conservation strategies greece

X

Conservation Strategies for Native Plant Species

and their Sustainable Exploitation: Case of the

Balkan Botanic Garden of Kroussia, N. Greece

Eleni Maloupa1 Nikos Krigas1,2 Katerina Grigoriadou1 Diamanto Lazari3

Georgios Tsoktouridis1*

1 Laboratory of Conservation and Evaluation of the Native and Floricultural Species-Balkan Botanic Garden of Kroussia, National Agricultural Research Foundation, P.O.

Box 60125, GR-570 01, Thermi, Thessaloniki, Greece 2 Laboratory of Systematic Botany & Phytogeography, Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-54124, Thessaloniki, Greece 3 Department of Pharmacognosy - Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, GR-54124, Thessaloniki, Greece

Corresponding author: * [email protected]

Keywords: GIS, ITS1, ITS2, propagation, molecular markers, native plants, Origanum dictamnus, sustainable cultivation, taxonomy

ABSTRACT

This study outlines a pioneer initiative concerning the native plant conservation that is considered as one of the most important plant refuges in

Europe. The Balkan Botanic Garden of Kroussia has designed a system built on people, infrastructure and a strategy of seven hierarchical and

complementary policies. To formulate and implement this strategy, we have created a flexible research team with scientists from different

disciplines that collaborate interdisciplinary. In this team, information flows horizontally, experience and expertise are joined and used collectively

and know-how is delivered vertically to all interested or involved parties. In the frame of this strategy, target plants are being initially explored,

located and collected from the wild and Important Plant Species are maintained, evaluated and studied. Explicitly documented living plant

collections are maintained with classical and innovative methods and species-specific propagation protocols for wild plants are being developed.

Environmental awareness is promoted and several educational activities on the native biodiversity are organized. Numerous plant conservation

actions are undertaken; all attempt to integrate the ex-situ with the in-situ plant conservation and contribute to the implementation of the targets

of the Global Strategy for Plant Conservation at local, regional and (inter-) national levels. Furthermore, evaluation of the medicinal, cosmetic,

flavouring, floricultural and ornamental value of selected native plants is performed, aiming at their sustainable exploitation in collaboration with

the state, the stakeholders and the market. This approach has the ultimate goal to deliver promising and unique new crops that are carefully

selected and designed, sustainably produced and managed, successfully launched and fair traded internationally.

1. TARGET AREA: NATURAL FEATURES AND THE RICHNESS OF THE GREEK FLORA

Greece has an exceptionally rich flora with more than 5,700 native taxa (species and subspecies); about 15-20% are unique, found nowhere

else in the world (Greek endemic taxa), presenting the highest degree of endemism for any comparable territory in Europe and the

Mediterranean region (Strid and Tan 1997).

This figure is all the more striking considering the relatively small area (132,000 km2) and the complex topography of the country. Much of

the land is wild, rugged, mountainous, with more than 40% of the national area lying above 500 m of altitude (including 314 mountains and 1,674

individual peaks above 1,000 m; Strid and Tan 1997). Varied climatic conditions may be found in Greece ranging from subtropical to subalpine

environments. The geology of the area is diverse and dissected, mainly with limestone massifs, serpentine regions, schistose and granite

mountains. A mosaic-like forest cover is estimated at ca. 18%, including forest, open woodland and scrub and at least 400 wetlands of various

sizes can be found in Greece. The Greek coastline stretches along more than 15,000 km, one of the longest in Europe, while about 3,000

islands and islets dispersed in the Aegean, Ionian and Cretan Seas, comprising ca. 19% of the land area of Greece.

The Greek territory can be divided into 13 different floristic regions, the borders of which almost follow natural geographic features such as

rivers and lowland areas between mountains (Fig. 1).

Abbreviations: ABS, Access and Benefit-Sharing; BBGK, Balkan Botanic Garden of Kroussia; CBD, Convention on Biological Diversity; GIS, Geographical

Information Systems; GSPC, Global Strategy for Plant Conservation; IPS, Important Plant Species; IPEN, International Plant Exchange Network; ITS1, Internal

Transcribed Sequence 1; MAT, Mutually Agreed Terms; MTAs, Material Transfer Agreements; PIC, Prior Informed Consent

Maloupa et al. Conservation strategies for native plants in Greece

Generally, mountain summits and islands seem to host the

rarest endemic wild plants. Such plants, confined as they are to

Greece, are a treasure of international importance, whose conserva-

tion will benefit future generations not just in Greece but in the whole

world; the rarity of many of the Greek endemic plants makes them

vulnerable to extinction through human activity, a loss not only to

Greece but to the world (IUCN Threatened Plants Committee

Secretariat 1982).

In total, four areas in Greece are considered as European

Centers of Plant Diversity and Endemism (CPDEu14-17; Akeroyd

and Heywood 1994), 10 areas have been declared as National

Parks, 10 wetlands are included in the Ramsar Convention, 575

areas are considered under protection and 270 sites have been pro-

posed to be included in the EU NATURA 2000 Network.

Nevertheless, the rich and unique native flora of Greece is being

threatened by global warming, fires, land reclamation, over-grazing

and current urban and tourism expansion.

Though Greece is no doubt a hot-spot for global biodiversity

(Akeroyd and Heywood 1994) due to its exceptionally rich and

unique flora, it has no long tradition in botanic gardens dedicated to

plant conservation.

2. THE ONLY NATIVE PLANTS POLICY

In-situ conservation of rare and endangered plant species is no

doubt indispensable and it may be achieved basically with habitat

and ecosystem conservation. Nevertheless, in-situ conservation is

also a difficult task to achieve since effective protection regimes are

not easily applied in the wild, they are costly and difficult to control and evaluate. The ex-situ conservation of rare plant taxa in botanic gardens is

an internationally acknowledged contribution to species conservation (CBD 1992), able to prevent the extinction of endangered species (e.g.

Diplotaxis siettiana, Lotus berthelotii, Lysimachia minoricensis) and able to guaranty their possible re-introduction in the wild. The majority of

botanic gardens feel proud of their botanic collections of rare or endangered plant species from every corner of the world and these displays are

often a major attraction for visitors.

The Balkan Botanic Garden of Kroussia, N. Greece (BBGK) is a newly established botanic garden founded in 2000, as an initiative of the

National Agricultural Research Foundation of Greece (NAGREF). Today it covers an area of 31 ha, located in Mt. Kroussia (Northern Greece), at

600 m of altitude, within a natural deciduous oak forest. Since its establishment, the BBGK in order to focus exclusively to the ex-situ

conservation of native plants of Greece and the Balkans has decided to leave aside the exotic plants and ornamental species commonly found in

other botanic gardens of Europe and the world.

All plants of the displays and the ex-situ conservation sections in the BBGK are native plant species originating in the wild (Fig. 2).

Fig. 1 Collection areas of native Important Plant Species (IPS: rare, endangered,

vulnerable, protected and endemic taxa of Greece and/or the Balkans, plants with

potential ornamental and/or medicinal value) currently in ex-situ conservation (white

circles) at the Balkan Botanic Garden of Kroussia (BBGK), N Greece and their distri-

bution across the different phytogeographical regions of Greece (Strid and Tan

1997). Every red dot in the map concerns at least one botanic expedition and col-

lection of at least one accession number of IPS (Krigas et al. 2007).

Fig. 2 Schematic representation of the Balkan Botanic Garden of Kroussia, N Greece.


3. THE IMPORTANT PLANT SPECIES POLICY Since 2001, BBGK has launched a series of

botanic expeditions to every different phytogeo-

graphic region of Greece in order to obtain wild

propagation material of native plants (Fig. 1;

Krigas et al. 2007).

With a special permit provided by the Ministry

of Rural Development and Foods, renewed every

year, the scientific staff of the BBGK is enabled to

collect wild plant material even from NATURA

2000 sites, Nature Reserves, National Parks and

other protected areas of Greece. The BBGK,

combining conservation principles and sustainable

plant exploitation policies, has formulated priorities

for the collection of propagation material of wild

plants. Five target-plant categories have been pri-

oritized:

(i) Greek endemic plants (plant species found

exclusively in Greece and nowhere else in the

world); This category includes (a) single-island en-

demics e.g. Origanum dictamnus, Campanula hie-

rapetrae and Ebenus cretica (Crete island), Limo-

nium arcuatum (Corfu island), Anchusa samothra-

cica (Samothraki island), Viola cephalonica (Fig. 3A; Mt. Aenos, Cephallonia island) etc, (b) single-mountain endemics e.g. Helichrysum sib-

thorpii (Fig. 3B) and Anthemis sibthorpii (Mt. Athos), Thymus plasonii (Mt. Chortiatis), Crocus hadriaticus subsp. parnassicus (Mt. Parnassos),

Achillea occulta (Mt. Koulochera), Centaurea cithaeronea (Mt. Kithaeronas) etc., (c) single-area narrow endemics e.g. Limonium antipaxorum

(Paxi and Antipaxi islands), Limonium ithacense (Cephallonia and Ithaca islands), Muscari cycladicum (Cyclades and Crete) etc, (d) regional

endemics (restricted to few phytogeographical regions) e.g. Crocus hadriaticus subsp. hadriaticus, Scaligeria moreana, Stachys ionica (Fig. 3D),

Campanula incurva (Fig. 3C) etc. and (e) national endemics (restricted to numerous phytogeographical regions of Greece) e.g. Anchusella

variegata, Cerastium candissimum, Dianthus corymbosus, etc.

