Analysis of metabolic variations

throughout growth and development of

adventitious roots in Silybum marianum L.
(Milk thistle), a medicinal plant
Mubarak Ali Khan, Bilal Haider Abbasi,
Naseer Ali Shah, Buhara Ycesan &
Huma Ali
Plant Cell, Tissue and Organ Culture
(PCTOC)
Journal of Plant Biotechnology
ISSN 0167-6857
Volume 123
Number 3
Plant Cell Tiss Organ Cult (2015)
123:501-510
DOI 10.1007/s11240-015-0854-8

1 23

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Plant Cell Tiss Organ Cult (2015) 123:501510
DOI 10.1007/s11240-015-0854-8

ORIGINAL ARTICLE

Analysis of metabolic variations throughout growth

and development of adventitious roots in Silybum marianum L.
(Milk thistle), a medicinal plant
Mubarak Ali Khan1 Bilal Haider Abbasi2 Naseer Ali Shah3 Buhara Yucesan4
Huma Ali5

Received: 20 May 2015 / Accepted: 13 August 2015 / Published online: 21 August 2015
Springer Science+Business Media Dordrecht 2015

Abstract Silybum marianum L. is a medicinal plant used in

the treatment for jaundice and liver diseases. In this study, an
adventitious root culture was developed for the production of
health promoting phytochemicals. Adventitious roots were
induced from nodal explants on solid Murashige and Skoog
medium supplemented with 1.0 mg l-1 of a-Naphthalene
acetic acid. Growth kinetics of the roots was investigated
every week, for 8 weeks of culture period. Highest fresh
biomass formation (153 mg l-1) was observed in 6-week old
cultures. Adventitious roots were harvested from different
growth stages as control (CTR), lag phase (LAG), logarithmic phase (LOG) or stationary phase (STN). Metabolite
profiling of the samples was investigated using electro spray
ionization time of flight mass spectrometry. Significant

phenylpropanoids such as cinnamic acid and di-hydro

kaempferol were predominantly found in LOG phase,
whereas the highest amount of malonic acid was detected in
STN as compared to other growth phases. More sucrose
content was detected in CTR, while the tryptophan content
was higher in LOG phase. Among the vital fatty acids,
prostaglandin A1 and phenyl acetic acid were at highest
levels in STN phase. However, more brassicasterols were
observed in LAG phase than other growth phases. Punicic
acid and lignan pinoresinol were detected abundantly in the
LOG phase. Biochemical characterization revealed significant correlations between silymarin content and DPPH as
well as TPC and TFC in the growth curve. Interestingly,
among all growth stages there was no correlation of PAL
activity with TFC and silymarin content.

Electronic supplementary material The online version of this

article (doi:10.1007/s11240-015-0854-8) contains supplementary
material, which is available to authorized users.

Keywords Silybum marianum L.
Silymarin

Adventitious roots
Mass spectrometry
Plant secondary
metabolites

& Mubarak Ali Khan

makhan@ciit.net.pk
& Bilal Haider Abbasi
bhabbasi@qau.edu.pk
1

Biotechnology Program, Department of Environmental

Sciences, COMSATS Institute of Information Technology
(CIIT), Abbottabad, Pakistan

Department of Biotechnology, Quaid-i-Azam University,

Islamabad 45320, Pakistan

Department of Biosciences, COMSATS Institute of

Information Technology, Islamabad, Pakistan

Department of Seed Science and Technology, Faculty of

Natural and Agricultural Sciences, Abant Izzet Baysal
University, 14280 Bolu, Turkey

Department of Biotechnology, Bacha Khan University,

Charsadda, KP, Pakistan

Abbreviations
PGR
Plant growth regulator
TPC
Total phenolic content
TFC
Total flavonoid content
PAL
Phenylalanine ammonia lyase
FRSA Free radical scavenging activity
TOF
Time of flight
ESI
Electro spray ionization
PCA
Principal component analysis

Introduction
Silybum marianum (L.) Gaertn. (milk thistle) belongs to
Asteracea family is an important medicinal herb used in
treatment of liver diseases with a history of use spanning

