ESTABLISHMENT AND DEVELOPMENT OF THE CATHERINE ’ S MOSS ATRICHUM UNDULATUM ( HEDW . ) P . BEAUV . ( POLYTRICHACEAE ) IN IN VITRO CONDITIONS

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. UDC 582.325.1:57.08

Beauv.] is among the largest European terrestrial moss species.It is widespread across Europe, and due to its size is widely used in moss biology research (e.g., Bequerel, 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 Lemmon, 1987;O n o et al., 1987;L i n d e m a n n et al., 1989; M i l e s and L o n g t o n, 1990; Stoneburner et al., 1992; R ü t t e n and Santarius, 1992;Santorius, 1993;I m u r a, 1994; M e y e r and Santorius, 1998; B e c k e t et al., 2000;Ligrone et al., 2002;G a n g et al., 2003;Bijelović et al., 2004;L i n et al., 2005;Sabovljević et al., 2005).However, cultivation of this species remains problematic, and it is therefore not easy to maintain controlled growth conditions while investigating biology of the Catherine's moss.Axenic culturing of bryophytes seems to be so complicated that many investigators gaveup the attempt.However, due to possible interaction with other organisms in non axenic conditions, sterile culturing is necessary for certain experimental procedures.
Moreover, bryophytes as models for plant biology investigations have great advantages, over vascular plants: (1) relatively simple structure compared to other higher plants, (2) haploid gametophyte of the dominant vegetative phase, and (3) lower chromosome numbers (Gang et al., 2003).Cells of bryophytes, especially in suspension culture, have been noted as ideal materials for morphogenetic, genetic, physiological, biochemical, and molecular studies (O n o et al., 1988).
According to F e l i x (1994), 31 liverworts, 18 mosses, and one hornwort have been used asexperimental objects in the sterile culture of bryophytes.Some new species have lately been added to the list of mosses (Sabovljević et al., 2002;Sabovljević et al., 2003; B i j e l o v i ć and Sabovljević, 2003).Systems of suspension culture have been established from 27 liverworts, 16 mosses, and one hornwort (Gang et al., 2003).However, progress in bryophyte tissue culture has not gone as fast as in culture of the cells of other higher plants, and the number of cases achieved still does not satisfy sufficiently the demands of various research fields (F e l i x, 1994).Many data are still controversial, and numerous species react in different ways or express different pathways of development under the same conditions (B i j e l o v i ć et al., 2004;Sabovljević et al., 2005).Also, very few data can be found on comparison of bryophyte development comparison in vitro and in vivo.

MATERIAL AND METHODS
Plant material was collected on roadside soil during investigation of Mt.Avala (Sabovljević and Cvetić, 2003).Voucher specimens are deposited in the BEOU herbarium of Belgrade University.
The collected material of A. undulatum was in the sporophyte phase, but with intact opercula.Cultures were initiated from almost mature spores, from unopened capsules that were taken for sterilization.After collection, the sporophytes were separated carefully from the gametophytes, placed in glasses, covered with cheese cloth, and rinsed with tap water for 30 minutes.Sporophytes were then disinfected for 7 minutes with a 13% solution of sodium hypochlorite.Finally, the sporophytes were rinsed three times in sterile deionized water.
The pH of the media was adjusted to 5.8 before autoclaving at 114°C for 25 minutes.
Cultures were kept at 25 ± 2°C, and light (16/8 hours of light to darkness) was supplied by cool-white fluorescent tubes at a photon fluency rate of 47 µmol/m 2 s.
Cultures were subcultured for a period of 4-6 weeks.
For analysis of MS mineral salts and sucrose influence, 10 mm long apical segments were transferred to nutrient media (MS 1 -MS 9 ).For each medium composition, approximately 40 transplants of A. undulatum were cultivated in four Petridishes.The influence of medium composition was quantified by measuring elongation of initial gametophyte explants and the index of multiplication.
Descriptive statistics and parametric statistical testing (ANOVA) were performed using Microcal ORIGIN 6.1 software.Morphological characteristics of in vivo and in vitro grown plants were compared.

