فصلنامه زیست شناسی گیاهی ایران
سال چهاردهم شماره 3 (پیاپی 53، پاییز 1401)

Wheat is the main source of carbohydrates and food source for human consumption. During growth period, it faces with various environmental stresses, including low micronutrients stress such as iron. One of the plant strategies to deal with environmental stresses, is the use of enzymatic antioxidant defense system. To investigate the effect of iron deficiency stress on the expression of genes carbonic anhydrase (CAR), peroxidase (PRX) and glutathione S-transferase (GTS) in Fe-efficient (Pishtaz) and -inefficient (Falat) bread wheat cultivars, a factorial greenhouse experiment was implemented in a completely randomized design with three replications. Cultivars were planted at two levels of iron: 1.4 (iron deficiency) and 10 milligrams of Fe per kilogram of soil (sufficient iron), and leaves and roots of the plants were sampled at two stages, one month after germination (vegetation) and 30% of heading (reproduction). The relative expression level of the mentioned genes was measured in the leaves and roots of the cultivars using Real time PCR technique. Results revealed the highest increase in carbonic anhydrase expression in iron deficiency conditions in the roots of Fe-inefficient cultivar (Falat) at vegetative stage, while the highest increase in the expression of peroxidase and glutathione S-transferase genes were observed in the leaves and roots of the Fe-efficient cultivar at the vegetative and reproductive stages, respectively, under iron deficiency conditions. Therefore, the increased expression of the genes encoding peroxidase and glutathione S-transferase enzymes in Fe-efficient cultivar shows that these cultivars use antioxidant enzymes more effectively to deal with soil iron deficiency stress. IntroductionWheat is the main source of carbohydrates and the most important source of food for human consumption. The plant faces various environmental stresses, including micronutrient deficiency stress, such as iron. One of the plant strategies to deal with environmental stresses is the use of an enzymatic antioxidant defense system. Peroxidase and glutathione S-transferase are antioxidant enzymes that plants use to deal with environmental stress. Peroxidase enzymes are a large group of iron-containing proteins that participate in electron transfer from biological membranes and oxidative reactions (Vlasits et al., 2010). These enzymes play a role in removing all kinds of oxidizing agents and free radicals produced under stress conditions. Plant peroxidases are the only class ІІІ peroxidase enzymes that work in the extracellular space to inhibit H2O2-induced damage (Glusac et al., 2019). Glutathione S-transferases are multifunctional enzymes with multiple substrates, which enable them to catalyze a wide range of reactions. These enzymes are used as hormones carrier, secondary metabolites, and other enzymes. In addition, these enzymes play a significant role in tolerance to biotic / abiotic stresses and regulation of intracellular homeostasis (Hernandez et al., 2020). Materials and MethodsTo investigate the effect of iron deficiency stress on the expression of genes encoding carbonic anhydrase, peroxidase, and glutathione S-transferase enzymes in iron-efficient (Pishtaz) and -inefficient (Falat) bread wheat cultivars, a factorial greenhouse experiment was carried out in a completely randomized design (CRD) with three replications. Cultivars were planted at two levels of iron: 1.4 (iron deficiency) and 10 (iron sufficiency) mg/kg soil, and leaves and roots of the plants were harvested in two stages, one month after germination (vegetation) and 30% of heading (reproduction). The relative expression level of the mentioned genes was measured in the leaves and roots of the cultivars using the Real-time PCR technique under iron deficiency conditions compared to the control conditions. The actin gene was used as a reference gene to normalize the data. After the Real-time PCR reactions, the melting curve analysis was implemented for each gene to ensure the specificity of the amplified products. Also, the specificity of the products was confirmed using agarose gel analysis. Then, the threshold cycle was extracted from the amplification curves using Rotor-Gene Q software, and the relative expression level of the genes was calculated using the ΔΔCT method (Pfaffi, 2001). The normality of the residuals and gene expression data was tested using the Kolmograph-Smirnov method implemented in MINITAB 19 software, and then data were subjected to analysis of variance. The comparison of the means was performed using the SNK test in SAS 2.9. Results and DiscussionThe results revealed the highest increase in carbonic anhydrase expression in the roots of the Fe-inefficient cultivar in the vegetative stage under iron deficiency conditions. Also, the expression level of this gene in the roots of this cultivar in the vegetative stage was significantly higher than that of Fe-efficient cultivar. No significant difference was observed between the expression levels of this gene in the leaves of two cultivars in the vegetative stage and in the roots and leaves of both cultivars in the reproductive stage. The highest increase in the expression of peroxidase and glutathione S-transferase genes under iron deficiency conditions was found in the leaves and roots of the Fe-efficient cultivar, respectively, in the vegetative and reproductive stages. Also, the level of expression of the glutathione S-transferase gene in the leaves of the Fe-efficient cultivar in the vegetative stage and the roots of the same cultivar in the reproductive stage had a significant difference compared to the leaves and roots of the Fe-inefficient cultivar. Oxidative stress caused by increasing in the level ofreactive oxygen species in iron deficiency conditions can lead to damage to cellular components such as proteins, DNA, and membrane lipids. Plants have developed antioxidant systems to deal with oxidative stress and regulate the levels of reactive oxygen species under stress conditions, which includes some antioxidant enzymes such as peroxidase and glutathione S-transferase. (Hernandez and Rodriguez, 2020). ConclusionThe results of the current investigation revealed the highest increase in the expression of the carbonic anhydrase gene under iron deficiency conditions in the roots of the Fe-inefficient cultivar (Falat) in vegetative stage, while the highest increase in the expression of peroxidase and glutathione S-transferase genes under iron deficiency conditions was observed in the leaves and roots of the Fe-efficient cultivar in the vegetative and reproductive stages, respectively. Therefore, the increased expression of genes encoding peroxidase and glutathione S-transferase enzymes in Fe-efficient bread wheat cultivars demonstrates that these cultivars use antioxidant enzymes more effectively to deal with soil iron deficiency stress.