(ii) Narrow Balkan endemics; This category includes native plants occurring around the boundaries of Greece with neighbouring Balkan

countries e.g. Lilium rhodopaeum, Centaurea pawlowskii, Marrubium thessalum, Stachys iva, etc.,

(iii) Other rare taxa found in Greece; This category includes native plant species of wider distribution than (i), (ii) or (iii) with one or only a few

scattered populations in Greece (e.g. Poa molinieri, Datisca canabina, Galanthus nivalis, Diathus crinitus, etc.),

(iv) Balkan (sub-) endemics; This category includes taxa found exclusively in the Balkan countries and/or extending to W Turkey and/or

parts of Italy with scattered populations e.g. Dianthus giganteus, Hypericum olympicum, Scabiosa crenata subsp. dellaportae, Thymus thracicus,

etc.) and

(v) Potentially ornamental and/or medicinal plants native to Greece and/or the Balkans (e.g. Geranium macrorrhizum, Coridothymus

capitatus, Digitalis grandiflora, Salvia officinalis, Crataegus monogyna, etc.).

All plant species of groups (i), (ii) and (iii) and numerous species of group (iv) have been characterized as Other Important Plant Species

(IPS) of the NATURA 2000 Network (in total 1,853 IPS for Greece, Kokkini et al. 1996).

To date, at least 60 botanic expeditions have been organized (Fig. 1) and more than 1,200 taxa (>20% of the Greek flora) have been

collected from the wild (ca. 2,300 accession numbers). All native taxa are currently cultivated and maintained ex-situ in BBGK. In total, ca. 40%

of them belong to target-plant groups (i), (ii), (iii), (iv) and another ca. 45% belong to group (v).

4. THE EXPLICIT PLANT DOCUMENTATION POLICY

4.1. Preparation of the botanic expeditions

A complete work about the flora of Greece is absent nowadays. Floristic data are widely scattered to numerous scientific papers and various

modern floras. The Flora Hellenica (Strid and Tan 1997, 2002), the only complete work for the flora of Greece, has produced hitherto only two

volumes (out of 10 expected). The Mountain Flora of Greece (Strid 1986; Strid and Tan 1991) covers exclusively mountain summits above ca.

1,500 m. The Flora Europaea (Tutin et al. 1968-1980) lack significant amount of information concerning the East Aegean Islands of Greece,

recently described taxa and updated distribution areas of plant species, while the Flora of Turkey and the East Aegean Islands (Davis 1965-

1985) covers only the flora of the East Aegean Islands of Greece. As a result, numerous areas of Greece are considered as floristically ill-

explored and therefore botanists may still encounter and describe taxa new to science (e.g. Constantinidis and Calpoutzakis 2005; Snogerup et

al. 2006).

Consequently, the scientists of the BBGK, prior to every botanic expedition scheduled to a specific area, have to:

(a) Review and study in advance all the published floristic literature concerning the target-area,

(b) Prepare working lists of the known IPS cited from this area,

Fig. 3 (A) Viola cephalonica, (B) Helichrysum sibthorpii, (C) Campanula incurva, (D) Stachys ionica in

their collection sites.


(c) Group target-plant species, target-sites and target-routes that have to be followed in the field,

(d) Assess the target-plant groups, sites and routes based on estimated cost vs. on the expected benefit.

4.2. Collection of data

Field and in-situ collection data are indispensable in many ways. Every botanic collection is considered unique by the combination of the

collector(s)-area-date information. Documentation takes place by using accession numbers (specific codes for different plants from different

localities), when only explicit and precise information is provided i.e. geographical coordinates and site description, specific location, region,

prefecture, and country; otherwise, no accession number is given to plant collections arriving with poor documentation and no subsequent

propagation is planned.

Habitat information for each plant individual collected from the wild (substrate, soil type, forest zone, habitat type, slope and altitude) is

valuable and necessary for future ex-situ conservation actions (Krigas et al. 2007). All these site and habitat characteristics mentioned above,

are documented in special collection forms in-situ, which accompany each accession number throughout the subsequent propagation.

4.3. Taxonomic identification and nomenclature

Taxonomic identification of herbarium specimens and collected propagation material from the wild is indispensable (Jarvis 2007). A plant species

without a name does not exist in terms of science and conservation (Taylor 2007). Each plant species has a unique scientific name; this is the

tag that allows to be found, counted, researched, and monitored, encoding and classify among other plants in the world; additionally this is the

index key that retrieves everything we know about it from various sources (Taylor 2007). The value of accessions in botanic gardens depends

critically on the correct identification and nomenclature, either they are utilized as conservation resources, either as reference material; the

consequences of inaccurate taxonomy could be very serious or even fatal (Heywood 2007).

Nomenclature of plant taxa conserved in the BBGK follows Strid and Tan (1997, 2002), for the taxa covered by Flora Hellenica, Strid (1986)

and Strid and Tan (1991), for the taxa included in the Mountain flora of Greece, and Tutin et al. (1968-1980) for all the rest.

4.4. Evaluation of the Important Plant Species (IPS)

The mother stock plants maintained in the nursery of the BBGK, are periodically evaluated according to the IUCN status (IUCN 2001), endemism

category, inclusion in Other Important Plant Species of EU NATURA 2000 Network (Kokkini et al. 1996), inclusion in national and/or international

catalogues and/or conventions (e.g. Greek Presidential Decrees, EU Directives and Annexes, Red Lists, CITES, Bern Convention, etc.). This is

exercised in order to identify the Important Plant Species (IPS) in the BBGKs mother plantations, meriting special ex-situ conservation

programmes as defined by the International Agenda of Botanic Gardens in Conservation (Wyse-Jackson and Sutherland 2000). Under this view,

four groups of IPS are identified as Priority Important Plant Species (Priority IPS; Table 2):

As Priority 1 IPS are designated (a) all taxa included in national and/or international catalogues and/or conventions (e.g. Greek Presidential

Degrees, EU Directives and Annexes, Red Lists, CITES, Bern Convention etc), regardless of endemism, as well as (b) narrow Greek endemics

(single-island, single-mountain, single-area or regional endemics), which are not included in national and/or international catalogues and/or

conventions.

Table 1 Ex-situ cultivation guidelines in the Balkan Botanic Garden of Kroussia regarding Important Plant Species (IPS) based on links of IPS collection data with

geodatabases (Krigas et al. 2007).

Source Type of variable Examples of attributes Guidelines for the ex-situ cultivation of IPS in BBGK

ESDB v.2 (EC 2004) Soil moisture Topsoil and subsoil available water capacity Selection of watering regimes

ESDB v.2 (EC 2004)

CORINE Soil

Classification (EC 1985)

Soil classes and types Textural class

FAO 1985 soil class

World Reference Base soil class

Dominant parent material

Selection of growing medium (texture, pH, drainage)

ESDB v.2 (EC 2004) Soil nutrient Topsoil and subsoil base saturation

Cation exchange capacity

Selection of growing medium and fertilization regime

ESDB v.2 (EC 2004) Soil limitations Depth to a gleyed horizon

Depth to rock

Depth of an obstacle to roots

Volume of stones

Selection of growing medium (texture, drainage)

and potting volume

WorldClim Database

(Guerin et al. 2002;

Hijmans et al. 2005)

Climate Mean minimum or maximum temperatures of the

coldest or the warmest month, respectively

Annual mean temperature range

Temperature seasonality

Mean diurnal temperature range

Precipitation of the driest month or the wettest month

Mean monthly precipitation

Annual precipitation

Mean precipitation of driest, wettest, coldest or

warmest quarter

Selection of appropriate growing sites and conditions in

greenhouse as well as in ex-situ cultivation

Selection of temperatures for seed germination

Selection of shading and ventilation

Variation of watering regimes per month

Digital Terrain Model

created

Topography Aspect

Slope

Altitude (elevation)

Customizing microclimate in ex-situ cultivation sites and

in positioning of plants in the displays

Mavromatis (1980) Vegetation zones 11 vegetation zones

CORINE Land cover

EC & ETC/LC (1999)

Land cover classes

and types

45 land-use classes and types

Selection of the ex-situ conservation sites

Selection of shading

Selection of species assemblages and sites for specific

plant displays


Priority 2 IPS include all taxa which they are not included in national and/or international catalogues and/or conventions but they (a) are

endemic to more than a single phytogeopraphical area of Greece, (b) are endemics of any other Balkan country and (c) are narrow endemics to

the boundary areas of neighbouring Balkan countries.

All taxa which are endemic to the Balkan Peninsula and they are not included in national and/or international catalogues and/or conventions

are designated as Priority 3 IPS.

