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Gibberellic Acid Increases Secondary MetaboliteProduction in
Echinacea purpurea Hairy Roots
Bilal H. Abbasi & Amanda R. Stiles &Praveen K. Saxena
& Chun-Zhao Liu
Received: 6 June 2012 /Accepted: 3 October 2012 /Published
online: 18 October 2012# Springer Science+Business Media New York
2012
Abstract Gibberellic acid (GA3) is reported to have diverse
effects on hairy root cultures ofmany plant species; therefore, the
effects of GA3 on the growth, secondary metaboliteproduction
(caffeic acid derivatives and lignin), phenylalanine ammonia lyase
(PAL) activ-ity, and free radical scavenging activity of
light-grown Echinacea purpurea L. hairy rootswere investigated.
Eight concentrations of GA3, ranging from 0.005 to 1.0 M, were
addedto shake flask cultures. The moderate GA3 concentration, 0.025
M, resulted in the highestconcentrations of cichoric acid, caftaric
acid, and chlorogenic acid, as well as increased PALactivity, cell
viability, and free radical scavenging activity, while higher and
lower GA3concentrations resulted in reduced levels compared to the
control (lacking GA3). Themoderate GA3 concentration also affected
root morphogenesis; supplementation with0.025 M GA3 resulted in the
development of thick, dense, purple-colored roots, whileroots
exposed to the higher and lower concentrations of GA3 were thin and
off-white. Thisstudy demonstrates that supplementation with GA3 may
be an excellent strategy to optimizethe production of secondary
metabolites from E. purpurea hairy root cultures; however, theGA3
concentration is a critical factor.
Keywords Echinacea purpurea . Hairy roots . Cichoric acid .
Anthocyanins . GA3 . Light
Introduction
Echinacea purpurea L. is a traditional herbal medicine used to
treat a wide range of medicalconditions, but is most commonly used
to prevent colds and upper respiratory infections by
Appl Biochem Biotechnol (2012) 168:20572066DOI
10.1007/s12010-012-9917-z
B. H. Abbasi : A. R. Stiles : C.-Z. LiuNational Key Laboratory
of Biochemical Engineering, Institute of Process
Engineering,Chinese Academy of Sciences, Beijing 100190, Peoples
Republic of China
P. K. SaxenaGosling Research Institute for Plant Preservation
and Department of Plant Agriculture,University of Guelph, Guelph,
Ontario, Canada N1G 2W1
C.-Z. Liu (*)National Key Laboratory of Biochemical Engineering,
Institute of Process Engineering,Chinese Academy of Sciences,
Beijing 100080, Peoples Republic of Chinae-mail:
[email protected]
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stimulating the immune system [1]. It is one of the most
accepted herbal medicines, with aworldwide market of approximately
US$1.3 billion per annum [2]. However, because it is anout-crossing
species, there is a significant amount of genetic, morphological,
and phyto-chemical diversity present among individual plants and
plant populations. As a result,commercial Echinacea products
display an exceptionally high degree of variability in thelevel of
various marker compounds, and clinical trials have produced
conflicting results. Toaddress this issue, the development of
methods to produce consistently high-quality plantmaterial with a
known chemical profile is needed.
Caffeic acid derivatives (CADs) are biologically active
components found in E. pur-purea. Cichoric acid is considered to be
the most important CAD for the medicinal value ofE. purpurea [2,
3]. It has demonstrated phagocytic, antihyaluronidase, and
antiviral activity,and it also inhibits HIV-1 integrase and
replicase [4]. CADs are currently extracted fromwild-grown E.
purpurea because its synthesis is difficult, expensive, and time
consuming.Therefore, the development of in vitro tissue culture
techniques for E. purpurea remains thefocus of considerable
research attention [5]. Hairy root cultures are a promising in
vitrosource for the consistent production of biomass and secondary
metabolites in many medic-inal plant species due to their
biochemical and genetic stability [2]. Several reports
onAgrobacterium-mediated transformation of E. purpurea are
available [2], but few demon-strate the capacity to produce CADs
[2, 3, 6]. In this work, E. purpurea hairy roots,generated from a
variety indigenous to South American and known to be a high
CADsproducer, especially of cichoric acid, were utilized.
