Main Article Content
Abstract
The establishment of axenic cultures and the propagation of the rare moss Entosthodon pulchellus (H. Philip.) Brugués (Funariaceae), as well as the optimisation of its ex situ growth conditions and rapid biomass production were the goals of this study. The results obtained also provide insights into the developmental biology of this moss in in vitro conditions, particularly regarding basal media contents with or without supplements of selected plant growth regulators or sugars, i.e. its nutritional needs. The procedure for establishing axenic cultures of this rare and threatened moss species is described. The optimisation protocol of the growth and production conditions is elaborated. The spore germinability of dried herbarium samples remained rather high in the tested laboratory conditions. Suitability tests on different media types showed KNOP basal media to be the best fit for the rapid biomass production of both secondary protonemata and leafy gametophores, without the addition of sugars or plant growth regulators. However, sugar, namely sucrose, induced rapid and massive protonemal development and can be used when this developmental stage is needed.
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Copyright (c) 2024 Marija Cosic, Djordje Bozovic, Michael Ignatov, Elena Ignatov, Milorad Vujicic, Aneta Sabovljevic, Marko Sabovljevic

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References
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- Ashton, N. W., Cove, D. J., 1990: Mutants as tools for the analytical dissection of cell differentiation in Physcomitrella patens gametophytes. In: Chopra, R. N., Bahtla, S. C. (eds.), Bryophyte development: physiology and biochemistry, 17–31. CRC Press, Boca Raton, FL.
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- Bopp, M., 1963: Development of the protonema and bud formation in mosses. Botanical Journal of Linnean Society 58, 305–309. https://doi.org/10.1111/j.1095-8339.1990.tb00899.x
- Ćosić, M.V., Sabovljević, M.S., Papp, B., Giba, Z.S., Šinžar-Sekulić, J.B., Sabovljević, A.D., Vujičić, M.M., 2022: Micropropagation of rare bryo-halophyte Hennediella heimii. Botanica Serbica 46,. 187–195. https://doi.org/10.2298/BOTSERB2202187C.
- Cvetić, T., Sabovljević, A., Sabovljević, M., Grubišić, D., 2007: Development of the moss Pogonatum urnigerum (Hedw.) P. Beauv. under in vitro culture conditions. Archives of Biological Sciences 59(1), 57–61. https://doi.org/10.2298/ABS0701057C
- Hallingbäck, T., Tan, B., 2010: Past and present activities and future strategy of bryophyte conservation. Phytotaxa 9, 266–274. https://doi.org/10.11646/phytotaxa.9.1.15.
- Hassid, W.Z., Putman, E.W., 1950: Transformation of sugars in plants. Annual Review of Plant Biology 1, 109–124. https://doi.org/10.1146/annurev.pp.01.060150.000545
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- Jadranin, B. Z., Ćosić, M. V., Božović, Đ. P., Vujičić, M. M., Ignatov, M. S., Ignatova, E. A., Sabovljević, A. D., Sabovljević, M. S., 2023: An insight into the biology of the rare and peculiar moss Pterygoneurum sibiricum (Pottiaceae): A conservation physiology approach. Plants 12(6), 1359. https://doi.org/10.3390/plants12061359
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- Klavina, L., 2014: Polysaccharides from lower plants: Bryophytes. In: Ramawat, K. G., Mérillon, J. M. (eds.), Polysaccharides, 146–158. Springer, Berlin, Germany.
- Löbel, S., Mair, L., Lönnell, N., Schröder, B., Snäll, T., 2018: Biological traits explain bryophyte species distributions and responses to forest fragmentation and climatic variation. Journal of Ecology 106(4): 1700-1713. https://doi.org/10.1111/1365-2745.12930
- Martinčič, A. 2024: New checklist and the red list of the mosses (Bryophyta) of Slovenia. Hacquetia 23(1), 69–118. https://doi.org/10.2478-hacq-2023-0006
- Mitra, G., Allsopp, A., 1959: Effects of kinetin, gibberellic acid and certain auxins on the development of shoot buds on the protonema of Pohlia nutans. Nature 183, 974–975. https://doi.org/10.1038/183974b0
- Ostendorf, A. K., van Gessel, N., Malkowsky, Y., Sabovljevic, M. S., Rensing, S. A., Roth-Nebelsick, A., Reski, R., 2021: Poliploidization within the Funariaceae – a key principle behind speciation, sporophyte reduction and the high variance of spore diameters? Bryophyte diversity and evolution 43(1), 164–179. https://doi.org/10.11646/bde.43.1.13
- R Core Team, R. A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2022. Available online: https://www.R-project.org/ (accessed on 25 December 2022).
