دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 14 4 2023 02 20 The Effect of Salicylic Acid and Nitric Oxide Pretreatment on ‎Reducing the Toxicity of Zinc Nanoparticles in Phlomis tuberosa تاثیر پیش‌تیمار سالیسیلیک‌اسید و نیتریک‌اکسید بر کاهش سمیت نانوذرات روی در گیاه فلومیس Phlomis tuberosa 1 20 27774 10.22108/ijpb.2023.136672.1310 FA الهام قاسمی فر گروه زیست‌شناسی، دانشگاه پیام نور، تهران، ایران قادر حبیبی گروه زیست‌شناسی، دانشگاه پیام نور، تهران، ایران Journal Article 2023 01 31 In this study, the effect of salicylic acid and sodium nitroprusside on the toxicity of zinc oxide nanoparticles (ZnONPs) and zinc sulfate (ZnSO4) in Phlomis tuberosa was investigated. The purpose of this research was to investigate new methods of increasing the tolerance threshold of Phlomis plants under the influence of zinc oxide and zinc sulfate nanoparticles toxicity. This experiment was conducted in the form of a completely randomized design with three replications in the form of pot cultivation in a perlite bed, and the plants were harvested after 21 days of treatment. The measured factors included total chlorophyll, carotenoid, total phenol, flavonoid, the activity of phenylalanine ammonia lyase, catalase, superoxide dismutase, and ascorbate peroxidase. The results of the analysis of the variance of the data in the laboratory showed that the application of 1000 mgL-1 of zinc oxide nanoparticles or zinc sulfate caused more toxicity. The interaction effect of salicylic acid and sodium nitroprusside pretreatments on zinc oxide nanoparticles in Phlomis seedlings was significant and salicylic acid pretreatment increased the amount of proline in the treatment of zinc oxide nanoparticles. Phenol antioxidants increased significantly under the effect of zinc oxide and zinc sulfate nanoparticles, which was associated with an increase in the activity of the phenylalanine ammonia lyase enzyme. Salicylic acid pretreatment showed the greatest effect in the Phlomis plant compared to the combination of salicylic acid and nitric oxide. Introduction Engineered zinc oxide nanoparticles (ZnONPs), due to their unique physicochemical properties, have many applications in industrial, optical, electrical, cosmetic, biomedical, pharmaceutical, and agricultural fields (Pullagurala et al., 2018; Mosquera-Sánchez et al., 2020). Despite the application of Zn nanoparticles in agriculture, some reports show that Timarin nanoparticles can lead to Zn accumulation and toxic effects in plants (Molnár et al., 2020; Zoufan et al., 2020). The toxicity mechanism of zinc oxide nanoparticles is better understood by using compounds that increase the tolerance and performance of plants during the treatment of nanoparticles. Salicylic acid, as a phenolic compound, plays a role in controlling plant growth, seed germination, photosynthesis and flowering (Arif et al., 2020). Under heavy metal stress, treatment with salicylic acid reduces the toxic effects of these metals and increases plant tolerance through changes in the antioxidant system (Zengin, 2014; Kotapati et al., 2017; Lu et al., 2018). When stresses cause the accumulation of excess reactive oxygen species, nitric oxide reacts with superoxide anions, thereby reducing the formation of other oxygen radicals (Domingos et al., 2015). There are few studies on the effects of nitric oxide on the tolerance of nanoparticle toxicity in plants, which confirms this hypothesis (Siddiqui et al., 2015). Tripathi et al. (2017) showed that sodium nitroprusside reduces the accumulation of zinc, reactive oxygen species and malondialdehyde and increases the amount of chlorophyll and the efficiency of photosynthesis in wheat shoots under the stress of zinc oxide nanoparticles. In this research, in order to better understand the effects of zinc oxide nanoparticles, the physiological and molecular reactions of the plant when exposed to zinc were compared to zinc sulfate. The importance of the role of salicylic acid and nitric oxide in reducing the tension of zinc oxide nanoparticles in plants was mentioned. These results showed that salicylic acid pretreatment alone is effective for Phlomis tuberosa plants under the stress of zinc oxide nanoparticles. Materials and Methods The seeds of Phlomis tuberos (Jerusalem sage) were collected from Madagh near Marand city, 65 km south of Tabriz (45°38'E and 38°22'N) at an altitude of 1800 meters in East Azarbaijan province (Northwest of Iran). The seeds were planted in cylindrical plastic pots containing perlite and then watered with 500 ml of Hoagland solution. For pre-treatment, the seeds of gerbera and phlomis were soaked in solutions of sodium nitroprusside (SNP, as NO donor) and salicylic acid (SA) at a concentration of 0.1 mM for 12 hours). Sixteen weeks after sowing, when the plants were about 10 cm (30 inches) tall, the pots were irrigated with 1000 ppm ZnONPs or ZnSO4 solution dissolved in tap water for 21 days (Zn treatment). Plants in the greenhouse with day and night temperatures of 25-30 degrees Celsius and 19-21 degrees Celsius respectively, day and night light periods of 16 and 8 hours respectively, relative humidity of 60-65% and daily light flux density of about 350-400 micromoles per square meter per second were tested during the maintenance period. After 21 days of Zn treatment, plants were harvested for morphological and physiological analysis. After the determination of fresh weight (FW), leaves were dried for 48 h at 70 °C for the determination of dry weight (DW). For the latter physiological analysis, samples were stored immediately in liquid N2 until assay. For the determination of leaf concentration of chlorophyll and carotenoids, samples were homogenized in the methanol according to Lichtenthaler and Wellburn (1983). Proline was calculated as described by Bates et