Industrial Crops & Products 164 (2021) 113382

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Industrial Crops & Products

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Sucrose-dependent production of biomass and low-caloric steviol

glycosides in adventitious root cultures of Stevia rebaudiana (Bert.)
Naveed Ahmad a, Abdur Rab a, Muhammad Sajid a, Nisar Ahmad b, *, Hina Fazal c,
Mohammad Ali b, Ulrika Egertsdotter d, e, **
a
Department of Horticulture, The University of Agriculture, Peshawar, Peshawar, 25120, Pakistan
b
Center for Biotechnology and Microbiology, University of Swat, Swat, 19200, Pakistan
c
Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex, Peshawar, 25120, Pakistan
d
G.W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 500 Tenth Street NW, 30332-0620, Atlanta, GA, USA
e
Department of Forest Genetics and Plant Physiology, UPSC, Swedish University of Agricultural Science, SE-901 83, Umeå, Sweden

A R T I C L E I N F O A B S T R A C T

Keywords: The market demand for steviol glycosides (SGs) is gradually increasing worldwide due to their low-caloric na­
Stevia ture. SGs are commonly added as a sweetener to various food items and are particularly useful for obese and
Sucrose feeding diabetic patients. Production of SGs is predominantly obtained from the plant Stevia (Stevia rebaudiana). Prop­
Adventitious root cultures
agation of Stevia plants for SGs production is limited due to short-term viability of seeds and high labor demands
Steviol glycosides
Polyphenolics
for shoot multiplication. Adventitious root cultures (ARC) offer an alternative production system to whole plants
for extraction of SGs. The overall objective of this study was to enhance the production of SGs in SRC of Stevia by
differential sucrose feeding (05− 50 g l− 1). A high concentration of sucrose (50 g l− 1) boosted biomass accu­
mulation while a low concentration (10 g l− 1) enhanced biosynthesis of major SGs (stevioside and rebaudioside-
A). A strongly positive correlation was found between biomass accumulation and phenolics and flavonoids
production respectively. Furthermore, 20 g l− 1 to 40 g l− 1 sucrose stimulated dulcoside-A, phenolics, flavonoids
and antioxidant activity. The results suggest that adventitious root cultures can be a viable option for large-scale
production of SGs for industrial applications.

1. Introduction alternatively suppress glycogenesis (Hatting et al., 2018). The heat

Stevia (Stevia rebaudiana) has attracted scientific and economic in­
terests as an alternative sweetener and commonly known as candy leaf
or honey leaf that biosynthesize steviol glycosides (SGs) in the leaves
(Chiew et al., 2016). The leaves of Stevia contain nine major steviol
glycosides: stevioside, rubusoside, steviobioside, dulcoside-A, and
rebaudioside-A, B, C, D, and F (Wölwer-Rieck, 2012). Among these,
dulcoside-A is sweeter than beet and sugar cane (Giri et al., 2014). SGs
are high potency, nontoxic secondary metabolites of potential impor­
tance in biopharmaceutical, food and bevereges industries (Yadav and
Guleria, 2012). SGs extracts, as antihyperglycemic agents (Modi et al.,
2014), can potentially act directly on β- cells and, thereby, enhance
insulin secretion without altering the normal channel activity of cAMP
and K + ATP level in the islets. Improved production of insulin may
stability (100 ◦ C) and its zero glycemic index indicate that it is zero
caloric and it has no toxic effect on human health (Dey et al., 2013).
Stevia has probably been used in traditional medicines; however, the
presence of nutritionally and pharmacologically important compounds
such as steviosides in this species has gained significant attention by
food and pharmaceutical industries (Singh et al., 2019). The medicinal
properties of this plant have recently been reviewed (Ahsan et al., 2020;
Zou et al., 2020; Chughtai et al., 2020; Singh et al., 2019; Ruiz-Ruiz
et al., 2017; Wang et al., 2020) which show its biological activities
including antihyperlipidimic, antihyperglycemic, antioxidant, neph­
roprotective, hypotensive, anti-inflammatory, antitumor, hep­
atoprotective, antimicrobial etc. This plant is the best natural alternative
source for diabetic patients. Many countries have issued administrative
standards to utilize stevia in foodstuffs in daily safe intake levels of

* Corresponding author.
** Corresponding author at: G.W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 500 Tenth Street NW, 30332-0620, Atlanta, GA,
USA.
E-mail addresses: ahmadn@uswat.edu.pk (N. Ahmad), Ulrika.Egertsdotter@me.gatech.edu (U. Egertsdotter).

