A new and fast method to obtain in vitro cultures of Huperzia selago (Huperziaceae) sporophytes, a club moss which is a source of huperzine A

Wojciech J. Szypuła, Paulina Mistrzak, Olga Olszowska


This study presents a protocol for a fast and effective in vitro axenic culture of Huperzia selago (Huperziaceae Rothm.) sporophytes, a club moss which is a source of huperzine A, an alkaloid of a considerable therapeutic potential extensively investigated for its uses as treatment for some neurodegenerative diseases. The proposed procedure allowed approximately tenfold shortening of the species developmental stages with the omission of the gametophyte stage while the sporophyte mass could be increased tenfold within a 6-month period. The cultures were established using vegetative propagules (bulbils) procured from sporophytes growing in the wild without degrading the habitats of this endangered plant species. Explants underwent surface and internal disinfection to eliminate the epiphytic and endophytic bacteria and fungi. In in vitro cultures, the optimum results were achieved using Moore (Mr) medium without growth regulators or supplemented with 0.015 mg/l IBA and 0.3 mg/l kinetin. These media ensured both viability of the propagules and their further development. The biomass growth index for H. selago sporophytes grown from propagules, determined at 3 months of culture (1 passage) on Mr medium with IBA and kinetin was 650%. At 6 months, the biomass growth index increased to 1114%. Vigorous growth of adventitious roots, especially on Mr medium with the addition of 0.25 mg/l NAA, and callus formation on shoot apices were observed. At 6 months of culture, some sporophytes obtained from the bulbils were used as the initiating material for shoot subcultures, which developed best on Mr medium with IBA and kinetin.


Huperzia selago; Lycopodium selago; Huperziaceae; Lycopodiaceae; huperzine A; selagine; regeneration

Full Text:



Rothmaler W. Pteridophyten-studien I. Feddes Repert. 1944;54(1):55–82. http://dx.doi.org/10.1002/fedr.19440540106

Rothmaler W. Über eininge Diphasium – Arten (Lycopodiaceae). Feddes Repert. 1962;66:234–236.

Bierhorst DW. Morphology of vascular plants. New York NY: The Macmillan Company; 1971.

Wikström N, Kenrick P. Phylogeny of epiphytic Huperzia (Lycopodiaceae): paleotropical and neotropical clades corroborated by rbcL sequences. Nord J Bot. 2000;20(2):165–171. http://dx.doi.org/10.1111/j.1756-1051.2000.tb01561.x

Ma X, Tan C, Zhu D, Gang DR. A survey of potential huperzine A natural resources in China: the Huperziaceae. J Ethnopharmacol. 2006;104(1–2):54–67. http://dx.doi.org/10.1016/j.jep.2005.08.042

Valentine DH, Moore DM. Lycopodiaceae L. In: Tutin TG, Burges NA, Chater AO, Edmondson JR, Heywood VH, Moore DM, et al., editors. Flora Europea. Cambridge: Cambridge University Press; 2007. p. 3–5. (vol 1).

Beitel JM, Mickel JT. The appalachian firmoss, a new species in the Huperzia selago (Lycopodiaceae) complex in eastern north America, with a new combination for the western firmoss. Am Fern J. 1992;82(2):41. http://dx.doi.org/10.2307/1547376

Ma X, Gang DR. The Lycopodium alkaloids. Nat Prod Rep. 2004;21(6):752–772. http://dx.doi.org/10.1039/B409720N

Ma X, Tan C, Zhu D, Gang DR, Xiao P. Huperzine A from Huperzia species-an ethnopharmacolgical review. J Ethnopharmacol. 2007;113(1):15–34. http://dx.doi.org/10.1016/j.jep.2007.05.030

Ma X, Gang DR. In vitro production of huperzine A, a promising drug candidate for Alzheimer’s disease. Phytochemistry. 2008;69(10):2022–2028. http://dx.doi.org/10.1016/j.phytochem.2008.04.017

Wu Q, Gu Y. Quantification of huperzine A in Huperzia serrata by HPLC-UV and identification of the major constituents in its alkaloid extracts by HPLC-DAD-MS-MS. J Pharm Biomed Anal. 2006;40(4):993–998. http://dx.doi.org/10.1016/j.jpba.2005.07.047

Szypuła W, Pietrosiuk A, Suchocki P, Olszowska O, Furmanowa M, Kazimierska O. Somatic embryogenesis and in vitro culture of Huperzia selago shoots as a potential source of huperzine A. Plant Sci. 2005;168(6):1443–1452. http://dx.doi.org/10.1016/j.plantsci.2004.12.021

