Industrial Crops & Products 148 (2020) 112278

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Industrial Crops & Products

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Establishment of cell suspension cultures of Ocimum basilicum L. and T

enhanced production of pharmaceutical active ingredients
Muhammed Akif Açıkgöz*
Ordu University, Department of Field Crops, Faculty of Agriculture, 52200, Ordu, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: Abiotic and biotic elicitors that activate the defense mechanisms of plants can be a good strategy for increasing
Abiotic and biotic elicitors bioactive compounds in plant tissue culture systems (cell, callus, hairy root, and organ cultures). The aim of this
Biologically active ingredients study was to determine the effects of cadmium chloride (CdCl2), silver nitrate (AgNO3) and yeast extract (YE)
Lamiaceae treatments applied at different concentrations to Ocimum bacilicum L. cell suspension cultures on the cell growth
Secondary metabolites
(cell dry weight and number), cell viability (%), total phenolic content (TPC) and flavonoid content (TFC) and
Sweet basil
pharmaceutical active ingredients. The TPCs and TFCs were determined by Folin-Ciocalteu and aluminum
chloride colorimetric assays, respectively. Antioxidant capacities were evaluated by 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) free radical assay. Polyphenols and terpenoids were identified by reversed phase-high-perfor-
mance liquid chromatography (RP-HPLC) and headspace solid-phase microextraction-gas-chromatography-
mass-spectrometry (HS-SPME–GC/MS), respectively. Antioxidant activity, TPCs, and TFCs were the highest in
the treatment of 200 mg/L YE among the tested elicitors. The analysis (RP-HPLC) showed that the highest
accumulation of chicoric acid (6.45 mg/g DW, 50 mg/L treatment) and rosmarinic acid (21.28 mg/g DW,
200 mg/L treatment) were obtained in yeast extract treatment which was 0.92 and 1.25 times greater than the
control. The optimum biosynthesis of rutin and isoquercetin was obtained in the treatment of 50 mg/L yeast
extract compared with the control culture, with an increase of 1.91 times (6.54 mg/g DW) and 1.86 times
(3.72 mg/g DW), respectively. The highest values of linalool and estragole compared with the control culture
were obtained from AgNO3 as 4.37 μg/g DW (25 μM treatment) and 3.30 μg/g DW (5 μM treatment), respec-
tively. These results show that CdCl2, AgNO3 and YE treatments may be a good strategy for increasing phar-
maceutical active ingredients in O. basilicum cell suspension cultures.

1. Introduction anti-ulcerogenic, cardiac stimulant (Bilal et al., 2012), antioxidant,

The genus Ocimum from the family Lamiaceae, the annual and
perennial herbs and shrubs, has close to 200 species (Joseph and
Vrundha, 2013). Ocimum species are called “king of herbs” because of
the breadth of their use in folk medicine, perfumery, pharmacy and
food industries (Brintnall and Molly, 1986). One of these species, sweet
basil (Ocimum basilicum L.), is an extremely valuable annual herb with
green, white or purple leaves, and their cultivars are grown commer-
cially in many countries. The leaves and flowering tops of the plant are
used fresh or dried in the food industry, confectionery products and
beverages as a flavoring agent (Makri and Kintzios, 2007), as well as in
traditional medicine as an antispasmodic, aromatic, carminative, di-
gestive, galactagogue, stomachic and tonic agent (Marwat et al., 2011).
In addition to all of these features, the sweet basil has been reported to
have the potentials as an antiseptic (Kosekia et al., 2002), analgesic,
antimicrobial (Koroch et al., 2017), antifungal (Piras et al., 2018), anti-
inflammatory (Złotek et al., 2016; Aye et al., 2019) and antituberculosis
(Siddiqui et al., 2012).
There are various pharmaceutical active ingredients including al-
kaloids, flavonoids, phenolics, and terpenoids in chemical structure of
O. basilicum. Therefore, it is so important for the food, cosmetic and
pharmaceutical industries. In general, estragole (methyl chavicol) and
linalool (Piras et al., 2018; Talebi et al., 2018; Alkuwayti et al., 2019) in
terpenoids, also, rosmarinic acid, chicoric acid, rutin and isoquercetin
(Lee and Scagel, 2009; Nguyen et al., 2010; Kwee and Niemeyer, 2011;
Vlase et al., 2014) in phenolic compounds have been reported to be the
dominant pharmaceutical active ingredients in basil. However, these
bioactive compounds have also been reported to vary depending on
genetics (Kwee and Niemeyer, 2011), environmental factors (Ahmed
et al., 2019), climate (Baldim et al., 2018), season (Hussain et al.,

⁎
Corresponding author.
E-mail address: makifacikgoz@gmail.com.

