Antioxidant and antimicrobial responses associated with in vitro salt stress of in vitro and in vivo grown Pistacia khinjuk stocks

P. khinjuk Stocks, known as Bıttım or Buttum in Turkey, is a member of the Anacardiaceae family. The essential oil of khinjuk pistachio has been used to treat various illnesses because of their anti-inflammatory, anticancer, antipyretic, antibacterial, anthelmintic, antiviral effects in various folk medicines. At the same time, fruits of khinjuk pistachio are used as edible wild fruits. In this study, it was aimed to determine and compare the antibacterial, antioxidant activities and total phenolic and flavonoid amounts of different parts (root, stem and leaf explants) of in vivo (grown naturally) and in vitro derived khinjuk pistachio plants under salt (NaCl) stress. Ethanol extracted explants were used for performing biological and chemical parameters. According to the results, generally, in vivo samples shows higher antioxidant and antimicrobial activity besides the higher number of phenolic compounds than their counterparts in vitro. We have also determined that the biological activity of in vitro salt elicited explants was higher than in vitro control explants. Generally, both female and male in vivo samples have higher antioxidants (DPPH, ABTS, CUPRAC) and antimicrobial activities than in vitro samples. The various plant parts (root, stem, leaf) belonging to both in vivo and in vitro samples have different biological activity level. In terms of antimicrobial activity, female plant extracts are more active than all other tested extracts. As a result, although increased salinity values significantly reduced antimicrobial activity, it is determined that 100 mM NaCl applications to in vitro leaf extracts exhibited moderate antimicrobial activity against S. aureus and C. albicans.

