https://orcid.org/0000-0003-4115-1932
Figures
Abstract
Saffron (Crocus sativus L.) has held significant cultural and medicinal value since the Greek-Minoan civilization. As a triploid spice with vegetative propagation from the Iridaceae family, the three-branch style of C. sativus flowers, known as saffron, constitutes the most economically valuable part of the plant, renowned for its diverse medicinal properties. This study explores the in vitro induction of stigma-like structures (SLSs) from various explants of the Ghaen ecotype flower. The study found that the optimal sampling time for the majority of explants was the third week of October. Ovary explants exhibiting a prolonged response to hormonal treatments for the production of SLSs. Furthermore, intact, and injury ovary explants were found to be the most effective explant types for inducing SLSs. The explants were cultured on MS, 1/2MS, LS and B5 basal media supplemented with various combinations and concentrations of plant growth regulators. The results indicated that the B5 medium, enriched with 5–10 mg/ L BAP and 5–10 mg/ L NAA was the most effective treatment for inducing SLSs in all types of explants. Quantitative and qualitative analyses of saffron compounds in SLSs indicated similarities with natural saffron, albeit at significant lower concentrations: crocin (up to 10.2 mg/g), picrocrocin (up to 4.8 mg/g), and safranal (up to 9.7 mg/g). The highest accumulation of the three studied secondary metabolites was observed in the SLSs of style (24.4 mg/g), stigma (28.3 mg/g), and ovary (21.4 mg/g) explants, respectively. This study introduces a comprehensive procedure for producing SLSs containing the three most important metabolites of saffron for the first time.
Citation: Mahmoudi P, Moieni A, Khayam Nekouei M, Mardi M, Hosseini Salekdeh G (2025) Induction of stigma-like structures in saffron (Crocus sativus L.): Exploring factors and metabolite analysis. PLoS ONE 20(1): e0317186. https://doi.org/10.1371/journal.pone.0317186
Editor: Zahra Lorigooini, Shahrekord University of Medical Science, ISLAMIC REPUBLIC OF IRAN
Received: July 7, 2024; Accepted: December 23, 2024; Published: January 13, 2025
Copyright: © 2025 Mahmoudi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Saffron (Crocus Sativus L.), stands out as the most significant and costly spice plant globally, representing the flagship species within the Crocus genus and Iridaceae family. This sterile triploid plant naturally reproduces vegetatively, by formation of daughter corms on a mother corm [1]. The crimson stigmas of C. sativus, referred to as Saffron, serve as repositories for plethora of valuable and unique components, including carotenoids and apocarotenoids, renowned for their therapeutic effects since the Greek-Minoan civilization [2–4]. Historically, saffron’s essential secondary metabolites, have rendered it a remedy for various human ailments, such as coughs, digestive dysfunctions, colic, insomnia, chronic uterine bleeding, scarlet fever, smallpox, colds, and asthma [5]. Recent investigations have explored its potential therapeutic effects on modern-world diseases like cancer and AIDS [6]. The key metabolites of saffron, namely crocin (C44H64O24), picrocrocin (C16H26O7), and safranal (C10H14O) impart its color, taste and aroma, respectively. Saffron is a water-efficient perennial herbaceous plant that remains dormant in the corm form for an extended period. Corms become active and initiate meiotic activity at the end of summer, with flowering commencing in autumn (October) after the summer heat subsides. The plant relies solely on vegetative reproduction, where buds on the corms give rise to the next generation, ensuring saffron’s survival. During the flowering season, the flowers precede the appearance of leaves. Each flower features three stamen, five petals, a triple-carpel ovary, three sepals, and three red stigma branches (Fig 1). The number of flowers per corm ranges from one to three, with mature forms exhibiting a 7–10 cm style length culminating in a three branches stigma. Each Saffron stigma weighs approximately 2 mg. To produce 1 kg of dried saffron, around 150 thousand flowers with a wet weight of 70 kg are required, necessitating about 2 to 3 thousand square meters of cultivated land to accommodate this floral abundance [7].
A) Mature flower, B) different parts of flower; stigmas, stamens, and petals [8].
Total world saffron production is estimated to be around 418,000 tons, with approximately 90% originating from Iran and the remaining 10% from countries such as, Afghanistan, Greece, Morocco, Spain and Italy [9]. The trade of saffron has experiences significant growth in recent years due to increased demand in the food, pharmaceutical, and hygiene industries. Consequently, there is a growing emphasis on enhancing stigma production with improved quality. Large scale cultivation of saffron faces various challenges, necessitating of new cultivation methods and chemical techniques for quality assessment and impurity prevention [10].
Additionally, saffron corms are susceptible to various bacterial, fungal and viral pathogens, which can persist even when the corms are removed from the soil for replanting. Despite employing hygienic practices, these pathogens commonly lead to necrosis in leaves, roots and corms, subsequently reducing flowering [11]. The in vitro production of SLSs, the economically significant organ of saffron, offers advantages such as reduced production time and the elimination of field-related requirements and certain saffron production processes. In vitro production of SLSs is achievable using suitable culture media, and in some cases, the crocin pigments and picrocrocin may exhibit similarity or lighter shades compared to natural stigmas [12].
Numerous studies have explored the in vitro formation of SLSs. The investigation was initiated by Himeno and Sano have started in 1987 and has been since pursued by various researchers to optimize the in vitro formation of SLSs [11–20]. These investigations have revealed that all parts of the ovary possess the ability to induce stigma formation. Additionally, the formation of SLSs is often linked to the conversion of amyloplasts to chromoplasts and the accumulation of carotenoids [20, 21].