One method for increasing phytochemical yield in in vitro
systems is the applica-tion of exogenously applied phytohormones
[7], which have been shown to affectphysiological and metabolic
processes in plant cell and organ cultures of severalspecies [7,
8]. The application of GA3 has resulted in a variety of growth
effects;cell elongation; the modulation of enzymes activities such
as phenylalanine ammonialyase (PAL), chlorophyllase, and
peroxidase; and changes in the metabolism andaccumulation of
anthocyanins, glutathione, flavonoids, lignin, proteins, and
starch[9]. The application of GA3 alone or in combination with
paclobutrazole and uni-conazole to in vitro-grown Echinacea plants
increased the accumulation of caftaricand cichoric acid in roots
(with negligible effects on their accumulation in shoots) [5].GA3
has also been shown to stimulate the growth of hairy roots in
various specieswith a varying degree of secondary metabolite
production [8, 10].
E. purpurea hairy roots grow well in hormone-free media [2, 6],
but the effects ofexogenous plant growth regulators have not yet
been tested. The goal of this study was toanalyze the effect of GA3
on the growth and CADs biosynthesis in Echinacea hairy rootcultures
over time and to evaluate the effect of GA3 on culture viability,
PAL enzymeactivity, lignin biosynthesis, and free radical
scavenging activity.
Materials and Methods
Hairy Root Culture and GA3 Treatment
Establishment of the E. purpurea hairy root cultures was
conducted as previously reported[2]. Briefly, hairy roots were
induced by transformation with Agrobacterium rhizogenesstrain ATCC
43057 (ATCC, Manassas, Virginia, USA) from leaf explants of
28-day-oldseedlings germinated from E. purpurea seeds (Richters,
Goodwood Ontario, Canada).Hairy root cultures were maintained at
251 C in the dark and subcultured at 21-day
2058 Appl Biochem Biotechnol (2012) 168:20572066
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intervals. The roots were cultured in 50 ml MS [11] basal media
with full strength salts and30 g/l sucrose, and the medium was
adjusted to a pH of 5.8 prior to autoclaving.
All experiments were performed in 250 ml Erlenmeyer flasks
containing 50 ml mediainoculated with 1 g of 21-day-old hairy roots
(approximately 2 cm long segments). Theflasks were incubated on a
rotary shaker set at 105 rpm and 251 C under continuous light(60
mol/m2/s). In the initial screening, GA3 was added to the culture
medium at a range ofconcentrations (0.005, 0.0075, 0.01, 0.025,
0.05, 0.1, 0.5, and 1.0 M), and three concen-trations were used for
later experiments (0.005, 0.025, and 1.0 M). However, for clarity,
forall experiments, data are presented for just three
concentrations (0.005, 0.025, and 1.0 M).
Analytical Methods
To determine fresh weight, the hairy root cultures were gently
pressed on filter paper toremove excess water and weighed.
Subsequently, the roots were dried in an oven at 60 C for24 h and
dry weights were recorded.
The PAL activity was determined according to the method of
Koukol and Conn [12] withone unit of activity corresponding to an
absorbance variation of 0.01. Cell viability wasestimated by the
reduction of 2,3,5-triphenyltetrazolium chloride [13]. The
viability indexwas defined as the absorbance measured per gram of
fresh tissue.
Anthocyanin analysis was conducted according to the method of
Harborne [14] using aShimadzu UVVIS spectrophotometer. Extraction
and quantification of CADs from thedried hairy root cultures were
carried out using the method described by Liu et al. [2]. Ligninwas
determined according to the method of Goering and Soest [15].
Eight to ten root cross sections were prepared from light- and
dark-grown hairy rootcultures using a modification of the method of
Rieger and Litvin [16]. Briefly, root sampleswere preserved in
formaldehyde, acetic acid, and ethanol (10:5:85, by volume) and
dehy-drated in ethanol prior to embedding in resin (Historesin).
Cross sections (810 m thick)were excised from 10 to 30 mm behind
the root tip to study differentiation. Roots werestained with
either aniline blue or safranin and photographed with a calibrated
lengthreference using the Nikon microscope at 2040
magnification.