civilizations from prehistoric times to the present day for various purposes due to their resin, fruit, leaves, and chemical components. The potential of antioxidant, anthelmintic, antimicrobial, anti-inflammatory, and cytotoxic effects of Pistacia species, primarily due to flavonoids and other secondary metabolites, have always attracted the attention of researchers. So far, there are many studies on the antimicrobial, antioxidant, antiinflammatory, cytotoxicity antiangiogenic, etc. properties of this genus in the literature.
For example, under the light of literature, a flavone showed high antioxidant activity was identified as well as apigenin, luteolin, and other flavonoids from the extracts of P. terebinthus fruits (Topçu, 2007). In another study, antimicrobial and antioxidant characteristics of the extracts of the resin of P. lentiscus has also been reported (Giaginis and Theocharis, 2011). Also, Gallotannin, a kind of tannin, has more antioxidant potential isolated from the leaf extract of P. weinmannifolia, called "Pistafolia A" (Wei et al., 2002). Furthermore, it was shown that the ethanol extracts of fruits and leaves of P. vera L. have a higher antioxidant effect than the resin, according to a study by Hosseinzadeh et al. (2012). It was also determined by the MIC test that P. terebinthus L. volatile components significantly inhibited the growth of different organisms such as Shigella dysenteriae, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa (Holley and Patel, 2005;Mohagheghzadeh et al., 2010). These studies mentioned above show that Pistacia species can use as an essential natural antioxidant and antimicrobial source. At the same time, it was reported that not only the seeds but also other parts of naturally grown P. khinjuk Stocks could be a source of phenolics and flavonoids compounds (Hacibekiroglu et al., 2015, Hatamnia et al., 2016Ahmed et al., 2017;Hazrati et al., 2020). However, many of chemical and biological activity studies were found in the literature on extracted explants for other economically important plant species (Esmat et al., 2012;Mirian et al., 2014;Tahvilian et al., 2016;Ahmed et al., 2017;Taghizadeh et al., 2018) there is no study about chemical and biological activity on in vitro grown plants under salt stress conditions. Although many studies confirm the negative effect of salinity on growth, it could be led to an increase in the production of secondary plant metabolites, antioxidant and antimicrobial effects. Moreover, the enhanced synthesis of these secondary metabolites under salt stress conditions is believed to protect the cellular structures from oxidative effects. To avoid oxidative damage resulting from salt stress, higher plants have developed different adaptive mechanisms through the biosynthesis of a cascade of antioxidants. Indeed, polyphenolic compounds participate in the defence against reactive oxygen species (ROS), which are inevitably produced when environmental stresses impair aerobic or photosynthetic metabolism. It has been proven that the amount of antioxidant and antimicrobial components increases in adverse environmental conditions in plant tissues (Salem et al., 2013).
In the light of the literature review about the effect of salt stress on Pistacia species, it has been found that the articles are mostly related to physiological studies, and generally, medium and high salt applications negatively affect growth and increase soluble sugar and proline content (Rahneshan et al., 2018). Considering the studies investigating the physiological responses of in vitro salt stress on the Pistacia genus, in a study on two different species (P. vera L. and P. atlantica Desf.), the seeds were cultured for 25 days at different NaCl concentrations (0, 40, 60, 80, 131, 158.5 and 240 mM). It was noted that low NaCl applications did not cause plant death in any sample, but under high salt conditions (158.5 and 240 mM), 20-25% mortality was observed. Besides, regarding salinity effects 60 and 80 mM NaCl levels caused significant reductions in stem growth and leaf number in P. vera species. However, salinity between 40 and 80 mM NaCl caused a reduction in the number of roots of both species. After 45 days of culture, fresh weights also decreased significantly, and it was observed that high NaCl applications (131-240 mM NaCl) caused a significant increase in proline content in both Pistacia species (Chelli-Chaabouni et al., 2010).
