Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International

Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
Journal of Botanic Gardens Conservation International

                                  Volume 9 • Number 1 • January 2012

         Botanic gardens -
   science, innovation and
       pushing boundaries
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
Volume 9 • Number 1      EDITORIAL SARA OLDFIELD   02     18
                                                                                                 EXCITING HORTICULTURAL
EDITORS                                                                                          CHALLENGES AT THE OMAN
                                                                                                 BOTANIC GARDEN
                                                                                                 IAN OLIVER, KHALID AL-FARSI,
                                                                                                 ABDULLAH AL-HOSNI, SALIM
                                                                                                 AL-MAKMARI, SARAH KNEEBONE

                                                                 PLANTS FOR THE FUTURE –
Suzanne Sharrock                  Sara Oldfield                  A FUTURE FOR OUR PLANET
Director of Global                Secretary General
Programmes                                                       TOWARDS A PROTOCOL FOR
                                                                 GENETIC MANAGEMENT OF EX
Cover Photo: Royal Botanic Gardens, Kew (BGCI)

Design: John Morgan, Seascape
                                                                 SITU LIVING PLANT COLLECTIONS    21
                                                                 STÉPHANIE SAMAIN &
                                                                 EDUARDO CIRES                   THE ASSISTED MIGRATION
                                                                                                 DEBATE – BOTANIC GARDENS
                                                                                                 TO THE RESCUE?
BGjournal is published by Botanic Gardens Conservation
                                                                                                 MARIA HÄLLFORS, ELINA VAARA &
International (BGCI). It is published twice a year and is sent                                   SUSANNA LEHVÄVIRTA
to all BGCI members. Membership is open to all interested
individuals, institutions and organisations that support the
aims of BGCI (see inside back cover for Membership
application form).

Further details available from:

• Botanic Gardens Conservation International, Descanso
  House, 199 Kew Road, Richmond, Surrey TW9 3BW
  UK. Tel: +44 (0)20 8332 5953, Fax: +44 (0)20 8332 5956
• BGCI-Russia, c/o Main Botanical Gardens,
  Botanicheskaya st., 4, Moscow 127276, Russia.
                                                                 BARCODE WALES: DNA
  Tel: +7 (095) 219 6160 / 5377, Fax: +7 (095) 218 0525,         BARCODING THE NATION’S           25
                                                                 NATIVE FLOWERING PLANTS AND
• BGCI-Netherlands, c/o Delft University of Technology
  Julianalaan 67, NL-2628 BC Delft, Netherlands
                                                                 CONIFERS NATASHA DE VERE        DREAMING OF SHEEP-EATING
  Tel: +31 15 278 4714 Fax: +31 15 278 2355                                                      PLANTS MATTHEW JEBB
• BGCI-Canarias, c/o Jardín Botánico Canario Viera y Clavijo,
  Apartado de Correos 14, Tafira Alta 35017,
  Las Palmas de Gran Canaria, Gran Canaria, Spain.
  Tel: +34 928 21 95 80/82/83, Fax: +34 928 21 95 81,

• BGCI-China, 723 Xingke Rd., Guangzhou 510650 China.
  Tel:(86)20-37252692. email:
• BGCI-Colombia, c/o Jardín Botánico de Bogotá,                   11
  Jose Celestino Mutis, Av. No. 61-13 – A.A. 59887,
  Santa Fe de Bogotá, D.C., Colombia. Tel: +57 630 0949,
  Fax: +57 630 5075, E-mail:,     GeoCAT - AN OPEN SOURCE          28         TOOL FOR RAPID RED LIST
• BGCI(US) Inc, c/o Chicago Botanic Garden,                      ASSESSMENTS                     GROWING THE SOCIAL ROLE OF
  1000 Lake Cook Road, Glencoe, Illinois 60022, USA.
  E-mail:,                         STEVEN BACHMAN & JUSTIN MOAT    BOTANIC GARDENS BGCI
BGCI is a worldwide membership organisation established in
1987. Its mission is to mobilise botanic gardens and engage
partners in securing plant diversity for the well-being of
people and the planet. BGCI is an independent organisation
registered in the United Kingdom as a charity (Charity Reg No
1098834) and a company limited by guarantee, No 4673175.
BGCI is a tax-exempt 501(c)(3) non-profit organisation in the
USA and is a registered non-profit organisation in Russia.
Opinions expressed in this publication do not necessarily
reflect the views of the Boards or staff of BGCI or of its

                                                                  14                              32
                                                                 FROM FREEZING TO THE FIELD—     SAFEGUARDING AUSTRALIA’S
                                                                 IN VITRO METHODS ASSISTING      FLORA THROUGH THE
                                                                 PLANT CONSERVATION              AUSTRALIAN SEED BANK
                                                                 VALERIE PENCE                   PARTNERSHIP LUCY SUTHERLAND

 01                                                                                                BGCI • 2012 • BGjournal • Vol 9 (1)
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International

       he study and cultivation of plant       botanic gardens that will be launched in

T      diversity by botanic gardens is
       firmly rooted in the past but
innovative new approaches look to the
                                               May this year with an ambitious action
                                               plan to restore 100 priority sites around
                                               the world. This scheme is a response to
future. Advances in science and                the urgent need to prevent the further
technology are providing new                   loss of ecosystem services and to
opportunities to understand plant              support international biodiversity policy
diversity and develop solutions to             objective. Availability of appropriate
current environmental problems. This           plant material is one of the prerequisites
issue of BGjournal celebrates innovation       for meeting this objective. Botanic
in botanic gardens – pushing the               gardens collectively hold around one
boundaries of scientific knowledge and         third of all plant species and thus
its practical application. It also considers   provide a vital store for restoration.
the engagement of gardens with their           The Australian Seed Bank Partnership
visitors to share the science and make         described by Sutherland is an exciting
environmental knowledge more                   new approach strengthening Australia’s
accessible and relevant to wider               capacity to restore and connect
audiences.                                     landscapes and ecosystems through
                                               seed-based restoration. The paper by
Scientific research has been an                Samain and Cires provides an example
important component of the work in             of how material in collections can be
botanic gardens for over 500 years.            evaluated to show its appropriateness
Documented plant collections continue          for restoration in the wild. The authors
to provide living laboratories and models      remind us that to be of conservation
of ecological interactions. From               value, living collections must be
observation of whole plants to genetic         genetically representative and they
and DNA analysis new techniques are            describe the first comprehensive study         grow all the native species of Oman is
enabling deeper understanding of the           of three plant groups sampled in the wild      described by Oliver, Al-Farsi, Al-Hosni,
plants in collections that have important      and in living collections.                     Al-Makmari and Kneebone.
implications for management of diversity
in the wild. The application of new            Restoration needs to take into account         I hope you enjoy the richness of ideas
techniques for identifying plant species       changing climatic conditions and the           presented in this issue of BGjournal!
is described by de Vere; for                   somewhat controversial question of             BGCI will strive to share the best of
documenting their conservation status          assisted migration is tackled by Hallfors,     botanic garden innovations, and at times
by Bachman and Moat, for conserving            Vaara and Lehvavirta in their article that     provocative, ideas. Please let us know
in vitro by Valerie Pence and                  highlights the responsibility, skills and      your thoughts and ideas for future
communicating with the public by               opportunities that botanic gardens have        debate as we collectively secure planet
Matthew Jebb.                                  in this emerging field.                        diversity for people and the planet.

