Acta agriculturae Slovenica, 118/3, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.3.1873 Original research article / izvirni znanstveni članek Influence of Ag nanoparticles on physiological and biochemical aspects of callus of Thymus species and Zataria multiflora Boiss. Nima MOSAVAT 1, 2, 3, Maryam YOUSEFIFARD 4, Pooran GOLKAR 5, 6, Rabia JAVED 7 Received September 12, 2020; accepted August 31, 2022. Delo je prispelo 12. septembra 2020, sprejeto 31. avgusta 2022 1 Department of Agricultural Biotechnology, Payame Noor University (PNU), Tehran, Iran 2 Institute of Biotechnology, School of Agriculture, Shiraz University, Shiraz, Iran 3 Corresponding author, e-mail: nima.mosavat@gmail.com 4 Department of Engineering and Technology, Payame Noor University (PNU), Tehran, Iran 5 Department of Natural Resources, Isfahan University of Technology, Isfahan, Iran 6 Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan, Iran 7 Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan Influence of Ag nanoparticles on physiological and biochemi- cal aspects of callus of Thymus species and Zataria multiflora Boiss. Abstract: Thymus species have found remarkable im- portance in food and medicine industries. The present study investigates the potential effect of Ag nanoparticle elicitors on proliferation of callus, and production of carvacrol and thymol in Zataria multiflora and three Thymus species. Firstly, callus was induced on Murashige and Skoog (MS) medium contain- ing 2 mg l−1 of 2, 4-dichlorophenoxy acetic acid (2,4-D) and 1 mg l−1 of kinetin (Kin)). Secondly, the effects of two differ- ent concentrations of Ag nanoparticles (4 and 8 mg l-1) were studied on callus growth and its secondary metabolites produc- tion. Results elucidated that after elicitation by 8 mg l-1 of Ag NPs, significantly the highest callus growth rate (CGR) (0.02 mm day-1), callus fresh mass (CFM) (0.99 g), and carvacrol (0.68 mg l-1) and thymol (11.09 mg l-1) content was achieved. Comparing different Thymus species, notably the greatest car- vacrol and thymol amount was obtained in .kotschyanus Boiss. & Hohen. and T. Daenesis Čelak. at 8 mg l-1 concentration of Ag NPs. Hence, it is evident that the stimulation by NPs is dose- dependent. This study has potential to be commercially applied for the enhancement of pharmaceutical compounds in different species of Thymus. Key words: Ag nanoparticles; Thymus species; Zataria multiflora; callus; carvacrol thymol Abbreviations: 2,4-D, 2, 4-dichlorophenoxyacetic acid; Kin, kinetin; PGRs, plant growth regulators; HPLC, high per- formance liquid chromatography; NPs, nanoparticles Vpliv nanodelcev srebra (Ag) na fiziološke in biokemične la- stnosti kalusa dveh vrst materine dušice (Thymus sp.) in vrste Zataria multiflora Boiss. Izvleček: Vrste iz rodu materine dušice (Thymus sp.) ima- jo velik pomen v prozivodnji hrane in zdravil. V raziskavi je bil preučevan potencialni učinek nanodelcev srebra kot eliciator- ja na rast kalusa, tvorbo karvakrola in timola pri vrsti Zataria multiflora in treh vrstah materine dušice. Najprej je bil na Mu- rashige in Skoog (MS) gojišču, ki je vsebovalo 2 mg l-1 2,4-D in 1 mg l-1 kinetina, vzgojen kalus. Potem je bil preučevan učinek dveh različnih koncentracij srebrovih nanodelcev (4 in 8 mg l-1) na rast kalusa in in tvorbo sekundarnih metabolitov. Rezultati, pridobljeni z visokotlačno tekočinsko kromatografijo (HPLC) so pokazali, da je bila po uporabi 8 mg l-1 srebrovih nanodelcev kot iliciatorjev dosežena značilno največja rast kalusa (CGR) (0,02 mm dan-1), največja sveža masa kalusa (CM) (0,99 g) in največja vsebnost karvakrola (0,68 mg l-1) in timola (11,09 mg l-1). V primerjavi različnih vrst materine dušice je bila dosežena največja vsebnost karvakrola in timola pri vrstah T. kotschyanus Boiss. & Hohen in T. daenesis Čelak pri koncentraciji srebrovih nanodelcev 8 mg l-1. Očitno je, da je stimulacijski učinek nano- delcev odvisen od doze. Izsledke raziskave bi lahko komercial- no uporabili za povečanje tvorbe zdravilnih spojin pri različl- nih vrstah materine dušice. Ključne besede: Ag nanodelci; vrste iz rodu Thymus; Za- taria multiflora; kalus; karvakrol; timol Okrajšave: 2,4-D: 2, 4-diklorfenoksi ocetna kislina; Kin: kinetin; PGRs: rastlinski rastni regulatorji; HPLC: visokotlačna tekočinska kromatografija; NPs: nanodelci Acta agriculturae Slovenica, 118/3 – 20222 N. MOSAVAT