Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-015-0785-4
ORIGINAL PAPER
Ectopic expression of SbNHX1 gene in transgenic castor (Ricinus
communis L.) enhances salt stress by modulating physiological
process
Manish Kumar Patel1 • Mukul Joshi1 • Avinash Mishra1 • Bhavanath Jha1
Received: 16 July 2014 / Accepted: 2 May 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract The SbNHX1 gene encodes a vacuolar Na?/H?
antiporter that involved in the maintenance of ion home-
ostasis and compartmentalization of excess Na? or K? ions
into the vacuole. Transgenic castor plants were developed
by an improved method of Agrobacterium mediated ge-
netic transformation using spermidine (1 mM) along with
acetosyringone (200 lM), which enhanced the transfor-
mation efficiency about twofolds from 2.76 to 5.91 %.
Transgenic plants were confirmed by PCR using gene
(SbNHX1, hptII and gus) specific primers. The single gene
integration event was confirmed by RTqPCR and Southern
hybridization. Transgenic lines CL7 and CL13 showed
high expression of the SbNHX1 gene compared to CL6 and
CL12, therefore selected for physio-biochemical analyses,
which were carried out under varying NaCl concentrations.
Higher chlorophyll, RWC, K? content, K?/Na? ratio and
lower electrolytic leakage, proline, MDA, Na? contents
compared to WT confirmed that ectopic expression of the
SbNHX1 gene enhances salt tolerance of transgenic plants
by modulating physiological process under stress condi-
tion. Though transgenic lines were affected under stress
conditions but performed better compared to WT plants.
The present study is the first report of engineering salt
Electronic supplementary material The online version of this
article (doi:10.1007/s11240-015-0785-4) contains supplementary
material, which is available to authorized users.
& Avinash Mishra
avinash@csmcri.org
& Bhavanath Jha
bjha@csmcri.org
1
Discipline of Marine Biotechnology and Ecology, CSIR-
Central Salt and Marine Chemicals Research Institute,
G. B. Marg, Bhavnagar 364002, Gujarat, India
tolerance in castor, so far. Transgenic castor may be uti-
lized for the cultivation in marginal salty land and thus
open up the possibility of releasing arable land, which is
presently under castor cultivation.
Keywords Abiotic stress  Genetic transformation 
Transgenic  Salicornia  Salt tolerance
Introduction
Castor (Ricinus communis L.), a member of the Euphor-
biaceae family, is a non-edible oilseed crop, widely culti-
vated in tropical and subtropical regions of the world, such
as India, Brazil, China, Thailand, Ethiopia and Philippines.
India is the largest producer of castor with an annual pro-
duction of about 2.6 million tonnes. Castor seeds contain
high oil content ranging between 42 and 58 %. Castor oil is
unsuitable for edible purpose because of high ratio
(84–90 %) of a monounsaturated hydroxy fatty acid rici-
noleate (12-hydroxy-octadeca cis-9-enoic acid; 18:1-OH)
(Weiss 2000). In recent years, castor oil has been used
extensively for medicinal and industrial purposes, such as
laxative, paint, varnishes, nylon type synthetic polymer,
cosmetics, resins, textile dyeing, hydraulic fluid, caulks and
high quality lubricants for high speed aero-engines and jet
turbine engines (Weiss 2000). Castor bean is a potential
source of biodiesel, however extremely high viscosity and
hygroscopicity of ricinoleic acid fascinate moisture and
produce high water content that hampers the use of castor
oil for biodiesel production (Conceicao et al. 2007). Rojas-
Barros et al. (2004) reported a natural castor mutant that
can be used for biodiesel production because it contains
high oleic acid and low ricinoleic acid. Recent studies and
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genetic improvements have increased the oil content in
castor, but plant growth and production intend to decline
because of abiotic and biotic stresses, such as high salinity,
drought, pathogens and insect pests (Dange et al. 2005;
Severino et al. 2012).
Plant development and productivity are adversely af-
fected due to increase in salinization of lands. It is one of
the irresistible forms of stress that limits the distribution of
fertile land around the world. Over 6 % of land throughout
the world is affected by salt, which is approximately 22 %
of the total agricultural land (FAO 2008). The threshold
level of salinity for castor growth and survival is
4–7 dS m-1 (40–70 mM), thereafter plants show reduction
in growth, photosynthesis and chlorophyll content with
increasing salinity (Zhou et al. 2010). Thus, salinity is one
of the major challenges for increased production of castor
(Severino et al. 2012).
Plants adopt numerous molecular mechanisms to pre-
vent themselves from the negative impact of salt stress.
The simplest strategy that plants employ for salt tolerance
is sodium exclusion mediated by Na?/H? antiporters and
sodium compartmentalization in large vacuoles for main-
taining low level of sodium in the cytosol (Apse et al.
1999). All NHX1 proteins are localized in the vacuolar
membrane and involved in ion homeostasis by using vac-
uolar membrane H?-ATPase and H?-pyrophosphatases
driven proton gradient (Apse et al. 1999). Over-expression
of Arabidopsis thaliana AtNHX1 conferred enhanced salt
tolerance in Arabidopsis (Apse et al. 1999) and several
other plant species, such as cotton (He et al. 2005), buck-
wheat (Chen et al. 2008) and poplar (Jiang et al. 2012).
Na?/H? antiporter has been isolated from different halo-
phytes, such as Mesembryanthemum crystallinum (Chau-
han et al. 2000), Atriplex gmelini (Hamada et al. 2001),
Thellungiella halophila (Wu et al. 2009), Salicornia
brachiata (Jha et al. 2011a), Karelinia caspica (Liu et al.
2012), Leptochloa fusca (Rauf et al. 2014) and showed
high expression under varying salt stress concentrations.
An efficient, rapid and reproducible in vitro regeneration
and genetic transformation system is a prerequisite for
developing a transgenic plant of any species. Previous
studies on regeneration of castor have been reported from
many meristematic explants (Sujatha and Reddy 1998).
Agrobacterium and microprojectile bombardment mediated
gene transfer methods are widely used for genetic trans-
formation. Till date, there were few reports of genetic
transformation via Agrobacterium (Sujatha and Sailaja
2005) and particle bombardment (Sailaja et al. 2008) in
castor.
Salicornia brachiata is an extreme halophyte and ex-
tensively explored for abiotic stress responsive genes (Jha
et al. 2011b; Chaturvedi et al. 2012; Tiwari et al. 2014;
Udawat et al. 2014). The plant requires NaCl for tissue
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Plant Cell Tiss Organ Cult
culture (Joshi et al. 2012), contains unique oligosaccha-
rides (Mishra et al. 2013), metabolites (Mishra et al. 2015),
sulphur rich seed storage proteins (Jha et al. 2012) and
eaten as salad green. Thus S. brachiata is a potential
indigenous candidate as the gene resource to be used for
developing abiotic stress tolerant transgenic plants (Jha
et al. 2011b; Joshi et al. 2013; Chaturvedi et al. 2014;
Singh et al. 2014a, b). In this study, Agrobacterium me-
diated genetic transformation of castor using spermidine
along with acetosyringone (for the vir gene induction) was
reported and salt tolerant transgenic castor was developed
using the SbNHX1 gene, cloned from an extreme halophyte
S. brachiata. It was observed that over-expression of the
SbNHX1 gene enhances salt stress by modulating physio-
logical processes of the transgenic plant.
Materials and methods
Plant material and explant preparation
Decoated mature castor seeds of a local cultivar (GCH-7)
were surface sterilized with 0.1 % (w/v) aqueous mercuric
chloride solution for 10 min and subsequently rinsed 6
times with sterile distilled water. Embryo explants were
aseptically dissected from endosperm and papery cotyle-
dons were removed carefully. Explants were pre-cultured
on solid Murashige and Skoog (MS; Murashige and Skoog
1962) medium (pH 5.8) supplemented with 0.88 lM BA,
0.7 % (w/v) agar and 3 % (w/v) sucrose. Embryos were
incubated at 25 °C for 1 week in the dark at controlled
laboratory conditions.
Genetic transformation and plant regeneration
Agrobacterium infection and co-cultivation
Genetic transformation was carried out via Agrobacterium
tumefaciens (EHA105 strain) harbouring the plasmid con-
struct pCAMBIA1301-SbNHX1, which contains the gusA
