مجله تحقیقات گیاهان دارویی و معطر ایران
سال چهلم شماره 3 (پیاپی 121، پاییز 1403)

 Nepeta, with 300 species, is one of the largest genera in the Lamiaceae family. Seventy-five species of this genus are distributed in Iran, many of which are endemic. Due to the medicinal importance and traditional uses of its various species, much research has been conducted to identify their secondary metabolites. Nepetalactone, cineole, and phenolic compounds have been reported as their main compounds, and most have medicinal, antimicrobial, and industrial properties. The amount of these compounds varies in different parts of the plant and different species and populations. Nepeta bornmulleriHausskn. ex Bornm. is endemic to Iran and is a type specimen from Kerman province. Although secondary metabolites have been studied in many species of this genus, more research is needed on N. bornmulleri. Therefore, the morphology, quantity, and quality of antioxidant compounds and essential oils were investigated in the plant's two vegetative and generative parts.

 Vegetative (stem and leaf) and generative (flower and fruit) parts of N. bornmulleri were collected in May 2023 from Rayen city (Kerman province) at an altitude of 2450 meters. The used generative parts were 80% flowers and 20% unripe fruits. The size of the plant, its vegetative form, and the structure of the inflorescence and flower were examined and photographed with the naked eye or stereomicroscope so that researchers could identify them quickly. Biochemical compounds, including phenolic compounds and essential oil, were studied in vegetative and generative parts. Total phenolic compounds, including total phenols, flavonoids, and anthocyanins from fresh samples, were studied by spectrophotometric method. The phenolic compounds and anthocyanins profile were investigated using dried samples with HPLC. The analysis of essential oil was done using dry samples with gas chromatography (GC) and gas chromatography coupled to a mass spectrometer (GC/MS). Essential oil extraction was done by distillation with water using Clevenger. The experiment was conducted in a completely random design, including two types of vegetative (stem and leaf) and generative (flower and fruit) organs with three replications.

 Morphological studies of N. bornmulleri showed that in the studied area, the height of the species is about 10-25 cm with hairy basal (rosette) leaves, apical and dense head-like inflorescence, purple-blue synpetal corolla with bilobate apex, synsepal calyx. The Androecium comprises four purple stamens of equal length (didynamous stamens) and an upper pistil with tetra-achene schizocarp fruit surrounded by persistent sepals. The total content of phenols, flavonoids, and anthocyanins in flowering branches (generative) was significantly higher than in leaves and stems (vegetative parts). The profile of phenolic and anthocyanin compounds by HPLC showed that ten phenolic compounds and three anthocyanin compounds were identified in the generative part, and six phenolic compounds and two anthocyanin compounds were identified in the vegetative part. The most phenolic compound observed in both vegetative and generative parts was luteolin, while malvidin-glucoside was recorded as the most anthocyanin compound. The amount of malvidin-glucoside in the generative part was twice that of the vegetative part. The percentage of essential oil in vegetative and generative parts was observed at 0.2 and 0.1, respectively. In the essential oils of vegetative and reproductive parts, respectively, 10 (69.41%) and 16 (89.29%) identified compounds and 9 (14.59%) and 4 (6.21%) unidentified compounds were reported, which in total 84% and 95.5% constituted the essential oil.The main components of the essential oil of the vegetative part include spatholenol (31.2%), 1,8-cineole (14.5%), hexadecanoic acid (10.6%), myrtenol (9.7%), caryophyllene oxide (6.4%). ) and trans-pinocarveol (4.4%). In the generative part, 1,8-cineole (31.7%), spatholenol (16.2%), myrtenol (14.14%), dihydro nepetalactone (6.92%), trans-pinocarveol (6.7%), pinocarovone (5.2%) and caryophyllene oxide (4.5%) were the main components of the essential oil.

 Based on the results, the quantity and quality of phenolic compounds, anthocyanin, and essential oil in the generative part were higher than in the vegetative part of the studied species. So, it is suggested that the generative parts of the plant be used to obtain antioxidant compounds. The amount of nepetalactone, which is significant in most species of Nepeta, was relatively low in this species, while spatholenol and 1, 8-cineole were the main compounds. Considering the numerous medicinal and industrial applications of the two dominant species combinations on the one hand and the exclusivity and limited distribution of the species on the other hand, it is essential to preserve its different populations.

Nigella sativa</em> L. is an important medicinal plant widely used in the food and pharmaceutical industries. Considering the increasing demand for medicinal plants worldwide and the need to produce cultivated and breeding varieties, it is necessary to study the genetic resources. The gain from selection for a trait depends on the relative importance of genetic factors in the occurrence of phenotypic differences among genotypes; in other words, the success of breeding programs depends on the genetic variation and heritability of traits. Therefore, this study investigated the genetic variation and inheritance of seed yield and yield component traits in different black cumin genotypes and identified desirable genotypes for use in breeding programs.

