Optimization of Regulatory Factors Affecting Biomass Growth and Crocin Production of Saffron Cells in Bioreactor

Optimization of Regulatory Factors Affecting Biomass Growth and Crocin Production of Saffron Cells in Bioreactor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimization of Regulatory Factors Affecting Biomass Growth and Crocin Production of Saffron Cells in Bioreactor Seyed Mahdi Ziaratnia, Somiyeh Amini, Khodayar Hemmati This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6857423/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Optimizing the media for plant cell cultures, particularly regarding the types and concentrations of growth regulators, can significantly enhance biomass and metabolite production in vitro . This study examined the effects of different basal media and plant growth regulators (PGRs) on cell growth and crocin production in saffron cell suspension cultures. Various media including SH, MS, and B 5 , in combination with specific PGRs, were tested for their efficacy in promoting cell biomass and crocin yield. Results identified that SH medium supplemented with NAA (1 mg/L) and BA (0.5 mg/L) was optimal for biomass growth, while IAA (1 mg/L) and KIN (0.5 mg/L) effectively boosted crocin production. The research continued by evaluating these factors in a bioreactor setup. For biomass enhancement, 50g of cells were inoculated into one liter of a chosen medium (SH, 3% sucrose, 0.5 mg/L BA, 1 mg/L NAA, 2.5 mM MES) with aeration at 0.5 vvm and further sucrose feeding. For crocin production, another setup used the same biomass with a medium of SH, 3% lactose, 0.5 mg/L Kin, 1 mg/L IAA, and no aeration, adjusting conditions similarly. Findings showed that cell growth index improved from 1.87 to 4.3, while crocin levels increased from 1.05 to 3.43 mg/g dry weight. Crocin content was quantified using spectrophotometry and HPLC, with image analysis employed to determine red color intensity. The study concludes that image analysis offers a cost-effective method for assessing crocin production, highlighting the effectiveness of bioreactors in enhancing both cell growth and metabolite yield. Image analysis cell suspension secondary metabolites Crocus sativus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Key messages The bioreactor serves as a highly efficient platform for cell proliferation and enhancing the biosynthesis of crocin metabolite in saffron ( Crocus sativus ) cell cultures in separated medium ingredients and growth regulators combinations. The image analysis a-index (red color intensity), is a precision, effectiveness and non-destructive method to estimate crocin content of the saffron cells. 1. Introduction Plant cell culture systems present an innovative and efficient alternative for producing valuable secondary metabolites, surpassing the limitations of traditional extraction methods from plant organs (Ferri and Tassoni, 2011 ). Despite the advantages, this technology faces challenges such as low metabolite accumulation in cells. Various strategies have been proposed to overcome this issue (Kolewe, 2011 ). One of the most important options to enhance the productivity of plant cell cultures is the optimization of the medium (Santos et al., 2016 ). Plant culture media contain various components including nutrients, vitamins, amino acids, and plant growth regulators, among which the type and concentration of growth regulators play a crucial role in cell growth and metabolites synthetize. In addition to hormonal compounds, researchers have proven that the type of culture medium also affects the aforementioned factors (Nagella and Murthy, 2010 ). Commonly used plant culture media include Murashige and Skoog (MS), Linsmaier and Skoog (LS), Nitsch and Nitsch (NN), Gamborg (B 5 ), and Schenk and Hildebrandt (SH) (Abobkar and Elshahed, 2012 ). In the past decade, several studies have highlighted the importance of optimizing culture media and hormonal compositions for increasing the efficiency of callus induction regeneration in various plant species (Ali et al., 2017 ; Javed et al., 2020 ; Gantait et al., 2018 ). Along to these efforts, extensive research has been conducted on optimizing callus induction in saffron ( Crocus sativus L.) using solid culture media influenced by different types and concentrations of auxin and cytokinin growth regulators (Sajjadi fard and Pazhohandeh, 2015 ; Safarnejad et al., 2016 ; Firoozi et al., 2019 ; Mereu et al., 2019 ). These studies have shown that the type of basal medium and growth regulators significantly affect saffron callus growth and secondary metabolites production, including carotenoids, phenolic compounds, and crocin (Moradi et al., 2018 ). Other researchers have shown that two-stage saffron callus culture effectively increases crocin content. In this approach, growth regulators are optimized for callus induction in the first stage, followed by suitable conditions for crocin production. In two-stage culture, crocin content increased by 295% compared to single-stage culture. They reported Gamborg’s medium enriched with 300 mg. L − 1 hydrolyzed casein as the best medium for callus growth and crocin production. They also found that the optimal growth regulator combination for maximum callus growth and crocin production was 1 mg. L − 1 benzyladenine (BA) + 2 mg. L − 1 naphthaleneacetic acid (NAA) and 0.5 mg. L − 1 BA + 2 mg.L − 1 indoleacetic acid (IAA) respectively (Chen et al., 2003 ). Amini et al. (2024) investigated the effects of saffron corm physiological age, basal culture media, and growth regulator types in different concentrations on callus induction and crocin production in solid medium. They could quantify the red color intensity of the callus as an index for measuring crocin by image analysis and screening the callus growth. The results of their research showed that if the initial callus formation of mature corms is performed on B 5 culture medium containing NAA (2 mg/l) + BA (1 mg/l) for 6 weeks and then the calli obtained are re-cultured on B 5 medium containing IAA (2 mg/l) + KIN (1 mg/l), the largest amount of cell mass with the highest intensity of red color (containing the metabolite crocin) will be produced (Amini et al., 2024). The researchers announced that Schenk and Hildebrandt (SH) basal medium with NAA (2 mg l − 1) and BA (1 mg l − 1) supplemented with 2.5 mM of MES as well as gradual increment of sucrose from 3 to 6% caused the highest cell biomass and crocin production. The results of saffron cell culture in the bioreactor also showed that, aeration was found to be an inhibiting factor for the production of crocin metabolite and natural pH fluctuation is a suitable condition for saffron biomass cell growth and crocin production (Amini et al., 2022 ). The results of the another research showed that among the different carbon sources, sucrose and lactose, at a concentration of 3% were found to be the best carbohydrates for the highest production of cell biomass and crocin metabolite, respectively. The elicitation results also showed that yeast extract at a concentration of 1.5% increased crocin to 1.5 times in 5 days after stimulation of the cells (unpublished). Despite existing researches, no reports have focused on optimizing culture medium type and plant growth regulators in saffron cell suspension culture. Therefore, this study is the first to optimize these factors to enhance cell growth and crocin production in saffron cell culture. Then, the results of this study were combined with the results obtained from other studies in the field of elicitor application, carbohydrate type, and bioreactor aeration, and their effects on biomass growth and crocin production in the bioreactor were evaluated. This study also compares different crocin measurement methods, including high-performance liquid chromatography (HPLC) and spectrophotometry with image analysis technique for red color intensity of cells indicating crocin content. 2. Materials and Methods 2.1. Callus Induction The callus was prepared under optimized conditions established in prior callus induction experiments conducted by Amini et al. (2024). The findings revealed that explants derived from mature saffron corms exhibit a significantly higher ability to produce callus and crocin pigments those from immature corms. Consequently, this research utilized mature corms harvested in late May for explant preparation. After removing outer shells, corms were disinfected with 70% EtOH for 1 min and then 1% sodium hypochlorite solution for 15 min. This step was followed by three times rinsing with sterilized distilled water, each time for 5 min. Under sterile conditions within a laminar flow hood, the sterilized corms were sliced into 1 cm×1 cm pieces with a thickness of 1 mm. To enhance callus induction and growth, explants cultured on B 5 medium enriched with NAA (2 mg. L − 1 ) and BA (1 mg. L − 1 ) for six weeks. Following this incubation period, the calli transferred to B 5 medium supplemented with 2 mg. L − 1 of IAA and 1 mg. L − 1 of KIN to increase crocin content. In both stages, the explants were stored in darkness at a controlled temperature of 22 ± 0.3°C. 2.2. Cell Suspension Establishment After the producing a desired volume of red color calli, the cell suspension establishment was implemented to homogenize the cells in terms of growth and acclimate them to a liquid culture medium. This step was performed with slight modifications according to the method presented by Amini et al ( 2022 ). An effective hormonal combination was chosen to enhance cell biomass in a liquid SH medium composed of NAA (2 mg. L − 1 ) + BA (1 mg. L − 1 ) and 3% sucrose. Subsequently, one-liter flasks containing 200 mL of this medium and 20 grams of finely processed cells were placed on a shaker operating at 120 rpm in complete darkness, maintained at a temperature of 22 ± 0.3°C. After two weeks, the cells cultivated in this medium were utilized for experimental treatments. 2.3. Evaluation of Culture Medium Type: After establishment of the cell suspension culture, the first experiment, involving the examination of the effects of three basal media on cell growth and crocin production, was conducted in flask system. For this purpose, MS, B 5 and SH basal medium with B 5 vitamins were selected and supplemented with NAA (2 mg. L − 1 ) + BA (1 mg. L − 1 ) and 3% sucrose. Each 100 ml flask with containing 20 ml of medium was inoculated with 1 gram of cells obtained from the cell suspension establishment stage. The flasks were maintained in darkness at 22 ± 0.3°C on an orbital shaker at 120 rpm. Weekly measurements included the pH of the culture medium and cell biomass growth (cell growth index). At the end of the growth period (end of the 14th week), the crocin content was measured and compared based on two systems of quantifications-spectrophotometry and HPLC. After statistical analysis of the recorded data, the most suitable medium for cell growth and crocin production in cell suspension culture was identified. 2.3.1. Measurement of Cell Growth Index in Suspension Culture: To measure cell growth, the method by Amini et al. ( 2022 ) was used, applying the Eq. 1: Equation 1: \(\:\text{G}\text{I}=\frac{W2-W1}{W1}\) Where GI: is the cell growth index, W 1 : is the initial cell weight at the start of the culture period, and W 2 : is the secondary cell weight at the time of measurement. 2.3.2. Drying and Extraction of Crocin from Cells: For crocin quantification, HPLC and spectrophotometry were used. The cells were dried and extracted using the method of Amini et al. ( 2022 ). The grown cells were separated from the liquid medium, dried in an oven at 35°C for 48 hours, then 0.05 g of dried cell powder was transferred to Falcon tubes with 5 mL of 50% ethanol and kept in darkness on a shaker at room temperature for 24 hours. The extract was separated from cell debris using centrifugation at 8000 rpm for 10 minutes. The extracted solution was filtered through a syringe filters (0.45µM) and stored in 4°C in darkness until HPLC or spectrophotometry analysis (Amini et al., 2022 ). 2.3.3. Crocin Measurement by Spectrophotometry: The extraction solution was applied on a spectrophotometer model CT-5700 with a quartz cell and the absorbance was recorded at 440 nm. Standard crocin solution (C44H64O24 Mr 976.98) was prepared in a serial dilution with the concentrations ranging from 25 to 200 mg. L − 1 using 50% ethanol as the solvent. The recorded absorbance at 440 nm was plotted to make a standard crocin. Ultimately, the crocin concentration in the cell extracts was calculated based on sample absorbance at 440 nm and equation derived from the standard curve (Eq. 2). Equation 2: Y = 0.128X + 0.0347 Where Y is the absorbance of the cell extract at 440 nm and X is the crocin concentration (mg. L − 1 ). Results were then reported as mg crocin per gram of dry cell weight. 