26 Thidiazuron-Induced Changes in Biomass Parameters, Total Phenolic Content, and Antioxidant Activity in Callus Cultures of Artemisia Absinthium L.

Appl Biochem Biotechnol (2014) 172:23632376
DOI 10.1007/s12010-013-0663-7
Thidiazuron-Induced Changes in Biomass Parameters,

Total Phenolic Content, and Antioxidant Activity in Callus
Cultures of Artemisia absinthium L.
Mohammad Ali & Bilal Haider Abbasi
Received: 22 September 2013 / Accepted: 28 November 2013 /

Published online: 28 December 2013
# Springer Science+Business Media New York 2013
Abstract Callus culture of Artemisia absinthium L. was established for enhanced production
of phenolics and higher antioxidant activity. Callus was induced from seed-derived leaf
explants, incubated on to MS media supplemented with thidiazuron (TDZ; 0.55.0 mg/l)
either alone or in combination with -naphthalene acetic acid (NAA; 1.0 mg/l). These callus
cultures were investigated for their growth kinetics, total phenolic content, and antioxidant
activity on weekly basis for a period of 49 days. Maximum dry biomass accumulation of
8.73 g/l was observed on day 42 in response to 1.0 mg/l TDZ and 1.0 mg/l NAA. Furthermore,
maximum level of total phenolic content of 8.53 mg GAE/g DW and highest 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity of 72.6 % were observed in calli
formed in response to 1.0 mg/l TDZ on day 42. The results showed a positive correlation
of total phenolic content and DPPH radical scavenging activity in most of the callus cultures of
A. absinthium L.
Keywords Artemisia . Callus . Phenolics . Antioxidant activity . Thidiazuron
Introduction
Artemisia absinthium L. (Wormwood) is a well-known traditional herb, mentioned in almost
all books of herbal medicine in the Western world [1]. This plant is a rich source of terpenes,
antioxidant phenolics, flavonoids, and other biologically active compounds [2]. The dry leaves
and stems contain, among others, 0.251.32 % essential oil, absinthin, anabsin, artemisinin,
anabsinthin, artabsin, and matricin [3]. The plant has traditionally been used as anti-helmintic,
choleretic, antiseptic, balsamic, depurative, digestive, diuretic, emmenagogue, and in treating
leukaemia and sclerosis [4].
Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors,
and other industrial materials either as a part of final product or as a raw material [5]. Among
different classes of secondary metabolites, plant polyphenols constitute the largest
M. Ali : B. H. Abbasi (*)

Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan
e-mail: bhabbasi@qau.edu.pk
2364
group of natural antioxidants [6]. Phenolics and flavonoids posses biological properties like antioxidant, anti-aging, anti-carcinogen, and protection from cardiovascular,
immune/autoimmune diseases, and brain dysfunctions, viz., Parkinsons, Alzheimers,
Huntingtons diseases, etc. [7, 8]. Phenolics are considered more potent antioxidants than
vitamin C, E and carotenoids [9].
There are various limitations in using wild plants as the sole source of secondary metabolites. The use of plant in vitro cultures is an alternative for obtaining secondary metabolites
that are either difficult to obtain by conventional techniques or whose production is economically not feasible [10]. The genus Artemisia has been exploited for enhanced production of
artemisinin; however, strategies should be adopted to enhance medicinally important phenolic
compounds by exploiting in vitro cultures of this genus. The aim of present study was to
investigate the interrelationship of biomass accumulation, total phenolic content, and
antioxidant activity in response to application of thidiazuron (TDZ), in callus cultures of
A. absinthium L.
Materials and Methods

