Crocus Species Research Articles

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus).

Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

Cytotoxic and Antioxidant Activities of Crocus pallasii subsp. haussknechtii Corms Extracts Compared with Crocus sativus

Background and objectivesThe goal of this study was to compare the cytotoxic and antioxidant activities of the corms extracts of two Crocus species (C. sativus L. and C. pallasii subsp. haussknechtii).Methodsn-Hexan, chloroform and methanol extracts of the corms were prepared. Cytotoxic activities of the extracts against human breast cancer cell lines MDA-MB-231 and MCF-7 were evaluated via MTT assay and dual acridine orange/ethidium bromide fluorescent staining test. Antioxidant activities of the extracts were assessed by DPPH method.ResultsThe results showed that the methanol extracts of the corms from both species had cytotoxic activity against the breast cancer cells. Cytotoxic activity of the corms extract of C. sativus L. was significantly higher than C. pallasii subsp. haussknechtii. n-Hexan and chloroform extracts from the corms of C. pallasii subsp. haussknechtii and Crocus sativus L. showed no anti-proliferative activity which indicated the polar nature of the cytotoxic agent(s) in the extracts. The results of dual acridine orange/ethidium bromide fluorescent staining test revealed that the methanol extracts of both species corms destroyed cancer cells through apoptosis. Moreover, the methanol corm extracts of the two species showed little antioxidant activity.ConclusionIt can be concluded that C. sativus L. may possess more significant apoptotic properties compared to C. pallasii subsp. haussknechtii.

Multi-species transcriptome analyses for the regulation of crocins biosynthesis in Crocus

BackgroundCrocins are soluble apocarotenoids that mainly accumulate in the stigma tissue of Crocus sativus and provide the characteristic red color to saffron spice, in addition to being responsible for many of the medicinal properties of saffron. Crocin biosynthesis and accumulation in saffron is developmentally controlled, and the concentration of crocins increases as the stigma develops. Until now, little has been known about the molecular mechanisms governing crocin biosynthesis and accumulation. This study aimed to identify the first set of gene regulatory processes implicated in apocarotenoid biosynthesis and accumulation.ResultsA large-scale crocin-mediated RNA-seq analysis was performed on saffron and two other Crocus species at two early developmental stages coincident with the initiation of crocin biosynthesis and accumulation. Pairwise comparison of unigene abundance among the samples identified potential regulatory transcription factors (TFs) involved in crocin biosynthesis and accumulation. We found a total of 131 (up- and downregulated) TFs representing a broad range of TF families in the analyzed transcriptomes; by comparison with the transcriptomes from the same developmental stages from other Crocus species, a total of 11 TF were selected as candidate regulators controlling crocin biosynthesis and accumulation.ConclusionsOur study generated gene expression profiles of stigmas at two key developmental stages for apocarotenoid accumulation in three different Crocus species. Differential gene expression analyses allowed the identification of transcription factors that provide evidence of environmental and developmental control of the apocarotenoid biosynthetic pathway at the molecular level.

Adding color to a century-old enigma: multi-color chromosome identification unravels the autotriploid nature of saffron (Crocus sativus) as a hybrid of wild Crocus cartwrightianus cytotypes.

Saffron crocus (Crocus sativus) is the source of the most expensive spice of the world, produced from manually harvested stigmas, thus serving as a cash crop for rural communities. However, despite its economic importance, its genome and chromosomes are poorly studied. C.sativus is a sterile triploid species harboring eight chromosome triplets, and propagated only as a clonal lineage by corms. Saffron's evolutionary origin, parental species and allo- or autotriploidy has been a matter of discussion for almost a century. We performed a survey sequencing of the saffron genome and selected cytogenetic landmark sequences consisting of major tandem repeats, which we used as probes in comparative multicolor fluorescent insitu hybridization (FISH). We tagged 92 chromosomal positions and resolved the chromosomal composition of saffron triplets. By comparative FISH of six Crocus species from 11 accessions, we demonstrate that C.sativus is an autotriploid hybrid derived from heterogeneous Crocuscartwrightianus cytotypes. The FISH reference karyotype of saffron is crucial for integrating genome sequencing data with chromosomes and for investigating the relationship among Crocus species. We provide an evolutionary model of the saffron emergence; the knowledge of the parental origin offers a route towards the resynthesis of C.sativus from C.cartwrightianus to broaden saffron's gene pool.