2.3.4. High-Performance Liquid Chromatography (HPLC): Crocin was identified and quantified by HPLC using a Waters 1525 binary HPLC pump, equipped with Waters 2489 UV/Visible Detector, and Breeze software. Column C18, ODS. 250 mm, 4.6 mm, particle size 5.0 µm was employed. Methanol (HPLC grade) with gradient flow rate (20 to 80%) at 1 ml min − 1 was used as mobile phase. Detection wavelength adjusted on 440 nm for crocin detection. Duration of the test was 60 min and the volume of the injection was 20 µl. Temperature was adjusted at 30°C. Standard crocin samples were prepared with ethanol at three different concentrations (0.024, 0.048, 0.072 mg. L − 1 ). The standard curve was obtained by injecting different concentrations into the HPLC system. Cell extracts were injected into the system, and the crocin concentration was calculated by comparing the peak area in the chromatogram with the standard crocin chromatogram (Amini et al., 2022 ). 2.4. Investigation on Plant Growth Regulators Type and Concentration: In the second part of this research, to study the effect of the type and concentration of plant growth regulators, the selected culture medium from the previous experiment was used as the basal. This medium was supplemented with four types of auxins group (IAA, IBA, NAA, 2,4-D) at three concentrations (1, 2, and 4 mg. L − 1 ) and two types of cytokinins (BA and Kin) at three concentrations (0.5, 1, and 2 mg.L − 1 ) with 3% sucrose. Cell cultures were performed in 100 mL flasks according to the previous experimental conditions. At the end of the experimental period, the red color intensity of the cells (a* index) was determined using image analysis by Image J software (Amini et al., 2024). After drying the cells (at 35°C) and extracting them, the crocin content was measured using spectrophotometry method presented in 2.3.3 section. In this part, we aimed to find a correlation between the red color intensity of the cells and crocin content by comparing the image analysis results (red color intensity) with the spectrophotometry results (crocin content). 2.4.1. Image Analysis Method: Image analysis was used to determine the red color intensity of cell masses. At the end of the culture period, the cell masses were placed on a scanner covered with a black box for darkness, and the cells were scanned. The Image J software (version 1.45s) and L*a*b* color space were used to determine the color parameter of the callus. The a* index indicating red color intensity (Mohd Jusoh et al., 2009). Selected regions within images were analyzed, and the a* index was calculated accordingly (Amini et al., 2024). 2.5. Final optimization in bioreactor system with fed-batch cultivation: This study integrated findings from the medium optimization and various plant growth regulators to enhance cell biomass production and crocin content using a bioreactor system. Fifty grams of cells obtained from the cell suspension stage were inoculated into one liter of the optimized culture medium, supplemented with MES (2.5 mM) and 3% sucrose. Gradual feeding with a 3% sucrose solution commenced on the seventh day, with aeration maintained at 0.5 vvm throughout the cultivation period (Amini et al., 2022 ). In the fifth week, 0.5% yeast extract was added to the medium, and the bioreactor was emptied after seven days to measure the cell growth index. For crocin optimization in bioreactor settings, 50 grams of cells were introduced into one liter of the selected medium with optimal hormonal treatment, supplemented as before with MES (2.5 mM) and 3% lactose. No aeration was applied during this culture phase (Amini et al., 2022 ). Gradual feeding of the medium with 3% sucrose began at the end of the first week, followed by the addition of 1.5% yeast extract in the fifth week, with crocin content measured 5 days after treatment. 3. Statistical Analysis Experiments in the flask system were arranged in a factorial experiment following a completely randomized design. The first experiment assessed three types of media (MS, SH, B 5 ) over 14 weeks. The second experiment evaluated four auxin types (NAA, IAA, IBA, 2,4-D) at three concentration levels (1, 2, and 4 mg/L), and two cytokinin types (BA, KIN) at concentrations of 0.5, 1, and 2 mg/L. Each experimental combination was conducted in quadrulicate. Statistical analyses were performed using SPSS software, with mean comparisons made utilizing Duncan’s multiple range test at a 95% confidence level. In the bioreactor system, additional statistical treatments will be detailed based on experimental outcomes. 4. Results 4.1. Assessment of Medium Type The results of the analysis of variance (ANOVA) indicated that different media significantly affected the cell growth index at a 95% confidence level. A comparison of the three culture media on the saffron cell growth index is presented in Fig. 1 . The results revealed that there was no statistically significant difference in the cell growth index between cells grown in SH and B 5 media, whereas there was a significant difference when compared with MS medium. The cell growth index in B 5 , SH, and MS media were reported as 2.18, 2.1, and 1.5, respectively (Fig. 1 ). Figure 2 shows the cell growth curve in different media over 14 weeks. As shown in this figure, the lag phase (initial slow cell growth phase) was not observed in any of the media, and the cells directly entered the exponential growth from the first week. Over all, in the first three weeks, the cell growth index was somehow similar in all three media. A significant increase in cell growth was observed in SH medium from the third week and in B 5 medium from the fourth week, in compared to MS medium. Cells grown in SH medium entered the cell death phase from the eleventh week, whereas, in B 5 medium started from the thirteenth week, and in MS medium from the twelfth week. The highest cell growth index was observed in SH and B 5 media in the eleventh week and in MS medium in the twelfth week. However, no significant difference in cell growth index was observed between the SH and B 5 media from the eighth week to the end of the experiment. Therefore, the most appropriate stage to end the saffron cell culture was considered to be the eight weeks from the culture time. The ANOVA results showed that the pH of the medium was influenced by the type of medium and the cell culture period. The results presented in Fig. 3 (a) indicate that the pH in SH and B 5 media did not differ significantly, whereas it was significantly higher than in MS medium. Crocin was detected in cells grown in the three culture media (SH, B 5 , and MS) using spectrophotometry. The results of this section also showed that the amount of crocin was significantly affected by the type of media (p ≤ 0.05). As shown in Fig. 3 (b), cells grown in SH medium produced the highest amount of crocin (1.42 mg. g − 1 dry cell weight), although there was no significant difference between the amount of crocin in SH and B 5 media (1.12 mg. g − 1 dry cell weight). The lowest amount of crocin was reported in cells grown in MS medium (0.8 mg. g − 1 dry cell weight). Since MS medium grown cells, the color was turned to brown, it was possible that they contained phenolic compounds with absorption wavelengths close to 440 nm (the wavelength absorbed by crocin), which can be considered as an error in the crocin measurement using the spectrophotometry. Therefore, to ensure the reliability of the spectrophotometry results, crocin was also measured using HPLC. A comparison of the results obtained from the spectrophotometry and HPLC methods is presented in Table 1 . The chromatograms resulting from the injection of extracts from cells grown in SH, B 5 , and MS media are shown in Figs. 4 , 5 , and 6 , respectively. Table 1 Comparison of crocin content in extracts from cells grown in different media using spectrophotometry and HPLC methods. Medium type HPLC Spectrophotometer SH 1.40 1.42 B 5 1.04 1.12 MS 0.34 0.8 The crocin content is presented in mg of crocin per g of cells dry weight. A comparative analysis of crocin measurements using spectrophotometry and high-performance liquid chromatography (HPLC) revealed consistent results when the cells retained their natural yellow color, with red cell masses visible, as observed in cultures grown in SH and B 5 media. This consistency indicates that spectrophotometry can be regarded as an inexpensive and reliable method for evaluating crocin levels in such conditions. Conversely, in samples where the cell color changed to brown or black, typical of those grown in MS medium, the results diverged. This color alteration may be attributed to the presence of phenolic compounds, which can interfere with crocin quantification using spectrophotometry. Therefore, in instances where cell coloration is compromised, HPLC is recommended as the preferred method for accurate crocin assessment. 4.2. Evaluation of Type and Concentration of Plant Growth Regulators The results of this section of the study indicated that in saffron suspension culture, the cell growth index was significantly affected by the type and concentration of plant growth regulators (p ≤ 0.05). A comparison of the mean cell growth index under the combined effect of different auxins and cytokinins is presented in Fig. 7 (a). The results showed that the combination of NAA from the auxins group with BA from the cytokinins group had the greatest impact on the cell growth index, resulting in the highest cell growth (1.07). The lowest cell growth (0.21) was observed in culture media containing 2,4-D combined with either KIN or BA. The relationship between the concentration of auxins group growth regulators and the cell growth index is shown in Fig. 7 (b). The results indicated that the application of 2 and 4 mg concentrations of these compounds in the culture medium resulted in less growth compared to the 1 mg concentration. Therefore, a lower concentration of auxins group growth regulators was reported as a more suitable option for saffron cell suspension culture. As shown in Fig. 7 (c), different concentrations of kinetin and benzyl adenine did not significantly affect the cell growth index. Therefore, it can be generally concluded that benzyl adenine at a concentration of 0.5 mg. L − 1 is a more suitable choice for saffron cell culture due to lower growth regulator consumption. A comparison between the spectrophotometry method for measuring the amount of crocin pigment in cells grown in saffron cell suspension culture and the image analysis method for calculating the intensity of their red color is presented in Figs. 8 and 9 . In Fig. 8 , the effect of different types of auxins group is compared. As shown in Fig. 8 (a), among the four hormones belonging to the auxins group, IAA produced the highest amount of crocin in the cells. This hormone (IAA) showed also the highest intensity of red color (a* value) in the cells (Fig. 8 (b)). In Fig. 9 , the effect of different types of cytokinins group at various concentrations on crocin production and the intensity of red color in cells is presented. The results in Fig. 9 (a) show that among benzyl adenine and kinetin, kinetin significantly increased the production of crocin metabolite. The average crocin production level in treatments containing kinetin was reported as 0.66 mg. g − 1 dry cell weight, while cells grown in treatments containing benzyl adenine showed 0.60 mg. g − 1 . The results regarding the role of cytokinin in crocin production using the image analysis (Fig. 9 -b) were completely consistent with the results obtained from the spectrophotometry (Fig. 9 -a). These results also showed that KIN had the greatest impact on producing red cells compared to BA. As shown in Fig. 9 -c, the concentration of cytokinins group also played a decisive role in crocin production. With a decrease in the concentration of these hormones in the medium, crocin production increased significantly. Similar results were obtained using the image analysis method, and culture media supplemented with kinetin at a concentration of 0.5 mg. L − 1 , which produced the highest intensity of red color in cells (Fig. 9 -d). Figure 10 , compares images of cell masses grown under different growth regulator treatments. As observed, the reddest cells grew in media supplemented with IAA and KIN compounds. 4.3. Optimization results in Bioreactor system: Using bioreactor system, the cell growth index reached to 4.3 and the amount of crocin from 1.05 mg/g increased to 3.43 mg. The chromatogram of crocin extraction from cells grown in a bioreactor is presented in Fig. 11 . It is possible to compare crocin produced in a bioreactor with crocin standard sample (Fig. 12 ). 