https://doi.org/10.1016/j.indcrop.2021.113382
Received 18 May 2020; Received in revised form 17 February 2021; Accepted 23 February 2021
Available online 3 March 2021
0926-6690/© 2021 Elsevier B.V. All rights reserved.
N. Ahmad et al.
4.0–4.4 mg/kg (as steviol equivalent) (Wang et al., 2020). It helps to
restores the normal pancreatic functions and nourishes the pancreas.
The stevioside also reduces blood glucose level mostly in type 2 diabetic
patients and approved by FDA as food supplement in USA, Asia and
Europe (Bondarev et al., 2003). Steviosides have been reported to inhibit
tumor promotion, increases the glomerular filtration rate and renal
plasma flow in normal and hypertensive rates. Obesity is one of the
major risk factors, which causes several diseases such as hypertension,
hyperlipidemia, diabetes, surgical risks, pulmonary and renal problems,
pregnancy complications and certain type of cancer. The administration
of Stevia or its metabolites could be one of the effective strategies in the
management and prevention of obesity and diabetes (Farhat et al.,
2019). Althoug debate exists about the benefits and potential side effects
of Stevia, many studies have recommended it as a safer alternative to
sugar for normal metabolic health (Wang et al., 2020).
The conventional methods of SGs production from Stevia plants is
hindered by poor propagation practices, multiple diseases, environ­
mental variations, reduced plant development and compromised
biosynthesis of secondary metabolites (Ahmad et al., 2014). Different in
vitro plant tissue culture techniques have been tested as production
systems for SGs (Dey et al., 2013; Aman et al., 2013). As compared to
field-grown plants, the development of adventitious root cultures pro­
vide a feasible platform for production of lagre-scale biomass and sec­
ondary metabolites. The production in adventitious cultures is uniform
and sometimes over-produces the metabolites of interests than intact
plants (Ali et al., 2013). Notably it has been demonstrated that biotic
and abiotic elicitors can increase the yields of bioactive compounds in
root cultures (Srivastava et al., 2016). Sucrose, pH, humidity, temper­
ature and light fluctuations also enhanced the biosynthesis of
high-valued bioactive compounds in cultures in vitro of many medicinal
species (Tariq et al., 2014; Ali and Abbasi, 2014; Barz et al., 1988; Ali
et al., 2016; Khan et al., 2017; Eilert, 1987).
An adventitious root culture (ARC) system is one of the major sources
of stimulated secondary metabolites production in plants (Dubrovsky
and Rost, 2003; Goel et al., 2009; Hao et al., 2020). Adventitious roots
are of particular interest due to their fast growth and consistency of
secondary metabolites production, compared to cell cultures (Sivaku­
mar, 2006). Additionally, ARCs are preferred over hairy root cultures
due to their simplicity and safety (Gaosheng and Jingming, 2012).
Cultivation of adventitious roots in bioreactor for plant bioactive com­
pounds has evolved as a technology of commercial importance (Paek
et al., 2009). Many pharmacologically active compounds including
anthraquinone and ginsenosides have been previously achieved by
exploiting ARC of Rubia tinctorum, Panax ginseng and Morinda citrifolia
(Sato et al., 1997; Kevers et al., 1999; Baque et al., 2010).
Various physiological processes are affected by sugars during growth
and development of plants (Businge and Egertsdotter, 2014). Never­
theless, specific forms of sugar control the biochemical pathways and
metabolic system in wild species as well as in aseptic cultures of plants
and play a key role in architecture development of various plant tissues
and one of the major carbon energy sources for various procesess (Cal­
amar and De-Klerk, 2002; Fazal et al., 2014). Apart from its role as
signaling molecule in sugar-response pathways, sucrose may be sensed
by other metabolic pathways as well (Baque et al., 2012). The sucrose
induced up-regulation of the genes coding for biosynthetic enzymes of
flavonoids has already been reported in the model plant Arabidopsis
(Solfanelli et al., 2006). Exposure of plants to stress induce the defense
mechanism and release toxic reactive species as well as non-toxic
