Genetic Resources of Saffron and Allies (Crocus spp.

J.A. Fernández
Laboratorio de Biotechnología, Instituto de Desarrollo Regional
Universidad de Castilla-La Mancha
Campus Universitario s/n
Albacete E-02071
Spain

Keywords: bank of germplasm, biodiversity, Crocus sativus, CROCUSBANK

Abstract
Saffron is the highest valuable food product and one of the oldest crops
and medicinal plants. Saffron crop is subject to strong genetic erosion. Until now
no international institution has taken the responsibility of the creation and
maintenance of a collection representative of the genetic variability of the crop
and its allies. Recently, the European Commission has approved an Agri Gen
Res 2005 project on “Genetic Resources of Saffron and Allies (Crocus spp.):
CROCUSBANK”, coordinated by this author. The objective of our consortium,
composed by 14 groups of 9 EU and non-EU countries, is to create, characterise
and exploit a germplasm collection (bank) in Crocus species, including Saffron
crocus. This collection is an urgent need for Saffron breeders and farmers, as
pointed out in the 1st ISSBB (Albacete, Spain). The present paper explains the
background, objectives, organization and technologies to be applied in the
development of the project.

IN MEMORIAN
Dr. Fikrat I. Abdullaev (1943-2006). A brave, enthusiastic, charismatic and sensitive
man; an original and wise scientist. Also a friend.

INTRODUCTION
Saffron is a Precious and Intriguing Species
Crocus species are members of the family Iridaceae. The plants in this family
are herbs with rhizomes, corms or bulbs. The family Iridaceae embraces about 60
genera and 1,500 species. The genus Crocus includes native species from Europe,
North Africa and temperate Asia, and is especially well represented in arid countries
of south-eastern Europe and Western and Central Asia. Among the 85 species
belonging to the genus Crocus, C. sativus L. (Saffron) is the most fascinating and
intriguing species (see Fernández, 2004). This is not only because it produces the well
known Saffron spice, but for the numerous mysteries surrounding its origins.
Questions as when it originated; the native area or areas; the ancestor species and the
mechanisms of origin; the wild or naturalized plants; the infertility and consequent
absence of fruit and seeds; remain to be explained. Greece (Crete) has been mentioned
as probable origin of this old crop with probably more than 4,500 years of age. If its
domestication occurred at more sites simultaneously or at different times is still not
resolved. This is because Saffron is not known to be wild or spontaneous and can only
be propagated by human help.
In addition to being the most precious spice in the world, Saffron possesses a
set of somewhat unique agronomic and eco-physiological characteristics including a
relatively low water use, growth and development during fall and winter, a very low
harvest index, a generative phase which is followed by the vegetative phase of
growth, and an economic yield which is produced prior to a significant vegetative
growth. Furthermore it has three exceptional quality attributes, i.e. aroma, flavour and
yellow dye, a set of characteristics bringing about its uniqueness for pharmaceutical,
food and textile industries. Between 100,000 to 200,000 flowers are required to yield
one kilogram of Saffron spice (about 900,000 dried stigmas). Medium yield are
around 10 kg of Saffron/hectare, but varying very much between countries, lands,
seasons, and agronomic practices. Saffron’s high price is due to the much direct
labour required for its cultivation, harvesting and handling. This fact has made
progressively uncompetitive this crop in the more developed countries, and
undoubtedly such tendency will extent to the currently producing leaders. Therefore,
there is need for increasing Saffron production and quality to cope with an increasing
demand and market differentiation. This will be achieved biologically by means of
plants with more flowers per plant, flowers with a higher number of stigmas,
increasing stigmas size or stigmas with an increased amount of dye and aroma. In a
further step, a new approach could take place, the consideration of C. sativus as a
source of phytochemicals and biopharmaceuticals.
C. sativus is an autumnal flowering geophyte with corms that are covered by a
tunic, dormant during summer, sprouting in autumn, and producing 1-4 flowers in a
cataphyll with linear leaves. The flower has an underground ovary, a style 9-10 cm
long, dividing at the top in three red trumpet-like stigmas (2.5 cm long) that once
dried form the commercial spice Saffron. Flowering spans from late autumn until
December according to climatic conditions. Cytological studies have indicate that
Saffron is a triploid species which genome shows 3n=24, x=8 chromosomes. Its
triploid condition allows vegetative multiplication, but not regular sexual
reproduction. This is because meiosis and gamete development in triploids are
irregular, resulting into many anomalies in sporogenesis and gametophyte
development.

Saffron is an Amphiploid with Probable Low Genetic Diversity

One important aspect of our issue is to ascertain the genetic diversity of C.
sativus. Saffron usually multiplies year by year by means of corms. Because corm
multiplication does not induce genome variations with the exception of some
mutation that in a triploid Saffron population are not easily detectable, all Saffron
should be similar one to the other. Some authors believed that Saffron was once
naturalized in small areas from where it was lost as a consequence of a change in land
use. C. sativus was generally assumed to be of autotriploid or hybrid origin. Now we
have several data that support the alloploidy of C. sativus being the parents C.
cartwrightianus and C. hadriaticus, both with 2n=16 and present currently in Greece
but not in overlapping areas. Other possible parents, e.g., C. thomasi, from Italy and
Croatia, C. mathewii from Turkey, and C. pallasii ssp. haussknechtii from Iran-Iraq-
Jordan, cannot be excluded. The complexity of the evolutionary history of the genus
Crocus suggests an intensive species hybridisation and explosive speciation in Crocus
evolution that could be on the basis of the origin of Saffron. We now are sure that
Saffron is an allopolyploid but the localization of the hybridisation event has not been
ascertained so far. If the event took part several times could have generated different
amphiploids and, in consequence, different Saffron lines. Saffron was introduced in
Western Europe in different historical moments: In the Iberian Peninsula by the Arabs
or even before (Romans); in Germany, Switzerland, France, Italy and Great Britain by
the Crusaders, and towards the east it was extended by a variety of cultures through
Transcaucasia, China, India and eventually Japan. Should we expect to find genetic
diversity then?
We can look the chromosomes as a first approach. The karyotype of C. sativus
has been studied by a number of authors, reporting that Saffron from different
countries (Azerbaijan, Iran, Italy, Turkey, France and England) was always 2n=3x=24
lacking karyologycal differences. However in the past literature other karyotypes
were described. Cytofluorimetric analysis on nuclear DNA, carried to detect genome
size and base pairs composition, revealed no differences in DNA content and
composition in Saffron corms cultivated in different countries (Italy, Israel, Spain,
Holland). Some assays with molecular markers (RAPDs) have revealed limited
genetic differences among Saffron samples from Italy, Iran, Greece and Spain.
Nevertheless, analysis of phenotype revealed differences in aspect flower size, tepal
shape and colour intensity with lobed tepal in plants from Israel and more intense
colour of tepals in plants from Sardinia (Italy). Variants of Saffron with an increased
number of stigmas, maintaining 2n=24 have been reported with a frequency of
1.2x10-6 of the rare type flowers. Such phenotypes are well known by farmers and
breeders but unfortunately they are not stable. Morphological differences with flowers
having higher number of style branches and stamens have been already described in
Saffron cultivation at L’Aquila (Italy). Phenotypic variants are also present in
Kashmir Saffron (F. Nehvi, personal communication).
Besides different commercial products are known that could suggest the
existence of different Saffron ecotypes or commercial varieties, the actual genetic
variability present in C. sativus at worldwide scale is currently unknown.
Nevertheless, there is a suspicion in Saffron breeders regarding the existence of scarce
genetic variability in this crop, but no serious effort has been carried out to ascertain
this important issue. They have been attempts with the objective to increase the
spectrum of variability for floral traits and recovering auto-hexaploids (6x=48) in
Saffron (to break the sterility barrier) by colchinization and to induce the genetic
variability in Saffron using physical irradiation in order to develop polyploid forms;
tetrafid, pentafid or hexafid stigmatic plants; and colour mutants. These attempts have
been unsuccessful so far (for review see Fernández, 2004).

