Fern Conservation: Spore, Gametophyte, and Sporophyte Ex Situ Storage, In Vitro Culture, and Cryopreservation

  • Daniel Ballesteros
  • Valerie C. Pence


Many species of ferns and lycophytes are threatened by habitat destruction, over-collecting, invasive species, and climate change, and plans to secure their in situ and ex situ conservation are urgently needed. However, there has not been a development of standard methods for fern ex situ storage and conservation, as there has been for seed plants. This is likely due to the fact that ferns have been less studied and are less understood than seed plants and that there has been less funding and institutional focus on them for conservation purposes when compared with domesticated or wild seed plants. Tissues from different stages of the fern and lycophyte life cycle can be used as a ready source of germplasm for ex situ conservation. Spore storage is generally the most efficient method for ex situ fern conservation, but there are also situations where storing gametophytes or sporophytes can be extremely useful. The longevity of spores at −20 °C, the standard seed banking temperature, may vary with the species. Storage in LN should improve longevity in spores and is likely necessary for non-spore tissues. Recent results of a study on the viability of spores, gametophytes, and sporophytes after 20 years of storage in liquid nitrogen is used as a case study to illustrate the potential of these methods. The state of current methods and needs for further research and application are discussed. While there is much more to learn, these tools are available for fern ex situ conservation and should be implemented on a broader scale to preserve rare fern taxa and provide materials for future restoration and research.


Cryopreservation Ex situ conservation Gametophyte Liquid nitrogen Spore 



We would like to thank Callihan A and Lindsey K for their contribution to the propagation and acclimation of diverse fern species at CREW and Thomas A and Stewart K for the work on the initial banking of spores and gametophytes. Also special thanks to Barber S, Pritchard H, Dickie J, Davies R (UK), Estrelles E (Spain), Mikula A (Poland), Magrini S (Italy), Walters C, Hill L, Wolkis D (USA), Reyes-Jaramillo I (Mexico), Randi M (Brazil), Sershen (South Africa), Huang YM (Taiwan), Cicuzza D, Bin W, Yang X-Y (China), Schnell J, McGill C (New Zealand), and Offord C (Australia) for their contributions in the development of Table 11.1 and Suggii N and From M for information on in vitro sporophyte collections. Fern spore, gametophyte, and sporophyte collections used in this work would not be possible without the help and the donations of multiple collectors and researchers from diverse institutions, including Bannister S, Yoshinaga A, Kawakami K, and staff of the Krohn Conservatory.


  1. Agrawal DC, Pawar SS, Mascarenhas AF (1993) Cryopreservation of spores of Cyathea spinulosa wall. Ex. Hook. F., an endangered tree fern. J Plant Physiol 142:124–126CrossRefGoogle Scholar
  2. Aguraiuja R (2010a) Reinforcement of the populations of the critically endangered endemic fern, Diellia pallid, Kaua’I, Hawwaiian Islands, U.S.A. In: Soorae PS (ed) Global re-introduction perspectives: additional case-studies from around the globe. IUCN/SSC Re-introduction Specialist Group. Abu Dhabi, UAE, pp 319–323Google Scholar
  3. Aguraiuja R (2010b) Conservation introduction of a locally extinct fern species in Estonia during 1998–2008. In: Soorae PS (ed) Global re-introduction perspectives: additional case-studies from around the globe. IUCN/SSC Re-introduction Specialist Group. Abu Dhabi, UAE, pp 345–348Google Scholar
  4. Alexander A, Dudas G, Herren G, Kolben G, Kuhara Y, Murdoch L, Slaven J, Paultre D, Rae N, Rayner J, Vileitaite J, Chamberlain M (2008) Testing the viability of spores held in the fern spore bank at the Royal Botanic Garden, Edinburgh. Newsletter of the Botanical Society of Scotland 91:29–31Google Scholar
