نشریه تحقیقات ژنتیک و اصلاح گیاهان مرتعی و جنگلی ایران
سال سی و یکم شماره 1 (پیاپی 61، بهار و تابستان 1402)

A study of indicator species to manage rangeland ecosystems and preserve the vegetation of natural areas in dry regions is necessary. The knowledge of the ecological requirements of the range species is important for planning and managing plant communities and protecting and exploiting their ecosystems. Golestan province, with its particular geographical position, is the habitat of many plant species. By identifying the factors affecting the growth and adaptation of these species, it is possible to avoid spending money and wasting time planning to improve and restore pastures. One of the valuable genera that grow in rangelands is Artemisia L., which is distributed in arid and semi-arid regions of the country and plays an essential role in soil protection, fodder supply for livestock and medicinal value.

In this study, 12 populations of four species of the genus Artemisia in the rangeland area in the north of Gonbad Kavos in Golestan province, Iran, were studied based on ecological, micro-morphological, and molecular evidence. The ecological studies were carried out using climate data and soil characteristics of the species' habitat. Also, the morphology of the species' pollen grain was investigated using a scanning electron microscope (SEM). The molecular relationships of four species (A. annua, A. sieberi, A. aucheri, and A. fragrans) were investigated using nuclear nrDNA ITS and chloroplast rpl32-trnL(UAG) sequences and their combinations.

The results of the ecological studies showed that many of the Artemisia species were grown in dry climates and in silty and silty loam soils. EC measurements indicated that the soils of area were saline and had poor organic matter. According to the results, many pollen grains of the species were medium-sized and tricolporate. In all studied species, according to the P/E ratio, the outline of the pollen grains, those seen in the polar view were circular, triangular, and equatorial with an elliptical outline and their exine decorations were echinate. The results of molecular studies indicated that all the species formed a monophyletic group with high support (PP=1.00, ML BS=100, MP BS=100). The combined analysis of data showed more supported relationships than the analyses of a part. The results of the cluster tree showed two separate clades. The annual species A. annua was placed in clade 1 and the rest of the studied species with high support (PP=0.88, ML BS=90, MP BS=94) were placed in clude 2.

The results of current research showed that the ecological, micro-morphological characteristics and molecular data were useful in the distinction between the Artemisia species. Such a study allows us to select suitable and adapted genes for the environment and transfer them to the crop species to produce stress-tolerant cultivars. Artemisia is widely distributed in Iran due to its ecological adaptation and high adaptation intensity in natural conditions. Unfortunately, the change in land use and the transformation of natural areas into agricultural lands have caused to shrink the steppic areas and led to limitation of the species formation and diversity. Examining the kinship relationships between species and the evolution of traits can be used to determine the probability of success in interspecies crosses. Therefore, the obtained information can help maintain and balance the Artemisia habitat.

Wheatgrass Elymus tauri (Boiss & Balansa) is a drought- and cold-resistant species that grows on rocky, stony slopes with steep slopes in Azerbaijan, Alborz and some parts of Zagros, Iran. It is a perennial plant. It has a strong root and is resistant to excessive livestock grazing. It is one of the delicious species of pasture for livestock grazing. The genetic diversity of grasses is affected by various factors such as human activities, the environment, pollination system, genetic drift, and population size. Therefore, ecogeographical factors are also important influencing factors in the populations’ structure and genetic diversity. The aim of this study was to investigate the effect of habitat height and ploidy level on the diversity of structural and functional traits of Elymus tauri populations in the northwest of Iran.

 The experimental materials included the seeds of 12 native populations of E. tauri, which were mainly collected from the pastures of East Azerbaijan and Ardabil provinces. At the time of collection, the altitude and geographical coordinates of locations were recorded using GPS. The collected seeds were sown in a research farm using a randomized complete block design in four replications in the northwest of Tabriz city, Iran, at 1350 m above sea level and on the edge of the Aji Chai River. The region’s climate is semi-arid, the texture of the station's soil is light loamy and sandy, and the amount of soil organic matter and chemical elements (NPK) was weak to moderate. The electrical conductivity was measured as EC=6.2 millimos per square centimeter. Soil pH (pH=7.55) was alkaline. Each plot contains four 6m lines. The distance between lines was 40 cm, and the distance between plants on each line was 40 cm. Irrigation was made during the plant growth period. The number of 10 plants from the two middle rows of each plot was randomly selected, and yield and morphological traits were measured and recorded. Genotypes were grouped based on habitat altitude and ploidy levels. A statistical analysis (T-test method) was made between two groups for habitat altitude.