This study investigated the genetic variation in 20 different black cumin genotypes (Iranian and foreign) regarding different agronomic traits as a randomized complete block design with three replications at the research farm of Shahrekord University. The genotypes were planted in the middle of March. Each genotype in every repetition was cultivated in four rows, each 1.5 meters long. The distance between the rows of cultivation was 30 cm, with seeds planted 15 cm apart within the row at a depth of 1-2 cm. The traits studied included seed yield, number of capsules per plant, number of seeds per capsule, thousand seed weight, capsule diameter, number of branches per plant, and plant height. Based on the expectation of mean square components, environmental, genetic, and phenotypic variance were calculated, and then the genotypic and phenotypic coefficient of variation and broad-sense heritability were estimated. Statistical analyses, including analysis of variance and comparison of the mean traits between genotypes, were performed using the least significant difference (LSD) test using SAS 9.0 software. Pearson correlation between traits and cluster analysis based on Ward's method according to squared Euclidean distance using R 3.6.1 software. Also, to ensure the correctness of grouping, multivariate tests and analysis of variance tests were performed based on a completely random, unbalanced design.

The analysis of variance showed a significant difference between the studied genotypes for all the agronomic traits (p<0.01</em>). The plant height in the studied genotypes varied from 22 cm (Nige7) to 58.4 cm (Nige1). The highest number of capsules per plant was observed in the Nige 53 genotype, with 20.45 capsules, and the lowest number of capsules per plant was observed in the Nige63 genotype, with 5.3 capsules. The number of branches per plant ranged from 3.9-11.5 per plant. The yield ranged from 263.56 kg/ha (Nige3) to 1409.39 kg/ha (Nige78 genotype). The phenotypic coefficient of variation varied from 48.58% (seed performance) to 5.20 (capsule diameter), and the genotypic coefficient of variation varied from 48.29% (seed performance) to 4.7 (capsule diameter). The highest genetic coefficient of variation (48.29%) belonged to the grain yield trait. The estimated heritability for the assessed traits ranged from 73.81% for number of branches per plant to 98.8% for seed yield. Heritability was 91.91% for the number of capsules per plant trait. According to the correlation coefficient results, seed yield per plant showed a positive and significant correlation with the number of capsules per plant (r=0.87**) and branches per plant (r=0.54*). In the cluster analysis, the studied genotypes were divided into four different groups, and the results of the multivariate analyses indicated appropriate grouping and significant differences in the amount of grain yield and yield components among the groups resulting from the cluster analysis.

The high genetic variation among the studied genotypes indicates the high efficiency of this germplasm for improving grain yield and various traits and the high efficiency of selection in black cumin. Genotypes Nige53, Nige78, and Nige60 had high grain yields compared to other genotypes, which can be used to improve and increase grain yield in black cumin.

Zataria multiflora</em> Boiss. is an important medicinal plant species in the Lamiaceae family, native to Iran and some neighboring countries. Water scarcity is a significant limiting factor for crop growth and production. Reduced efficiency of chemical fertilizer uptake under drought conditions and their adverse environmental effects necessitates using sustainable agricultural practices. Mycorrhizal fungi, through their symbiotic with plants, provide essential nutrients to plants and mitigate the adverse effects of biotic and abiotic stresses on plants. This study investigates the influence of mycorrhizal symbiosis under drought stress and non-stress conditions on morpho-physiological characteristics of eight Z. multiflora</em> ecotypes.

This study was conducted on eight Z. multiflora</em> ecotypes collected from Safashahr, Neyriz, Khanekhore, and Fasa (located in Fars province), Faridan, Baharestan, Kalat Ghazi (located in Isfahan province), and Qasreghand (located in Sistan and Baluchestan province). Two-year-old plants of these ecotypes were subjected to two levels of drought stress, consisting of 50% and 90% soil field capacity, representing drought stress and non-drought stress conditions, respectively. Mycorrhizal fungi were applied at two levels (with and without) in a factorial experiment based on the completely randomized design with three replications, conducted in a greenhouse of the Agriculture Faculty, Shahrekord University, Iran. Drought stress was applied through reduced watering, and arbuscular mycorrhizal fungi were inoculated in holes at a depth of 4-5 cm around the roots in the pots. The symbiosis was confirmed through root staining. Morpho-physiological traits, including plant length, width, crown area, leaf area, dry plant yield, and chlorophyll "a" and "b" content, were measured. After variance analysis, the means were compared using the LSD test. Pearson correlation coefficients were used to determine trait correlations, and stepwise regression was employed to identify traits affecting dry plant yield.

