AFLP and MS-AFLP Analysis of the Variation within Saffron Crocus (Crocus sativus L.) Germplasm

The presence and extent of genetic variation in saffron crocus are still debated, as testified by several contradictory articles providing contrasting results about the monomorphism or less of the species. Remarkably, phenotypic variations have been frequently observed in the field, such variations are usually unstable and can change from one growing season to another. Considering that gene expression can be influenced both by genetic and epigenetic changes, epigenetics could be a plausible cause of the alternative phenotypes. In order to obtain new insights into this issue, we carried out a molecular marker analysis of 112 accessions from the World Saffron and Crocus Collection. The accessions were grown for at least three years in the same open field conditions. The same samples were analysed using Amplified Fragment Length Polymorphism (AFLP) and Methyl Sensitive AFLP in order to search for variation at the genetic (DNA sequence) and epigenetic (cytosine methylation) level. While the genetic variability was low (4.23% polymorphic peaks and twelve (12) effective different genotypes), the methyl sensitive analysis showed the presence of high epigenetic variability (33.57% polymorphic peaks and twenty eight (28) different effective epigenotypes). The pattern obtained by Factorial Correspondence Analysis of AFLP and, in particular, of MS-AFLP data was consistent with the geographical provenance of the accessions. Very interestingly, by focusing on Spanish accessions, it was observed that the distribution of the accessions in the Factorial Correspondence Analysis is not random but tends to reflect the geographical origin. Two clearly defined clusters grouping accessions from the West (Toledo and Ciudad Real) and accessions from the East (Cuenca and Teruel) were clearly recognised.     

西红花FT同源基因的表达及功能分析

FT（FLOWERING LOCUS T）及其同源基因作为三大开花途径整合子之一,被认为是调控植物开花的重要基因。为了深入研究FT同源基因的功能以及西红花（Crocus sativus L.）开花的分子机理,对已报道的3个西红花FT同源基因（CsatFT1、CsatFT2和CsatFT3）进行分离及分析。gDNA包含长度分别为835、1 642和1 132 bp的完整开放阅读框（ORF）,均具有4个外显子和3个内含子;cDNA包含长度分别为528、525和540 bp的ORF,分别编码175、174和179个氨基酸;系统进化分析表明,CsatFT1、CsatFT2、CsatFT3分别和同为单子叶植物的水仙（Narcissus chinensis）NtFT、麝香百合（Lilium longiflorum）LlFT和洋葱（Allium cepa）AcFT1表现出较近的遗传距离。qRT-PCR分析结果显示,小球茎膨大阶段前期,CsatFT1、CsatFT2、CsatFT3在叶片中表达水平最高,侧根中次之,子球茎、主根中极低几乎检测不到;小球茎膨大阶段后期,CsatFT1、CsatFT2、CsatFT3都在子球茎中表达水平较高,在顶芽中几乎检测不到;室内储藏开花阶段,CsatFT1、CsatFT2、CsatFT3在柱头中表达水平最高,叶中次之,花瓣和花药中较低几乎检测不到。通过观测转基因烟草（Nicotiana tabacum）和转基因拟南芥（Arabidopsis thaliana）植株表型发现,CsatFT1,CsatFT2和CsatFT3均具有促进植物提早开花的功能。     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

RAPD Analysis in Crocus sativus L. Accessions and Related Crocus Species

In the present paper a Random Amplified Polymorphic DNA (RAPD) investigation was carried out on DNAs from five Crocus sativus L. (saffron) accessions cultivated in different countries and on six closely related Crocus species. Aims of the study are to check whether cultivated saffron has maintained a constant genomic organisation and to clarify its relationships with possible ancestor species. For the fifteen primers, which produced positive results, DNAs of saffron corms from different accessions present the same amplification pattern, in accordance with the similar DNA content and base composition pointed out in previous studies. The amplification of the seven Crocus species DNAs with twenty-one primers provided 217 repeatable and interpretable fragments, which were scored for presence/absence and employed for a cluster analysis. Results indicated that C. sativus is very closely related to C. cartwrightianus and also similar to C. thomasii. This result, concurring with part of the previous evidence, would rule out the hypothesis of close relationships between C. sativus and C. pallasii.     

Effect of Irrigation Water Salinity and Deficit Irrigation on Soil Ions Variation and Uptake by Saffron (Crocus sativus L.) Under Two Planting Methods

Journal of Plant Growth Regulation - A 2-years field experiment in a split-split plot arrangement was considered to investigate the effects of different irrigation water salinity [0.45 (well water,...     