polyphenolics to cope with stressers (Ahmad et al., 2014; Baque et al.,
2012; Lee et al., 2006). Higher sucrose treatments can potentially
up-regulate the synthesis of bioactive compounds, like trans-caffeic
acids (Kim et al., 2011a). Sucrose-induced production of industrially
important compounds and other antioxidants are widely documented in
previous reports (Ali et al., 2013; Ferri et al., 2011).
Notwithstanding, with previous reports on field propagation-
methods of Stevia and its micropropagation (Sharma et al., 2016;
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Industrial Crops & Products 164 (2021) 113382
Singh et al., 2017; Bhingradiya et al., 2019), limited information is
available on the effects of various elicitors on establishment of adven­
titious rooting system for large-scale production of SGs and others me­
tabolites. The current report was aimed to test the effect of differential
sucrose feeding on growth parameters of adventitious roots (fresh and
dry biomass, growth kinetics), biosynthesis of secondary cell products
(polyphenolics, Steviol glycosides) as well as antioxidant potential for
various industrial applications.
2. Materials and methods
2.1. Adventitious root culture development
Fresh viable seeds of Stevia rebaudiana (Paraguay) were procured
from the nursery of the University of Agriculture, Peshawar, in April
2015. The black-coated seeds were grown in vitro for stock plantlets
development following the method of Aman et al. (2013). From
seed-derived plantlets, 1− 2 cm root segments (4 roots) were excised
after 30 days of seed inoculation and transfered to 100 mL Erlenmeyer
flasks supplemented with full strength (FS; 35 mL) and half strength (HS;
35 mL) liquid Murashige and Skoog (MS) (1962) media. Both the HS-MS
and FS-MS media were augumented with various concentrations of NAA
or IBA (0.5, 1.0, 1.5, 2.0 mg l− 1). Plant growth regulators (PGRs)-free
medium (MS0) was used as control. The cultured flasks were placed onto
orbital shaker at 120 rpm for 30 days at 25 ◦ C in dark.
The establishment of adventitious roots under different sucrose
concentrations (5− 50 g l− 1; increment 5 g l− 1) was investigated by
inoculating approximately 20 g of root segments per litre of liquid HS-
MS media, supplemented with 0.5 mg l− 1 NAA and each sucrose con­
centration. Each treatment was carried out in 30 flasks (100 mL each;
33− 35 ml media). The cultures inoculated with 0.5 mg l− 1 NAA and 30 g
l-1 sucrose was considered control. The samples were placed on an
orbital shaker (Gallenkamp, England) in darkness for 30 days to estab­
lish homogenous cultures. The contents of 3 flasks of each treatment
were used to determine fresh biomass (FB) and dry biomass (DB) with an
increment of 3-day for a total 30-day culture period; 2,2-diphenyl-1-pic­
rylhydrazyl (DPPH) radical scavenging activity (DRSA), polyphenolics
and steviol glycosides were investigated at the end of log phase after 18
days.
2.2. Measurements of biomass
For fresh biomass determination and establishment of growth ki­
netics at 3-day interval (30-day culture), the stevia root contents were
removed from 3 flasks at each time-point (10 time-points), were care­
fully washed with autoclaved distilled water and excess water was
minimized using sterilized filter paper before weighing. The samples
were subsequently dried for 24 h at 50 ◦ C to determine dry biomass. A
growth curve was established based on the accumulated fresh biomass
during the lag phases, log phases and stationary phases of growth
kinetics.
2.3. Extract preparation
For extraction, 20 mg of each dried sample obtained in response to
various treatments was ground to yield fine powder and transferred to a
falcon tube containing HPLC grade ethanol (20 mL). All samples were
incubated at 4 ◦ C for 7 days with regular vortexing and finally centri­
fuged (5000 rpm for 10− 15 min) to obtained supernatant for the anal­
ysis of secondary metabolites as described below.
2.4. Estimation of total phenolic content
The total phenolic content (TPC) in the treated adventitious roots
was investigated using the method of Singleton and Rossi (1965) with
minor modification. Briefly, 0.1 mL of each sample extract was mixed
N. Ahmad et al. Industrial Crops & Products 164 (2021) 113382