The ex situ collections of plant material      The creation of new botanic gardens
and expertise within botanic gardens are       allows the opportunity for research to
increasingly being used to support             be fully integrated with garden design,
species reintroduction and ecological          management of living collections,
restoration programmes. BGCI is                education and training from the outset.
currently involved in the development of       Exciting progress is the development of        Sara Oldfield
the Ecological Restoration Alliance of         the Oman Botanic Garden that aims to           Secretary General, BGCI

   02                                                                                       BGCI • 2012 • BGjournal • Vol 9 (1) • 02
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
Authors: Stéphanie Samain and Eduardo Cires


There is an urgent need for worldwide assessment of                                                              Introduction

genetic diversity of ex situ living plant collections and                                                           t is estimated that one third of all plant

comparison with wild populations, especially for                                                                I   species are threatened with extinction
                                                                                                                    and yet plants continue to be
                                                                                                                 underrepresented in conservation
threatened species. The Botanical Garden of Ghent                                                                debates and policies. However, global
                                                                                                                 initiatives, such as the Global Strategy
University, Belgium is addressing this need.                                                                     for Plant Conservation (GSPC) are
                                                                                                                 aiming to bring a significant change. As
                                                                                                                 stated in Target 8 of the GSPC, at least
                                                                                                                 75% of threatened plant species should
                                                                                                                 be present in ex situ collections.
                                                                                                                 Botanical gardens play a key role in
                                                                                                                 conservation of plant diversity, but only
                                                                                                                 about 30% of globally threatened plant
                                                                                                                 species are currently held in their living
                                                                                                                 collections (Oldfield, 2010). To be of
                                                                                                                 conservation value, living collections
                                                                                                                 must be genetically representative.
                                                                                                                 Although information on genetic diversity
                                                                                                                 in ex situ collections is scarce, it is
                                                                                                                 thought that diversity is low in
                                                                                                                 collections of numerous species.
                                                                                                                 Moreover, besides the many studies and
                                                                                                                 initiatives for specific taxa, our
                                                                                                                 knowledge about the genetic diversity of
                                                                                                                 threatened plant species in general is
                                                                                                                 quite limited. This lack of insight into
                                                                                                                 genetic diversity of threatened wild plant
                                                                                                                 species contrasts with the broad
                                                                                                                 knowledge about crops and their wild
                                                                                                                 relatives. This fact is also reflected by
                                                                                                                 GSPC Target 9, which specifically states
                                                                                                                 that 70% of the genetic diversity of
                                                                                                                 crops, including their wild relatives and
                                                                                                                 other socio-economically valuable plant
                                                                                                                 species, should be conserved. We still
                                                                                                                 have a long way to go to understanding
                                                                                                                 genetic diversity in threatened plants,
                                                                                                                 conserving an important part of this
                                                                                                                 diversity in ex situ collections, defining
                                                                                                                 which percentage of genetic diversity
Esteban Martínez climbing a tree to collect a newly discovered Hydrangea species on the volcano Tacaná in        might be feasible and/or desirable to aim
Chiapas state in southeast Mexico near the border with Guatemala. Stems with leaves of this species are          for, and finally making this available for
visible on all the trunks (Marie-Stéphanie Samain).                                                              conservation and restoration projects.

   03                                                                                                       BGCI • 2012 • BGjournal • Vol 9 (1) • 03-06
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
Angiosperm groups with different life
                                                                                                        histories and growth forms. A range of
                                                                                                        genomic tools will be applied to wild and
                                                                                                        ex situ individuals of selected species of
                                                                                                        the flagship groups Hydrangea, Magnolia
                                                                                                        and Cactaceae to enable rapid
                                                                                                        characterization of genetic variation,
                                                                                                        providing 1) support for specific
                                                                                                        conservation actions, and 2) general
                                                                                                        guidelines and a protocol on genetic
                                                                                                        management for acquiring accessions for
                                                                                                        ex situ collections, in order to be used by
                                                                                                        collectors and botanical gardens
                                                                                                        worldwide in the framework of the
                                                                                                        achievement of Target 8 of the GSPC.

                                                                                                        Sampling and lab work

A happy team of botanists and local children after discovering a new Hydrangea species on the volcano   The three plant groups being studied are
Tacaná in Chiapas state in southeast Mexico near the border with Guatemala (Paco Najarro).              considered as priority groups for BGCI,
                                                                                                        the IUCN/SSC Global Trees Specialist
Setting the stage                                       habitat destruction narrowing genetic           Group and the International Organization
                                                        diversity and subsequent evolution              for Succulent Plant Study (IOS), with
The GSPC 2011-2020 states that:                         (= genetic erosion), 2) collection bias         whom we cooperate. These groups are
                                                        (‘easy’ localities, selection of                rather well-studied taxonomically and
                                                        morphological variation which is not            are also very important in the

“     Without plants there is no
life. The functioning of the
planet, and our survival, depends
                                                        necessarily reflected by genetic variation),
                                                        3) cultivation bottleneck (accessions
                                                        dying because of unsuitable conditions).
                                                                                                        horticultural trade, with especially
                                                                                                        Cactaceae also suffering from illegal
                                                                                                        harvest and trade. Despite the fact that
                                                        Hence, the establishment of a protocol          these groups are a priority for
upon plants.
Achieving Target 8 of the GSPC depends
on the existence of genetically
                                                        guiding genetic management during the
                                                        different steps (wild population – ex situ
                                                        collection – reintroduction) is essential for
                                                        implementation of Target 8 of the GSPC
                                                                                                        conservation, the genetic diversity within
                                                                                                        ex situ collections is almost entirely
                                                                                                        unknown, and has not been compared
                                                                                                        with wild populations. There is clearly an
representative collections, and these are               and, ultimately, for safeguarding the           urgent need for this research.
essential for recovery and restoration                  future of our planet.
work. Our focus should therefore be on
assessing and ensuring the conservation                 Towards an assessment for
value of ex situ collections (Sharrock et               flagship groups: Hydrangea,
al., 2010).                                             Magnolia and Cactaceae

Assessments of a representative sample                  The Botanical Garden and the Research
of plant species will provide a basis for               Group Spermatophytes of Ghent
an initial estimation of a baseline, and to             University (Belgium) are increasingly
monitor progress towards this target.                   involved in conservation of the plant
Therefore, toolkits under this target need              groups which are housed in their living
to include protocols for genetic                        collections and studied in ongoing
management of ex situ collections and                   projects, such as Peperomia (Samain et
consequent reintroductions.                             al., 2010), Hydrangea (Red List project in
                                                        cooperation with BGCI, including a
One of the suggested milestones to serve                specific conservation project in Mexico),
as a step towards the 2020 target is that               and Aristolochia (Rivera Hernández &
ex situ collections of all critically                   Samain, 2011). Our strategy, to combine
endangered species should be                            fundamental botanical research with
genetically representative of the species               conservational efforts, has also led to the
(SBSTTA, 2010). However, preliminary                    establishment of a project, initiated in
observations and data suggest that this                 2011, which will be continued and
currently is not the case in many                       accelerated thanks to the support of the
collections, as the genetic diversity from              Fondation Franklinia. Within this project
wild population to ex situ collections                  we aim to compare genetic diversity
‘gradually’ decreases (= genetic                        between wild populations and ex situ            Collecting Hydrangea aspera with local botany students
bottleneck) as a consequence of 1)                      collections of three unrelated                  on mountain Taiping Shan, Taiwan ( Eduardo Cires).

