33 (2): 137-140 (2009) Original Scientifi c Paper Axenically culturing the bryophytes: a case study of the moss Dicranum scoparium Hedw. (Dicranaceae, Bryophyta) Milorad Vujičić, Aneta Sabovljević and Marko Sabovljević✳ Institute of Botany and Garden Jevremovac, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia ABSTRACT: Th e aim of this study was to establish axenic culture of the moss Dicranum scoparium a counterpart of rare and widly endangered D. viride. Th e media contents, as well as light and temperature were varied to fi nd the optimal conditions for spore germination, protonema growing, bud formation and gametophyte development. Th e best contitions for micropropagation or axenically bryo-farming is to grow D. scoparium on the MS medium enriched with sucrose (1.5%), at 18-20°C independent of light length condition. Key words: moss, Dicranum scoparium, in vitro, axenical culture Received 30 September 2009 Revision accepted 30 November 2009 UDK 582.32.085.2 INTRODUCTION One of the species in high risk of extinction is Dicranum viride (Sull. & Lesq. in Sull.) Lindb. (species of Bryophytes, although the second largest group of terrestrial community interest listed in the Habitat Directive 92/43 plants, received much less attention in conservation EEC under annex II, Bern Convention Species, appendix and protection in comparison to vascular plants and 1). It is ranged in northern hemisphere but rare all over. In higher animals. However, bryophytes are documented to Europe, this species is threatened all over (ECCB 1995). In decrease in nature aff ected by habitat devastation directly Serbia, D. viride is estimated by available data as vulner- or indirectly. able (VU) (Sabovljević et al., 2004). Th us, for some species there is urgently need of the Since this species is very rare, hardly accessible in active protection and conservation with aim to save some enough amounts, a more spread counterpart of it was used species from extinction. to study axenically establishing, growing and propagation For many of the species in threat of extinction, there of Dicranum species with aim to apply the knowledge are very few data on their biology which so diminish the acquired to the D. viride, when starting project of its active attempts to work on their conservation. conservation and protection and material propagation for By defi nition, material for the initiation of ex situ spreading in nature. collection (i.e. in vitro culture, in vitro farming) of Th e problem of establishing in vitro culture of threatened species is rare (Rowntree & Ramsay 2009). bryophytes, even they were the fi rst plants grown in in Consequently, it is important that technique used have a vitro condition (Servettaz 1913) remain up to date. high success rate and to be applicable to related species at Besides the problems with bryophyte establishment in least. Th e importance of the development of the methods axenic culture, it is offt en problem of material availability, of in vitro and/or ex situ maintainig of bryophytes was genetic variability of material, disposal of axenic organizms already elaborated elswhere (e.g. Rowntree & Ramsay living on bryophytes and low level of species biology 2005; Rowntree 2006). knowledge. ✳correspondence: [email protected] © 2009 Institute of Botany and Botanical Garden Jevremovac, Belgrade 138 vol. 33 (2) Th e problems, perspective and potential usefulness of Combination 1: 16/8 hours of light to darkness, at in vitro bryophyte farming overpass the issue of bryophyte 25 ± 2°C. ex situ conservation and can be found only in few studies Combination 2: 8/16 hours of light to darkness, at (Bijelović & Sabovljević 2003; Sabovljević et al. 2003, 20 ± 2°C. 2006, 2009; Bijelović et al. 2004; Duckett et al. 2004; Combination 3: 16/8 hours of light to darkness, at Cvetić et al. 2005; González et al. 2006; Mallón et 20 ± 2°C. al. 2006; Rowntree 2006; Sabovljević & Sabovljević Combination 4: 16/8 hours of light to darkness, at 2008). 18 ± 2°C. MATERIALS AND METHODS Light was supplied by cool-white fl uorescent tubes at a photon fl uency rate of 47 μmol/m2s. Cultures were Th e complete developed plants (both sporophyte and subcultured for a period of 4-6 weeks. For analysis of gametophyte) of D. scoparium from the Tara Mt. (Western condition set infl uence to development 10 mm long Serbia) were collected in the May 2007. Collected materials apical segments (gametophyte) or spores were transferred were stored in plastic bag till laboratory and then in to nutrient media. For each medium composition, paper bag at room temperature until manipulation and approximately 40 transplants of D. scoparium or 0.5 ml establishing axenical culture. Th e collected material of D. of water containing spores from one unopened capsules scoparium were chosen so that the sporophytes contain were cultivated in four Petri-dishes. Th e infl uence of tested also ripen unopened capsules with intact opercula. Th e environmental condition was quantifi ed by measuring voucher