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502

more than centuries (Khan et al. 2013). It is of a great

value for its unique compound called silymarin having
several pharmacological activities including anti-inflammatory, anti-hepatitis, anti-viral and antioxidant (Khan
et al. 2014). Silymarin is an isomeric flavonoid mixture
containing silybin, silibinin, silidianin and silychristin
isolated from milk thistle (Abbasi et al. 2010). These
flavanolignans are well known for their antioxidant
capacity due to their redox properties, and it has been
assumed that a diet rich in flavonoids is inversely proportional to cell aging, lipid peroxidation and cancer
(Ferreira et al. 2010). Unfortunately, the quality and
quantity of these metabolites are strictly species dependent; thus show a high diversity with respect to the
physiological age, plant tissue type and environmental
conditions (Khan et al. 2015). The biotechnological production of the natural compounds is an attractive
approach, alternative to the conventional extraction protocols using whole plant material without disturbing natural habitats (Ali et al. 2013). Especially, plant cell and
organ culture systems provide an insight through the
production of value-added plant-specific metabolites
(Ahmad et al. 2014). Establishment of adventitious root
culture can serve as a promising source of nutritionally
and pharmaceutically important metabolites. The main
advantages of adventitious root culture are faster growth
rate, easier maintenance and handling with high homogeneity in genetic makeup (Nagarajan et al. 2011). It has
also advantageous over hairy root culture since adventitious roots do not produce any toxic chemicals like opines
(Cui et al. 2010). Flavonoids have a plethora of diverse
functions in plants ranging from physiological processes
such as pigmentation, seed coat and pollen tube development to the plant responses against abiotic and biotic
stresses (Stracke et al. 2007; Misra et al. 2010). Moreover,
due to their health-promoting effects including antioxidative, anticancer, anti-inflammatory, cardioprotective,
and neuroprotective activities in mammalian tissues, flavonoids are also considered to be of pharmaceutical
interest (Ferreira et al. 2010).
Electro spray ionization (ESI) mass spectrometry is a
rapid, highly sensitive and soft ionization technique which
mainly produces the protonated molecular species for a
wide range of compounds; thereby making data interpretation easier (Smedsgaard and Nielsen 2005). During
adventitious root cultivation, morphogeny of the cells in
each growth phase as mentioned in this study is likely
influenced by expression of some hardly visible key
metabolites; thus, phytochemical profiling seems crucial
for our understanding on metabolic pattern of the root
growth.

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In the present study, we established adventitious root

culture system, and investigated the key metabolites
responsible for the growth of adventitious roots through
ESI/TOFMS analysis. Furthermore, total phenolic and
flavonoid contents, antioxidant potential, PAL activity and
silymarin content were also evaluated during the growth
cycle of adventitious roots of milk thistle.

Materials and methods

In vitro seed germination
Mature seeds of S. marianum were collected from the main
campus of Quaid-e-Azam University Islamabad in 2012.
The seeds were rinsed quickly with 70 % (v/v) ethanol for
5 min prior to surface sterilization with 0.1 % (w/v) freshly
prepared mercuric chloride solution (HgCl2) for 3 min.
After rinsing three times with sterile distilled water, all the
seeds were placed into germination medium as described in
Khan et al. (2013) under in vitro conditions.
Adventitious root induction
Leaf explants (*25 mm2), cotyledon explants (*25 mm2),
root segments (*5 mm) and nodal explants (*5 mm) were
excised from 4 weeks old in vitro germinated seedlings, and
were cultured on solid MS (Murashige and Skoog 1962)
medium supplemented with 0.5 mg l-1 NAA in Petri plates.
Due to high morphogenetic potential, nodal explants were
selected for subsequent experiments. To study the effects of
plant growth regulators on adventitious root formation,
nodal explants were incubated on MS media containing
4.0 % sucrose (w/v) and 0.8 % (w/v) plant agar in 150 ml
conical flask supplemented with various concentrations (0.5,
1.0, 1.5 or 2.0 mg l-1) of indole-3-butyric acid (IBA),
indole-3-acetic acid (IAA) or NAA. The pH of media was
adjusted to 5.8 prior to autoclaving (121 C, 20 min at
1 atm.). All cultures were incubated in a growth chamber at
16 h photoperiod with a light irradiance of *40 lmol
m-2 s-1, and temperature was maintained at 25 1 C
with 70 % relative humidity. In all set of experiments, PGR
free solid MS medium was used as a control treatment. After
4 weeks of cultivation, the frequency of adventitious roots
(%), mean number of roots and average biomass were
recorded respectively.
Growth kinetics of adventitious root culture
To establish adventitious root culture, 4-week-old fresh
adventitious roots were transferred to MS liquid media

