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Genetic Resources of Saffron and Allies ( Crocus spp.)

J.A. FernndezLaboratorio de Biotechnologa, Instituto de Desarrollo RegionalUniversidad de Castilla-La ManchaCampus Universitario s/nAlbacete E-02071Spain

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

AbstractSaffron is the highest valuable food product and one of the oldest crops

and medicinal plants. Saffron crop is subject to strong genetic erosion. Until nowno international institution has taken the responsibility of the creation andmaintenance of a collection representative of the genetic variability of the cropand its allies. Recently, the European Commission has approved an Agri GenRes 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, characteriseand exploit a germplasm collection (bank) in Crocus species, including Saffroncrocus. This collection is an urgent need for Saffron breeders and farmers, aspointed out in the 1 st ISSBB (Albacete, Spain). The present paper explains thebackground, objectives, organization and technologies to be applied in thedevelopment of the project.

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

INTRODUCTIONSaffron is a Precious and Intriguing Species

Crocus species are members of the family Iridaceae . The plants in this familyare herbs with rhizomes, corms or bulbs. The family Iridaceae embraces about 60genera and 1,500 species. The genusCrocus includes native species from Europe, North Africa and temperate Asia, and is especially well represented in arid countriesof south-eastern Europe and Western and Central Asia. Among the 85 species

belonging to the genusCrocus , C. sativus L. (Saffron) is the most fascinating andintriguing species (see Fernndez, 2004). This is not only because it produces the wellknown Saffron spice, but for the numerous mysteries surrounding its origins.Questions as when it originated; the native area or areas; the ancestor species and themechanisms of origin; the wild or naturalized plants; the infertility and consequentabsence of fruit and seeds; remain to be explained. Greece (Crete) has been mentionedas probable origin of this old crop with probably more than 4,500 years of age. If itsdomestication occurred at more sites simultaneously or at different times is still notresolved. 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 aset of somewhat unique agronomic and eco-physiological characteristics including arelatively low water use, growth and development during fall and winter, a very low

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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 vegetativegrowth. Furthermore it has three exceptional quality attributes,i.e. aroma, flavour andyellow 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 yieldone kilogram of Saffron spice (about 900,000 dried stigmas). Medium yield arearound 10 kg of Saffron/hectare, but varying very much between countries, lands,seasons, and agronomic practices. Saffrons high price is due to the much directlabour required for its cultivation, harvesting and handling. This fact has made progressively uncompetitive this crop in the more developed countries, andundoubtedly such tendency will extent to the currently producing leaders. Therefore,there is need for increasing Saffron production and quality to cope with an increasingdemand 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 afurther step, a new approach could take place, the consideration of C. sativus as asource of phytochemicals and biopharmaceuticals.

C. sativus is an autumnal flowering geophyte with corms that are covered by atunic, dormant during summer, sprouting in autumn, and producing 1-4 flowers in acataphyll with linear leaves. The flower has an underground ovary, a style 9-10 cmlong, dividing at the top in three red trumpet-like stigmas (2.5 cm long) that oncedried form the commercial spice Saffron. Flowering spans from late autumn untilDecember according to climatic conditions. Cytological studies have indicate thatSaffron is a triploid species which genome shows 3n=24, x=8 chromosomes. Itstriploid condition allows vegetative multiplication, but not regular sexualreproduction. This is because meiosis and gamete development in triploids areirregular, resulting into many anomalies in sporogenesis and gametophytedevelopment.

Saffron is an Amphiploid with Probable Low Genetic DiversityOne 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 cormmultiplication does not induce genome variations with the exception of somemutation that in a triploid Saffron population are not easily detectable, all Saffronshould be similar one to the other. Some authors believed that Saffron was oncenaturalized in small areas from where it was lost as a consequence of a change in landuse.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 parentsC.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 andCroatia,C. mathewii from Turkey, andC. pallasii ssp. haussknechtii from Iran-Iraq-Jordan, cannot be excluded. The complexity of the evolutionary history of the genusCrocus suggests an intensive species hybridisation and explosive speciation inCrocusevolution that could be on the basis of the origin of Saffron. We now are sure thatSaffron is an allopolyploid but the localization of the hybridisation event has not beenascertained so far. If the event took part several times could have generated differentamphiploids and, in consequence, different Saffron lines. Saffron was introduced inWestern Europe in different historical moments: In the Iberian Peninsula by the Arabs

or even before (Romans); in Germany, Switzerland, France, Italy and Great Britain bythe Crusaders, and towards the east it was extended by a variety of cultures through

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Transcaucasia, China, India and eventually Japan. Should we expect to find geneticdiversity then?