5. Discussion Although numerous studies have investigated the optimization of saffron callus induction under the influence of medium type and hormonal combinations (Amini et al., 2023 , 2024; Ziaratnia & Amini 2021 ; Sajjadifard and Pazhouhandeh, 2015; Safarnejad et al., 2016 ; Firoozi et al., 2019 ; Mereu et al., 2019 ), but there has been limited research conducted on optimizing saffron cell suspension culture in liquid medium (Amini et al., 2021, 2022 ). This study was conducted for the first time with the aim of investigating the effect of basal medium and plant growth regulators (PGRs) types on cell mass growth and crocin metabolite production. The results of the first part of the research showed the type of medium significantly affect these factors. It was clearly indicated that SH and B 5 media are more suitable than MS medium for increasing cell mass growth. Additionally, SH medium was the best choice for producing the highest amount of crocin metabolite. Since the types and concentrations of other organic material, including growth regulators, carbohydrates, and vitamins, were similarly used in all three media, the observed variation in results can be attributed to the different compositions such as mineral salts. Given that both SH and B 5 media have lower salt concentrations compared to MS medium (Alizadeh, 2011), the findings from this study indicate that elevated mineral salt concentration hinder saffron cell growth and reduce crocin production. Additionally, cells cultivated in MS medium suffered significant damage, evident by their browning, likely resulting from the high salt levels present in this medium. Although MS medium has demonstrated effectiveness in inducing callus formation on solid media (Amini et al., 2023 ), its liquid form may contribute to its unsuitability for saffron cell culture, as it increases nutrient availability for cells in comparison to solid media (Sanchez-Ramos et al., 2020 ). In this research, no significant difference was observed between B 5 and SH media regarding crocin metabolite production, in compared to the MS medium. Due to the lower mineral element consumption and economic efficiency, SH medium can be chosen for further studies. Amini et al. ( 2023 ) reported that saffron corm cultivation on B 5 medium produced the highest red color intensity, indicating crocin levels in calli. Chen et al. ( 2003 ) also identified B 5 medium as the best option for saffron callus induction and crocin production. The findings from the second experiment of the study revealed that not only the type of hormones but also their concentration significantly impacts biomass growth and the production of crocin metabolite. In this part, image analysis technique employed to assess the red color intensity of the cells. To explore the correlation between the red color intensity of the cells and the amount of crocin produced, the spectrophotometry method utilized across all treatments. Amini et al. (2024) highlighted that crocin, as a red pigment, imparts a distinctive red color intensity to saffron calli, which can serve a reliable indicator of crocin presence. This correlation enable differentiation based on color intensity can be employed to quantitatively assess the red color intensity of the cells. They used an image analysis technique to assess the intensity of the cells' red color (Amini et al., 2024). Image analysis is a widely used non-destructive, cost-effective technology for rapid and accurate evaluation of desired factors in modern agriculture and food industries (Anup Vibhute and Bodhe, 2012). In this study, this technique using a scanner, computer, and ImageJ software was employed to assess the red color intensity (a*-index) of saffron cells from cell images. Subsequently, a-index and obtained crocin levels by spectrophotometry were compared across different treatments. These results fulfill one of the goals of this study that intended to determine whether image analysis can effectively replace spectrophotometry in identifying crocin-containing cells? The correlation between image analysis results (cell red color intensity) and spectrophotometry (crocin levels) indicated that image analysis could be used to screen crocin-containing cell masses with lower cost and time consumed associated with spectrophotometry and HPLC methods. However, image analysis only allows visual comparison of red color intensity, making it suitable for initial cell screening, while spectrophotometry and HPLC can quantitatively measure crocin levels. Previously, image analysis has been used to measure cell growth indices, color, cell density, and nature in suspension cultures of other plants. Researchers have described image analysis for evaluating these factors as non-destructive, simple, and efficient (Ibaraki and Kenji, 2001 ). Amini et al. ( 2023 ) also highlighted the usefulness of image analysis for determining saffron corm-derived callus growth indices. Although they measured callus red color intensity, they did not assess the relationship between red color intensity and crocin levels in saffron calli. This study not only evaluated crocin metabolite levels in saffron cell suspension culture under various media and hormones for the first time, but also examined the relationship between crocin levels and cell red color intensity. The correlation between spectrophotometry results (crocin levels) and image analysis a-index (red color intensity) demonstrates the precision and effectiveness of image analysis in this context. This research also contributes to existing literature by examining the impact of factors such as sucrose concentration, method of sucrose addition, and the concentration of MES buffer on saffron cell growth and crocin production (Amini et al., 2022 ). Unpublished data from our team indicate that yeast extract can enhance both biomass and crocin production, dependent on its concentration. Our results affirm the critical role of sucrose in promoting biomass and highlight the importance of lactose for crocin synthesis. Utilizing a bioreactor system, we successfully increased the cell growth index from 1.8 to 4.3, which surpasses previous reports indicating a maximum growth index of less than 1.5 in saffron cell cultures (Amini et al., 2022 ). Additionally, crocin production in this study reached 3.43 mg/g of dry cell weight, a significant improvement over prior findings of 2.0 mg/g (Amini et al., 2022 ). 6. Conclusion The findings of this study underscore the importance of both the type and concentration of basal culture medium and plant growth regulators in enhancing cell biomass growth and crocin production in saffron. Moreover, the optimized bioreactor system has demonstrated the potential to significantly elevate both biomass and crocin yields by incorporating these optimal factors. Building on this inquiry and prior research concerning the optimization of culture media regarding minerals, carbohydrates, PGRs, and elicitors, future investigations may further enhance biomass and crocin production through the exploration of various vitamins within the culture media. By advancing our understanding of these factors, we can optimize saffron cultivation methods and enhance the production of this economically significant metabolite. The results of this study demonstrated that there is a correlation between spectrophotometry results (crocin levels) and image analysis a-index (red color intensity), hence, it could be concluded that the image analysis is a precision, effectiveness and non-destructive method to estimate crocin content of the saffron cells. Abbreviations BA: Benzyladenine B 5 : Gamborg’s B 5 Medium HPLC: High Performance Liquid Chromatography IAA: Indole-3-acetic acid KIN: Kinetin MES: Morpholino-Ethane-Sulfonic acid buffer MS: Murashige and Skoog Medium NAA: Naphthaleneacetic acid PGRs: Plant Growth Regulators SH: Schenk and Hildebrandt Medium Declarations Conflict of interest Authors hereby declare that there is no conflict of Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contributions In this experiment, Ziaratnia and Hemmati designed the experiments. Amini carried out all the experiments, analyzed data and wrote the paper. All authors read and approved the manuscript. Acknowledgment: We extend our sincere gratitude to the Research Institute of Food Science and Technology (RIFST) for their financial support. We also thank Dr. Javad Feizy, a faculty member of this institute, for his valuable scientific guidance. Data Availability Statement: The data will be available on reasonable request. References Abobkar IMS, Elshahed AM (2012) Recent advances in plant in vitro culture. page number: 222 Ahmed SA, Baig MMV (2014) Biotic elicitor enhanced production of psoralen in suspension cultures of P soralea corylifolia L. Saudi J Biol Sci 21:499–504. https://doi.org/https://doi.org/10.1016/j.sjbs.2013.12.008 Ali H et al (2017) Optimization of callus induction and regeneration system for tomato ( Solanum lycopersicum L.) under different hormonal conditions. J Plant Biochem Biotechnol 26(4):439–448 Amini S, Hemmati Kh, Ziaratnia SM (2024) Image analysis for detection of crocin content of saffron corm derived cells under different physiological and chemical conditions. Res Innov Food Sci Technol 13(2):65–78. https://doi.org/10.22101/jrifst.2023.375070.1414 Amini S, Ziaratnia SM, Rajabzadeh G (2023) Improvement of Friable Callus Induction of Crocus sativus L. and Establishment of a Cell Suspension Culture System with High Biomass. J Hortic Postharvest Res 6(4):397–408. https://doi.org/10.22077/jhpr.2023.6850.1338 Amini S, Ziaratnia SM, Hemmati K (2022) Optimization of conditions for increasing of saffron cell biomass and crocin production in stirred bioreactor. Plant Cell Tissue Organ Cult 149(10). https://doi.org/10.1007/s11240-022-02233-4 Amini S, Ziaratnia SM (2021) The Effect of Developmental Stages of Corm, Types of Medium and Hormones in Callus Induction of Saffron. Journal of Horticulture and Postharvest Research 4(Special Issue: Recent Advances in Saffron. 43–56. https://doi.org/10.22077/jhpr.2021.3684.1166 Anup Vibhute, Bodhe SK (2012) Applications of image processing in agriculture: A Survey. Int J Comput Appl 52:34–39 Asgarpanah J, Darabi-Mahboub E, Mahboubi A et al (2013) In-Vitro evaluation of Crocus sativus L. petals and stamens as natural antibacterial agents against food-borne bacterial strains. Iran J Pharm Sci 9:69–82 Behbahani M, Shanehsazzadeh M, Hessami MJ (2011) Optimization of callus and cell suspension cultures of Barringtonia racemosa (Lecythidaceae family) for lycopene production. Sci Agric 68:69–76. https://doi.org/10.1590/s0103-90162011000100011 Castro AH et al (2015) Influence of nitrate and ammonium concentrations on callus induction and plant regeneration in Rice ( Oryza sativa L). Plant Cell Tissue Organ Cult 123(3):641–650 Chen S, Wang X, Zhao B et al (2003) Production of crocin using Crocus sativus callus by two-stage culture system. Biotechnol Lett 25:1235–1238. https://doi.org/10.1023/A:1025036729160 Chen S, Zhao B, Wang X et al (2004) Promotion of the growth of Crocus sativus cells and the production of crocin by rare earth elements. Biotechnol Lett 26:27–30. https://doi.org/10.1023/B:BILE.0000009455.31817.a5 Ebrahimzadeh H, Radjabian T, Karamian R (2000) In vitro production of floral buds and stigma-like structures on floral organs of Crocus sativus L. Pakistan J Bot 32:141–150 Ferri M, Tassoni A (2011) Chitosan as elicitor of health beneficial secondary metabolites in in vitro plant cell cultures. Handb Chitosan Res Appl 389–413 Firoozi B, Zare N, Sofalian O, Sheikhzade-Mosadegh P (2019) In vitro indirect somatic embryogenesis and secondary metabolites production in the saffron: Emphasis on ultrasound and plant growth regulators. Tarim Bilim Derg 25:1–10. https://doi.org/10.15832/ankutbd.538973 Gamborg O, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151–158 Gantait S et al (2018) Influence of basal media and plant growth regulators on in vitro callus induction and plant regeneration in Momordica charantia L. Biocatal Agric Biotechnol 15:266–273 Gregory MJ, Menary RC, Davies NW (2005) Effect of drying temperature and air flow on the production and retention of secondary metabolites in Saffron. J Agric Food Chem 53:5969–5975. https://doi.org/10.1021/jf047989j Ibaraki Y, Kenji K (2001) Application of image analysis to plant cell suspension cultures. Comput Electron Agric 30:193–203. https://doi.org/10.1016/S0168-1699(00)00164-2 Ismail H, Kayani SS, Kayani S-I et al (2019) Optimization of cell suspension culture of transformed and untransformed lettuce for the enhanced production of secondary metabolites and their pharmaceutical evaluation. 