In vitro seed propagation constraints of Mexican lady’s slipper orchid, Pichouaxtle, Cypripedium irapeanum.
DOI:
https://doi.org/10.15517/895ark56Keywords:
Ceratobasidiaceae, Epulorhiza, mycorrhizal incompatibility, orchid mycorrhiza, symbiotic germination, TulasnellaceaeAbstract
Cypripedium irapeanum known in Mexico as "Pichohuaxtle" in Nahuatl, is the most emblematic Mexican lady’s slipper orchid and one of the most studied cypripedioid species in Mesoamerica. Despite its protected status in Mexico, habitat loss driven by land-use change has severely reduced its populations. Ex situ conservation approaches are urgently needed to safeguard its genetic diversity, yet seed germination in the genus remains challenging due to innate dormancy and specific nutritional requirements. We collected seeds from two Mexican ecosystems where this species is distributed —Quercus and Tropical Dry Forests— and isolated mycorrhizal fungi from adult plants to examine both symbiotic and asymbiotic cultures. We found limitations in seed production due to flower ripening, ineffective pollination, or parasitism. Furthermore, germination was achieved after a cold treatment, and the best medium to obtain seedlings in vitro was Norstog medium. Two mycorrhizal fungi, isolated from adult plants belonging to the family Tulasnellaceae, formed a clade with previous isolates from American slipper orchids. One of these isolates promoted germination but later produced fungal incompatibility in vitro. Our study contributes to clarify previously undescribed processes in the biology of this highly appreciated and vulnerable species, supporting future efforts to propagate it from seed and obtain successful cultures.
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Alejo-Viderique, A., Ortega-Larrocea, M. P., Rojas-Oropeza, M. & Cabirol, N. (2025). Mycorrhizal fungi and Fusarium species associated with vanilla in traditional management systems in Papantla, Veracruz, Mexico. Botanical Sciences, 103(3), 776–793. https://doi.org/10.17129/botsci.3704
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389–3402. https://doi.org/10.1093/nar/25.17.3389
Barsberg, S., Rasmussen, H. N. & Kodah, N. (2013). Composition of Cypripedium calceolus (Orchidaceae) seeds analyzed by attenuated total reflectance IR spectroscopy: In search of understanding longevity in the ground. American Journal of Botany, 100(10), 2066–2073. https://doi.org/10.3732/ajb.1200646
Bell, J., Yokoya, K., Kendon, J. P. & Sarasan, V. (2020). Diversity of root-associated culturable fungi of Cephalanthera rubra (Orchidaceae) in relation to soil characteristics. PeerJ, 8, e8695. https://doi.org/10.7717/peerj.8695
Bidartondo, M. I. & Read, D. J. (2008). Fungal specificity bottlenecks during orchid germination and development. Molecular Ecology, 17(16), 3707–3716. https://doi.org/10.1111/j.1365-294X.2008.03848.x
Clements, M. A., Muir, H. & Cribb, P. J. (1986). A Preliminary Report on the Symbiotic Germination of European Terrestrial Orchids. Kew Bulletin, 41(2), 437. https://doi.org/10.2307/4102957
Colin Rivera, J. & Ortega-Larrocea, M. P. (2012). Colonización micorrízica de Cypripedium irapeanum La Llave & Lex. (Orchidaceae) en un bosque de Quercus. In: M. de los A. Tellez-Velasco (Ed.), Conservacion de orquideas en Mexico (pp. 186–192). México, Instituto de Biología, UNAM.
Curtis, J. T. (1943). Germination and Seedling Development in Five Species of Cypripedium L. American Journal of Botany, 30(3), 199–206.