https://doi.org/10.1016/j.indcrop.2020.112278
Received 24 October 2019; Received in revised form 22 January 2020; Accepted 23 February 2020
0926-6690/ © 2020 Elsevier B.V. All rights reserved.
M.A. Açıkgöz
2008), growth and development periods (Verma et al., 2012), plant
parts (Chalchat and Özcan, 2008) pre- and post-harvest cultural pro-
cesses (Złotek et al., 2016; Talebi et al., 2018; Alkuwayti et al., 2019)
and extraction methods (Coelho et al., 2018).
All these factors make the production of bioactive compounds and
plants by classical methods both difficult and costly. In addition, being
homogeneity and high purity of these compounds are extremely im-
portant for the cosmetic, pharmaceutical and food industries (Rao and
Ravishankar, 2002). Therefore, plant tissue culture systems (cell, callus,
hairy root, and organ cultures) areare used very often in production of
bioactive compounds for reasons such as being reliable and predictable,
independent of geographical, seasonal and environmental factors,
modifying or eliminating unwanted tastes and smells and obtaining
products of certain quality and standards (Karuppusamy, 2009; El-
Salam et al., 2015). There is no doubt that the best strategy for in-
creasing bioactive compounds in plant tissue culture systems is biotic
and abiotic elicitors (Theis and Lerdau, 2003). Biotic elicitors are
polysaccharides (chitin, pectin, etc.) (Mathew and Sankar, 2014;
Açıkgöz, 2017; Krishnan and Siril, 2018) and micro-organisms (yeast
extract, bacteria, etc.) (Mathew and Sankar, 2012; Açıkgöz, 2017;
Gupta et al., 2018), however, abiotic elicitors are physical, chemical,
and hormonal factors (drought stress, heat stress, salinity stress, os-
motic stress, light, silver nitrate, cadmium chloride, salicylic acid, jas-
monate, etc.) (Akula and Ravishankar, 2011; Mathew and Sankar,
2012; Liang et al., 2013; Nadeem et al., 2019; Açıkgöz et al., 2019).
In studies conducted using plant tissue culture systems in O. basi-
licum, a large number of bioactive compounds have been reported to be
successfully increased through elicitors, including berberine (El-Salam
et al., 2015), betulinic acid, ursolic acid and oleanolic acid (Pandey
et al., 2015; Nazir et al., 2019; Pandey et al., 2019), 1,8-cineole (Duran
et al., 2019) rosmarinic acid, caffeic acid, chicoric acid and eugenol
(Hakkim et al., 2007; Pandey et al., 2015; Nadeem et al., 2019; Nazir
et al., 2019), cyanidin and peonidin (Nazir et al., 2019). In addition,
some researchers have reported that cell suspension culture within
plant tissue culture systems is more effective in increasing bioactive
compounds than callus and other cultures due to its advantages such as
easy applicability, rapid response to elicitors and rapid division of cells
(Phillips et al., 1995; Szabo et al., 1999; Kintzios et al., 2003; Georgiev
et al., 2007; Mathew and Sankar, 2011, 2014).
When recent studies on increasing bioactive compounds in plant
tissue culture systems in O. basilicum are examined in detail, it is seen
that researchers are more focused on methyl jasmonate, chitosan, sal-
icylic acid and hormone (GA3, 2,4-D, NAA and TDZ) as an elicitor,
callus and hairy root cultures (Hakkim et al., 2007; Pandey et al., 2015;
Duran et al., 2019; Nadeem et al., 2019; Nazir et al., 2019). However, as
far as we know, there is only one study conducted by Guirgis et al.
(2007) on the application of yeast extract elicitor in callus culture in O.
basilicum. In studies conducted in cell suspension culture in O. basilicum,
the treatments of elicitor are seen to be limited to mechanical stress,
methyl jasmonate and chitosan (Strazzer et al., 2011; Mathew and
Sankar, 2012, 2014; Pandey et al., 2019), and there are no studies of
cadmium chloride, silver nitrate and yeast extract elicitors, which have
been successfully used in cell suspension culture of other species up to
now.
Therefore, the purpose of the current research was to investigate the
effects of cadmium chloride, silver nitrate and yeast extract elicitors on
the accumulation of pharmaceutical active ingredients using cell sus-
pension cultures in O. basilicum and determine effective elicitor con-
centrations.
2. Materials and methods
2.1. Seed collection, germination and obtaining aseptic plantlets
O. basilicum seeds were obtained from the the Department of Field
Crops, Faculty of Agriculture of Gaziosmanpaşa University, Tokat,
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Industrial Crops & Products 148 (2020) 112278
Turkey. Ocimum basilicum seeds were used to obtain aseptic seedlings.
The seeds were sterilized 30 min in 1.95 % sodium hypochloride solu-
tion and after rinsing with sterile distilled water, the pH was adjusted to
5.6–5.8 and added to Murashige and Skoog (MS) medium with 1.0 mg/
L GA3, 3% sucrose and 0.8 % agar. Magenta plates were kept in
25 ± 2 °C for 16/8 h light/dark conditions in the climate chamber.
2.2. Establishment callus and cell suspension culture
Aseptic seedlings (15−18 cm, 8 weeks after incubation) cut leaves
(approximately 1 cm2) were used as an explant source for the formation
of callus cultures. Leaf explants were planted in MS growth medium
with 2.5 mg/L NAA (naphthalene-acetic acid) +0.5 mg/L KIN (kinetin),
3% sucrose and 0.8 % agar, kept in climate room at 25 ± 2 °C for 16/
8 h light/dark conditions. The calluses, which were taken to subcultures
twice with three weeks interval (the same hormone combination and
growth medium), were placed in 250 mL of conical flasks containing
100 mL of liquid growth medium in the sterile cabin six weeks later.
Samples were kept in conditions of 25 ± 2 °C for 16/8 h (light/dark)
over 4 weeks (taken to sub-culture at every 2 weeks) on the shaker with
an average speed of 105 rpm. Fresh cells were then weighed to be 2.0 g
in the sterile cabinet and placed in MS nutrient media containing
2.5 mg/L NAA +0.5 mg/L KIN, 3% sucrose. Then, abiotic (cadmium
chloride and silver nitrate) and biotic (yeast extract) elicitors, which
activate the cells' defense systems, were applied in different con-
centrations. Then, cell suspension cultures were kept in 16/8 h light/
dark conditions at 25 ± 2 °C in the climate chamber on the shaker with
an average speed of 105 rpm.
2.3. Elicitor treatments
After preparing stock solutions for cadmium chloride (CdCl2) and
silver nitrate (AgNO3) elicitors, CdCl2 and AgNO3 were adjusted in 4
different concentrations (5, 25, 50 and 100 μM). Stock solution for the
yeast extract (YE) elicitor was prepared according to Hahn and
Albersheim (1978). In short, the yeast extract was dissolved in 20 g/
100 mL distilled water, mixed with 400 mL of ethanol and kept in the
refrigerator for 4 days (4 °C). Then the remaining precipitate was dis-
solved again in 100 mL distilled water and mixed with 400 mL ethanol.
The adhesive precipitate was dissolved in 50 mL distilled water and
adjusted in 5 different concentrations (5, 25, 50, 100 and 200 mg/L).
Before to the cell suspension cultures was added, the pH of the elicitor
solutions was adjusted to 5.8 and filter sterilization (0.2 μm, a micro-
filter) was performed. From each elicitor application, a total of 5
samples were taken at the end of the second, fourth, sixth, eighth, and
tenth day and kept at −20 °C until extraction. In addition, 5 mL of each
were allocated for the determination of cell count and cell dry weight.
2.4. Sample extraction
The extraction process of samples was done using an ultrasonic bath
(ULT AG 440, Germany) designed with 35 kHz fixed frequency and
power density (180 W). For each application, frozen cell samples were
weighed 100 mg and 10 mL of methanol was added. The solution was
then kept at 45 °C in the ultrasonic bath for 2 h and the solution was
centrifuged for 10 min at 8000 rpm 3-times at 30-min intervals. The
supernatants were collected and stored at 4 °C until to be necessary for
analysis.
2.4.1. Determination of the cell dry weight, cell number, and cell viability
Cell growth was determined by measuring the average number of