We can look the chromosomes as a first approach. The karyotype of C. sativushas been studied by a number of authors, reporting that Saffron from differentcountries (Azerbaijan, Iran, Italy, Turkey, France and England) was always 2n=3x=24lacking karyologycal differences. However in the past literature other karyotypeswere described. Cytofluorimetric analysis on nuclear DNA, carried to detect genomesize and base pairs composition, revealed no differences in DNA content andcomposition in Saffron corms cultivated in different countries (Italy, Israel, Spain,Holland). Some assays with molecular markers (RAPDs) have revealed limitedgenetic differences among Saffron samples from Italy, Iran, Greece and Spain. Nevertheless, analysis of phenotype revealed differences in aspect flower size, tepalshape and colour intensity with lobed tepal in plants from Israel and more intensecolour of tepals in plants from Sardinia (Italy). Variants of Saffron with an increasednumber 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 flowershaving higher number of style branches and stamens have been already described inSaffron cultivation at LAquila (Italy). Phenotypic variants are also present inKashmir Saffron (F. Nehvi, personal communication).

Besides different commercial products are known that could suggest theexistence of different Saffron ecotypes or commercial varieties, the actual geneticvariability present inC. sativus at worldwide scale is currently unknown. Nevertheless, there is a suspicion in Saffron breeders regarding the existence of scarcegenetic variability in this crop, but no serious effort has been carried out to ascertainthis important issue. They have been attempts with the objective to increase thespectrum of variability for floral traits and recovering auto-hexaploids (6x=48) inSaffron (to break the sterility barrier) by colchinization and to induce the geneticvariability 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 Fernndez, 2004).

THE RELEVANCE OF SAFFRON CROPSaffron is Highly Valuable

Medicinal and aromatic plants have been increasing in importance to societycontinuously for the past 100 years. Saffron is made from the dried stigmas of theSaffron 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, lessextensively, as a textile dye or perfume. However, due to its analgesic and sedative properties folk herbal medicines have used Saffron for the treatment of numerousillnesses for centuries. Saffron is considered to be the highest priced spice in theworld (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 CountriesSaffron 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 worlds Saffron production isestimated in 205 tons per year, Iran is said to produce 80 percent of this total,i.e. 160

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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 10t mostly dedicated to Indias self-consumption. Greek production (4 t) is locatedexclusively 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 toillustrate 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) 100kg; Turkey (Davutobasi, Saffranbulli) 10 kg; France (Gtinais, Quercy) 4/5 kg andSwitzerland (Mund) 1 kg are nearly nominal. Other countries as Azerbaijan producenegligible amounts of Saffron. An illustrating fact: In early 1970s Saffron cultivationin Spain and Iran were 6,000 and 3,000 ha, respectively, while at present the surfaceareas are 77 ha in Spain and near 50,000 ha in Iran. Only 20 years ago Spain and Iranwere 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 atown called Saffron Walden) and Cambridgeshire. After having been the leaders of Saffron production at commercialisation at a worldwide scale for centuries, nowadaysEuropean countries only produce a scarce 3 %, even tough the quality and the prestigein the marker still correspond to the European brands. The European Union hasawarded the designation "appellation of origin" to the "Azafrn de la Mancha", theGreek "red Saffron" under the name "Krokos Kozanis", and the Italian "Zafferanodell'Aquila". The reasons of Saffron decadency are various. An intensive (andexpensive) hand labour of up to 15 working days per kilogram of dry Saffron spice isrequired for flower picking and stigma separation. To the high cost of this labour itshould be added the very uncomfortable stooping position of the flower pickers, andthe very short picking period which comprises the early morning hours of the 20-30days of duration of the flowering season. The mechanisation of flower picking in fieldgrown Saffron has proved difficult.

All Saffron producers in the EU, also soon Turkey, suffer from increasinglabour costs. Iran can increase its production to more than 200 t whereas India couldoffer its Kashmir Saffron to the world market in growing amounts. China will becomea massive maker and there are serious projects of Saffron production in Afghanistanand 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, mostlyCarthamus tinctoriusor Curcuma. Hence, the Saffron world market panorama is al least uncertain. Nevertheless, although the tendency of Saffron diminution has been constant, they aresymptoms of a revival in Saffron crop in Europe. France, for instance, has shown theemergence of new associations of Saffron farmers ("Les safraniers du Gtinais" in1987, 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

TheCrocus genus is known mainly for the cultivated speciesC. sativus , whichis of prime economic importance. However, there are also other species belonging to