De Pauw, M. A. & Remphrey, W. R. (1993). In vitro germination of three Cypripedium species in relation to time of seed collection, media, and cold treatment. Canadian Journal of Botany, 71, 879–885. DOI 71. 879-885. 10.1139/b93-100
Faleiro, F. V., Nemésio, A. & Loyola, R. (2018). Climate change likely to reduce orchid bee abundance even in climatic suitable sites. Global Change Biology, 24(6), 2272–2283. https://doi.org/10.1111/gcb.14112
Fay, M. F., Feustel, M., Newlands, C. & Gebauer, G. (2018). Inferring the mycorrhizal status of introduced plants of Cypripedium calceolus (Orchidaceae) in northern England using stable isotope analysis. Botanical Journal of the Linnean Society, 186(4), 587–590. https://doi.org/10.1093/botlinnean/box104
Gardes, M. & Bruns, T. (1993). ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology, 2, 113–118.
Givnish, T., Spalink, D., Lyon, S., Hunter, S., Zuluaga, A., Iles, W., Clements, M., Kalin, M., Leebens-Mack, J., Endara, L.,Kriebel, R., Neubig, K., Whitten, W., Williams, N. & Cameron, K. (2015). Orchid phylogenomics and multiple drivers of their extraordinary diversification. Proceedings. Biological sciences / The Royal Society, 282. 10.1098/rspb.2015.1553.
Guindon, S., Dufayard, J. F. F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology, 59(3), 307–321. https://doi.org/10.1093/sysbio/syq010
Guo, Y. Y., Luo, Y. B., Liu, Z. J. & Wang, X. Q. (2012). Evolution and biogeography of the slipper orchids: Eocene vicariance of the conduplicate genera in the old and new world tropics. PLoS ONE, 7(6). https://doi.org/10.1371/journal.pone.0038788
Hernández-Apolinar, M., Gutiérrez-Paredes, C. C., Sánchez-Gallen, I., Aguirre, E. & Pérez-García, E. A. (2012). Ecological aspects of Cypripedium irapeanum La Llave & Lex., an endangered Mexican orchid species. The Slipper Orchid Alliance Newsletter, 13(4), 1–5.
Hsu, R. C. & Lee, Y. I. (2012). Seed development of Cypripedium debile Rchb. f. in relation to asymbiotic germination. Hortscience, 47(10),1495–1498.
Huh, Y. S., Lee, J. K., Nam, S. Y., Hong, E. Y., Paek, K. Y. & Son, S. W. (2016a). Effects of altering medium strength and sucrose concentration on in vitro germination and seedling growth of Cypripedium macranthos Sw. Journal of Plant Biotechnology, 43, 132–137. DOI: http://dx.doi.org/10.5010/JPB.2016.43.1.132
Huh, Y. S., Lee, J. K., Nam, S. Y., Paek, K. Y. & Suh, G. U. (2016b). Improvement of asymbiotic seed germination and seedling development of Cypripedium macranthos Sw. with organic additives. Journal of Plant Biotechnology, 43, 138–145. DOI: http://dx.doi.org/10.5010/JPB.2016.43.1.138
Jacquemyn, H., Deja, A., de hert, K., Cachapa Bailarote, B. & Lievens, B. (2012). Variation in mycorrhizal associations with tulasnelloid fungi among populations of five Dactylorhiza species. PLoS ONE, 7(8), e42212. https://doi.org/10.1371/journal.pone.0042212
Jiang, H., Chen, M. C. & Lee, Y. I. (2017). In vitro germination and low-temperature seed storage of Cypripedium lentiginosum P. J. Cribb & S. C. Chen, a rare and endangered lady’s slipper orchid. Scientia Horticulturae, 225, 471–479. http://dx.doi.org/10.1016/j.scienta.2017.07.040
Katoh, K., Misawa, K., Kuma, K. I. & Miyata, T. (2002). MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transformation. Nucleic Acids Research, 30(14), 3059–3066. https://doi.org/10.1093/nar/gkf436
Katoh, K. & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772–780. https://doi.org/10.1093/molbev/mst010
Kaur, S. (2023). In vitro asymbiotic propagation of the vulnerable slipper orchid Cypripedium cordigerum D. Don. European Journal of Environmental Sciences, 13(82), 90–95. https://doi.org/10.14712/23361964.2023.10
Lee, Y., Lee, N., Yeung, E. & Chung, M. C. (2005). Embryo Development of Cypripedium formosanum in Relation to Seed Germination In Vitro. Journal of the American Society for Horticultural Science, 130(5), 747–753. DOI:10.21273/JASHS.130.5.747
