Plant Cell, Tissue and Organ Culture

, Volume 87, Issue 2, pp 191–201 | Cite as

Development of novel methods for the initiation of in vitro bryophyte cultures for conservation

  • J. K. RowntreeEmail author
Original Paper


The value of ex situ plant collections for conservation is increasingly recognised. The Royal Botanic Gardens, Kew (RGB, Kew) has been pioneering methods for the development of such collections with the establishment of the Millennium Seed Bank and a project for the ex situ conservation of threatened UK bryophytes (mosses, liverworts, hornworts). A broad range of bryophytes are currently held in a tissue culture collection at RBG, Kew on sucrose-free ¼ or ½ Murashige & Skoog or Knops minimal medium. The efficacy of the novel sterilising agent Sodium dichloroisocyanurate (NaDCC) was tested on a range of taxa, utilising a variety of bryophytic initiation material. Concentrations of 1% (w/v) for 3 min and 0.5% (w/v) for 2 min, without the addition of detergent, were found to be successful in initiating cultures from sporophytes and leafy gametophores respectively. Initiation of cultures from wild-collected sporophytes was more successful than from wild-collected gametophores. However, pre-culturing of gametophore material was found to enhance success rate of procedures. Transferring visibly clean material away from contamination post-initiation was also determined to increase protocol success rate. The ability of the biocide Plant Preservative MixtureTM (PPMTM) to control fungal and bacterial contamination in bryophyte protonemal cultures was also tested, but not found to be suitable. However, results obtained indicated that contamination may confer resistance on the moss against the phytotoxic effects of the biocide. Methods developed have wider applicability for the establishment of in vitro collections of other threatened plants.


Threatened plants Sodium dichloroisocyanurate Plant Preservative MixtureTM Bryophytes Gametophore Sporophyte Protonema 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



The author thanks English Nature, Scottish Natural Heritage, Conservation Council for Wales for providing funding for the project, the lead partners for the UK species Biodiversity Action Plans (Countryside Council for Wales, English Nature, Natural History Museum, Plantlife, Royal Botanic Gardens, Kew Scottish Natural Heritage) for allowing collection and J. G. Duckett, A. D. Headley, N. G. Hodgetts, D. T. Holyoak, D. Long, R. Porley, G. Rothero, F. J. Rumsey and R. Wood for collecting and supplying and verifying the bryophyte material. Thanks also to M. M. Ramsay and two anonymous reviewers for comments on an earlier draft.