In another study, the salinity tolerance of pistachio (Pistacia vera L.), embryos were investigated. The embryos developed from mature seeds were isolated and cultured in vitro and subjected to different NaCl concentrations (0, 42.8, 85.5, 171.1 and 256.6 mM) for 30 days. According to the results in vitro germination of embryonic axes was not affected by the salt concentration. However, the germinated embryo survival rates decreased from 100% for the control to 62.9% for the highest salt concentration (256.6 mM) (Benmahioul et al., 2009).
Another study (Ayaz Tilkat et al., 2019) reported that the effect of different sodium chloride (NaCl) concentrations (0,50,100,150,200, 250 mM) on growth and physiological parameters of Pistacia lentiscus L. seedlings raised in in vitro condition for four weeks was investigated. According to the study, the morphological, physiological and biochemical changes that occurred in the seedlings were measured and recorded after exposure to salt stress. The results indicate that the visible leaf damage of Lentisk seedlings is affected by high salt concentrations. High salinity concentrations significantly reduce root and stem lengths, relative water content (RWC), total chlorophyll, Chl a, Chl b and carotenoid values after the culture periods. At 250 mM salt concentration, root and stem growth were found to be stopped entirely. Consequently, the parameters that over the 50 mM salt concentrations are caused in a decrease in the activity of the antioxidant enzyme peroxidase (POD).
There is only one study in the literature investigating the physiological effects of salt stress applied in vitro in khinjuk pistachio (Ayaz Tilkat et al., 2017). In this study, mature khinjuk pistachio seeds cultured in in-vitro conditions were exposed to different salt (NaCl) parameters (0, 50, 100, 150, 200, 250 mM). The seeds germinated in PGR-free MS (Murashige and Skoog, 1962) medium containing different concentrations of NaCl were exposed to salt stress for four weeks. As a result, it was determined that there is a positive correlation between soluble carbohydrate values and POD activities due to the increased NaCl concentration (Ayaz Tilkat et al., 2017).
All these studies aside, in this context, the current study aims to investigate and compare the antioxidant and antimicrobial properties of the root, stem, and leaf extracts obtained from the plantlets germinated under different salt concentrations in vitro, and the same parts extract from in vivo grown plants. Antimicrobial activity screening was determined using the disc diffusion method, and minimal inhibitory concentration (MIC) values were defined. The antioxidant activity potential of the extracts was evaluated by different methods, namely, Folin-Ciocalteu (FCR), 1,1-diphenyl-2-picrylhydrazyl (DPPH) 2,2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity tests and cupric ion reducing antioxidant capacity (CUPRAC) method.
Since there is no study in the literature about antimicrobial and antioxidant activities of in vivo and in vitro samples of khinjuk pistachio, the topic is highly original, and the results will be reported for the first time.

Materials and Methods
Biological material In this study, leaves, roots, and stems of mature male and female P. khinjuk Stocks trees raised in Gaziantep Pistachio Research Institute were used for obtaining in vivo extracts, and mature seeds of this trees were used as in-vitro salt stress experiments for antioxidant and antimicrobial activity studies. The protocols developed by Tilkat et al. (2005) were modified for in vitro micropropagation of seeds. The diagram of all the processes in the study was shown in Figure 1. The root, stem, and leaves originated mature female, and male khinjuk pistachio was harvested from the orchards of Gaziantep Pistachio Research Institute in July 2019, then dried at room temperature in the dark, pounded using pestles, and wooden mortars separately for using in the extraction procedures.

