CONSERVATION-RELATED PROBLEMS OF PTERIDOPHYTES IN POLAND

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2 2006 BOTANICAL GUIDEBOOKS No. 29 CONSERVATION-RELATED PROBLEMS OF PTERIDOPHYTES IN POLAND Edited by Halina Pl^KOS-MlRKOWA Elzbieta ZKNKTELER POLISH ACADEMY OF SCIENCES K R A K O W W. SZAFER INSTITUTE OF BOTANY

3 2006 BOTANICAL GUIDEBOOKS No. 29 Botanical Guidebooks (continuation of Polish Botanical Studies, Guidebook Series) is an irregularly issued series publishing guides to Polish botanical institutions, collections and collectors, botanical literaiure, and directories as well as botanical field-guides and basic information regarding various branches of botany. Editor-in-Chief: Co-Editor: Editorial Assistant: Editorial Board: Make-up Editor: Cover Design: Zbigniew MIREK Jan J. WOJCICKI Magdalena ZARZYKA-RYSZKA Krystyna GRODZINSKA Andrzej JANKUN Leon STUCHLIK Marian WYSOCKI Maciej PIHRZCHALA Editorial Office: W. Szafer Institute of Botany. Polish Academy of Sciences. Lubicz 46, Krakow, Poland Tel. [ , Fax: [ , This volume is published with the financial support of Ministry of Science and Higher Education W. Szafer Foundation for Polish Botany, & W. Szafer Institute of Botany Polish Academy of Sciences Copyright W. Szafer Institute of Botany. Polish Academy of Sciences Published, sold and distributed by W. Szafer Institute of Botany. Polish Academy of Sciences, Lubicz 46, Krakow, Poland: Issued: 2006 Printed in Poland ISBN: ISBN: ISSN:

4 CONTENTS II. Pii:.K(>s-M[RKO\\A. E. ZHNK/ILLFR: Conservation-related problems of pteridophyles in Poland - Foreword A. PAC > s \ Taxonomic problems in The genus Diphasiastrtsm in Poland and Central Europe D. WKOBLI: Phytosociological eharacieristies and variability of Equtietum tclmaicia in Poland.1. Bon/i \R( /\. A. KK/I s: Relative age of Phyllitix scolopcndnum phytoccnoscs as determined by population structure analysis E. ZhNKTlihR, K. SZPOTKOWSKI: Clonal growth of Thelypteris palustris populations at three contrasting sites H. Pli'Kos-MiKKOWA: Pteridophytes of the Polish Tatra Mountains- distribution, threat and conservation F. S/CYI.SM\K: Asplcuium serpentine ferns in Poland threats and conservation imperatives B. WOZIVVODA: Distribution of protected ferns in Central Poland A. KCXZLR.,1. KUR/YNSKI: An attempt ;ji active protection of the great horsetail Equisetum tc'/natu'iti threatened by a road construction project A. K \7\ni RC / \K: Role of gibberellie acid and ethylene in controlling sex determination in gametophytes of the homosporous fern Anemiaphyllltidis A. MIKU.A, J. J. RYBCYVKSKI: Preliminary studies on eryopreservation and strategics ofcyathea australix gametophyte development in vitro K. KROMLK, J. \ \ISVM -J \ci.\fka. K. KLMPINSK\ T. NO\\AK, L. ZOLMMV. D. POTURAI \ K. S\: In vitro propagation and ex \iiit preservation of endangered ferns from Lower Silesia K.. KEMPINSKA, K. K.ROMER, E, ZKNK [ I,LI R: Influence of tocopherol on gainetogencsis ofasplenium cuneifolium and Asplenium adulterinum W. Szvi'i'i \ (). ()i s/owsk\ M. Fi RMANO\ /;/ vitro culture of Lycopodiaceae (club mosses).

