Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland

Bhavanath Jha; Avinash Mishra; Anupama Jha; Mukul Joshi

doi:10.1371/journal.pone.0071136

Abstract

Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na⁺/H⁺ antiporter gene that compartmentalises excess Na⁺ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.

Citation: Jha B, Mishra A, Jha A, Joshi M (2013) Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland. PLoS ONE 8(8): e71136. https://doi.org/10.1371/journal.pone.0071136

Editor: Jean Peccoud, Virginia Tech, United States of America

Received: May 29, 2013; Accepted: July 2, 2013; Published: August 5, 2013

Copyright: © 2013 Jha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The financial assistance received from CSIR (www.csir.res.in), New Delhi (CSC0102: TapCoal) is duly acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Jatropha is a second generation biofuel resource valued for its high oil content, low seed cost, land reclamation and easy adaptation to different kinds of marginal and semi marginal lands. The broad potential of this plant and multiple uses of different plant parts have made this species quite profitable for cultivation [1], [2]. As fossil fuels pose a great threat to energy security and also adversely affect the environment, initiatives are being taken for the partial replacement of fossil fuels by biofuels [3]. Recently, attention has been drawn to the high oil content (up to 50%) of its seeds that can be easily processed to partially or fully replace petroleum based diesel fuel [3], [4]. Jatropha is a non-food crop that separates this plant from fuel vs food controversy. Its oil properties include flash point 235°C and calorific value 39.63 MJ kg⁻¹ that make this oil suitable as biofuel [1]. Jatropha oil contains 45.79% oleic acid (18∶1), 32.27% linoleic acid (18∶2), 13.37% palmitic acid (16∶0) and 5.43% stearic acid (18∶0) that is comparable with peanut, palm and corn oil [5]. Jatropha ranks next to oil palm in oil production per hectare, encouraging its cultivation worldwide [6]. There are concerns that Jatropha investors may drive its cultivation from marginal or degraded lands towards agricultural lands in order to reduce financial risk [7] but this possibility was recently ruled out [8].

Jatropha is well distributed in India [9]; this encourages its use as an alternative source for energy security in the country. India has a plan to expand biodiesel production and substitute 20% of diesel consumption by 2020 [10]. The land required to achieve this substitution target ranges from 4.24–66.98 million hectares (Mha) depending on the potential yield of the plant varieties and further its improvement programs. This target is feasible because of the extent of available wastelands in India [11]. CSIR-CSMCRI has been recognized worldwide and is actively working on Jatropha elite accessions’ selection, cultivation, genetic improvement and biodiesel production [6], [12].

Over 800 Mha of land throughout the world are affected by salt [13]. In India, the total arable land area is about 184 Mha, out of which 8.6 Mha is salt affected [14]. Salinity imposes various constraints on plant growth and yield from osmotic stress created due to high concentrations of Na⁺ and Cl^- ions [15]. There are some major effects of salt stress, i.e. water potential reduction, ionic imbalance or disturbances in ion homeostasis and ion toxicity, which inhibits enzymatic functions in key biological processes [15], [16].Salt tolerance depends on a range of physiological, biochemical and molecular adaptations activated by gene(s). The adaptive response to salinity is multigenic in nature, however a single gene can also increase the salt tolerance of a plant species [17], [18], [19]. Antiporters Na⁺/H⁺ are expressed by functional gene(s) and play an important role in plant salt tolerance. The basic strategy of salt tolerance is maintenance of Na⁺ homeostasis in the cytosol [15]; however it varies with the plant species. Ion homeostasis is carried out either by sequestration of excess sodium into the vacuoles via vacuolar Na⁺/H⁺ antiporters (NHX1) energized by the proton gradient generated by vacuolar membrane H⁺-ATPase and H⁺-pyrophosphatases [15], [17], or by active exclusion through Na⁺/H⁺ antiporters (SOS1) located on the plasma membrane [18], [19]. Therefore, the ability to maintain lower Na⁺ and Cl^- in the cytosol may be a key determinant of salt tolerance.

The previous study that vacuolar NHX proteins are capable of Na⁺ and H⁺ exchange across the tonoplast [15], [17], has been modified based on the biochemistry of the NHX proteins. Recently it was reported that NHX proteins do not discriminate between Na⁺ and K⁺ or have a preference for K⁺ transport [20], [21]. These reports suggest that NHX1 and NHX2 are vacuolar Na⁺–K⁺/H⁺ exchangers essential for active K⁺ uptake at the tonoplast, osmotic adjustment, turgor regulation, stomata function and cell expansion [22], [23].

Jatropha growth and yield are adversely affected by increasing salt concentration beyond 30–50 mM [24], [25], [26]. Therefore, Jatropha is considered to be a moderately salt-tolerant plant and its salinity tolerance is comparable with major crops such as soybean or wheat [27]. Development of transgenic Jatropha plants by the overexpression of selected salt-responsive gene(s) is a better choice for genetic improvement of the plant for enhanced salt tolerance and fastening the breeding of improved plants. There are several methods available for genetic transformation of plants but the widely used methods are the vector-mediated method using Agrobacterium and direct gene transfer via microprojectile bombardment. Both these methods have their advantages and limitations [28], [29]. We have reported an efficient microprojectile bombardment mediated transformation protocol for Jatropha [12] that has been used in this study.

Nevertheless, generating transgenic cultivars is not only limited by the success of the transformation process but also proper incorporation of the new stress tolerance. Recently, transgenic Jatropha plants were developed for trait improvement [30], [31]. Assessment of transgenic plants under salt stress conditions and understanding the physiological effects of inserted genes at the whole plant level remain as major challenges to overcome [32], [33]. A salt-tolerant SbNHX1 gene (GenBank: EU448383) (Figure 1) encoding an active vacuolar Na⁺/H⁺ antiporter has been cloned and characterized from an extreme halophyte Salicornia brachiata in our laboratory [34]. The core objective of the present work is developing transgenic Jatropha with the SbNHX1 gene for enhanced salinity tolerance. Salt tolerant transgenic Jatropha plants can be further utilized for sustainable cultivation in salt-affected marginal areas.