THE RELEVANCE OF SAFFRON CROP

Saffron is Highly Valuable
Medicinal and aromatic plants have been increasing in importance to society
continuously for the past 100 years. Saffron is made from the dried stigmas of the
Saffron flower, a triploid sterile plant that is vegetatively propagated by means of
bulbs (or corms). Saffron is mostly used as spice and food colorant and, less
extensively, as a textile dye or perfume. However, due to its analgesic and sedative
properties folk herbal medicines have used Saffron for the treatment of numerous
illnesses for centuries. Saffron is considered to be the highest priced spice in the
world (on average, 500 $ every Saffron kg). Its high value makes Saffron the object of
frequent adulteration and fraud (see Fernandez and Abdullaev, 2004).

Saffron is a Crop in Danger of Extinction in Many Countries

Saffron is currently being cultivated more or less intensely in Iran, India,
Greece, Morocco, Spain, Italy, Turkey, France, Switzerland, Israel, Pakistan,
Azerbaijan, China, Egypt, United Arab Emirates, Japan and recently in Australia
(Tasmania), Afghanistan and even Iraq. While the world’s Saffron production is
estimated in 205 tons per year, Iran is said to produce 80 percent of this total, i.e. 160
tons. Khorasan province alone accounts for 46,000 hectares and 137 t of the above-
mentioned totals, respectively. The Kashmir region in India produces between 8 to 10
t mostly dedicated to India’s self-consumption. Greek production (4 t) is located
exclusively in Macedonia (Kozani) and controlled by a single cooperative. Morocco
produces between 0.8 and 1 t (Table 1). These figures do not pretend to be accurate
(an impossible task for several reasons). They just want to be good enough to
illustrate the reader.
Saffron production has decreased rapidly in many European countries. Spain,
the traditionally world leader and most reputed Saffron producer for centuries,
nowadays makes about 0.3/0.5 t. Productions of Italy (Sardinia, Aquila, Cascia) 100
kg; Turkey (Davutobasi, Saffranbulli) 10 kg; France (Gâtinais, Quercy) 4/5 kg and
Switzerland (Mund) 1 kg are nearly nominal. Other countries as Azerbaijan produce
negligible amounts of Saffron. An illustrating fact: In early 1970s Saffron cultivation
in Spain and Iran were 6,000 and 3,000 ha, respectively, while at present the surface
areas are 77 ha in Spain and near 50,000 ha in Iran. Only 20 years ago Spain and Iran
were producing the same quantity, about 35 to 40 t.
Saffron crop disappeared in other European countries such as Germany,
Austria and England; here it was grown in great quantities in Essex (especially near a
town called Saffron Walden) and Cambridgeshire. After having been the leaders of
Saffron production at commercialisation at a worldwide scale for centuries, nowadays
European countries only produce a scarce 3 %, even tough the quality and the prestige
in the marker still correspond to the European brands. The European Union has
awarded the designation "appellation of origin" to the "Azafrán de la Mancha", the
Greek "red Saffron" under the name "Krokos Kozanis", and the Italian "Zafferano
dell'Aquila". The reasons of Saffron decadency are various. An intensive (and
expensive) hand labour of up to 15 working days per kilogram of dry Saffron spice is
required for flower picking and stigma separation. To the high cost of this labour it
should be added the very uncomfortable stooping position of the flower pickers, and
the very short picking period which comprises the early morning hours of the 20-30
days of duration of the flowering season. The mechanisation of flower picking in field
grown Saffron has proved difficult.
All Saffron producers in the EU, also soon Turkey, suffer from increasing
labour costs. Iran can increase its production to more than 200 t whereas India could
offer its Kashmir Saffron to the world market in growing amounts. China will become
a massive maker and there are serious projects of Saffron production in Afghanistan
and Iraq. A grey market of Saffron has developed in some countries in the Caucasus,
trading Iranian Saffron through doubtful channels without quality control. Countries
in North Africa are the primary origin of forged Saffron, mostly Carthamus tinctorius
or Curcuma. Hence, the Saffron world market panorama is al least uncertain.
Nevertheless, although the tendency of Saffron diminution has been constant, they are
symptoms of a revival in Saffron crop in Europe. France, for instance, has shown the
emergence of new associations of Saffron farmers ("Les safraniers du Gâtinais" in
1987, and “Les Safraniers du Quercy” in 1999) after decades or even centuries of
abandon of the crop. Other initiatives are flowering in Italy (Sicily) and in many other
countries outside Eurasia such as Australia (Tasmania), New Zeeland, Argentina,
Chile, Bolivia, even USA (Pennsylvania).

Other Crocuses are also Economically Important

The Crocus genus is known mainly for the cultivated species C. sativus, which
is of prime economic importance. However, there are also other species belonging to
this genus, which are highly prized for their colourful flowers, and thus used
extensively in specialized gardening (Table 2). These are horticultural varieties of C.
vernus, C. versicolor and C. aureus, amongst others. Most of the Crocus species grow
naturally in the fields between shrubs and grass or light woodlands. The plants in this
family are herbs with rhizomes, corms or bulbs. The genus Crocus includes about 80
species distributed from south-western Europe, through central Europe to Turkey and
south-western parts of Asia, as far east as western China (Mathew, 1982).

Saffron is Subject to Strong Genetic Erosion

As explained, the lost of land surface dedicated to Saffron crop in many areas
has resulted in a corresponding genetic erosion, the situation being dramatic at the
present time. Traditional plant breeding techniques are based on a bulk selection of
the best samples among natural or cultivated populations; genetic breeding with wild
ancestral species; and spontaneous or induced mutations. Sterility in Saffron limits the
application of conventional breeding approaches for its further improvement. Besides
different commercial products are known that could suggest the existence of different
Saffron ecotypes or commercial varieties, the actual genetic variability present in C.
sativus at worldwide scale is currently unknown (Fernandez, 2004). They have been
efforts by Indian researchers to increase genetic variability in Saffron using non-
conventional breeding techniques, such as induced mutagenesis employing physical
irradiation and induction of polyploidy by colchinisation. Nevertheless, the
preliminary results of induced genetic variability are not completely hopeful and
probably would require further work.

A CROP OF ADDED-VALUE FOR EUROPE

Saffron must be considered as an endangered crop of added value deserving
scientific interest in Europe for various reasons.

