Crocus Species Research Articles

Morphological Trait Variations and Flower Color Differences in Wild Crocus Species

This study investigates the phenotypic diversity of spring-flowering Crocus species native to Serbia by analyzing their morphological traits and flower color variations. Detailed phenotypic characterization was performed on seven species: C. alexandri, C. chrysanthus, C. heuffelianus, C. randjeloviciorum, C. rujanensis, C. variegatus, and C. weldenii. The study examines how morphological parameters of tested species diverged from average values during a hot and dry winter, resulting in smaller plants, earlier and shorter flowering periods, and reduced flower size, regardless of species. Hierarchical clustering based on morphological traits grouped the species into three clusters: the first cluster included C. alexandri, C. chrysanthus, C. rujanensis, and C. weldenii, characterized by the highest number of leaves and the lowest number of flowers; the second consisted of C. randjeloviciorum, distinguished by shorter plant height and the greatest number of flowers; and the third cluster comprised C. heuffelianus and C. variegatus, notable for the tallest plants, widest corms and leaves, and longest perigon segments. Grouping based on petal color components (L*, a*, b*, chroma, and hue) formed five distinct clusters, demonstrating differences in flower color. These findings are valuable for taxonomic classification, conservation measures, and breeding programs, which ultimately support the conservation of biodiversity and the promotion of ornamental plant cultivation. Flower color in particular has proven to be a reliable species indicator, as it remains constant despite the year and/or habitat. The use of a colorimeter can speed up the identification of species in the field and provides researchers and conservationists with a practical tool.

Choosing the optimal technology for obtaining Crocus alatavicus extract with a high content of flavonoids and carotenoids

In recent years, there has been a significant increase in demand for herbal medicines, due to the growing interest of consumers in natural and safe alternatives to synthetic medicines. In response to these trends, manufacturers are focusing on developing new formulas and extraction technologies to meet the increased demand for herbal medicines. Modern extraction methods make it possible to efficiently extract secondary metabolites from plant raw materials, maintaining an almost complete chemical composition and ensuring a high yield of extractives. The possibility of regulating the concentration of extracted biologically active substances during the process opens up new prospects for the use of plant raw materials as basic pharmaceutical substances. It was found that all the studied plant species of the genus Crocus L. demonstrate antioxidant, anti-inflammatory and cardioprotective activity. Crocus alatavicus, which is a representative of the flora of Kazakhstan, is of particular interest. The purpose of this study is to select the optimal extraction method for obtaining extracts with the maximum content of flavonoids and carotenoids from the aboveground part of Crocus alatavicus. The study of the technological parameters of obtaining an extract from this plant active substance is of practical importance for the further development of medicines and the formation of a scientific justification for their use.

Phytochemical characterization of callus cultures from the endangered plant Crocus scepusiensis (Rehm. & Woł.) Borbás ex Kulcz.

Crocus scepusiensis (Rehm. & Woł.) Borbás ex Kulcz., a critically endangered herbaceous plant which serves as a valuable source of bioactive compounds found across Europe and Asia. The aim of this study was to produce a calli from two different plant parts (leaf and shoot tip) for the critically endangered C. scepusiensis through tissue culture techniques, characterize the resulting calli through chemical profiling, with a focus on identifying key phytoconstituents, and lay the groundwork for future research on the biological activities of these calli extracts. Leaf disc and micro shoot tip explants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA) to induce organogenic calli. The resulting calli exhibited distinct biochemical profiles. Moreover, a phytochemical analysis was conducted to compare the metabolite composition of callus 1 (derived from leaf discs) and callus 2 (derived from shoot tips). Callus 1 displayed a higher total phenolic content (30.3558 ± 1.3564 mg (GAE)/g) compared to callus 2 (29.1543 ± 0.9754 mg (GAE)/g). Similarly, callus 1 exhibited a greater total flavonoid content (26.0089 ± 1.8029 mg (RE)/g) than callus 2 (18.4464 ± 1.4797 mg (RE)/g). Liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry (LC-PDA-ESI-MS/MS) analysis revealed the presence of 26 and 25 constituents in callus 1 and 2, respectively. Fourteen and thirteen of these identified compounds have been previously reported in other Crocus species, with 22 constituents common to both calli. Twelve constituents were reported here in Crocus for the first time as far as we know.

