From Spore to Sporophyte: How to Proceed In Vitro

  • V. Menéndez
  • R. Arbesú
  • M. Somer
  • A. Revilla
  • H. Fernández


In vitro culture techniques represent an excellent tool for enabling our mind to understand those processes occurring along the life cycle of plants and also for realising successful benefits of their applications. This chapter summarises the in vitro culture of ferns from spore to sporophyte, giving details of how to proceed for achieving optimal performance of each phase involved in the life cycle, defining the cultural requirements leading to a greater production of these plants.


Sexual Organ Fern Spore Leptosporangiate Fern Fertile Frond Sporophyte Production 


  1. Amaki, W., and Higuchi, H. 1991. A possible propagation system of Nephrolepis, Asplenium, Pteris, Adiantum,and Rhumora through tissue culture. Act. Hort. 300: 237–243.Google Scholar
  2. Beck, M.J., and Caponetti, J.D. 1983. The effects of kinetin and naphtalenacetic acid in vitro shoot multiplication and rooting in the fishtail fern. Am. J. Bot. 70:1–7.CrossRefGoogle Scholar
  3. Bhambie, S., and Gupta, B. 1994. Induced apogamy and apospory in Adiantum capillus-veneris L. J. Ind. Bot. Soc.73:25–28.Google Scholar
  4. Bordonneau, M., and Tourte, Y. 1987. Potentialites ontogenetique des tissues gametophytiques chez le Pilularia globulifera L. C. R. Acad. Aci. Paris Ser. III, 304:437–440.Google Scholar
  5. Camloh, M., Gogala, N., and Rode, J. 1994. Plant regeneration from leaf explants of the fern Platycerium bifurcatum in vitro. Sci. Hortic. 56:257–266.CrossRefGoogle Scholar
  6. Camloh, M., Ravnikar, M., and Zel, J. 1996. Jasmonic acid promotes division of fern protoplasts, elongation of rhizoids and early development of gametophytes. Physiol. Plant. 97:659–664.CrossRefGoogle Scholar
  7. Chang, H.C., Agrawal, D.C., Kuo, C.L., Wen, J.L., Chen, C.C., and Tsay, H.S. 2007. In vitro culture of Drynaria fortunei, a fern species source of Chinese medicine “Gu-Sui-Bu”. In Vitro Cell. Dev. Biol. Plant 43:133–139.CrossRefGoogle Scholar
  8. Chou, H.M., Huang, Y.M., Wong, S.L., Hsieh, T.H., Hsu, S.Y., and Chiou, W.L. 2007. Observations of gametophytes and juvenile sporophytes of Archangiopteris somai Hayata (Marattiaceae), an endangered fern in Taiwan. Botanical Stud. 48:205–213.Google Scholar
  9. Cox, J., Bhatia, P., and Ashwath, N. 2003. In vitro spore germination of the fern Schizaea dichotoma. Sci. Hort. 97:369–378.CrossRefGoogle Scholar
  10. Deberg, P. 1994. In vitro culture of ornamentals. In Plant Cell and Tissue Culture, ed. I.K. Vasil and T.A. Thorpe, pp. 561–573. Kluwer Academic Publishers, Dordrecht, the Netherlands.Google Scholar
  11. DeSoto, L, Quintanilla, L.G., and Méndez M. 2008. Environmental sex determination in ferns: effects of nutrient availability and individual density in Woodwardia radicans. J. Ecol. 96:1319–1327.CrossRefGoogle Scholar
  12. Dyer, A.F. 1979. The culture of fern gametophytes for experimental investigation. In: The Experimental Biology of Ferns, ed. A.F. Dyer, pp.253–305. Academic Press, London.Google Scholar
  13. Fay, M.F. 1994. In what situation is in vitro culture appropriate to plant conservation? Biodivers. Conserv. 3:176–183.CrossRefGoogle Scholar