In order to investigate the effects of nanoparticles) nano Fe-Si oxide) and biofertilizers (Azospirillum lipoferum and Pseudomonas putida) on chlorophyll fluorescence components and some physiological traits of triticale at different levels of irrigation, an experimental as the factorial study was conducted based on randomized complete block design with three replications. The studied factors included three levels of irrigation (full irrigation as control, irrigation withholding at 50% of booting and heading stages as severe, and moderate water limitation, respectively), application of biofertilizers (Azospirillum  lipoferum and Pseudomonas putida) in four levels, and nanoparticles foliar application (nano iron oxide and nano silicon oxide) at four levels. Both applications of plant growth-promoting rhizobacteria and nanoparticle foliar application under full irrigation increased chlorophyll index (50.23%), relative water content (43.97%), stomatal conductance (36.78%), quantum yield (47.38%), maximum fluorescence (34.84%), variable fluorescence (98.49%), and grain yield (43.28%) in comparison to no application of biofertilizers and nanoparticles under irrigation withholding at booting stage. Both applications of plant growth-promoting rhizobacteria and nanoparticles increased the chlorophyll index, relative water content, stomatal conductance, quantum yield, maximum fluorescence, variable fluorescence and grain yield in both levels of full irrigation and severe water limitation, but decreased electrolyte leakage and minimum fluorescence. In addition, severe water limitation or irrigation withholding at the booting stage increased chlorophyll index (7.74%), relative water content (10.99%), stomatal conductance (22.46%), quantum yield (15.2%), maximum fluorescence (26.54%), variable fluorescence (75.61%) and grain yield (28.59%) in comparison to no application of biofertilizers and nanoparticles at the same irrigation level. Generally, the application of biofertilizers and nanoparticles showed a better performance at different irrigation levels due to the improvement of physiological traits.
Triticale is a human-made crop, being a hybrid by cross-fertilization of wheat (Triticum spp.) and rye (Secale spp.). In general, triticale combines the high yield potential of wheat with the biotic and abiotic stress tolerance of rye, making it more suitable for production in marginal areas (acidic, saline, or soils with heavy metal toxicity) (Cantale et al. 2016). In arid and semi-arid regions, drought stress as the main factor and salinity stress as a secondary factor decrease plant growth and yield. Water limitation can damage pigments and plastids, and educe chlorophyll index, stomatal conductance, quantum yield, and relative water content. Several strategies have been developed in order to decrease the toxic effects caused by environmental stresses on plant growth. Among them, the use of bio-fertilizers such as plant growth-promoting rhizobacteria (PGPR) and also nanoparticles such as nano iron-silicon oxide plays a very important role in yield improvement. Inoculation of plants with native suitable microorganisms may decrease the deleterious effects of environmental stresses and increase stress resistance of plants by various mechanisms, including synthesis of phytohormones such as auxins, cytokinin and gibberellins, solubilization of minerals like phosphorus, production of siderophores and increase in nutrient uptake. There is little information on the effects of nanoparticles and biofertilizers on chlorophyll fluorescence components and some physiological traits of triticale at different irrigation levels. Therefore, the objective of the present study was to evaluate the effect of nanoparticles (nano Fe-Si oxide) and biofertilizers (Azospirillum  lipoferum and  Pseudomonas putida) on chlorophyll fluorescence components and some physiological traits of triticale at different levels of irrigation.
 