Rethinking Pliny’s “Sicilian Crocus”: Ecophysiology, Environment, and Classical Texts

Classical scholars have long held that the saffron in widespread use throughout the ancient Mediterranean was Crocus sativus (Iridaceae), a sterile triploid descendant of the wild Crocus cartwrightianus, and indeed use of Crocus sativus in antiquity has been extensively borne out both by iconographic and phylogenetic studies. Two principal scholars of the Roman world, Dioscorides the physician and Pliny the natural historian, disagreed radically over the virtues and commercial value of saffron crocus from Sicily, with one praising its quality, and the other excoriating it. This study draws on ecophysiology, classical texts, environmental archeology, and phytochemistry to explain this disagreement and its implications. It explores the potential impact of microclimate on crocus cultivation in the ancient Mediterranean and proposes a new species identification for Sicilian crocus: Crocus longiflorus. The identification of Crocus longiflorus as “Sicilian saffron” offers an important corrective to the assumption that Crocus sativus was the sole crocus species of commercial value in the ancient Mediterranean and renews attention to the economic potential and utility of an indigenous southern Italian species overlooked in classical and later scholarship.

First report of saffron latent virus in Crocus sativus from Hungary.

In early April of 2018 we sampled asymptomatic autumn flowering Crocus plants (Fig. S1.) in a private collection in Hajdú-Bihar county, Hungary. From each species (Cr. kotschyanus subsp. kotschyanus, Cr. sativus, Cr. speciosus) 200 mg leaf sample was collected from 5 neighboring shoot, which were treated as one sample. ELISA tests were carried out in duplicates using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) on the samples (Agdia, Elkhart, IN, USA). A sample was considered positive if the absorbance was at least three times greater than that of the negative control. Only one sample tested positive; the absorbance values of Cr. sativus leaves were 0.013 and 0.014, while the negative controls were 0.002 and 0.003, respectively. The samples were further tested by RT-PCR for potyviruses (Salamon and Palkovics 2005), tomato spotted wilt virus (TSWV) (Nemes and Salánki 2020) and nepovirus subgroup A (Digiaro et al. 2007). Total nucleic acid was extracted with the phenol-chloroform method of White and Kaper (1989), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) using random hexamer primer. The samples were negative for TSWV and nepovirus subgroup A, but a single PCR product of ~ 1700 nucleotide (nt) was amplified with potyvirus specific primers and cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). The 1726 nt long insert sequence, including the complete coat protein region was determined and deposited in the NCBI GenBank database (Accession No: OR425160). Digestion of the original PCR products with restriction enzyme SacI yielded only the predicted restriction fragments (364 / 1362 bp), indicating the presence of only a single potyvirus in the infected sample. BLASTn analysis of the CP cistron revealed the highest nt identities to saffron latent virus (SaLV) Iranian isolates (GenBank AccNo.: MN990394 - 85.44%, MN990415 - 85.39% and RefSeq: NC_036802 - 84.05%). For phylogenetic analyses MEGA11 (Tamura et al. 2021) was used. The resulting Maximum Likelihood tree (Fig. S2) showed that all Iranian SaLV isolates grouped together, while the Hungarian isolate is on an adjacent branch, separate from other virus species, and supported with 100% bootstrap values. From these results, it appears that the Hungarian isolate has been separated from the Iranian clade, and has evolved separately as a distinct lineage. We were unable to fulfill Koch's postulates as all available Crocus sativus plants were infected with SaLV. Latent potyvirus infection of Crocus species, by bean yellow mosaic virus (BYMV), iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV) and turnip mosaic virus (TuMV) has been reported by Grilli Caiola and Faoro (2011). SaLV was first reported from Iran (Parizad et al. 2017), but to our knowledge has never been reported from Europe or from any current EPPO member state. Since Crocus species can be asymptomatic virus reservoirs, it is important that any certification scheme for production should require laboratory tests to prove the health of the plants; or advise growers to keep possible high value susceptible crops such as breeding material and nuclear stocks at a distance from crocuses to mitigate virus transmission between stocks. It is also advisable to grow infected lots far from healthy stocks and protected wild hosts. To our knowledge, this is the first report of SaLV from Hungary and from Europe.

