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     

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.     

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.     

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.     

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.     

A glycoconjugate from corms of saffron plant (Crocus sativus L.) inhibits root growth and affects in vitro cell viability

A glycoconjugate has been characterized from saffron corms (Crocus sativus L.) that inhibits the growth of roots of Nicotiana tabacum and Arabidopsis thaliana, at concentrations ranging from 1-100 micrograms m-3. Roots of seedlings grown in the presence 0.1 microgram m-3 glycoconjugate showed bulging of epidermal cells, whereas at 10 micrograms m-3, roots were completely devoid of hairs. At 100 micrograms m-3 glycoconjugate the cell walls of the root vascular tissues were thicker and, overall, the vascular tissue was enlarged. In addition, this glycan is cytotoxic to isolated tobacco cells and protoplasts, with 50% cell death induced by 0.5 and 2 micrograms m-3 glycoconjugate, respectively. Morphological and biochemical changes induced by the exposure to the glycoconjugate included cell size decrease, loss of regular cell shape, cytoplasm collapse, and release of intracellular proteins. This molecule at low concentrations (0.1 microgram m-3) mimics the effects of Yariv phenylglycosides and of mutant Arabidopsis which present defective or missing arabinogalactan-proteins (AGPs) in roots, indicating the glycoconjugate might interact with cell surface AGPs.     

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.     

Dehydration and viability of saffron crocus (Crocus sativus)

Saffron pollen reaches the shedding stage with a high water content which averages 58% under 18-20°C and RH 55-70%. Because of the precocious incision of anther stomium and the apertures of pollen exine, when the flower opens the pollen is soon exposed to dehydration. As a result, it goes from the highly hydrated stage to 15% water within 2 h from flower opening, and to the minimum 7% within 10 h. The transition to dry state results in a progressive loss of the plasmamembrane integrity, but this is restored after gradual rehydration. On average, 50% of the total pollen grains shows capacity for semi-vivo germination regardless of the dehydration degree. Pollen loses the competence for germination within 3 weeks from shedding. The results are discussed in comparison with fertile Crocus species.     

The antileukemic effects of saffron (Crocus sativus L.) and its related molecular targets: A mini review

Saffron (Crocus sativus L.), and its main constituents, crocin, and crocetin have shown promising effects as an antileukemic agent in animal models and cell culture systems. Saffron retards the growth of cancer cells via inhibiting nucleic acid synthesis and enhancing antioxidative system. It can induce apoptosis and chemosensitivity via inhibiting multidrug resistance proteins. Saffron also induces differentiation pathways via inhibiting promyelocytic leukemia/retinoic acid receptor-α, histone deacetylase1, and tyrosyl DNA phosphodiesterase-1 as well. The present review highlights the most recent findings on the antileukemic effects of saffron and its underlying molecular targets. The emerging evidence suggests that saffron has a selective toxicity effect against leukemic cells while is safe for the normal cells.     

Catalytic properties of three L-lactate dehydrogenases from saffron corms (Crocus sativus L)

Three L-lactate dehydrogenase isoenzymes were detected in saffron corms, using potassium ferricyanide as the electron acceptor. Their pH optima were 5.5, 7.5 and 9.5, respectively. All three dehydrogenases were substrate-inhibited by ferricyanide, but at different concentrations; maximum enzymatic activity was observed for 250, 100 and 600 M ferricyanide, at pH 5.5, 7.5 and 9.5, respectively. Catalytic efficiency, calculated per mg corm extract protein, was 1.9, 1.0 and 0.4 min^-1, respectively at pH 5.5, 7.5 and 9.5. Pseudo first order rate constant was also different under the three pH conditions. Malate was an inhibitor for the isoenzyme active at pH 9.5, but had no effect on the others.     

Effect of polymer/nanosilver composite packaging on long-term microbiological status of Iranian saffron (Crocus sativus L.)

