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Improved salt tolerance and delayed leaf senescence in transgenic cotton expressing the Agrobacterium IPT gene


The manipulation of cytokinin contents via Agrobacterium-mediated transformation is an efficient tool for delaying leaf senescence and improving the resistance to environmental stresses. In the present study, cotton transformants harbouring the Agrobacterium tumefaciens isopentenyl transferase (IPT) gene under the control of the promoter of Gossypium hirsutum cysteine proteinase (Ghcysp) were generated. PCR and Southern blot analysis indicated that the foreign DNA fragment was successfully integrated into the cotton genome. The chlorophyll and cytokinin contents, and ROS-scavenging enzymatic activities were significantly increased in transgenic cotton lines, which resulted in a significant delay in leaf senescence. The growth characteristics of transgenic cotton lines resembled the non-transgenic lines except delaying premature senescence and the lint yield and fiber quality of transgenic lines were improved. In addition, the transgenic lines had higher biomasses, IPT transcripts, and endogenous cytokinin contents compared with those of non-transgenic lines under 200 mM NaCl stress.

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cetyltrimethyl-ammonium bromide


days after emergence


days after planting






isopentenyl transferase


leaves of the tenth node on main stem


reactive oxygen species






  • Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K.: Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. — Plant Cell 15: 63–78, 2003.

    PubMed  Article  CAS  Google Scholar 

  • Barry, G.F., Rogers, S.G., Fraley, R.T., Brand, L.: Identification of a cloned cytokinin biosynthetic gene. — Proc. nat. Acad. Sci. USA 81: 4776–4780, 1984.

    PubMed  Article  CAS  Google Scholar 

  • Calderini, O., Bovone, T., Scotti, C., Pupilli, F., Piano, E., Arcioni, S.: Delay of leaf senescence in Medicago sativa transformed with the ipt gene controlled by the senescencespecific promoter SAG12. — Plant Cell Rep. 26: 611–615, 2007.

    PubMed  Article  CAS  Google Scholar 

  • Casanova, E., Trillas, M.I., Moysset, L., Vainstein, A.: Influence of rol genes in floriculture. — Biotechnol. Adv. 23: 3–39, 2005.

    PubMed  Article  CAS  Google Scholar 

  • Chuck, G., Lincoln, C., Hake, S.: KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. — Plant Cell 8: 1277–1289, 1996.

    PubMed  Article  CAS  Google Scholar 

  • Cowan, A.K., Freeman, M., Bjorkman, P.O., Nicander, B., Sitbon, F., Tillberg, E.: Effects of senescence-induced alteration in cytokinin metabolism on source-sink relationships and ontogenic and stress-induced transitions in tobacco. — Planta 221: 801–814, 2005.

    PubMed  Article  CAS  Google Scholar 

  • Dong, H.Z., Niu, Y.H., Li, W.J., Zhang, D.M.: Effects of cotton rootstock on endogenous cytokinins and abscisic acid in xylem sap and leaves in relation to leaf senescence. — J. exp. Bot. 59: 1295–1304, 2008.

    PubMed  Article  CAS  Google Scholar 

  • Fricke, W., Akhiyarova, G., Wei, W., Alexandersson, E., Miller, A., Kjellbom, P.O., Richardson, A., Wojciechowski, T., Schreiber, L., Veselov, D., Kudoyarova, G., Volkov, V.: The short-term growth response to salt of the developing barley leaf. — J. exp. Bot. 57: 1079–1095, 2006.

    PubMed  Article  CAS  Google Scholar 

  • Gan, S., Amasino, R.M.: Inhibition of leaf senescence by autoregulated production of cytokinin. — Science 270: 1986–1988, 1995.

    PubMed  Article  CAS  Google Scholar 

  • Gan, S., Amasino, R.M.: Making sense of senescence (molecular genetic regulation and manipulation of leaf senescence). — Plant Physiol. 113: 313–319, 1997.