5 Botanical Guidebooks 29: IN VITRO CULTURE OF LYCOPODIACEAE (CLUB MOSSES) WOJCIKCH SZYPULA, OlGA OLSZOWSKA, MlROSLAWA FURMANOWA SZYPULA W., OLSZOWSKA O., FURMANOWA M. In vitro culture of Lycopodiaceae (club mosses). Botanical Guidebooks 29: , Abstract. Club mosses in Poland are under strict protection. They arc represented by nine species, two of which, Diphasiastrwn issleri and D, tristachyum, arc listed in the Polish 'red book'; two others, Lycopodiella immdaia and D. zeilleri, arc on the Polish 'red list.' The remaining Polish club mosses are locally threatened with extinction. In recent years the pharmaceutical industry has become increasingly interested in huperzine alkaloids found in some club mosses. Excessive harvesting of sporophytes as a source of huperzine has resulted in a marked decline of these plants in some countries. Studies now being conducted focus on the use of in vitro culture for propagation of plant material for the pharmaceutical industry. In vitro cultures of club mosses may also contribute to ex sint protection of these plants. The most important stage in culture of//, selago sporophytes described in this paper is elimination of microbiological endophytic contamination. The best results were achieved with short-term antibiotic treatment combined with longterm antimyeotic treatment. The first stage consisted of 12^48 h exposure of shoots and 7-day exposure of gcmmulcs to antibiotics and fungicides. In the second stage, cxplants were incubated for 4 weeks on medium with the addition of a systemic fungicide. After the first stage of disinfection, % of the shoots did not show any contamination in the first 4 weeks of culture. The highest survival rate (81%) was seen on stabilizing MS medium containing no sucrose, half-strength mineral salts, agar (5.5 g 1"') and activated charcoal (1 g 1 '). In the second stage of sterilization, shoots were incubated on 1/2 MS medium with the addition of fungicide (Benazol 50 WP). Up to 95% of the shoots obtained in subsequent passages showed no endophytic contamination. After external and internal disinfection the gemmulcs were placed on 1/2 MS. Knudson (Kn) and Moore (Mr) media. Gemmulcs incubated for 4 weeks at 4 C in the dark grew vigorously after transfer to Mr

6 164 and Kn media, and developed sporophytcs. The survival rate was 65%. The systemic fungicide Topsin M 500 SC tested in short- and long-term antibiotic treatment produced side effects, causing death of shoots. The addition of IAA or kinetin to the medium caused rapid withering of shoots and gemmulcs. Key words: Lycapodiaceae, club mosses, Huperzia seltigo, gemmulcs, in vitro culture, hupcrxine, sclagine. Wojciech Szypula, Olga Olsiowska, Mimslawa Furmanowa, Department of Biology and Pharmaceutical Botany, Medical University of Warsaw, ul. Banacha I, Warsaw, Poland: wajciecho@pocxta.fin; kzbb@fann.qfnwaw.edu.pl INTRODUCTION Club mosses in Poland are represented by nine species, accounting for only 0.3% of ca 3000 taxons of the native flora. They are legally protected. The Polish Red Data Book of Plants (Kazmierczakowa, Zarzycki 2001) lists two species, Diphasiastmm issleri (critically endangered, CR) and Dtphasiostrum tristachyum (endangered, EN). According to Zarzycki and Szcl^g (2006), Lycopodiella innna'ata, Dlphasiastritm zeilleri, and in some localities Huperzia selago are also threatened with extinction. Diphasiastntm aspinum, another club moss found in Poland, grows at less than 100 sites (Zarzycki et al. 2002), while such common species as Diphasiastrum complanatum, Lycopodium clavatitm and Lycopodiltm annotinitm arc destroyed and threatened with extinction by improper forestry practices and collection for use in popular floral decorations (Piekos-Mirkowa, Mirck 2003). Excessive harvesting of these plants for the pharmaceutical industry is the latest major threat that may lead to the extinction of some Lycopodium species. Phytochemical and pharmacological studies of Lycopodium alkaloids have confirmed the therapeutic properties of some of them. Iluperzine A (selagine. IltipA) found in different species of Huperzia and Phlegmariurus is a valuable alkaloid with a high therapeutic index in the treatment of symptoms of Alzheimer's disease (Ma, Gang 2004). This alkaloid is also a precursor used in the synthesis of ZT-1, a substance whieh is undergoing clinical trials for treatment of Alzheimer's disease (Ma, Gang 2004). Excessive harvesting of club mosses as a source of these alkaloids has resulted in a considerable decline of their population numbers in China (Ma et al. 2006). The likelihood of the same occurring in the very near future in other countries of Asia is very high. In view of the long life cycle of club mosses, their exceptionally slow growth, and the relatively high cost of chemical synthesis of huperzine alkaloids, researchers have undertaken biotechnological studies on in vitro propagation of elub mosses to meet the needs of the pharmaceutical industry. Effective propagation of club mosses in vitro also