et al. 1 INTRODUCTION Nanobiotechnology has enormous applications in diverse fields including agriculture, cosmetics, pharma- ceutics, and food industry) Kim et al., 2017; Rastogi et al., 2017). Nanoparticles (NPs) are employed as elicitors of various cell signaling pathways in metabolism of plants (Kim et al., 2017; Marslin, et al., 2017). NPs have been found to play crucial role in enhancement of plant sec- ondary metabolites (SMs) by imposing oxidative stress and increasing cell membrane permeability (Jasim et al., 2017; Marslin et al., 2017; Ahmad et al., 2020). The effects of NPs on growth rate of plants (Sharma et al., 2012), ger- mination of seeds (Zaka et al., 2016), production of SMs (Marslin et al., 2017; Mosavat et al., 2019; Zaka et al., 2016; Golkar et al., 2021) and plant physiology (Jasim et al., 2017; Sharma et al., 2012) have been studied recently. Although there are few studies related to effect of NPs on callus culture development, physiology and second- ary metabolism (Dykman & Shchyogolev, 2017; Kokina et al., 2013; Marslin et al., 2017; Sanzari et al., 2019; Zu- verza-Mena et al., 2017), still this research domain needs to be explored further. Silver (Ag) NPs possess unique properties in terms of toxicity and alteration of yield, de- velopment, antioxidant activities, and SMs production of plants due to their high catalysis and reactivity (Rastogi et al., 2017; Sadak 2019). Furthermore, the influence of Ag NPs on the callus cultures of Solanum nigrum L. (Ewais et al., 2015), Prunella vulgaris L. (Fazal et al., 2019) and Caralluma tuberculata N.E. Brown (Ali et al., 2019) has recently been studied. Thymus L., belonging to Lamiaceae family, has world-wide distribution (Sajed et al., 2013). However, it is dominantly present in Asia, Europe and North Africa (Zarshenas & Krenn, 2015). The essential oils and SMs enhance the commercial value of flowers and leaves of Thymus making it a valuable crop in cosmetics, pharma- ceutics, and food and agriculture industry (Miraj & Ki- ani, 2016). A thyme-like plant, Zataria multiflora Boiss., belonging to Lamiaceae family, is wild plant found in only southern and central Pakistan, Afghanistan, and Iran (Sajed et al., 2013). Tissue culture propagation of thyme plant is well-known because it is a wide source of ingredi- ents of pharmacology. Both Thymus sp. and Z. multiflora possess anti-cancerous, anti-inflammatory, anti-oxidant, anti-bacterial, anti-fungal, and anti-spasmodic proper- ties (Mathela et al., 2010; Sajed et al., 2013). Naturally occurring terpenoid thymol (2-isopropyl-5-methyl phe- nol) and its phenol isomer, carvacrol/cymophenol are the phenolic compounds that play an important role in inducing these properties to Thymus sp. and Z. multiflora (Kianersi et al., 2021; Mathela et al., 2010). Some other properties like their use as additive in perfumes, deodor- ant, toothpastes, soaps, etc. and as important flavoring agent in foods are also attributed to these compounds (Sajed et al., 2013). The defense system of plants is activated by chemi- cal, biological or physical elicitors (Asadollahei et al., 2022; Zhao et al., 2005). The gene expression is then modulated that transcribes the formation of SMs (Ajun- gla et al., 2009). A more efficient method could be the use of callus cultures for extracting SMs naturally from Thymus sp. and Z. multiflora in a sustained manner (Ramakrishna & Ravishankar, 2011). Previously, there is no report concerning the carvacrol and thymol produc- tion by imposing Ag NPs in cell cultures of Thymus sp. and Z. multiflora. Hence, the current study investigates proliferation of callus and SMs production in three Thy- mus species, i.e., T. vulgaris L., T. daenensis Čelak, and T. kotschyanus Boiss. & Hohen as well as Z. multiflora Boiss. after Ag NPs exposure. 