Li, J., Liu, Z., Salazar, G. A., Bernhardt, P., Perner, H., Tomohisa, Y., Jin, X., Chung, S. & Luo, Y. (2011). Molecular phylogeny of Cypripedium (Orchidaceae: Cypripedioideae) inferred from multiple nuclear and chloroplast regions. Molecular Phylogenetics and Evolution, 61(2), 308–320. https://doi.org/10.1016/j.ympev.2011.06.006
Liu, H., Jacquemyn, H., He, X., Chen, W., Huang, Y., Yu, S., Lu, Y. & Zhang, Y. (2021). The impact of human pressure and climate change on the habitat availability and protection of Cypripedium (Orchidaceae) in Northeast China. Plants, 10(1), 1–15. https://doi.org/10.3390/plants10010084
Ma, M., Tan, T. K. & Wong, S. M. (2003). Identification and molecular phylogeny of Epulorhiza isolates from tropical orchids. Mycological Research, 107(9), 1041–1049. https://doi.org/10.1017/S0953756203008281
Mcgough, H. N., Roberts, D. L., Brodie, C. & Gardens, R. B. (2006). CITES y las zapatillas de Venus. Una introducción a las zapatillas de Venus amparadas por la Convención sobre el Comercio Internacional de Especies Amenazadas. UK: Royal Botanic Gardens, Kew. Retrieved from https://www.yumpu.com/es/document/read/14398272/cites-y-las-zapatillas-devenus-royal-botanic-gardens-kew [Accessed 26, XI, 2025]
Meng, Y. Y., Fan, X. L., Zhou, L. R., Shao, S. C., Liu, Q., Selosse, M. A. & Gao, J. Y. (2019). Symbiotic fungi undergo a taxonomic and functional bottleneck during orchid seeds germination: a case study on Dendrobium moniliforme. Symbiosis, 79(3), 205–212. https://doi.org/10.1007/s13199-019-00647-x
Moreno-Camarena, M. & Ortega-Larrocea, M. D. P. (2022). Mesoamerican Cypripedium, mycorrhizal contributions to promote their conservation as critically endangered species. Plants, 11(12), 1554. https://doi.org/10.3390/plants11121554
Murashige, T. & Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15(3), 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nicolè, F., Brzosko, E. & Till-Bottraud, I. (2005). Population viability analysis of Cypripedium calceolus in a protected area: Longevity, stability and persistence. Journal of Ecology, 93(4), 716–726. https://doi.org/10.1111/j.1365-2745.2005.01010.x
Norstog, K. (1970). Induction of embryolike structures by kinetin in cultured barley embryos. Developmental Biology, 23(4), 665–670. https://doi.org/10.1016/0012-1606(70)90146-6
Oliva, A. P. & Arditti, J. (1981). Seed Germination of North American Orchids. II. Native California and Related Species of Aplectrum, Cypripedium, and Spiranthes. Botanical Gazette, 142(4), 442–453. https://doi.org/10.1086/337245
Park, H. B., An, J., Bae, K. H., Hong, S. H., Park, H. J., Kim, S., Lee, C.W., Lee, B. D., Baek, J. H., Kim, N. Y. & Hwang, J. E. (2023). Asymbiotic seed germination and in vitro seedling development of the endangered orchid species Cypripedium guttatum. Plants, 12, 3788. https://doi.org/10.3390/plants12223788
Pedersen, H. A., Rasmussen, H. N., Kahandawala, I. M. & Fay, M. F. (2012). Genetic diversity, compatibility patterns and seed quality in isolated populations of Cypripedium calceolus (Orchidaceae). Conservation Genetics, 13(1), 89–98. https://doi.org/10.1007/s10592-011-0267-0
Perner, H., Zhou, R., Perner, W., Jiang, H. & Lee, Y. I. (2022). Cypripedium subtropicum embryo development and cytokinin requirements for asymbiotic germination. Botanical Studies, 63(28), 1–9. https://doi.org/10.1186/s40529-022-00359-4
Porras-Alfaro, A. & Bayman, P. (2007). Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth. Mycologia, 99(4), 510–525. https://doi.org/10.3852/mycologia.99.4.510
Rankou, H. & Salazar Chávez, G. A. (2014). Cypripedium irapeanum. The IUCN Red List of Threatened Species 2014: e.T43316630A43327669. Retrieved from https://doi.org/10.2305/IUCN.UK.2014-1.RLTS.T43316630A43327669.en [Accessed 26. XI. 2025].