7 W. SZYPULA ET AL.: In vitro culture of Lycopodiaccae 1 65 provides insight into their biology and has applications in conservation. So far, several studies have been published describing micropropagation of club mosses. Freeberg and Wetmore (1957) described gamctophyte culture and the apogarnous development of sporophytes of Dipha.siastrum complunatitm, Lycopodium cernuum and Huperzia se/ago. Atmane et al. (2000) obtained cultures of callus and sporophytes grown from somatic embryos of Lycopodiella immdata. Cultures of gametophytes of Lycopodium digitatum (Whittier 1981), L. lucidulum (Whittier, Webster 1986) and L, obscurum (Whittier 1977) have been reported. Szypula et al. (2005) described somatic embryogenesis and obtained cultures of sterile shoots and sporophytes grown from somatic embryos of H. selago. They also performed phytochemical analyses to determine the presence of HupA in tissues obtained from sporophytes grown from somatic embryos and collected from the natural habitat in the Beskid Sla.ski Mts and Beskid Zywiecki Mts. The results of that study confirmed the need to develop other club moss micropropagation methods to shorten and simplify the complex and multistage process of initial explant disinfection. Studies conducted since 2003 in the Department of Biology and Pharmaceutical Botany of the Medical University of Warsaw involve in vitro culture of gametophytes and sporophytes grown from gemmules and shoots of H. selago. Another stage of the study consists of phytochemical, qualitative and quantitative analyses of alkaloids found in tissues obtained by in vitro culture and collected from different natural habitats. MATERIAL AND METHODS Shoots and gemmules of Huperzia selago collected in the Babia Gora National Park in August and September 2004 were used to initiate in vitro sporophyte cultures. Apical fragments (5 cm long) of shoots with developing gemmules were excised. Explants of H. selago for. laxum (Fig. 1A) were obtained from populations growing in the Szumiqca Woda valley, being a complex of West Carpathian subalpine spruce forest Plagiothecio-Piceelum, and in Hala Czarna glade in fertile Carpathian beech wood Dentario gjanduhsae- Fageium. Shoots and gemmules of H. selago for. imbrication (Fig. IB) were collected from populations growing on the slopes of two mountains, Diablak and K^pa, in a complex of dwarf mountain pine Pineium mugu carpaticum and in acidophilic alpine meadow Junco trtfidi-festucetum airoidis (Matuszkiewiez 2001). After transfer to the laboratory, the shoots and gemmules (Fig. 2) were cleaned of remnants of forest litter, soil and leaves of other plants, rinsed in lap water for a few hours, and stored in a refrigerator for 3 days at 4 C. After separation from the shoots, some of the gemmules were placed in Petri dishes lined with cotton wool moistened with tap water and refrigerated at 4 C for 4 weeks. Removal of epiphytic and cndophytic microorganisms from the initial explants was the next stage, employing a method developed for decontamination of cultures of ferns in vitro (Zcnkteler 2000), modified for in vitro culture of club mosses. This multistage process consisted of short-term antibiotic treatment of the explants, surface disinfection, and long-term antimycotic treatment in vitro.

8 166 Fig. 1. Huperzia selago sporophyies in their natural habitat in the Babia Gora National Park. A - Huperzia selago for. laxum growing in Hala Czama glade in fertile Carpathian beeeh wood Dentario glandiihsae-fagetum; B - Huperzia selago for. imbricatum growing in acidophilic alpine meadow Jvnca trifidi-festucetum airoidis.