Download:

Figure 1. Schematic map of plant expression gene construct pCAMBIA1301-SbNHX1.

Expression of SbNHX1 is driven by the cauliflower mosaic virus 35S promoter.

https://doi.org/10.1371/journal.pone.0071136.g001

Results

Microprojectile Bombardment Mediated Transformation

Five days pre-cultured embryo axes were bombarded at optimized parameters, i.e. 1 µm microcarrier size, 1100 and 1350 psi He pressure, with target distances of 9 and 12 cm, respectively [12]. After bombardment, 3–5 transformed embryos per shot per plate were selected randomly after 24 h and transient gus expression was assessed. Most of the explants showed discrete blue spots or regions (Figure 2a). Bombarded embryo axes were regenerated into transgenic plants after hygromycin selection (Figure 2b–f) as reported previously [12]. Putative transgenic plants with rooted shoots were transplanted into pots for hardening and acclimatization (Figure 2g and Table S1). Regenerated young leaves from putative transformed plants were assayed for constitutive expression of the gus gene using non-transformed leaves as control (Figure 2h–i).

Download:

Figure 2. Genetic transformation of J. curcas via microprojectile bombardment.

(A) Transient gus expression on embryo axis after 24 h of bombardment. Selection of transformants (B) during 1^st round (8 days old), (C) during 3^rd round (60 days old) on hygromycin. Regeneration of transformants; (D) multiple shoot induction, (E) multiple shoot regeneration (120 days old), (F) rooting (200 days old), (G) hardening of transgenic plant in plastic jar (250 days old). GUS assay of (H) non transformed leaf and (I) leaf of transgenic plant.

https://doi.org/10.1371/journal.pone.0071136.g002

Molecular Analysis of Transgenic Plants

Genetic transformation was confirmed by PCR amplification of 172 bp (internal fragment), 963 bp and 157 bp (internal fragment) of the SbNHX1 gene, selectable marker hptII gene and reporter gus gene, respectively (Figure 3a–c). Real time quantitative PCR (RTqPCR) was performed to determine the copy number using a single copy JcKASIII gene as the internal control. Transgenic lines JL2, JL8 and JL19 showed gus to JcKASIII ratio 0.746, 2.922 and 1.274 respectively (Figure 3d). RTqPCR analysis revealed that transgenic lines JL2 and JL19 had single copy insertion while transgenic line JL8 showed a triple copy insertion. Melt curve analysis of the internal control JcKASIII gene and the transgenic lines individually showed single peaks that confirmed the absence of any dimer or other contamination. Single band amplification of the gus gene as well as the control gene was further confirmed by 1.2% agarose gel electrophoresis. The expression of the SbNHX1 gene was further confirmed by reverse transcriptase PCR (Rt-PCR). Transgenic lines showed high expression of the gene whereas it was absent in WT plants (Figure 3e).

Download:

Figure 3. Molecular confirmation of transgenic Lines.

PCR amplification of (A) SbNHX1 gene (172 bp internal fragment), (B) hptII gene (963 bp) and (C) gus gene (157 bp internal fragment). Lane M is 100 bp Molecular weight marker ladder (Bangalore Genei, India); lane+is pCAMBIA1301-SbNHX1 (positive control). (D) Real time qPCR analysis to determine the ratio of gus to JcKASIII gene in transgenic lines. Three lines L2, L8 and L19 have a ratio of 0.74, 2.92 and 1.27, respectively. (E) Reverse transcriptase PCR of SbNHX1 transgenic lines. Amplification was observed in transgenic lines while there was no amplification from WT; JcKASIII gene was used as an internal control.

https://doi.org/10.1371/journal.pone.0071136.g003

Physiological and Biochemical Analysis

Leaf senescence assay, chlorophyll content and growth assay.

The salt stress tolerance of T0 transgenic lines was studied by leaf disc senescence assay and chlorophyll estimation. Leaf discs from WT and T0 transgenic lines JL2, JL8 and JL19 were floated separately on 0, 50, 100, 150 and 200 mM NaCl for 8 days. Salinity-induced decreases in chlorophyll were lower in the SbNHX1-overexpressing lines compared to WT plants (Figure 4). The damage caused by salt stress was visualized by the degree of bleaching of leaf tissues. It was evident that the transgenic plants (JL2 and JL8) had a better ability to tolerate salinity stress. However, no significant difference in leaf senescence or chlorophyll content between WT and JL19 was observed. The chlorophyll content in the WT plants reduced significantly with increasing salt concentration while the transgenic lines (JL2 and JL8) retained higher amounts of chlorophyll than WT up to 200 mM NaCl (Figure 5a). Two months old transgenic lines and WT were transferred to SEM supplemented with 200 mM NaCl and kept for 21 days. Transgenic lines showed better growth, while WT showed bleaching in the leaves and growth was hindered (Figure 6).

Download:

Figure 4. Leaf disc senescence assay of transgenic J. curcas for salt tolerance.

Leaf discs of JL2, JL8 and JL19 transgenic lines and WT respectively were floated in 0 mM, 50 mM, 100 mM, 150 and 200 mM NaCl solution for 8 days.

https://doi.org/10.1371/journal.pone.0071136.g004

Download:

Figure 5. Physiological and biochemical analysis of transgenic lines.

(A) Chlorophyll content assay of leaf of transgenic lines JL2, JL8, JL19 and WT at 0, 50, 100, 150 and 200 mM NaCl concentrations. (B) Na⁺ and (c) K⁺ ion content analysis; (D) Electrolyte leakage and (E) MDA content analysis of transgenic lines (JL2, JL8 and JL19) and WT at 0 and 200 mM of NaCl. Graph represents mean ± SD value and similar letters show significant difference at P<0.05.

https://doi.org/10.1371/journal.pone.0071136.g005

Download:

Figure 6. Effect of 200 mM NaCl on in vitro growing transgenic lines JL2 and JL8, and WT after 21 days.

https://doi.org/10.1371/journal.pone.0071136.g006

Ion content analysis.