  5. Aragón CF, Pangua E (2004) Spore viability under different storage conditions in four rupicolous Asplenium L. taxa. Am Fern J 941:28–38CrossRefGoogle Scholar
  6. Arcand NN, Ranker TA (2008) Conservation biology. In: Ranker TA, Haufler CH (eds) Biology and evolution of ferns and lycophytes. Cambridge University Press, Cambridge, pp 257–283CrossRefGoogle Scholar
  7. Ashcroft CJ, Sheffield E (2000) The effect of spore density on germination and development in Pteridium, monitored using a novel culture technique. Am Fern J 90:91–99CrossRefGoogle Scholar
  8. Ashmore SE, Hamilton KN, Offord CA (2011) Conservation technologies for safeguarding and restoring threatened flora: case studies from Eastern Australia. In Vitro Cell Dev Biol Plant 47:99–109CrossRefGoogle Scholar
  9. Bacchetta G, Bueno Sánchez A, Fenu G, Jiménez-Alfaro B, Mattana E, Piotto B, Virevaire M (eds) (2008) Conservación ex situ de plantas silvestres. Principado de Asturias/La Caixa, Principado de Asturias, SpainGoogle Scholar
  10. Ballesteros D (2010) Conservation of fern spores. In: Kumar A, Fernández H, Revilla-Bahillo A (eds) Working with ferns. Issues and applications. Springer, New York, pp 165–172Google Scholar
  11. Ballesteros D, Pence VC (2017) Survival and death of seeds during liquid nitrogen storage: a case study on seeds with short lifespans. CryoLetters 38(4):278–289Google Scholar
  12. Ballesteros D, Walters C (2007a) Water properties in fern spores: sorption characteristics relating to water affinity, glassy states and storage stability. J Exp Bot 58:1185–1196CrossRefPubMedGoogle Scholar
  13. Ballesteros D, Walters C (2007b) Calorimetric properties of water and triacylglycerols in fern spores relating to storage at cryogenic temperatures. Cryobiology 55:1–9CrossRefPubMedGoogle Scholar
  14. Ballesteros D, Walters C (2011) Detailed characterization of mechanical properties and molecular mobility within dry seed glasses: relevance to the physiology of dry biological systems. The Plant J 68:607–619CrossRefPubMedGoogle Scholar
  15. Ballesteros D, Estrelles E, Walters C, Ibars AM (2011) Effect of storage temperature on green spore longevity for the ferns Equisetum ramosissimum and Osmunda regalis. CryoLetters 32(2):89–98PubMedGoogle Scholar
  16. Ballesteros D, Estrelles E, Walters C, Ibars AM (2012) Effects of temperature and desiccation on ex situ conservation of non-green fern spores. Am J Bot 99:721–729CrossRefPubMedGoogle Scholar
  17. Ballesteros D, Hill LM, Walters C (2017) Variation of desiccation tolerance and longevity in fern spores. J Plant Physiol 211:53–62CrossRefPubMedGoogle Scholar
  18. Barnicoat H, Cripps R, Kendon J, Sarasan V (2011) Conservation in vitro of rare and threatened ferns—case studies of biodiversity hotspot and island species. In Vitro Cell Dev Biol Plant 47:37–45CrossRefGoogle Scholar
  19. Beri A, Bir SS (1993) Germination of stored spores of Pteris vittata L. Am Fern J 833:73–78CrossRefGoogle Scholar
  20. Bertrand AM, Albuerne MA, Fernández H, González A, Sánchez-Tamés R (1999) In vitro organogenesis of Polypodium cambricum. Plant Cell Tissue Organ Cult 57:65–69CrossRefGoogle Scholar
  21. Camloh M (1999) Spore age and sterilization affects germination and early gametophyte development of Platycerium bifurcatum. Am Fern J 89:124–132CrossRefGoogle Scholar
  22. Camloh M, Ambrozic-Dolinsek J (2010) In vitro regeneration systems of Platycerium. In: Fernández H, Kumar A, Angeles-Revilla A (eds) Working with ferns: issues and applications. Springer Science & Business Media, New York, pp 111–125Google Scholar
  23. Chapman AD (2009) Numbers of living species in Australia and the world. Australian Biodiversity Information Services, Toowoomba, AustraliaGoogle Scholar
  24. Colli Aurea MT, Perez Sonia CJG (1999) The effect of red light on the germination of a brazilian pteridophyte. Brazil Arch Biol Technol 422:169–174Google Scholar