An experiment as factorial was conducted based on a randomized complete block design with three replications at the research farm of the Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili in 2021. The experimental factors included irrigation in three levels (full irrigation as control, irrigation withholding at 50% of booting and heading stages as severe, and moderate water limitation, respectively (BBCH 43 and 55 respectively), the application of biofertilizers in four levels (no application as control, application of Azospirillum, Pseudomonas, both application Azospirillum + Pseudomonas) and nanoparticles foliar application at four levels (foliar application with water as control, nano iron oxide foliar application (1 g.L-1), nano silicon oxide (50 mg.L-1), both application nano iron-silicon oxide). Psedomunas and Azospirillum were isolated from the rhizospheres of wheat by the Research Institute of Soil and Water, Tehran, Iran. For inoculation, seeds were coated with gum Arabic as an adhesive and rolled into the suspension of bacteria until uniformly coated. The strains and cell densities of microorganisms used as PGPR in this experiment were 1×108 colony forming units (CFU). In each plot, there were 5 rows with 2 m long. In each experimental plot, two beside rows and 0.5 m from the beginning and end of planting lines were removed as margin, and measurements were done on 0.2 m2 for grain yield. The used nano silicon-iron oxide had an average particle size of less than 30 nm and the special surface of particles was more than 30 m2.g-1. They were the product of Nanomaterial US Research which was provided by Pishgaman Nanomaterials Company of Iran. Nano silicon oxide and nano silicon oxide powder were added to deionized water and placed on ultrasonic equipment (100 W and 40 kHz) on a shaker for better solution. Foliar application of nano silicon and nano iron oxide was done in two stages of period growth BBCH 21 and 30. Chlorophyll Index was calculated by a chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Japan). RWC and EL were measured according to the method of Kostopoulou et al. (2010) and Farooq and Azam (2006), respectively. Quantum yield was measured on flag leaves by the uppermost fool expanded leaf using a fluorometer (chlorophyll fluorometer; Optic Science-OS-30 U.S.A.). Analysis of variance and mean comparisons were performed using software package SAS v9.12. The main effects and interactions were compared by LSD (least significant difference) test at the 0.05 probability level, using the SAS version 9.1. 
 
The results showed that both applications of plant growth-promoting rhizobacteria and nanoparticle foliar application under full irrigation increased chlorophyll index (50.23%), relative water content (43.97%), stomatal conductance (36.78%), quantum yield (47.38%), maximum fluorescence (34.84%), variable fluorescence (98.49%), and grain yield (43.28%) in comparison to no application of biofertilizers and nanoparticles under irrigation withholding at booting stage. Both applications of plant growth-promoting rhizobacteria and nanoparticles increased chlorophyll index, relative water content, stomatal conductance, quantum yield, maximum fluorescence, variable fluorescence, and grain yield at both levels of full irrigation and severe water limitation, but decreased electrolyte leakage and minimum fluorescence. In addition, severe water limitation or irrigation withholding at the booting stage increased chlorophyll index (7.74%), relative water content (10.99%), stomatal conductance (22.46%), quantum yield (15.2%), maximum fluorescence (26.54%), variable fluorescence (75.61%), and grain yield (28.59%) in comparison to no application of biofertilizers and nanoparticles at the same level from irrigation levels.
 
Based on the results of the study, the application of biofertilizers and nanoparticles showed a better performance at different irrigation levels due to the improvement of physiological traits. In other words, the effects of the simultaneous application of PGPR and nanoparticle inoculation are more positive than the single application of PGPB and nanoparticles. Therefore, one of the suitable methods to improve plants' resistance to environmental stress is the application of PGPB and nanoparticles, which play a very important role in yield and growth improvement.

In desert areas, a variety of environmental stresses, including dust, affect the biomass and nutritional value of forage plants. In order to improve the value of fodder in dry areas, in the present study, the effect of plant growth-promoting rhizobacteria (Bacillus pumilus and Zhihengliuella halotolerans) on some morphological and physiological characteristics and ionic content of Seidlitzia rosmarinus was investigated under dust stress (control and 1.5 g/m2/month) in a factorial experiment based on a completely randomized design with three replications for 5 months in greenhouse conditions. The results showed that dust stress had a negative effect on the morpho-physiological characteristics of Seidlitzia rosmarinus. Z. halotolerans strain compared to B. pumilus had a greater effect on the improvement of Seidlitzia rosmarinus under dust stress so that Z. halotolerans increased 192% of potassium, 20% of nitrogen, 12% of iron, 48% of calcium, 37% of magnesium, 31 % of manganese, 30% of dry biomass, 74% of proline, 18% of soluble sugars, and 52% of sodium. The results of this research showed that the effect of bacteria on the species of Seidlitzia rosmarinus can be dependent on the type of bacteria. The results also showed that the plant growth-promoting rhizobacteria can increase the seedling quality index under dust stress conditions and help us to increase the fodder species in arid and semi-arid ecosystems.
 