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supplemented with 1.0 mg l-1 NAA. For each treatment,

500 mg fresh root was cultured into 40 ml media in 250 ml
flasks. All cultures were incubated under 24 h dark with a
continuous shaking in gyrator shaker at 110 rpm at room
temperature. Data on biomass accumulation was recorded
weekly by weighing adventitious roots in sterile conditions.
Duration of the study was 8 weeks, and the flasks in triplicate were used in all experiments.
Metabolite profiling
The samples were denoted on the basis of growth stage
during adventitious root culture as CTR (Control: fresh
adventitious roots used as inoculum for root suspension
culture), LAG phase (adventitious roots harvested on day
14 of root suspension culture), LOG phase (adventitious
roots harvested on day 35 of root suspension culture), STN
phase (Stationary; adventitious roots harvested on day 52
of root suspension culture). For each growth stage, three
replicates were collected. All samples were transferred to
air tight vials, flash frozen in liquid nitrogen, and stored at
-80 C for phytochemicals extraction.
Extraction of metabolites
Extraction was carried out according to an earlier protocol established by Overy et al. (2005). Briefly, 1000 ll
of solvent mixture A (methanol:chloroform:water,
2.5:1:1 at -20 C) was added to the centrifuge tube
(2 ml) containing fine powder (10 mg) of each sample.
Samples were mixed with vortex for 25 s, and kept on ice
for 5 min prior to centrifugation at 14 9 103 rpm for
5 min at 4 C. The supernatant was collected and transferred into a new pre-chilled storage tube (2 ml) and
labeled as supernatant A. The remaining pellet was reextracted with 500 ll of the pre-chilled solvent B
(methanol:chloroform, 1:1 at -20 C) followed by slight
mixing and centrifugation at 14 9 103 rpm for 5 min at
4 C to obtain the supernatant B. In the next step,
supernatant A and B were combined together, and the
supernatant (methanolic extract) was decanted into a new
pre-cooled microcentrifuge tube. Then organic layer was
separated from aqueous layer by adding 250 ll chilled
distilled water into the mixture followed by centrifugation for 2 min. Both aqueous and organic phases were
then stored at -80 C until analysis.
Electrospray ionization time of flight mass spectrometry
(ESI-TOF MS)
ESI-TOF MS was performed on a LCT spectrometer (API
Q-Star, Waters Corporation, Milford, USA.) based on the
methods described by Davey et al. (2008) and Walker

503

(2011). The mass spectrometer was operated at a resolution

of 4000 (FWHM) in positive mode with a capillary voltage
of 4800 V, extraction cone at 3 V and sample cone at 20 V
with a range finder lens voltage of 75 V chosen for
detection of masses from 50 to 800 Da. Source temperature
was 110 C, and desolvation temperature was 120 C.
Flow rates were 100 l h-1 for nebulisation and 400 l h-1
for desolvation. Spectra were collected in centroid mode at
a rate of one spectrum s-1 with 180 summed over a 3-min
period without background subtraction or smoothing.
Samples were loaded using a syringe pump (Razel, Connecticut, USA) at a flow rate of 20 ll min-1.
Data processing and metabolite identification
For each sample run, the summation of 180 centroid mode
spectra were exported from MassLynx data systems as text
file peak lists. These were imported into Microsoft Excel
(Microsoft Corp, USA), and an in-house macro program
was used to compare the accurate masses of three technical
replicate analyses of each sample. For the identification of
real masses within these profiles from background noise,
three replicates of mass spectra from each individual
sample were obtained. Once the peak was selected as a true
peak, the mean of the three masses over the three replicate
scans was used as the accurate mass, and this value along
with the corresponding average intensity made up the
metabolite profile. Finally the text files contained mass
spectra of respective samples were analyzed by SimcaP ? (version 12.0). Principal component analysis (PCA)
was carried out using Pareto scaled 0.2 Da binned data sets
in Simca-P v12.0 software (Umetrics, Sweden). Identification of putatively known metabolites was performed
through the comparison of monoisotopic masses likely to
be present in extracts, including [M?H]?, [M-H]- and
[M?Na]? against the list of metabolites in the plantcyc
database (http://pmn.plantcyc.org/).