The results of the analysis of variance showed that ecotype, drought stress, and mycorrhizal application had highly significant effects on plant length, plant width, crown area, leaf area, dry plant yield, and chlorophyll "a" and "b" content. The significance of the two- and three-way interaction effects of ecotypes with drought stress and mycorrhizal application in all studied traits indicates the different responses of ecotypes to drought stress and mycorrhizal fungus application. Comparing the means of the two drought stress levels indicated a significant reduction in all traits under stress conditions. Mycorrhizal application in non-stress conditions significantly increased the traits. Using mycorrhizal fungi under drought stress conditions compensated for part of the reduction caused by drought stress in the traits, resulting in an increase ranging from 14% (plant width) to 210% (dry plant yield) compared to drought stress conditions. The comparison of ecotypes in each combination of stress levels and mycorrhizal application levels suggests that plant length and width, crown area, leaf area, and plant dry yield increased. The intensity of response or change in these ecotypes to drought stress and mycorrhizal application varies. The correlation coefficients and stepwise regression results indicate the critical role of leaf area and plant length in dry plant yield under both non-drought and drought stress conditions.

In this restudy, high variability was observed in the evaluated traits such as plant length and width, canopy area, leaf area, plant dry yield, and chlorophyll "a" and "b" levels in the Z. multiflora</em> ecotypes. On the other hand, the different reactions of these ecotypes to drought stress indicate the high genetic potential of this native plant in Iran. Therefore, it can be used to select superior genotypes. Considering the increasing and significant effect of most of the characteristics studied, including yield due to the application of mycorrhizal fungus, this fungus can be used in producing Z. multiflora</em> under standard cultivation conditions and drought stress.

Medicinal plants produce bioactive compounds with high antioxidant value. Due to the importance of Withania coagulans</em> in the pharmaceutical industry and also due to the cutting of this shrub species and its use in the pharmaceutical industry using traditional methods, the risk of extinction of this valuable species will increase, so we should look for the method - to be alternatives to increase the production of secondary metabolites of this plant. Using elicitors as a technique in vitro increases plant production of effective compounds (secondary metabolites). It has been reported that fungal stimulants are also used to induce the production or increase of secondary metabolites in medicinal plants. Therefore, in this study, the effect of cellulase elicitor on the growth index and biochemical and physiological traits of Withania coagulans</em> (Stocks) Dunal) was investigated in cell suspension culture.

Seeds of Withania coagulans</em> were collected from the Mehrestan region of Saravan city, and after disinfection, they were cultivated in ½ MS medium to produce explant. Production explants were cultured in an MS culture medium containing auxin and cytokinin hormones (2mg/l 2, 4, D, and 0.5 mg/l Kinetin) to produce callus. Three sub-cultures of production called into MS culture medium containing transfer previous regulating hormones were done. Investigated treatments include six elicitor treatments: 1: Control 2: Elicitor with a concentration of 200 μg/ml and 24 hours of exposure to the Elicitor, 3: Elicitor with a concentration of 200 μg/ml and 48 hours of exposure to the Elicitor, 4: Elicitor with a concentration of 200 μg/ml and 72 hours of exposure Elicitor, 5: Elicitor with a concentration of 7.5 μg/ml and 14 days of exposure to the Elicitor and 6: Elicitor with a concentration of 10 μg/ml and 14 days of exposure to the cellulase elicitor in a completely randomized design with three replications in the central laboratory of the university of Zabol was applied. Investigated traits were: callus growth index, antioxidant activity, proline content, soluble carbohydrates, malondialdehyde, total alkaloid, and activity of antioxidant enzymes (catalase, ascorbate peroxidase, guaiacol peroxidase, superoxide dismutase, and polyphenol oxidase).

The effect of different cellulase treatments on all traits was significant except polyphenol oxidase enzyme activity. In general, different elicitor treatments improved all the measured traits except the content of total carbohydrates. However, the increase in the trait depended on the concentration and period of exposure to the Elicitor. So, in most of the measured traits, the elicitor treatment with a concentration of 10 micrograms/ml and a period of 14 days caused an increase in all treatments. All cellulase elicitor treatments increased the antioxidant activity of Withania coagulans</em> cells in cell suspension culture. The highest antioxidant activity was observed in the sixth and fifth treatments and the lowest in the second and first treatments (control). The addition of Elicitor in the early stage of cell growth in cell suspension culture (eighth day) despite the lower concentrations of Elicitor (7.5 and 10 μg/ml) leads to a further increase in callus growth, antioxidant activity, Proline, and total alkaloid increased then the addition of Elicitor in exponential phase, despite the high concentration of Elicitor (200 μg/ml). A higher concentration of elicitors causes a hypersensitive response, which leads to cell death; hence, an optimal Elicitor level is required to induce growth.

