Journal of Medicinal Plants Biotechnology

Abstract

Abstract Thyme is one of the most important medicinal plants which grows naturally in wide areas of range lands in Iran and simultaneously is cultured widely in Iran and other parts of the world. As Iran has placed in arid and semi-arid regions of the world, with actually low precipitation, doing projects in order to reveal the mechanisms or increase the drought tolerance of crop and medicinal plants has great value. Drought stress decreases the yield and quality of medicinal plants as well as crops. To investigate the mechanisms of drought tolerance of thyme plants and to evaluate the possibility of increasing of drought tolerance in these valuable medicinal plants, activity of some antioxidant enzymes and total soluble proteins were been analyzed in Thymus vulgaris L.in a complete randomized design experiment. After reaching to the adequate stage of growth, seedlings were grown in pots containing suitable soil, were subjected to different levels of drought stress (80% as control, 60% and 40% irrigation of field capacity(FC) as treatments). Results show that, drought stress had significant effect on total protein content as well as the activity of antioxidant enzymes. At 60% FC treatment and especially at 40% FC the amount of total soluble proteins drastically decreased. However; the activity of catalase increased at 60% FC treatment. The activity of peroxidase did not change significantly at 60% treatment but its activity decreased at 40% treatment. As a conclusion; it can be suggested that, one of the drought tolerance mechanisms of thyme is increasing of the activity of antioxidant enzymes especially the activity of catalase.  

Abstract The increasing tendency of human societies to use herbal medicines has caused an increase in the demand for effective medicinal plants. Despite many advances in the field of artificial synthesis of effective plant substances, extraction from plant sources is still the only way to obtain many valuable medicinal substances. A group of alkaloids are natural drugs that are known as sedatives and painkillers; Like morphine and codeine, which are obtained from poppy, they are the most common painkillers. Currently, the country's annual need for morphine is 30 tons, which will increase with the increase in population. 70% of Iran's needs are supplied by importing concentrated poppy straw (CPS) and 30% from opium sap etc. About 5000 types of alkaloids are known in 15% of plants belonging to 150 plant families, among which tropane alkaloids such as hyoscyamine, atropine and scopolamine are widely used in medicine. One of the main uses of poppy alkaloids in the pharmaceutical industry is They are in painkillers. Today, the industrial production of tropane alkaloids through new techniques such as plant cell and tissue culture, somatic hybridization, metabolic engineering, large-scale cultivation and its commercialization using bioreactors has attracted a lot of attention. This article examines poppy alkaloids and their biosynthetic pathways, which can be useful in clarifying the future direction of research in this field.

Abstract Medicinal plants have been a source of medicinal compounds and other useful products since ancient times. Due to some limitations, the traditional methods of supplying medicinal compounds are not able to meet the ever-increasing needs of the market, and therefore the use of alternative methods is inevitable. Metabolic engineering is a logical and efficient solution to increase the production of medicinal compounds and reduce related costs. In addition to improving the production of desired metabolites and reducing the production of unwanted metabolites, metabolic engineering also leads to the production of new metabolites in plants. Currently, many methods are used to manipulate metabolic pathways, among which CRISPR/Cas technology has a special place due to its high accuracy and ease of use. In this article, the importance of medicinal plants and their metabolites has been discussed first, and then the necessity of using metabolic engineering to increase the medicinal compounds of these plants has been pointed out. In the following, we have explained the CRISPR/Cas technology and how it works in gene editing. Then, the application of this technology in manipulating the metabolic pathways of medicinal plants is discussed, focusing on three major groups of plant metabolites, namely terpenoids, alkaloids and phenylpropanoids. The next section deals with some of the challenges in the application of this technology in the metabolic engineering of medicinal plants, and proposed solutions. At the end, the future prospects of using this technology in metabolic improvements of medicinal plants are briefly described.

