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Light-induced biochemical variations in secondary metabolites production
and antioxidant activity in callus cultures of Stevia rebaudiana (Bert.)

Naveed Ahmad, Abdur Rab, Nisar Ahmad

PII: S1011-1344(15)30059-2
DOI: doi: 10.1016/j.jphotobiol.2015.11.015
Reference: JPB 10202

To appear in:

Received date: 22 September 2015


Accepted date: 27 November 2015

Please cite this article as: Naveed Ahmad, Abdur Rab, Nisar Ahmad, Light-induced
biochemical variations in secondary metabolites production and antioxidant activity in
callus cultures of Stevia rebaudiana (Bert.), (2015), doi: 10.1016/j.jphotobiol.2015.11.015

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Research article for Journal of Photochemistry and Photobiology B: Biology

Light-induced biochemical variations in secondary

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metabolites production and antioxidant activity in callus
cultures of Stevia rebaudiana (Bert.)

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Naveed Ahmad1, Abdur Rab1, Nisar Ahmad2

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1
Department of Horticulture, The University of Agriculture, Peshawar, Peshawar-25120, Pakistan.

2
Centre for Biotechnology and Microbiology, University of Swat, Swat 19200 Pakistan
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*Correspondence:
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Dr. Nisar Ahmad


Assistant Professor
Center for Biotechnology and Microbiology (CB&M),
University of Swat, Swat-19200, Pakistan
Cell: +92-332-9959234
E.mail: ahmadn@uswat.edu.pk, nisarbiotech@gmail.com
Web (Official): http://www.uswat.edu.pk/index.php/departments/microbiology-biotechnology/teaching-
faculty/
Details: http://scholar.google.com.pk/citations?user=uQRMhb8AAAAJ&hl=en&oi=ao

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Abstract

Stevia rebaudiana (S. rebaudiana) is very important specie with a worldwide medicinal and
commercial uses. Light is one of the major elicitor that fluctuate morphogenic potential and

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biochemical responses. In the present study, we investigated the effect of various spectral lights
on biomass accumulation and secondary metabolites production in callus cultures of S.

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rebaudiana. Leaf explants were placed on Murashige and Skoog (MS) medium and exposed to
various spectral lights. 6-benzyle adenine (BA) and 2, 4-dichlorophenoxy acetic acid (2, 4-D; 2.0

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mg l-1) was used for callus induction. The control light (16/8 hr) produced optimum callogenic
response (92.73%) than other colored lights. Compared to other colored lights, control grown

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cultures displayed maximum biomass accumulation (5.78 g l-1) during a prolong log phase at 18th
day of growth kinetics. Cultures grown under blue light enhanced total phenolics content (TPC;
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102.32 g/g DW), total flavonoids content (TFC; 22.07 g/g DW) and total antioxidant capacity
(TAC; 11.63 g/g DW). Contrary, green and red lights improved reducing power assay (RPA;
0.71 Fe(II) g -1 DW) and DPPH-radical scavenging activity (DRSA; 80%). Herein, we concluded
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that the utilization of colored lights is a promising strategy for enhanced production of
antioxidant secondary metabolites in callus cultures of S. rebaudiana.
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Key words: Stevia rebaudiana; light; callus; phenolics/flavonoids; peroxidase; antioxidant


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Introduction

Stevia rebaudiana is one of the medicinal and emerging commercial herbs in Asteraceae family
that is cultivated worldwide [1]. The medicinal importance of S. rebaudiana can be judge from

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yerba mate (herbal remedy for heartburn) that is used by Guarani tribes of Paraguay and Brazil
since long time [2]. During plant development, the leaves accumulate steviosides which add 300

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times more sweetness than normal sugar [3, 4]. Among different compounds in the leaves of S.
rebaudiana, stevioside is one of the sweetest compounds present in larger quantities and now-a-

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days commonly used in various commercial products [5]. As compared to glucose, there are no
receptors for steviosides content that is why it is considered as zero caloric. Therefore,