Its Origin and Historical Background

The origin of Saffron crop is uncertain. C. sativus is cultivated for its spice for
at least 3,500 years in Egypt and Middle East. The name Crocus finds its origins in
the Greek word krokos, Saffron, which in turn derives from the Semitic word karkom,
one of the oldest names for this plant. Some archaeological and historical studies
indicate that domestication of Saffron dates back to 2,000–1500 years BC. This is
derived from documents reproducing the plant or showing people collecting the crop.
However the sites where the first Saffron plants appeared differ according to the
opinion of various authors. Vavilov placed Saffron into IV plant origin centre of
Middle East (Minor Asia, Transcaucasia, and Turkestan); whereas more recent
contributions indicate that the process of Saffron domestication has to be identified on
Crete during the Late Bronze Age. Historical records detail the use of Saffron date to
ancient Egypt, Persia, Greece and Rome, where it was used as a medicinal plant, as a
dye, in perfume, and as a spice and colorant for culinary purpose. Saffron use reached
its highest point in the Iberian Peninsula by the Arabs. During the Middle Age Saffron
crop was extended for rest of Europe due to the Crusaders, and was settled in
Switzerland, France and Italy, reaching Great Britain in the 14th century. Towards the
east, Saffron crop was preserved for diverse civilizations in the current territories of
Iran, Azerbaijan and Kashmir. Saffron was firstly exported to China in 13th century as
healthy food, and again in 16th century as medicine. It reached Japan at the beginning
of 17th century. Since then, Japanese people have used Saffron almost exclusively as
health product.
Tradition and Quality
Besides the European countries only produce a scarce 3 % of world Saffron
market, the quality and the prestige in the marker still correspond mostly to the
European brands. La Mancha Saffron has been generally the highest prized, and “La
Mancha” region was during centuries the world biggest producer. Currently the
Spanish manufacturers and dealers control 70 % of the world’s Saffron market.
The European Saffron under Protected Geographical Indication (PGI) status
are:
• "Krokos Kozanis"(EC Reg. 378/1999), Greece (Kozani)
• “Azafrán de la Mancha" (EC Reg. 464/2001), Spain (La Mancha)
• "Zafferano dell'Aquila" (EC Reg. 2081/92), Italy (L’Aquila)
• “Munder Safran AOC”, Mund (Switzerland)
• France have two associations of Saffron farmers that pursue the same PGI
status:
• "Les safraniers du Gâtinais" in 1987
• “Les Safraniers du Quercy” in 1999
As mentioned Saffron crop is in severe danger of extinction in Europe at the
present time. What used to be the “King of Spices” suffers from a worldwide
devaluation. Amazingly, very little has been done to protect the Saffron crop in
Europe. Recently, in 2002, the Regional Government of Castilla-La Mancha (Spain)
spent 150,000 euros/year in direct financial support to Saffron producers, ranging
between 1,200-1,800 euros/ha with a maximum of 3,000 euros per farmer.
Apparently, such benefit has not continued in the subsequent years. Anyhow, the
genetic erosion has been intense and now it now it is urgent to protect what remains of
the European Saffron germplasm. On the other hand, Saffron is considerate to be the
highest priced spice in the world. In fact, Saffron is one of the most valuable food
products (the red gold), and its highest quality is still associated to the European
brands. Although not exclusively, the European production has the added value of
prestige and cultural significance of the product (gastronomy, history, art).

Food Safety
Consumers are confused with the differences in quality between Saffron of
different origins and the subsequent fluctuation of prices. The quality of Saffron is
certified in the international trade market following the ISO 3632 Normative since
1993. The most important parameter is colouring strength, calculated from UV-Vis
measurements at 440 nm in aqueous extracts of this spice. Such measurements are
related to the total carotenoid content. This regulation is currently under controversy
since leave aside the most important organoleptic properties of Saffron (odour,
flavour) and does not prevent fraud. Saffron commands a rather high value in the
international spice trade that results in its frequent adulteration by artificial colorants
and by mixing genuine stigmas of Saffron flower with other parts of plants (e.g. some
species of grass) artificially coloured. Molecular tools will be ideal for checking
purity of product, even after processing, act as markers for adulteration (either with
dyes or with other plant species).

Alternative Uses
Saffron is mostly used as spice and food colorant and, less extensively, as a
textile dye or perfume. However, due to its analgesic and sedative properties,
traditional Eurasian herbal medicines have used Saffron for the treatment of numerous
diseases. Saffron has been indeed one of the oldest plants used as medicine (see the
frescoes of Thera, Greece). The renewed importance of Saffron as nutraceutical is
being sustained by an increasing number of evidences. Saffron has been claimed to
have effects on senile dementia, retina-degeneration, immunomodulation, as well as
antimicrobial, antidepressant, antitumour or cardiovascular protective properties.
Although these effects required more pharmacological/nutraceutical trials and
significantly additional scientific substation, we support the potential health
application of this plant, with a profile similar to green-tea, ginseng, etc.
The application of the agroresource refining concept to the other parts of the
Saffron plant (style, petals, stamens, leaves, stem and corms) is actually underway in
order to valorise all the organs of the plant. This Saffron by-product valorisation is in
agreement to the European Agricultural Policy that tries to encourage alternative uses
for agricultural matters. Some of our research groups are focused in obtaining
pharmaceutical, aromatic and dye products for health and cosmetic industries
(Fernández and Abdullaev, 2004).

Sustainability
Saffron is a low water demanding plant well adapted to arid or semiarid lands;
it has a low input of fertilizers (if any) and chemicals. Actually, Saffron is still
cultivated almost in the same manner for 3,000 years. This environmentally sound
crop is one of the ancients of Europe. Its unique eco-physiology amongst many crops
makes this plant appropriate for marginal soils, mainly but not exclusively in the
Mediterranean area. The concept of a sustainable EU agriculture fits perfectly with
the Saffron crop.

WHAT TO DO TO PREVENT SAFFRON DECLINE

The majority of the germplasm collections include crops with a high economic
value like cereals, legumes, fruits and forage species. However, is rare the presence of
species with neutraceutical, therapeutic or medical applications, aromatic plants,
ornamental and spice producing plants in public collections. The limited genetic
variation suspected for this sterile crop, exclusively propagated vegetatively and
subject of rapid genetic erosion in the last century, would have required the creation
of a collection of landraces, ecotypes or simply accessions of C. sativus. However, no
germplasm collection of Saffron in Europe neither in the world has been created so
far. The creation of such collection will contribute not only to slow down the intense
genetic erosion but also will make available a wide variety of Crocus genotypes of
potential carriers of interesting genes for plant breeders, e.g. resistance to biotic or
abiotic stresses, reserve accumulation, biosynthesis of secondary metabolites, etc.
In order to ensure the future of Saffron crop it is necessary to improve
cultivation techniques, plant material, quality evaluation methods, and to develop a
wide range of Saffron uses particularly those related to human well-being. The
worldwide increase in utilization of Saffron as natural product requires new biological
and economical development, and co-operative programs on technological and
medicinal studies. Production and processing of ‘medicinal’ and aromatic plants
should be one of the successful branches of horticulture in the future. To fulfil these
requirements, however, production systems need to be modernised and high quality
material must be provided to farmers. The creation of a Bank of Germplasm in
crocuses could help in that purpose
 The 1st International Symposium on Saffron Biology and Biotechnology held
in Albacete (Spain) and organised under the auspices of the International Society For
Horticultural Science and the University of Castilla-La Mancha, counted with the
participation of one hundred researchers, technologists and businessmen from 15
countries (Spain, Iran, Mexico, India, Greece, Italy, France, Japan, Switzerland, USA,
Turkey, Hungary, Denmark, Canada, and Azerbaijan). The meeting, chaired by this
author provided a worldwide overview of research in Saffron achieved in the last
decades, including basic biology, agronomy, genetics and breeding, chemistry,
industrial production, pharmacognosy, pharmacology, and economics. Attention was
also given to the scientific and practical problems and the challenges for Saffron plant
in the XXI century. One of the statements of the Working Group on Saffron Biology
and Biotechnology (M04), created at this meeting inside the Section of Medicinal and
Aromatic Plants of the ISHS, was to promote the creation of a Bank of Germplasm
and Gene Banks in Saffron to preserve genetic biodiversity in Saffron and related
species (Fernández and Abdulalev, 2004).