Cellulase elicitor improved the growth index and antioxidant potential of Withania coagulans </em>through physiological and biochemical traits. According to the obtained results, the use of cellulase elicitor can be considered an important strategy to increase the growth, effective compounds, and antioxidant properties of Withania coagulans</em> for commercial production.

Creeping savory is a medicinal and perennial plant that grows in the north and northwest of Iran. The essential oil of this plant has antibiotic properties and is used in herbal medicines, food preparation, and health products. Salinity stress has adverse effects on photosynthetic processes and plant growth and yield. Salicylic acid protects plants against stress by regulating many physiological and enzymatic processes. So far, not much information has been published about the effect of salicylic acid on the physiological process, morphologically, and yielding traits of creeping savory under salinity stress conditions.

 This factorial experiment was implemented in the greenhouse of the Kermanshah Agricultural and Natural Resources Research Center based on a Completely Randomized Design including four levels of salinity (0-50-100-150 mM) and two levels of salicylic acid (0 and 2 mM). Chlorophyll fluorescence (Fv/Fm) was measured with a Hansatech, UK Pocket PEA device. The chlorophyll index (SPAD) was measured with a SPAD-502Plus device, Minolta, Japan. Leaf proline content and soluble protein were measured based on Bradford method using a Bio Tek PowerWave XS2 Microplatereader, USA. Various morphological and yield traits such as plant height, leaf area (by a Light Box device, ADC, UK), leaf fresh weight, root fresh weight, and shoot fresh weight (g) were measured. Leaf dry weight, root dry weight, and shoot dry weight were weighed after drying the samples at 75°C for 48 hours. Relative water content (RWC) was calculated. Leaf electrical conductivity (µS/cm) was measured with an EC COND 3110, WTW (Germany). Analysis of variance and comparison of means (Dunkan test) were performed using IBM SPSS Statistics (Ver. 26).

 The highest plant height (92.7 cm), leaf area (0.8 cm2</sup>), shoot fresh weight (26.9 g), and shoot dry weight (9.15 g) were obtained at 0 mM NaCl + 2 mM SA. The highest leaf fresh weight (13.5 mg), leaf dry weight (2.5 mg), quantum yield of photosystem II (0.80), and photosynthetic index (37.0) were observed at 50 mM NaCl + 2 mM SA. The highest root fresh weight (27.3 grams), root dry weight (4.3 grams), and the highest relative water content (91.7 percent) were obtained at 0 mM NaCl. The highest proline (12.7 μg/g) was observed at 150 mM NaCl and the highest soluble protein (1.1 mg/g) was observed at 100 mM NaCl + 2 mM SA. The use of 2 mM salicylic acid, under salinity stress conditions, increased plant height (16.4%), leaf area (18.6%), leaf fresh weight (17.3%), shoot fresh weight (35.4%), shoot dry weight (35.8%), relative water content (8.4%) and soluble protein by 41.4%, but decreased proline content (41.4%) and electrical conductivity (49.4%). Applying 2 mM salicylic acid has a significant effect on root fresh weight, maximum quantum yield of photosystem II and chlorophyll index in mild salinity conditions. Also, SA increased leaf dry weight in mild salt stress but decreased it in severe salinity conditions.

This research showed that applying salicylic acid under salinity stress causes changes in some morphophysiological, photosynthetic, and biochemical characteristics of creeping savory. Increasing the salinity levels decreased some photosynthetic, physiological, vegetative, and yield traits, but the content of some osmotic regulators, such as proline and protein, was increased. The application of 2 mM salicylic acid improved some of the adverse effects of salinity in creeping savory at different salinity levels, enhancing growth and yield traits in the salicylic acid-treated plants. Applying two mM salicylic acid increases the tolerance of creeping savory against salinity stress by increasing the osmotic protectants and inducing the activity of antioxidant systems. Based on the research results, growing this plant in soils with a salinity of more than 100 mM is not recommended. Also, in the case of planting creeping savory in saline soils (less than 100 mM), to increase plant growth and farmers' income, it is recommended to apply two mM salicylic acid as a foliar spray.

Alfalfa, Medicago</em> sativa</em> L. from the leguminous family, is among the best fodder plants due to its richness in protein, calcium, vitamins, palatability, and low percentage of cellulose, which is called green gold. Alfalfa</em> is the first crop that was domesticated and cultivated as fodder. Alfalfa leaf weevil, Hypera</em> postica </em>Gyllenhal, is one of the important pests of alfalfa fields in Iran. Its main damage is related to the larval stage, caused by feeding on the plant leaves, especially in the first cut. The most common method of controlling this pest is using chemical pesticides, which cause pesticide residues, the death of natural enemies, resistance, environmental pollution, and the emergence of secondary pests. Therefore, research is necessary today to replace other compounds, such as botanical pesticides. In this research, the effects of Neemarin extracted from the neem plant (Azadirachta indica</em> A. Juss) and Matrine extracted from Sophora flavescens</em> (Ait.) on second instar larvae and adult insects of alfalfa weevils were investigated in laboratory and field conditions.