Szypuła WJ, Kiss AK, Pietrosiuk A, Świst M, Danikiewicz W, Olszowska O. Determination of Huperzine A in Huperzia selago plants from wild population and obtained in in vitro culture by high performance liquid chromatography using a chaotropic mobile phase. Acta Chromatogr. 2011;23(2):339–352. http://dx.doi.org/10.1556/AChrom.23.2011.2.11

Ayer WA, Browne LM, Orszanska H, Valenta Z, Liu JS. Alkaloids of Lycopodium selago. On the identity of selagine with huperzine A and the structure of a related alkaloid. Can J Chem. 1989;67(10):1538–1540. http://dx.doi.org/10.1139/v89-234

Zarzycki K, Trzcińska-Tacik H, Różański W, Szeląg Z, Wołek J, Korzeniak U. Ecological indicator values of vascular plants of Poland. Cracow: W. Szafer Institute of Botany, Polish Academy of Sciences; 2002.

Colling G. Red list of the vascular plants of Luxembourg. Luxembourg: Ferrantia; 2005.

Piessens K, Hermy M. Does the heathland flora in north-western Belgium show an extinction debt? Biol Cons. 2006;132(3):382–394. http://dx.doi.org/10.1016/j.biocon.2006.04.032

Whittier P, Webster TR. Gametophytes of Lycopodium lucidulum from axenic culture. Am Fern J. 1986;76(2):48. http://dx.doi.org/10.2307/1547558

Whittier P. Germination of spores of the Lycopodiaceae in axenic culture. Am Fern J. 1998;88(3):106. http://dx.doi.org/10.2307/1547683

Whittier DP, Storchova H. The gametophyte of Huperzia selago in culture. Am Fern J. 2007;97(3):149–154. http://dx.doi.org/10.1640/0002-8444(2007)97[149:TGOHSI]2.0.CO;2

Szypuła WJ. A fast method to obtain in vitro culture of Huperzia selago gametophyte, a club moss which is a source of huperzine A and other alkaloids. In: Proceedings of the 56th meeting of the Polish Botanical Society. Olsztyn: Polish Botanical Society; 2013. p. 167–168.

Callaghan TV, Svensson BM, Headley A. The modular growth of Lycopodium annotinum. Fern Gaz. 1986;13(2):65–76.

Freeberg JA, Wetmore RH. Gametophytes of Lycopodium as grown in vitro. Phytomorphol. 1957;7:204–217.

Freeberg JA. The apogamous development of sporelings of Lycopodium cernuum L., L. complanatum var. flabelliforme Fernald and L. selago L. in vitro. Phytomorphol. 1957;7:217–229.

Freeberg JA. Lycopodium prothalli and their endophytic fungi as studies in vitro. Am J Bot. 1962;49(5):530. http://dx.doi.org/10.2307/2439425

Atmane N, Blervacq AS, Michaux-Ferriere N, Vasseur J. Histological analysis of indirect somatic embryogenesis in the marsh clubmoss Lycopodiella inundata (L.) holub (Pteridophytes). Plant Sci. 2000;156(2):159–167. http://dx.doi.org/10.1016/S0168-9452(00)00244-2

Whittier DP. Gametophytes of Lycopodium obscurum as grown in axenic culture. Can J Bot. 1977;55(5):563–567. http://dx.doi.org/10.1139/b77-067

Whittier DP. Gametophytes of Lycopodium digitatum (formerly L. complanatum var. flalellifare) as grown in axenix culture. Bot Gaz. 1981;142:519–524. http://dx.doi.org/10.1086/337254

Szypuła WJ, Olszowska O, Furmanowa M. In vitro culture of Lycopodiaceae (club mosses). In: Piękoś-Mirkowa H, Zenkteler E, editors. Conservation-related problems of Pteridophytes. Cracow: W. Szafer Institute of Botany, Polish Academy of Sciences; 2006. p. 163–175. (vol 29).

Ishiuchi K, Park JJ, Long RM, Gang DR. Production of huperzine A and other Lycopodium alkaloids in Huperzia species grown under controlled conditions and in vitro. Phytochemistry. 2013;91:208–219. http://dx.doi.org/10.1016/j.phytochem.2012.11.012

Gola EM. Reproductive strategies of Huperzia. In: Szczęśniak E, Gola EM, editors. Club mosses, horsetails and ferns in Poland: resources and protection. Wrocław: Polish Botanical Society, Institute of Plant Biology, University of Wrocław; 2008. p. 5–14.