2 MATERIALS AND METHODS The seeds of Thymus species, i.e., T. vulgaris, T. dae- nensis, T. Kotschyanus and Zataria multiflora (two acces- sions) (Table 1) were deposited at Botanic Herbarium of Research Institute for Biotechnology and Bioengineer- ing, Isfahan University of Technology (IUT), Isfahan, Iran, after collection from different geographical regions and identification by using Flora Iranica (Rechinger, 1982). Their characteristics is shown in Table 1. The seeds of four different species were surface sterilized with 70 % (v/v) ethanol for 1 min, followed by the addition of 3 % (v/v) sodium hypochlorite for 20 min, and then rinsing in sterile distilled water thrice. After surface steriliza- tion, the seeds were grown in Murashige and Skoog (MS) (1962) medium (Duchefa, Netherland). These were incu- bated for germination and growth of plantlets. The leaflet explants from about 1-month old plantlets were cultured in MS medium containing 2,4-D (2 mg l-1) and Kin (1 mg l-1) supplemented with 3  % (w/v) sucrose (Sigma- Aldrich, USA), 0.8 % (w/v) agar (Sigma-Aldrich, USA) and 0.1 mg l-1 myoinositol for callus induction. The pH was adjusted at 5.7. The samples were exposed to 16h/8h (light/dark) photoperiod for a period of 2 months at 23 ± 2 ℃. Silver (Ag) nano-powder was purchased from US Research Nanomaterials Inc., Houston, TX, USA hav- ing an average size of 30–50 nm and purity of 99.99 %. The nanoparticles were characterized by x-ray diffraction (XRD) and scanning electron microscopy (SEM) tech- niques by following the protocols of Javed et al. ( 2016). XRD was performed using Carlo ERBA Model EA 1108 analyzer and the instrument for getting SEM image was Acta agriculturae Slovenica, 118/3 – 2022 3 Influence of Ag nanoparticles on physiological and biochemical aspects of callus of Thymus species and Zataria multiflora Boiss. Hitachi S4800 (Japan). These NPs were added to MS me- dium after filter sterilization. The 2-months old friable callus (0.25 g) was transferred to MS containing 2,4-D (2 mg l-1) and Kin (1 mg l-1) under Ag NPs stress of 4 and 8 mg l-1. This callus material was placed at 23 ± 2 ℃ under a photoperiod of 16h/8h. After incubation for 21 days of callus with Ag NPs, the callus growth rate (CGR) and callus fresh mass (CFM) was calculated. CGR was measured according to Afshar et al. (2016) every 7 days in 21 days period. Later on, the quantity of carvacrol and thymol was obtained by high performance liquid chromatography (HPLC). The method of Castro et al. (2016) was utilized for preparing callus extracts. The process involved dry- ing of 200 mg of callus from each treatment in an oven at 50 ℃ for 24 h, and then soaking it in 5 ml of diethyl ether for a period of 24 h. In order to prevent the evapo- ration of diethyl ether, the vials were kept closed and ex- traction was performed in a cold room. After adding the 80 % of methanol (1 ml) to remaining solid material, the extracts were filtered (0.22 μm pore size) into clean vi- als and prepared for injection to HPLC instrument. The HPLC (SY-8100 series, Beijing Beifan-Ruili Analytical Instrument, China) was performed by UV-VIS detec- tor, a flow rate of 0.9 ml min-1, injection volume of 20 μl at 28 °C, C18 column (25 cm × 4.6 mm, partial size 5 μm), mobile phase methanol-water (80:20; v/v), and flow rate of 0.9 ml min-1. The detection was carried out at 280 nm of wavelength and a pressure of 12 atm. The UV spectra of phenolic compounds were recorded at 280 and 320 nm. The content of carvacrol and thymol were determined based on the calibration curve of standard compounds, including carvacrol (Sigma-Aldrich, USA) and thymol (Sigma-Aldrich, USA). For this purpose, 4 concentrations (10, 25, 50, and 100 mg l-1) of carvacrol and 3 concentrations (25, 100, and 400 mg l-1) of thymol were examined by HPLC. The retention time for car- vacrol and thymol were appeared at 3-4 min and 14-15 min, respectively. After calibration of the standards with HPLC, the quantities of carvacrol and thymol in different samples were calculated. 2.1 STATISTICAL ANALYSIS The experimentation was conducted with three rep- lications in completely randomized design and the statis- tics was determined using two-way analysis of variance (ANOVA). LSD test (p ≤ 0.05) in SAS software (SAS 9.1 Inc. USA) was applied to determine significant difference among the treatments. 