cells (n) and cell dry weight (g/L). Cell count was determined by the
following formula using the Nageotte counting circle according to
Moroff et al. (1994).
M.A. Açıkgöz
40
n= ∑ c·20 · df
i=1
n: average number of cells
c: number of cells counted in each rectangle
df: dilution factor
The cell dry weight was obtained by weighing the filtered cells after
they were kept in a drying oven (60 °C at 24 h). Cell viability was made
according to Laloue et al. (1980) using the technique of Trypan blue
staining. In short, 50 μL cell suspension culture, 125 μL Trypan blue and
75 μL phosphate buffer that keep the cells alive in the counting phase
and to increase staining efficiency were added to the Eppendorf tubes.
After vortexing, the counting process was made in the light microscope
and the cell viability rate was determined as a percentage (%).
2.4.2. Determination of total phenolic and total flavonoid content
Total phenolic content (TPC) was measured using the Folin-
Ciocalteu method as defined by Velioglu et al. (1998) with slight
modifications. Briefly, 20 μL of the sample was mixed with 90 μL of
Folin-Ciocalteu reagent and incubated for 60 min at room conditions
and the mixture was diluted with 90 μL of sodium carbonate and the
absorbance was measured at 725 nm. Gallic acid served as the standard
for plotting calibration curve, and the results were shown as mg gallic
acid equivalents (GAE)/g of DW.
Total flavonoids content (TFC) was evaluated by the protocol of
Fazal et al. (2016) with minor modifications. Briefly, 10 μL of AlCl3 and
potassium acetate was mixed with 20 μL of the sample. Then, each
sample was diluted with 160 μL of distilled water and incubated for
30 min. Spectrophotometrical readings were executed at 415 nm using
a microplate reader. Quercetin standard calibration curve was used for
TFC and the values were shown as mg quercetin equivalents (QE)/g of
DW.
2.4.3. DPPH (2,2-diphenyl-1-picrylhydrazyl) activity
Free radical-scavenging capacity was tested by the DPPH assay with
the help of the method of Amarowicz et al. (2004). Briefly, 10 mg of the
cell sample was dissolved in 4 mL of methanol then added to a me-
thanol solution of DPPH (1 mM, 0.5 mL). The mixture was vortexed and
kept in dark condition (60 min). Then the absorbance values of reaction
mixtures were measured at 517 nm using UV–vis spectrophotometer.
The radical-scavenging activities of the samples, expressed as percen-
tage scavenging capacity of DPPH, were calculated according to the
formula: % scavenging DPPH free radical = (CA−SA)/CA x 100, where
SA is the sample absorbance and CA is the control absorbance.
2.4.4. Reversed-phase high-performance liquid chromatography (RP-
HPLC) analysis
The phenolic compound's contents of the samples were determined
by Reversed-Phase High-Performance Liquid Chromatography (RP-
HPLC) analysis that was carried out using the Shimadzu Prominence
Modular LC20A system via C18 column 250 × 4.6 mm id, 5 μm. The
mobile phase includes acetonitrile and 1% aq. acetic acid 1: 9. The
column was controlled at 28 °C, the injection was adjusted to 20 μL. The
solution was prepared by changing the proportion of solvent B (acet-
onitrile) to solvent A (acetic acid). The solvent B proportion in the so-
lution was alteredchanged from 10 % to 40 % gradually during the first
28 min, from 40 % to 60 % up to 39 min, and from 60 % to 90 % up to
50 min. The mobile phase composition was changed compared with the
first condition in 55 min and kept for 10 min, the same procedure was
done for the other samples. The analysis time for each sample was
65 min and HPLC chromatograms were formed using photodiode array
UV detector at the wavelengths of 272, 280 and 310 nm according to
the absorption maxima of analyzed compounds. Each phenolic com-
pound was defined by the addition of standards under the same con-
ditions and by the comparison of retention times.
The amount of each phenolic compounds was fixed using 5 point
3
Industrial Crops & Products 148 (2020) 112278
calibration curves of each standards ranging from 6.25–400 μg/mL with
a correlation coefficient of at least 0.9998 (chicoric acid, y = 105846x
+ 102.38, r2 = 0.9998; rosmarinic acid, y = 365706x + 280.17, r2 =
0.9998; rutin, y = 520849x + 3642, r2 = 0.9998 and isoquercetin, y
= 124652x + 9862, r2 = 0.9998).
2.4.5. Headspace solid-phase microextraction-gas-chromatography-mass-
spectrometry (HS-SPME–GC/MS) analysis
The linalool and estragole content was measured with Headspace
Solid-phase Microextraction-Gas-Chromatography-Mass-Spectrometry
(HS-SPME–GC/MS) (Innovatech Labs, LLC, USA) using a Shimadzu
QP2010 Ultra integrated with a Shimadzu AOC-5000 plus autosampler
(Shimadzu Scientific Inst., USA). The column was an RTX-5MS capillary
column (30 m × 0.32 mm ID, 0.50 μm with Integra-guard column).
Helium was used as the carrier gas with a constant flow mode at a flow
rate of 1 mL/min. Injection temperature of 250 °C, injection volume of
0.5 mL, ionization voltage of 70 eV, the temperature of 100 °C, and
heating period of 10 min were used as standard. For the analysis, li-
nalool and estragole stock solution were prepared in ethyl acetate.
Then, 9 calibration points ranging from 0.75 to 100 μg/mL were pre-
pared from the stock standard solutions and n-tridecane (100 μg/mL).
These solutions were used to construct individual terpene calibration
curves, and by using these curves, the linalool and estragole content
were expressed as μg/g DW.
2.5. Statistical analysis
All of the experiments were performed in triplicates and the results
were presented as mean ± SD. The data were subjected to analysis of
variance (ANOVA) using statistical analysis system software (SPSS)
version 22.0. The significant differences were calculated using the least
significance difference (LSD) and the differences were considered sta-
tistically significant at P < 0.05.
3. Results and discussion
3.1. Cell dry weight, cell number, and cell viability
Effect of cadmium chloride (CdCl2), silver nitrate (AgNO3) and yeast
extract (YE) elicitor treatments on cell dry weight (g/L), cell number,
and cell viability (%) are shown in Figs. 1–3. The cell dry weight, cell
number, and cell viability decreased sharply due to the increased con-
centration levels in the overall elicitor treatments. In CdCl2 treatment at
the end of the tenth day, cell dry weight decreased from 7.89 g/L to
2.38 g/L, cell number from 83.000–32.450, and cell viability from
95.1%–61.4%. Cadmium chloride treatment was followed by AgNO3
treatment with cell dry weight of 2.38 g/L, cell count of 39.500 and cell
viability of 70.1 % at the end of the tenth day. YE was the treatment
with minimal decreases in cell dry weight, number, and viability. The
lowest cell dry weight, number, and viability of the three elicitors were
detected in the treatment of 100 μM CdCl2, which was harvested at the
end of tenth day as 2.38 g/L, 32.450 and 61.4 %, respectively.
It has been reported by many researchers that elicitor treatments are
very effective in cell growth and in the enhancement of bioactive
compounds. The age of cell culture (Namdeo, 2007; Kang et al., 2009),
the duration of exposure to elicitors and the type of elicitor play im-
portant roles in increasing the effectiveness of these treatments (Kubeš
et al., 2014; Nazir et al., 2019). In previous studies, some researchers
have reported that CdCl2, AgNO3 and YE treatments inhibit cell growth
similar to the results in this study (Zhao et al., 2010; Cai et al., 2013;
Sivanandhan et al., 2014; Zaker et al., 2015; Gonçalves et al., 2019).
However, some researchers have reported that appropriate concentra-
tions of AgNO3 and YE elicitors can be promoted in a positive way (Yan
et al., 2006; Deepthi and Satheeshkumar, 2016; Singh et al., 2017;
Nadeem et al., 2018; Roy and Bharadvaja, 2019). On the other hand,
some studies have shown that the concentration selected in elicitor
M.A. Açıkgöz Industrial Crops & Products 148 (2020) 112278