Analysis of biochemical parameters

Fresh (FW) and dry weight (DW) of biological samples,
determination of electrical conductivity (EC) of residual
media were evaluated according to Baque et al. (2010).
Total phenolic content (TPC) and total flavonoid content
(TFC), were determined according to Velioglu et al. (1998)
and Chang et al. (2002), respectively. The radical scavenging activity of adventitious root cultures was determined by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)
according to Abbasi et al. (2010). Phenylalanine ammonialyase (PAL, EC 4.3.1.5) activity and silymarin content
were determined by methods described in Khan et al.
(2013).

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Experimental design and data analysis

any auxin used) resulted in a rise in biomass accumulation

(for fresh and dry biomass, ca. 60 % increase in IBA, ca.
90 % in IAA); however, a tremendous increase in biomass
was observed when NAA only was taken into account
(14.8 to 61.2 mg fresh- and 0.9 to 4.6 mg dry biomass for
NAA; Table 1). On the other hand, increase in NAA
concentrations (from 1.0 to 2.0 mg l-1) clearly showed a
significant decline in terms of root number per explant
(compare 1.0 with 1.5 or 2.0 mg l-1 NAA producing 34.3,
21.4 or 10.9 roots per explant, respectively). Morphological
observations revealed that the resulting adventitious roots
were thick, numerous and mostly shorter while few were
longer without lateral root formation on MS medium
containing 1.0 mg l-1 NAA. Although few adventitious
roots with profound lateral branching were induced on MS
media supplemented with 1.5 mg l-1 IAA. For IAA and
IBA at 1.5 mg l-1, frequency of rooting, mean number of
root as well as biomass of the roots produced from nodal
explants were significantly higher than other concentrations tested (0.5 or 1.0 mg l-1). Similarly, Lee et al. (2011)
observed optimum adventitious root production in Aloe
vera at low concentrations of NAA (0.51.0 mg l-1). It is
conspicuously established that addition of NAA to solid/
liquid medium foster adventitious root induction in a
variety of medicinal herbs. Enhanced biomass in hairy
roots of S. marianum was observed clearly on MS medium
supplemented with 0.5 mg l-1 NAA (Hasanloo et al.
2008). The efficiency of NAA for the production of
adventitious roots is due to its rapid absorption by plant
cells during in vitro growth conditions as compared to
other auxins. Peeters et al. (1991) reported that the uptake
of NAA is six times faster than IAA in tobacco explants.

All experiments were conducted in a factorial experimental

design with three replicates and were repeated twice. Mean
values of the treatments were subjected to the analysis of
variance (ANOVA), and significant difference was shown
with Duncans Multiple Range Test (DMRT) using SPSS
(Windows version 7.5.1, SPSS Inc., Chicago).