All taxa that are found exclusively in the Balkan countries and/or extending to W Turkey and/or parts of Italy with scattered populations and

they are not included in national and/or international catalogues and/or conventions are designated as Priority 4 IPS.

Other plants native to Greece and/or the Balkans with potential ornamental and/or medicinal value, which are not included to the previously

mentioned categories, are designated as Priority 5 Plant Species.

4.5. Species-specific baseline cultivation guidelines for IPS using GIS

All collection data of taxonomically iden-

tified and evaluated as IPS are being in-

corporated in a GIS (Geographical Infor-

mation Systems) environment (Krigas et

al. 2007). The collection data are conse-

quently combined and/or verified with

data from ecological databases (Fig. 4);

this includes several topographic and

habitat features (altitude, slope, aspect,

vegetation zones, habitat types), climatic

conditions (minimum, maximum and

mean temperature per month, accumu-

lated temperature, minimum, maximum

and mean monthly and annual precipita-

tion, isothermality, Emberger ombrother-

mic quotient) and soil properties (soil

water capacity, soil types and dominant

parent material).

This approach (Krigas et al. 2007) is

being used to: (i) consider the in-situ eco-

logical amplitude of different IPS popula-

tions, (ii) inform and guide the ex-situ

growing conditions preferred and/or toler-

ated by each IPS, (iii) minimise the risk of

cultivation failure, (iv) select the best pos-

sible different treatments, (v) help or-

ganize better ecological groupings of

mother plants in the nurseries of BBGK

and (vi) provide species-specific baseline

cultivation guidelines for each IPS (Table

1; Figs. 5-6; Mouflis et al. 2007). This

approach may additionally facilitate gap

analysis of the botanic expeditions orga-

nized so far, permits better schedule of

next expeditions of BBGK, analyses and

reveals gaps in representation of IPS

from different altitudes, phytogeographic

and climatic regions of Greece, and fin-

ally is aimed to permit assessment of the conservation strategy and actions of BBGK.

4.6. Distribution of plant material and the International Plant Exchange Network Policy

The BBGK produces yearly an Index Seminum distributed in other botanic gardens worldwide. All available plant accession numbers maintained

in ex-situ conservation in BBGK are numbered according to the International Plant Exchange Network (IPEN) (www.bgci.org). The IPEN policy

has been adopted by botanic gardens in Europe as well as by the BBGK in order to meet the provisions of 15 of the Convention on Biological

Diversity (CBD 1992) in receiving, storing, and supplying plant material, including free exchange only for non-commercial purposes and free

exchange only between botanic gardens. Otherwise, regarding institutions not sharing this policy and their request to access the phytogenetic

resources of Greece, an Access and Benefit-Sharing (ABS) policy is adopted by BBGK; this requires a Code of Conduct endorsing bilateral

agreements with Mutually Agreed Terms (MAT), Prior Informed Consent (PIC) and Material Transfer Agreements (MTAs), before the distribution

of the plant material.

Fig. 4 Schematic representation of the link created in a GIS environment between the collection data of IPS and

the data from ecological databases. (Table 1; Krigas et al. 2007)

Viola_cephThym_hol

Limo_ithSileCepCep

TmeanTmax

Tmin

17,917,7

15,0

9,2

32,232,3

29,5

23,96,7

6,3

3,9

-1,6-5,0

5,0

15,0

25,0

35,0

Temperature profiles

TmeanTmaxTmin

oC

Fig. 5 Temperature profiles (Tmean, Tmax, Tmin) regarding four selected IPS of Cephalonia island (Viola

cephalonica (Fig. 3A), Thymus holosericeus, Limonium ithacense, Silene cephallenia subsp. cephallenia. (Mouflis et

al. 2007).


Table 2 Example of evaluation of 50 native plant taxa (species or subspecies) of Greece that are currently maintained in ex-situ conservation at the Balkan Botanic

Garden of Kroussia aiming at assigning their potential commercial value according to conservation priorities, aromatic and pharmaceutical properties, impressive

botanical features, ornamental and/or horticultural interest, propagation methods and cultivation regimes.

Use Impressive botanical features Taxon (species or

subspecies)

Family

Co

nse

rvat

ion

pri

orit

y

Aro

mat

ic p

rop

ertie

s

Ph

arm

aceu

tica

l pro

per

ties

Gar

den

orn

amen

tal

Cu

t fl

ow

er

Po

tted

pla

nt

Pro

pag

atio

n

Irri

gat

ion

Typ

e o

f so

il

Lar

ge

flow

ers

Den

se in

flore

scen

ce

Oth

er

Lea

ves

Po

st h

arve

st

/ tra

nsp

ort

atio

n

char

acte

rist

ics

Acanthus balcanicus Acanthaceae 3 T M Acid/ Clay Large d $ Achillea ageratifolia

subsp. ageratifolia

Asteraceae 2 A1 P1 S/iv L Lime Everlasting flowers

Glaucous b

$ Achillea chrysocoma Asteraceae 2 A P S/iv L Lime Hairy c Achillea occulta Asteraceae 1 A1 P1 iv L Lime Everlasting

flowers

e

Adonis cyllenea Ranunculaceae 1 P1 T L Lime Large b Anthemis sibthorpii Asteraceae 1 A1 P1 S/C L Lime Everlasting

flowers

a

Biebersteinia orphanidis Biebersteiniaceae 1 A P1 T M Sandy -Clay

Large b #Campanula hierapetrae Campanulaceae 1 P1 S/iv L Acid Velutinous a #Campanula incurva Campanulaceae 1 P1 S/iv M Acid Pubescent a #Campanula pelviformis Campanulaceae 1 P1 S/iv L Acid Hispid b Centaurea cithaeronea Asteraceae 1 A1 P1 iv L Lime Everlasting

flowers

Glaucous b

Centaurea pawlowskii Asteraceae 1 A1 P1 S M Lime Coloured bracts c #Cerastium

candidissimum

Caryophyllaceae 2 A C L Lime Glaucous c #Crocus cartwrightianus Iridaceae 1 A1 P T M Lime Long orange

stigmas

b

#Crocus hadriaticus

subsp. hadriaticus

Iridaceae 1 A1 P1 T M Lime Coloured corolla throat

b

$ Crocus veluchensis Iridaceae 3 A1 P1 T M Lime Orange stamens and stigma

b

$ Dianthus crinitus Caryophyllaceae 4 A1 P1 S/C/iv L Lime Dissected petals Glaucous a Dianthus fruticosus

subsp. occidentalis

Caryophyllaceae 1 A1 P1 S/C/iv L Lime Toothed petals Fleshy b $ Dianthus

haematocalyx

Caryophyllaceae 2 A1 P1 S/C/iv L Lime Cushion form b $ Digitalis lanata Scrophulariaceae 4 P S H Acid Coloured stems Hairy b $ Digitalis viridiflora Scrophulariaceae 4 P S M Acid b # Ebenus cretica Fabaceae 1 P C L Lime d Erodium hartvigianum Geraniaceae 1 A1 P1 T L Lime Long fruits Hairy c Erysimum naxense Brassicaceae 1 P1 S L Lime Densely arranged

leaves

d

Fritillaria pelinea Liliaceae 1 A1 P1 T L Lime Glaucous-green stems

Glaucous-

green shiny

c

$ Gentiana verna subsp.

balcanica

Gentianaceae 4 A1 P1 S H Acid Coloured calyces, compact form

a

$ Haberlea rhodopensis Gesneriaceae 1 Iv M Acid b # Helichrysum sibthorpii Asteraceae 1 A1 P1 T M Lime Everlasting

flowers

Glaucous b

Heptaptera

colladonioides

Apiaceae 1 S L Lime Large d Hypericum rumeliacum

subsp. rumeliacum

Clusiaceae 3 A P C M Lime Densely arranged leaves

b

$ Inula ensifolia Asteraceae 1 A1 P1 T M Sandy/ Clay

b $ Iris reichenbachii Iridaceae 3 A1 P1 T M Sandy/

Clay

Compact form b $ Jasione heldreichii Campanulaceae 4 T M Lime b Lilium rhodopaeum Liliaceae 1 A1 P1 S M Lime Orange long

stamens

Long, Shiny c

$ Linaria peloponnesiaca Scrophulariaceae 2 P1 T M Lime e Muscari cycladicum Liliaceae 2 P1 T L Lime d # Origanum dictamnus Lamiaceae 1 A P C/iv L Lime Hairy a $ Origanum onites Lamiaceae 4 A P C L Lime a $ Paeonia mascula

subsp. hellenica

Paeoniaceae 1 A P T M Lime Large b $ Pancratium maritimum Amaryllidaceae 4 P S/C L Sandy Glaucous-green

stems

Glaucous-

green

b


5. THE DNA BARCODING POLICY

5.1. Molecular markers for plants

Molecular markers have been developed to characterize/distinguish closely related plant species, moreover to investigate the genetic diversity

and find the genetic relationship of plants grown in different geographical areas. Potential target genes, which are conserved in all plants, play

significant role in plant species evolution. These genes/genomic areas are used to define polymorphisms among species and to develop

molecular markers to characterize and find the genetic relationships among them.