Abstract Medicinal plants have been associated with human life throughout history and have been used as food, fodder and medicine. One of these plants is wild celery, which is one of the endemic plants of the Zagros region of Iran, which, like other plants, undergoes some biotic and abiotic stresses during its growth in these regions. Wild celery is resistant to temperature, humidity and drought stress in its growing place, because over time it has adapted to the conditions of the Zagros region. There is no salt in the natural growth place of this plant. Regional pests have also been observed in the main habitats of this plant. By knowing the ecological conditions of its growing place as a model from nature, wild celery can be cultivated in agricultural conditions. leucoptera scitella zell, Hyadaphis foeniculi and Eupithecia sp are regional pests of wild celery. When wild celery is cultivated in the field, the most important weeds in its fields are mentioned Avena ludoviciana Dur, Hordeum murinum L., Hordeum spontaneum C. Koch, Secale cereal L, Phalaris minor Retz., Bromus tectorum L., Malva sylvestris L., and Alhagi camelorum Fisch.

Abstract Anghuzeh is one of the important medicinal plants of Apiaceae family. Anghuzeh is very important in traditional Iranian medicine and there is a great global demand for it. This plant has many applications in the pharmaceutical field and has been used in the treatment of diseases for a long time. The results of the research of Angouzeh plant conducted in this field show the anti-diabetic, anti-cancer, antiviral, antibacterial and antioxidant effects. The applications of this plant in the food industry are very important, so this plant is used as an additive, flavoring or spice in the preparation and processing of various foods. Anghuzeh also has many applications in the cosmetics industry. The importance of Anghuzeh is due to the valuable gum that is obtained from cutting its roots. In recent years, due to the considerable profit from the sale of gum, the harvesting of this plant from nature has increased, and this issue leads to the extinction of the Anghuzeh generation. Anghuzeh gum contains special compounds. Essential oil is one of the main active ingredients in gum, which is used in other related industries after extraction and purification. At present, Anghuzeh gum is sold in raw form in Iran. It is hoped that with proper planning and the creation of suitable conversion industries for Anghuzeh, it will be possible to create good added value (valuation) for the country while creating employment.

Abstract Helicobacter pylori, a Gram-negative bacterium, is a major causative agent of peptic ulcers and gastrointestinal cancers. To neutralize gastric acid and ensure survival in the harsh stomach environment, H. pylori relies on urease enzyme activity, which converts urea into ammonia. Hence, urease inhibitors play a critical role in combating H. pylori and managing associated diseases, including gastritis and other gastrointestinal disorders. This study evaluated the urease-inhibitory and antioxidant activities of Cydonia oblonga Mill. (quince) fruit and seed gel extract, aiming to identify natural sources of effective urease inhibitors. The urease inhibition potential was assessed using the Berthelot method (targeting jack bean urease), while antioxidant activity was measured via the 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) free radical scavenging assay. Both extracts were prepared with 80% ethanol, and inhibitory effects were quantified through enzyme inhibition percentages and IC₅₀ values for urease, alongside DPPH radical scavenging percentages. The results revealed that the seed gel extract exhibited stronger urease inhibition (IC₅₀ = 136.6 ± 7.5 μg/mL) compared to the fruit extract (IC₅₀ = 435.3 ± 10.8 μg/mL). Antioxidant activity was higher in the fruit extract (85.23%) than in the seed gel (79.66%). These findings highlight Cydonia oblonga through its suitable antioxidant, and urease-inhibitory activity for developing safer therapeutic strategies against H. pylori infection and preventing relevant complications.

