Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland
Identifieur interne : 000173 ( Pmc/Corpus ); précédent : 000172; suivant : 000174Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland
Auteurs : Bhavanath Jha ; Avinash Mishra ; Anupama Jha ; Mukul JoshiSource :
- PLoS ONE [ 1932-6203 ] ; 2013.
Abstract
Url:
DOI: 10.1371/journal.pone.0071136
PubMed: 23940703
PubMed Central: 3733712
Links to Exploration step
PMC:3733712Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Developing Transgenic <italic>Jatropha</italic> Using the <italic>SbNHX1</italic> Gene from an Extreme Halophyte for Cultivation in Saline Wasteland</title><author><name sortKey="Jha, Bhavanath" sort="Jha, Bhavanath" uniqKey="Jha B" first="Bhavanath" last="Jha">Bhavanath Jha</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mishra, Avinash" sort="Mishra, Avinash" uniqKey="Mishra A" first="Avinash" last="Mishra">Avinash Mishra</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Jha, Anupama" sort="Jha, Anupama" uniqKey="Jha A" first="Anupama" last="Jha">Anupama Jha</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Joshi, Mukul" sort="Joshi, Mukul" uniqKey="Joshi M" first="Mukul" last="Joshi">Mukul Joshi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23940703</idno><idno type="pmc">3733712</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733712</idno><idno type="RBID">PMC:3733712</idno><idno type="doi">10.1371/journal.pone.0071136</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000173</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000173</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Developing Transgenic <italic>Jatropha</italic> Using the <italic>SbNHX1</italic> Gene from an Extreme Halophyte for Cultivation in Saline Wasteland</title><author><name sortKey="Jha, Bhavanath" sort="Jha, Bhavanath" uniqKey="Jha B" first="Bhavanath" last="Jha">Bhavanath Jha</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mishra, Avinash" sort="Mishra, Avinash" uniqKey="Mishra A" first="Avinash" last="Mishra">Avinash Mishra</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Jha, Anupama" sort="Jha, Anupama" uniqKey="Jha A" first="Anupama" last="Jha">Anupama Jha</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Joshi, Mukul" sort="Joshi, Mukul" uniqKey="Joshi M" first="Mukul" last="Joshi">Mukul Joshi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Jatropha</italic> is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including <italic>Jatropha</italic>. <italic>SbNHX1</italic> is a vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter gene that compartmentalises excess Na<sup>+</sup> ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the <italic>SbNHX1</italic> gene from an extreme halophyte, <italic>Salicornia brachiata</italic>. Transgenic plants of <italic>Jatropha curcas</italic> with the <italic>SbNHX1</italic> 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 <italic>Jatropha</italic> 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 <italic>Jatropha</italic> using the <italic>SbNHX1</italic> gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under <italic>Jatropha</italic> cultivation for agriculture purposes. Apart from this, transgenic <italic>Jatropha</italic> can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Divakara, Bn" uniqKey="Divakara B">BN Divakara</name></author><author><name sortKey="Upadhyaya, Hd" uniqKey="Upadhyaya H">HD Upadhyaya</name></author><author><name sortKey="Wani, Sp" uniqKey="Wani S">SP Wani</name></author><author><name sortKey="Gowda, Cll" uniqKey="Gowda C">CLL Gowda</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pandey, Vc" uniqKey="Pandey V">VC Pandey</name></author><author><name sortKey="Singh, K" uniqKey="Singh K">K Singh</name></author><author><name sortKey="Singh, Js" uniqKey="Singh J">JS Singh</name></author><author><name sortKey="Kumar, A" uniqKey="Kumar A">A Kumar</name></author><author><name sortKey="Singh, B" uniqKey="Singh B">B Singh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ilham, Z" uniqKey="Ilham Z">Z Ilham</name></author><author><name sortKey="Saka, S" uniqKey="Saka S">S Saka</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Berchmans, Hj" uniqKey="Berchmans H">HJ Berchmans</name></author><author><name sortKey="Hirata, S" uniqKey="Hirata S">S Hirata</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chhetri, Ab" uniqKey="Chhetri A">AB Chhetri</name></author><author><name sortKey="Tango, Ms" uniqKey="Tango M">MS Tango</name></author><author><name sortKey="Budge, Sm" uniqKey="Budge S">SM Budge</name></author><author><name sortKey="Watts, Kc" uniqKey="Watts K">KC Watts</name></author><author><name sortKey="Islam, Mr" uniqKey="Islam M">MR Islam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fairless, D" uniqKey="Fairless D">D Fairless</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Achten, Wmj" uniqKey="Achten W">WMJ Achten</name></author><author><name sortKey="Maes, Wh" uniqKey="Maes W">WH Maes</name></author><author><name sortKey="Aerts, R" uniqKey="Aerts R">R Aerts</name></author><author><name sortKey="Verchot, L" uniqKey="Verchot L">L Verchot</name></author><author><name sortKey="Trabucco, A" uniqKey="Trabucco A">A Trabucco</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dyer, Jc" uniqKey="Dyer J">JC Dyer</name></author><author><name sortKey="Stringer, Lc" uniqKey="Stringer L">LC Stringer</name></author><author><name sortKey="Dougill, Aj" uniqKey="Dougill A">AJ Dougill</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sunil, N" uniqKey="Sunil N">N Sunil</name></author><author><name sortKey="Sivaraj, N" uniqKey="Sivaraj N">N Sivaraj</name></author><author><name sortKey="Anitha, K" uniqKey="Anitha K">K Anitha</name></author><author><name sortKey="Abraham, B" uniqKey="Abraham B">B Abraham</name></author><author><name sortKey="Kumar, V" uniqKey="Kumar V">V Kumar</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Ravindranath, Nh" uniqKey="Ravindranath N">NH Ravindranath</name></author><author><name sortKey="Lakshmi, Cs" uniqKey="Lakshmi C">CS Lakshmi</name></author><author><name sortKey="Manuvie, R" uniqKey="Manuvie R">R Manuvie</name></author><author><name sortKey="Balachandra, P" uniqKey="Balachandra P">P Balachandra</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Joshi, M" uniqKey="Joshi M">M Joshi</name></author><author><name sortKey="Mishra, A" uniqKey="Mishra A">A Mishra</name></author><author><name sortKey="Jha, B" uniqKey="Jha B">B Jha</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Blumwald, E" uniqKey="Blumwald E">E Blumwald</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, Hx" uniqKey="Zhang H">HX Zhang</name></author><author><name sortKey="Blumwald, E" uniqKey="Blumwald E">E Blumwald</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Apse, Mp" uniqKey="Apse M">MP Apse</name></author><author><name sortKey="Aharon, Gs" uniqKey="Aharon G">GS Aharon</name></author><author><name sortKey="Snedden, Wa" uniqKey="Snedden W">WA Snedden</name></author><author><name sortKey="Blumwald, E" uniqKey="Blumwald E">E Blumwald</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shi, H" uniqKey="Shi H">H Shi</name></author><author><name sortKey="Ishitani, M" uniqKey="Ishitani M">M Ishitani</name></author><author><name sortKey="Kim, C" uniqKey="Kim C">C Kim</name></author><author><name sortKey="Zhu, Jk" uniqKey="Zhu J">JK Zhu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shi, H" uniqKey="Shi H">H Shi</name></author><author><name sortKey="Lee, Bh" uniqKey="Lee B">BH Lee</name></author><author><name sortKey="Wu, Sj" uniqKey="Wu S">SJ Wu</name></author><author><name sortKey="Zhu, Jk" uniqKey="Zhu J">JK Zhu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jiang, Xy" uniqKey="Jiang X">XY Jiang</name></author><author><name sortKey="Leidi, Eo" uniqKey="Leidi E">EO Leidi</name></author><author><name sortKey="Pardo, Jm" uniqKey="Pardo J">JM Pardo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bassil, E" uniqKey="Bassil E">E Bassil</name></author><author><name sortKey="Tajima, H" uniqKey="Tajima H">H Tajima</name></author><author><name sortKey="Liang, Yc" uniqKey="Liang Y">YC Liang</name></author><author><name sortKey="Ohto, Ma" uniqKey="Ohto M">MA Ohto</name></author><author><name sortKey="Ushijima, K" uniqKey="Ushijima K">K Ushijima</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Barragan, V" uniqKey="Barragan V">V Barragán</name></author><author><name sortKey="Leidi, Eo" uniqKey="Leidi E">EO Leidi</name></author><author><name sortKey="Andres, Z" uniqKey="Andres Z">Z Andrés</name></author><author><name sortKey="Rubio, L" uniqKey="Rubio L">L Rubio</name></author><author><name sortKey="Luca, Ad" uniqKey="Luca A">AD Luca</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bassil, E" uniqKey="Bassil E">E Bassil</name></author><author><name sortKey="Coku, A" uniqKey="Coku A">A Coku</name></author><author><name sortKey="Blumwald, E" uniqKey="Blumwald E">E Blumwald</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Silva, En" uniqKey="Silva E">EN Silva</name></author><author><name sortKey="Ribeiro, Rv" uniqKey="Ribeiro R">RV Ribeiro</name></author><author><name sortKey="Ferreira Silva, Sl" uniqKey="Ferreira Silva S">SL Ferreira-Silva</name></author><author><name sortKey="Viegas, Ra" uniqKey="Viegas R">RA Viégas</name></author><author><name sortKey="Silveira, Jag" uniqKey="Silveira J">JAG Silveira</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Diaz L Pez, L" uniqKey="Diaz L Pez L">L Díaz-López</name></author><author><name sortKey="Gimeno, V" uniqKey="Gimeno V">V Gimeno</name></author><author><name sortKey="Lid N, V" uniqKey="Lid N V">V Lidón</name></author><author><name sortKey="Sim N, I" uniqKey="Sim N I">I Simón</name></author><author><name sortKey="Martinez, V" uniqKey="Martinez V">V