Arch. Biol. Sci., Belgrade, 58 (2), 87-93, 2006. ESTABLISHMENT AND DEVELOPMENT OF THE CATHERINE’S MOSS ATRICHUM UNDULATUM (HEDW.) P. BEAUV. (POLYTRICHACEAE) IN IN VITRO CONDITIONS 1 ANETA SABOVLJEVIĆ1, 2, TIJANACVETIĆ andM. SABOVLJEVIĆ1, 3 1Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia and Montenegro 2Institute of Botany, University of Cologne, 50931 Cologne, Germany 3AG Bryology, Nees Institute of Botany, University of Bonn, 53115 Bonn, Germany Abstract - The effect of sucrose and mineral salts on morphogenesis of the Catherine’s moss (Atrichum undulatum)in in vitro culture was tested. In vitro culture of this species was established from disinfected spores on Murashige and Skoog (MS) medium. Apical shoots of gametophytes were used to investigate the influence of sucrose and mineral salts on protonemal and gametophyte growth and multiplication. Paper also treats morpho-anatomical characteristics of plants grown in nature and plants derived from in vitro culture. Key words: Brzophytes, morphogenesis, Catherine’s moss, growth, multiplication UDC 582.325.1:57.08 INTRODUCTION higher plants, (2) haploid gametophyte of the dominant vegetative phase, and (3) lower chromosome numbers The Catherine’s moss [Atrichum undulatum (Hedw.) P. (Gang et al., 2003). Cells of bryophytes, especially in Beauv.] is among the largest European terrestrial moss suspension culture, have been noted as ideal materials for species. It is widespread across Europe, and due to its morphogenetic, genetic, physiological, biochemical, and size is widely used in moss biology research (e.g., Be- molecular studies (O n o et al., 1988). querel, 1906; G e m m e l l, 1953; W o r d, 1960; Sitte, 1963; W o l t e r s, 1964; B r o w n and Lem- According to F e l i x (1994), 31 liverworts, 18 mon, 1987; O n o et al., 1987; L i n d e m a n n et al., mosses, and one hornwort have been used asexperimen- 1989; M i l e s and L o n g t o n, 1990; Stoneburn- tal objects in the sterile culture of bryophytes. Some new er et al., 1992; R ü t t e n and Santarius, 1992; species have lately been added to the list of mosses (Sa- Santorius, 1993; I m u r a, 1994; M e y e r and bovljević et al., 2002; Sabovljević et al., Santorius, 1998; B e c k e t et al., 2000; Ligro- 2003; B i j e l o v i ć and Sabovljević, 2003). ne et al., 2002; G a n g et al., 2003; Bijelović et Systems of suspension culture have been established al., 2004; L i n et al., 2005; Sabovljević et al., from 27 liverworts, 16 mosses, and one hornwort 2005). However, cultivation of this species remains prob- (Gang et al., 2003). However, progress in bryophyte lematic, and it is therefore not easy to maintain controlled tissue culture has not gone as fast as in culture of the cells growth conditions while investigating biology of the Catherine’s moss. Axenic culturing of bryophytes seems of other higher plants, and the number of cases achieved to be so complicated that many investigators gaveup the still does not satisfy sufficiently the demands of various attempt. However, due to possible interaction with other research fields (F e l i x, 1994). Many data are still con- organisms in non axenic conditions, sterile culturing is troversial, and numerous species react in different ways necessary for certain experimental procedures. or express different pathways of development under the same conditions (B i j e l o v i ć et al., 2004; Sa- Moreover, bryophytes as models for plant biology bovljević et al., 2005). Also, very few data can be investigations have great advantages, over vascular found on comparison of bryophyte development compar- plants: (1) relatively simple structure compared to other ison in vitro and in vivo. 87 88 A. SABOVLJEVIĆ, T.CVETIĆ andM. SABOVLJEVIĆ MATERIAL AND METHODS of light to darkness) was supplied by cool-white fluores- cent tubes at a photon fluency rate of 47 µmol/m2s. Plant material was collected on roadside soil during Cultures were subcultured for a period of 4-6 weeks. investigation of Mt. Avala (Sabovljević and Cvetić, 2003). Voucher specimens are deposited in For analysis of MS mineral salts and sucrose influ- the BEOU herbarium of Belgrade University. ence, 10 mm long apical segments were transferred to nu- trient media (MS -MS ). For each medium composition, The collected material of A. undulatum was in the 1 9 approximately 40 transplants of A. undulatum were culti- sporophyte phase, but with intact opercula. Cultures were vated in four Petridishes. The influence of medium com- initiated from almost mature spores, from unopened cap- position was quantified by measuring elongation of ini- sules that were taken for sterilization. After collection, tial gametophyte