   04                                                                                                      BGCI • 2012 • BGjournal • Vol 9 (1)
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
Characteristic                        RFLP                     Microsatellite                    AFLP                      ISSR                PCR sequencing

             Genomic abundance                      High                         Medium                      Very high                  Medium                     Very high
           Part of genome surveyed         Low copy coding regions             Whole genome                Whole genome               Whole genome               Whole genome
           Amount of DNA required                   High                           Low                       Medium                       Low                        Low

            Type of polymorphism              Single base changes,            Changes in length        Single base changes,        Single base changes,       Single base changes,
                                               insertion, deletion               of repeats             insertion, deletion         insertion, deletion        insertion, deletion
            Level of polymorphism                  Medium                           High                      Very high                    High                    Very high
                  Inheritance                     Codominant                    Codominant                    Dominant                  Dominant                       –
                  Ease of use                   Labour intensive                    Easy                   Difficult initially             Easy                      Easy
             Automation possible                     Low                            High                       Medium                    Medium                      High
              Cost of automation                     High                           High                         High                    Medium                      High
              Development costs                    Medium                           High                         Low                       Low                       High
          Reproducibility (reliability)              High                           High                         High                 Medium to high                 High
           Level of training required                Low                        Low/Medium                     Medium                      Low                       High
          Cloning and/or sequencing                  Yes                            Yes                           No                        No                        Yes
             Radioactive detection                  Yes/No                         Yes/No                       Yes/No                      No                      Yes/No
           Proprietary rights status                  No                     No (some licensed)                Licensed                 Licensed              No (some licensed)

       Table 1. Comparative assessment of some of the salient characteristics of different molecular genetic screening techniques: RFLP (or Restriction fragment length polymorphism),
       Microsatellites or simple sequence repeats (SSRs), AFLP (or Amplified fragment length polymorphism), ISSR (or Inter Simple Sequence Repeat) and PCR sequencing.

            “    Around one-third of the                                 much reduced, but we believe it is not                  Field work has already been or will be
                                                                         useful to include samples without                       performed in countries and areas where
            globally threatened species that                             documented origin, as their value, for                  the Research Group has extensive field
            are found in botanic garden                                  example in reintroduction projects, is                  experience and local cooperation
                                                                         highly doubtful. Additionally, Magnolia                 partners (Latin America and East Asia).
            collections are recorded in only                             and Hydrangea individuals are plants                    Within the wild populations, a

            one collection.                                              with life spans of several hundred years,               representative sample of individuals will
                                                                         predating fragmentation events, so we                   be collected, meanwhile in the ex situ
                                                                         will obtain reliable genetic information                collections we aim to include an equal
            Global and/or local Red Lists are                            that may help genetic restoration                       number of accessions from as many
            available for each group (Magnolia:                          projects (e.g. López et al., 2008).                     gardens as possible. In addition, our
            Cicuzza et al., 2007; Cactaceae: e.g.                                                                                work on Hydrangea will also help to build
            IUCN, 2011) or are being prepared                            Availability in botanical garden collections            new living collections, e.g. in Mexico,
            (Hydrangea s.l.). These groups represent                     has been checked via the PlantSearch                    supported by the Mohamed Bin Zayed
            different unrelated families of                              module on the BGCI website                              Species Conservation Fund.
            Angiosperms, with different habitat                          ( or               Molecular tools provide valuable data on
            preferences and a range of life history                      via direct and ongoing communication                    diversity through their ability to detect
            traits. Within each group, taxa are                          with gardens. A final selection criterion is            variation at the DNA level. Identification
            selected based on: 1) presence in a                          that most of the species are ‘Vulnerable’,              is of fundamental importance in diversity
            representative number of public and                          ‘Endangered’ or ‘Critically Endangered’                 studies in a variety of different ways. For
            accessible private ex situ collections                       according to IUCN Red List categories                   evaluation of species diversity, it is
            (botanical gardens, arboreta, etc.), 2)                      and criteria, hence they are priority                   essential that individuals can be
            well-documented origin of the                                species for conservation and for use in                 classified accurately. The identification of
            accessions, and 3) availability of material                  recovery and restoration work.                          taxonomic units and endangered
            verified by specialists. Because of the                      Furthermore, the genetic diversity of these             species, whose genetic constitution is
            second criterion, the quantity of useful                     rare species will be compared with some                 distinct from their more abundant
            accessions available for the research is                     more common or widespread species.                      relatives, is important in the
                                                                                                                                 development of appropriate
                                                                                                                                 conservation strategies. Taking
                                                                                                                                 advantage of current molecular
                                                                                                                                 techniques, relatively quick assessments
  Try another molecular                         Did we detect enough                     Can we divide the population            of genetic diversity in plants can be
                                   No                                          Yes
  marker and/or primers                          genetic variability?                     into k independent groups              performed using well-sampled material
                                                                                                    a priori?
                                                                                                                                 and ready-made protocols, as long as
                                                                                                                                 the most limiting factor, funding, is
                                                        Yes                                                                      available. For each flagship species,
Flow chart representing the                                                                                                      different molecular techniques (e.g.
main strategies to assess                                                                                                        RFLP, microsatellites, AFLP, ISSR, PCR
the genetic diversity in the                      Are these groups                                                               sequencing) are currently being tested
                                   Yes           consistent with the            No
flagship groups studied.                                                                   Reinforcing the sampling              and/or developed, with comparisons of
                                                genetic parameters?
                                                                                                                                 cost versus data yielded, ease of use,
                                                                                             Asian populations                   and applicability for conservation
                                                                                            American populations

                       Identification of priority                          Choose those n
                      populations to be ex situ                        populations which best
                              conserved                                 represent each group                                        BGCI • 2012 • BGjournal • Vol 9 (1)
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
research (Table 1). For the elaboration of              conserved), 4) indication of the number
these techniques, either in-house                       of samples that needs to be collected in
experience has been developed or                        the wild to obtain a genetically
collaboration with experienced research                 representative collection, 5) testing the
groups has been set up.                                 hypothesis that endangered species with
                                                        narrow distribution are genetically
Outlook – Connecting the dots                           limited, 6) conservation of flagship
                                                        species, and finally, 7) reports and
To our knowledge, this is the first                     publications in cooperation with e.g.
comprehensive study of this kind, with a                BGCI, the IUCN/SSC Global Trees
constant evaluation of the different                    Specialist Group and the IOS.
techniques, in order to assess the                      Last but not least, it has already become
progress of the project (see Figure 1).                 clear during this pilot year 2011 that this   Marie-Stéphanie Samain preparing herbarium
The expected results can be subdivided                  study will lead to further international      specimens of a new Hydrangea species of Los
in two levels: i) specific results for each             cooperation with many interested              Tuxtlas, state of Veracruz, Mexico (Esteban Martínez).
group: they will provide insight into                   gardens and researchers.
genetic diversity and history, providing a                                                            , López, D.M., Bock, B.C. and Bedoya,
basis for further conservation work; ii)                                                                G. 2008. Genetic structure in remnant
comparison of different unrelated groups
with different life histories and growth
forms: this approach will lead to a
                                                        “    We expect to form a
                                                        scientific network studying
                                                        genetic diversity in plants for
                                                                                                        populations of an endangered Andean
                                                                                                        Magnolia. Biotropica 40: 375-379.