A World-Scale Task
Note that while the world’s total annual Saffron production is estimated at
about 200 tons per year, Iran with more than 47,000 hectares of land under Saffron
cultivation is said to produce more then 80 percent of this total. India is the second
producer. Any serious effort to create a bank germplasm in Saffron would be enriched
with the participation of researchers, agronomists or merchants from these countries,
simply because they must keep a significant amount of the global genetic variability
present in Saffron crop, together with a wide knowledge of its agronomy and uses. It
must be strongly taken into account that having the support of research groups from
countries that are putative commercially competitsors is highly valuable. This
confidence is based on the environment of collaboration and frankness amongst
scientists generated in the 1st ISSBB. A new big step has been done with the 2 nd
ISSBT carried out at the Ferdowsi University of Mashhad (Iran) in October 2006
which contents are included in the current Acta Horticulturae volume.
It is not an overstatement to say that this Bank of Germplasm extents its
obvious European interest to a wider scale, and must be the basis of the ‘World
Saffron & Crocus Collection’ for everybody’s use.

A FIRST MILESTONE: THE CROCUSBANK PROJECT

Agricultural Policies
Environmental concerns play a vital role in the agricultural policy of the
European Union (EU). By managing a large part of the European Union's territory,
agriculture preserves many specific genes, species and habitats. The EU set up
measures in support of the agricultural biodiversity, to contribute reaching the 2010
target of halting the loss of biodiversity.
In the Biodiversity Action Plan for Agriculture (Council Regulation (EC) No
870/2004) it was proposed to launch a new EU programme on the conservation,
characterisation, collection and utilisation of genetic resources in agriculture
(2004-2006). This programme promotes genetic diversity and the exchange of
information including close co-ordination between Member States and between the
Member States and the European Commission for the conservation and sustainable
use of genetic resources in agriculture. It also facilitates co-ordination in the field of
international undertakings on genetic resources in agriculture, in particular within the
Convention on Biological Diversity (www.biodiv.org/default.shtml) the International
Treaty on Plant Genetic Resources for Food and Agriculture (www.fao.org/AG/cgrfa/
itpgr.htm) and the FAO's Global Plan of Action for the Conservation and Sustainable
Utilisation of Plant Genetic Resources for Food and Agriculture
(www.fao.org/waicent/search/5_dett_FAO.asp?
cgiar=&calling=simple_s_result&publication=1&webpage=2&photo=3&press=5&vi
deo=9&lang=en&pub_id=57930).
The principle of “The conservation and sustainable use of genetic resources in
agriculture are essential to the sustainable development of agricultural production and
of rural areas” prompt the Saffron germplasm main stakeholders in the European
regions and third states around the world to joint a consortium involving crocus
relatives to propose the so-called CROCUSBANK project on “GENETIC
RESOURCES OF SAFFRON AND ALLIES (CROCUS SPP)”, aiming to contribute
to the Biodiversity Action Plan for Agriculture. The CROCUSBANK project will
play a part to the new “Programme on the Conservation, Characterisation, Collection
and Utilisation of Genetic Resources in Agriculture. AGRI GEN RES” launched by
the European Commission in 2005. The recent approval of this 4-years length project
constitutes a big success for all Saffron researchers taking into account the strong
competition of several projects dealing with animal and plant species.
The objective of this project is to create, characterise and exploit a germplasm
collection (bank) in Crocus species, including Saffron crocus. These are the goals.
1. Collection, multiplication, conservation and documentation of Crocus genetic
resources. This collection has two main goals: First, to collect and reproduce Saffron
bulbs, coming from all the countries that cultivate Saffron, for direct use of this plant
material in selection programmes all over the world; and second, to create a collection
of Saffron allies for conservation, since they are endangered and threatened taxa and
populations in Crocus, and for research in taxonomy and evolution, genetics,
physiology, ecology and agronomy. The Crocus species are exploitable sources of
resistances and other agronomical interesting traits to be transferred to Saffron,
through appropriate breeding programmes and technological tools. Resistance to fungi
and other stresses are actually priorities in Saffron breeding.
The general objective can be divided into the following actions:
• Exploration and collection of germplasm of Saffron and related species. The
collection of Crocus material will be carried out by means of requests to
different regional centres growing the plants and visiting specific locations at
appropriate date to collect both cultivated Saffron and wild species. The taxa
included in this project shape a proposal of maximum achievements. The
reality of the field work is taken into account regarding the number of Crocus
species and subspecies that we are able to collect.
• Elaboration of a list of descriptors for the characterisation of the genus Crocus
and primary characterisation of the collected material.
• Multiplication of the collected plant material for its conservation in the Bank
of Plant Germplasm of Cuenca (Spain). Conservation methods based on tissue
culture techniques will be used when required.
• Elaboration of an effective documentation system, with the passport and
characterization data of the accessions, in order to guaranty an appropriate
management of the Crocus germplasm collection.
• To make available this material to potential users by distribution of corms,
tissue culture and DNA samples.
2. Characterization and evaluation of Crocus genetic resources. We will elaborate
a list of descriptors for the characterization of the genus Crocus and primary
characterization of the collected material. For the characterization/evaluation of the
material we will take into consideration phenotypic characters with good heritability
at different structural and physiological levels and include both simple, single-gene
autoapomorphic characters and complex quantitative traits: Morphological (floral
features, corm size); Phenological (flowering and relationship of climate, latitude and
altitude); Cytological (chromosome numbers, genome size, ploidy level and
identification of hybrids); Phytochemical (Saffron chemical composition; metabolic
profiling); Molecular (DNA analysis); and Physiological (abiotic stresses and
pathogen responses).
3. Application of the Crocus germplasm information and banked accessions.
• Rationalization of collections, identifying duplicates in order to optimise the
management of the bank.
• Definition of valuable germplasm for Saffron breeding.
• Identification of ecologically rare and important species/genotypes in the
natural environment.
• Identification of valuable species, cultivars and hybrids for the horticultural
industry.
• Comparative genomics with model and crop species to identify universal
features and valuable genes for agronomy.

The Consortium
The CROCUSBANK consortium (Table 3) is composed by partners from 6
European Union countries and 3 partners from third countries where rich resources of
Saffron and Crocus relatives are found. All of them have long experience in research
on complementary aspects of the Saffron and/or Crocus biology, agronomy, and
biotechnological applications. The consortium tries to joint a wide representation of
the effort carry out currently on Saffron research at international level, and the
partner’s geographical distribution comprise the Saffron producing areas as well as
the main habitats of the Crocus species. The non-presence of Iranian and Indian
groups is explained in terms of administration inconveniences. The AGRI GEN RES
programme does not subsidize groups from third-countries, which nevertheless are
free to participate without funding, as partners from Azerbaijan, Egypt, and Turkey
have done. Besides, having the collaboration of Iranian and Indian scientists and
institutions is highly desirable and we look forward to accomplish it.
All scientific, financial and administrative aspects of CROCUSBANK will be
co-ordinated by this author at the University of Castilla-La Mancha (Spain), who will
represent the consortium in the European Commission. The aim is to collect and
reproduce Saffron bulbs, coming from the EC countries that still cultivate Saffron
(Spain, Greece, Italy, France, and Hungary), but also from Turkey, Azerbaijan,
Morocco, India and Iran. This plant material, once adapted and reproduced, could be
used in selection programmes all over the world. Secondly, the collection of Saffron
allies (both seeds and/or corms) will be made for conservation, since some Crocus
taxa are threatened, and for research in genetics, plant breeding and basic biology.
The creation of the mother collection in Spain will be followed by replicas in other
countries in the coming years, once the bank is well established.