reporter gene and selectable hptII gene, both controlled by
cauliflower mosaic virus (CaMV) 35S promoter (Jha et al.
2011a). Agrobacterium culture was grown in Luria–Bertani
(LB) medium containing 25 mg L-1 rifampicin and
50 mg L-1 kanamycin until the absorbance reached up to
0.6 at 600 nm (OD600 = 0.6). Bacterial cells were pelleted
by centrifugation at 27009g for 10 min at 4 °C. The pellet
was resuspended in ‘ MS media (50 ml) containing 2 %
sucrose, 200 lM acetosyringone (AS) and 1 mM sper-
midine (SPD). The radicle part was removed from the pre-
cultured explants, embryo axes were injured by sterile
needle in the meristematic region and infected with 50 ml
Agrobacterium suspension for 30 min, including a gentle
Plant Cell Tiss Organ Cult
shaking at 10 g to improve the infection. Excess
Agrobacterium cells were blotted out and explants were co-
cultivated on MS medium in the dark at 25 °C for 3 days.
Selection and regeneration of transformants
Following co-cultivation, infected explants were washed
four times with half strength (‘) MS medium containing
400 mg L-1 cefotaxime for 5 min each and further rinsed
in ‘ MS medium for 10 min to remove the antibiotic.
Explants were dried on sterile filter paper and transferred to
MS media supplemented with 0.88 lM BA ? 2.27 lM
TDZ and 400 mg L-1 cefotaxime. After 10 days, explants
were subjected to three rounds of selection on selection
media (SM; MS media supplemented with 2.22 lM
BA ? 2.32 lM KN ? Cefotaxime 400 mg L-1) with 20,
25 or 30 mg L-1 hygromycin, respectively. After selec-
tion, putative transformed shoots were transferred to shoot
induction medium (SIM; MS medium supplemented with
2.22 lM BA ? 1.14 lM TDZ and 400 mg L-1 cefo-
taxime) for 15 days. Regenerated shoots were transferred
to shoot proliferation medium (SPM; MS medium sup-
plemented with 2.22 lM BA ? 2.32 lM KN and
400 mg L-1 cefotaxime) for 30 days. Proliferated shoots
were transferred to shoot elongation medium (SEM; MS
medium supplemented with 1.33 lM BA ? 2.32 lM KN
and 400 mg L-1 cefotaxime) for further 30 days. Elon-
gated shoots were transferred to root induction medium
(RIM; ‘ MS salts with B5 vitamins ? 1.47 lM
IBA ? 0.6 mg L-1 AgNO3 ? 20 mg L-1 PVP and
400 mg L-1 cefotaxime) for rooting. After 30–40 days,
rooted plants were transferred to plastic pots (5 9 10 cm)
containing autoclaved soil and vermiculite mixture (1:1).
The pots were covered with transparent plastic bags and
plantlets were maintained under high humidity for 2 weeks
and then gradually exposed to culture room conditions for
acclimatization. All cultures were maintained under con-
trolled laboratory conditions; 16/8 h (light/dark) photope-
riod at 25 ± 2 °C with cool white fluorescent lamp of
35 lmol m-2 s-1 light intensity.
Histochemical GUS assay
Transient b-glucuronidase gene expression was observed in
transformed embryo axes after 24 h of transformation.
Regenerated putative transgenic shoots (T0 plants) were
assessed for constitutive gus expression after 3, 6 and
9 weeks of selection. Histochemical GUS assay was per-
formed by incubating tissues in GUS assay buffer (1 g L-1
X-Gluc with 0.05 M Na2HPO4, 0.5 mM K3Fe(CN)6,
0.5 mM K4Fe(CN)6, 10 mM EDTA and 0.1 % (v/v) Triton
X-100) for 12 h at 37 °C. The tissue was subsequently de-
stained in 70 % alcohol and GUS spots were examined
under a binocular stereomicroscope (Olympus, Japan).
Molecular analysis
PCR analysis
The genomic DNA of putative transgenic lines of castor
was isolated using CTAB method (Doyle and Doyle 1987).
Transformation was confirmed by PCR using SbNHX1 (F:
50 -ATG TGG TCA CAG TTG AGC TC-30 and R: 50 -CTA
TGT TCT GTC TAG CAA AT TG-30 ), reporter gene gus
(F: 50 -GAT CGC GAA AAC TGT GGA AT-30 and R: 50 -
TGA GCG TCG CAG AAC ATT AC-30 ) and hygromycin
selection marker hptII gene specific primers (F: 50 -TTC
TTT GCC CTC GGA CGA GTG-30 and R: 50 -ACA GCG
TCT CCG ACC TGA TG-30 ) with an initial denaturation
temperature of 94 °C for 5 min, subsequent 35 cycles of
94 °C denaturation for 1 min, 60 °C annealing for 1 min,
72 °C extension for 2 min followed by final extension at
72 °C for 7 min.
Real time quantitative PCR
The real time quantitative PCR (RTqPCR) assay was car-
ried out in the Real-Time iQ5 Cycler (Bio-Rad, USA) to
calculate gene copy number. Genomic DNA concentration
was determined by NanoDrop Spectrophotometer and di-
luted to 1, 10 and 100 ng lL-1 concentrations. The RcFAH
gene exists as single copy in the castor genome (Chan et al.
2010), therefore selected as internal control and standard in
RTqPCR for the determination of numbers of trans-gene.
RTqPCR conditions were optimized for gus gene using
primers (GusRTF: 50 -CTA TGC CGG AAT CCA TCG-30 ,
GusRTR: 50 -GCA TCA CGC AGT TCA ACG-30 ) and the
RcFAH gene (GenBank: XM002528081) using primers
(OHXF: 50 -CGT ATG TCG CTT GGC TGG T-30 , OHXR:
50 -TGT TAG AAT GGT GGC GGC-30 ). The RTqPCR
reaction was performed using 0.25 lM primers (gus or
RcFAH) in a 20 ll reaction mixture using QuantiFast
SYBR Green PCR reaction kit (Qiagen, USA). The opti-
mized RTqPCR conditions were an initial denaturation at
95 °C for 5 min; subsequent 40 cycles at 94 °C for 30 s,
60 °C for 30 s and 72 °C for 45 s followed by a cycle at 95
and 60 °C for 1 min each. The RTqPCR specificity was
checked through a melt curve analysis and 1.5 % agarose
gel electrophoresis. The experiment was repeated three
times independently with two replicates each time. Stan-
dard curve was plotted using threshold cycle (Ct) value of
the endogenous gene (RcFAH) and the transgene (gus) to
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determine RTqPCR efficiencies (Shepherd et al. 2009) as
follows:
Efficiency ¼ 10ð1=slopeÞ  1 ð1Þ
The efficiency values were put in the following formula
(Shepherd et al. 2009) to determine the copy number ratio
of the gus gene to RcFAH gene.
ðCt gene of interestÞ
1 þ Efficiencygene of interest
Ratio ¼ ðCt controlÞ
ð2Þ
1 þ Efficiencycontrol
Southern hybridization
Gene copy number and integration were further analyzed
by Southern blot analysis. For this, genomic DNA (30 lg)
was isolated from transformants, digested with HindIII and
BamHI, separated by a 0.7 % agarose gel electrophoresis
and transferred to Hybond N? membrane (Amersham
Pharmacia, UK) by the capillary method using alkaline
transfer buffer (0.4 N NaOH, 1 M NaCl). The purified
pCAMBIA1301-SbNHX1 plasmid and DNA from a non-
transformed plant were used as a positive and negative
control, respectively. The membrane was air-dried and
DNA transferred to the membrane was fixed by UV cross-
linking using 56 mJ cm-2 energy for 1 min in a UVC 500
cross-linker (Amersham Biosciences, UK). The blot was
hybridized with PCR-generated probe for the SbNHX1
gene, labelled with DIG-11-dUTP and amplified from the
plasmid construct pCAMBIA1301-SbNHX1 using PCR
DIG probe synthesis kit (Roche, Germany). The hybridized
membrane was detected by using CDP-Star chemilumi-
nescent as a substrate, following the manufacturer’s in-
structions (Roche, Germany) and signals were visualized
with X-ray film after 20 min.
Subcellular localization of a SbNHX1:RFP translational
fusion protein
The full length SbNHX1 cDNA was amplified with gene
specific primers (F: 50 -CACC ATG TGG TCA CAG TTG
AGC TC-30 and R: 50 -CTA TGT TCT GTC TAG CAA AT
TG-30 ) and fused with RFP (red fluorescent protein) using
Gateway technology (Walhout et al. 2000). The amplified
blunt-end PCR product (SbNHX1) was first cloned into
pENTER/D-TOPO entry vector (Invitrogen, USA) fol-
lowed by further cloning into destination vector pSITE-
4CA by the LR recombination reaction using LR Clonase
II enzyme mix (Invitrogen, USA). Fusion gene construct
(RFP:SbNHX1) was transformed into onion epidermal
cells by particle bombardment (PDS-1000/He Biolistic,
Biorad, USA). The pSITE-4CA (RFP) vector was used as
control. Transformed onion epidermal cells were observed
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Plant Cell Tiss Organ Cult
for transient expression of RFP using an epifluorescence
microscope (Axio Imager, Carl Zeiss AG, Germany).
Over-expression analysis
The total RNA was isolated from WT and transgenic plant
samples (CL6, CL7, CL12 and CL13) and over-expression of
the SbNHX1 gene was confirmed by semi-quantitative
Reverse transcriptase-PCR (Rt-PCR) analysis. The cDNA
was prepared using ImProm-II Reverse Transcription Sys-
tem kit (Promega, USA). Semi-quantitative Rt-PCR analysis
