AOBPreview published online on July 30, 2007
Annals of Botany, doi:10.1093/aob/mcm141
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Formation of Specialized Propagules Resistant to Desiccation and Cryopreservation in the Threatened Moss Ditrichum plumbicola (Ditrichales, Bryopsida)
1 Micropropagation Unit, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3AB, UK
2 School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK
3 Faculty of Life Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK
4 Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
* For correspondence. E-mail jkrowntree{at}mac.com
Received: 24 January 2007 Returned for revision: 19 March 2007 Accepted: 25 May 2007
Background and Aims: Successful cryopreservation of bryophytes is linked to intrinsic desiccation tolerance and survival can be enhanced by pre-treatment with abscisic acid (ABA) and sucrose. The pioneer moss Ditrichum plumbicola is naturally subjected to desiccation in the field but showed unexpectedly low survival of cryopreservation, as well as a poor response to pre-treatment. The effects of the cryopreservation protocol on protonemata of D. plumbicola were investigated in order to explore possible relationships between the production in vitro of cryopreservation-tolerant asexual propagules and the reproductive biology of D. plumbicola in nature.
Methods: Protonemata were prepared for cryopreservation using a four-step protocol involving encapsulation in sodium alginate, pre-treatment for 2 weeks with ABA and sucrose, desiccation for 6 h and rapid freezing in liquid nitrogen. After each stage, protonemata were prepared for light and electron microscopy and growth on standard medium was monitored. Further samples were prepared for light and electron microscopy at intervals over a 24-h period following removal from liquid nitrogen and re-hydration.
Key Results: Pre-treatment with ABA and sucrose caused dramatic changes to the protonemata. Growth was arrested and propagules induced with pronounced morphological and cytological changes. Most cells died, but those that survived were characterized by thick, deeply pigmented walls, numerous small vacuoles and lipid droplets in their cytoplasm. Desiccation and cryopreservation elicited no dramatic cytological changes. Cells returned to their pre-dehydration and cryopreservation state within 2 h of re-hydration and/or removal from liquid nitrogen. Regeneration was normal once the ABA/sucrose stimulus was removed.
Conclusions: The ABA/sucrose pre-treatment induced the formation of highly desiccation- and cryopreservation-tolerant propagules from the protonemata of D. plumbicola. This parallels behaviour in the wild, where highly desiccation-tolerant rhizoids function as perennating organs allowing the moss to endure extreme environmental conditions. An involvement of endogenous ABA in the desiccation tolerance of D. plumbicola is suggested.
Key words: ABA, asexual propagules, cell-biology, cryopreservation, desiccation tolerance, Ditrichum plumbicola, extremophiles, protonemata, sucrose