  25. Constantino S, Santamaria LM, Hodson E (2000) Storage and in vitro germination of tree fern spores. Bot Garden Microprop News 24:58–60Google Scholar
  26. De Brum FMR, Randi AM (2006) Germination of spores and growth of gametophytes and sporophytes of Rumohra adiantiformis (Forst.) Ching (Dryopteridaceae) after spore cryogenic storage. Rev Bras Bot 29:489–495CrossRefGoogle Scholar
  27. De Long JM, Prange RK (2008) Fiddlehead fronds: nutrient rich delicacy. Chronica Hort 48:12–15Google Scholar
  28. Dolinsek JA, Camloh M (1997) Gametophytic and sporophytic regeneration from bud scales of the fern Platycerium bifurcatum (Cav.) C.Chr. in vitro. Ann Bot 80:23–28CrossRefGoogle Scholar
  29. Dong S, Zuo Z, Yan Y, Xiang J (2017) Red list assessment of lycophytes and ferns in China. Biodivers Sci 25(7):765–773CrossRefGoogle Scholar
  30. Dyer AF (1979) The culture of fern gametophytes for experimental investigation. In: Dyer AF (ed) The experimental biology of ferns. Academic Press, LondonGoogle Scholar
  31. Dyer AF (1994) Natural soil spore banks—can they be used to retrieve lost ferns? Biodivers Conserv 3:160–175CrossRefGoogle Scholar
  32. Fabre J, Dereuddre J (1990) Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot tips. CryoLetters 11:413–426Google Scholar
  33. FAO (2014) Genebank standards for plant genetic resources for food and agriculture, Rev. edn, Rome. Food and Agriculture Organization of the United Nations, RomeGoogle Scholar
  34. Farrar DR (1967) Gametophytes of four tropical fern genera reproducing independently of their sporophytes in the southern Appalachians. Science 155:1266–1267CrossRefPubMedGoogle Scholar
  35. Farrar DR (2016) Vittaria appalachiana continues to provide insight into the biology of ferns: a commentary on two studies recently published in American Journal of Botany. Am J Bot 103:593–595CrossRefPubMedGoogle Scholar
  36. Fernández H, Revilla MA (2003) In vitro culture of ornamental ferns. Plant Cell Tissue Organ Cult 73:1–13CrossRefGoogle Scholar
  37. Fernández H, Bertrand A, Sánchez-Tamés R (1996) Micropropagation and phase change in Blechnum spicant and Pteris ensiformis. Plant Cell Tissue Organ Cult 44:261–265CrossRefGoogle Scholar
  38. Fernández H, Kumar A, Revilla A (eds) (2010) Working with ferns: issues and applications. Springer Science & Business Media, New YorkGoogle Scholar
  39. Ford MV (1992) Growing ferns from spores in sterile culture. In: Ide JM, Jermy AC, Paul AM (eds) Fern horticulture: past, present and future perspectives. Intercept, Andover, pp 295–297Google Scholar
  40. From MM (2010) Conservation status and re-introduction of Bermuda’s Governor Laffan fern—an endangered endemic. In: Sourae PS (ed) Global Re-introduction Perspectives—2010: additional Case Studies from around the Globe. IUCN/SSC Re-introduction Specialist Group, Abu Dhabi, UAE, pp 316–318Google Scholar
  41. Guillon JM, Fievet D (2003) Environmental sex determination in response to light and biased sex ratios in Equisetum gametophytes. J Ecol 91:49–57CrossRefGoogle Scholar
  42. Higuchi H, Amaki W (1989) Effects of 6-benzylaminopurine on the organogenesis of Asplenium nidus L. through in vitro propagation. Sci Hortic 37:351–359CrossRefGoogle Scholar
  43. Higuchi H, Amaki W, Suzuki S (1987) In vitro propagation of Nephrolepis cordifolia Presl. Sci Hortic 32:105–113CrossRefGoogle Scholar
  44. Hirai D, Sakai A (1999) Cryopreservation of in vitro-grown axillary shoot-tip meristems of min (Mentha spicata L.) by encapsulation vitrification. Plant Cell Rep 19:150–155CrossRefGoogle Scholar
  45. Houser DC, From M, Landry M, Copeland A, Kellar R (2016) Systematics of Diplazium laffanianum (Athyriaceae), a fern species endemic to Bermuda. Am Fern J 106:206–222CrossRefGoogle Scholar
  46. Huang Y-M, Lee P-H, Fu C-H, Chang Y-H (2014) The relation between spore maturity and viability of tree ferns. J Natl Taiwan Mus 67(2):45–58Google Scholar