was performed using cDNA (1 ll) as a template, SbNHX1
gene as a target and RcFAH gene as an internal control. The
Rt-PCR was performed using SbNHX1 (Rt-NHX1F: 50 -ATG
GTG TTT GGG TTG CTG A-30 and Rt-NHX1R; 50 -CTG
CTT CGT CTT GGT TGT CC-30 ) and RcFAH gene specific
primers (OHXF and OHXR) and the amplification products
were analysed with 1.2 % agarose gel.
Physio-biochemical analyses
Leaf disc assay and chlorophyll estimation
Leaf senescence assay was carried out to analyze trans-
genic plants for their salt tolerance (Joshi et al. 2013).
Healthy leaves from transgenic lines (T0 generation) and
WT of similar age were detached, leaf discs of 5 mm di-
ameter were punched out and floated in 5 ml sterilized
distilled water containing different concentrations of sodi-
um chloride (0, 50, 100, 150 or 200 mM) for 8 days. Leaf
discs were maintained under 16/8 h light/dark photoperiod
with white fluorescent lamp of 35 lmol m-2 s-1 light in-
tensity at 25 ± 2 °C. The effect of salt concentrations on
leaf discs was assessed by observing phenotypic changes.
Leaf discs of WT and transgenic lines treated with dif-
ferent concentrations of NaCl were homogenized in 80 %
acetone and incubated for 1–2 h with shaking at 15 g in the
dark. The homogenate was centrifuged at 10009g for
5 min. The absorption of the supernatant was recorded (at
645 and 665 nm) and chlorophyll content was calculated
per gram fresh weight of the tissue (Arnon 1949) as
follows:
Chlorophyll content ðmg=g tissueÞ
½ð20:2  OD645nm Þ þ ð8:02  OD665nm Þ  Volume
¼
Weight of tissues in gram
Salt stress assay
Transgenic lines (CL7 and CL13) and WT plants were
assessed for salt tolerance. For this, plants were grown in
plastic pot, irrigated with 200 mM NaCl at 2 days interval
Plant Cell Tiss Organ Cult
and effect of salt was observed after 7 days. Leaves were
harvested from treated and untreated plants (WT and
transgenic lines, CL7 and CL13) and different physio-
biochemical analyses (relative water, proline, malondi-
aldehyde and ion contents, and electrolyte leakage) were
performed.
Electrolyte leakage analysis
Leaves were harvested from treated and untreated plants
(WT and transgenic lines, CL7 and CL13) and washed
thoroughly with deionised water to remove surface-adhered
electrolytes. Samples were immersed in close vials con-
taining 10 ml deionized water and incubated at 25 °C on a
rotary shaker for 24 h. Subsequently, the initial electrical
conductivity (EC) of the solution (Lt) was measured using
conductivity meter (SevenEasy, Mettler Toledo AG 8603,
Switzerland). Leaf Samples were autoclaved at 120 °C for
20 min, cooled up to 25 °C and final electrical conductivity
(L0) of the solution was measured. The electrolyte leakage
was determined as follows:
Electrolyte leakage ð%Þ ¼ ðLt =L0 Þ  100
Relative water content analysis
Similar size of leaves was used for the determination of
relative water content (RWC). Leaves were harvested from
treated and untreated plants (WT and transgenic lines, CL7
and CL13) and their fresh weight (FW) were recorded
immediately. Leaves were submerged into the deionised
water for overnight and turgid weight (TW) was recorded.
Finally, leaves were kept at 80 °C for 48 h and dry weight
(DW) was recorded. RWC was estimated in percentage as
follows (Barrs and Weatherley 1962):
RWC ð%Þ ¼ ðFWDWÞ=ðTWDWÞ  100
Proline content analysis
Free proline contents of treated and untreated leaves (WT
and transgenic lines, CL7 and CL13) were extracted in 3 %
sulphosalicylic acid by centrifugation at 50009g for
30 min. The extract was treated with acetic acid and nin-
hydrin followed by boiling for 1 h. Proline content was
calculated by a standard curve, prepared against known
concentration of proline measured at 520 nm absorbance
(Bates et al. 1973).
Malondialdehyde content analysis
Lipid peroxidation was estimated by quantifying MDA
contents. Leaf samples (2 g) from WT and transgenic lines
(CL7 and CL13) were homogenized in 2 ml trichloroacetic
acid (0.1 %, TCA), centrifuged at 10,0009g for 10 min
and supernatant were collected. Two set of solution were
prepared. Solution 1 (?TBA) contained Thiobarbituric
acid (0.65 % w/v) prepared in TCA (20 % w/v), whereas
solution 2 (-TBA) was 20 % w/v TCA (TBA was ex-
cluded). A measure of 1 ml solution (1 or 2) was added to
1.0 ml extract. The mixtures were incubated at 95 °C for
30 min and samples were cooled on ice, centrifuged at
10,0009g for 5 min. The absorbance of supernatants was
measured at 440, 532 and 600 nm. Lipid peroxidation was
estimated by determining the concentration of malondi-
aldehyde (MDA) produced by TBA reaction using fol-
lowing formula (Hodges et al. 1999):
A ¼ ½ðAbs532þTBA Abs600þTBA Þ
 ðAbs532TBA Abs600TBA Þ
B ¼ ½ðAbs440þTBA Abs600þTBA Þ  0:0571
MDA ðnmol g1 Þ ¼ ½ðA  B=157000Þ
 106 =Weight of tissues in gram
Ion content analysis
For ion content analysis, treated and untreated leaves
tissues (WT and transgenic lines, CL7 and CL13) were
dried at 70 °C for 48 h in hot air oven and dry weight was
measured. Dried tissues were digested with 4 ml perchloric
acid and nitric acid solution (3:1) for 8–10 h. Digested
samples were dried on the hot plate, diluted to 25 ml with
deionised water and filtered through 0.2-mm filter. Ion
contents were measured by inductively coupled plasma
optical emission spectrometer (Optima 2000DV,
Perkin Elmer, Germany).
Statistical analysis
All data were expressed as mean ± SE and subjected to
analysis of variance (ANOVA) to determine the significance
of difference between the means of WT and transgenic
plants of each treatment group. A Tukey HSD multiple
comparison of mean test was used, significant differences
were considered at P \ 0.01 and indicated by similar letters.
Results
Genetic transformation and regeneration
of transgenic plants
Transgenic castor plants were developed by Agrobac-
terium mediated genetic transformation using embryo
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Fig. 1 Agrobacterium mediated transformation of castor using
embryo axis as explant: a embryo axis explant used for transforma-
tion; transient gus expression of explants with b high, c medium and
d low intensity. Selection of transformants after e 1st round (30 days
old), f 2nd round (50 days old) and g 3rd round (75 days old) on
axes as explants. The effect of AS alone or with SPD on
the transformation was assessed by means of transient gus
expression efficiency. Out of 20 explants per treatment,
number of explants showing transient gus expression
(high, medium or low) was calculated (Fig. 1 and Table
S1). Among different AS concentrations, 15 explants out
of 20, showed gus expression, in which 9, 2 and 4 ex-
plants showed high, medium and low gus intensity, re-
spectively. The maximum gus expression efficiency
(75 %) was observed with 200 lM AS after 72 h.
Therefore, 200 lM AS was selected for experiments with
different concentrations of SPD. About 100 % gus ex-
pression was observed with a combination of 200 lM AS
and 1 mM SPD, co-cultivated for 72 h, in which 15, 4
and 1 explants showed high, medium and low gus in-
tensity, respectively (Figure S1 and Table S1). A total of
1016 embryo axis explants were used for the genetic
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Plant Cell Tiss Organ Cult
hygromycin. Regeneration of transformants; h shoot induction
(90 days old), i multiple shoot proliferation (120 days old), j shoot
elongation (180 days old), k rooting (220 days old) and l hardening
(270 days old) of transgenic plant in plastic pot. m GUS assay of
whole leaf of transgenic plant
transformation and the transformation efficiency was
calculated as the number of GUS and PCR positive
plants, survived after third round of selection with respect
to total explants transformed (Table 1). Average trans-
formation efficiency of 2.76 % was calculated, when
transformation was done with 200 lM AS alone, whereas
5.91 % efficiency was achieved with the combination of
AS and SPD (200 lM AS ? 1 mM SPD) (Table 1).
After co-cultivation, transformants were regenerated to
transgenic plants after hygromycin selection (Fig. 1).
Regenerated leaves from transgenic lines were assayed for
the constitutive expression of gus gene and 50 % of the
total regenerated transgenic plants were found GUS
positive (Fig. 1). Putative transgenic plants were trans-
ferred to plastic pots for hardening and total 4 transgenic
lines (CL6, Cl7, CL12 and CL13) were obtained after
hardening.
Plant Cell Tiss Organ Cult

