Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 15-22, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1447 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Sherzad R. Abdull, Sahar H. Rashid, Bakhtiar S. Ghafoor, Barzan S. Khdhir (2022) Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana. Caryologia 75(2): 15-22. doi: 10.36253/caryologia-1447 Received: November 04, 2021 Accepted: May 24, 2022 Published: September 21, 2022 Copyright: © 2022 Sherzad R. Abdull, Sahar H. Rashid, Bakhtiar S. Gha- foor, Barzan S. Khdhir. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana Sherzad R. Abdull, Sahar H. Rashid*, Bakhtiar S. Ghafoor, Barzan S. Khdhir Technical College of Applied Science, Sulaimani Polytechnic University, Iraq *Corresponding author. E-mail: Sahar.rashid@spu.edu.iq Abstract. Nowadays, overproduction of secondary metabolites in remedial herbs through giving biotic/abiotic stresses is an interesting area of research. In the current study, the influences of various concentrations of silver nanoparticles (Ag NPs) were evaluated on several morphological and physio-biochemical traits, such as the steviol glycosides level in Stevia. The findings showed that the herbs incubated with 400-ppm Ag NPs own the highest dry and fresh weight of shoot, while those incubated with 80- up to 200- ppm Ag NPs own the highest steviol glycosides content. As a result, we successfully improve the content of stevioside glycoside up to 1.75-fold by apply- ing the 80- up to 200-ppm Ag NPs in Stevia medicinal plant. Moreover, our findings revealed that low concentrations the Ag NPs lead to an increase of glutathione content and total antioxidant capacity, and a decrease of MDA, whereas treatments at higher concentrations induced adverse effects for the plant. As a result, the treatment with Ag NPs low concentrations had a favorable efficacy on physio-biochemical and morpho- logical characteristics of Stevia. These achievements are very promising, because they revealed a considerable capability for the Ag NPs application in enhancing the second- ary metabolites in Stevia remedial herb. The present study is the first case assessing the desirable influences of Ag NPs on the Stevia, in regard with shifting of biosynthetic pathway of steviol glycosides in a concentration-dependent manner. Keywords: Ag Nanoparticles, Medicinal Plant, Stevia, Steviol Glycosides. INTRODUCTION Stevia (Stevia Rebaudiana Bertoni) is a medicinal perennial herb sweet in taste. This herb is a member of Asteraceae family and native to Paraguay as well as Brazil (Shivanna et al. 2012). Stevia gives rise to steviosides and rebaudiosides as zerocalorie diterpene glycosides, and naturally keeps safe from obesity, hypertension, and diabetes mellitus (Thiyagarajan and Ven- katachalam 2012; Goyal et al. 2010; Geuns 2003). Stevia can be propagated through tissue culture techniques for producing elite varieties (Yucesan et al. 2016). The less efficiency of stem cutting and poor seeds germination actually led to difficulties for in vitro large-scale propagation of this herb (Hendaw- 16 Sherzad R. Abdull, Sahar H. Rashid, Bakhtiar S. Ghafoor, Barzan S. Khdhir ey et al. 2015). Up to now a number of approaches have been established to achieve an increased content of sec- ondary metabolites from the leaf tissues of Stevia (Javed et al. 2017a; si et al. 2020; Liu et al. 2021). Biotic and abiotic elicitors own the potential of bringing about the larger level of secondary metabolites as well as sweetening compounds through modifica- tion of metabolic cycles (Sabzehzari and Naghavi 2018, 2019; Sabzehzari et al. 2020, 2019; Gupta et al. 2015; Peng et al. 2021; Ma et al. 2021). The elicitors, however, are beneficial up to particular threshold levels because being cytotoxic at much higher concentration (Javed et al. 2017a; Hendawey et al. 2015; Chen et al. 2021; Bi et al. 2021). The cytotoxic influences of various nanoparti- cles as abiotic elicitors have been recorded in a variety of crops/plants (Javed et al. 2017b, c; Shaw and Hossain 2013; Lin and Xing 2008; Lee et al. 2008, 2010; Spanò et al. 2020). In terms of Stevia, only a few types of nanoparticles have been evaluated. For instance, Rezaizad et al. (2019) observed that the plants incubated with 200 ppm TiO2 NPs had the lowest MDA extent and the highest ste- viol glycosides content, while those incubated with 400 ppm TiO2 NPs had the highest fresh and dry weights of shoot. Accordingly, the