Results and discussion

Adventitious root induction
In preliminary studies, leaf, root, cotyledon or nodal
explants were cultured into solid MS medium supplemented with 0.5 mg l-1 NAA for adventitious roots formation. Of the explants tested, nodal explants were
effective for root induction (56 % of rooting); however,
leaf explants failed to induce viable roots (data not shown).
This could be due to the different morphogenic responses
derived from different explants at same growth treatments
(Abbasi et al. 2010). Of the treatments concerning adventitious root formation and biomass accumulation,
1.0 mg l-1 NAA was the most effective with a frequency
of 73.4 % rooting and producing 34.3 roots per explant as
compared to 38.2 or 28.5 % of rooting and 12.7 or 13.2
roots per nodal explant produced from MS medium containing 1.0 mg l-1 IAA or IBA, respectively (see Table 1).
Similarly, biomass accumulation of the adventitious roots
was also directly dependent on auxin concentrations.
Increase in auxin concentration from 0.5 to 1.0 mg l-1 (for

Table 1 Effect of different auxins at different concentrations on induction of adventitious roots from nodal explants of S. marianum
S.no.

MS?PGRS (mg l-1)

Root induction frequency (%)

Roots per explant (mean)

Fresh bio mass (g/l)

Dry bio mass (g/l)

MS (0)

2
3
4
5

IBA (0.5)
IBA (1.0)
IBA (1.5)
IBA (2.0)

21.3 1.1

b,c

28.5 1.6

36.4 2.2

a,b

23.2 1.4

b,c
b

8.4 0.9

b,c

12.7 1.2

17.8 1.8

a,b

13.2 1.4

a,b

6.3 0.4

b,c

30.1 2.1

b,c

3.7 1.8b

48.3 3.8

a,b

4.8 0.6a,b

50.6 4.1

a,b

5.3 0.3a

36.2 2.3

3.1 0.7b,c

IAA (0.5)

26.2 1.5

7
8

IAA (1.0)
IAA (1.5)

38.2 2.4a,b
45.1 3.1a,b

13.2 1.4a,b
15.4 1.4b

19.3 1.1c
31.5 2.2b,c

1.4 0.8b
2.9 1.9b,c

IAA (2.0)

22.4 1.2b,c

6.5 0.7b,c

17.3 1.2c

1.2 0.9b,c

39.6 2.9

a,b

20.2 1.2

a,b

14.8 1.1

0.9 0.2c

34.3 2.2

61.2 5.4

5.9 1.4a

NAA (0.5)

11

NAA (1.0)

73.4 4.3

12

NAA (1.5)

47.2 2.3a,b

NAA (2.0)

b,c

13

23.6 1.6

Values are mean per explant standard error of three replicates

Values with different letter/s are significantly different at P \ 0.01

123

21.4 1.4a,b
10.9 1.1

58.3 5.1a
38.1 2.8

0.8 0.3

cd

10

10.1 0.9

cd

4.6 0.4a,b
2.4 0.4c

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Growth kinetics of adventitious root culture

Adventitious roots established in suspension culture displayed a relatively quick growth curve which was characterized by lag phase at first week, followed by log phase for
4 weeks (from 2nd to 5th week), and a subsequent stationary phase for last three weeks of cultivation (from 6th
to 8th week). Maximum fresh biomass (FBM) and dry
biomass (DBM) of 167 5.91 and 10.73 0.384 mg l-1,
respectively, were observed on 42th day (Fig. 1). FBM was
almost seven times higher than the initial inoculum culture.
Similarly, Kim et al. (2004) observed FBM of adventitious
roots in Panax gingeng on MS liquid medium by using
2.0 mg l-1 NAA. A linear decrease in culture volume was
inversely correlated with an increase in fresh weight of
roots depending on the number of sub cultivations (Wu
et al. 2008). Gradual decrease in electrical conductivity
(EC) of residual medium with consumption of nutrients by
adventitious roots was clearly observed between 3rd and
8th week of cultivation (Fig. 1). At initial phase, there was
only a minor increase in EC that might be due to excretion
of metabolites to the medium. The decrease in EC of
residual medium strongly confers the increase in biomass
of adventitious root; therefore, it can be correlated with
intake of nutrients (PO4-, NH3?, NO3-, etc.) from the
culture medium (Liu et al. 2006).
Metabolic analysis of adventitious root culture
Mass spectra and principal component analysis (PCA)
The raw mass spectrum profiles of both aqueous and
organic fractions differed in peaks detected at varying signals/intensities (Fig. S1&S2; Supplementary). The spectra