Abstract Achillea genus is belonging to Asteraceae. The composition of the active ingredients of plants in different climatic conditions show very different constituent in phytochemical propertiesand , In order to investigating quantity and quality of essential oils of three species of achillea in Zanjan provences plant samples collected from northern heights of Zanjan After selecting a suitable plants, after harvesting the whole vegetative body of the plant was harvested in the full bloom time and after drying in room, and its essential oil was extracted by distillation . Then, the components of the essential oil were identified and the amount of the components was identified using a gas chromatography device connected to a mass spectrometer. The results showed that the essential oil of Achillea wilhelmsii C. Koch had a yellow color with a yield of 0.89%. The results of GC-MS showed that the essential oil of this plant is composed of 106 substances in which 15 representative compounds 54% were total essential oil. In the study of essential oil of Achillea tenuifolia Lam, it was found that 20 substances constituted 54.37% of essential oil. Also, the essential oil of Achillea millefolium was white with a yield of 0.74%, GC-MS results showed that this essential oil The plant in the region is composed of 105 substances, of which 10 compounds represented 50.78% of the total essential oil Which 47 compounds accounted for 81.05% of the essential oil.

Abstract Salinity stress, as one of the major challenges in agriculture, disrupts water and nutrient uptake by accumulating sodium and chloride ions in the soil, leading to reduced plant growth and yield. This stress damages plant cells through oxidative stress and disrupts metabolic processes. In this study, the effects of different salinity levels (0, 2.5, and 5 dS/m) and salicylic acid foliar application (0, 0.5, and 1 mM) on purslane (Portulaca oleracea) were investigated using a completely randomized factorial design with three replications. The results showed that salinity reduced plant growth and negatively affected chlorophyll a and b content. However, the application of salicylic acid (1 mM) mitigated these adverse effects by improving potassium uptake. Although salinity increased sodium and chloride accumulation, salicylic acid improved plant tolerance by reducing oxidative stress and maintaining photosynthetic efficiency. In conclusion, this study demonstrates that salicylic acid can serve as an effective strategy to enhance purslane's resistance under saline conditions.

Abstract Catharanthus roseus is an important medicinal plant containing alkaloids such as vinblastine and vincristine, which have strong cell division inhibitory activity and can serve as a colchicine alternative in polyploidization programs. This study aimed to evaluate the effects of aqueous and ethanolic leaf extracts of C. roseus on cell division in meristematic cells of onion (Allium cepa) roots. Extracts at four concentrations (0, 20, 50, and 100%) were applied to onion bulbs. The experiment was conducted as a completely randomized factorial design with three replications. Traits assessed included root length and number, rooting time, mitotic and phase indices, chromosome counts, and cytological abnormalities such as multinucleation and chromatid bridges. Results showed that increasing extract concentration reduced root length, and rooting was delayed in the ethanolic extract, with the greatest delay observed at 100% concentration. The highest prophase index was recorded at 20% extract for both types, while the highest telophase index occurred at 100% ethanolic extract. Mitotic index peaked at 20% and 50% ethanolic extracts. Chromosomal abnormalities, including stickiness, breakage, and duplication, were mainly observed in 50% ethanolic extract, whereas chromatid bridges appeared only in 50% aqueous extract. These findings indicate that C. roseus alkaloids can influence mitosis and may be applied in breeding programs and polyploidization of medicinal plants.

Abstract This study investigated the effect of different levels of charcoal obtained from acacia wood on some growth characteristics, essential oil content, and nutrient absorption of sage plants under cadmium stress. The experimental treatments included different levels of charcoal (0, 1, and 2% by weight) and cadmium stress at three levels of 0, 30, and 60 mg/kg of soil, which was factorial in the form of a completely randomized design with 4 replications in the research greenhouse of the Department of Horticultural Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad. The results showed that increasing the concentration of cadmium caused a significant decrease in the growth of sage plants in terms of height, stem diameter, number of sub-branches, leaf area, number of leaves, biomass, and root growth. Cadmium stress also caused a decrease in essential oil content and the amount of nitrogen, phosphorus, and potassium elements, and increased iron, manganese, and cadmium in the aerial parts of the sage plant. The use of charcoal reduced the harmful effects of cadmium on growth by reducing the transfer of cadmium from roots to aerial organs and improving the absorption of nutrients. In plants treated with increasing charcoal concentration, the amount of essential oil in the flowering branches of sage significantly increased. The greatest effect of charcoal on morphological traits, percentage of essential oil, and concentration of nutrients was observed in the highest concentration used (2% by weight) and in plants under severe cadmium stress.

