Propagation in vitro and ex situ cultivation of Woodsia alpina (Bolton

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Propagation in vitro and ex situ cultivation
of Woodsia alpina (Bolton) S.F. Gray
KRYSTYNA KROMER1, ANDRZEJ RAJ2, LUDWIK ¯O£NIERZ3,
DOROTA P OTURA£A 1
Botanical Garden, University of Wroc³aw, Sienkiewicza 23, PL-50-335 Wroc³aw,
Poland; e-mail: [email protected]
1)
2)
Karkonoski National Park, Cha³ubiñskiego 23, PL-58-570 Jelenia Góra, Poland
Department of Botany and Plant Ecology, Wroc³aw University of Environmental
and Life Sciences, Pl. Grunwaldzki 24a, PL-50-363 Wroc³aw, Poland;
e-mail: [email protected]
3)
Institute of Natural Sciences, Wroc³aw University of Environmental and Life
Sciences, Pl. Grunwaldzki 24a, PL-50-363 Wroc³aw, Poland
3)
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
K ROMER K., R AJ A., ¯ O£NIERZ L., P OTURA£A D. 2008. 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. 15–28.
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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 270 000 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
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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 10–15 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; http://hardyfernlibrary.com). It has a short erect rhizome, compact and stiff; fronds usually are shorter than 10 cm. It sporulates in summer
and in early autumn.
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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 (10.07.2003) and August (6.08.2004) 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).
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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 121oC. Flasks with spores were incubated in different temperatures and light regimes: 25 oC and 16-h light photoperiod at the irradiance of 14.2 ìmol·m2·s-1; 10oC at the irradiance of 0.15 ìmol·m2·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.
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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
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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
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2
3
4
22
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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 -40oC 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)
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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)
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Propagation-in-vitro-and-ex-situ-cultivation-of-W.-alpina-(Bolton)-S.F.-Gray
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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.
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Fig. 8. Plants of W. alpina in the first year of cultivation (phot. D. Potura³a)
Ryc. 8. Roœliny 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)
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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.