Laboratory studies were conducted in a completely randomized design, and field evaluation was conducted in the form of completely randomized blocks in the spring of 2023 in Naghadeh City. The botanical insecticide Matrine, with its commercial form on Rui Agro and Neemarin extracted from the neem plant, was used in the experiments. Bioassays were performed on the second instar larvae and adults by immersing the host leaf in the concentrations of both compounds, and the mortality was recorded 24 and 48 hours after the treatment. After determining the LC50 and LC25 of Matrine and Neemarin, their mixed effects on second-instar larvae and adult insects were evaluated. This study used treatments including LC50 Matrine, LC50Neemarin, LC25 Neemarin + LC25 Matrine, and the control treatment on second instar larvae and adults. Also, the effect of sublethal concentration (LC25) on metamorphosis (transformation of the larval stage to pupa and transformation of the pupal stage to the adult insect) was studied. The field experiments were analyzed in three alfalfa fields treated with Neemarin (2000, 3000, and 4000 ppm), Matrine (200, 400, and 600 ppm), and control (water). Each treatment was evaluated in three repetitions using the Henderson-Tilton method.

The results of probit analysis showed that calculated LC50</sub> of the two botanical insecticides, Neemarin and Matrine on adult insects and second instar larvae of alfalfa after 48 hours were 544.65, 50.23, 45.86 and 3.76 ppm, respectively. Also, the effect of LC25</sub> of both biopesticides showed that the greatest inhibition in the transformation of larva to pupa is related to Neemarin (30%) and in the transformation of pupa to adult is related to Matrine (51.50%). In the field experiments, the highest mortality in adults was observed in the concentration of 600 ppm Matrine with 53.33% and in the second instar larvae related Neemarin (2000 ppm) with 27.33% mortality.

These investigations showed that Matrine pesticide has a higher lethality effect than Neemarin in laboratory and field conditions on adults and second-instar larvae of alfalfa leaf weevil. The effect of a sublethal dose of Neemarin on pest metamorphosis was more effective. Therefore, both pesticides can be used to manage this important pest.

 Adventitious root cultures of medicinal plants represent a bountiful reservoir of valuable secondary metabolites.  The esteemed medicinal plant Echinacea</em> purpurea</em> (L.) Monech is highly sought after for its phytochemical properties, with a traditional use of its roots in herbal medicines and dietary supplements as an immune system stimulant for treating inflammatory, viral, and respiratory diseases. However, the heterogeneous synthesis of bioactive compounds in natural environments and the limitations of conventional plant production methods necessitate exploring alternative approaches for obtaining active plant substances and organs. Among various in</em> vitro</em> cultivation techniques, the cultivation of differentiated tissues, specifically roots, consistently demonstrates the ability to yield higher quantities of metabolites than undifferentiated tissues such as callus and suspension cells. 

 The primary aim of this study was to accurately determine the ideal combination of plant explants and plant growth regulators that would facilitate the successful establishment of adventitious root suspension cultures. Three distinct plant explants, namely leaf, petiole, and root, were carefully evaluated in this study. These explants were subjected to varying concentrations (0, 0.5, 1, 1.5, and 2 mg.L-1) of two types of plant growth regulators: indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA). After six weeks, the number of adventitious roots developed by the explants under different concentrations of plant growth regulators was measured. The impact of different sucrose concentrations (10, 20, 30, 40, 50, and 60 grams per liter) in the culture medium was investigated over a four-week cultivation period. Various dynamic characteristics of root growth, including fresh weight, dry weight, volume of the remaining medium, electrical conductivity, and pH, were closely examined. Furthermore, phytochemical properties such as total phenol, total flavonoid, and antioxidant activity were assessed. To administer the sucrose treatments, 0.35 grams of adventitious roots were placed inside 250 ml Erlenmeyer flasks containing 50 ml of half MS culture medium, 1 mg.L-1IBA, and varying concentrations of sucrose, maintaining a pH of 5.8. Measurements were taken from three flasks of each treatment weekly. The experiments followed a Completely Randomized Factorial Design, with statistical analysis conducted using SPSS software.