Abstract An experiment was conducted to investigate the effects of water deficit stress (field capacity (FC), 50%, and 30% of FC) and foliar application of gamma-aminobutyric acid (GABA) and spermidine (control and 3 mg L⁻¹), as well as their combined application, on growth and selected physiological traits of basil. The highest plant biomass was observed under irrigation at FC + simultaneous foliar application of spermidine and GABA. The main effects of water deficit and foliar treatments significantly influenced chlorophyll, H2O2, malondialdehyde, proline, elemental content, and essential oil content of the plant. The highest chlorophyll a, calcium, magnesium, phosphorus, and potassium content were observed under irrigation at FC. Irrigation at FC and 50% FC significantly increased chlorophyll b and nitrogen contents. Irrigation at 30% FC resulted in elevated levels of H2O2, malondialdehyde, and proline accumulation. All three foliar treatments led to increased chlorophyll b, nitrogen and potassium contents. Simultaneous foliar application of both compounds contributed to a reduction in H2O2 and malondialdehyde levels, while enhancing chlorophyll a, proline, phenolic compounds, calcium, magnesium, phosphorus, and essential oil contents. Essential oil content was notably increased under irrigation at 50% FC. Sixteen constituents were identified in the basil essential oil, with methyl chavicol (38.42–42.38%) being the predominant compound, particularly prominent in the treatment combining irrigation at 30% FC with foliar application of both compounds. The results of the present study demonstrate the positive effect of concurrent application of both compounds on the evaluated physiological and biochemical traits of basil under water deficit conditions.

Abstract In light of the growing demand for medicinal plants and their increasing significance in global society, this study examined the essential oils composition and phenolic content in the flowers and leaves of Stachys schtschegleevi. Plant samples were collected from the Jolfa region, and the essential oils were extracted using a Clevenger apparatus. The chemical composition of the essential oils was analyzed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The results showed that the percentage of essential oil in the flower organs was 39% higher than in the leaves. Analysis of the essential oils identified 61 compounds in the flowers (98.39% of the total) and 49 compounds in the leaves (98.73% of the total). The dominant compounds in the flower organs were germacrene D (20.69%), α-cadinol (9.51%), α-muurolene (7.16%), n-hexadecanoic acid (5.93%), thymol (5.41%), spathulenol (4.77%), α-pinene (4.62%) and β-pinene (4%). The dominant compounds in Pulak leaf essential oil were found to be α-pinene (12.98%), germacrene D (11.92%), α-cadinol (7.9%), thymol (6.91%), n-hexadecanoic acid (4.82%), spathulenol (4.44%) and α-muurolene (4.22%). Evaluation of the phenolic compounds showed that the total phenol and flavonoid content in the flowers (81.8 and 3.5 mg/ g of dry extract, respectively) was higher than in the leaves (56.1 and 3 mg/g of dry extract, respectively). Therefore, due to its higher essential oil percentage and greater richness in phenolic compounds, the flower organ of Stachys schtschegleevi is considered more suitable than the leaf organ for industrial applications, particularly in the field of essential oil extraction.

Abstract The excessive and improper use of antimicrobial agents has led to the emergence of drug-resistant strains. Therefore, It necessitates the discovery and implementation of new sources to combat microbial pathogens. This study aimed to evaluate the antibacterial effect of ethanolic extract of Ziziphus spina-christi leaves against four bacteria; Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Corynebacterium glutamicum. Initial screening of the antibacterial activity of the extract was performed using the disk diffusion method. Subsequently, the minimum inhibitory concentration (MIC) was determined using the microdilution method, and the minimum bactericidal concentration (MBC) was identified through microbial culture. According to the results, the highest antibacterial effect of the leaf extract was observed against Corynebacterium glutamicum. The microdilution test showed that the MIC for all bacteria, except Escherichia coli, was 75 mg/mL. The lowest survival rate was observed in Corynebacterium glutamicum and Staphylococcus epidermidis. The MBC for these two bacteria was determined to be 150 mg/mL, while for Escherichia coli, it was 300 mg/mL. The findings of this research demonstrated that Ziziphus leaf extract has notable antibacterial activity, especially against the studied Gram-positive bacteria, and could be considered a natural source for developing of new antibacterial drugs.