Obtaining of in-vitro explants under salt stress
For all the stages of surface sterilization of seeds, culture initiation, and proliferation of seedlings, the protocol developed by Tilkat et al. (2005) was modified and used. The seeds were surface sterilized by immersion in a 20% (w/v) commercial bleach solution (NaOCl) for 20 min. In this context, the seed coats were then removed, and the kernels were washed three times with sterile distilled water before inoculating onto the MS basal medium. The plant growth regulator (PGR)-free MS basal medium was supplemented with 100 mg/l l-ascorbic acid, 3% sucrose (w/v), and solidified with agar (0.7%, w/v). The media were adjusted to pH 5.7 by using hydrochloric acid (HCl) at 0.1 N and sodium hydroxide (NaOH) at 0.5 N before autoclaving (120 °C for 20 min). Cultures were maintained at 25 ± 2 °C with a 16 h photoperiod (40 µmol m -2 s -1 ). Almost 1 cm long shoots of in vitro cultivated P. khinjuk Stocks, were taken from axenic stock cultures and transferred into MS medium containing different NaCl concentrations (0, 50, 100, 200 mM) with a control group for a culture period (28 days). Leaf, root, and stem parts of seedlings obtained in vitro were isolated separately after four weeks to use in the extraction procedures.

Plant extraction
In vivo and in vitro, plant samples were dried in the dark at room temperature and powdered. The material was macerated with ethanol a few times, and then the solvent was evaporated, and the crude extract was obtained.

DPPH method
In DPPH method different concentrations (10, 25, 50, 100 µg/ml) of extracts prepared. 4 ml of 0.004% DPPH/ethanol solution and 1 ml of extract was mixed in the test tube and were kept at room temperature for 30 min. After the incubation period, the absorbance (A) was measured at 517 nm. The inhibition % (I%) was calculated by the following equation: Inhibition % = [Acontrol-Asample/Acontrol] × 100

ABTS method
In ABTS method, different concentrations (10, 25, 50, 100 µg/ml) of extracts prepared. 7 mM ABTS solution was adjusted to 0.7 absorbances at 734 nm. 4 ml of ABTS solution and 1 ml of the extract have mixed in the test tube and were kept at room temperature for 30 min. After the incubation period, the absorbance (A) was measured at 734 nm. The inhibition (I%) was calculated by the following equation: Inhibition % = [Acontrol-Asample/Acontrol] × 100

CUPRAC method
In CUPRAC method, different concentrations (10, 25, 50, 100 µg/ml) of extracts prepared. 1 ml of CuCl2 solution, 1 ml of neocuproine solution and 1 ml of NH4CH3CO2 solution were mixed in the test tube. 1 ml of extract was added to tube content. After the 60 minutes of the incubation period, the absorbance was measured at 450 nm. The absorbance values of the samples were evaluated against the control. The increased absorbance value indicates increased activity.
The total phenolic and flavonoid content The total phenolic and flavonoid content of the extracts was determined using the Folin-Ciocalteu reagent (FCR) (Slinkard and Singleton, 1977) and by the aluminium nitrate method (Moreno et al., 2000), respectively. Total phenolic and flavonoid contents in the crude extracts expressing as Gallic acid and quercetin equivalents. 4.6 ml of different concentrations of gallic acid was incubated with 0.1 ml FCR for 3 min. 0.3 ml of 2% Na2CO3 solution was added to a test tube and incubated for 2 hours. After then the absorbance was measured at 760 nm. A standard curve was plotted, and the amount of total phenolic content was calculated according to the following equations: Absorbance = 0.0356 gallic acid (μg) -0.0047 (R2 = 0.9970) 4.8 ml of different concentrations of quercetin was incubated with 0.1 ml of 1 M CH3CO2K for 60 min. 0.1 ml of 10% Al (NO3)3 solution was added to the test tube and incubated for 40 min. After then the absorbance was measured at 415 nm. A standard curve was plotted, and the amount of total phenolic content was calculated according to the following equations:

Antimicrobial activity
The antimicrobial activity of the extracts was evaluated by the disc diffusion method (NCCLS, 1997) and minimum inhibitory concentrations (MIC) (NCCLS, 2009). Five microorganisms (Escherichia coli ATCC25922, Pseudomonas aeroginosa ATCC27853, Staphylococcus aureus ATCC25923, Streptococcus pyogenes ATCC19615 and Candida albicans ATCC10231) were used for antimicrobial assays. Ampicillin and nystatin were used as positive controls. The nutrient broth medium was inoculated with each microorganism and incubated for 12-16 hours at 37 °C. 100 µl of the test microorganisms prepared overnight culture was spread into plates containing nutrient agar. Then 15 µl of 100 mg/ml concentrations of all extracts were prepared and impregnated on sterile paper discs placed in plates. Inhibition zone diameters were measured after 24 hours' incubation at 37 °C for bacteria and 48 hours' incubation at 30 °C for yeast. The active extracts were then processed to determine the MIC value. 100 µl serial dilutions of extracts, 90 µl broth, and 10 µl microorganism overnight cultures (turbidity equal to 0.5 McFarland) were pipetted into 96 well sterile plates. After 24 hours incubation at the appropriate temperature, the wells were evaluated. 100 µl was taken from the lowest concentration well without visible growth and spread on solid medium. After incubation at the appropriate temperature and time mentioned above, MIC values were determined according to the number of colonies on the plate.

Statistical analysis
All the experiments were conducted using a completely randomized block design, and they were performed in triplicate. Statistical analysis was performed using SPSS 25.0 Statistical program. One-way ANOVA was used for the analysis and mean values were compared by employing Tukey's test at p ≤ 0.05 probability level.

Results
The modified in vitro micropropagation protocol developed by Tilkat et al. (2005) was applied to P. khinjuk Stocks seeds to initiate shoot cultures, and successful results were obtained ( Figure 2). In general, it was seen that healthy shoot growth was obtained from seeds belonging to the control group, whereas seeds elicited by different salt concentrations show weaker shoot growth. The observations indicate that the shoot growth of seeds was reduced by salt application (50, 100, and 200 mM). Parallel to increasing salt concentrations, stem number, and root-stem-leaf dry weights were decreased; especially chlorosis was detected in shoots. Antioxidant and antimicrobial activity result of in vitro seedlings, which are elicited in 3 different salt concentrations and in vivo leaves, roots, and stems of male and female P. khinjuk Stocks were separately investigated and presented in tables below.
We observed a strong positive correlation between TPC in the plant and its antioxidant activity, which suggests that phenolic compounds significantly contribute to this antioxidant activity.
Assessment of DPPH activity, generally in vivo male and female samples were showed higher activity than in vitro elicited seedling explants. Applied minimum salt concentration to in vitro male root was showed higher activity (56.80 ± 1.50/10 µg) compare from other genotype samples. Between in vitro elicited seedling samples was observed highest activity in 50 mM NaCl leaf sample (39.67 ± 0.51/10 µg) (Table 1). When evaluated in terms of antioxidant activity in the root part, there is a significant increase compared to the control together with salt concentration increases. In vivo samples showed very high activity in the ABTS test system, which is far above both in vivo samples and positive controls. In both female and male plants (Table 2.), in vivo derived stem extracts showed relatively low activity compared to leaves and roots. Most of the in vitro samples were exhibited activity close to positive controls. Besides this, in vitro root extract has a deficient activity compared to stem and leaf extracts (Table 2.) In terms of in vitro plants compared to control, the highest values stand out as 99.26 ± 0.13 (control 55.88 ± 1.39) at 100 µg/ml 200 mM NaCl in root; 87.26 ± 1.07 (control 71.77 ± 0.65) at 10 µg/ml 50 mM NaCl in stem and 98.70 ± 0.07 (control 69.00 ± 0.73) at 10 µg/ml 50 mM NaCl in leaf.  Table 3 shows the results of antioxidant activity by the CUPRAC method of in vivo and in vitro samples as absorbance value. Increased absorbance refers to increased activity. Comparing in vivo and in vitro samples, we generally see that in vivo samples exhibit higher activity. Furthermore, in vivo samples showed higher activity than positive controls. In terms of in vitro plants compared to control, the highest values stand out as 1.189 ± 0.01 (control 0.143 ± 0.01) at 100 µg/ml 200mM NaCl in root; 3.117 ± 0.05 (control 1.199 ± 0.02) at 100 µg/ml 50 mM NaCl in stem and 1.706 ± 0.01 (control 1.029 ± 0.02) at 50 µg/ml 50 mM NaCl in leaf.
The activity order of the in vitro samples was found to be leaf> stem> root in parallel with DPPH method findings. As we have seen in Table 4, total phenolic and flavonoid content results of all samples are given as equivalent to gallic acid and quercetin, respectively. According to Table 4, in vivo samples showed higher phenolic content than in vitro samples, whereas all samples showed similar values in terms of flavonoids. The activity and total phenolic content order in the in vivo samples was found to be as root> leaf> stem; and in the in vitro samples, leaf> stem> root. Regarding the total phenolic and flavonoid content, our results showed significantly higher values in in vivo samples as compared to in vitro samples. In vitro samples were found to respond differently depending on the concentration of salt applied.
Increased activity and increased phenolic content were determined depending on increased salt concentration in root samples. This phenomenon is not obtained for stem and leaf samples. While the activity and phenolic content increased at 50 mM NaCl concentration compared to the control, then a decrease in activity was detected according to increasing salt concentration. Many studies are indicating the changes in phenolic compounds amount of plants under salt stress (Yuan et al., 2010;Falleh et al., 2012;Arzani and Ashraf, 2016;Golkar and Taghizadeh, 2018;Golkar et al., 2019).
To evaluate the results of the chemical analysis as a summary; in vivo plant extracts were determined to have higher chemical activity compared to extracts from different parts of plants germinated in MS medium with low or no salt content. However, it has been determined that when the amount of extract and salt concentration is increased, in vitro extracts may exhibit higher chemical activity than the control extracts.  Table 5. shows the antimicrobial activities of the in vivo and in vitro plant extracts. Inactive samples are not included in the table. The in vivo plant extracts found to be active in terms of antimicrobial effect, especially, female parts are more active than male parts. The inhibition zone diameters of in vivo samples range from 8 mm to 16mm, and the MIC values from 100 µg/ml to 800 µg/ml. The best activity was 16 mm inhibition zone diameter and 100 µg/ml MIC value recorded by female leaf extract against C. albicans and by male root extract against P. aeruginosa.
We could not see the same activity in in vitro samples. Most of the in vitro samples did not exhibit any activity. However, 100 mM NaCl leaf extract exhibited moderate antimicrobial activity (inhibition zone diameter between 12-20 mm) against S. aureus (17 ± 0.6 zone diameter, 50 ± 4.0 MIC value) and C. albicans (15 ± 0.0 zone diameter, 100 ± 5.0 MIC value). While antimicrobial activity was not observed in root control, 100 and 200 mM NaCl applications induced the activity against E. coli and P. aeruginosa. Similarly, we see that 50, 100 and 200 mM NaCl applications on the leaf increase the antimicrobial activity compared to control.