Seed Germination
Seeds of A. absinthium L. were obtained from National Agriculture Research Centre, Islamabad,
Pakistan. Following a single wash with running tap water, seeds were immersed in ethanol
(3 min), followed by treatment with mercuric chloride (2 min), and then washed with autoclaved
water and dried on sterilized filter papers. Murashige and Skoog basal medium (MS0, 1962;
Phytotechnology Labs, USA) containing 3 % sucrose and 0.8 % (w/v) agar (Phytotechnology
Labs, USA) was used for seed germination.
Callus Induction and Biomass Accumulation
For callus induction, approximately 1.5 cm of the leaf sections from 28-day-old seedderived plantlets were inoculated on MS [11] media supplemented with varying
concentration of TDZ (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/l) either alone or in
combination with -naphthalene acetic acid (NAA; 1.0 mg/l). Callus formation was
observed on weekly basis through visual observations. All calli were subcultured on
the same media after 20 days. To investigate biomass accumulation, 1 g of each
40 days old callus, formed in response to the above mentioned plant growth regulators (PGRs) types and concentrations, was subcultured on fresh media with same
composition of PGRs. Each experiment was carried out in 20 flasks, containing 20 g
of inoculated calli as starting culture/treatment. Fresh weight (FW) and dry weight
(DW) accumulation were recorded with an interval of 7 days for a period of 49 days.
Triplicate flasks were used in all experiments.
Analytical Methods
To investigate biomass accumulation, calli were harvested from the media and weighed for
FW determination and then oven dried (60 C, 24 h) for DW determination.
The callus samples were extracted as described by Ali et al. [12]. Briefly, each finely
ground dried callus sample (100 mg) was mixed with 80 % (v/v) methanol (10 ml). The
mixtures were sonicated (10 min; Toshiba, Japan) three times with a resting period of 30 min

160
TDZ0.5
TDZ1.0
TDZ2.0
TDZ3.0
TDZ4.0
TDZ5.0
140
120
Fresh weight (g/l)
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100
80
60
40
20
0
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42
49
Culture time (days)

10
TDZ0.5
TDZ1.0
TDZ2.0
TDZ3.0
TDZ4.0
TDZ5.0
9
8
Dry weight (g/l)
6
5
4
3
2
1
0
14
21
28
35
42
49
Culture time (days)

Fig. 1 a Fresh callus biomass and b dry callus biomass of A. absinthium L. on MS medium supplemented with
TDZ (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/l). Values are meanSE of three replicates
in between and centrifuged (8,000 rpm, 10 min). The supernatant was either collected and
stored at 4 C or immediately used for analysis.
2366

200
160
140
Fresh weight (g/l)
TDZ0.5+NAA1.0
TDZ1.0+NAA1.0
TDZ2.0+NAA1.0
TDZ3.0+NAA1.0
TDZ4.0+NAA1.0
TDZ5.0+NAA1.0
180
120
100
80
60
40
20
0
14
21
28
35
42
49
Culture time (days)

10
TDZ0.5+NAA1.0
TDZ1.0+NAA1.0
TDZ2.0+NAA1.0
TDZ3.0+NAA1.0
TDZ4.0+NAA1.0
TDZ5.0+NAA1.0
9
8
Dry weight (g/l)
6
5
4
3
2
1
0
14
21
28
35
42
49
Culture time (days)

Fig. 2 a Fresh callus biomass and b dry callus biomass of A. absinthium L. on MS medium supplemented with
TDZ (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/l) and NAA (1.0 mg/l). Values are meanSE of three replicates
Phenolics were quantified by using FolinCiocalteu reagent according to the protocol

of Velioglu et al. [13]. Absorbance was measured at 725 nm by using UV/VisDAD
2367
Total Phenolic Content (mg GAE/g DW)
10
TDZ 0.5
TDZ 1.0
TDZ 2.0
TDZ 3.0
TDZ 4.0
TDZ 5.0
9
8
7
6
5
4
3
2
1
0
0
14
21
28
35
42
49
Culture time (days)
Total Phenolic Content (mg GAE/g DW)
10
TDZ0.5+NAA1.0
TDZ1.0+NAA1.0
TDZ2.0+NAA1.0
TDZ3.0+NAA1.0
TDZ4.0+NAA1.0
TDZ5.0+NAA1.0
9
8
7
6
5
4
3
2
1
0
0
14
21
28
35
42
49
Culture time (days)

Fig. 3 a Total phenolic content (milligrams GAE per gram DW) in callus cultures of A. absinthium L. on MS
medium supplemented with TDZ and b TDZ+NAA. Values are meanSE of three replicates
spectrophotometer (Halo DR-20, UVVis spectrophotometer, Dynamica Ltd., Victoria, Australia).