Abstract Medicinal plants are recognized as significant sources of natural antioxidants due to their bioactive compounds, particularly phenolics and flavonoids, which have shown potential in the prevention and treatment of various diseases. These compounds are widely studied for their notable antioxidant, anti-inflammatory, and anticancer properties. The efficiency and profile of extracted phytochemicals are highly influenced by extraction parameters, including solvent type, solvent concentration, and extraction time. This study aimed to evaluate the effects of different extraction methods, namely maceration and ultrasound, along with solvent type and concentration (ethanol and methanol at 50% and 70%), and extraction durations (5 and 10 minutes), on the total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of extracts from Portulaca oleracea and the leaves and fruits of Ziziphus spina-christi. A factorial experiment was conducted based on a completely randomized design with two replications for statistical analysis. The results revealed that the highest phenolic content was obtained from P. oleracea extract using the maceration method with 70% methanol for 5 minutes. Similarly, the highest flavonoid content was observed in Z. spina-christi leaf extract under the same conditions. Regarding antioxidant activity, the most potent effect was recorded in the leaf extract of Z. spina-christi using 70% ethanol and an extraction time of 10 minutes. These findings highlight the importance of optimizing extraction conditions to enhance the recovery of beneficial bioactive compounds from medicinal plants, thereby improving the antioxidant potential of the resulting extracts.

Abstract In order to evaluate the physiological and biochemical responses of different ecotypes of caper (Capparis spinosa L.) seedlings to drought stress, an experiment was conducted under laboratory conditions in 2023 at the Seed Quality Analysis Laboratory of the Seed and Plant Certification and Registration Research Institute, Karaj, Iran, using a completely randomized design with three replications. In this study, seeds of nine native caper ecotypes collected from Ardabil Province (Moghan), Alborz (Karaj), Ilam Province (Mehran), North Khorasan Province (Bojnord), Kermanshah Province (Dalahoo), and Khuzestan Province (Ramhormoz, Hendijan, Mahshahr, and Ahvaz) were evaluated. Drought stress was imposed using polyethylene glycol (PEG 6000) at an osmotic potential of −6 bar. The results indicated that drought stress significantly increased malondialdehyde content from approximately 6.5 to 11.3 nmol g⁻¹ FW and hydrogen peroxide from 3.1 to 6.0 µmol g⁻¹ FW, while total chlorophyll content decreased from 0.35 to 0.21 mg g⁻¹ FW. In contrast, proline content (12.7–45.8 µg g⁻¹ FW), total phenolic content (2.6–3.6 mg gallic acid g⁻¹ FW), and antioxidant capacity (38–47% DPPH inhibition) showed significant increases under drought conditions. Comparison among ecotypes revealed that under non-stress conditions, the Karaj and Moghan ecotypes exhibited greater physiological stability, whereas under drought stress, the Mahshahr and Hendijan ecotypes showed the lowest oxidative damage indices and the highest accumulation of protective compounds, and were therefore identified as drought-tolerant ecotypes. Overall, the results highlight the key role of antioxidant defense mechanisms and osmotic adjustment in the adaptation of caper plants to drought stress conditions.