general overview of the genetic diversity                                                             , Oldfield, S. 2010. Plant conservation:
available in wild populations and ex situ               conservation purposes under the                 facing tough choices. Bioscience 30:
collections, as well as of the specific                                                                 778-779.
problems involved with decreasing
genetic diversity from the wild to the
collection, information which will be
                                                        auspices of BGCI.
                                                        Please feel free to contact us if you
                                                                                                      , Rivera Hernández, J.E. and Samain,
                                                                                                        M.S. 2011. Where has Aristolochia
extremely useful for implementation of                  would like to cooperate, exchange               tricaudata (Aristolochiaceae) gone?
Target 8 of the GSPC.                                   experiences, or if you have comments            New record of a critically endangered
                                                        or questions.                                   species in Oaxaca, Mexico. Revista
Some important specific outcomes are                                                                    Mexicana de Biodiversidad 82: 281-
listed here: 1) assessment of percentage                References                                      286.
of genetic diversity present in ex situ
collections worldwide for the three plant               , Cicuzza, D., Newton, A. and Oldfield,       , Samain, M.S., Dugardin, C. and
groups under study, 2) identification of                  S. 2007. The red list of Magnoliaceae.        Goetghebeur, P. 2010. Peperomia
priority populations for ex situ                          Fauna & Flora International.                  reference collection: an ex situ living
conservation, 3) definition of a                          Cambridge, UK.                                plant collection for scientific research.
genetically representative collection                                                                   Proceedings of the 4th Global Botanic
(compared to Target 9 of the GSPC                       , IUCN, 2011. IUCN red list of                  Gardens Congress, June 2010.
stating that 70% of the genetic diversity                 threatened species.
of crops and other socio-economical              (Accessed       , SBSTTA, 2010. Progress in the
valuable plant species should be                          January 2012).                                implementation of Global Strategy for
                                                                                                        Plant Conservation and development
                                                                                                        of a consolidated update beyond
                                                                                                        2010. UNEP/CBD/SBSTTA/14/INF/16.

                                                                                                      , Sharrock, S., Hird, A., Kramer, A. and
                                                                                                        Oldfield, S. (Comp.) 2010. Saving
                                                                                                        plants, saving the planet: Botanic
                                                                                                        Gardens and the implementation of
                                                                                                        GSPC Target 8. BGCI, Richmond, UK.

                                                                                                      Marie-Stéphanie Samain and
                                                                                                      Eduardo Cires
                                                                                                      Ghent University
                                                                                                      Research Group
                                                                                                      Spermatophytes & Botanical
                                                                                                      K.L. Ledeganckstraat 35
                                                                                                      B-9000 Gent
                                                                                                      E-mail: MarieStephanie.Samain
Mountain Wawu Shan, Sichuan, China, location of several Hydrangea specimens (Eduardo Cires).

   06                                                                                                    BGCI • 2012 • BGjournal • Vol 9 (1)
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
Author: Natasha de Vere


  DNA-based identification systems can help to
  identify and monitor species and provide a
  platform for a wide range of applications where
  accurate species identification is required.

                                                                                                         DNA-barcoding at the NBGW laboratory.

       he ability to identify plant species   Correct identification is also vital in order   DNA barcoding uses short sections of DNA

T      is fundamental to our
       understanding of the world around
us. To conserve plants, their habitats and
                                              for us to use plants for food, medicine or
                                              materials. Identification of plants often
                                              relies on morphological examination, but
                                                                                              to act as a unique identifier for species. The
                                                                                              aim of DNA barcoding is to have global
                                                                                              agreement on the regions of DNA and
ecosystems we need to be able to              around the world there is a shortage of         protocols used for different groups of living
identify and monitor species.                 taxonomic experts able to identify              things in order to create an international
                                              species. Beyond this, it is often desirable     resource for species identification. To begin
                                              to be able to identify species from             with, reference DNA databases are

“    Objective 1 of the Global
Strategy for Plant Conservation
is that: “plant diversity is well
                                              material where morphological
                                              approaches are difficult or impossible to
                                              use; for example, from pollen, roots,
                                                                                              developed using species identified by a
                                                                                              taxonomic expert; unknown DNA
                                                                                              sequences can then be compared to these
                                              seeds, or fragments of tissue. In these         to make an identification. Open Science is
understood, documented and                    situations DNA-based identification             key, DNA barcodes, their associated
                                              systems can be used and in 2003 Paul            information and protocols for their
               ”                              Hebert coined the term ‘DNA barcoding’
                                              (Hebert et al., 2003).
                                                                                              development should be available to
                                                                                              everyone, from researchers, regulatory
                                                                                              authorities to the general public.

   07                                                                                   BGCI • 2012 • BGjournal • Vol 9 (1) • 07-10
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
barcodes need to have access to this
                                                                                           information, along with a scan of the
                                                                                           herbarium voucher. Where fresh material
                                                                                           is collected for DNA barcoding an
                                                                                           associated herbarium voucher must
                                                                                           always be made. The only exception to
                                                                                           this is for endangered species where
                                                                                           creating a voucher is not possible on
                                                                                           conservation grounds; in this case a
                                                                                           photograph is used instead. We also
                                                                                           need to DNA barcode more than one
                                                                                           specimen for each species to allow for
                                                                                           errors and any variation between
                                                                                           individuals within the species. The level
                                                                                           of variation within the species should be
                                                                                           low however, as the idea behind DNA
                                                                                           barcoding is to use regions of DNA that
Tephroseris integrifolia ssp. maritima.                                                    differ between species but which are the
                                                                                           same within a species. For our Barcode
There are now initiatives throughout the     bryophytes and at the National Botanic        Wales project we aim to DNA barcode at
world for DNA barcoding species from all     Garden of Wales, we are DNA barcoding         least three samples for each species
of the kingdoms of life, such as the         the native flowering plants and conifers      using the DNA barcode markers rbcL
International Barcode of Life initiative     of Wales, with our partners at the            and matK.
(IBOL), which works across 25 countries      National Museum Wales (Hollingsworth
and aims to DNA barcode 5 million            et al., 2011).                                Over the last three years we have
specimens from 500,000 species within                                                      sampled 4,272 plant specimens, 3,637
five years. The Consortium of the                                                          from the National Museum Wales
Barcode of Life (CBOL) is devoted to
promoting DNA barcoding as a global
standard for DNA-based species
                                             “    Botanic gardens are
                                             increasingly using DNA barcoding
                                             as an identification tool for plants
                                                                                           herbarium (NMW) and 635 freshly
                                                                                           collected from throughout Wales. We
                                                                                           have 5,723 DNA barcodes, 3,304 for
identification and the Barcode of Life                                                     rbcL and 2,419 for matK. Of the 1,143
Data System (BOLD) provides a key
repository for DNA barcodes and their
associated data (Ratnasingham and
Hebert, 2007). In 2009, the Plant Working
                                             in their collections.