al., 1973. Soluble sugar concentrations were determined according to the method of Quentin et al. (2015). The resulting precipitate was used for starch analysis following the method of Magne et al. (2006). The activity of phenylalanine ammonialyase was determined by measuring the absorbance of cinnamic acid with spectrophotometry at a wavelength of 290 nm based on the modified method of Zucker (1965). Superoxide dismutase (SOD, EC 1.15.1.1) activity was estimated according to the method of Giannopolitis and Ries (1977). Nitric oxide concentration was measured using the modified method described by Wu et al. (2016). The digested plant solution was diluted in distilled water, and zinc content was estimated by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, INTEGRA XL2, GBC, Australia). HPLC analysis was performed using an Agilent 1290 Santa Clara, CA, USA HPLC high-performance liquid chromatography system with a diode array detector (DAD) according to Sinrod et al. (2019). Experiments were done in a complete randomized block design with 3 replications. Statistical analysis was carried out using Sigma Stat (3.5) with the Tukey test (P<0.05). Results and Discussion This research showed that the effect of pretreatment of seeds with salicylic acid, nitric oxide and the combination of salicylic acid and nitric oxide at a concentration of 0.1 mM on the physiological and biochemical parameters of Phlomis under the influence of the application of zinc oxide nanoparticles and zinc sulfate at a concentration of 1000 mM Gram per liter was studied and the following general results were obtained: Zinc oxide and zinc sulfate nanoparticles at a concentration of 1000 ppm caused a significant decrease in plant growth due to the increase in zinc content, lipid peroxidation, hydrogen peroxide and oxidative stress. The amount of sinapyl alcohol, chlorogenic acid, ascorbic acid and cinnamic acid compounds of salicylic acid pretreated leaves showed a significant increase under the influence of zinc oxide nanoparticles. The greatest effect of priming seeds with salicylic acid under the influence of zinc stress with the accumulation of phenolic compounds (sinapil-alcohol, chlorogenic-acid, ascorbic-acid, cinnamic-acid) and the amount of flavonoid, activity of antioxidant enzymes (superoxide dismutase), increased proline Obtained. Salicylic acid may have an important function in increasing the integrity of membranes by controlling the function of the antioxidant system. Conclusion We showed that both ZnONPs and ZnSO4 adversely decreased growth, due to enhanced levels of Ag and significant lipid peroxidation. Strong amelioration of Zn-induced stress was achieved under SA priming. These findings provide valuable information for the development of sustainable strategies in order to reduce the negative impact of ZnONPs and ZnSO4 on crops. در پژوهش حاضر، اثر سالیسیلیک‌اسید و نیتروپروسایدسدیم بر میزان سمیت نانوذرات اکسیدروی (ZnONPs) و سولفات‌روی (ZnSO4) در فلومیس (Phlomis tuberosa) بررسی شد. آزمایش حاضر در قالب طرح کاملاً تصادفی با سه تکرار به‌شکل کشت گلدانی در بستر پرلیت انجام شد. پس از 21 روز تیمار، گیاهان برداشت و شاخص‌های کلروفیل کل، کاروتنوئید، فنول کل، فلاونوئید، فعالیت آنزیم فنیل‌آلانین‌آمونیالیاز، کاتالاز، سوپراکسیددیسموتاز و آسکوربات‌پراکسیداز اندازه‌گیری شدند. نتایج تجزیه واریانس داده‌ها در آزمایشگاه نشان دادند اِعمال 1000 میلی‌گرم‌در‌لیتر نانوذرات اکسیدروی یا سولفات روی می‌تواند سمیت بیشتری ایجاد کند. اثر متقابل پیش‌تیمارهای سالیسیلیک‌اسید و نیتروپروساید سدیم بر نانوذرات اکسیدروی در گیاهچه‌های فلومیس معنادار بود و پیش‌تیمار سالیسیلیک‌اسید مقدار پرولین در تیمار نانوذرات اکسیدروی را افزایش داد. افزایش آنتی‌اکسیدان‌های فنول تحت‌تأثیر نانوذرات اکسیدروی و سولفات‌روی معنادار و با افزایش فعالیت آنزیم فنیل‌آلانین‌آمونیالیاز همراه بود. پیش‌تیمار سالیسیلیک‌اسید در گیاه فلومیس در مقایسه با ترکیب سالیسیلیک‌اسید و نیتریک‌اکسید بیشترین تأثیر را نشان داد. هدف پژوهش حاضر، بررسی روش‌های جدید افزایش آستانۀ تحمل گیاه فلومیس به سمیت نانوذرات اکسیدروی و سولفات‌روی بود.

https://ijpb.ui.ac.ir/article_27774_5f509355759b9c40db4f7f3f851a7718.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 14 4 2023 02 20 Development of Male and Female Reproduction Organs in ‎Moldavian Dragonhead, Dracocephalum moldavica L. (Lamiaceae)‎ مطالعۀ تکوین اندام زایشی نر و ماده در گیاه بادرشبو ‏Dracocephalum moldavica L.‎‏ (تیرۀ نعنائیان) ‏ 21 38 27791 10.22108/ijpb.2023.137260.1316 FA معصومه ماهوت فروشها گروه زیست‌شناسی، دانشکدۀ علوم زیستی، دانشگاه آزاد اسلامی، واحد تهران شمال، تهران، ایران سایه جعفری مرندی گروه زیست‌شناسی، دانشکدۀ علوم زیستی، دانشگاه آزاد اسلامی، واحد تهران شمال، تهران، ایران گلناز تجدد گروه زیست‌شناسی، دانشکده علوم زیستی، دانشگاه آزاد اسلامی، شعبه تهران شمال، تهران، ایران مریم پیوندی گروه زیست‌شناسی، دانشکده علوم زیستی، دانشگاه آزاد اسلامی، شعبه تهران شمال، تهران، ایران Journal Article 2023 04 05 Due to its great medicinal value, the Moldavian dragonhead, belonging to Lamiaceae, is naturally distributed in Europe, Siberia, and Central Asia and planted in different Iranian provinces. This research studied the development of male/female reproductive organs. Flowers and buds in different stages of development were fixed and preserved in order to prepare microtome sections using appropriate solutions and embed them in paraffin. Hematoxylin-eosin was used to stain the slices. Based on the results, despite the relative simultaneity of the formation of stamen primordium and carpel, stamens develop faster than ovules. Nurses’ cells in secretory and plasmodium types are rectangular, spindle-shaped, and often mononuclear or more. The microspore’s tetrads had tetrahedral ornamentations, and mature pollen grains were hexacolpate with reticulate ornamentation of the exine. The ovules were anatropous, unitegmic, and tenuinucellate. The functional megaspore has a