  1. Basile DV, Basile MR (1988) Procedures used for the axenic culture and experimental treatment of bryophytes. In: Glime JM (ed) Methods in bryology. Proceedings of the bryological methods workshop, Mainz. The Hattori Botanical Laboratory, Nichinan, Japan, pp 1–16Google Scholar
  2. Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, a revised edition. Elsevier, Amsterdam, The NetherlandsGoogle Scholar
  3. Blockeel TL, Long DG (1998) A check-list and census catalogue of British and Irish bryophytes. British Bryological Society, Cardiff, UKGoogle Scholar
  4. Church JM, Hodgetts NG, Preston CD, Stewart NF (2001) British red data books – mosses and liverworts. JNCC, PeterboroughGoogle Scholar
  5. Duckett JG, Burch J, Fletcher PW, Matcham HW, Read DJ, Russell AJ, Pressel S (2004) In vitro cultivation of bryophytes: a review of practicalities, problems, progress and promise. J Bryol 26:3–20CrossRefGoogle Scholar
  6. George MW, Tripepi RR (2001) Plant Preservative Mixture (TM) can affect shoot regeneration from leaf explants of chrysanthemum, European birch, and rhododendron. Hortscience 36(4):768–769Google Scholar
  7. IUCN (2004) 2004 Red List of Threatened Species. Downloaded on 20th January 2006 Scholar
  8. IUCN SCC Bryophyte Specialist Group (2002) 2000 IUCN World Red List of Bryophytes. Downloaded on 20th January 2006 Scholar
  9. Jules ES, Shaw AJ (1994) Adaptation to metal-contaminated soils in populations of the moss, Ceratodon purpureus – vegetative growth and reproductive expression. Am J Bot 81(6):791–797CrossRefGoogle Scholar
  10. Knop W (1865) Quantitative Untersuchungen ueber die Ernahrungsprozesse der Pflanzen. Landwirtschaftlichen Versuchsstationen 7:93–107Google Scholar
  11. Kraj W, Dolnicki A (2003) The influence of PPM upon the sterility of the in vitro cultures in European beech (Fagus sylvatica L.). Acta Soc Bot Pol 72(4):303–307Google Scholar
  12. Lal M (1984) The culture of bryophytes including apogamy, apospory, parthenogenesis and protoplasts. In: Dyer AF, Duckett JG (eds) The experimental biology of bryophytes. Academic Press, London, pp 97–115Google Scholar
  13. Long RD (1997) Photoautotrophic micropropagation – a strategy for contamination control? In: Cassells AC (ed) Pathogen and microbial contamination management in micropropagation, vol 12. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 267–278Google Scholar
  14. Maunder M, Havens K, Guerrant EO Jr, Falk DA (2004) Ex situ methods: a vital but underused set of conservation resources. In: Guerrant EO Jr, Havens K, Maunder M (eds) Ex situ plant conservation – supporting species survival in the wild. Island Press, Washington, DC, pp 3–20Google Scholar
  15. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3):473–497Google Scholar
  16. Newmaster SG, Bell FW, Vitt DH (1999) The effects of glyphosate and triclopyr on common bryophytes and lichens in northwestern Ontario. Can J For Res 29:1101–1111CrossRefGoogle Scholar
  17. Niedz RP, Bausher MG (2002) Control of in vitro contamination of explants from greenhouse- and field-grown trees. In Vitro Cell Dev Biol Plant 38(5):468–471CrossRefGoogle Scholar
  18. Nowak J, Asiedu SK, Bensalim S, Richards J, Stewart A, Smith C, Stevens D, Sturz AV (1997) From laboratory to applications: challenges and progress with in vitro dual cultures of potato and beneficial bacteria. In: Cassells AC (ed) Pathogen and microbial contamination management in micropropagation, vol 12. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 321–329Google Scholar
  19. Parkinson M, Prendergast M, Sayegh AJ (1996) Sterilisation of explants and cultures with sodium dichloroisocyanurate. Plant Growth Regul 20(1):61–66CrossRefGoogle Scholar
  20. Paul AL, Semer C, Kucharek T, Ferl RJ (2001) The fungicidal and phytotoxic properties of benomyl and PPM in supplemented agar media supporting transgenic arabidopsis plants for a Space Shuttle flight experiment. Appl Microbiol Biotechnol 55(4):480–485PubMedCrossRefGoogle Scholar
  21. Plant-Cell-Technology (2006) PPM: A powerful tool to prevent or eliminate microbial contamination in plant tissue culture. Downloaded on 19th June 2006 Scholar