The results of the T-test analysis between the two altitude ranges showed there were significant differences between the two altitudes in terms of the tillers number, the length of the second leaf, the fresh and dry weight of the plant, the number of fertile tillers, the number of infertile tillers, the length of the spike, the length of the flag leaf and number of spikelets (P<0.05). The means of populations in the upper areas were higher than those in the lower areas. Grouping based on altitude showed that populations at altitudes less than 1600 m above sea level were diploid, while populations at altitudes higher than 1700 m were tetraploid. The cluster analysis and principal component analysis (PCA) results confirmed the grouping of populations based on habitat altitude and ploidy levels. Also, the biplot of PC1 vs. PC2 confirmed the grouping of cluster analysis.

This plant had better adaptation in high altitude habitats, so it has grown better in cold conditions than in mild temperate and plain areas. The higher mean values of many traits in the tetraploid populations confirmed the correction of the grouping. Considering that this species grows more in cold regions, it was suggested to use polyploid populations from higher altitude areas to improve breeding varieties.

The Chalghoza pine (Pinus gerardiana) is grown in the eastern and southeastern regions of Afghanistan and has an important role in the socio-economic progress of rural communities. It serves various purposes, such as providing pine nuts, fuel wood, medicinal plants, grazing areas, and shelter for livestock. In this research, the genetic diversity and structure of different Chalghoza pine populations were examined using molecular markers known as start codon targeted (SCoT) and inter primer binding site (iPBS) markers. Due to the excessive harvesting of its nuts and a decline in its population, the IUCN categorizes the Chilgoza pine as a nearing threat.

39 Chalghozeh pine genotypes were collected from various regions across five provinces in Afghanistan, namely Khost, Paktia, Laghman, Kunar, and Nuristan. To extract genomic DNA, a modified CTAB procedure was employed, utilizing megagametophyte tissue from 4-5 seeds per genotype. For this research, six primers each for SCoT and iPBS markers were utilized. To analyze the data and determine genetic relationships, cluster analysis was conducted using the unweighted pair-group method arithmetic averages.

In this study, 12 SCoT and iPBS primers were utilized, resulting in the generation of 48 and 55 bands for SCoT and iPBS markers, respectively. The percentage of polymorphism was estimated at 20.8% for SCoT markers and 29.1% for iPBS markers. The average values of PIC (polymorphic information content) were determined as 0.026 for SCoT markers and 0.045 for iPBS markers, indicating a higher differentiation power of iPBS markers compared to SCoT markers. The genetic similarity coefficient revealed a relatively low genetic diversity among the populations examined. The UPGMA dendrogram, based on the similarity coefficient, demonstrated that the genotypes did not cluster according to their collection sites. This outcome can be attributed to the species' highly cross-pollinating nature and limited distribution range in the studied area. The observed number of alleles (NA) was 1.08 and 1.09, while the effective number of alleles (NE) was determined as 1.075 for both SCoT and iPBS markers. The Shannon's information index (I) was calculated as 0.055 and 0.060 for SCoT and iPBS markers, respectively. The expected heterozygosity (HE) values were estimated as 0.039 for SCoT markers and 0.042 for iPBS markers. The analysis of molecular variance (AMOVA) indicated that the genetic diversity within populations was higher than among populations. Through a Bayesian model-based STRUCTURE analysis, two groups (K=2) were identified among the five populations of Pinus gerardiana, and admixture was observed within individuals. 

The minimal variations observed in total diversities and levels of population differentiation among the five Chalghoza pine populations suggest that the genetic structure of these populations aligns with the species' long-lived perennial nature and regional distribution. These findings have implications for the conservation and cultivation of this economically significant tree, providing valuable insights for its management and preservation.

 Grasses are important forage crops, which are used for forage production, pasture establishment, rangeland renovation, and soil conservation. Perennial cold-season grasses have high forage production, are resistant to adverse environmental stress and have broad adaptability in various environments. They are cultivated for various purposes of agriculture and soil protection. The perennial cold-season grasses with relatively deep roots have a long life in suitable conditions. These plants are able to establish and grow with good productivity in poor to moderate soils. The purpose of this research was to identify and select the best genotypes of four perennial cold-season grass species, in terms of physiological response under normal irrigation and drought stress. 