At zero NaCl, transgenic lines and WT plants exhibited approximately similar Na⁺ content in leaf tissues. However, at 200 mM NaCl, there was significant increase in Na⁺ in all transgenic lines than in the WT plants (Figure 5b). These findings indicated that the transgenic lines accumulated more Na⁺ than the WT under the same stress conditions. In the case of K⁺, all the transgenic lines and WT showed non-significantly higher content at zero NaCl. However, at 200 mM NaCl, K⁺ decreased in both transgenic and WT plants. Although this decrease was also non-significant, WT exhibited higher K⁺ content than transgenic lines (Figure 5c). We also analysed the effect of NaCl on Ca²⁺ content (data not shown), but it was slightly and non-significantly increased at 200 mM NaCl. Transgenic lines showed slightly higher Ca²⁺ content than WT at 200 mM NaCl.

Electrolyte leakage and MDA content analysis.

All the transgenic lines and WT exhibited approximately similar electrolyte leakage at zero NaCl while reduced electrolyte leakage was observed in transgenic lines during salt stress compared to WT but this decrease was non-significant (Figure 5d). Transgenic lines accumulated significantly less MDA than WT in response to salt stress, which was indicative of reduced oxidative damage in plants overexpressing SbNHX1 (Figure 5e).

Discussion

Plant tolerance to salt stress is a multigenic trait and requires the coordinated action of several genes, but it is evident from several reports that overexpression of a single gene can also impart salt tolerance to plants [17], [19], [35], [36]. Before proceeding to genetic transformation with any important gene and engage in further improvement strategies, optimization of an efficient regeneration and genetic transformation protocol is a crucial criterion to produce the required number of improved plants in a limited area. In the present study, we have successfully transformed Jatropha with the salt-responsive SbNHX1 gene for its enhanced salt tolerance via microprojectile bombardment (Table S1). The optimized microprojectile bombardment protocol [12] was applied for bombardment of Jatropha embryo axes with the recombinant binary plasmid construct pCAMBIA1301-SbNHX1. RTqPCR is recently being used to determine copy numbers of integrated genes because of its high sensitivity and requirement for very small amounts of genomic DNA; this technique has been used in many recent reports [36], [37]. In our results, transgenic lines JL2 and JL19 showed single copy gene insertion while JL8 showed three copies. In the gene expression study by semi-quantitative Rt-PCR, JL8 showed higher expression than JL2 and JL19. The integration of three gene copies could be the possible reason for this higher expression.

In one of the two most recent reports on transgenic Jatropha for trait improvement, Qu et al. [30] developed marker-free transgenic plants with increase in high quality seed oil by down-regulating the seed-specific JcFAD2-1 gene. In another report, transgenic Jatropha plants overproducing glycine betaine were developed for enhanced drought tolerance [31], but this study is at the primary level and needs to be studied further to obtain any concrete outcome. To date, several important plants have been transformed with NHX1 gene cloned from different plant sources and its overexpression shown to enhance salt tolerance [35], [38].

Physiological analysis of transgenic plants represents the extent of expression of an integrated gene responsible for a trait. The strategy to accumulate Na⁺ inside vacuoles is used by many plants to survive under salt stress. An active vacuolar antiporter utilizes the proton motive force generated by vacuolar ATPases and pyrophosphatases to sequester excess Na⁺ into the vacuole [15]. SbNHX1 contains 11 strong transmembrane domains spanning in vacuolar membrane similar to some of the other halophytes and that actively regulates the ion homeostasis [34]. The response of the SbNHX1-transgenic lines JL2, JL8 and JL19 was assessed, and in a leaf senescence assay of T0 plants we studied the effect of NaCl on WT and transgenic lines in a dose-dependent manner (Figure 4). Transgenic lines showed better responses than WT, and chlorophyll content was found to be higher at 100, 150 and 200 mM NaCl concentrations after 8 days. However, at 50 mM NaCl there was no visible adverse effect on WT compared to transgenic lines and chlorophyll amount was on a par with that under conditions of zero NaCl (Figure 5a). Similarly, transgenic tobacco plants overexpressing SbNHX1 showed better growth and chlorophyll content than WT plants at 100–300 mM NaCl concentrations [34]. Recently, Liu et al. [39] isolated KcNHX1 and KcNHX2 genes from the halophyte Karelinia caspica and showed that RNAi silencing of KcNHX1 reduced the salt tolerance of K. caspica plants at 200 mM NaCl in a leaf disc assay, and chlorophyll content was decreased.In the ion content analysis, higher Na⁺ content and less K⁺ was observed in transgenic leaf tissues under salt stress. This can be attributed to ion homeostasis due to the activity of the vacuolar Na⁺/H⁺ antiporter SbNHX1. Significant increase in Na⁺ and reduction in K⁺ due to overexpression of AtNHX1 during salt stress has been reported in tomato [16], rapeseed [40], wheat [41], tall fescue [42] and buckwheat [43]. The growth of transgenic plants was not significantly affected by increased salinity, suggesting that K⁺ content was not compromised in the transgenic plants.

The transgenic lines (Jl2 and JL8) showed enhanced tolerance at 200 mM NaCl (Figure 6). The results show the positive effect of the Na⁺/H⁺ antiporter SbNHX1 in T0 plants and their enhanced salt tolerance. The PgNHX1 gene from Pennisetum glaucum has been reported to confer a high level of salinity tolerance when overexpressed in Brassica juncea [35] and tomato [38], and transgenic plants could withstand higher salt stress than WT in pot. Considering all the results, present study clearly demonstrate that overexpression of SbNHX1 improved the osmoregulatory capacity of transgenic lines by alleviating the toxic effect of Na⁺ due to its enhanced sequestration into the vacuole.

Conclusions

In conclusion, we developed the functional transgenic J. curcas plants overexpressing SbNHX1 and showing enhanced salt tolerance. Transgenic lines (JL2 and JL8) showed better response than WT up to 200 mM NaCl with respect to chlorophyll content, leaf senescence assay, ion content, electrolyte leakage and MDA content. The transgenic lines, JL2 and JL8 showed enhanced tolerance at 200 mM NaCl. To the best of our knowledge, this is the first report on enhanced salt tolerance in Jatropha. Salt tolerant transgenic Jatropha plants can be used for cultivation in salt-affected marginal areas for their sustainable management with biofuel production.