505

of aqueous fractions were dominated by some common

peaks at m/z 242 and m/z 146; however, many uncommon
peaks were also detected with a highest peak at m/z 96 in
STN. Nevertheless, the mass spectra in organic fractions
were dominated by uncommon peaks only. The highest
peaks at m/z 178.045, m/z 233.89, m/z 178.044 and m/z
178.11 were detected in CTR, LAG, LOG and STN,
respectively. The common peaks represent the masses in the
biological samples which are thoroughly expressed during
growth of adventitious roots, whereas the uncommon peaks
represent the rare masses which are responsible for separating one growth stage from the rest in the growth cycle
(Walker 2011).
These mass spectrum profiles were then converted into
text files by using in-house software and were then subjected to PCA analysis to determine the putatively known
metabolites responsible for growth and development of
adventitious roots. PCA loading scatter plots distinguished
the samples on the basis of differences in growth stages
during root formation, and enabled the detection of several
bin masses responsible for separating samples from different growth stages (Fig. 2a, b). The principal components
PC1 and PC2 accounted for 76 % variation in aqueous
fractions and 52 % variation in organic fractions. The
loadings scatter plot for a PCA analysis can provide a list
of the metabolite bins, as to whether they show an
increasing or decreasing pattern in content. The bins which
are closest to the origin in the loading plot are the bins that
changed the least. Conversely, the bins furthest away from
the origin are those that changed most, suggesting that
these bins contain compounds which might be useful for
the differential responses at different growth stages during
adventitious root formation (Khan et al. 2015).

Bin masses

Fig. 1 Growth kinetics of Adventitious root culture of Silybum

marianum L. in relation to electrical conductivity of residual medium
on MS medium supplemented with 1.0 mg l-1 NAA. Values are
mean standard error of three replicates

The bin masses having high PCA loadings derived from

four different growth stages were selected, and their total
ion counts/intensities were summed up (Fig. 3a, b;
S3A&B, Supplementary). Bin mass 159 was found in the
loading scatter plot influencing the separation of the STN
lines and bin mass 337 was found contributing in the
separation of the LAG lines in all aqueous fractions.
Similarly, the organic fractions of the biological samples
showed bin mass 148 separating the LOG lines from the
other plant lines (Fig. 2a, b). Bin masses 214 and 377
contributed high score scatter loadings of aqueous and
organic fractions respectively; nevertheless, these bins
were not identified by the in-house bin program. Therefore,
those aforesaid bins that were not identified by the in-house
bin program were ignored at all, and the selected bins
identified by the in-house bin program were compared

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Fig. 2 PCA Loading Scatter plots of biological samples evaluated by SIMCA-P ? (12.0). a Aqueous fractions and b Organic fractions

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507

Evaluation of key metabolites in different growth stages

during root culture

100

80

Total Ion counts (%)

Ctr
Lag
Log
Stn

90

ab

70

ab
b

60

50

bc

40
30

bc

20

bc
bc

bc
c

c
c

10

4
7.

1.
30

43

9
28

14

Bin Masses
Fig. 3 Percentage of total ion counts of the bins (putatively identified
metabolites) in CTR, LAG, LOG and STN. Bin masses were selected
from the loading plots sorted by PCA. a Aqueous fractions and
b Organic fractions. Data represents the values of the mean standard error from three replicates. Column bars sharing the same letter/s
are similar otherwise differ significantly at P \ 0.05

among the biological samples. Bin masses 159 and 337.2

were detected with sequential increase in total ion counts/
average intensity from CTR to LOG (Fig. 3a, b; S3A&B,
Supplementary). However, increased signals of the bin
masses 149 and 381.2 were found in LOG followed by
STN and LAG while the signals decline was detected in
CTR. Bin mass 365.2 was found to be at sequential decline
in peak signals from CTR to STN. Average intensities
calculated also depicted the same behavior. Increased signals of the bin mass 437.4 were found to be higher in LAG
phase among all the bins tested in the organic fractions
(Fig. 3a, b; S3A&B, Supplementary).

