Jacopo Calevo, Department of Life Sciences and Systems Biology, University of Turin (Viale Mattioli 25, 10125, Turin, Italy); e-mail:
Miriam Bazzicalupo, Department of Earth, Environment and Life Sciences, University of Genoa (Corso Europa 26, 16132, Genoa, Italy).

Reference to article

Calevo J., Bazzicalupo M. 2020. Less is more: low-cost in vitro propagation of an Endangered Italian orchid. Nature Conservation Research 5(Suppl.1): 172–177.

Section Short Communications

Orchids are among the most widely distributed plants but also the most threatened by biotic and abiotic factors. Their interactions with specific symbiotic fungi during germination in natural environments make their propagation challenging for conservation biologists. In this study, in the framework of the European conservation project LIFEorchids, the germination protocol for the endangered Mediterranean species Orchis patens was optimised, and the effect of controlled constant temperature in growth chambers with fluctuations of temperature occurring in natural environments was compared. Seed viability was also evaluated by using a double-staining technique. Minimum, maximum and average daily temperatures were recorded for six months, from July to December, and germination percentages, as well as developmental stages of seeds, were annotated, for both conditions, every month. Even if germination was obtained under both conditions, the effect of temperature fluctuations was evident, by increasing the germination percentage of seeds from about 10% under controlled conditions to 59%. The latter result was almost comparable with seed viability data, indicating a strong impact of temperature fluctuations in breaking seed dormancy. Our results are a confirmation that temperature oscillations play a more important role than the average temperature in seed germination and suggest avoiding a constant temperature to optimise germination protocols for European orchid species.


asymbiotic germination, ex situ conservation, Orchidaceae, Orchis patens, temperature, viability

Artice information

Received: 06.04.2020. Revised: 23.06.2020. Accepted: 11.07.2020.

