Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 108, Issue 2, pp 237–249 | Cite as

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

  • Marek Petřivalský
  • Pavla Vaníčková
  • Markéta Ryzí
  • Božena Navrátilová
  • Jana Piterková
  • Michaela Sedlářová
  • Lenka Luhová
Original Paper


The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.


Cucumis sativus Protoplast Regeneration Nitric oxide Reactive oxygen species 







Calcofluor White


4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate


Fluorescein diacetate




2′,7′-Dichlorofluorescein diacetate


Indole-3-butyric acid


NG-nitro-l-arginine methyl ester




2-(N-Morpholino)ethanesulphonic Acid


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


1-Naphthaleneacetic acid


Nitric oxide


Nitric oxide synthase


Reactive nitrogen species


Reactive oxygen species


Sodium nitroprusside



The cooperation of the Olympus Czech Group (Prague, Czech Republic) is greatly appreciated. This research was supported by the Czech Ministry of Education, Youth and Sports through research grants Kontakt ME 08048 and MSM 6198959215.


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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Marek Petřivalský
    • 1
  • Pavla Vaníčková
    • 1
  • Markéta Ryzí
    • 1
  • Božena Navrátilová
    • 2
  • Jana Piterková
    • 1
  • Michaela Sedlářová
    • 2
  • Lenka Luhová
    • 1
  1. 1.Department of Biochemistry, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  2. 2.Department of Botany, Faculty of SciencePalacký UniversityOlomoucCzech Republic

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