3 Biotech 9 Jan S, Wani AA, Kamili AN, Kashtwari M (2014) Distribution, chemical composition and medicinal importance of Saffron ( Crocus sativus L). Afr J Plant Sci 8:537–545 Javed SB et al (2020) Efficient callus induction and regeneration in Faba Bean ( Vicia faba L.) Using Gamborg's B 5 Medium. Plants 9(4):439 Jedinak A, Farago J, Psenakova I, Maliar T (2004) Approaches to flavonoid production in plant tissue cultures. Biol - Sect Cell Mol Biol 59:697–710 Kolewe M (2011) Development of plant cell culture processes to produce natural product pharmaceuticals: characterization, analysis, and modeling of plant cell aggregation 1–168 Mereu A, Dorsaf K, Scarpa G (2019) In vitro culture of Saffron: Hormones influence on the dvelopment of new shoots and callus. Plant Cell Biotechnol Mol Biol 20:511–520 Molina RV, Renau-Morata B, Nebauer SG et al (2010) Greenhous saffron culture temperature effects on flower emergence and vegetative growth of the plants. Acta Horticulturae. International Society for Horticultural Science (ISHS), Leuven, Belgium, pp 91–94 Moradi A, Zarinkamar F, Caretto S, Azadi P (2018) Influence of thidiazuron on callus induction and crocin production in corm and style explants of Crocus sativus L. Acta Physiol Plant 40. https://doi.org/10.1007/s11738-018-2760-2 Moradi A, Zarinkamar F, De Domenico S et al (2020) Salycilic acid induces exudation of crocin and phenolics in saffron suspension-cultured cells. Plants 9. https://doi.org/10.3390/plants9080949 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497 Nagella P, Murthy HN (2010) Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresour Technol 101:6735–6739. https://doi.org/10.1016/j.biortech.2010.03.078 Oliveira Y, Lima A (2016) Role of nitrate and ammonium on pH stability and callus induction in Cassava ( Manihot esculenta Crantz). J Plant Nutr 39(2):237–247 Safarnejad A, Alamdari SBL, Darroudi H, Dalir M (2016) The effect of different hormones on callus induction, regeneration and multiplication of saffron ( Crocus sativus l.) corms. Saffron Agron Technol 4:143–154. https://doi.org/10.22048/jsat.2016.17364 Sajjadi fard M, Pazhohandeh M (2015) Study on effect of type of explant and hormone on callus induction and regeneration in Saffron ( Crocus sativus ). Saffron Agron Technol 3:195–202 Sanchez-Ramos M, Alvarez L, Romero-Estrada A, Bernabe-Antonio A, Marquina-Bahena S, Cruz-Sosa F (2020) Establishment of a cell suspension culture of Ageratina pichinchensis (Kunth) for the improved production of anti-inflammatory compounds. Plants 9(10):1398. https://doi.org/10.3390/plants9101398 Santos RB, Abranches R, Fischer R et al (2016) Putting the Spotlight back on plant suspension cultures. Front Plant Sci 7. https://doi.org/10.3389/fpls.2016.00297 Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199 Sharaf-Eldin M, Elkholy S, Fernandez J-A et al (2008) Bacillus subtilis FZB24 affects flower quantity and quality of saffron ( Crocus sativus ). Planta Med 74:1316–1320. https://doi.org/10.1055/s-2008-1081293 Winson KWS, Chew BL, Sathasivam K, Subramaniam S (2020) The establishment of callus and cell suspension cultures of Hylocereus costaricensis for the production of betalain pigments with antioxidant potential. Ind Crops Prod 155:112750. https://doi.org/10.1016/j.indcrop.2020.112750 Yang B, Guo Z, Liu R (2005) Crocin synthesis mechanism in Crocus sativus . Tsinghua Sci Technol 10:567–572. https://doi.org/10.1016/S1007-0214(05)70119-X Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6857423","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476186176,"identity":"325272a9-22af-47ea-bbe6-3876f2d3b0ee","order_by":0,"name":"Seyed Mahdi Ziaratnia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIie3RsUoDMRzH8d8RuCwB1xSKbyD8oRBx8V7lxBdo6SIUNOBwyz2M3R3+JcMtgnOXYhE6dbguBeEG/xbdbK5jh3yHPyHwIQkBUqkzjQ9TIfsAXf7uUT+xQhSBRjCnEBwIkMv4I5GuvF7z1+vt47VWm9nDmIoCat1ivDpKHKNc1Jt7e/Ocu+Ub0V2NfGRB0xhhNqwsBbilp66Uizl5SxkhmV90/CRE7yeeqDDQ+x6iEAwHIcZlQrIapueUkCMMuRm8BDMd+J+3yMKWMdJUn7stzy7ovZnvfEeFrqp523bHifzgfzsRkEqlUqkT+gY7GUrpm2bEBAAAAABJRU5ErkJggg==","orcid":"","institution":"Research Institute of Food Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Seyed","middleName":"Mahdi","lastName":"Ziaratnia","suffix":""},{"id":476186177,"identity":"30d114b7-e732-474a-a298-8b239a5fd104","order_by":1,"name":"Somiyeh Amini","email":"","orcid":"","institution":"Gorgan University of Agricultural Sciences and Natural Resources","correspondingAuthor":false,"prefix":"","firstName":"Somiyeh","middleName":"","lastName":"Amini","suffix":""},{"id":476186178,"identity":"45d765d5-d04e-4b40-a530-d7d0403e0f22","order_by":2,"name":"Khodayar Hemmati","email":"","orcid":"","institution":"Gorgan University of Agricultural Sciences and Natural Resources","correspondingAuthor":false,"prefix":"","firstName":"Khodayar","middleName":"","lastName":"Hemmati","suffix":""}],"badges":[],"createdAt":"2025-06-09 21:14:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6857423/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6857423/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85845995,"identity":"7fc3a872-a65a-48a9-851c-7936d7bdae27","added_by":"auto","created_at":"2025-07-02 09:40:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44473,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different culture media on the cell growth index of saffron in suspension culture. Growth regulators in all three media included NAA (2 mg. L\u003csup\u003e-1\u003c/sup\u003e) and BA (1 mg. L\u003csup\u003e-1\u003c/sup\u003e). (Identical letters indicate no significant difference) (p≤0.05).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/e82d41d760d8fc9010546756.jpg"},{"id":85848742,"identity":"fa0c41cf-a424-4619-b0fe-1b53ceeb7fc6","added_by":"auto","created_at":"2025-07-02 10:04:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96209,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different culture media on the cell growth index of saffron over 14 weeks. Growth regulators in all three media included NAA (2 mg. L\u003csup\u003e-1\u003c/sup\u003e) and BA (1 mg. L\u003csup\u003e-1\u003c/sup\u003e) (p≤0.05).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/137026b6ed2df138a2eda349.jpg"},{"id":85846889,"identity":"a7c4a692-f08e-4fad-9364-fba840809839","added_by":"auto","created_at":"2025-07-02 09:48:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49560,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different culture media on (a) the pH of the medium and (b) the amount of crocin in cells grown in each culture medium. The amount of crocin was measured using spectrophotometry. Growth regulators in all three culture media included NAA (2 mg. L\u003csup\u003e-1\u003c/sup\u003e) and BA (1 mg.L\u003csup\u003e-1\u003c/sup\u003e) (Identical letters indicate no significant difference) (p≤0.05).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/4c90c0bfdd65883ae4334211.jpg"},{"id":85848372,"identity":"3db62c73-0a4c-4cca-b75f-5a6fe969f1d1","added_by":"auto","created_at":"2025-07-02 09:56:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15327,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatogram of crocin in cells grown in SH medium.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/7ddfdbfddb8d8fd47f18b958.jpg"},{"id":85846892,"identity":"f1b39adb-b077-4c03-b4e8-d5fe843351f9","added_by":"auto","created_at":"2025-07-02 09:48:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16781,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatogram of crocin in cells grown in B\u003csub\u003e5\u003c/sub\u003e medium.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/e47ac0a6493078384c5b587e.jpg"},{"id":85846007,"identity":"45548204-b181-4bec-a439-8b79508664cc","added_by":"auto","created_at":"2025-07-02 09:40:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":17664,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatogram of crocin in cells grown in MS medium.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/aed615c7a123d00ef74f93a5.jpg"},{"id":85848376,"identity":"42fd3858-4459-4483-bc28-b1b587228a2b","added_by":"auto","created_at":"2025-07-02 09:56:41","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52089,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different types of auxins and cytokinins group at various concentrations on the cell growth index of saffron in suspension culture: (a) Interaction effect of different types of auxins and cytokinins group; (b) Different concentrations of auxins group; (c) Interaction effect of different types of cytokinins group at various concentrations (Identical letters indicate no significant difference) (p≤0.05).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/986a4d8420a7b67c2830d95f.jpg"},{"id":85846013,"identity":"da788a98-a623-4f60-9add-8a2953494719","added_by":"auto","created_at":"2025-07-02 09:40:41","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":45019,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the effect of different types of auxin group on crocin content. (a) Crocin was measured by spectrophotometry; (b) the intensity of red color in cells (index a) measured by image analysis. Identical letters indicate no significant difference (p≤0.05).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/7aac54396a1f31be95570ade.jpg"},{"id":85846009,"identity":"5228d7d7-0fb5-4d7a-a4c9-d5889000fa18","added_by":"auto","created_at":"2025-07-02 09:40:41","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":72438,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different types of cytokinin group hormones on crocin metabolite levels by different quantification methods. (a) using spectrophotometry; (b) Assessment the cell red color intensity (a-index) using image analysis; (c) Evaluation of the effect of cytokinin concentrations on crocin metabolite levels using spectrophotometry; (d) Evaluation of the effect of cytokinin concentrations in cell red color intensity (a-index) using image analysis. Identical letters indicate no significant difference (p≤0.05).\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/c78ba78a871ab1fbb5bcfa8a.jpg"},{"id":85846895,"identity":"252429c9-7c7d-4c6f-9ff5-ac56c1a334e3","added_by":"auto","created_at":"2025-07-02 09:48:41","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":96551,"visible":true,"origin":"","legend":"\u003cp\u003eVisual comparison of different treatments containing various growth regulators on the color of saffron cells grown in cell culture.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/3a41fe9e5cc39fa2404d1c19.jpg"},{"id":85846014,"identity":"e7c51791-fc5c-4e17-806c-259f6555b45d","added_by":"auto","created_at":"2025-07-02 09:40:41","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":13023,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of crocin extracted from cells grown in the bioreactor (optimized to increase crocin production).\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/c58834f793fdd8b89a88eb8b.jpg"},{"id":85848378,"identity":"bcbd62b9-064d-43ec-a411-368668e8a3a5","added_by":"auto","created_at":"2025-07-02 09:56:41","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":41079,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of crocin standard sample.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/05c2201c768199369ac1c637.jpg"},{"id":88402880,"identity":"bba57a2c-754d-4d04-8c8d-cfef88ba8961","added_by":"auto","created_at":"2025-08-06 07:17:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1385852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6857423/v1/8ebf6bf9-234d-47ca-9bff-f1980207b23f.pdf"}],"financialInterests":"","formattedTitle":"Optimization of Regulatory Factors Affecting Biomass Growth and Crocin Production of Saffron Cells in Bioreactor","fulltext":[{"header":"Key messages","content":"\u003cp\u003eThe bioreactor serves as a highly efficient platform for cell proliferation and enhancing the biosynthesis of crocin metabolite in saffron (\u003cem\u003eCrocus sativus\u003c/em\u003e) cell cultures in separated medium ingredients and growth regulators combinations. The image analysis a-index (red color intensity), is a precision, effectiveness and non-destructive method to estimate crocin content of the saffron cells.