The calibration curve (050 g/ml, R2 =0.968) was plotted by using gallic acid as standard,
2368
and the total phenolic content (TPC) was expressed as gallic acid equivalents (GAE) per
gram of DW.
For antioxidant activity determination, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free
radical scavenging assay (FRSA) was exploited [14]. Briefly, absorbance of the mixture was
recorded at 517 nm by spectrophotometer. For background correction, a methanolic solution of
DPPH that had decayed and showed no purple color (2 mg of butylated hydroxyanisole (BHA)
dissolved in 4 ml of methanol with 0.5 ml of DPPH solution added) was used instead of pure
methanol. The radical scavenging activity was calculated by the following formula and
expressed as percent DPPH discoloration:
% scavenging DPPH free radical 100 1AE=AD
where AE is absorbance of the solution when an extract was added at a particular concentration
and AD is the absorbance of the DPPH solution with nothing added.
Experimental Design and Data Analysis
All experiments were conducted in a completely randomized design and were repeated twice.
Each treatment was consisted of three replicates. Mean values of various treatments were
subjected to analysis of variance, and significant difference was separated using Duncans
multiple range test. SPSS (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine
the significance at P<0.05.
Results and Discussion

Effects of Plant Growth Regulators on Biomass Accumulation and Growth Kinetics
When used alone, 1.0 mg/l TDZ induced fresh callus biomass (FCB) of 130 g/l and dry callus
biomass (DCB) of 8.1 g/l on day 42 of culture (Fig. 1a, b). However, among all the
concentrations and combination of PGRs tested, 1.0 mg/l TDZ and 1.0 mg/l NAA resulted
in maximum FCB of 163 g/l and DCB of 8.73 g/l on day 42 of culture. It suggests that 1.0 mg/l
NAA in combination with TDZ has positive effect on callus biomass accumulation as
compare to 1.0 mg/l TDZ alone, which is also evident from the previous reports
describing the synergistic effect of auxins and cytokinins on callus formation frequency
in A. absinthium L. [1517]. Similar reports are also available for other medicinal plant
species [1823]. In this study, maximum FCB and DCB were recorded on day 42 of
culture in response to lower concentrations (0.5, 1.0, and 2.0 mg/l) of TDZ either alone
or in combination with NAA (1.0 mg/l). Further increase in TDZ concentration significantly
reduced the callus formation frequency, and maximum callus biomass was recorded on day
35 of culture.
A lag phase of 7 days was observed for fresh and dry callus biomass accumulation in
response to PGRs tested. Maximum biomass profiles were observed on day 42 or day 35 of
culture, depending upon the concentrations and combinations of PGRs used.
Fig. 4 Antioxidant activity (percent), total phenolic content (milligrams GAE per gram DW) and dry biomass b
(grams per liter) in calli formed in response to a TDZ 0.5 mg/l, b TDZ 1.0 mg/l, c TDZ 2.0 mg/l, d TDZ 3.0 mg/l,
e TDZ 4.0 mg/l, and f TDZ 5.0 mg/l. Values are meanSE of three replicates. Columns with similar alphabets
are not significantly different at P<0.05
2369
12
90
RSA
DBM
TPC
80
60
50
de
cd
e
e
10
10
9
bc
cd
40
ab
a
c
4
30
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0
14
21
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35
42
16
16
Total Phenolic Content (mg GAE/g)
11
Dry Biomass (g/l)
Radical Scavenging Activity (%)
ab
70
20
12
49
Time (days)
90
RSA
DBM
TPC
60
50
30
20
40
b b
12
11
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de
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8
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15
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Dry Biomass (g/l)
80
49
Time (days)
80
RSA
DBM
TPC
70
bcd
50
40
cd
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b
c
a
a
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Dry Biomass (g/l)
60
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Time (days)
35
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11
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2370