  47. Ibars AM, Estrelles E (2012) Recent developments in ex situ and in situ conservation of ferns. Fern Gaz 19(3):67–86Google Scholar
  48. Ibars AM, Estrelles E (2015) Protocolo de propagación de helechos. Botanic as PPECTS 1 1:1–5Google Scholar
  49. Ibars AM, Gómez Serrano MA, Mayoral O, Estrelles E (2011) Prioridades para la conservación en el ámbito de los helechos en Castilla-La Mancha. In: Hernandez JE, Herranz JM (eds) Protección de la diversidad vegetal y de los recursos fitogenéticos de Castilla-La Mancha. Instituto de Estudios Albacetenses- Botánico de Castilla-La Mancha. Serie I Estudio, Albacete, Spain, pp 191–213Google Scholar
  50. IUCN (2017) Table 3a: Status category summary by major taxonomic group (plants). In: The IUCN red list of threatened species, version 2017–2. Last updated 14 Sept 2017, Accessed 29 Sept 2017
  51. Jha TB, Mukherjee S, Basak A, Adhikari J (2013) In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring. Plant Biotechnol Rep 7:239–245CrossRefGoogle Scholar
  52. Kamachi H, Noguchi M (2012) Negative gravitropism in dark-grown gametophytes of the fern Ceratopteris richardii. Am Fern J 102:147–153CrossRefGoogle Scholar
  53. Khan S, Raziq M, Kayani HA (2008) In vitro propagation of bird’s nest fern (Asplenium nidus) from spores. Pakistan J Bot 40:91–97Google Scholar
  54. Kuriyama A, Sugawara Y, Matsushima H, Takeuchi M (1990) Production of sporophytic structures from gametophytes by cytokinin in Equisetum Arvense. Naturwissenschaften 77(1):31–32CrossRefGoogle Scholar
  55. Li D, Pritchard HW (2009) The science and economics of ex situ plant conservation. Trends Plant Sci 14:614–621CrossRefPubMedGoogle Scholar
  56. Li Y, Shi L (2015) Effect of maturity level and desiccation process on liquid nitrogen storage of green spores of Osmunda japonica. Plant Cell Tissue Organan Cult 120:531–538CrossRefGoogle Scholar
  57. Li X, Han J-D, Fang Y-H, Bai S-N, Rao G-Y (2017) Expression analyses of embryogenesis-associated genes during somatic embryogenesis of Adiantum capillus-veneris L. in vitro: new insights into the evolution of reproductive organs in land plants. Front Plant Sci 8:1–12Google Scholar
  58. Liu Y, Wujisguleng W, Long C (2012) Food uses of ferns in China: a review. Acta Soc Bot Pol 81(4):263–270CrossRefGoogle Scholar
  59. Maeda M, Ito M (1981) Isolation of protoplasts from fern prothallia and their regeneration to gametophytes. Bot Mag Tokyo 94:35–40CrossRefGoogle Scholar
  60. Makowski D, Rybczyński JJ, Mikula A, Klimaszewska K (2015) A simple way to overcome the recalcitrance of the water fern Ceratopteris thalictroides (L.) Brongn. to cryopreservation. Acta Soc Bot Pol 84:385–388CrossRefGoogle Scholar
  61. Makowski D, Tomiczak K, Rybczyński JJ, Mikula A (2016) Integration of tissue culture and cryopreservation methods for propagation and conservation of the fern Osmunda regalis L. Acta Physiol Plant 38:1–12CrossRefGoogle Scholar
  62. Mannan M, Maridass M, Victor B (2008) A review on the potential uses of ferns. Ethnobotanical Leaflets 12:281–285Google Scholar
  63. Marimuthu J, Manickam VS (2011) Ex situ conservation of two threatened ferns of the Western Ghats through in vitro spore culture. J Threat Taxa 3:1919–1928CrossRefGoogle Scholar
  64. Martin KP, Sini S, Zhang CL, Slater A, Madhusoodanan PV (2006) Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.) Plant Cell Rep 25:1300–1307CrossRefPubMedGoogle Scholar
  65. Matsuura T, Sugimura K, Miyamoto A, Tanaka H, Tanaka N (2014) Spatial characteristics of edible wild fern harvesting in mountainous villages in northeastern Japan using GPS tracks. Forests 5:269–286CrossRefGoogle Scholar
  66. Mehltreter K, Walker LR, Sharpe JM (eds) (2010) Fern ecology. Cambridge University Press, CambridgeGoogle Scholar