The full text of the article

Baskin C.C., Baskin J.M. 2001. Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. San Diego: Academic Press. 666 p.
Baskin C.C., Baskin J.M. 2003. When breaking seed dormancy is a problem: try a move-along experiment. Native Plants Journal 4(1): 17–21. DOI: 10.3368/npj.4.1.17
Bilz M., Kell S.P., Maxted N., Lansdown R.V. 2011. European Red List of Vascular Plants. Luxembourg: Publications Office of the European Union. 144 p.
Calevo J., Massa G., Maglio M. 2014. Elenco delle Orchidaceae del Parco Regionale di Portofino. GIROS Notizie 57: 35–39.
Calevo J., Giovannini A., Cornara L., Peccenini S., Monroy F. 2017a. Orchis patens Desf.: seed morphology of an endangered Mediterranean orchid. Plant Biosystems 151(5): 770–774. DOI: 10.1080/11263504.2017.1297335
Calevo J., Monroy F., Peccenini S., Cornara L., Giovannini A. 2017b. First time in vitro asymbiotic germination of Orchis patens Desf.: a preliminary study on an endangered Mediterranean orchid. GIROS Orchidee Spontanee d'Europa 60(1): 94–104.
Calevo J., Giovannini A., Cornara L., Peccenini S. 2017c. Asymbiotic seed germination of hand-pollinated terrestrial orchids. Acta Horticolturae 1155: 415–418. DOI: 10.17660/ActaHortic.2017.1155.61
Calevo J., Voyron S., Portigliatti A., Monroy F., Giovannini A., Cornara L., Giordani P., Girani A., Zocco D., Cristaldi L., Palazzolo P., Colombo S., Marzorati M., Moravec J., Perotto S., Girlanda M. 2018. Orchids for LIFE: the LIFE17NAT/IT/000596 project for the conservation of orchids and their habitat. GIROS Orchidee Spontanee d'Europa 61(2): 277–288.
Calevo J., Voyron S., Ercole E., Girlanda M. 2020. Is the Distribution of Two Rare Orchis Sister Species Limited by Their Main Mycobiont? Diversity 12(7): 262. DOI: 10.3390/d12070262
Darwin C. 1862. On the various contrivances by which British and foreign orchids are fertilized by insects, and on the good effects of intercrossing. London: John Murray. 365 p.
Dixon K.W. 2009. Pollination and restoration. Science 325(5940): 571–573. DOI: 10.1126/science.1176295
Ercole E., Rodda M., Girlanda M., Perotto S. 2015. Establishment of a symbiotic in vitro system between a green meadow orchid and a Rhizoctonia-like fungus. Bioprotocol 5: e1482.
Euro+Med. 2006. Euro+Med PlantBase – the information resource for Euro-Mediterranean plant diversity. Available from
Fay M.F. 1994. In what situations is in vitro culture appropriate to plant conservations? Biodiversity and Conservation 3(2): 176–183. DOI: 10.1007/BF02291887
Girlanda M., Selosse M.A., Cafasso D., Brilli F., Delfine S., Fabbian R., Ghignone S., Pinelli P., Segreto R., Loreto F., Cozzolino S., Perotto S. 2006. Inefficient photosynthesis in the Mediterranean orchid Limodorum abortivum is mirrored by specific association to ectomycorrhizal Russulaceae. Molecular Ecology 15(2): 491–504. DOI: 10.1111/j.1365-294X.2005.02770.x
Han C.Y., Long C.L. 2010. Dormancy, germination and storage of Magnolia ingrata seeds. Seed Science and Technology 38(1): 252–256. DOI: 10.15258/sst.2010.38.1.28
Johnson T., Kane M. 2012. Effects of temperature and light on germination and early seedling development of the pine pink orchid (Bletia purpurea). Plant Species Biology 27(2): 174–179. DOI: 10.1111/j.1442-1984.2011.00347.x
Kretzschmar H., Eccarius W., Dietrich H. 2007. The Orchid Genera Anacamptis, Orchis and Neotinea. Phylogeny, Taxonomy, Morphology, Biology, Distribution, Ecology and Hybridization. Bürgel: EchinoMedia Verlag. 544 p.
Leake J.R. 1994. The biology of myco-heterotrophic ('saprophytic') plants. New Phytologist 127(2): 171–216. DOI: 10.1111/j.1469-8137.1994.tb04272.x
Leitch I.J., Fay M.F. 2008. Plant genome horizons: Michael Bennett's contribution to genome research. Annals of Botany 101(6): 737–746. DOI: 10.1093/aob/mcn045
Magrini S., Barreca D., Zucconi L. 2019a. A rapid double-staining technique to improve seed viability testing in terrestrial orchids. Plant Biosystems 153(6): 877–882. DOI: 10.1080/11263504.2019.1587541
Magrini S., De Vitis M., Torelli D., Santi L., Zucconi L. 2019b. Seed banking of terrestrial orchids: evaluation of seed quality in Anacamptis following 4-year dry storage. Plant Biology 21(3): 544–550. DOI: 10.1111/plb.12936
Malmgren S. 1996. Orchid propagation. Theory and Practice. In: North American Native Terrestrial Orchids. Propagation and Production Conference Proceedings. Germantown: C. Allen. P. 63–71.
Orsenigo S., Bacchetta G., Calevo J., Castello M., Cogoni D., Gennai M., Licht W., Montagnani C., Perrino E.V., Pinna S.M., Silletti G.N., Vela E., Viciani D., Vidali M., Wagensommer R.P., Zappa E., Fenu G. 2016. Global and Regional IUCN Red List Assessments: 1. Italian Botanist 1: 61–85. DOI: 10.3897/italianbotanist.1.8647
Pierce S., Belotti J. 2011. The Conservation of Terrestrial Orchids: from the Alps to the Po Plain of Lombardy. Lombardy: POB and CFA. 89 p.
Poff K.E., Sharma J., Richards M. 2016. Cold-Moist Stratification Improves Germination in a Temperate Terrestrial Orchid. Castanea 81(4): 292–301. DOI: 10.2179/16-098
Rost T.L., Barbour M.G., Stocking C.R., Murphy T.M. 2006. Plant biology. Stamford, Connecticut: Thomson Corporation. 624 p.
Sharma J., Zettler L.W., Van Sambeek J.W., Ellersieck M.R., Starbuck C.J. 2003. Symbiotic seed germination and mycorrhizae of federally threatened Platanthera praeclara (Orchidaceae). American Midland Naturalist 149(1): 104–120. DOI: 10.1674/0003-0031(2003)149[0104:SSGAMO]2.0.CO;2
Stewart S.L., Kane M.E. 2006. Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell, Tissue and Organ Culture 86(2): 147–158. DOI: 10.1007/s11240-006-9098-y
Swarts N.D., Dixon K.W. 2009. Terrestrial orchid conservation in the age of extinction. Annals of Botany 104(3): 543–556. DOI: 10.1093/aob/mcp025
Swarts N.D., Dixon K.W. 2017. Conservation methods for terrestrial orchids. Plantation: J. Ross Publishing. 240 p.
Valletta A., Attorre F., Bruno F., Pasqua G. 2008. In vitro asymbiotic germination of Orchis mascula L. Plant Biosystems 142(3): 653–655. DOI: 10.1080/11263500802411205
Wraith J., Pickering C. 2018. Quantifying anthropogenic threats to orchids using the IUCN Red List. Ambio 47(3): 307–317. DOI: 10.1007/s13280-017-0964-0