Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 107, Issue 3, pp 541–552 | Cite as

Overexpression of TaNHX2 enhances salt tolerance of ‘composite’ and whole transgenic soybean plants

Original Paper


Salinity is a major factor resulting in extensive loss of agricultural production. Genetic transformation has become a powerful tool for studying gene function and for improving crop salt tolerance. In this study, a TaNHX2 gene was transformed into a plant cloning vector under the control of cauliflower mosaic virus 35S promoter, and then introduced into Agrobacterium rhizogenes strain K599. Explants of soybean were transformed with A. rhizogenes and ‘composite’ plants consisting of wild-type shoots and transgenic hairy roots overexpressing TaNHX2 were produced. When exposed to salt stress, ‘composite’ plants displayed high salinity tolerance at 171 mM NaCl in vermiculite and in solid medium supplemented with up to 200 mM NaCl, whereas control plants displayed chlorosis and died within 15 days under above treatment conditions. We subsequently obtained soybean plants overexpressing TaNHX2 through A. tumefaciens-mediated transformation and studied four homozygous lines of TaNHX2. Transgenic lines displayed an enhanced salt tolerance in plant biomass and flower number per plant, compared with wild type plants grown on sand culture containing 150 mM NaCl. Furthermore, transgenic plants of line C12-11 showed longer survival, less growth inhibition and greater number of flowers than wild type plants. Taken together, these results indicated that TaNHX2 gene could enhance salt tolerance of soybean, and A. rhizogenes-mediated transformation system could be used as a complementary tool of A. tumerfaciens-mediated transformation to rapidly investigate candidate gene function in soybean.


TaNHX2 Soybean Salt tolerance Transformation Agrobacterium rhizogenes Agrobacterium tumefaciens 



Cauliflower mosaic virus


Co-cultivation medium




Murashige and Skoog basal nutrient salts with B5 vitamins


Reverse transcription-PCR


Salt damage index (%)