Crocus sativus L. (saffron) is a valuable plant which is native to Iran. Saffron is the dried stigmata of the flowering part of the plant that is usually contaminated with different bacteria and fungi through production process. Antimicrobial properties of silver nanoparticles are well recognized. To survey the effects of nanosilver packaging on microbiological status of spiked, saffron samples over a six month period were chosen. Saffron samples from five regions of Khorasan province were purchased and de novo frequencies of microbial contaminants were determined using standard procedures. Totally 35 g of saffron was spiked with known numbers of four bacterial and two fungal species and packaged into one gram packets. The packaging materials consisted of polyethylene polymers containing 0, 400, 800, 1200 or 4000 ppm nanosilver (as Ag). Total and differential numbers of spiked microorganisms in the packaged saffrons were enumerated at initial and at six time points of seven, 14, 28, 64, 90 and 180 days. Baird-Parker agar (BP agar), Kenner Fecal (KF), Salmonella–Shigella agar (SS agar), Violet Red Bile Glucose Agar (VRBGA), and Sabouraud Dextrose agar (SD agar) media were used for enumeration of the six spiked microorganisms including Staphylococcus aureus, Enterococcus faecalis, Salmonella Enteritidis, Enterobacter species and Escherichia coli, Fusarium oxysporum and Aspergillus flavus, respectively. Direct antibacterial activity of the composites was also determined. De novo frequencies of microorganisms in five saffron samples were at acceptable levels with dominance of fungi species. Nanosilver embedded packages accelerated the reduction in live microbial numbers in saffron samples and the efficacy was the best in packages containing 4000 ppm nanosilver particles. Nanosilver packaging can significantly reduce microbial burden of saffron.     

Production of a cytotoxic proteoglycan using callus culture of saffron corms (Crocus sativus L.).

Saffron corms have been demonstrated to contain a proteoglycan that inhibits growth of human tumor cells. In this study, we show strong evidence indicating that callus cultures of saffron corm also synthesize such glycoconjugate. This compound is cytotoxic against human cervical epithelioid carcinoma cells (IC50 = 7 mg ml-1), and consists of approximately 90% carbohydrate and 10% protein. In order to optimize the glycoconjugate purification to complete functional and therapeutic studies, we have designed an improved method consisting of anion-exchange chromatography and reversed-phase HPLC, which can be easily scaled-up.     

Intestinal formation of trans-crocetin from saffron extract (Crocus sativus L.) and in vitro permeation through intestinal and blood brain barrier

Aims: Extracts of saffron (Crocus sativus L.) have traditionally been used against depressions. Recent preclinical and clinical investigations have rationalized this traditional use. Trans-crocetin, a saffron metabolite originating from the crocin apocarotenoids, has been shown to exert strong NMDA receptor affinity and is thought to be responsible for the CNS activity of saffron. Pharmacokinetic properties of the main constituents from saffron have only been described to a limited extent. Therefore the present in vitro study aimed to determine if crocin-1 and trans-crocetin are able to pass the intestinal barrier and to penetrate the blood brain barrier (BBB). Additionally, the intestinal conversion of glycosylated crocins to the lipophilic crocetin had to be investigated. Experiments with Caco-2 cells and two different porcine BBB systems were conducted. Further on, potential intestinal metabolism of saffron extract was investigated by ex vivo experiments with murine intestine.Methodology: In vitro Caco-2 monolayer cell culture was used for investigation of intestinal permeation of crocin-1 and trans-crocetin. In vitro models of porcine brain capillary endothelial cells (BCEC) and blood cerebrospinal fluid barrier (BCSFB) were used for monitoring permeation characteristics of trans-crocetin through the blood brain barrier (BBB). Intestine tissue and feces homogenates from mice served for metabolism experiments.Results: Crocin-1, even at high concentrations (1000 µM) does not penetrate Caco-2 monolayers in relevant amounts. In contrast, trans-crocetin permeates in a concentration-independent manner (10–114 µM) the intestinal barrier by transcellular passage with about 32% of the substrate being transported within 2 h and a permeation coefficient of P_app 25.7 × 10⁻⁶ ± 6.23 × 10⁻⁶ cm/s. Trans-crocetin serves as substrate for pGP efflux pump. Trans-crocetin permeates BBB with a slow but constant velocity over a 29 h period (BCEC system: P_app 1.48 × 10⁻⁶ ± 0.12 × 10⁻⁶ cm/s; BCSFB system P_app 3.85 × 10⁻⁶ ± 0.21 × 10⁻⁶ cm/s). Conversion of glycosylated crocins from saffron extract to trans-crocetin occurs mainly by intestinal cells, rather than by microbiological fermentation in the colon.Conclusion: The here described in vitro studies have shown that crocins from saffron are probably not bioavailable in the systemic compartment after oral application. On the other side the investigations clearly have pointed out that crocins get hydrolyzed in the intestine to the deglycosylated trans-crocetin, which subsequently is absorbed by passive transcellular diffusion to a high extend and within a short time interval over the intestinal barrier. Crocetin will penetrate in a quite slow process the blood brain barrier to reach the CNS. The intestinal deglycosylation of different crocins in the intestine is mainly due to enzymatic processes in the epithelial cells and only to a very minor extent due to deglycosylation by the fecal microbiome. On the other side the fecal bacteria degrade the apocarotenoid backbone to smaller alkyl units, which do not show any more the typical UV absorbance of crocins. As previous studies have shown strong NMDA receptor affinity and channel opening activity of trans-crocetin the use of saffron for CNS disorders seems to be justified from the pharmacokinetic and pharmacodynamic background.     