Martínez</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Niu, G" uniqKey="Niu G">G Niu</name></author><author><name sortKey="Rodriguez, D" uniqKey="Rodriguez D">D Rodriguez</name></author><author><name sortKey="Mendoza, M" uniqKey="Mendoza M">M Mendoza</name></author><author><name sortKey="Jifon, J" uniqKey="Jifon J">J Jifon</name></author><author><name sortKey="Ganjegunte, G" uniqKey="Ganjegunte G">G Ganjegunte</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fujimaki, H" uniqKey="Fujimaki H">H Fujimaki</name></author><author><name sortKey="Kikuchi, N" uniqKey="Kikuchi N">N Kikuchi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tzfira, T" uniqKey="Tzfira T">T Tzfira</name></author><author><name sortKey="Citovsky, V" uniqKey="Citovsky V">V Citovsky</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Altpeter, F" uniqKey="Altpeter F">F Altpeter</name></author><author><name sortKey="Baisakh, N" uniqKey="Baisakh N">N Baisakh</name></author><author><name sortKey="Beachy, R" uniqKey="Beachy R">R Beachy</name></author><author><name sortKey="Bock, R" uniqKey="Bock R">R Bock</name></author><author><name sortKey="Capell, R" uniqKey="Capell R">R Capell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qu, J" uniqKey="Qu J">J Qu</name></author><author><name sortKey="Mao, Hz" uniqKey="Mao H">HZ Mao</name></author><author><name sortKey="Chen, W" uniqKey="Chen W">W Chen</name></author><author><name sortKey="Gao, Sq" uniqKey="Gao S">SQ Gao</name></author><author><name sortKey="Bai, Yn" uniqKey="Bai Y">YN Bai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tsuchimoto, S" uniqKey="Tsuchimoto S">S Tsuchimoto</name></author><author><name sortKey="Cartagena, J" uniqKey="Cartagena J">J Cartagena</name></author><author><name sortKey="Khemkladngoen, N" uniqKey="Khemkladngoen N">N Khemkladngoen</name></author><author><name sortKey="Singkaravanit, S" uniqKey="Singkaravanit S">S Singkaravanit</name></author><author><name sortKey="Kohinata, T" uniqKey="Kohinata T">T Kohinata</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Flowers, Tj" uniqKey="Flowers T">TJ Flowers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bhatnagar Mathur, P" uniqKey="Bhatnagar Mathur P">P Bhatnagar-Mathur</name></author><author><name sortKey="Vadez, V" uniqKey="Vadez V">V Vadez</name></author><author><name sortKey="Sharma, Kk" uniqKey="Sharma K">KK Sharma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jha, A" uniqKey="Jha A">A Jha</name></author><author><name sortKey="Joshi, M" uniqKey="Joshi M">M Joshi</name></author><author><name sortKey="Yadav, Ns" uniqKey="Yadav N">NS Yadav</name></author><author><name sortKey="Agarwal, Pk" uniqKey="Agarwal P">PK Agarwal</name></author><author><name sortKey="Jha, B" uniqKey="Jha B">B Jha</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rajagopal, D" uniqKey="Rajagopal D">D Rajagopal</name></author><author><name sortKey="Agarwal, P" uniqKey="Agarwal P">P Agarwal</name></author><author><name sortKey="Tyagi, W" uniqKey="Tyagi W">W Tyagi</name></author><author><name sortKey="Singla Pareek, Sl" uniqKey="Singla Pareek S">SL Singla-Pareek</name></author><author><name sortKey="Reddy, Mk" uniqKey="Reddy M">MK Reddy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jha, B" uniqKey="Jha B">B Jha</name></author><author><name sortKey="Sharma, A" uniqKey="Sharma A">A Sharma</name></author><author><name sortKey="Mishra, A" uniqKey="Mishra A">A Mishra</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Casu, Re" uniqKey="Casu R">RE Casu</name></author><author><name sortKey="Selivanova, A" uniqKey="Selivanova A">A Selivanova</name></author><author><name sortKey="Perroux, Jm" uniqKey="Perroux J">JM Perroux</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bhaskaran, S" uniqKey="Bhaskaran S">S Bhaskaran</name></author><author><name sortKey="Savithramma, Dl" uniqKey="Savithramma D">DL Savithramma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, L" uniqKey="Liu L">L Liu</name></author><author><name sortKey="Zeng, Y" uniqKey="Zeng Y">Y Zeng</name></author><author><name sortKey="Pan, X" uniqKey="Pan X">X Pan</name></author><author><name sortKey="Zhang, F" uniqKey="Zhang F">F Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, Hx" uniqKey="Zhang H">HX Zhang</name></author><author><name sortKey="Hodson, Jn" uniqKey="Hodson J">JN Hodson</name></author><author><name sortKey="Williams, Jp" uniqKey="Williams J">JP Williams</name></author><author><name sortKey="Blumwald, E" uniqKey="Blumwald E">E Blumwald</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xue, Zy" uniqKey="Xue Z">ZY Xue</name></author><author><name sortKey="Zhi, Dy" uniqKey="Zhi D">DY Zhi</name></author><author><name sortKey="Xue, Gp" uniqKey="Xue G">GP Xue</name></author><author><name sortKey="Zhang, H" uniqKey="Zhang H">H Zhang</name></author><author><name sortKey="Zhao, Yx" uniqKey="Zhao Y">YX Zhao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, J" uniqKey="Zhao J">J Zhao</name></author><author><name sortKey="Zhi, D" uniqKey="Zhi D">D Zhi</name></author><author><name sortKey="Xue, Z" uniqKey="Xue Z">Z Xue</name></author><author><name sortKey="Liu, H" uniqKey="Liu H">H Liu</name></author><author><name sortKey="Xia, G" uniqKey="Xia G">G Xia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, Lh" uniqKey="Chen L">LH Chen</name></author><author><name sortKey="Zhang, B" uniqKey="Zhang B">B Zhang</name></author><author><name sortKey="Xu, Zq" uniqKey="Xu Z">ZQ Xu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jefferson, Ra" uniqKey="Jefferson R">RA Jefferson</name></author><author><name sortKey="Kavanagh, Ta" uniqKey="Kavanagh T">TA Kavanagh</name></author><author><name sortKey="Bevan, Mw" uniqKey="Bevan M">MW Bevan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, J" uniqKey="Li J">J Li</name></author><author><name sortKey="Li, Mr" uniqKey="Li M">MR Li</name></author><author><name sortKey="Wu, Pz" uniqKey="Wu P">PZ Wu</name></author><author><name sortKey="Tian, Ce" uniqKey="Tian C">CE Tian</name></author><author><name sortKey="Jiang, Hw" uniqKey="Jiang H">HW Jiang</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Fan, L" uniqKey="Fan L">L Fan</name></author><author><name sortKey="Zheng, S" uniqKey="Zheng S">S Zheng</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Arnon, Di" uniqKey="Arnon D">DI Arnon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lutts, S" uniqKey="Lutts S">S Lutts</name></author><author><name sortKey="Kinet, Jm" uniqKey="Kinet J">JM Kinet</name></author><author><name sortKey="Bouharmont, J" uniqKey="Bouharmont J">J Bouharmont</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hodges, Dm" uniqKey="Hodges D">DM Hodges</name></author><author><name sortKey="Delong, Jm" uniqKey="Delong J">JM DeLong</name></author><author><name sortKey="Forney, Cf" uniqKey="Forney C">CF Forney</name></author><author><name sortKey="Prange, Rk" uniqKey="Prange R">RK Prange</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23940703</article-id><article-id pub-id-type="pmc">3733712</article-id><article-id pub-id-type="publisher-id">PONE-D-13-22634</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0071136</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Agriculture</subject><subj-group><subject>Biofuels</subject><subj-group><subject>Biodiesel</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology</subject><subj-group><subject>Biotechnology</subject><subj-group><subject>Plant Biotechnology</subject><subj-group><subject>Transgenic Plants</subject></subj-group></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Developing Transgenic <italic>Jatropha</italic> Using the <italic>SbNHX1</italic> Gene from an Extreme Halophyte for Cultivation in Saline Wasteland</article-title><alt-title alt-title-type="running-head">Salt Tolerant <italic>Jatropha</italic></alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Jha</surname><given-names>Bhavanath</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mishra</surname><given-names>Avinash</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Jha</surname><given-names>Anupama</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Joshi</surname><given-names>Mukul</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib></contrib-group><aff id="aff1"><addr-line>Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Peccoud</surname><given-names>Jean</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Virginia Tech, United States of America</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>bjha@csmcri.org</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: BJ. Performed the experiments: MJ AJ. Analyzed the data: MJ AM. Wrote the paper: MJ AM BJ.</p></fn></author-notes><pub-date pub-type="collection"><year>2013</year></pub-date><pub-date pub-type="epub"><day>5</day><month>8</month><year>2013</year></pub-date><volume>8</volume><issue>8</issue><elocation-id>e71136</elocation-id><history><date date-type="received"><day>29</day><month>5</month><year>2013</year></date><date date-type="accepted"><day>2</day><month>7</month><year>2013</year></date></history><permissions><copyright-year>2013</copyright-year><copyright-holder>Jha et al</copyright-holder><license><license-p>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.</license-p></license></permissions><abstract><p><italic>Jatropha</italic> is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including <italic>Jatropha</italic>. <italic>SbNHX1</italic> is a vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter gene that compartmentalises excess Na<sup>+</sup> ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the <italic>SbNHX1</italic> gene from an extreme halophyte, <italic>Salicornia brachiata</italic>. Transgenic plants of <italic>Jatropha curcas</italic> with the <italic>SbNHX1</italic> 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 <italic>Jatropha</italic> 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 <italic>Jatropha</italic> using the <italic>SbNHX1</italic> gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under <italic>Jatropha</italic> cultivation for agriculture purposes. Apart from this, transgenic <italic>Jatropha</italic> can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.