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this genus, which are highly prized for their colourful flowers, and thus usedextensively in specialized gardening (Table 2). These are horticultural varieties of C.vernus , C. versicolor andC. aureus , amongst others. Most of theCrocus species grownaturally in the fields between shrubs and grass or light woodlands. The plants in thisfamily are herbs with rhizomes, corms or bulbs. The genusCrocus includes about 80species distributed from south-western Europe, through central Europe to Turkey andsouth-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 wildancestral species; and spontaneous or induced mutations. Sterility in Saffron limits theapplication of conventional breeding approaches for its further improvement. Besidesdifferent commercial products are known that could suggest the existence of differentSaffron ecotypes or commercial varieties, the actual genetic variability present inC.

sativus at worldwide scale is currently unknown (Fernandez, 2004). They have beenefforts by Indian researchers to increase genetic variability in Saffron using non-conventional breeding techniques, such as induced mutagenesis employing physicalirradiation 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 EUROPESaffron must be considered as an endangered crop of added value deserving

scientific interest in Europe for various reasons.

Its Origin and Historical BackgroundThe 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 nameCrocus finds its origins inthe Greek wordkrokos , Saffron, which in turn derives from the Semitic wordkarkom ,one of the oldest names for this plant. Some archaeological and historical studiesindicate that domestication of Saffron dates back to 2,0001500 years BC. This isderived from documents reproducing the plant or showing people collecting the crop.However the sites where the first Saffron plants appeared differ according to theopinion of various authors. Vavilov placed Saffron into IV plant origin centre of Middle East (Minor Asia, Transcaucasia, and Turkestan); whereas more recentcontributions indicate that the process of Saffron domestication has to be identified onCrete during the Late Bronze Age. Historical records detail the use of Saffron date toancient Egypt, Persia, Greece and Rome, where it was used as a medicinal plant, as adye, in perfume, and as a spice and colorant for culinary purpose. Saffron use reachedits highest point in the Iberian Peninsula by the Arabs. During the Middle Age Saffroncrop was extended for rest of Europe due to the Crusaders, and was settled inSwitzerland, France and Italy, reaching Great Britain in the 14th century. Towards theeast, 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 ashealthy food, and again in 16th century as medicine. It reached Japan at the beginningof 17th century. Since then, Japanese people have used Saffron almost exclusively ashealth product.

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Tradition and QualityBesides the European countries only produce a scarce 3 % of world Saffron

market, the quality and the prestige in the marker still correspond mostly to theEuropean brands. La Mancha Saffron has been generally the highest prized, and LaMancha region was during centuries the world biggest producer. Currently theSpanish manufacturers and dealers control 70 % of the worlds Saffron market.

The European Saffron under Protected Geographical Indication (PGI) statusare:

"Krokos Kozanis"(EC Reg. 378/1999), Greece (Kozani) Azafrn de la Mancha" (EC Reg. 464/2001), Spain (La Mancha) "Zafferano dell'Aquila"(EC Reg. 2081/92), Italy (LAquila) Munder Safran AOC, Mund (Switzerland) France have two associations of Saffron farmers that pursue the same PGI

status:

"Les safraniers du Gtinais" in 1987 Les Safraniers du Quercy in 1999As 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 worldwidedevaluation. Amazingly, very little has been done to protect the Saffron crop inEurope. 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, thegenetic 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 thehighest 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 iscertified in the international trade market following the ISO 3632 Normative since1993. The most important parameter is colouring strength, calculated from UV-Vismeasurements at 440 nm in aqueous extracts of this spice. Such measurements arerelated to the total carotenoid content. This regulation is currently under controversysince leave aside the most important organoleptic properties of Saffron (odour,flavour) and does not prevent fraud. Saffron commands a rather high value in theinternational spice trade that results in its frequent adulteration by artificial colorantsand by mixing genuine stigmas of Saffron flower with other parts of plants (e.g. somespecies of grass) artificially coloured. Molecular tools will be ideal for checking purity of product, even after processing, act as markers for adulteration (either withdyes or with other plant species).

Alternative UsesSaffron 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,

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traditional Eurasian herbal medicines have used Saffron for the treatment of numerousdiseases. Saffron has been indeed one of the oldest plants used as medicine (see thefrescoes of Thera, Greece). The renewed importance of Saffron as nutraceutical is being sustained by an increasing number of evidences. Saffron has been claimed tohave effects on senile dementia, retina-degeneration, immunomodulation, as well asantimicrobial, antidepressant, antitumour or cardiovascular protective properties.Although these effects required more pharmacological/nutraceutical trials andsignificantly additional scientific substation, we support the potential healthapplication of this plant, with a profile similar to green-tea, ginseng, etc.