Selection of the appropriate molecular markers for species identity is very difficult for plants. This is due to the large genome of plants and

not unique high plasticity genomic regions they contain. Depending on the intimacy of species individuals, the molecular marker selection in

BBGK focus on using the matK and ndhF genes at family level, while the nrDNA ITS1 and ITS2 regions for genus and species level. To

differentiate individual plants at subspecies level, or within a population having undistinguishable phenotypes, the MADS-box gene pistillata and

three plastid DNA regions of the rbcL gene will be attempted to apply. There are also many other useful molecular markers described in the

literature applied in particular species, which may potentially be used in cases that we could not find genetic differences in some taxa.

The most widely used molecular markers for plants focus on the cpDNA rapidly evolving genes, such as matK and ndhF (Soltis and Soltis

1998), the phytochrome genes PhyA and PhyC (Mathews and Donoghue 1999), the RPB2 gene (encoding the RNA Polymerase II gene)

(Denton et al. 1998), two loci, Adc1 and Adc2, of the arginine decarboxylase gene Adc, (Galloway et al. 1998), the granule-bound starch

synthase gene (waxy) (Mason-Gamer et al. 1998), a portion of the exon 1 of the phytochrome B gene PhyB (Mathews et al. 2000), a low copy

nuclear gene encoding 4-coumarate: coenzyme A ligase (4CL) in the lignin biosynthesis pathway (Wang et al. 2000), the alcohol dehydrogenase

genes Adh1 and Adh2 (Sang et al. 1997), the chloroplast-expressed glutamine synthase gene ncpGS (Emshwiller and Doyle 1999), a portion of

the intron of a MADS-box gene pistillata and the cpDNA trnL intron (Bailey and Doyle 1999), the vicilin gene (Whitlock and Baum 1999), the

single-copy nuclear genes Glb1 (Hilton and Gaut 1999) and cl (Hanson et al. 1996), three plastid DNA regions of the rbcL gene, the trnL-F

intron/intergenic spacer and the rps16 intron (Goldblatt et al. 2003) and many other molecular markers were also used and found in literature

with very little significance. Additionally, the nrDNA ITS1 and ITS2 regions, which have been widely used at interspecific level in plant

phylogenetic studies (Baldwin et al. 1995) and it is nowadays applied in thousands of species.

5.2. DNA barcoding for plants through the nrDNA ITS1 and ITS2 regions

Nucleotide sequence information of multicopy genes provide accurate evidence of divergence in closely related organisms and have been widely

used for phylogenetic inference. This is the main reason that internal transcribed sequence (ITS) information of the nrDNA ITS1 and ITS2

regions were selected in BBGK to apply to the different accession numbers of native Greek plants. The dynamics of DNA sequences constitute

worldwide attention and significance for species identification and origin. Therefore these sequences can be used as DNA barcodes and

accompany every individual plant or vegetative clone propagated in the nursery or tissue culture laboratories of BBGK, stimulating the creation

Table 2 (Cont.)

Use Impressive botanical features Taxon (species or

subspecies)

Family

Co

nse

rvat

ion

pri

orit

y

Aro

mat

ic p

rop

ertie

s

Ph

arm

aceu

tica

l pro

per

ties

Gar

den

orn

amen

tal

Cu

t fl

ow

er

Po

tted

pla

nt

Pro

pag

atio

n

Irri

gat

ion

Typ

e o

f so

il

Lar

ge

flow

ers

Den

se in

flore

scen

ce

Oth

er

Lea

ves

Po

st h

arve

st

/ tra

nsp

ort

atio

n

char

acte

rist

ics

Pterocephalus perennis

subsp. bellidifolius

Dipsacaceae 3 P S/C M Acid Compact form c Satureja cuneifolia Lamiaceae 2 A P C L Lime c $ Saxifraga rotundifolia

subsp. chrysosplenifolia

Saxifragaceae 2 A1 P1 T M Clay Dotted petals Rounded b Silene orphanidis Caryophyllaceae 1 A1 P1 S L Lime Compact form c Stachys ionica Lamiaceae 1 A P1 C M Clay/

Alkaline

Glaucous-green,

hairy

c

Staehelina uniflosculosa Compositae 2 P? C L Lime Everlasting flowers

Large b

$ Thymus sibthorpii Lamiaceae 4 A P1 C L Lime d Thymus thracicus Lamiaceae 4 A P1 C L Lime c Verbascum

xanthophoeniceum

Scrophulariaceae 4 A1 P1 T M Acid Rosette d Viola cephalonica Violaceae 1 A1 P1 S M Lime b Empty cells concern absence of information. Taxa marked with (#) concern narrow Greek endemics that according to the Royal Horticultural Society Plant Finder have

been or are currently purchased in nurseries of Great Britain, while taxa (sub-) endemic to the Balkans are marked with ($).Conservation priorities 1: Taxa included in

national and/or international catalogues and/or conventions (e.g. Greek Presidential Degrees, EU Directives and Annexes, Red Lists, CITES, Bern Convention etc),

regardless of their endemism and/or narrow Greek endemics (single-island, single-mountain or single-phytogeographic area endemics). 2: Taxa endemic to more than a

single phytogeopraphical area of Greece or of other Balkan country or endemic to the boundary areas of neighbouring Balkan countries. 3: Taxa endemic to the Balkan

Peninsula. 4: Taxa found mainly in the Balkan countries and/or extending to W Turkey and/or parts of Italy or other taxa rare in Greece. A: Species with aromatic

properties. A1: Known aromatic properties in other species of the genus. P: Species with pharmaceutical properties. P?: under investigation. P1: Known

pharmaceutical properties in other species of the genus. Propagation S: seed, C: cuttings, Iv: in vitro, T: testing in process. Irrigation L: low. M: medium, H: high. Post

harvest / transportation characteristics: refer to desirable for the market characteristics based on observations during maintenance. a: very good, b: good, c:

medium, d: not good, e: bad.


of a database of documented reference sequences in a universal library worldwide, to which comparisons of unidentified taxa can be made.

In 2003, researchers at the University of Guelph in Ontario, Canada, proposed DNA barcoding as a way to identify species (see

www.barcoding.si.edu). DNA barcoding follows the same principle like a basic taxonomic practice of associating a name with an exact reference

collection, in conjunction with a functional understanding of species concepts (i.e., interpreting discontinuities in interspecific variation) (Kress et

al. 2005). In other words, it is a standardized genetic approach that uses short but specific DNA tags, bar codes, from a uniform locality on the

genome presented in all living organisms, in order to distinguish one species from another. The aim and the benefits are subject to problems

related to biodiversity and the tree of life (Stoeckle et al. 2004). The most important ones which meet the objectives and standards of BBGK

focus on: (i) distinguishing species that look alike, enabling a more accurate view of biodiversity, (ii) reducing ambiguity because a sequence of

four discrete nucleotides CATG along a uniform locality on genomes, providing a digital identifying feature which supplements the more

analogue gradations of words, shapes and colours, (iii) making expertise go further by facilitating plant identification, and (iv) contributing to a

more democratized access to a standardized library of barcodes, that will empower many more people to call by name the species worldwide,

making possible the identification of species whether abundant or rare, native or invasive, engendering appreciation of biodiversity locally and

globally.

M ean m onth ly m in im um tem perature

-1,4

0

2,3

6

9,3

12

9 ,6

6,3

3

0

21 ,2

1 8,7

15,6

12

8,8

-1,6

11,9

6,7

6 ,8

8,6

11 ,2

14,5

18,1

20 ,8

-5

0

5

10

15

20

25

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

S ep

O ct

Nov

Dec

C

Vio la_cep Thym _hol

Lim o_ ith

SileC epCep

M ean m on thly m ax im um t t

57,1

11

16,4

20,7

23,8

20

14,8

10,1

6

32,2

28,5

24,1

19

15

4,6

23,9

32,2

28,7

24,5

20

16,11 4,113,4

0

5

10

15

20

25

30

35

Jan

F eb

M ar

A pr

M ay

Jun

Jul

A ug

Sep

O ct

N ov

D ec

C

V iola_cep

T hym _hol

L im o_ith

S ileC epC ep

Fig. 6 Differences in monthly temperature profiles (mean minimum and maximum temperature), mean precipitation of the wettest and driest quarter of the year, Emberger

pluviothermic quotient and variation of the mean monthly precipitation regarding four selected IPS of Cephalonia island (Viola cephalonica (Fig. 3A), Thymus holosericeus,

Limonium ithacense, Silene cephallenia subsp. cephallenia, Mouflis et al. 2007).


Generally, four individual steps are required in

order to barcode each plant individual. The first

step is the specimen under investigation, which all

of them are currently maintained in living collec-

tions in the BBGK. The second step is the experi-

ments in the laboratory, which are subject to DNA

extraction, PCR for amplification of target genes,

purification steps and sequencing the obtained

sequences for each specimen. There are a large

number of protocols in the literature to apply and

work with even difficult samples, but there is not

any universal protocol yet to apply for every spe-

cimen and therefore, it is necessary to be deve-

loped. The third step is to deposit these sequen-

ces in a Database with free worldwide access.