Fig. 1. The effect of abiotic and biotic elicitor treatments on cell dry weight (g/L). The result are expressed as means ± standard deviation (n = 3). Error bars show
standard deviation values.

Fig. 2. The effect of abiotic and biotic elicitor treatments on cell number. The result are expressed as means ± standard deviation (n = 3). Error bars show standard
deviation values.

Fig. 3. The effect of abiotic and biotic elicitor treatments on cell viability (%). The result are expressed as means ± standard deviation (n = 3).

(CdCl2, AgNO3, and YE) treatments are important in cell viability added to the hairy root culture in Salvia miltiorrhiza positively promote
(Ahmed and Baig, 2014), and elicitor treatments at high concentrations, growth and increase root dry weight. Furthermore, in the study of
as in this study, cause the death of cells (Deepthi and Satheeshkumar, Perovskia abrotanoides adventitious root cultures, Zaker et al. (2015)
2016). Yan et al. (2006) reported that the 15 μm Ag+ and 200 mg/L YE found that 25 and 50 μM of Ag+ and 50 and 100 mg/L of YE treatment

4
M.A. Açıkgöz Industrial Crops & Products 148 (2020) 112278

Fig. 4. The effect of abiotic and biotic elicitor treatments on total phenolic content (μg gallic acid equivalents/g dry weight). The result are expressed as means
± standard deviation (n = 3). Error bars show standard deviation values.

Fig. 5. The effect of abiotic and biotic elicitor treatments on total flavonoid content (μg quercitin equivalent/g dry weight). The result are expressed as means ±
standard deviation (n = 3). Error bars show standard deviation values.

Fig. 6. The effect of abiotic and biotic elicitor treatments on % scavenging 2, 2-diphenyl-1-picrylhydrazyl free radical. The result are expressed as means ± standard
deviation (n = 3). Error bars show standard deviation values.

5
M.A. Açıkgöz Industrial Crops & Products 148 (2020) 112278

Fig. 7. RP-HPLC chromatogram of mix standards. 1 = chicoric acid, 2 = rosmarinic acid, 3 = rutin, 4 = isoquercetin.

Table 1
Chicoric acid and rosmarinic acid recorded in Ocimum basilicum L. cell suspension cultures subjected to different cadmium chloride (CdCl2), silver nitrate (AgNO3)
and yeast extract concentrations for various days. The result are expressed as means ± standard deviation (n = 3). Mean followed by the same letter in the same
column do not differ statistically at P < 0.05 according to LSD test.
Chicoric acid (mg/g dry weight) Rosmarinic acid (mg/g dry weight)

Elicitors treatments

Concentrations day CdCl2 (μM) AgNO3 (μM) Yeast (mg/L) CdCl2 (μM) AgNO3 (μM) Yeast (mg/L)

0 2 3.35 ± 0.91e 3.35 ± 0.91d 3.35 ± 0.91ı 9.47 ± 0.11f 9.47 ± 0.11f 9.47 ± 0.11m
4 3.77 ± 0.10d 3.77 ± 0.10c 3.77 ± 0.10g 10.02 ± 0.78e 10.02 ± 0.78e 10.02 ± 0.78l
6 3.90 ± 0.07d 3.90 ± 0.07c 3.90 ± 0.07g 10.40 ± 2.44de 10.40 ± 2.44d 10.40 ± 2.44l
8 4.05 ± 0.35c 4.05 ± 0.35c 4.05 ± 0.35g 10.52 ± 0.56d 10.52 ± 0.56d 10.52 ± 0.56l
10 4.30 ± 0.56c 4.30 ± 0.56b 4.30 ± 0.56f 10.53 ± 1.02d 10.53 ± 1.02d 10.53 ± 1.02l
5 2 5.28 ± 0.20b 3.98 ± 0.14c 4.08 ± 0.85f 10.52 ± 0.40d 9.78 ± 0.11e 11.20 ± 0.70k
4 5.84 ± 0.18a 4.35 ± 0.19b 4.77 ± 0.33e 10.85 ± 0.30d 10.02 ± 0.78e 11.30 ± 0.64k
6 5.97 ± 0.12a 4.48 ± 0.57b 4.90 ± 1.09e 11.27 ± 0.08c 10.40 ± 0.44d 11.82 ± 1.28j
8 5.45 ± 0.03b 4.71 ± 3.40a 4.92 ± 0.35e 12.35 ± 1.74b 10.52 ± 0.56d 12.04 ± 0.72j
10 5.21 ± 0.85b 4.96 ± 0.44a 5.10 ± 0.18d 12.86 ± 2.36a 10.53 ± 1.02d 12.35 ± 0.80j
25 2 3.37 ± 0.08e 2.88 ± 1.20f 5.35 ± 0.85d 5.74 ± 0.18j 11.30 ± 0.37c 16.10 ± 1.08ı
4 3.40 ± 0.30e 2.45 ± 0.90g 5.45 ± 0.33d 5.41 ± 0.91j 12.00 ± 0.62b 16.42 ± 1.22ı
6 3.45 ± 0.56e 2.41 ± 0.25g 5.70 ± 0.09c 4.27 ± 0.80l 12.38 ± 0.35b 17.31 ± 0.65h
8 3.32 ± 0.43e 2.42 ± 0.30g 5.92 ± 0.35c 4.00 ± 2.24l 13.20 ± 0.88a 17.30 ± 0.13h
10 3.08 ± 0.15e 2.48 ± 0.28g 6.08 ± 0.18c 3.95 ± 1.08l 13.40 ± 1.30a 17.56 ± 0.32h
50 2 2.35 ± 0.08f 3.02 ± 0.62e 6.45 ± 1.20b 8.33 ± 1.58g 7.25 ± 0.50g 18.24 ± 0.40g
4 2.00 ± 0.10f 3.30 ± 0.75d 6.51 ± 0.90b 8.18 ± 0.42g 6.46 ± 0.56h 19.05 ± 0.41f
6 1.75 ± 0.09g 3.38 ± 0.52d 6.67 ± 0.25a 8.42 ± 0.09g 6.10 ± 0.08h 19.76 ± 1.13e
8 1.42 ± 0.40h 3.12 ± 0.36e 6.83 ± 0.30a 7.95 ± 0.66h 6.22 ± 1.47h 20.11 ± 0.96d
10 1.15 ± 0.51h 3.00 ± 0.03e 6.90 ± 0.28a 7.00 ± 1.64ı 6.53 ± 1.83h 20.08 ± 0.43d
100 2 0.80 ± 0.70ı 2.56 ± 0.72g 4.43 ± 0.27f 4.75 ± 2.01k 6.29 ± 0.38h 20.26 ± 0.13d
4 0.77 ± 0.18ı 2.25 ± 0.09h 4.85 ± 0.85e 3.10 ± 0.57m 6.10 ± 2.15h 20.58 ± 0.32c
6 0.90 ± 0.02ı 1.84 ± 0.48ı 4.98 ± 0.25e 2.36 ± 0.70n 5.84 ± 0.45ı 20.71 ± 0.40c
8 0.91 ± 0.02ı 1.56 ± 0.35ı 5.30 ± 0.75d 2.25 ± 0.43n 5.68 ± 0.96ı 20.90 ± 0.58c
10 0.85 ± 0.03ı 1.42 ± 0.05ı 5.39 ± 1.44d 2.04 ± 0.28n 5.43 ± 1.05ı 20.98 ± 1.02c
200 2 4.85 ± 0.05e 21.28 ± 0.64b
4 4.52 ± 0.10f 21.57 ± 0.09b
6 4.34 ± 0.38f 22.10 ± 2.08a
8 4.00 ± 0.49g 22.30 ± 1.65a
10 3.90 ± 0.42g 22.53 ± 1.40a