 Leaf explants exhibited superior root formation to root and petiole explants. The average number of adventitious roots in leaf explants reached 3.37, while petiole and root explants produced only 0.23 and no adventitious roots, respectively. The most effective root formation occurred in MS medium with 1 mg.L-1 IBA, resulting in an average of 4 roots per explant. Hormone concentrations of 0 and 2 mg.L-1 did not induce root formation. In the fourth week of cultivation, the highest fresh weight recorded was 29.8 grams per liter, marking a remarkable 3.4-fold increase from the initial inoculation weight. The culture medium's electrical conductivity and pH decreased as the cultivation progressed. Cultivation weeks did not significantly impact total flavonoid production and related antioxidant activity in adventitious roots. However, the cultivation period significantly affected the total phenol content at 57.56 mg of gallic acid per gram of dry weight. Altogether, lower sucrose concentrations proved more efficient for biomass production, bioactive compound content, and antioxidant activity. The highest biomass accumulation and total phenol content occurred at 3% sucrose. In comparison, in the fourth week of cultivation, the highest total flavonoid content and the highest free radical inhibition percentage were observed at 2% and 1% sucrose, respectively. Notably, a negative correlation was observed between the electrical conductivity of the remaining culture medium and root biomass in lower sucrose concentrations as the culture duration increased.

In light of the immense potential harbored by adventitious root suspension cultures of E. purpurea</em> as an alternative source for the production of secondary metabolites, particularly caffeic acid derivatives, the discoveries made in this research have the potential to advance the field of mass production for active root organs, thereby facilitating the synthesis of these precious compounds through the implementation of appropriate laboratory bioreactor systems.

 The medicinal plant Ocimum basilicum</em> L. is one of the most important medicinal plants. It is native to tropical regions from Central Africa to Southeast Asia and has antimicrobial, antioxidant, analgesic, and anti-inflammatory properties. Drought stress is one of the most important abiotic factors that can severely affect plant growth and yield. This study aimed to investigate the effect of mycorrhizal fungus on some physiological and biochemical properties of the medicinal plant O. basilicum</em> L. under drought stress.

 This experiment was conducted in the research greenhouse of Islamic Azad University, Kermanshah branch in 2022. The experiment was performed as a factorial experiment in a Random completely blocked design with two factors (mycorrhiza fungus) in three levels including control (without inoculation with fungus), G. etunicatum</em> and G. mosseae</em>, water drought stress at three levels (without stress, 75 and 50% of field capacity) were performed in four replications. The traits measured in the experiment include, the number of leaves, relative water content, ion leakage, photosynthetic pigments, proline, carbohydrate, antioxidant activity, phenol content and clonization percentage.

 The results of analysis of variance analysis showed that the effect of mycorrhizal fungus on all the traits measured in the experiment was significant, and also, the effect of drought stress on all the investigated traits was significant except for the amount of phenol and total chlorophyll. The interaction effect of two factors, mycorrhizal fungus and different levels of drought stress, on the characteristics of ion leakage and carotenoid content was significant. Mean comparisons of data showed that the highest number of leaves  (70.5) was observed in the treatment of G. mosseae</em> fungus, and the lowest number of leaves (58) was in the treatment without fungus. The highest relative water content of leaves (79.059%) was in non-stress conditions, which had a significant difference with the stress conditions of 75 and 50% of crop capacity. The highest electrolyte leakage percentage (33.02%) in the stress condition was 50% of the field capacity in the control treatment, which was not significantly different in the same condition as the G. etunicatum</em> fungus treatment. Inoculation with mycorrhizal fungus increased the content of total chlorophyll so that the highest amount of total chlorophyll (0.96 mg/g of fresh weight) was obtained in the treatment of G. mosseae</em>, which was not significantly different from G. etunicatum</em> and also inoculation with mycorrhizal fungi increased the content of chlorophyll a and b by 91.8%. Inoculation with mycorrhizal fungus increased the proline content by 25.33%, too. The highest antioxidant activity (21.13%) was obtained in treating G. mosseae</em>fungus. The lowest amount of essential oil was observed in the 50% of the field capacity treatment, 25.96% less than the control treatment. Drought stress decreased the percentage of root colonization in G. mosseae</em> by 55.77% and in G. etunicatum</em> by 57.93%.

Mycorrhizal fungus improved the morphological and physiological characteristics in green basil plant.symbiosis with G. mosseae</em> fungus under drought stress conditions with increasing proline and antioxidant activity, improved physiological traits and resistance to drought stress in green basil plant.

Plants are exposed to various environmental stresses during their growth period in nature and under field conditions. Due to its medicinal and therapeutic properties, Silybum marianum L. Gaertn. has been used since ancient times for the treatment of liver and gallbladder diseases. This study aimed to investigate the response of Silybum marianum to plant growth regulators under different soil moisture conditions.