While most studies have focused on SLSs production from different flower organs, a few have identified the most suitable explant concerning both physiological age and the sampling time for SLSs production. Furthermore, there is no clear guidance on achieving the highest efficiency in SLSs production across various flower parts. Due to factors such as the slow growth rate of saffron, vulnerability to viral and bacterial contamination, labor-intensive picking, and substantial manpower requirements, exploring alternative methods for faster and easier saffron production and processing, is imperative. The feasibility may be realized through more comprehensive studies on SLSs production. Accordingly, this study aims to investigate: 1. The effects of different flower organs and their physiological age (sampling time) on SLSs induction and production, 2. The effects of different hormone treatments and culture media on SLSs induction in each type of explant, and 3. Quantity and quality of three secondary metabolites in SLSs. The research aims to provide the first report on determining the optimal sampling time and hormone combination for various explants to produce the highest number of SLSs (through direct and indirect regeneration). Additionally, it examines and compares the ploidy level and the amount of important secondary metabolites in in vitro SLSs and field stigmas for the first time.
Material and methods
Plant material and surface sterilization
Saffron corms of the Ghaen ecotype, bearing blossomed floral buds, were systematically collected three times per week over 12 weeks, spanning from mid-August to mid-November, from the experimental field of the Agricultural Biotechnology Research Institute of Iran (ABRII).
To minimize the time between collection and laboratory transfer, the corms were swiftly transported. Following the removal of the outer layer, the corms underwent thorough washing with tap water and hand wash liquid for 15 minutes. Given the sensitivity of flower parts, traditional long-term sterilization methods were impractical. In all treatments, following washing with tap water and hand wash liquid, the samples were incubated at 28°C for 24 hours to eliminate possible viral infection. Then, the corms were underwent surface sterilization, involving immersion in 70% ethanol, sodium hypochlorite, and nano-silver solution [22] for different durations (Table 1). Subsequent to each step, the corms were briefly soaked in sterile distilled water, with a final stage involving placement on sterilized filter paper for drying. Then, the buds were dissected from the corms. The flower parts including stigma (red, orange and white base on sampling time), intact ovary, injured ovary (incurred via scalpel), style and the stamens were utilized as explants. Ten days after plant material sterilization, the effectiveness of the treatments was assessed by measuring the percentage of uninfected explants and the number of surviving explants.
Media composition and culture conditions
The explants were subjected to culture on Murashige and Skoog (MS), 1/2MS [23], Linsmaier and Skoog (LS) [24] and B5 medium (B5) [25] basal media, supplemented with various combinations and concentrations of plant growth regulators (cytokinine and auxin) provided from Sigma-Aldrich, USA. All media included 30 g/L sucrose and were solidified with 3 g/L phytagel. Before autoclaving, the pH of all media was adjusted to 5.7–5.8 using HCl/NaOH. The culture media and tools were autoclaved at 121°C for 20, and 30 minutes, respectively. The media were dispensed into disposable Petri dishes (60 × 15 mm) [26].
Sterile explants were placed in Petri dishes, with three explants per dish. The petri dishes were sealed with Parafilm and then placed in a controlled growth chamber at 22°C for inducing SLSs. Subculturing of cultures were occurred at 28-day intervals. Each treatment was replicated three times, and each replication consisted of three Petri dishes.
The initial experiment aimed to identify the optimal sampling time for various flower parts. Experiments were conducted by selecting explants at different physiological ages from mid-August to mid-November. The growth stage of the explants was meticulously monitored under a stereomicroscope. The experiment was carried out based on a factorial experiment in a completely randomized design considering two factors, explant type across six levels (stigma, style, intact ovary, injured ovary, petal, and stamens) and sampling time (third and fourth weeks of August, four weeks in September, four weeks in October and the first two weeks in November). Each treatment was replicated three times, and each replication consisted of three Petri dishes.
In the subsequent year, a second experiment was conducted to assess the impact of plant growth regulators (PGRs) on the induction of SLSs. The experiment utilized the MS medium, supplemented with various combinations and concentrations of commonly used auxins and cytokinines. The auxins included naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and 2, 4-dichlorophenoxyacetic acid (2, 4-D), and cytokinins included benzylaminopurine (BAP) and kinetin (Kin), resulting in a total of 216 PGRs combinations. The experiment was carried out based on a factorial experiment in a completely randomized design considering two factors, explant type across six levels (stigma, style, intact ovary, injured ovary, petal, and stamens) and PGRs combinations in 216 levels, with 3 replications. The frequency of SLSs formation and the number of SLSs per explant were meticulously recorded approximately 90 days after the initial inoculation, which included three subcultures.
A third experiment was conducted to investigate the influence of culture medium on the induction and formation of SLSs. The media were supplemented with the most effective hormone combinations identified in the second experiment. This experiment employed a factorial design in a completely randomized setup, encompassing two factors: the type of culture medium at four levels (MS, ½ MS, LS, and B5) and the optimal hormone combinations (5 mg/L of NAA and 5 mg/L BAP and 10 mg/L of NAA and 10 mg/L Kin), at 2 levels. The experiment was conducted using injured ovaries as the explants. Each treatment was replicated three times, and each replication consisted of three Petri dishes.
Statistical analysis
The data obtained from the experiments were subjected to statistical analysis using SPSS Version 18 software [27]. The first experiment’s non-normal data were analyzed using the non-parametric Kruskal–Wallis method. For the second and third experiments, which were conducted based on a factorial experiment employing a Completely Randomized Design (CRD), the data were analyzed with three replications for each scenario.
Flow cytometry
To assess the ploidy level of the stigma-like structures, flow cytometric analysis was conducted to compare the nuclear DNA content between stigma cells of field plants and in vitro stigma-like structures [28]. Each sample, comprising approximately 40–50 mg of stigmas, underwent chopping using a sharp razor blade for approximately 60 s. Subsequently, the homogenate was mixed with 0.5 mL of buffer solution (Nuclei extraction buffer, Partec) and filtered through a 50-μm nylon filter to eliminate large debris. Nuclei were stained using 80 μg mL−1 propidium iodide (PI; Fluka, Buchs, Switzerland), and 80 μg mL−1 Ribonuclease (RNase) (Sigma, St Louis, MO, USA) was added to the nuclear suspension to prevent the staining of double-stranded RNA. Samples were incubated on ice and analyzed within 10 min. The amount of DNA was determined by examining at least 10,000 cells [29].