specimen was deposited in the Belgrade University elongation of initial gametophyte explants and the index of Herbarium BEOU2158. multiplication, protonemal development (both secondary Cultures initiation was tried from almost mature spores, and primary) and new bud formation. from unopened capsules that were taken for sterilization and young gametophyte vegetative shoots (i.e. not bearing RESULTS AND DISCUSSION sex organs or sporophytes). Aft er collection, the chosen sporophytes or gametophytes were separated carefully Surface sterilization of sporophytes was eff ective at from the impurity and each other, placed in glasses, concentration of 7, 10, 13% and 15% solution of sodium covered with cheese cloth, and rinsed with tap water for 30 hypochlorite for fi ve minutes. Concentrations of 10- minutes. Sporophytes and gametophyte shoots were then 15% of sodium hypochlorite solution for fi ve minutes disinfected for 5 minutes with a 3, 5, 7, 10, 13% or 15% period were lethal for gametophyte shoots. Even, if the solution of sodium hypochlorite. Finally, the sporophytes period of sterilization were shortened, the green part of and gametophyte shoots were rinsed three times in sterile plants could not survive applied concentrations. In lower deionized water. sodium hypochlorite concentration gametophyte shoots As a basal medium for establishment of in vitro survived but also contaminants, xenical organisms living culture, we used Murashige and Skoog (1962) (MS) on mosses. medium containing Murashige and Skoog mineral salts Th us, contaminated starting material produced and vitamins, 100 mg/l inositol, 0.70% (w/v) agar (Torlak secondary protonema and aft er ca. one week period it purifi ed, Belgrade), and 3% sucrose and BCD medium (see was overgrown with fungi, algae or bacteria and could Sabovljević et al. 2009 for the media details). In order to not be used for further developmental researches. So, the observe the infl uence of sucrose and/or mineral salts on further developmental studies were focused on plants the morphogenesis of this species, the following medium development deriving from spores on diff erent media. compositions were used: Th e highest percentage (95%) of spores germinated MS1: half strength of MS mineral salts, sugar free; on basal MS medium (supplemented with 3% sucrose). MS2: half strength of MS mineral salts, 1.5% sucrose; Spore germination of D. scoparium occurred 22 days MS3: half strength of MS mineral salts, 3% sucrose; aft er establishing in vitro culture. Spores germinated MS4: MS mineral salts, sugar free; ineff ectively of light duration but prefer slightly lower MS5: MS mineral salts, 1.5% sucrose; temperature. At temperature of 25 ± 2°C, the germination MS6: MS mineral salts, 3% sucrose; rate is estimated up to 60%. BCD1: BCD mineral salts, 1.5% sucrose; Visible protonema can be noticed already 30 days BCD2: BCD mineral salts, 3% sucrose; aft er spore germination, and it grows rapidly at 18°C and BCD3: BCD mineral salts, sugar free; 20±2°C. Media containing sugar slightly increased the Th e pH of the media was adjusted to 5.8 before germination speed, but aft er germination sugar available autoclaving at 114°C for 25 minutes. in substrate seems to play role in bud induction on Th e temperature and light duration varied in combined protonema since protonema developed on media without with sets of media: sugar remain in that stage for a long period. M. Vujičić et al.: Axenically culturing the bryophytes: a case study of the moss Dicranum scoparium Hedw. (Dicranaceae, Bryophyta) 139 Fig. 1. Protonema of Dicranum scoparium with buds Fig. 2. A detail of Dicranum scoparium gemetophyte indicated by arrows. developed in in vitro condition one should use less sugar in growing substrate, and such material is applicable for micro-propagantion on the same media where secondary protonema, followed by bud formation and gametophyte plantlets appear in highest percentage. CONCLUSION Surface sterilization is easier to achieve on sporophyte than to gametophyte. Th e best conditions for micro-propagation or axenically bryo-farming is to grow D. scoparium on the MS medium enriched with sucrose (1.5%), at 18-20°C independent of light length condition. Th e results obtained can be used for axenically culturing D. viride, a rare and Fig. 3. Dicranum scoparium gemetophyte developed in endangered counterpart of tested species. in vitro condition Acknowledgement – Th is work was supported by Serbian Ministry of Science and Technological Development – Four weeks aft er bud development, buds started to grow grants No. 143015 and 143031. to fully developed gametophytes (Fig. 1). Th e number of developed buds was not high, and they remained for a long REFERENCES time in the bud phase without growing to fully developed gametophytes. Bijelović A & Sabovljević M. 2003. Callus induction and Th e best medium for the plantlets regeneration both plant regeneration in the moss Aloina aloides (Schultz.) from primary and secondary protonema was MS5.