Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 115, Issue 2, pp 139–150 | Cite as

Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn.

  • Milana Trifunović
  • Aleksandar Cingel
  • Ana Simonović
  • Slađana Jevremović
  • Marija Petrić
  • Ivana Č. Dragićević
  • Václav Motyka
  • Petre I. Dobrev
  • Lenka Zahajská
  • Angelina Subotić
Original Paper


Cytokinin oxidase/dehydrogenase (CKX) is the only known enzyme involved in cytokinin catabolism. Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were introduced separately into a binary cloning vector, immobilized into Agrobacterium tumefaciens strain GV3101, and introduced into root explants of centaury (Centaurium erythraea Rafn.). The integration of each transgene was confirmed by genomic PCR. Of the total transformed explants, 30 and 28.2 % of the transformants carried AtCKX1 and AtCKX2 transgenes, respectively. Of these transformants, 50 % exhibited expression of the AtCKX1 transgene, while 64 % of transformants exhibited expression of the AtCKX2 transgene. For all analysed AtCKX transgenic centaury lines, as well as for untransformed control plants, CKX activity was higher in roots than in shoots. Expression of AtCKX in most transgenic lines contributed to enhanced levels of CKX activity in root tissues; whereas, only a few lines demonstrated increased CKX activity in shoot tissues compared to those of control plants. Moreover, overexpression of AtCKX resulted in reduced morphogenetic potential in transgenic plants, but did not significantly affect biomass production in comparison to untransformed control plants.


Centaurium erythraea Rafn. Agrobacterium tumefaciens Genetic transformation qRT-PCR AtCKX genes CKX activity 



Cytokinin oxidase/dehydrogenase


Neomycin phosphotransferase


Hygromycin phosphotransferase


Luria–Bertani broth medium


Murashige and Skoog medium


Reverse transcription



The bacterial strains A. tumefaciens GV3101/pBinHTX-AtCKX1 and GV3101/pBinHTX-AtCKX were kindly provided by dr Thomas Schmülling and dr Tomáš Werner (Freie Universität Berlin, Germany). The authors thank Marie Korecká for her invaluable technical support. This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant No. ON173015) and the Czech Science Foundation (P506/11/0774).