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003ePlant cell culture systems present an innovative and efficient alternative for producing valuable secondary metabolites, surpassing the limitations of traditional extraction methods from plant organs (Ferri and Tassoni, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Despite the advantages, this technology faces challenges such as low metabolite accumulation in cells. Various strategies have been proposed to overcome this issue (Kolewe, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). One of the most important options to enhance the productivity of plant cell cultures is the optimization of the medium (Santos et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Plant culture media contain various components including nutrients, vitamins, amino acids, and plant growth regulators, among which the type and concentration of growth regulators play a crucial role in cell growth and metabolites synthetize. In addition to hormonal compounds, researchers have proven that the type of culture medium also affects the aforementioned factors (Nagella and Murthy, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Commonly used plant culture media include Murashige and Skoog (MS), Linsmaier and Skoog (LS), Nitsch and Nitsch (NN), Gamborg (B\u003csub\u003e5\u003c/sub\u003e), and Schenk and Hildebrandt (SH) (Abobkar and Elshahed, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In the past decade, several studies have highlighted the importance of optimizing culture media and hormonal compositions for increasing the efficiency of callus induction regeneration in various plant species (Ali et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Javed et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gantait et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Along to these efforts, extensive research has been conducted on optimizing callus induction in saffron (\u003cem\u003eCrocus sativus\u003c/em\u003e L.) using solid culture media influenced by different types and concentrations of auxin and cytokinin growth regulators (Sajjadi fard and Pazhohandeh, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Safarnejad et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Firoozi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mereu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These studies have shown that the type of basal medium and growth regulators significantly affect saffron callus growth and secondary metabolites production, including carotenoids, phenolic compounds, and crocin (Moradi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Other researchers have shown that two-stage saffron callus culture effectively increases crocin content. In this approach, growth regulators are optimized for callus induction in the first stage, followed by suitable conditions for crocin production. In two-stage culture, crocin content increased by 295% compared to single-stage culture. They reported Gamborg\u0026rsquo;s medium enriched with 300 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e hydrolyzed casein as the best medium for callus growth and crocin production. They also found that the optimal growth regulator combination for maximum callus growth and crocin production was 1 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e benzyladenine (BA)\u0026thinsp;+\u0026thinsp;2 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e naphthaleneacetic acid (NAA) and 0.5 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BA\u0026thinsp;+\u0026thinsp;2 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indoleacetic acid (IAA) respectively (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmini et al. (2024) investigated the effects of saffron corm physiological age, basal culture media, and growth regulator types in different concentrations on callus induction and crocin production in solid medium. They could quantify the red color intensity of the callus as an index for measuring crocin by image analysis and screening the callus growth. The results of their research showed that if the initial callus formation of mature corms is performed on B\u003csub\u003e5\u003c/sub\u003e culture medium containing NAA (2 mg/l)\u0026thinsp;+\u0026thinsp;BA (1 mg/l) for 6 weeks and then the calli obtained are re-cultured on B\u003csub\u003e5\u003c/sub\u003e medium containing IAA (2 mg/l)\u0026thinsp;+\u0026thinsp;KIN (1 mg/l), the largest amount of cell mass with the highest intensity of red color (containing the metabolite crocin) will be produced (Amini et al., 2024). The researchers announced that Schenk and Hildebrandt (SH) basal medium with NAA (2 mg l\u0026thinsp;\u0026minus;\u0026thinsp;1) and BA (1 mg l\u0026thinsp;\u0026minus;\u0026thinsp;1) supplemented with 2.5 mM of MES as well as gradual increment of sucrose from 3 to 6% caused the highest cell biomass and crocin production. The results of saffron cell culture in the bioreactor also showed that, aeration was found to be an inhibiting factor for the production of crocin metabolite and natural pH fluctuation is a suitable condition for saffron biomass cell growth and crocin production (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The results of the another research showed that among the different carbon sources, sucrose and lactose, at a concentration of 3% were found to be the best carbohydrates for the highest production of cell biomass and crocin metabolite, respectively. The elicitation results also showed that yeast extract at a concentration of 1.5% increased crocin to 1.5 times in 5 days after stimulation of the cells (unpublished).\u003c/p\u003e \u003cp\u003eDespite existing researches, no reports have focused on optimizing culture medium type and plant growth regulators in saffron cell suspension culture. Therefore, this study is the first to optimize these factors to enhance cell growth and crocin production in saffron cell culture. Then, the results of this study were combined with the results obtained from other studies in the field of elicitor application, carbohydrate type, and bioreactor aeration, and their effects on biomass growth and crocin production in the bioreactor were evaluated. This study also compares different crocin measurement methods, including high-performance liquid chromatography (HPLC) and spectrophotometry with image analysis technique for red color intensity of cells indicating crocin content.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Callus Induction\u003c/h2\u003e \u003cp\u003eThe callus was prepared under optimized conditions established in prior callus induction experiments conducted by Amini et al. (2024). The findings revealed that explants derived from mature saffron corms exhibit a significantly higher ability to produce callus and crocin pigments those from immature corms. Consequently, this research utilized mature corms harvested in late May for explant preparation. After removing outer shells, corms were disinfected with 70% EtOH for 1 min and then 1% sodium hypochlorite solution for 15 min. This step was followed by three times rinsing with sterilized distilled water, each time for 5 min. Under sterile conditions within a laminar flow hood, the sterilized corms were sliced into 1 cm\u0026times;1 cm pieces with a thickness of 1 mm. To enhance callus induction and growth, explants cultured on B\u003csub\u003e5\u003c/sub\u003e medium enriched with NAA (2 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and BA (1 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for six weeks. Following this incubation period, the calli transferred to B\u003csub\u003e5\u003c/sub\u003e medium supplemented with 2 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of IAA and 1 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of KIN to increase crocin content. In both stages, the explants were stored in darkness at a controlled temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell Suspension Establishment\u003c/h2\u003e \u003cp\u003eAfter the producing a desired volume of red color calli, the cell suspension establishment was implemented to homogenize the cells in terms of growth and acclimate them to a liquid culture medium. This step was performed with slight modifications according to the method presented by Amini et al (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). An effective hormonal combination was chosen to enhance cell biomass in a liquid SH medium composed of NAA (2 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;+\u0026thinsp;BA (1 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 3% sucrose. Subsequently, one-liter flasks containing 200 mL of this medium and 20 grams of finely processed cells were placed on a shaker operating at 120 rpm in complete darkness, maintained at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C. After two weeks, the cells cultivated in this medium were utilized for experimental treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Evaluation of Culture Medium Type:\u003c/h2\u003e \u003cp\u003eAfter establishment of the cell suspension culture, the first experiment, involving the examination of the effects of three basal media on cell growth and crocin production, was conducted in flask system. For this purpose, MS, B\u003csub\u003e5\u003c/sub\u003e and SH basal medium with B\u003csub\u003e5\u003c/sub\u003e vitamins were selected and supplemented with NAA (2 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;+\u0026thinsp;BA (1 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 3% sucrose. Each 100 ml flask with containing 20 ml of medium was inoculated with 1 gram of cells obtained from the cell suspension establishment stage. The flasks were maintained in darkness at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C on an orbital shaker at 120 rpm. Weekly measurements included the pH of the culture medium and cell biomass growth (cell growth index). At the end of the growth period (end of the 14th week), the crocin content was measured and compared based on two systems of quantifications-spectrophotometry and HPLC. After statistical analysis of the recorded data, the most suitable medium for cell growth and crocin production in cell suspension culture was identified.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Measurement of Cell Growth Index in Suspension Culture:\u003c/h2\u003e \u003cp\u003eTo measure cell growth, the method by Amini et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) was used, applying the Eq.\u0026nbsp;1:\u003c/p\u003e \u003cp\u003eEquation 1: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{G}\\text{I}=\\frac{W2-W1}{W1}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWhere GI: is the cell growth index, W\u003csub\u003e1\u003c/sub\u003e: is the initial cell weight at the start of the culture period, and W\u003csub\u003e2\u003c/sub\u003e: is the secondary cell weight at the time of measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Drying and Extraction of Crocin from Cells:\u003c/h2\u003e \u003cp\u003eFor crocin quantification, HPLC and spectrophotometry were used. The cells were dried and extracted using the method of Amini et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The grown cells were separated from the liquid medium, dried in an oven at 35\u0026deg;C for 48 hours, then 0.05 g of dried cell powder was transferred to Falcon tubes with 5 mL of 50% ethanol and kept in darkness on a shaker at room temperature for 24 hours. The extract was separated from cell debris using centrifugation at 8000 rpm for 10 minutes. The extracted solution was filtered through a syringe filters (0.45\u0026micro;M) and stored in 4\u0026deg;C in darkness until HPLC or spectrophotometry analysis (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Crocin Measurement by Spectrophotometry:\u003c/h2\u003e \u003cp\u003eThe extraction solution was applied on a spectrophotometer model CT-5700 with a quartz cell and the absorbance was recorded at 440 nm. Standard crocin solution (C44H64O24 Mr 976.98) was prepared in a serial dilution with the concentrations ranging from 25 to 200 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using 50% ethanol as the solvent. The recorded absorbance at 440 nm was plotted to make a standard crocin. Ultimately, the crocin concentration in the cell extracts was calculated based on sample absorbance at 440 nm and equation derived from the standard curve (Eq.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eEquation 2: Y\u0026thinsp;=\u0026thinsp;0.128X\u0026thinsp;+\u0026thinsp;0.0347\u003c/p\u003e \u003cp\u003eWhere Y is the absorbance of the cell extract at 440 nm and X is the crocin concentration (mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Results were then reported as mg crocin per gram of dry cell weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. High-Performance Liquid Chromatography (HPLC):\u003c/h2\u003e \u003cp\u003eCrocin was identified and quantified by HPLC using a Waters 1525 binary HPLC pump, equipped with Waters 2489 UV/Visible Detector, and Breeze software. Column C18, ODS. 250 mm, 4.6 mm, particle size 5.0 \u0026micro;m was employed. Methanol (HPLC grade) with gradient flow rate (20 to 80%) at 1 ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used as mobile phase. Detection wavelength adjusted on 440 nm for crocin detection. Duration of the test was 60 min and the volume of the injection was 20 \u0026micro;l. Temperature was adjusted at 30\u0026deg;C. Standard crocin samples were prepared with ethanol at three different concentrations (0.024, 0.048, 0.072 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The standard curve was obtained by injecting different concentrations into the HPLC system. Cell extracts were injected into the system, and the crocin concentration was calculated by comparing the peak area in the chromatogram with the standard crocin chromatogram (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Investigation on Plant Growth Regulators Type and Concentration:\u003c/h2\u003e \u003cp\u003eIn the second part of this research, to study the effect of the type and concentration of plant growth regulators, the selected culture medium from the previous experiment was used as the basal. This medium was supplemented with four types of auxins group (IAA, IBA, NAA, 2,4-D) at three concentrations (1, 2, and 4 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and two types of cytokinins (BA and Kin) at three concentrations (0.5, 1, and 2 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with 3% sucrose. Cell cultures were performed in 100 mL flasks according to the previous experimental conditions. At the end of the experimental period, the red color intensity of the cells (a* index) was determined using image analysis by Image J software (Amini et al., 2024). After drying the cells (at 35\u0026deg;C) and extracting them, the crocin content was measured using spectrophotometry method presented in 2.3.3 section. In this part, we aimed to find a correlation between the red color intensity of the cells and crocin content by comparing the image analysis results (red color intensity) with the spectrophotometry results (crocin content).