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A Simple Method to Obtain Microbial-Free in Vitro Moss Cultures Hanan Shaaban*, Hanaa Shabbara, Mohamed Farag and Wagieh El Saadawi

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A Simple Method to Obtain Microbial-Free in Vitro Moss Cultures Hanan Shaaban*, Hanaa Shabbara, Mohamed Farag and Wagieh El Saadawi ISSN: 1110-7413 Taeckholmia 36 (2016): 17-25 eISSN: 2357-044X A Simple Method to Obtain Microbial-free in vitro Moss Cultures Hanan Shaaban*, Hanaa Shabbara, Mohamed Farag and Wagieh El saadawi Botany Department, Faculty of Science, Ain Shams University, Abbasia, Cairo, Egypt. *Corresponding author: [email protected] Abstract Developing a suitable method for moss sterilization is a crucial step for successful in vitro culture. Different chemical and physical methods have been tried by many authors for more than a century but with low axenic culture percentages. An easy and economic (no use of chemicals or antibiotics) cultivation method is developed here, which proved high success with three moss species starting from gametophores. Key words Axenic cultivation, mosses, Bryum, Philonotis, sterilizing agents Introduction Trials for propagation of mosses in pure cultures started a long time ago by Bryophyta is one of the earliest pioneer bryologists (e.g. Vö chting 1885, inhabitants of terrestrial environments. Becquerel 1906) and are still going on till They survived and tolerated for ages, now (e,g. Rowntree and Ramsay 2009, various climatic conditions. However, Rowntree et al. 2011, Carey et al. 2015), harvesting large quantities of bryophytes however the obtained results of survival for greening and different applications percentages were poor or at least not can seriously damage the natural habitat ( satisfactory, particularily when the in Peck and Muir 2001).To avoid or at least vitro culture was started from minimize such a serious environmental gametophore tissues and not from spores. problem an alternative solution exists in the in vitro culture technique which Initiating axenic in vitro cultures facilitates growing and regenerating from field grown plants requires surface- plants. In vitro culture of plants including sterilization of plant part inocula to get rid bryophytes furnishes,therefore a good of superficial contaminants (Basile 1972). solution for nature conservation. In As surface sterilization of mature capsules addition it gives us opportunities to - before dehiscing- is effective, axenic increase our knowledge about certain cultures of mosses are most easily aspects of plant biology, development established from spores (Duckett et al. (Duckett et al. 2004, Victoria et al. 2011) 2004, Lal 1984, Reski 1998, Hohe and and biotechnological research (Beike et Reski 2005). Unfortunately, starting al. 2010). cultures from spores is impractical for many species, especially dioicous taxa (ca. 60% of recorded moss taxa); Received on 09/03/2016 and accepted on 28/03/2016 Hanan Shaaban et al. Hedenäs and Bisang 2011, which mostly cultures of different moss species starting never produce capsules (Glime 2013). from gametophores remain to be a current Alternatively, gametophores have to be challenge. This work represents one of used for producing axenic cultures of the these trials, however distinguished majority of mosses. However, surface because efficient sterilization is sought sterilization of moss gametophore is without using chimicals or antibiotics. usualy ineffective in establishing axenic cultures. Gametophores are very delicate Materials and Methods and the phylloids are in general tinny, Fresh gametophores of three moss mono-layered, inhabitated by various taxa collected from natural habitats in xenic organisms like bacteria, algae, Cairo, Egypt, were used in this protozoa, fungi and others (Vujičić et al. experiment. These taxa were Bryum 2009, Sabovljevic et al. 2012). As a result, argenteum Hedw. Bryum subapiculatum vegetative fragments may be either killed Hampe (Bryaceae) and Philonotis hastata by the sterilization procedure or by (Duby) Wijk& Margad (Bartramiaceae). microbes ( Duckett et al. 2004). B. argenteum gametophores (given Generally, there are many previously herbarium no. MF 03) were collected in described techniques for surface- October 2013, from Kobri El-Kobba, sterilizing of small parts of moss underground Metro station, ca. 30 4 gametophyte, as washing machine (Basile 58.10 N and 31 17 32.29 E, where 1972, Basile and Basile 1988), ultrasound they grow in shade, on a wet vertical side (Cano 1996), and chemicals ( e.g. sodium of a cemented pavement. B. dichloro-isocyanurate (SDICN) subapiculatum gametophores (MF10) (Parkinson et al. 1996, Niedz and Bausher were collected in October 2013 from the 20002, Sarasan et al. 2006, Liang et al. Botanical Garden of the Faculty of 2010). The survival percentages of Science, Ain shams University, ca. 30 4 sterilized gametophores were 2-11% with 40.75 N and 31 16 56.03 E, where sodium hypochloride (NaOCl) and 37- they grew exposed to sun, on a wet 66% with sodium dichloroisocyanurate cemented layer of a sewage drain. (SDICN) (Sabovljevic et al. 2012) and Philonotis hastata gametophores (MF14) 70% with combination between 70% were collected in November 2013, from ethanol for 5 sec. and 5% (NaOCl), 30 Kobri El-Kobba, underground Metro sec. Station, ca. 30 5 4.30 N and 31 17 (Liang et al. 2010) [23]. Mercuric 42.71 E, where they grew exposed to Chloride was also used for sterilization of sun, on a wet vertical side of a cemented protonema, however it was very harsh and pavement. Both P. hastata and B. the method has low survival percentage argenteum carried easily detachable (Chen et al. 2009). Recently, an agar bulbils. embedding method with antibiotic, The present developed technique is significantly reduced the presence of modified from that of (Chen et al. 2009) . bacterial contaminants in gametophyte The following steps were carried out: tissue (Carey et al. 2015). 