The present study was conducted in the agricultural year 1401-1400 at the Forests and Rangelands Research Institute research farm located in Karaj, Iran. The research farm is 5 kilometers southeast of Karaj city at a geographical latitude of 35 degrees and 48 minutes north and a longitude of 51 minutes east, with an elevation of 1320 meters above sea level. The study was carried out in a randomized complete block design with three replications. The experimental treatments included different levels of field capacity (90%, 75%, 50%, 25% FC) and foliar application with plant growth regulators such as salicylic acid (75-150 mg.l-1), spermine (70-140 mg.l-1), brassinosteroid (C28) (1-1.2 µM), and water (control). Soil moisture levels were determined daily using the weighing method and TDR (Time Domain Reflectometry) in the range between field capacity and 25% soil moisture content. Irrigation was conducted according to the desired treatments throughout the growth period based on the plotted graph and with the help of TDR. Foliar spraying was performed seven days apart in three stages, five months after seed sowing in April. At the end of the study, traits including average leaf number, leaf area, canopy diameter, number of lateral branches, root length, root collar diameter, days to leaf emergence, fruit antioxidant capacity, fruit color index, seed ash percentage, leaf sugar content, thousand seed weight, fruit yield, and oil content were measured.

 The study showed that the reduction of field capacity (FC) reduced growth and yield traits in the plant compared to the control treatment. However, the foliar application of plant growth regulators improved growth traits effectively. For instance, the leaf number in the 25% field capacity treatment was almost halved compared to the control treatment (90% field capacity). Foliar spraying with brassinosteroids prevented a significant decrease in leaf number in the plant under different field capacity levels. The highest leaf area was observed in the treatment with 140 mg.l-1spermine + 75% field capacity. Foliar spraying with spermine and brassinosteroids had a significant effect on improving growth traits under stress conditions. Foliar spraying with spermine or brassinosteroids resulted in the highest oil content in the fruit under 50% field capacity conditions. Under 75% field capacity, foliar spraying with brassinosteroids (1 µM) increased fruit yield in the plant by 71% compared to the control treatment at the same stress level and by 5.5% compared to the control treatment under 90% field capacity conditions. The correlation analysis between color indices and fruit antioxidant capacity showed a positive and significant correlation between the percentage of fruit antioxidant capacity and brightness index (L), indicating that fruits with darker colors had higher antioxidant capacity. However, the redness index (a) and yellowness index (b) had a negative and significant correlation with fruit antioxidant capacity.

The research findings have shown that reducing field capacity reduces plants' vegetative and reproductive growth parameters. However, foliar spraying plants with plant growth regulators improves plant growth conditions under water-limited conditions. In general, based on the research results, foliar spraying of plants with spermine at a concentration of 140 mg.l-1 helps improve the growth and performance characteristics, increasing the plant's resistance to moisture stress conditions in the soil.

 As a member of the Lamiaceae family native to Europe, Asia, and North Africa, Vitex agnus-castus is a popular medicinal plant. Despite much research, it is always essential to verify the safety of this valuable species, as it is among the world's best-selling plants. Several species in the genus Vitex do not exhibit recognizable morphologies, confusing their identification. The medicinal use of V. agnus-castus differs from that of other species. For effective pharmaceutical performance, the identification of this species is essential. This study used morphological, micromorphological, and molecular approaches to identify this medicinal plant.

 A total of 17 populations of the target species were investigated in Maraveh Tappeh, a city in the Golestan province located in the eastern region. Pollen samples were collected directly from the natural habitat of the target area. After acetolysis, 30-40 pollen grains were photographed from the polar and equatorial views with a light microscope and 40 and 100 magnification and with the help of a Canon digital camera. In order to study the seed morphology, fully ripe fruits were collected from each studied population during the fruiting season. Twenty seeds from each population were kept for photography with light microscopy and Scanning electron microscopy (SEM). Electron micrographs were prepared from suitable seeds and pollen at the Razi Metallurgical Research Center (RMRC) using an SEM electron microscope. Leaf cells from herbarium samples were extracted from DNA using a DNA extraction kit. Plastid trnL-trnF sequences and nrDNA ITS region sequences were used as barcodes. Polymerase chain reaction (PCR) was performed in 20 microliters with desired primers and a specific temperature program in a thermocycler. After performing the polymerase chain reaction, in order to ensure the amplification of fragments, the final product was electrophoresed. Strong single bands were sent to Codon Genetics Company in Tehran for sequencing.