A comparative assessment of DNA fingerprinting assays of ISSR and RAPD markers for molecular diversity of Saffron and other Crocus spp. in Iran

Genetic diversity among 25 accessions of Crocus L. (Iridaceae) representing 5 accession of cultivated C. sativus, and 20 accessions belonging to wild species namely, C. speciosus, C. cancellatus, C. haussknechtii, C. michelsonii, and two unknown accessions, obtained from different geographical locations of Iran were studied using 7 ISSR and 10 RAPD primers. Out of 78 bands generated from 7 ISSR primers, 71 were polymorphic (90.2%), whereas out of 94 bands generated from 10 RAPD primers 83 were polymorphic (88.3%). The average PIC value obtained with ISSR and RAPD markers was 0.366 and 0.324, respectively. Marker index (ISSR = 3.71; RAPD = 2.68) and resolving power (ISSR = 5.31; RAPD = 4.12) indicated that the ISSR markers were relatively more efficient than RAPD assay in respect of revealing the genetic diversity of Crocus species. Based on percentage of polymorphic bands; resolving power; polymorphic information content; and Marker Index, the A1 (ISSR) and OPB-12 (RAPD) primers were the most informative ones. Clusters obtained from ISSR, RAPD and a combination of both data sets did match the actual taxonomic classification. The mantel test between two Jaccard’s similarity matrices gave r = 0.862, showing very good fit correlation in between ISSR and RAPD based similarities. The analysis of molecular variance showed that the total polymorphism was largely because of within-group variance (ISSRs 57%; RAPDs 64%; Combined RAPDs + ISSRs 60%). Principal coordinates analysis of the pooled data of both the markers, also supports their UPGMA dendrogram. The results will help in future genetic improvement programme of Crocus species.

Study of phylogenic relationships of some wild and crop species of Iranian Crocus by ITS nuclear loci

ITS (Internal Transcription Spacer of nuclear ribosomal DNA) regions method was applied to identify and study the phylogenetic relationships existing between 41 individuals of Crocus, including 8 species collected in Iran (4 crop and 8 wild individuals) and 16 species (2 crop and 27 wild individuals) already registered in GenBank (NCBI). PCR amplifications were performed using the primer designed on the nucleotide sequence of nuclear region (ITS). A total of 12 sequences were obtained and registered in NCBI database. In particular, 4 of these sequences were not already present in the scientific library. Nucleotide polymorphic sites were counted for ITS (N = 97). The results showed that, the Cytosine base is the most abundant nucleotide. Relatively high conservation was observed in the ITS regions of the populations (0.761). Each sample could be distinguished from the others in the phylogenic trees developed based on the data obtained by ITS barcode genes. In general, ITS sequence indicating high resolution at the species level is very suitable for phylogenetic studies in the Crocus family. Phylogenetic analysis demonstrated the genetic relationship between crop saffron and wild Crocus species. According to the results of this study, the wild species of C. cartwrightianus and C. oreocreticus were considered as the closest relatives of the saffron. The results of the study also indicated that, the different ecotypes of C. sativus may have evolved through independent events probably due to the geographic and environmental pressures

Phytochemical and genetic characterization of styles of wild Crocus species from the island of Crete, Greece and comparison to those of cultivated C. sativus

The aim of this study was to contribute to the characterization of Crocus taxa using morphological, phytochemical and genetic analysis. The styles of C. cartwrightianus, C. oreocreticus and C. laevigatus, collected in the island of Crete were compared to those of C. sativus cultivated at the region of Western Macedonia (Greece). Phytochemical analysis was done using GC–MS and HPLC methods, while ISSR markers were used for their genetic characterization. Safranal was the major volatile component of the styles of C. sativus, 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde of C. cartwrightianus and C. oreocreticus, and isophorone of C. laevigatus. C. sativus had the highest content of crocins and picrocrocin, while C. laevigatus the lowest (only 5% of C. sativus' quantity) and negligible amount of picrocrocin. According to both the genetic and the chemical analysis, C. cartwrightianus is more related to C. oreocreticus, while C. sativus and C. laevigatus are more distinct. Concordance between the two different types of data was also confirmed by the Mantel test (r = 0.932, P = .68). This is the first thorough screening of secondary metabolites (volatile and non-volatile) and also genetic and morphological characters of wild Crocus styles simultaneously, that contributes to the identification and valorisation of genotypes with similar to C. sativus traits which may be introduced as new cultivars through breeding.