inhibited root growth, while 5 μM of Ag+ and 200 mg/L of YE treat-
ment increased root growth.
3.2. Total phenolic and flavonoid content
Total phenolic and flavonoid contents of samples obtained from cell
cultures treated with elicitor are shown in Figs. 4 and 5. Accordingly,
the total phenolic content in CdCl2 treatment varies between
14.12–18.75 (μgGAE/g DW) and the total flavonoid content varies
between 1.32–2.92 (μgQE/g DW). In AgNO3 and YE treatments, the
total phenolic and flavonoid contents are 13.10–17.51 (μgGAE/g DW)
and 1.32–3.00 (μgQE/g DW), 14.03–21.77 (μgGAE/g DW) and
1.32–3.71 (μgQE g/DW). The highest total phenolic content among
elicitor treatments was determined as 21.77 (μgGAE/g DW) in the
treatment of 200 m/L YE harvested at the end of the tenth day; the
lowest total phenolic content is 13.10 (μgGAE/g DW) in the treatment
of 100 μM AgNO3 harvested at the end of the second day; the highest
total flavonoid content is 3.71 (μgQE/g DW) in the treatment of 200 m/
L YE harvested at the end of the tenth day; the lowest was also detected
in the control culture without elicitor treatment harvested at the end of
the second day.
In this study, it was found that abiotic (CdCl2 and AgNO3) and biotic
(YE) elicitor treatments may increase the total amount of phenolic and
flavonoids. Many previous studies confirm the results obtained. In these
studies, it has been reported that the use of CdCl2 and AgNO3 in low
concentrations and YE at high concentrations may increase the total

6
M.A. Açıkgöz Industrial Crops & Products 148 (2020) 112278

Table 2
Rutin and isoquercetin recorded in Ocimum basilicum L. cell suspension cultures subjected to different cadmium chloride (CdCl2), silver nitrate (AgNO3) and yeast
extract concentrations for various days. The result are expressed as means ± standard deviation (n = 3). Mean followed by the same letter in the same column do
not differ statistically at P < 0.05 according to LSD test.
Rutin (mg/g dry weight) Isoquercetin (mg/g dry weight)

Elicitors treatments

Concentrations day CdCl2 (μM) AgNO3 (μM) Yeast (mg/L) CdCl2 (μM) AgNO3 (μM) Yeast (mg/L)

h j m ı h
0 2 2.25 ± 0.05 2.25 ± 0.05 2.25 ± 0.05 1.30 ± 0.03 1.30 ± 0.03 1.30 ± 0.03k
4 2.59 ± 0.11g 2.59 ± 0.11h 2.59 ± 0.11l 1.58 ± 0.10h 1.58 ± 0.10f 1.58 ± 0.10j
6 2.95 ± 0.16f 2.95 ± 0.16g 2.95 ± 0.16k 1.72 ± 0.40h 1.72 ± 0.40e 1.72 ± 0.40ı
8 3.16 ± 0.45f 3.16 ± 0.45f 3.16 ± 0.45j 1.85 ± 0.15g 1.85 ± 0.15d 1.85 ± 0.15ı
10 3.38 ± 0.42e 3.38 ± 0.42e 3.38 ± 0.42ı 1.92 ± 0.18g 1.92 ± 0.18d 1.92 ± 0.18h
5 2 3.20 ± 0.32e 3.06 ± 0.05f 4.89 ± 0.19g 2.10 ± 0.39f 2.74 ± 0.83c 1.70 ± 0.60ı
4 3.68 ± 0.45d 3.30 ± 0.56e 5.23 ± 0.30f 2.18 ± 0.30f 3.46 ± 0.21b 1.84 ± 0.27ı
6 3.62 ± 0.10d 3.32 ± 0.01e 5.30 ± 0.60f 2.30 ± 0.13e 3.78 ± 0.74a 2.03 ± 0.40h
8 3.50 ± 0.08d 3.15 ± 0.17f 5.25 ± 0.85f 2.24 ± 0.15e 3.75 ± 0.19a 1.87 ± 1.46ı
10 3.48 ± 0.52d 3.26 ± 0.25e 5.18 ± 0.27f 2.36 ± 0.46e 3.77 ± 0.68a 1.52 ± 0.81j
25 2 4.72 ± 0.63c 4.52 ± 0.74c 5.88 ± 0.05e 3.20 ± 0.03d 1.74 ± 0.43e 2.75 ± 0.34f
4 4.96 ± 0.15b 4.79 ± 0.61b 6.30 ± 0.19c 3.57 ± 0.90c 1.46 ± 0.22g 2.88 ± 0.12e
6 5.02 ± 0.28b 4.98 ± 0.35b 6.72 ± 0.63a 3.80 ± 0.49b 1.45 ± 0.57g 3.03 ± 0.72d
8 5.18 ± 0.21a 5.22 ± 0.10a 6.59 ± 0.40b 3.98 ± 0.05a 1.38 ± 0.63g 2.92 ± 0.10e
10 5.30 ± 0.48a 5.10 ± 0.92a 6.85 ± 0.23a 3.79 ± 0.17b 1.40 ± 0.75g 2.98 ± 0.09d
50 2 2.20 ± 0.13h 3.77 ± 0.90d 6.54 ± 0.10b 0.98 ± 0.92j 1.30 ± 0.91h 3.72 ± 0.41a
4 2.22 ± 0.20h 3.31 ± 0.74e 6.45 ± 0.09b 0.80 ± 0.66k 1.17 ± 0.70h 3.60 ± 0.40a
6 2.40 ± 0.57g 3.20 ± 0.09e 6.50 ± 0.46b 0.82 ± 0.41k 1.10 ± 0.16ı 3.47 ± 0.35b
8 2.18 ± 0.50h 3.02 ± 0.13f 6.40 ± 0.30c 0.78 ± 0.30k 1.19 ± 0.25h 3.58 ± 0.14a
10 2.20 ± 0.27h 3.08 ± 0.18f 6.69 ± 0.32a 0.85 ± 0.18k 1.20 ± 0.20h 3.52 ± 0.20b
100 2 1.86 ± 0.88ı 2.65 ± 0.42h 6.10 ± 0.58d 0.32 ± 0.43l 0.94 ± 0.05j 2.91 ± 0.10e
4 1.82 ± 0.20ı 2.41 ± 0.30ı 5.88 ± 0.50e 0.27 ± 0.15l 0.95 ± 0.19j 2.79 ± 0.53f
6 1.75 ± 0.05ı 2.46 ± 0.03h 6.00 ± 0.09d 0.28 ± 0.03l 0.74 ± 0.74k 2.95 ± 0.74e
8 1.80 ± 0.05ı 2.30 ± 0.41ı 6.43 ± 0.11b 0.25 ± 0.02l 0.86 ± 0.26j 2.84 ± 0.02f
10 1.58 ± 0.62j 2.38 ± 0.70ı 6.35 ± 0.85c 0.27 ± 0.10l 0.85 ± 0.08j 2.52 ± 0.08g
200 2 4.86 ± 0.40h 3.13 ± 0.10d
4 5.02 ± 0.30g 3.32 ± 0.23c
6 5.19 ± 0.15f 3.40 ± 0.16b
8 4.97 ± 0.03g 3.49 ± 0.09b
10 5.01 ± 0.67g 3.46 ± 0.30b