- Reski, R., Abel, W. O., 1985: Induction of budding on chloronemata and caulonemata of the moss Physcomitrella patens, using isopentenyladenine. Planta 165, 354–358. https://doi.org/10.1007/BF00392232
- Rowntree, J.K., Pressel, S., Ramsay, M.M., Sabovljevic, A. & Sabovljevic, M., 2011: In vitro conservation of European bryophytes. In Vitro Cellular and Developmental Biology – Plant 47(1), 55–64. https://doi.org/10.1007/s11627-010-9326-3
- Sabovljevic, M., Vujicic, M., Pantovic, J., Sabovljevic, A., 2014: Bryophyte conservation biology: in vitro approach to the ex situ conservation of bryophytes from Europe. Plant Biosystems 148(4), 857–868. https://doi.org/10.1080/11263504.2014.949328
- Sabovljević, A., Sabovljevic, M., Grubisic, D., Konjevic, R., 2005: The effect of sugars on development of two moss species (Bryum argenteum and Atrichum undulatum) during in vitro culture. Belgian Journal of Botany 138, 79–84. https://doi.org/10.2307/20794569
- Sabovljević, A., Sabovljević, M., Jocković, N., 2009: In vitro culture and secondary metabolite isolation in bryophytes. In: Jain, S. M., Saxena, P.K. (eds.), Methods in molecular biology, protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, vol. 547, 117–128. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-287-2_10
- Sabovljević, M., Papp, B., Sabovljević, A., Vujičić, M., Szurdoki, E., Segarra-Moragues, J. G., 2012a: In vitro micropropagation of rare and endangered moss Entosthodon hungaricus (Funariaceae). Bioscience Journal 28, 632–640.
- Sabovljević, M., Vujičić, M., Šinžar-Sekulić, J., Segarra-Moragues, J. G., Bapp, B., Skorić, M., Dragačević, L., Sabovljević, A., 2012b: Reviving, in vitro differentiation, development and micropropagation of the rare and endangered moss Bruchia vogesiaca (Bruchiaceae). HortScience 47, 1347–1350. https://doi.org/10.21273/HORTSCI.47.9.1347
- Sabovljević, M. S., Vujičić, M. M., Sabovljević, A. D., 2014: Plant growth regulators in bryophytes. Botanica Serbica 38(1), 99–107.
- Sabovljević, M. S., Ćosić, M. V., Jadranin, B. Z., Pantović, J. P., Giba, Z. S., Vujičić, M. M., Sabovljević, A. D., 2022: The conservation physiology of bryophytes. Plants 11(10), 1282. https://doi.org/10.3390/plants11101282
- Sakakibara, K., Nishiyama, T., Sumikawa, N., Kofuji, R., Murata, T., Hasebe, M., 2003: Involvement of auxin and a homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens. Development 130, 4835–4846. https://doi.org/10.1242/dev.00644
- Sarla, Chopra, R. N., 1987: Effect of some auxins and antiauxins on protonemal growth and bud formation in Bryum pallescens Schleich. ex Schwaegr. grown in vitro. Plant Science 51, 251–256. https://doi.org/10.1016/0168-9452(87)90200-7
- Singh, V. J., Gupta, D., Sahu, V., Asthana A., K., 2017: In vitro growth pattern of moss Drummondia stricta (Mitt.) Müll. Hal. (Orthotrichaceae) in different hormonal concentrations. Recent Advances in Botanical Science 1, 1–7. https://doi.org/10.2174/9789811433788120010005
- von Schwartzenberg, K., 2009: Hormonal regulation of development by auxin and cytokinin in moss. In: Knight, C., Perroud, P.-F., Cove, D. (eds.), The Moss Physcomitrella patens, Volume 36, pp. 246–281. Wiley Online Library: Hoboken, NJ, USA.
- Vujičić, M., Sabovljević, A., Sabovljević, M. 2009: Axenically culturing the bryophytes: a case study of the moss Dicranum scoparium Hedw. (Dicranaceae, Bryophyta). Botanica Serbica 33(2), 137–140.