  14. Fernández, H., Bertrand, A.M., and Sánchez-Tamés R. 1993. In vitro regeneration of Asplenium nidus L. from gametophytic and sporophytic tissue. Sci Hort. 56:71–77.CrossRefGoogle Scholar
  15. Fernández, H., Bertrand, A.M., and Sánchez-Tamés, R. 1996a. Influence of tissue culture conditions on apogamy in Dryopteris affinis ssp. affinis. Plant Cell Tissue Organ Cult. 45:93–97.CrossRefGoogle Scholar
  16. Fernández, H., Bertrand, A.M., and Sánchez-Tamés, R. 1996b. Micropropagation and phase change in Blechnum spicant and Pteris ensiformis. Plant Cell Tissue Organ Cult. 44:261–265.CrossRefGoogle Scholar
  17. Fernández, H., Bertrand, A.M., and Sánchez-Tamés, R. 1997a. Gemmation in Osmunda regalis L. gametophyte cultured in vitro. Plant Cell Rep. 16:358–362.CrossRefGoogle Scholar
  18. Fernández, H., Bertrand, A., and Sánchez-Tamés, R. 1997b. Plantlet regeneration in Asplenium nidus L. and Pteris ensiformis L. by homogenization of BA treated rhizomes. Sci. Hort. 68:243–247.CrossRefGoogle Scholar
  19. Fernández, H., Bertrand, A., and Sánchez-Tamés, R. 1997c. Gametophyte culture in vitro and antheridiogen activity in Blechnum spicant. Plant Cell Tissue Organ Cult. 50:71–74.CrossRefGoogle Scholar
  20. Fernández, H., Bertrand, A., and Sánchez-Tamés, R. 1999. Biological and nutritional aspects involved in fern multiplication. Plant Cell Tissue Organ Cult. 56:211–214.CrossRefGoogle Scholar
  21. Fernández, H., and Revilla, M.A. 2003. In vitro culture of ornamental ferns. Plant Cell Tissue Organ Cult. 73:1–13.CrossRefGoogle Scholar
  22. Fernández, H., Arbesú, R., Nistal, A.M., Menéndez, M., and Revilla, M.A. 2007. Preservation and characterization by confocal microscopy of fern spores. Adv. Hort. Sci. 21:203–206.Google Scholar
  23. Hamilton, R.G., and Lloyd, R.M. 1991. Antheridiogen in the wild: The development of fern communities. Funct. Ecol. 5:804–809.CrossRefGoogle Scholar
  24. Harper, K. 1976. Asexual multiplication of Leptosporangiatae ferns through tissue culture. M.Sc. University of California.Google Scholar
  25. Haufler, C.H., and Ranker, T.A. 1985. Differential antheridiogen response and evolutionary mechanisms in Cystopteris. Am. Bot. 72:659–665.CrossRefGoogle Scholar
  26. Hicks G., and von Aderkas P. 1986. A tissue culture of the ostrich fern Matteuccia struthiopteris L. Todaro. Plant Cell Tissue Organ Cult. 5:199–204.CrossRefGoogle Scholar
  27. Higuchi H., Amaki W., and Suzuki S. 1987. In vitro propagation of Nephrolepis cordifolia Prsel. Sci. Hort. 32:105–113.CrossRefGoogle Scholar
  28. Higuchi H., and Amaki W. 1989. Effects of 6-benzyladenine on the organogenesis of Asplenium nidus L. through in vitro propagation. Sci. Hort. 37:351–359.CrossRefGoogle Scholar
  29. Hirsch, A.M. 1975. The effect of sucrose on the differentiation of excised fern leaf tissue into either gametophytes or sporophytes. Plant Physiol. 56:390–393.CrossRefPubMedGoogle Scholar
  30. Hua, W., Ping-Ting, C., Li-Ping, Y., and Long-Qing, C. 2009. An efficient method for surface sterilization and sowing fern spores in vitro culture. Am. Fern J. 99:227–231.Google Scholar
