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Bryophyte Ex Situ Conservation at the Royal Botanic Gardens,Kew: Past, Present and Future Margaret M Ramsay, Dr Jennifer K Rowntree, Paul P

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Bryophyte Ex Situ Conservation at the Royal Botanic Gardens,Kew: Past, Present and Future Margaret M Ramsay, Dr Jennifer K Rowntree, Paul P Bryophyte Ex Situ Conservation at the Royal Botanic Gardens,Kew: Past, Present and Future Margaret M Ramsay, Dr Jennifer K Rowntree, Paul P. Smith, Dr Sylvia Pressel, Dr David G. Long Global Strategy for Plant Conservation 2011-2020 • Target 8: At least 75 per cent of threatened plant species in ex situ collections, preferably in the country of origin, and at least 20 per cent available for recovery and restoration programmes. Ex situ Conservation • Ex situ & in vitro collections important part of integrated conservation programmes • BUT in general focus Millennium Seedbank Partnership on vascular , seed- holds largest and most diverse bearing plants collection of wild species in the world Advantages of ex situ conservation for bryophytes • Maintain basal storage ‘insurance’ collection representing genetic diversity • Supply material for re-establishment trials minimising sampling from wild • Resource for auto-ecological research and underlying biological processes • Provide source of uncontaminated DNA for molecular screening UK Bryophytes The UK has an exceptional bryophyte flora (c. 1000 species) - 53% of European bryophyte flora - Atlantic bryophyte communities of Rhynchostegium rotundifolium - global importance Critically endangered Conservation Biotechnology at RBGKew Ex Situ Conservation Project for UK Bryophytes: Phase 1 and 2 A collaborative project with UK statutory conservation agencies to develop and evaluate experimental protocols for the collection, in vitro propagation, cryogenic storage and re- establishment of threatened UK bryophytes Bartramia stricta Outputs of Ex Situ Project • 21 Priority species in cryo-storage • Material deposited in DNA bank • Re-introduction trial initiated • Peer-reviewed scientific papers • Techniques developed for: – Initiation & propagation in aseptic culture – ‘weaning’ of ex situ material – cryopreservation protocols Phase 3 • Funding from Natural England for 3 years to apply propagation and cryopreservation techniques to secure addition priority taxa in the existing ex situ collection. • 111 UK BAP priority bryophytes (majority with ex situ conservation a priority action http://www.ukbap.org.uk/PrioritySpecies.aspx?g roup=6 ) Photo-autotrophic Culture • Bryophytes are autotrophic in culture • Grown on ½ or ¼ MS , Knops no sucrose • Gelrite used as a gelling agent for species susceptible to agar impurities. • Sealing Petri dishes with micropore tape allows air exchange Culture Initiation: Sporophytes •Initiation from sporophytes generally more successful than leafy gametophores •Sporophyte availability depends on fruiting season and for some species has yet to be recorded. •Sterilising agent Sodium dichloroisocyanurate effective at low concentrations Buxbaumia viridis Wiessia levieri (Endangered) Culture Initiation: gametophoric tissue • Gametophoric tissue much more delicate than sporophytes and often directly contaminated •0.05% SDICN for 3 minutes,or 0.1% for 2 minutes. NO TWEEN •Vacuum filtration effective •Preculturing gametophore material enhances success rate (2nd sterilisation step ) Entosthodon pulchellus (Near Threatened) Splachnum vasculosum ( Vulnerable ) Cryopreservation • Storage in liquid nitrogen (-196°C) secures conservation collections on a long term basis • Suspension of cellular metabolic activities minimises genetic drift • Cost and space efficient Cryopreservation of Bryophytes Sphagnum re - growing after O.gracile re-growing after cryopreservation cryopreservation Rowntree J. K., and Ramsay M.M. (2009) How bryophytes came out of the cold: successful cryopreservation of threatened species. Biodivers Conserv 18:1413-1420 Encapsulation/dehydration Encapsulation in alginate-coated 2 weeks pretreatment with filter paper strips 5% sucrose 10 µM ABA 6 hrs dehydration in laminar flow bench Rapid direct freezing in Liquid Nitrogen, vials stored in boxes