The capacity of prepared extracts to scavenge free radicals
(1,1-diphenyl-2-picrylhydrazyl,DPPH) was monitored according to the
method of Amarowicz et al. [17]. Briefly, 2.0 mg ofhairy root
extracts was dissolved in 4 ml of methanol and added to a
methanolic solution ofDPPH (1 mM, 0.5 ml). The mixture was vortexed
for 15 s and then left at room temperature for30min. The absorbance
of the resulting solutionwas measured at 517 nm. Amethanolic
DPPHsolution (2 mg of BHA dissolved in 4 ml of methanol with 0.5 ml
of DPPH solution) that haddecayed (no longer exhibited a purple
color) was used for the background correction. Theradical
scavenging activity was calculated as a percentage of DPPH
discoloration using theequation: % scavenging DPPH free
radicals0100(1AE/AD), in which AE was the absor-bance of the
solution when the extract had been added and AD was the absorbance
of theDPPH solution with no added extract.
Statistical Analyses
Triplicate flasks were used in all experiments, and the
experiments were repeated twice. Alldata were the meanstandard
deviation. The data were subjected to a one-way analysis
ofvariance. Tukey-HSD test was used for calculation of significant
differences. SPSS (forWindows, standard version 7.5.1 by SPSS Inc.
Chicago) was used to determine the signif-icance at p
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Results and Discussion
GA3 is reported to have a physiological effect at concentrations
ranging from 109 to 105M
[18]; therefore, we studied the effect of GA3 on Echinacea hairy
root cultures at eightconcentrations ranging from 0.005 to 1.0 M.
The GA3 concentrations ranging from 0.01 to0.1 M increased the
secondary metabolite production, PAL activity, and cell viability
incomparison with the control. The concentration of 0.025 M GA3 had
the strongest effect;therefore, for clarity, the data are shown for
the control (lacking GA3), the lowest concentration(0.005 M), a
moderate concentration (0.025 M), and the highest concentration
(1.0 M).
Effects of GA3 on the Growth and Morphology of E. purpurea Hairy
Roots
The application of GA3 has been shown to affect hairy root
cultures of several plant species[10, 19], and an earlier study by
Jones et al. [5] demonstrated that changes in GA3metabolism
influence the production of cichoric acid and caftaric acid in
Echinacea plantletsgrown in vitro. In the current study, we
evaluated the effects of GA3 on growth kinetics andsecondary
metabolite production in E. purpurea hairy root cultures. The hairy
roots treatedwith the moderate GA3 concentration (0.025 M) achieved
a higher biomass than theuntreated control (Fig. 1a), and both the
low (0.005 M) and moderate (0.025 M) GA3concentrations resulted in
a lower biomass than the untreated control. Moderate
concen-trations of GA3 similarly increased biomass in hairy root
cultures of other plant species [10]and increased shoot and root
biomass in potted maize plants [20].
There was a direct relationship between the culture biomass and
cell viability: as the biomassincreased, the cell viability index
increased (Fig. 1a, b). At the high GA3 concentration(1.0 M), the
culture viability was higher after day 45; this may indicate that
higher concen-trations of GA3 reduce the growth rate and alter the
timing of the log phase (Fig. 1b). Lowviability is a sign of
reduced metabolic activity associated with mitochondrial function
andrespiration, which ultimately leads to cell death [21]. Cell
viability is also linked to stress-enhanced cell permeabilization.
Thus, continued research on the relationship between rootmorphology
and physiology is important to improve our understanding of root
dynamics.
The GA3 concentration impacted the anatomy of the E. purpurea
hairy roots; thin rootswere produced at the high GA3 concentration
(1.0 M), thicker roots were produced at themoderate GA3
concentration (0.025 M) (Fig. 2), and the root diameter at the low
GA3concentration (0.005 M) was not statistically different from the
control (data not shown).GA3 impacts cell elongation and expansion,
and this may account for the changes in growthand root diameter.
Similarly, Baluska et al. [22] observed a significant effect of GA3
on rootgrowth and cell size in treated maize roots, and a reduction
in root diameter in response toGA3 has also been reported in
several grape cultivars [23]. These observations may indicatea
common initial response to GA3 treatment, including a slackening of
the cell wall whichresults in a decrease in the overall growth
rate, depending on the physiological age of thecells. When the
cells are dynamic, the cell wall slackening permits rapid
development,whereas when they are strained, the metabolic activity
cannot keep pace with the transientextension, resulting in a drop
in turgor pressure in the stressed cells.