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steviosides are very useful for diabetic patients and also helpful in weight reduction.
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Various in vitro cultures have been exploited for steviosides production [6, 5, 7]. Callus culture
is one of the effective substitutes than whole micropropagation for accumulation of secondary
metabolites. However, the biosynthetic pathways of these secondary metabolites are markedly
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influenced by various elicitors. The addition/exposure of these elicitors to culture media may
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modulate the production of secondary metabolites. Mostly, the abiotic and biotic stresses alter
the accumulation of bioactive compounds in higher quantities as compared to naturally growing
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plants [8].
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Among various elicitors, light quality/quantity greatly influence plant architecture development,
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morphogenetic responses and synthesis of valuable bioactive compounds [9]. Optimum light
intensity and selective wavelength enhanced the production of diosgenin in callus cultures of
Dioscorea deltoidea [10]. However, the physiological and morphological responses of plants
towards light quality are greatly varied depending upon the plant species [11]. Tariq et al. [9]
have reported significant variation in both morphogenic and biochemical attributes in callus
cultures of Artemisia absinthium subjected to various monochromatic lights. It is well known
fact that light play a key role in primary and secondary metabolism and various plant
developmental processes [12-14]. Many reports have suggested that light sources directly
stimulated the production of important secondary metabolites including anthocyanins,
artemisinin, caffeic acid derivatives and flavonoids [15-17, 12]. The inhibitory effects of light on
nicotine and shikonin production were also reported [18]. Besides its synergistic/antagonistic

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effects on secondary metabolites, light also play a key role in regulating the secretion mechanism
of secondary metabolites (Kim et al., 1988).

Medicinal plants are small natural factories that synthesize and release various bioactive

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compounds under specific conditions. These plant based compounds are gaining more attention
as a potential drugs, nutraceuticals, pharmaceuticals and food additives [19]. Plants under stress

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conditions either release antioxidative enzymes or phenolics and flavonoids as defense system
[20]. Plant polyphenols represent the principal group of natural antioxidants among various

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classes of secondary metabolites that are considered to be more valuable as compared to
carotenoids and vitamins [21]. Plant based flavonoids are well reputed for their antioxidant

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properties due to their redox capacity. It has been implicit that a flavonoids rich diet inversely
effect lipid peroxidation, cell aging and cancer [22]. These phenolics and flavonoids play a key
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role in scavenging toxic reactive oxygen species (ROS) [23]. These compounds have numerous
uses in pharmacological activities like antioxidant, anti-carcinogenic, to cure cardiovascular
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diseases and promote immune system [24]. In vitro cultures are amongst the best options for
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production of antioxidant compounds [25]. Antioxidants are compounds having the ability to
block or minimize the oxidation process through inhibition of the initiation of oxidizing chain
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reactions [26].
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To the best of our knowledge, there are no previous reports regarding the effect light illumination
of specific wavelength on callus induction, biomass accumulation and production of antioxidant
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secondary metabolites in S. rebaudiana. Therefore, the main objective of the present study was
to evaluate the proliferation of callus cultures under various sources of illumination having
different wavelengths, suitability of specific spectrum for particular biochemical response and
correlation between total phenolics, flavonoids and antioxidant activities.

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Materials and methods

Leaf explants collection and sterilization

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Fresh leaves were excised from 50-days old nursery grown plants of S. rebaudiana at Ground
and Garden Nursery, Department of Horticulture, The University of Agriculture Peshawar.

