ISSN: 1110-7413 Taeckholmia 36 (2016): 17-25 eISSN: 2357-044X

A Simple Method to Obtain Microbial-free in vitro 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, , 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 , 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 (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. Therefore 100% present work, due to the attention paid to success is highly expected, using the two points during the in vitro cultivation: present technique, if the re-culture step is 1. Avoidance of sterilizing the cultured repeated more than once. and the recultured gametophores by chemicals and 2. The use of repeatedly Thus the technique used in the present cleaned gametophyte tissues for work not only avoids probabilities of inoculation on nutrient media. Cleaning gametophores damage (by sterilizing was achieved by washing several times agents and/or microbes) but also produces with SD H20 which is considered a luxurious growth. Because contamination modification of the technique that has was absent in most jars, gametophores been used before by (Chen et al. 2009), had full chance to obtain their required who used chemicals for the sterilization of nutrients from the culture medium and the re-cultured gametophores. grow to form large colonies establishing a good biomass (see figs. 1, 2, 3).

Table (1). Percentages of survival of Bryum argenteum, Bryum subapiculatum and Philonotis hastata in liquid and on solid media: nutrient free medium (NFM), half strength Murashig and skoog (HsMS) medium, full strength Murashig and Skoog (FsMS) medium and nutrient free Agar (NFA) medium.

% of survival Species Liquid media Solid media NFM HsMS FsMS NFA HsMS FsMS

B. argenteum 100% 100% 100% 100% 67% 67% B. subapiculatum 100% 100% 100% 33% 67% 67% P. hastata 100% 100% 33% 100% 67% 100%

20 A Simple Method to Obtain Microbial-free in vitro Moss Cultures

Fig. 1: Axenic cultures of Bryum argentum developed on solid media (NFA, HsMS & FsMS) after incubation for 10 weeks.

Fig. 2: Axenic cultures of Bryum subapiculatum developed on solid media (NFA, HsMS & FsMS) after incubation for 10 weeks.

21

Hanan Shaaban et al.

Fig. 3: Axenic cultures of Philonotis hastata developed on solid media (NFA, HsMS & FsMS) after I ncubation for 10 weeks. Although the changes from distended 2006), the caulonema allows the spore to filamentous protonema to developing plant to colonize on available gametophore buds can require substrate more rapidly (Cuming and increasingly more specialized conditions Cuming 2008). (Duckett et al. 2004, Glime 2007), yet the Protonemata of both B. argenteum and three species studied here exhibited P. hastata started to develop in the 1st normal development on all nutrient media week when cultured on HsMS and FsMS as well as on NFM (solid or liquid) solid and liquid media, which agree with without any spicified requirements. This the results obtained for Physcomitrella might be attributed to the high light patens (Hedw.) Bruch & Schimp. (Decker intensity used in the plant growth and Reski 2004), B. argenteum (Liang et chamber (9200 lux). It may be mentioned, al. 2010), and Entosthodon hungaricus in this context, that high light intensity (Boros) Loeske (Sabovljevic et al. 2012). and temperature promoted branching of The present results, therefore, reflect the caulonema, proliferated potential bud success of the developed modified sites and provided a thicker mat in technique. Hymenostylium, Campylopus and Physcomitrella (Glime, B.C. Knoop On the other hand, the development of 1986, Mehta 1988). B. subapiculatum protonema was in the 1st week only when cultured on HsMS solid Gametophores were observed earlier medium but delayed to the 2nd week with on NFA solid medium than with the other HsMS liquid medium and on FsMS solid nutrient media, especially in case of B. and liquid media. Comparing growth rates argenteum. This might be attributed to of the three mosses in response to various that developed protonemata were mainly nutrient concentrations showed that caulonemata filaments in NFA solid HsMS solid and liquid media were better medium. As caulonema grows more for the growth than other media. It is rapidly than chloronema (Cove et al. known that moss species prefer dilute 22

A Simple Method to Obtain Microbial-free in vitro Moss Cultures culture solutions to concentrated ones Beike, A.K. , Decker, E.L., Frank, W., (Awasthi et al. 2010). The concentrated Lang, D. , Scheebaum, M.V, Zimmer, media may even cause poor growth A.D. and Reski,R. 2010. Applied (Awasthi et al. 2012). Bryology– Bryotechnology, Tropical Bryology 31: 22-32. Thus, the modified technique developed in the present work for in vitro Cano, M.J., Ros, R.M. and Guerra, J. cultivation of B. argenteum, B. 1996. In vitro culture of Pterigoneurum subapiculatum and P. hastata is an easy, compactum (Musci, Pottiaceae): control inexpensive and successful technique of taxonomical characters. , Cryptogamie which in worth extension for propagation, Bryologie 17: 67-70. conservation and molecular studies of Carey, S.B. , Payton, A.C. and other species of mosses. McDaniel, S.F. 2015. A method for References eliminating bacterial contamination from in vitro moss cultures, Applications in Awasthi, V. Nath,V. and Asthana, A.K. plant sciences. 2010. In vitro study on micropropagation and reproductive behaviour of moss Chen, Y.-Y. , Lou, Y.-X., Guo, S.-L and Bryoerythrophyllum recurvirostrum Cao, T. 2009. Successful Tissue Culture (Hedw.) Chen. The International Journal of the Medicinal Moss Rhodobryum of Plant Reproductive Biology, 2: 31-37. giganteum and Factors Influencing Proliferation of Its Protonemata, Annales Awasthi, V., Nath, V. , Pande, N. and Botanici Fennici 46 :516-524. Asthana, A.K.. 2012. Morphogenetic Studies and In vitro Propagation of Two Cove, D., Bezanilla, M. , Harries,P. and Mosses: Philonotis thwaitesii Mitt. and Quatrano, R. 2006. Mosses as model Brachythecium plumosum (Hedw.) systems for the study of metabolism and B.S.G., Taiwania, 57: 27-36. development, Annual Review of plant biology 57: 497-520. Basile, D.V. 1972. Method for Surface- Sterilizing Small Plant Parts. , Bulletin of Cuming, A.C. and Cuming, A.C. 2008. the Torrey Botanical Club 99 : 313-316. Mosses as model organisms for developmental, cellular, and molecular Basile, D.V. , Basile, M.R. 1988. biologyBryophyte Biology, Cambridge Procedures used for the axenic culture University Press. and experimental treatment of bryophytes. , in: G. J.M. (ed.) Methods in Decker,E.L. and Reski,R. 2004. The Bryology: Proceedings of the bryological moss . Current opinion in plant methods workshop, The Journal of the biology 7: 166-170. Hattori Botanical laboratory, Mainz., pp. Duckett, J.G., Burch, J., Fletcher, 1–16. P.W., Matcham, H.W., Read, D.J., Becquerel, P. 1906. Germination des Russell, A.J. and Pressel, S. 2004. In spores d’ Atrichum undulatum et d’ vitro cultivation of bryophytes: a review Hypnum velutinum. Nutrition et of practicalities, problems, progress and developpement de leurs protonema dans promise. Journal of Bryology 26 : 3-20. des milieux sterilises. Revue générale de Glime, J.M. 2007. Bryophyte Ecology. Botanique 18: 49–67. Michigan Technological University and 23

