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Plant Physiology and Biochemistry 49 (2011) 1259e1263

Contents lists available at SciVerse ScienceDirect

Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Simultaneous determination of different endogenetic plant growth regulators in

common green seaweeds using dispersive liquideliquid microextraction method
Vishal Gupta a, Manoj Kumar a, Harshad Brahmbhatt b, C.R.K. Reddy a, *, Abhiram Seth c, Bhavanath Jha a
a

Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientic and Industrial Research (CSIR), Bhavnagar 364021, India
Analytical Sciences, Central Salt and Marine Chemicals Research Institute, Council of Scientic and Industrial Research (CSIR), Bhavnagar 364021, India
c
Aquagri Processing Pvt Ltd, 18, Anand Lok, New Delhi 110049, India
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 18 July 2011
Accepted 11 August 2011
Available online 22 August 2011

A simple and rapid HPLC-based method was developed for simultaneous determination of major classes
of plant growth regulators (PGRs) in Monostroma and different species of Ulva. The plant growth regulators determined included gibberellic acid (GA3), indole-3-acetic acid (IAA), abscisic acid (ABA), indole3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75,
3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobile
phase (60:40 methanol and 0.6% acetic acid in water), column temperature (35
C) and ow rate (1 ml/
min). This method presented an excellent linearity (0.2e100 mg/ml) with limit of detection (LOD) as
0.2 mg/ml for ABA, 0.5 mg/ml for KR and salicylic acid, and 1 mg/ml for IAA, IBA and GA3. The precision and
accuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plant
matrix was compensated after spiking and the resultant recoveries estimated were in the range of 80
e120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimized
in this study determined IBA along with IAA for the rst time in the seaweed species investigated except
Ulva linza where the former was not detected. In all the species studied, ABA level was detected to be the
highest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, sample
preparation and analysis time were substantially reduced while allowing determination of more classes
of PGRs simultaneously.
2011 Elsevier Masson SAS. All rights reserved.

Keywords:
Plant growth regulator
Green seaweed
High performance liquid chromatography
Dispersive liquideliquid micro-extraction

1. Introduction
Plant growth regulators (PGRs) are structurally diverse group of
naturally occurring substances that interacts with each other in
a complex manner to regulate almost every aspect of plant life [1].
They are highly functional and even at trace quantities trigger
a variety of basic physiological processes including cell division,
enlargement and differentiation, organogenesis, seed dormancy
and germination, leaf senescence and abscission as well as defence
against abiotic and biotic stresses [2]. Endogenic cytokinins
together with their riboside conjugate and auxins have been
reported from seaweed species such as Porphyra perforata,
Sargassum muticum [3e5], Laminaria japonica [6], Dictyota humifusa, Ulva fasciata [7], Undaria pinnatida [8] and Caulerpa paspaloides [9]. Recently, Yokoya et al. [10] quantied cytokinin, auxin
and abscisic acid in common red algae from Brazilian coast. The

* Corresponding author. Tel.: 91 278 256 5801/256 3805x614; fax: 91 278 256
7562/256 6970.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0981-9428/$ e see front matter 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2011.08.004

inherent growth regulating substances including both PGRs and

minerals in seaweed were attributed to their application as fertilizer in agriculture [11]. The foliar spray of seaweed extract or its
direct application to root has shown wide range of benecial effects
on terrestrial crops ranging from early seed germination, improved
crop performance, increased resistance toward abiotic and biotic
stress and enhanced post harvest shelf-life of perishable products
[12,13]. As a result, the seaweed industry has emerged with a new
sector of phycosupplement with an estimated global market value
of US$53 million [14]. Nowadays, the seaweed-based fertilizers are
gaining importance over petrochemical based fertilizers because of
the detrimental effects caused by petrochemical based fertilizers on
soil productivity upon prolonged use. The commonly used
seaweed-based fertilizers in the agriculture and horticulture are
mainly prepared out of brown seaweeds namely Ascophyllum
nodosum, Ecklonia maxima, Macrocystis pyrifera [12] owing to their
round the year availability. However, the foliar spray of red seaweed
Kappaphycus alvarezii sap on soyabean has resulted in an increase
of crop yields as high as 46% over control [15]. The analysis of this
sap for PGRs has revealed the presence of IAA, GA3, kinetin and
zeatin [16] in addition to several micro and macronutrients [17].