Rasmussen, H. N., Dixon, K. W., Jersáková, J. & Těšitelová, T. (2015). Germination and seedling establishment in orchids: A complex of requirements. Annals of Botany, 116(3), 391–402. https://doi.org/10.1093/aob/mcv087
Rasmussen, H. N. & Pedersen, H. Æ. (2012). Cypripedium calceolus germination in situ: seed longevity, and dormancy breakage by long incubation and cold winters. European Journal of Enviromental Sciences, 1(2), 69–70. https://doi.org/10.14712/23361964.2015.49
Seaton, P. & Ramsay, M. (2005). Growing orchids from seed. Kewgrowing. Richmond, Royal Botanic Gardens, Kew, U.K.
Shefferson, R. P., Bunch, W., Cowden, C. C., Lee, Y. I., Kartzinel, T. R., Yukawa, T., Downing, J. & Jiang, H. (2019). Does evolutionary history determine specificity in broad ecological interactions? Journal of Ecology, 107(4), 1582–1593. https://doi.org/10.1111/1365-2745.13170
Shefferson, R. P., Mizuta, R. & Hutchings, M. J. (2017). Predicting evolution in response to climate change: The example of sprouting probability in three dormancy-prone orchid species. Royal Society Open Science, 4(1), 160647. https://doi.org/10.1098/rsos.160647
Shefferson, R. P., Taylor, D. L., Weiß, M., Garnica, S., McCormick, M. K., Adams, S., Gray, H. M., McFarland, J. W., Kull, T., Tali, K., Yukawa, T., Kawahara, T., Miyoshi, K. & Lee, Y. I. (2007). The evolutionary history of mycorrhizal specificity among lady’s slipper orchids. Evolution, 61(6), 1380–1390. https://doi.org/10.1111/j.1558-5646.2007.00112.x
Shefferson, R. P., Weiß, M., Kull, T. & Taylor, D. L. (2005). High specificity generally characterizes mycorrhizal association in rare lady’s slipper orchids, genus Cypripedium. Molecular Ecology, 14(2), 613–626. https://doi.org/10.1111/j.1365-294X.2005.02424.x
Shimura, H. & Koda, Y. (2004). Micropropagation of Cypripedium macranthos var. rebunense through protocorm-like bodies derived from mature seeds. Plant Cell, Tissue and Organ Culture, 78(3), 273–276. https://doi.org/10.1023/B:TICU.0000025641.49000.b5
Shimura, H. & Koda, Y. (2005). Enhanced symbiotic seed germination of Cypripedium macranthos var. vebunense following inoculation after cold treatment. Physiologia Plantarum, 123(3), 281–287. https://doi.org/10.1111/j.1399-3054.2005.00462.x
Shimura, H., Matsuura, M., Takada, N. & Koda, Y. (2007). An antifungal compound involved in symbiotic germination of Cypripedium macranthos var. rebunense (Orchidaceae). Phytochemistry, 68(10), 1442–1447. https://doi.org/10.1016/j.phytochem.2007.03.006
Shimura, H., Sadamoto, M., Matsuura, M., Kawahara, T., Naito, S. & Koda, Y. (2009). Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid. Mycorrhiza, 19(8), 525–534. https://doi.org/10.1007/s00572-009-0251-4
Sigma-Aldrich. (1990). Phytamax™ Orchid Maintenance Medium [Powder]. Retrieved from https://www.sigmaaldrich.com/catalog/product/sigma/p6668?lang=es®ion=MX
Soto-Arenas, M. A. & Solano-Gómez, R. A. (2007). Ficha técnica de Cypripedium irapeanum. In M. A. Soto-Arenas & R. Solano Gómez (Eds.), Información actualizada sobre las especies de orquídeas del PROY-NOM- 059-ECOL-2000 (Instituto Chinoin, pp. 1–12).