Methods

Plant Material and Culture Conditions

Mature decoated seeds of Jatropha curcas CP9 variety were surface sterilized with 2% (v/v) sodium hypochlorite for 15 min and washed 4–6 times with sterile distilled water. Embryo explants were dissected out from endosperm aseptically and pre-cultured for 5 days on MS (Murashige and Skoog) basal medium supplemented with 2.22 µM BAP (6-benzylaminopurine) +2.46 µM IBA (indole-3-butyric acid), 0.8% (w/v) agar and 3% (w/v) sucrose. All cultures were maintained under controlled laboratory conditions at 25±2°C under a 16/8 h light/dark photoperiod with a cool white fluorescent lamp with a light intensity of 35 µmol m⁻² s⁻¹.

Plasmid Construct

The Binary plasmid vector pCAMBIA1301 (CAMBIA, Australia) containing the SbNHX1 gene under the control of the CaMV35S promoter was named the pCAMBIA1301-SbNHX1 gene construct (Figure 1) [34], which was used for microprojectile bombardment.

Microprojectile Bombardment Mediated Transformation

Plasmid construct pCAMBIA1301-SbNHX1 was isolated using a plasmid Miniprep kit (Qiagen, Germany) following the manufacturer’s protocol. Preparation of microcarriers, arrangement of explants and microprojectile bombardment conditions were the same as reported previously [12]. Bombardments were performed on 40–50 five days pre-cultured embryo axes per plate with a biolistic gene gun (PDS 1000/He, Bio-Rad).

Selection and Regeneration of Transformants

After bombardment, the explants were kept in the dark at 25°C for 24 h and then transferred to shoot induction medium (SIM). Regeneration was carried out by following our previous reported protocol [12]. After 5–7 days, explants were transferred to selection medium (SIM) containing 5 mg/l hygromycin. For effective selection, the explants were transferred to shoot regeneration medium with increasing concentrations (6 and 7 mg/l) of hygromycin. After three cycles of selection, putative transformed shoots were transferred for approx. 40–60 days to shoot elongation medium for further shoot elongation.

For rooting, elongated shoots were transferred to root induction medium. After 4–6 weeks, plantlets with rooted shoots were transplanted into autoclaved red soil: sand: FYM (2∶2∶1) mix in small pots (5×10 cm) covered with transparent plastic lids and maintained under high humidity for 7–10 days, and thereafter gradually exposed to culture room conditions. Established plantlets were transferred to pots containing a mixture of red soil: sand: FYM (2∶2∶1) in green house conditions [12].

Histochemical GUS Assay

Transient gus expression in embryo axis and leaves was assessed [44] after 24 h of transformation and randomly 3–5 explants were subjected to GUS assay for primary confirmation. Leaves of transgenic lines (4–5 months after transformation) were assayed for constitutive expression of the gus gene. GUS assay was done by incubating the tissues in freshly prepared GUS assay buffer (1 mg/ml 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 12–16 h at 37°C in the dark. Thereafter tissues were destained with 70% alcohol to examine blue regions.

PCR Amplification and Determination of Copy Number by Real Time Quantitative PCR

The genomic DNA of transformants was isolated and concentration was determined with a NanoDrop Spectrophotometer. Transformation was confirmed by PCR with SbNHX1 gene specific (RT-NHX1F 5′-ATG GTG TTT GGG TTG CTG A-3′ and RT-NHX1R 5′-CTG CTT CGT CTT GGT TGT CC-3′); gus (reporter gene) specific (gusRTF: 5′-CGA CTG GGC AGA TGA ACA T-3′ and gusRTR: 5′-CTG TAA GTG CGC TTG CTG AG-3′) and hptII (hygromycin selection marker gene) specific (hptIIF: 5′-TTC TTT GCC CTC GGA CGA GTG-3′ and hptIIR: 5′-ACA GCG TCT CCG ACC TGA TG-3′) primers.

The genomic DNA was diluted to 1, 10 and 100 ng/µl concentrations and the real time quantitative PCR (RTqPCR) reaction was run in a Real Time iQ5 Cycler (Bio-Rad, USA). RTqPCR conditions were optimized for the gus gene primers (gusRTF and gusRTR) and JcKASIII gene (GenBank: DQ987701) primers (JcKAS1F: 5′-GCA CTT GGC TGC AAA ACA AAT-3′, JcKAS1R: 5′-CGT CCA GTC AAC ATA TCG AG-3′). The JcKASIII gene was used as an internal control because this is a single copy gene in the Jatropha genome [45]. The PCR reactions were carried out using 0.25 µM primers for both genes in a 20 µl reaction using the QuantiFast SYBR Green PCR reaction kit (Qiagen, USA). The following PCR conditions were maintained during RTqPCR: 95°C for 5 min (1 cycle), 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s (40 cycles), 95°C for 1 min (1 cycle) and 60°C for 1 min (1 cycle). At the end of the PCR cycles, the products were subjected to melt curve analysis. The amplified product was run on a 1.5% agarose gel to confirm the expected size. The experiments were repeated twice independently with three replicates each time. Standard curves were plotted using threshold cycle (CT) value to determine reaction efficiencies [46] as follows:

The efficiency values were put in the following equation [46] to determine the copy number ratio of gus to JcKASIII:

Semiquantitative Rt-PCR Analysis

The total RNA was isolated from WT and transgenic plant samples using RNeasy Plus Mini Kit (Qiagen, Germany) and quantified with Nanodrop spectrophotometer (USA). The cDNA was prepared using 2 µg of total RNA using ImProm-II Reverse Transcription System kit (Promega, USA). Semiquantitative Reveres transcriptase-PCR analysis was performed using cDNA (1 µl) as template, SbNHX1 gene as target and JcKASIII gene as an internal control using SbNHX1 and JcKASIII specific primers (RT-NHX1F & RT-NHX1R; JcKAS1F & JcKAS1R). The PCR reactions were carried out in 1×PCR buffer supplemented with 200 µM dNTPs, 1.25 U Taq DNA polymerase and 5 pmol of each gene-specific primers with following conditions: an initial denaturation at 94°C for 3 min, 30 cycles at 94°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec, followed by a final extension step at 72°C for 5 min. Rt-PCR experiments were repeated three times, and the amplification products were analysed with 1.2% agarose gel electrophoresis.