with 2.5 mL of 2 N Folin-Ciocalteus reagent (Sigma Aldrich) for 4 min,
and then 2 mL of 20 % Na2CO3 was added to the mixture. The reaction
mixtures were kept in dark for 2 h and absorbance was taken at 765 nm
on a spectrophotometer (Shimadzu-1650, Japan). A calibration curve
was established using different concentrations of a commercial standard
of gallic acid (GA; Sigma; 1.0− 100 μg/mL, R2 = 0.9878). The total
phenolic contents of the root extracts were expressed in GA equivalent-
mg/g-DW.
Total phenolic content = (AR-AB) / (CFr × DFr)
DRSA. Accuratly, 2.0 mL of treated roots extract was combined with 4.0
mL DPPH solution in a 15 mL falcon tube and finally placed in in dark for
30 min to avoid unwanted reaction. The absorbance of each treated
sample was checked at 517 nm. The DRSA was shown in % DRSA using
the following equation
DRSA (%) = 100 × (1 – ARE/AFR)
Where ARE represents absorbance of root extracts and AFR represents
absorbance of free radical solution.

Where AR represents root extract absorbance, AB represents absorbance

of blank, CFr represents conversion factor and DFr represents dilution
factor.
The values for total phenolic production (TPP) were calculated by
multiplying total phenolic contents and corresponding dry biomass
profiles as following:
Total phenolic production (mg/l) = Total phenolic content (mg/g) × Dry
biomass (g/l)
2.5. Estimation of total flavonoid content
The total flavonoids content (TFC) in adventitious roots was inves­
tigated using the method of Ahmad et al. (2014) with minor modifica­
tion. Briefly, each 0.25 mL root extract was mixed with 0.2 mL AlCl3 (5%
w/v) and each mixture was diluted with ethanol to make a final volume
of 25 mL. To prepare blank sample, each 0.25 mL of root extract was
mixed with a single drop of acetic acid and a final volume of 25 mL was
prepared by dilution. The absorbance of all samples was recorded at 415
nm after 40 min. The same procedure was used to prepare rutin cali­
beration curve (1.0–100 μg/ml, R2 = 0.9866) in ethanolic solutions.
Results of flavonoids were presented as Rutin-equivalent-mg/g-DW.
The values for total flavonoid production (TFP) were calculated by
multiplying total flavonoid contents and corresponding dry biomass
profiles as following:
Total flavonoid production (mg/l) = Total flavonoid content (mg/g) × Dry
biomass (g/l)
2.7. Quantification of steviol glycosides
The quantification of SGs (stevioside, rebaudioside-A and dulcoside-
A) was achieved by following the method of Aman et al. (2013). The
HPLC system used in this study was Perkin-Elmer HPLC (Series 200)
system (USA) with 150 × 4.6 mm C-18 column (ODS). During quanti­
fication, the mobile phase was used as water (HPLC grade-A; 25 %) and
acetonitrile (B; 75 %; Sigma Aldrich). The injection volume was kept 10
μL and injected to the system at 1.0 mL min− 1 flow rate. The SGs stan­
dard (Sigma, USA), prepared as 200 μg/ml in HPLC grade water, was run
on the HPLC system for determination of the retention time of stevio­
sides, rebaudioside-A and dulcoside-A. The contents of SGs were
determined by comparing their relative retention times with the reten­
tion time of standard, and were expressed as mg/g of dry weight.
The values for steviol glycosides production were calculated by
multiplying steviol glycosides contents and corresponding dry biomass
profiles as following:
Steviol glycosides production (mg/l) = Steviol glycosides content (mg/g) ×
Dry biomass (g/l)
2.8. Statistical analysis
Completely Randomized Design (CRD) was used during the
deveoplment of ARC of Stevia and each sucrose-mediated treatment was
repeated rwice in triplicates. One-way ANOVA (Analysis of variance) for
mean values, least significant differences (LSD) and standard errors (±
SE) was determined Statistix software (8.1 versions). The graphical
presentation of mean values was made using Origin Lab (8.5) software.

2.6. Antioxidant activity

3. Results and discussion
The free radical 2,2-Diphenyl-1-Picryl Hydrazyl (DPPH) was used for
Generally, the quantitatively compromised accumulation of medic­
the investigation of antioxidant activity as DPPH-radical scavenging
inally important secondary metabolites in wild plants is one of the major
activity (DRSA) in sucrose-treated adventitious roots of S. rebaudiana.
limitations, impeding the utilization of wild medicinal plants as the only
The protocol of Ahmad et al. (2010) was used for the determination of
source to meet the huge industrial demand (Ali et al., 2017). The
problem that more than 70 % drugs isolated from wild plants are prone
Table 1
to destructive harvesting (Ved et al., 1998) may be solved by biotech­
Pearson correlation coefficient (r) of secondary metabolites with dry weight
nological approaches, such as micropropagation and cell or root cultures
(DW) and DPPH radical scavenging activity (DRSA).
(Borroto et al., 2008). Conventional propagation practices are not
Parameters Pearson r
considered ideal for Stevia due to limited availability of healthy mother
Total Phenolic Content 0.701 plants (Khalil et al., 2014), low germination frequency of seeds
Total phenolic production 0.9133 (Abdullateef et al., 2015), fungal diseases such as powdery mildew and
Total flavonoid content 0.5522
Total flavonoid production 0.9106
root rot (Basharat et al., 2020), environmental variations and reduced
Stevioside content − 0.1990 plant development and secondary metabolites (Ahmad et al., 2014). In
Dry weight vs:
Stevioside production 0.8255 vitro culture technonology has been reported superior over field-grown
Rebaudioside content − 0.3053 Stevia plants for enhanced production of steviol glucosides (Gupta et al.,
Rebaudioside A production 0.7681
2015, 2010). Furthermore, the high density growth and accumulation of
Dulcoside content 0.3029
Dulcoside production 0.3753 secondary metabolites in root cultures make them suitable system for
Total phenolic content 0.4453 bioreactor scale culture (Wilson, 1997). The superiority of biotechno­
DPPH Radical scavenging activity vs:
Total phenolic production 0.3939 logical methods, including adventitious root cultures, for enhanced
Total flavonoid content 0.6379 steviol glycosides biosynthesis has been excellently reviewed (Modi and
Total flavonoid production 0.5211
Kumar, 2018; Basharat et al., 2020; Kazmi et al., 2019).