Coordinated Implementation Plan

The action comprises three basic tasks: collection, conservation, evaluation
and utilisation of Crocus genetic diversity. The first duty will be completed by the
participation of 12 groups of the consortium. Each group has the responsibility to
collect the quoted plant material using botanic gardens, personal contacts, other
collections and explorations to the field. The Crocus wild species that are included in
the list are the complete genus (Table 5).
Explorations and collection per area are designed on the basis of the
geographical proximity of both Saffron production areas and Crocus spp. population
habitats. The programme of collection of Crocus taxa is planned on a basis of
maximums, in order to obtain different subspecies and populations of each species.
Partial results are not considered a failure since the purpose is to obtain as many
accessions as possible, representative of the species range and diversity, according to
the financial and human resources available.
The conservation, multiplication, maintenance, documentation and
management of the collection will be carried out by the JCCM Group, centre
specialised in the management of germplasm banks of the Castilla-La Mancha region
in Spain, amongst them, the dedicated to “aromatic and medicinal plants”.
According with the objectives of the project, translated into workpakages,
various overlapping groups are established. The first one, named “Saffron Collectors
Group (SCG)” is integrated by partners 0, 4, 5, 6, 7, 8, 9, 11 and 12, under the
supervision of partner 0 (UCLM, Spain). The second is the “Crocus Collectors Group
(CCG)” composed by partners 0, 4, 5, 7, 8, 9, 10, 11, 12 and 13, under the command
of partner 11 (NAGREF, Greece). The partners 1, 4, 5, and 10 constitute the
“Descriptors Group (DG)” whose manager is partner 4 (UPVLC, Spain). The
“Multiplication and Conservation Group (MCG)” is organized by partners 0, 1, and
10, with partner 1 (JCCM, Spain) as manager. The characterization and evaluation
task is approached by two groups: (1) morphological, physiological and agronomical
level, with partners 0, 1, 4, 5, 7, and 10, leaded by partner 1, and (2) chemical and
molecular level, with partners 0, 2, 3, 6, 8, 9, 10, 11, 12 and 13, under the
organization of partner 2 (AUA, Greece). These groups are called CEGA and CEGC,
respectively. The coordination of the complete characterisation task will be made by
the project coordinator (partner 0). The application of the germplasm information and
banked accessions is implemented by the “Application Group (AG)” integrated by
partners 0, 1, 3 and 10, leaded by the latest (ULEIST, UK).The management and
coordination of the project) is the responsibility of the project coordinator (partner 0)
assisted by the Project Management Committee, PMC (partners 1, 2, 4, 10 and 11).
Partner 0 (UCLM, Spain) is the overall project coordinator, whereas partner 1 (JCCM,
Spain) is the manager of the collections and the central information system.
SCG activities consist in the collection of Saffron corms from different EU
and non-EU countries, in an attempt to store the maximum genetic variability present
in this crop. The wide commercial experience and good knowledge of Saffron
cropping areas of the French company (TJMT) (partner 6) will be extremely
convenient for this achievement. CCG task is the collection of Crocus taxa by means
of requests to different collection holders or visiting specific locations at appropriate
date to collect either cultivated or wild specimens (seeds and/or bulbs). It is of
particular interest to collect a wide representation of taxa phylogenetically close to
Saffron (i.e. Crocus series of the genus) for their interest in Saffron breeding. These
species are present in Greece and its islands. The Greek sub-coordinator (partner 3)
has a long experience in C. sativus studies and incorporates experienced geneticists
and plant breeders. Included in partner 10 we gain the participation of the Crocus
Group (historically) a splinter group from the British Iris Society, that is an
assemblage of persons with a shared interest in the genus Crocus. The group was
created in 1974 and includes botanists, Crocus growers and people dedicated to
computing, seed exchange and other duties (www.thealpinehouse.fsnet.co.uk/crocus
%20pages/). Anthony Goode is coordinator of the Crocus Group and will be
subcontracted by partner 10. He is an expert in cropping conditions and techniques for
a big number of Crocus. His valuable practical knowledge is of major interest for the
Crocus germplasm project.
The experience of partner 4 (DG coordinator) in Saffron experimental
cultivation will be useful for the elaboration of descriptors and primary
characterization of the plant material. The activities of MCG are the core of the
project. Partner 1, its coordinator, has wide experience in the management and
conservation of genetic resources in Spain. Lastly, CEGA and CEGC, coordinated by
partner 1 (Spain) and partner 2 (Greece), respectively, must initiate the
characterisation of the material at different levels, with the aim of continuing this task
in the coming years. Partner 10 (UK) will be the manager of the application task
(definition of valuable germplasm for Saffron breeding, identification of important
accessions in ecology, horticulture, agronomy, industry, etc. Partner 11 (Greece) has a
long experience in the management of genetic resources in this country.

MAIN METHODOLOGIES
Exploration and Recollection
1. Preparation and Execution of Expeditions. With the aim to obtain genetic
material (corms) of cultivated Saffron, as well as seeds and occasionally corms of
other Crocus species in their areas of distribution to be part of the germplasm bank, a
previous work of planning of expeditions will be carried out. For cultivated species
(some of them used in gardening) we will contact plant nurseries, botanic gardens and
private cultivators. In the case of wild species we will make a complete bibliographic
search on the different species in order to determine their chorology, dates and
locations where the material could be found. Recollections will include natural
habitats, crop lands or even local markets. The distribution of areas by groups will be
tentatively as described in the next table, taken into account that changes will made on
the way. The distribution of geographic areas, the taxa and the groups responsible of
the exploration are explained in Table 3.
The exploration of Saffron corms will start at early 2007, with the aim to carry
out their sowing at the end of spring, and will be maintained through the years of
extent of the project. Since wild species can only be detected in flowering time each
group responsible of a geographic area should carry out four collecting campaigns of
about two weeks per campaign during the autumns of 2007 and 2008, and the springs
of 2008 and 2009, in order to collect Crocus species flowering in spring or autumn.

2. Elaboration of protocols for collecting material. The protocols of recollection of

cultivated and wild species will be elaborated previously. These protocols must detail
the information that must be compiled in the moment of recollection and associated to
each germplasm sample: botanical data, ecological and geographical data, uses, crop
techniques and any other observation of interest. They also will include the methods
of collecting samples and identification of each of them.

Conservation and Multiplication

Most of the wild Crocus species are markedly entomophilous, fertile and seed
producers. Nevertheless we will use the same method as for Saffron for conservation
of accessions; i.e. the maintenance of corms in vegetative form, in order to guarantee
the genetic stability of material, avoiding hybridisation between accessions.
 The corms obtained in the campaigns of recollection will be multiplied in the
field with the aim of produce a suitable minimum amount that guaranties their long-
term conservation in the germplasm bank and the supply to potential users. For the
case of accessions of wild Crocus with scarce material, this will be previously
multiplied in acclimatised greenhouse with the substrate of the collecting zone, in
order to make sure the propagation of some material under the best conditions before
the transference to the field, to minimize the risk of loss. In some cases the
conservation/propagation in the greenhouse will continue.
Multiplication will be carried out in a fenced parcel in the experimental farm
of the Bank of Plant Germplasm of Cuenca (partner 1), with a medium altitude
between 950 an 1000 meters, medium temperature of 11.5 ºC, and a precipitation
ranging from 550 to 600 mm mainly concentrated in spring and autumn. The edaphic
characteristics in this parcel are typical of the area, sandy loams, alkaline pH
(7.6-8.4), normal electric conductivity (<400 mmhos/cm) and a low content in organic
matter (1-2.5 %). Conventional labouring will be used to prepare soil for seeding
(disk harrow, cultivator and rotary harrow). Each accession will be seeded in a 12 cm
furrow; with 20 cm between plants and 40 cm amongst furrows. Weeding out will be
carried out when required. The collection will be kept in the field indefinitely but
every two years the corms of half furrow will be collected and reseeded to obtain
supplying material. The other half furrow will be treated in the same way the next
campaign.
In cases of Crocus accessions with low fertility, difficulties for conventional
multiplication, or in cases of endangered and threatened taxa and populations we will
develop methods for in vitro conservation.