  1. Abou-Alaiwi WA, Potlakayala SD, Goldman SL, Josekutty PC, Karelia DN, Rudrabhatla SV (2012) Agrobacterium-mediated transformation of the medicinal plant Centaurea montana. Plant Cell Tiss Org Cult 109:1–8CrossRefGoogle Scholar
  2. Angenon G, Dillen W, Montagu MV (1994) Antibiotic resistance markers for plant transformation. In: Schilperoort RA, Gelvin SB (eds) Plant molecular biology manual. Springer, Netherlands, pp 125–137CrossRefGoogle Scholar
  3. Bertani G (1951) Studies of lysogenesis I: the mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300PubMedGoogle Scholar
  4. Bhau BS, Wakhlu AK (2001) Effect of some antibiotics on the in vitro morphogenetic response from callus cultures of Coryphantha elephantides. Biol Plant 44:19–24CrossRefGoogle Scholar
  5. Bilyeu KD, Cole JL, Laskey JG, Riekhof WR, Esparza TJ, Kramer MD, Morris RO (2001) Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol 125:378–386CrossRefPubMedGoogle Scholar
  6. Boszoradova E, Libantova J, Matusikova I, Poloniova Z, Jopcik M, Berenyi M, Moravcikova J (2011) Agrobacterium-mediated genetic transformation of economically important oilseed rape cultivars. Plant Cell Tiss Org Cult 107:317–323CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Chevallier A (2000) Encyclopedia of herbal medicine. Dorling Kindersley, LondonGoogle Scholar
  9. Crane C, Wright E, Dixon RA, Wang ZY (2006) Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens-transformed roots and Agrobacterium rhizogenes-transformed hairy roots. Planta 223:1344–1354CrossRefPubMedGoogle Scholar
  10. Dean C, Jones J, Favreau M, Dunsmuir P, Bedbrook J (1988) Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Res 16:9267–9283CrossRefPubMedGoogle Scholar
  11. Deroles SC, Ledger SE, Miller RM, Davies KM, Given NK (1993) Tranformation of Eustoma grandiflorum (Lisianthus). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, series: plant protoplasts and genetic engineering III, Vol 22, Springer, Berlin, pp 202–212Google Scholar
  12. Dutta I, Kottackal M, Tumimbang E, Tajima H, Zaid A, Blumwald E (2013) Sonication-assisted efficient Agrobacterium-mediated genetic transformation of the multipurpose woody desert shrub Leptadenia pyrotechnica. Plant Cell Tiss Organ Cult 112:289–301CrossRefGoogle Scholar
  13. Fang Y, Spector DL (2010) Live cell imaging of plants. Cold Spring Harb Protoc 2010:1–4Google Scholar
  14. Feng DS, Wang HG, Zhang XS, Kong LR, Tian JC, Li XF (2008) Using an inverse PCR method to clone the wheat cytokinin oxidase/dehydrogenase gene TaCKX1. Plant Mol Biol Rep 26:143–155CrossRefGoogle Scholar
  15. Gajdošová S, Spíchal L, Kamínek M, Hoyerová K, Novák O, Dobrev PI, Galuszka P, Klíma P, Gaudinová A, Žižková E, Hanuš J, Dančák M, Trávníček B, Pešek B, Krupička M, Vaňková R, Strnad M, Motyka V (2011) Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants. J Exp Bot 62:2827–2840CrossRefPubMedGoogle Scholar
  16. Galuszka P, Frébort I, Werner T, Yamada M, Strnad M, Schmülling T, Frébort I (2004) Cytokinin oxidase/dehydrogenase genes in barley and wheat cloning and heterologous expression. Eur J Biochem 271:3990–4002CrossRefPubMedGoogle Scholar
  17. Galuszka P, Popelková H, Werner T, Frébortvá J, Pospíšilová H, Mik V, Köllmer I, Schmülling T, Frébort I (2007) Biochemical characterization of cytokinin oxidases/dehydrogenases from Arabidopsis thaliana expressed in Nicotiana tabacum L. J Plant Growth Regul 26:255–267CrossRefGoogle Scholar
  18. Gašić K, Hernandez A, Korban SS (2004) RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction Plant Mol Biol Rep 22:437a–437gGoogle Scholar
  19. Gaudinová A, Dobrev PI, Šolcová B, Novák O, Strnad M, Friedecký D, Motyka V (2005) The involvement of cytokinin oxidase/dehydrogenase and zeatin reductase in regulation of cytokinin levels in pea (Pisum sativum L.) leaves. J Plant Growth Regul 24:188–200CrossRefGoogle Scholar
  20. Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51:223–256CrossRefPubMedGoogle Scholar
  21. Hartmann A, Senning M, Hedden P, Sonnewald U, Sonnewald S (2011) Reactivation of meristem activity and sprout growth in potato tuber require both cytokinin and gibberellin. Plant Physiol 155:776–796CrossRefPubMedGoogle Scholar
  22. Houba-Hérin N, Pethe C, d’ Alayer J, Laloue M (1999) Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts. Plant J 17:615–626CrossRefPubMedGoogle Scholar
  23. Janković T, Krstić D, Šavikin-Fodulović K, Menković N, Grubišić D (1997) Comparative investigation of secoiridoids compounds of Centaurium erythreae growth in nature and cultured in vitro. Pharm and Pharmacol Lett 7:30–32Google Scholar