9 W. SZYPULA ET AL.: In vitro culture of Lycopodiaceae 167 Fig. 2. Gemmule of Hupcrzia xelago after transfer to the laboratory; bar = 20 mm. The shoots were exposed for 12, 24, 36 and 48 h to an antibiotic and fungicide bath to which three components were added: 4 g 1 ' Benazol 50 WP (active substance: benomyl, a benzimidazole compound) or 4 g 1 ' Topsin M 500 SC (active substance: methyl thiophanatc), 30 mg 1 ' nystatin and 30 nig l~' streptomycin. The controls were shoots rinsed several times in sterile tap water (Table I). Each sample consisted of ca \0 cxplants. Gemmules incubated 4 weeks in a refrigerator (Fig. 3) were placed in Petri dishes lined with blotting paper moistened with water to which the systemic fungicide Benazol (2 g I'1) and the topical fungicide Dithanc M-45 (2 g 1~') were added, and incubated for 7 days in a culture room at 25 C (short-term antirnycotic treatment). Gemmules previously incubated in a refrigerator only for 3 days were placed in Pctri dishes lined with blotting paper moistened with water to which the fungicide Benzol 50 WP (2 g 1 ') was added, and then kept in a refrigerator at 4 C for 7 days. The control Table. 1. Variants* of short-term antibiotic treatment of Huperzia selago shoots and MS medium. Duration of immersion of shoots in dilutions of Benazol 50 WP (4 g Tl), nystatin (30 mg 1"') and streptomycin (30 mg 1"') I2h 24 h 36 h 48 h 12h 24 h 36 h 48 h control Modifications of 1/2 MS medium (C - activated charcoal, Sue. - sucrose) 1/2 MS, C - Ig r1, Sue g t1 1/2 MS. C- Igl '.Sue. -2(1 gl ' 1/2 MS, C- lgl1,suc.-20gr1 1 2 MS. C Igl '.Sue. 20 gl 1/2 MS, C-lgT1 1/2 MS, C-lgl ' i :MS. c- igi 1/2 MS, C- Igl 1/2 MS, C - Ig I"1 % of shoots showing no microbial contamination after 4 weeks of culture %of viable shoots after 4 weeks of culture JU *For each variant 100 shoots were cultured.

10 170 Botanical Guidebooks, 2006, No. 29 Fig. 5. Development of Huperzia selago shoots on 1/2 MS medium after 4-month incubation; bar = 25 mm. medium with added activated charcoal (1 g 1 ') and normal sucrose content (20 g I"1) was 12 h; 64% of the shoots remained viable after 4 weeks of culture (Table 1). After the same length of time, shoots soaked for 24, 36 and 48 h and transferred to the same medium showed lower viability, ranging from 12% to 32% (Table 1). For the first 4 weeks of culture there was no growth of shoots in any of the above cultures. After subsequent passaging on fresh 1/2 MS medium, the shoots began to grow and branched dichotomously (Fig. 5). After another 3 months, some of them developed nodular structures, which underwent somatic embryogenesis (Szyputa et al. 2005). Some shoots were passaged on 1/2 MS medium with the addition of 0.1, 0.5 or 1.0 mg 1 ' IA A. The addition of auxin did not prove beneficial, and the shoots died after two weeks of culture. In the second and subsequent passages after short-term antibiotic therapy, some of the shoots demonstrated contamination with endophytic microorganisms. These were mostly fungi forming white or black mycelia growing from within the shoot and gradually covering all the shoot and the medium (Fig. 4). Isolates of these fungi were transferred to PDA medium (Zenkteler 2000), and later studied to identify the species. As contamination regularly appeared in successive passages, long-term antimycotic treatment was applied In vitro. Immediately after the initial stages of sterilization, some of the shoots were transferred to medium to which a systemic fungicide, either Benazol 50 WP or Topsin M 500 SC, was added. The best results were obtained with Benazol 50 WP at a concentration of 2 g 1"', as 95% of the

11 W. SZYPULA i;i AL.: In vitro culture of Lycopodiaueae 171 shoots showed no trace of contamination on the medium in the succeeding weeks of culture. Adding Topsin M 500 SC to the medium produced rapid death of the shoots. In cultures of gemmulcs in vitro, 1/2 MS, Mr and Kn media were used with or without growth regulators, and with or without Bcnazol 50 WP (2 g 1"'). After 4 weeks of incubation on medium without fungicide, 40% of the gemmules showed endophytic contamination. After 4 weeks of antimycotic treatment on medium with 50 WP, fungal contamination was seen in only 5% of the gemmulcs. In the control sample, cultured for 4 weeks on 1/2 MS and Mr medium without Benazol 50 WP, 95% of the explants showed signs of contamination. The type and composition of the medium used to culture gemmules and the sporophytcs that grew from them had a considerable effect on the survival and further growth of the explants. High survival rates (95% and 80% respectively) at 4 weeks were seen in explants cultured on 1/2 MS medium with 50 g I ' sucrose and on stabilizing medium, thai is, MS with the addition of 1 g 1"' activated charcoal and 2 g 1 ' Benazol 50 WP. Gemmules cultured on 1/2 MS medium with the addition of g 1 ' sucrose showed moderate survival of 25^40%. On all of these media, however, gemmule development was arrested; they did not grow during a further 3 months of culture and then gradually withered. Gemmules incubated on 1/2 MS medium with the addition of 5, 10, 15, 20 or 25 ug 1~' kinetin died from week 2 of culture on. The best development was seen in gemmules incubated for 4 weeks in a refrigerator at 4 C on blotting paper soaked with water. Fig. 6. Young sporophyte of Huperiia selago developed from gemmule after 2';J week of incubation on Mr medium; bar = 20 mm.