</p></abstract><funding-group><funding-statement>The financial assistance received from CSIR (<ext-link ext-link-type="uri" xlink:href="http://www.csir.res.in">www.csir.res.in</ext-link>), 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.</funding-statement></funding-group><counts><page-count count="10"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p><italic>Jatropha</italic> 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 <xref ref-type="bibr" rid="pone.0071136-Divakara1">[1]</xref>, <xref ref-type="bibr" rid="pone.0071136-Pandey1">[2]</xref>. 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 <xref ref-type="bibr" rid="pone.0071136-Ilham1">[3]</xref>. 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 <xref ref-type="bibr" rid="pone.0071136-Ilham1">[3]</xref>, <xref ref-type="bibr" rid="pone.0071136-Berchmans1">[4]</xref>. <italic>Jatropha</italic> 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<sup>−1</sup> that make this oil suitable as biofuel <xref ref-type="bibr" rid="pone.0071136-Divakara1">[1]</xref>. <italic>Jatropha</italic> 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 <xref ref-type="bibr" rid="pone.0071136-Chhetri1">[5]</xref>. <italic>Jatropha</italic> ranks next to oil palm in oil production per hectare, encouraging its cultivation worldwide <xref ref-type="bibr" rid="pone.0071136-Fairless1">[6]</xref>. There are concerns that <italic>Jatropha</italic> investors may drive its cultivation from marginal or degraded lands towards agricultural lands in order to reduce financial risk <xref ref-type="bibr" rid="pone.0071136-Achten1">[7]</xref> but this possibility was recently ruled out <xref ref-type="bibr" rid="pone.0071136-Dyer1">[8]</xref>.</p><p><italic>Jatropha</italic> is well distributed in India <xref ref-type="bibr" rid="pone.0071136-Sunil1">[9]</xref>; 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 <xref ref-type="bibr" rid="pone.0071136-Ministryof1">[10]</xref>. 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 <xref ref-type="bibr" rid="pone.0071136-Ravindranath1">[11]</xref>. CSIR-CSMCRI has been recognized worldwide and is actively working on <italic>Jatropha</italic> elite accessions’ selection, cultivation, genetic improvement and biodiesel production <xref ref-type="bibr" rid="pone.0071136-Fairless1">[6]</xref>, <xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref>.</p><p>Over 800 Mha of land throughout the world are affected by salt <xref ref-type="bibr" rid="pone.0071136-FAO1">[13]</xref>. In India, the total arable land area is about 184 Mha, out of which 8.6 Mha is salt affected <xref ref-type="bibr" rid="pone.0071136-FAO2">[14]</xref>. Salinity imposes various constraints on plant growth and yield from osmotic stress created due to high concentrations of Na<sup>+</sup> and Cl<sup>-</sup> ions <xref ref-type="bibr" rid="pone.0071136-Blumwald1">[15]</xref>. There are some major effects of salt stress, <italic>i.e.</italic> water potential reduction, ionic imbalance or disturbances in ion homeostasis and ion toxicity, which inhibits enzymatic functions in key biological processes <xref ref-type="bibr" rid="pone.0071136-Blumwald1">[15]</xref>, <xref ref-type="bibr" rid="pone.0071136-Zhang1">[16]</xref>.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 <xref ref-type="bibr" rid="pone.0071136-Apse1">[17]</xref>, <xref ref-type="bibr" rid="pone.0071136-Shi1">[18]</xref>, <xref ref-type="bibr" rid="pone.0071136-Shi2">[19]</xref>. Antiporters Na<sup>+</sup>/H<sup>+</sup> 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<sup>+</sup> homeostasis in the cytosol <xref ref-type="bibr" rid="pone.0071136-Blumwald1">[15]</xref>; however it varies with the plant species. Ion homeostasis is carried out either by sequestration of excess sodium into the vacuoles via vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporters (NHX1) energized by the proton gradient generated by vacuolar membrane H<sup>+</sup>-ATPase and H<sup>+</sup>-pyrophosphatases <xref ref-type="bibr" rid="pone.0071136-Blumwald1">[15]</xref>, <xref ref-type="bibr" rid="pone.0071136-Apse1">[17]</xref>, or by active exclusion through Na<sup>+</sup>/H<sup>+</sup> antiporters (SOS1) located on the plasma membrane <xref ref-type="bibr" rid="pone.0071136-Shi1">[18]</xref>, <xref ref-type="bibr" rid="pone.0071136-Shi2">[19]</xref>. Therefore, the ability to maintain lower Na<sup>+</sup> and Cl<sup>-</sup> in the cytosol may be a key determinant of salt tolerance.</p><p>The previous study that vacuolar NHX proteins are capable of Na<sup>+</sup> and H<sup>+</sup> exchange across the tonoplast <xref ref-type="bibr" rid="pone.0071136-Blumwald1">[15]</xref>, <xref ref-type="bibr" rid="pone.0071136-Apse1">[17]</xref>, has been modified based on the biochemistry of the NHX proteins. Recently it was reported that NHX proteins do not discriminate between Na<sup>+</sup> and K<sup>+</sup> or have a preference for K<sup>+</sup> transport <xref ref-type="bibr" rid="pone.0071136-Jiang1">[20]</xref>, <xref ref-type="bibr" rid="pone.0071136-Bassil1">[21]</xref>. These reports suggest that NHX1 and NHX2 are vacuolar Na<sup>+</sup>–K<sup>+</sup>/H<sup>+</sup> exchangers essential for active K<sup>+</sup> uptake at the tonoplast, osmotic adjustment, turgor regulation, stomata function and cell expansion <xref ref-type="bibr" rid="pone.0071136-Barragn1">[22]</xref>, <xref ref-type="bibr" rid="pone.0071136-Bassil2">[23]</xref>.</p><p><italic>Jatropha</italic> growth and yield are adversely affected by increasing salt concentration beyond 30–50 mM <xref ref-type="bibr" rid="pone.0071136-Silva1">[24]</xref>, <xref ref-type="bibr" rid="pone.0071136-DazLpez1">[25]</xref>, <xref ref-type="bibr" rid="pone.0071136-Niu1">[26]</xref>. Therefore, <italic>Jatropha</italic> is considered to be a moderately salt-tolerant plant and its salinity tolerance is comparable with major crops such as soybean or wheat <xref ref-type="bibr" rid="pone.0071136-Fujimaki1">[27]</xref>. Development of transgenic <italic>Jatropha</italic> 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 <italic>Agrobacterium</italic> and direct gene transfer via microprojectile bombardment. Both these methods have their advantages and limitations <xref ref-type="bibr" rid="pone.0071136-Tzfira1">[28]</xref>, <xref ref-type="bibr" rid="pone.0071136-Altpeter1">[29]</xref>. We have reported an efficient microprojectile bombardment mediated transformation protocol for <italic>Jatropha</italic><xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref> that has been used in this study.</p><p>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 <italic>Jatropha</italic> plants were developed for trait improvement <xref ref-type="bibr" rid="pone.0071136-Qu1">[30]</xref>, <xref ref-type="bibr" rid="pone.0071136-Tsuchimoto1">[31]</xref>. 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 <xref ref-type="bibr" rid="pone.0071136-Flowers1">[32]</xref>, <xref ref-type="bibr" rid="pone.0071136-BhatnagarMathur1">[33]</xref>. A salt-tolerant <italic>SbNHX1</italic> gene (GenBank: EU448383) (<xref ref-type="fig" rid="pone-0071136-g001">Figure 1</xref>) encoding an active vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter has been cloned and characterized from an extreme halophyte <italic>Salicornia brachiata</italic> in our laboratory <xref ref-type="bibr" rid="pone.0071136-Jha1">[34]</xref>. The core objective of the present work is developing transgenic <italic>Jatropha</italic> with the <italic>SbNHX1</italic> gene for enhanced salinity tolerance. Salt tolerant transgenic <italic>Jatropha</italic> plants can be further utilized for sustainable cultivation in salt-affected marginal areas.</p><fig id="pone-0071136-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0071136.g001</object-id><label>Figure 1</label><caption><title>Schematic map of plant expression gene construct pCAMBIA1301-<italic>SbNHX1</italic>.</title><p>Expression of <italic>SbNHX1</italic> is driven by the cauliflower mosaic virus 35S promoter.</p></caption><graphic xlink:href="pone.0071136.g001"></graphic></fig></sec><sec id="s2"><title>Results</title><sec id="s2a"><title>Microprojectile Bombardment Mediated Transformation</title><p>Five days pre-cultured embryo axes were bombarded at optimized parameters, <italic>i.e.</italic> 1 µm microcarrier size, 1100 and 1350 psi He pressure, with target distances of 9 and 12 cm, respectively <xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref>. After bombardment, 3–5 transformed embryos per shot per plate were selected randomly after 24 h and transient <italic>gus</italic> expression was assessed. Most of the explants showed discrete blue spots or regions (<xref ref-type="fig" rid="pone-0071136-g002">Figure 2a</xref>). Bombarded embryo axes were regenerated into transgenic plants after hygromycin selection (<xref ref-type="fig" rid="pone-0071136-g002">Figure 2b–f</xref>) as reported previously <xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref>. Putative transgenic plants with rooted shoots were transplanted into pots for hardening and acclimatization (<xref ref-type="fig" rid="pone-0071136-g002">Figure 2g</xref> and <xref ref-type="supplementary-material" rid="pone.0071136.s001">Table S1</xref>). Regenerated young leaves from putative transformed plants were assayed for constitutive expression of the <italic>gus</italic> gene using non-transformed leaves as control (<xref ref-type="fig" rid="pone-0071136-g002">Figure 2h–i</xref>).