Saffron (Crocus sativus L.) and health outcomes: a meta-research review of meta-analyses and an evidence mapping study

BackgroundAlthough a number of systematic reviews and meta-analyses of saffron (Crocus sativus L.) have been published, no study has comprehensively summarized the clinical evidence from meta-analyses, or assessed the reporting or methodological quality of these reviews.PurposeThe present meta-research study was designed to fill the gaps in knowledge to inform future studies and allow enhanced clinical decision-making on saffron.MethodsThe PubMed, Cochrane Library, Embase, and CNKI databases were systematically searched from inception to April 3 rd, 2021, for meta-analyses of clinical trials that assessed the efficacy and safety of saffron. PRISMA 2009 and AMSTAR-2 were employed to assess the reporting and methodological quality of meta-analyses identified in the search, respectively. The present study was registered on PROSPERO with registration number CRD42020220274.ResultsNineteen eligible systematic reviews with meta-analyses published in English were identified from 235 records. These meta-analyses were published in 12 peer-reviewed journals from 2013 to 2021. The heterogeneous results indicated that saffron significantly reduced fasting blood glucose, waist circumference, diastolic blood pressure, concentrations of total cholesterol and low-density lipoprotein cholesterol, and improved symptoms of depression, cognitive function and sexual dysfunction compared with controls (mainly placebos). Common side effects of saffron consumption included nausea, dry mouth, poor appetite, and headache, but no serious adverse reactions were reported. Primary analysis and sensitivity analysis confirmed that the reporting and methodological quality of reviews included in the study were highly correlated (p < 0.001). The quality of meta-analyses of saffron requires improvement by including a structured abstract, a prospective protocol and registration, explanation of the study designs within each study that is reviewed, the searches, risk of bias assessment, literature selection, and reporting of funding sources.ConclusionThe available evidence indicates that saffron is a safe plant for administration as a medicine and can improve diverse clinical outcomes, but the scientific quality of the published systematic reviews needs to be improved. Moreover, the clinical effects of saffron need to be confirmed through high-quality randomized trials in multiple countries with large sample sizes.     

Improved Al tolerance of saffron (Crocus sativus L.) by exogenous polyamines

The aluminum (Al) tolerance of saffron (Crocus sativus L.) in hydroponics and the method of improving Al tolerance were investigated. Compared with the Al-free control, saffron root elongation was decreased by 59.3 and 75% at 0.05 and 0.2 mM Al stress, respectively. At 0.5 mM Al stress, the root elongation was inhibited completely. Addition of 1 mM polyamines improved saffron root growth markedly at 0.2 mM Al stress. Putrescine (Put) showed better amelioration effect than spermidine (Spd) and spermine (Spm). The root elongation in Put treatment was only 15% lower than that of Al-free control. The alleviation of Al rhizotoxicity by polyamines might be attributed to lower Al content in the root tips, and subsequent less lipid peroxidation and oxidative stress. Higher activities of amine oxidases and Hydrogen peroxide (H₂O₂) content might decrease the effects of Spd and Spm on alleviating oxidative damage compared with that of Put.     

In vitro proliferation of saffron (Crocus sativus L.) stigma

Excised young intact stigmas plus ovaries of Crocus sativus L. were cultured on Linsmaier-Skoog media supplemented with either a cytokinin or an auxin alone or in combinations. Benzyladenine and kinetin at concentrations of 0.1, 1, and 5 mgl^-1 supported growth, and crocin was biosynthesized in the stigmas in vitro. Auxins had little effect. Young excised single stigmas or half ovaries were also cultured so as to form stigma-like structures in order to explore a possible new approach to industrial production of the spice, saffron. On Linsmaier-Skoog and Nitsch media supplemented with kinetin at concentration of 1 or 5 mgl^-1 and alpha-naphthalene acetic acid or indole-butyric acid at concentration of 0.1 or 10 mgl^-1 in combinations, stigma-like structures appeared directly and indirectly (through meristematic tissue), grew and matured. The maximum number of structures were 75 per half ovary. Three kinds of yellow pigments including crocin were tentatively identified by TLC in the stigma-like structures as was the case for the in vivo grown natural stigma, although the contents were lower.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-acetic acid - IBA indole-butyric acid - NAA alpha-naphthalene-acetic acid - TLC thin layer chromatography     

Comparative evaluation of extraction methods for total proteins from Crocus sativus L. (Saffron)

Broadly speaking proteomic studies are one of the various techniques of utmost importance for understanding complex biological processes that occur under inductive conditions and revealing the multidimensional aspects of Crocus sativus in biological systems. In order to get an insight into the molecular changes and to characterize the variations in protein expression of C. sativus, a detailed proteomic analysis on one-dimensional gel electrophoresis is one of the basic steps to accomplish. We have compared total protein profiles of C. sativus extracted by three different recipes and analyzed on 10% sodium dodecyl sulfate polyacrylamide gels. Gels were subjected to densitometric analysis for further characterization. Among three different protocols NP-40 extraction buffer recipe resulted in the extraction of proteins most efficiently with minimum background and streaking. There was maximum solubilization of proteins with high efficiency. Such a profile can be used for high precision analysis of differential protein expression. This work is an attempt to assist researchers in effective extraction of proteins from C. sativus. As a researcher faces a perplexing array of choices as where to start we describe a method based on our collective analysis of the different protein protocols. This paper presents a method that could be applied at the outset of any proteomic study.     