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.     

Variation in Saffron (Crocus sativus L.) accessions and Crocus wild species by RAPD analysis

Crocus, a genus of Iridaceae is mostly grown in areas with Mediterranean environment as the best region. Saffron (C. sativus) is a perennial plant and is cultivated as an industrial crop in several regions of Iran. In this research, five accessions of cultivated Saffron from five areas in Khorasan, and Esfahan including Gonabad, Ferdows, Torbat-e-Heyidariye, Estahbanat, Gopayegan were used. Other nine species of saffron were grown naturally in Iran; so we collected two wild species (C. caspius and C. speciosus) from north of Iran (Gilan Province). RAPD markers were used to classify these species and to find their relationships. In the results of this study, the cluster analysis showed two distinct groups. Also, the maximum similarity was seen between C. caspius and C. speciosus (0.82) and the minimum was between Estahbanat, Ferdows accessions and C. speciosus (0.33). Finally, this method as a convenience procedure could be used to separate different accessions and species of Crocus as well.     

离体培养藏红花细胞合成的藏红花色素稳定性

研究pH、温度、光照、氧化剂、还原剂、食品添加剂和金属离子对离体培养的藏红花细胞合成的藏红花色素稳定性的影响,并与藏红花柱头色素进行比较。结果表明:藏红花细胞合成的色素与柱头色素均为水溶性色素,呈亮黄色,对食品添加剂(食盐、柠檬酸、蔗糖、苯甲酸钠)和H2O2表现出良好的稳定性。与柱头色素相比,藏红花细胞合成的色素对强酸(pH1)、高温(80、100℃)和光照(日光灯光、紫外光)表现出更好的稳定性,对VC和Cu2+(0.001mol/L)的稳定性略差。稀碱溶液(0.47mol/LNa2CO3、0.1mol/LNaOH)、Na2SO3、Fe2+(0.050mol/L)和Fe3+能使细胞合成的色素色强增强。     

Evaluation of Crocus sativus L. (saffron) on male erectile dysfunction: A pilot study

In this study, the effect of Crocus sativus (saffron) was studied on male erectile dysfunction (ED). Twenty male patients with ED were followed for ten days in which each morning they took a tablet containing 200 mg of saffron. Patients underwent the nocturnal penile tumescence (NPT) test and the international index of erectile function questionnaire (IIEF-15) at the start of the treatment and at the end of the ten days. After the ten days of taking saffron there was a statistically significant improvement in tip rigidity and tip tumescence as well as base rigidity and base tumescence. ILEF-15 total scores were significantly higher in patients after saffron treatment (before treatment 22.15±1.44; after treatment 39.20±1.90, p<0.001). Saffron showed a positive effect on sexual function with increased number and duration of erectile events seen in patients with ED even only after taking it for ten days.