A REVIEW OF GENUS CROCUS: PHENOLIC, NUTRIENT, MINERAL, ESSENTIAL OIL CONTENTS AND BIOLOGICAL ACTIVITIES

Plants are a rich natural resource that serve many uses for people. Humans have long relied on plants for a wide range of needs, including sustenance, housing, and health care. People have been looking to nature for medical solutions since ancient times. He accomplished this by experimenting with and repurposing various organic materials. Plants are the most often utilized of these natural resources. Because of the potential for adverse reactions to synthetic medications, there has been a shift in recent years toward a greater reliance on natural goods. As such, it is crucial to learn which plants are active in certain biological processes. Our research focused on the phenolic, mineral, nutritional, and essential oil content of Crocus genus members as well as their biological activity. A number of crocus species have been demonstrated to have medicinal and nutritional value. Antioxidant, antibacterial, antiviral, and anticancer research has also seen a surge in recent years. Essential oils and phenolic chemicals are suspected to be present in this plant, making it a potential natural source.

Chemical composition of essential oils from Crocus ancyrensis (Herbert) Maw Spreading In Çorum (Türkiye) Region

Crocus ancyrensis is a yellow-flowered Crocus species and is in the same family and genus with saffron. Although various studies have been conducted on the antioxidant capacity and essential oil content characterization of saffron (Crocus sativus), there is no literature information about the C. ancyrensis plant, which is known as Crocus among the people. The members of Crocus family contain many valuable components including antioxidants, phenolic compounds and essential oils. The essential oils obtained from this family is a complex mixture of more than 30 components, which are primarily terpenes and their derivatives. These mixtures are used in paint, medicine, and food applications especially in the cosmetics sector. In the study, the essential oils of the Crocus ancyrensis plant were extracted with the clevenger system and characterized by GC-MS analyses. As a result, 23 volatile components were identified. 2-Hexenal, 1-ethylbutyl Hydroperoxide, 2-nitro-Hexane, β-Isophorone, α-Isophorone, 2-Caren-10-al and Eugenol are found as the main components of Crocus ancyrensis plant extract. Due to the antioxidant, antimicrobial, antifungal, anticancer and odorant properties of some of the identified components, C. ancyrensis can be used as a medicinal aromatic plant in various fields, especially in the cosmetics and perfume industry.

Identifying potential adaptive SNPs within combined DNA sequences in Genus Crocus L. (Iridaceae family): A multiple analytical approach

The genus Crocus L. of Iridaceae family contains about 160 species and is considered as a complex group of plant taxa with regard to evolutionary and phylogenetic events. Inter-specific hybridization and gene flow contribute to species genetic homogeneity in one hand and high within species genetic variability and species genetic content overlaps caused species resolution a problem. In spite of extensive molecular phylogenetic studies in this genus, nothing is known about DNA sequences or Single nucleotide polymorphisms (SNPs) which are of adaptive nature. Moreover, nothing is known about which geographical or environmental factors plays role in species local adaptation and speciation events within Crocus L. genus. Therefore, the present study was conducted to answer the above said questions. We used a combined molecular data set of internal transcribed spacer (ITS) nuclear gene and trnL-F intergenic spacer (trnL-F) sequences of chloroplast genome. A multiple analytical method of Canonical correlation (CCA), Redundency analysis (RDA), and Latent Factor Mixed Model (LFMM)identified a few potential adaptive SNPs. Moreover, population criterions like Tajimas’ D, molecular clock test, as well as skyline-plot revealed a smooth and continuous genetic changes for most of the Crocus species, but the occurrence of a sudden deep nucleotide substitution for Crocus taxa of Iran. The impact of latitude was significantly higher on nucleotide changes compared to that of longitudinal distribution of Crocus species.

Untargeted Metabolomics Analysis of Crocus cancellatus subsp. damascenus (Herb.) B. Mathew Stigmas and Their Anticarcinogenic Effect on Breast Cancer Cells.