Leaf Senescence Assay

Leaf disc assay was performed to analyse transgenic plants for their salt tolerance according to procedures described by Fan et al. [47]. Healthy leaves (third leaf from the top) from WT and transgenic plants (T0 generation) of similar age were detached. Leaf discs (10–12) 5 mm in diameter were punched out and floated in 5 ml sterilized distilled water with different concentrations of sodium chloride (0, 50, 100, 150 and 200 mM) for 8 days. The leaf discs were kept under 16 hours white light (35 µmol m⁻² s⁻¹)/8 hours dark at 25±2°C. The effects of this treatment on leaf discs were assessed by observing phenotypic changes. The experiment was repeated twice.

Chlorophyll Estimation

Leaf discs of WT and the three transgenic lines, JL2, JL8 and JL19, treated with different concentrations of NaCl as described earlier were used for chlorophyll estimation. Treated leaf discs were homogenized thoroughly in 80% acetone and incubated for 1–2 h with shaking at 120 rpm in the dark. The homogenate was centrifuged at 3000 rpm for 5 min in the dark. The OD of the supernatant was taken at 645 and 665 nm, and chlorophyll was calculated per gram fresh weight of tissue [48].

Effect of Salt on in vitro Growing Plants

Two months old in vitro growing WT and transgenic lines JL2 and JL8 were assessed for salt tolerance. Plants were grown in SEM supplemented with 200 mM NaCl and effect of salt was observed after 21 days.

Ion Contents

For the analysis of ion contents, 0.5 g of fresh young leaves tissues were dried at 70°C for 48 h in an oven and dry weight was measured. The dried tissues were digested for 8–10 h with 4 ml perchloric acid and nitric acid solution (3∶1). Digested samples were dried on the hot plate, diluted to 25 ml with deionised water and filtered through 0.2-µm filter. Ion contents were measured by inductively coupled plasma optical emission spectrometer (Optima 2000DV, PerkinElmer, Germany).

Electrolyte Leakage

Electrolyte leakage was measured according to Lutts et al. [49]. Leaf discs of similar size were collected from plants for each treatment and washed thoroughly with deionised water to remove surface-adhered electrolytes. The samples were kept in closed vials containing 10 ml of deionised water and incubated at 25°C on a rotary shaker for 24 h. Subsequently, the electrical conductivity (EC) of the solution (L_t) was determined using conductivity meter (SevenEasy, Mettler Toledo AG 8603, Switzerland). Samples were autoclaved (120°C for 20 min), cooled up to 25°C and electrical conductivity (L₀) was determined. The electrolyte leakage was defined as follows:

Lipid Peroxidation

Lipid peroxidation was estimated by following the method described by Hodges et al. [50]. Leaf material (0.2 g) was homogenised in 5 ml of aqueous (80% v/v) acetone solution. In one set, 1 ml solution containing 0.65% (w/v) Thiobarbituric acid (TBA) and 20% (w/v) Trichloroacetic acid (TCA) was added to 1 ml extract, however in second set TBA was excluded. The mixtures were incubated at 90°C for 30 min and cooled to room temperature. Samples were centrifuged at 4,000 rpm for 3 min and the absorbance of the supernatants at 440 nm, 532 nm and 600 nm was determined. Concentration of malondialdehyde (MDA) produced by TBA reaction was determined according to the following equations:(1)(2)(3)

Each experiment was carried out in three replicates. To determine the significance of difference between the means of WT and transgenic plants of each treatment group, data was subjected to analysis of variance (ANOVA) and were expressed as mean ± SD. A Tukey HSD multiple comparison of mean test was used. When significant differences were found, P<0.05 was considered as significant. Significantly different mean values were indicated by similar letters.

Supporting Information

Table S1.

Transformation efficiency and overall regeneration efficiency after microprojectile bombardment of embryo axes with pCAMBIA1301-SbNHX1 gene construct.

https://doi.org/10.1371/journal.pone.0071136.s001

(DOC)

Author Contributions

Conceived and designed the experiments: BJ. Performed the experiments: MJ AJ. Analyzed the data: MJ AM. Wrote the paper: MJ AM BJ.