Abstract To investigate the effect of proline and gamma-aminobutyric acid on the quantitative and qualitative traits of medicinal plant chamomile (Matricaria chamomilla L.) under water deficit stress, an experiment was conducted according to split plots design with a completely randomized block arrangement with four replications during the spring and summer of 2024 in the research greenhouse at the University of Zanjan. The main factor consisted of two irrigation levels (70 and 100% of field capacity) and the subplots included foliar applications of two levels of proline (0.5 and 1 mM), gamma-aminobutyric acid (0.5 and 1 mM) and control treatment (distilled water).
The results showed that the highest chlorophyll a content (1.40 mg/g fresh weight) was achieved under 100% field capacity irrigation combined with 1 mM proline. Foliar application of 0.5 mM proline under 70% irrigation level resulted in the highest phenolic content (4.78 mg gallic acid/g dry weight) and the greatest total antioxidant activity (51.75%). The maximum flavonoid content (1.49 mg quercetin/g dry weight) was observed with 0.5 M GABA treatment. Additionally, the highest thousand-seed weight (148 mg) was recorded under full irrigation (100% field capacity) with 1 mM proline, compared to the control. Overall, the combined application of 0.5 mM proline and GABA was most effective in mitigating the adverse effects of drought stress on chamomile.

Abstract Modern tissue culture and genetic transformation techniques play a significant role in manipulating biosynthetic pathways and enhancing the production of secondary metabolites in medicinal plants. Although a standardized method for the genetic transformation of Hyoscyamus niger has not yet been established, by optimizing factors influencing Agrobacterium tumefaciens-mediated transformation—one of the most effective methods for genetic modification—it is possible to increase the frequency of regeneration and transformation in plant cells. In this study, with the aim of developing an efficient protocol to improve gene transfer efficiency in H. niger, three main factors were evaluated: the A. tumefaciens strain (EHA105, GV3101, and LBA4404), explant type (leaf and petiole), and transformation method (agroinoculation, agroinfection, agroinjection, and agrosonication). Optimization of gene transfer conditions revealed that petiole explants inoculated with the LBA4404 strain using agrosonication and agroinoculation methods exhibited the highest rates of callus and embryo formation, respectively. However, histochemical gusA staining indicated that among the hygromycin-resistant plantlets, only those derived from leaf explants inoculated with the LBA4404 strain carrying the pCAMBIA1304 vector via agroinfection showed positive X-gluc staining and contained blue pigments, achieving the highest transformation rate at 21.74%. The successful integration of gusA and hptII genes in putative transgenic plantlets was confirmed through PCR and RT-PCR analyses. Therefore, by investigating key factors affecting A. tumefaciens-mediated transformation, an efficient genetic transformation protocol for H. niger can be established.

Abstract Rosemary (Rosmarinus officinalis L.) is a woody, evergreen perennial medicinal plant of the Lamiaceae family that is rich in phenolic, flavonoid, and terpenoid compounds. Rosemary is known for its analgesic, anti-rheumatic, and carminative properties and is traditionally used to alleviate respiratory disorders and epilepsy. The importance of rosemary in the pharmaceutical, cosmetic, and healthcare industries, together with its adaptability to diverse environmental conditions, has attracted considerable research interest. Therefore, this study aimed to evaluate the effect of harvest time on biomass production, phytochemical characteristics, essential oil yield, and essential oil composition of rosemary. This study was conducted as a randomized complete block design (RCBD), with three harvest times (March, June, and July), and five replications during the 2022–2023 growing seasons at a rosemary farm in Gotvand, Khuzestan Province, Iran. The evaluated traits included fresh and dry biomass, total phenolic content, total flavonoid content, antioxidant capacity, essential oil content, essential oil yield, and essential oil composition. Essential oil constituents were identified using GC–MS. Analysis of variance showed that harvest time significantly affected fresh and dry biomass, total flavonoid content, and essential oil content, but had no significant effect on total phenolic content or antioxidant capacity. GC–MS analysis identified 30 compounds, 13 of which were major constituents. Harvesting in July maximized essential oil yield, indicating that this harvest time is optimal for rosemary cultivation under the climatic conditions of Khuzestan Province.