    PubMed  CAS  Google Scholar 

  • Ghanem, M.E., Albacete, A., Martinez-Andujar, C., Acosta, M., Romero-Aranda, R., Dodd, I.C., Lutts, S., Perez-Alfocea, F.: Hormonal changes during salinity-induced leaf senescence in tomato (Solanum lycopersicum L.). — J. exp. Bot. 59: 3039–3050, 2008.

    PubMed  Article  CAS  Google Scholar 

  • Havlova, M., Dobrev, P.I., Motyka, V., Storchova, H., Libus, J., Dobra, J., Malbeck, J., Gaudinova, A., Vankova, R.: The role of cytokinins in responses to water deficit in tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under 35S or SAG12 promoters. — Plant Cell Environ. 31: 341–353, 2008.

    PubMed  Article  CAS  Google Scholar 

  • Hofgen, R., Willmitzer, L.: Storage of competent cells for Agrobacterium transformation. — Nucl. Acids Res. 16: 9877–--, 1988.

    PubMed  Article  CAS  Google Scholar 

  • Jordi, W., Schapendonk, A., Davelaar, E., Stoopen, G.M., Pot, C.S., De Visser, R., Van Rhijn, J.A., Gan, S., Amasino, R.M.: Increased cytokinin levels in transgenic PSAG12-ipt tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning. — Plant Cell Environ. 23: 279–289, 2000.

    Article  CAS  Google Scholar 

  • Khan, R.S., Nakamura, I., Mii, M.: Production and selection of marker-free transgenic plants of Petunia hybrida using sitespecific recombination. — Biol. Plant. 54: 265–271, 2010.

    Article  CAS  Google Scholar 

  • Lin, Y.J., Cao, M.L., Xu, C.G., Chen, H., Wei, J., Zhang, Q.F.: Cultivating rice with delaying leaf-senescence by PSAG12-IPT gene transformation. — Acta bot. sin. 44: 1333–1338, 2002.

    CAS  Google Scholar 

  • Liu, J., Wu, Y.H., Yang, J.J., Liu, Y.D., Shen, F.F.: Protein degradation and nitrogen remobilization during leaf senescence. — J. Plant Biol. 51: 11–19, 2008.

    Article  CAS  Google Scholar 

  • Livak, K.J., Schmittgen, T.D.: Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCt method. — Methods 25: 402–408, 2001.

    PubMed  Article  CAS  Google Scholar 

  • Munné-Bosch, S., Alegre, L.: Die and let live: leaf senescence contributes to plant survival under drought stress. — Funct. Plant Biol. 31: 203–216, 2004.

    Article  Google Scholar 

  • Munns, R.: Salinity, growth and phytohormones. — In: Läuchli, A, Lüttge, U. (ed.): Salinity: Environment — Plants — Molecules. Pp. 271–290. Kluwer Academic Publishers, Dordrecht 2002.

    Google Scholar 

  • Nakashima, K., Fujita, Y., Katsura, K., Maruyama, K., Narusaka, Y., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K.: Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis. — Plant mol. Biol. 60: 51–68, 2006.

    PubMed  Article  CAS  Google Scholar 

  • Narusaka, Y., Nakashima, K., Shinwari, Z.K., Sakuma, Y., Furihata, T., Abe, H., Narusaka, M., Shinozaki, K., Yamaguchi-Shinozaki, K.: Interaction between two cisacting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. — Plant J. 34: 137–148, 2003.

    PubMed  Article  CAS  Google Scholar 

  • Nguyen, T.T., Dix, P.J., Nugent, G.D.: Transformation of potato via Agrobacterium coated microparticle bombardment. — Biol. Plant. 54: 141–144, 2010.

    Article  Google Scholar 

  • Noodén, L.D., Guiamét, J.J., John, I.: Senescence mechanisms. — Physiol. Plant. 101: 746–753, 1997.

    Article  Google Scholar 

  • Pic, E., De la Serve, B.T., Tardieu, F., Turc, O.: Leaf senescence induced by mild water deficit follows the same sequence of macroscopic, biochemical, and molecular events as monocarpic senescence in pea. — Plant Physiol. 128: 236–246, 2002.

