Abstract
This study presents a protocol for a fast and effective in vitro axenic culture of <em>Huperzia selago</em> (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 <em>H. selago</em> 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.
Highlights
Huperzia selago (L.) Bernh. ex Schrank et Mart. (=Lycopodium selago L.), fir club moss (Fig. 1) is the only European lycopod of the Huperziaceae Rothm. family distinguished by Rothmaler [1,2] following the taxonomic revision Lycopodiaceae sensu lato
Good results were achieved using Mr medium supplemented with 0.25 mg/l 1-naphthaleacetic acid (NAA) as at 8 weeks of culture 30% of the explants began to develop into sporophytes (Tab. 1)
Mr medium supplemented with other combinations and concentrations of growth regulators was found to be unsuitable since only a limited number of explants developed into sporophytes at 8 weeks of culture (Tab. 1)
Summary
Huperzia selago (L.) Bernh. ex Schrank et Mart. (=Lycopodium selago L.), fir club moss (Fig. 1) is the only European lycopod of the Huperziaceae Rothm. family distinguished by Rothmaler [1,2] following the taxonomic revision Lycopodiaceae sensu lato. The Huperziaceae family is thought to include approximately 150 to 400 species with a worldwide distribution, though predominantly found in the tropical regions [3,4,5]. In Europe, Huperzia selago is commonly found in a widespread territory stretching from the Scandinavia to the northern Mediterranean countries [6]. It grows in the boreal and temperate regions of Asia [5] and North America [7]. A number of alkaloids have been isolated from Huperzia selago plant [8]. Huperzine A (selagine, HupA), one of these alkaloids, is an effective, reversible and selective acetylcholinesterase (AChE) inhibitor, undergoing clinical
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