In vitro Multiplication
To establish the in vitro conservation of the distinct genotypes of Saffron and
some wild Crocus collected in the development of our project, we will apply the
methodology reported by Piqueras et al. (1999). Briefly, healthy corms will be
selected from the collection and will be used as source of tissues as starting material
in two developmental stages: (i) vegetative dormancy previous to sprouting and (ii)
vegetative growing; in order to evaluate the impact of the physiological stage of
corms in response to in vitro morphogenesis. The Saffron micropropagation protocol
includes the following steps: Initiation of the culture and explant selection,
Multiplication, Multiplication of meristematic nodules in liquid medium (optional),
Elongation and rooting, Acclimatization to ex vitro conditions and formation of
corms, Control of genetic stability and clonal uniformity of the regenerated material.

Elaboration of a List of Descriptors and Characterization of the Collection

Due to the local and minority character of Saffron crop there is no list of
descriptors published by the International Plant Genetic Resources Institute (IPGRI,
http://www.ipgri.cgiar.org/index.htm). For that reason, one of the basic goals of this
project will be the elaboration of such a list. We will try to construct a list of
descriptors valid for the genus Crocus, based on the taxonomic criteria for the genus,
but also including the categories present in other IPGRI list of descriptors. The
descriptor resulting of this project must be as complete as possible. The primary
characterisation assays (morphological and agronomical traits) will be basically
carried out in the experimental farm of Bank of Plant Germplasm of Cuenca during
three consecutive years and with an identical design to the multiplication parcel (12
cm furrows; 20 cm between plants and 40 cm amongst furrows). A portion of the
corms will be collected each year for characterisation. The size of the samples will
depend on the variability observed for the characters evaluated. Corms (and/or seeds
in the case of fertile crocuses) extracted from the field of characterisation, together
with those of the field of conservation/multiplication, will serve as supply to the
potential petitioners in this campaign. The parcel of characterisation will be laboured
as that of multiplication, but both will be located on separated zones of the fence, and
will serve as replicas of security for contingencies.

Documentation
All the information associated to the Crocus collection will be included in a
relational database in order to guaranty its correct management and availability for the
potential users. Such database will be designed attending to recommendations and
rules internationally established for documentation in plant germplasm collections and
will be accessible via Internet through the Website of the Servicio de Investigación y
Tecnología Agraria de Castilla-La Mancha (Spain)
(www.jccm.es/agricul/paginas/desarrollorural/investigacion/CISITA.htm).
The data to be included in the database will be divided in the following
categories:
1. Passport data. Which will be referred to geographical and ecological indicators of
the recollection site. They would have been recorded in the recollection instant.
2. Management data. That will include the information generated in the process of
conservation, multiplication and supply.
3. Characterisation data. Including accession identification, place of
characterisation, characterisation data s. str. e the nature of an active collection and in
its management a registry of incomes and outcomes of plant material will be carried
out.
The collection will have the nature of an active collection and in its
management a registry of incomes and outcomes of plant material will be carried out.

Molecular and Cytological Characterization

The objective is to provide molecular and molecular cytogenetics markers for
the characterization of Crocus germplasm and application in selection of improved
cultivars for Saffron production. Thus, the molecular characterization of Crocus will
allow:
• To identify clearly the off types in some collections
• To appreciated the extend of diversity of various geographic or genetic groups
cultivated and wild.
• To clarify the genetic relationships between these groups (heterozygosity of
varieties).
A range of appropriate molecular marker are available for such purposes:
Microsatellites (SSRs), inter-SSR and inter-retroelement methods, and gene based
markers (PCR and hybridization based) will be used. A wide range of accessions of
both cultivated Saffron and wild species will be used for characterization, and one or
two outgroups will be included in analysis (Romulea and Iris) to allow robust rooting
of phylogenies.
Standard primers for phylogenetic analysis will be used to amplify gene
segments from nuclear, chloroplast and mitochondrial genes. Molecular-based trees
will be constructed to infer relationships between groups and accessions. Molecular-
based analysis will show how much diversity is present in the genus; this data will be
in a form that it can be used to develop conservation policy.
In addition to estimating genetic diversity, these tools can be utilized to
establish molecular markers for phenotypic traits of interest. Genes which are widely
conserved between species, with a focus on those associated with biotic and abiotic
stress tolerance, will be amplified and sequenced from a range of accessions, using
conserved sets of PCR primers. Genes associated with corm development and
dormancy will be isolated and characterized from a range of accessions. Gene
expression analyses, will allow to found more efficient alleles. Genes associated to
flavour and other associated quality traits important in Crocus commercialization.
Several genes directly involved in flavour have been identified in C. sativus, and
several biochemical markers are used to determine the quality of flavour and aroma in
Saffron, we would like to correlated molecular and biochemical flavour traits.

Phytochemical Characterization
.......Saffron major constituents are crocins, picrocrocin and safranal, which are
responsible for its colour, taste and aroma, respectively. Quality control of Saffron is
based on chemical analysis that should discriminate such different compounds.
Except for the method currently recommended by the International Standardization
Organization, namely UV-Vis Spectrophotometry, information on the chemical
composition is derived by HPLC combined with spectroscopic detection means (diode
array, MS). Chemical composition of the polar fraction extracted from the stigmas of
wild Crocus species will be characterized by the means of UV-Vis and HPLC-UV
techniques. Further investigation on the chemical composition of stigmas of species
with particular commercial interest will be based on the examination of other extracts
(petroleum ether, diethyl ether, acetone, methanol, water). A typical UV-Vis profile of
C. sativus extracts will be constructed for each of the different extracts and will then
be used as a reference one for the characterization of the wild species. UV-Vis spectra
derivatives of the individual extracts are also expected to highlight any deviation from
the typical profile of C. sativus. In all cases, the quality parameters suggested by ISO
3632-2 (2003) such as colouring strength, bitterness and safranal content will be also
determined. Main target of this work is finally the identification of constituents that
could be a discriminative key for the characterization of different Crocus species to
appreciate the extend of diversity of various geographic or genetic groups cultivated
and wild.
The essential oils (EO) content of the Crocus stigmas will be determined and
the chemical composition of the produced oil will be characterized by using
chromatographic and spectroscopic techniques such as GC-FID, GC-MS FT-IR, FT-
Raman and UV-Vis. The hydrodistillation and the microsteam distillation - extraction
to organic solvent will be used as reference methods for the production of EO. The
ultrasound based assisted extraction and the microwaves based distillation will be the
innovator methods for the EO production. These methods are expected to limit the
time demand for the oil isolation receiving similar quality oil with the conventional
methods and in some cases to improve the quality of the produced oil, especially by
receiving enriched in active components extracts. The influence of temperature,
solvent, frequency and time to the EO production will be taken into account in order
to obtain the highest EO yield. Experiments will be performed with different solvents
in variable extractable times, using ultrasound or microwaves apparatus, which will
be functioning at specific frequency. Extractions based on ultrasound will be
performed at room temperature, while the distillations based on microwaves will be
rapid, so the creation of by-products, due to oxidation processes, will be avoided.
Main target of this work is finally an essential oil product with high quality
chemical profile. In addition, the possibility of the essential oil discrimination
between different plant species, subspecies, or chemotypes by their FT-IR and FT-
Raman spectra has been examined.