authors suggested that the treat- ment with TiO2 NPs leads to a positive influence on phy- tochemical and morphological attributes in Stevia herb. Javed et al. (2018) observed that total reducing power (TRP), total antioxidant capacity (TAC), scavenging activity of free radical (DPPH), and total phenolic con- tent (TPC) were highest at 10 ppm of CuO NPs, while the highest level of total flavonoid content (TFC), DPPH, and TPC were registered at 100 ppm concentration of ZnO NPs. Their results clearly showed that CuO NPs are more cytotoxic to Stevia plant when compared to ZnO NPs. Thus, the authors proposed a promising way for future research using CuO or ZnO NPs for increasing commercially significant secondary metabolites in vari- ous medicinal herbs. In terms of Ag NPs, Kaveh et al. (2013) observed that exposure to higher concentration of these nano- particles (up to 20 ppm) led to a decrement of the bio- mass in Arabidopsis. Similarly, Dimkpa et al. (2013) recorded that Ag NPs treatment decreased the roots and shoots length in a dose-dependent way in wheat. Nair and Chung (2014a) also recorded that Ag NPs treat- ment decreased root and shoot weight and root elonga- tion in rice. Al-Huqail et al. (2018) registered a decrease in the total protein content, total chlorophyll content, fresh weight, and root and shoot elongation after expo- sure to Ag NPs in Lupinus termis. Patlolla et al. (2012) reported that Ag NPs treatment enhanced the micro- nuclei and chromosomal aberrations and declined the mitotic index in root tips of broad bean, proposing that mitosis and cell cycle in root tips was disrupted by sil- ver nanoparticles. However, there are several studies that documented the positive effect of silver nanoparticles on plant growth and development in a plant-dependent manner (Reviewed in Yan and Chen, 2019). However, there is no report on the effect of silver nanoparticles on Stevia plant. Based on what has been mentioned about the value of Stevia secondary metabolites and the effect of silver nanoparticles, the present study was focused on the evaluation of the photocatalytic efficacy of Ag NPs on the phytochemical as well as morphological charac- teristics of Stevia plant under controlled conditions. MATERIAL AND METHODS Plant material and growth conditions The seeds of Stevia were provided by the College of Agriculture and Natural Resources, University of Teh- ran, Iran. The current research was carried out through a completely randomized design with three replications. After germination, the seedlings were moved to pot comprising pittmoss and perlite (1:1) (each pot includ- ing three samples), and then put in growth chambers for 10 days under 18-h light at 25°C to grow and take root. The seedlings were moderately irrigated on the first day, and then watered every two days through 50% Hoagland solutions. The treatments in this study were performed at increasing concentrations (0, 20, 40, 60, 80, 100, 200, 400 and 800) of Ag NPs. After being developed, the leaves of all plants were sprayed by Ag NPs on the 11th day. The subsequent spraying operations were performed one week later, followed by the last spraying operations two weeks later. At the end of the treatments some mor- phological and physio-biochemical traits like the dry and fresh weights of shoot, MDA level as a measure of membrane lipid peroxidation, glutathione content, total antioxidant capacity and the steviol glycosides content were assayed in the Stevia leaves. Morphological evaluation To estimate the fresh weights, the shoots (leaves as well as stems) were washed, cut into pieces, and even- tually the shoot fresh weights were registered in g. To measure the dry weights, the samples were dried at 60°C by uing an oven, and ultimately the weights were regis- tered in g. 17Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana Physio-biochemical analysis The estimation of the level of malondialdehyde (MDA), as output derived from lipid peroxidation, has been performed by the method described by Cakmak and Horst (1991). The absorbance at 532 and 600 nm of cell extracts was recorded and the average of the readings in triplicate was utilized for estimating the level of MDA by 155 mM-1cm-1 extinction coefficient as follows: malon- dialdehyde (nM)= ΔA(532-600)/1.56*105. The glutathione content (GSH) was calculated through the procedure as elucidated by Moron et al. (1979). For evaluation of total antioxidant