</p><fig id="pone-0071136-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0071136.g002</object-id><label>Figure 2</label><caption><title>Genetic transformation of <italic>J. curcas</italic> via microprojectile bombardment.</title><p>(A) Transient <italic>gus</italic> expression on embryo axis after 24 h of bombardment. Selection of transformants (B) during 1<sup>st</sup> round (8 days old), (C) during 3<sup>rd</sup> 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.</p></caption><graphic xlink:href="pone.0071136.g002"></graphic></fig></sec><sec id="s2b"><title>Molecular Analysis of Transgenic Plants</title><p>Genetic transformation was confirmed by PCR amplification of 172 bp (internal fragment), 963 bp and 157 bp (internal fragment) of the <italic>SbNHX1</italic> gene, selectable marker <italic>hptII</italic> gene and reporter <italic>gus</italic> gene, respectively (<xref ref-type="fig" rid="pone-0071136-g003">Figure 3a–c</xref>). Real time quantitative PCR (RTqPCR) was performed to determine the copy number using a single copy <italic>JcKASIII</italic> gene as the internal control. Transgenic lines JL2, JL8 and JL19 showed <italic>gus</italic> to <italic>JcKASIII</italic> ratio 0.746, 2.922 and 1.274 respectively (<xref ref-type="fig" rid="pone-0071136-g003">Figure 3d</xref>). 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 <italic>JcKASIII</italic> gene and the transgenic lines individually showed single peaks that confirmed the absence of any dimer or other contamination. Single band amplification of the <italic>gus</italic> gene as well as the control gene was further confirmed by 1.2% agarose gel electrophoresis. The expression of the <italic>SbNHX1</italic> 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 (<xref ref-type="fig" rid="pone-0071136-g003">Figure 3e</xref>).</p><fig id="pone-0071136-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0071136.g003</object-id><label>Figure 3</label><caption><title>Molecular confirmation of transgenic Lines.</title><p>PCR amplification of (A) <italic>SbNHX1</italic> gene (172 bp internal fragment), (B) <italic>hptII</italic> gene (963 bp) and (C) <italic>gus</italic> gene (157 bp internal fragment). Lane M is 100 bp Molecular weight marker ladder (Bangalore Genei, India); lane+is pCAMBIA1301-<italic>SbNHX1</italic> (positive control). (D) Real time qPCR analysis to determine the ratio of <italic>gus</italic> to <italic>JcKASIII</italic> 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; <italic>JcKASIII</italic> gene was used as an internal control.</p></caption><graphic xlink:href="pone.0071136.g003"></graphic></fig></sec><sec id="s2c"><title>Physiological and Biochemical Analysis</title><sec id="s2c1"><title>Leaf senescence assay, chlorophyll content and growth assay</title><p>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 <italic>SbNHX1</italic>-overexpressing lines compared to WT plants (<xref ref-type="fig" rid="pone-0071136-g004">Figure 4</xref>). 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 (<xref ref-type="fig" rid="pone-0071136-g005">Figure 5a</xref>). 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 (<xref ref-type="fig" rid="pone-0071136-g006">Figure 6</xref>).</p><fig id="pone-0071136-g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0071136.g004</object-id><label>Figure 4</label><caption><title>Leaf disc senescence assay of transgenic <italic>J. curcas</italic> for salt tolerance.</title><p>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.</p></caption><graphic xlink:href="pone.0071136.g004"></graphic></fig><fig id="pone-0071136-g005" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0071136.g005</object-id><label>Figure 5</label><caption><title>Physiological and biochemical analysis of transgenic lines.</title><p>(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<sup>+</sup> and (c) K<sup>+</sup> 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 <italic>P</italic><0.05.</p></caption><graphic xlink:href="pone.0071136.g005"></graphic></fig><fig id="pone-0071136-g006" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0071136.g006</object-id><label>Figure 6</label><caption><title>Effect of 200 mM NaCl on <italic>in vitro</italic> growing transgenic lines JL2 and JL8, and WT after 21 days.</title></caption><graphic xlink:href="pone.0071136.g006"></graphic></fig></sec><sec id="s2c2"><title>Ion content analysis</title><p>At zero NaCl, transgenic lines and WT plants exhibited approximately similar Na<sup>+</sup> content in leaf tissues. However, at 200 mM NaCl, there was significant increase in Na<sup>+</sup> in all transgenic lines than in the WT plants (<xref ref-type="fig" rid="pone-0071136-g005">Figure 5b</xref>). These findings indicated that the transgenic lines accumulated more Na<sup>+</sup> than the WT under the same stress conditions. In the case of K<sup>+</sup>, all the transgenic lines and WT showed non-significantly higher content at zero NaCl. However, at 200 mM NaCl, K<sup>+</sup> decreased in both transgenic and WT plants. Although this decrease was also non-significant, WT exhibited higher K<sup>+</sup> content than transgenic lines (<xref ref-type="fig" rid="pone-0071136-g005">Figure 5c</xref>). We also analysed the effect of NaCl on Ca<sup>2+</sup> content (data not shown), but it was slightly and non-significantly increased at 200 mM NaCl. Transgenic lines showed slightly higher Ca<sup>2+</sup> content than WT at 200 mM NaCl.</p></sec><sec id="s2c3"><title>Electrolyte leakage and MDA content analysis</title><p>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 (<xref ref-type="fig" rid="pone-0071136-g005">Figure 5d</xref>). Transgenic lines accumulated significantly less MDA than WT in response to salt stress, which was indicative of reduced oxidative damage in plants overexpressing <italic>SbNHX1</italic> (<xref ref-type="fig" rid="pone-0071136-g005">Figure 5e</xref>).</p></sec></sec></sec><sec id="s3"><title>Discussion</title><p>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 <xref ref-type="bibr" rid="pone.0071136-Apse1">[17]</xref>, <xref ref-type="bibr" rid="pone.0071136-Shi2">[19]</xref>, <xref ref-type="bibr" rid="pone.0071136-Rajagopal1">[35]</xref>, <xref ref-type="bibr" rid="pone.0071136-Jha2">[36]</xref>. 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 <italic>Jatropha</italic> with the salt-responsive <italic>SbNHX1</italic> gene for its enhanced salt tolerance via microprojectile bombardment (<xref ref-type="supplementary-material" rid="pone.0071136.s001">Table S1</xref>). The optimized microprojectile bombardment protocol <xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref> was applied for bombardment of <italic>Jatropha</italic> embryo axes with the recombinant binary plasmid construct pCAMBIA1301-<italic>SbNHX1</italic>. 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 <xref ref-type="bibr" rid="pone.0071136-Jha2">[36]</xref>, <xref ref-type="bibr" rid="pone.0071136-Casu1">[37]</xref>. 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.</p><p>In one of the two most recent reports on transgenic <italic>Jatropha</italic> for trait improvement, Qu <italic>et al.</italic><xref ref-type="bibr" rid="pone.0071136-Qu1">[30]</xref> developed marker-free transgenic plants with increase in high quality seed oil by down-regulating the seed-specific <italic>JcFAD2-1</italic> gene. In another report, transgenic <italic>Jatropha</italic> plants overproducing glycine betaine were developed for enhanced drought tolerance <xref ref-type="bibr" rid="pone.0071136-Tsuchimoto1">[31]</xref>, 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 <italic>NHX1</italic> gene cloned from different plant sources and its overexpression shown to enhance salt tolerance <xref ref-type="bibr" rid="pone.0071136-Rajagopal1">[35]</xref>, <xref ref-type="bibr" rid="pone.0071136-Bhaskaran1">[38]</xref>.</p><p>Physiological analysis of transgenic plants represents the extent of expression of an integrated gene responsible for a trait. The strategy to accumulate Na<sup>+</sup> 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<sup>+</sup> into the vacuole <xref ref-type="bibr" rid="pone.0071136-Blumwald1">[15]</xref>. SbNHX1 contains 11 strong transmembrane domains spanning in vacuolar membrane similar to some of the other halophytes and that actively regulates the ion homeostasis <xref ref-type="bibr" rid="pone.0071136-Jha1">[34]</xref>. The response of the <italic>SbNHX1</italic>-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 (<xref ref-type="fig" rid="pone-0071136-g004">Figure 4</xref>). 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 (<xref ref-type="fig" rid="pone-0071136-g005">Figure 5a</xref>). Similarly, transgenic tobacco plants overexpressing <italic>SbNHX1</italic> showed better growth and chlorophyll content than WT plants at 100–300 mM NaCl concentrations <xref ref-type="bibr" rid="pone.0071136-Jha1">[34]</xref>. Recently, Liu <italic>et al.</italic><xref ref-type="bibr" rid="pone.0071136-Liu1">[39]</xref> isolated <italic>KcNHX1</italic> and <italic>KcNHX2</italic> genes from the halophyte <italic>Karelinia caspica</italic> and showed that RNAi silencing of <italic>KcNHX1</italic> reduced the salt tolerance of <italic>K. caspica</italic> plants at 200 mM NaCl in a leaf disc assay, and chlorophyll content was decreased.In the ion content analysis, higher Na<sup>+</sup> content and less K<sup>+</sup> was observed in transgenic leaf tissues under salt stress. This can be attributed to ion homeostasis due to the activity of the vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter SbNHX1. Significant increase in Na<sup>+</sup> and reduction in K<sup>+</sup> due to overexpression of AtNHX1 during salt stress has been reported in tomato <xref ref-type="bibr" rid="pone.0071136-Zhang1">[16]</xref>, rapeseed <xref ref-type="bibr" rid="pone.0071136-Zhang2">[40]</xref>, wheat <xref ref-type="bibr" rid="pone.0071136-Xue1">[41]</xref>, tall fescue <xref ref-type="bibr" rid="pone.0071136-Zhao1">[42]</xref> and buckwheat <xref ref-type="bibr" rid="pone.0071136-Chen1">[43]</xref>. The growth of transgenic plants was not significantly affected by increased salinity, suggesting that K<sup>+</sup> content was not compromised in the transgenic plants.