 Pollen grains of all species are small (12-28 micrometers). According to Ertman's classification, Ghazan ghayeh pollen grains are prolate spheroidal (elongated spherical), and Ghoshe Tappeh pollen grains are subprolate (semi-elongated). All pollen grains are tricolporate. Ghazan ghayeh ornamentation is micro-perforated, while Ghoshe Tappeh ornamentations are reticulated-micro-perforated. With an average length of 3.84 mm and width of 1.52 mm, Ghazan ghayeh seeds are the largest. They are all almond-shaped, but in the Ghazan Ghayeh population, the outer surface of the seed is hollow; in the Ghoshe Tappeh population, the surface is wrinkled and striated. The trnL-trnF gene locus was found to have a multiplication success rate of 85% in the examined plants. There were 516 nucleotides in the amplified fragment in this species. For final registration, the sequence was sent to the GenBank. A 675 nucleotide fragment amplified from this species' ITS marker was sequenced, and the chromatograms were compared with the NCBI database. The gene bank has been notified of the sequence determination. The results showed the highest similarity (98%) with the Vitex agnus-castus species reported from America.

 Local herbal medicines are gaining popularity in different countries and play a significant role in treating diseases today. In order to make medicinal plants more accessible, we need to pay more attention to trust, marketing, and consumption. Barcoding and molecular approaches serve this purpose effectively. To compare plant samples available on the market with natural medicinal plants using DNA barcoding of correctly identified medicinal species. Hence, the sequences used in this study are essential for barcoding Vitex agnus-castus. The medicinal species has been correctly identified and can be used as a standard for evaluating the plants available on the market.

Limited research exists on the relationship between different root structures and the content of steviol glycosides in various genotypes of the Stevia plant (Stevia rebaudiana</em> Bertoni) under varying levels of drought stress. This study aimed to assess the relationship between root characteristics and the content of Rebaudioside-A (Reb-A) and Stevioside (Stev) in two Stevia genotypes under drought stress.

This study evaluated the relationship between root attributes, yield characteristics, and the content of Reb-A and Stev in two Stevia genotypes (Chinese and Indian) under hydroponic conditions. Seven drought levels (-2, -4, -6, -8, -10, and -12 bars using polyethylene glycol 6000) were applied in 2023. The experiment consisted of two completely randomized design trials with three replications each. To begin the experiment, pots filled with a 1:1 ratio of vermiculite and perlite were prepared, and three uniform seedlings were planted in early April. Throughout the growth period and the application of drought stress using Hoagland solution, all pots were initially irrigated with distilled water until the establishment of plants. Once plant establishment was ensured, drought treatments were administered by irrigating once every three days with 400 mL of Hoagland solution with varying osmotic potentials based on the designated treatments. After 65 days from planting, all plants were carefully removed from the pots, and their aboveground and underground parts were separated to measure the desired traits. Steviol glycosides were quantified using high-performance liquid chromatography (HPLC) with two C18 columns and a UV-Vis detector at a wavelength of 202 nm. Finally, the results pertaining to each genotype were analyzed and presented separately.

The results indicated that both genotypes experienced significant reductions in yield and growth characteristics, including plant height, leaf number, leaf fresh weight, leaf yield, and total biomass yield, under drought stress conditions. Compared to the control, all mentioned traits decreased notably under stress. At the -12 bar level, the reduction in leaf yield traits was 59.84% and 87.9% in the Chinese and Indian genotypes, respectively, while total biomass yield decreased by 52.51% and 64.19%, respectively. The Indian genotype exhibited a higher average leaf yield at non-drought stress levels, whereas the Chinese genotype showed a higher yield at higher drought stress levels (beyond -8 bars). This suggests that the Chinese genotype displays greater tolerance in terms of leaf yield under higher stress levels compared to the Indian genotype. Root-related traits, including fresh and dry weight, volume, length, and density, showed the highest averages in the Chinese genotype at the -4 bar level and in the Indian genotype at the -2 bar level. In both genotypes, partial drought stress (-2 bars) resulted in increased average content of Stev when compared to the non-stress treatment, with the highest content of Reb-A and Stev observed at this stress level in both the Chinese and Indian genotypes (2.58% and 2.58%, respectively, and 7.8% and 5.96%, respectively). According to the stepwise regression results, root area, leaf fresh weight, and total biomass yield were identified as influential traits in increasing the combined content of Reb-A and Stev.

Based on the findings, severe drought stress resulted in a decline in growth and yield traits, root characteristics, and the content and yield of the combined Reb-A and Stev. The synthesis of these two glycosides as metabolites in Stevia plants peaked at the partial drought stress level (-2 bars), highlighting their pivotal role as influential metabolites in imparting stress tolerance. The Chinese genotype exhibited the highest resistance to drought stress, displaying superior yield and content of combined Reb-A and Stev compared to the Indian genotype in certain instances. Overall, this study illustrates that severe drought stress directly diminishes the yield and content of Reb-A and Stev in Stevia plants. In contrast, certain root-related and yield traits can serve as reliable indicators of drought tolerance in this plant.