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Leaves were gently dipped in autoclaved distilled water to retain viability. These leaf explants
were surface decontaminated according to the protocol of Aman et al. [6]. Healthy leaves were

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exposed to 70% ethanol (1 min) and 0.2% mercuric chloride (2 min) to remove surface
contaminants. Subsequently, leaves were rinsed three times with sterile distilled water to

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minimize the content of mercuric chloride and ethanol. These surface sterilized explants were
dried on sterilized filter paper to remove surplus water.
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Establishment of callus cultures under different colored lights
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To study the effect of various spectral lights on callogenic frequency, approximately, 3-4 mm2
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leaf pieces were inoculated on Murashige and Skoog (MS; Phytotechnology Lab, USA) [27]
media augmented with BA (2.0 mg l-1) and 2, 4-D (2.0 mg l-1) (from experiment of Aman et al.
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2014). MS-media without plant growth regulators (PGRs) was used as control (MS0). The MS-
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media was supplemented with 3% sucrose (Merck), solidified with 8 g l-1 agar (Agar Technical
LP0013, Oxoid, Hampshire, England), the pH was adjusted to 5.8 by using pH meter (Eutech
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Instruments pH 510, Singapore) and finally all the media were autoclaved (Systec VX 100,
Germany) at 121C for 25 min. Different monochromatic colored lights used were; green lights
(40W Litex; 480-670 nm), yellow lights (36 W, Philips Ltd.; 530-780 nm), blue lights (220 V; 50
Hz, Keliang Ltd.; 380-560 nm) and red lights (25 W, BINXIANG; 610-715 nm), while white
fluorescent tube lights (20 W, Toshiba FL20T9D/19; 380-780 nm) with 16/8 h photoperiod and
light intensity ranges from ~40-50 mol m-2 s-1 were used as control. These cultures were
maintained in a growth room at temperature of 251C. Each treatment was divided into three
independent experiments. Each experiment was designed on Completely Randomized Design
(CRD). After 30 days of callus establishment, the averages were randomly recorded using each
replication as % callus induction.

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Callus growth kinetics and biomass accumulation

During growth kinetics, data was collected from 30 days old culture with 3 days interval. Growth
curve was established for rapidly growing callus biomass in response to each colored light. After

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30 days of culture establishment, fresh callus was harvested from solid media for determination
of fresh weight (FW). After FW determination, the callus was dried at 50C in an oven (Thermo

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Scientific; Germany) and subsequently the dried weight (DW; Sortorious digital balance;
Germany) was determined (figure 1). Fresh and dry weights of calli were expressed in gram/litre

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(g l-1).

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Analytical methods

The dried calli obtained from various colored lights were powder by using a motar and pestle for
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extract preparation. The total phenolics content (TPC) was determined according to the protocol
of Ahmad et al. [23]. During phenolics determination, 0.03 ml of extract and 0.1 ml of Folin-
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Ciocalteus reagent was slowly mixed with 2.55 ml sterile water. Before incubation in dark for 30
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min, the mixture was centrifuged at 10,000 rpm for 15 min. The supernatant was collected and
passed through 45 m membrane filter paper in UV visible spectrophotometer cuvette
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(Shimadzu-1650; Japan). Gallic acid (Sigma; 1.0-10 mg/ml) was used for standard curve
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establishment. The absorbance of each callus extract and gallic acid was monitored at 760 nm.
Results were expressed as GAE mg/g DW of callus. The total flavonoids content (TFC) was
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determined according to the protocol of Ahmad et al. [23]. For flavonoids determination, 0.25 ml
extract, 0.075 ml AlCl3 (5% w/v) and 0.5 ml NaOH were slowly mixed with 1.25 ml sterile
water. The mixture was centrifuged at 10,000 rpm for 15 min and then kept in dark for 30 min.
The absorbance of each sample was measured at 510 nm with a UV-visible spectrophotometer.
Rutin (Sigma; 1.0-10 mg/ml) was used for establishment standard calibration curve. The total
flavonoids content was expressed as RE mg/g DW of callus.