Parsley (Petroselinum crispum (Mill.) Fuss) [30], with an approximate nuclear DNA weight of 46.4 picograms, served as an internal standard plant [31]. Fluorescence intensity was measured using a flow cytometer (Partec Gmbh, Munster, Germany) with UV-Laser and High-Pressure Mercury (HBO)-Lamp).
Metabolite extraction
Field saffron stigmas and in vitro SLSs (20 mg) were dried and then suspended in 1 ml of methanol–water (50:50, v/v). The suspension was magnetically stirred for 24 h at 4°C in the dark. After extraction, samples underwent centrifugation at 30,000 ɡ for 35 min to remove plant residues. The supernatant was collected and filtered through a nylon membrane (Acrodisc 13, 0.45 μm pore size, 13 mm diameter, Waters, Milford, MA, USA) [32]. Prior to quantitative chromatographic analysis, 500 μL of 2-nitroaniline (0.5 mg/ml) was added as an internal standard to 500 μL of each tested sample [33].
Picrocrocin purification
Picrocrocin purification was conducted using preparative High-Performance Liquid Chromatography (HPLC) (Waters Delta Prep 4000). A volume of 500 mL of saffron methanol extract (20 mg/mL 80% Ethanol) was directly injected into a 20 mL/min stream. An HPLC linear gradient was employed, ranging from 90:10 (H2O: acetonitrile) to 50:50 over 25 min. Picrocrocin peak elution was monitored at 250 nm. The fraction was concentrated by rotary evaporation, and water was removed through solvent exchange using a C-18 Sep-pack. High-resolution mass spectrometry was employed to confirm both molecular weight and molecular formula [34]. A solution of 1 mg purified picrocrocin in 1 mL of HPLC-grade methanol was prepared. Subsequently, the solution was filtered through a 0.22 μm nylon filter to remove every particulate matter. Then, 5 μL of the picrocrocin solution was injected into the mass spectrometer via the electrospray ionization (ESI) source [35]. The molecular weight and molecular formula of picrocrocin were determined by identifying the peak corresponding to the molecular ion (M+) and analyzing the isotopic pattern of the molecular ion peak, respectively [36]. The collected picrocrocin was determined to be 92% pure [37].
HPLC
Equal volumes (10 μL) from each replicate were manually injected into the HPLC system (Knauer, pump K-1001) equipped with a C18 column (250 x 4.6 mm, pore size 5 μm; Teknokroma), a pump (Model 600E) and UV–Vis detection. A linear gradient of methanol (50–50% v/v) in water (15% acetonitrile v/v) was employed as a mobile phase, with a flow rate of 1.0 ml min-1, and a maximum elution time of 25 min at room temperature [36].
A linear gradient of methanol, ranging from an initial concentration of 50% (v/v) to a final concentration of 50% (v/v) in water, was applied as the mobile phase. The water used in the mobile phase contained 15% acetonitrile (v/v). The gradient was programmed with a flow rate of 1.0 ml min-1, and a maximum elution time of 25 min at room temperature.
Two standards, safranal (88%) and crocins (96%), were obtained from Sigma–Aldrich (St. Louis, MO). Picrocrocin was purified as previously described. HPLC-grade methanol (Methanol (MeOH): Ultraviolet (UV) cutoff 205 nm) and HPLC-grade acetonitrile (Acetonitrile (CAN): UV cutoff 190 nm) were supplied by Fisher Chemicals (Pittsburgh, PA).
The quantifications of crocins (440 nm), safranal (310 nm), and picrocrocin (250 nm) were determined on the basis of peak area with comparison to the above-mentioned standards. The internal standard was detected at the above three mentioned wavelengths [32]. Concentrations of crocins, safranal and picrocrocin were measured on a dry weight basis (mg/g). The analyses of each sample were repeated three times.
Results and discussion
Plant material surface sterilization
Due to the harsh effects of the sterilization stages on the flower buds, a significant portion of our first-year experiment samples did not survive, primarily due to 1) infections caused by inadequate sterilization in some treatments, and 2) severe sterilization processes in other treatments. To address these challenges, various sterilization treatments were examined to minimize infection and maximize the viability of explants.
The results indicated that a treatment involving a 5-second exposure to 70% ethanol, followed by immersion in a 5% sodium hypochlorite solution containing tween 20 (two drops) for 10 seconds, application of Nano-silver solution for 10 minutes, and a final rinse with sterile distilled water three times proved to be the best and most effective method for surface sterilization of saffron flower buds (Table 1). The duration of sodium hypochlorite treatment was found to have a significant impact on explant contamination. Optimizing the treatment time to 10 seconds was crucial for minimizing contamination while ensuring explant survival. Previous studies have explored various sterilization methods for different parts of the saffron flower [3, 19, 38]. However, this study demonstrated that incorporating a Nano-silver solution into the sterilization process improved effectiveness and led to enhanced samples survival.
In vitro SLSs induction
The effect of sampling time on the in vitro formation of SLSs.
The results of the first experiment, revealed distinct optimal sampling times for the various flower parts of saffron, including the stamens, stigma, style, ovary, and petal. Each flower part has a unique physiological age, which determines its ideal sampling time. Figs 2 and 3 illustrate the most suitable sampling time for each flower component. The ovary explants exhibited prolonged viability compared to other explants, indicating their superior responsiveness to hormone treatments. This extended viability contributes to a lengthened period of SLSs production, making ovaries a favorable choice for future studies. The data suggests that the third week of October is the overall optimal sampling time for all types of explants. However, it is essential to recognize that this timing is subject to variation based on geographical location and specific environmental conditions. The optimal physiological age for each explant may differ depending on flower type and environmental factors [39]. Our findings align with previous research [40–42], indicating that younger explants generally exhibit superior performance in SLSs formation. This study is the first to report the appropriate physiological age for each explant type based on their effectiveness in producing SLSs.
The sampling times that not shown in this figure did not produce any SLS.