micropillar position, and the embryo sac development belongs to the Polygonum type. Introduction The origin of Dracocephalum Moldavica L., known as "Zarrin Giah" in Persian, is in the mountainous regions of Central Asia (Botanica, 2009). This plant is mostly perennial and rarely annual or semi-shrub (Dastmalchi et al., 2007). The Dracocephalum species, which naturally grows in Europe, Siberia, and Central Asia, is cultivated in Iran in western and eastern provinces, Mazandaran, Yazd, and Tehran (Mozaffarian, 2013; Dastmalchi et al., 2007). The Lamiaceae family, commonly known as the mint family, has always been of interest to researchers due to its significant medicinal properties. The formation of the male and female reproductive structures, pollen grain formation, and various stages of embryo formation in plants of this family have been studied by various researchers (Daskalova, 2004; Tajadod et al., 2015; Jafari Marandi et al., 2017). The anatomical structure of vegetative organs, secretory tissue structure, trichomes, and the nectary tissue structure of D. Moldavica have been studied by some researchers (Dmitruk et al., 2019, 2018; Liapunova, 1975). However, to date, no information has been provided regarding the anatomical structure of the male reproductive organs and their development. Considering the importance of developmental studies in biological, agricultural, horticultural, and botanical sciences, studying the development of this valuable medicinal plant can contribute to the advancement of biological knowledge and increased productivity. Materials and Methods After the plants grew to the flowering stage, samples were collected from the buds and flowers at different stages and at the appropriate time. The samples were fixed in FAA70 solution, which contained formalin, glacial acetic acid, and 70% ethanol in a 5:5:90 ratio for 16 hours to preserve the buds and flowers. Then, dehydration of the samples was performed with different concentrations of ethanol. The samples were embedded in paraffin and sectioned with a thickness of 8 micrometers using a microtome (Cut 4040, Co., Mainz, Germany) for studying the developmental stages. Staining was carried out using hematoxylin-eosin based on the Yeung's method (1984). More than 10 samples were studied for each developmental stage, and examination of the samples and preparation of images was conducted using a Nikon light microscope (Nikon, Tokyo, Japan). Results and Discussion According to the results, despite the relative simultaneity of primordium formation and the cotyledon, cotyledon growth occurs more rapidly than ovule development. The growth of cotyledons and petals occurs closely spaced, and pollen grains mature before the flower blooms, while the ovules are still in the ovule sac formation stage. Unlike plants such as mango (Jonoubi et al., 2015; Mohsenzadeh et al., 2012), where the epidermal layer of cotyledons disintegrates during maturation, in D. Moldavica, as in other members of the mint family like basil, thyme, catnip, and marjoram (Kameli et al., 2018; Jafari Marandi et al., 2017; Tahmasebi et al., 2016; Tajadod et al., 2015), the mechanical layer, along with the epidermal layer, remains until the end of flowering and the maturation of pollen grains. Pollen sacs exist in four-pollinium and various shapes, including rectangular, double, single-core, or more, and are of exine secretion or plasmoid type. Contrary to plants such as mint (Jafari Marandi et al., 2017), catnip (Daskalova, 2004), and species of Brassicaceae like Brassica jordanoffri (Yankova-Tsvetkoya et al., 2016) and Arabidopsis thaliana (Basiri et al., 2021), where two-nucleate cells of the middle layer are reported, in this medicinal plant, the middle layer cells are polygenomic. The type of ovule in this plant is anatropous, bitegmic, and with a thin integument. The functional megaspore is of the monosporic type and ultimately becomes a megaspore mother cell. The structural features of the hypostase, androecium, and the nectary conductor tissue, as well as the pollen tube conductive tissue, are observed in this plant. Conclusion Pollen grains of D. Moldavica reach maturity before flowering. The middle layer that exists until the formation of immature pollen grains is lost before the formation of the top layer. During flowering, cotyledonary and mechanical layers remain intact. The top layer is of secretory and amoeboid type and exists as mononucleate, binucleate, or more. Ovaries in this plant have four pollen sacs with various shapes. Pollen grains are hexacolpate and exine is reticulate. The ovule is anatropous, bitegmic, and with a thin integument. The functional megaspore is monosporic, and eventually, the megaspore mother cell becomes a pollen sac. The structure of the hypostase, androecium, and the nectary conductor tissue, as well as the pollen tube conductive tissue, are present in this plant. گیاه بادرشبو (متعلق به تیرۀ نعنائیان) به‌طور طبیعی در اروپا، سیبری و آسیای مرکزی پراکنش دارد و به‌علت داشتن ارزش دارویی فراوان، در استان‌های مختلف ایران کاشته می‌شود. در پژوهش حاضر، تکوین اندام زایشی نر و مادۀ گیاه بادرشبو مطالعه شد. غنچه‌ها و گل‌ها در مراحل مختلف نمو به‌منظور تهیۀ برش‌های میکروتومی با محلول‌‌های مناسب تثبیت، نگهداری و در پارافین قالب‌گیری شدند. از هماتوکسیلین- ائوزین برای رنگ‌آمیزی برش‌ها استفاده شد. بر اساس نتایج، باوجود هم‌زمانی نسبی تشکیل پریموردیوم پرچمی و برچه، نمو پرچم‌ها سریع‌تر از تخمک انجام می‌شود. بساک‌ها چهارکیسه‌گرده‌ای هستند و لایۀ تاپی به شکل‌های مستطیلی، دوکی‌شکل، تک‌هسته‌ای یا بیشتر و به دو حالت ترشحی و پلاسمودیومی وجود دارد. تترادهای میکروسپوری از نوع تتراهیدرال هستند، دانۀ گرده شش‌شیاره و اگزین دارای تزئینات مشبک است. تخمک از نوع واژگون، تک‌پوششی با دیوارۀ نازک است. سلول عملکردی با موقعیت سُفتی و کیسۀ رویانی تیپ پلی‌گونوم از دیگر ویژگی‌های این گونه است.