Based on the values of total ions count (%), and average

intensity of selected bins in all biological samples, putatively known metabolites were obtained from in-house bin
program (Fig. 4; Table S1, Supplementary). Significant
phenylpropanoids from shikimate pathway in plants such
as cinnamic acid and di-hydro kaempferol (DHK), were
found to be at high amount in LOG phase. These phenolic
acids are produced as intermediary products of phenylpropanoid metabolic pathway, and can be anticipated for
stimulating role in biomass formation during in vitro root
culture in present study. They are also reported for their
role in oxidative phosphorylation and photophosphorylation, stimulation of RNA synthesis, bud development as
well as prevention of senescence due to their strong
antioxidant nature (Rathod et al. 2014). Both cinnamic acid
and di-hydro kaempferol activate antioxidant enzymes
SOD and POD against reactive oxygen species (ROS),
under stress conditions (Szalai and Janda 2009).When
taken into account total ion counts (Fig. 4), malonic acid
showed a growing trend from CTR to STN (from 0.8 to
4.8 %). Among the organic acids, malonic acid is proven as
a preferential plant defensive chemical against a variety of
abiotic stresses (Kim 2002).
The highest sucrose level was detected in CTR with a
sequential decline through different growth stages, and
lowest value was detected in STN. Generally, sucrose is the
most frequently used carbohydrate acting as a signaling
molecule, osmoticum and a source of carbon and energy in
culture media for following in vitro morphogenetic processes such as adventitious rooting, callus organogenesis
and somatic embryogenesis (Khan et al. 2015; Yucesan
et al. 2015). The amount of tryptophan accumulated at
LOG phase was significantly higher than other growth
phases during adventitious root culture (Fig. 4). Since
auxin plays a key role during in vitro rooting in majority of
plant species, its de-novo biosynthesis is controlled by
concomitant production of tryptophan through multiple
routes. Usually it acts as a key precursor for auxin (indole3-acetic acid, IAA) biosynthesis through the tryptophandependent pathway (Michalczuk et al. 1992). From our
results, we extrapolated that the accumulation of tryptophan in LOG phase results in more biomass accumulation
underlying the essential role of auxin during development
of adventitious roots. Comparison of phenyl-acetic acid
(PAA) among the biological samples revealed its highest
level in the STN followed by LOG (Fig. 4). Although PAA
is a non-indolic, active endogenous auxin present at
physiologically different levels in higher plants, the biological significance of PAA is not completely clear.
However, with possession for an auxin-like activity, PAA

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Fig. 4 Comparison of the key

metabolites in CTR, LAG, LOG
and STN lines. Putatively
known metabolites were
obtained from in-house bin
program on the basis of their
distribution with total ions count
(%) in the biological samples.
Data represents the values of the
mean standard error from
three replicates. Column bars
sharing the same letter/s are
similar otherwise differ
significantly at P \ 0.05

regulates the auxin transport, and has also been shown to

influence morpho-regulation (Korasick et al. 2013). Among
the vital fatty acids, prostaglandin A1 was detected at highest
signal (2.5 %) in STN phase. Prostaglandins (PGs) are cyclic
fatty acids, generally called as oxylipins and are considered
as signal compounds in plants and animals. They accumulate
as a consequence of oxidative stress. Notably prostaglandin
A1 has an important role in the induction of genes involved in
plant defense and biosynthesis of secondary metabolites
(Loeffler et al. 2005). Besides stress modulation, PGs also
play significant roles in other physiological processes
including cell division, elongation, membrane polarization,
vascular and reproductive development, proton pumping and
photo morphogenesis (Thoma et al. 2003). The highest
amount of brassicasterols was detected in LAG phase than
other growth lines (Fig. 4). As the name suggests, brassicasterols are predominately found in the Brassicacea family,
and are reported as plant steroids essential for normal plant
growth and development (Bishop and Yokota 2001). In
contrast to malonic acid and PAA, higher amount of punicic
acid was found in the LOG phase during growth kinetics of
adventitious root culture (Fig. 4). Puccinic acid has some
structural similarities (such as carbon composition, atomic
arrangement and the number of carbon double bonds) to the
conjugated linoleic and a-linolenic acids (Viladomiu et al.
2013). Recently, puccinic acid has increasingly attracted
scientific interest because of its several potential health
benefits including antioxidant, antitumor, immunomodulatory, anti-atherosclerotic and serum lipid-lowering activities