amount of phenolics and flavonoids as in this study (Cai et al., 2013;
Dowom et al., 2017; Nadeem et al., 2018). Cells can react to some
elicitor treatments at low concentration and in a short time, while may
react to other elicitor treatments with high concentration and over a
longer period of time. In the study of Thymus lottocephalus shoot cul-
tures, Gonçalves et al. (2019) reported that 50 μM AgNO3 and 500 mg/
L YE treatments increased the total phenolic content by 12.5 % and 23
%, and the total flavonoid content by 3.87 % and 11.9 % respectively at
the end of 6 weeks, while another study found that for increasing the
total phenolic content, the Ag+ treatment was more effective at fourth
day after administration and at a lower concentration (15 μM) (Yan
et al., 2006).
3.3. DPPH (2, 2-diphenyl-1-picrylhydrazyl) activity
Evaluating the antioxidant potential of elicitor treated cell cultures
is important in determining the therapeutic potential of these cells.
DPPH assays, which are commonly used to evaluate the antioxidant
potential of cell cultures, were therefore performed, the results are
shown in Fig. 6. There were increases in radical scavenging capacity
due to increased concentration and elicitor exposure time of cells in the
treatment of YE, while the concentration of 200 mg/L was the most
effective treatment. Among elicitor treatments, the strongest radical
scavenging capacity (90.12 %) was observed in the treatment of
200 mg/L YE (at the end of the day 10), the weakest radical scavenging
capacity (78.5 %) was also found in cells without elicitor treatment (at
the end of the day 2). The highest increase in radical scavenging ca-
pacity was 11.4 % in the cells harvested at the end of the second day
(200 mg/L, YE treatment). The highest increases in radical scavenging
capacity in CdCl2 and AgNO3 treatments were observed at the
concentrations of 5 μM and 25 μM, respectively (Fig. 6.). Furthermore,
parallelism between TPC, TFC and radical scavenging capacity was
found to be in line with previous studies (Nadeem et al., 2018;
Gonçalves et al., 2019). These differences of antioxidant activation seen
in cell suspension cultures have been shown in previous studies to be
related to the type and concentration of phenolic acids and flavonoids
in cells (Sabir et al., 2012; Xu et al., 2016). Therefore, these differences
between antioxidant capacities can be explained by differences in the
phenolic acids and flavonoids content that occur during elicitor treat-
ment (Krishnan et al., 2015; Sarkate et al., 2017).
In addition, previous research has shown that the duration of ap-
plication of CdCl2, AgNO3 and YE elicitor treatments and elicitor levels
may be effective in the change of TPC, TFC, and radical scavenging
capacity. Gonçalves et al. (2019) studied on Thymus lottocephalus shoot
cultures and reported that 50 μM AgNO3 and 500 mg/L YE treatments
significantly increased radical scavenging capacity after 6 weeks com-
pared with the control culture, however Cai et al. (2013) studied on
Vitis vinifera cell suspension cultures and reported that 24 h after 25 μM
Ag treatment, it significantly reduced radical scavenging capacity
compared with the control culture, while Cd treatment of 5 and 50 μM
increased it.
3.4. Reversed-phase high-performance liquid chromatography (RP-HPLC)
analysis
The quantification of chicoric acid, rosmarinic acid, rutin, and iso-
quercetin were evaluated by RP-HPLC analysis and the values were
expressed as mg/g DW, and RP-HPLC chromatogram of the standards
was indicated in Fig. 7. The effects of elicitor treatments (CdCl2, AgNO3,
and YE) applied to O. basilicum cell suspension cultures on the