- Vujičić, M., Sabovljević, A., Sabovljević, M., 2011: Axenically culturing the bryophytes: Establishment and propagation of the moss Hypnum cupressiforme Hedw. (Bryophyta, Hypnaceae) in in vitro conditions. Botanica Serbica 35, 71–77.
- Vujičić, M. M., Sabovljević, A. D., Šinžar-Sekulić, J., Skorić, M., Sabovljević, M., 2012: In vitro development of the rare and endangered moss Molendoa hornschuchiana (Hook.) Lindb. ex Limpr. (Pottiaceae, Bryophyta). Hort Science 47, 84–87. https://doi.org/10.21273/HORTSCI.47.1.84
- Zanatta, F., Engler, R., Collart, F., Broennimann, O., Mateo, R.G., Papp, B., Munoz, J., Baurain, D., Guisan, A., Vanderpoorten, A., 2020: Bryophytes are predicted to lag behind future climate change despite their high dispersal capacities. Nature Communications 11, 5601. https://doi.org/10.1038/s41467-020-19410-8
References
Ashton, N. W., Grimsley, N. H., Cove, D. J., 1979: Analysis of gametophyte development in the moss Physcomitrella patens, using auxin and cytokinin resistant mutants. Planta 144, 427–435. https://doi.org/10.1007/BF00380118
Ashton, N. W., Cove, D. J., 1990: Mutants as tools for the analytical dissection of cell differentiation in Physcomitrella patens gametophytes. In: Chopra, R. N., Bahtla, S. C. (eds.), Bryophyte development: physiology and biochemistry, 17–31. CRC Press, Boca Raton, FL.
Bijelović, A., Sabovljević, M., Grubišić, D., Konjević, R., 2004: Phytohormone influence on the morphogenesis of two mosses Bryum argenteum Hedw. and Atrichum undulatum (Hedw.) P. Beauv. Israel Journal of Plant Sciences 52, 31–36. https://doi.org/10.1560/RX30-U7QH-DEMQ-GL7P
Bopp, M., 1963: Development of the protonema and bud formation in mosses. Botanical Journal of Linnean Society 58, 305–309. https://doi.org/10.1111/j.1095-8339.1990.tb00899.x
Ćosić, M.V., Sabovljević, M.S., Papp, B., Giba, Z.S., Šinžar-Sekulić, J.B., Sabovljević, A.D., Vujičić, M.M., 2022: Micropropagation of rare bryo-halophyte Hennediella heimii. Botanica Serbica 46,. 187–195. https://doi.org/10.2298/BOTSERB2202187C.
Cvetić, T., Sabovljević, A., Sabovljević, M., Grubišić, D., 2007: Development of the moss Pogonatum urnigerum (Hedw.) P. Beauv. under in vitro culture conditions. Archives of Biological Sciences 59(1), 57–61. https://doi.org/10.2298/ABS0701057C
Hallingbäck, T., Tan, B., 2010: Past and present activities and future strategy of bryophyte conservation. Phytotaxa 9, 266–274. https://doi.org/10.11646/phytotaxa.9.1.15.