Phylogeny of the saffron-crocus species group, Crocus series Crocus (Iridaceae)

Phylogenetic relationships among the taxa of Crocus series Crocus are still unclear, preventing the understanding of species diversity and the evolution of the important spice saffron (Crocus sativus). Therefore, we analyzed sequences of two chloroplast (trnL-trnF, matK-trnK) and three nuclear (TOPO6, ribosomal DNA ETS and ITS) marker regions to infer phylogenetic relationships among all species belonging to series Crocus. Our phylogenetic analyses resolved the relationships among all taxa of the series. Crocus hadriaticus and the former C. pallasii subspecies appeared polyphyletic. The latter deserve elevating the subspecies to species rank, while for C. hadriaticus a detailed study of species boundaries is necessary. Multi-locus and also genome-wide single nucleotide polymorphism data obtained through genotyping-by-sequencing placed C. sativus within C. cartwrightianus with no indication that other Crocus species contributed to the evolution of the triploid. Our analyses thus made an autotriploid origin of C. sativus from C. cartwrightianus very likely.

Transcriptome analysis in tissue sectors with contrasting crocins accumulation provides novel insights into apocarotenoid biosynthesis and regulation during chromoplast biogenesis

Crocins, the red soluble apocarotenoids of saffron, accumulate in the flowers of Crocus species in a developmental and tissue-specific manner. In Crocus sieberi, crocins accumulate in stigmas but also in a distinct yellow tepal sector, which we demonstrate contains chromoplast converted from amyloplasts. Secondary metabolites were analysed by LC-DAD-HRMS, revealing the progressive accumulation of crocetin and crocins in the yellow sector, which were also localized in situ by Raman microspectroscopy. To understand the underlying mechanisms of crocin biosynthesis, we sequenced the C. sieberi tepal transcriptome of two differentially pigmented sectors (yellow and white) at two developmental stages (6 and 8) by Illumina sequencing. A total of 154 million high-quality reads were generated and assembled into 248,099 transcripts. Differentially expressed gene analysis resulted in the identification of several potential candidate genes involved in crocin metabolism and regulation. The results provide a first profile of the molecular events related to the dynamics of crocetin and crocin accumulation during tepal development, and present new information concerning apocarotenoid biosynthesis regulators and their accumulation in Crocus. Further, reveals genes that were previously unknown to affect crocin formation, which could be used to improve crocin accumulation in Crocus plants and the commercial quality of saffron spice.

Production and Characterization of Tissue Cultures of Four Crocus Species from the Carpathian Basin

AbstractWe aimed to produce tissue cultures and plant regeneration from endangered Crocus species: C. scepusiensis, C. tommasinianus, C. vittatus (“Verni” series of the genus) and C. banaticus. For initiation of cultures we used a plant growth regulator (PGR) combination used for in vitro culture of saffron and its relatives: 10 mg L-1 α-naphthaleneacetic acid (NAA) and 1 mg L-1 6-benzyladenine (BA). Shoot tips of young seedlings (C. scepusiensis) and corms (for the rest of species) were used as explants. C. scepusiensis explants developed into organogenic calli. On media with decreased NAA and with or without increased BA concentration, calli produced stigma-like structures and/or shoots and whole plants. In the other species, callus initiation medium induced callus formation with abundant somatic embryos. In C. tommasinianus, embryos developed shoots when auxin content of medium was decreased. In C. banaticus, a decrease of auxin with or without an increase in cytokinin content led to shoot or whole plant regeneration, as in C. scepusiensis. In the case of C. vittatus and C. banaticus, initiation and/or maintenance of cultures on indole-3-butyric acid (IBA) and increased sucrose concentration stimulated whole plant regeneration and in vitro cormlet development. C. scepusiensis and the rest of cultures (organogenic vs. embryogenic) differed at the biochemical level: C. scepusiensis cultures had higher (yet still low) enzymatic antioxidant (catalase, peroxidase) activities. With respect to catalase isoenzyme patterns, C. banaticus was different from the rest of cultures, demonstrating its distinct taxonomical position. Besides germplasm preservation use of the present cultures, they have a potential biotechnological value.