Such Databases are found available at: (i) The

International Nucleotide Sequence Database

Collaborative, (ii) the GenBank in the U.S.A., (iii)

the European Molecular Biology Lab in Germany,

(iv) the DNA Data Bank of Japan and (v) the

Barcode of Life Database (BOLD). The forth step

applies to data analysis, where first the sequences

of the specimens are annotated and then they are

identified or compared to all closest relatives that are located in the database.

To improve species concepts, there is a need to develop a more sophisticated approach to barcoding, which would ideally include

sequences from multiple (perhaps six to eight) independent markers, a multi-locus barcode, and specific inference tools that could be used to

explore species limits and identify genetic gaps. This second type of barcode would improve the information, which DNA barcodes depend on

(Chase et al. 2005).

Currently the BBGK maintains more than 1,200 different species and more than 2,300 accession numbers (>20% of the native flora of

Greece). Identification of these species is many times difficult and a time-consuming process. Furthermore, there is a need to reveal the genetic

identity of different accession numbers for many reasons i.e. uniqueness of specimens from different native plant populations originating in

different phytogeographical regions, possible sustainable exploitation of selected accessions, explicit plant documentation, and copyright.

Therefore a molecular DNA-based procedure has been adopted in the laboratories of BBGK.

Currently there are three projects subject to the phylogenetic relationships by using the nrDNA ITS1 and ITS2 molecular markers. The first

one concerns the wild carnations Dianthus crinitus (Fig. 7A), D. corymbosus (Fig. 7B), D. gracilis subsp. gracilis (Fig. 7C), D. gracilis subsp.

drenowskianus (Fig. 7D), D. giganteus, D. deltoides, D. petraeus, D. haematocalyx, D. monadelphus subsp. pallens, and 23 more unidentified

accessions belonging to wild Dianthus spp. The second one is an EU financed project for saffron and its wild allies, investigating cultivated

clones of Crocus sativus from different phytogeographical regions with different accession numbers of wild relatives of Crocus sativus such as C.

cartwrightianus and C. hadriaticus, collected from different areas of Greece (see www.crocusbank.org). The third project is related to different

accessions of plants belonging to Satureja sensu stricto (35 accessions of species belonging to Satureja montana group) and representatives of

other closely related genera like Micromeria, Calamintha and Acinos all belonging to Satureja sensu lato. Preliminary results from this project

have revealed high levels of interspecific divergence in ITS1 and ITS2 ribosomal regions (Tsoktouridis et al. 2007).

In the future the aim is to extend and cover all the IPS and further to apply to the whole collection of taxa maintained in the BBGK. Data will

be deposited in the NCBI GeneBank DNA sequence database and the sequences will be compared with the existed ones in order to possibly

confirm and/or identify the accession numbers examined or to contribute new reference material to the database for future comparisons by other

scientists. In addition it is aimed that such an action would act as a unique documentation of the phytogenetic resources evaluated and managed

by the BBGK.

6. THE PROPAGATION OF THE IPS FIRST POLICY

6.1. Maintenance of IPS mother plants in the BBGK

In the BBGK, information from the literature and particular ecological databases (Krigas et al. 2007, Mouflis et al. 2007) are used in order to

provide the cultivation guidelines for the IPS (Table 1), aiming at decreasing the transplanting sock and increasing cultivation and propagation

success (Table 1; Figs. 5-6). In addition, IPS mother plants with similar ecological requirements are grouped accordingly, in order to save time

and labour.

All IPS are maintained on farm or in pots in the full equipped extensive mother plantations and nurseries of BBGK under ideal conditions,

which are as similar as feasibly possible to the wild habitat of each taxon studied (Maloupa et al. 2003b).

Although great consideration is constantly given to climatic conditions and soil properties, similar to the natural habitat of the plants,

sometimes looses of few mother plants are unavoidable especially regarding the mountainous plants and the (sub)alpine species. In this case,

the maintenance of problematic species has been overcome by in vitro culture methods. For instance, the single-mountain Greek endemic

Achillea occulta (Contstantinidis and Kalpoutzakis 2005), which grows in semi-shade, hidden limestone rock hollows, could not adapt in the

C

A

D

B

Fig. 7 (A) Dianthus crinitus, (B) D. corymbosus, (C) D. gracilis subsp. gracilis, (D) D. gracilis subsp.

drenowskianus


environments of the nursery (sea level). Therefore, apical meristems from mother plants have been successfully established in vitro in MS

(Murashige and Skoog 1962) medium supplemented with 4 M 6-benzyladenine (BA), 0.5 M indole-3-butyric acid (IBA), 2% sucrose, 0.6% agar and pH 5.8. Cultures were kept at 222oC and 16-h photoperiod under cool white fluorescent light (40 mol m-2 s-1). Explants produced

adventitious shoots and they were divided in small shoot clusters which formed roots in MS medium supplemented with 10 M IBA. Rooted plantlets were planted in trays filled with a peat-perlite 1:1(v/v) mixture and they were maintained in a glasshouse under a 90% RH fog-system

and 50% shading for 10 days. In the following 10 days, RH was reduced (5%/day), while light intensity was gradually increased. Plants were

acclimatized and transferred from the nursery to BBGK at 600m altitude where the climatic conditions were more suitable for their development.

Plant material at the nursery is now maintained only in vitro until the elimination of the problems described above (Fig. 8).

Emphasis is also given to the hygienic state of the stock mother plants. In some taxa, a water stress is recommended in order to eliminate

pathogen infections prior propagation (Hadidi 1998; Smith 2002, 2005). Viruses and virus-like diseases are very common in native species

(Dovas et al. 2002; Oshima et al. 2004) and the danger of spreading could always become catastrophic, especially in some species like

Dianthus spp. (Smyrnioudis et al. 2001; Terzakis et al. 2002). Thus, all the initial propagation material is obtained from mother plants treated with

exceptional care followed the standards of BBGK.

6.2. Propagation of the IPS in the BBGK

The major priority of the BBGK is to support, promote and contribute to the combined in-situ and ex-situ conservation and management of the

IPS. Propagation and mainly the asexual reproduction of the IPS constitute the most significant stage prior to their evaluation for sustainable

exploitation in order to improve human well being (Maloupa et al. 2003a, 2003b, 2003c, 2005).

The plants collected from nature comprise the initial source provided for the production of hundred thousands of plants, in a very short

period of time; therefore, an error in this procedure could be very critical (Hartman et al. 2002). The production of certified propagation material of

the IPS requires a broad-spectrum of research strategies and methods that have been extensively described and successfully applied for other

cultivated plants (George 1993, 1996; Pierik 1997; Hartman et al. 2002).

At the Laboratory-BBGK, each mother plant collected from the natural environment is treated as a clone; it is asexually propagated and

Fig. 8 In vitro propagation of Achillea occulta (up), Aubrieta erubescens (middle) and Dianthus crinitus (bottom) in the BBGK.


considered as the fundamental propagation material for the reproduction of the IPS. The selection of certain clones requires specific criteria

according to the standards followed by the BBGK. This certified propagation material provided for the reproduction, should be derived from a

selection procedure in order to supply products of high quality. These criteria strictly focus on:

a. The characteristics of the plants related to their sustainable exploitation (high production and quality of essential oils, potential

exploitation in floriculture or landscaping etc),

b. True to type, certified for the genetic identity and stability, of the selected clone (by using molecular DNA markers, Section 5),

c. Disease free, to ensure a tolerable limit (if not absence) of pathogens (Smith et al. 2002),

d. Yield, necessary to evaluate the selected material in order to verify that the unique characteristics are maintained after cultivation

treatments, achieving the maximum economic benefit, and

e. Asexual reproduction of the selected clones.

The BBGK currently maintains 2,300 accession numbers belonging to 1,200 native plant taxa (more than 20% of the native Greek flora). In

total, 326-selected Priority 1, 2, 3 and 4 IPS are maintained (176 taxa as stock mother plants and 150 taxa as seeds preserved within adjusted

environments). All of them have been or are currently subject to propagation research in the nursery and laboratories of BBGK (Table 2). Already

ca. 45% of them (139 taxa) have been successfully propagated and species-specific mass multiplication protocols have been developed (Table

2). Taking into account their ornamental characteristics and/or aromatic-medicinal properties, these taxa could be possibly considered as

commercially valuable plants for sustainable cultivations (Table 2). Additionally, ca. 100 propagation protocols and cultivation protocols have

already been developed regarding Priority 5 IPS (ca. 45% of the total mother plants maintained in the nurseries of BBGK).