explants and the index of multiplication. the sporophytes were separated carefully from the gametophytes, placed in glasses, covered with cheese Descriptive statistics and parametric statistical test- cloth, and rinsed with tap water for 30 minutes. Sporo- ing (ANOVA) were performed using Microcal ORIGIN phytes were then disinfected for 7 minutes with a 13% 6.1 software. solution of sodium hypochlorite. Finally, the sporophytes were rinsed three times in sterile deionized water. Morphological characteristics of in vivo and in vit- ro grown plants were compared. As a basal medium for establishment of in vitro cul- ture, we used M u r a s h i g e and S k o o g (1962) (MS) medium containing Murashige and Skoog mineral salts RESULTS and vitamins, 100 mg/l inositol, 0.70% (w/v) agar (Tor- lak purified, Belgrade), and 3% sucrose. In order to ob- Surface sterilization of sporophytes was effective, serve the influence of sucrose and/or mineral salts on the and a high percentage of spores germinated on basal MS morphogenesis of this species, the following medium medium (supplemented with 3% sucrose). Spore germi- compositions were used: nation of A. undulatum occurred one month after estab- lishing in vitro culture. Protonema developed 15 days aft- 1) MS : 1/10 strength of MS mineral salts; 1 er spore germination, and bud formation occurred two months after spore germination. The number of devel- 2) MS2: 1/10 strength of MS mineral salts, 1.5% sucrose; oped buds was not high, and they remained for a long time in the bud phase without growing to fully developed 3) MS3: 1/10 strength of MS mineral salts, 3% sucrose; gametophytes. Four weeks after bud development, buds started to grow to fully developed gametophytes. 4) MS4: half strength of MS mineral salts; To study the influence of sucrose and MS mineral 5) MS : half strength of MS mineral salts, 1.5% sucrose; salts on morphogenesis of A. undulatum, gametophyte 5 shoots were used in the following experiments. For each medium composition (MS – MS ), approximately 40 6) MS : half strength of MS mineral salts, 3% sucrose; 1 9 6 transplants of A. undulatum were cultivated in four Pet- ridishes (Fig.2). 7) MS7: MS mineral salts; According to our results, that higher sucrose con- 8) MS : MS mineral salts, 1.5% sucrose; 8 centrations (MS3, MS6,andMS8) tended to have a posi- tive effect on shoot elongation, but these differences were 9) MS9: MS mineral salts, 3% sucrose (basal medium). not statistically significant at the 0.05 level (Fig. 1A). The pH of the media was adjusted to 5.8 before au- The media MS7 andMS9 were effective as well, al- toclaving at 114°C for 25 minutes. though plants remain significantly smaller than the native counterparts (Fig. 3). Cultures were kept at 25 ± 2°C, and light (16/8 hours DEVELOPMENT OF ATRICHUM UNDULATUM 89 A. Analyses of the index of multiplication (Fig. 1B) showed a statistically significant difference between 3.0 plants grown on media with no sucrose and media sup- 2.5 plied with 1.5% sucrose. The presence of sucrose pro- moted multiplication in any mineral composition 2.0 (MS1<MS2,MS4<MS5,MS7<MS8). High sucrose con- 1.5 centration combined with dilute mineral solution (MS3, x [mm]x D MS ) had the same positive effect on multiplication as 1.0 6 moderate sucrose concentration, but when combined with 0.5 high mineral concentration, high sucrose had no effect at 0.0 all (MS7/MS9). MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 MS9 medium composition Nevertheless, our results suggest that both elonga- tion and multiplication of A. undulatum shoots are in B. most casesstimulated by sucrose presence in the medi- 7 um, even if the latter did not contain mineral salts (MS3 and MS ).According to our results (Fig.1), 3% sucrose 6 6 was more effective than 1.5% sucrose, except in MS9 me- 5 dium. 4 3 The highest index of multiplication of plants devel- 2 oped on MS8 medium (Fig. 1B) indicates that gameto- index of multiplication 1 phytes of A. undulatum could develop and even multiply better on media supplemented with a lower amount of su- 0 MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 MS9 crose (1.5%), while in this case a higher concentration of medium composition sucrose (3%) promoted protonema development. Simi- larly, it was shown that Bryum argenteum also multiplies much better on a medium supplemented with 1.5% su- Fig. 1. Effect of different mineral salts and sucrose content of agarose nutrient media on elongation (A) and multiplication (B) of Atrichum undu- crose compared to media with higher sucrose concentra- latum shoot explants. Δ x presents growth of gametophytes after 6 weeks. tions (Sabovljević et al., 2005).