</p><p>The transgenic lines (Jl2 and JL8) showed enhanced tolerance at 200 mM NaCl (<xref ref-type="fig" rid="pone-0071136-g006">Figure 6</xref>). The results show the positive effect of the Na<sup>+</sup>/H<sup>+</sup> antiporter <italic>SbNHX1</italic> in T0 plants and their enhanced salt tolerance. The <italic>PgNHX1</italic> gene from <italic>Pennisetum glaucum</italic> has been reported to confer a high level of salinity tolerance when overexpressed in <italic>Brassica juncea</italic><xref ref-type="bibr" rid="pone.0071136-Rajagopal1">[35]</xref> and tomato <xref ref-type="bibr" rid="pone.0071136-Bhaskaran1">[38]</xref>, 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<sup>+</sup> due to its enhanced sequestration into the vacuole.</p><sec id="s3a"><title>Conclusions</title><p>In conclusion, we developed the functional transgenic <italic>J. curcas</italic> plants overexpressing <italic>SbNHX1</italic> 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 <italic>Jatropha</italic>. Salt tolerant transgenic <italic>Jatropha</italic> plants can be used for cultivation in salt-affected marginal areas for their sustainable management with biofuel production.</p></sec></sec><sec sec-type="methods" id="s4"><title>Methods</title><sec id="s4a"><title>Plant Material and Culture Conditions</title><p>Mature decoated seeds of <italic>Jatropha curcas</italic> 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<sup>−2</sup> s<sup>−1</sup>.</p></sec><sec id="s4b"><title>Plasmid Construct</title><p>The Binary plasmid vector pCAMBIA1301 (CAMBIA, Australia) containing the <italic>SbNHX1</italic> gene under the control of the CaMV35S promoter was named the pCAMBIA1301-<italic>SbNHX1</italic> gene construct (<xref ref-type="fig" rid="pone-0071136-g001">Figure 1</xref>) <xref ref-type="bibr" rid="pone.0071136-Jha1">[34]</xref>, which was used for microprojectile bombardment.</p></sec><sec id="s4c"><title>Microprojectile Bombardment Mediated Transformation</title><p>Plasmid construct pCAMBIA1301-<italic>SbNHX1</italic> 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 <xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref>. Bombardments were performed on 40–50 five days pre-cultured embryo axes per plate with a biolistic gene gun (PDS 1000/He, Bio-Rad).</p></sec><sec id="s4d"><title>Selection and Regeneration of Transformants</title><p>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 <xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref>. 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.</p><p>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 <xref ref-type="bibr" rid="pone.0071136-Joshi1">[12]</xref>.</p></sec><sec id="s4e"><title>Histochemical GUS Assay</title><p>Transient <italic>gus</italic> expression in embryo axis and leaves was assessed <xref ref-type="bibr" rid="pone.0071136-Jefferson1">[44]</xref> 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 <italic>gus</italic> gene. GUS assay was done by incubating the tissues in freshly prepared GUS assay buffer (1 mg/ml X-Gluc with 0.05 M Na<sub>2</sub>HPO<sub>4</sub>, 0.5 mM K<sub>3</sub>Fe(CN)<sub>6</sub>, 0.5 mM K<sub>4</sub>Fe(CN)<sub>6</sub>, 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.</p></sec><sec id="s4f"><title>PCR Amplification and Determination of Copy Number by Real Time Quantitative PCR</title><p>The genomic DNA of transformants was isolated and concentration was determined with a NanoDrop Spectrophotometer. Transformation was confirmed by PCR with <italic>SbNHX1</italic> 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′); <italic>gus</italic> (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 <italic>hptII</italic> (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.</p><p>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 <italic>gus</italic> gene primers (gusRTF and gusRTR) and <italic>JcKASIII</italic> gene (GenBank: DQ987701) primers (JcKAS1F: <named-content content-type="gene">5′-GCA CTT GGC TGC AAA ACA AAT-3′</named-content>, JcKAS1R: 5′-CGT CCA GTC AAC ATA TCG AG-3′). The <italic>JcKASIII</italic> gene was used as an internal control because this is a single copy gene in the <italic>Jatropha</italic> genome <xref ref-type="bibr" rid="pone.0071136-Li1">[45]</xref>. 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 <xref ref-type="bibr" rid="pone.0071136-Shepherd1">[46]</xref> as follows:<disp-formula id="pone.0071136.e001"><graphic xlink:href="pone.0071136.e001"></graphic></disp-formula></p><p>The efficiency values were put in the following equation <xref ref-type="bibr" rid="pone.0071136-Shepherd1">[46]</xref> to determine the copy number ratio of <italic>gus</italic> to <italic>JcKASIII</italic>:<disp-formula id="pone.0071136.e002"><graphic xlink:href="pone.0071136.e002"></graphic></disp-formula></p></sec><sec id="s4g"><title>Semiquantitative Rt-PCR Analysis</title><p>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, <italic>SbNHX1</italic> gene as target and <italic>JcKASIII</italic> gene as an internal control using <italic>SbNHX1</italic> and <italic>JcKASIII</italic> 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.</p></sec><sec id="s4h"><title>Leaf Senescence Assay</title><p>Leaf disc assay was performed to analyse transgenic plants for their salt tolerance according to procedures described by Fan et al. <xref ref-type="bibr" rid="pone.0071136-Fan1">[47]</xref>. 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<sup>−2</sup> s<sup>−1</sup>)/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.</p></sec><sec id="s4i"><title>Chlorophyll Estimation</title><p>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 <xref ref-type="bibr" rid="pone.0071136-Arnon1">[48]</xref>.</p></sec><sec id="s4j"><title>Effect of Salt on <italic>in vitro</italic> Growing Plants</title><p>Two months old <italic>in vitro</italic> 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.</p></sec><sec id="s4k"><title>Ion Contents</title><p>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).</p></sec><sec id="s4l"><title>Electrolyte Leakage</title><p>Electrolyte leakage was measured according to Lutts et al. <xref ref-type="bibr" rid="pone.0071136-Lutts1">[49]</xref>. 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<sub>t</sub>) 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 (L<sub>0</sub>) was determined. The electrolyte leakage was defined as follows:<disp-formula id="pone.0071136.e003"><graphic xlink:href="pone.0071136.e003"></graphic></disp-formula></p></sec><sec id="s4m"><title>Lipid Peroxidation</title><p>Lipid peroxidation was estimated by following the method described by Hodges et al. <xref ref-type="bibr" rid="pone.0071136-Hodges1">[50]</xref>. 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:<disp-formula id="pone.0071136.e004"><graphic xlink:href="pone.0071136.e004"></graphic><label>(1)</label></disp-formula><disp-formula id="pone.0071136.e005"><graphic xlink:href="pone.0071136.e005"></graphic><label>(2)</label></disp-formula><disp-formula id="pone.0071136.e006"><graphic xlink:href="pone.0071136.e006"></graphic><label>(3)</label></disp-formula></p><p>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, <italic>P</italic><0.05 was considered as significant. Significantly different mean values were indicated by similar letters.</p></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0071136.s001"><label>Table S1</label><caption><p><bold>Transformation efficiency and overall regeneration efficiency after microprojectile bombardment of embryo axes with pCAMBIA1301-</bold><bold><italic>SbNHX1</italic></bold><bold> gene construct.</bold></p><p>(DOC)</p></caption><media xlink:href="pone.0071136.s001.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ref-list><title>References</title><ref id="pone.0071136-Divakara1"><label>1</label><mixed-citation publication-type="journal"><name><surname>Divakara</surname><given-names>BN</given-names></name>, <name><surname>Upadhyaya</surname><given-names>HD</given-names></name>, <name><surname>Wani</surname><given-names>SP</given-names></name>, <name><surname>Gowda</surname><given-names>CLL</given-names></name> (<year>2010</year>) <article-title>Biology and genetic improvement of <italic>Jatropha curcas</italic> L.: A review</article-title>. <source>Appl Energ</source><volume>87</volume>: <fpage>732</fpage>–<lpage>742</lpage></mixed-citation></ref><ref id="pone.0071136-Pandey1"><label>2</label><mixed-citation publication-type="journal"><name><surname>Pandey</surname><given-names>VC</given-names></name>, <name><surname>Singh</surname><given-names>K</given-names></name>, <name><surname>Singh</surname><given-names>JS</given-names></name>, <name><surname>Kumar</surname><given-names>A</given-names></name>, <name><surname>Singh</surname><given-names>B</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title><italic>Jatropha curcas</italic>: A potential biofuel plant for sustainable environmental development</article-title>. <source>Renew Sust Energy Rev</source><volume>16</volume>: <fpage>2870</fpage>–<lpage>2883</lpage></mixed-citation></ref><ref id="pone.0071136-Ilham1"><label>3</label><mixed-citation publication-type="journal"><name><surname>Ilham</surname><given-names>Z</given-names></name>, <name><surname>Saka</surname><given-names>S</given-names></name> (<year>2010</year>) <article-title>Two-step supercritical dimethyl carbonate