6.3. In vitro production of elite pre-basic material and IPS mother plants

In vitro culture of any tissue used as explant material (meristem, bud, shoot, node, etc.) makes possible vegetative cloning from axillary buds, by

the adventitious shoot multiplication method (Daunay et al. 2007). Propagation is carried out under aseptic conditions, free from pathogens,

yielding millions of plants within a year. The plant material derived from in vitro cultures become the elite stock plant material, from which mother

plantations of IPS are established for massive reproduction.

Currently, in the tissue culture laboratory of BBGK are mainly maintained Priority 1 IPS, which either could not adjust and grow at the

nursery conditions or they were not even possible to be propagated using conventional methods. Furthermore, the massive reproduction of

socio-economically valuable Priority 5 IPS is studied. Characteristic examples are Aubrieta erubescens, Astragalus maniaticus, Campanula

incurva, Centaurea cithaeronea, C. subsericans, Crithmum maritimum, Dianthus spp., Melissa officinalis, Origanum dictamnus etc. (Fig. 8).

Further propagation of the selected clones of the IPS is carried out asexually, using mainly softwood cuttings from the elite mother plants

(Table 2). The propagation material is preserved by the BBGK with the possibility to be provided for research in other research institutes. These

elite IPS materials are being also characterized genetically by using the ITS1 and ITS2 molecular markers in order to certify true to type

clone/species identity. However, the elite certified IPS becomes the best value initial explant material for sustainable commercial utilization and

therefore, many competitive companies are interested to establish collaboration with the BBGK.

7. THE COMBINED EX-SITU AND IN-SITU PLANT CONSERVATION POLICY

The Global Strategy for Plant Conservation (GSPC 2002) acknowledges 16 main targets in order to halt biodiversity loss by 2010 and, the

Convention on Biological Diversity prioritizes the in-situ conservation of rare and endangered plant species and their back up by ex-situ

conservation (CBD 1992). If the steady decline of plant diversity is to be halted in Europe as well, a thorough understanding of the flora is

needed, including listing and assessment of wild plant species, their abundance, and monitoring of changes in their distribution and status

(Council of Europe 2002).

During the last years, BBGK has undertaken efforts in order to contribute for the implementation of the Global Strategy for Plant

Conservation targets (GSPC) at local, national, regional and (inter-) national levels (Table 3).

7.1. Local scale actions

At the local level, BBGKs major plant display sections are dedicated to the ex-situ and the in-situ conservation of native plants (15 ha and 16 ha,

respectively). All propagated material of IPS is hosted in the ex-situ conservation sections of BBGK, where plants are arranged thematically and

ecologically (Fig. 9). Additionally, attention has been given in incorporating the issue of native biodiversity within the environmental activities in

order to increase public awareness (Krigas et al. 2006; Maloupa et al. 2006; Krigas et al. 2007). In order to reveal distribution changes due to

construction works in the garden or trampling from visitors and spreading of involuntarily introduced invasive species, population monitoring of ca.

300 plant species is in process in-situ at the 16 ha of BBGKs natural oak forest which hosts ca. 10% of the regional flora. Furthermore,

numerous micro-reserves are being created in-situ for wild orchids protected from (inter-) national legislation as well as for regional endemic

species (Fig. 10; Maloupa et al. 2007). Labeling of wild plant populations is made evident along the Path of Biodiversity within the oak forest by

using plant identity tags (ca. 500 tags for populations of 126 plant taxa, Fig. 10). Additionally, regular transplanting of orchids from trampled sites

into protected display areas is being exercised (Maloupa et al. 2007).

7.2. Regional scale actions

At the regional level, the in-situ population monitoring, backed up by ex-situ conservation actions, is currently in process at different

phytogeographic regions of Greece (Maloupa et al. 2007): (a) in Mt Athos, NE Greece, concerning the single-mountain endemics Silene

orphanidis, Helichrysum sibthorpii, Anthemis sibthorpii, and Aubrieta erubescens, the regional endemic Fritillaria euboica, as well as the rare in

Greece Galanthus nivalis subsp. nivalis, and (b) in Mt Aenos National Park and Cephalonia, Ionian Islands, SW Greece, concerning some

single-area and regional endemic plant species (e.g. Teucrium halacsyanum, Dianthus fruticosus subsp. occidentalis, Stachys ionica, S. parolinii,

Silene cephallenia subsp. cephallenia, Viola cephalonica etc). Additionally, in-situ population monitoring, investigation of the genetic variability


Table 3 In-situ and ex-situ conservation actions of the Balkan Botanic Garden of Kroussia (BBGK) contributing to the implementation of the principal targets of The

Global Strategy for Plant Conservation (GSPC) at local, national, regional, and international levels. Local level (LL): actions within and/or around the grounds of BBGK

(31 ha). National level (NL): actions of BBGK in collaboration with other institutes and/or organizations of Greece or actions in different administrative and/or

phytogeopraphic areas of Greece. Regional (RL): actions of BBGK in collaboration with adjacent countries and/or targeted in borderline areas of Greece. International

level (IL): actions of BBGK in collaboration with institutes of other countries and/or international organizations. BBGKs principal target is the documentation and

conservation of the Important Plant Species (IPS) of Greece and the Balkans, including single-mountain endemics, single-area endemics, Greek endemics, and local

Balkan endemics, other rare, threatened and/or endangered taxa, and Balkan subendemics with scattered distribution (Maloupa and Krigas 2007).

Target number (#) and short description in GSPC

(2002)

BBGKs

contribution level

In-situ /

Ex-situ

BBGKs activities realized and/or in process

# 1: A widely accessible working list of known plant

species as a step towards a complete world flora

LL, NL In-situ Synthesis of existing floristic knowledge scattered in various sources

and compilation of working lists of known IPS and their distribution

for 10 areas in different phytogeographical regions of Greece

(including a national park, 14 Natura 2000 sites, 8 islands, and 3

mountain areas)

# 2: A preliminary assessment of the conservation

status of all known plant species at national, regional

and international levels

LL, NL, RL, IL In-situ GIS mapping, monitoring and preliminary assessment of the size, the

exact location and the distribution of known wild populations of

globally and/or nationally threatened and endangered plant species in

Mt Athos, Mt Aenos National Park and the Ionian Islands

Fieldwork searching for new populations

Recording of ecological and habitat preferences

# 3: Development of models with protocols for plant

conservation and sustainable use

LL, NL, RL, IL Ex-situ Plant propagation protocols for 109 plant taxa

Cultivation protocols for 101 plant taxa

In vitro cultivation protocols

Establishment of propagation and conservation priorities

# 4: At least 10% of each of the Worlds ecological

regions effectively conserved

LL, NL In-situ In -situ conservation section in BBGK

Guided in -situ conservation in Cephalonia Botanica and in Chios

Botanic Garden of the East Aegean Islands

# 5: Protection of 50% of the most important areas for

plant diversity assured

LL, NL In-situ Identification of the most important sites for IPS diversity in a

mountain, a national park, 2 islands and 3 botanic gardens

GIS mapping of IPS

Labelling of plant populations in the wild in 3 botanic gardens

Delimitation and protection of populations of wild orchids and endemic

species in 3 botanic gardens

# 6: At least 30% of production lands managed

consistent with the conservation of plant diversity

LL

In-situ Fine scale pilot application of different forest management actions in

BBGKs natural oak forest

# 7: 60% of the worlds threatened species conserved

in -situ

LL, NL Ex-situ

In-situ

Propagation, hardening and cultivation protocols of rare and

threatened plant species, aiming to potential future re-introduction in

the wild

# 8: 60% of threatened plant species in accessible ex -

situ collections preferably in the country of origin and

10% of them included in recovery and restoration

programmes

LL, NL, RL, IL Ex -situ >2,300 accession numbers of >1,200 taxa (ca.20%) of the Greek flora

in ex -situ conservation in BBGK

Official call and request for the repatriation of IPS and native species

of Greece from other botanic gardens

Integration of all living collections of BBGK in BGCIs website in

preparation

# 9: 70% of the genetic diversity of crops and other

major socio-economically valuable plant species

conserved and associated indigenous and local

knowledge maintained

LL, NL, RL, IL Ex-situ Documentation, characterization and evaluation of Crosus genetic

resources, including saffron and its allies (Crocusbank, EU Project)

Pilot cultivation of 22 accession numbers of the protected vulnerable

Cretan endemic Origanum dictamnus for essential oils research and

production of natural cosmetics

Pilot cultivation of Melissa officinalis and Crithmum maritimum for

essential oils research and production of natural cosmetics

# 10: Management plans in place of at least 100 major

alien species that threaten plants, plant communities

and associated habitats and ecosystems

LL, NL In -situ Identification and assessment of invasive alien species in numerous

Greek cities (in collaboration with the Laboratory of Systematic Botany

& Phytogeography, School of Biology, Aristotle University of

Thessaloniki)

# 11: No species of wild flora endangered by

international trade

LL, NL Ex-situ IPEN numbering in all accession numbers of BBGK

# 12: 30% of plant-based products derived from

sources that are sustainably managed

- - -

# 13: The decline of plant resources, and associated

indigenous and local knowledge, innovations and

practices that support sustainable livelihoods, local

food security and health care, halted

LL In-situ Ethnobotanical survey in villages of Mt Kroussia (in collaboration with

the Department of Pharmacognosy, School of Pharmacy, Aristotle

University of Thessaloniki)

# 14: The importance of plant diversity and the need for

its conservation incorporated into communication,

educational and public-awareness programmes

LL, NL, RL, IL In-situ

Ex-situ

Bi-lingual thematic calendars of native plant species produced yearly,

distributed in Europe

Leaflets, presentations, posters, fliers

Environmental games, Environmental Trail, Path of Biodiversity,

Educational Field Work, Experiential Conservation of Wild Orchids, for

primary, secondary and tertiary education target groups

Organization of official events open to public and policy-makers,

yearly

Design and 3D modelling of the Botanic Garden of Environmental

Awareness, in Thermi, Thessaloniki


and ex-situ conservation of Greek endemic Crocus spp. (C. cartwrightianus, C. hadriaticus) which are considered as socio-economically valuable

wild relatives of saffron is in process, through a multilateral EU funded project (see www.crocusbank.org).