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Image Analysis Method:\u003c/h2\u003e \u003cp\u003eImage analysis was used to determine the red color intensity of cell masses. At the end of the culture period, the cell masses were placed on a scanner covered with a black box for darkness, and the cells were scanned. The Image J software (version 1.45s) and L*a*b* color space were used to determine the color parameter of the callus. The a* index indicating red color intensity (Mohd Jusoh et al., 2009). Selected regions within images were analyzed, and the a* index was calculated accordingly (Amini et al., 2024).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Final optimization in bioreactor system with fed-batch cultivation:\u003c/h2\u003e \u003cp\u003eThis study integrated findings from the medium optimization and various plant growth regulators to enhance cell biomass production and crocin content using a bioreactor system. Fifty grams of cells obtained from the cell suspension stage were inoculated into one liter of the optimized culture medium, supplemented with MES (2.5 mM) and 3% sucrose. Gradual feeding with a 3% sucrose solution commenced on the seventh day, with aeration maintained at 0.5 vvm throughout the cultivation period (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the fifth week, 0.5% yeast extract was added to the medium, and the bioreactor was emptied after seven days to measure the cell growth index.\u003c/p\u003e \u003cp\u003eFor crocin optimization in bioreactor settings, 50 grams of cells were introduced into one liter of the selected medium with optimal hormonal treatment, supplemented as before with MES (2.5 mM) and 3% lactose. No aeration was applied during this culture phase (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Gradual feeding of the medium with 3% sucrose began at the end of the first week, followed by the addition of 1.5% yeast extract in the fifth week, with crocin content measured 5 days after treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Statistical Analysis","content":"\u003cp\u003eExperiments in the flask system were arranged in a factorial experiment following a completely randomized design. The first experiment assessed three types of media (MS, SH, B\u003csub\u003e5\u003c/sub\u003e) over 14 weeks. The second experiment evaluated four auxin types (NAA, IAA, IBA, 2,4-D) at three concentration levels (1, 2, and 4 mg/L), and two cytokinin types (BA, KIN) at concentrations of 0.5, 1, and 2 mg/L. Each experimental combination was conducted in quadrulicate. Statistical analyses were performed using SPSS software, with mean comparisons made utilizing Duncan\u0026rsquo;s multiple range test at a 95% confidence level. In the bioreactor system, additional statistical treatments will be detailed based on experimental outcomes.\u003c/p\u003e"},{"header":"4. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Assessment of Medium Type\u003c/h2\u003e \u003cp\u003eThe results of the analysis of variance (ANOVA) indicated that different media significantly affected the cell growth index at a 95% confidence level. A comparison of the three culture media on the saffron cell growth index is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results revealed that there was no statistically significant difference in the cell growth index between cells grown in SH and B\u003csub\u003e5\u003c/sub\u003e media, whereas there was a significant difference when compared with MS medium. The cell growth index in B\u003csub\u003e5\u003c/sub\u003e, SH, and MS media were reported as 2.18, 2.1, and 1.5, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the cell growth curve in different media over 14 weeks. As shown in this figure, the lag phase (initial slow cell growth phase) was not observed in any of the media, and the cells directly entered the exponential growth from the first week. Over all, in the first three weeks, the cell growth index was somehow similar in all three media. A significant increase in cell growth was observed in SH medium from the third week and in B\u003csub\u003e5\u003c/sub\u003e medium from the fourth week, in compared to MS medium. Cells grown in SH medium entered the cell death phase from the eleventh week, whereas, in B\u003csub\u003e5\u003c/sub\u003e medium started from the thirteenth week, and in MS medium from the twelfth week. The highest cell growth index was observed in SH and B\u003csub\u003e5\u003c/sub\u003e media in the eleventh week and in MS medium in the twelfth week. However, no significant difference in cell growth index was observed between the SH and B\u003csub\u003e5\u003c/sub\u003e media from the eighth week to the end of the experiment. Therefore, the most appropriate stage to end the saffron cell culture was considered to be the eight weeks from the culture time.\u003c/p\u003e \u003cp\u003eThe ANOVA results showed that the pH of the medium was influenced by the type of medium and the cell culture period. The results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e (a) indicate that the pH in SH and B\u003csub\u003e5\u003c/sub\u003e media did not differ significantly, whereas it was significantly higher than in MS medium.\u003c/p\u003e \u003cp\u003eCrocin was detected in cells grown in the three culture media (SH, B\u003csub\u003e5\u003c/sub\u003e, and MS) using spectrophotometry. The results of this section also showed that the amount of crocin was significantly affected by the type of media (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e (b), cells grown in SH medium produced the highest amount of crocin (1.42 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry cell weight), although there was no significant difference between the amount of crocin in SH and B\u003csub\u003e5\u003c/sub\u003e media (1.12 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry cell weight). The lowest amount of crocin was reported in cells grown in MS medium (0.8 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry cell weight).\u003c/p\u003e \u003cp\u003eSince MS medium grown cells, the color was turned to brown, it was possible that they contained phenolic compounds with absorption wavelengths close to 440 nm (the wavelength absorbed by crocin), which can be considered as an error in the crocin measurement using the spectrophotometry. Therefore, to ensure the reliability of the spectrophotometry results, crocin was also measured using HPLC. A comparison of the results obtained from the spectrophotometry and HPLC methods is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The chromatograms resulting from the injection of extracts from cells grown in SH, B\u003csub\u003e5\u003c/sub\u003e, and MS media are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of crocin content in extracts from cells grown in different media using spectrophotometry and HPLC methods.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpectrophotometer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003csub\u003e\u003cb\u003e5\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eThe crocin content is presented in mg of crocin per g of cells dry weight.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis of crocin measurements using spectrophotometry and high-performance liquid chromatography (HPLC) revealed consistent results when the cells retained their natural yellow color, with red cell masses visible, as observed in cultures grown in SH and B\u003csub\u003e5\u003c/sub\u003e media. This consistency indicates that spectrophotometry can be regarded as an inexpensive and reliable method for evaluating crocin levels in such conditions.\u003c/p\u003e \u003cp\u003eConversely, in samples where the cell color changed to brown or black, typical of those grown in MS medium, the results diverged. This color alteration may be attributed to the presence of phenolic compounds, which can interfere with crocin quantification using spectrophotometry. Therefore, in instances where cell coloration is compromised, HPLC is recommended as the preferred method for accurate crocin assessment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Evaluation of Type and Concentration of Plant Growth Regulators\u003c/h2\u003e \u003cp\u003eThe results of this section of the study indicated that in saffron suspension culture, the cell growth index was significantly affected by the type and concentration of plant growth regulators (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). A comparison of the mean cell growth index under the combined effect of different auxins and cytokinins is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003e (a). The results showed that the combination of NAA from the auxins group with BA from the cytokinins group had the greatest impact on the cell growth index, resulting in the highest cell growth (1.07). The lowest cell growth (0.21) was observed in culture media containing 2,4-D combined with either KIN or BA. The relationship between the concentration of auxins group growth regulators and the cell growth index is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003e (b). The results indicated that the application of 2 and 4 mg concentrations of these compounds in the culture medium resulted in less growth compared to the 1 mg concentration. Therefore, a lower concentration of auxins group growth regulators was reported as a more suitable option for saffron cell suspension culture.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003e (c), different concentrations of kinetin and benzyl adenine did not significantly affect the cell growth index. Therefore, it can be generally concluded that benzyl adenine at a concentration of 0.5 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is a more suitable choice for saffron cell culture due to lower growth regulator consumption.\u003c/p\u003e \u003cp\u003eA comparison between the spectrophotometry method for measuring the amount of crocin pigment in cells grown in saffron cell suspension culture and the image analysis method for calculating the intensity of their red color is presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e. In Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the effect of different types of auxins group is compared. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e (a), among the four hormones belonging to the auxins group, IAA produced the highest amount of crocin in the cells. This hormone (IAA) showed also the highest intensity of red color (a* value) in the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e (b)).\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the effect of different types of cytokinins group at various concentrations on crocin production and the intensity of red color in cells is presented. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e (a) show that among benzyl adenine and kinetin, kinetin significantly increased the production of crocin metabolite. The average crocin production level in treatments containing kinetin was reported as 0.66 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry cell weight, while cells grown in treatments containing benzyl adenine showed 0.60 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The results regarding the role of cytokinin in crocin production using the image analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e-b) were completely consistent with the results obtained from the spectrophotometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e-a). These results also showed that KIN had the greatest impact on producing red cells compared to BA.