Discussion
Under stress conditions, including salinity, the speed of reactive oxygen species (ROS) will increase, and the resulting stress can cause harmful oxidation of various plant components. To prevent oxidation, ROS concentration in the plant cells is kept in check by several scavenging antioxidant compounds (Apel and Hirt, 2004).
Various adaptation tolls have been developed by higher plants for extreme conditions through cascade biosynthesis for both enzymatic and non-enzymatic antioxidants to protect themselves against ROS production by detoxification systems under salinity stress (Arzani and Ashraf, 2016;Golkar and Taghizadeh, 2018). Phenolic compounds are among such antioxidants, and several studies have reported changes in their concentrations in plants upon salinity stress (Yuan et al., 2010;Falleh et al., 2012;Golkar and Taghizadeh, 2018;Golkar et al., 2019). This change was observed as both increasing (Ksouri et al., 2007) and decreasing depending on the stress levels (Navarro et al., 2006). Besides, it has been reported that these phenolic components are in different concentrations in different parts of the same plant, with their increases positively correlated with increases in antioxidant activity (Hatamnia et al., 2016;Golkar and Taghizadeh, 2018;Golkar et al., 2019). The results of these previous studies are comparable to those obtained in our current study.
Previous research has indicated that plants react to salinity by increasing their antioxidant activity potentials (Hernandez and Almansa, 2002;Falleh et al., 2012;Javed and Gürel, 2019) through the metabolites commonly found in them that demonstrate antioxidant activity and are effective defence mechanisms against oxidative stress from free radicals (Sacchetti et al., 2005). Moreover, the increases in these metabolites also enhance tolerance levels in plants against oxidative stress coming from increases in salinity (Ksouri et al., 2007).
Several different methods were developed to measure the antioxidant capacity, which has a different substrate, function, polarity, source, and mechanism. Besides, the extraction method, parts of the plant, age of the plant, and the plant's environmental conditions are important factors for measuring antioxidant activity (Brewer, 2011;Esmaeili et al., 2015). In this study, three methods by which to measure antioxidant activity in plants 2,2-azino-bis (ethylbenzene-thiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and cupric ion reducing antioxidant capacity (CUPRAC) were used, the best results of which were obtained from the ABTS measurements.
Pistacia species contains several different bioactive compounds, such as triterpenes, essential oils, and phenolic compounds, and different ratios of these compounds are found in different parts of the plant, such as its resin, stem, leaves, and fruit (Kaliora et al., 2004;Assimopoulou, 2005;Tilkat et al., 2018); consequently, we attributed the differences in antioxidant activities determined from our results to these concentration differences. The overall evaluation of our results indicates that plant samples in vivo demonstrated higher antioxidant activity and phenolic ingredients than their counterparts in vitro. Esmaeili et al. (2015) have examined the total phenolic content (TPC), flavonoid content (TFC), and antioxidant activity in vitro, in vivo, and callus tissue from Trifolium pratense. All TPC, TFC, and antioxidant activities were in the order of in vitro > callus > in vivo. Parsaeimehr et al. (2010) have demonstrated results comparable to those in our in vitro and in vivo studies. Similarly, we have deduced that in vivo samples have higher potential than in vitro samples regarding the total phenolic and flavonoid content, antioxidant and antimicrobial activity.
In the literature, the studies comparing mostly between wild plants and plants grown in an in vitro culture medium are encountered. However, there is no study comparing antioxidant and antimicrobial activity between wild plants and plants treated with salt in vitro up to now. So, this is the first study to be performed in this sense.
Variation in the amount and diversity of secondary plant metabolites under abiotic stress has been reported by several studies (Navarro et al., 2006;Valifard et al., 2014). The antimicrobial activity most probably affiliated to the chemical structure as well as the amount of the most abundant compounds and interaction of major and minor active compounds (Dorman and Deans, 2000;Delaquis et al., 2002;Mazari et al., 2010).
Salinity could affect the antimicrobial activity as it has significant effects on the quality and quantity of the different composition of secondary metabolites. According to our results, the antimicrobial activity exhibited diversity with organs type, and salt stress level and these results may be explained by the possible variations of the chemical structure of P. khinjuk plant grown under NaCl stress. In many studies comparing antimicrobial activities of in vitro and in vivo samples, different results have been obtained indicating that activity increases, decreases or remains constant (Taran et al., 2010;Cuce et al., 2017;Ahmed et al., 2018;Taghizadeh et al., 2018). Contrary to our results, Salem et al. (2014) reported that salinity reduces antimicrobial activity in Carthamus tinctorius plant. When we compare in vivo and in vitro control samples; it is clear that the in vivo samples are more active, while the activity appears to be increased up to 100 mM NaCl when comparing salt-eluted samples with in vitro control samples. We consider that the activity has decreased as a result of damage in the physiological mechanism by the in vitro elicitation. Briefly, in terms of antimicrobial activity, female plant extracts are more active than all other tested extracts.
As a result, although increased salinity values significantly reduced antimicrobial activity, it is determined that 100 mM NaCl applications to in vitro leaf extracts exhibited moderate antimicrobial activity against S. aureus and C. albicans.

Conclusions
Our study aimed to reveal the antioxidant and antimicrobial activities of the different plant parts (root, stem and leaf) of the Pistacia khinjuk Stocks plant both in vitro and in vivo, and to determine whether these activities increase or not, especially with salt elicitations.
As a result of our study, it was determined that the antioxidant and antimicrobial activities of all in vivo samples had close or higher activity than the standard BHT, BHA, AA, and in vivo samples had higher activity in terms of both parameters compared to in vitro samples. Changes in activities were found in positive correlation with the increase or decrease in the total amount of phenolic and flavonoid substances. It is clear that salinity can change the metabolic profile of plants, and this metabolic profile change causes them to differ in their antimicrobial and antioxidant activities.

Authors' Contributions
EAT: In vitro propagation of the plant, NaCl elicitation to in vitro plants, experimental design and preparation of the manuscript. NH: Antimicrobial studies as well as preparation of the manuscript. İSK: Conducted antioxidant studies and preparation of the manuscript. VS: Conducted the statistical analysis and preparation of manuscript according to journal rules. All authors read and approved the final manuscript.