  31. Huang, Y.M, Chou, H.M., and Chiou, W.L. 2004. Density affects gametophyte growth and sexual expression of Osmunda cinnamomea (Osmundaceae: Pteridophyta). Ann. Bot. 94:229–232.CrossRefPubMedGoogle Scholar
  32. Klekowski, E.J. Jr 1969. Reproductive biology of the Pteridophyta. II. Theoretical considerations. Bot. J. Linn. Soc. 62:347–359.CrossRefGoogle Scholar
  33. Klekowski, E.J. Jr 1970. Reproductive biology of the Pteridophyta. IV. An experimental study of mating systems in Ceratopteris thalictroides (L.) Brong. Bot. J. Linn. Soc. 63:153–169.CrossRefGoogle Scholar
  34. Knop, W. 1865. Quantitative untersuchungen uber die ernahrungsprozesse der pflanzen. Landwirsch Vers Stn. 7:93–107.Google Scholar
  35. Knudson, L. 1946. A nutrient solution for the germination of orchid seed. Bull. Am. Orchid Soc. 15:214–217.Google Scholar
  36. Korpelainen, H. 1994. Growth, sex determination and reproduction of Dryopteris filix-mas (L.) Schott gametophytes under shifting nutritional conditions. Bot. J. Linn. Soc. 114:357–366.CrossRefGoogle Scholar
  37. Kshirsagar, M.R., and Mehta, A.R. 1978. In vitro studies in ferns: growth and differentiation in rhizome callus of Pteris vittata. Phytomorphology 28: 51–58.Google Scholar
  38. Kuriyama, A., Kobayashi, T., Hayashi, S., and Maeda, M. 2004. Medium composition for the ­production of sporophytes of the fern Adiantum capillus-veneris. J. Jap. Soc. Hort. Sci. 73:580–582.CrossRefGoogle Scholar
  39. Kwa, S.H., Wee, Y.C., and Loh, C.S. 1988. Production of aposporous gametophytes from Drymoglossum piloselloides (L.) Presl. fronds strips. Plant Cell Rep. 7:142–143.CrossRefGoogle Scholar
  40. Kwa, S.H., Wee, Y.C., and Loh, C.S. 1990. Production of first- and second-generation aposporous gametophytes from Pyrrosia piloselloides (L.) Price frond strips cultured in vitro. Plant Cell Rep. 8:530–533.CrossRefGoogle Scholar
  41. Kwa, S.H., Wee, Y.C., Lim, T.M., and Kumar, P.P. 1995. IAA-induced apogamy in Platycerium coronarium (Koening) Desv. Gametophytes cultured in vitro. Plant Cell Rep 14:598–602.CrossRefGoogle Scholar
  42. Kwa, S.H., Wee, Y.C., Lim, T.M., and Kumar, P.P. 1997. Morphogenic plasticity of callus reinitiated from cell suspension cultures of the fern Platycerium coronarium. Plant Cell Tissue Organ Cult. 50:75–82.CrossRefGoogle Scholar
  43. Loescher W.H., and Albrecht C.N. 1979. Development in vitro of Nephrolepsis axaltata cv. Bostoniennsis runner tissues. Physiol Plant. 47:250–254.CrossRefGoogle Scholar
  44. Martin, K.P., Sini, S., Zhang, C.L., Slater, A., and Madhusoodanan, P.V. 2006. Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.). Plant Cell Rep. 25:1300–1307.CrossRefPubMedGoogle Scholar
  45. Materi, D.M., and Cumming, B.G. 1991. Effects of carbohydrate deprivation on rejuvenation, apospory, and regeneration in ostrich fern (Matteuccia struptiopteris) sporophyte. Can. J. Bot. 69:1241–1245.CrossRefGoogle Scholar
  46. Mehra, P.N., and Sulklyan, D.S. 1969. In vitro studies on apogamy, apospory and controlled differentiation of rhizome segments of the fern Amelopteris prolifera (Retz.) Copel. Bot. J. Linn. Soc. 62:431–443.CrossRefGoogle Scholar
  47. Menéndez, V., Revilla, M.A., and Fernández, H. 2006. Growth and gender in the gametophyte of Blechnum spicant L. Plant Cell Tissue Organs Cult. 86:47–53.CrossRefGoogle Scholar