A new Serbian endemic species of the genus Crocus (Iridaceae)

Recent research within the genus Crocus (Iridaceae) let us doubt that Crocus adamii Gay from Serbia represents the same taxon as C.adamii s. str. of the locus classicus in the Caucasian Mountains. The latter belongs to a group of crocuses, which is distributed from the Anatolian Diagonal, a mountain belt in inner Anatolia, to Iran and the Caucasian Mountains. To infer ( i ) if the Serbian C. adamii represents a new species and ( ii ) its taxonomical and phylogenetic affiliation within the genus we combined morphological and molecular investigations. The results show the presence of two morphologically and molecularly differentiated lineages, which both share a close relationship e.g. to C. alexandrii , C. chrysanthus , and C. weldenii but not to C. adamii s.str., which indicates a new species. As a result we here describe C. randjeloviciorum to honor the Serbian botanists Novica and Vladimir Ranđelovic.

Greek PDO saffron authentication studies using species specific molecular markers

Saffron, the spice produced from the red stigmas of the flower of Crocus sativus L. is a frequent target of fraud and mislabeling practices that cannot be fully traced using the ISO 3632 trade standard specifications and test methods. A molecular approach is proposed herein as a promising branding strategy for the authentication of highly esteemed saffron brands such as the Greek Protected Designation of Origin (PDO) “Krokos Kozanis”. Specific ISSR (inter-simple sequence repeat) markers were used to assess for the first time, the within species variability of several populations of C. sativus L. from the cultivation area of “Krokos Kozanis” as well as the potential differences with the band pattern produced by other Crocus species. Then, species-specific markers were developed taking advantage of an advanced molecular technique such as the HRM analysis coupled with universal DNA barcoding regions (trnL) (Bar-HRM) and applied to saffron admixtures with some of the most common plant adulterants (Calendula officinalis, Carthamus tinctorius, Gardenia jasminoides, Zea mays and Curcuma longa). The sensitivity of the procedure was tested for turmeric as a case study whereas HPLC-fluorescence determination of secondary metabolites was also employed for comparison. The overall results indicated that the Bar-HRM approach is quite effective in terms of specificity and sensitivity. Its effectiveness regarding the detection of turmeric was comparable to that of a conventional HPLC method (0.5% vs 1.0%, w/w). Yet, the proposed DNA-based method is much faster, cost-effective and can be used even by non-geneticists, in any laboratory having access to an HRM-capable real-time PCR instrumentation. It can be, thus, regarded as a strong analytical tool in saffron authentication studies.

Evolutionarily distinct carotenoid cleavage dioxygenases are responsible for crocetin production in Buddleja davidii

Crocetin, one of the few colored apocarotenoids known in nature, is present in flowers and fruits and has long been used medicinally and as a colorant. Saffron is the main source of crocetin, although a few other plants produce lower amounts of this apocarotenoid. Notably, Buddleja davidii accumulates crocetin in its flowers. Recently, a carotenoid dioxygenase cleavage enzyme, CCD2, has been characterized as responsible for crocetin production in Crocus species. We searched for CCD2 homologues in B. davidii and identified several CCD enzymes from the CCD1 and CCD4 subfamilies. Unexpectedly, two out of the three CCD4 enzymes, namely BdCCD4.1 and BdCCD4.3, showed 7,8;7',8' activity in vitro and in vivo over zeaxanthin. In silico analyses of these enzymes and CCD2 allowed the determination of key residues for this activity. Both BdCCD4 genes are highly expressed during flower development and transcripts levels parallel the accumulation of crocins in the petals. Phylogenetic analysis showed that BdCCD4.2 grouped with almost all the characterized CCD4 enzymes, while BdCCD4.1 and BdCCD4.3 form a new sub-cluster together with CCD4 enzymes from certain Lamiales species. The present study indicates that convergent evolution led to the acquisition of 7,8;7',8' apocarotenoid cleavage activity in two separate CCD enzyme families.