In desert areas, environmental stresses affect the biomass of plants which can be used as forage. Seidlitzia rosmarinus Bunge ex Boiss forms an important part of the flora of the desert areas and plays an essential role in protecting the soil and feeding camels and sheep. One of the important environmental stresses accompanying drought and salinity is dust both on a local scale and on a regional scale. Dust particles are placed on the leaves, young branches, and the surface of the trunk of plants for a long time. This fact applies more to desert environments, due to the low rainfall that prevents the removal of dust particles from the surface of the leaves and other parts of the plant. Dust can cause harmful effects on photosynthetic activity and cause serious damage to the plant (Najafi Zilaie et al., 2022). Plants under these stresses will experience a series of morphological, physiological, and biochemical changes. In recent years, the use of plant growth-promoting rhizobacteria as a leading strategy for reducing the effects of environmental stresses has attracted the attention of researchers. This research was designed and implemented with the aim of investigating the effect of inoculation of two plant growth-stimulating bacteria, Bacillus pumilus and Zhihengliuella halotolerans, on some morphological and physiological characteristics and ionic content of Seidlitzia rosmarinus under dust. The results of this research can help us in the afforestation of this species under dust in the conditions of climate change in arid and semi-arid ecosystems.
 
Three-month-old seedlings of the same size were inoculated by plant growth-promoting bacteria under dust stress in a two-factor factorial experiment with a randomized complete block design in three replications in a period of 5 months. The strains of Bacillus pumilus and Zhihengliuella halotolerans were provided by previously purified research by Amini et al. (2022). Inoculation was applied twice to the plant roots. Only nutrient broth solution without bacteria was applied to the control plants. Dusting was done using a simulator. The amount of falling dust equal to 1.5 grams per square meter per month was applied once a week. After completing the treatments, 8-month-old seedlings were sampled for physiological, morphological, and ionic content tests.
 
The results showed that dust stress had a negative effect on the morpho-physiological characteristics of Seidlitzia rosmarinus. Z. halotolerans strain compared to B. pumilus had a greater effect on the improvement of Seidlitzia rosmarinus under dust stress so that Z. halotolerans increased 192% of potassium, 20% of nitrogen, 12% of iron, 48% of calcium, 37% of magnesium, 31 % of manganese, 30% of dry biomass, 74% of proline, 18% of soluble sugars, and 52% of sodium. The results showed that dust treatment alone reduced the seedling quality index by 17%. The seedling quality index in the inoculation of Z. halotolerans and B. pumilus bacteria with seedlings in conditions with dust and without dust was not significant compared to the control. The results of this research showed that the effect of bacteria on the species of Seidlitzia rosmarinus can be dependent on the type of bacteria. The results also showed that the plant growth-promoting rhizobacteria can increase the seedling quality index under dust stress conditions and help us to increase the fodder species in arid and semi-arid ecosystems.
 
It is concluded that the plant growth-stimulating bacteria improved the morphological, physiological, and biochemical characteristics of Seidlitzia rosmarinus seedlings under dust stress compared to the control treatment. Meanwhile, the Z. halotolerans strain (that had been isolated from the Seidlitzia rosmarinus) had a greater effect than B. pumilus in improving the negative effects of dust on most of the measured characteristics and this makes clear the importance of bacteria-plant interaction. It can be concluded that the rhizosphere of plants is a suitable source for isolating growth-promoting bacteria and they can promise to increase the production of fodder and improve the tolerance of plants to air pollution. However, it is necessary to carry out additional field research in the habitat of these desert species in order to prove the performance and efficiency of these bacterial strains as suitable biofertilizers to deal with dust-stress conditions.

