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