  1. Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285:1256–1258PubMedCrossRefGoogle Scholar
  2. Bhuiyan MSU, Min SR, Jeong WJ, Sultana S, Choi KS, Song WY, Lee Y, Lim YP, Liu JR (2011) Overexpression of a yeast cadmium factor 1 (YCF1) enhances heavy metal tolerance and accumulation in Brassica juncea. Plant Cell Tiss Organ Cult 105:85–91CrossRefGoogle Scholar
  3. Blumwald E, Aharon GS, Apse MP (2000) Sodium transport in plant cells. Biochim Biophys Acta 1465:140–151PubMedCrossRefGoogle Scholar
  4. Brini F, Hanin M, Mezghani I, Berkowitz GA, Masmoudi K (2007) Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants. J Exp Bot 58:301–308PubMedCrossRefGoogle Scholar
  5. Cao D, Hou W, Song S, Sun H, Wu C, Gao Y, Han T (2009) Assessment of conditions affecting Agrobacterium rhizogenes-mediated transformation of soybean. Plant Cell Tiss Organ Cult 96:45–52CrossRefGoogle Scholar
  6. Chen H, An R, Tang J, Cui X, Hao F, Chen J, Wang X (2007) Over-expression of a vacuolar Na+/H+ antiporter gene improves salt tolerance in an upland rice. Mol Breed 19:215–225CrossRefGoogle Scholar
  7. Cho H, Farrand S, Noel G, Widholm J (2000) High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta 210:195–204PubMedCrossRefGoogle Scholar
  8. Collier R, Fuchs B, Walter N, Kevin Lutke W, Taylor CG (2005) Ex vitro composite plants: an inexpensive, rapid method for root biology. Plant J 43:449–457PubMedCrossRefGoogle Scholar
  9. Flores T, Karpova O, Su X, Zeng P, Bilyeu K, Sleper DA, Nguyen HT, Zhang ZJ (2008) Silencing of GmFAD3 gene by siRNA leads to low alpha-linolenic acids (18:3) of fad3-mutant phenotype in soybean [Glycine max (Merr.)]. Transgenic Res 17:839–850PubMedCrossRefGoogle Scholar
  10. Gao F, Xiong A, Peng R, Jin X, Xu J, Zhu B, Chen J, Yao Q (2010) OsNAC52, a rice NAC transcription factor, potentially responds to ABA and confers drought tolerance in transgenic plants. Plant Cell Tiss Organ Cult 100:255–262CrossRefGoogle Scholar
  11. Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proc Nat Acad Sci USA 96:1480–1485PubMedCrossRefGoogle Scholar
  12. Godfray HC, Beddington JR, Crute IR, Haddad L, Lawrence D, Muir JF, Pretty J, Robinson S, Thomas SM, Toulmin C (2010) Food security: the challenge of feeding 9 billion people. Science 327:812–818PubMedCrossRefGoogle Scholar
  13. He C, Yan J, Shen G, Fu L, Holaday AS, Auld D, Blumwald E, Zhang H (2005) Expression of an Arabidopsis vacuolar sodium/proton antiporter gene in cotton improves photosynthetic performance under salt conditions and increases fiber yield in the field. Plant Cell Physiol 46:1848–1854PubMedCrossRefGoogle Scholar
  14. He C, Yang A, Zhang W, Gao Q, Zhang J (2010) Improved salt tolerance of transgenic wheat by introducing betA gene for glycine betaine synthesis. Plant Cell Tiss Organ Cult 101:65–78CrossRefGoogle Scholar
  15. James C (2010) Global status of commercialized biotech/GM Crops: 2010. ISAAA Brief No. 42. ISAAA, IthacaGoogle Scholar
  16. Jian B, Hou W, Wu C, Liu B, Liu W, Song S, Bi Y, Han T (2009) Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics. BMC Plant Biol 25:69–78Google Scholar
  17. Jin T, Chang Q, Li W, Yin D, Li Z, Wang D, Liu B, Liu L (2010) Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa. Plant Cell Tiss Organ Cult 100:219–227CrossRefGoogle Scholar
  18. Kereszt A, Li D, Indrasumunar A, Nguyen CD, Nontachaiyapoom S, Kinkema M, Gresshoff PM (2007) Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat Protoc 2:948–952PubMedCrossRefGoogle Scholar
  19. Kronzucker HJ, Britto DT (2011) Sodium transport in plants: a critical review. New Phytol 189:54–81PubMedCrossRefGoogle Scholar
  20. Lam HM, Xu X, Liu X, Chen W, Yang G, Wong FL, Li MW, He W, Qin N, Wang B, Li J, Jian M, Wang J, Shao G, Sun SS, Zhang G (2010) Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection. Nat Genet 42:1053–1059PubMedCrossRefGoogle Scholar
  21. Leidi EO, Barragan V, Rubio L, El-Hamdaoui A, Ruiz MT, Cubero B, Fernandez JA, Bressan RA, Hasegawa PM, Quintero FJ, Pardo JM (2010) The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato. Plant J 61:495–506PubMedCrossRefGoogle Scholar
  22. Li W, Wong F, Tsai S, Phang T, Shao G, Lam H (2006) Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells. Plant Cell Environ 29:1122–1137PubMedCrossRefGoogle Scholar
  23. Li J, Todd T, Trick H (2010a) Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants. Plant Cell Rep 29:113–123PubMedCrossRefGoogle Scholar
  24. Li T, Zhang Y, Liu H, Wu Y, Li W, Zhang H (2010b) Stable expression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1, and salt tolerance in transgenic soybean for over six generations. Chin Sci Bull 55:1127–1134CrossRefGoogle Scholar
  25. Li Y, Zhang Y, Feng F, Liang D, Cheng L, Ma F, Shi S (2010c) Overexpression of a Malus vacuolar Na+/H+ antiporter gene (MdNHX1) in apple rootstock M.26 and its influence on salt tolerance. Plant Cell Tiss Organ Cult 102:337–345CrossRefGoogle Scholar