80
12
RSA
DBM
TPC
70
50
d
e
cd
cd
b
ab
6
a
30
bc
Dry Biomass (g/l)
ab
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20
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49
Time (days)
80
RSA
DBM
TPC
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a
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bcd
ab
Dry Biomass (g/l)
cd
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a
60
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Time (days)
80
RSA
DBM
TPC
c
cd
cd
40
30
a
8
50
ab
bc
ab
6
ab
ab
20
10
8
7
6
5
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cd
2
10
1
0
0
0
14
21
28
Time (days)
Fig. 4 (continued)
35
42
49
60
11
10
Dry Biomass (g/l)
70
2371
Starting with the inoculum culture of 20 g/l, more than doubling in FCB with the values
55.7, 44.3, and 51.3 g/l was observed on day 14 while maximum FCB with the values 112,
130, and 118 g/l was recorded on day 42 of culture in response to 0.5, 1.0, and 2.0 mg/l of
TDZ, respectively. On the other hand, more than twofold increase in FCB was found to be 56,
60.7 and 43.3 g/l on day 21 of culture and maximum accumulation recorded was 103, 90, and
67.7 g/l on day 35 in response to 3.0, 4.0, and 5.0 mg/l of TDZ, respectively (Fig. 1a).
Furthermore, twofold increase in DCB accumulation was not found strictly linked to particular
day/days and maximum DCB with the values 7.63, 8.1, and 6.8 g/l was observed on day 42 in
response to 0.5, 1.0, and 2.0 mg/l of TDZ, respectively, followed by 5.04, 5.0, and 4.5 g/l in
response to 3.0, 4.0, and 5.0 mg/l, respectively (Fig. 1b).
Biomass accumulation in response to combination of different concentrations of TDZ
and 1.0 mg/l NAA was also investigated. Maximum values of FCB 153, 163, and 144 g/l
and DCB 8.17, 8.73, and 7.52 g/l accumulation were recorded on day 42 of culture in
response to 0.5, 1.0, and 2.0 mg/l of TDZ (in combination with 1.0 mg/l), respectively.
On the other hand, maximum levels of FCB 111, 98.3, and 75.8 g/l and DCB 5.44, 5.30
and 4.83 g/l in response to the respective concentrations 3.0, 4.0, and 5.0 mg/l of TDZ
(in combination with 1.0 mg/l) were recorded on day 35 of culture (Fig. 2a, b). These
results suggest that higher concentrations of TDZ alone and in combination with NAA
decrease the callus formation frequency as well as reduce the log phase for biomass
accumulation in callus cultures of A. absinthium L.
Total Phenolic Content and Its Dependence on Biomass Accumulation
Differential TPC profiles displayed by callus cultures of A. absinthium L. were found to be
dependent upon PGR concentration and type and age of the callus. Maximum levels of TPC in
response to different concentrations of TDZ alone were found on day 42 of culture, except for
TDZ 2.0 mg/l where it was observed on day 35 (Fig. 3a). In response to combinations of TDZ
and NAA, maximum TPC levels were observed on day 35 of culture except for 1.0 mg/l TDZ
and 1.0 mg/l NAA where it was observed on day 42 (Fig. 3b).
When correlated with dry biomass accumulation, maximum TPC was found to be
either growth dependent or independent in response to TDZ lower concentrations (0.5 and
1.0 mg/l) and higher concentrations (3.0, 4.0, and 5.0 mg/l), respectively, while maximum
TPC in response to 2.0 mg/l was observed during the log phase of growth. On the other
hand, maximum total phenolic content in response to lower concentrations of TDZ
(0.5 and 3.0 mg/l) and higher concentrations (3.0, 4.0, and 5.0 mg/l) were observed in log
phase and stationary phase, respectively. Exceptionally, maximum TPC in response to
2.0 mg/l was also found in stationary phase of growth. These results suggest that a mixed
pattern of phenolics accumulation is followed by calli of A. absinthium L. in response to
different concentration of TDZ either alone or in combination with 1.0 mg/l NAA, in
relation to DBM.
The importance of TDZ as an effective plant growth regulator for plant morphogenesis has
been reviewed several times [2427]. Additionally, TDZ had been reported for the production
of economically important secondary metabolites in some plant species [28].
Antioxidant Activity and Its Relationship with Biomass Accumulation and Total Phenolic
Content