method for biodiesel production from <italic>Jatropha curcas</italic> oil</article-title>. <source>Bioresour Technol</source><volume>101</volume>: <fpage>2735</fpage>–<lpage>2740</lpage><pub-id pub-id-type="pmid">19932022</pub-id></mixed-citation></ref><ref id="pone.0071136-Berchmans1"><label>4</label><mixed-citation publication-type="journal"><name><surname>Berchmans</surname><given-names>HJ</given-names></name>, <name><surname>Hirata</surname><given-names>S</given-names></name> (<year>2008</year>) <article-title>Biodiesel production from crude <italic>Jatropha curcas</italic> L. seed oil with a high content of free fatty acids</article-title>. <source>Bioresour Technol</source><volume>99</volume>: <fpage>1716</fpage>–<lpage>1721</lpage><pub-id pub-id-type="pmid">17531473</pub-id></mixed-citation></ref><ref id="pone.0071136-Chhetri1"><label>5</label><mixed-citation publication-type="journal"><name><surname>Chhetri</surname><given-names>AB</given-names></name>, <name><surname>Tango</surname><given-names>MS</given-names></name>, <name><surname>Budge</surname><given-names>SM</given-names></name>, <name><surname>Watts</surname><given-names>KC</given-names></name>, <name><surname>Islam</surname><given-names>MR</given-names></name> (<year>2008</year>) <article-title>Non-edible plant oils as new sources for biodiesel production</article-title>. <source>Int J Mol Sci</source><volume>9</volume>: <fpage>169</fpage>–<lpage>180</lpage><pub-id pub-id-type="pmid">19325741</pub-id></mixed-citation></ref><ref id="pone.0071136-Fairless1"><label>6</label><mixed-citation publication-type="journal"><name><surname>Fairless</surname><given-names>D</given-names></name> (<year>2007</year>) <article-title>The little shrub that could – maybe</article-title>. <source>Nature</source><volume>449</volume>: <fpage>652</fpage>–<lpage>655</lpage><pub-id pub-id-type="pmid">17968401</pub-id></mixed-citation></ref><ref id="pone.0071136-Achten1"><label>7</label><mixed-citation publication-type="journal"><name><surname>Achten</surname><given-names>WMJ</given-names></name>, <name><surname>Maes</surname><given-names>WH</given-names></name>, <name><surname>Aerts</surname><given-names>R</given-names></name>, <name><surname>Verchot</surname><given-names>L</given-names></name>, <name><surname>Trabucco</surname><given-names>A</given-names></name>, <etal>et al</etal> (<year>2010</year>) <article-title><italic>Jatropha</italic>: From global hype to local opportunity</article-title>. <source>J Arid Environ</source><volume>74</volume>: <fpage>164</fpage>–<lpage>165</lpage></mixed-citation></ref><ref id="pone.0071136-Dyer1"><label>8</label><mixed-citation publication-type="journal"><name><surname>Dyer</surname><given-names>JC</given-names></name>, <name><surname>Stringer</surname><given-names>LC</given-names></name>, <name><surname>Dougill</surname><given-names>AJ</given-names></name> (<year>2012</year>) <article-title><italic>Jatropha curcas</italic>: Sowing local seeds of success in Malawi? In response to Achten et al. (2010)</article-title>. <source>J Arid Environ</source><volume>79</volume>: <fpage>107</fpage>–<lpage>110</lpage></mixed-citation></ref><ref id="pone.0071136-Sunil1"><label>9</label><mixed-citation publication-type="journal"><name><surname>Sunil</surname><given-names>N</given-names></name>, <name><surname>Sivaraj</surname><given-names>N</given-names></name>, <name><surname>Anitha</surname><given-names>K</given-names></name>, <name><surname>Abraham</surname><given-names>B</given-names></name>, <name><surname>Kumar</surname><given-names>V</given-names></name>, <etal>et al</etal> (<year>2009</year>) <article-title>Analysis of diversity and distribution of <italic>Jatropha curcas</italic> L. germplasm using Geographic Information System (DIVA-GIS)</article-title>. <source>Genet Resour Crop Evol</source><volume>56</volume>: <fpage>115</fpage>–<lpage>119</lpage></mixed-citation></ref><ref id="pone.0071136-Ministryof1"><label>10</label><mixed-citation publication-type="other">Ministry of New and Renewable Energy (2009) National Policy on Biofuels. Available: <ext-link ext-link-type="uri" xlink:href="http://mnes.nic.in">http://mnes.nic.in</ext-link> Accessed 2012 Jan 25.</mixed-citation></ref><ref id="pone.0071136-Ravindranath1"><label>11</label><mixed-citation publication-type="journal"><name><surname>Ravindranath</surname><given-names>NH</given-names></name>, <name><surname>Lakshmi</surname><given-names>CS</given-names></name>, <name><surname>Manuvie</surname><given-names>R</given-names></name>, <name><surname>Balachandra</surname><given-names>P</given-names></name> (<year>2011</year>) <article-title>Biofuel production and implications for land use, food production and environment in India</article-title>. <source>Energ Policy</source><volume>39</volume>: <fpage>5737</fpage>–<lpage>5745</lpage></mixed-citation></ref><ref id="pone.0071136-Joshi1"><label>12</label><mixed-citation publication-type="journal"><name><surname>Joshi</surname><given-names>M</given-names></name>, <name><surname>Mishra</surname><given-names>A</given-names></name>, <name><surname>Jha</surname><given-names>B</given-names></name> (<year>2011</year>) <article-title>Efficient genetic transformation of <italic>Jatropha curcas</italic> L. by microprojectile bombardment using embryo axes</article-title>. <source>Ind Crop Prod</source><volume>33</volume>: <fpage>67</fpage>–<lpage>77</lpage></mixed-citation></ref><ref id="pone.0071136-FAO1"><label>13</label><mixed-citation publication-type="other">FAO (2008) Land and Plant Nutrition Management Service. Available: <ext-link ext-link-type="uri" xlink:href="http://www.fao.org/ag/agl/agll/spush/">http://www.fao.org/ag/agl/agll/spush/</ext-link> Accessed 2010 Dec 21.</mixed-citation></ref><ref id="pone.0071136-FAO2"><label>14</label><mixed-citation publication-type="other">FAO (2005) Global network on integrated soil management for sustainable use of salt-affected soils. Available: <ext-link ext-link-type="uri" xlink:href="http://www.fao.org/ag/agl/agll/spush/">http://www.fao.org/ag/agl/agll/spush/</ext-link> Accessed 2008 July 28.</mixed-citation></ref><ref id="pone.0071136-Blumwald1"><label>15</label><mixed-citation publication-type="journal"><name><surname>Blumwald</surname><given-names>E</given-names></name> (<year>2000</year>) <article-title>Sodium transport and salt tolerance in plants</article-title>. <source>Curr Opin Cell Biol</source><volume>12</volume>: <fpage>431</fpage>–<lpage>434</lpage><pub-id pub-id-type="pmid">10873827</pub-id></mixed-citation></ref><ref id="pone.0071136-Zhang1"><label>16</label><mixed-citation publication-type="journal"><name><surname>Zhang</surname><given-names>HX</given-names></name>, <name><surname>Blumwald</surname><given-names>E</given-names></name> (<year>2001</year>) <article-title>Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit</article-title>. <source>Nat Biotechnol</source><volume>19</volume>: <fpage>765</fpage>–<lpage>768</lpage><pub-id pub-id-type="pmid">11479571</pub-id></mixed-citation></ref><ref id="pone.0071136-Apse1"><label>17</label><mixed-citation publication-type="journal"><name><surname>Apse</surname><given-names>MP</given-names></name>, <name><surname>Aharon</surname><given-names>GS</given-names></name>, <name><surname>Snedden</surname><given-names>WA</given-names></name>, <name><surname>Blumwald</surname><given-names>E</given-names></name> (<year>1999</year>) <article-title>Salt tolerance conferred by over expression of a vacuolar Na<sup>+</sup>/H<sup>+</sup> antiport in <italic>Arabidopsis</italic></article-title>. <source>Science</source><volume>285</volume>: <fpage>1256</fpage>–<lpage>1258</lpage><pub-id pub-id-type="pmid">10455050</pub-id></mixed-citation></ref><ref id="pone.0071136-Shi1"><label>18</label><mixed-citation publication-type="journal"><name><surname>Shi</surname><given-names>H</given-names></name>, <name><surname>Ishitani</surname><given-names>M</given-names></name>, <name><surname>Kim</surname><given-names>C</given-names></name>, <name><surname>Zhu</surname><given-names>JK</given-names></name> (<year>2000</year>) <article-title>The <italic>Arabidopsis thaliana</italic> salt tolerance gene <italic>SOS1</italic> encodes a putative Na<sup>+</sup>/H<sup>+</sup> antiporter</article-title>. <source>Proc Natl Acad Sci USA</source><volume>97</volume>: <fpage>6896</fpage>–<lpage>6901</lpage><pub-id pub-id-type="pmid">10823923</pub-id></mixed-citation></ref><ref id="pone.0071136-Shi2"><label>19</label><mixed-citation publication-type="journal"><name><surname>Shi</surname><given-names>H</given-names></name>, <name><surname>Lee</surname><given-names>BH</given-names></name>, <name><surname>Wu</surname><given-names>SJ</given-names></name>, <name><surname>Zhu</surname><given-names>JK</given-names></name> (<year>2003</year>) <article-title>Over-expression of a plasma membrane Na<sup>+</sup>/H<sup>+</sup> antiporter gene improves salt tolerance in <italic>Arabidopsis thaliana</italic></article-title>. <source>Nat Biotechnol</source><volume>21</volume>: <fpage>81</fpage>–<lpage>85</lpage><pub-id pub-id-type="pmid">12469134</pub-id></mixed-citation></ref><ref id="pone.0071136-Jiang1"><label>20</label><mixed-citation publication-type="journal"><name><surname>Jiang</surname><given-names>XY</given-names></name>, <name><surname>Leidi</surname><given-names>EO</given-names></name>, <name><surname>Pardo</surname><given-names>JM</given-names></name> (<year>2010</year>) <article-title>How do vacuolar NHX exchangers function in plant salt tolerance?