Table 1 Transformation efficiency of castor using AS (200 lM) alone and in-combination with SPD (1.0 mM) during Agrobacterium infection
Transformed embryo Acetosyringone (200 lM)
Selection (hygromycin) Regenerated plants, GUS positive PCR positive Transformation
after selection efficiency (%)
1st 2nd 3rd

270 123 80 41 22 8 8 2.96

207 103 50 32 10 5 5 2.42
308 152 91 52 28 10 10 3.25
231 110 67 35 16 5 5 2.16
Total: 1016 488 288 160 76 28 28 2.76
Transformed embryo Acetosyringone (200 lM) ? spermidine (1.0 mM)
Selection (hygromycin) Regenerated plants, GUS positive PCR positive Transformation
after selection efficiency (%)
1st 2nd 3rd

270 150 116 73 30 15 15 5.56

207 107 72 35 19 11 11 5.31
308 200 140 103 42 22 22 7.14
231 138 82 44 29 12 12 5.19
Total: 1016 595 410 255 120 60 60 5.91

Molecular analysis of transgenic plants
The gene integration in regenerated transgenic plants was
confirmed by the presence of SbNHX1, selectable marker
hptII and reporter gus genes (Fig. 2a) using PCR amplifi-
cation of expected bands of sizes 1.68, 0.96 and 1.28 kb,
respectively (Fig. 2b–d). Copy number of the integrated
gene was determined by RTqPCR using RcFAH gene as an
internal control (Figure S2) and single copy of gene insertion
was observed in all transgenic lines (CL6, CL7, CL12 and
CL13 obtained after hardening). The melt curve analyses of
genes RcFAH and gus showed single peaks that confirmed
the absence of any dimer or other contamination (Figure S2).
Single band amplification of the gus gene as well as control
gene was confirmed with 1.2 % agarose gel electrophoresis.
Copy number of the gene was further confirmed by Southern
blot analysis and single gene copy insertion was detected in
all transgenic lines CL6, CL7, CL12 and CL13 (Fig. 2e, f).
The over-expression of the SbNHX1 gene was confirmed by
reverse transcriptase PCR (Rt-PCR). Transgenic lines (CL7
and CL13) showed high expression of the gene, whereas low
expression was observed in CL6 and CL12 transgenic lines
(Fig. 2g).
Subcellular localization of SbNHX1:RFP fusion
protein
The subcellular localization study was performed with
pSITE-4CA construct (control) expressing RFP alone and
the RFP:SbNHX1 fusion construct (Fig. 3a) by transient
expression assays using onion epidermal cells (Fig. 3b). In
vivo subcellular localization analysis revealed that the
SbNHX1 protein is a vacuolar NHX (Fig. 3b). Red
fluorescence signals were observed uniformly distributed in
the entire cell region of the onion cells transformed with
RFP alone, whereas vacuolar membrane localized signals
were detected in cells which are transformed with
RFP:SbNHX1 fusion construct (Fig. 3b).
Physio-biochemical analyses of transgenic plants
Based on the SbNXH1 gene expression level (Fig. 2g), two
independent transgenic lines; CL7, and CL13 were selected
for the further physio-biochemical analyses.
Leaf disc assay and chlorophyll content
Leaf disc assay showed salt tolerance of WT and T0
transgenic lines (CL7 and CL13) up to 50 mM NaCl. At
high salt concentration, transgenic lines were also affected
but showed improved growth and enhanced salt tolerance
compared to WT (Fig. 4a). Similar effect of salt concen-
tration was also observed on chlorophyll content (Fig. 4b).
The chlorophyll content of WT and transgenic lines were
not affected up to 50 mM NaCl concentration, thereafter
reduced significantly in WT with increasing salt concen-
trations. The chlorophyll content did not reduce drastically
in transgenic lines compared to WT. Furthermore, trans-
genic line CL13 showed enhanced salt tolerance compared
to transgenic line CL7.