The DPPH radical scavenging activity in callus cultures of A. absinthium L. was
determined with an interval of 7 days for a period of 49 days. In response to lower
2372
concentrations of TDZ (0.5 and 1.0 mg/l), a positive correlation of antioxidant activity,
total phenolic content, and dry biomass accumulation was observed. The calli developed on MS medium supplemented with 0.5 and 1.0 mg/l of TDZ displayed maximum corresponding values for antioxidant activity (72.3 and 72.6 %), total phenolic
content (8.45 and 8.53 mg GAE/g DW), and dry biomass accumulation (7.63 and
8.10 g/l) on day 42 of culture (onset of stationary phase) (Fig. 4a, b). While biomass
dependent and total phenolic content independent antioxidant potential was observed
at higher concentrations of TDZ (4.0 and 5.0 mg/l) (Fig. 4e, f). Contrarily, total
phenolic content dependent and biomass independent antioxidant activity was observed in calli obtained in response to TDZ and 1.0 mg/l NAA, except for the
biomass accumulation at higher concentrations of TDZ (3.0, 4.0, and 5.0 mg/l). These
results suggested the involvement of phenolics in antioxidant activity of calli formed
in response to lower concentrations of TDZ. However, a mixed pattern of antioxidant
activity, total phenolic content, and biomass was observed in response to higher
concentrations of TDZ. Maximum antioxidant activity (61.3 %) and maximum dry
biomass (7.54 g/l) were observed on day 42 (Onset of stationary phase), while
maximum TPC (5.04 mg GAE/g DW) was recorded on day 35 (log phase) of culture
in callus formed in response to 3.0 mg/l TDZ (Fig. 4d), which could be linked with
the assumption that antioxidants other than phenolics are produced in response to
some PGRs. On the other hand, the calli developed in response to 4.0 and 5.0 mg/l of
TDZ showed maximum corresponding values of antioxidant activity (61.2 and
60.8 %) and dry biomass (5.0 and 4.5 g/l) on day 35 (onset of stationary phase)
while maximum TPC (7.14 and 6.81 mg GAE/g DW) on day 42 of culture (stationary phase)
(Fig. 4e, f).
Antioxidant activity was found to be TPC dependent in almost all cultures in
response to combination of TDZ and NAA (Fig. 5). Callus cultures developed in
response to lower concentrations of TDZ (0.5, 2.0, and 3.0 mg/l) and 1.0 mg/l NAA
displayed maximum corresponding values for antioxidant potential (61.4, 60.7, and
58.8 %) and total phenolic content (7.35, 7.14, and 6.88 mg GAE/g DW) on day 35
of culture. The remaining cultures showed a mixed pattern regarding the correlation of
antioxidant activity with TPC and biomass accumulation. With increase in TDZ concentration to 5.0 mg/l, the maximum antioxidant activity (49 %) was found to be TPC
and biomass accumulation independent, suggesting the role of antioxidants other than
phenolics in the log phase of culture. Previously, we have reported phenolics dependent
antioxidant activity in callus and cell suspension cultures of A. absinthium L [12].
Several reports are available on the positive correlation of phenolics and antioxidant
activities in different plants [2933]. Earlier studies have been undertaken on the
investigations of total phenolic content in callus culture of various medicinal plants
[3436]. Furthermore, a strong relationship has been observed between the phenolic
compounds produced by the in vitro cultures of different plants and their antioxidant activities
[3739]. These studies suggested the involvement of phenolics as major antioxidants in
some plants.
Fig. 5 Antioxidant activity (percent), total phenolic content (milligrams GAE per gram DW) and dry biomass b
(grams per liter) in calli formed in response to a TDZ 0.5 mg/l+NAA 1.0 mg/l, b TDZ 1.0 mg/l+NAA 1.0 mg/l,
c TDZ 2.0 mg/l+NAA 1.0 mg/l, d TDZ 3.0 mg/l+NAA 1.0 mg/l, e TDZ 4.0 mg/l+NAA 1.0 mg/l, and f TDZ
5.0 mg/l+NAA 1.0 mg/l. Values are meanSE of three replicates. Columns with similar alphabets are not
significantly different at P<0.05
2373
14
80
RSA
DBM
TPC
60
ab
c
11
ab
bc
50
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70
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RSA
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a
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2374