To optimize the bioassay system of auxin hormones by identifying their effect on the growth of Arabidopsis plants under in vitro conditions, Arabidopsis seeds were germinated vertically in ½ MS medium. After 5 days, seedlings with the same root growth were transferred to ½MS medium culture containing IAA, IBA, NAA, and 2,4-D hormones with concentrations of 0, 0.01, 0.03, and 0.05 mg/ L. After 10 days, plant growth characteristics were measured using image analysis plus the fresh and dry weight of seedlings in each treatment. The different hormones had different effects on the growth. Increasing the concentration of IAA, IBA, and NAA hormones caused the reduction of the rootlets’ induction. The use of different concentrations of 2,4-D hormone prevented the growth of roots. Higher concentrations resulted in the production of callus in the roots. The concentration of 0.01 mg/L of the examined auxin hormones mostly had the highest rate of leaf formation and biomass production. This bioassay method can distinguish the effects of different auxin hormones on different aspects of plant growth. The results can help in selecting the required auxin hormone in various activities and make it possible to identify unknown auxin compounds. IntroductionPlant hormones perform different biological functions in different tissues according to the growth stage or in response to environmental conditions. Evidence shows that the response of the plant to different types of auxin is different from each other and the activity of each of these hormones causes the activation of a range of different physiological processes in the plant. On the other hand, all lateral root growth stages are dependent on the amount of intracellular auxin hormone accumulation. Despite the similarity, these hormones are functionally different due to differences in structure which affect their signal transmission and downstream physiological pathways. Therefore, it is necessary to optimize a bioassay system to identify and differentiate the auxin hormones based on their biological effects, so we tried measuring the performance of different concentrations of auxin hormones. Materials and MethodsThe effects of different levels of auxin on Arabidopsis thaliana (Columbia cultivars) seedlings were investigated and ½MS medium culture containing 1% sucrose and 0.75% agar. The pH of the medium culture was set at 5.8 ± 0.1 and autoclaved at 121°C for 20 minutes. The seeds were disinfected with 1% sodium hypochlorite and sterile distilled water for 10 minutes and were cultured in ½MS without hormones. The petri dishes were placed vertically in the growth room for 5 days. The seedlings with 1 cm roots length were selected and in the 4 hormonal treatments, medium cultures were sub-cultured including IBA, 2,4-D, IAA, and NAA in three concentrations of 0.01, 0.03, and 0.05 mg/L, as well as the control treatment without hormones with three replications. Hormones were added to the sterile medium culture after passing through a 2 μm filter. After 10 days, the number of leaves, root length, the number of lateral roots, and fresh weight were measured. The growth level of the shoot and root and the level of greenness of seedling leaves were performed using image analysis and ImageJ software, dry weight of the seedlings was measured after exposure to 72°C for 48 hours. All treatments were analyzed using one-way ANOVA. The mean comparison of treatments was performed by LSD test with a minimum significant difference at a 5% probability level using JMP software (version 8.0). Graphs were also drawn using Excel software.  Results and DiscussionThe presence of auxin in different concentrations inhibits the root length growth and the control seedlings showed the highest root length growth. Seedlings grown at the high concentrations of NAA and in the presence of 2,4-D had the lowest root length with an average of one cm. Seedlings grown in NAA hormone treatment with a concentration of 0.01 mg/L had the highest rootlets number. However, the roots formed in the presence of 2,4-D and high concentrations of NAA produced callus and did not grow normally. The root area in the presence of IBA was more than IAA. The number of seedling leaves grown at 0.01 mg/L concentration of hormones was equal to or more than the higher concentrations of these hormones. The lowest number of leaves belonged to the seedlings grown in 0.03 mg/L NAA and 0.05 mg/L 2,4-D treatments. IAA concentration had a direct relationship with leaf area and with the increase in the concentration of this hormone the leaf area increased, but the use of IBA had the opposite effect on leaf area and decreased with increasing its concentration. The highest leaf greenness was observed in three concentrations of 2,4-D and then in the control. The use of other auxin in the medium culture inhibited the production of chlorophyll in the cells. After the use of auxin in Arabidopsis plants, the fresh and dry weight of the seedlings grown in the presence of the concentration of 0.01 mg/L of NAA had the highest fresh and dry weight. ConclusionThe effect of auxin hormones on different aspects of plant growth was different from each other. IAA increased the number of rootlets and low concentration and formed many rootlets on the main root. High concentrations led to an increase in the number of lateral roots, but the root length significantly decreased. Also, 2,4-D stimulated callus production in the roots. The presence of IBA in the medium culture decreased the average root length. The difference in the morphological effects of different auxin hormones can be considered a road map that will provide the possibility of identifying the auxin hormone in unknown extracts in future research.