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V. Gupta et al. / Plant Physiology and Biochemistry 49 (2011) 1259e1263

Further eld trials of this sap on other agricultural crops has also
shown encouraging results over control and showed increased
yields of 30e40% in sugarcane, 26% in potato, 16% in barley and
gram, and 13e15% in corn (Abhiram Seth personal communication).
The genus Ulva with its worldwide distribution, wider adaptability to diverse environmental conditions, higher growth rates
and amenability for depolymerization makes it an attractive feedstock for developing fertilizer and bio-renery products. The
removal of moisture from the biomass indeed is the bottleneck
toward the economy of energy industry therefore an integrated
process involving extraction of liquid sap having agriculture
applications could make overall process much competitive.
Development of such innovative processes will also be advantageous in utilizing the massive biomass of Ulva produced during
sudden outbreak as green tides or blooms recently reported in
China [18], the Baltic Sea [19] and Chile [20].
The analytical techniques employed for PGR analysis include
high pressure liquid chromatography (HPLC), gas chromatographymass spectrometry (GC-MS), liquid chromatographyemass spectrometry (LC-MS), capillary electrophoresis etc. Of these, HPLC with
its inherent advantages such as wide application, short analysis
time and high separation efciency merits its choice as a better tool
over others. However, intracellular traces of PGRs and coexistence
of many interfering substances make the analysis difcult and
necessitate for sample preparation and purication prior to analysis. The most commonly used pre-treatment techniques are
liquid-liquid extraction (LLE), solid phase extraction (SPE), column
chromatography and solid phase microextraction (SPME). Of these
techniques, dispersive liquideliquid microextraction (DLLME)
method developed by Razaee et al. [21] is relatively new and has
several advantageous over others such as 1) sample enrichment, 2)
dispenses the need of purication through column, 3) low organic
solvent consumption and 4) overall short operation time. The
DLLME method together with HPLC coupled to ultraviolet detector
(UV) has been employed for the analysis of polycyclic aromatic
hydrocarbons [22] and psycotropic drugs [23]. Lu et al. [1] for the
rst time successfully demonstrated DLLME-HPLC-FLD as a powerful pre-concentration method sensitive for detecting auxins.
However, the requirement of rapid methodologies for sample
derivatization has shifted the bottleneck for sample throughput
from analysis to sample preparation. Therefore the objective of this
investigation was to optimize a method for simultaneous determination of broad classes of plant growth regulators such as ABA,
IAA, IBA, GA3, KR along with salicylic acid in common macrophytic
marine green algae Monostroma and Ulva.
2. Results and discussion
The interaction of different endogenetic hormone response
pathways plays a crucial role in regulating various developmental
and physiological processes. Therefore a method determining
simultaneously different classes of endogenetic plant growth
regulators (PGRs) will facilitate the understanding of various
cellular, physiological and biochemical responses. In this study,
a simple and rapid HPLC based method was optimized for simultaneous determination of major classes of PGRs such as auxins (IAA
and IBA), ABA, GA3 in common green seaweeds. The resolution of
hydrophobic PGRs in HPLC analysis was found to signicantly be
affected by mobile phase composition and concentration. The
distinct separation of these PGRs was attained in mobile phase
constituted with methanol: 0.6% acetic acid (60:40). The modied
mobile phase composition was distinct and simple compared to the
binary mixtures used in majority of the studies in seaweed PGR
analysis [7,10,24,25]. Also the modied composition was different
from those reported for PGR analysis in terrestrial plants [26,27]. In