Comparative expression analysis of senescence gene CsNAP and B-class floral development gene CsAP3 during different stages of flower development in Saffron (Crocus sativus L.)

Physiology and Molecular Biology of Plants - Crocus sativus, a monocot triploid species belonging to the Iridaceae family, is cultivated for its red stigmatic lobes of the carpel that constitute...     

In vitro corm production in the saffron crocus (Crocus sativus L.)

Means to increase the reproductive capacity of Crocus sativus L., in vitro, are described. Cytokinins and auxin were found to be essential for development of bud explants. Ethylene and ethaphon pretreatments inhibited leaf development but induced corm production. Microsurgery of the apical bud combined with ethylene pretreatment increased both sprouting and corm production.     

Cloning and Characterization of FLOWERING LOCUS T-Like Genes from the Perennial Geophyte Saffron Crocus (Crocus sativus)

The transition to flowering is one of the most important developmental decisions made by plants. At the molecular level, many genes coordinate this transition. Among these, genes encoding for phosphatidylethanolamine-binding proteins (PEBPs) play important roles in regulating flower time and the fate of inflorescence meristem. To investigate the role of PEBPs in an industrially important crop cultivated for its nutritional and medicinal properties, the monocotyledonous species Crocus sativus L., we have isolated three FLOWERING LOCUS T (FT)-like genes designated as CsatFT1-like, CsatFT2-like, and CsatFT3-like. The isolated genes maintain the exon/intron organization of FT-like genes and encode proteins similar to the members of the PEBP family. Phylogenetic and amino acid analysis at critical positions confirmed that the isolated sequence belongs to the FT clade of the PEBP family phylogeny distinctly from the TERMINAL FLOWER 1 (TFL1) and MOTHER OF FT AND TFL1 clades. Expression analysis indicated differences in the expression of the three FT-like genes in different organs and different expressions during the day–night diurnal clock. Additionally, analysis of isolated promoter sequences using computational methods reveals the preservation of common binding motifs in FT-like promoters from other species, thus suggesting their importance among plant species.     

Thermal stability of catalases active in dormant saffron (Crocus sativus L.) corms

Catalase activity was detected in crude extract prepared from dormant saffron (Crocus sativus L.) corms. The activity was independent of pH in the range 6.0 – 11.0. Thermostability studies suggested the presence of three isoenzymes with transition temperatures of 30°C, 45°C and 60°C, respectively, as given by Arrhenius plots. When stained for catalase activity gel electropherograms of extract revealed 3 distinct bands with apparent molecular weight of 323,000, 295,000 and 268,000, respectively. Thus it appeared that at least three isoenzymes of catalase were present in dormant saffron corms.     

Profile of polyamines during sprouting and growth of saffron (Crocus sativus L.) Corms

The profile of free and conjugated polyamines putrescine, spermidine, and spermine was studied at the onset of sprouting and during various stages of vegetative growth in saffron (Crocus sativus L.) corms. Polyamines were extracted from the shoot meristems and estimated by high performance liquid chromatography. Free putrescine was not detected at the onset of sprouting, whereas free spermidine and spermine levels increased rapidly on sprouting and decreased during further stages of corm development. The levels of conjugated polyamines were several times higher than the free forms indicating their possible role in the developmental processes. A comparison of polyamine levels of vegetative and floral corms showed higher titers of free polyamines in vegetative and conjugated polyamines in floral corms.     

In vitro development of microcorms and stigma like structures in saffron (Crocus sativus L.)

Saffron is an important spice derived from the stigmas of Crocus sativus, a species belonging to the family Iridaceae. Due to its triploid nature it is sterile and is not able to set seeds, so it is propagated only by corms. The natural propagation rate of most geophytes including saffron is relatively low. An in vitro multiplication technique like micropropagation has been used for the propagation of saffron. In the present study, various explants were cultured on different nutrient media supplemented with various concentrations of plant growth regulators to standardize the best media combination for obtaining optimum response with respect to corm production and development of Stigma Like Structures (SLS). Highest response (60 %) was observed with half ovaries on G-5 media supplemented with 27 μM NAA and 44.4 μM BA followed by 55 % on LS media with 27 μM NAA and 44.4 μM BA. Maximum size (1.3 g) of microcorms were obtained from apical buds on the LS media supplemented with 21.6 μM NAA and 22.2 μM. Stigma Like Structures were developed from half ovary explants both directly and indirectly. Maximum number (120 indirectly and 20 directly) and size (5.2 cm) of SLS were obtained in G-5 medium supplemented with 27 μM NAA and 44.4 μM BA followed by 100 indirectly and 20 directly and 4.5 cm long on LS medium supplemented with 27 μM NAA and 44.4 μM BA.     