Hanan Shaaban et al. the International Association of Niedz, R.P. and Bausher, M.G. 2002. Bryologists. Control of in vitro contamination of explants from greenhouse-and field- Glime, J.M. 2013. Sexual Strategies. in: grown trees, In Vitro Cellular & J.M. Glime (ed.) Bryophyte Ecology. Developmental Biology-Plant 38: 468- Michigan Technological University and 471. the International Association of Bryologists. Peck, J.E., Muir, P.S. 2001. Harvestable epiphytic bryophytes and their Glime, J.M. and Knoop, B.C. 1986. accumulation in central western Oregon. Spore germination and protonemal The Bryologist 104: 181-190. development of Fontinalis squamosa. The Journal of the Hattori Botanical Reski, R. 1998. Development, genetics laboratory 61: 487-497. and molecular biology of mosses. Botanica Acta 111: 1-15. Hedenäs, L. and Bisang, I. 2011. The overlooked dwarf males in mosses— Rowntree, J. 2006. Development of unique among green land plants, novel methods for the initiation of in vitro Perspectives in plant ecology. Evolution bryophyte cultures for conservation, Plant and Systematics, 13: 121-135. cell, tissue and organ culture 87:191-201. Hohe, A. and Reski, R. 2005. From Rowntree, J. and Ramsay, M. 2009. axenic spore germination to molecular How bryophytes came out of the cold: farming. Plant cell reports 23: 513-521. successful cryopreservation of threatened species. Biodiversity and Conservation Lal, M. 1984. The culture of bryophytes 18: 1413-1420. including apogamy, apos- pory, parthenogenesis and protoplasts. , in: Rowntree, J.K., Pressel, S., Ramsay, A.F. Dyer, J.G. Duckett (Eds.) The M.M., Sabovljevic, A. and M. Experimental Biology of Bryophytes. , Sabovljevic. 2011. In vitro conservation Academic Press., London., pp. 97-115. of European bryophytes, In Vitro Cellular & Developmental Biology-Plant, 47: 55- Liang, S.F., Sun, Y. and Zhu, R.-L. 64. 2010. In vitro micropropagation of Bryum argenteum Hedw. Crypt Bryol 31: 233- Sabovljevic, M.S., Papp, B. , 239. Sabovljević, A., Vujičić, M., Szurdoki, E. and J.G. Segarra-Moragues 2012. In Mehta, P. 1988. In vitro studies on spore vitro micropropagation of rare and germination, protonemal differentiation endangered moss Entosthodon and bud formation in three mosses grown hungaricus (Funariaceae) = in vitro. The Journal of the Hattori Micropropagação in vitro do musgo raro Botanical laboratory 64:401-410. Entosthodon hungaricus, ameaçado de Parkinson, M., Prendergast, M. and extinção (Funariaceae). Bioscience Sayegh, A. 1996. Sterilisation of explants Journal, 28. and cultures with sodium Sarasan, V., Cripps, R., Ramsay, dichloroisocyanurate, Plant Growth M.M., Atherton, C., McMichen, M., Regulation, 20: 61-66. Prendergast, G. and J.K. Rowntree 2006. Conservation in vitro of threatened 24

A Simple Method to Obtain Microbial-free in vitro Moss Cultures plants—progress in the past decade, In Vitro Cellular & Developmental Biology- Plant 42: 206. Victoria, F.C., Oliveira, A.C. and Peters, J.A. 2011. Establishment of the moss Polytrichum juniperinum Hedw. under axenic conditions. Bioscience Journal, Uberlandia 27:673-676. Vö chting, H. 1885. Ü ber die Regeneration der Marchantiaceen. , Jb Wiss Bot 16 : 367–414. Vujičić, M., Sabovljević, A. and M. Sabovljević. 2009. Axenically culturing the bryophytes: a case study of the moss Dicranum scoparium Hedw. Dicranaceae, Bryophyta. Botanica Serbica 33: 137-1.

25