  67. Mikula A, Jata K, Rybczynksi JJ (2009) Cryopreservation strategies for Cyathea australis (R.Br.) Domin. CryoLetters 30:429–439PubMedGoogle Scholar
  68. Mikula A, Makowski D, Walters C, Rybczynski JJ (2010) Exploration of cryo-methods to preserve tree and herbaceous fern gametophytes. In: Fernández H, Kumar A, Angeles-Revilla A (eds) Working with ferns: issues and applications. Springer Science & Business Media, New York, pp 173–192Google Scholar
  69. Mikula A, Tomiczak K, Makowski D, Niedzielski M, Rybczynski JJ (2015) The effect of moisture content and temperature on spore aging in Osmunda regalis. Acta Physiol Plant 37:229–240CrossRefGoogle Scholar
  70. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  71. Ong BL, Ng ML (1998) Regeneration of drought-stressed gametophytes of the epiphytic fern, Pyrrosia piloselloides (L.) price. Plant Cell Rep 18:225–228CrossRefGoogle Scholar
  72. Page CN, Dyer AF, Lindsay S, Mann DG (1992) Conservation of Pteridophytes: the ex situ approach. In: Ide JM, Jermy AC, Paul AM (eds) Fern horticulture: past, present and future perspectives. Intercept, Andover, pp 269–278Google Scholar
  73. Pence VC (2000a) Cryopreservation of in vitro grown fern gametophytes. Am Fern J 90:16–23CrossRefGoogle Scholar
  74. Pence VC (2000b) Survival of chlorophyllous and nonchlorophyllous fern spores through exposure to liquid nitrogen. Am Fern J 90:119–126CrossRefGoogle Scholar
  75. Pence VC (2001) Cryopreservation of shoot tips of Selaginella uncinata. Am Fern J 91:37–40CrossRefGoogle Scholar
  76. Pence VC (2004) Ex situ conservation methods for bryophytes and pteridophytes. In: Guerrant Jr. EO, Havens K, Maunder M (eds) Ex situ plant conservation. Supporting species survival in the wild. Island Press,Washington, DC, pp 206–227Google Scholar
  77. Pence VC (2008a) Ex situ conservation of ferns and lycophytes – approaches and techniques. In: Ranker TA, Hauffler CH (eds) Biology and evolution of ferns and lycophytes. Cambridge University Press, Cambridge, pp 284–300CrossRefGoogle Scholar
  78. Pence VC (2008b) Cryopreservation of bryophytes and ferns. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 117–140CrossRefGoogle Scholar
  79. Pence VC (2014) In vitro propagation and cryopreservation of the endangered filmy fern, Trichomanes punctatum subsp. floridanum (Hymenophyllaceae). Fern Gaz 19:307–317Google Scholar
  80. Pence VC (2015) Propagation and cryopreservation of Asplenium scolopendrium var. americanum, the American hart’s-tongue fern. Am Fern J 105:211–225CrossRefGoogle Scholar
  81. Pence VC, Philpott M, Culley TM, Plair B, Yorke SR, Lindsey K, Vanhove A-C, Ballesteros D (2017) Survival and genetic stability of shoot tips of Hedeoma todsenii R.S. Irving after long-term cryostorage. In Vitro Cellular and Developmental Biology Plant 53(4):328–338CrossRefGoogle Scholar
  82. Pennisi E (2010) Tending the global garden. Science 329:1274–1277CrossRefPubMedGoogle Scholar
  83. Pérez-García B, Reyes-Jaramillo I (1993) Helechos: propagación y conservación. Ciencias 30:10–17Google Scholar
  84. Possley J (2014) Fern conservation in a biodiversity hotspot. The Palmetto 31(2):1–9Google Scholar
  85. Pritchard HW, Nadarajan J, Ballesteros D, Thammasiri K, Prasongsom S, Malik SK, Chaudhury R, Kim H-H, Lin L, Li W-Q, Yang X-Y, Popova E (2017) Cryobiotechnology of tropical seeds – scale, scope and hope. Acta Hortic 1167:37–47CrossRefGoogle Scholar
  86. Proctor MCF, Oliver MJ, Wood AJ, Alpert P, Stark LR, Cleavitt NL, Mishler BD (2007) Desiccation-tolerance in bryophytes: a review. Bryologist 110:595–621CrossRefGoogle Scholar
  87. Quintanilla LG, Amigo J, Pangua E, Pajaron S (2002) Effect of storage method on spore viability in five globally threatened fern species. Ann Bot 904:461–467CrossRefGoogle Scholar
  88. Raine ACA, Sheffield E (1997) Establishment and maintenance of aseptic culture of Trichomanes speciosum gametophytes from gemmae. Am Fern J 87:87–92CrossRefGoogle Scholar