Safranal, crocin, crocetin, and picrocrocin are major known compounds in the stigma extract of Crocus sativus with various medicinal properties. Crocus cancellatus is another Crocus species that grows extensively in Iran's various regions, such as the Kurdistan province. The predominant metabolites and biological properties of C. cancellatus have not yet been investigated. The ingredients of the stigma ethanol extract of C. cancellatus were investigated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS). The ROIMCR approach was performed to analyze the LC-MS full scan data sets. This method searches the MS regions of interest (ROI) data in the m/z domain and analyses the results using the multivariate curve-resolution alternating least squares (MCR-ALS) chemometrics technique for simultaneous resolution of two extracts. Also, the antiproliferative properties of C. cancellatus against MDA-MB-231 and MCF-7 cancer cells were examined by MTT, dual acridine orange/ethidium bromide test, Annexin V-FITC/PI, and zymography. The GC-MS and LC-MS untargeted metabolomics data analysis of the extract indicated the presence of cytotoxic agents including safranal, crocin, picrocrocin, and crocetin in the stigma ethanol extract of C. cancellatus. Biological tests showed that the viability of MDA-MB-231 and MCF-7 cancer cells is decreased following C. cancellatus treatment in a time- and dose-dependent way in both monolayer and 3D cell cultures. The MCF-7 cell spheroids had greater resistance to the cytotoxic activity of the extract in 3D cell culture than the MDA-MB-231 cell spheroids. The morphological changes of the cells treated with C. cancellatus stigmas extract were indicative of apoptosis. Zymography analysis revealed a similar trend of matrix metallopeptidase-2 (MMP-2) and matrix metallopeptidase-9 (MMP-9) activity in the treated cells with C. cancellatus extract in comparison with doxorubicin treatment as a positive control. The findings of this research indicate that the ethanolic extract of C. cancellatus stigmas was a good source of bioactive metabolites with anticancer activity.

An integrated approach for the characterization of wild Crocus species adopting phenotypical and phytochemical traits

Crocus is a taxonomically complex genus, characterized by high intra- and inter-specific variability. Five wild Crocus taxa - Crocus biflorus, C. longiflorus, C. neapolitanus, C. siculus and C. thomasii from three different areas of Southern Italy (Calabria, Basilicata and Sicily) have been investigated. We considered both flower morphological traits (inner and outer perigone segments, style branches and anthers) and the chemical profile of specialised metabolites (apocarotenoids: crocins; flavonoids: flavonols) in style branches, which were determined by high-performance liquid chromatography, coupled with a diode array detector and mass spectrometry (HPLC-UV-DAD-ESI-MS). Saffron (C. sativus) was also included as the ‘control’ species. The length of perigone tube, outer and inner tepals, anthers and style branches gave the highest contribution to the discrimination of different taxa. Concerning the specialised metabolite profiles, 20 flavonols and 24 crocins have been identified and quantified in the Crocus extracts and used to discriminate among samples, confirming that Crocus taxa can be considered as an important natural source of these substances. The chemical profiles of the different populations showed some distinctive qualitative and quantitative patterns that contributed to a certain degree of discrimination among species, in fact, flavonoids content range is comprised between 2.7 and 145.9 mg/g, whereas crocins range between 19.8 and 604.0 mg/g. It is thus hypothesized that the combination of morphological and phytochemical screenings may be an effective methodology to characterize wild Crocus genotypes from Southern Italy, also in comparison to C. sativus (saffron).

Genome skimming approach reveals the gene arrangements in the chloroplast genomes of the highly endangered Crocus L. species: Crocus istanbulensis (B.Mathew) Rukšāns.

Crocus istanbulensis (B.Mathew) Rukšāns is one of the most endangered Crocus species in the world and has an extremely limited distribution range in Istanbul. Our recent field work indicates that no more than one hundred individuals remain in the wild. In the present study, we used genome skimming to determine the complete chloroplast (cp) genome sequences of six C. istanbulensis individuals collected from the locus classicus. The cp genome of C. istanbulensis has 151,199 base pairs (bp), with a large single-copy (LSC) (81,197 bp), small single copy (SSC) (17,524 bp) and two inverted repeat (IR) regions of 26,236 bp each. The cp genome contains 132 genes, of which 86 are protein-coding (PCGs), 8 are rRNA and 38 are tRNA genes. Most of the repeats are found in intergenic spacers of Crocus species. Mononucleotide repeats were most abundant, accounting for over 80% of total repeats. The cp genome contained four palindrome repeats and one forward repeat. Comparative analyses among other Iridaceae species identified one inversion in the terminal positions of LSC region and three different gene (psbA, rps3 and rpl22) arrangements in C. istanbulensis that were not reported previously. To measure selective pressure in the exons of chloroplast coding sequences, we performed a sequence analysis of plastome-encoded genes. A total of seven genes (accD, rpoC2, psbK, rps12, ccsA, clpP and ycf2) were detected under positive selection in the cp genome. Alignment-free sequence comparison showed an extremely low sequence diversity across naturally occurring C. istanbulensis specimens. All six sequenced individuals shared the same cp haplotype. In summary, this study will aid further research on the molecular evolution and development of ex situ conservation strategies of C. istanbulensis.