capacity, 100 μL stock solution of each speci- men (5 mg/mL in dimethyl sulfoxide) was combined with 1000 μL reagent solution, including 0.7 M of sulfuric acid, 5 mM of ammonium molybdate, and 30 mM of sodium phosphate. The reaction admixture was maintained for an hour and a half at 95°C, followed by cooling at 25°C. The absorbance of specimens was recorded at 695 nm through micro plate reader in triplicates. Vitamin C was utilized as standard. The data was represented as μg AA/mg (i.e., mg ascorbic acid equivalent) (Ali et al. 2015). Measurement of steviol glycosides To calculate the steviol glycosides content in leaves, 100mg of dry leaves was incubated in 10 ml methanol for 15 min. The residual solvent was removed and the solid residue was solubilized in 5ml water/acetonitrile mix (20:80). The extract (20μL/ml) was injected into the HPLC column (Cosmosoil 5 NH2-MS with particle size of 5 μm, 4.5 mm in diameter, and 15 cm in length) linked to the HPLC (Rezaizad et al. 2019).The mov- ing phase of 80% of acetonitrile as well as 20% distilled water was set at a rate of 1 ml/min. Statistical analysis The q data was analyzed through SPSS Ver. 20. In variance analysis, p=0.05 was taken into consideration as significant level. RESULTS AND DISCUSSION Influence of Ag NPs on the dry and fresh weights of shoot, membrane lipid peroxidation, glutathione con- tent, total antioxidant capacity and the steviol glycosides content was assayed in the Stevia plants. The findings revealed that Ag NPs own a considerable positive effica- cy on the analyzed traits. Influence of Ag NPs on the dry and fresh weights of shoot A comparison of mean dry and fresh weight of shoot in Ag NPs-treated Stevia plants indicated that the 0 ppm Ag NPs (control) sample has the lowest shoot weight, whereas the 400-ppm concentration of Ag NPs has the highest (Figures 1, 2). The fresh and dry weights were increased up to 5- and 3-fold in the 400-ppm Ag NPs-treated Stevia plants when compared to control, respectively. In order to explain our observations, we can suppose that Ag NPs can stimulate root strength and enhance the capability of the root to uptake nutri- ents and water, finally leading to an increment in the fresh and dry weights of the shoot, as reported by Van- nini et al. (2013) in Eruca sativa. Moreover, it was found that the using of Ag NPs in the early developmental stages has ability to increases the carbon fixation effi- ciency and photosynthesis rate in the plants, resulting in enhancing of the yield of dry matter (Rezaizad et al., 2019). However, the 800-ppm Ag NPs treatment led to a considerable decrease in dry and fresh weight of shoot in treated Stevia plants. Similarly, in Sorghum bicolor, 0 0,5 1 1,5 2 2,5 3 0 20 40 60 80 100 200 400 800 Fr es h W ei gh ts o f S ho ot s (g ) Concentrations of Ag nanoparticles (ppm) 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 0 20 40 60 80 100 200 400 800 D ry W ei gh ts o f S ho ot s (g ) Concentrations of Ag nanoparticles (ppm) Figure 1. Efficacy of various concentrations of Ag nanoparticles on fresh weight of shoots in Stevia plant. Figure 2. Efficacy of various concentrations of Ag nanoparticles on dry weight of shoots in Stevia plant. 18 Sherzad R. Abdull, Sahar H. Rashid, Bakhtiar S. Ghafoor, Barzan S. Khdhir Krishnaraj et al. (2012) observed at low concentrations of Ag NPs, an increase in the shoot and root length and weight, while at high concentrations a decline in the length and weight. Influence of Ag NPs on the MDA The production rate of free oxygen radicals has a close relationship with the strength of the membrane (Shivanna et al. 2012). Malondialdehyde, as a reactive oxygen species leads to peroxidation of membrane lipid, enhancing membranes permeability, whereas declining membranes strength. Thus, the content of MDA serves as a measure of the lipid peroxidation, offering an image of cell damages (Yan and Chen, 2019). However, the 200- up to 800-ppm Ag NPs treat- ments resulted in an increase in MDA level in treated Stevia plants (Figures 3). Similarly, Thiruvengadam et al. (2015) evaluated the effect of Ag NPs exposure in turnip seedlings, observing that a higher concentration of Ag NPs led to overproduction of superoxide radicals and increased the lipid peroxidation; hydrogen peroxide pro- duction was also enhanced after exposure to silver nano- particles. Nair and Chung (2014b) recorded that exposure to Ag NPs leads to an increment in lipid