Hassid, W.Z., Putman, E.W., 1950: Transformation of sugars in plants. Annual Review of Plant Biology 1, 109–124. https://doi.org/10.1146/annurev.pp.01.060150.000545
Hodgetts, N., Calix, M., Englefield, E., Fettes, N., Garcia Criado, M., Patin, L., Nieto, A., Bergamini, A., Bisang, I., Baisheva, E., Campisi, P., Cogoni, A., Hallingback, T., Konstantinova, N., Lockhart, N., Sabovljevic, M., Schnyder, N., Schrock, C., Sergio, C., Sim Sim, M., Vrba, J., Ferreira, C.C., Afonina, O., Blockeel, T., Blom, H., Caspari, S., Gabriel, R., Garcia, C., Garilleti, R., Gonzalez Mancebo, J., Goldberg, I., Hedenas, L., Holyoak, D., Hugonnot, V., Huttunen, S., Ignatov, M., Ignatova, E., Infante, M., Juutinen, R., Kiebacher, T., Kockinger, H., Kučera, J., Lonnell, N., Luth, M., Martins, A., Maslovsky, O., Papp, B., Porley, R., Rothero, G., Soderstrom, L., Ştefǎnuţ, S., Syrjanen, K., Untereiner, A., Vaňa, J., Vanderpoorten, A., Vellak, K., Aleffi M., Bates, J., Bell, N., Brugues, M., Cronberg, N., Denyer, J., Duckett, J., During, H.J., Enroth, J., Fedosov, V., Flatberg, K.-I., Ganeva, A., Gorski, P., Gunnarsson, U., Hassel, K., Hespanhol, H., Hill, M., Hodd, R., Hylander, K., Ingerpuu, N., Laaka-Lindberg, S., Lara, F., Mazimpaka, V., Mežaka, A., Muller, F., Orgaz, J.D., Patino, J., Pilkington, S., Puche, F., Ros, R.M., Rumsey, F., Segarra-Moragues, J.G., Seneca, A., Stebel, A., Virtanen, R., Weibull, H., Wilbraham, J., Żarnowiec, J., 2019a: A miniature world in decline: European Red List of Mosses, Liverworts and Hornworts. Brussels, Belgium: IUCN. ISBN 978-2-8317-1993-1 (PDF), 978-2-8317-1994-8 (print). https://doi.org/10.2305/IUCN.CH.2019.ERL.2.en
Hodgetts, N., Blockeel, T., Konstantinova, N., Papp, B., Schnyder, N., Schröck, C., Sergio, C. & Untereiner, A., 2019b: Entosthodon pulchellus (Europe assessment). The IUCN Red List of Threatened Species 2019: e.T85841585A87794990. Accessed on 18 May 2024.
Jadranin, B. Z., Ćosić, M. V., Božović, Đ. P., Vujičić, M. M., Ignatov, M. S., Ignatova, E. A., Sabovljević, A. D., Sabovljević, M. S., 2023: An insight into the biology of the rare and peculiar moss Pterygoneurum sibiricum (Pottiaceae): A conservation physiology approach. Plants 12(6), 1359. https://doi.org/10.3390/plants12061359
Jin, C., Huixia, K., Shubin, D., 2020: Population genetic structure and gene flow of rare and endangered Tetraena mongolica Maxim. reveald by reduced representation sequencing. BMC Plant Biology 20: 391. https://doi.org/10.1186/s12870-020-02594-y
Klavina, L., 2014: Polysaccharides from lower plants: Bryophytes. In: Ramawat, K. G., Mérillon, J. M. (eds.), Polysaccharides, 146–158. Springer, Berlin, Germany.
Löbel, S., Mair, L., Lönnell, N., Schröder, B., Snäll, T., 2018: Biological traits explain bryophyte species distributions and responses to forest fragmentation and climatic variation. Journal of Ecology 106(4): 1700-1713. https://doi.org/10.1111/1365-2745.12930
Martinčič, A. 2024: New checklist and the red list of the mosses (Bryophyta) of Slovenia. Hacquetia 23(1), 69–118. https://doi.org/10.2478-hacq-2023-0006
Mitra, G., Allsopp, A., 1959: Effects of kinetin, gibberellic acid and certain auxins on the development of shoot buds on the protonema of Pohlia nutans. Nature 183, 974–975. https://doi.org/10.1038/183974b0
Ostendorf, A. K., van Gessel, N., Malkowsky, Y., Sabovljevic, M. S., Rensing, S. A., Roth-Nebelsick, A., Reski, R., 2021: Poliploidization within the Funariaceae – a key principle behind speciation, sporophyte reduction and the high variance of spore diameters? Bryophyte diversity and evolution 43(1), 164–179. https://doi.org/10.11646/bde.43.1.13
R Core Team, R. A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2022. Available online: https://www.R-project.org/ (accessed on 25 December 2022).