The results of this experiment substantiated the distinct capacities of flower explants in forming SLSs. The findings underscored that the physiological age and degree of differentiation of explants were the two essential factors influencing their in vitro responses. The younger flower parts demonstrated superior performance, attributed to the ongoing mitotic cell division in their meristematic tissues, a phenomenon further enhanced by the application of auxin and cytokinin [42]. Saffron exhibits protandrous flowering, with the ovary being younger than the stamens within the closed flower bud. This likely accounts for the more favorable response of the ovary and style compared to the stamen in the initial weeks of saffron flower differentiation, as observed in the first experiment. Different parts of the saffron flower undergo rapid organelle differentiation to coincide with the production and accumulation of colored pigments (Fig 4).
(A): Comparison of stamens and stigmas in terms of physiological age and pigments amount in October. Stamens are completely yellow (a) and longer than the stigma, while the stigmas are not yet red and their size is shorter (b). (B): Petal and stigma comparison in terms of physiological age and pigment amount. Stigmas are still yellow (a), the petals are colorless and smaller (b), indicating the petals as the youngest part of the saffron flower. (C): Saffron flower at the beginning of November. Stigmas and stamens are mature (a), while the petals (b) remain colorless, small and younger; (D): A mature saffron flower in November. Stigmas are longer than the stamens and petals. Petals are purple and fully developed.
In this study, a detailed examination of flower buds before opening revealed the maturity of stigmas and stamens, while the petals of young flowers remained colorless. Notably, young petals and stamens exhibited responsiveness to certain treatments even before complete differentiation. SLSs were generated from the base of the young meristem, the point of connection between them. Additionally, in contrast to mature stigmas, the colorful end of the style produced SLSs under the influence of high concentrations of cytokinin and auxin.
The effects of PGRs on the in vitro formation of SLSs.
In the second experiment, the impact of common plant growth regulators, including auxins (NAA, IBA, and 2, 4-D) and cytokinins (BAP and Kin), on the induction of SLSs in saffron was investigated. The most effective induction of SLSs was observed in MS medium supplemented with 5 mg/L of NAA and 5 mg/L BAP and also, 10 mg/L of NAA and 10 mg/L Kin in injured ovaries, resulting in a success rate of 28%. Intact ovaries treated with the same hormone combination yielded a slightly lower induction rate of 27%. Table 2 shows the best hormonal compositions for each explant. Fig 5 shows SLSs induction in different explants.
Style (a), intact ovary (b), and injured ovary (c).
Hormone treatment results consistently demonstrated that the most successful SLSs induction across all flower parts was achieved with treatments containing NAA and BAP (Table 2). Similar hormone combinations have been reported for SLSs development on LS medium using half ovary explants [3], young ovary and half ovary explants [3, 42, 43], and floral bud explants [15, 17].
Substituting NAA with IBA or 2, 4-D significantly reduced the percentage of SLSs induction, highlighting the importance of NAA in the process. Among the various auxin and cytokinin combinations tested, the lowest SLSs induction rates were observed with different concentrations of IBA + Kin and 2, 4-D + Kin. Conversely, MS medium supplemented with NAA and BAP yielded the most favorable response, surpassing other auxin and cytokinin combinations and concentrations. These findings suggest that both the type and ratio of specific auxins and cytokinins are critical factors for successful SLS induction in saffron. Similar observations have been reported in other medicinal plants, such as Calotropis gigantea [44] and C. sativus [19, 45]. Furthermore, our study revealed that a concentration range of 5–10 mg/L NAA and 5–10 mg/L BAP is necessary for SLS formation in different saffron flower parts. Our research aligns with previous studies on the role of NAA and BAP in the formation of in vitro SLSs in saffron. For instance, Zeng et al. (2003) demonstrated that different concentrations of NAA and BAP can induce SLSs formation in saffron, and they also found that this combination can directly induce SLS from explants, bypassing callus formation [17]. Otsuka et al. (1992) discovered that using NAA and BAP in combination with alanine can induce SLSs in saffron [46]. Studies by Namera et al. (1987), Koyama et al. (1988), Lu et al. (1992), and Loskutov et al. (1999) have also explored the impact of different concentrations of auxin and cytokinin on SLSs formation in saffron flower explants, providing insight into the optimal concentrations needed to induce this process [12, 47–49].
Among the studied explants, stamens and stigmas showed the least success in SLS induction (Fig 3). The prolonged viability of ovary explants compared to other explants made them more suitable for SLS production, especially in response to plant growth regulators treatments. Similarly, Hosseinzadeh Namin et al. (2010) found that ovary explants had a longer viability compared to other explants and were more responsive [19].
The effects of culture media on the in vitro formation of SLSs.
The results of the third experiment indicate that the B5 medium exhibited superior performance in SLSs production compared to the MS, 1/2 MS, and LS media, respectively. The results of mean comparison indicated that the B5 medium was the most suitable medium for inducing SLSs in most explants. In contrast, the LS medium was not a suitable, and in many treatments, the explants turned black and did not respond to hormonal treatments after being placed in the LS medium. For the explants of style and ovary, the B5 medium appeared to be the most suitable medium compared to the other three media. These results are along with the findings of other researchers who have also reported the superiority of B5 medium in SLS production [50]. The increased SLSs production in B5 medium is likely due to its favorable mineral and organic nutrients. B5 culture medium differs from MS culture medium in the amounts of macro- and micronutrients used. B5 culture medium contains higher amounts of (NH4)2SO4 and Ca (NO3)2·4H2O. Overall, the macronutrients content of B5 medium is lower than that of MS medium (4530 mg l-1 in MS and 3184 mg l-1 in B5). The micronutrients content is also higher in MS medium (103.33 mg l-1 in MS and 81.15 mg l-1 in B5). Additionally, B5 medium lacks glycine, but contains 10 times more nicotinic acid and pyridoxine, and 100 times more thiamine than MS medium. Our findings support the idea that a diverse array of basal media, concentrations, and combinations of various additives can effectively initiate and produce colored SLSs on half-ovary explants, aligning with prior studies [14, 43, 51].