1. Freshly collected small moss samples However, trials for the development of were carefully cleaned mechanically an easy, convenient, effective and, more from sticking soil particles and other successful method for obtaining axenic impurities and rinsed with distilled 18 A Simple Method to Obtain Microbial-free in vitro Moss Cultures water and then vortexed three times 8. The gametophores of each species were for 10 minutes each. then divided into six groups. The first 3 2. Individual gametophores were picked groups were cultured on 3 different up under stereomicroscope (Novex solid media and the other 3 groups were model, Netherlands), then washed and cultured in 3 different liquid media as rinsed with sterile distilled water (SD one gametophore per each jar in three H2O) three times for five minutes each. replicates (i.e. a total of 54 jars for the 3. Washed gametophores of each species three species). The composition of the were divided into two groups; the first three different solid media was as group was propagated in 250 ml follows: conicals, containing 100 ml SD H2O, i. Nutrient free agar NFA (9 jars for the while the second one was propagated in 3 species). 250 ml transparent plastic containers, ii. Half strength Murashige and Skoog containing a thin film (5 mm thick) of miniral salts HsMS (1962) (9 jars sterilized water-saturated soil (30 g for the three species). soil; particles of the size 300 µm). iii. Full strength MS mineral salts; FsMS 4. The former group was kept on the lab (9 jars for the 3 species). bench under low light intensity for four The composition of the three different to six weeks. The latter group was liquid media was as follows: incubated in a plant growth chamber i. Nutrient free liquid medium; (Vision scientific plant growth NFM(Strelized distilled water) (9 chamber model V3-DM; Vision jars for the 3 species). Scientific Company Ltd, Korea) for ii. Half strength Murashige and Skoog four to six weeks, under controlled mineral salts HsMS (9 jars for the conditions of adjusted light/ dark three species). periods (16/8 hrs) using white iii. Full strength MS mineral salts; FsMS illumination with intensity of 9200 Lux (9 jars for the 3 species). and fluctuant temperature of 25/ 9. The 54 jars were incubated in the plant 18°C±2 for light and dark periods, growth chamber under the previous respectively. described conditions (of illumination 5. The growth medium (water or soil) on and temperature) and externally which new gametophores were observed weekly for ten weeks. The developed faster was considered more protonemata and gametophores were suitable for the corresponding species. examined microscopically afterwards 6. Newly developed gametophores of B. i.e. after incubation for ten weeks. argenteum and P. hastata grew faster 10. The percentages of survived on water-saturated soil whereas those gametophores i.e. the axenic cultures of B. subapiculatum grew faster in SD (growth without any contamination) H2O. All gametophores were carefully were calculated. excised under aseptic conditions. 7. The excised gametophores were Observations and Discussion gathered, washed twice with SD H2O Cultures in 45 jars out of 54 jars (i.e., while shaking, each time, for 5 min. 83.3 %) were axenic. The percentages of they were then spread on dry sterile survival with the used technique were filter paper. mostly higher in liquid media than on solid media (Table 1). The percentages of 19 Hanan Shaaban et al. survival were 67-100% for B. argenteum, In the present work, when the new (33-100%) for B. subapiculatum and (33- branches were excised and re-cultured on 100%) for P. hastata (Table 1). nutrient media, it was noticed that they grew more successfully than their These results show almost 100 % progenitors. This agrees with results success for the axenic cultivation of obtained by (Rowntree 2006) who gametophores in different types of liquid reported that Ditrichum plumbicola media (NFM, HsMS,FsMS) for the three Crundw. gametophyte grew significantly mosses under investigation. These results better when harvested from recultured are highly promising, compared with the non-axenic cultures than from the wild percentages (5% -70%) of survival of collected material. This might be gametophores reported by some authors interpreted on the basis that pre-culturing e.g., (Sabovljevic et al. 2012, Liang et al. partially acclimatizes plants to culture 2010) using different sterilizing agents. conditions, and reduces the number and These results have been achieved, in the type of contaminants.
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