                                             Barcode Wales
                                                                     ”                     species of Wales we have DNA
                                                                                           barcoded 98% using rbcL and 90% with
                                                                                           rbcL and matK. Our first scientific
                                                                                           publication describing our results and
Group of CBOL proposed two sections of       Wales has 1,143 native and                    protocols will be available soon (de Vere
genes within the chloroplast genome,         archaeophyte (species introduced before       et al., in press) and all of our DNA
rbcL and matK, for plant DNA barcoding,      1500 AD) flowering plants and conifers,       barcodes, along with their associated
with the suggestion that more markers        found within 455 genera, 95 families and      collection information and scans of their
may be required (CBOL Plant Working          34 orders. In order to create a reference     herbarium vouchers will be accessible
Group, 2009). In December 2011 the           database of DNA barcodes for these, it        shortly on the Barcode of Life Data
nuclear gene ITS was announced as the        is vital to start with correctly identified   System in the Plants of Wales project.
official barcode for fungi during the 4th    source material. Taxonomist, Dr Tim
International Barcode of Life Conference     Rich, from the National Museum Wales is
in Adelaide, Australia.                      responsible for checking every species
                                             that we DNA barcode. We have
Projects are now underway to DNA             concentrated on using herbarium
barcode the world's plant species. For       specimens for DNA barcoding. The
example, the New York Botanic Garden         herbaria of the world provide an
is DNA barcoding the world's trees,          incredibly valuable resource of plant
whilst the Universities of Adelaide and      species that have been collected and
British Columbia are working on grasses.     identified by expert taxonomists. We
Some projects are concentrating on           have developed special protocols for
floristic regions: Korea University is       extracting DNA from herbarium
working on the flora of Korea, the           specimens to make full use of this
University of Johannesburg on the flora      resource.
of the Kruger National Park, the
Smithsonian Institute on tropical forestry   For the resulting DNA barcodes to be of
plots and institutions throughout China      most use, it is very important that along
are working together to DNA barcode          with the DNA sequence there is a full
their flora. The Royal Botanic Garden        record of when, where and by whom the
Edinburgh is DNA barcoding British           plant was collected. All users of the DNA     The herbarium at the National Museum of Wales.

    08                                                                                        BGCI • 2012 • BGjournal • Vol 9 (1)
Botanic gardens - science, innovation and pushing boundaries - Journal of Botanic Gardens Conservation International
The Barcode Wales project provides a          al., 2011). DNA sequences obtained for     Many of our applications use ‘next
valuable resource for researchers wanting     DNA barcoding have also been used to       generation’ DNA sequencing as this
to identify species using DNA-based           create phylogenetic trees for              allows us to analyse samples containing
approaches. Our DNA barcodes for Wales        phylogenetic community ecology             mixtures of plant species. Our PhD
also provide a stock of barcodes that can     research (Kress et al., 2009).             student, Hannah Garbett, co-supervised
be incorporated into other projects. The                                                 by Dr Tatiana Tatarinova (Glamorgan
1,143 species of Wales represents 77% of      At the National Botanic Garden of Wales    University) is developing bioinformatic
the native flowering plants of the UK. We     we have started to develop applications    tools that will help to analyse these large
have just begun DNA barcoding the rest        that use our DNA barcodes for Wales in     and complicated datasets. As well as
of the UK flora, working with the Royal       collaboration with partners around the     using our DNA barcodes for species
Botanic Garden Edinburgh.                     world. We have worked with Dr Sandra       identification we are working with Prof.
                                              Ronca (Aberystwyth University) and Prof.   Pete Hollingsworth (Royal Botanic
                                              Mike Wilkinson (University of Adelaide)    Garden Edinburgh) to create the first

“    Once the native flora of the
UK is complete we will begin on
                                              to track the movements of pollinators in
                                              threatened habitats by DNA barcoding
                                              pollen found on their bodies. We are
                                                                                         complete national phylogeny of the
                                                                                         flowering plants of the UK.
the non-native species.

                                    ”         working with Dr Joel Allainguillaume
                                              (University of the West of England) to
                                              use DNA barcoding to carry out
                                                                                         Public engagement

                                                                                         DNA barcoding is not only a resource for
                                              ecological surveys and Dr Neil Loader      scientists; it is also an excellent way to
The range of applications using plant         (Swansea University) on reconstructing     engage people with their native flora and
DNA barcoding is as broad as our              landscapes from plant remains in peat      plant sciences research. At the National
imaginations, with some great                 cores. We are also using our DNA           Botanic Garden of Wales we are
applications already being developed.         barcodes for human health in a project     particularly interested in projects that link
Ecological applications include               with Jenny Hawkins and Prof. Les Baillie   arts and science. We have worked with
understanding below-ground biodiversity       of the Welsh School of Pharmacy (Cardiff   botanical artist, Emma Tuck, on a
by DNA barcoding roots (Kesanakurti et        University). For her PhD research, Jenny   contemporary art exhibition and
al., 2011), reconstructing past               is collecting honey samples from           community artist, Pod Clare, on a
landscapes using plant remains                throughout the UK and testing their        Barcode Wales mosaic created by
(Sonstebo et al., 2010) and identifying       ability to fight the hospital infections   community groups from throughout
invasive species (Bleeker et al., 2008).      MRSA and Clostridium difficile. She will   Wales. We are currently working with
DNA barcoding has been used to verify         then DNA barcode the honey to find out     Prof. Andrea Liggins (Swansea
the contents of plant products including      what plant species the bees visited to     University) on a photographic exhibition
medicinal plants (Chen et al., 2010),         make it. We hope to use this to pinpoint   to be shown in botanic gardens and
berries (Jaakola et al., 2010), olive oil     active phytochemicals donated by the       universities throughout China on native
(Kumar et al., 2011), tea (Stoeckle et al.,   plant species that contribute to the       Welsh plants and their DNA barcodes.
2011) and kitchen spices (De Mattia et        honey’s anti-microbial properties.

Collecting specimens for DNA barcoding.

   09                                                                                       BGCI • 2012 • BGjournal • Vol 9 (1)
Barcode Wales is also a mechanism for
training the next generation of plant
scientists. The workforce assembling the
DNA barcodes for the Barcode Wales
and Barcode UK projects are
undergraduate students who spend a
year at the Garden as part of their
degree. Their work is supplemented with
work experience students from A-level to
postgraduate who spend from two
weeks to a few months at the Garden.