peroxidation and hydrogen peroxide production in rice root and shoot in a dose-dependent way. Nair and Chung (2014a) docu- mented that lipid peroxidation increases after exposure to silver nanoparticles in Arabidopsis. De La Torre-Roche et al. (2013) also documented that Ag NPs exposure with concentration at 500 up to 2000 ppm caused a considera- ble increase in MDA level in soybean. Overall, our results revealed that the 100-ppm Ag NPs treatment can decline the level of MDA, whereas Ag NPs treatment with a con- centration above 100-ppm represents an adverse effect likely because of damaging to thylakoid membrane struc- ture (Nair and Chung 2014a; Shivanna et al. 2012). Influence of Ag NPs on the glutathione content In light of our findings, glutathione content was enhanced in Stevia plants after 100-ppm Ag NPs treat- ments when compared to the 0 ppm (control) con- centration. However, the 200- up to 800-ppm Ag NPs treatments led into a decrease in glutathione content in treated Stevia plants. The glutathione extent was increased up to 2-fold in the 100-ppm Ag NPs-treat- ed herbs when compared to control conditions (Fig- ures 4). Similarly, Nair and Chung, (2014b) reported an increased glutathione content after exposure Arabidop- sis thaliana seedlings to the high concentration of Ag NPs. The enhanced level of glutathione may be resulted from the increased glutathione biosynthesis, expression of glutathione S-transferase and glutathione reductase genes, and sulfur assimilation after exposure to Ag NPs (Nair and Chung, 2014b). After silver NPs exposure, a considerable increase in shoots glutathione content was observed, proposing that plant employs glutathione to decrease the effect of ROS derived from the high con- centration of silver nanoparticles (Mirzajani et al. 2013). These outputs are in coincidence with Jiang et al. (2014), who showed that silver own an important function in the increase of antioxidant potentials like glutathione content in Spirodela polyrhiza plant. In fact, antioxidants such as glutathione present a key role in detoxification of toxic metal ions (Pompella et al. 2003). Indeed glu- tathione is a key antioxidant in crops/plants, and pre- serves significant cellular components from reactive oxy- gen species (Singh and Sinha, 2005). Influence of Ag NPs on the total antioxidant capacity We found that total antioxidant capacity enhanced in Stevia plants after incubation with the 20- up to 200-ppm Ag NPs concentrations in contrast to the con- 0 5 10 15 20 25 0 20 40 60 80 100 200 400 800 M em br an e lip id p er ox id at io n nm .g -1 f. w Concentrations of Ag nanoparticles (ppm) Figure 3. Efficacy of various concentrations of Ag nanoparticles on the MDA (membrane lipid peroxidation) in Stevia plant. 0 0,5 1 1,5 2 2,5 0 20 40 60 80 100 200 400 800 G lu ta th io ne c on te nt (μ m ol / g FW ) Concentrations of Ag nanoparticles (ppm) Figure 4. Efficacy of various concentrations of Ag nanoparticles on glutathione content in Stevia plant. 19Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana trol conditions. However, the 400- and 800-ppm Ag NPs treatment resulted in a decrease in total antioxi- dant capacity in the Stevia herbs. The total antioxidant capacity was increased up to 1.65-fold in the 100- and 200-ppm Ag NPs-treated Stevia plants when compared to control (Figures 5). In line with our findings, Jiang et al. (2014) reported Ag NPs can induce accumulation of ROS, and alter antioxidant system in the Spirodela polyrhiza aquatic plant. Qian et al. also observed that Ag NPs accumulated in the leaves of Arabidopsis and changed the transcription of aquaporin and antioxidant genes, proposing that Ag nanoparticles can alter the balance between antioxidant as well as oxidant systems (Qian et al. 2013). Influence of Ag NPs on the stevioside glycoside content Analysis of the stevioside glycoside content revealed that 80- up to 200-ppm Ag NPs concentrations led into the highest stevioside glycoside content, whereas spraying the Ag NPs at 400- and 800-ppm concentra- tions lead to a decrease in percentage weight of this compound (Figures 6). As a result, we successfully improve the content of stevioside glycoside up to 1.75- fold by applying the 80- up to 200-ppm Ag NPs in Ste- via medicinal plant. To the best of our knowledge, no research has ever been documented the Ag NPs applica- tion on Stevia medicinal herb so far. However, previous findings applying