In our study, different parts of saffron flower were utilized as explants for establishing SLSs formation. The significant impact of explant type on the organogenesis has been reported in various plants, including C. sativus [52], Cnidium officinale [53], and Dendrocalamus latiflorus [54], among others.
In general, it appears that the combination and ratio of auxin and cytokinin play crucial roles in determining the number and type of SLSs produced. The results indicate that as this ratio decreases, there is a reduction in callus formation, leading to direct SLSs formation. Conversely, an increase in this ratio resulting in indirect SLSs formation. Furthermore, it was observed that direct SLSs formation occurs exclusively in different parts of the pistil (ovary, style, and stigma), with no direct SLSs produced in any other part of saffron flower. It is noteworthy that direct SLSs, in terms of quality, are more favorable compared to indirect SLSs, on the other hand in terms of quantity, indirect SLSs demonstrate a greater ability for commercial-scale production.
The number of SLSs per explant in this study ranged from 0 to 24 (Fig 2). Kohda et al. (1993) observed a frequency of 16 or more SLSs per explant (half-ovary) after culture for 4 months [51], while Loskutov et al. (1999) observed 8–12 SLSs on B5 or LS medium supplemented with a wide range of levels of NAA or IBA and BA [49]. Additionally, Koyama et al. (1988) reported a frequency of 10–20 SLSs per explant (injured ovary) on LS medium [12]. Previous studies and our research findings indicate that SLSs can be produced from the stigma and ovary of saffron flowers. Our investigation, however, is the first to report SLSs induction from other flower explants, including the style, petal, and stamen.
Flow cytometric analysis
Results from examining the ploidy level of the in vitro SLSs using flow cytometry showed no difference in the ploidy level of field stigmas and in vitro SLSs; all of them were triploid.
HPLC analysis of the crocin, picrocrocin, and safranal
Based on our analysis, there were significant differences in the biochemical composition (crocin, picrocrocin, and safranal) between saffron in vivo stigma and in vitro SLSs. Table 3 presents the values of the detected compounds in the in vitro regenerated SLSs and in the in vivo stigma, along with previous reports on the same components in the SLSs and field stigma. The highest concentration of all three studied secondary metabolites was found in field-grown saffron. Among the in vitro regenerated SLSs, the SLSs directly regenerated on the style explant had the highest amount of the secondary metabolites (24.4 mg/g), followed by SLSs directly regenerated from stigma and ovary explants (23.8 and 21.4 mg/g, respectively). The lowest amount was observed in SLSs indirectly regenerated from ovary explant (19.3 mg/g). The results indicate that the SLSs obtained from flower style, had the highest accumulation of the studied secondary metabolites. Divergent reports exist regarding the production of apocarotenoids in SLSs, with some studies indicating lower apocarotenoid levels in SLSs compared to natural stigmas [3, 16] while others suggest similar production, under in vitro and in vivo conditions [11]. Interestingly, there is a report where a higher amount of crocin was found in SLSs compared to natural stigmas [49] (Table 3).Various studies have reported different quantities of important secondary metabolites in saffron in vitro SLSs. The differences in these reports may be attributed to factors such as the initial explant, and the type of SLS regeneration, whether direct or indirect. In our study, we selected the Saffron SLSs obtained through direct and indirect regeneration from the top three treatments for examination using HPLC. The SLSs regenerated directly were longer, fewer in number, and red, similar to in vivo saffron, while the indirectly regenerated SLSs were more numerous, shorter, and dark orange (Fig 6). In general, the indirect regeneration system produced a greater number of SLSs.
Direct (right) and indirect (left) regeneration.
Standard deviations are shown in the parentheses.
The direct regenerated SLSs displayed a biochemical profile more closely resembling that of field saffron stigmas compared to the indirect ones. Compared to direct SLS regeneration, the indirect regeneration through the callus stage seems to induce more changes in the regenerated SLS. This is likely due to the genetic instability induced during the callogenesis phase. The callogenesis involves dynamic changes in cells, potentially leading to significant differences compared to their original state [55]. This crucial period can provide insights into the variations observed between indirectly and directly regenerated SLS. The callus phase may act as a catalyst for genetic and epigenetic modifications, ultimately resulting in heightened genetic instability. Subculturing the calli could further amplify this instability due to the accumulation of mutations or chromosomal alterations over time. These findings align with other studies suggesting that callogenesis plays a regulatory role in the expression of genes involved in the biosynthetic pathways of secondary metabolites [56].
It’s important to compare field stigma and in vitro SLSs in a proper manner. Since metabolite accumulation in the field increases with development, it is essential to consider the developmental stage when comparing the measured metabolites in the field stigma and in vitro SLSs. Notably, the amount of stigma carotenoids increases before the flowers open, reaching its peak. Subsequently, after the flower opens, the amount of these metabolites decreases, and environmental conditions such as high temperature (40°C) and relative humidity (75%) can further affect the color, aroma and flavor of saffron [57, 58]. Therefore, sampling immediately after flowering represents the optimal time for measuring important metabolites. Differences in the observed values in field stigmas (Table 3) may be due to the sample’s origin, different drying processes with varying time periods, and storage conditions in each country [31]. These factors can influence the concentration of glycosidic carotenoids due to their thermal sensitivity and susceptibility to light [59]. The observed reduction or lack of response in relatively older or advanced stage explants may result from qualitative and quantitative changes in DNA, cell cycle status, endogenous hormonal levels, and genotype [60]. One of the reasons for the notable differences in metabolites amounts in this report appears to be the attention to explant type as well as the consideration of the type of SLS regeneration (direct or indirect). Based on the previous reports various factors can increase the production of secondary metabolites in saffron tissue culture including sucrose, iron, copper, potassium, gibberellin, polyamines, and a combination of sucrose and iron [61]. Moreover, elicitors can be employed as an effective tool to enhance the production of secondary metabolites in saffron. Recently, studies have shown that salicylic acid (SA), polyethylene glycol (PEG), and ultrasonic waves can be used as effective elicitors to increase the production of crocin and safranal in saffron suspension cultures. These elicitors stimulate the expression of genes controlling crocin and safranal biosynthesis and increase the activity of related enzymes, leading to an increase in the production of these secondary metabolites. Treatment with salicylic acid and polyethylene glycol increased crocin and safranal production. The use of elicitors can be a viable approach to enhance the production of secondary metabolites in saffron and can contribute to the development of new methods for producing these valuable metabolites [61].