</article-title><source>Plant Signal Behav</source><volume>5</volume>: <fpage>792</fpage>–<lpage>795</lpage><pub-id pub-id-type="pmid">20495345</pub-id></mixed-citation></ref><ref id="pone.0071136-Bassil1"><label>21</label><mixed-citation publication-type="journal"><name><surname>Bassil</surname><given-names>E</given-names></name>, <name><surname>Tajima</surname><given-names>H</given-names></name>, <name><surname>Liang</surname><given-names>YC</given-names></name>, <name><surname>Ohto</surname><given-names>MA</given-names></name>, <name><surname>Ushijima</surname><given-names>K</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>The <italic>Arabidopsis</italic> Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction</article-title>. <source>Plant Cell</source><volume>23</volume>: <fpage>3482</fpage>–<lpage>3497</lpage><pub-id pub-id-type="pmid">21954467</pub-id></mixed-citation></ref><ref id="pone.0071136-Barragn1"><label>22</label><mixed-citation publication-type="journal"><name><surname>Barragán</surname><given-names>V</given-names></name>, <name><surname>Leidi</surname><given-names>EO</given-names></name>, <name><surname>Andrés</surname><given-names>Z</given-names></name>, <name><surname>Rubio</surname><given-names>L</given-names></name>, <name><surname>Luca</surname><given-names>AD</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in <italic>Arabidopsis</italic></article-title>. <source>Plant Cell</source><volume>24</volume>: <fpage>1127</fpage>–<lpage>1142</lpage><pub-id pub-id-type="pmid">22438021</pub-id></mixed-citation></ref><ref id="pone.0071136-Bassil2"><label>23</label><mixed-citation publication-type="journal"><name><surname>Bassil</surname><given-names>E</given-names></name>, <name><surname>Coku</surname><given-names>A</given-names></name>, <name><surname>Blumwald</surname><given-names>E</given-names></name> (<year>2012</year>) <article-title>Cellular ion homeostasis: emerging roles of intracellular NHX Na<sup>+</sup>/H<sup>+</sup> antiporters in plant growth and development</article-title>. <source>J Exp Bot</source><volume>63</volume>: <fpage>5727</fpage>–<lpage>5740</lpage><pub-id pub-id-type="pmid">22991159</pub-id></mixed-citation></ref><ref id="pone.0071136-Silva1"><label>24</label><mixed-citation publication-type="journal"><name><surname>Silva</surname><given-names>EN</given-names></name>, <name><surname>Ribeiro</surname><given-names>RV</given-names></name>, <name><surname>Ferreira-Silva</surname><given-names>SL</given-names></name>, <name><surname>Viégas</surname><given-names>RA</given-names></name>, <name><surname>Silveira</surname><given-names>JAG</given-names></name> (<year>2010</year>) <article-title>Comparative effects of salinity and water stress on photosynthesis, water relations and growth of <italic>Jatropha curcas</italic> plants</article-title>. <source>J Arid Environ</source><volume>74</volume>: <fpage>1130</fpage>–<lpage>1137</lpage></mixed-citation></ref><ref id="pone.0071136-DazLpez1"><label>25</label><mixed-citation publication-type="journal"><name><surname>Díaz-López</surname><given-names>L</given-names></name>, <name><surname>Gimeno</surname><given-names>V</given-names></name>, <name><surname>Lidón</surname><given-names>V</given-names></name>, <name><surname>Simón</surname><given-names>I</given-names></name>, <name><surname>Martínez</surname><given-names>V</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>The tolerance of <italic>Jatropha curcas</italic> seedlings to NaCl: An ecophysiological analysis</article-title>. <source>Plant Physiol Biochem</source><volume>54</volume>: <fpage>34</fpage>–<lpage>42</lpage><pub-id pub-id-type="pmid">22377428</pub-id></mixed-citation></ref><ref id="pone.0071136-Niu1"><label>26</label><mixed-citation publication-type="journal"><name><surname>Niu</surname><given-names>G</given-names></name>, <name><surname>Rodriguez</surname><given-names>D</given-names></name>, <name><surname>Mendoza</surname><given-names>M</given-names></name>, <name><surname>Jifon</surname><given-names>J</given-names></name>, <name><surname>Ganjegunte</surname><given-names>G</given-names></name> (<year>2012</year>) <article-title>Responses of <italic>Jatropha curcas</italic> to salt and drought stresses</article-title>. <source>Int J Agron</source><volume>2012</volume>: <fpage>1</fpage>–<lpage>7</lpage></mixed-citation></ref><ref id="pone.0071136-Fujimaki1"><label>27</label><mixed-citation publication-type="journal"><name><surname>Fujimaki</surname><given-names>H</given-names></name>, <name><surname>Kikuchi</surname><given-names>N</given-names></name> (<year>2010</year>) <article-title>Drought and salinity tolerances of young <italic>Jatropha</italic></article-title>. <source>Int Agrophys</source><volume>24</volume>: <fpage>121</fpage>–<lpage>127</lpage></mixed-citation></ref><ref id="pone.0071136-Tzfira1"><label>28</label><mixed-citation publication-type="journal"><name><surname>Tzfira</surname><given-names>T</given-names></name>, <name><surname>Citovsky</surname><given-names>V</given-names></name> (<year>2006</year>) <article-title><italic>Agrobacterium</italic>-mediated genetic transformation of plants: biology and biotechnology</article-title>. <source>Curr Opin Biotechnol</source><volume>17</volume>: <fpage>147</fpage>–<lpage>154</lpage><pub-id pub-id-type="pmid">16459071</pub-id></mixed-citation></ref><ref id="pone.0071136-Altpeter1"><label>29</label><mixed-citation publication-type="journal"><name><surname>Altpeter</surname><given-names>F</given-names></name>, <name><surname>Baisakh</surname><given-names>N</given-names></name>, <name><surname>Beachy</surname><given-names>R</given-names></name>, <name><surname>Bock</surname><given-names>R</given-names></name>, <name><surname>Capell</surname><given-names>R</given-names></name>, <etal>et al</etal> (<year>2005</year>) <article-title>Particle bombardment and the genetic enhancement of crops: myths and realities</article-title>. <source>Mol Breed</source><volume>15</volume>: <fpage>305</fpage>–<lpage>327</lpage></mixed-citation></ref><ref id="pone.0071136-Qu1"><label>30</label><mixed-citation publication-type="journal"><name><surname>Qu</surname><given-names>J</given-names></name>, <name><surname>Mao</surname><given-names>HZ</given-names></name>, <name><surname>Chen</surname><given-names>W</given-names></name>, <name><surname>Gao</surname><given-names>SQ</given-names></name>, <name><surname>Bai</surname><given-names>YN</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>Development of marker-free transgenic <italic>Jatropha</italic> plants with increased levels of seed oleic acid</article-title>. <source>Biotechnol Biofuels</source><volume>5</volume>: <fpage>10</fpage><pub-id pub-id-type="pmid">22377043</pub-id></mixed-citation></ref><ref id="pone.0071136-Tsuchimoto1"><label>31</label><mixed-citation publication-type="journal"><name><surname>Tsuchimoto</surname><given-names>S</given-names></name>, <name><surname>Cartagena</surname><given-names>J</given-names></name>, <name><surname>Khemkladngoen</surname><given-names>N</given-names></name>, <name><surname>Singkaravanit</surname><given-names>S</given-names></name>, <name><surname>Kohinata</surname><given-names>T</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>Development of transgenic plants in <italic>Jatropha</italic> with drought tolerance</article-title>. <source>Plant Biotechnol-Nar</source><volume>29</volume>: <fpage>137</fpage>–<lpage>143</lpage></mixed-citation></ref><ref id="pone.0071136-Flowers1"><label>32</label><mixed-citation publication-type="journal"><name><surname>Flowers</surname><given-names>TJ</given-names></name> (<year>2004</year>) <article-title>Improving crop salt tolerance</article-title>. <source>J Exp Bot</source><volume>55</volume>: <fpage>307</fpage>–<lpage>319</lpage><pub-id pub-id-type="pmid">14718494</pub-id></mixed-citation></ref><ref id="pone.0071136-BhatnagarMathur1"><label>33</label><mixed-citation publication-type="journal"><name><surname>Bhatnagar-Mathur</surname><given-names>P</given-names></name>, <name><surname>Vadez</surname><given-names>V</given-names></name>, <name><surname>Sharma</surname><given-names>KK</given-names></name> (<year>2008</year>) <article-title>Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects</article-title>. <source>Plant Cell Rep</source><volume>27</volume>: <fpage>411</fpage>–<lpage>424</lpage><pub-id pub-id-type="pmid">18026957</pub-id></mixed-citation></ref><ref id="pone.0071136-Jha1"><label>34</label><mixed-citation publication-type="journal"><name><surname>Jha</surname><given-names>A</given-names></name>, <name><surname>Joshi</surname><given-names>M</given-names></name>, <name><surname>Yadav</surname><given-names>NS</given-names></name>, <name><surname>Agarwal</surname><given-names>PK</given-names></name>, <name><surname>Jha</surname><given-names>B</given-names></name> (<year>2011</year>) <article-title>Cloning and characterization of the <italic>Salicornia brachiata</italic> Na<sup>+</sup>/H<sup>+</sup> antiporter gene <italic>SbNHX1</italic> and its expression by abiotic stress</article-title>. <source>Mol Biol Rep</source><volume>38</volume>: <fpage>1965</fpage>–<lpage>1973</lpage><pub-id pub-id-type="pmid">20853145</pub-id></mixed-citation></ref><ref id="pone.0071136-Rajagopal1"><label>35</label><mixed-citation publication-type="journal"><name><surname>Rajagopal</surname><given-names>D</given-names></name>, <name><surname>Agarwal</surname><given-names>P</given-names></name>, <name><surname>Tyagi</surname><given-names>W</given-names></name>, <name><surname>Singla-Pareek</surname><given-names>SL</given-names></name>, <name><surname>Reddy</surname><given-names>MK</given-names></name>, <etal>et al</etal> (<year>2007</year>) <article-title><italic>Pennisetum glaucum</italic> Na+/H+ antiporter confers high level of salinity tolerance in transgenic <italic>Brassica juncea</italic></article-title>. <source>Mol Breed</source><volume>19</volume>: <fpage>137</fpage>–<lpage>151</lpage></mixed-citation></ref><ref id="pone.0071136-Jha2"><label>36</label><mixed-citation