A C

B

ED

C

G F

Fig. 9 Ex-situ conservation sections with different micro-environments in the BBGK. (A, B) Aromatic-medicinal plants, (C) Stone-dwellers, (D, E) Aquatic and

hydrophillus (hydrophilic?) plants, (F) Educational herb garden, (G) Mediterranean stone garden.

Table 3 (Cont.)

Target number (#) and short description in GSPC

(2002)

BBGKs

contribution level

In-situ / Ex-

situ

BBGKs activities realized and/or in process

# 15: The number of trained people working with

appropriate facilities in plant conservation increased,

according to national needs, to achieve targets of this

strategy

LL, NL - Increasing number of researchers, agriculturalists, horticulturalists and

technicians in BBGK

Seminars concerning capacity building for the staff of BBGK

# 16: Networks for plant conservation activities

established or strengthened at national, regional and

international levels

NL, RL, IL In-situ

Ex-situ

In collaboration with Botanic Garden Conservation International

(BGCI), efforts to establish a National Network of Greek Botanic

Gardens of small size in different phytogeographic areas of Greece,

dedicated to the combined ex-situ and in-situ conservation.


7.3. (Inter-)national scale actions At the international level, BBGK being an active member of Botanic Garden Conser-

vation International (BGCI, see www.bgci.org) and is currently undertaking efforts

towards the establishment of a National Network of Greek Botanic Gardens dedi-

cated to the combined in-situ and ex-situ plant conservation of the native Greek and

Balkan flora (Maloupa et al. 2007). These efforts are aiming to deliver the message to

all institutions and stakeholders involved in plant conservation. The concept of this

initiative is subject to the establishment of numerous new, small-scale botanic gar-

dens across different phytogeographical regions of Greece, which will dedicate

accordingly to the ex-situ conservation of the endemic flora of different regions. In

addition, BBGKs conservation actions of IPS attempt to meet targets 1, 2, 3, 7, 8, 9,

14, 16 of the Global Strategy for Plant Conservation (GSPC 2002), contributing to the

effective conservation of globally threatened plant species, implemented at local and

regional levels (Maloupa et al. 2007).

8. THE EVALUATION FOR SUSTAINABLE EXPLOITATION POLICY

8.1. The importance of medicinal plants and natural products

Natural products have been investigated and utilized to alleviate disease since early

human history. Natural products were considered as a valuable source of drug leads,

and the testing of natural product extracts was widely practiced in the pharmaceutical

industry. In more recent times, natural products have continued to be significant

sources of drugs and leads. Their dominant role is evident since approximately 60%

of anticancer compounds and 75% of drugs for infectious diseases are either natural

products or derivatives of natural products (Shu 1998; Newman et al. 2003). Plants

continue to provide us with new chemical entities (lead molecules) for the develop-

ment of drugs against various pharmacological targets, including cancer, HIV/AIDS,

malaria, Alzheimer's disease and pain. The fact that many currently used drugs have being derived from natural sources (including paclitaxel,

camptothecin-derived analogues, artemether, galanthamine, tiotropium to name a few), suggests that there might be compounds in the plant

kingdom with promising clinical use.

The most interesting agents that are identified as natural products come directly from the biodiversity, i.e., the richness in variety of

organisms and genomes in the ecosphere. There are literally millions of natural chemical structure types resulting from natures combinational

chemistry effort supplying almost unimaginable chemical diversity, which yields stereochemically complex structures with diverse functional

groups, molecules ideal for interacting spherically with biological target molecules. There are undoubtedly many more unique and potent

biologically active natural products waiting to be discovered.

In addition to medicinal plants, many aromatic plants are used for the preparation of herbal drinks, in cooking or for phyto-cosmetic uses

(Sideritis spp., Origanum spp., Rosa spp., etc.). More opportunities for natural products discovery and development concern agrochemicals,

cosmetics, new chemicals and nutraceuticals. However, wild plants have played an important role in complementing staple foods to provide a

balanced diet by supplying trace elements, vitamins, and minerals, and may do so again in the future. Their interest as a source of nutra-

ceuticals has been highlighted in recent studies (Heinrich et al. 2005).

In the case of native plants of Greece, Alkanna tinctoria has been known from ancient times for its wound healing properties. Native A.

tinctoria plants, with alkannin and shikonin, two enantiomeric hydroxynaphthoquinone red pigments found in their roots, have been the base for

the development of a famous ointment which is approved and marketed in Greece and abroad as a drug of botanical origin, as well as for other

pharmaceutical and cosmetic preparations. Although Greece has been blessed with a diversity of flora, most of it remains unexploited regarding

species chemical constituents and biological potentials.

The BBGK, in collaboration with the Department of Pharmacognosy-Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki,

are directed towards the study of bioactive molecules found in IPS. The prime concern is to discover or develop pharmacologically active agents,

which might be interesting for clinical practice in the future. Up to date results show that a significant number of IPS appear to contain

compounds with interesting biological activities i.e. antioxidant, antivirus, antibacterial etc. (Skaltsa et al. 2003; Mata et al. 2006; Sylignaki et al.

2006; Charami et al. 2007; Karalaki et al. 2007; Kreziou et al. 2007; Mata et al. 2007a, 2007b; Papaioannou et al. 2007).

Furthermore, IPS with potential high economic value are under special investigation. In this approach, two types of plant material are used

and analyzed: (a) plant material collected in-situ from wild populations and (b) plant material originating at the same populations and sites,

accordingly, which has been asexually propagated and ex-situ cultivated in the BBGK (at sea level and at 600m). This approach concern several

aromatic-medicinal plants (e.g. Crithmum maritimum, Origanum dictamnus, Melissa officinalis, Staehelina uniflosculosa, Geranium macrorrhizum,

Thymus spp., Sideritis spp., Achillea spp., Scutellaria spp.) and is adopted aiming to evaluate and/or compare their quantitative and qualitative

composition (essential oils and biologically active secondary metabolites) under different cultivation regimes.

In the last few years, Greek and foreign companies have developed applied research projects, aiming at the exploitation of the unique

characteristics of the native Greek aromatic-medicinal species, many of which are confined to Greece only (Korres 2005; Grigoriadou et al.

2006). These research projects have been extensively considered by the BBGK and are carried out with the joint collaboration of Research

Institutes, Universities, National Research Centers and industry in applied research programs, financed by different sources. The aim for all of

Fig. 10 In-situ conservation actions in the BBGK.


these research projects focus on contributing to the integrated conservation and management of the aromatic-medicinal plants as well as other

major socio-economically valuable native Greek species (Maloupa et al. 2005).

The most representative examples of these research projects concern Origanum dictamnus and Melissa officinalis (both Lamiaceae) and

Crithmum maritimum (Apiaceae) (Maloupa et al. 2003c, 2005; Grigoriadou et al. 2006), all plants with exceptional characteristics and valuable

properties, which could potentially play an important role at the local economy (see www.bgci.org/cultivate/article/400/).

In addition, this initiative seems perfectly harmonized with the key role of Botanic Gardens, ensuring that plant recourses are not only

conserved but also used sustainably for the benefit of all people, in order to improve human well-being (Wyse-Jackson and Sutherland 2000;

GSPC 2002).

8.2. Evaluation of native plants prior commercialization

There is a rich base of information from many developmental projects and case studies that have been explored on various aspects on new crop

development. This information has been assembled in a non-unique improvised mode, using a range of methods at different levels, focusing

mainly on different elements, which are mostly related to product production, processing and marketing systems.

A universal standardized evaluation system is required to be established worldwide, in order to document and compare cases using

consistent terms and definitions for particular range of variables, following a data matrix as the basis for the comparative analysis (Armitage

1996).

The ultimate goal is to provide guidance for action-oriented interventions based on new crop development, moreover to identify conditions

and types of cases that are amenable to development interventions, as well as to flag types of cases that may not be good investments.

Exploratory data analysis will be used to outline patterns, gradients of variability, clusters of cases and key variables associated with them.