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e-c, the concentration of cytokinins group also played a decisive role in crocin production. With a decrease in the concentration of these hormones in the medium, crocin production increased significantly. Similar results were obtained using the image analysis method, and culture media supplemented with kinetin at a concentration of 0.5 mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which produced the highest intensity of red color in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e-d).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e, compares images of cell masses grown under different growth regulator treatments. As observed, the reddest cells grew in media supplemented with IAA and KIN compounds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Optimization results in Bioreactor system:\u003c/h2\u003e \u003cp\u003eUsing bioreactor system, the cell growth index reached to 4.3 and the amount of crocin from 1.05 mg/g increased to 3.43 mg. The chromatogram of crocin extraction from cells grown in a bioreactor is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e. It is possible to compare crocin produced in a bioreactor with crocin standard sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eAlthough numerous studies have investigated the optimization of saffron callus induction under the influence of medium type and hormonal combinations (Amini et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, 2024; Ziaratnia \u0026amp; Amini \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sajjadifard and Pazhouhandeh, 2015; Safarnejad et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Firoozi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mereu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), but there has been limited research conducted on optimizing saffron cell suspension culture in liquid medium (Amini et al., 2021, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This study was conducted for the first time with the aim of investigating the effect of basal medium and plant growth regulators (PGRs) types on cell mass growth and crocin metabolite production. The results of the first part of the research showed the type of medium significantly affect these factors. It was clearly indicated that SH and B\u003csub\u003e5\u003c/sub\u003e media are more suitable than MS medium for increasing cell mass growth. Additionally, SH medium was the best choice for producing the highest amount of crocin metabolite. Since the types and concentrations of other organic material, including growth regulators, carbohydrates, and vitamins, were similarly used in all three media, the observed variation in results can be attributed to the different compositions such as mineral salts. Given that both SH and B\u003csub\u003e5\u003c/sub\u003e media have lower salt concentrations compared to MS medium (Alizadeh, 2011), the findings from this study indicate that elevated mineral salt concentration hinder saffron cell growth and reduce crocin production. Additionally, cells cultivated in MS medium suffered significant damage, evident by their browning, likely resulting from the high salt levels present in this medium. Although MS medium has demonstrated effectiveness in inducing callus formation on solid media (Amini et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), its liquid form may contribute to its unsuitability for saffron cell culture, as it increases nutrient availability for cells in comparison to solid media (Sanchez-Ramos et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this research, no significant difference was observed between B\u003csub\u003e5\u003c/sub\u003e and SH media regarding crocin metabolite production, in compared to the MS medium. Due to the lower mineral element consumption and economic efficiency, SH medium can be chosen for further studies. Amini et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that saffron corm cultivation on B\u003csub\u003e5\u003c/sub\u003e medium produced the highest red color intensity, indicating crocin levels in calli. Chen et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) also identified B\u003csub\u003e5\u003c/sub\u003e medium as the best option for saffron callus induction and crocin production.\u003c/p\u003e \u003cp\u003eThe findings from the second experiment of the study revealed that not only the type of hormones but also their concentration significantly impacts biomass growth and the production of crocin metabolite. In this part, image analysis technique employed to assess the red color intensity of the cells. To explore the correlation between the red color intensity of the cells and the amount of crocin produced, the spectrophotometry method utilized across all treatments. Amini et al. (2024) highlighted that crocin, as a red pigment, imparts a distinctive red color intensity to saffron calli, which can serve a reliable indicator of crocin presence. This correlation enable differentiation based on color intensity can be employed to quantitatively assess the red color intensity of the cells. They used an image analysis technique to assess the intensity of the cells' red color (Amini et al., 2024). Image analysis is a widely used non-destructive, cost-effective technology for rapid and accurate evaluation of desired factors in modern agriculture and food industries (Anup Vibhute and Bodhe, 2012). In this study, this technique using a scanner, computer, and ImageJ software was employed to assess the red color intensity (a*-index) of saffron cells from cell images. Subsequently, a-index and obtained crocin levels by spectrophotometry were compared across different treatments. These results fulfill one of the goals of this study that intended to determine whether image analysis can effectively replace spectrophotometry in identifying crocin-containing cells? The correlation between image analysis results (cell red color intensity) and spectrophotometry (crocin levels) indicated that image analysis could be used to screen crocin-containing cell masses with lower cost and time consumed associated with spectrophotometry and HPLC methods. However, image analysis only allows visual comparison of red color intensity, making it suitable for initial cell screening, while spectrophotometry and HPLC can quantitatively measure crocin levels. Previously, image analysis has been used to measure cell growth indices, color, cell density, and nature in suspension cultures of other plants. Researchers have described image analysis for evaluating these factors as non-destructive, simple, and efficient (Ibaraki and Kenji, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Amini et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also highlighted the usefulness of image analysis for determining saffron corm-derived callus growth indices. Although they measured callus red color intensity, they did not assess the relationship between red color intensity and crocin levels in saffron calli. This study not only evaluated crocin metabolite levels in saffron cell suspension culture under various media and hormones for the first time, but also examined the relationship between crocin levels and cell red color intensity. The correlation between spectrophotometry results (crocin levels) and image analysis a-index (red color intensity) demonstrates the precision and effectiveness of image analysis in this context.\u003c/p\u003e \u003cp\u003eThis research also contributes to existing literature by examining the impact of factors such as sucrose concentration, method of sucrose addition, and the concentration of MES buffer on saffron cell growth and crocin production (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Unpublished data from our team indicate that yeast extract can enhance both biomass and crocin production, dependent on its concentration. Our results affirm the critical role of sucrose in promoting biomass and highlight the importance of lactose for crocin synthesis. Utilizing a bioreactor system, we successfully increased the cell growth index from 1.8 to 4.3, which surpasses previous reports indicating a maximum growth index of less than 1.5 in saffron cell cultures (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, crocin production in this study reached 3.43 mg/g of dry cell weight, a significant improvement over prior findings of 2.0 mg/g (Amini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eThe findings of this study underscore the importance of both the type and concentration of basal culture medium and plant growth regulators in enhancing cell biomass growth and crocin production in saffron. Moreover, the optimized bioreactor system has demonstrated the potential to significantly elevate both biomass and crocin yields by incorporating these optimal factors. Building on this inquiry and prior research concerning the optimization of culture media regarding minerals, carbohydrates, PGRs, and elicitors, future investigations may further enhance biomass and crocin production through the exploration of various vitamins within the culture media. By advancing our understanding of these factors, we can optimize saffron cultivation methods and enhance the production of this economically significant metabolite. The results of this study demonstrated that there is a correlation between spectrophotometry results (crocin levels) and image analysis a-index (red color intensity), hence, it could be concluded that the image analysis is a precision, effectiveness and non-destructive method to estimate crocin content of the saffron cells.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eBA:\u003c/strong\u003e Benzyladenine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003csub\u003e5\u003c/sub\u003e:\u003c/strong\u003e Gamborg’s B\u003csub\u003e5\u003c/sub\u003e Medium\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC:\u003c/strong\u003e High Performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIAA:\u003c/strong\u003e Indole-3-acetic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKIN:\u003c/strong\u003e Kinetin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMES:\u003c/strong\u003e Morpholino-Ethane-Sulfonic acid buffer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMS:\u003c/strong\u003e Murashige and Skoog Medium\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAA:\u003c/strong\u003e Naphthaleneacetic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePGRs:\u003c/strong\u003e Plant Growth Regulators\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSH:\u003c/strong\u003e Schenk and Hildebrandt Medium\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003e Authors hereby declare that there is no conflict of\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eIn this experiment, Ziaratnia and Hemmati designed the experiments. Amini carried out all the experiments, analyzed data and wrote the paper. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment:\u003c/h2\u003e \u003cp\u003eWe extend our sincere gratitude to the Research Institute of Food Science and Technology (RIFST) for their financial support. We also thank Dr. Javad Feizy, a faculty member of this institute, for his valuable scientific guidance.\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003eThe data will be available on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbobkar IMS, Elshahed AM (2012) Recent advances in plant in vitro culture. page number: 222\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed SA, Baig MMV (2014) Biotic elicitor enhanced production of psoralen in suspension cultures of P\u003cem\u003esoralea corylifolia\u003c/em\u003e L. Saudi J Biol Sci 21:499\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.sjbs.2013.12.008\u003c/span\u003e\u003cspan address=\"10.1016/j.sjbs.2013.12.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli H et al (2017) Optimization of callus induction and regeneration system for tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) under different hormonal conditions. J Plant Biochem Biotechnol 26(4):439\u0026ndash;448\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmini S, Hemmati Kh, Ziaratnia SM (2024) Image analysis for detection of crocin content of saffron corm derived cells under different physiological and chemical conditions. Res Innov Food Sci Technol 13(2):65\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22101/jrifst.2023.375070.1414\u003c/span\u003e\u003cspan address=\"10.22101/jrifst.2023.375070.1414\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmini S, Ziaratnia SM, Rajabzadeh G (2023) Improvement of Friable Callus Induction of Crocus sativus L. and Establishment of a Cell Suspension Culture System with High Biomass. J Hortic Postharvest Res 6(4):397\u0026ndash;408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22077/jhpr.2023.6850.1338\u003c/span\u003e\u003cspan address=\"10.22077/jhpr.2023.6850.1338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmini S, Ziaratnia SM, Hemmati K (2022) Optimization of conditions for increasing of saffron cell biomass and crocin production in stirred bioreactor. Plant Cell Tissue Organ Cult 149(10). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-022-02233-4\u003c/span\u003e\u003cspan address=\"10.1007/s11240-022-02233-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmini S, Ziaratnia SM (2021) The Effect of Developmental Stages of Corm, Types of Medium and Hormones in Callus Induction of Saffron. Journal of Horticulture and Postharvest Research 4(Special Issue: Recent Advances in Saffron. 43\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22077/jhpr.2021.3684.1166\u003c/span\u003e\u003cspan address=\"10.22077/jhpr.2021.3684.1166\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnup Vibhute, Bodhe SK (2012) Applications of image processing in agriculture: A Survey. Int J Comput Appl 52:34\u0026ndash;39\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsgarpanah J, Darabi-Mahboub E, Mahboubi A et al (2013) In-Vitro evaluation of \u003cem\u003eCrocus sativus\u003c/em\u003e L. petals and stamens as natural antibacterial agents against food-borne bacterial strains. Iran J Pharm Sci 9:69\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehbahani M, Shanehsazzadeh M, Hessami MJ (2011) Optimization of callus and cell suspension cultures of \u003cem\u003eBarringtonia racemosa\u003c/em\u003e (Lecythidaceae family) for lycopene production. Sci Agric 68:69\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/s0103-90162011000100011\u003c/span\u003e\u003cspan address=\"10.1590/s0103-90162011000100011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastro AH et al (2015) Influence of nitrate and ammonium concentrations on callus induction and plant regeneration in Rice (\u003cem\u003eOryza sativa\u003c/em\u003e L). Plant Cell Tissue Organ Cult 123(3):641\u0026ndash;650\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Wang X, Zhao B et al (2003) Production of crocin using \u003cem\u003eCrocus sativus\u003c/em\u003e callus by two-stage culture system. Biotechnol Lett 25:1235\u0026ndash;1238. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1025036729160\u003c/span\u003e\u003cspan address=\"10.1023/A:1025036729160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Zhao B, Wang X et al (2004) Promotion of the growth of \u003cem\u003eCrocus sativus\u003c/em\u003e cells and the production of crocin by rare earth elements. Biotechnol Lett 26:27\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/B:BILE.0000009455.31817.a5\u003c/span\u003e\u003cspan address=\"10.1023/B:BILE.0000009455.31817.a5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbrahimzadeh H, Radjabian T, Karamian R (2000) In vitro production of floral buds and stigma-like structures on floral organs of \u003cem\u003eCrocus sativus\u003c/em\u003e L. Pakistan J Bot 32:141\u0026ndash;150\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerri M, Tassoni A (2011) Chitosan as elicitor of health beneficial secondary metabolites in in vitro plant cell cultures. Handb Chitosan Res Appl 389\u0026ndash;413\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFiroozi B, Zare N, Sofalian O, Sheikhzade-Mosadegh P (2019) In vitro indirect somatic embryogenesis and secondary metabolites production in the saffron: Emphasis on ultrasound and plant growth regulators. Tarim Bilim Derg 25:1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15832/ankutbd.538973\u003c/span\u003e\u003cspan address=\"10.15832/ankutbd.538973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGamborg O, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151\u0026ndash;158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGantait S et al (2018) Influence of basal media and plant growth regulators on in vitro callus induction and plant regeneration in \u003cem\u003eMomordica charantia\u003c/em\u003e L. Biocatal Agric Biotechnol 15:266\u0026ndash;273\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGregory MJ, Menary RC, Davies NW (2005) Effect of drying temperature and air flow on the production and retention of secondary metabolites in Saffron. J Agric Food Chem 53:5969\u0026ndash;5975. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf047989j\u003c/span\u003e\u003cspan address=\"10.1021/jf047989j\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIbaraki Y, Kenji K (2001) Application of image analysis to plant cell suspension cultures. Comput Electron Agric 30:193\u0026ndash;203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0168-1699(00)00164-2\u003c/span\u003e\u003cspan address=\"10.1016/S0168-1699(00)00164-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsmail H, Kayani SS, Kayani S-I et al (2019) Optimization of cell suspension culture of transformed and untransformed lettuce for the enhanced production of secondary metabolites and their pharmaceutical evaluation. 3 Biotech 9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJan S, Wani AA, Kamili AN, Kashtwari M (2014) Distribution, chemical composition and medicinal importance of Saffron (\u003cem\u003eCrocus sativus\u003c/em\u003e L). Afr J Plant Sci 8:537\u0026ndash;545\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaved SB et al (2020) Efficient callus induction and regeneration in Faba Bean (\u003cem\u003eVicia faba\u003c/em\u003e L.) Using Gamborg's B\u003csub\u003e5\u003c/sub\u003e Medium. Plants 9(4):439\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJedinak A, Farago J, Psenakova I, Maliar T (2004) Approaches to flavonoid production in plant tissue cultures. Biol - Sect Cell Mol Biol 59:697\u0026ndash;710\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolewe M (2011) Development of plant cell culture processes to produce natural product pharmaceuticals: characterization, analysis, and modeling of plant cell aggregation 1\u0026ndash;168\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMereu A, Dorsaf K, Scarpa G (2019) In vitro culture of Saffron: Hormones influence on the dvelopment of new shoots and callus. Plant Cell Biotechnol Mol Biol 20:511\u0026ndash;520\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolina RV, Renau-Morata B, Nebauer SG et al (2010) Greenhous saffron culture temperature effects on flower emergence and vegetative growth of the plants. Acta Horticulturae. International Society for Horticultural Science (ISHS), Leuven, Belgium, pp 91\u0026ndash;94\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradi A, Zarinkamar F, Caretto S, Azadi P (2018) Influence of thidiazuron on callus induction and crocin production in corm and style explants of \u003cem\u003eCrocus sativus\u003c/em\u003e L. Acta Physiol Plant 40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11738-018-2760-2\u003c/span\u003e\u003cspan address=\"10.1007/s11738-018-2760-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradi A, Zarinkamar F, De Domenico S et al (2020) Salycilic acid induces exudation of crocin and phenolics in saffron suspension-cultured cells. Plants 9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants9080949\u003c/span\u003e\u003cspan address=\"10.3390/plants9080949\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473\u0026ndash;497\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagella P, Murthy HN (2010) Establishment of cell suspension cultures of \u003cem\u003eWithania somnifera\u003c/em\u003e for the production of withanolide A. Bioresour Technol 101:6735\u0026ndash;6739. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biortech.2010.03.078\u003c/span\u003e\u003cspan address=\"10.1016/j.biortech.2010.03.078\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira Y, Lima A (2016) Role of nitrate and ammonium on pH stability and callus induction in Cassava (\u003cem\u003eManihot esculenta\u003c/em\u003e Crantz). J Plant Nutr 39(2):237\u0026ndash;247\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSafarnejad A, Alamdari SBL, Darroudi H, Dalir M (2016) The effect of different hormones on callus induction, regeneration and multiplication of saffron (\u003cem\u003eCrocus sativus\u003c/em\u003e l.) corms. Saffron Agron Technol 4:143\u0026ndash;154. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22048/jsat.2016.17364\u003c/span\u003e\u003cspan address=\"10.22048/jsat.2016.17364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSajjadi fard M, Pazhohandeh M (2015) Study on effect of type of explant and hormone on callus induction and regeneration in Saffron (\u003cem\u003eCrocus sativus\u003c/em\u003e). Saffron Agron Technol 3:195\u0026ndash;202\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-Ramos M, Alvarez L, Romero-Estrada A, Bernabe-Antonio A, Marquina-Bahena S, Cruz-Sosa F (2020) Establishment of a cell suspension culture of \u003cem\u003eAgeratina pichinchensis\u003c/em\u003e (Kunth) for the improved production of anti-inflammatory compounds. Plants 9(10):1398. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants9101398\u003c/span\u003e\u003cspan address=\"10.3390/plants9101398\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantos RB, Abranches R, Fischer R et al (2016) Putting the Spotlight back on plant suspension cultures. Front Plant Sci 7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2016.00297\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2016.00297\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharaf-Eldin M, Elkholy S, Fernandez J-A et al (2008) Bacillus subtilis FZB24 affects flower quantity and quality of saffron (\u003cem\u003eCrocus sativus\u003c/em\u003e). Planta Med 74:1316\u0026ndash;1320. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-2008-1081293\u003c/span\u003e\u003cspan address=\"10.1055/s-2008-1081293\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinson KWS, Chew BL, Sathasivam K, Subramaniam S (2020) The establishment of callus and cell suspension cultures of Hylocereus costaricensis for the production of betalain pigments with antioxidant potential. Ind Crops Prod 155:112750. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.indcrop.2020.112750\u003c/span\u003e\u003cspan address=\"10.1016/j.indcrop.2020.112750\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang B, Guo Z, Liu R (2005) Crocin synthesis mechanism in \u003cem\u003eCrocus sativus\u003c/em\u003e. Tsinghua Sci Technol 10:567\u0026ndash;572. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1007-0214(05)70119-X\u003c/span\u003e\u003cspan address=\"10.1016/S1007-0214(05)70119-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Image analysis, cell suspension, secondary metabolites, Crocus sativus","lastPublishedDoi":"10.21203/rs.3.rs-6857423/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6857423/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOptimizing the media for plant cell cultures, particularly regarding the types and concentrations of growth regulators, can significantly enhance biomass and metabolite production \u003cem\u003ein vitro\u003c/em\u003e. This study examined the effects of different basal media and plant growth regulators (PGRs) on cell growth and crocin production in saffron cell suspension cultures. Various media including SH, MS, and B\u003csub\u003e5\u003c/sub\u003e, in combination with specific PGRs, were tested for their efficacy in promoting cell biomass and crocin yield. Results identified that SH medium supplemented with NAA (1 mg/L) and BA (0.5 mg/L) was optimal for biomass growth, while IAA (1 mg/L) and KIN (0.5 mg/L) effectively boosted crocin production. The research continued by evaluating these factors in a bioreactor setup. For biomass enhancement, 50g of cells were inoculated into one liter of a chosen medium (SH, 3% sucrose, 0.5 mg/L BA, 1 mg/L NAA, 2.5 mM MES) with aeration at 0.5 vvm and further sucrose feeding. For crocin production, another setup used the same biomass with a medium of SH, 3% lactose, 0.5 mg/L Kin, 1 mg/L IAA, and no aeration, adjusting conditions similarly. Findings showed that cell growth index improved from 1.87 to 4.3, while crocin levels increased from 1.05 to 3.43 mg/g dry weight. Crocin content was quantified using spectrophotometry and HPLC, with image analysis employed to determine red color intensity. The study concludes that image analysis offers a cost-effective method for assessing crocin production, highlighting the effectiveness of bioreactors in enhancing both cell growth and metabolite yield.\u003c/p\u003e","manuscriptTitle":"Optimization of Regulatory Factors Affecting Biomass Growth and Crocin Production of Saffron Cells in Bioreactor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 09:40:36","doi":"10.21203/rs.3.rs-6857423/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c031d428-e93a-45ef-b9ea-f869de46af93","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-06T07:09:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-02 09:40:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6857423","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6857423","identity":"rs-6857423","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}