in Dewar Thawing and Recovery Regeneration rates from frozen> 68 % of all species tested; half had regeneration rates of 100% European red list Revised British red Species World red list ECCB (1995) list (Hodgetts 2011) UK priority species in Aplodon wormskjoldii Critically Endangered cryostorage post Bartramia stricta Critically Endangered Bryum calophyllum Vulnerable Phase 1 & 2 of Ex Bryum schleicheri Critically Endangered Extinct/Regionally Bryum uliginosum situ Project extinct Bryum warneum Near Threatened Buxbaumia viridis Vulnerable Near Threatened Cyclodictyon laetevirens Endangered Ditrichum cornubicum Critically Endangered Endangered Endangered Ditrichum plumbicola Vulnerable Near threatened ? Jamesoniella undulifolia Vulnerable Endangered Vulnerable Leptodontium gemmascens Near Threatened Micromitrium tenerum Vulnerable Endangered Orthodontium gracile Endangered Vulnerable Orthotrichum pallens Endangered Rhynchostegium Critically Endangered rotundifolium Seligeria carnicolica Critically Endangered Sematophyllum demissum Vulnerable Tortula cernua Endangered Weissia multicapsularis Endangered Critically Endangered 21 species , 29 collections. Weissia rostellata Near threatened? Desiccation Tolerance •Many bryophytes exhibit desiccation tolerance •Survival post-cryopreservation enhanced with pre-treatment with ABA and sucrose. •Cytological studies of pioneer moss Ditrichum plumbicola provided insights into its reproductive biology Ditrichum plumbicola Rowntree JK, Duckett JG ,Mortimer CL, Ramsay MM , Pressel S ( 2007) Formation of specialized propagules resistant to desiccation and cryopreservation in the threatened moss Ditrichum plumbicola ditrichales, bryopsida) Orthodontium gracile Rowntree J.K., Cowan R.S., Leggett M., Ramsay M.M. and Fay M.F. (2010) Which moss is which? Identification of the threatened moss Orthodontium gracile using molecular and morphological techniques . Conservation Genetics 11(3): 1033-1042 Genetic Diversity • Maintaining genetic diversity • Sampling protocols • Genetic Changes, Somaclonal Variation • Effect of life cycle? • Impact of vegetative reproduction European Network • Dissemination and development of protocols including training of personnel • Co-ordinate collection of priority species across Europe • Collections duplicated at multiple locations and include components of tissue culture , cryopreservation and spore collections Rowntree ,J.,K. Pressel,S., Ramsay,M.M., Sabovljevic,A., Sabovljevic,M. (2011) In vitro conservation of European bryophytes. In Vitro Cell.Dev.Biol.—Plant (2011) 47:55–64 EBEsCoNet http://www.ebesconet.org UK Bryophyte Genebank • Feasibility planning with NHM, RBGE and BBS for a comprehensive ex situ programme for UK bryophytes • Aim to systematically collect and store at least half of the UK bryophyte flora • To carry out research to improve storage protocols and to enable the use of bryophyte collections in restoration and reintroduction programmes Collection Programme • Use of amateur networks ‘citizen science’ • Expertise needed for species selection and co-ordination of collecting • Implications of bryophyte reproductive biology • Genetic diversity sampling Implications of Bryophyte Reproductive Biology Breeding System No of Species % of Species Dioecious 609 57.6 Normally dioecious, rarely monoecious 4 0.4 Normally monoecious, rarely dioecious 8 0.8 Monoecious or dioecious 25 2.3 Monoecious 395 37.4 Gametangia not known 16 1.5 Sporophyte Frequency No of Species % of Species Abundant 265 25.1 Frequent 189 17.9 Occasional 140 13.2 Rare 248 23.5 Not in Britain or Ireland but found elsewhere 151 14.3 Sporophytes not known 64 6.0 Research Questions • Spore storage methods ( -20°C and Cryopreservation) • Axenic culture/cryopreservation of liverworts • Liverwort/fungal symbioses • DNA banking/barcoding • Dessication Biology Archive Cryostorage New facilities at MSB – one vessel can store 37400 cryovials in long-term archival storage THANK YOU ! Acknowledgements • Natural England for funding Phase 1, 2 & 3 • Scottish Natural Heritage, Countryside Council for Wales for funding Phases 1& 2 • Staff and students of CBU past and present , especially Dr Jane Burch and Carla Mortimer .
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