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

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.

Huperzine A and Huperzine B Production by Prothallus Cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart

“…As demonstrated in the present study, the use of MS medium rich in reduced nitrogen produced the least increase in biomass growth. Similarly, MS medium proved not suitable for sporophyte cultures [25]. The fastest gametophyte biomass growth was observed on W/S media without growth regulators.…”

“…Up to now, club moss gametophytes from natural populations have been only infrequently described and in very rare cases they were obtained in vitro, but they have not been effectively used for the biosynthesis of secondary metabolites [23]. The first studies on the biosynthesis of HupA were conducted by Szypuła et al [17,20,25] and Ma and Gang [24]. Szypuła et al [17] presented a protocol for the establishment and maintenance of in vitro culture of Huperzia selago sporophytes from shoot explants (Figure 1).…”

“…Importantly, spore disinfection according the protocol developed specifically for the present study allowed tissue cultures free from bacterial and fungal contamination to be established and maintained. Achieving axenic cultures of the sporophytes of H. selago and other club mosses is the most challenging step [17,23,25]. Club mosses are mycorrhizal plants and their aboveground tissues are colonized by endophytic bacteria and fungi.…”

“…Importantly, H. selago is a rich source of HupA, since its sporophytes contain much more of the alkaloid than found in Huperzia serrata native to Asia [17,20,23]. The preliminary studies of HupA production in in vitro culture of H. selago gametophytes indicate that this is a more efficient method of obtaining secondary metabolites than sporophyte culture [17,23,25]. So far, there have been no studies on the biosynthesis of alkaloids or any other metabolites using in vitro culture of gametophytes (prothallus).…”

Original article Assessment of antioxidative activity of alkaloids from Huperzia selago and Diphasiastrum complanatum using in vitro systems

“…Moreover, studies by Szypuła et al [46][47][48] have shown that H. selago is a rich source of HupA and other alkaloids, which are much more abundant in this plant than in the Chinese club moss H. serrata.…”