The application of the agroresource refining concept to the other parts of theSaffron plant (style, petals, stamens, leaves, stem and corms) is actually underway inorder to valorise all the organs of the plant. This Saffron by-product valorisation is inagreement to the European Agricultural Policy that tries to encourage alternative usesfor agricultural matters. Some of our research groups are focused in obtaining pharmaceutical, aromatic and dye products for health and cosmetic industries(Fernndez and Abdullaev, 2004).

SustainabilitySaffron 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 stillcultivated almost in the same manner for 3,000 years. This environmentally soundcrop is one of the ancients of Europe. Its unique eco-physiology amongst many cropsmakes this plant appropriate for marginal soils, mainly but not exclusively in theMediterranean area. The concept of a sustainable EU agriculture fits perfectly withthe Saffron crop.

WHAT TO DO TO PREVENT SAFFRON DECLINEThe 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 geneticvariation suspected for this sterile crop, exclusively propagated vegetatively andsubject of rapid genetic erosion in the last century, would have required the creationof a collection of landraces, ecotypes or simply accessions of C. sativus . However, nogermplasm collection of Saffron in Europe neither in the world has been created sofar. The creation of such collection will contribute not only to slow down the intensegenetic 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 awide range of Saffron uses particularly those related to human well-being. Theworldwide increase in utilization of Saffron as natural product requires new biologicaland economical development, and co-operative programs on technological andmedicinal studies. Production and processing of medicinal and aromatic plantsshould be one of the successful branches of horticulture in the future. To fulfil theserequirements, however, production systems need to be modernised and high qualitymaterial must be provided to farmers. The creation of a Bank of Germplasm in

crocuses could help in that purpose

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The 1st International Symposium on Saffron Biology and Biotechnology heldin 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 15countries (Spain, Iran, Mexico, India, Greece, Italy, France, Japan, Switzerland, USA,Turkey, Hungary, Denmark, Canada, and Azerbaijan). The meeting, chaired by thisauthor provided a worldwide overview of research in Saffron achieved in the lastdecades, including basic biology, agronomy, genetics and breeding, chemistry,industrial production, pharmacognosy, pharmacology, and economics. Attention wasalso given to the scientific and practical problems and the challenges for Saffron plantin the XXI century. One of the statements of the Working Group on Saffron Biologyand Biotechnology (M04), created at this meeting inside the Section of Medicinal andAromatic Plants of the ISHS, was to promote the creation of a Bank of Germplasmand Gene Banks in Saffron to preserve genetic biodiversity in Saffron and relatedspecies (Fernndez and Abdulalev, 2004).

A World-Scale Task Note that while the worlds total annual Saffron production is estimated at

about 200 tons per year, Iran with more than 47,000 hectares of land under Saffroncultivation 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 enrichedwith 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. Itmust be strongly taken into account that having the support of research groups fromcountries that are putative commercially competitsors is highly valuable. Thisconfidence is based on the environment of collaboration and frankness amongstscientists generated in the 1st ISSBB. A new big step has been done with the 2ndISSBT carried out at the Ferdowsi University of Mashhad (Iran) in October 2006which contents are included in the current Acta Horticulturae volume.

It is not an overstatement to say that this Bank of Germplasm extents itsobvious European interest to a wider scale, and must be the basis of the WorldSaffron & Crocus Collection for everybodys use.

A FIRST MILESTONE: THE CROCUSBANK PROJECTAgricultural 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 upmeasures in support of the agricultural biodiversity, to contribute reaching the 2010target of halting the loss of biodiversity.

In the Biodiversity Action Plan for Agriculture (Council Regulation (EC) No870/2004) 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 theMember States and the European Commission for the conservation and sustainableuse of genetic resources in agriculture. It also facilitates co-ordination in the field of

international undertakings on genetic resources in agriculture, in particular within theConvention on Biological Diversity (www.biodiv.org/default.shtml) the International

http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=EN&numdoc=32004R0870&model=guichetihttp://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=EN&numdoc=32004R0870&model=guichetihttp://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=EN&numdoc=32004R0870&model=guichetihttp://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=EN&numdoc=32004R0870&model=guicheti

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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 SustainableUtilisation 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&video=9&lang=en&pub_id=57930).The principle of The conservation and sustainable use of genetic resources inagriculture are essential to the sustainable development of agricultural production andof rural areas prompt the Saffron germplasm main stakeholders in the Europeanregions and third states around the world to joint a consortium involving crocusrelatives to propose the so-called CROCUSBANK project on GENETICRESOURCES OF SAFFRON AND ALLIES (CROCUS SPP), aiming to contributeto the Biodiversity Action Plan for Agriculture. The CROCUSBANK project will play a part to the new Programme on the Conservation, Characterisation, Collectionand Utilisation of Genetic Resources in Agriculture. AGRI GEN RES launched bythe European Commission in 2005. The recent approval of this 4-years length projectconstitutes a big success for all Saffron researchers taking into account the strongcompetition of several projects dealing with animal and plant species.