Despite numerous reports on hard germination and growth of Bunium persicum, there is no published study on B. cylindricum. Although the amount of essential oils and phenolics is high in B. persicum, they are significant in B. cylindricum too. Plant development from seed cultivation to seed production took 3-4 years including the formation of cotyledon leaves, a uni- or bi-pinnate leaf, one tri-pinnate leaf, and two or more tri-pinnate leaves (rosette) with long petiole in the fourth year. During development, corm size increases, and in flowering years, along with rosette leaf formation, flowering stems are also formed and their number depends on corm size. Flowers were protandrous, without calyx with thin petals containing secretory epidermis. Tapetum is the secretory type and pollen is tricolporate, prolate (P/E 1.7), small size (24µm), ovate with striate-regulated sculpture. The Ovary is two-carpellate, inferior with apical placentation which its two cylindric-linear mericarps are separated acropetally from the carpophore at maturity. IntroductionBunium (Apiaceae) has about 14-17 species in Iran. Secondary metabolites of some species of Bunium, especially B. persicum, have been studied. The antimicrobial, antioxidant, anti-inflammatory, anti-diabetic, and other therapeutic properties of their essential oils have been reported. The presence of numerous essential oils in B. cylindricum indicates its medicinal importance. In some cases, the same names are used for different species, or different names are used for different populations of one species. In addition, some people mix fruits (seeds) of different species of Bunium and sell them as B. persicum in the markets. Therefore, the correct identification of species and their cultivation is important, particularly for species that are marketed. There are several reports about hard germination and cultivation and growth problems of B. persicum. However, there is no published study on the cultivation, life cycle, and vegetative and reproductive structures of B. cylindricum, except general descriptions in plant Flora's books. Kazemivash et al. (2020) reported that in B. persicum, the tapetum layer is secretory type; pollen grains are ovate and bi-porate; gynoecium is two-carpellate with lower ovary and schizocarpic fruits. Apiaceae pollen has been divided into five types: sub-rhomboidal (P/E=1–1.5), sub-circular (1–1.5), oval (1.5–2), sub-rectangular (2), and equatorially constricted (≥ 2). Fruit is a dry schizocarp composed of 2 mericarps that split apart at maturity acropetally. Mericarps have secretory ducts that cause good aroma and flavor in many plants of this family law. In the present study, species cultivation and seed formation were done for the first time. Further, the structure and development of vegetative and reproductive structures were studied and investigated. Materials and MethodsThe ripe seeds (mericarps) of B. cylindricum were collected from Sarduiyeh, Kerman province, Iran. They are cultivated in petri dishes, pots, and in plant habitat in a garden. Seed germination, the life cycle of the species, and different organ formation were studied until flower and seed/fruit formation. Fresh structures (corms, leaves, inflorescences, flowers, and fruits) in different developmental stages were studied and photographed using stereo and light microscopes or the naked eye. The pollen structure, P/E ratio, and pollen sculpture were studied using light and Scanning Electron Microscopy (SEM). Fruit size and the number of fruits in the umbellate were surveyed too. For anatomical studies, fresh flowers were fixed in FAA (formaldehyde: acetic acid: 70% ethanol, 5:5:90, v/v/v) for 24 h, washed with distilled water 4 times (each time for 5 min), dehydrated in 30%, 50%, 70%, 80%, and 100% ethanol series and embedded in paraffin. Then, 5 μm sections were cut by a rotary microtome (Micro-Tec, Germany), put on glass slides, stained by hematoxylin and eosin, observed by a light microscope, and photographed. The mean of seed germination, the polar and equatorial axis of pollen, and their ratio (P/E), as well as the average length of umbel rays and fruit, were measured. Results and DiscussionBunium is one of the medicinal plants with high economic and export value. There are no published studies on the cultivation, seed germination, and development of B. cylindricum. The study of the life cycle of B. cylindricum showed that the species produces flowers and fruits (seeds) 3-4 years after cultivation. In the first year, cotyledon leaves and a small underground part (corm) are formed, and its size increases during the following years producing compound rosette leaves and shoots. After cotyledon leaves, a uni- or bi-pinnate compound leaf with long petiole, then in the next year one tri-pinnate leaf and finally with increasing tuber size, tri-pinnate leaves (≥2) and flowering shoots are formed. There are several studies indicating hard plantation and germination of Bunium using different treatments for breaking dormancy without studying the next developmental stages till flowering. The present study optimized cultivation, seed germination, and flower and seed production in B. persicum for the first time. The abundance of flowers in broad umbellate inflorescences and secretory structures of pistils and petals help to attract pollinators confirming Linder (1998) who reported the effective role of inflorescences in pollination. Flowers were pentamerous, protandrous, without calyx with thin petals containing secretory epidermis. Tapetum was secretory type and pollen grains were tricolporate, prolate (P/E 1.7), small size (24µm), ovate with striate-regulated sculpture according to Punt et al. (2007), Cerceau-Larrival and Roland-Heydacker (2013), Başer et al. (2021), and Kadluczka et al. (2022). Studies in B. persicum indicated secretory tapetum and bi-porate. The gynoecium is bicarpelate producing a schizocarp fruit in which its two mericarps are separated at maturity. Its distinctive flavors are due to fruit secretory cavities.  ConclusionThere are several studies reporting hard plantation and seed germination of Bunium genus, especially B. persicum. In this study, cultivation, seed germination, and seed production of B. cylindricum were optimized. Seed germination was ≥ 90% and germinated seedlings produced fruits (seed) in the fourth year. Flowers were pentamerous, protandrous, without calyx with thin petals containing secretory epidermis. Pollen grains were tricolporate, prolate, small-sized, and ovate with striate-regulated sculpture. The fruit is a schizocarp consisting of two fused carpels that separate at maturity into two mericarps, each containing a single seed. Secretory ducts are distributed in the fruit pericarp.