Reski, R., Abel, W. O., 1985: Induction of budding on chloronemata and caulonemata of the moss Physcomitrella patens, using isopentenyladenine. Planta 165, 354–358. https://doi.org/10.1007/BF00392232
Rowntree, J.K., Pressel, S., Ramsay, M.M., Sabovljevic, A. & Sabovljevic, M., 2011: In vitro conservation of European bryophytes. In Vitro Cellular and Developmental Biology – Plant 47(1), 55–64. https://doi.org/10.1007/s11627-010-9326-3
Sabovljevic, M., Vujicic, M., Pantovic, J., Sabovljevic, A., 2014: Bryophyte conservation biology: in vitro approach to the ex situ conservation of bryophytes from Europe. Plant Biosystems 148(4), 857–868. https://doi.org/10.1080/11263504.2014.949328
Sabovljević, A., Sabovljevic, M., Grubisic, D., Konjevic, R., 2005: The effect of sugars on development of two moss species (Bryum argenteum and Atrichum undulatum) during in vitro culture. Belgian Journal of Botany 138, 79–84. https://doi.org/10.2307/20794569
Sabovljević, A., Sabovljević, M., Jocković, N., 2009: In vitro culture and secondary metabolite isolation in bryophytes. In: Jain, S. M., Saxena, P.K. (eds.), Methods in molecular biology, protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, vol. 547, 117–128. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-287-2_10
Sabovljević, M., Papp, B., Sabovljević, A., Vujičić, M., Szurdoki, E., Segarra-Moragues, J. G., 2012a: In vitro micropropagation of rare and endangered moss Entosthodon hungaricus (Funariaceae). Bioscience Journal 28, 632–640.
Sabovljević, M., Vujičić, M., Šinžar-Sekulić, J., Segarra-Moragues, J. G., Bapp, B., Skorić, M., Dragačević, L., Sabovljević, A., 2012b: Reviving, in vitro differentiation, development and micropropagation of the rare and endangered moss Bruchia vogesiaca (Bruchiaceae). HortScience 47, 1347–1350. https://doi.org/10.21273/HORTSCI.47.9.1347
Sabovljević, M. S., Vujičić, M. M., Sabovljević, A. D., 2014: Plant growth regulators in bryophytes. Botanica Serbica 38(1), 99–107.
Sabovljević, M. S., Ćosić, M. V., Jadranin, B. Z., Pantović, J. P., Giba, Z. S., Vujičić, M. M., Sabovljević, A. D., 2022: The conservation physiology of bryophytes. Plants 11(10), 1282. https://doi.org/10.3390/plants11101282
Sakakibara, K., Nishiyama, T., Sumikawa, N., Kofuji, R., Murata, T., Hasebe, M., 2003: Involvement of auxin and a homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens. Development 130, 4835–4846. https://doi.org/10.1242/dev.00644
Sarla, Chopra, R. N., 1987: Effect of some auxins and antiauxins on protonemal growth and bud formation in Bryum pallescens Schleich. ex Schwaegr. grown in vitro. Plant Science 51, 251–256. https://doi.org/10.1016/0168-9452(87)90200-7
Singh, V. J., Gupta, D., Sahu, V., Asthana A., K., 2017: In vitro growth pattern of moss Drummondia stricta (Mitt.) Müll. Hal. (Orthotrichaceae) in different hormonal concentrations. Recent Advances in Botanical Science 1, 1–7. https://doi.org/10.2174/9789811433788120010005
von Schwartzenberg, K., 2009: Hormonal regulation of development by auxin and cytokinin in moss. In: Knight, C., Perroud, P.-F., Cove, D. (eds.), The Moss Physcomitrella patens, Volume 36, pp. 246–281. Wiley Online Library: Hoboken, NJ, USA.
Vujičić, M., Sabovljević, A., Sabovljević, M. 2009: Axenically culturing the bryophytes: a case study of the moss Dicranum scoparium Hedw. (Dicranaceae, Bryophyta). Botanica Serbica 33(2), 137–140.
Vujičić, M., Sabovljević, A., Sabovljević, M., 2011: Axenically culturing the bryophytes: Establishment and propagation of the moss Hypnum cupressiforme Hedw. (Bryophyta, Hypnaceae) in in vitro conditions. Botanica Serbica 35, 71–77.
Vujičić, M. M., Sabovljević, A. D., Šinžar-Sekulić, J., Skorić, M., Sabovljević, M., 2012: In vitro development of the rare and endangered moss Molendoa hornschuchiana (Hook.) Lindb. ex Limpr. (Pottiaceae, Bryophyta). Hort Science 47, 84–87. https://doi.org/10.21273/HORTSCI.47.1.84
Zanatta, F., Engler, R., Collart, F., Broennimann, O., Mateo, R.G., Papp, B., Munoz, J., Baurain, D., Guisan, A., Vanderpoorten, A., 2020: Bryophytes are predicted to lag behind future climate change despite their high dispersal capacities. Nature Communications 11, 5601. https://doi.org/10.1038/s41467-020-19410-8