12 172 Botanical Guidebooks, 2006, No. 29 Fig. 7. Sporophytc ofhuperzia selago developed from the gemmule after 6 months of culture on Mr medium; bar = 5 cm. At that time they grew a root and a shoot with the first leaves proper (Fig. 3). Gemrnule development was not arrested, and after transfer to Mr and Kn media they grew vigorously (Fig. 6). Their survival rate after 4 weeks of culture was 60%. Young sporophytes were passaged to fresh medium every 3 months. After 6 months the club mosses developed 3-6 lateral shoots and a root system (Fig. 7). After subsequent passages, some shoots 2-3 cm in length were cut off the sporophytes. These were used as initial material for starting sterile shoot cultures on Kn and Mr media. After 12 months of culture some of the sporophytes were used for comparative phytochemical studies of alkaloids by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). DISCUSSION Our findings demonstrate that effective elimination of plant tissue contamination by endophytic bacteria and fungi is a key stage in the initiation of in vitro culture of Huperzia selago (Fig. 4). Our knowledge of saprophytic, parasitic and symbiotic fungi populating the tissues of club mosses is fairly limited. As with ferns, all literature data concern the problems of rnycorrhizas (Zenkteler 2000). As early as the end of the 19th century there were attempts to identify the systematic position of mycorrhizas found in the prothalli of different club moss species. A review (Pacyna 1972) of the literature on the spores and prothalli of club mosses shows that until the early 1970s that research focused on the prothalli and the germination of spores in cultures in vitro. Studies were done to identify the systematic position of the mycorrhizas and their role in the development of club moss prothalli.

13 W. SzYPUiA CT AL.: In vitro culture of Lycopodiaceae 173 The state of this knowledge has not changed much since then. Endophytic bacteria isolated from in vitro cultures of Huperzia selago shoots were identified as belonging to the genus Acetobacter (Szypula et al. 2005). Studies are in progress to identifythe systematic position of some endophytic fungi isolated from tissues of//, selago sporophytes. The presence of bacteria and fungi inside club moss tissues often makes it very difficult to use them as explants to initiate in vitro cultures. Contamination considerably prolongs the time required to start a sterile culture in vitro. In Poland and most other European countries, club mosses are protected and not infrequently threatened with extinction. As a result, plant material for in vitro studies is difficult to obtain - another good reason to develop effective methods of explant sterilization. Endophytic contamination may be eliminated by a disinfection process employing a combination of several antibiotics (e.g., streptomycin and penicillin) and benzimidazole fungicides such as Benazol. A short-term antibiotic treatment in which explants are exposed to a combination of antibiotics and fungicides for several hours (Zenkteler 2000) together with a long-term antimycotic treatment proved effective: up to 95% of the shoots showed no signs of contamination after several weeks of culture. The gemmules that spontaneously developed in the apical parts of H. selago were better material than shoots for initiating in vitro cultures. They may be obtained from the natural habitat without excising shoots of rare and protected plants. Gemmules may be collected at the stage of their full development (best in spring) from naturally growing populations without damage to the mother sporophytes. The gemmules of//, selago are easier to disinfect externally and internally than the shoots. After a short-term antimycotic treatment followed by incubation on medium with the addition of a systemic fungicide, as many as 95% of the explants were free of endophytic contaminants. Incubation for 4 weeks at a lower temperature (4 C) in the dark on blotting paper soaked with sterile tap water was found to be the most important stage in initiation of gemmule cultures in vitro. At that stage the gemmules developed a root and the first leaves proper (Fig. 3). When transferred to medium after short-term antimycotic treatment, they grew well and developed sporophytes a few centimeters long (Figs 6, 7). Since club mosses are protected ex situ, in vitro cultures of them may play a very important role, though further studies are required to develop methods allowing rapid germination of spores after a few weeks of culture. In vitro cultures of sterile prothalli may produce sporophytes either through apogamous development (Freeberg 1957) or following fertilization of the ovum. The process of somatic embryogenesis described for cultures of Lycopodiella inundata (Atmane et al. 2000) and Huperzia selago (Szypula et al. 2005) may yield sterile sporophytes as a source of valuable and therapeutically useful alkaloids. The most important hindrance to practical application of club moss culturing is the difficulty in ac-