  24. Janković T, Krstić D, Šavikin-Fodulović K, Menković N, Grubišić D (2000) Xanthone compounds of Centaurium erythraea grown in nature and cultured in vitro. Pharm and Pharmacol Lett 10:23–25Google Scholar
  25. Jones RJ, Schreiber BMN (1997) Role and function of cytokinin oxidase in plants. J Plant Growth Regul 23:123–134CrossRefGoogle Scholar
  26. Jorgensen RA, Cluster PD, English J, Que Q, Napoli CA (1996) Chalcone synthase co-suppression in Petunia flowers: comparison of sense versus antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol Biol 31:957–973CrossRefPubMedGoogle Scholar
  27. Kim JB (2005) Development of efficient regeneration and transformation systems in Alstroemeria Dissertation. Wageningen University, NetherlandsGoogle Scholar
  28. Kumarasamy Y, Nahar L, Cox PJ, Jaspars M, Sarker SD (2003) Bioactivity of secoiridoid glucosides from Centaurium erythraea. Phytomedicine 10:344–347CrossRefPubMedGoogle Scholar
  29. Laloue M, Fox JE (1989) Cytokinin oxidase from wheat: partial purufication and general properties. Plant Physiol 90:899–906CrossRefPubMedGoogle Scholar
  30. Massonneau A, Houba-Hérin N, Pethe C, Madzak C, Falque M, Mercy M, Kopecny D, Majira A, Rogowsky P, Laloue M (2004) Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs. J Exp Bot 55:2549–2557CrossRefPubMedGoogle Scholar
  31. McGaw BA, Horgan R (1983) Cytokinin catabolism and cytokinin oxidase. Phytochemistry 22:1103–1105CrossRefGoogle Scholar
  32. Milojević J, Lj Tubić, Nolić N, Mitić N, Ćalić-Dragosavac D, Vinterhalter B, Zdravković-Korać S (2012) Hygromycin promotes somatic embryogenesis in spinach. Plant Cell Tiss Organ Cult 109:573–579CrossRefGoogle Scholar
  33. Mok DWS, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118CrossRefPubMedGoogle Scholar
  34. Morris RO, Bilyeu KD, Laskey JG, Cheich N (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255:328–333CrossRefPubMedGoogle Scholar
  35. Motyka V, Faiss M, Strnad M, Kamínek M, Schmülling T (1996) Changes in cytokinin content and cytokinin oxidase activity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol 112:1035–1043PubMedGoogle Scholar
  36. Motyka V, Vaňková R, Čapková V, Petrášek J, Kamínek M, Schmülling T (2003) Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. Physiol Plantarum 117:11–21CrossRefGoogle Scholar
  37. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479CrossRefGoogle Scholar
  38. Piatczak E, Wielanek M, Wysokinska H (2005) Liquid culture system for shoot multiplication and secoiridoid production in micropropagated plants Centaurium erythraea Rafn. Plant Sci 168:431–437CrossRefGoogle Scholar
  39. Piatczak E, Krolocka A, Wysokinska H (2006) Genetic transformation of Centaurium erythraea Rafn. by Agrobacterium rhizogenes and production of secoiridoids. Plant Cell Rep 25:1308–1315CrossRefPubMedGoogle Scholar
  40. Raspor M, Motyka M, Žižková E, Dobrev P, Trávníčková A, Zdravković-Korać S, Simonović A, Ninković S, Dragićević I (2012) Cytokinin profiles of AtCKX2-overexpressing potato plants and the impact of altered cytokinin homeostasis on tuberization in vitro. J Plant Growth Regul 31:460–470CrossRefGoogle Scholar
  41. Schmülling T, Werner T, Riefler M, Krupková E, Bartrina y Manns I (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116:241–252CrossRefPubMedGoogle Scholar
  42. Shilpa KS, Kumar VD, Sujatha M (2010) Agrobacterium-mediated genetic transformation of safflower (Carthamus tinctorius L.). Plant Cell Tiss Organ Cult 103:387–401CrossRefGoogle Scholar
  43. Simonović A, Trifunović M, Raspor M, Cingel A, Bogdanović M, Dragićević M, Subotić A (2012) Dimethyl sulfoxide improves sensitivity and specificity of RT-PCR and qRT-PCR amplification of low-expressed transgenes. Arch Biol Sci 64:865–876CrossRefGoogle Scholar
  44. Šmehilová M, Galuszka P, Bilyeu KD, Jaworek P, Kowalska M, Šebela M, Sedlářová M, English JT, Frébort I (2009) Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehydrogenase enzymes from maize. J Exp Bot 60:2701–2712CrossRefPubMedGoogle Scholar
  45. Subotić A, Budimir S, Grubišić D, Momčilović I (2003/2004) Direct regeneration of shoots from hairy root cultures of Centaurium erythraea inoculated with Agrobacterium rhizogenes. Biol Plant 47:617–619Google Scholar
  46. Subotić A, Janković T, Jevremović S, Grubišić D (2006) Plant tissue culture and secondary metabolites productions of Centaurium erythraea Rafn., a Medical plant. In: Teixeira da Silva JA (ed) Floriculture, ornamental and plant biotechnology: advances and topical issues Vol. II, 1st edn., Global Science Books, London, pp 564–570Google Scholar