In this research, the best genotypes of each species of desert wheatgrass (Agropyron desertorum),bromegrass (Bromus inermis), cocksfoot (Dactylis glomerata) and tall fescue (Festuca arundinacea) were selected in previous trials for both seed and forage yield. For each species, three genotypes were evaluated in a field experiment under normal irrigation at 100% and 50% field capacity (FC), using a complete randomized block design with three repetitions in Agriculture and Natural Resources Research of East Azerbaijan in 2021. Drought stress was applied and at the vegetative growing stage, some key physiological traits such as cell membrane stability (CMS), protein content, content of pigments (Chlorophyll and carotenoid) and water potential under both drought stress and control were evaluated . 

The results showed that genotype Fa-G1 in Festuca arundinacea, genotype Bi-G3 in Bromus inermis, genotype Dg-G1 in Dactylis glomerata and genotype Ad-G1 in Agropyron desertorum were more tolerant to drought stress compared to other genotypes within species. 

The response of plants to drought stress was different depending on the type of plant. It was suggested that the leaf osmotic potential index could be used as one of the most important physiological indicators in drought stress conditions to identify drought-tolerant genotypes. Generally, Agropyron desertorum, Bromus inermis, Festuca arundinacea, and Dactylis glomerata are the most resistant to drought stress.

Anise Pimpinella anisum L. is a valuable medicinal plant that 2-3% of its seed weight is essential oils, and about 80-90% of its essential oil is trans-antol. This research was carried out to optimize the indirect organogenesis of P. anisum under in vitro conditions. 

In the first experiment, different methods of seed germinating and disinfection treatments for decontamination of samples were investigated. In the second experiment, the effect of different combinations of BA, Kin and NAA on the direct regeneration of hypocotyl bud explants was investigated. In the third experiment, callus production was investigated from explants of root, hypocotyl, hypocotyl bud, cotyledon leaf and normal leaf, using different concentrations of 2,4-D. In the fourth experiment, the effect of a narrower range of 2,4-D concentration on the callus formation of petiole and root explants was evaluated. In the fifth experiment, the produced calli was exposed to different concentrations of BA and NAA for regeneration. All experiments were conducted in a completely randomized design with three replications. Data analysis was done using Minitab software, and the mean comparison was made using the Tukey method (p<0.05). 

The best germination was obtained at a value of 70% in 24 hours of seed soaking in water (hydro priming) and disinfection with 70% alcohol and 1.5% sodium hypochlorite in 1/2 MS culture medium. In direct regeneration, stem-less leaves were produced. The best direct regeneration response in terms of petiole length was related to treatments of 9 (0.5 mg/L BA plus 0.5 mg/L Kin plus 0.5 mg/L NAA), 14 (0.5 mg per liter Kin plus 1mg/L NAA), 17 (0.25 mg/L BA plus 0.25 mg/L Kin plus 1mg/L NAA) and 19 (1mg/L BA plus 1mg/L Kin plus 1mg/L NAA) with about 4 cm. The highest leaf number (47 leaves) was obtained using the treatments of 14 and 17. In the third experiment, petiole and root explants had the best callus formation. In the fourth experiment, using the treatment of (0.5 mg/L BA +0.75 mg/L of 2, 4-D), the highest rate of 97.22% callus formation was observed in petiole explants. In the fifth experiment, the callus obtained from the previous stage induced the highest shoot with a value of 40% in the control treatment without hormones. Some branches were rooted, and the resulting seedlings were transferred to the greenhouse after adaptation.

 It was concluded that for the regeneration and breeding of anise plant, the indirect regeneration of the callus is more suitable than the direct regeneration method. Also, the petiole was a suitable organ as the initial explant. The 1.5% sodium hypochlorite was the best disinfectant for seed decontamination. Hormonal treatment of 0.75 mg/L of 2, 4-D +0.5 mg/L of BA induced the highest rate (97.22%) of callus in a petiole explant. Therefore, it could be used as a successful hormonal compound for callus formation in anise. The production of branches and roots from callus in the control treatment indicated that it was possible to obtain a whole plant without using any plant growth regulators. The results of this research can be used in micropropagation studies, genetic engineering, cell suspension culture and production of secondary metabolites and other projects related to anise improvement and molecular studies.