Previously, light was reported to induce purple coloration in E.
purpurea hairy roots dueto the accumulation of anthocyanins [6]. In
the present study, GA3 had a considerable effecton the accumulation
of light-induced anthocyanins; anthocyanin accumulation was
en-hanced in cultures grown in 0.025 M GA3 while cultures grown at
0.005 and 1.0 Mwere lighter in color compared to the control (Fig.
3). These data indicate that GA3 inhibitedanthocyanins accumulation
at higher concentrations. The impact of GA3 on color has also
2060 Appl Biochem Biotechnol (2012) 168:20572066
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been reported for other plant species such as Vitis [21].
Formation of anthocyanins, whichmitigated light-induced oxidative
stress, was also reported in plant cell cultures; Ilan andDougall
[24] demonstrated that GA3-treated carrot cell cultures did not
exhibit chalconesynthase activity, which is necessary for
anthocyanin biosynthesis. However, Teszlak et al.[21] found that in
grape, the anthocyanin content was significantly increased by
GA3application. These studies indicate that GA3-induced anthocyanin
accumulation is speciesand concentration specific. In the current
study, fluctuations in the levels of anthocyaninaccumulation led us
to quantify the levels of antioxidative enzyme activity in order
toexamine the interaction of GA3 and light-induced oxidative
stress. Highly differentiatedroots possess higher levels of
secondary metabolites [6].
0 5 10 15 20 25 30 35 40 45 50 550
4
8
12
16
c
bbb
c
d
eeefef
bcb
c
deed
c
bcab
bcbc
efe
de
c
bc
abab
Dry
biom
ass
(g/l)
Days
Control 0.005 0.025 1.0
aa
0 5 10 15 20 25 30 35 40 45 50 550
2
4
6
8
10
cdcd
bcccd
ddde
bbcdcd
dde
ee
ef
bbb
ded
cbc b
ab
Control 0.005 0.025 1.0
Viab
ility
of
cu
lture
s
Days
b
a
Fig. 1 a Growth kinetics and b cell viability of E. purpurea
hairy root cultures grown in MS media with orwithout GA3
supplementation for 50 days. Values are the means of triplicate
samplesstandard deviation.Means with different letters are
statistically different from the control at p0.05 according to
Tukeys HSD test
Appl Biochem Biotechnol (2012) 168:20572066 2061
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Effects of GA3 on CADs Production
In some plant species, GA3 is reported to affect the
concentrations of phenolic substances[9, 23]; therefore, we
measured the levels of CADs, phenolic substances derived from
phenyl-propanoid metabolism, in the hairy root cultures [6, 25].
GA3 affected the accumulation ofcichoric acid, caftaric acid, and
chlorogenic acid but did not affect caffeic acid (Fig.
4ad).Exposing the cultures to the moderate GA3 concentration (0.025
M) significantly enhancedthe accumulation of the principal CAD,
cichoric acid (Fig. 4a), and the maximum level ofcichoric acid
accumulated as the hairy roots entered the late stationary phase of
growth. Cellsmanufacture primary compounds that are required for
the biosynthesis of cell componentsduring the exponential growth
phase [2], and when nutrients are exhausted and waste productsare
excreted by cells into the medium, the metabolism switches from
primary to secondarymetabolism [10]. Therefore, biologically active
metabolites are commonly not produced insignificant quantities
until the culture enters stationary phase. In a study by Jones et
al. [5], theconcentrations of CADs in the Echinacea plantlets were
not significantly different fromcontrols toward the end of the
treatment period, potentially indicating degradation of GA3
oracclimatization of the tissues to the inductive stimulus. In a
previous study, the highest
Control 0.005 M 0.025 M 1.0 M
Fig. 2 Effects of GA3 supplementation in MS media on the anatomy
and diameter of E. purpurea hairy rootsat day 35. Bar0300 m
460 480 500 520 540 560 580 6000.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
Abs
orb
ance
Wavelength
0.025
Control0.005
1.0
GA3 ( M)Fig. 3 Absorption spectra ofanthocyanins extracted from
45-day-old E. purpurea hairy rootsgrown with or without GA3
sup-plementation in MS media
2062 Appl Biochem Biotechnol (2012) 168:20572066
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concentrations of cichoric acid were also present in the
purple-colored flowers and E. purpurearoot stock [2, 6], which is
in accordance with the data presented in the current study.