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 Plant Cell Tiss Organ Cult

PstI KpnI XbaI PstI

LB NOS(A)n hptII CaMV35S CaMV35S SbNHX1 35S (A)n CaMV35S uidA NOS(A)n RB
a
M PC WT CL4 CL5 CL6 CL7 CL9 CL10 CL11 CL12 CL13 CL15 PC CL6 CL7 CL12 CL13 WT

b PC WT CL6 CL7 CL12 CL13

M PC WT CL4 CL5 CL6 CL7 CL9 CL10 CL11 CL12 CL13 CL15

c
WT Cl6 CL7 CL12 CL13
M PC WT CL4 CL5 CL6 CL7 CL9 CL10 CL11 CL12 CL13 CL15

RcFAH

d g SbNHX1

Fig. 2 Molecular analysis of transgenic plants. a Schematic map of
plant expression gene construct pCAMBIA1301-SbNHX1. PCR
confirmation of the b SbNHX1 gene (1.68 kb), c gus gene (1.28 kb)
and d hptII gene (0.96 kb). Lane M 1 kb molecular weight marker
ladder; lane PC positive control; lane WT wild type plant and lane CL
transgenic lines. Southern hybridization of putative transgenic plants
using genomic DNA digested with e HindIII and f BamHI. All the
four selected lines showed single copy insertion of the gene. g Over-
expression of the SbNHX1 gene in transgenic lines compared to wild-
type control plants analyzed by semi-quantitative Rt-PCR, where the
RcFAH gene was used as internal gene control

Salt stress assay under unstressed condition (0 mM NaCl), however sig-

Transgenic lines (CL7 and CL13) and WT plants were
assessed for salt tolerance in soil condition for which, both
transgenic and WT plants were irrigated with 200 mM
NaCl solutions (at 2 days interval). Transgenic lines (CL7
and CL13) showed enhanced tolerance at 200 mM NaCl
compared to WT plants (Fig. 5). The result shows a posi-
tive effect of the Na?/H? antiporter on salt endurance.
Electrolyte leakage and relative water content
The electrolyte leakage and relative water content (RWC)
were found almost similar in transgenic and WT plants
nificant (P [ 0.01) reduction in electrolyte leakage was
observed in transgenic lines (CL7 and CL13) during salt
stress (200 mM NaCl) compared to WT plant (Fig. 6a).
Similarly, transgenic lines showed higher RWC compared
to WT plants under salt stress condition (Fig. 6b).
Proline and MDA content
Both transgenic lines (CL7 and CL13) and WT plants ex-
hibited approximately similar proline and MDA contents at
unstressed conditions (0 mM NaCl). Transgenic lines
showed lower accumulation of proline content under stress
condition compared to WT plants (Fig. 6c). Lower content

123
Plant Cell Tiss Organ Cult

a
Vector RFP RB 35SP 35SP TL RFP TER Ocs P nptII OCST LB

RFP-SbNHX1 RB 35SP 35SP TL RFP SbNHX1 TER Ocs P nptII OCST LB

b Bright Field RF Field Merge

RFP (Control)
RFP-SbNHX1 gene construct

Fig. 3 Subcellular localization of SbNHX1:RFP translational fusion expression analysis of onion epidermal cells transformed with vector
protein in onion epidermal cells. a Schematic map of pSITE-4CA alone and recombinant gene construct
(RFP) vector and SbNHX1:RFP gene construct. b Transient

of MDA is an indicative of reduced oxidative damage,
which was observed in transgenic lines compared to WT
plants (Fig. 6d).
Ion content analysis
Transgenic (CL7 and CL13) and WT plants exhibited al-
most similar Na? and K? contents as well as K?/Na? ratio
under controlled conditions (i.e. unstressed; 0 mM NaCl).
There was significant increase of Na? in WT plants com-
pared to transgenic plants under stress treatment. Though
high Na? content was also detected in transgenic plants
(CL7 and CL13) under stress condition compared to their
respective control plants (transgenic plant under unstressed
condition; 0 mM NaCl), but the increase was insignificant
compared to increase of Na? accumulation in WT

123
 Plant Cell Tiss Organ Cult

A B
NaCl WT CL7 CL13
(mM) 1.2
WT CL7 CL13

Chlorophyll content [mg g-1 FW]

1.0
0

a b c 0.8

0.6

50 0.4

d e f
0.2

0.0
100 0mM 50mM 100mM 150mM 200mM
NaCl concentration
g h i

150

j k l

200

m n o

Fig. 4 Leaf disc assay and chlorophyll content analysis. Leaf disc solution for 8 days. Chlorophyll content analysis (B). Chlorophyll
assay of transgenic castor for assessing salt tolerance (A). Leaf discs content of WT and transgenic lines CL7 and CL13 were estimated at
of WT and transgenic lines CL7 and CL13 were floated in a–c 0 mM, 0, 50, 100, 150 and 200 mM NaCl. Graph represents mean and SE of
d–f 50 mM, g–i 100 mM, j–l 150 mM and m–o 200 mM NaCl chlorophyll content in the leaves

plants under stress conditions, however among plants,

CL13 showed maximum K? content followed by CL7 and
WT plants (Fig. 7b). In the case of K?/Na? ratio, all
transgenic lines and WT showed high ratio at unstressed
condition (0 mM NaCl). However, at 200 mM NaCl, ratio
decreased in both transgenic and WT plants but transgenic
lines exhibited higher ratio than WT (Fig. 7c).

Discussion

Fig. 5 Effect of salt on transgenic castor lines and WT plant.
Selected transgenic line and WT plant were irrigated with 200 mM
NaCl solutions in plastic pot and effect of salt was observed after
7 days
(Fig. 7a). The result indicates that transgenic lines accu-
mulated less Na? compared to WT. A significant accu-
mulation of K? was observed in transgenic lines and WT
Antiporters are attributed to ion homeostasis and the
strategy to accumulate Na? or K? inside vacuoles is used
by many plants, especially halophytes like Salicornia
brachiata to survive under salt stress condition. The
SbNHX1 gene was found actively involved in the regula-
tion of ion homeostasis (Jha et al. 2011b; Joshi et al. 2013).
In this study, salt tolerant transgenic castor was developed
using the SbNHX1 gene cloned from extreme halophyte S.