A new species of Crocus (Iridaceae) from southern Albania (SW Balkan Peninsula)

A new Crocus species (Iridaceae) for southern Albania, Crocus novicii sp. nov. (Iridaceae), is described and illustrated. We here provide diagnostic morphological characteristics, results of molecular analyses, detailed descriptions and illustrations of this new species and compare it with its relatives C. jablanicensis, C. cvijicii and C. veluchensis. Our molecular analysis is based on two chloroplast (matK–trnK, rps16–trnQ) and three nuclear markers (nrETS, nrITS, TOPO6B exon3–exon6) and includes representatives of all related species (C. cvijicii, C. dalmaticus, C. jablanicensis, C. rujanensis, C. sieberi subsp. atticus, and C. veluchensis). Morphologically, C. novicii can be distinguished from its relatives by its white flower with lilac coloring at the base of the perigone and its higher number of leaves (> 3). Although it is morphologically close to C. jablanicensis, molecular analysis has revealed a close affiliation to C. veluchensis. Crocus novicii is diploid with 2n = 20 chromosomes. The new species is named after the Serbian botanist Novica Ranđelović to honor his important work on the genus Crocus on the Balkan Peninsula.

A Matter of Contrast: Yellow Flower Colour Constrains Style Length in Crocus species.

Most flowers display distinct colour patterns comprising two different areas. The peripheral large-area component of floral colour patterns attracts flower visitors from some distance and the central small-area component guides flower visitors towards landing sites. Whereas the peripheral colour is largely variable among species, the central colour, produced mostly by anthers and pollen or pollen mimicking floral guides, is predominantly yellow and UV-absorbing. This holds also for yellow flowers that regularly display a UV bull’s eye pattern. Here we show that yellow-flowering Crocus species are a noticeable exception, since yellow-flowering Crocus species–being entirely UV-absorbing–exhibit low colour contrast between yellow reproductive organs and yellow tepals. The elongated yellow or orange-yellow style of Crocus flowers is a stamen-mimicking structure promoting cross-pollination by facilitating flower visitors’ contact with the apical stigma before the flower visitors are touching the anthers. Since Crocus species possess either yellow, violet or white tepals, the colour contrast between the stamen-mimicking style and the tepals varies among species. In this study comprising 106 Crocus species, it was tested whether the style length of Crocus flowers is dependent on the corolla colour. The results show that members of the genus Crocus with yellow tepals have evolved independently up to twelve times in the genus Crocus and that yellow-flowering Crocus species possess shorter styles as compared to violet- and white-flowering ones. The manipulation of flower visitors by anther-mimicking elongated styles in Crocus flowers is discussed.

Molecular genetic evidence for allotetraploid hybrid speciation in the genus Crocus L. (Iridaceae)

Although dysploidy and polyploidisation events are known to be important drivers in the evolutionary history of the genus Crocus, only a few examples of natural hybrid origins have so far been documented. Here, we describe the phylogenetic affinities of five taxa in the Crocus vernus species complex from the Carpathian Basin in Central Europe. Genetic variability was evaluated using chloroplast DNA sequences of the accD-psaI intergenic spacer and amplified fragment length polymorphism (AFLP) in combination with karyological observations. Genetic diversity and differentiation of the seven investigated Crocus species were also evaluated using AFLP data. We created a phylogenetic hypothesis using both sequences and AFLP fingerprinting data under maximum parsimony (MP). AFLPs were also analysed by means of multivariate statistics using principal coordinate (PCoA) analysis and Bayesian clustering (BC) to test for hybridity. Both the AFLP and plastid phylogenetic trees separated the taxa into two groups: (1) a ‘Balkan’ clade, and; (2) an ‘Adriatic’ clade. The Balkan clade contained Crocus heuffelianus samples and one Crocus vittatus from Croatia as well as Crocus tommasinianus samples from Hungary; the Adriatic clade included Crocus vittatus and Crocus vernus samples from Hungary and Croatia as well as Crocus neapolitanus from Italy. A hard incongruence was found in the placement of the Slovakian Crocus scepusiensis which clustered inside the Balkan clade on our plastid tree, while it fell in the Adriatic clade on the AFLP tree. The same populations occupied intermediate position on the PCoA plot. The BC analysis assigned all Crocus scepusiensis specimens as F1 hybrids, while Crocus vernus and Crocus heuffelianus were assigned as parental species. Together with our cytological investigation that determined a 2n=18 chromosome number of Crocus scepusiensis, we conclude an allopolyploid hybrid origin for this Northern Carpathian taxon as the result of a cross between members of the 2n=10 Balkan clade (e.g., Crocus heuffelianus from Transylvania) and the 2n=8 Adriatic clade (Crocus vernus). A similar origin is postulated for the North Balkan Crocus vittatus (2n=18), which was clustered as an F1 hybrid in the BC analysis; thus, parallel evolution may have taken place in the northern and southern part of the Carpathian Basin beginning from the same parental species but leading to different allopolyploid derivatives.

