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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
on vascular , seed-
bearing plants Millennium Seedbank Partnership
holds largest and most diverse
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
global importance Rhynchostegium rotundifolium -
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
Buxbaumia viridis
•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
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
O.gracile re-growing after cryopreservation Sphagnum 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 18:1413-1420
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
Thawing and Recovery
Regeneration rates from
frozen> 68 % of all species
tested; half had regeneration
rates of 100%
Species World red list European red list
ECCB (1995)
Revised British red
list (Hodgetts 2011)
Aplodon wormskjoldii Critically Endangered
Bartramia stricta Critically Endangered
Bryum calophyllum Vulnerable
Bryum schleicheri Critically Endangered
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
rotundifolium Critically Endangered
Seligeria carnicolica Critically Endangered
Sematophyllum demissum Vulnerable
Tortula cernua Endangered
Weissia multicapsularis Endangered Critically Endangered
Weissia rostellata Near threatened?
UK priority species in
cryostorage post
Phase 1 & 2 of Ex
situ Project
21 species , 29 collections.
Desiccation Tolerance
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)
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
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