References

1. Divakara BN, Upadhyaya HD, Wani SP, Gowda CLL (2010) Biology and genetic improvement of Jatropha curcas L.: A review. Appl Energ 87: 732–742.
- View Article
- Google Scholar
2. Pandey VC, Singh K, Singh JS, Kumar A, Singh B, et al. (2012) Jatropha curcas: A potential biofuel plant for sustainable environmental development. Renew Sust Energy Rev 16: 2870–2883.
- View Article
- Google Scholar
3. Ilham Z, Saka S (2010) Two-step supercritical dimethyl carbonate method for biodiesel production from Jatropha curcas oil. Bioresour Technol 101: 2735–2740.
- View Article
- Google Scholar
4. Berchmans HJ, Hirata S (2008) Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids. Bioresour Technol 99: 1716–1721.
- View Article
- Google Scholar
5. Chhetri AB, Tango MS, Budge SM, Watts KC, Islam MR (2008) Non-edible plant oils as new sources for biodiesel production. Int J Mol Sci 9: 169–180.
- View Article
- Google Scholar
6. Fairless D (2007) The little shrub that could – maybe. Nature 449: 652–655.
- View Article
- Google Scholar
7. Achten WMJ, Maes WH, Aerts R, Verchot L, Trabucco A, et al. (2010) Jatropha: From global hype to local opportunity. J Arid Environ 74: 164–165.
- View Article
- Google Scholar
8. Dyer JC, Stringer LC, Dougill AJ (2012) Jatropha curcas: Sowing local seeds of success in Malawi? In response to Achten et al. (2010). J Arid Environ 79: 107–110.
- View Article
- Google Scholar
9. Sunil N, Sivaraj N, Anitha K, Abraham B, Kumar V, et al. (2009) Analysis of diversity and distribution of Jatropha curcas L. germplasm using Geographic Information System (DIVA-GIS). Genet Resour Crop Evol 56: 115–119.
- View Article
- Google Scholar
10. Ministry of New and Renewable Energy (2009) National Policy on Biofuels. Available: http://mnes.nic.in. Accessed 2012 Jan 25.
11. Ravindranath NH, Lakshmi CS, Manuvie R, Balachandra P (2011) Biofuel production and implications for land use, food production and environment in India. Energ Policy 39: 5737–5745.
- View Article
- Google Scholar
12. Joshi M, Mishra A, Jha B (2011) Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes. Ind Crop Prod 33: 67–77.
- View Article
- Google Scholar
13. FAO (2008) Land and Plant Nutrition Management Service. Available: http://www.fao.org/ag/agl/agll/spush/. Accessed 2010 Dec 21.
14. FAO (2005) Global network on integrated soil management for sustainable use of salt-affected soils. Available: http://www.fao.org/ag/agl/agll/spush/. Accessed 2008 July 28.
15. Blumwald E (2000) Sodium transport and salt tolerance in plants. Curr Opin Cell Biol 12: 431–434.
- View Article
- Google Scholar
16. Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19: 765–768.
- View Article
- Google Scholar
17. 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.
- View Article
- Google Scholar
18. Shi H, Ishitani M, Kim C, Zhu JK (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na⁺/H⁺ antiporter. Proc Natl Acad Sci USA 97: 6896–6901.
- View Article
- Google Scholar
19. Shi H, Lee BH, Wu SJ, Zhu JK (2003) Over-expression of a plasma membrane Na⁺/H⁺ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21: 81–85.
- View Article
- Google Scholar
20. Jiang XY, Leidi EO, Pardo JM (2010) How do vacuolar NHX exchangers function in plant salt tolerance? Plant Signal Behav 5: 792–795.
- View Article
- Google Scholar
21. Bassil E, Tajima H, Liang YC, Ohto MA, Ushijima K, et al. (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.
- View Article
- Google Scholar
22. Barragán V, Leidi EO, Andrés Z, Rubio L, Luca AD, et al. (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.
- View Article
- Google Scholar
23. Bassil E, Coku A, Blumwald E (2012) Cellular ion homeostasis: emerging roles of intracellular NHX Na⁺/H⁺ antiporters in plant growth and development. J Exp Bot 63: 5727–5740.
- View Article
- Google Scholar
24. Silva EN, Ribeiro RV, Ferreira-Silva SL, Viégas RA, Silveira JAG (2010) Comparative effects of salinity and water stress on photosynthesis, water relations and growth of Jatropha curcas plants. J Arid Environ 74: 1130–1137.
- View Article
- Google Scholar
25. Díaz-López L, Gimeno V, Lidón V, Simón I, Martínez V, et al. (2012) The tolerance of Jatropha curcas seedlings to NaCl: An ecophysiological analysis. Plant Physiol Biochem 54: 34–42.
- View Article
- Google Scholar
26. Niu G, Rodriguez D, Mendoza M, Jifon J, Ganjegunte G (2012) Responses of Jatropha curcas to salt and drought stresses. Int J Agron 2012: 1–7.
- View Article
- Google Scholar
27. Fujimaki H, Kikuchi N (2010) Drought and salinity tolerances of young Jatropha. Int Agrophys 24: 121–127.
- View Article
- Google Scholar
28. Tzfira T, Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 17: 147–154.
- View Article
- Google Scholar
29. Altpeter F, Baisakh N, Beachy R, Bock R, Capell R, et al. (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breed 15: 305–327.
- View Article
- Google Scholar
30. Qu J, Mao HZ, Chen W, Gao SQ, Bai YN, et al. (2012) Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid. Biotechnol Biofuels 5: 10.
- View Article
- Google Scholar
31. Tsuchimoto S, Cartagena J, Khemkladngoen N, Singkaravanit S, Kohinata T, et al. (2012) Development of transgenic plants in Jatropha with drought tolerance. Plant Biotechnol-Nar 29: 137–143.
- View Article
- Google Scholar
32. Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55: 307–319.
- View Article
- Google Scholar
33. Bhatnagar-Mathur P, Vadez V, Sharma KK (2008) Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects. Plant Cell Rep 27: 411–424.
- View Article
- Google Scholar
34. Jha A, Joshi M, Yadav NS, Agarwal PK, Jha B (2011) Cloning and characterization of the Salicornia brachiata Na⁺/H⁺ antiporter gene SbNHX1 and its expression by abiotic stress. Mol Biol Rep 38: 1965–1973.
- View Article
- Google Scholar
35. Rajagopal D, Agarwal P, Tyagi W, Singla-Pareek SL, Reddy MK, et al. (2007) Pennisetum glaucum Na+/H+ antiporter confers high level of salinity tolerance in transgenic Brassica juncea. Mol Breed 19: 137–151.
- View Article
- Google Scholar
36. Jha B, Sharma A, Mishra A (2011) Expression of SbGSTU (Tau class glutathione S-transferase) gene isolated from Salicornia brachiata in tobacco for salt tolerance. Mol Biol Rep 38: 4823–4832.
- View Article
- Google Scholar
37. Casu RE, Selivanova A, Perroux JM (2012) High-throughput assessment of transgene copy number in sugarcane using real-time quantitative PCR. Plant Cell Rep 31: 167–177.
- View Article
- Google Scholar
38. Bhaskaran S, Savithramma DL (2011) Co-expression of Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter and Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic tomato. J Exp Bot 62: 5561–5570.
- View Article
- Google Scholar
39. 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 39: 7193–7202.
- View Article
- Google Scholar
40. Zhang HX, Hodson JN, Williams JP, Blumwald E (2001) Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proc Natl Acad Sci USA 98: 12832–12836.
- View Article
- Google Scholar
41. Xue ZY, Zhi DY, Xue GP, Zhang H, Zhao YX, et al. (2004) Enhanced salt tolerance of transgenic wheat (Triticum aestivum L.) expressing a vacuolar Na⁺/H⁺ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na⁺. Plant Sci 167: 849–859.
- View Article
- Google Scholar
42. Zhao J, Zhi D, Xue Z, Liu H, Xia G (2007) Enhanced salt tolerance of transgenic progeny of tall fescue (Festuca arundinacea) expressing a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis. J Plant Physiol 164: 1377–1383.
- View Article
- Google Scholar
43. Chen LH, Zhang B, Xu ZQ (2008) Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na⁺/H⁺ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum). Transgenic Res 17: 121–132.
- View Article
- Google Scholar
44. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907.
- View Article
- Google Scholar
45. Li J, Li MR, Wu PZ, Tian CE, Jiang HW, et al. (2008) Molecular cloning and expression analysis of a gene encoding a putative β-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from Jatropha curcas.. Tree Physiol 28: 921–927.
- View Article
- Google Scholar
46. Shepherd CT, Lauter ANM, Scott MP (2009) Determination of transgene copy number by real-time quantitative PCR. In: Scott MP, editor. Methods in molecular biology: transgenic maize. Humana, New York. 129–134.
47. Fan L, Zheng S, Wang X (1997) Antisense suppression of phospholipase Dα retards abscisic acid and ethylene promoted senescence of post harvest Arabidopsis leaves. Plant Cell 9: 2183–2196.
- View Article
- Google Scholar
48. Arnon DI (1949) Copper enzymes in isolated chloroplasts polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1–15.
- View Article
- Google Scholar
49. Lutts S, Kinet JM, Bouharmont J (1996) NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann Bot 78: 389–398.
- View Article
- Google Scholar
50. 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.
- View Article
- Google Scholar