The Barcode Wales project provides the          Emma Tuck’s Decode exhibition at NBGW.
most complete coverage of DNA
barcodes of any national flora, offering a        and Wilkinson, M.J. (in press) DNA          Proceedings of the National Academy
platform for a wide range of applications         barcoding the native flowering plants       of Sciences of the United States of
where accurate species identification is          and conifers of Wales. Plos One.            America 106: 18621-18626.
required. It also provides a hub for multi-
disciplinary research across the arts,          , Hebert, P.D.N., Cywinska, A., Ball,       , Kumar, S., Kahlon, T., and Chaudhary,
sciences and social sciences and a                S.L., and DeWaard, J.R. 2003.               S. 2011. A rapid screening for
vehicle for training plant science                Biological identifications through DNA      adulterants in olive oil using DNA
students and volunteers. The DNA                  barcodes. Proceedings of the Royal          barcodes. Food Chemistry 127: 1335-
barcodes and their associated                     Society of London Series B-Biological       1341.
information are Open Access to make a             Sciences 270: 313-321.
resource available to everyone.                                                             , Ratnasingham, S., and Hebert, P.D.N.
                                                , Hollingsworth, P.M., Graham, S.W.,          2007. BOLD: The Barcode of Life Data
References                                        and Little, D.P. 2011. Choosing and         System (
                                                  Using a Plant DNA Barcode. Plos One         Molecular Ecology Notes 7: 355-364.
, Bleeker, W., Klausmeyer, S.,                    6: e19254.
  Peintinger, M., and Dienst, M. 2008.                                                      , Sonstebo, J.H., Gielly, L., Brysting, A.K.,
  DNA sequences identify invasive alien         , Jaakola, L., Suokas, M., and                Elven, R., Edwards, M., Haile, J.,
  Cardamine at Lake Constance.                    Haggman, H. 2010. Novel approaches          Willerslev, E., Coissac, E., Rioux, D.,
  Biological Conservation 141: 692-698.           based on DNA barcoding and high-            Sannier, J., Taberlet, P., and Brochmann,
                                                  resolution melting of amplicons for         C. 2010. Using next-generation
, CBOL Plant Working Group. 2009.                 authenticity analyses of berry species.     sequencing for molecular reconstruction
  A DNA barcode for land plants.                  Food Chemistry 123: 494-500.                of past Arctic vegetation and climate.
  Proceedings of the National Academy                                                         Molecular Ecology Resources 10: 1009-
  of Sciences of the United States of           , Kesanakurti, P.R., Fazekas, A.J.,           1018.
  America 106: 12794-12797.                       Burgess, K.S., Percy, D.M.,
                                                  Newmaster, S.G., Graham, S.W.,            , Stoeckle, M.Y., Gamble, C.C., Kirpekar,
, Chen, S., Yao, H., Han, J., Liu, C.,            Barrett, S.C.H., Hajibabaei, M., and        R., Young, G., Ahmed, S., and Little, D.P.
  Song, J., Shi, L., Zhu, Y., Ma, X., Gao,        Husband, B.C. 2011. Spatial patterns        2011. Commercial Teas Highlight Plant
  T., Pang, X., Luo, K., Li, Y., Li, X., Jia,     of plant diversity below-ground as          DNA Barcode Identification Successes
  X., Lin, Y., and Leon, C. 2010.                 revealed by DNA barcoding. Molecular        and Obstacles. Sci. Rep. 1: 42.
  Validation of the ITS2 Region as a              Ecology 20: 1289-1302.
  Novel DNA Barcode for Identifying                                                         Natasha de Vere
  Medicinal Plant Species. Plos One 5:          , Kress, W.J., Erickson, D.L., Andrew       National Botanic Garden
  e8613.                                          Jones, F., Swenson, N.G., Perez, R.,      of Wales
                                                  Sanjur, O., and Bermingham, E. 2009.      Llanarthne, Carmarthenshire,
, De Mattia, F., Bruni, I., Galimberti, A.,       Plant DNA barcodes and a community        SA32 8HG, UK
  Cattaneo, F., Casiraghi, M., and Labra,         phylogeny of a tropical forest            Email: ndevere@gardenofwales.
  M. 2011. A comparative study of                 dynamics plot in Panama.        
  different DNA barcoding markers for
  the identification of some members of
  Lamiacaea. Food Research                        Further information
  International 44: 693-702.                      National Botanic Garden of Wales
, de Vere, N., Rich, T.C.G., Ford, C.R.,          Consortium for the Barcode of Life
  Trinder, S.A., Long, C., Moore, C.W.,           International Barcode of Life Initiative
  Satterthwaite, D., Davies, H.,                  Barcode of Life Data System
  Allainguillaume, J., Ronca, S.,                 4th International Barcode of Life Conference
  Tatarinova, T., Garbett, H., Walker, K.

   10                                                                                         BGCI • 2012 • BGjournal • Vol 9 (1)
Authors: Steven Bachman and Justin Moat

GeoCAT -
The combination of botanical collection data
and the latest web mapping technology is
providing better tools to support the work
of conservation scientists. The Royal
Botanic Gardens, Kew have developed
GeoCAT – the Geospatial Conservation
Assessment Tool - to help ease and
speed up the process of Red List
assessment for plants.

Plants and the Red List                         There are as many as 380,000
                                                species of plants presently known and
       he IUCN Red List is well known to        around a thousand new species are

T      many as the most authoritative
       source of information on extinction
risk for the world’s organisms. The annual
                                                described as new to science every year1.
                                                There are more species in a single family
                                                of plants such as grasses (Poaceae) than
release highlights the familiar creatures:
the polar bear, the orang-utan, the rhino,
the gorilla - the celebrity species of the
                                                there are all known mammals (5,488). With
                                                that in mind it is no wonder that so far
                                                only around 4% of the world’s flora have
                                                                                               “    Only around 4% of the
                                                                                               world’s plants have been
                                                                                               assessed for their conservation
conservation world. And each year we are        been assigned a category of threat on the
reminded again that many species are
slipping toward extinction, usually as a
result of pressures from human activities.
                                                global Red List. Although significant
                                                activity on Red Listing for plants is taking
                                                place around the world, often in botanic
                                                                                               status at the global level.
                                                                                               resources into practical conservation
But when was the last time you remember         gardens, the sheer scale of the task is        effort if we don’t know where or what to
a species of plant being mentioned? All         daunting.                                      focus on? The good news is that
too often plants fall under the radar when                                                     progress is being made. Not only is raw
it comes to the Red List, although they are     Despite attempts from the botanical            information on plants becoming
being assessed and they are as                  community to accelerate the production         increasingly available through data
threatened as the ‘better known’ groups         of Red List assessments, such as the           sharing platforms such as the Global
such as mammals (see the Sampled Red            ambitious Target 2 of the Global Strategy      Biodiversity Information Facility (GBIF),
List Index – Plants Under Pressure a            for Plant Conservation2 ‘an assessment         but automated and semi-automated
global assessment 2010). So why is there        of conservation status of all known plant      tools are now available to allow
such a shortfall? The sheer number of           species’ we are still falling short. The       scientists and experts to harness that
species, in comparison to the number of         implications of this are very significant.     data to answer important questions like
available scientists, is the main problem.      How can we even begin to focus our             – how endangered is my species?

   11          1                                         BGCI • 2012 • BGjournal • Vol 9 (1) • 11-13
With these powerful analytic tools we
can more rapidly assess species and
more effectively prioritise conservation
action. With the development of tools
such as GeoCAT, the target of assessing
all species may just become more

Introducing GeoCAT

The Royal Botanic Gardens, Kew has
long been associated with the Red List
process, being key players in the
development of the original Red Data
books of endangered plants and
continuing with major contributions of
plant assessments to the present day
Red List. The Geographical Information
Systems (GIS) Unit at Kew has a more                 Screenshot from GeoCAT – illustrating multiple data sources, and the metadata window allowing you to view
specific interest in the application of GIS          the underlying data for each point.
techniques for Red List assessment, in
particular the geographic components of              GeoCAT is a rapid assessment tool that                 Geographic range measures
an assessment. Innovations within the                utilises primary plant occurrence data to              used in the Red List
field of GIS over the last few decades               produce measurements relating to the
have allowed for the first time the                  geographic range of a species. These                   One aspect of Red Listing that has been
production of tools to aid Red List                  are then compared with the IUCN Red                    particularly challenging so far is the
assessment such as the CAT                           List categories and criteria and provide               calculation of geospatial measurements
(Conservation Assessment Tools)                      evidence to support a full Red List                    included in the criteria – in other words
project3. Released in 2007, CAT is an                assessment. It is important to note that it            criteria related to the geography of a
extension to the ArcView GIS software                is an expert driven system i.e. it is                  species. Geospatial aspects of the Red
system and allows automatic calculation              assumed that the user of the tool has                  List criteria mostly relate to Criterion B -
of measures used in Red List                         good knowledge of the species being                    Geographic Range, but also appear as
assessment. With the recent explosion of             assessed as well as a thorough                         elements throughout the other
web mapping innovations such as                      understanding of the Red List categories               categories A – D. Two different measures
Google Earth and Google Maps it was                  and criteria (IUCN 2001).                              of geographic range: extent of
clear that these conservation tools
needed to be brought into this new era,
which led to the development of GeoCAT.