3 RESULTS AND DISCUSSION Callus growth and development as well as forma- tion of secondary metabolic compounds is positively or negatively influenced by supplementing the growth medium with abiotic or biotic stress elicitors (Ajungla et al., 2009; Zaka et al., 2016). The contents of SMs show significant changes under elicitation of callus by different stresses (Fazal et al., 2016; Mosavat et al., 2019; Sanzari et al., 2019). NPs, specifically the metallic oxide NPs like ZnO, CuO, TiO2 act as oxidative abiotic stress elicitors (Lala 2021). According to Al-jibouri et al. (2012), thymol amount was increased by proline in Origanum vulgare L. Similarly, the production of significantly the highest con- tent of hyperforin in Hypericum perforatum L. (Sharafi et al., 2013), rebaudioside A and stevioside in Stevia rebau- diana Bertoni (Javed, et al., 2018), and proline in Triti- cum aestivum L. (Barbasz, et al., 2016) under ZnO NPs elicitation has been previously documented. Ag NPs show significant effect on the production of SMs in callus culture, resulting in their ultimate increase (Fazal et al., 2016). For instance, Ali et al. (2019) reported that various concentrations of Ag NPs significantly af- fected the callus proliferation and substantially increased the callus biomass and SMs in Caralluma tuberculata. In another study, Fazal et al. (2019) reported the positive ef- fects of Ag NPs and Au NPs on the production of bio- mass and SMs in the cell culture of Prunella vulgaris L. XRD of Ag NPs is given in Figure 1 which shows 100  % phase purity by the sharpness of peaks. Similar XRD pattern was obtained by Kim et al. (2006). The Species Abbreviation Origin Genotype code Latitude (m) Longitude (m) Altitude (m) Zataria multiflora (1) Zm (1) Dehbala, Yazd, Iran RIBB/ZM01/2016 31°59’ N 54°11’ E 2600 Zataria multiflora (2) Zm (2) Abadeh, Fars, Iran RIBB/ZM02/2016 31°45’ N 51°21’ E 2030 Thymus vulgaris Tv Marvdasht, Fars, Iran RIBB/TV01/2016 35°56’ N 52°10’ E 1620 Thymus daenensis Td Aligoodarz, Lorestan, Iran RIBB/TD01/2016 33°24’ E 49°41’ E 2022 Thymus kotschyanus Tk Lahijan, Gilan, Iran RIBB/TK01/2016 37°12’ N 50°14’ E 396 Table 1: The geographical origins of Thymus sp. and Z. multiflora with their geographical traits collected from Iran Acta agriculturae Slovenica, 118/3 – 20224 N. MOSAVAT et al. spherical shape of Ag NPs was illustrated by SEM image given in Figure 2 which is coinciding with the results of Elumalai et al. (2010). The size, shape, surface, concentration and chemi- cal composition of NPs cause stimulatory or inhibitory effects on the growth of callus cells (Al-Jibouri et al., 2012). The synthesis and accumulation of SMs in cells is enhanced by an increased surface area of NPs as a result of reduced size and transport of NPs in to the cells apo- plastically which increases electrostatic interactions be- tween living cell membranes (Javed et al., 2017). Table 2 and Figure 3 shows the effect of Ag NPs on callus growth of Thymus sp. and Zataria multiflora. Compact calli with white and greenish colour were obtained after 10 days upon control culture (no AgNPs), whereas friable watery calli with white, greenish or yel- lowish colour were observed after 10-13 days upon cul- ture supplemented with AgNPs (Figure 4.) A significant effect is produced on callus traits by different concentrations of Ag NPs in this study that is coherent with the reports about effects of Ag NPs on Figure 1: X-ray diffractogram (XRD) of Ag nanoparticles Figure 2: Scanning electron micrograph (SEM) of Ag nano- particles Nanoparticle Concentration (mg l-1) Color and Texture CGRІ (mm day-1) CFM II (g)7 14 21 Mean Ag 8 Green, friable 0.045 0.06 0.015 0.022 a 0.99 a Ag 4 Green, friable 0.07 0.06 0.032 0.015 b 0.85 ab Control - White to yellow, soft 0.02 0.01 0.006 0.012 c 0.61 b Table 2: Effect of different concentrations of Ag NPs on color, texture, growth rate, and fresh mass of callus cultures of Thymus sp. and Zataria multiflora Mean values followed by the same letter in each column are not significantly different at p<0.05 (Least Significant Difference Test). І: CGR: Callus growth rate, II: CFM: Callus fresh mass Fig 3: Effect of Ag nanoparticles on callus growth rate (A) and callus fresh mass (B) content of different Thymus species and Z. multiflora under callus culture Acta agriculturae Slovenica, 118/3 – 2022 5 Influence of Ag nanoparticles on physiological and biochemical aspects of callus of Thymus species and Zataria multiflora Boiss. callus culture of Solanum nigrum L. (Ewais et al., 2015) which gain friable watery calli with greenish or yellow- ish colour were observed after 10-13 days upon culture supplemented with AgNPs and effects of TiO2 NPs on the callus of Hordeum vulgare L. (Mandeh et al., 2012). The higher TiO2 NPs concentration influenced callogen- esis of Hordeum vulgare explants in this study. Also ac- cording to Kokina et al. (2013) elicitation with Ag and Au NPs shows positive effects on callus width and length in Linum