14 K74 Botanical Guidebook*. 2006, No. 29 climatizing sporophytes transferred from in vitro cultures to soil. Plants placed in pots of soil taken from their natural stations did not grow and quickly withered. Our attempts to grow H. selago sporophytes in vivo were unsuccessful, as were the few attempts in studies described in the literature (Ma, Gang 2004). REFERENCES ATMANF N., BLERVACQ A. S., MICHAUX-FERRIERE N., VASSEUR J Histological analysis of indirect somatic embryogcncsis in the Marsh clubmoss Lycopodiella inundata (L.) Holub (Pteridophytes). Plant Science 156: FREIEBERG J. A The apogamous development of sporclings oflycopodiltm ccrnmim L., L. coinplanatnm var. flabelliforme Fetnald and L. selago L. in vitro. Phytomorphologyl: FREEBERG J. A., WETMORE R. H Gametophytes of Lycopodium as grown in vitro. Phytomorphology 7: KAZMIERCZAKOWA R., ZARZYCKI K. (cds) Polish Red Data Book of Plants. Pteridophytes and Flowering Plants. Instytul Botaniki im W. Szafera. Instytut Ochrony Przyrody, Polska Akademia Nauk, Krakow (in Polish with English summary). KNUDSON L Nonsymbiotic germination of orchid seeds. Botanical Gazelle 73: MA X., GANG R The Lycopodium alkaloids. Natural Product Report 21: MA X., TAN CH., ZHU D., GANG D. R A survey of potential hupcrzine a natural resources in China: The Huperziaceae. Journal ofethnopharmacology 104: MATUSZKIEWICZ W Przewodnik do oznaczania zbiorowisk roslinnych Polski. Wydawnictwo Naukowe PWN, Warszawa. MURASHIGF T., SKOOG F A revised medium for rapid growth and bioassays with tobacco tissue culture. Plan! Physiology 15: PACYNA A Zarodniki i przedrosla widtakow, Wiadomofai Botaniczne 16(1): PIE.KOS-MIRKOWA H., MIREK Z Atlas Roslin Chronionych. Flora Polski. Multico Oficyna Wydawnicza, Warszawa. SZYPULA W., PlETROSlUK A., SuCHOCKl P., OLSZOWSKA O., FURMANOWA M., KAZIM1ERSICA O Somatic embryogencsis and in vitro culture of Huperzia selago shoots as a potential source of huperzinc A. Plant Science 168: WHITTIKR D. P Gametophytes of Lycopodium obscurum as grown in axenic culture. Canadian Journal of Botany 55: WHITTIFR D. P Gametophytes of Lycopodium Jigitatum (fomierly L. complanatum var. flabeliiforme) as grown in Axenix Culture. Botanical Gazette 142: WHITHER D. P., WEBSTER T. R Gametophytes of Lycopodium lucidulum from Axenic Culture. American Fern Journal 76(2):

15 W. SZYPLILA ET AL.: In vitro culture oi" Lycopodiaccac 175 ZARZYCKI K., SZELAG Z Red list of the vascular plants in Poland. In: Z. MIRKK, K. ZARZYCKI, W. WOJEWODA, Z. SZLLAG (cds), Red list of plants and fungi in Poland, W. Szafer Institute of Botany. Polish Academy of Sciences, Krakow, pp ZARZYCKI K., TRZCINSKA-TACIK H., ROZANSKI W., SZELAG Z., WOLEK J.. KORZENIAK U Ecological Indicator Values of Vascular Plants of Poland. W. Szafer Institute of Botany, Polish Academy of Sciences, Krakow. ZENKTELER E Systems of vegetative propagation of fern in vivo and in vitro. Wydawnictwo Uniwersytetit im. A. Mickiewicza, Poznari, sei: Biologia 62: (in Polish with English summary).