GeoCAT (accessible at this URL: is a web based tool
developed through a collaboration of the Royal
Botanic Gardens, Kew, IUCN and ViBRANT that
supports the Red Listing process. If you want a
quick way to find out more you can watch the video
The GeoCAT home page -
                                                     Screenshot of extent of occurrence (EOO) and area of occupancy (AOO) analysis. When the analysis is
                                                     enabled the results are displayed showing area in km2 and corresponding Red List category.

   12             depth/gis/resources-and-publications/tools/cat/
                                                                                                                BGCI • 2012 • BGjournal • Vol 9 (1)
occurrence (EOO) and area of                  • Upload your own data - where                  assessment. Perhaps then we will see
occupancy (AOO) are used in the Red             occurrence data has already been              plants in the spotlight for the next edition
List criteria. Extent of occurrence (EOO)       gathered, for example, in a specimen          of the Red List.
is a measure of the geographic range            database or spreadsheet, it is
size of a species. IUCN guidelines              possible to import this data directly to      References
suggest a minimum convex polygon                the map editor. Data simply needs to
(MCP) can be used to calculate this             be converted to comma separated               , Bachman, S., Moat, J., Hill, A.W.,
value. A MCP is defined as the smallest         values format following a standard              de la Torre, J. and Scott, B. 2011.
polygon in which no internal angle              structure.                                      Supporting Red List threat
exceeds 180˚ and contains all sites of                                                          assessments with GeoCAT: geospatial
occurrence. This value represents the         • Manual add - you may have specific              conservation assessment tool.
spread of risk for a species such that          knowledge regarding the distribution            ZooKeys 150: 117–126 doi:
species with a large extent will be more        of a species. The user can add points           10.3897/zookeys.150.210
robust to threatening processes. An             directly to the map with a single click.
alternative approach for depicting a                                                          , Brummitt, N., Bachman, S.P. and
species range is to show the area where       With data on the map the user can then            Moat, J. 2008. Using the Red List as
a species occurs or area of occupancy         edit by adjusting points, removing them           a barometer for plant diversity.
(AOO). A simple way to measure this           and by examining the original data e.g.           Endangered Species Research.
value is to overlay the distribution with a   inspecting a Flickr image to see if it really     doi: 10.3354/esr0013
grid and sum the area of square grid          is the species it is labelled as. From here,
cells the species occupies                    at the click of a button the analysis can       , IUCN. 2001. IUCN Red List Categories
                                              be enabled. Based on the points on the            and Criteria: Version 3.1. IUCN
How it works - calculating                    map, the extent of occurrence (EOO) and           Species Survival Commission. IUCN,
geographic range of species for               area of occupancy (AOO) values are                Gland, Switzerland and Cambridge,
Red List assessments                          instantly calculated and the values are           UK: 30 p
                                              compared with the thresholds set in the
GeoCAT is driven by primary occurrence        IUCN Criteria. For example if the extent        , Plants under pressure a global
data. For hundreds of years botanists         is less than 5,000 km2 then it meets the          assessment. The first report of the
have been travelling to the ends of the       threshold for the Endangered (EN)                 IUCN Sampled Red List Index for
earth to take cuttings from plants as         category. From here a simple report can           Plants. Royal Botanic Gardens, Kew,
scientific specimens. Herbarium               be generated and the data can be saved            UK. 2010
specimens are verifiable records that         or exported to other formats such as     
show where and when a particular              KML for visualisation in other packages            conservation/search-rescue/mapping-
species was collected. By combining           such as Google Earth. As mentioned                 plants/plants-at-risk/index.htm
these records at the species level we         previously it should be noted that this
immediately have an indication of the         does not represent a full Red List              Steve Bachman and Justin Moat
geographic range of a species and it is       assessment, it provides evidence that           Royal Botanic Gardens Kew,
this data that GeoCAT analyses to             fulfils part of the criteria.                   Richmond,
determine the two measures of extent of                                                       Surrey
occurrence (EOO) and area of occupancy        Impact and benefits                             TW9 3AB,
(AOO). A benefit of GeoCAT is that as                                                         UK
well as data being provided by the user       GeoCAT is a good example of the                 Email:
e.g. from a database of their own             innovative work presently being carried
specimens, it can also be imported from       out in botanic gardens, in this case to
existing online sources such as GBIF and      directly support plant conservation.
Flickr. The images in Flickr need to be       Specifically GeoCAT provides the
labelled with the species name and            potential to speed up the Red Listing
‘geotagged’ or ‘georeferenced’ i.e.           process for plants and offers hope that
assigned a latitude and longitude co-         global targets such as the Global
ordinate so we know where it was taken.       Strategy for Plant Conservation may be
                                              reached. By utilising existing data it is an
The data can be entered in three ways:        evidence based approach that can be
                                              used immediately as much occurrence
• Import from online sources - existing       data is already available. The tool is
  sources of primary data can be              open and free to use so there are no
  queried. By adding a search term e.g.       restrictions in terms of accessibility aside
  a scientific name or a common name          from a connection to the internet. It
  you can query two sources of online         provides a platform that can be built on
  data: GBIF and Flickr. Any matching         in the future to make other aspects of
  record that has been georeferenced          the Red List criteria automated. In short,
  can be added directly to the map            it could be the first step towards a fully
  editor.                                     automated data driven Red List

   13                                                                                            BGCI • 2012 • BGjournal • Vol 9 (1)
Author: Valerie Pence


                                             When seeds are few and cuttings difficult, tissue culture
                                             can provide an alternative conservation pathway

                                             Above: The rockhouse habitat that is home to the Cumberland sandwort experimental population (Valerie Pence)
                                             Left: A storage tank in CREW’s CryoBioBank (CREW).