Theaccumulation of caftaric acid was enhanced only with 0.025 M
GA3, while 1.0 M GA3inhibited the production of caftaric acid
compared to 0.005 M GA3 and the control (Fig. 4b).The maximum
accumulation of both caftaric acid and cichoric acid occurred on
day 45.Chlorogenic acid accumulation was stimulated by both the low
(0.005 M) and moderate(0.025 M) GA3 concentrations (Fig. 4c), while
the accumulation in response to the high GA3concentration was
similar to the control until day 40 and then increased.
Sharaf-Eldin et al. [9]studied the effects of GA3 on chlorogenic
acid and cynarin in Cynara cardunculus leaves andconcluded that GA3
affected chlorogenic acid more than cynarin. Exposure to GA3 did
not havea significant effect on caffeic acid accumulation in the
present study (Fig. 4d).
Lignin is present in nearly all nonaquatic higher plants as an
essential component of thedifferentiated cell wall. It is lignin
which provides roots with strength and rigidity. BecauseGA3 is
known to promote cell elongation and increase cell wall plasticity,
we investigated itseffect on lignification in the hairy root
cultures. At the moderate GA3 concentration(0.025 M), the lignin
concentration was significantly higher than the control, while
thehigher and lower GA3 concentrations were not significantly
different (Fig. 5). The timecourse indicated that the enhanced
lignification occurred primarily following the exponential
0 5 10 15 20 25 30 35 40 45 50 550
8
16
24
32
40
ef efe
ded
cdc
c
bc
cddde
efefefeff
ef ee de d
d cdbc
bbc
b
ede
d cdc
bcbc
aa
Control 0.005 0.025 1.0
Cich
oric
acid
(mg/
g DW
)
Days
aa
0 5 10 15 20 25 30 35 40 45 50 550
4
8
12
de de de
ccc
cddede de
de dd d
cdc
cc
d dcd cd
c cbc
bb b
cd cdc c
c bcb
aab
Control 0.005 0.025 1.0
Cafta
ric ac
id (m
g/g
DW)
Days
b
a
0 5 10 15 20 25 30 35 40 45 50 550
4
8
12
d d dd cd
cd
d dcd
cdcdd
dddddede d
d dcd cd
c c
bcb
cbc bc
b ab aba
Control 0.005 0.025 1.0
Chlo
roge
nic
acid
(mg/
g DW
)
Days
c
a
0 5 10 15 20 25 30 35 40 45 50 550.00
0.01
0.02 Control 0.005 0.025 1.0
Caffe
ic ac
id (m
g/g
DW
)
Days
d
Fig. 4 Time course of the accumulation of caffeic acid
derivatives in E. purpurea hairy root cultures grown inMS media
with or without GA3 supplementation for 50 days: a cichoric acid; b
caftaric acid; c chlorogenicacid; and d caffeic acid. Values are
means of triplicate samplesthe standard deviation. Means with
differentletters are statistically different from control at p0.05
according to Tukeys HSD test
Appl Biochem Biotechnol (2012) 168:20572066 2063
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growth phase, but GA3-enhanced lignification had no direct
relation to GA3-promotedgrowth (Fig. 1a). Neves et al. [26]
demonstrated that in soybean, enhanced lignin productionsolidifies
the cell wall and restricts root growth.