Influence of nutrient media on callus induction, somatic embryogenesis and plant regeneration in selected Turkish crocus species

Callus induction, somatic embryogenesis and plant regeneration were initiated in selected five species of Turkish crocus using three diffrent explants (leaf, stem and corm) cultured on four different media (MS, GB5, LS and CHE). The highest frequencies of callus induction (100%) and shoot regeneration (70%, with 7.2 shoots/callus) were found in the crocus species Crocus oliveri ssp. Oliveri, using the MS medium containing 5% (w/v) sucrose supplemented with (4mg/L NAA+4mg/L TDZ) and (2mg/L IAA+2mg/L TDZ+2mg/L BAP). When the embryogenic calli were transferred into the four nutrient media containing (2mg/L IAA+2mg/L TDZ) and 100mg/L ABA, these further developed into cotyledonary embryos. Maximum number of somatic embryos (2.9 embryos per leaf explant, with a frequency 46.6%) was obtained in C. oliveri ssp. Oliveri. During subculture using the half strength media, cotyledonary embryos gradually developed into plantlets.

Somatic Embryogenesis in Crocus sativus L.

Saffron (Crocus sativus L.) is one of the most important species in Crocus genus because of its effective usage. It is not only a very expensive spice, but it has also a big ornamental plant potential. Crocus species are propagated by corm and seed, and male sterility is the most important problem of this species. Hence, somatic embryogenesis can be regarded as a strategic tool for the multiplication of saffron plants. In this chapter, the production of saffron corms via somatic embryogenesis is described.

The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme.

The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD2 (AIG94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom. Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCDs, namely CaCCD1, CaCCD2 and CaCCD4a/b and CaCCD4c were first cloned and characterized. CaCCD2 was localized in plastids, and a longer CCD2 version, CsCCD2L, was also localized in this compartment. The activity of CaCCD2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCDs was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. CaCCD2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the CaCCD1 activity in Crocus ancyrensis flowers. The specific CCD2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast.

Diversity and relationships of Crocus sativus and its relatives analysed by inter-retroelement amplified polymorphism (IRAP).

Saffron (Crocus sativus) is a sterile triploid (2n = 3x = 24) cultivated species, of unknown origin from other diploid and polyploid species in the genus Crocus (Iridaceae). Species in the genus have high morphological diversity, with no clear phylogenetic patterns below the level of section Crocus series Crocus. Using DNA markers, this study aimed to examine the diversity and relationships within and between species of Crocus series Crocus. Eleven inter-retroelement amplified polymorphism (IRAP) primers were used in 63 different combinations with 35 single-plant accessions of C. sativus and related Crocus species in order to determine genetic variability and to conduct phylogenetic analysis. A total of 4521 distinct polymorphic bands from 100 bp to approx. 4 kb were amplified; no fragment specific to all accessions of a single species was amplified. The polymorphic information content (PIC) values varied from approx. 0·37 to approx. 0·05 (mean 0·17 ± 0·1) and the major allele frequency had a mean of 0·87. High levels of polymorphism were identified between accessions of the six species of Crocus series Crocus related to C. sativus, with further variation between the species. In contrast, no polymorphisms were seen among 17 C. sativus accessions obtained in the region from Kashmir through Iran to Spain. In contrast to the intraspecific variability seen in other Crocus species, C. sativus has minimal genetic variation, and it is concluded that the triploid hybrid species has most probably arisen only once. The data show that saffron is an allotriploid species, with the IRAP analysis indicating that the most likely ancestors are C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics, and show the need for conservation and collection of wild Crocus.