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Plant Cell Tiss Organ Cult

a 100 acegh b 100

WT CL7 CL13 a WT CL7 CL13

Relative water content [%]

90 90
Electrolyte leakage [%] 80 bdfh 80
70 70 a
60 g 60
50 50
40 40
30 ab ef 30
cd
20 20
10 10
0 0
0 mM 200 mM 0 mM 200 mM

c d
1000
Proline content [μg mg-1 FW]

MDA content [nmol g-1 FW]

1.2 WT CL7 CL13 WT CL7 CL13
ace 900
1.0 800
700
0.8 bdf 600
500
0.6
400
0.4 300
ab 200
0.2 cd ef
100
0.0 0
0 mM 200 mM 0 mM 200 mM

Fig. 6 Physiological analyses of transgenic castor lines under (WT) and transgenic lines (CL7 and CL13) treated with salt (200 mM
different stress condition. Comparison of a electrolyte leakage, NaCl). Graph represents the mean ± SE followed by similar letters
b relative water content, c proline and d MDA contents of wild type are significantly different according to Tukey HSD at P \ 0.01

brachiata. Physiological analyses of transgenic plants were
used to understand the efficacy of an integrated gene and its
involvement to modulate the physiological traits.
Transgenic castor plants, over-expressing the SbNHX1
gene were developed via A. tumefaciens mediated genetic
transformation using embryo axes as explants (Fig. 1). In
this study, juvenile embryo axis was chosen as explant
because it is acquiescent to higher transformation effi-
ciency (Sujatha and Reddy 1998; Sujatha and Sailaja
2005; Sailaja et al. 2008). Optimization of an efficient
regeneration and genetic transformation protocol is a key
step before proceeding to further biotechnological pro-
grammes of any crop (Singh et al. 2010; Joshi et al. 2011;
Pandey et al. 2013; Tiwari et al. 2015). Different pa-
rameters have been used earlier for castor transformation
(Sujatha and Sailaja 2005). The condition optimized for
transformation in this study was Agrobacterium cell cul-
ture of 0.6 OD600, 200 lM AS, 1 mM SPD, a co-culti-
vation time of 72 h and wounding using a needle (Figure
S1). SPD is a polyamine and reported to enhance the
Agrobacterium tumefaciens transformation frequency by
inducing the vir genes (Kumar and Rajam 2005). SPD is
commonly used in microprojectile bombardment mediated
genetic transformation. Recently, 200 lM AS was used
along with 1 mM SPD during Agrobacterium infection to
enhance the transformation efficiency in banana (Chong-
Pérez et al. 2012).
There were some reports on the improvement of disease
tolerance in castor (Dange et al. 2005), but limited infor-
mation is available for the improvement of abiotic stress
tolerance. Salt stress is one of the major factor among
different abiotic stresses, which negatively affects the
growth of castor plant (Zhou et al. 2010). Salt stress tol-
erance of a plant is a multigenic trait, which is controlled
by the coordinated action of several genes. But it is also
reported that over-expression of a single gene can confer
salt tolerance to the plant (Apse et al. 1999; Jha et al.
2011b; Chaturvedi et al. 2014; Singh et al. 2014a, b). It
may take into account that the protein produced in trans-
genic plants by over-expression of only one gene probably
interacts with other proteins of plant, contributing for tol-
erance to salt stress, which is a very complex process. In
the present study, we have successfully developed salt
tolerant transgenic castor over-expressing the salt-respon-
sive SbNHX1 gene via optimized Agrobacterium mediated
transformation method. This protocol yielded transgenic
plants with 5.91 % transformation efficiency, which is
higher than previous reports (Sujatha and Sailaja 2005;
Sailaja et al. 2008) for castor. RTqPCR is recently being
used to study functional gene abundance (Yousuf et al.
2012; Keshri et al. 2013; Yousuf et al. 2014a, b; Keshri
et al. 2015) and as an alternative to Southern hybridization
to determine copy numbers of integrated genes (Joshi et al.
2013). Four transgenic lines CL6, CL7, CL12 and CL13

123
 Plant Cell Tiss Organ Cult

a 20 transgenic lines CL7 and CL13 was assessed under salt

WT CL7 CL13 ace stress in a dose-dependent manner and compared with WT
Na+ content [mg g-1 DW]

16 plants (Figs. 4, 5, 6). Higher chlorophyll content, potassi-

bdf
um ion content, K?/Na? and water retention capacity
12 (RWC) of transgenic plants exhibited better physiology of
plants under stressed conditions. Moreover, lower accu-
8 mulation of proline and MDA contents revealed that
transgenic castor lines have higher osmoprotectants and
4 low lipid peroxidation, respectively compared to WT plants
ef under stress condition. These results evident the better
ab cd
0 physiological status of transgenic plants compared to WT
0 mM 200 mM
under stress. Additionally, significant increase of Na? and
b 40 reduction of K? in WT plants were observed compared to
WT CL7 CL13 c
transgenic plants, over-expressing the SbNHX1 gene under
35
stress condition (Fig. 7). Accumulation of less Na? but
K+ content [mg g-1 DW]

30 b
more K? in transgenic plants than WT plants under salt
25 stress suggested that salinity resistance could be due to the
a
20 optimised K? compartmentalization. It was observed that
AtNHX1 protein can transport Na? and K? with equal
15
c affinity and it transported K? for osmoregulation under
10 a b
saline stress (Venema et al. 2002). It was also found that
5 AtNHX1 and AtNHX2 were actively involved in K?
0 homeostasis than Na? sequestration (Bassil et al. 2011).
0 mM 200 mM Similarly, AtNHX1 gene over-expression provided NaCl
c 10 tolerance to tomato by accumulating K? in vacuoles
ab WT CL7 CL13
(Barragán et al. 2012). Previously, Jatropha and poplar
8 over-expressing SbNHX1 and AtNHX1, respectively
showed similar results, moreover WT Jatropha also showed
K+/Na+ Ratio

6 bleaching in leaves under stress treatments compared to

4 a
2 AtNHX1 gene during salt stress. It was also observed that
0 Na?, K?/H? antiport activity, involved in K? homeostasis
0 mM 200 mM
Fig. 7 Sodium and potassium ion contents in transgenic castor lines
under salt stress condition. Comparison of a Na?, b K? contents and
c K?/Na? ratio in WT and transgenic lines
showed single copy gene insertion analyzed by RTqPCR,
which was further confirmed by Southern analysis (Fig. 2).
The SbNHX1 was classified as a vacuolar NHX gene based
protein topology and the presence of functional domains
(Jha et al. 2011a). However, a direct evidence of the vac-
uolar localization was shown in the present study (Fig. 3).
It was observed that AtNHX1 and AtNHX2 were localize to
the vacuole (Bassil et al. 2011). Recently, transient ex-
pression of AtNHX3 and AtNHX4 displayed expression at
the vacuolar membrane (McCubbin et al. 2014).
Chlorophyll content, electrolyte leakage, RWC, proline
and MDA contents are considered notable indicators for
the physiological status of plants. Physiological response of
transgenic plants (Jiang et al. 2012; Joshi et al. 2013).
b Similar results have been reported in buckwheat (Chen
et al. 2008) and cotton (He et al. 2005) over-expressing the
antiporters AtNHX1 and AtNHX2 together account for the
and Arabidopsis NHX double mutant shows increased Na?
content compared to WT under 100 mM NaCl stress
(Barragán et al. 2012).
Furthermore, transgenic lines (CL7 and CL13) and WT
plants were also assessed for salt tolerance in soil condition
for which, both transgenic and WT plants were irrigated
with 200 mM NaCl solutions (at 2 days interval). The re-
sult further confirmed that transgenic plants, over-ex-
pressing the SbNHX1 gene had better growth compared to
WT plant and showed salt endurance (Fig. 5). The
PgNHX1 gene from Pennisetum glaucum has been reported
to confer salinity tolerance when over-expressed in Bras-
sica juncea and transgenic plants could tolerate higher salt
stress than the WT in a leaf disc assay and in pot (Ra-
jagopal et al. 2007). Salt tolerance of a halophyte Karelinia
caspica was shown to decrease by RNAi silencing of
KcNHX1 (Liu et al. 2012). Recently, it was observed that
over-expression of TaNHX3 enhances salt stress tolerance