The objective of this project is to create, characterise and exploit a germplasmcollection (bank) inCrocus species, including Saffron crocus. These are the goals.1. Collection, multiplication, conservation and documentation of Crocus geneticresources. 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 plantmaterial in selection programmes all over the world; and second, to create a collectionof Saffron allies for conservation, since they are endangered and threatened taxa and populations inCrocus , and for research in taxonomy and evolution, genetics, physiology, ecology and agronomy. TheCrocus 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 fungiand 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. Thecollection of Crocus material will be carried out by means of requests todifferent regional centres growing the plants and visiting specific locations atappropriate date to collect both cultivated Saffron and wild species. The taxaincluded in this project shape a proposal of maximum achievements. Thereality 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 Crocusand 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 tissueculture techniques will be used when required.

Elaboration of an effective documentation system, with the passport andcharacterization data of the accessions, in order to guaranty an appropriatemanagement 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 elaboratea list of descriptors for the characterization of the genusCrocus and primary

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characterization of the collected material. For the characterization/evaluation of thematerial we will take into consideration phenotypic characters with good heritabilityat different structural and physiological levels and include both simple, single-geneautoapomorphic characters and complex quantitative traits: Morphological (floralfeatures, corm size); Phenological (flowering and relationship of climate, latitude andaltitude); Cytological (chromosome numbers, genome size, ploidy level andidentification of hybrids); Phytochemical (Saffron chemical composition; metabolic profiling); Molecular (DNA analysis); and Physiological (abiotic stresses and pathogen responses).3. Application of the Crocu s germplasm information and banked accessions.

Rationalization of collections, identifying duplicates in order to optimise themanagement 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 horticulturalindustry. Comparative genomics with model and crop species to identify universal

features and valuable genes for agronomy.

The ConsortiumThe CROCUSBANK consortium (Table 3) is composed by partners from 6

European Union countries and 3 partners from third countries where rich resources of Saffron andCrocus relatives are found. All of them have long experience in researchon 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 partners geographical distribution comprise the Saffron producing areas as well asthe main habitats of theCrocus species. The non-presence of Iranian and Indiangroups is explained in terms of administration inconveniences. The AGRI GEN RES programme does not subsidize groups from third-countries, which nevertheless arefree to participate without funding, as partners from Azerbaijan, Egypt, and Turkeyhave done. Besides, having the collaboration of Iranian and Indian scientists andinstitutions is highly desirable and we look forward to accomplish it.

All scientific, financial and administrative aspects of CROCUSBANK will beco-ordinated by this author at the University of Castilla-La Mancha (Spain), who willrepresent the consortium in the European Commission. The aim is to collect andreproduce 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 beused in selection programmes all over the world. Secondly, the collection of Saffronallies (both seeds and/or corms) will be made for conservation, since someCrocustaxa 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 PlanThe 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

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collect the quoted plant material using botanic gardens, personal contacts, other collections and explorations to the field. TheCrocus wild species that are included inthe list are the complete genus (Table 5).

Explorations and collection per area are designed on the basis of thegeographical proximity of both Saffron production areas andCrocus spp. populationhabitats. 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 manyaccessions as possible, representative of the species range and diversity, according tothe financial and human resources available.

The conservation, multiplication, maintenance, documentation andmanagement of the collection will be carried out by the JCCM Group, centrespecialised in the management of germplasm banks of the Castilla-La Mancha regionin 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 CollectorsGroup (SCG) is integrated by partners 0, 4, 5, 6, 7, 8, 9, 11 and 12, under thesupervision 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 commandof partner 11 (NAGREF, Greece). The partners 1, 4, 5, and 10 constitute theDescriptors Group (DG) whose manager is partner 4 (UPVLC, Spain). TheMultiplication and Conservation Group (MCG) is organized by partners 0, 1, and10, with partner 1 (JCCM, Spain) as manager. The characterization and evaluationtask is approached by two groups: (1) morphological, physiological and agronomicallevel, with partners 0, 1, 4, 5, 7, and 10, leaded by partner 1, and (2) chemical andmolecular level, with partners 0, 2, 3, 6, 8, 9, 10, 11, 12 and 13, under theorganization of partner 2 (AUA, Greece). These groups are called CEGA and CEGC,respectively. The coordination of the complete characterisation task will be made bythe 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 andcoordination 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 EUand non-EU countries, in an attempt to store the maximum genetic variability present

