The high mountain pottioid moss Molendoa hornschuchiana (Hook) Lindb. ex Limpr. is a very rare and critically endangered bryophyte species in Europe in need for ex situ conservation. A 25-year-old herbarium sample was used to revive and propagate this species for further reintroduction and introduction to potential natural habitats. The reviving of “dead” herbarium specimen was achieved by disposing of axenical organisms as well as adjusting condition for developing secondary protonema, bud inductions, and optimization of gametophyte propagation in vitro condition.
The influence of exogenously added growth regulators on the morphogenesis of this species was studied. The plants were cultured in the two basic types of media, viz., BCD and half-strength Murashige and Skoog (MS) supplemented with different concentrations (0.01–0.3 μM) of indole-3-butyric acid (IBA) and N6-benzyladenine (BA) under a 16-h photoperiod. The influence of growth regulators on gametophores multiplication in vitro as well as on protonemal diameter was recorded. Well-developed gametophores were obtained on BCD medium, whereas on half-strength MS medium, secondary protonema was produced, both on hormone-free and supplemented substrate exclusively. Based on multiplication index in vitro, maximum development of gametophores was realized on BCD medium supplemented with 0.3 μM IBA and 0.1 μM BA. However, the widest diameter of secondary protonema was obtained on BCD medium enriched with low concentration of both BA (0.01 and 0.03 μM) and constant concentration of IBA (0.03 μM). Chemical names used: indole-3-butyric acid (IBA), N6-benzyladenine (BA), Murashige and Skoog medium (MS).
Rindera umbellata (Waldst. & Kit.) Bunge is a rare, critically endangered and horticulturally appealing plant with unexplored pharmaceutical potential. Its distribution is restricted to sandy habitats, whereas propagation in nature is limited by fungal infections of the seeds. To initiate its ex situ conservation and provide material for metabolomic studies, we have introduced R. umbellata into in vitro culture using immature embryos as primary explants. A 72% of the embryos germinated on growth regulator-free medium. The optimization of growth conditions was based on varying carbohydrates (sucrose, glucose, and fructose) in the medium. In vitro growth and development of R. umbellata plants were significantly affected by both the type and concentration of the applied sugars. For most recorded parameters, including leaf elongation, biomass production, rooting percentage, and the number and length of roots, 0.1 m sucrose was optimal. The highest percentage of explants with developed buds was achieved on 0.06 m sucrose (38.77%) or 0.3 m glucose (27.43%). The plantlets obtained on 0.1 m sucrose were successfully acclimatized to greenhouse and field conditions with survival rates of 71.43% and 42.86%, respectively. To our best knowledge, this is the first publication dedicated to this species.
This study provides the results of the developmental biology of the highly rare and endangered moss species Bruchia vogesiaca (recorded in less than 30 localities in the Northern Hemisphere, mainly western, central, and southwestern Europe). The aim of the study was to achieve the fully developed gametophyte and to propagate it for the purpose of conservation, reintroduction, and introduction to potential habitats free from xenic contamination. These gametophytes will be used for the study of genetics and genomics of this species. The micropropagation of B. vogesiaca was successfully applied on BCD medium supplemented with 0.1 μM BA and on BCD supplemented with 0.3 μM IBA and 0.3 μM BA for numerous gametophore production. The highest production of secondary protonema was achieved on MS/2 S/2 medium enriched with 0.1 or 0.3 μM IBA and 0.3 μM BA. Rather successfully applied micropropagation of this threatened moss species enables better knowledge of its biology and is of great value for its conservation biology and developmental research. Chemical names used: indole-3-butyric acid (IBA), N6-benzyladenine (BA), Murashige and Skoog medium (MS).