Bryophyte Ex Situ Conservation at the Royal Botanic Gardens,Kew: Past, Present and Future

Transcription

1 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

2 Global Strategy for Plant Conservation 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.

3 Ex situ Conservation Millennium Seedbank Partnership holds largest and most diverse collection of wild species in the world Ex situ & in vitro collections important part of integrated conservation programmes BUT in general focus on vascular, seedbearing plants

4 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

5 UK Bryophytes The UK has an exceptional bryophyte flora (c species) - 53% of European bryophyte flora - Atlantic bryophyte communities of global importance Rhynchostegium rotundifolium - Critically endangered

6 Conservation Biotechnology at RBGKew

7 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 reestablishment of threatened UK bryophytes Bartramia stricta

8 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

9 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 roup=6 )

10 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

11 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

12 Wiessia levieri (Endangered)

13 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 (2 nd sterilisation step )

14 Entosthodon pulchellus (Near Threatened)

15 Splachnum vasculosum ( Vulnerable )

16 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

17 Cryopreservation of Bryophytes Sphagnum re - growing after cryopreservation O.gracile re-growing after cryopreservation Rowntree J. K., and Ramsay M.M. (2009) How bryophytes came out of the cold: successful cryopreservation of threatened species. Biodivers Conserv 18:

18 Encapsulation/dehydration Encapsulation in alginate-coated filter paper strips 2 weeks pretreatment with 5% sucrose 10 µm ABA 6 hrs dehydration in laminar flow bench Rapid direct freezing in Liquid Nitrogen, vials stored in boxes in Dewar

19 Thawing and Recovery Regeneration rates from frozen> 68 % of all species tested; half had regeneration rates of 100%

20 UK priority species in cryostorage post Phase 1 & 2 of Ex situ Project Species Aplodon wormskjoldii Bartramia stricta Bryum calophyllum Bryum schleicheri Bryum uliginosum World red list European red list ECCB (1995) Revised British red list (Hodgetts 2011) Critically Endangered Critically Endangered Vulnerable Critically Endangered Extinct/Regionally 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 Rhynchostegium rotundifolium Seligeria carnicolica Endangered Critically Endangered Critically Endangered Sematophyllum demissum Vulnerable 21 species, 29 collections. Tortula cernua Endangered Weissia multicapsularis Endangered Critically Endangered Weissia rostellata Near threatened?

21 Desiccation Tolerance Ditrichum plumbicola 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 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)

22

23 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):

24 Genetic Diversity Maintaining genetic diversity Sampling protocols Genetic Changes, Somaclonal Variation Effect of life cycle? Impact of vegetative reproduction

25 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

26 EBEsCoNet

27 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

28 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

29 Implications of Bryophyte Reproductive Biology Breeding System No of Species % of Species Dioecious Normally dioecious, rarely monoecious Normally monoecious, rarely dioecious Monoecious or dioecious Monoecious Gametangia not known Sporophyte Frequency No of Species % of Species Abundant Frequent Occasional Rare Not in Britain or Ireland but found elsewhere Sporophytes not known

30 Research Questions Spore storage methods ( -20 C and Cryopreservation) Axenic culture/cryopreservation of liverworts Liverwort/fungal symbioses DNA banking/barcoding Dessication Biology

31 Archive Cryostorage New facilities at MSB one vessel can store cryovials in long-term archival storage

32 THANK YOU!

33 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