DPPH-radical scavenging activity (DRSA) in different callus cultures exposed to colored lights
was determined according to the protocol of Ahmad et al. [26]. Briefly, 5 mg of each callus
extract was independently dissolved in 20 ml HPLC grade methanol. The DPPH solution was
prepared by taking 0.25 mg DPPH powder in 20 ml methanol. The DPPH solution was diluted
four times. Afterward, 1.0 ml of each callus methanolic solution was mixed with 2.0 ml of DPPH

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solution. The mixture was incubated in dark for 30 min to scavenge maximum radicals. After
incubation, the absorbance of the solution was monitored on 517 nm at room temperature by
using a UV-visible spectrophotometer (Shimadzu-1650PC, Japan). The DRSA in each sample
was calculated as percentage of DPPH discoloration using the following equation;

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DRSA (%) = 100 (1 AP/AD)

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Where AP represents absorbance of shoots extract at 517 nm and AD is the absorbance of the

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DPPH solution without extract

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The TAC was determined according to the method of Piatczak et al. [28]. The TAC was
expressed as ascorbic acid milligram equivalent per gram of DW. The reducing power assay
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(RPA) was determined according to Pulido et al. [29]. The assay was calculated against known
value of FRAP, ferrous sulphate and the calibration curve was established from 0-2000 M
concentrations. The reducing power assay was expressed in mol Fe(II) g -1 of DW.
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Statistical analysis
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Analysis of replicated values, standard errors (), and least significant difference (LSD) were
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carried out by using Statistix software (8.1 versions) and Origin Lab (8.5) software was used for
graphical presentation.
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Results and Discussion

Effect of different colored lights on callogenic frequency

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Elicitation has been one of the most efficient strategies to improve in vitro culture development
and production of desirable secondary metabolites [30, 11]. Light is one of the important elicitors

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that play a key role in photosynthesis, plant architectural development and plant morphogenesis
[31]. Practically, fluorescent tubes are the major source of light energy for in vitro cultures

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development [9]. Selective wavelength and optimum intensity of light stimulate the production
of important secondary metabolites in various cultures of medicinal plants [32, 33]. Previous

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studies confirmed that light quality directly affect morphological and physiological responses
depending upon plant species [9, 11].
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In this study, leaf explants were placed on MS-media augmented with the combination of 2, 4-D
and BA (2.0 mg l-1). These cultured flasks were then kept under different colored monochromatic
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lights for callogenic response. The control light produced optimum callogenic response (92.73%)
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than other colored lights. The yellow light induced 88.34% callogenesis followed by blue
(76.4%) and green (75.12%) lights. However, the red light was found less effective in callus
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induction (64.34%) from leaf explants of S. rebaudiana (figure 2 and 3). The current data are in
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agreement with the reports of Tariq et al. [9] that white light enhanced callus development (90%)
from leaf explants of Artemisia absinthium L. Ali and Abbasi [11] also observed higher biomass
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accumulation under white light in cell suspension culture of Artemisia absinthium L. The
possible mechanism underlying callus development is that white light provide optimum energy
for mass proliferation than other colored lights. Therefore, we suggest that white light may be the
best option for callogenesis and establishment of cell suspension culture in S. rebaudiana as
compared to other colored lights.

Callus growth kinetics and biomass accumulation under different monochromatic lights

The callus biomass accumulation was recorded for a period of 30 days with 3 days interval under
the influence of various monochromatic lights (figure 4). A shorter lag phases were noted for all
applied colored lights. Most of the colored lights and control have shown an elongated log
phases which started from day 3 and reached up to 18 days. However, a sudden increased in

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biomass accumulation was observed from day 12 to 18 days during log phases. As compared to
lag and log phases, all the applied colored lights and control have shown decline phases from day
21 to 30 days. Among various monochromatic lights tested, the maximum biomass accumulation
(2.7 g l-1) was displayed by red light during log phase (day 18) of growth kinetics (figure 4).