Biosynthesis of phenolic acids involves the induction of PAL,
the enzyme that catalyzesthe first metabolic step in
phenylpropanoid metabolism and the production of
secondarymetabolites [27]. The reported correlation between
application of GA3, PAL activity, andsynthesis of phenylpropanoids
such as anthocyanins and lignin [6, 28] led us to investigatethe
effect of GA3 on PAL in the E. purpurea hairy root cultures. The
moderate GA3concentration (0.025 M) significantly enhanced PAL
activity compared to the control,while the other GA3 concentrations
significantly decreased it (Fig. 6). This indicates that
theincrease in anthocyanins, CAD accumulation, and lignin content
are related to PAL activity
0 5 10 15 20 25 30 35 40 45 50 550
5
10
cc
cdcdcdcdcddddee
de dd cd
cd cdc
cc c
cd cdc
c bcbc b
aa
Control 0.005 0.025 1.0
Lign
in (%
DW
)
Days
a
Fig. 5 Time course of lignin bio-synthesis in E. purpurea
hairyroot cultures grown in MS mediawith or without GA3
supplemen-tation for 50 days. Values aremeans of triplicate
samplesthestandard deviation. Means withdifferent letters are
statisticallydifferent from control at p0.05according to Tukeys HSD
test
0 5 10 15 20 25 30 35 40 45 50 550
300
600
900
1200
1500
cc
c
c
cdde
e
ef
e
e
cdc
b bb
fef
e
e
d
cb
a
Control 0.005 0.025 1.0
PAL
(U/g
FW
)
Days
a
Fig. 6 Time course of phenylala-nine ammonia lyase (PAL)
activ-ity in E. purpurea hairy rootcultures grown in MS media
withor without GA3 supplementationfor 50 days. Values are means
oftriplicate samplesthe standarddeviation. Means with
differentletters are statistically differentfrom control at p0.05
accordingto Tukeys HSD test
2064 Appl Biochem Biotechnol (2012) 168:20572066
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in E. purpurea hairy roots, a finding that has been demonstrated
in several other E. purpureastudies [6]. GA3 treatment in pea
plants and Jatropa curcas also increased PAL activity [29]but
inhibited it in others [30], although the combination of GA3 with
uniconazole lessenedthis inhibitory effect [31].
DPPH scavenging activity has been shown to correlate with
cichoric acid and caftaricacid accumulation; however, chlorogenic
acid, caffeic acid, and anthocyanins also contribute(Fig. 7).
Pellati et al. [32] and Tsai et al. [33] found that cichoric acid
had a higher freeradical scavenging activity due to the presence of
two adjacent hydroxyl groups on each ofits phenolic rings, while
chlorogenic acid and caffeic acid have lower scavenging activitydue
to the presence of only one phenolic ring for the hydroxyl groups.
Taveira et al. [34]found antioxidant activity in Brassica shoots
due to the presence of phenolics.
In summary, the supplementation of the light-grown hairy root
cultures with a moderateconcentration of GA3 (0.025 M) resulted in
increases in culture biomass, cell viability,secondary metabolite
production, PAL activity, free radical activity, and root
morphology,while lower and higher levels of GA3 supplementation
resulted in a decrease in all parameterstested compared to the
control. Therefore, the use of GA3 supplementation may function as
aneffective method to optimize the production of secondary
metabolites from E. purpurea hairyroot cultures; however, it is
clear that the GA3 concentration is a critical factor.
Acknowledgments This work was funded by the National Natural
Science Foundation of China (no.21150110459), the Knowledge
Innovation Program of the Chinese Academy of Sciences (nos.
YZ-06-03 &Y227051304), the Chinese Academy of Sciences
Fellowship for Young International Scientists (no.2011Y1GA01), the
Chinese Academy of Sciences Visiting Professorship for Senior
International Scientists (no.2011T1G05), and the Gosling Research
Institute for Plant Preservation, Canada. Abbasi BH
acknowledgesfinancial support of Higher Education Commission of
Pakistan for providing financial assistance for PhD.
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0 5 10 15 20 25 30 35 40 45 50 550
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75
100
cdcd
bcb
ee
ded cd
cd
bc
bbcbc
dede
cdcd
bc
b ab
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bab ab
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2066 Appl Biochem Biotechnol (2012) 168:20572066
Gibberellic Acid Increases Secondary Metabolite Production in
Echinacea purpurea Hairy RootsAbstractIntroductionMaterials and
MethodsHairy Root Culture and GA3 TreatmentAnalytical
MethodsStatistical Analyses
Results and DiscussionEffects of GA3 on the Growth and
Morphology of E. purpurea Hairy RootsEffects of GA3 on CADs
Production
References