copper, silicon, iron, as well as TiO2 NPs on this herb showed that the higher concentrations of copper and iron nanoparticles lead to the higher lev- els of stevioside when compared to control (Rezaizad et al. 2019). For silicon NPs, the findings indicated that the highest stevioside content is generated at low concentra- tions (0.20 up to 2 mg/L) and the least extent of stevio- side glycoside is produced at the 4 up to 8 mg/L concen- trations of silicon NPs (Hendawey et al. 2015). CONCLUSION Many studies showed the adverse effect of silver nanoparticles on various crops/plants at molecular, cel- lular, physiological, and morphological level (Reviewed in Yan and Chen 2019; Wang et al. 2021; Yin et al. 2021; Zhao et al. 2021). However, a number of researches recorded the positive effect of Ag NPs on plants growth and development, and on physio-biochemical param- eters (Vannini et al. 2013; Krishnaraj et al. 2012; Jia et al. 2020; Shi et al. 2021; Zheng et al. 2021; Zhu et al. 2021). These contradictory findings reveal the complexity of the response of crops/plants to silver nanoparticles, which are not only determined through the features of Ag NPs (Ag chemical form, surface coating, shape, concentra- tion, size, etc.), but are also dependent on the plant sys- tems utilized (developmental stage, organ, tissue, species, etc.) and experimental methodologies (exposure time, exposure procedure, medium, etc.). We firstly reported the Ag NPs, at low concentrations, had a favorable effica- cy on physio-biochemical and morphological character- istics of Stevia herb, while the high concentrations had an adverse effect. REFERENCES Ali, A., Phull, A.R., Zia, M., Shah, A.M.A., Malik, R.N. 2015. 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Genetic Diversity And Relationships Among Salvia Species By Issr Markers; Genetika-Belgrade, 53(2): 559-574. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 2 - 2022 Firenze University Press Cytogenetic Studies of Six Species in Family Araceae from Thailand Piyaporn Saensouk1, Surapon Saensouk2,*, Rattanavalee Senavongse2 Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana Sherzad R. Abdull, Sahar H. Rashid*, Bakhtiar S. Ghafoor, Barzan S. Khdhir Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified polymorphism Wei Cao1, Xiao Chen2,*, Zhiwei Cao3 Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae) Xiaoju Zhang1, Li Bai2,*, Somayeh Esfandani-Bozchaloyi3 SCoT molecular markers are efficient in genetic fingerprinting of pomegranate (Punica granatum L.) cultivars Shiva Shahsavari1, Zahra Noormohammadi1,*, Masoud Sheidai2,*, Farah Farahani3, Mohammad Reza Vazifeshenas4 First record of nucleus migration in premeiotic antherial cells of Saccharum spontaneum L. (Poaceae) Chandra Bhanu Singh1, Vijay Kumar Singhal2, Manish Kapoor2,* Genetic Characterization of Salicornia persica Akhani (Chenopodiaceae) Assessed Using Random Amplified Polymorphic DNA Zhu Lin1,*, Hamed Khodayari2 Comparative chromosome mapping of repetitive DNA in four minnow fishes (Cyprinidae, Cypriniformes) Surachest Aiumsumang1, Patcharaporn Chaiyasan2, Kan Khoomsab3, Weerayuth Supiwong4, Alongklod Tanomtong2 Sumalee Phimphan1,* Classical chromosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand Weera Thongnetr1, Surachest Aiumsumang2, Alongklod Tanomtong3, Sumalee Phimphan2,* Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate Made Pharmawati1,*, Ni Nyoman Wirasiti1, Luh Putu Wrasiati2 Chromosomal description of three Dixonius (Squamata, Gekkonidae) from Thailand Isara Patawang1, Suphat Prasopsin2, Chatmongkon Suwannapoom3, Alongklod Tanomtong4, Puntivar Keawmad5, Weera Thongnetr6,* First Report on Classical and Molecular Cytogenetics of Doi Inthanon Bent-toed Gecko, Cyrtodactylus inthanon Kunya et al., 2015 (Squamata: Gekkonidae) in Thailand Suphat Prasopsin1, Nawarat Muanglen2, Sukhonthip Ditcharoen3, Chatmongkon Suwannapoom4, Alongklod Tanomtong5, Weera Thongnetr6,* Evaluation of genetic diversity and Gene-Pool of Pistacia khinjuk Stocks Based On Retrotransposon-Based Markers Qin Zhao1,*, Zitong Guo1, Minxing Gao1, Wenbo Wang1, Lingling Dou1, Sahar H. Rashid2 A statistical overview to the chromosome characteristics of some Centaurea L. taxa distributed in the Eastern Anatolia (Turkey) Mikail Açar1,*, Neslihan Taşar2 Cytotoxicity of Sunset Yellow and Brilliant Blue food dyes in a plant test system Elena Bonciu1, Mirela Paraschivu1,*, Nicoleta Anca Șuțan2, Aurel Liviu Olaru1