The objective is to create typologies of cases, identify conditions associated with particular kinds of development and conservation outcomes

and identify/test hypotheses about new crop development (Becker 2004).

This chapter provides some guidelines and describes the categories of information and the necessary steps required to take under

consideration in order to select promising plant taxa for commercialization. Interventions, and outcomes of new crop development provide

definitions and rationales for the various descriptors used. The evaluation parameters are described within five individual interconnected stages.

Stage I: Targeting the market

It is very important to initially characterize the plant material collected from the wild and to make a brief estimation of the sector that is

targeting in the market. Depending on the characteristics of each taxon it is necessary to be associated with a market category of promotion

according to the relative importance and use (Roh and Lawson 1996; Armitage 1996; Daly 2005; Johnston 2005). These major categories, which

lead to corresponded industries include:

a. Ornamental horticulture and floriculture; this sector holds a huge number of species, cultivars and hybrids, therefore, it is very

difficult to establish a new flower crop in a relative short period of time. There is too much competition among the species promoted but it is

also necessary to enrich the market with new taxa in order to maintain and increase the consumption. The related ornamental groups

include cut flowers, potted plants (usually for indoor use), dried flower compositions, amenity and landscaping. Many promising examples of

such plants originating in the wild Greek flora can be found in Table 2.

b. Cosmetology; this sector includes plants with aromatic properties for the production of fragrances, as well as taxa that provide

substances extracted and mixed with crmes, lotions and other cosmetics. This is a profitable category, due to the high demand of products

based on natural extracts, therefore, new valuable crops provide high income to farmers in rural areas. Many promising examples of such

plants originating in the wild Greek flora concern Origanum dictamnus, Crithmum maritimum, Melissa officinalis, etc.

c. Pharmaceuticals; this sector focus on taxa with very particular chemical substances, which mainly used for therapeutic/medicinal

purposes. These taxa automatically become valuable and research focus on the production of high quality plants used as the initial raw

material for drugs. In this case, advanced technology and systems can be used throughout the production process, because of the

importance and value of the raw materials. Many promising examples of such plants originating in the wild Greek flora can be found in Table

2.

d. Herbs and spices; this sector is nowadays of great interest because people prefer on consuming more fresh and natural healthy

products in their diet. Interesting examples originating in the wild Greek flora concern Origanum dictamnus, O. onites, Thymus thracicus, Th.

sibthorpii, Satureja cuneifolia, Crocus spp., etc. Most of the products are available in open markets as row material, which usually is not

certified. The production of culinary herbs in diverse agroforestry systems may provide small-scale farmers on competitively priced products

for local and potential export markets (Runham 1996). The increased demand and knowledge of people require an organized system that

provides certified and branded name products.

e. Sustainability and conservation; this category concerns taxa with very particular characteristics. Usually they are not

commercialized but maintained for research and conservation reasons. Plant species used for sustainability include taxa that are aimed to

be re-introduced in the wild and they are part of a countrys biodiversity. These taxa are massively propagated for plantations in nature

under particular circumstances such as fires, flooding problems, highway constructions and sinking of the ground, where there are

governmental programs to sustain or re-introduce the regional flora (Runham 1996). Conservation of threatened species, which tend to

extinct from the wild, is subject from most Botanic Gardens, which are aiming to rescue and maintain this genetic material and germplasm of

plants. Moreover, national and international research programs support the conservation, propagation of these endangered species and

further re-introduction to nature. Such actions of the BBGK are described in short in Table 3 and many examples of such species originating

in the wild Greek flora can be found in Table 2.

Stage II: Basic marketing information

Basic value chain issues and information in marketing is required in order to select the potential taxa for commercialization. It is very difficult


to explore and analyze multiple marketing parameters that may affect the introduction of new species in the market. The reason is that many

scientists who are specialized in the marketing of agricultural products should exclusively work on the establishment of new crops in the market,

increasing the cost and final price of the new products.

However, there is a lot of research developed on a variety of agricultural products from Universities. Research topics are considered from

time to time depending on the market. In this case, a joint collaboration program will help to promote the native IPS of Greece and the Balkans in

the market. Moreover, postgraduate research dissertations at MSc or PhD level would be potentially applied on native flora, aiming to

dynamically lead the IPS into the market. As the size of the Greek market and sales are limited, the native plants are often not appreciated by

the local consumers and therefore, it would be much more plausible to promote them into the foreign markets.

The size of the market in the European Union is promising to support the native plants from Greece produced with sustainable methods

since Greek native plants collected from the wild have already been on sale. From the native species mentioned in Table 2, a total of 54% are

being purchased in nurseries of Great Britain (18% are narrow Greek endemics and 36% are Balkan endemics).

It is not astonishing that many rare plants, which are endemic to Greece or the Balkans, can be found and are actually being sold in several

markets. For instance, everyone could easily find in the Royal Horticulture Societys Plant Finder (www.rhs.org.uk/rhsplantfinder/plantfinder.asp)

and purchase at least 115 Greek endemic taxa (ca. 10% of the endemic flora of Greece; Maloupa and Krigas 2007). Many among them are

included in the Red Data Book of Rare and Threatened Plant of Greece (Phitos et al. 1995), Annexes II and/or IV of the EU Directive 92/43

(Dafis et al. 1996) and WCMC lists (1991). To name but a few: the Cretan endemic Origanum dictamnus, the regional Greek endemic

Campanula incurva and the single-mountain (Mt. Athos) local Greek endemic Helichrysum sibthorpii (the latter is also included in the Bern

Convention). This fact brings about some legal and authority issues: Although clearly stated in 15 of the CBD (1992), no official agreement has

been made with the country of origin, regarding access to its phytogenetic resources, fair trade and benefit sharing. Furthermore, nowadays it is

recognized by most authorities that the collection of rare species is a significant problem in Greece and the Balkans (Polunin 1980). Either

advent of botanical interest tours or independently organized tours, the threat from the naturalist, amateur botanist, gardener or plant hobbyist

who uproots plants for his own collection, whether of live plants in a garden or pressed specimens in a private herbarium, is internationally

deplored (IUCN Threatened Plants Committee Secretariat 1982).

Apart from the legal and authority issues mentioned above, it is necessary to consider additionally the basic information adapted to regional

or national standards. The suggested topics for consideration are subject to: (a) Market characteristics (location, size, distance, etc), (b) Number

of participants in the market, (c) Role of participants in the market, (d) Production group processes, (e) Number of people with capability and

skills (production capacity and schedule) (Johnston 2005).

Stage III: Production

Important keys to success in the high value horticulture crop production are the low cost of production, high quality and the likely returns. A

chain of value issues takes part during the production process aiming to yield the best for the market and the consumer. The vast number of

different species commercialized requires special treatments for each taxon, therefore, a lot of research and many people are needed to

optimize every production line. This procedure (Roh and Lawson 1996; Armitage 1996; Daly 2005; Johnston 2005) focus on particular stages

which are subject to (see Table 2 and Figs. 4-6 for examples originating in the native flora of Greece):

(a) Propagation by seed or vegetative in vivo or in vitro, (b) Ability to produce planting material, (c) Climatic requirements and limitations, (d)

Soil and nutritional requirements (soil type, drainage, nutrient availability), (e) Production systems and their relative costs, (f) Qualitative growth

characteristics (flowers/inflorescences, roots, habit/sprouts, leaves, fruits/seeds, inner substances, habitat, diversity, hybridization, stability,

durability, special sensibilities, etc.), (g) Controlled flowering, flowering percentage and photoperiod, (h) Plant health (pathogens, pests and

weeds), (i) Total cropping time, yield and supply period, and (j) Price per unit.

Stage IV: Postharvest and transportation

Postharvest requirements focus mainly on packaging, maintenance and transportation (see Table 2 for examples originating in the native

flora of Greece). Packaging is depended on the size/shape of plants or flowers, delicacy and the market of sale. For auctions and wholesale

markets, packaging is of decisive significance; alternatively packing which presents the products require means and features like transparent foil,

text with the name, origin and indications for use etc., that increase the final cost of the products (von Hentig 1996).

Maintenance is the most diverse parameter to estimate. There are different requirements for each type of product and it is very difficult to

hold simultaneously different products within appropriate conditions. The main factors that affect maintenance include humidity, temperature,

chemical treatments and stress of the plants. The durability of the plants/flowers is connected also with the transportation; in many cases

controlled environments are necessary for fragile products that also increase the durability. The way of transportation and distance from the

producers is also very critical, affecting the final price and competence of the products in the market. Postharvest manipulations for herbs and

spices, are much easier and simple, due to most of them are sold as dried materials.

Stage V: Economics

Economics is a critical factor in producers adoption of new crops and technologies. Aspects of economic benefits include the maintenance

or restoration of the productivity of land or the provision of low cost alternatives to fertilizer and soil conditioners, direct economic benefits to

farmers by the addition of products or the diversification of the range of farm outputs and capital reserves in the form of new crops that accrue

value over time and can be harvested as needed (Arnold 1983).

The cost-benefit analysis can be used to determine which of a combination of systems is the most efficient (Becker 2004)

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