StatSoft, Inc. (2004). Statistica (Version 7.0). StatSoft.
Stewart, S. L. (ed.) (2004). Symbiotic Techniques in Orchid Seed Germination (1st edition). Stewart, Scott L.
Stewart, S. L. & Zettler, L. W. (2002). Symbiotic germination of three semi-aquatic rein orchids (Habenaria repens, H. quinquiseta, H. macroceratitis) from Florida. Aquatic Botany, 72(1), 25–35. https://doi.org/10.1016/S0304-3770(01)00214-5
Taniguchi, H., Katsumi, M., Yamamoto, Y., Tatsumi, Y., Sano, C. M., Choi, Y. E. & Sano, H. (2008). In vitro proliferation and genetic diversity of Cypripedium macranthos var. rebunense. Plant Biotechnology, 25(4), 341–346. https://doi.org/10.5511/plantbiotechnology.25.341
Valdés, M., Bautista Guerrero, H., Martínez, L. & Víquez, R. (2011). The root colonizing fungi of the terrestrial orchid Cypripedium irapeanum. Lankesteriana, 11(1), 15–21. http://www.redalyc.org/articulo.oa?id=44339820003
Whitridge, H. O. (2004). Mycorrhizal Fungi Associated with the Clustered Lady’S Slipper Orchid (Cypripedium fasciculatum). Southern Oregon University.
Whitridge, H. & Southworth, D. (2005). Mycorrhizal Symbionts of the Terrestrial Orchid Cypripedium fasciculatum. Selbyana, 26(1–2), 328–334. https://doi.org/10.2307/41760223
Yuan, L., Yang, Z. L., Li, S. Y., Hu, H. & Huang, J. L. (2010). Mycorrhizal specificity, preference, and plasticity of six slipper orchids from South Western China. Mycorrhiza, 20(8), 559–568. https://doi.org/10.1007/s00572-010-0307-5
Zeng, S., Wu, K., Teixeira da Silva, J. A., Zhang, J., Chen, Z., Xia, N. & Duan, J. (2012). Asymbiotic seed germination, seedling development and reintroduction of Paphiopedilum wardii Sumerh., an endangered terrestrial orchid. Scientia Horticulturae, 138, 198–209. https://doi.org/10.1016/j.scienta.2012.02.026
Zeng, S., Zhang, Y., Teixeira da Silva, J. A., Wu, K., Zhang, J. & Duan, J. (2014). Seed biology and in vitro seed germination of Cypripedium. Critical Reviews in Biotechnology, 34(4), 358–371. https://doi.org/10.3109/07388551.2013.841117
Zhang, Y., Lee, Y. I., Deng, L. & Zhao, S. (2013). Asymbiotic germination of immature seeds and the seedling development of Cypripedium macranthos Sw., an endangered lady’s slipper orchid. Scientia Horticulturae, 164, 130–136. https://doi.org/10.1016/j.scienta.2013.08.006
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Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México
Grant numbers PAPIME PE115024;DGAPA-PAPIIT IT-201422 -
Consejo Nacional de Humanidades, Ciencias y Tecnologías
Grant numbers SEP-CONACYT-ANUIES-ECOS-NORD-FRANCIA 299021