https://ijpb.ui.ac.ir/article_27791_0a35ab2edf61fb6c317ed0cd47353ca8.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 14 4 2023 02 20 The Effects of Vermicompost and Methanol on the Trend of Changes of Chlorophyll Fluorescence Components and Some Physiological Traits of Triticale under Salinity Stress تأثیر ورمی‌کمپوست و متانول بر روند تغییرات اجزای فلورسانس کلروفیل و برخی صفات فیزیولوژیکی تریتیکاله تحت تأثیرتنش شوری 39 70 27919 10.22108/ijpb.2023.138576.1332 FA خدیجه صورآذر‏ گروه مهندسی تولید و ژنتیک گیاهی، دانشکدۀ کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران رئوف سید شریفی گروه مهندسی تولید و ژنتیک گیاهی، دانشکدۀ کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران Journal Article 2023 07 31 In order to investigate the effects of salinity, vermicompost, and methanol on the trend of changes of chlorophyll fluorescence components and some physiological traits of triticale, this experimental study was conducted as factorial based on randomized complete block design with three replications in greenhouse research of the Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili in 2022. Factors experiments included salinity in three levels (no salinity as control, salinity 50 and 100 mM by NaCl), application of vermicompost at two levels (without vermicompost as control and application of vermicompost), and methanol foliar application in three levels (foliar application with water as control, foliar application 15 and 30% volume). The results showed that the highest salinity level (100 mM) and both vermicompost and foliar application of 30% volume of methanol increased chlorophyll index (27%), nitrogen index (20.5%), stomatal conductance (38.9%), relative water content (26.7%), maximum fluorescence (27.6%), variable fluorescence (70.7%), and quantum yield (33.8%) compared to no application of vermicompost and methanol under the same of salinity level. In addition, the highest grain yield (5.4 g per plant) was obtained due to the application of vermicompost and methanol 30% volume under no-stress conditions. Based on the results of this study, it seems that the application of vermicompost and methanol can be used to mitigate the adverse effects of salinity stress due to the improvement of chlorophyll fluorescence components and some physiological traits in triticale. Introduction Triticale is a human-made crop, being a hybrid by cross-fertilization of wheat (Triticum spp.) and rye (Secale spp.). This plant can act as a soil improver, as its extensive root system binds erosion-prone soil and provides a good substrate for conversion into subsoil organic carbon by soil microbes. Salinity stress is one of the most destructive environmental stresses, which affects almost every aspect of the physiology and biochemistry of a plant, resulting in a reduction in its yield. On the other hand, salinity can damage the photosynthesis of plants and decrease chlorophyll content, relative water content, stomata conductance, and quantum yield. Several strategies have been suggested in order to improve yield under environmental stresses in plants, among them the application of methanol and vermicompost play a key role in yield improvement. Methanol can increase the concentration of CO2 in a plant and help photosynthesis rate and vermicompost increase growth due to access to nutrients under salinity stress. A better understanding of physiological responses under salinity stress may help in programs in which the objective is to improve the salinity resistance of crops. Therefore, the aim of this study was to investigate the effects of methanol and vermicompost on some physiological and biochemical (i.e., chlorophyll index, electrical conductivity, quantum yield, stomata conduction, and relative water content) responses of triticale under salinity stress. Materials and Methods The experiment was conducted as factorial based on a randomized complete block design with three replications in greenhouse research of the Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili in 2022. Factors experiments included salinity in three levels (no salinity as control, salinity 50 and 100 mM by NaCl), application of vermicompost at two levels (without vermicompost as control and application of vermicompost), and methanol foliar application in three levels (foliar application with water as control, foliar application 15 and 30% volume). Vermicompost was purchased from the Gilda Corporation and soils were treated based on the manufacturer’s protocol. The triticale cultivar "Sanabad" was used in the experiment with a plant density of 400 seeds m-2. In this study, chlorophyll index, nitrogen index, stomatal conductance, EC, RWC, maximum fluorescence, variable fluorescence, quantum yield, and grain yield were investigated. Stomatal conductance was measured by Prometer (Decagon Devices, Inc.) and chlorophyll index was measured using a chlorophyll meter (SPAD-502 Minolta, Japan). Electrical conductivity (EC) is measured using an electrical-conductivity meter (Mi 180 Bench Meter model). The quantum yield was measured by the uppermost fool expanded leaf using a fluorometer (chlorophyll fluorometer; Optic Science-OS-30 USA). The relative water content was measured according to the following formula: Where, RWC, FW, DW, and TW are relative water content, fresh weight, dry weight, and turgid weight, respectively. At plant maturity, grain yield in each pot was harvested by five plants per pot. Analysis of variance and mean comparisons were performed using SAS9.4 computer software packages. The main effects and interactions were tested using the least significant difference (LSD) test at the 0.05 probability level. Results and Discussion The results showed that the highest salinity level (100 mM) and both vermicompost and foliar application of 30% volume of methanol increased chlorophyll index (27%), nitrogen index (20.5%), stomatal conductance (38.9%), relative water content (26.7%), maximum fluorescence (27.6%), variable fluorescence (70.7%), and quantum yield (33.8%) compared to no application of vermicompost and methanol under the same of salinity level. Moreover, the highest grain yield (5.4 g per plant) was obtained due to the application of vermicompost and methanol 30% volume under no-stress conditions. Conclusion Based on the results of the present study, it seems that the application of vermicompost and methanol can be used to mitigate the adverse effects of salinity stress due to the improvement of chlorophyll fluorescence components and some physiological traits in triticale. به‌منظور بررسی تأثیر شوری، ورمی‌کمپوست و متانول بر چگونگی تغییرات اجزای فلورسانس کلروفیل و برخی صفات فیزیولوژیکی تریتیکاله، آزمایشی به‌صورت فاکتوریل در قالب طرح پایه بلوک‌های کامل تصادفی در سه تکرار در گلخانه تحقیقاتی دانشگاه محقق اردبیلی در سال 1401 اجرا شد. فاکتورهای مورد بررسی شامل شوری در سه سطح (عدم اعمال شوری به‌عنوان شاهد، شوری 50 و 100 میلیمولار با نمک کلریدسدیم)، کاربرد ورمی‌کمپوست در دو سطح (بدون ورمی‌کمپوست به‌عنوان شاهد و کاربرد ورمیکمپوست) و محلول‌پاشی متانول در سه سطح (محلول‌پاشی با آب ب‌عنوان شاهد و محلول‌پاشی 15 و 30 درصد حجمی) بود. نتایج نشان داد که بالاترین سطح شوری (100 میلی‌مولار) و کاربرد توأم ورمیکمپوست و متانول 30 درصد حجمی شاخص کلروفیل (27%)، شاخص نیتروژن (5/20%)، هدایت روزنه‌ای (9/38%)، محتوای نسبی آب (7/26%)، فلورسانس بیشینه (6/27%)، فلورسانس متغیر (7/70%) و عملکرد کوانتومی (8/33%) را در مقایسه با عدم کاربرد ورمی‌کمپوست و متانول در همین سطح از شوری افزایش داد. همچنین، بیشترین عملکرد دانه در اثر کاربرد ورمی‌کمپوست و متانول 30 درصد حجمی در شرایط عدم اعمال تنش (4/5 گرم در بوته) بدست آمد. براساس نتایج این بررسی به نظر می‌رسد که کاربرد ورمی‌کمپوست و متانول می‌تواند برای تعدیل تأثیرات سوء ناشی از تنش شوری به علّت بهبود اجزای فلورسانس کلروفیل و برخی صفات فیزیولوژیکی تریتیکاله مورد استفاده قرار گیرد.