Response to Stress
1. Evaluation for Salt Stress. The work aims to individuate landraces and/or wild
genotypes of interest for utilising in marginal areas characterised by soil salinity and
develop local communities. A core collection of Crocus spp. of about 100 accessions
(o more) will be evaluated in different soil salinity levels. 120 corms per accession of
about 2-3 g weight will be planted in containers (10 x 1.2 m) in protected conditions
and will be irrigated with nutrient solutions with different doses of salt (by addition of
0, 1.5 and 3.0 g l-1 of NaCl). The experimental design is factorial; salt dose represent
the main factor and the accession the secondary one. The substrate utilised will be
peat and perlite (1:1 in volume). The corms will be “seeded” at single row at 30 cm
between rows and 10 cm along the row. The corms will be kept in the substrate
indefinitely. For each plant will be registered the date of the first flower in anthesis,
the number of flowers harvested per plant, fresh and dry style weight. Sample of dried
styles will be send to chemical research unit to analyse the main qualitative
compounds.

2. Pathogenesis Response. The work intend to check Crocus accessions against fungi
that have been reported as Saffron pathogens in different geographic areas: Fusarium
monliforme (corm rot, Kashmir), Rhizoctonia crocorum [sin. R. violacea] (violet root
rot in Greece and Spain) , Phoma crocophyla (Spain), Fusarium oxysporum f. sp.
gladioli (Italy), F. oxysporum f.sp. tuberosi (Spain), Penicillium cyclopium (Italy),
Burkholderia gladioli (soft rot, Argentina), amongs others. The final objective is no
find genetic resistances against these pathogens in Saffron cultivars or Crocus
genotypes. A Crocus spp. collection of about 50-100 accessions will be evaluated.
The infections of plant material will be performed using 106/ml fungal
zoospores during 72 h. Treatment solutions will be diluted in 0.1 % Tween 80 from 1
mM stock solutions prepared in water and then applied on healthy roots. The number
of infected corms in each accession will be determined by presence of brown to dark
brown sunken and irregular patches below the corm scales. Rot lesions are usually 1
mm deep having raised margins. The rot symptoms are mostly located in root and bud
regions. In severe cases the entire corm turns into black powdery mass outer fibrous
scales in position. In some corms white or yellowish white fungal mass is observed.

Application of the Crocus Germplasm Information and Banked Accessions

The deliverables from collection to evaluation will have applications at
applied and fundamental levels. This application task aims to provide some
demonstrator results using the accessions and information, and by including some
preliminary application-oriented work, to assist with focussing aspects of the other
work packages. We have selected diverse but representative target applications of the
germplasm and markers, where relatively little works can give significant and fast
results. Firstly, what proportion of the germplasm collection consists of duplicates?
Secondly, how much diversity is present in Saffron, is the CROCUSBANK collection
likely to represent most of the diversity and how does Saffron diversity relate to
Crocus diversity? Are there ecologically rare but important species/genotypes in the
natural environment (results that have consequences for tourism industry and
development; for genetic resources; sustainable land use and biodiversity; treaty
commitments eg Rio Convention on Biodiversity? Thirdly, do the molecular markers
confirm the postulated origin and closest diploid relative(s) of Saffron, and how can
genes from Saffon and Crocus species be used in comparative genomics with other
model species? Finally, do the data from collection to evaluation allow selection of
the most valuable germplasm for Saffron selection, identification of valuable species,
cultivars and hybrids for the horticultural industry, and help with searching for
valuable agronomic genes? These are the specific goals:
• Identification of accessions of Saffron within the collection those are genetically
identical. Duplicates within germplasm collections are costly to curate, but it is
undesirable to discard unique accessions. Some molecular markers from WP04
will show the frequency of duplicates within the collection, allowing them to be
discarded. Passport data combined with duplicate frequency will estimate how
representative the collection is of the complete range of Saffron.
• Quantification of diversity in Saffron and Crocus. It is essential to measure levels
of biodiversity of plants throughout Europe. We will measure the level of
molecular diversity of the Crocus species in the collection, and compare this with
other wild plants. The results will also allow us to recommend optimal in situ
conservation strategies to complement the germplasm bank.
• Genomics in Saffron. The origin of Saffron from wild Crocus species is not fully
proven, and we will check that the molecular markers are consistent with
published suggestions. We will also investigate the structure of genes which are
conserved across all plant species, and define how the Crocus/Saffron genome is
related to other species.
• Identification of germplasm for exploitation by breeders and growers. Based on
the morphological markers, agronomic characters, cytogenetics and molecular
studies, we will identify accessions and species which are suggested for
consideration for commercial exploitation and making of hybrids.
Saffron is the highest valuable food product and one of the oldest crops and
alleged medicinal plants, also a significant part of the Eurasian legacy. This crop is
well integrated into the EU agriculture policy, because its added-value from
processing, sustainable and ecologically friendly cultivation methods, perennial nature
making optimum use of water resources and minimizing erosion, and its character of
niche crop for poorer rural communities. Until now, no EU or non-EU institution had
taken the responsibility of the creation and maintenance of a collection representative
of the genetic variability of the crop and its allies. At a world scale level there was not
a Crocus collection institutionally protected and at the disposal of potential users. No
descriptor list has been published in saffron. The CROCUSBANK project will change
this status quo. The elaboration and publication through the IPGRI of a complete list
of descriptors for characterisation of Crocus becomes a duty. Data generated will be
the first ones to be available, together with the plant material itself. We will create a
database following the international standards for the management of germplasm
collections.
The CROCUSBANK collection has two main goals: First, to collect and
reproduce saffron bulbs, coming from all the countries that cultivate saffron, for direct
use of this plant material in selection programmes all over the world; and second, to
create a collection of saffron allies for conservation, since they are endangered and
threatened taxa and populations in Crocus, and for research in taxonomy and
evolution, genetics, physiology, ecology and agronomy. This Crocus species are
exploitable sources of resistances and other agronomical interesting traits to be
transferred to saffron, through appropriate breeding programmes and technological
tools.

ACKNOWLEDGEMENTS
The author is grateful to his collaborators at the IDR-Biotechnology Lab,
doctors Lourdes Gómez-Gómez, Ángela Rubio, Manuel Álvarez-Ortí, Raquel
Castillo, and Oussama Ahrazem for their daily work and dedication. His also
emphatically thanks Dr. Marta Roldán for her crucial assistance as expert in managing
EU projects and the members of the CROSCUSBANK consortium for their support in
preparing the project proposal and their encouragement with the endeavour of
conservation of Saffron genetic resources. These researchers are: Dr. Marcelino De-
Los-Mozos, Prof. Moschos Polissiou, Dr. Maria Tsimidou, Prof. Jose-Luis Guardiola,
Prof. Hasan Vurdu, Mr. Jean-Marie Thiercelin, Dr. Ferdinando Branca, Prof. George
Boberly, Dr. Thierry Talou, Prof. Pat Heslop-Harrison, Dr. Eleni Maloupa, Prof.
Khalil Gasimov, and Dr. Mahmoud Sharaf-Eldin, together with the members of their
respective teams.
The author wants to give a tribute to Dr. Fikrat Abdullaev to whom memory
the CROCUSBANK project is dedicated.