The thinning of the ozone layer due to nitrogen oxides and halogenated hydrocarbons has caused an increase in ultraviolet radiation on the planet. This radiation can affect many physiological and anatomical characteristics of plants. This study aimed to investigate the effect of UV-B radiation on the morphological, physiological, and molecular parameters of the peppermint plant (Mentha piperita L.) and the effect of salicylic acid (SA) on reducing the harmful effects of this radiation. 

The eight treatments, including 0.0SA, 0.5mM SA, 1mM SA, 2mM SA, UV-B, SA0. 5+UV-B, SA1+UV-B, and SA2+UV-B were evaluated using a completely randomized design with three replications. Treatments were applied four weeks after planting. Different concentrations of SA were applied as a foliar spray three times at an interval of 10 days. UV-B rays were applied for eight days (40 min each day). Different morpho-physiological parameters were measured. For the gene expression assay, leaf samplings were done one day after the UV treatment stop (named first stage; 18 days after SA spraying) and after recovery time (named second stage; 30 days after SA spraying). 

Compared to the control plants, UV-B radiation caused a significant decrease in the roots' fresh and dry weight. The highest root fresh and dry weights were observed in the SA1mM and the lowest in the UV-B treatments. Under the influence of UV-B rays, the number of leaves significantly decreased in peppermint compared to the control. The highest number of leaves was observed in the SA0.5mM, and the lowest in the UV-B treatment. The amount of anthocyanin under UV-B rays increased significantly compared to control plants. The highest amount of anthocyanin was observed in SA1+ UV-B and the lowest in control plants. The amount of soluble protein in shoots and roots significantly decreased under UV-B rays compared to the amount in the control plants. The highest amount of soluble protein was observed in the shoots in the SA2mM and the roots in the SA2+ UV-B, and the lowest amount was observed in the shoots and roots under UV-B treatment. The amount of proline increased significantly in the shoots and roots under UV-B radiation compared to control plants. The highest amount of proline was observed in shoots and roots in the SA0.5+ UV-B and the lowest amount was observed in the control plants. LS gene expression increased in recovery time compared to the first stage. 

The results showed that the application of UV-B rays to the peppermint plant caused a decrease in the fresh and dry weight of the root, the number of leaves, soluble protein, and LS gene expression and a significant increase in the amount of anthocyanin and proline. Treatment using salicylic acid reduced the damage caused by applying the UV-B rays to the plants.

Flower yield in Rosa damascena is a complex trait that is affected by many traits and environmental factors. Therefore, studying the relationships among traits can be an essential step in selection to improve flower yield. Path analysis by identifying the main components of yield with a high direct effect plays an important role in the selection success, especially indirect selections through yield components. Due to the lack of resources in this regard, the present research aims to identify and determine the effective traits in flower yield, estimate the relationships among traits, and analyze the causality to identify their direct and indirect effects on flower yield via path analysis. 

The germplasm used in this research included 49 genotypes of Rosa damascena Mill.) belonging to different regions of Iran. One-year saplings of the genotypes were planted in April 2004 in the Zaleh research station, Sanandaj (Kurdistan province, Iran), based on the randomized complete block design (RCBD) with three replications and three plants in each plot with three meters of planting distance. Irrigation of the field was carried out in drip form for 15 days. The investigated morphological traits (plant height, number of flowers, etc.) in 2008 were measured on a 5-year old plant, and for the phenological traits (days until leaf opening, flower opening, etc.) the average of three years (2006 to 2008) was used. Data variance analysis was done based on RCBD design. Phenotypic correlation among traits was estimated, and path analysis was done in three steps of estimation of direct, indirect and residual effects of traits on flower yield.

The results showed significant differences (p≤0.01) among the genotypes for studying traits. The correlations of phonological traits with flower yield (FY) were significantly negative, while its relationships with morphological traits were significantly positive. The results of path analysis showed that the number of flowers per plant (FN) and fresh flower weight (with the direct effects of P=0.97** and P=0.33**, respectively, and residual effects of R=0.195) were the main components of flower yield. The total green surface of the plant (TGS) and plant crown diameter (with the direct effects of P=0.56** and P=0.28*, respectively, and R=0.395) were the main components of FN. Also, the number of leaves per plant and average leaf area (with the direct effects of P=1.02** and P=0.085*, respectively, and R=0.237) were the main components of TGS. 