Treu R, Laursen GA, Stephenson SL, Landolt JC, Densmore R. Mycorrhizae from Denali National Park and preserve, Alaska. Mycorrhiza. 1995;6(1):21–29. http://dx.doi.org/10.1007/s005720050101

Higgins KL, Arnold AE, Miadlikowska J, Sarvate SD, Lutzoni F. Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages. Mol Phylogenet Evol. 2007;42(2):543–555. http://dx.doi.org/10.1016/j.ympev.2006.07.012

Budziszewska J, Szypuła WJ. Influence of site conditions on the diversity of endophytic fungi of clubmoss species Huperzia selago (L.) Bernh. ex Schrank et Mart. Pol J Ecol. 2010;58(4):612–629.

Budziszewska J, Szypuła WJ, Wilk M, Wrzosek M. Paraconiothyrium babiogorense sp. nov., a new endophyte from fir club moss Huperzia selago (Huperziaceae). Mycotaxon. 2011;115(1):457–468. http://dx.doi.org/10.5248/115.457

Schmid E, Oberwinkler F. Mycorrhiza-like interaction between the achlorophyllous gametophyte of Lycopodium clavatum L. and its fungal endophyte studied by light and electron microscopy. New Phytol. 1993;124(1):69–81. http://dx.doi.org/10.1111/j.1469-8137.1993.tb03798.x

Wang B, Qiu YL. Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza. 2006;16(5):299–363. http://dx.doi.org/10.1007/s00572-005-0033-6

Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473–497. http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x

Chée RP, Leskovar DI, Cantliffe DJ. Optimizing embryogenic callus and embryo growth of a synthetic seed system for sweetpotato by varying media nutrient concentrations. J Am Soc Hortic Sci. 1992;117(4):663–667.

Knudson L. Nonsymbiotic germination of orchid seeds. Bot Gaz. 1922;73(1):1–25. http://dx.doi.org/10.1086/332956

Knop W. Quantitative Untersuchungen űber den Ernährungsprocek der Pflanze Landwirtsch. Vers Stn. 1865;7:93–107

Lloyd GB, McCown BH. Commercially-feasible micropropagation of mountain laurel Kalmia latifolia, by use of shoot-tip culture. Proc Int Plant Prop Sci. 1980;30:421–427.

Street HE, Henshaw GG. Introduction and employed in plant tissue culture. In: Willmer EN, editor. Cells and tissue culture. London: Academic Press; 1966. p. 459–532. (vol 3).

Williams S. A contribution to the experimental morphology of Lycopodium selago, with special reference to the development of adventitious shoots. Trans R Soc Edinb. 1934;57(3):711–737. http://dx.doi.org/10.1017/S0080456800016938

DeMaggio AE. Organization in a gametophyte callus of Lycopodium and its morphogenetic implications. Proc Natl Acad Sci USA. 1964;52(3):854–859. http://dx.doi.org/10.1073/pnas.52.3.854

Headley AD, Callaghan TV. Modular growth of Huperzia selago (Lycopodiaceae: Pteridophyta). Fern Gaz. 1990;13(7):361–372.

Tilquin JP. Note on apomixes in ferns. Acta Soc Bot Pol. 1981;50:217–222.

Walker TG. Some aspects of agamospory in ferns-the Braithwaite system. Proc R Soc Edinb. 1985;86B:59–66. http://dx.doi.org/10.1017/S026972700000796X

Bell PR. The phase change in ferns. Acta Soc Bot Pol. 1982;50:307–314.

Bell PR. Apospory and apogamy: implications for understanding the plant life cycle. Int J Plant Sci. 1992;153:123–136. http://dx.doi.org/10.1086/297070

Zenkteler E. Systems of vegetative propagation of fern in vitro and in vivo. Poznań: Adam Mickiewicz University Press; 2000.

DOI: https://doi.org/10.5586/asbp.2013.034

Journal ISSN:
  • 2083-9480 (online)
  • 0001-6977 (print; ceased since 2016)
This is an Open Access journal, which distributes its content under the terms of the Creative Commons Attribution License, which permits redistribution, commercial and non-commercial, provided that the content is properly cited.
The journal is a member of the Committee on Publication Ethics (COPE) and aims to follow the COPE’s principles.
The journal publisher is a member of the Open Access Scholarly Publishers Association.
The journal content is indexed in Similarity Check, the Crossref initiative to prevent scholarly and professional plagiarism.
Polish Botanical Society