Bryophyte Ex Situ Conservation at the Royal Botanic Gardens,Kew: Past, Present and Future - Margaret M Ramsay, Dr Jennifer K Rowntree, Paul
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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. 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
exchangeCulture 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 viridisWiessia 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 18:1413-1420Encapsulation/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 DewarThawing 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
Bryum calophyllum
Critically Endangered
Vulnerable
Phase 1 & 2 of Ex Bryum schleicheri Critically Endangered
situ Project Bryum uliginosum
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 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) 47:55–64EBEsCoNet 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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