3
N. Ahmad et al. Industrial Crops & Products 164 (2021) 113382

Fig. 1. Influence of differential sucrose feeding on morphological features of adventitious roots as 05 (a), 10 (b), 15 (c), 20 (d), 25 (e), 30 (f), 35 (g), 40 (h), 45 (i)
and 50 g l− 1 (j) in Stevia rebaudiana.

3.1. Establishment of adventitious root culture system observed when HS-MS media was supplemented with NAA (0.5 mg l− 1).
In contrast, other concentrations of NAA reduced calli-induced devel­
Fresh and viable roots obtained from seed-derived plantlets were opment of adventitious roots in Stevia. The 0.5 mg l− 1 NAA induced-
transferred to HS and FS-liquid MS media augumented with various adventitious roots were exploited for stock ARC development and sub­
concentrations of either IBA or NAA (0.5–2.0 mg l− 1) alone for devel­ sequent experiments (Table 1).
opment of adventitious roots in S. rebaudiana. Different concentrations
of IBA (0.5–2.0 mg l− 1) were found least effective in terms of adventi­
tious rooting in both HS and FS-MS media (Table 1, Supplementary).
However, maximum adventitious roots development (90 %) was

4
N. Ahmad et al.
Fig. 2. Influence of 05, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l− 1 sucrose on
growth kinetics and biomass accumulation in adventitious root cultures of
Stevia rebaudiana. Mean difference of three replicates of each sample was
analyzed by ANOVA using Statistix 8.1 software (USA) and mean values with
SE (±) are significant (p ≤ 0.05).
3.2. Effect of sucrose on root culture morphology and biomass
accummulation
Root morphology (Fig. 1) and biomass production in adventitious
root cultures of S. rebaudiana were highly dependent on sucrose con­
centrations. The roots formed in response to lower concentrations of
sucrose (5− 15 g/l) were whitish in colour, loosely arranged and less-
branched. Higher sucrose concentrations (20− 50 g/l) induced roots
were yellowish, dense and highly branched. Growth curves based on
fresh biomass accumulation were characterized through log phase upto
18th day of roots inoculation (Fig. 2). Here, the least growth accumu­
lation was observed when inoculum roots were exposed to 5.0 g l− 1 and
10 g l− 1 sucrose, that displayed 15 days lag phase, followed by a stunted
log phase. The lowest sucrose augemnetation (5 g l− 1) produced the least
FB and DB (37.71 g l− 1 and 2.86 g l− 1; Fig. 3). The highest FB and DB
(174.42 g l-1 and 11.14 g l− 1) at 18th day of log phase was observed in
Fig. 3. Influence of 05, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l− 1 sucrose feeding on both fresh and dry biomass biosynthesis in Stevia rebaudiana adventitious roots.
Replicated mean values including standard errors represented by alphabets are non-significant at P ≤ 0.05.
5
Industrial Crops & Products 164 (2021) 113382
liquid media containing the highest sucrose concentration (50 g l− 1),
followed by stationary phase at 24th day of culture (Fig. 2). Interest­
ingly, the second-best biomass accumulation was displayed by the
control cultures at 30 g l− 1 sucrose, which was higher than the biomass
obtained in response to 35− 45 g l− 1. The ARC exposed to 35− 45 g l− 1
sucrose displayed a stationary phase (18–24 days), followed by a decline
in growth (Fig. 2).
It has been observed that lower sucrose augmentation to culture
media is not suitable for biomass biosynthesis in cell suspension cultures
of Artemisia absinthium (Ali et al., 2016) and Prunella vulgaris (Fazal
et al., 2016a). Cui et al. (2010) previously observed biomass increase by
the augmentation of 50 g l− 1 sucrose in ARC of Hypericum perforatum.
The current results are in agreement with the report of Wu et al. (2006),
who observed elevated biomass accumulation by the addition of 30 g l− 1
and 50 g l− 1 sucrose to ARC of Echinacea angustifolia. The incremental
growth of adventitious roots may be due to higher sucrose demand for
growth at the differentiation phase and structural integrity (Tremblay
and Tremblay, 1995).
3.3. Total phenolics and flavonoids and their correlation with biomass
In this study, the sucrose concentration in the culture media was
correlated with phenolics (TPC) and flavonoids (TFC) biosynthesis in
adventitious roots of S. rebaudiana. The maximum TPC and TFC (155.00
mg/g DW and 94.78 mg/g-DW) were exhibited by the adventitious roots
exposed to 30 g l− 1 sucrose concentration. However, further increase in
sucrose augmentation lead to a linear decrease in the biosynthesis of
phenolics and flavonoids. The culture media augmented with 05 g l-1
sucrose (lowest level) displayed least biosynthesis of TPC and TFC