addition to mobile phase, the column temperature and ow rate

were the other determinants that offer better resolution of the
analytes. The separation column temperature and pump ow rate
was optimized as 35
C and 1.0 ml/min respectively. The standards
were detected at all the three wavelengths selected under
the optimized conditions and quantication was performed at
208 nm for GA3 and at 265 nm for other PGRs. The retention time of
the standard analytes such as ABA, IAA, IBA, GA3, salicylic acid and
KR was 3.91, 3.3, 4.95, 2.75, 5.39 and 6.59 min respectively
(Supplementary Fig. 1).
The linear range along with the limit of detection (LOD) for the
target compounds are summarized in Table 1. The LOD was found to
be lowest for ABA (0.2 mg/ml) followed by KR, salicylic acid (0.5 mg/
ml), and IAA, IBA and GA3 (1.0 mg/ml) (Table 1). The linear tting of
the repeated data was found signicant at p  0.01 and the evaluation results of the linear model by regression variance analysis are
summarized in Table 2. The results revealed a good linear t. The
precision in the analysis was validated with inter/intra-day analysis
in replicate and the relative deviation observed was in the range of
0.21e0.92%. The samples were found stable when stored at 20
C
even for a month and the observed relative reduction was 1.72% for
all the targeted compounds except KR which was not recovered
after storage (data not shown). The overall assay procedure has
shown a short separation time of 7 min only. Therefore this optimized method reduced the sample preparation and required
analysis time in addition to simultaneous proling of more classes
of PGRs. These merits make this method a choice for high
throughput analysis over the other extraction and analysis strategies employed for seaweed PGR analysis [7,10].
The method precision and accuracy was found to be comparable
with those of the methods previously developed for simultaneous
analysis of PGRs from various plant matrices [1,2,28]. Recently, Lu
et al. [29] developed a method employing SPE strategy followed by
the analysis on pressurized capillary electrochromatography that
showed LOD as lower as 0.2 mg/ml for endogenic (IAA) and ectogenic hormones (BA, IAA, IPA, NAA and KT). The other extraction
method based on SPME also showed LODs in the range of
0.121e0.442 mg/ml for IAA, ABA and IBA [2]. The comparison of the
method investigated in this study with other reported methods for
PGR detection is illustrated in Table 3. The LODs for the sample
analyzed herein were in the similar range as reported in some of
the earlier reported methods thus demonstrating another advantage of dispensing the need of expensive cartridges. The earlier
developed methods were evaluated at a few real samples however
the method optimized in this study was evaluated on different
green seaweed species thereby giving precision to the analysis.
Matrix effects were also compensated in this method to attain
accuracy in quantication. Signal recoveries were in the range of
80-92% for PGRs except GA3 for which the signal recovery was 120%
in Ulva reticulata. The LOD and signal recovery was inferior in this
study compared to the report of Lu et al. [1] in which the same
sample enrichment method was employed for analysis of auxins in
Chlorella vulgaris with FLD detection system. The method exploited

Table 1
Analytical performance data for major endogenetic plant growth regulators.
Analyte

Linear range
(mg/ml)

Regression equation

LOD
(mg/ml)

ABA
GA3
IAA
IBA
KR
Salicylic acid

0.2e100
0.2e100
0.2e100
0.2e100
0.2e100
0.2e100

y 26,547x  10,400
y 53,554x 11,074
y 96,168x  17,311
y 74,274x  72,997
y 15,770x  46,378
y 69,670x 40,625

0.995
0.999
0.995
0.990
0.976
0.971

0.5
1
1
1
0.5
0.5

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V. Gupta et al. / Plant Physiology and Biochemistry 49 (2011) 1259e1263

Table 2
Evaluation of linear model for the endogenetic plant growth regulators quantied by
regression variance analysis at p  0.01. SSE is the residual sum of squares.
PGRs

