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

Abstract

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.

Keywords

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

Abbreviations

CKX

Cytokinin oxidase/dehydrogenase

nptII

Neomycin phosphotransferase

hptII

Hygromycin phosphotransferase

LB

Luria–Bertani broth medium

MS

Murashige and Skoog medium

RT

Reverse transcription

Notes

Acknowledgments

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).

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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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