Conclusion
In the present study, we investigated the production of in vitro SLSs as an alternative method for saffron stigma production. The B5 medium, enriched with 5–10 mg/ L BAP and 5–10 mg/ L NAA was the most effective treatment for inducing SLSs in all types of explants. The intact, and injury ovary explants were found to be the most effective explant types for SLSs formation. The highest accumulation of the three studied secondary metabolites was observed in the SLSs of style, stigma, and ovary explants, respectively. A biochemical comparison of regenerated SLSs with field stigmas revealed that the in vitro SLSs can produce the most important secondary metabolites of saffron, with concentrations similar to those of field stigma. Besides the type of explant, the method of regeneration can also impact the number of SlSs and the concentration of secondary metabolites. Therefore, choosing the appropriate explant and SlSs regeneration method is crucial for optimizing SLSs regeneration as an alternative method for producing secondary metabolites of saffron.
This study found that the highest concentrations of metabolites were obtained in the directly regenerated SLSs. However, it also demonstrated that these SLSs can be used as explants to produce new SLSs. If the method is optimized to produce the maximum number of SLSs per explant, the yield of metabolites can be increased through successive subcultures.
The amounts of three metabolites examined in indirectly regenerated SLSs were slightly less than those of the directly regenerated SLSs. However, the indirect regeneration system has the potential for mass production of SLSs, thereby increasing the production of metabolites. Therefore, it is necessary to optimize the conditions for mass production of callus from desirable explants and conduct research to produce the maximum number of SLSs on the calli. Additionally, it is essential to study the effects of different elicitors to increase the biosynthesis of the mentioned metabolites in the SLSs.
Recommendations
This study highlights the potential of in vitro production of SLSs as an alternative method for producing saffron stigma. The findings indicate that the optimal sampling time for most explants is the third week of October. Other researchers are encouraged to re-examine this timing based on their specific geographical regions.
Future perspectives
Biochemical analysis indicated that SLSs regenerated directly from the style explants produced the highest concentrations of, picrocrocin, and safranal, closely resembling the quality of field-grown saffron stigmas. Although the indirect regeneration system produces slightly lower metabolite concentrations, it has significant potential for mass production. It is essential to balance the quality and quantity of SLSs produced. Future research should optimize in vitro conditions for direct and indirect regeneration to maximize the yield of SLSs and secondary metabolites. More detailed studies are recommended to refine various factors including modifications to the B5 culture medium. This may involve investigating zeatin and thidiazuron and adjusting the levels of macroelements and microelements. Additionally, it is important to consider incubating conditions, such as the temperature and container types, along with subculturing techniques to ensure a consistent, high-quality product. Utilizing precursors and elicitors may also enhance the production of the targeted secondary metabolites, making in vitro production methods more viable for commercial applications.
Supporting information
S1 Table. The hormonal composition’s effects on the number of SLSs produced in the intact ovary.
https://doi.org/10.1371/journal.pone.0317186.s001
(DOCX)
S2 Table. The hormonal composition’s effects on the number of SLSs produced in the injured ovary.
https://doi.org/10.1371/journal.pone.0317186.s002
(DOCX)
Acknowledgments
The authors gratefully acknowledge the Research Deputy of Tarbiat Modares University.
References
- 1. Pandey D K, Nandy S, Mukherje A, Abhijit D. Chapter 10-Advances in bioactive compounds from Crocus sativus (saffron): Structure, bioactivity and biotechnology. Studies in Natural Products Chemistry. 2020; 66: 273–304.
- 2. Musazadeh V, Zarezadeh M, Faghfouri A H, Keramati M, Ghoreishi Z, Farnam A. Saffron, as an adjunct therapy, contributes to relieve depression symptoms: An umbrella meta-analysis. Pharmacological Research. 2022; 175: 105963. pmid:34757208
- 3. Mir J, Ahmed N, Singh D B, Wani S H, Shafia Zaffer M H, Shabeena Y, et al. In vitro stigma like structure and stigma development in saffron. 2015; 28 (2): 55–58.
- 4. D’Agostino N, Pizzichini D, Chiusano M L, Giuliano G. An EST database from saffron stigmas. BMC Plant Biology. 2007; 7:1–8.
- 5. Basker D, Neghbi M. Uses of saffron. Economic Botany. 1983; 37:228–236.
- 6. Rashid M, Birm H, Ashktorab H. Saffron, Its Active Components and Their Association with DNA and Histone Modification: A Narrative Review of Current Knowledge. Nutrients. 2022; 14 (16). pmid:36014823
- 7. Aytekin A, Acikgoz A O. Hormone and microorganism treatments in the cultivation of saffron (Crocus sativus L.). Plants. Molecules. 2008; 13:1135–1147.
- 8. Khorasani S, Ghandehari Yazdi AP, Saadatfar A, Kamali Rousta L, Nejatian M, Abarian M, et al. Valorization of saffron tepals for the green synthesis of silver nanoparticles and evaluation of their efficiency against foodborne pathogens. Waste and Biomass Valor. 2022; 13: 4417–4430.
- 9. Cardone L, Castronuovo D, Perniola M, Cicco N, Candido V. Saffron (Crocus sativus L.), the king of spices: An overview. Scientia Horticulturae. 2020; 272: 109–560.
- 10. Mohammadi H, Reed M. Chapter 22-Saffron marketing: challenges and opportunities. Saffron Science, Technology and Health. 2020; 45: 357–365.