  22. Porley RD, Hodgetts N (2005) Mosses & liverworts. HarperCollins, London, UKGoogle Scholar
  23. Proctor MCF (2000) Physiological ecology. In: Shaw AJ, Goffinet B (eds) Bryophyte biology. Cambridge University Press, Cambridge, pp 225–247Google Scholar
  24. Ramsay MM, Burch J (2001) Ex situ techniques in support of UK bryophyte conservation. Novit Bot Univ Carol 15:27–33Google Scholar
  25. Ramsay MM, Jackson AD, Porley RD (2000) A pilot study for the ex situ conservation of UK bryophytes. EuroGard 2000 – II European Botanic Gardens Congress. Las Palmas de Gran Canaria, Canary Islands, Spain Google Scholar
  26. Rincon E (1993) Growth responses of six bryophyte species to different light intensities. Can J Bot 71:661–665CrossRefGoogle Scholar
  27. Rowntree JK, Ramsay MM (2005) Ex situ conservation off bryophytes: progress and potential of a pilot project. Bol Soc Esp Briol 26–27:17–22Google Scholar
  28. Rowntree JK, Sheffield E, Burch J (2005) Growth and development of mosses are inhibited by the common herbicide asulam. Bryologist 108(2):287–294CrossRefGoogle Scholar
  29. Rowntree JK, Lawton KF, Rumsey FJ, Sheffield E (2003) Exposure to Asulox inhibits the growth of mosses. Ann Bot (Lond.) 92(4):547–556Google Scholar
  30. Sabovljevic M, Bijelovic A, Dragicevic I (2003) In vitro culture of mosses:Aloina aloides (K.F. Schultz) Kindb., Brachythecium velutinum (Hedw.) B.S.G., Ceratodon purpureus (Hedw.) Brid., Eurhynchium praelongum (Hedw.) B.S.G. and Grimmia pulvinata (Hedw.) Sm. Turk. J Bot 27:441–446Google Scholar
  31. Sarasan V, Cripps R, Ramsay MM, Atherton C, McMichen M, Prendergast G, Rowntree JK (2006) Conservation in vitro of threatened plants – progress in the last decade. In Vitro Cell Dev Biol Plant 42(3):206–214CrossRefGoogle Scholar
  32. Sargent ML (1988) A guide to the axenic culturing of a spectrum of bryophytes. In: Glime JM (ed) Methods in bryology. Proceedings of the bryological methods workshop, Mainz. The Hattori Botanical Laboratory, Nichinan, Japan, pp 17–24Google Scholar
  33. Såstad SM, Bakken S, Pedersen B (1998) Propagation of Sphagnum in axenic culture – a method for obtaining large numbers of cloned gametophores. Lindbergia 23:65–73Google Scholar
  34. Scheffe H (1959) The analysis of variance. John Wiley and Sons, New YorkGoogle Scholar
  35. Smith AJE (2004) The moss flora of Britain & Ireland. Cambridge University Press, Cambridge, UKGoogle Scholar
  36. Sokal I, Kuta E, Przywara L (1997) Callus induction and gametophyte regeneration in moss cultures. Acta Biol Cracov Ser Bot 39:35–42Google Scholar
  37. Sokal RR, Rohlf FJ (1995) Biometry. W.H. Freeman & Co., New YorkGoogle Scholar
  38. SYSTAT-Software-Inc. (2004) SYSTAT. 10 SYSTAT statistics I. Richmond, CA, USAGoogle Scholar
  39. Tanner C (2004) The Millenium Seed Bank. Curtis’s Bot Mag 21(1):91–94CrossRefGoogle Scholar
  40. UK Biodiversity Group (1999) Tranche 2 Action Plans. Volume III – plants and fungi. UK Biodiversity Group & English Nature, PeterboroughGoogle Scholar
  41. UNEP (1992) Convention on Biological Diversity (CBD). Text and Annexes. CBD Secretariat, Montreal, Canada Scholar
  42. UNEP (2002) Global Strategy for Plant Conservation. Decision VI/9 UNEP/CBD/COP/6/20 CBD Secretariat, Montreal, Canada Scholar
  43. Vitt DH (2000) Peatlands: ecosystems dominated by bryophytes. In: Shaw AJ, Goffinet B (eds) Bryophyte biology. Cambridge University Press, Cambridge, pp 17–24Google Scholar
  44. Walter KS, Gillett HJ (1998) 1997 IUCN Red List of Threatened Plants. IUCN, Gland, Switzerland & Cambridge, UKGoogle Scholar
  45. Whipps JM (2001) Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52(suppl 1):487–511PubMedGoogle Scholar
  46. Wiklund K, Rydin H (2004) Ecophysiological constraints on spore establishment in bryophytes. Funct Ecol 18(6):907–913CrossRefGoogle Scholar
  47. Zar JH (1999) Biostatistical analysis. Prentice-Hall, Upper Saddle River, New JerseyGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.Micropropagation UnitRoyal Botanic Gardens, KewRichmondUK

Personalised recommendations