    PubMed  Article  CAS  Google Scholar 

  • Pogány, M., Koehl, J., Heiser, I., Elstner, E.F., Barna, B.: Juvenility of tobacco induced by cytokinin gene introduction decreases susceptibility to Tobacco necrosis virus and confers tolerance to oxidative stress. — Physiol. mol. Plant Pathol. 65: 39–47, 2004.

    Article  Google Scholar 

  • Prochazkova, D., Sairam, R.K., Srivastava, G.C., Singh, D.V.: Oxidative stress and antioxidant activity as the basis of senescence in maize leaves. — Plant Sci. 161: 765–771, 2001.

    Article  CAS  Google Scholar 

  • Robson, P.R., Donnison, I.S., Wang, K., Frame, B., Pegg, S.E., Thomas, A., Thomas, H.: Leaf senescence is delayed in maize expressing the Agrobacterium IPT gene under the control of a novel maize senescence-enhanced promoter. — J. Plant Biotechnol. 2: 101–112, 2004.

    Article  CAS  Google Scholar 

  • Shen, F.F., Yu, S.X., Han, X.L., Fan, S.L.: Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum. — Chin. Sci. Bull. 49: 2601–2607, 2004.

    Article  CAS  Google Scholar 

  • Sunilkumar, G., Rathore, K.S.: Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. — Mol. Breed. 8: 37–52, 2001.

    Article  CAS  Google Scholar 

  • Sykorova, B., Kuresova, G., Daskalova, S., Trckova, M., Hoyerova, K., Raimanova, I., Motyka, V., Travnickova, A., Elliott, M.C., Kaminek, M.: Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield. — J. exp. Bot. 59: 377–387, 2008.

    PubMed  Article  CAS  Google Scholar 

  • Synková, H., Wilhelmová, N., Šesták, Z., Pospíšilová, J.: Photosynthesis in transgenic plants with elevated cytokinin content. — In: Pessarakli, M. (ed.): Handbook of Photosynthesis. Pp. 541–552. Marcel Dekker, New York 1997.

    Google Scholar 

  • Tapia, G., Verdugo, I., Yanez, M., Ahumada, I., Theoduloz, C., Cordero, C., Poblete, F., Gonzalez, E., Ruiz-Lara, S.: Involvement of ethylene in stress-induced expression of the TLC1.1 retrotransposon from Lycopersicon chilense Dun. — Plant Physiol. 138: 2075–2086, 2005.

    PubMed  Article  CAS  Google Scholar 

  • Thomas, J.C., McElwain, E.F., Bohnert, H.J.: Convergent induction of osmotic stress-responses: abscisic acid, cytokinin, and the effects of NaCl. — Plant Physiol. 100: 416–423, 1992.

    PubMed  Article  CAS  Google Scholar 

  • Van Staden, J., Cook, E.L., Noodén, L.D.: Cytokinins and senescence. — In: Noodén, L.D., Leopold, A. (ed.): Senescence and Aging in Plants. Pp. 281–328. Academic Press, London 1988.

    Google Scholar 

  • Xu, Y.J., Wang, D.Y., Zhu, Y.X.: Expression of thylakoid membrane localized PPF1 in transgenic Arabidopsis affects chloroplast development. — Acta bot. sin. 44: 1314–1320, 2009.

    Google Scholar 

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This research was supported by the China Key Development Project for Basic Research (Grant 2007CB116208), and the China Major Projects for Transgenic Breeding (Grant Nos. 2008ZX005-004, 2008ZX08005-002, 2009ZX08005-020). Y.D. Liu and Z.J. Yin contributed equally to this work.

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Correspondence to F. F. Shen.

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Liu, Y.D., Yin, Z.J., Yu, J.W. et al. Improved salt tolerance and delayed leaf senescence in transgenic cotton expressing the Agrobacterium IPT gene. Biol Plant 56, 237–246 (2012).

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Additional key words

  • Gossypium hirsutum
  • isopentenyl transferase
  • cytokinins
  • NaCl
  • senescence-specific promoter