- 11. Ebrahimzadeh H, Rajabian T, Karamian R. In vitro production of feloral buds and stigma-like structure on floral organ of Crocus sativus L. Pakistanian Journal of Botany. 2000; 23(1): 141–150.
- 12. Koyama A, Ohmori Y, Fujioka N, Miyagawa H, Yamasaki K, Kohda H, Formation of stigma-like structure and pigment in cultured tissue of Crocus sativus L. Plant Medicin. 1988; 54: 375–376.
- 13. Hidetaka H, Katsuyoshi E, Makoto N. Induction of callus from pisitils of Crocus sativus L. and Production of Color Compound in the Callus. Plant Tissue Culture Letters. 1988; 5(2): 72–77.
- 14. Fakhari F, Evans PK. Morphogenetic potential of cultured floral explants of Crocus chrysanthus. Journal of Experimental Botany. 1989; 40 (216): 809–812.
- 15. Fakhari F, Evans P K. Morphogenetic potential cultured explant of Crocus sativus L. for the in vitro production of saffron. Journal of Experimental Botany. 1990; 41 (1): 47–52.
- 16. Sarma K S, Sharada K, Maesato K, Hara T, Sonoda Y. Chemical and sensory analysis of saffron produced through cultures of Crocus sativus L. Plant Cell Tissue Organ Culture. 1991; 26: 11–16.
- 17. Zeng Y, Yan F, Tang L, Chen F. Increased crocin production and induction frequency of stigma-like-structure from floral organs of Crocus sativus L. by precursor feeding. Plant Cell, Tissue and Organ Culture. 2003; 72(2): 185–191.
- 18. Zhao T, Tang L, Xiao H B, YAN C, Chen F. Optimization on Regeneration Conditions of Style-stigma-like Structure in Crocus sativus L. Journal-Sichuan University Natural Science Edition. 2005; 42(6): 1238.
- 19. Hosseinzadeh Namin M, Ebrahimzadeh H, Ghareyazie B, Radjabian T, Hosseinzadeh Namin H. Initiation and origin of stigma-like structure (SLS) on ovary and style explants of saffron in tissue culture. ACTA Biological Cracoviensia. 2010; 52(1): 55–60.
- 20. Kareem M, Nabi B, Kashtwari M, Wani A, Dhar K. Preliminary report on development of proper stigmas and stigma-like structures in saffron under in vitro conditions. Biological Sciences. 2019; 89: 1213–1217.
- 21. Castillo R, Fernandes J A, Gomez-Gomez L. Implications of carotenoid biosynthetic genes in apocarotenoid formation during the stigma development of Crocus sativus L. and Its closer relatives. Plant Physiology. 2005; 139: 674–676.
- 22. Tung HT, Van HT, Bao HG, Khai HD, Luan VQ, Phong TH, Nhut DT. Silver nanoparticles enhanced efficiency of explant surface disinfection and somatic embryogenesis in Begonia tuberous via thin cell layer culture. Vietnam Journal of Biotechnology. 2021; 19(2): 337–347.
- 23. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15 (3): 473–497.
- 24. Linsmaier E M. Skoog F. Organic Growth Factor Requirements of Tobacco Tissue Cultures. Physiologia Plantarum. 1965; 18: 100–127. https://doi.org/10.1111/j.1399-3054.1965.tb06874.x
- 25. Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension culture of soybean root cells. Exprimentar Cell Research. 1968; 50: 15–158.
- 26.
Singh MP, Kumar S. Plant tissue culture. APH Publishing; 2009.
- 27.
SPSS, Inc., 2009, Chicago, IL, www.spss.com
- 28. Nirmal Babu K, Divakaran M, Raj RP, Anupama K, Peter KV, Sarma YR. Biotechnological approaches in improvement of spices: a review. Plant Biology and Biotechnology: Volume II: Plant Genomics and Biotechnology. 2015; 487–516.
- 29. Yokoya K, Roberts AV, Mottley J, Lewis R, Brandham PE. Nuclear DNA amounts in roses. Annals of Botany. 2000; 85(557–561).
- 30. Temsch EM, Koutecký P, Urfus T, Šmarda P, Doležel J. Reference standards for flow cytometric estimation of absolute nuclear DNA content in plants. Cytometry Part A. 2022; 101(9): 710–724. pmid:34405937
- 31. Jowkar A, Kermani M, Kafi M, Mardi M, Hoseini ZS, Koobaz P. Cytogenetic and flow cytometry analysis of Iranian Rosa spp. Floriculture Ornamental Biotech. 2009; 3(1): 71–74.
- 32. Lozano P, Castellar MR, Simancas MJ, Iborra L. A quantitative high-performance liquid chromatographic method to analyse commercial saffron (Crocus sativus L.) products. Journal of Chromatography A. 1999; 830:477–483.
- 33. Caballero-Ortega H, Rogelio Pereda M, Abdullaev F. HPLC quantification of major active components from 11 different saffron (Crocus sativus L.). Food Chemistry. 2007; 3: 1126–1131.
- 34. Yilmaz A, Nyberg NT, Mølgaard P, Asili J, Jaroszewski JW. 1 H NMR metabolic fingerprinting of saffron extracts. Metabolomics. 2010; 6: 511–517.
- 35. Koulakiotis NS, Pittenauer E, Halabalaki M, Tsarbopoulos A, Allmaier G. Comparison of different tandem mass spectrometric techniques (ESI‐IT, ESI‐and IP‐MALDI‐QRTOF and vMALDI‐TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L. Rapid Communications in Mass Spectrometry. 2012; 26(6): 670–8.
- 36. Kabiri M, Rezadoost H, Ghassempour A. A comparative quality study of saffron constituents through HPLC and HPTLC methods followed by isolation of crocins and picrocrocin. Lwt. 2017; 84: 1–9.
- 37. Lage M, Cantrell L CH. Quantification of saffron (Crocus sativus L.) metabolites crocins, picrocrocin and safranal for quality determination of the spice grown under different environmental Moroccan conditions. Scientia Horticulturae. 2009; 121: 366–373.