Morphogenic Potential of Cultured Floral Explants of Crocus sativus L. for the In Vitro Production of Saffron

Explants of immature ovaries, stigmas, anthers and petals ofCrocus sativus were cultured on White's media supplemented witheither NAA and zeatin or 2,4-D and BAP in various combinations.The formation of stigma-like structures occurred on the explantsor on the callus derived from the explants, but this was onlyobserved when NAA and zeatin were used. Formation of stigma-likestructures were observed on anthers, petals, stigmas and half-ovaries,with the best result being obtained on explants consisting ofhalf-ovaries cultured on medium containing NAA at 40 mg dm^–3and zeatin at 4.0 mg dm^–3. These stigmas developed anintense orange pigment and grew to 3.0 cm in length and hada strong saffron aroma. A preliminary comparison using thinlayer chromatography of the yellow pigments produced by thestigma-like structures grown in vitro and those grown naturallyshowed the pigment composition to be similar. Key words: Crocus sativus L., explants, NAA, zeatin, saffron     

Embryo origin and development in Crocus sativus L. (Iridaceae)

Abstract

Fruit and seed set were obtained in the triploid Crocus sativus (saffron) either by hand crossing with pollen of the diploid C. cartwrightianus or by free pollination. The morphology of the capsules and seeds was similar in both Crocus species. The embryo is comparatively small, whereas the endosperm is formed of numerous layers of cells with hemicellulose-thickened walls and a cytoplasm containing few lipid and protein reserves. Seed germination gave rise to a white prophyll from which a green leaf emerged. At the base of this leaf, a small corm without tunics developed, which, after leaf desiccation, became dormant. These corms sown separately in pots were again capable of growth and emitted a single long green leaf. Vegetative development of new corms was similar in the diploid and hybrid saffron plants. In sites of past saffron cultivation the beds were found to contain degenerated corms and corms at different development stages. The absence of fruit and seed set in emasculated and self- and out-pollinated plants suggests that saffron does not form apomictic embryos.     

Genetic Diversity of Radish (Raphanus sativus L.) Germplasm Resources Revealed by AFLP and RAPD Markers

Genetic diversity of 56 radish accessions, representing nearly all the typical types and origins of cultivated radish germplasms conserved in the National Mid-term Genebank for Vegetables of China, was assessed with amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers. A total of 72 and 128 polymorphic bands were generated by the 12 selected RAPD primers and eight AFLP primer combinations respectively. A moderate correlation with the value of r = 0.66 was observed between AFLP and RAPD markers. The total 200 polymorphic bands were integrated to assess the genetic diversity of 56 radish accessions. The Jaccard similarity coefficients between the accessions varied from 0.30 to 0.83 with the mean of 0.54. Cluster analysis classified the germplasms into three groups of var. hortensis Becker, var. sativus, and var. niger Kerner. The three-dimensions scatter plot of principle coordinate analysis (PCA) further divided var. hortensis Becker germplasms into two separate groups. The results indicated that the genetic diversity harbored among var. hortensis Becker germplasms was very abundant, which could be further exploited for radish genetic improvement.     

Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.)

Background

Somatic embryogenesis (SE) is a complex biological process that occurs under inductive conditions and causes fully differentiated cells to be reprogrammed to an embryo like state. In order to get a better insight about molecular basis of the SE in Crocus sativus L. and to characterize differentially accumulated proteins during the process, a proteomic study based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry has been carried out.

Results

We have compared proteome profiles of non-embryogenic and embryogenic calli with native corm explants. Total soluble proteins were phenol-extracted and loaded on 18 cm IPG strips for the first dimension and 11.5% sodium dodecyl sulfate-polyacrylamide gels for the second dimension. Fifty spots with more than 1.5-fold change in abundance were subjected to mass spectrometry analysis for further characterization. Among them 36 proteins could be identified, which are classified into defense and stress response, protein synthesis and processing, carbohydrate and energy metabolism, secondary metabolism, and nitrogen metabolism.

Conclusion

Our results showed that diverse cellular and molecular processes were affected during somatic to embryogenic transition. Differential proteomic analysis suggests a key role for ascorbate metabolism during early stage of SE, and points to the possible role of ascorbate-glutathione cycle in establishing somatic embryos.