https://ijpb.ui.ac.ir/article_27919_80a73454fcd185bb49ab6b9c88f5ced2.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 14 4 2023 02 20 Investigating Specific Diversity of Aquatic Plant Ceratophyllum L. in Iran Using Morphological Characters and ISSR and SRAP Molecular Markers بررسی تنوع گونه ای گیاه آبزی Ceratophyllum L. در ایران با صفات ریخت‌شناسی و نشانگرهای مولکولی ISSR و SRAP 71 86 28055 10.22108/ijpb.2023.139564.1339 FA حانیه بهادری گروه زیست‌شناسی گیاهی و جانوری، دانشکدۀعلوم و فناوری‌های زیستی، دانشگاه اصفهان، اصفهان، ایران سعید افشارزاده گروه زیست‌شناسی گیاهی و جانوری، دانشکدۀعلوم و فناوری‌های زیستی، دانشگاه اصفهان، اصفهان، ایران شبنم عباسی گروه آموزش زیست‌شناسی، دانشگاه فرهنگیان، صندوق پستی 889-14665، تهران، ایران Journal Article 2023 10 23 The genus Ceratophyllum L. is a submerged aquatic plant of the Ceratophyllaceae family, with six species recognized worldwide. So far, two species, C. demersum and C. submersum, have been reported for the flora of Iran. Considering the importance of this plant from an environmental and ecological point of view, its taxonomic and molecular studies have been carried out in this study to investigate the species diversity of this genus. Following the identification of species by assessing morphological features in individuals from different populations, 7 ISSR primers and 3 pairs of SRAP primers were used to confirm the identifications and study the genetic diversity of distinct populations. Molecular investigations showed the number of 5 genetic groups, whose intra-population diversity (52%) was higher than inter-population diversity (48%) with P<0.05. Furthermore, the data revealed that the populations of this plant in Iran have relatively the same genetic pool. Based on this, it can be concluded that several elements, including the connectedness of aquatic ecosystems, particularly in the north of the country, as well as the high adaptability of this submerged plant with aquatic habitats, can be effective in producing such conditions. Recent droughts have increased the sexual reproduction of this plant, and as a result, the species diversity within the populations has increased. Introduction Aquatic plants (macrophytes) are important components of freshwater ecosystems that affect the functioning and diverse services of the ecosystem. Aquatic plants grow along the edge of the lake and are both a protective and nourishing component of the ecosystem for healthy lakes. Ceratophyllum has about 2 to 30 species that have global distribution. The determination of genetic relationships of plant materials is important in terms of choosing the right parental combination for producing suitable populations, designing effective breeding programs, responding to long-term selection and reducing genetic vulnerability. The study of genetic relationships is a prerequisite for the improvement of agricultural plants and also a prerequisite for the preservation of genetic resources. Considering that in the Ceratophyllum, the separation of species is done by intermediate morphological characteristics, the study of the genetic diversity of different populations of this species in Iran using molecular markers seems necessary. In addition, considering the recent droughts in the country and the destruction of many aquatic habitats, the study of this aquatic plant can provide useful knowledge for the protection of the country's environment. Therefore, in this study, some of the important morphological traits of the samples, the relationships of the species of this genus, as well as their genetic diversity were investigated using molecular markers. Materials and Methods In this study, 50 individuals and 29 populations of two species, C. submersum and C. demersum, were examined. The modified CTAB method was used to extract DNA from 50 individuals from 29 studied aquatic plant populations. Seven ISSR primers and three SRAP primer pairs were selected. For data analysis and statistical analysis of populations, individuals were scored based on the presence of band (1) and the absence of band (0). Using NTSYSpc software and the Jaccard and Nei similarity coefficient, cluster analysis was performed and two dendrograms were obtained. GenAlex software was used for principal component analysis and STRUCTURE software was used to examine the genetic structure of populations. Results and Discussion The morphological studies of this plant showed an herbaceous perennial state. The stem of this plant has multiple nodes and internodes, and the stem mostly branches in the upper 1/3. In the C. demersum species, they have 1 or 2 divisions and in the C. submersum species, they have 3 or 4 dichotomy divisions. The shape of the leaves is thin, long, pointed, and fork-like, which are stringy or linear. In both dendrograms, the samples from the north of the country and the central region of the country (Isfahan) are almost separated, but a kind of genetic connection can be seen between the samples from the north of the country and the center of the country. Through humans and also through water birds, they show a kind of genetic connection. Regarding the C. demersum and C. submersum species that exist in Iran, it seems that the C. demersum species has a wider genetic treasure in the aquatic habitats of the country and the species C. submersum has been found only in a region in the north of the country (Izbaran, Amirkalaye, and Bandar Anzali) in our sampling. Therefore, it seems that C. demersum is ecologically more compatible with various environmental factors and the C. submersum species exists only in limited ecological water niches in the country due to the lack of suitable living conditions. The results of the principal component analysis were in agreement with the cluster analysis and showed some kind of connection and continuity between populations. The results of AMOVA showed 52% genetic diversity within populations and 48% genetic diversity between populations. The results of the Mantel test indicated that there is no relationship between the genetic distance and the geographical distance (R= 