- 38. Lu W, Tong X, Zhang Q, Gao W. Study on in vitro Regeneration of Style-Stigma-Like Structure in Crocus sativus L. J Integr Plant Biology. 1992; 34(4).
- 39. Tahiri A, Mazri MA, Karra Y, Ait Aabd N, Bouharroud R, Mimouni A. Propagation of saffron (Crocus sativus L.) through tissue culture: a review. The Journal of Horticultural Science and Biotechnology. 2022; 98(1): 10–30.
- 40. Hyouta H, Matsushima H, Sano K. Scanning electron microscopic study on the in vitro organogenesis of saffron stigma and style-like structures. Plant Scienec. 1998; 58: 93–102.
- 41. Taheri-Dehkordi A, Naderi R, Martinelli F. A robust workflow for indirect somatic embryogenesis and cormlet production in saffron (Crocus sativus L.) and its wild allies; C. caspius and C. speciosus. Heliyon. 2020; 6 (12).
- 42. Sarma KS, Maesato K, Hara T, Sonoda Y. In vitro production of stigma-like structures from stigma explants of Crocus sativus L. Journal of Experimental Botany. 1990; 41(6): 745–748.
- 43. Himeno H, Sano K. Synthesis of crocin, picrocrocin and safranal by saffron stigma-like structures proliferated in vitro. Agricultural and Biological Chemistry. 1987; 51: 2395–2400.
- 44. Tripathi P, Awasthi SH, Kanojiya S, Tripathi V, Mishra D. Callus culture and in vitro biosynthesis of cardiac glycosides from Calotropis gigantea (L.) Ait. In vitro Cellular and Developmental Biology. 2013; 49: 455–460.
- 45. Vahedi M, Kalantari S, Salami SA. Factors affecting callus induction and organogenesis in saffron (Crocus sativus L.). Plant Tissue Culture and Biotechnology. 2014; 24(1): 1–9.
- 46. Otsuka M, Saimoto H, Murata Y, Kawashima M. Method for Producing Saffron Stigma-Like Tissue and Method for Producing Useful Components from Saffron Stigma-Like Tissue. United States Patent N 5, 1992; 085, 995.
- 47. Lu WL, Tong XR, Zhang Q, Gao W. Study on in vitro regeneration of style-stigma-like structure in Crocus sativus L. Acta Botanica Siniea. 1992, 34: 251–256.
- 48. Namera S, Sano K, Himeno M. Effects of NAA and BAP on stigma-like structure formation in saffron flowers. Journal of Plant Physiology. 1987, 130(2):151–156.
- 49. Loskutov AV, Beninger CW, Ball TM, Hosfield GL, Nair M, Sink KC. Optimization of in vitro conditions for stigma-like-structure production from half-ovary explants of Crocus sativus L. In vitro Cellular and Developmental Biology-Plant. 1999; 35: 200–205.
- 50. Mir JI, Nazeer A, Shabir HW, Rizwan R, Hidayatullah M, Muneer A Sh. In vitro development of microcorms and stigma like structures in saffron (Crocus sativus L.). Physiology and Molecular Biology of Plants. 2010; 16: 369–373.
- 51. Kohda H, Yamasaki K, Koyama A, Miyagawa H, Fujioka N, Omori Y, et al. Process for culturing saffron stigma tissues. United States Patent N. 1993; 5: 217,897.
- 52. Da Silva JT, Kulus D, Zhang X, Zeng S, Ma G, Piqueras A. Disinfection of explants for saffron (Crocus sativus) tissue culture. Environmental and Experimental Biology. 2016; 14(4): 183–198.
- 53. Adil M, Ren X, Kang DI, Jeong BR. Effect of explant type and plant growth regulators on callus induction, growth and secondary metabolites production in Cnidium officinale Makino. Molecular Biology Reports. 2018; 45(6); 1919–1927.
- 54. Ye S, Cai C, Ren H, Wang W, Xiang M, Tang X, et al. An efficient plant regeneration and transformation system of Ma bamboo (Dendrocalamus latiflorus Munro) started from young shoot as explant. Frontiers in Plant Science. 2017; 8:1298.
- 55. Neelakandan AK, Wang K. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Reports. 2012; 31: 597–620. pmid:22179259
- 56. Nasrollahi V, Mirzaie-Asl A, Piri K, Nazeri S, Mehrabi R. The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponins in liquorice (Glycyrrhiza glabra). Phytochemistry. 2014; 103: 32–37. pmid:24768283
- 57. Cid-Pérez T S, Nevárez-Moorillón GV, Ochoa-Velasco CE, Navarro-Cruz AR, Hernández-Carranza P, Avila-Sosa R. The relation between drying conditions and the development of volatile compounds in saffron (Crocus sativus). Molecules. 2021; 26(22); 6954–6965.
- 58. Alonso GL, Varon R, Gomez R, Navarro F, Salinas MR. Autoxidation in saffron at 40 C and 75% relative humidity. Journal of Food Science. 1990; 55(2); 595–596.
- 59. Tarantilis PA, Tsoupras G, Polissiou M. Determination of saffron (Crocus sativus L.) components in crude plant extract using high-performance liquid chromatography-UV-visible photodiode-array detection-mass spectrometry. Journal of Chromatography A. 1995; 699: 107–118.
- 60. Chakravarthi D, Indukuri V, Goparaju UA, Yechuri V. Effect of genotype, explant and hormonal concentration on in vitro response of eggplant. Notulae Scientia Biologicae. 2010; 2(3): 77–85.
- 61. Moradi A, Zarinkamar F, De Domenico S, Mita G, Di Sansebastiano GP, Caretto S. Salycilic acid induces exudation of crocin and phenolics in saffron suspension-cultured cells. Plants. 2020; 9 (8): 949. pmid:32731416
- 62. Visvanath S, Ravishankar GA, Venkataraman LV. Induction of crocin, crocetin, picrocrocin, and safranal synthesis in callus cultures of saffron: Crocus sativus L. Biotechnology and Applied Biochemistry (USA). 1990; 12(3).