SSE

Degree of
freedom

The mean squares

F-value

ABA
GA3
IAA
IBA
KR
Salicylic acid

3.127  1012
4.772  1010
4.164  1011
3.872  1011
5.799  1012
1.967  1014

7
5
5
5
6
6

668369.29
97697.27
288587.13
278313.43
983118.76
5.726

2032.14
4148.68
1373.08
901.85
307.57
228.70

the natural uorescence properties of indole and naphthalene

derivatives of IAA, IBA and NAA therefore selectively detected
auxins only. However, the same method with slight modications
in this study has shown its versatility for simultaneous analysis of
major hormones in their natural state, dispensing the need of
derivatization of extract prior to analysis or purication through
expensive columns.
The PGRs thereby detected were further identied using electron spray ionization mass spectroscopy (ESI-MS). The precursor
and product ions transition specic for each PGR were identied
using standard compounds. The main product ions obtained after
the molecular fragmentation of standard compounds were used as
diagnostic product ions. The precursor ion (m/z) peak at 265, 175,
203, 346 and 138 matched well with the standard compounds ABA,
IAA, IBA, GA3 and SA respectively when analyzed on ESI- mode. The
m/z peak for KR was recorded at 348 with reference to the standard
when analyzed on ESI mode. The product ion scan spectrum for
IAA gave the ion [M-COOH] and its diagnostic transition (m/z) was
175.18 / 130.12. Similarly, IBA and SA showed ionized structure
as [M-COOH] and their precursor ion transitions (m/z) were
203.01 /158.03 and 138.14 / 93.08 respectively. The stable
product ion for GA3 was [M-C14H22O] and its mass fragmentation
was 346.36 / 253.0 and the corresponding ionized state for ABA
was [M-C5H8O2] with mass fragmentation 265.23 / 164.98. The
diagnostic ion state for KR analyzed on positive mode corresponded
to the [M-C5H9ONH] with precursor-to-product ion transition
(m/z) as 348.16 / 216.12.
The auxins quantied in the present study were IAA and IBA
(Table 4). IAA is the most prevalent auxin in plants but very little is
known about their metabolism and their conjugate intermediates
in seaweeds. Of the few reports on auxins from seaweeds, IAA along
with indole-3-acetamide (IAM), an intermediate of IAA synthesis
through tryptophan mediated pathway was quantied [7,10].
Interestingly, IAA along with another auxin IBA was detected in all
species investigated in this study except Ulva linza. Terrestrial
plants investigated for auxins, other than IAA, showed in-vivo
synthesis of phenyl acetic acid, halogenated IAA as 4-Cl-IAA and IBA
[30]. The plant growth regulator IBA, considered as natural plant
hormone, has shown growth inducing properties more effective

1261

than IAA even at lower concentrations. But its metabolism through

IAA dependent or independent pathway is being debated. One of
the possible synthesis routes for IBA may be through acetylation of
IAA by a microsomal membrane fraction in the presence of acetyl
CoA and ATP [31]. The concentration of IAA in green seaweed
U. fasciata was reported to be 800 pmol/g DW [7], 8.47 mg/kg Fwt in
Undaria pinnatida [8] and 1 mg/g Fwt in Caulerpa sp. [1]. In another
study, IAA content of 90e95 mg/kg Fwt was determined in kelp
extract using HPLC as analytical technique [32]. For unicellular
green alga Chlorella vulgaris, IAA content was reported as 37 ng/g
Fwt [1]. The estimation of IAA together with IBA in this study
(Table 4) apparently ascribed to the sensitivity of the DLLME
method toward auxins as also summarized by Lu et al. [1]. Also, the
hormonal content estimated for U. fasciata (Table 4) in the present
study was signicantly higher over the same studied by Stirk et al.
[7]. Although we are tempted to attribute such high values to the
efciency of method employed, it could be even due to natural
occurrence of higher amounts in the sample itself, which needs to
be investigated.
The trend of endogenous ABA was similar to that of auxins and
was highest for U. reticulata than U. linza and followed by U. taeniata,
U. lactuca, U. fasciata and Monostroma oxyspermum (Table 4).
GA3 was present in the range of 2.26  0.20 to 8.46  0.29
nmole/g Fwt and was below limit of detection for M. oxyspermum.
Gibberellins are known for the regulation of plant growth and
mainly the stem elongation in higher plants [33]. GA3 was detected
and quantied by Prasad et al. [16] in the sap of K. alvarezii which is
shown to have plant growth promoting capacity. The resultant
quantication was 30.85 ppm by HPLC and 27.87 ppm by ESI-MS
therefore signied the HPLC based analysis. In another study for
plant growth regulator analysis, Giannarelli et al. [34] showed
HPLC-MS/MS as a better quantication tool than GC/MS analysis. In
seaweeds, most of the earlier studies on hormone analysis were
restricted to the detection and quantication of auxins, cytokinines
and abscisic acid [7,10,24]. The GA3 in Ulva spp. detected in the
present study although in trace amount highlighted the potential of
the preconcentration method opted in this study.
The cytokinin in the form of riboside conjugate of kinetin was
quantied in all the seaweed species but its relative ratio was
lowest over the other PGRs studied. The endogenous KR content
ranged from 0.76  0.02 to 3.35  0.53 nmole/g Fwt. Cytokinin and
its conjugates have been shown to control numerous events in cell
division, enlargement and differentiation, chloroplast and vascular
tissue development, shoot growth, owering and senescence [35].
Recently, nineteen types of cytokinins including its isoprenoid and
aromatic forms were detected in a number of seaweed species [24].
The hormone kinetin or its conjugate which were earlier considered as un-natural was detected in marine unicellular algae [36].
The same was also identied in plant cell extract (coconut chunk)
by modied method of HPLC/UV/EC [37] and its riboside conjugate
in coconut water using LC-tandem mass spectrometry [38]. Prasad