(17.77 and 23.20 mg/g-DW). As evident by previous reports, signifi­
cantly lower levels of phenolics and flavonoids in ARCs of Stevia in
response to different treatments have been reported (Alvarado-Orea
et al., 2020; Kazmi et al., 2019; Ahmad et al., 2020; Idrees et al., 2018;
Ghazal et al., 2018; Ahmad et al., 2018). However, the elevated levels of
phytochemicals obtained in the current study may be attributed to
different explants source, inoculum age and differential treatments. The
production of total phenolics and total flavonoids, as previously re­
ported by Ali and Abbasi (2014) for suspension cultures of Artemisia
absinthium, was found in strongly positive correlatation with biomass
N. Ahmad et al.
Fig. 4. The dry biomass (g l− 1) correlation with: a) total phenolic content (mg Gallic acid/ gram dry weight) and total phenolic production (mg Gallic acid/ litre
dry weight).
accumulation, suggesting the feasibility of biomass-dependent produc­
tion of phenolics and flavonoids in adventitious root cultures of Stevia
for commercial production (Fig. 4).
Cultured cells have been shown to have a higher potential than intact
plants to accumulate important metabolites like polyphenols and fla­
vonoids (Ali et al., 2013). This may be attributed to increased stress from
the in vitro culture conditions, triggering higher production of such
stress-related compounds. Media components, like sucrose, are funda­
mental for higher yield in cultures and their addition has shown to in­
fluence the developmental and metabolic processes in plants through
the expression of specific genes (Koch, 1996; Lee and Shuler, 2000). The
current study revealed that maximum biosynthesis of phenolics and
flavonoids was achieved in ARC with the incremental sucrose addition
in culture media. It has been previously observed that fed-batch cultures
(sucrose regulated) of Panax notoginseng and Artemisia absinthium dis­
played maximum biomass gain and biosynthesis of metabolites of ine­
terst than batch cultures of these species (Zhang and Zhong, 1997; Ali
et al., 2016). Furthermore, carbohydrate sources like sugar enhanced
the flavonoids biosynthesis by activating the enzymatic activity of
phenylalanine ammonia-lyase, which play a key role in
6
Industrial Crops & Products 164 (2021) 113382
phenypropanoids biosynthesis (Wong et al., 1974; Thimann et al.,
1950). The current results are in agreement with previous reports where
higher sucrose concentration stimulated the biosynthesis of flavonoids
in the in vitro cultures of high-valued medicinal plants (Tan et al., 2010;
Antognoni et al., 2007).
3.4. Radical scavenging activity and its correlation with phenolics and
flavonoids
The presence of natural antioxidants in wild plants and plant-based
cultures make them potent scavengers of toxic reactive oxygen species
(Ahmad et al., 2010). Stevia and other plants have widely been tested for
their in vitro antioxidant efficacy which is attributed to the presence of
polyphenols. (Kim et al., 2006; Shukla et al., 2009). Previously, several
studies have reported positive correlation of biological activities (anti­
oxidant) and secondary metabolites (polyphenolics) in Stevia (Tadhani
et al., 2007; Kim et al., 2011b; Rao et al., 2014). Moreover, Shukla et al.
(2012) and Criado et al. (2014) have described the role of SGs in the
antioxidant potential of S. rebaudiana. The current results revealed that
DRSA in ARC of Stevia was not found to be significantly affected by
N. Ahmad et al.
Fig. 5. Influence of differential sucrose feeding on biosynthesis of polyphenolics content and antioxidant activity in adventitious root cultures of Stevia rebaudiana.
Mean difference of three replicates of each sample was analyzed by ANOVA using Statistix 8.1 software (USA) and mean values with SE (±) are significant (p ≤ 0.05).
differential sucrose feeding; however, lowest sucrose (5 g l− 1)
augmented roots displayed exceptionally least response. The root cul­
tures supplemented with 5 g l− 1 and 20 g l− 1 sucrose exhibited lowest
(46.5 %) and highest (94.43 %) DRSA, respectively (Fig. 5).
With increase in sucrose concentration, phenolics and flavonoids
accumulation was significantly increased in ARC of S. rebaudiana
(Fig. 5). The addition of 30 g l− 1 sucrose to culture media enhanced the
bioaccumulation of total phenolics and flavonoids in higher levels
(155.00 mg/g-DW and 94.78 mg/g-DW). However, the addition of 20 g
l− 1 sucrose enhanced the DRSA (94.43 %), as compared to other sucrose
concentrations (Fig. 5). In this study, DRSA was not found to be stricktly