in this crop. The wide commercial experience and good knowledge of Saffroncropping areas of the French company (TJMT) (partner 6) will be extremelyconvenient for this achievement. CCG task is the collection of Crocus taxa by meansof requests to different collection holders or visiting specific locations at appropriatedate 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 toSaffron (i.e.Crocus series of the genus) for their interest in Saffron breeding. Thesespecies are present in Greece and its islands. The Greek sub-coordinator (partner 3)has a long experience inC. sativus studies and incorporates experienced geneticistsand plant breeders. Included in partner 10 we gain the participation of theCrocusGroup (historically) a splinter group from the British Iris Society, that is an

assemblage of persons with a shared interest in the genusCrocus . The group wascreated in 1974 and includes botanists,Crocus growers and people dedicated to

http://www.thealpinehouse.fsnet.co.uk/crocus%20pages/Crocus%20Group.htmhttp://www.thealpinehouse.fsnet.co.uk/crocus%20pages/Crocus%20Group.htmhttp://www.thealpinehouse.fsnet.co.uk/crocus%20pages/Crocus%20Group.htmhttp://www.thealpinehouse.fsnet.co.uk/crocus%20pages/Crocus%20Group.htm

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The corms obtained in the campaigns of recollection will be multiplied in thefield 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 thecase of accessions of wildCrocus with scarce material, this will be previouslymultiplied in acclimatised greenhouse with the substrate of the collecting zone, inorder to make sure the propagation of some material under the best conditions beforethe transference to the field, to minimize the risk of loss. In some cases theconservation/propagation in the greenhouse will continue.

Multiplication will be carried out in a fenced parcel in the experimental farmof the Bank of Plant Germplasm of Cuenca (partner 1), with a medium altitude between 950 an 1000 meters, medium temperature of 11.5 C, and a precipitationranging from 550 to 600 mm mainly concentrated in spring and autumn. The edaphiccharacteristics in this parcel are typical of the area, sandy loams, alkaline pH(7.6-8.4), normal electric conductivity (

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corms will be collected each year for characterisation. The size of the samples willdepend on the variability observed for the characters evaluated. Corms (and/or seedsin 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 labouredas that of multiplication, but both will be located on separated zones of the fence, andwill serve as replicas of security for contingencies.

DocumentationAll the information associated to theCrocus 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 andrules internationally established for documentation in plant germplasm collections andwill be accessible via Internet through the Website of theServicio de Investigacin yTecnologa 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 followingcategories: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 inits 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 itsmanagement a registry of incomes and outcomes of plant material will be carried out.

Molecular and Cytological CharacterizationThe objective is to provide molecular and molecular cytogenetics markers for

the characterization of Crocus germplasm and application in selection of improvedcultivars for Saffron production. Thus, the molecular characterization of Crocus willallow:

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 basedmarkers (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 rootingof phylogenies.

Standard primers for phylogenetic analysis will be used to amplify genesegments from nuclear, chloroplast and mitochondrial genes. Molecular-based treeswill be constructed to infer relationships between groups and accessions. Molecular-

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based analysis will show how much diversity is present in the genus; this data will bein a form that it can be used to develop conservation policy.

In addition to estimating genetic diversity, these tools can be utilized toestablish molecular markers for phenotypic traits of interest. Genes which are widelyconserved between species, with a focus on those associated with biotic and abioticstress tolerance, will be amplified and sequenced from a range of accessions, usingconserved sets of PCR primers. Genes associated with corm development anddormancy will be isolated and characterized from a range of accessions. Geneexpression analyses, will allow to found more efficient alleles. Genes associated toflavour and other associated quality traits important inCrocus commercialization.Several genes directly involved in flavour have been identified inC. sativus , andseveral biochemical markers are used to determine the quality of flavour and aroma inSaffron, 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 StandardizationOrganization, namely UV-Vis Spectrophotometry, information on the chemicalcomposition is derived by HPLC combined with spectroscopic detection means (diodearray, 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-UVtechniques. Further investigation on the chemical composition of stigmas of specieswith 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 spectraderivatives of the individual extracts are also expected to highlight any deviation fromthe typical profile of C. sativus . In all cases, the quality parameters suggested by ISO3632-2 (2003) such as colouring strength, bitterness and safranal content will be alsodetermined. Main target of this work is finally the identification of constituents thatcould be a discriminative key for the characterization of different Crocus species toappreciate the extend of diversity of various geographic or genetic groups cultivatedand wild.