[ref1] 1. Divakara BN, Upadhyaya HD, Wani SP, Gowda CLL (2010) Biology and genetic improvement of Jatropha curcas L.: A review. Appl Energ 87: 732–742.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Pandey VC, Singh K, Singh JS, Kumar A, Singh B, et al. (2012) Jatropha curcas: A potential biofuel plant for sustainable environmental development. Renew Sust Energy Rev 16: 2870–2883.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Ilham Z, Saka S (2010) Two-step supercritical dimethyl carbonate method for biodiesel production from Jatropha curcas oil. Bioresour Technol 101: 2735–2740.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Berchmans HJ, Hirata S (2008) Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids. Bioresour Technol 99: 1716–1721.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Chhetri AB, Tango MS, Budge SM, Watts KC, Islam MR (2008) Non-edible plant oils as new sources for biodiesel production. Int J Mol Sci 9: 169–180.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Fairless D (2007) The little shrub that could – maybe. Nature 449: 652–655.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref7] 7. Achten WMJ, Maes WH, Aerts R, Verchot L, Trabucco A, et al. (2010) Jatropha: From global hype to local opportunity. J Arid Environ 74: 164–165.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref8] 8. Dyer JC, Stringer LC, Dougill AJ (2012) Jatropha curcas: Sowing local seeds of success in Malawi? In response to Achten et al. (2010). J Arid Environ 79: 107–110.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref9] 9. Sunil N, Sivaraj N, Anitha K, Abraham B, Kumar V, et al. (2009) Analysis of diversity and distribution of Jatropha curcas L. germplasm using Geographic Information System (DIVA-GIS). Genet Resour Crop Evol 56: 115–119.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref10] 10. Ministry of New and Renewable Energy (2009) National Policy on Biofuels. Available: http://mnes.nic.in. Accessed 2012 Jan 25.

[ref11] 11. Ravindranath NH, Lakshmi CS, Manuvie R, Balachandra P (2011) Biofuel production and implications for land use, food production and environment in India. Energ Policy 39: 5737–5745.
View Article
Google Scholar

[30] View Article

[31] Google Scholar

[ref12] 12. Joshi M, Mishra A, Jha B (2011) Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes. Ind Crop Prod 33: 67–77.
View Article
Google Scholar

[33] View Article

[34] Google Scholar

[ref13] 13. FAO (2008) Land and Plant Nutrition Management Service. Available: http://www.fao.org/ag/agl/agll/spush/. Accessed 2010 Dec 21.

[ref14] 14. FAO (2005) Global network on integrated soil management for sustainable use of salt-affected soils. Available: http://www.fao.org/ag/agl/agll/spush/. Accessed 2008 July 28.

[ref15] 15. Blumwald E (2000) Sodium transport and salt tolerance in plants. Curr Opin Cell Biol 12: 431–434.
View Article
Google Scholar

[38] View Article

[39] Google Scholar

[ref16] 16. Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19: 765–768.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref17] 17. 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.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref18] 18. Shi H, Ishitani M, Kim C, Zhu JK (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na⁺/H⁺ antiporter. Proc Natl Acad Sci USA 97: 6896–6901.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref19] 19. Shi H, Lee BH, Wu SJ, Zhu JK (2003) Over-expression of a plasma membrane Na⁺/H⁺ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21: 81–85.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref20] 20. Jiang XY, Leidi EO, Pardo JM (2010) How do vacuolar NHX exchangers function in plant salt tolerance? Plant Signal Behav 5: 792–795.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref21] 21. Bassil E, Tajima H, Liang YC, Ohto MA, Ushijima K, et al. (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.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref22] 22. Barragán V, Leidi EO, Andrés Z, Rubio L, Luca AD, et al. (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.
View Article
Google Scholar

[59] View Article

[60] Google Scholar

[ref23] 23. Bassil E, Coku A, Blumwald E (2012) Cellular ion homeostasis: emerging roles of intracellular NHX Na⁺/H⁺ antiporters in plant growth and development. J Exp Bot 63: 5727–5740.
View Article
Google Scholar

[62] View Article

[63] Google Scholar

[ref24] 24. Silva EN, Ribeiro RV, Ferreira-Silva SL, Viégas RA, Silveira JAG (2010) Comparative effects of salinity and water stress on photosynthesis, water relations and growth of Jatropha curcas plants. J Arid Environ 74: 1130–1137.
View Article
Google Scholar

[65] View Article

[66] Google Scholar

[ref25] 25. Díaz-López L, Gimeno V, Lidón V, Simón I, Martínez V, et al. (2012) The tolerance of Jatropha curcas seedlings to NaCl: An ecophysiological analysis. Plant Physiol Biochem 54: 34–42.
View Article
Google Scholar