Literature Cited
Council Regulation (EC) No 870/2004 of 24 April 2004 establishing a Community
programme on the conservation, characterisation, collection and utilisation of
genetic resources in agriculture and repealing Regulation (EC) No 1467/94.
Official Journal L 162 , 30/04/2004 P. 0018 – 0028.
Fernández, J.A. 2004. Biology, biotechnology and biomedicine of Saffron. Recent
Res. Devel. Plant Sci. 2:127-159.
Fernández, J.A. and Abdullaev, F. (eds.). 2004. Proceedings of the First International
Symposium on Saffron Biology and Biotechnology. Acta Horticulturae 650.
Mathew, B. 1982. The Crocus. A Revision of the Genus Crocus (Iridaceae). B.T.
Batsford Ltd. London].
Piqueras, A., Han, B.H., Escribano, J., Rubio, C., Hellín, E. and Fernández, J.A.1999.
Development of cormogenic nodules and microcorms by tissue culture, a new tool
for the multiplication and genetic improvement of saffron. Agronomie 19:603-610.
Table 1. Estimate of current Saffron world production (various sources)
Quantities Countries Qualities or Zones

150/170 MT IRAN Sarghol, Poushal, Dasteh

8/10 MT INDIA (Kashmir) Mongra, Lutcha

4/6 MT GREECE Kozani

0,8/1 MT MOROCCO Talliouine

0,3/0,5 MT SPAIN La Mancha, Valle del Jiloca

100 kg ITALY Sardegna, Aquila, Cascia

10 kg TURKEY Saffranbulli

4/5 kg FRANCE Gâtinais, Quercy

1 kg SWITZERLAND Mund
Table 2. Some cultivars of ornamental crocuses
C. laevigatus ‘Fontenayi’
C. pulchellus ‘Albus’, ‘Zephyr’
C. sativus cartwrightianus ‘Albus’
C. speciosus ‘Albus’, ‘Artabir’, ‘Cassiope’, ‘Oxonian’, ‘Aitchinsonii’,
‘Conqueror’
C. chrysanthus ‘Advance’, ‘Ard Schenk’, ‘Audabe’, ‘Blue Bird’, ‘Blue Pearl’,
‘Cream Beauty’, ‘Dorothy’, ‘E.P.Bowles’, ‘Fuscotinctus’, ‘Gipsy Girl’,
‘Goldilocks’, ‘Herald’, ‘Jeannine’, ‘Ladykiller’, Miss Vain’, ‘Prins Claus’,
‘Romance’, ‘Saturnus’, ‘Skyline’, ‘Snowbunting’, ‘Sunspot’, ‘Uschak Orange’,
‘Zwananburg Bronze’, ‘Zenith’
C. biflorus ssp. biflorus ‘Parkinsonii’
C. biflorus ssp. weldenii ‘Albus’, ‘Fairy’
C. etruscus ‘Rosalind’, ‘Zwanenburg’
C. flavus ssp. flavus ‘Aureus’
C. imperati ‘De Jager’
C. korolkowii ‘Kiss of Spring’
x luteus ‘Stellaris’ (C. angustifolius x C. flavus)
C. sieberi ‘Albus’, ‘Firefly’, ‘Hubert Edelsten’, ‘Ronald Ginns’, ‘Tricolor’,
‘Violet Queen’
C. tommasinianus ‘Albus’, ‘Barr’s Purple’, ‘Lilac Beauty’, ‘Pictus’, ‘Roseus’,
‘Ruby Giant’, ‘Whitewell Purple’
C. vernus ‘Albiflorus’, ‘Graecus’, ‘Harlem Gem’, ‘White Star’, ‘Enchantress’,
‘Flower Record’, ‘Jeanne d’Arc’, ‘Mammoth Yellow’, ‘Pickwick’, ‘Purpureus
Grandiflorus’, ‘Queen of the Blues’, ‘Remembrance’, ‘Vanguard’, ‘Glory of
Sassenheim’, ‘Negroboy’, ‘Paulus Potter’, ‘Twinborn’, ‘White Christmas’
C. versicolor ‘Picturatus’
Table 3. Partnership of the CROCUSBANK project
Nº Country Short Institution Expertise/role
Name
0 Spain UCLM Universidad de Genetics and Plant
Castilla-La Mancha Breeding. Biochemistry.
Biotechnology.
Pathogenesis
1 Spain JCCM CIA de Albaladejito. Germplasm Bank.
Junta de Agronomy. Plant
Comunidades de Breeding. Phenology.
Castilla la Mancha
2 Greece AUA Agricultural Chemical Analysis
University of Athens (molecular modelling),
3 Greece AUTH Aristotle University Chemical Analysis
of Thessaloniki (quality). Genetics and
Plant Breeding
4 Spain UPVLC Universidad Plant Physiology.
Politécnica de Phenology. Agronomy
Valencia
5 Turkey GU Gazi University Botany. Morphology
6 France TJMT Tradimpex JM Commercial. Sensorial
Thiercelin SA analysis (spice)
7 Italy DOFATA Università di Catania Plant Breeding.
-UNICT Evaluation for abiotic
stress.
8 Hungary UD University of Botany. Biotechnology.
Debrecen Molecular markers
9 France INPT National Polytechnic Chemical analysis
Institute of Toulouse (aromatic, dying, other
chemicals)
10 UK ULEIST University of Molecular Cytogenetics.
Leicester Genomics.
11 Greece NAGREF National Institute of Botany, Genetic
Agricultural Research Resources
12 Azerbaijan ANAS Azerbaijan National Botany. Pharmacology
Academy of Sciences
13 Egypt NRC National Research Biochemistry. Chemical
Centre analysis (bioactivity)
Table 4. Crocus taxonomy. Brian Mathew subdivided the genus in two subgenera:
Crocus and Crociris. C. sativus is classified into subgenera Crocus, Section
Crocus, Series Crocus (Mathew, 1982)
Subgenus Section Series Species
Crocus Crocus Verni C. vernus, C. tommasinianus, C. kosaninii, C. etruscus, C. baytopiorum
Scardici C. scardicus, C. pelistericus
Versicolores C. versicolor, C. imperati, C. malyi, C. corsicus, C. minimus, C. cambessedesii
Longiflori C. longiflorus, C. serotinus, C. medius, C. niveus, C. goulimyi
Kotschyani C. kotschyanus, C. vallicola, C. gilanicus, C. autranii, C. scharojanii, C. ochroleucus,
C. karduchorum
Crocus C. sativus, C. cartwrightianus, C. thomasii, C. hadriaticus, C. asumaniae, C.
moabiticus, C. oreocreticus, C. pallasii, C. mathewii,
Nudiscapus Reticulati C. reticulatus, C. sieberi, C. dalmaticus, C. robertianus, C. abantensis, C. ancyrensis,
C. cvijicii, C. gargaricus, C. angustifolius, C. sieheanus, C. rujanensis, C. cancellatus,
C. hermoneus
Biflori C. biflorus, C. chrysanthus, C. danfordiae, C. almehensis, C. cyprius, C.
hartmannianus, C. aerius, C. pestalozzae, C. caspius, C. kerndorffiorum, C. paschei, C.
wattiorum, C. adanensis, C. leichtlinii
Orientales C. alatavicus, C. korolkowii, C. michelsonii
Flavi C. flavus, C. olivieri, C. antalyensis, C. candidus, C. vitellinus, C. graveolens, C.
hyemalis
Aleppici C. aleppcius, C. veneris, C. boulosii
Carpetani C. carpetanus, C. nevadensis
Intertextii C. fleischeri
Speciosi C. speciosus, C. pulchellus
Laevigatae C. laevigatus, C. tournefortii, C. boryi
Crociris C. batanicus