123

(Carvalho et al. 2010). Similarly, pinoresinol had also higher

total ion counts in the LOG phase that other growth phases in
all the biological samples (Fig. 4). Pinoresinol is a type of
lignans derived from the enantio-selective dimerization of
two coniferyl alcohol units (Nakatsubo et al. 2008). However, the exact roles of lignans in plant are not clear, although
it has been suggested that they are involved in plant defense
(Naoumkina et al. 2010).
Biochemical parameters in developmental stages
during adventitious root culture
DPPH-free radical scavenging activity was found to be
highest in STN phase (78 %) while lowest in LAG phase
(31 %).Throughout the growth curve, TFC and TPC were
detected at maximum levels (3.5 mg GAE/g DW and
1.9 mg QE/g DW) in log phase respectively, whereas
silymarin content was observed at highest level
(3.08 0.14 mgg-1 DW) in stationary phase (Fig. 5, 6).
PAL activity test of the biological samples showed its
higher level in the LAG phase and least in STN phase.
Interestingly, the different growth stages exhibited significant variations in PAL activity and silymarin content
(Fig. 6). Biochemical parameters during growth of adventitious root culture in present study revealed significant
correlations (r = 0.96, P = 0.044) between silymarin
content and DPPH as well as TPC and TFC (r = 0.97,
P = 0.032) at P \ 0.05 (Table 2). However, among all
growth stages, there was no correlation between PAL

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Plant Cell Tiss Organ Cult (2015) 123:501510

509

Fig. 5 Antioxidant potential (% FRSA), total phenolic content (mg

Quercetin/g dry weight) and total flavonoid content (mg Gallic acid/g
dry weight) in adventitious root culture with respect to growth curve
(DW). Values are mean standard error of three replicates. Column
bars sharing the same letter/s are similar otherwise differ significantly
at P \ 0.05

activity with TFC and silymarin content. Therefore, an

understanding of the biosynthesis of flavonoids during the
process of adventitious rooting is important because of
their role in develotpmental physiology and defense
mechanisms. Generally, biosynthesis of flavonoids occurs
through the derivation of their carbon skeletons from two
basic compounds: malonyl-CoA and p-coumaroyl-CoA in
the phenylpropanoid pathway (Heller and Forkmann 1988).
The first committed step for biosynthesis of the phenylpropanoid skeleton in plants is the deamination of Lphenylalanine to yield trans-cinnamic acid and ammonia.
This reaction is catalyzed by phenylalanine ammonia lyase
(PAL, EC 4.3.1.5), and is often regarded as a key step in
the biosynthesis of these flavonoids (Khan et al. 2013). The
inverse correlation of PAL activity with TFC and silymarin
content in our study can be explained in a way that the
specific growth stage during development of adventitious
roots do affect the PAL activity, as the root growth progresses from one phase to another. Notwithstanding, the
level of PAL activity depends on the genotype, developmental stage and the organ or tissue type of the plant
(Swedan 2013). Moreover, PAL turnover may also be
responsible for the biosynthesis of a wide range of
phenylpropanoid compounds, such as cinnamic acid
malonic acid, DHK and lignin (Khan et al. 2015).
Acknowledgments Financial Support of National Research Programme for Universities (NRPU), Higher Education Commission
(HEC) of Pakistan is appreciated here. Dr. Khan M.A. acknowledges
Financial Support of Indigenous Scholarship Programme for his Ph.D.
by HEC.

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Fig. 6 Phenylalanine ammonia lyase activity (Ug FW) and silymarin content (mg g-1 DW) in adventitious root culture with respect
to growth curve. Values are mean standard error of three
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differ significantly at P \ 0.05

Table 2 Pearson correlation for biochemical activities at various

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