123
Plant Cell Tiss Organ Cult
in tobacco by improving physiological processes (Lu et al.
2014).
Conclusion
In conclusion, introgression of the SbNHX1 gene mod-
ulates physiology of transgenic plants and thus imparts
enhanced stress endurance. Transgenic castor plants were
developed using optimized and improved protocol with
5.91 % transformation efficiency. Transgenic lines CL7
and CL13 contained higher chlorophyll, RWC, K? con-
tents and K?/Na? ratio, furthermore lower electrolytic
leakage, proline, MDA and Na? contents were observed in
transgenic lines compared to WT under stress condition. In
the present study, transgenic lines showed improved
growth and enhanced salt tolerance compared to control
plants at 200 mM NaCl stress. The transgenic castor with
improved salt tolerance may be utilized for the cultivation
in salt-affected marginal areas for sustainable land man-
agement, oil production and other industrial applications.
Acknowledgments CSIR-CSMCRI Communication No. PRIS-035/
2013. The financial support received from Council of Scientific and
Industrial Research (CSIR), New Delhi [SIMPLE; BSC0109] is
thankfully acknowledged. Dr. Santlal Jaiswar is duly acknowledged
for providing technical support in the microscopic analysis of local-
ization study. Authors are grateful to Arabidopsis Biological Re-
source Centre, Ohio State University, USA for providing the pSITE-
4CA vector.
References
Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt
tolerance conferred by over expression of a vacuolar Na?/H?
antiport in Arabidopsis. Science 285:1256–1258
Arnon DI (1949) Copper enzymes in isolated chloroplasts polyphenol
oxidase in Beta vulgaris. Plant Physiol 24:1–15
Barragán V, Leidi EO, Andrés Z, Rubio L, De Luca A, Fernández JA,
Cubero B, Pardo JM (2012) Ion exchangers NHX1 and NHX2
mediate active potassium uptake into vacuoles to regulate cell
turgor and stomatal function in Arabidopsis. Plant Cell
24:1127–1142
Barrs HD, Weatherley PE (1962) A re-examination of the relative
turgidity technique for estimating water deficits in leaves. Aust J
Biol Sci 15:413–428
Bassil E, Tajima H, Liang YC, Ohto MA, Ushijima K, Nakano R,
Esumi T, Coku A, Belmonte M, Blumwald E (2011) The
Arabidopsis Na?/H? antiporters NHX1 and NHX2 control
vacuolar pH and K? homeostasis to regulate growth, flower
development, and reproduction. Plant Cell 23:3482–3497
Bates LS, Waldern R, Teare ID (1973) Rapid determination of free
proline for water stress studies. Plant Soil 39:205–207
Chan AP, Crabtree J, Zhao Q, Lorenzi H, Orvis J, Puiu D (2010) Draft
genome sequence of the oilseed species Ricinus communis. Nat
Biotechnol 28:951–956
Chaturvedi AK, Mishra A, Tiwari V, Jha B (2012) Cloning and
transcript analysis of type 2 metallothionein gene (SbMT-2) from
extreme halophyte Salicornia brachiata and its heterologous
expression in E. coli. Gene 499:280–287
Chaturvedi AK, Patel MK, Mishra A, Tiwari V, Jha B (2014) The
SbMT-2 gene from a halophyte confers abiotic stress tolerance
and modulates ROS scavenging in transgenic tobacco. PLoS
ONE 9(10):e111379
Chauhan S, Forsthoefel N, Ran Y, Quigley F, Nelson DE, Bohnert HJ
(2000) Na?/myo-inositol symporters and Na?/H? antiport in
Mesembryanthemum crystallinum. Plant J 24:511–522
Chen LH, Zhang B, Xu ZQ (2008) Salt tolerance conferred by
overexpression of Arabidopsis vacuolar Na?/H? antiporter gene
AtNHX1 in common buckwheat (Fagopyrum sculentum). Trans-
genic Res 17:121–132
Chong-Pérez B, Reyes M, Rojas L, Ocaña B, Pérez B, Kosky RG,
Angenon G (2012) Establishment of embryogenic cell suspen-
sion cultures and Agrobacterium-mediated transformation in
banana cv. ‘Dwarf Cavendish’ (Musa AAA): effect of sper-
midine on transformation efficiency. Plant Cell, Tissue Organ
Cult 111:79–90
Conceicao MM, Fernandez VJ, Bezerra AF, Silva MCD, Santos IMG,
Silva FC (2007) Dynamic kinetic calculations of castor oil
biodiesel. J Therm Anal Calorim 87:865–869
Dange SRS, Desal AG, Patel SI (2005) Diseases of castor. In: Saharan
GS, Mehta N, Sangwan MS (eds) Diseases of oilseed crops.
Indus Publishing Company, New Delhi, pp 211–234
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small
quantities of fresh leaf tissue. Phytochem Bull 19:11–15
FAO (2008) FAO land and plant nutrition management service. http://
www.fao.org/ag/agl/agll/spush
Hamada A, Shona M, Xia T, Ohta M, Hayashi Y, Tanaka A,
Hayakawa T (2001) Isolation and characterization of a Na?/H?
antiporter gene from the halophyte Atriplex gmelini. Plant Mol
Biol 46:35–42
He C, Yan J, Shen G, Fu L, Holaday AS, Auld D, Blumwald E, Zhang
H (2005) Expression of an Arabidopsis vacuolar sodium/proton
antiporter gene in cotton improves photosynthetic performance
under salt conditions and increases fiber yield in the field. Plant
Cell Physiol 46:1848–1854
Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving
the thiobarbituric acid-reactive-substances assay for estimating
lipid peroxidation in plant tissues containing anthocyanin and
other interfering compounds. Planta 207:604–611
Jha A, Joshi M, Yadav NS, Agarwal PK, Jha B (2011a) Cloning and
characterization of the Salicornia brachiata Na?/H? antiporters
gene SbNHX1 and its expression by abiotic stress. Mol Biol Rep
38:1965–1973
Jha B, Sharma A, Mishra A (2011b) Expression of SbGSTU (tau class
glutathione S-transferase) gene isolated from Salicornia brachi-
ata in tobacco for salt tolerance. Mol Biol Rep 2011(38):
4823–4832
Jha B, Singh NP, Mishra A (2012) Proteome profiling of seed storage
proteins reveals the nutritional potential of Salicornia brachiata
Roxb., an extreme halophyte. J Agric Food Chem 60:4320–4326
Jiang C, Zheng Q, Liu Z, Xu W, Liu L, Zhao G, Long X (2012)
Overexpression of Arabidopsis thaliana Na?/H? antiporter gene
enhanced salt resistance in transgenic poplar (Populus eu-
ramericana ‘Neva’). Trees 26:685–694
Joshi M, Mishra A, Jha B (2011) Efficient genetic transformation of
Jatropha curcas L. by microprojectile bombardment using
embryo axes. Ind Crops Prod 33:67–77
Joshi M, Mishra A, Jha B (2012) NaCl plays a key role for in vitro
micropropagation of Salicornia brachiata, an extreme halophyte.
Ind Crops Prod 35:313–316
Joshi M, Jha A, Mishra A, Jha B (2013) Developing transgenic
jatropha using the SbNHX1 gene from an extreme halophyte for
cultivation in saline wasteland. PLoS ONE 8(8):e71136
123