  89. Rajasekharan PE (2015) Gene banking for ex situ conservation of plant genetic resources. In: Bahadur B, Rajam MV, Sahijram L, Krishnamurthy KV (eds) Plant biology and biotechnology Vol II: Plant genomics and biotechnology. Springer, New Delhi, pp 445–459CrossRefGoogle Scholar
  90. Reed BM (ed) (2008) Plant cryopreservation: a practical guide. Springer, New YorkGoogle Scholar
  91. Rogers SMD, Banister S (1992) Micropropagation of Notholaena. Hortscience 27:1224–1225Google Scholar
  92. Rogge GD, Viana AM, Randi AM (2000) Cryopreservation of spores of Dicksonia sellowiana: an endangered tree fern indigenous to South and Central America. CryoLetters 21:223–230PubMedGoogle Scholar
  93. Sara SC, Manickam VS (2007) In vitro developmental ontogeny and life cycle of a rare fern species – Thelypteris confluens (Thunb.) Morton. Indian J Biotechnol 6:372–380Google Scholar
  94. Shukla SP, Khare PB (2012) In vitro shoot regeneration via caulogenesis in fern, Pteris vittata L. J Environ Biol 33:683–687PubMedGoogle Scholar
  95. Simabukuro EA, Dyer AF, Felippe GM (1998) The effect of sterilization and storage conditions on the viability of the spores of Cyathea delgadii. Am Fern J 88:72–80CrossRefGoogle Scholar
  96. Somer M, Arbesu R, Menéndez V, Revilla MA, Fernández H (2009) Sporophyte induction studies in ferns in vitro. Euphytica 171:203–210CrossRefGoogle Scholar
  97. Srinivasan JC, Kaufman PB (1978). Sex expression in Equisetum scirpoides in vitro. Phytomorphology 28:331–5Google Scholar
  98. Stamps RH (2006) Irrigation and nutrient management practices for commercial leatherleaf fern production in Florida. University of Florida, IFAS, FloridaGoogle Scholar
  99. Taha RM, Haron NW, Wafa SN (2011) Morphological and tissue culture studies of Platycerium coronarium, a rare ornamental fern species from Malaysia. Am Fern J 101:241–251CrossRefGoogle Scholar
  100. Templeman TS, DeMaggio AE, Stetler DA (1987) Biochemistry of fern spore germination: globulin storage proteins in Matteuccia struthiopteris L. Plant Physiol 85:343–349CrossRefPubMedPubMedCentralGoogle Scholar
  101. Towill LR, Ikuma H (1973) Photocontrol of the germination of Onoclea spores. Plant Physiol 51:973–978CrossRefPubMedPubMedCentralGoogle Scholar
  102. Vertucci CW, Roos EE, Crane J (1994) Theoretical basis of protocols for seed storage III. Optimum moisture contents for pea seeds stored at different temperatures. Ann Bot 74(5):531–540CrossRefGoogle Scholar
  103. Walters C, Hill LM, Wheeler LJ (2005) Dying while dry: kinetics and mechanisms of deterioration in desiccated organisms. Integr Comp Biol 45:751–758CrossRefPubMedGoogle Scholar
  104. Whittier DP (1973) The effect of light and other factors on spore germination in Botrychium dissectum. Can J Bot 51:1791–1794CrossRefGoogle Scholar
  105. Whittier DP, Pintaud JC (1999) Spore germination and early gametophyte development in Stromatopteris. Am Fern J 89:142–146CrossRefGoogle Scholar
  106. Wilkinson T (2002) In vitro techniques for the conservation of Hymenophyllum tunbrigense (L.) Sm. Fern Gaz 16:458Google Scholar
  107. Winarto B, Teixeira da Silva JA (2012) Improved micropropagation protocol for leatherleaf fern (Rumohra adiantiformis) using rhizomes as donor explants. Sci Hortic 140:74–80CrossRefGoogle Scholar
  108. Yu R, Zhang G, Li H, Cao H, Mo X, Gui M, Zhou X, Jiang Y, Li S, Wang J (2017) In vitro propagation of the endangered tree fern Cibotium barometz through formation of green globular bodies. Plant Cell Tissue Organ Cult 128:369–379CrossRefGoogle Scholar

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© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Center for Conservation and Research of Endangered WildlifeCincinnati Zoo & Botanical GardenCincinnatiUSA
  2. 2.Comparative Seed Biology Group, Comparative Plant and Fungal Biology DepartmentRoyal Botanic Gardens KewRichmondUK

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