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However, the control white light has shown 2 folds increase in biomass accumulation (5.78 g l -1)
than red light at day 18 of growth kinetics (figure 4). Furthermore, blue (2.025 g l-1), yellow

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(1.95) and green (1.8 g l-1) lights accumulate significantly similar biomass but comparatively

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lower than control white light. The current results showed that white light is more effective for
callogenesis and biomass accumulation than colored lights. We did not found specific reports on

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the effect of colored lights on biomass accumulation in callus cultures of S. rebaudiana.
However, Tariq et al. [9] observed positive response of white light on callogenesis than other
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colored lights in Artemisia absinthium. Similarly, Ali and Abbasi [11] documented that cell
culture grown under white lights have shown maximum biomass accumulation than colored
lights in Artemisia absinthium. Moreover, various studies confirmed that these responses vary
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considerably depending upon plant species and light quality [13, 34].
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Total phenolics and flavonoids accumulation


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In this study, we evaluated the effect of different colored lights on total phenolics and total
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flavonoids accumulation in callus cultures of S. rebaudiana. Callus cultures grown under blue
lights enhanced the accumulation of TPC (102.32 g/g DW) as compared to control (33.27 g/g
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DW). TPC have shown a positive correlation with TFC production. Blue light enhanced the
accumulation of TFC (22.07 g/g DW) in higher quantities than other colored lights and control
respectively (figure 5). Cultures maintained under green light produced 58.12 g/g DW of TPC
and 12.26 g/g DW of TFC followed by yellow and red lights. Phenolics production in callus
cultures have shown a positive addiction with blue light, then green, yellow, red and control light
as shown in figure 5. The similar pattern was also observed for TFC, which showed a strong
correlation with TPC accumulation. Plants produce different defense responses against various
stresses including biotic and abiotic elicitors [35]. In response to these stresses, plants released a
variety of low molecular weight antioxidant secondary metabolites including phenolics and
flavonoids [36]. Among various physical elicitors, light is well known elicitor that directly
affects the morphology, physiology and production of secondary metabolites during plants

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development [25]. The effect of colored lights on secondary metabolites production in callus
cultures of S. rebaudiana is little known. However, the effect of colored lights on secondary
metabolites production is widely reported in many medicinal plant species [9, 11]. Tariq et al. [9]
reported that callus cultures of A. absinthium maintained under white light accumulate maximum

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content of phenolics and flavonoids than colored lights. Ali and Abbasi [11] also documented
that white light enhanced total phenolics and total flavonoids content in cell cultures of A.

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absinthium. The variation in data may be due plant species and the exposure time to colored

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lights. The transformation efficiency of secondary metabolites also depends on light quality. It
may be possible that blue light enhanced the transformation efficiency to produce higher

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quantities of phenolics and flavonoids in current study.

Phenolics content and its correlation with antioxidant activities


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Here, we observed a strong correlation of phenolics and flavonoids accumulation with
antioxidant activities. As we discussed earlier that blue light enhanced phenolics and flavonoids
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content (figure 6-7). Similarly the blue light enhanced TAC (11.63 g/g DW) as compared to
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-1
control (figure 6). Contrary, green light enhanced reducing power assay (RPA; 0.71 Fe(II) g
DW) as compared to other treatments. The DRSA and TAC have shown maximum dependency
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on phenolics and flavonoids accumulation (figure 6-7). It means that maximum antioxidant
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activities in callus cultures are due to phenolics accumulation. The red lights also influenced the
DRSA (80%) as compared to control. These results suggest that blue and red lights are very
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effective for accumulation of secondary metabolites in callus cultures of S. rebaudiana. Up to


some extent phenolics and flavonoids showed a positive correlation with antioxidant activities.
Many available reports indicated a significant correlation of phenolics production and
antioxidant activities in various medicinal plants [37-40].

Conclusion

Among various elicitors, the application of colored lights is one the important elicitation strategy
to enhance biomass accumulation and production of bioactive compounds. In this study control
white light improved callogenic frequency than other colored lights. The control was followed by
yellow, blue and green lights. During growth kinetics, the red light enhanced biomass

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accumulation but was lower than control cultures. However, the blue light improved phenolics
and flavonoid contents than control. The TPC showed a linear correlation with TFC and total
antioxidant capacity. However, green and red lights enhanced reducing power assay and DRSA.
These results suggest that the application of colored lights is a promising approach for enhanced

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production of antioxidant secondary metabolites.