0.019 and P = 0.320), which is due to the fact that in the principal component analysis, we saw the lack of separation of the populations. The molecular analysis of Cerarophyllum intraspecies relationships through STRUCTURE analysis showed 5 genetic groups and demonstrated the relationship between individuals of different populations, whose gene pool was relatively similar. Conclusion According to analyses done, it can be said that the gene pool of Ceratophyllum population in Iran is somewhat distorted and there is a relationship and genetic similarity between different populations and only some populations in the north of the country are more uniform. As was stated, various factors can be effective in creating such conditions. A high intra-population genetic diversity has been shown in the populations of this study. جنس Ceratophyllum L. یک گیاه آبزی غوطه‌ور از خانواده Ceratophyllaceae است که شش گونه آن در سراسر جهان شناخته شده است. تاکنون دو گونه C. demersum و C. submersum برای فلور ایران گزارش شده‌اند. باتوجه به اهمیت این گیاه از نظر زیست‌محیطی و اکولوژیکی، پژوهش های تاکسونومی و مولکولی آن برای آشنایی با تنوع گونه ای این جنس انجام شد. پس از شناسایی گونه ها با ارزیابی ویژگی‌های ریخت‌شناسی در افرادی از جمعیت‌های مختلف، از 7 آغازگرISSR و 3 جفت آغازگر SRAP برای تأیید هویت‌ها و بررسی تنوع ژنتیکی جمعیت‌های متمایز استفاده شد. بررسی‌های مولکولی تعداد 5 گروه ژنتیکی را نشان داد که تنوع درون جمعیتی آنها (52%) از تنوع بین جمعیتی (48%) با P<0.05 زیادتر بود. علاوه بر این، داده‌ها نشان داده‌اند که جمعیت‌های این گیاه در ایران دارای خزانه ژنتیکی نسبتاً مشابهی است. بر این اساس می‌توان نتیجه گرفت که عوامل متعددی از جمله پیوستگی اکوسیستم‌های آبی به‌ویژه در شمال کشور و همچنین سازگاری بالای این گیاه غوطهور با زیستگاه‌های آبی می‌توانند در ایجاد چنین شرایطی مؤثر باشد. خشکسالی‌های اخیر در کشور سبب افزایش تولید مثل جنسی این گیاه شده و در نتیجه تنوع گونه ای درون جمعیت‌ها افزایش یافته است.

https://ijpb.ui.ac.ir/article_28055_3bff09fe2ac38c723409274f0779a2bf.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 14 4 2023 02 20 Optimizing the Induction of Somatic Embryogenesis in Tomato (Solanum lycopersicum L.) Variety Money Maker بهینه‌سازی القای جنین‌زایی سوماتیکی در گوجه‌فرنگی (Solanum lycopersicum L.) رقم Money Maker 87 100 28219 10.22108/ijpb.2024.138740.1333 FA اسما زاهدی گروه بیوتکنولوژی و به نژادی گیاهی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران نسرین مشتاقی گروه بیوتکنولوژی و به نژادی گیاهی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران عبدالرضا باقری گروه بیوتکنولوژی و به نژادی گیاهی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران امین میرشمسی کاخکی گروه بیوتکنولوژی و به نژادی گیاهی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران Journal Article 2023 08 12 The factorial experiment conducted in this study aimed to induce somatic embryogenesis in tomato cultivar Money Maker using different culture media and growth regulator combinations. The experiment was done in a completely randomized design with three replications. Three types of culture media, namely MS, B5, and MS + Vitamin B5, were used in combination with three hormonal combinations: 2,4-D (2 mg/L) + BA (1.5 mg/L), 2,4-D (8 mg/L) + BA (6 mg/L), and IAA (2 mg/L) + BA (5 mg/L). Additionally, three types of explants (cotyledon, hypocotyl and root) were utilized to induce somatic embryos. To promote embryo maturation, the explants were transferred to a hormone-free culture medium. The results revealed that the highest number of immature embryos was obtained from cotyledon explants cultured in MS medium with the hormonal combination of 2,4-D (2 mg/L) + BA (1.5 mg/L) and in hypocotyl explants in MB medium supplemented with the hormonal combination of 2,4-D (8 mg/L) + BA (6 mg/L), with an average of 20.33 embryos per explant. Also, the highest percentage of embryogenesis (93.33%) was in hypocotyl explants at MS culture medium with the hormonal combination of IAA (2 mg/L) + BA (5 mg/L). After two weeks of cultivation, the presence of globular embryos was visible on the surface of the explants under binoculars. By transferring the explants to light conditions and subsequently to a hormone-free culture medium, the embryos further developed and matured. Introduction Tomatoes belong to the Solanaceae family. In recent years, there has been an increasing interest among scientists in utilizing tomato as a model plant due to its importance. Somatic embryogenesis, a process in which embryos develop from somatic or non-reproductive cells, represents a means of inducing totipotency in plant cells. Standardizing successful regeneration methods for Agrobacterium-mediated gene transfer to somatic embryos in tomato can establish a sustainable transfer approach. During the induction of somatic embryogenesis, one of the major factors influencing cell behavior is the presence of high concentrations of auxin in the culture medium. Cytokinin, in combination with auxins, stimulates cell division, which both act synergistically. By investigating the induction of somatic embryos under treatments of auxin and cytokinin hormones in vitro, this research contributes to the advancement of propagation techniques for stress-tolerant varieties and genetic improvement in tomato under laboratory conditions. In conclusion, the study highlights the potential of somatic embryogenesis as a powerful tool for plant propagation and genetic manipulation in tomato. The optimization of hormone treatments and culture conditions can enhance the efficiency and success rate of somatic embryogenesis, leading to improved techniques to produce stress-tolerant varieties and the advancement of genetic improvement strategies in tomato. Materials and methods The seeds were surface sterilized with 1.5% sodium hypochlorite and sterile distilled water for 10 minutes and were cultured on MS medium without hormones. 