The essential oils (EO) content of theCrocus stigmas will be determined andthe 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 - extractionto organic solvent will be used as reference methods for the production of EO. Theultrasound based assisted extraction and the microwaves based distillation will be theinnovator methods for the EO production. These methods are expected to limit thetime demand for the oil isolation receiving similar quality oil with the conventionalmethods and in some cases to improve the quality of the produced oil, especially byreceiving 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 solventsin variable extractable times, using ultrasound or microwaves apparatus, which will

be functioning at specific frequency. Extractions based on ultrasound will be

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performed at room temperature, while the distillations based on microwaves will berapid, 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 qualitychemical 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 Stress1. Evaluation for Salt Stress. The work aims to individuate landraces and/or wildgenotypes of interest for utilising in marginal areas characterised by soil salinity anddevelop 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 conditionsand 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 representthe 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 substrateindefinitely. 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 driedstyles will be send to chemical research unit to analyse the main qualitativecompounds.

2. Pathogenesis Response . The work intend to check Crocus accessions against fungithat have been reported as Saffron pathogens in different geographic areas: Fusariummonliforme (corm rot, Kashmir),Rhizoctonia crocorum [ sin. R. violacea] (violet rootrot 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 nofind genetic resistances against these pathogens in Saffron cultivars or Crocusgenotypes. ACrocus spp. collection of about 50-100 accessions will be evaluated.

The infections of plant material will be performed using 106/ml fungalzoospores during 72 h. Treatment solutions will be diluted in 0.1 % Tween 80 from 1mM 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 1mm 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 fibrousscales in position. In some corms white or yellowish white fungal mass is observed.

Application of the Crocus Germplasm Information and Banked AccessionsThe deliverables from collection to evaluation will have applications at

applied and fundamental levels. This application task aims to provide somedemonstrator 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 thegermplasm and markers, where relatively little works can give significant and fastresults. Firstly, what proportion of the germplasm collection consists of duplicates?

Secondly, how much diversity is present in Saffron, is the CROCUSBANK collectionlikely to represent most of the diversity and how does Saffron diversity relate to

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Crocus diversity? Are there ecologically rare but important species/genotypes in thenatural environment (results that have consequences for tourism industry anddevelopment; for genetic resources; sustainable land use and biodiversity; treatycommitments eg Rio Convention on Biodiversity? Thirdly, do the molecular markersconfirm the postulated origin and closest diploid relative(s) of Saffron, and how cangenes from Saffon andCrocus 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 isundesirable to discard unique accessions. Some molecular markers from WP04will show the frequency of duplicates within the collection, allowing them to bediscarded. Passport data combined with duplicate frequency will estimate howrepresentative the collection is of the complete range of Saffron.

Quantification of diversity in Saffron and Crocus. It is essential to measure levelsof biodiversity of plants throughout Europe. We will measure the level of molecular diversity of the Crocus species in the collection, and compare this withother wild plants. The results will also allow us to recommend optimal in situconservation 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 areconserved across all plant species, and define how the Crocus/Saffron genome isrelated to other species.

Identification of germplasm for exploitation by breeders and growers. Based onthe 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 andalleged medicinal plants, also a significant part of the Eurasian legacy. This crop iswell integrated into the EU agriculture policy, because its added-value from processing, sustainable and ecologically friendly cultivation methods, perennial naturemaking 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 hadtaken the responsibility of the creation and maintenance of a collection representativeof the genetic variability of the crop and its allies. At a world scale level there was nota Crocus collection institutionally protected and at the disposal of potential users. Nodescriptor list has been published in saffron. The CROCUSBANK project will changethis status quo. The elaboration and publication through the IPGRI of a complete listof descriptors for characterisation of Crocus becomes a duty. Data generated will bethe first ones to be available, together with the plant material itself. We will create adatabase following the international standards for the management of germplasmcollections.

The CROCUSBANK collection has two main goals: First, to collect andreproduce saffron bulbs, coming from all the countries that cultivate saffron, for directuse of this plant material in selection programmes all over the world; and second, tocreate a collection of saffron allies for conservation, since they are endangered andthreatened taxa and populations inCrocus , and for research in taxonomy and

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evolution, genetics, physiology, ecology and agronomy. ThisCrocus species areexploitable sources of resistances and other agronomical interesting traits to betransferred to saffron, through appropriate breeding programmes and technologicaltools.

ACKNOWLEDGEMENTSThe author is grateful to his collaborators at the IDR-Biotechnology Lab,

doctors Lourdes Gmez-Gmez, ngela Rubio, Manuel lvarez-Ort, RaquelCastillo, and Oussama Ahrazem for their daily work and dedication. His alsoemphatically thanks Dr. Marta Roldn for her crucial assistance as expert in managingEU 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. GeorgeBoberly, 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 memorythe CROCUSBANK project is dedicated.

Literature CitedCouncil 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.

Fernndez, J.A. 2004. Biology, biotechnology and biomedicine of Saffron. RecentRes. Devel. Plant Sci. 2:127-159.