The Pharmacological Activities of Crocus sativus L.: A Review Based on the Mechanisms and Therapeutic Opportunities of its Phytoconstituents.

Crocus species are mainly distributed in North Africa, Southern and Central Europe, and Western Asia, used in gardens and parks as ornamental plants, while Crocus sativus L. (saffron) is the only species that is cultivated for edible purpose. The use of saffron is very ancient; besides the use as a spice, saffron has long been known also for its medical and coloring qualities. Due to its distinctive flavor and color, it is used as a spice, which imparts food preservative activity owing to its antimicrobial and antioxidant activity. This updated review discusses the biological properties of Crocus sativus L. and its phytoconstituents, their pharmacological activities, signaling pathways, and molecular targets, therefore highlighting it as a potential herbal medicine. Clinical studies regarding its pharmacologic potential in clinical therapeutics and toxicity studies were also reviewed. For this updated review, a search was performed in the PubMed, Science, and Google Scholar databases using keywords related to Crocus sativus L. and the biological properties of its phytoconstituents. From this search, only the relevant works were selected. The phytochemistry of the most important bioactive compounds in Crocus sativus L. such as crocin, crocetin, picrocrocin, and safranal and also dozens of other compounds was studied and identified by various physicochemical methods. Isolated compounds and various extracts have proven their pharmacological efficacy at the molecular level and signaling pathways both in vitro and in vivo. In addition, toxicity studies and clinical trials were analyzed. The research results highlighted the various pharmacological potentials such as antimicrobial, antioxidant, cytotoxic, cardioprotective, neuroprotective, antidepressant, hypolipidemic, and antihyperglycemic properties and protector of retinal lesions. Due to its antioxidant and antimicrobial properties, saffron has proven effective as a natural food preservative. Starting from the traditional uses for the treatment of several diseases, the bioactive compounds of Crocus sativus L. have proven their effectiveness in modern pharmacological research. However, pharmacological studies are needed in the future to identify new mechanisms of action, pharmacokinetic studies, new pharmaceutical formulations for target transport, and possible interaction with allopathic drugs.

Phytochemical Analysis and Antioxidant Activity of Crocus speciosus Leaves

The numerous studies indicate leaves of plants are a rich source of bioactive compounds that can be a valuable source of compounds used in the pharmaceutical and cosmetic industries. Aim of this study was to investigate the chemical composition and the antioxidant property of Crocus speciosus leaves. Primary phytochemical screening of C. speciosus leaves revealed the presence of some following compound categories such as phenolic compounds, aminoacids, saponins, proteins, tannins, triterpenoids, glycosides, polysaccharides. The total flavonoids and phenolic compounds content were determined spectrophotometrically and by HPLC-DAD and HPLC-MS. Antiradical activity was determined by ABTS radical-cation scavenging method, spectrophotometrically. The total amount of flavonoids in C. speciosus leaves was 1.07 ± 0.02 mg RE/g (p < 0.05), the total amount of phenolic compounds was 0.41 ± 0.01 mg GAE/g (p < 0.05). By HPLC-DAD-MS analysis the presence of the mangiferin, chlorogenic acid, isoorientin, kaempferol, hyperoside, and isoquercitin was established for the first time in Crocus leaves. The antiradical activity of C. speciosus leaves extracts was 150.08 ± 4.5 μmol/g (p < 0.05) and its was mainly attributed to phenolic compounds content. The high amounts of flavonoids and antiradical activity in C. speciosus leaves suggests promising phytochemical and pharmacological study of this Crocus species.

A New Glycosyltransferase Enzyme from Family 91, UGT91P3, Is Responsible for the Final Glucosylation Step of Crocins in Saffron (Crocus sativus L.).

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.