  26. Li M, Lin X, Li H, Pan X, Wu G (2011) Overexpression of AtNHX5 improves tolerance to both salt and water stress in rice (Oryza sativa L). Plant Cell Tiss Organ Cult. doi:10.1007/s11240-011-9979-6 Google Scholar
  27. Mittler R, Blumwald E (2010) Genetic engineering for modern agriculture: challenges and perspectives. Ann Rev Plant Biol 61:443–462CrossRefGoogle Scholar
  28. Muhammad AL, Ye GN, Weeden NF, Reisch BI (1994) A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis. Plant Mol Biol Rep 12:6–13CrossRefGoogle Scholar
  29. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Ann Rev Plant Biol 59:651–681CrossRefGoogle Scholar
  30. Ohta M, Hayashi Y, Nakashima A, Hamada A, Tanaka A, Nakamura T, Hayakawa T (2002) Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice. FEBS Lett 532:279–282PubMedCrossRefGoogle Scholar
  31. Olhoft PM, Flagel LE, Donovan CM, Somers DA (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735PubMedGoogle Scholar
  32. Paz MM, Shou H, Guo Z, Zhang Z, Banerjee A, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136:167–179CrossRefGoogle Scholar
  33. Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K (2006) Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep 25:206–213PubMedCrossRefGoogle Scholar
  34. Phang TH, Shao G, Lam HM (2008) Salt tolerance in soybean. J Integr Plant Biol 50:1196–1212PubMedCrossRefGoogle Scholar
  35. Rech EL, Vianna GR, Aragao FJ (2008) High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat Protoc 3:410–418PubMedCrossRefGoogle Scholar
  36. Rengasamy P (2006) World salinization with emphasis on Australia. J Exp Bot 57:1017–1023PubMedCrossRefGoogle Scholar
  37. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang XC, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183PubMedCrossRefGoogle Scholar
  38. Shao GH, Song JZ, Liu HL (1986) Preliminary studies on the evaluation of salt tolerance in soybean varieties. Acta Agron Sin 6:30–35Google Scholar
  39. Subramanyam K, Sailaja KV, Subramanyam K, Rao DM, Lakshmidevi K (2011) Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annum L.). Plant Cell Tiss Organ Cult 105:181–192CrossRefGoogle Scholar
  40. Tester M, Langridge P (2010) Breeding technologies to increase crop production in a changing world. Science 327:818–822PubMedCrossRefGoogle Scholar
  41. Vickers CE, Schenk PM, Li D, Mullineaux PM, Gresshoff PM (2007) pGFPGUSPlus+, a new binary vector for gene expression studies and optimising transformation systems in plants. Biotechnol Lett 29:1793–1796PubMedCrossRefGoogle Scholar
  42. Wei Q, Guo Y, Cao H, Kuai B (2011) Cloning and characterization of an AtNHX2-like Na+/H+ antiporter gene from Ammopiptanthus mongolicus (Leguminosae) and its ectopic expression enhanced drought and salt tolerance in Arabidopsis thaliana. Plant Cell Tiss Organ Culture 105:309–316CrossRefGoogle Scholar
  43. Xue Z, Zhi D, Xue G, Zhang H, Zhao Y, Xia G (2004) Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L.) expressing a vacuolar Na+/H+ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na+. Plant Sci 167:849–859CrossRefGoogle Scholar
  44. Xue RG, Zhang B, Xie HF (2007) Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress. Plant Cell Tiss Organ Cult 89:177–183CrossRefGoogle Scholar
  45. Yoshida K, Miki N, Momonoi K, Kawachi M, Katou K, Okazaki Y, Uozumi N, Maeshima M, Kondo T (2009) Synchrony between flower opening and petal-color change from red to blue in morning glory, Ipomoea tricolor cv. Heavenly Blue. Proc Jpn Acad Series B Phy Biol Sci 85:187–197CrossRefGoogle Scholar
  46. Yu JN, Huang J, Wang ZN, Zhang JS, Chen SY (2007) An Na+/H+ antiporter gene from wheat plays an important role in stress tolerance. J Biosci 32:1153–1161PubMedCrossRefGoogle Scholar
  47. Zeng P, Vadnais DA, Zhang Z, Polacco JC (2004) Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill]. Plant Cell Rep 22:478–482PubMedCrossRefGoogle Scholar
  48. Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19:765–768PubMedCrossRefGoogle Scholar
  49. Zhang Z, Xing A, Staswick P, Clemente T (1999) The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tiss Organ Cult 56:37–46CrossRefGoogle Scholar
  50. Zhang HX, Hodson JN, Williams JP, Blumwald E (2001) Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proc Natl Acad Sci USA 98:12832–12836PubMedCrossRefGoogle Scholar
  51. Zhou G, Chang R, Qiu L (2010) Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis. Plant Mol Biol 72:357–367PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and AgroecologyChinese Academy of SciencesHarbinPeople’s Republic of China
  2. 2.The National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop ScienceThe Chinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  3. 3.The Graduate University of Chinese Academy of SciencesBeijingPeople’s Republic of China

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