Fernndez, J.A. and Abdullaev, F. (eds.). 2004. Proceedings of the First InternationalSymposium on Saffron Biology and Biotechnology. Acta Horticulturae 650.

Mathew, B. 1982. TheCrocus . A Revision of the GenusCrocus (Iridaceae). B.T.Batsford Ltd. London].

Piqueras, A., Han, B.H., Escribano, J., Rubio, C., Helln, E. and Fernndez, J.A.1999.Development of cormogenic nodules and microcorms by tissue culture, a new toolfor the multiplication and genetic improvement of saffron. Agronomie 19:603-610.

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Table 1. Estimate of current Saffron world production (various sources)Quantities Countries Qualities or Zones

150/170 MT IRAN Sarghol, Poushal, Dasteh8/10 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 Gtinais, Quercy

1 kg SWITZERLAND Mund

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Table 2. Some cultivars of ornamental crocusesC. laevigatus FontenayiC. pulchellus Albus, ZephyrC. sativus cartwrightianus AlbusC. speciosus Albus, Artabir, Cassiope, Oxonian, Aitchinsonii,ConquerorC. 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, ZenithC. biflorus ssp.biflorus ParkinsoniiC. biflorus ssp.weldenii Albus, FairyC. etruscus Rosalind, ZwanenburgC. flavus ssp. flavus Aureus

C. imperati De JagerC. korolkowii Kiss of Springx luteus Stellaris (C. angustifolius x C. flavus )C. sieberi Albus, Firefly, Hubert Edelsten, Ronald Ginns, Tricolor,Violet QueenC. tommasinianus Albus, Barrs Purple, Lilac Beauty, Pictus, Roseus,Ruby Giant, Whitewell PurpleC. vernus Albiflorus, Graecus, Harlem Gem, White Star, Enchantress,Flower Record, Jeanne dArc, Mammoth Yellow, Pickwick, PurpureusGrandiflorus, Queen of the Blues, Remembrance, Vanguard, Glory of Sassenheim, Negroboy, Paulus Potter, Twinborn, White ChristmasC. versicolor Picturatus

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Table 3. Partnership of the CROCUSBANK project N Country Short

NameInstitution Expertise/role

0 Spain UCLM Universidad de

Castilla-La Mancha

Genetics and Plant

Breeding. Biochemistry.Biotechnology.Pathogenesis

1 Spain JCCM CIA de Albaladejito.Junta de

Comunidades deCastilla la Mancha

Germplasm Bank.Agronomy. Plant

Breeding. Phenology.

2 Greece AUA AgriculturalUniversity of Athens

Chemical Analysis(molecular modelling),

3 Greece AUTH Aristotle Universityof Thessaloniki

Chemical Analysis(quality). Genetics and

Plant Breeding4 Spain UPVLC Universidad

Politcnica deValencia

Plant Physiology.Phenology. Agronomy

5 Turkey GU Gazi University Botany. Morphology6 France TJMT Tradimpex JM

Thiercelin SACommercial. Sensorial

analysis (spice)7 Italy DOFATA

-UNICTUniversit di Catania Plant Breeding.

Evaluation for abioticstress.

8 Hungary UD University of Debrecen

Botany. Biotechnology.Molecular markers

9 France INPT National PolytechnicInstitute of Toulouse

Chemical analysis(aromatic, dying, other

chemicals)10 UK ULEIST University of

Leicester Molecular Cytogenetics.

Genomics.11 Greece NAGREF National Institute of

Agricultural ResearchBotany, Genetic

Resources12 Azerbaijan ANAS Azerbaijan National

Academy of SciencesBotany. Pharmacology

13 Egypt NRC National ResearchCentre

Biochemistry. Chemicalanalysis (bioactivity)

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Table 4.Crocus taxonomy. Brian Mathew subdivided the genus in two subgenera:Crocus and Crociris . C. sativus is classified into subgeneraCrocus , SectionCrocus, SeriesCrocus (Mathew, 1982)

Subgenus Section Series Species

Verni C. vernus, C. tommasinianus, C. kosaninii , C. etruscus, C. baytopiorumScardici C. scardicus, C. pelistericusVersicolores 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 Crocus

Crocus C. sativus , C. cartwrightianus, C. thomasii, C. hadriaticus, C. asumaniae, C.moabiticus, C. oreocreticus, C. pallasii, C. mathewii,

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. boulosiiCarpetani C. carpetanus, C. nevadensis

Intertextii C. fleischeriSpeciosi C. speciosus, C. pulchellus

Nudiscapus

Laevigatae C. laevigatus, C. tournefortii, C. boryiCrociris C. batanicus

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