Distribution patterns and conservation status of Crocus species in Iran, one of the diversity centers of Crocus in the Middle East

Abstract Assessing distribution patterns of valuable taxa plays an important role in the biodiversity conservation of these taxa. The genus Crocus L. (Iridaceae) comprises about 100 species that are distributed mainly in the Mediterranean region and western Asia. The present study purposed to assess the distribution patterns of Iranian Crocus species (including C. sativus L. and 8 wild species) and their conservation status in Iran as one of diversity centers of Crocus in the Middle East. A set of geographic distribution data was compiled through field studies, and reviews of herbarium specimens, iNaturalist, and various Flora. Localities were marked on geo-referenced maps (1/106) of Iran using ArcView version 3.2 (Esri, 2000). The distribution patterns of the taxa were mapped per 0.25° × 0.25° universal transverse Mercator grid cells (25 km2 with the exception of boundary area). Based on the species distribution (SDI) (33%) and species specialization (SSI) (44%) indices, those Iranian Crocus species that are threatened are mainly distributed in Irano-Turanian region. The mountainous ecosystems of Almeh and Western Alborz are important distribution centers of these taxa. Iranian Crocus species with SSI &lt; 0.5 (C. almehensis, C. gilanicus, C. michelsonii, and C. caspius) have the highest conservation value.

The Evaluations and Comparisons of Nuclear and Chloroplast DNA Regions Based on Species Identification and Phylogenetic Relationships of Crocus L. Taxa

The genus Crocus L. (Iridaceae) consists of about 200 species distributed from western Europe and north west Africa to western China. The genus is taxonomically very problematic and day after day it becomes more difficult to understand the relationships between Crocus species because of the identification of new species. Recently, the studies based on DNA sequences are frequently used for the solution of taxonomic problems, the determination of species diversity and especially the evaluation of phylogenetic relationships among the studied taxa. The use of the regions belonging to nuclear DNA and especially chloroplast genome and the combinations of these regions are frequently recommended in many studies. However, there is no information about suitable barcoding regions that can be used for all plant groups. This study focuses on determination of the most succesful barcoding regions, understanding the species discrimination abilities and finally evaluation of phylogenetic relationships among the Crocus taxa using one region from nuclear DNA (ITS1-5.8S rRNA gene-ITS2) and four regions from chloroplast DNA (1. trnK-matK IGS, 2. partial psbA gene-psbA/trnH IGS-partial trnH gene, 3. rpoC1 gene, 4. partial trnL gene-trnL/trnF IGS-partial trnF gene). Sequence informations for related DNA regions were obtained from NCBI. As a result, it can be stated that the region which is only covering ITS1-5.8S rRNA gene-ITS2 separated the all taxa in species level and it is more beneficial for the phylogenetically evaluation of Crocus taxa. Furthermore, dendrogram show taxonomic problems belonging to some species in the genus such as C. biflorus taxa.

Ongoing Evolution in the Genus Crocus: Diversity of Flowering Strategies on the Way to Hysteranthy.

Species of the genus Crocus are found over a wide range of climatic areas. In natural habitats, these geophytes diverge in the flowering strategies. This variability was assessed by analyzing the flowering traits of the Spanish collection of wild crocuses, preserved in the Bank of Plant Germplasm of Cuenca. Plants of the seven Spanish species were analyzed both in their natural environments (58 native populations) and in common garden experiments (112 accessions). Differences among species observed in the native habitats were maintained under uniform environmental conditions, suggesting a genetic basis for flowering mechanisms. Two eco-morphological types, autumn- and spring-flowering species, share similar patterns of floral induction and differentiation period in summer. The optimal temperature for this process was 23 °C for both types. Unlike Irano-Turanian crocuses, spring-flowering Spanish species do not require low winter temperatures for flower elongation. Hysteranthous crocuses flower in autumn prior to leaf elongation. We conclude that the variability in flowering traits in crocuses is related to the genetic and environmental regulation of flower primordia differentiation and elongation prior to emergence above the soil surface. The elucidation of the physiological differences between eco-morphological types of crocuses: synanthous with cold requirements and synanthous and hysteranthous without cold requirements, unlocks a new approach to the flowering evolution of geophytes in Mediterranean regions. Crocus species can serve both as a new model in the study of the molecular basis of hysteranthy and for the purposes of developing the molecular markers for desirable flowering traits.