dependent on phenolics and flavonoids, suggesting the involvement of
antioxidant metabolites other than phenolics and flavonoids, as has
previously been reported for cell suspension cultures of Artemisia
absinthium (Ali et al., 2014) and callus cultures of Prunella vulgaris (Fazal
et al., 2016b).
3.5. Effect of differential sucrose feeding on biosynthesis of steviol
glycosides
In this study, 10 g l− 1 sucrose induced the highest levels of SGs,
including stevioside (73.97 mg/g-DW) and rebaudioside-A (24.57 mg/g-
DW), in ARC of Stevia. A prominent reduction in stevioside and
rebaudioside-A (25.58 mg/g-DW and 10.02 mg/g-DW) was noted with
20 g l− 1. Nonetheless, the SGs (stevioside and rebaudioside-A) again
linearly increased with increasing sucrose concentrations up to 40 g l− 1,
followed by a subsequent reduction at 45− 50 g l− 1 sucrose (Fig. 6a, 6b).
Furthermore, the highest dulcoside-A content (12.24 mg/g-DW) was
observed in root cultures exposed to 40 g l− 1 sucrose (Fig. 6c). Inter­
estingly, the current study reports highest levels of SGs in ARCs of Stevia
in response to differential sucrose feeding compared to other published
studies which may be attributed to difference in initial explants source,
plant growth regulators, nutrient composition and culture conditions.
For instance, Kazmi et al. (2019) have shown maximum stevioside and
rebaudiosides contents as 4.2 ± 0.18 mg/g-DW and 6.5 ± 0.34
mg/g-DW, respectively, in leaf explants derived ARC of Stevia. Simi­
larly, Alvarado-Orea et al. (2018) have reported 50.8 μg/g-DW of SGs
(stevioside + rebaudioside A) in ARC of Stevia in response to hydrogen
peroxide elicitation. In one of our previous studies, highest levels of
stevioside (79.48 mg/g-DW) and rebaudioside A (13.10 mg/g-DW) were
7
Industrial Crops & Products 164 (2021) 113382
detected in ARC of Stevia with pH level 5.1. These contrasting results
suggest the role of differential molecular events involved in SGs
biosynthesis in response to varying explants source and culture condi­
tions. Overall, stevoiside and rebaudioside-A contents showed negative
correlation with sucrose-induced biomass accumulation (correlation
coefficients: SC and DW= -0.199, RC and DW= -0.305); however, ste­
vioside and rebaudioside-A production displayed strongly positive cor­
relation with biomass accumulation in response to increase in sucrose
concentration (correlation coefficients: SP and DW = 0.826, RP and DW
= 0.768) (Fig. 6).
The biosynthesis of SGs is however not proportional to the biomass
increase and has an optimum at 10 g l− 1 sucrose for stevioside and
rebauside-A, and sucrose 40 g l− 1 for dulcoside-A, resulting in the
highest measured total stevioside production (>70 mg/g-DW) in root
cultures as compared to control steviosides/g DW (40 mg/g-DW). This is
comparable to production of steviol glycosides in recent report of
adventitious root cultures of Stevia (Ahmad et al., 2018, 2020).
Furthermore, it was demonstrated that the total contents of phenolics
and flavonoids are proportionally stimulated by increased sucrose con­
centration in the medium from 5 to 30 g l-1. The DPPH- scavenging was
not affected by the sucrose content above 5 g l-1, indicating that com­
pounds other than phenolics and flavonoids may be responsible for the
DPPH- scaneving effect. The levels of DPPH scavenging were relatively
high/low compared to control cultures.
The industrial and pharmacological importance of medicinal species
mostly depends on the quality and quantity of extractable levels of high-
valued active compounds (Selmar and Kleinwächter, 2013). It is,
therefore, mandatory to optimize and comprehened the role of
stress-mediated elicitors in the production of bioactive secondary cell
products such as altering growth conditions, light, sucrose, pH and many
other physical or chemical inducers (Vasconsuelo and Boland, 2007;
Zhao et al., 2005). Fowler (1983) has previously reported that sucrose
augmentation to culture media play a key role in the biosynthesis of
secondary metabolites in many medicinal species. The current results
revealed that changing sucrose concentration in the culture media alters
the biosynthesis and production of SGs (stevioside, rebaudioside-A and
dulcoside-A) in ARC of Stevia. Yadav and Guleria (2012); Richman et al.
(1999) and Brandle and Telmer (2007) broadly identified the enzymes
and genes responsible for SGs biosynthetic pathways and further studied
steviol glycoside production. Transcript expression profiles of genes
N. Ahmad et al. Industrial Crops & Products 164 (2021) 113382