usitatissimum L. The positive effects of different NPs on callus growth of Prunella vulgaris L. is reported by Fazal et al. (2019). Callus growth traits affected by ZnO NPs in Solanum lycopersicum Mill. have also been reported (Alharby et al., 2016). The effects of elicitation by Ag NPs on production of carvacrol and thymol under in vitro conditions are presented in Figure 5. The production of thymol and car- vacrol was determined at 8 mg l-1 concentration of Ag NPs and control treatment. The carvacrol (0.68 mg l-1) and thymol (11.09 mg l-1) quantity was enhanced under 8 mg l-1 of Ag NPs. The chromatographic separation of the methanolic extracts in Zataria multiflora for carvacrol and thymol by HPLC is given (Figure 6). Asadollahei et al. (2022) employed different concentrations of CuNPs in in vitro culture medium and observed significant rise in thymol and carvacrol content compared to control in Za- taria multiflora. This study elucidated that the selection of appropriate plant species and suitable elicitor is crucial for increasing the production of bioactive compounds as well as antioxidants of Zataria multiflora. This can be done by inducing expression changes in the biosynthetic pathways of thymol and carvacrol. In fact, the gene ex- pression patterns of the pathways of formation of thymol and carvacrol were greatly influenced by the Ag NPs in our study which is the phenomenon well explained by the studies of Kianersi et al. (2021). The interactive effects of NPs and genotypes/species of Thymus and Zataria multiflora for production of SMs Fig 4: Friable calli with greenish or yellowish colour of Thymus species and Z. multiflora after culture treatment with AgNPs (a) and under control culture (b) Fig 5: Effect of 8 mg l-1 Ag NPs on in vitro production of carvacrol and thymol in Thymus sp. and Zataria multiflora Fig 6: Representative HPLC chromatograms of thymol and carcacrol of Z. multiflora. Peak identifications were performed by matching retention time and UV spectra against commer- cially available reference compounds Acta agriculturae Slovenica, 118/3 – 20226 N. MOSAVAT et al. have been presented in Table 3. The exposure of Ag NPs has significantly increased the content of two SMs of cal- lus compared to control. Moreover, in vitro synthesis of thymol was notably greater than that of carvacrol which is also evident from the molecular studies of Kianersi et al. (2021). The highest content of carvacrol (1.06 mg l-1) was observed at 8 mg l-1 concentration of Ag NPs in T. kotschyanus, whereas the least amount (0.10 mg l-1) was observed in control treatment of T. daenensis. Further- more, the highest concentration of thymol was obtained at 8 mg l-1 of Ag NPs in callus of T. daenesis (19.75 mg l-1), while the least thymol content (3.95 mg l-1) was achieved in T. daenesis under control condition. This result can be well supported by the phenom- enon that NPs trigger thymol synthetic pathways and/or transcription factors more than the carvacrol pathways (Mosavat et al., 2019). Taking into account of concur- rent studies, the phenolics and flavonoids production is activated by ZnO NPs in seedlings of Brassica nigra L. (Zafar et al., 2016) Additionally, the significant rise in hy- perforin content in cell suspension culture of Hypericum perfolatum L. under ZnO NPs stress is reported (Sharafi et al., 2013). A complex variety of elicitation effects on in vitro synthesis of SMs is obtained using different types of elicitors (Goswami et al., 2017; Marslin et al., 2017; Syu et al., 2014), plant tissues (Ajungla et al., 2009), and physi- ochemical environment of various species (Shakya et al., 2019). 4 CONCLUSION The formation of callus from Thymus species and Zataria multiflora was performed in the presence of Ag NPs elicitors. Addition of abiotic elicitors, i.e., Ag NPs (8 mg l-1) to the MS medium played a vital role in enhanc- ing the thymol and carvacrol content in the callus cul- tures of different Thymus species and Zataria multiflora. In other words, Ag nano-elicitors applied to the in vitro callus cultures of Thymus species and Zataria multiflora in our study resulted in increase in SMs production at a concentration of 8 mg l-1. Our finding opens the way for studies involving relationship between chemical elicitors and formation & accumulation of thymol/carvacrol. 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