                                             primarily at species for which traditional              (Sarasan, et al., 2006). The PRD
                                             approaches are not adequate. Thus,                      collaborates with a number of other
                                             when seeds are few and cuttings are not                 institutions, including botanical gardens
                                             workable, tissue culture can provide an                 in the Center for Plant Conservation
                                             alternative method for propagation.                     network (Center for Plant Conservation
                                             When seeds are lacking or are                           2012), government agencies, NGOs, and
                                             “recalcitrant” (sensitive to the drying                 research stations. These institutions
                                             necessary for traditional seed-banking),                often identify plants for which in vitro
                                             freezing tissues from in vitro cultures can             approaches could be useful and then
                                             be an additional tool for banking valuable              provide the starting material (seeds or
                                             genetic material from rare taxa. The PRD                shoot tips) to CREW for propagation.
                                             has developed protocols for the in vitro
Introduction                                 propagation and/or preservation of over

        ff the main public path of the
                                             50 rare species, providing materials for
                                             restoration projects and tissues for long-              “     Over 100 botanic gardens
                                                                                                     around the world have tissue

O       Cincinnati Zoo & Botanical
        Garden, in the corner of the park,
                                             term storage in liquid nitrogen in CREW’s
                                                                                                     culture and micropropagation
one can find the Carl H. Lindner Family
Center for Conservation and Research of
Endangered Wildlife (CREW). From this
small, two-story building, plant and
                                             Propagation for restoration

                                             Propagation is a key element in
                                                                                                     (BGCI GardenSearch database, 2012)

animal research is being conducted that      providing plants for recovery projects.                 The Autumn buttercup (Ranunculus
is impacting rare species across the         For many endangered species,                            aestivalis) is one such species, known
country, and beyond. CREW’s Plant            traditional propagation by seed or                      from one site in south central Utah in the
Research Division (PRD)’s particular         cuttings can meet propagation needs,                    Sevier River Valley. When it became
focus is using in vitro (tissue culture)     but for species with few or no seeds or                 obvious that the population was
methods for propagating and preserving       few individuals, plant tissue culture can               declining, The Nature Conservancy
endangered plants. The work is directed      be used to supplement these methods                     bought the property in order to protect

   14                                                                                         BGCI • 2012 • BGjournal • Vol 9 (1) • 14-17
Todsen’s pennyroyal growing at White Sands
                                                                                           Missile Base (Doug Winget).

                                                                                           and northern Tennessee. Like the
                                                                                           Autumn buttercup, CREW initiated
                                                                                           several genetically different tissue
                                                                                           culture lines from seeds that had been
                                                                                           collected by researchers at the Missouri
                                                                                           Botanical Garden. Shoots were
                                                                                           produced, rooted, and plants transferred
                                                                                           back to soil.

                                                                                           In order to demonstrate the feasibility of
                                                                                           using tissue culture propagated plants
                                                                                           for the recovery of this species, an
                                                                                           experimental out-planting was made at a
                                                                                           rockhouse site in southern Kentucky in
                                                                                           collaboration with the U.S. Forest
                                                                                           Service. Plants were planted in different
                                                                                           areas of the rockhouse, in order to
                                                                                           evaluate the microhabitats within the
                                                                                           rockhouse, which differed in light and
                                                                                           moisture levels. Over the course of six
                                                                                           years, plants at several of the microsites
                                                                                           have grown and reproduced well,
                                                                                           indicating the viability of these methods,
                                            were initiated from each seedling. As          should they be needed to help preserve
                                            with most tissue culture systems, the          the species in the future. A genetic
                                            shoots can be maintained and multiplied        study is also underway to analyze the
                                            in culture by dividing and sub-culturing       genetic diversity of this new,
                                            the shoots onto fresh medium every 6-8         experimental population.
                                            weeks. By changing the components of
                                            the medium, the shoots can be                  By combining the laboratory strengths of
                                            stimulated to form roots, and once             CREW with the field expertise of
                                            rooted, the shoots are then ready to be        collaborators in botanical gardens,
                                            moved back into soil. This is a clonal         governmental agencies, and non-profit
                                            method of propagation, but each genetic        organizations, the work at CREW is
                                            line initiated from a separate seed is         being integrated into conservation
                                            maintained separately, creating a              efforts for species recovery and
                                            collection of genetically distinct lines in    demonstrating that tissue culture
                                            tissue culture.                                propagation can be an important tool for
                                                                                           conservation and restoration.
                                            When the plants were rooted, they were
                                            sent to the Arboretum at Flagstaff, where
                                            they were carefully removed from their
                                            culture tubes and acclimatized to soil
                                            and to real-life conditions in a
                                            greenhouse. At that point they were
Cumberland sandwort flowering in culture    ready to face life out in the wild. In 2007,
(Valerie Pence).                            a group of buttercups, originally
                                            propagated at CREW, was planted back
the plant. Over the past decade, five       out at the Sevier Preserve, and, while
organizations, including CREW, The          none of the plants from the original
Arboretum at Flagstaff, The Nature          population have been seen in recent
Conservancy, U.S. Fish & Wildlife           years, a group of the CREW-propagated
Service, and Utah State University, have    plants are surviving and flowering.
come together in a partnership to work
on studying and restoring this species      Another such species is the Cumberland
(Pence et al., 2008). A small number of     sandwort (Minuartia cumberlandensis), a
seeds were collected at the site and sent   small plant that grows only in sandstone
to CREW where they were germinated          rockhouse habitats in the Daniel Boone
in vitro, and shoot propagating cultures    National Forest of southern Kentucky           The Autumn buttercup (Valerie Pence).

   15                                                                                         BGCI • 2012 • BGjournal • Vol 9 (1)
Resources for the Future                              of state and federally endangered              banking (Pence, 2011). For these plants,
                                                      species from Kentucky, Indiana, and            banking tissues from in vitro cultures in
In a similar fashion, the PRD is utilizing in         Ohio, including such species as Short’s        liquid nitrogen can provide an alternative
vitro methods to provide tissues for                  goldenrod (Solidago shortii) and Running       method for long-term germplasm
long-term liquid nitrogen storage, when               buffalo clover (Trifolium stoloniferum).       storage. At CREW, multiple genotypes of
seed banking is not a workable option                 Because not all endangered plants are          several species are being collected, put
for a species (Engelmann, 2004). This                 seed plants, there is a Pteridophyte           into culture, and then cryopreserved, in
work centers on the CryoBioBank (CBB),                (Fern) Bank and a Bryophyte (Moss)             order to maintain a back-up to the
CREW’s liquid nitrogen storage facility.              Bank, which contain spores, as well as         genetic diversity of these species,
The plant collections, or “Frozen                     tissues of gametophytes and                    should they decline or be lost in the wild.
Garden” within the CryoBioBank include                sporophytes. The Endangered Plant              These include three endangered
seeds, spores, and tissues of about 150               Tissue Bank contains samples of tissues        pawpaws endemic to Florida, the Four-
plant species in four distinct collections,           from in vitro cultures of endangered           petal pawpaw (Asimina tetramera),
all stored at -196oC (-320oF).                        plants that are grown at CREW. Material
                                                      is continually added to the collection,        The small Cumberland sandwort flower
Seeds and spores are prepared for                     and some samples are over 20 years old.        (Brian Jorg).
freezing by simple drying, but tissues,               Recently, samples of pollen from the
such as shoot tips and embryos, require               American chestnut (Castanea dentata)
more elaborate procedures.                            were shown to be viable after 15 years of
Concentrated solutions of sugar and other             storage in liquid nitrogen, while shoot
chemicals, known as cryoprotectants,                  tips of the Cumberland sandwort were
remove water from the tissues and protect             recovered after 10 years in liquid
the cells from damage during the freezing             nitrogen.
and thawing processes. The extremely
low temperatures of liquid nitrogen                   Perhaps one of the most important uses
provide stability for the tissues,                    of the CryoBioBank is to preserve
maintaining them in a state of suspended              tissues from endangered species that fall
animation for decades.                                into the category of “exceptional”
                                                      species. These are plants that either
The CryoBioBanks's Frozen Garden is                   produce few or no seeds, or their seeds
made up of four distinct collections.                 are sensitive to drying and cannot be put
The Regional Seed Bank includes seeds                 through the rigors of normal seed-

A second planting of CREW propagated Autumn buttercups took place in 2010 in Utah (Valerie Pence).

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