A robust workflow for indirect somatic embryogenesis and cormlet production in saffron (Crocus sativus L.) and its wild allies; C. caspius and C. speciosus

Saffron (Crocus sativus L.) and its wild relatives, Crocus caspius and Crocus speciosus are of considerable significance in the pharmaceutical, nutraceutical, and ornamental bulbs industry. Towards the ultimate goal of the conservation of wild Crocus species and establishment of an efficient workflow for in vitro production of Crocuses, efficient protocols were developed for disinfection and in vitro production of cormlets in C. sativus and its wild allies C. caspius and C. speciosus. Moreover, the differential expression of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene was evaluated as a potential molecular marker during embryogenesis between embryogenic and non-embryogenic calli. A highly efficient disinfection recipe and a low-cost TDZ-free protocol have been successfully developed for in vitro cormlet production in three Crocus species. MS medium containing 10.18 μM 2, 4-D + 4.44 μM BAP was most efficiently induced callus and somatic embryo formation. The highest conversion frequency and maximum cormlet weight were achieved in MS containing 5.37 μM NAA + 8.88 μM BAP. The SERK expression was significantly much higher in embryogenic calli than non-embryogenic in all Crocus species. The current low-cost and easy-to-use recipe suggests a promising in vitro propagation workflow for mass production of uniform pathogen-free cormlets of Crocus species, as well as a platform to better conservation of wild Crocus species and effective gene and genome editing using CRISPR-Cas9 in future studies.

Evolutionary and phylogenic relationships of wild and crop species of Iranian Saffron by DNA barcoding

DNA barcoding method was applied to identify and study the phylogenic relationships existing between 13 species of saffron collected in Iran including 4 crop and 9 wild species. PCR amplifications were performed using primers designed on the nucleotide sequence of three plastid barcode genes, comprising two protein encoding genes (rbcL and matK) and an intragenic spacer (trnH-psbA), and a nuclear region (ITS). A total of 52 sequences were obtained and registered in NCBI database. In particular, 21 of these sequences were not present in the scientific library. Nucleotide polymoprhic sites were counted for each barcode gene (rbcL, n = 16; matK, n = 15; trnH-psbA, n = 46; ITS, n = 71). Each sample could be distinguished from the others in the phylogenic trees developed based on the data obtained by single barcode gene. In addition, a phylogenic tree based only on plastid information (trnH-psbA + rbcL + matK) and another created on the data resulting from both nuclear and plastid genomes (trnH-psbA + rbcL + matK + ITS) was also generated. In general, ITS sequence, indicating high resolution at the genus and species level, appeared as the best barcode sequence of the present study. Phylogenic analysis demonstrated the genetic relationship between crop saffron and wild Crocus species. According to the results of this study, among 13 available sample, the wild species are Crocus cancellatus L. and Crocus sp. Eslamabad were hypothesized as the closest species to the Iranian saffron. The present investigation also indicated that the different ecotypes of C. sativus L. may have evolved through independent events probably due to geographic and environmental pressures.

Flowering Persistence and Pollinator Attraction of Early-spring Bulbs in Warm-season Lawns

Early-spring flowering bulbs can increase biodiversity while adding color to lawns and other grassy areas. However, few studies have investigated whether bulbs can flower and persist in warm-season lawns or provide feeding habitat for pollinating insects. Thirty early-spring flowering bulbs, including species of Anemone, Chionodoxa, Crocus, Eranthis, Hyacinthus, Ipheion, Iris, Leucojum, Muscari, and Narcissus, were established in bermudagrass (Cynodon dactylon L. Pers) and buffalograss [Buchloe dactyloides (Nutt.) J.T. Columbus] lawns in late autumn 2015 in Fayetteville AR. Bulbs were assessed over three growing seasons for flowering characteristics, persistence, and their ability to attract pollinating insects. A growing degree day model was also developed to predict peak flowering times in our region. Numerous bulb entries produced abundant flowers in bermudagrass and buffalograss lawns in the first year after planting, but persistence and flower production were reduced in both the second and third years of the trial. Five bulbs persisted for multiple years in both turfgrass species and continued to produce flowers, including Crocus flavus Weston ‘Golden Yellow’ (crocus), Leucojum aestivum L. (spring snowflake), Narcissus (daffodil) ‘Baby Moon’, Narcissus ‘Rip Van Winkle’, and Narcissus ‘Tete-a-Tete’. Several bulbs, primarily crocuses and Muscari spp. (grape hyacinth), were also observed to attract pollinating insects, principally honey bees (Apis mellifera). These results demonstrate that some early-spring bulbs can persist in competitive warm-season turfgrasses, while providing pollinator forage, but species and cultivar selection is critical for long-term success.