publication-type="journal"><name><surname>Jha</surname><given-names>B</given-names></name>, <name><surname>Sharma</surname><given-names>A</given-names></name>, <name><surname>Mishra</surname><given-names>A</given-names></name> (<year>2011</year>) <article-title>Expression of <italic>SbGSTU</italic> (Tau class glutathione S-transferase) gene isolated from <italic>Salicornia brachiata</italic> in tobacco for salt tolerance</article-title>. <source>Mol Biol Rep</source><volume>38</volume>: <fpage>4823</fpage>–<lpage>4832</lpage><pub-id pub-id-type="pmid">21136169</pub-id></mixed-citation></ref><ref id="pone.0071136-Casu1"><label>37</label><mixed-citation publication-type="journal"><name><surname>Casu</surname><given-names>RE</given-names></name>, <name><surname>Selivanova</surname><given-names>A</given-names></name>, <name><surname>Perroux</surname><given-names>JM</given-names></name> (<year>2012</year>) <article-title>High-throughput assessment of transgene copy number in sugarcane using real-time quantitative PCR</article-title>. <source>Plant Cell Rep</source><volume>31</volume>: <fpage>167</fpage>–<lpage>177</lpage><pub-id pub-id-type="pmid">21953330</pub-id></mixed-citation></ref><ref id="pone.0071136-Bhaskaran1"><label>38</label><mixed-citation publication-type="journal"><name><surname>Bhaskaran</surname><given-names>S</given-names></name>, <name><surname>Savithramma</surname><given-names>DL</given-names></name> (<year>2011</year>) <article-title>Co-expression of <italic>Pennisetum glaucum</italic> vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter and <italic>Arabidopsis</italic> H<sup>+</sup>-pyrophosphatase enhances salt tolerance in transgenic tomato</article-title>. <source>J Exp Bot</source><volume>62</volume>: <fpage>5561</fpage>–<lpage>5570</lpage><pub-id pub-id-type="pmid">21841179</pub-id></mixed-citation></ref><ref id="pone.0071136-Liu1"><label>39</label><mixed-citation publication-type="journal"><name><surname>Liu</surname><given-names>L</given-names></name>, <name><surname>Zeng</surname><given-names>Y</given-names></name>, <name><surname>Pan</surname><given-names>X</given-names></name>, <name><surname>Zhang</surname><given-names>F</given-names></name> (<year>2012</year>) <article-title>Isolation, molecular characterization, and functional analysis of the vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter genes from the halophyte <italic>Karelinia caspica</italic></article-title>. <source>Mol Biol Rep</source><volume>39</volume>: <fpage>7193</fpage>–<lpage>7202</lpage><pub-id pub-id-type="pmid">22311041</pub-id></mixed-citation></ref><ref id="pone.0071136-Zhang2"><label>40</label><mixed-citation publication-type="journal"><name><surname>Zhang</surname><given-names>HX</given-names></name>, <name><surname>Hodson</surname><given-names>JN</given-names></name>, <name><surname>Williams</surname><given-names>JP</given-names></name>, <name><surname>Blumwald</surname><given-names>E</given-names></name> (<year>2001</year>) <article-title>Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation</article-title>. <source>Proc Natl Acad Sci USA</source><volume>98</volume>: <fpage>12832</fpage>–<lpage>12836</lpage><pub-id pub-id-type="pmid">11606781</pub-id></mixed-citation></ref><ref id="pone.0071136-Xue1"><label>41</label><mixed-citation publication-type="journal"><name><surname>Xue</surname><given-names>ZY</given-names></name>, <name><surname>Zhi</surname><given-names>DY</given-names></name>, <name><surname>Xue</surname><given-names>GP</given-names></name>, <name><surname>Zhang</surname><given-names>H</given-names></name>, <name><surname>Zhao</surname><given-names>YX</given-names></name>, <etal>et al</etal> (<year>2004</year>) <article-title>Enhanced salt tolerance of transgenic wheat (<italic>Triticum aestivum</italic> L.) expressing a vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na<sup>+</sup></article-title>. <source>Plant Sci</source><volume>167</volume>: <fpage>849</fpage>–<lpage>859</lpage></mixed-citation></ref><ref id="pone.0071136-Zhao1"><label>42</label><mixed-citation publication-type="journal"><name><surname>Zhao</surname><given-names>J</given-names></name>, <name><surname>Zhi</surname><given-names>D</given-names></name>, <name><surname>Xue</surname><given-names>Z</given-names></name>, <name><surname>Liu</surname><given-names>H</given-names></name>, <name><surname>Xia</surname><given-names>G</given-names></name> (<year>2007</year>) <article-title>Enhanced salt tolerance of transgenic progeny of tall fescue (<italic>Festuca arundinacea</italic>) expressing a vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter gene from <italic>Arabidopsis</italic></article-title>. <source>J Plant Physiol</source><volume>164</volume>: <fpage>1377</fpage>–<lpage>1383</lpage><pub-id pub-id-type="pmid">17561307</pub-id></mixed-citation></ref><ref id="pone.0071136-Chen1"><label>43</label><mixed-citation publication-type="journal"><name><surname>Chen</surname><given-names>LH</given-names></name>, <name><surname>Zhang</surname><given-names>B</given-names></name>, <name><surname>Xu</surname><given-names>ZQ</given-names></name> (<year>2008</year>) <article-title>Salt tolerance conferred by overexpression of <italic>Arabidopsis</italic> vacuolar Na<sup>+</sup>/H<sup>+</sup> antiporter gene <italic>AtNHX1</italic> in common buckwheat (<italic>Fagopyrum esculentum</italic>)</article-title>. <source>Transgenic Res</source><volume>17</volume>: <fpage>121</fpage>–<lpage>132</lpage><pub-id pub-id-type="pmid">17541720</pub-id></mixed-citation></ref><ref id="pone.0071136-Jefferson1"><label>44</label><mixed-citation publication-type="journal"><name><surname>Jefferson</surname><given-names>RA</given-names></name>, <name><surname>Kavanagh</surname><given-names>TA</given-names></name>, <name><surname>Bevan</surname><given-names>MW</given-names></name> (<year>1987</year>) <article-title>GUS fusions: <italic>β</italic>-glucuronidase as a sensitive and versatile gene fusion marker in higher plants</article-title>. <source>EMBO J</source><volume>6</volume>: <fpage>3901</fpage>–<lpage>3907</lpage><pub-id pub-id-type="pmid">3327686</pub-id></mixed-citation></ref><ref id="pone.0071136-Li1"><label>45</label><mixed-citation publication-type="journal"><name><surname>Li</surname><given-names>J</given-names></name>, <name><surname>Li</surname><given-names>MR</given-names></name>, <name><surname>Wu</surname><given-names>PZ</given-names></name>, <name><surname>Tian</surname><given-names>CE</given-names></name>, <name><surname>Jiang</surname><given-names>HW</given-names></name>, <etal>et al</etal> (<year>2008</year>) <article-title>Molecular cloning and expression analysis of a gene encoding a putative β-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from <italic>Jatropha curcas.</italic></article-title>. <source>Tree Physiol</source><volume>28</volume>: <fpage>921</fpage>–<lpage>927</lpage><pub-id pub-id-type="pmid">18381272</pub-id></mixed-citation></ref><ref id="pone.0071136-Shepherd1"><label>46</label><mixed-citation publication-type="other">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.</mixed-citation></ref><ref id="pone.0071136-Fan1"><label>47</label><mixed-citation publication-type="journal"><name><surname>Fan</surname><given-names>L</given-names></name>, <name><surname>Zheng</surname><given-names>S</given-names></name>, <name><surname>Wang</surname><given-names>X</given-names></name> (<year>1997</year>) <article-title>Antisense suppression of phospholipase Dα retards abscisic acid and ethylene promoted senescence of post harvest <italic>Arabidopsis</italic> leaves</article-title>. <source>Plant Cell</source><volume>9</volume>: <fpage>2183</fpage>–<lpage>2196</lpage><pub-id pub-id-type="pmid">9437863</pub-id></mixed-citation></ref><ref id="pone.0071136-Arnon1"><label>48</label><mixed-citation publication-type="journal"><name><surname>Arnon</surname><given-names>DI</given-names></name> (<year>1949</year>) <article-title>Copper enzymes in isolated chloroplasts polyphenol oxidase in <italic>Beta vulgaris</italic></article-title>. <source>Plant Physiol</source><volume>24</volume>: <fpage>1</fpage>–<lpage>15</lpage><pub-id pub-id-type="pmid">16654194</pub-id></mixed-citation></ref><ref id="pone.0071136-Lutts1"><label>49</label><mixed-citation publication-type="journal"><name><surname>Lutts</surname><given-names>S</given-names></name>, <name><surname>Kinet</surname><given-names>JM</given-names></name>, <name><surname>Bouharmont</surname><given-names>J</given-names></name> (<year>1996</year>) <article-title>NaCl-induced senescence in leaves of rice (<italic>Oryza sativa</italic> L.) cultivars differing in salinity resistance</article-title>. <source>Ann Bot</source><volume>78</volume>: <fpage>389</fpage>–<lpage>398</lpage></mixed-citation></ref><ref id="pone.0071136-Hodges1"><label>50</label><mixed-citation publication-type="journal"><name><surname>Hodges</surname><given-names>DM</given-names></name>, <name><surname>DeLong</surname><given-names>JM</given-names></name>, <name><surname>Forney</surname><given-names>CF</given-names></name>, <name><surname>Prange</surname><given-names>RK</given-names></name> (<year>1999</year>) <article-title>Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds</article-title>. <source>Planta</source><volume>207</volume>: <fpage>604</fpage>–<lpage>611</lpage></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Agronomie/explor/SisAgriV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000173 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000173 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Agronomie
|area= SisAgriV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:3733712
|texte= Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:23940703" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a SisAgriV1
| This area was generated with Dilib version V0.6.28. |  |