Keshri J, Mishra A, Jha B (2013) Microbial population index and
community structure in saline-alkaline soil using gene targeted
metagenomics. Microbiol Res 168:165–173
Keshri J, Yousuf B, Mishra A, Jha B (2015) The abundance of
functional genes, cbbL, nifH, amoA and apsA and bacterial
community structure of intertidal soil from Arabian Sea.
Microbiol Res Online. doi:10.1016/j.micres.2015.02.007
Kumar SV, Rajam MV (2005) Polyamines enhance Agrobacterium
tumefaciens vir gene induction and T-DNA transfer. Plant Sci
168:475–480
Liu L, Zeng Y, Pan X, Zhang F (2012) Isolation, molecular
characterization, and functional analysis of the vacuolar Na?/
H? antiporter genes from the halophyte Karelinia caspica. Mol
Biol Rep 9:7193–7202
Lu W, Guo C, Li X, Duan W, Ma C, Zhao M et al (2014)
Overexpression of TaNHX3, a vacuolar Na?/H? antiporter gene
in wheat, enhances salt stress tolerance in tobacco by improving
related physiological processes. Plant Physiol Biochem 76:17–28
McCubbin T, Bassil E, Zhang S, Blumwald E (2014) Vacuolar Na?/
H? NHX-type antiporters are required for cellular K? home-
ostasis, microtubule organization and directional root growth.
Plants 3(3):409–426
Mishra A, Joshi M, Jha B (2013) Oligosaccharide mass profiling of
nutritionally important Salicornia brachiata, an extreme halo-
phyte. Carbohydr Polym 92:1942–1945
Mishra A, Patel MK, Jha B (2015) Non-targeted metabolomics and
scavenging activity of reactive oxygen species reveal the
potential of Salicornia brachiata as a functional food. J Funct
Foods 13:21–31
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bio-assays with tobacco tissue cultures. Physiol Plant 15:
473–497
Pandey S, Mishra A, Patel MK, Jha B (2013) An efficient method for
Agrobacterium-mediated genetic transformation and plant re-
generation in cumin (Cuminum cyminum L.). Appl Biochem
Biotechnol 171:385–392
Rajagopal D, Agarwal P, Tyagi W, Singla-Pareek SL, Reddy MK,
Sopory SK (2007) Pennisetum glaucum Na?/H? antiporters
confers high level of salinity tolerance in transgenic Brassica
juncea. Mol Breed 19:1–9
Rauf M, Shahzad K, Ali R, Ahmad M, Habib I, Mansoor S et al
(2014) Cloning and characterization of Na?/H?antiporter
(LfNHX1) gene from a halophyte grass Leptochloa fusca for
drought and salt tolerance. Mol Biol Rep 41:1669–1682
Rojas-Barros P, De Haro A, Muñoz J, Fernández-Martı́nez JM (2004)
Isolation of a natural mutant in castor bean (Ricinus communis
L.) with high oleic/low ricinoleic acid content. Crop Sci
44:76–80
Sailaja M, Tarakeswari M, Sujatha M (2008) Stable genetic
transformation of castor (Ricinus communis L.) via particle
gun-mediated gene transfer using embryo axes from mature
seeds. Plant Cell Rep 27:1509–1519
Severino LS, Cordoba-Gaona OJ, Zanotto MD, Auld DL (2012) The
influence of the caruncle on the germination of castor seed under
high salinity or low soil water content. Seed Sci Technol
40:139–143
Shepherd CT, Lauter ANM, Scott MP (2009) Determination of
transgene copy number by real-time quantitative PCR. In: Scott
MP (ed) Methods in molecular biology: transgenic maize.
Humana, New York, pp 129–134
Singh N, Mishra A, Joshi M, Jha B (2010) Microprojectile
bombardment mediated genetic transformation of embryo axes
123
Plant Cell Tiss Organ Cult
and plant regeneration in cumin (Cuminum cyminum L.). Plant
Cell Tissue Org Cult 103:1–6
Singh N, Mishra A, Jha B (2014a) Over-expression of the peroxiso-
mal ascorbate peroxidase (SbpAPX) gene cloned from halophyte
Salicornia brachiata confers salt and drought stress tolerance in
transgenic tobacco. Mar Biotechnol 16:321–332
Singh N, Mishra A, Jha B (2014b) Ectopic over-expression of
peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt
stress tolerance in transgenic peanut (Arachis hypogaea). Gene
547:119–125
Sujatha M, Reddy TP (1998) Differential cytokinin effects on the
stimulation of in vitro shoot proliferation from meristematic
explants of castor (Ricinus communis L.). Plant Cell Rep
17:561–566
Sujatha M, Sailaja M (2005) Stable genetic transformation of castor
(Ricinus communis L.) via Agrobacterium tumefaciens-mediated
gene transfer using embryo axes from mature seeds. Plant Cell
Rep 23:803–810
Tiwari V, Chaturvedi AK, Mishra A, Jha B (2014) The transcriptional
regulatory mechanism of the peroxisomal ascorbate peroxidase
(pAPX) gene cloned from an extreme halophyte, Salicornia
brachiata. Plant Cell Physiol 55:201–217
Tiwari V, Chaturvedi AK, Mishra A, Jha B (2015) An efficient
method of Agrobacterium-mediated genetic transformation and
regeneration in local Indian cultivar of groundnut (Arachis
hypogaea) using grafting. Appl Biochem Biotechnol 175:
436–453
Udawat P, Mishra A, Jha B (2014) Heterologous expression of an
uncharacterized universal stress protein gene (SbUSP) from the
extreme halophyte, Salicornia brachiata, which confers salt and
osmotic tolerance to E. coli. Gene 536:163–170
Venema K, Quintero FJ, Pardo JM, Donaire JP (2002) The
Arabidopsis Na?/H? exchanger AtNHX1 catalyzes low affinity
Na? and K? transport in reconstituted liposomes. J Biol Chem
277:2413–2418
Walhout A, Temple G, Brasch M, Hartley J, Lorson MA, van den
Heuvel S, Vidal M (2000) GATEWAY recombinational cloning:
application to the cloning of large numbers of open reading
frames or ORFeomes. Methods Enzymol 328:575–592
Weiss A (2000) Oilseed crops. Blackwell Science, Oxford
Wu CX, Gao XH, Kong XQ, Zhao YX, Zhang H (2009) Molecular
cloning and functional analysis of a Na?/H? antiporter gene
ThNHX1 from a halophytic plant Thellungiella halophila. Plant
Mol Biol Rep 27:1–12
Yousuf B, Keshri J, Mishra A, Jha B (2012) Application of targeted
metagenomics to explore abundance and diversity of CO2-fixing
bacterial community using cbbL gene from the rhizosphere of
Arachis hypogaea. Gene 506:18–24
Yousuf B, Kumar R, Mishra A, Jha B (2014a) Differential distribu-
tion and abundance of diazotrophic bacterial communities across
different soil niches using gene targeted clone library approach.
FEMS Microbiol Lett 360:117–125
Yousuf B, Kumar R, Mishra A, Jha B (2014b) Unravelling the carbon
and sulphur metabolism in coastal soil ecosystems using
comparative cultivation-independent genome-level characterisa-
tion of microbial communities. PLoS ONE 9(9):e107025
Zhou G, Ma BL, Li J, Feng C, Lu J, Qin P (2010) Determining
salinity threshold level for castor bean emergence and stand
establishment. Crop Sci 50:2030–2036