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Ahmad et al. figures legends

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Ahmad et al. figure 1: Fresh weight, dry weight and extractive values of callus cultures exposed
to different spectral lights. Data was collected from three replications. Mean values ( S.E) with

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common alphabets are significantly different at P < 0.05.

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Ahmad et al. figure 2: Effect of different spectral lights on callus morphological features in S.
rebaudiana (a) red light induced callus (b) blue light (c) yellow light (d) green light and (e)

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control white light.

Ahmad et al. figure 3: Spectral lights induced variation in callogenic frequency (%) from leaf
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explants in S. rebaudiana. Mean data was collected from triplicate experiments after 30 days of
explant incubation. Bars with common alphabets are significantly different at P > 0.05.
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Ahmad et al. figure 4: Spectral lights induced variation in biomass accumulation during growth
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kinetics of callus cultures. Mean data was collected from three triplicates with 3 days interval for
a period of 30 days. Mean values are significantly different at P > 0.05.
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Ahmad et al. figure 5: Effect of different spectral lights on total phenolics and flavonoids
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content in callus cultures of S. rebaudiana. Mean data was collected from three replications. Bars
with common alphabets are significantly different at P > 0.05.
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Ahmad et al. figure 6: Correlation of total phenolics content with antioxidant activities in callus
cultures of S. rebaudiana. Mean data was collected from three replications. Bars with common
alphabets are significantly different at P > 0.05.

Ahmad et al. figure 7: Correlation of total flavonoids content with antioxidant activities in
callus cultures of S. rebaudiana. Mean data was collected from three replications. Bars with
common alphabets are significantly different at P > 0.05.

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5 5
FW

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DW a
30

Extract callus weight (FW-g l )


EW 4 4

-1
Dry callus weight (FW-g l )
-1
Fresh callus weight (FW-g l )
-1

a
b

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3 3
20
b

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c 2 2
c
c
10
c c
c
1 1

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a
b b
0 c
c 0 0

Green Yellow
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Blue Red White

Light treatments
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Figure 1
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a b c d e

Figure 2

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110

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100
a
ab
90

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Callus induction (%)

80 b
b

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70
c

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60

50
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40
Red Blue Yellow Green White

Light treatments
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Figure 3
P
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AC

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Yellow
6 Blue
Red
Green
5 White

Growth kinetics (FW-g/l)


4

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3

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2

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1

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0 3 6 9 12 15 18 21 24 27 30

Culture period (days)


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Figure 4
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P TE
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120 120
TPC
TFC
a
100 100

Total phenolics content (g/g-DW)

Total flavonoids content (g/g-DW)


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80 80

60 b 60
bc

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bc
40 c 40

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a
20 20
b
c c d
0 0

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Green Yellow Blue red White

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Figure 5
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Figure 6
Total phenolics content (g/g-DW) Total phenolics content (g/g-DW) Total phenolics content (g/g-DW)
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0
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80
100
120
0
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TPC
TAC

Green
TPC
TPC

PRA
c

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DRSA

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b

Yellow
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a
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a

Blue
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80

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80
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-20
100
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120

100
Power reducing assay (mol Fe(II)/g-DW) Total antioxidant capacity (g/g-DW) DPPH radical scavenging activity (%)

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Figure 7
Total flavonoids content (g/g-DW) Total flavonoids content (g/g-DW) Total flavonoids content (g/g-DW)
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0
5
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b
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RPA
TFC

TFC
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a
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DRSA

c
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bc
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a

a
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a

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c
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Power reducing assay (mol Fe(II)/g-DW) Total antioxidant capacity (g/g-DW) DPPH radical scavenging activity (%)

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Research Highlights

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White light enhanced callus induction frequency in Stevia rebaudiana.

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White light displayed maximum biomass accumulation in log phases.

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Blue lights enhanced the production of phenolics and flavonoids content.

Blue lights improved total antioxidant capacity.

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Green and red lights promoted reducing power assay and DPPH activity.
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Most of the colored lights enhanced natural antioxidants.
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