowledge, this is the first paper describing the possibility of propagation of Woodsia alpina in tissue culture. Since the species can be reproduced only by spores and this propagation is difficult, the presented here method is the only available way of preservation and preventing the extinction of the small populations of this species.

12 26 Krystyna-Kromer,-Andrzej-Raj,-Ludwik- o³nierz,-dorota-potura³a 26 Fig. 8. Plants of W. alpina in the first year of cultivation (phot. D. Potura³a) Ryc. 8. Ro liny W. alpina w pierwszym roku uprawy (fot. D. Potura³a) Fig. 9. Alpine cliff fern in a hotbed in the second year of cultivation (phot. D. Potura³a) Ryc. 9. Rozrzutka alpejska w inspekcie w drugim roku uprawy (fot. D. Potura³a)

13 27 Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray 27 References ATKINSON L.R A new germination pattern for the Hymenophyllaceae. Phytomorphology 12: BROWN D.F.M A monographic study of the fern genus Woodsia. Nova Hedvigia 16: FABISZEWSKI J., PIÊKOŒ-MIRKOWA H Woodsia alpina. In: KA MIERCZAKOWA R., ZARZYCKI K. (eds), Polska czerwona ksiêga roœlin, Wyd. 2. IB PAN, Kraków, p FERNANDEZ H., REVILLA M.A In vitro culture of ornamental ferns. Plant Cell, Tissue and Organ Culture 73: GENTRY A.H Endemism in tropical versus temperate plants communities. In: SOULE M.E. (ed.), Conservation biology: Science of Scarcity and Diversity. Sinauer Press, Sunderland, MA, p GOFF G.F., DAWSON G.A., ROCHOW J.J Site examination for threatened and endangered plant species. Environmental Management 6: MURAHIIGE T., SKOOG F A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 47: NAYAR B.K., KAUR S Gametophytes of homosporous ferns. The Botanical Review 37(3): NILSSON C., GRELSSON C., JOHANSSON M.M., SPERENS U Can rarity and diversity be predicted in vegetation along river banks? Biological Conservation 44: PAGE C.N Ecological strategies in fern evolution: a neopteridological overview. Review of Paleobotany and Palynology 119: PIÊKOŒ-MIRKOWA H., DELIMAT A Occurrence of Woodsia alpina (Athyriaceae) in the Tatra Mts. Polish Botanical Journal 47(1): RAGHAVAN V Developmental biology of fern gametophytes. Cambridge University Press, Cambridge, 361 pp. STOKEY A.G., ATKINSON L. R The gametophyte of Grammitidaceae. Phytomorphology 8: WAGNER F.S Evidence for the origin of the hybrid cliff fern, Woodsia alpina (Aspleniaceae: Athyrioideae). Syst. Bot. 12: WINDHAM M.D Chromosomal and electrophoretic studies of the genus Woodsia in North America. Amer. J. Bot. 74: 715. ZAJ C A., ZAJ C M. (eds) Distribution Atlas of Vascular Plants in Poland. Laboratory of Computer Chorology, Institute of Botany, Jagiellonian University, Krakow, 594 pp. ZAGO D ON P.P., ZAGO D ON K Charakterystyka wyst¹pienia oligoceñskiego bazaltoidu w Ma³ym Kotle Œnie nym (Karkonosze). Przegl¹d Geologiczny 54: ZARZYCKI K., SZEL G Z Red list of the vascular plants in Poland. In: Mirek Z., Zarzycki K., Wojewoda W., Szel¹g Z., Red list of plants and fungi in Poland. W. Szafer Insatitute of Botany Intitute. Polish Academy of Sciences, Kraków, p

14 28 Krystyna-Kromer,-Andrzej-Raj,-Ludwik- o³nierz,-dorota-potura³a 28 Rozmna anie in vitro oraz uprawa ex situ Woodsia alpina (Bolton) S.F. Gray Woodsia alpina jest gatunkiem o zasiêgu wokó³biegunowym, obejmuj¹cym Europê, Azjê i Amerykê Pó³nocn¹. W Europie wystêpuje na pó³nocy oraz w Pirenejach, Apeninach, Alpach, Sudetach i Karpatach. W Polsce rozrzutka alpejska jest arktyczno-alpejskim reliktem glacjalnym. W Sudetach i Karpatach roœnie na pojedynczych stanowiskach. Po polskiej stronie Sudetów znana jest z jednego stanowiska w Ma³ym Œnie nym Kotle. Inne podawane stanowiska to po³udniowa czêœæ Œnie ki i masyw Pradziada (czeskie Sudety). W Tatrach podawana jest z trzech stanowisk. Jest to gatunek wysokogórski, roœnie w szczelinach skalnych, w Sudetach na bazaltach, a w Tatrach na dolomitach triasowych i granitach. Gatunek ten w wielu krajach uznawany za zagro ony i gin¹cy, w Polsce umieszczony w Polskiej Czerwonej Ksiêdze z kategori¹ CR (Fabiszewski, Piêkoœ-Mirkowa 2001). Z uwagi na to, e rozrzutka alpejska jest coraz rzadsza w Europie i Ameryce Pó³nocnej, podjêto próby jej ratowania przez zastosowanie rozmna ania w kulturach in vitro i opracowania zasad tworzenia stanowisk zastêpczych. Sporofile pobrano ze stanowiska w Ma³ym Œnie nym Kotle. Badano wp³yw terminu pobierania sporofili ze œrodowiska naturalnego na kie³kowanie zarodników w kulturze in vitro. Wykazano, e zarodniki sporofili pobranych pod koniec lipca kie³kowa³y bardzo s³abo. Z partii wysiewów lipcowych po 7 miesi¹cach kultury skie³kowa³ zaledwie jeden zarodnik. PóŸniejszy o 2 tygodnie termin zbioru zarodników da³ nieco lepsze wyniki. Zarodniki zebrane w roku 2004 kie³kowa³y bardzo nierównomiernie, a czas kie³kowania wyd³u a³ siê od 1,5 do 9 miesiêcy. Po up³ywie roku skie³kowa³o zaledwie 6% wysianych zarodników. Po wykszta³ceniu inicja³ów ryzoidów i protonemy nastêpowa³ dalszy rozwój gametofitów od stadium nitkowatego do sercowatego. Gametofity namna a³y siê w kulturze in vitro g³ównie przez rozga³êzianie i tworzenie przedroœli potomnych. Gametangia powstawa³y na przedroœlach znajduj¹cych siê w ró nych etapach rozwoju. Na gametofitach sercowatych powstawa³y rodnie i plemnie, a na nitkowatych przedroœlach jedynie plemnie. Liczba sporofitów pojawiaj¹cych siê na po ywce 1/2 MS by³a niewielka. Merystemy wierzcho³kowe sporofitów powsta³ych w wyniku zap³odnienia proliferowa³y tworz¹c zielone cia³a globularne, z których regenerowa³y kolejne pokolenia rosn¹cych zwarcie sporofitów. Kêpy sporofitów czêsto br¹zowia³y i traci³y liœcie, chocia wiele z nich wznawia³o wzrost po okresie spoczynku. Roœliny wysadzano w szklarni w odkwaszony torf, a po aklimatyzacji przenoszono do inspektu. Koñczy³y one wegetacjê w sierpniu, a w roku nastêpnym odnawia³y wzrost tylko te, które wykszta³ci³y dostatecznie du e k³¹cza.