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

Abstract

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.

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Abbreviations

BAP:

6-Benzylaminopurine

cPTIO:

2-(4-Carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

CW:

Calcofluor White

DAF-FM DA:

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

FDA:

Fluorescein diacetate

GSNO:

S-nitrosoglutathione

H2DCF DA:

2′,7′-Dichlorofluorescein diacetate

IBA:

Indole-3-butyric acid

L-NAME:

NG-nitro-l-arginine methyl ester

L-NMMA:

NG-monomethyl-l-arginine

MES:

2-(N-Morpholino)ethanesulphonic Acid

MTT:

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

NAA:

1-Naphthaleneacetic acid

NO:

Nitric oxide

NOS:

Nitric oxide synthase

RNS:

Reactive nitrogen species

ROS:

Reactive oxygen species

SNP:

Sodium nitroprusside

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Acknowledgments

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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Correspondence to Marek Petřivalský.

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Petřivalský, M., Vaníčková, P., Ryzí, M. et al. The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts. Plant Cell Tiss Organ Cult 108, 237–249 (2012). https://doi.org/10.1007/s11240-011-0035-3

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Keywords

  • Cucumis sativus
  • Protoplast
  • Regeneration
  • Nitric oxide
  • Reactive oxygen species