Abstract Taxanes are diterpenoid compounds with medicinal properties, obtained from various yew species (Taxus baccata). Among them, paclitaxel (Taxol) is the most important and is widely used in cancer chemotherapy. Due to the extremely low yield of taxol in yew plants, alternative production methods are necessary. Callus and plant cell culture present an environmentally friendly alternative for producing taxol and related taxanes. To enhance taxane production in cultured callus, various strategies such as elicitation can be employed. In this study, calli were treated with the elicitors chitosan (10, 30, and 50 mg/L) and jasmonic acid (50, 100, and 150 µM) in triplicate. The calli were maintained in B5 medium supplemented with 4 mg/L 2,4-D, 1 mg/L kinetin, 0.5 mg/L gibberellin, 10 mL/L antioxidant, 30 g/L sucrose, and 7 g/L agar, and incubated in darkness at 25°C for 21 days. The levels of five major taxanes: taxol, 10-deacetylbaccatin III, baccatin III, 10-deacetyltaxol, and cephalomannine were quantified using HPLC. The results indicated that the highest taxol and 10-deacetylbaccatin III contents were achieved with 100 µM jasmonic acid treatment. The maximum baccatin III yield was observed with 50 mg/L chitosan, while 10-deacetyltaxol production was highest with 50 µM jasmonic acid. Interestingly, the highest cephalomannine accumulation was found in the control (untreated) calli.

Abstract This study aimed to elucidate the multilayered regulatory network controlling tanshinone biosynthesis in Salvia miltiorrhiza, focusing on microRNA–gene interactions, transcription factors, and cis-regulatory elements. mRNA sequences of three key pathway genes (CPS1, KSL1, and CYP) were retrieved from the NCBI database, and microRNA sequences were collected from miRBase. Target prediction was performed using psRNATarget with an Expectation threshold of 5. Network architecture were visualized in Cytoscape. Promoter sequences (~2000 bp upstream) were extracted and analyzed for transcription factor binding sites using PlantRegMap and PlantCARE, with results processed for graphical representation in TBtools. A total of 252 microRNAs from 28 families were identified as targeting these three genes (CPS1: 84, KSL1: 90, CYP: 79). Dominant families included miR781/miR319/miR482 for CPS1, miR156/miR902 for KSL1, and miR2630/miR399 for CYP. Several microRNAs (e.g., miR7492 and miR482) targeted multiple pathway genes simultaneously, indicating cooperative regulatory activity and the potential for enhanced post-transcriptional repression. Transcription factor binding site analysis revealed high-frequency interactions (KSL1: 387, CPS1: 240, CYP: 159) involving WRKY, ERF, MYB, bZIP, and Dof families. Promoters were enriched in cis-elements responsive to light, ABA, MeJA, and gibberellin. These findings demonstrate that tanshinone biosynthesis is governed by a coordinated transcriptional and post-transcriptional network, allowing dynamic modulation and co-regulation in response to environmental and hormonal signals. The identified genes and microRNAs represent promising targets for experimental validation (e.g., ChIP-qPCR, EMSA) and metabolic engineering strategies, including CRISPR and RNAi, to enhance tanshinone production.

Abstract Medicinal plants are used in various cosmetic, food, pharmaceutical, and medical industries. Artichoke (Cynara scolymus L.) is a perennial medicinal plant that has antioxidant, anticancer, antiinflammatory, antimicrobial, lipid-lowering, and heart and liver-protecting properties due to its phenolic compounds (such as cynarin and caffeoylquinic), flavonoids, inulin, and volatile organic compounds (e.g., methyl salicylate, camphene, and alpha-terpinene). Tissue culture of medicinal plants is widely used to produce secondary metabolites. This study investigated the callus formation of artichoke from explants (leaflet, crown, and rootlet) in culture medium containing NAA (0, 3, and 6 mg L-1) and 2,4-D (0, 0.5, and 1 mg L-1) hormones. The results showed that callus formation occurred in all cultured explants. According to the results of callus weight and dimensions, the best explant for producing callus was the rootlet, and the leaflet had the weakest callus formation. Considering the largest callus dimensions (average 14.3 cm) in rootlet explants cultured in medium containing 6 mg L-1 NAA, rootlet explants and NAA hormone can be suggested for optimizing artichoke tissue culture and callus formation.