14-day-old tomato seedlings were used to obtain explants (cotyledon leaves, cotyledon axis and root) for somatic embryo induction. The explants were cultured on three different types of culture media: MS, B5, and MS + Vitamin B5 (MB). The culture media were supplemented with specific hormonal compounds: 2,4-D (2 mg/L) + BA (1.5 mg/L), 2,4-D (8 mg/L) + BA (6 mg/L), and IAA (2 mg/L) + BA (5 mg/L) 30 g/L sucrose and 8 g/L agar. The cultures were incubated at 25°C for 2 weeks of darkness and one week of light. After three weeks, the explants containing somatic embryos were transferred to MS, B5, and MB culture media without hormones for one week to promote embryo maturation. Microscopic images were taken from embryonic calli. The stages of embryo induction and development were documented. The following traits were measured at the end of the experiment: callus formation percentage, embryogenesis percentage, and the number of embryos in each explant. The experiment was conducted with three replications. The data were analyzed by JMP software. The average values were compared using the LSD test at a significance level of 5%. Results and discussion Somatic embryogenesis was successfully induced in tomato explants under different hormonal treatments and culture media conditions. Callus formation was observed in the explants starting from the second week of cultivation. The highest percentage of callus formation (100%) was achieved in all three types of culture media when using cotyledon explants and the hormonal combinations of 2,4-D (1 mg/L) + BA (1.5 mg/L) and 2,4-D (8 mg/L) + BA (6 mg/L). The formation of embryos was mainly observed on the explants from the 14th day of cultivation. The highest percentage of embryogenesis (90.33%) was observed in hypocotyl explants cultured on MS culture medium with the hormonal combination of IAA (2 mg/L) + BA (5 mg/L). The highest number of immature embryos (20.33) was produced in cotyledon explants cultured on MS culture medium with the hormonal combination of 2,4-D (2 mg/L) + BA (1.5 mg/L) and in hypocotyl explants, the highest number of immature embryos was obtained in the MB medium with the hormonal combination of 2,4-D (6 mg/L) + BA (8 mg/L). After three weeks, the growth of the embryos stopped at the globular and heart-shaped stage. The calluses were initially light green, but with time, they turned brown and lost their embryonic tissue. However, in the hormonal treatment of IAA (2 mg/L) + BA (5 mg/L), globular embryos were observed on the surface of the explant after two weeks of culture. By transferring the explant to light and then to a hormone-free culture medium, the embryos underwent further developmental stages and reached maturity. This treatment resulted in the highest number of somatic embryos in hypocotyl explants. Conclusion The results of the experiment indicate that the hormonal treatment of IAA (2 mg/L) + BA (5 mg/L) was particularly effective in promoting the formation of embryonic callus and somatic embryogenesis in tomato hypocotyl explants. The successful production of somatic embryos can provide possibilities for further investigations into artificial seed production in tomato plants. پژوهش حاضر با هدف القای جنینزایی سوماتیکی با تنظیم‌کننده‌های رشدی در گیاه گوجه فرنگی انجام شد. بدین منظور آزمایش فاکتوریل در قالب طرح کامل تصادفی با سه تکرار طراحی شد. در این آزمایش از سه نوع محیط کشت MS ، B5 و MS + Vitamin B5 با ترکیب‌های هورمونی 2,4-D (2 میلی‌گرم در لیتر)+ BA (5/1 میلی‌گرم در لیتر)، 2,4-D (8 میلی‌گرم در لیتر)+ BA (6 میلی‌گرم در لیتر) و IAA (2 میلی‌گرم در لیتر) + BA (5 میلی‌گرم در لیتر) و سه نوع ریزنمونه کوتیلدون، هیپوکوتیل و ریشه استفاده شد. نتایج نشان داد بیشترین تعداد جنین نابالغ تولید شده، در ریزنمونه کوتیلدون در محیط کشت MS با ترکیب هورمونی 2,4-D (2 میلی‌گرم در لیتر)+ BA (5/1 میلی‌گرم در لیتر) و در ریزنمونه هیپوکوتیل در محیط کشت MB با ترکیب هورمونی 2,4-D (8 میلی‌گرم در لیتر)+ BA (6 میلی‌گرم در لیتر) (33/20 عدد در هر ریزنمونه) بود. بیشترین درصد جنین زایی در محیط کشت MS با ترکیب هورمونی IAA (2 میلی‌گرم در لیتر) + BA (5 میلی‌گرم در لیتر) و در ریزنمونه هیپوکوتیل (33/93%) بود. با انتقال ریزنمونه به روشنایی و سپس محیط کشت بدون هورمون، جنین‌ها مراحل تکاملی را طی کرده و به بلوغ رسیدند. نتایج بدست آمده نشان داد، امکان تولید جنین سوماتیکی در حجم انبوه در گیاه گوجه فرنگی وجود دارد و می‌توان از این جنین‌ها در تولید بذر مصنوعی استفاده کرد.

https://ijpb.ui.ac.ir/article_28219_07807b666fbaf4301ff4b387e1473594.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 14 4 2023 02 20 Floristic and soil study of the Malat water pond in Langarud city مطالعه فلوریستیکی و خاک‌شناسی محدوده آب‌بندان ملاط شهرستان لنگرود 101 124 28204 10.22108/ijpb.2024.138139.1324 FA مریم مفیدنژاد دانشکده علوم پایه، دانشگاه گیلان، رشت، ایران اصغر زمانی دانشکده علوم پایه، دانشگاه گیلان، رشت، ایران کورش کمالی مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی گیلان، سازمان تحقیقات، ‌آموزش و ترویج کشاورزی، رشت، ایران Journal Article 2023 06 26 Identification of plant species in each region is a reflection of its various ecological factors. Malat Storage pool of Langarud city is a stable water ecosystem in the north of the country, which plays an important role in supplying water to paddy fields. In this study, floristic composition, life form, geographical distribution of plants and soil characteristics of Malat Storage pool area of Langarud city were investigated. First, plant samples were collected from this Storage pool, surrounding lowlands and plains and forest areas overlooking it. Then, soil samples were taken from the surface horizon of habitats and some of their physical and chemical properties were determined. Based on the results, a total of 250 plant samples were collected, which belonged to 60 families, 127 genera and 146 species. The plant families Poaceae with 22 genera and Asteraceae with 11 genera formed the most populous plant family in terms of genera. Also, Poaceae and Fabaceae had the highest species richness with 24 and 15 species. The highest percentage of plant species in the region had the predominant life form of therophyte (29.7%) and hemicryptophyte (26.2%) and the highest frequency of chorological type was Pluriregional vegetative elements (31.5%). Examining the properties of the soil showed the uniformity of the soil texture (medium to light class) and the absence of salinity with electrical conductivity less than 