Thus, FN and FFW can be used in indirect selections for FY improvement in Rosa damascena. The other studied traits affected FY indirectly via FN mainly (phonological traits negatively and morphological traits positively). Although the correlation of the height and crown diameter of the plant with FY was positive and very significant, the direct effects of the supplementary stages and the causality analysis sequence showed that the crown diameter (horizontal growth) is more effective than its height (vertical growth) in the reproductive processes of the rose. In other words, the genotypes with higher flower yield usually rise up to a certain height and spread leaves after that.

Elderberry (Elaeagnus angustifolia ˊAnabiˊ) from the Elaeagnaceae family is an essential medicinal plant in Iran. Considering that E. angustifolia has not been given serious attention in Urmia County, Iran for its cultivation, exploitation, and use in the food and pharmaceutical industries of the country. Also, due to its unknown medicinal value in this region, the study aimed to evaluate the phytochemical, antioxidant, and morphological diversity of genotypes in the Urmia region, Iran.  

 In this study, ten genotypes of Elaeagnus angustifolia ˊAnabiˊ were evaluated to investigate the diversity among genotypes based on fruit length, fruit weight, total phenol, flavonoid, ascorbic acid content, and antioxidant activity. The color of the fruit was measured by Hunter Lab device. Total phenol, flavonoid, and ascorbic acid contents were measured using Folin–Ciocalteu, aluminum chloride, and 2,6-Dichlorophenol-indophenol. The antioxidant activity was measured using 2,2-Diphenyl-1-picrylhydrazyl (DPPH). Cluster analysis (Ward method) and Pearson correlation were made using morphological and phytochemical traits. 

The results of this research showed significant differences between genotypes regarding morphological traits such as fruit weight. The maximum value of the fruit length, with a value of 22.43 mm, was related to G2. The highest fruit weight, with a value of 3.18 g, was related to G5. Fruit weight had the most significant effect on fruit yield. Color is one of the most important morphological characteristics of the fruit. In measuring fruit color, the highest amount of L* with a value of 39.6 was related to G2, the highest amount of a* with a value of 24.7, was related to G7, and the highest amount of b* with a value of 19.2 was related to G2. The highest amount of Chroma, with a value of 27.94, was observed in G10. The highest amount of Hue, with a rate of 55.63, was related to G2. The highest amount of total phenol, flavonoids, and ascorbic acid content was observed with 33.58 mg GAE/100 g FW in genotype 10 (G10), 21.11 mg QUE/100 g FW in genotype 10 (G10) and 42.5 mg AA/g FW in genotype 1 (G1), respectively. Also, the antioxidant capacity using DPPH varied between 21.11% and 33.81%. Cluster analysis using morphological and phytochemical data splinted genotypes into two groups. The genotypes of the first group (G1, G6, G7, G8, G9, and G10) had the highest mean values of phenol, flavonoids, ascorbic acid, and antioxidant activity. Correlation results among phytochemical properties showed that antioxidant activity positively and significantly correlated with total phenol, flavonoid and ascorbic acid content.

In general, this research showed that different genotypes, especially genotypes available in cluster one, had significantly higher amounts of antioxidant capacity and phenolic compounds that can be used in the food and drug industry. According to the results, the genotype (G10) was suggested as the best genotype among the genotypes of group one.

 Water shortage is one of the most important environmental factors that may reduce the growth and yield of many agricultural crops. Silicon, as the second most abundant element in the earth's crust, plays a vital role in resisting biotic and abiotic stresses in plants. Considering the increasing problems of water scarcity in Iran and the need to know about drought tolerant plants and the treatments to help increase this tolerance, this research aimed to study the effect of silicon on yield, morpho-physiological, and phytochemical characteristics of hyssop (Hyssopus officinalis) under irrigation regimes in a pot experiment in the Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. 

A factorial experiment was conducted in a randomized complete block design with three replications (each replication included two pots). Drought stress levels included: [(D1) 100% field capacity (FC), (D2) 75% FC, (D3) 50% FC, and (D4) 25% FC)] during three months. Different silicon levels of [(S1) zero (control), (S2) 0.75, (S3) 1.5, and (S4) 2.25 mg/liter] were also sprayed four times, once every three weeks. At full flowering stage, the tested traits including root and shoot length, fresh and dry weight of root and shoot, leaf area, chlorophyll and carotenoid content, total phenol, total flavonoid, antioxidant activity, percentage of essential oil were evaluated. 