  47. Subotić A, Jevremović S, Trifunović M, Petrić M, Milošević S, Grubišić D (2009) The influence of gibberellic acid and paclobutrazol on induction of somatic embryogenesis in wild type and hairy root cultures of Centaurium erythraea—Gillib. Afr J Biotechnol 8:3223–3228Google Scholar
  48. Sun SB, Meng LS (2010) Genetic transformation of Gentiana dahurica Fish. by Agrobacterium tumefaciens using zygotic embryo-derived callus. Acta Physiol Plant 32:629–634CrossRefGoogle Scholar
  49. Valvekens D, Montagu MV, Lijsebettens MV (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540CrossRefPubMedGoogle Scholar
  50. Van der Krol AR, Lenting PE, Veenstra J, Van der Meer IM, Koes RE, Gerats AGM, Mol JNM, Stuitje AR (1988) An antisense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333:866–869CrossRefGoogle Scholar
  51. Van der Sluis WG (1985a) Secoiridoids and xanthones in the genus Centaurium Hill (Gentianaceae)—a pharmacognostical study. Drukkerij Elinkwijk bv, UtrechtGoogle Scholar
  52. Van der Sluis WG (1985b) Chemotaxonomical investigations of the Genera Blackstonia and Centaurium (Gentianaceae). Plant Syst Evol 149:253–286CrossRefGoogle Scholar
  53. Van Leeuwen W, Ruttink T, Borst-Vrenssen AWM, van der Plas LHW, van der Krol AR (2001) Characterization of position-induced spatial and temporal regulation of transgene promoter activity in plants. J Exp Bot 52:949–959CrossRefPubMedGoogle Scholar
  54. Vaseva-Gemisheva I, Lee D, Karanov E (2005) Response of Pisum sativum cytokinin oxidase/dehydrogenase expression and specific activity to drought stress and herbicide treatments. Plant Growth Regul 46:199–208CrossRefGoogle Scholar
  55. Vinterhalter B, Momčilović I, Vinterhalter D (2000) High biomass producing root cultures of Gentiana punctata L. Tranformed with Agrobacterium tumefaciens C58C1 (pArA4b). Arch Biol Sci 52:83–87Google Scholar
  56. Von Schwartzenberg K, Nunez MF, Blaschke H, Dobrev PI, Novák O, Motyka V, Strnad M (2007) Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins. Plant Physiol 145:786–800CrossRefGoogle Scholar
  57. Wang Y, Luo JP, Wei ZJ, Zhang JC (2009) Molecular cloning and expression analysis of a cytokinin oxidase (DhCKX) gene in Dendrobium huoshanense. Mol Biol Rep 36:1331–1338CrossRefPubMedGoogle Scholar
  58. Wang CH, Hsu SY, Chen PY, To KY (2012) Transformation and characterization of transgenic Bidens pilosa L. Plant Cell Tiss Organ Cult 109:457–464CrossRefGoogle Scholar
  59. Weigel D, Glazebrook J (2002) Arabidopsis: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  60. Wen L, Tan B, Guo WW (2012) Estimating transgene copy number in precocious trifoliate orange by TaqMan real-time PCR. Plant Cell Tiss Organ Cult 109:363–371CrossRefGoogle Scholar
  61. Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA 98:10487–10492CrossRefPubMedGoogle Scholar
  62. Werner T, Motyka V, Laucou V, Stems R, Van Onckelen H, Schmülling T (2003) Cytokinin deficient transgenic Arabidopsis plant show multiple developmental alterations indicating opposite function of cytokinins in the regulation of shoot and root meristem sctivity. Plant Cell 15:2532–2550CrossRefPubMedGoogle Scholar
  63. Werner T, Köllmer I, Bartrina I, Holst K, Schmülling T (2006) New insights into the biology of cytokinin degradation. Plant Biol 8:371–381CrossRefPubMedGoogle Scholar
  64. Yang SH, Yu H, Goh CJ (2002) Isolation and characterization of the orchid cytokinin oxidase DSCKX1 promoter. J Exp Bot 53:1899–1907CrossRefPubMedGoogle Scholar
  65. Yang SH, Yu H, Goh CJ (2003a) Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Mol Biol 51:237–248CrossRefPubMedGoogle Scholar
  66. Yang SH, Yu H, Xu Y, Goh YC (2003b) Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1. FEBS Lett 555:291–296CrossRefPubMedGoogle Scholar
  67. Zhou X, Cao G, Lin R, Sun Y, Li W (1994) A rapid and efficient DNA extraction method of genus Fagopyrum for RAPD analysis. In: Javornik B, Bohanec B, Kreft I (eds) Proceedings of impact of plant biotechnology on agriculture. Biotechnical Faculty, Ljubljana, pp 171–175Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Milana Trifunović
    • 1
  • Aleksandar Cingel
    • 1
  • Ana Simonović
    • 1
  • Slađana Jevremović
    • 1
  • Marija Petrić
    • 1
  • Ivana Č. Dragićević
    • 2
  • Václav Motyka
    • 3
  • Petre I. Dobrev
    • 3
  • Lenka Zahajská
    • 4
  • Angelina Subotić
    • 1
  1. 1.Institute for Biological Research “Siniša Stanković”University of BelgradeBelgradeSerbia
  2. 2.Faculty of BiologyUniversity of BelgradeBelgradeSerbia
  3. 3.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPrague 6Czech Republic
  4. 4.Isotope Laboratory, Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPrague 4Czech Republic

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