  48. Miller, J.H. 1968. Fern gametophyte as experimental material. Bot. Rev. 34:361–440.CrossRefGoogle Scholar
  49. Mohr, H. 1962. The influence of visible radiation on the germination of archegoniate spores and the growth of the fern protonema. J. Linn. Soc. Bot. 58:287–296.CrossRefGoogle Scholar
  50. Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  51. Padhya, M.A., and Mehta, A.R. 1982. Propagation of fern (Nephrolepis) through tissue culture. Plant Cell Rep. 1:261–263.CrossRefGoogle Scholar
  52. Peck, J.H., Peck, C.J., and Farrar, D.R. 1990. Influences of life history attributes on formation of local and distant fern populations. Am. Fern J. 80:126–142.CrossRefGoogle Scholar
  53. Pierik, R.M.L. 1991. Commercial aspects of micropropagation. In Horticulture- New Technologies and Applications, J. Prakash and R.M.L. Pierik, (eds.), pp. 141–153. Kluwer Academic Publishers Dordrecht, the Netherlands.Google Scholar
  54. Raghavan, V. 1989. Developmental Biology of Fern Gametophytes. Cambridge University Press, Cambridge, UK.CrossRefGoogle Scholar
  55. Schneller, J.J., Haufler, C.H., and Ranker, T.A. 1990. Antheridiogen and natural gametophyte populations. Am. Fern J. 80:143–152.CrossRefGoogle Scholar
  56. Somer, M., Arbesú, R., Menéndez, V., Revilla, M.A., and Fernández, H. 2010. Sporophytes induction studies in ferns in vitro. Euphytica 171: 203–210.CrossRefGoogle Scholar
  57. Steeves, T.A. 1962. Morphogenesis in isolated fern leaves. In Regeneration, 20th Growth Symposium, D. Rudnick (ed.), Ronald Press, New York, pp. 117–151.Google Scholar
  58. Teng, W.L., and Teng, M.C. 1997. In vitro regeneration patterns of Platycerium bifurcatum leaf suspension culture. Plant Cell Rep 16:820–824.CrossRefGoogle Scholar
  59. Tyron, R.M., and Vitale, G. 1977. Evidence for antheridiogen production and its mediation of a mating system in natural populations of fern gametophytes. Bot. J. Linn. Soc. 74:243–249.CrossRefGoogle Scholar
  60. von Aderkas, P. 1986. Enhancement of apospory in liquid culture of Matteuccia strupthiopteris. Ann. Bot. 57:505–510.Google Scholar
  61. Wagner, W.H., and Smith A.R. 1993. Pteridolophytes. In Flora of North America North of Mexico, Flora of North America Editorial Committee (ed.), pp.247–266. Oxford University Press. New York.Google Scholar
  62. Whittier, D.P. 1975. The influence of osmotic conditions on induced apogamy in Pteridium ­gametophytes. Phytomorphology 25:246–249.Google Scholar
  63. Whittier, D.P. 2008. Red light inhibition of spore germination in Lycopodium clavatum. Am. Fern J. 98:194–198.CrossRefGoogle Scholar
  64. Whittier, D.P., and Steeves, T.A. 1960. The induction of apogamy in the bracken fern. Can. J. Bot. 38:925–930.CrossRefGoogle Scholar
  65. Whittier, D.P., and Steeves, T.A. 1962. Further studies on induced apogamy in ferns. Can. J. Bot. 40:1525–1531.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • V. Menéndez
  • R. Arbesú
  • M. Somer
  • A. Revilla
  • H. Fernández
    • 1
  1. 1.Area Plant Physiology, Departamento de Biología de Organismos y SistemasUniversidad de OviedoOviedoSpain

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