The Moringa (Moringa oleifera L.) is a medicinal plant with medicinal properties and many valuable metabolites including flavonoids. Due to the medicinal and therapeutic importance of this plant, this research was conducted with the aim of studying the effect of elicitors such as methyl jasmonate, salicylic acid, and phenylalanine on biochemical properties and the production of valuable secondary metabolites from its callus tissue in vitro. According to the results obtained, it was found that higher concentrations of salicylic acid and methyl jasmonate led to a significant increase in peroxidase enzyme activity in the treated calluses. The callus samples treated with methyl jasmonate and phenylalanine showed significantly higher catalase enzyme activity compared to the control treatment at most concentrations and treatment durations. The highest level of flavonoids (0.628 mg g-1) was related to the culture medium containing 200 mg L-1 methyl jasmonate for 96 h. Regarding the duration of treatment with plant growth stimulants, for most treatments, the amount of anthocyanin production increased with the growing duration of elicitors treatment. Based on the results, increasing the salicylic acid concentration from 50 to 100 and 200 mg L-1, particularly over a 48-h period, resulted in a rise in rutin production in the Moringa callus. On the other hand, the highest amount of quercetin (5.38 mg g-1) in the culture medium with 200 mg L-1 methyl jasmonate for 96 hours and the highest amount of kaempferol (3.45 mg g-1) in the culture medium containing 100 mg L-1 salicylic acid was observed for 96 h.

Moringa, Moringa oleifera L, is a member of the Moringaceae family (Mashamaite et al., 2022). Due to the high medicinal and food value of this plant, it is known as the tree of the century. All constituent parts of Moringa, encompassing its roots, stems, leaves, and seeds, bear both medicinal and dietary utility, serving as a pivotal reservoir of vitamins and minerals (Mohlala et al., 2023). The leaves, in particular, exhibit pivotal medicinal attributes, owing to the presence of notable bioactive compounds such as saponins, tannins, steroids, flavonoids, coumarins, quinones, phenolic compounds, and alkaloids, which are proficiently employed in cancer prevention and treatment endeavors (Kurtulbaş et al., 2022). Prominently, a comprehensive range of quercetins, rutin, kaempferol, gallic acid, moringin, and the extensive Niazimicin assemblage represents the principal assortment of compounds derived from the diverse organs of this botanical specimen (Syeda & Riazunnisa, 2020). By virtue of its potent antioxidant property, quercetin, abundantly discovered within the leaf and seed samples of M. oleifera, orchestrates metal chelation and free radical inhibition (Bhaskar et al., 2022). There are various methods to increase the production of secondary metabolites as valuable compounds in medicinal plants, which include the use of agents in cell and plant tissue culture in vitro. The induction of callus cells in M. oleifera plant is largely influenced by temperature, nutrients, pH, and the addition of ascorbic acid and plant growth regulators in the culture medium. Stress induction increases the production of plant secondary metabolites. Stimulants are divided into biological and non-biological categories (Arya et al., 2021). Among the widely used stimulating factors used in most tissue culture programs are fungal carbohydrates, yeast extract, methyl jasmonate, salicylic acid, and amino acids such as phenylalanine and Polyamines such as chitosan (Raj & Saudagar, 2019). Common plant hormones such as salicylic acid and jasmonic acid are key markers for the expression of genes involved in plant defense systems (Patel et al., 2020; Shafighi et al., 2022). Considering the unique properties of the M. oleifera plant and its importance in the pharmaceutical and food industries, as well as the importance of new methods of plant tissue culture to produce and increase the amount of plant secondary metabolites, the present research aimed at increasing the production of biochemical compounds and valuable secondary metabolites such as rutin, quercetin and kaempferol from the callus tissue obtained from the leaf explants of this plant, using methyl jasmonate, salicylic acid, and phenylalanine as stimulating agents in different concentrations and time durations.
 