RESULTS
Surface sterilization of sporophytes was effective, and a high percentage of spores germinated on basal MS medium (supplemented with 3% sucrose).Spore germination of A. undulatum occurred one month after establishing in vitro culture.Protonema developed 15 days after spore germination, and bud formation occurred two months after spore germination.The number of developed buds was not high, and they remained for a long time in the bud phase without growing to fully developed gametophytes.Four weeks after bud development, buds started to grow to fully developed gametophytes.
To study the influence of sucrose and MS mineral salts on morphogenesis of A. undulatum, gametophyte shoots were used in the following experiments.For each medium composition (MS 1 -MS 9 ), approximately 40 transplants of A. undulatum were cultivated in four Petridishes (Fig. 2).
According to our results, that higher sucrose concentrations (MS 3, MS 6 ,andMS 8 ) tended to have a positive effect on shoot elongation, but these differences were not statistically significant at the 0.05 level (Fig. 1A).
The media MS 7 andMS 9 were effective as well, although plants remain significantly smaller than the native counterparts (Fig. 3).
Analyses of the index of multiplication (Fig. 1B) showed a statistically significant difference between plants grown on media with no sucrose and media supplied with 1.5% sucrose.The presence of sucrose promoted multiplication in any mineral composition (MS 1 <MS 2 ,MS 4 <MS 5 ,MS 7 <MS 8 ).High sucrose concentration combined with dilute mineral solution (MS 3 , MS 6 ) had the same positive effect on multiplication as moderate sucrose concentration, but when combined with high mineral concentration, high sucrose had no effect at all (MS 7 /MS 9 ).Nevertheless, our results suggest that both elongation and multiplication of A. undulatum shoots are in most casesstimulated by sucrose presence in the medium, even if the latter did not contain mineral salts (MS 3 and MS 6 ).According to our results (Fig. 1), 3% sucrose was more effective than 1.5% sucrose, except in MS 9 medium.
The highest index of multiplication of plants developed on MS 8 medium (Fig. 1B) indicates that gametophytes of A. undulatum could develop and even multiply better on media supplemented with a lower amount of sucrose (1.5%), while in this case a higher concentration of sucrose (3%) promoted protonema development.Similarly, it was shown that Bryum argenteum also multiplies much better on a medium supplemented with 1.5% sucrose compared to media with higher sucrose concentrations (Sabovljević et al., 2005).

DEVELOPMENT OF ATRICHUM UNDULATUM 89
A.  According to the scarce data published to date, sugars have a positive effect on protonemal growth and development of mosses under in vitro conditions (Chopraand K u m r a, 1988).Also, literature data indicate that sugar has a positive effect on bud induction and shoot formation in in vitro culture of some moss species (M i t r a and A l l s o p p, 1959).
Data on mineral nutrition of bryophytes are lacking.It is assumed that bryophytes have macronutrient and micronutrient requirements closely similar to those of vascular plants (B a t e s, 2000; Z e c h m e i s t e r et al., 2002).
However, an additional problem in consideration of bryophyte nutrient needs is the specific nature of mineral uptake and transport of mineral elementsin bryophyte tissue (B r o w n and B a t e s, 1990), which are significantly altered when minerals present are bound in an organic complex (V u k o j e v i ć et al., 2004).
Atrichum undulatum grown on a medium supplemented with sugars developed until the juvenile phase and remained in this stage with further subculturing (Sabovljević et al., 2005), like plants on a medium supplemented with phytohormones (B i j e l o v i ć et al., 2004).On a medium that was supplemented just with mineral salts (without sugars), plants in axenic culture developed into fully developed plant, but never reached the size of native counterparts.There were no significant morphological differences between plants from nature and from in vitro culture other than size differences.Phyloids grown in in vitro culture formed fully developed lamellae on costa, similar to plants grown in nature.However, plants developed in axenic culture under any growth conditions have tenderer constitution of phyloids and lamellae (Figs. 4,5,6A,and 6B).

CONCLUSION
Our attempt to achieve initiation and propagation of both protonema and gametophytes of A. undulatum was   successful.The optimal medium for gametophyte development contained MS salts and 1.5% sucrose.A higher sucrose concentration (3%) inhibited full plant development (plants remain in juvenile stage) and promoted protonemal development.
Further experiments should be performed in order to elucidate effects of mineral composition and carbon availability on the development of different moss species, since results so far are sparse and controversial.У раду су дати упоредни подаци о морфоанатомским карактеристикама биљака одраслих у природи и биљака одраслих у контролисаним условима in vitro културе.

Fig. 1 .
Fig. 1.Effect of different mineral salts and sucrose content of agarose nutrient media on elongation (A) and multiplication (B) of Atrichum undulatum shoot explants.Δ x presents growth of gametophytes after 6 weeks.

Fig. 3 .
Fig. 3. Atrichum undulatum grown in in vitro culture on MS medium without sucrose and with full mineral salts.Plants remain small.

Fig. 4 .
Fig. 4. Cross section of the phyloid of Atrichum undulatum grown in nature, with fully developed filaments.

Fig. 5 .
Fig. 5. Cross section of the phyloid of Atrichum undulatum grown in nature, juvenile leaf with incompletely developed filaments

Fig
Fig. 6B.Cross section of the phyloid of Atrichum undulatum grown in in vitro conditions, with fully developed but tender filaments.