1 (dS/m). The range of soil pH ranged from neutral (6.78) to strongly acidic (4.03), the range of organic carbon was in the medium to optimal class, and the amount of N-P-K was in the medium to rich class. The present floristic study can provide the basis for more comprehensive studies and management practices in this touristic area. Introduction Floristic studies are the base of ecological investigations, prediction of the future situation and management application in each region (Azarnivand and Zare Chahouki, 2010). Because of the diverse climatic conditions of Iran, different ecosystems and as a result a very rich and unique biodiversity is observed in the country. So, 65 families, 359 genera and 8112 species of vascular plants occur in Iran, of which 2597 are endemic (approximately 32%) (Noroozi et al., 2019). Floristic research in Iran has a long history due to its species richness (Jafari and Zarifian, 2016). Also, in the north of Iran, floristic studies have been conducted in diverse habitats, including aquatic ecosystems (Asri and Moradi, 2004; Ghahreman et al., 2004; Tavakoli et al., 2013; Yari et al., 2018; Moradi et al., 2020; Hosseinalizadeh Ahangar and Jafari, 2021). Soil, as one of the elements of the ecosystem, has a major role in the change and diversity of plant species, and on the other hand, the plant type also has a significant role in the change and transformation of the physical, chemical and biological characteristics of soils. Malat water pond located in the south of Langarud city as small reservoir for regulating and storing water play an important role in supplying and compensating the water shortage of paddy fields downstream. The aims of the current study are collection, identification and introduction of plant species in order to provide a floristic list of the area of water ponds on the southern edge of Langarud city. Materials and Methods The collection of samples was conducted during early spring to late autumn 2022. Then drying and preparing the samples was done. The samples were identified using some floristic references (Rechinger, 1963-2015; Davis, 1965-1988; Bor, 1968; Ghahreman, 1978-2022; Assadi, 1993-2022; Mozaffarian, 2018). The chorological types (Zohary, 1973; Takhtajan, 1986) and life forms (Raunkiaer, 1934) of taxa were determined. For soil assessment, four height ranges soil profile were collected and their A horizon was investigated for some physical and chemical properties such as soil texture, electrical conductivity of saturated extract, reaction of saturated soil extract, soil organic carbon, total soil nitrogen, soil absorbable phosphorus and potassium. Results and Discussion According to results, 147 plant taxa were identified belonging to 60 families, 127 genera and 146 species. Poaceae with 22 genera and 24 species was the biggest family of the region. This result is in accordance with some studies on the similar regions in Iran (e.g., Hosseinalizadeh Ahangar and Jafari, 2021). Also, Rumex L., Trifolium L. and Rubus L. (each with three species) were the biggest genera of the study area. Regarding life form, therophytes (29.7%) were the dominant group of the area. This observation can be the reflection of some conditions of the region such as the dominance of drought in some times of the year (Habibi et al., 2013), human disturbances through some actions such as overgrazing and tourism. Investigating the chorological types of the species, revealed that Pluriregional (PL, 30.9%) elements were the biggest group in the area. This could be reflection of the favorable environmental and biological conditions for the growth and expansion of different plants with different biological requirements. Examining the soil characteristics of the studied area showed that the soil texture, salinity and lime content were uniform in the altitude ranges from -20 to 325 meters above sea level. The soil texture in medium to light class, electrical conductivity was less than 1 dS/m. Conclusion North of Iran, due to having suitable temperature, regular rainfall and proximity to the sea, has provided suitable conditions for the diversity of plant species. In the studied area, the presence of species of the Poaceae and Fabaceae families is very important for their fodder value and soil protection against water and wind erosion, while the presence of some identified species of the Asteraceae family in the area indicates the destruction of vegetation, livestock grazing, and human manipulations. Many of the identified plants are very important for the people of the region in terms of medicinal and edible properties. شناسایی گونه‌های گیاهی هر منطقه بازتابی از عوامل مختلف بوم‌شناختی آن است. آب‌بندان ملاط شهرستان لنگرود یک بوم‌سازگان آبی پایدار در شمال کشور است که نقش مهمی‌ در تامین آب شالیزارها دارد. در این مطالعه، شناسایی ترکیب فلوریستیک، شکل زیستی، پراکنش جغرافیایی گیاهان و خاک‌شناسی محدوده آب‌بندان ملاط شهرستان لنگرود بررسی شد. ابتدا، جمع‌آ‌وری نمونه‌های گیاهی از این آب‌بندان، مناطق پست و جلگه‌ای اطراف و مناطق جنگلی مشرف بر آن انجام گرفت. سپس از افق سطحی رویشگاه‌ها نمونه خاک برداشت و برخی خصوصیات فیزیکی و شیمیایی آن‌ها تعیین شد. براساس نتایج حاصل، از مجموع 250 نمونه گیاهی جمع‌آوری شده، 60 خانواده، 127 جنس و 146 گونه گیاهی شناسایی شد. خانواده‌های گیاهی Poaceae با 22 جنس و Asteraceae با 11 جنس پرجمعیت‌ترین خانواده گیاهی از نظر جنس را تشکیل دادند. همچنین Poaceae و Fabaceae با 24 و 15 گونه دارای بیشترین غنای گونه‌ای بودند. بیشترین درصد گونه‌های گیاهی منطقه دارای شکل زیستی غالب تروفیت (7/29%) و همی‌کریپتوفیت (2/26%) و بیشترین فراوانی پراکنش جغرافیایی، عناصر رویشی چندناحیه‌ای (5/31%) بودند. بررسی خصوصیات خاک رویشگاه این گیاهان، نشان از یکنواختی بافت خاک (کلاس متوسط تا سبک) و نبود شوری با هدایت الکتریکی کمتر از یک دسی‌زیمنس برمتر (dS/m) بود. دامنه pH خاک از خنثی (78/6) تا به شدت اسیدی (03/4)، دامنه کربن آلی در کلاس متوسط تا بهینه و میزان N-P-K در کلاس متوسط تا غنی قرار داشتند. مطالعه فلوریستیکی منطقه ضمن افزایش شناخت آن، زمینه لازم را برای مطالعات جامع‌تر و اعمال مدیریت در این منطقه توریستی فراهم می‌کند.

https://ijpb.ui.ac.ir/article_28204_9393d317bafddc61091dc8f6c3027b07.pdf