Table 3
Comparison of the method optimized in this study with the previously reported methods for detection of plant growth promoting substances. ABA: Abscisic acid, BA: Benzyl
adenine, GA3: Gibberellic acid, IAA: Indole acetic acid, IBA: Indole butyric acid, IPA: Indole propionic acid, KR: Kinetin riboside, KT: Kinetin-6-furfurylaminopurine, NAA:
Naphthaline acetic acid, SA: Salicylic acid, NR: Not required.
Analytical method

HPLC

Pressurized CEC

HPLC

HPLC

HPLC

Detector
Column ltration
Mobile phase
LOD (mg/ml)
PGRs detected

UV
NR
Single
0.2e1.0
IAA, IBA, ABA,
GA3, KR, SA
This study

UV
SPE
Single
0.1e0.9
IAA, IPA, NAA,
BA, KT
29

Fluorescence
NR
Single
0.001e0.002
IAA, IBA, IPA,
NAA
1

UV
SPE
Binary mixture
0.1e2.0
ABA, IAA, GA3

UV
SPME
Binary mixture
0.05e0.44
ABA, IAA, IBA, NAA

28

Reference

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V. Gupta et al. / Plant Physiology and Biochemistry 49 (2011) 1259e1263

Table 4
Recoveries of endogenetic plant growth regulators from different green seaweed species. The results showed the amount of hormones in nmole/g FW of the extracted samples.
Different letters above the quantied values indicate signicant differences at probability p  0.05 according to one-way ANOVA.
Analytes

Seaweed samples
U. fasciata

U. lactuca

U. linza

U. reticulata

U. taeniata

M. oxyspermum

ABA
GA3
IAA
IBA
KR
Salicylic acid

19.68  2.15c
2.26  0.5c
13.37  0.57c
3.29  0.63c
1.91  0.52bc
3.27  1.44c

34.85  1.75b
3.43  1.68bc
28.12  1.32ab
4.38  0.81b
2.83  1.63b
5.86  2.72b

39.74  5.68b
5.58  0.23ab
31.67  3.74ab
>LOD
3.35  0.53a
6.02  1.02b

68.28  2.09a
8.46  0.29a
35.24  1.59a
11.16  0.85a
3.32  1.75ab
14.85  0.94a

38.5  1.29b
5.88  1.63ab
22.04  0.62ab
5.08  1.02b
1.52  0.22c
2.91  1.23c

11.35  0.48d
>LOD
19.29  0.41b
3.24  0.29d
0.76  0.03d
2.20  1.26c

et al. [16] also conrmed the presence of kinetin riboside in

K. alvarezii sap.
The method optimized in this study also estimated the quantity
of salicylic acid along with other PGRs. Seaweed thalli contained
salicylic acid as high as 14.85  0.94 nmole/g Fwt in U. reticulata
and lowest as 2.2  0.26 nmole/g Fwt in M. oxyspermum (Table 4).
3. Conclusion
This study reveals a rapid, sensitive and accurate method for
simultaneous proling of major classes of plant growth regulators
in common marine macrophytic green algae. The method validation revealed reproducibility and a linear t analysis. The major
merits of this method include high enrichment efciency, reduced
sample preparation and analysis time. This developed method will
facilitate analysis of phytohormones even at trace amounts in
complex matrices of seaweeds leading to a greater understanding
of various cellular, physiological and biochemical responses and
processes. Further, the phytohormones detected in the algal extract
could possibly be used in formulations of liquid fertilizer for
enhancing the productivity of agriculture crops.
4. Materials and methods

transferred to micro-centrifuge tube and mixed with 2 ml of

extraction buffer (methanol: water: formic acid as 15:4:1), vortexed
at high speed and then extracted overnight at 20
C. This mixture
was centrifuged at 5000 rpm and supernatant was collected,
whereas the residue was further extracted for another an hour.
Both the fractions of supernatants were mixed and processed for
purication and enrichment by DLLME procedure described by Lu
et al. [1]. The concentrated samples were placed into a vial insert
tted with polymeric feet. Each vial insert was then placed in a 2 ml
sample vial and positioned in a sample tray for HPLC analysis.
4.4. HPLC analysis
Chromatographic separation of PGRs was performed on Waters
alliance model (2695 separation module) equipped with photodiode array detector (Waters 2996). A Luna-C18 reversed-phase
column (5.0 mm, 4.6  150 mm, Phenomenex, USA) maintained at
35
C was used as the separation channel. Isocratic elution was
performed at 35
C with mobile phase consisting of methanol: 0.6%
acetic acid (60:40 v/v %) at a constant ow rate of 1.0 ml/min. The
injection volume for standards and samples were 50 ml. UV detection was carried out at three different wavelengths such as 208, 254
and 265 nm. The resultant peaks were further conrmed through
spiking with pure compounds.