7
M.A. Açıkgöz
Fig. 8. HS-SPME–GC/MS chromatogram of linalool and estragole standards.
accumulation of rosmarinic acid, chicoric acid, rutin and isoquercetin
are presented in Table 1 and 2. According to this, the amount of chi-
coric and rosmarinic acid was between 0.77–5.97 (mg/g DW) and
2.04–12.86 (mg/g DW) in CdCl2 treatment, 1.42–4.96 (mg/g DW) and
5.43–13.40 (mg/g DW) in AgNO3 treatment, and 3.35–6.90 and
9.47–22.53 (mg/g DW) in YE treatment, respectively. The highest
chicoric and rosmarinic acid content in elicitor treatments was obtained
from YE treatment as 6.90 and 22.53 mg/g DW, respectively, (50 mg/L
and 100 mg/L, at the end of the day 10). Chicoric and rosmarinic acid
accumulation increased with the treatment of CdCl2 of 5 μM and AgNO3
of 5 and 25 μM compared with the control culture, while chicoric and
rosmarinic acid accumulation decreased in the other treatments of both
elicitors (Table 1).
Chicoric and rosmarinic acid, one of the most important phenolic
compounds found in Ocimum genus, can be produced quickly and easily
in plant cell culture systems, in some cases, chicoric and rosmarinic acid
may have more production levels in plant tissue culture systems (cell,
callus, hairy root, and organ cultures) than the main plant (Petersen
and Simmonds, 2003). To date, many studies have been conducted in
plant tissue culture systems to increase these bioactive compounds by
elicitor treatments in Ocimum species (Bais et al., 2002; Duran et al.,
2019; Nadeem et al., 2019; Nazir et al., 2019). In particular, many
researchers reported that AgNO3 and YE treatments in Salvia miltior-
rhiza hairy root cultures increase the accumulation of rosmarinic acid
(Yan et al., 2006; Xiao et al., 2010) and that the accumulation of
8
Industrial Crops & Products 148 (2020) 112278
chicoric acid in Echinacea purpurea increases incubation temperature,
photoperiod and ultrasound treatments (Wu et al., 2007; Liu et al.,
2012). On the other hand, studies conducted on O. basilicum tissue
culture systems have reported that chicoric and rosmarinic acid accu-
mulation can be significantly increased with plant growth regulators
(PGRs) and spectral light treatments (Nadeem et al., 2019; Nazir et al.,
2019). Park et al. (2016) studied on Agastache rugosa cell culture and
reported that the accumulation of rosmarinic acid increased with
30 mg/L AgNO3 by 10.12 times and with 750 mg/L YE therapy by 18.5
times compared with the control culture. Another study of Salvia virgate
shoot cultures reported that Ag+ (2.5 mg/L, at the end of fifth day) was
the most effective treatment for the accumulation of rosmarinic acid,
among Ag+, methyl jasmonate, and YE elicitors (Dowom et al., 2017).
Rutin, also known as vitamin P, quercetin-3-rutinoside or sophorin,
is the polyphenol that prevails in O. basilicum (Vlase et al., 2014;
Mousavi et al., 2019). The highest rutin and isoquercetin biosynthesis
have been obtained from YE treatment. Compared with the control
culture, rutin biosynthesis was increased by 1.6 times (6.54 mg/g DW)
in YE treatment and 1.9 times (3.42 mg/g DW) in isoquercetin bio-
synthesis (Table 2). However, in elicitor treatments, isoquercetin bio-
synthesis was found to be at maximum level in 25 μM treatments of
CdCl2 and AgNO3, and in 25 μM treatments of CdCl2 and 5 μm treat-
ments of AgNO3. With CdCl2 treatment, a 1.1-fold (4.72 mg/g DW)
increase in rutin biosynthesis and a 1.5-fold (3.20 mg/g DW) increase in
isoquercetin biosynthesis compared with control culture were achieved.
M.A. Açıkgöz Industrial Crops & Products 148 (2020) 112278

Table 3
Linalool and estragol recorded in Ocimum basilicum L. cell suspension cultures subjected to different cadmium chloride (CdCl2), silver nitrate (AgNO3) and yeast
extract concentrations for various days. The result are expressed as means ± standard deviation (n = 3). Mean followed by the same letter in the same column do
not differ statistically at P < 0.05 according to LSD test.
Linalool (μg/g dry weight) Estragol (μg/g dry weight)

Elicitors treatments

Concentrations day CdCl2 (μM) AgNO3 (μM) Yeast (mg/L) CdCl2 (μM) AgNO3 (μM) Yeast (mg/L)

f ı g d e
0 2 1.16 ± 0.23 1.16 ± 0.23 1.16 ± 0.23 2.20 ± 0.17 2.20 ± 0.17 2.20 ± 0.17e
4 1.54 ± 0.10e 1.54 ± 0.10g 1.54 ± 0.10e 2.71 ± 0.30c 2.71 ± 0.30d 2.71 ± 0.30c
6 1.72 ± 0.02e 1.72 ± 0.02f 1.72 ± 0.02d 2.77 ± 0.02c 2.77 ± 0.02d 2.77 ± 0.02c
8 1.81 ± 0.31d 1.81 ± 0.31f 1.81 ± 0.31c 2.80 ± 0.09c 2.80 ± 0.09c 2.80 ± 0.09b
10 1.95 ± 0.17d 1.95 ± 0.17e 1.95 ± 0.17c 2.88 ± 0.18c 2.88 ± 0.18c 2.88 ± 0.18b
5 2 2.96 ± 0.05c 2.08 ± 0.36e 1.64 ± 0.13d 3.15 ± 0.58b 3.30 ± 0.62b 1.96 ± 0.96f
4 3.20 ± 0.10b 2.57 ± 0.40d 1.80 ± 0.20c 3.42 ± 0.71a 3.44 ± 0.51a 2.05 ± 0.11f
6 3.31 ± 0.15b 2.95 ± 0.15c 1.85 ± 0.57c 3.48 ± 0.09a 3.41 ± 0.42a 2.12 ± 0.18f
8 3.59 ± 0.02a 3.11 ± 0.09c 1.96 ± 0.13c 3.50 ± 0.40a 3.38 ± 0.34a 2.08 ± 0.80f
10 3.82 ± 0.02a 3.09 ± 0.15c 1.02 ± 0.20h 3.47 ± 0.35a 3.49 ± 0.03a 2.00 ± 0.67f
25 2 1.10 ± 0.74f 4.37 ± 0.43b 1.20 ± 0.17g 1.81 ± 0.33e 1.75 ± 0.40g 2.35 ± 0.25e
4 1.21 ± 0.22f 4.41 ± 0.25b 1.18 ± 0.15g 1.68 ± 0.10e 1.79 ± 0.12f 2.28 ± 0.42e
6 1.28 ± 0.20f 4.60 ± 0.09a 1.05 ± 0.28g 1.55 ± 0.19f 1.68 ± 0.16g 2.35 ± 0.30e
8 1.24 ± 0.11f 4.71 ± 0.43a 1.13 ± 0.21g 1.45 ± 0.45f 1.55 ± 0.10h 2.48 ± 1.21d
10 1.25 ± 0.50f 4.70 ± 0.49a 1.00 ± 0.42h 1.32 ± 0.07g 1.70 ± 0.57g 2.75 ± 1.23c
50 2 0.72 ± 0.08g 1.35 ± 0.11h 2.65 ± 0.19b 0.76 ± 0.02h 1.87 ± 0.13f 3.10 ± 0.95a
4 0.68 ± 0.09g 1 32 ± 0.26h 2.79 ± 0.15b 0.53 ± 0.05ı 1.95 ± 0.05f 3.18 ± 0.87a
6 0.60 ± 0.13g 1.16 ± 0.41ı 2.98 ± 0.20a 0.35 ± 0.30j 1.92 ± 0.39f 3.09 ± 1.57a
8 0.61 ± 0.61g 1.00 ± 0.10ı 3.00 ± 0.50a 0.35 ± 0.29j 1.79 ± 0.50f 3.17 ± 0.70a
10 0.55 ± 0.25g 0.92 ± 0.15j 3.10 ± 0.27a 0.38 ± 0.15j 1.80 ± 0.49f 2.96 ± 0.27b
100 2 0.34 ± 0.05h 0.70 ± 0.09k 1.25 ± 0.58f 0.30 ± 0.25j 1.26 ± 0.14ı 2.12 ± 0.88f
4 0.25 ± 0.20h 0.72 ± 0.20k 1.40 ± 0.20f 0.26 ± 0.08j 1.28 ± 0.20ı 2.48 ± 2.17d
6 0.25 ± 0.06h 0.80 ± 0.19j 1.38 ± 0.27f 0.25 ± 0.02j 1.50 ± 0.01h 2.51 ± 2.05d
8 0.23 ± 0.45h 0.86 ± 0.24j 1.50 ± 0.36e 0.27 ± 0.03j 1.40 ± 0.09h 2.60 ± 1.22d
10 0.24 ± 0.06h 0.77 ± 0.05j 1.59 ± 0.25e 0.18 ± 0.03k 1.42 ± 0.07h 2.58 ± 0.76d
200 2 1.56 ± 0.08e 1.50 ± 0.59h
4 1.50 ± 0.20e 1.88 ± 0.28g
6 1.68 ± 0.15d 1.95 ± 1.23f
8 1.80 ± 0.12c 2.30 ± 1.00e
10 1.75 ± 0.03d 2.27 ± 0.97e