The results showed that different irrigation regimes had a significant effect on all the measured traits except aerial weight and internode length. So, drought stress decreased the fresh and dry weight of the root, the leaf area, and the number of branches. Whereas, drought stress increased the amount of total phenol and the percentage of essential oil. Also, the silicon effects were significant on all the measured traits except the number of flowers. Increasing the silicon concentration caused an increase in the plant leaf area and the essential oil percentage.  

Increasing the concentration of silicon in foliar spraying caused an increase in the yield components of the hyssop plant under water stress conditions. At the same time, this treatment was able to increase the production of plant secondary metabolites, including total phenolic compounds, flavonoids, essential oil percentage, and as a result, increase the percentage of free radical inhibition. These results indicate the improving effects of silicon on hyssop performance as a medicinal plant under drought stress conditions. According to the result, the concentration of silicon was 1.5 mg/liter was most effective.

 Withania somnifera, known as Indian ginseng, is a small evergreen shrub that grows in tropical and subtropical regions and belongs to the Solanaceae family. The major constituents of W. somnifera are withanolides, including withafrin A, withanolide D, withanolide A, and withanones. Withanolide A and withanones are primarily synthesized in the roots, while withafrin A and withanones are mainly produced in the leaves. The genes responsible for the appropriate metabolic pathways, such as farnesyl pyrophosphate (FPP), cycloartenol synthase (CAS), sterol methyltransferase (SMT1), and hydroxymethylglutaryl coenzyme A reductase (HMGR) along with non-coding RNAs are essential for the biosynthesis of these compounds. These non-coding RNAs play critical roles in controlling gene expression and enzyme activity. In general, miRNAs are a class of small regulatory RNAs which negatively regulate gene expression at the post-transcriptional level by binding target mRNAs for mRNA cleavage or inhibition of mRNA translation. Many investigations have shown that miRNAs play an essential role in various biological and metabolic processes in plants. This study was aimed to the identification and investigation of the expression of some conserved microRNAs involved in the biosynthesis pathway of Withanolides in W. somnifera. 

For HPLC analysis, a high-performance liquid chromatography system coupled with an ELSD detector, model C-650, was employed. Conserved miRNAs in W. somnifera were identified using BLASTn with a criterion of up to four mismatches and an E-value < 10, based on RNA-seq data and homology analysis conducted by C-mii software. The secondary structure of several miRNAs was determined using Mfold software. The target genes of these potential miRNAs were predicted using psRNATarget, applying the following criteria: In order to determine the function of the target genes, the BLASTx program was used against the protein database with the following parameters: E-value > 1.0E-5, a range of missing central bases between 10 and 11 nucleotides. Samples were collected from leaf and root tissues of three genotypes of W. somnifera (W1 - 7629245, W3 - 7629241, and W4 - 7629244) during the flowering stage. Three technical and biological replicates were used for data collection. The expression levels of miRNA5021 and its target gene SMT1 and miRNA5140 were analyzed using real-time PCR. The 2-ΔΔ CT method was employed to analyze the expression data. 

According to the statistical analysis of the HPLC data, there was no statistically significant difference in the amounts of withaferin-A among the three genotypes (W1, W3, and W4). Among the examined tissues, the leaf of W3 exhibited the highest amount, while the root of W1 had the lowest amount. To identify the miRNAs in W. somnifera, the transcriptome sequences were compared to plant miRNAs using the miRbase website for BLAST homology search. The results obtained from the RT-qPCR method showed that the relative expression levels of miR5021 in W1 roots were 1.16, 1.37, and 1.17 times higher compared to the control (W1 leaf tissue), while they were 1.3 and 3.7 times lower in W3 and W4 leaves, respectively. In the roots of the three genotypes of W. somnifera, the expression of the SMT1 gene decreased by 7.7, 2.8, and 4.0 fold, respectively. However, in the leaves of genotypes W3 and W4, the expression increased by 2.48 and 1.82 times, respectively, compared to the control. The statistical analysis for miRNA5021 and miRNA5140 revealed that in the leaves of W3, the relative expression of miR5140 was 1.57, while in W4, it remained unchanged. In the roots of W1, W3, and W4, there was a decrease in miR5140 expression by seven, nine, and three times, respectively, compared to the control. 

In general, regarding the regulatory role of the identified miRNAs in the present study, these genes could be used to better and more accurately identify the withanolides biosynthetic pathway.