Callus tissue samples of M. oleifera plant were obtained from leaf explants grown in an MS base medium containing 2 mg/L 2,4-D and 0.5 mg/L BAP (Riyathong et al., 2010). Plant growth stimulants (methyl-jasmonate, salicylic acid, and phenylalanine) in concentrations of zero (control), 50, 100, and 200 mg/l during time periods in an MS base culture medium containing 2 ml 2,4-D and 0.5 mg/liter of BAP were utilized. Biochemical compounds such as peroxidase (MacAdam et al., 1992), catalase (Chanes & Mahely, 1996), proline (Bates, 1973), flavonoid (Chang et al., 2002) and anthocyanin (Wagner, 1979) along with metabolites secondary substances such as rutin, quercetin, and kaempferol (Hurst et al., 1983) were measured in callus tissue samples collected after 24, 48, and 96 hours after applying the stimuli. The experiment was conducted in a completely randomized design with three replications. Data variance analysis and average data comparison using SPSS ver. 26 were done.

The results of variance analysis of the data demonstrated that the activity level of peroxidase and catalase enzymes, the amount of amino acid accumulation of proline, total flavonoid, anthocyanin, the amount of rutin, quercetin, and kaempferol in the callus samples of M. oleifera plant in vitro was significantly (p < 0.01) affected by the type of stimulus used, the duration of use, and the interaction between the type of stimulus and the duration of its use. The highest amount of peroxidase enzyme activity was related to the culture medium containing 100 or 200 mg/l of methyl jasmonate and 200 mg/l of phenylalanine for a period of 96 hours. According to the obtained results, by raising the concentration of plant growth stimulants (methyl jasmonate and salicylic acid) from 50 to 100 and 200 mg/liter in different periods of time, the peroxidase enzyme activity did not change significantly. On the other hand, the highest amount of peroxidase enzyme activity (455.886 protein-µmol H2O2 min-1 mg-1) was related to the culture medium containing 200 mg/liter of phenylalanine for a period of 96 hours. The obtained results are consistent with the results reported by Shabani et al., (2009). The highest amount of proline amino acid accumulation (2.19 μg/mg) was observed in the treatment of callus samples with 200 mg/L of methyl-jasmonate for a period of 96 hours. In regards to the temporal extent of utilizing stimulants in any and all culture mediums that encompass methyl jasmonate, phenylalanine, or salicylic acid, the quantity of proline accumulation detected in callus obtained subsequent to a treatment period of 96 hours is notably greater. This phenomenon becomes apparent within the time frames of 24 and 48 hours. The highest amount of flavonoid (0.628 mg/g of callus) was observed in the treatment of 200 mg/l of methyl jasmonate for 96 hours. Zhang et al. (2009) stated that the flavonoid content of blackberry callus tissue increases remarkably in the treatment with methyl jasmonate and salicylic acid, which is consistent with the results obtained in this study. Also, the highest amount of anthocyanin (12.42 μmol/g of callus weight) was related to the treatment of 200 mg/l phenylalanine for 96 hours. According to the results obtained in this research, utilizing methyl jasmonate, phenylalanine, and salicylic acid as stimulants (especially in higher concentrations and time durations) significantly increased the amount of rutin production in callus tissue samples, which is consistent with the results obtained by Ishikawa et al. (2007). The highest amounts of rutin, quercetin, and kaempferol were respectively (12.86, 5.38 and 3.45 mg per gram of callus fresh weight) related to the culture medium containing 50 mg/liter salicylic acid for 48 hours, the culture medium containing 200 mg/liter methyl-jasmonate for 96 hours, and the culture medium containing 100 mg/liter salicylic acid for 96 hours. 

Recent advances in various fields of biotechnology have made it possible to revive new methods of plant tissue culture and produce valuable compounds at a commercial level. The employment of plant biotechnological techniques, particularly the generation of plant compounds in a controlled environment, presents the potential to manufacture items possessing therapeutic properties, irrespective of the variability in climate and seasons, thereby ensuring year-round availability. Today, many studies have been conducted and are being conducted on the medicinal and nutritional value of the Moringa plant (M. oleifera). Due to the high value of this plant in terms of medicine and treatment of diseases such as cancer in the traditional medicine of different nations, suitable and alternative methods for producing its effective compounds are very important. The use of plant tissue culture and esters is an effective method to produce valuable compounds of this plant in vitro. According to the results obtained in this study, treating callus with higher concentrations (100 and 200 mg/L) of methyl jasmonate, salicylic acid, and phenylalanine increases the possibility of increasing the production of plant secondary metabolites (rutin, quercetin, and kaempferol) in this plant.