On the other hand, with AgNO3 treatment, a 1.0-fold (4.52 mg/g DW)
increase in rutin biosynthesis and a 1.4-fold (3.46 mg/g DW) increase in
isoquercetin biosynthesis compared with control culture were achieved.
The results showed that CdCl2, AgNO3 and YE treatments were effective
in rutin and isoquercetin biosynthesis in line with previous studies
(Azeez and Ibrahim, 2013; Zhao et al., 2014). On the other hand, the
researchers reported that rutin and isoquercetin synthesized in plant
tissue culture systems could be produced at higher levels than organs
growing in natural conditions, as in this study (Kuo et al., 2015). In
some of these studies, the amount of rutin was increased 9.5 times by
using the hairy root culture compared with the roots of the wild plant
(Kim et al., 2009). In another study, researchers reported that they
increased rutin biosynthesis by 1.2–3.3 times and isoquercetin bio-
synthesis by 1.5–13 times with biotic elicitors added to cell cultures in
eight different Hypericum species (Bálintová et al., 2019). In contrast to
these studies, Tusevski et al. (2016), in their research of Hypericum
perforatum callus cultures, reported that rutin biosynthesis remained
lower compared with wild growing plants, while isoquercetin was not
detected in callus cultures.
3.5. Headspace solid-phase microextraction-gas-chromatography-mass-
spectrometry (HS-SPME–GC/MS) analysis
The quantification of linalool and estragole (methyl chavicol) were
evaluated by HS-SPME–GC/MS analysis, and the values were expressed
as μg/g DW, also Fig. 8. indicated the HS-SPME–GC/MS chromatogram
of standards. The effects of elicitor treatments (CdCl2, AgNO3 and YE)
applied to O. basilicum cell suspension cultures on linalool and estragole
biosynthesis are given in Table 3. Accordingly, the highest amount of
linalool and estragole was obtained from AgNO3 treatment with an
increase of 2.8 times (from 1.16 to 4.37 μg/g DW) and 0.5 times
(2.20–3.30 μg/g DW), respectively (Table 3). This was followed by
CdCl2 treatment with an increase of 1.6 times (from 1.16 to 2.96 μg/g
DW) and 0.4 times (2.20–3.15 μg/g DW), respectively. However, lina-
lool and estragole were found to be at the maximum level in 5 μM
treatments of CdCl2 in elicitor treatments, also, estragole and linalool
biosynthesis were found to be at maximum levels in 5 and 25 μM
treatments of AgNO3, respectively. Although the effect of YE elicitor on
linalool and estragole biosynthesis was low compared with other eli-
citor treatments, the 50 mg/L treatment was the most effective in li-
nalool and estragole biosynthesis.
Previous studies have shown that elicitor treatment can be used
effectively in linalool and estragole biosynthesis (Tavares et al., 2004;
Kazemi et al., 2016; Roda et al., 2019). Furthermore, some researchers
have reported that linalool and estragole, which are not present in the
wild plant organs in plant tissue culture systems, can be synthesized
with elicitor treatments (Trettel et al., 2018; Coskun et al., 2019; El-
Kader et al., 2019). On the other hand, as far as we know, no studies
have been conducted on the effects of CdCl2, AgNO3 and YE treatments
on linalool and estragole biosynthesis in O. basilicum cell suspension
cultures. However, other elicitor treatments (chitosan, methyl jasmo-
nate, methyl salicylate plant growth regulators and CuSO4) in O. basi-
licum plant tissue culture systems have been studied in terms of their
effects on linalool and estragole biosynthesis. In some of these studies,
elicitor treatments increased linalool and estragole biosynthesis by
0.5–1.1 times and 0.1–1.3 times, (Deschamps and Simon, 2006; Gupta
et al., 2018; Monfort et al., 2018), respectively, while in others they
caused a decrease by 8.4 times (linalool biosynthesis, CuSO4 treatment)
(Trettel et al., 2018). Cells with different growth stages in plant tissue
culture systems have various levels of mRNA and proteins (Chong et al.,

9
M.A. Açıkgöz
2005). Therefore, they may react differently to elicitor treatments,
which causes bioactive compounds to accumulate at different levels
(Kang et al., 2010). Therefore, the doses and application times of eli-
citor treatments should be adjusted very well in the triggering of signal
molecules in cells. Some concentrations of AgNO3 and CdCl2 elicitor
treatments used in this study were found to be more successful in in-
creasing linalool and estragole accumulation than previous studies.
4. Conclusion
As far as we know, the effects of CdCl2, AgNO3 and YE treatments on
cell growth (cell dry weight and number), cell viability (%), total
phenolic content (TPC) and flavonoid content (TFC) and pharmaceu-
tical ingredient in O. basilicum cell suspension cultures were in-
vestigated for the first time. Cadmium chloride was the treatment in
which cell dry weight, number, and viability were mostly decreased,
also YE in which they were the least decreased. TPCs, TFCs, and anti-
oxidant activity were the highest in the treatment of 200 mg/L YE
among the elicitors. Compared with the control culture, the highest
accumulation of chicoric acid (6.45 mg/g DW, 50 mg/L treatment) and
rosmarinic acid (21.28 mg/g DW, 200 mg/L treatment) was obtained
from the yeast extract treatment. The optimum biosynthesis of rutin
and isoquercetin was obtained the highest from the treatment of 50 mg/
L yeast extract compared with the control culture. Estragole and lina-
lool's highest values compared with the control culture were obtained
from AgNO3 treatments of 5 and 25 μM as 3.30 and 4.37 μg/g DW,
respectively. These results show that CdCl2, AgNO3 and YE treatments
can be used effectively and efficiently in the biosynthesis of pharma-
ceutical active ingredients in O. basilicum cell suspension cultures.
Author statement
Muhammed Akif Açıkgöz planned to work, designed, conducted the
experiments and wrote the manuscript.
Declaration of Competing Interest
Authors declare no conflict of interest.
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