4.1. Reagents and chemicals

4.5. Quantication of PGRs
The standards of plant growth regulators (PGRs) such as ABA,
IAA, IBA, GA3, KR and salicylic acid (purity > 99%) were purchased
from SigmaeAldrich (USA). The stock solution of the above standards (1 mg/ml) were individually prepared in methanol and
stored at 40
C. Working concentration (50 ppm) of individual
standard was obtained by diluting each with methanol. A mixture
of all the standards (50 ppm) was also made to analyze the peak
separation and assignment of individual peak to the respective
standard.

Young thalli of green seaweeds U. fasciata, U. lactuca, U. taeniata

and U. linza were collected from Veraval (N 20
54.870 ; E 70
20.830 ),
U. reticulata from Okha (N 22
27.040 ; E 69
03.58) situated on the
west coast of India. Seaweed species M. oxyspermum was laboratory
grown culture in authors laboratory. The thalli of all the seaweed
samples were harvested manually during low tide and then
transported to the laboratory in wet tissue towels in an ice box.
They were then thoroughly cleaned with brush in autoclaved
seawater to remove attached epiphytes and dirt.

The repeatability of the retention times of each standard was

determined with three different injections. The calibration curve
was plotted between peak area of standard compound and
different concentrations of standard compounds ranging from
0.2 mg/ml to 100 mg/ml. The linear range, correlation coefcient (R)
and limit of detection (LOD) were measured from the calibration
curve.
The quantitative analysis of PGRs in the real samples was performed on the basis of the external standard method. Under the
optimized conditions, different concentrations of standard
mixtures were spiked in the plant matrix (M. oxyspermum) to
compensate the matrix effect. The linear range, correlation coefcient, and LOD were determined for all the compounds. The LOD
was based on signal to noise ratio (S/N) > 3. The signal recovery was
determined by the formula (AS/AB)  100, where AS is area of the
peak for spiked sample and AB is area of peak in blank plant matrix.
The repeatability of the analysis was determined by the intraday
and interday precision with sample injections in replicate of three.
Further the precision of the analysis was validated with curve
tting test for linearity and regression variance.

4.3. Sample preparation

4.6. ESI-MS measurement

About 200 mg of seaweed thalli was accurately weighed and

ground to ne powder in liquid nitrogen. The powdered tissue was

Water Q-Tof Micromass instrument equipped with an electrospray ionization interface, MCP detector, and Water MassLynx

4.2. Seaweed samples

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V. Gupta et al. / Plant Physiology and Biochemistry 49 (2011) 1259e1263

software (version 4.0) was used. The eluent of each hormone from
HPLC was manually collected and introduced into the mass spectrometer by direct injection. Mass fragmentation pattern of ABA,
IAA, IBA, GA3 and SA was recorded on ESI negative mode (ESI-),
whereas ESI positive mode (ESI) was employed for KR. The mass
spectrometer was operated at the parameters optimized by Prasad
et al. [16].
4.7. Statistical analysis
Each experiment was conducted in triplicates. The repeated data
was evaluated for linear tting followed by F test for regression
analysis using software Origin 8.0. Further, the quantication data
was reported as mean  SD and analysis of variance (ANOVA) was
performed for the comparison of the results. Any statistical property p  0.01 was considered as signicant.
Acknowledgments
The nancial support received from the FP7 programme of
European Commission under grant agreement no. 241383 and CSIR
for facilities is gratefully acknowledged. Mr. Vishal and Mr. Manoj
would like to thank CSIR for awarding Senior Research Fellowship.
We would also like to thank Dr. P. Paul, Head, Analytical Sciences for
kindly permitting us to use ESI-MS and HPLC facility.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.plaphy.2011.08.004.
References
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plants using dispersive liquideliquid microextraction followed by high
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