Fig. 6. Influence of differential sucrose feeding on biosynthesis of steviol glycosides content and production in adventitious root cultures of Stevia rebaudiana: (a)
stevioside, (b) rebaudioside-A and (c) dulcoside-A. Mean difference of three replicates of each sample was analyzed by ANOVA using Statistix 8.1 software (USA) and
mean values with SE (±) are significant (p ≤ 0.05).

8
N. Ahmad et al.
involved in the steviol glycosides biosynthesis pathway (CDPS, KS, KO,
UGT85C2, UGT76G1) increased in a dose-dependent manner and a
maximum increase was observed in response to 5% sucrose treatment
(Guleria et al., 2011).
Sucrose in the culture medium plays a dual role as carbon source and
as osmotic agent. It has distinct affects on morphological and
biochemical pathways. Furthermore, the cell lines of different species
may respond distinctly to same sucrose treatment (Ali et al., 2016).
Generally, sucrose induces normal growth and development at op­
timum concentration (3%) in cultured cells; however, it seems to alter
biosynthetic pathways involved in secondary metabolism indepen­
dently. Inhibition of biomass at higher sucrose concentrations might be
due to higher osmotic pressure (Baque et al., 2012), resulting in poor
nutrient uptake due to high medium viscosity (Suan See et al., 2011).
Wu et al. (2006) reported that the addition of 50 and 90 g/l sucrose
enhanced biomass of adventitious roots cultures in Echinacea angustifolia
but reduced the production of secondary metabolites, as has been
described in our study.
In this study, SGs biosynthesis in ARC of Stevia was found to be in­
dependent of biomass accumulation in response to higher sucrose con­
centration which suggests the involvement of independent sugar-
responsive genes for biomass accumulation. Moreover, the interaction
of endogenous and exogenous sucrose concentration may also affect SGs
gene expression profiles (Guleria et al., 2011). In accordance with our
results, Ghorbani et al. (2017) have also found lower sucrose concen­
tration (100 mM) desirable for SGs biosynthesis, following higher
expression of UGT76G1 and UGT85C2 genes in the pathway.
4. Conclusions
In conclusion, considerable variations were observed in biomass
accumulation and secondary metabolites production in response to
differential sucrose feeding. It is suggested that HS-MS media
augmented with 0.5 mg l− 1 NAA was the most suitable medium for
maximum development (90 %) of adventitious root cultures in Stevia.
The media containing similar NAA concentration along with 5− 15 g l-1
sucrose induced whitish and less-branched roots while 20− 50 g/l su­
crose concentrations induced yellowish, dense and highly branched
adventitious roots. In conclusion, ARC of Stevia can offer an alterntive
culture system for biomass-dependent improved biosynthesis of steviol
glycosides and industrially valuable phenolics and flavonoids, due to
stimulated biomass accumulation in response to higher concentrations
of sucrose.
CRediT authorship contribution statement
Naveed Ahmad: Formal analysis, Data curation, Writing - original
draft. Abdur Rab: Supervision. Muhammad Sajid: Validation. Nisar
Ahmad: Conceptualization, Methodology. Hina Fazal: Formal analysis.
Mohammad Ali: Software, Validation. Ulrika Egertsdotter: Writing -
review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2021.113382.
9
Industrial Crops & Products 164 (2021) 113382
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