[68] View Article

[69] Google Scholar

[ref26] 26. Niu G, Rodriguez D, Mendoza M, Jifon J, Ganjegunte G (2012) Responses of Jatropha curcas to salt and drought stresses. Int J Agron 2012: 1–7.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref27] 27. Fujimaki H, Kikuchi N (2010) Drought and salinity tolerances of young Jatropha. Int Agrophys 24: 121–127.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref28] 28. Tzfira T, Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 17: 147–154.
View Article
Google Scholar

[77] View Article

[78] Google Scholar

[ref29] 29. Altpeter F, Baisakh N, Beachy R, Bock R, Capell R, et al. (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breed 15: 305–327.
View Article
Google Scholar

[80] View Article

[81] Google Scholar

[ref30] 30. Qu J, Mao HZ, Chen W, Gao SQ, Bai YN, et al. (2012) Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid. Biotechnol Biofuels 5: 10.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref31] 31. Tsuchimoto S, Cartagena J, Khemkladngoen N, Singkaravanit S, Kohinata T, et al. (2012) Development of transgenic plants in Jatropha with drought tolerance. Plant Biotechnol-Nar 29: 137–143.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref32] 32. Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55: 307–319.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref33] 33. Bhatnagar-Mathur P, Vadez V, Sharma KK (2008) Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects. Plant Cell Rep 27: 411–424.
View Article
Google Scholar

[92] View Article

[93] Google Scholar

[ref34] 34. Jha A, Joshi M, Yadav NS, Agarwal PK, Jha B (2011) Cloning and characterization of the Salicornia brachiata Na⁺/H⁺ antiporter gene SbNHX1 and its expression by abiotic stress. Mol Biol Rep 38: 1965–1973.
View Article
Google Scholar

[95] View Article

[96] Google Scholar

[ref35] 35. Rajagopal D, Agarwal P, Tyagi W, Singla-Pareek SL, Reddy MK, et al. (2007) Pennisetum glaucum Na+/H+ antiporter confers high level of salinity tolerance in transgenic Brassica juncea. Mol Breed 19: 137–151.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref36] 36. Jha B, Sharma A, Mishra A (2011) Expression of SbGSTU (Tau class glutathione S-transferase) gene isolated from Salicornia brachiata in tobacco for salt tolerance. Mol Biol Rep 38: 4823–4832.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref37] 37. Casu RE, Selivanova A, Perroux JM (2012) High-throughput assessment of transgene copy number in sugarcane using real-time quantitative PCR. Plant Cell Rep 31: 167–177.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref38] 38. Bhaskaran S, Savithramma DL (2011) Co-expression of Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter and Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic tomato. J Exp Bot 62: 5561–5570.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref39] 39. 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 39: 7193–7202.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref40] 40. Zhang HX, Hodson JN, Williams JP, Blumwald E (2001) Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proc Natl Acad Sci USA 98: 12832–12836.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref41] 41. Xue ZY, Zhi DY, Xue GP, Zhang H, Zhao YX, et al. (2004) Enhanced salt tolerance of transgenic wheat (Triticum aestivum L.) expressing a vacuolar Na⁺/H⁺ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na⁺. Plant Sci 167: 849–859.
View Article
Google Scholar

[116] View Article

[117] Google Scholar

[ref42] 42. Zhao J, Zhi D, Xue Z, Liu H, Xia G (2007) Enhanced salt tolerance of transgenic progeny of tall fescue (Festuca arundinacea) expressing a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis. J Plant Physiol 164: 1377–1383.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref43] 43. Chen LH, Zhang B, Xu ZQ (2008) Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na⁺/H⁺ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum). Transgenic Res 17: 121–132.
View Article
Google Scholar

[122] View Article

[123] Google Scholar

[ref44] 44. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref45] 45. Li J, Li MR, Wu PZ, Tian CE, Jiang HW, et al. (2008) Molecular cloning and expression analysis of a gene encoding a putative β-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from Jatropha curcas.. Tree Physiol 28: 921–927.
View Article
Google Scholar

[128] View Article

[129] Google Scholar

[ref46] 46. Shepherd CT, Lauter ANM, Scott MP (2009) Determination of transgene copy number by real-time quantitative PCR. In: Scott MP, editor. Methods in molecular biology: transgenic maize. Humana, New York. 129–134.

[ref47] 47. Fan L, Zheng S, Wang X (1997) Antisense suppression of phospholipase Dα retards abscisic acid and ethylene promoted senescence of post harvest Arabidopsis leaves. Plant Cell 9: 2183–2196.
View Article
Google Scholar

[132] View Article

[133] Google Scholar

[ref48] 48. Arnon DI (1949) Copper enzymes in isolated chloroplasts polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1–15.
View Article
Google Scholar

[135] View Article

[136] Google Scholar

[ref49] 49. Lutts S, Kinet JM, Bouharmont J (1996) NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann Bot 78: 389–398.
View Article
Google Scholar

[138] View Article

[139] Google Scholar

[ref50] 50. 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.
View Article
Google Scholar

[141] View Article

[142] Google Scholar

Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland

Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland

Correction

Figures

Abstract

Introduction

Results

Microprojectile Bombardment Mediated Transformation

Molecular Analysis of Transgenic Plants

Physiological and Biochemical Analysis

Leaf senescence assay, chlorophyll content and growth assay.

Ion content analysis.

Electrolyte leakage and MDA content analysis.

Discussion

Conclusions

Methods

Plant Material and Culture Conditions

Plasmid Construct

Microprojectile Bombardment Mediated Transformation

Selection and Regeneration of Transformants

Histochemical GUS Assay

PCR Amplification and Determination of Copy Number by Real Time Quantitative PCR

Semiquantitative Rt-PCR Analysis

Leaf Senescence Assay

Chlorophyll Estimation

Effect of Salt on in vitro Growing Plants

Ion Contents

Electrolyte Leakage

Lipid Peroxidation

Supporting Information

Table S1.

Author Contributions

References