80
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60
RSA
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ab
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a b
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Dry Biomass (g/l)
ab
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Time (days)
Fig. 5 (continued)
35
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50
2375
Conclusions
The present study showed that phenolics production was positively regulated in callus cultures
of A. absinthium L. in response to TDZ. A linear relationship of total phenolic content and
anti-oxidative potential was observed in most of the calli formed. The results suggest the
exploitation of callus cultures of A. absinthium L. by treatment with other PGRs and elicitors
to enhance total phenolic content and antioxidant activity and subsequently to be used for the
establishment of suspension cultures.
Acknowledgments Financial support from Higher Education Commission (HEC) is gratefully acknowledged.
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26 Thidiazuron-Induced Changes in Biomass Parameters, Total Phenolic Content, and Antioxidant Activity in Callus Cultures of Artemisia Absinthium L.

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26 Thidiazuron-Induced Changes in Biomass Parameters, Total Phenolic Content, and Antioxidant Activity in Callus Cultures of Artemisia Absinthium L.

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Appl Biochem Biotechnol (2014) 172:23632376

Thidiazuron-Induced Changes in Biomass Parameters,

Received: 22 September 2013 / Accepted: 28 November 2013 /

M. Ali : B. H. Abbasi (*)

Appl Biochem Biotechnol (2014) 172:23632376

Materials and Methods

Appl Biochem Biotechnol (2014) 172:23632376

Fresh weight (g/l)

Culture time (days)

Dry weight (g/l)

Culture time (days)

Appl Biochem Biotechnol (2014) 172:23632376

Fresh weight (g/l)

Culture time (days)

Dry weight (g/l)

Culture time (days)

Phenolics were quantified by using FolinCiocalteu reagent according to the protocol

Appl Biochem Biotechnol (2014) 172:23632376

Total Phenolic Content (mg GAE/g DW)

Culture time (days)

Total Phenolic Content (mg GAE/g DW)

Culture time (days)

spectrophotometer (Halo DR-20, UVVis spectrophotometer, Dynamica Ltd., Victoria, Australia).

Appl Biochem Biotechnol (2014) 172:23632376

Results and Discussion

Appl Biochem Biotechnol (2014) 172:23632376

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Total Phenolic Content (mg GAE/g)

Appl Biochem Biotechnol (2014) 172:23632376

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Total Phenolic Content (mg GAE/g)

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Appl Biochem Biotechnol (2014) 172:23632376

Appl Biochem Biotechnol (2014) 172:23632376

Appl Biochem Biotechnol (2014) 172:23632376

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)

Appl Biochem Biotechnol (2014) 172:23632376

Radical Scavenging Activity (%)

Total Phenolic Content (mg GAE/g)

Total Phenolic Content (mg GAE/g)

Dry Biomass (g/l)

Radical Scavenging Activity (%)