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Genotoxic effects due to in vitro culture and H2O2 treatments in Petunia × hybrida cells monitored through DNA diffusion assay, FPG-SCGE and gene expression profile analyses

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Abstract

In the present work, Petunia × hybrida leaf discs maintained on regeneration medium for 8 days were used to assess the effects of genotoxic stress induced by in vitro culture. The investigation was carried out by comparing the response of intact leaves excised from Petunia × hybrida plantlets grown in vitro and the regenerating leaf discs. In situ detection by histochemical staining and alkaline-Single Cell Gel Electrophoresis (SCGE) analysis demonstrated that both reactive oxygen species accumulation and DNA damage were enhanced in explants cultured in vitro. Significant up-regulation of the PhOGG1 (8-oxoguanine DNA glycosylase/lyase), PhAPX (ascorbate peroxidase) and PhMT2 (metallothionein) genes involved in DNA repair and antioxidant defence was observed in the explants cultured in vitro, compared to intact leaves. The Petunia × hybrida leaf discs were exposed to increasing (0, 100, 150, 200 and 400 mM) doses of the model genotoxic agent hydrogen peroxide (H2O2) and then analysed. The DNA diffusion assay highlighted the dose- and time-dependent fluctuations of programmed cell death/necrosis events in response to H2O2. Leaf discs treated with increasing H2O2 concentration and untreated controls were analysed by FPG-SCGE to assess the level of oxidative DNA damage at different time points following treatments. The PhOGG1, PhAPX and PhMT2 genes were significantly up-regulated in response to H2O2, reaching the highest transcript levels with the 150 mM dose. Based on the reported data, these genes might be used as molecular indicators of the genotoxic stress response in Petunia × hybrida cells.

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Abbreviations

APX:

Ascorbate peroxidase

BA:

Benzyladenine

BER:

Base excision repair

DAB:

3,3′-Diaminobenzidine

DAPI:

4′,6-Diamino-2-phenylindole

DSB:

Double strand break

FPG:

Formamidopyrimidine-DNA glycosylase

MT:

Metallothionein

8-oxo-Dg:

7, 8-Dihydro-8-oxoguanine

Ph:

Petunia × hybrida

NBT:

Nitroblue tetrazolium

PCD:

Programmed cell death

OGG1:

8-Oxoguanine DNA glycosylase/lyase

QRT-PCR:

Quantitative real-time polymerase chain reaction

ROS:

Reactive oxygen species

SCGE:

Single cell gel electrophoresis

SSB:

Single strand break

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Acknowledgments

Authors would like to thank Dr. Raffaele Langhella and Albani Vincenzo e Ruggeri Italina s.s.a., via Fontanatetta 158, 00053 Civitavecchia (RO) for supplying the P. ×  hybrida genotype. This research was supported by the ‘MUTAFLOR’ project from the Italian Ministry of Agriculture-MiP.A.F.

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Authors and Affiliations

  1. Dipartimento di Chimica, Università di Pavia, Via Taramelli 12, 27100, Pavia, Italy

    L. Ventura & A. Buttafava

  2. Dipartimento di Biologia e Biotecnologie ‘Lazzaro Spallanzani’, Laboratori di Genetica e Microbiologia ‘A. Buzzati-Traverso’, Università di Pavia, Via Ferrata1, 27100, Pavia, Italy

    A. Macovei, M. Donà, S. Paparella, D. Carbonera & A. Balestrazzi

  3. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per la Floricoltura e le Specie Ornamentali (CRA-FSO), Corso Inglesi 508, 18038, Sanremo, IM, Italy

    A. Giovannini

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  1. L. Ventura
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Correspondence to A. Balestrazzi.

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Communicated by Y. Wang.

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Ventura, L., Macovei, A., Donà, M. et al. Genotoxic effects due to in vitro culture and H2O2 treatments in Petunia × hybrida cells monitored through DNA diffusion assay, FPG-SCGE and gene expression profile analyses. Acta Physiol Plant 36, 331–341 (2014). https://doi.org/10.1007/s11738-013-1415-6

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  • DOI: https://doi.org/10.1007/s11738-013-1415-6

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