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ORIGINAL PAPER
Genotoxic effects due to in vitro culture and H
2
O
2
treatments
in Petunia 3hybrida cells monitored through DNA diffusion
assay, FPG-SCGE and gene expression profile analyses
L. Ventura •A. Macovei •M. Dona
`•
S. Paparella •A. Buttafava •A. Giovannini •
D. Carbonera •A. Balestrazzi
Received: 27 May 2013 / Revised: 20 August 2013 / Accepted: 9 October 2013 / Published online: 20 October 2013
ÓFranciszek Go
´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako
´w 2013
Abstract In the present work, Petunia 9hybrida 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 9hybrida 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 9hybrida leaf discs were
exposed to increasing (0, 100, 150, 200 and 400 mM)
doses of the model genotoxic agent hydrogen peroxide
(H
2
O
2
) and then analysed. The DNA diffusion assay
highlighted the dose- and time-dependent fluctuations of
programmed cell death/necrosis events in response to
H
2
O
2
. Leaf discs treated with increasing H
2
O
2
concentra-
tion 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 H
2
O
2
, 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 9hybrida cells.
Keywords DNA repair FPG-SCGE Hydrogen
peroxide In vitro culture Mutation breeding
Petunia 9hybrida
Abbreviations
APX Ascorbate peroxidase
BA Benzyladenine
BER Base excision repair
DAB 3,30-Diaminobenzidine
DAPI 40,6-Diamino-2-phenylindole
DSB Double strand break
FPG Formamidopyrimidine-DNA glycosylase
MT Metallothionein
8-oxo-Dg 7, 8-Dihydro-8-oxoguanine
Ph Petunia 9hybrida
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
Communicated by Y. Wang.
L. Ventura A. Buttafava
Dipartimento di Chimica, Universita
`di Pavia, Via Taramelli 12,
27100 Pavia, Italy
A. Macovei M. Dona
`S. Paparella D. Carbonera
A. Balestrazzi (&)
Dipartimento di Biologia e Biotecnologie ‘Lazzaro Spallanzani’,
Laboratori di Genetica e Microbiologia ‘A. Buzzati-Traverso’,
Universita
`di Pavia, Via Ferrata1, 27100 Pavia, Italy
e-mail: alma.balestrazzi@unipv.it
A. Giovannini
Consiglio per la Ricerca e la Sperimentazione in Agricoltura,
Unita
`di Ricerca per la Floricoltura e le Specie Ornamentali
(CRA-FSO), Corso Inglesi 508, 18038 Sanremo, IM, Italy
123
Acta Physiol Plant (2014) 36:331–341
DOI 10.1007/s11738-013-1415-6
Introduction
Plant cells subjected to in vitro culture are exposed to stress
factors, such as wounding at the excision site of tissue
explants and phytohormones, and their response relies on
adaptation mechanisms that include genetic reprogram-
ming as well as modifications of physiological processes
(Pasternak et al. 2000). An interesting aspect of in vitro
culture is the accumulation of ex novo mutations, generally
referred to as ‘somaclonal variation’ (Larkin and Scowcroft
1981; Sato et al. 2011), that are exploited by ornamental
breeders in combination with chemical/physical agents to
increase genetic variability (Jain 2010).
Tissue damage and other suboptimal in vitro factors
inevitably lead to reactive oxygen species (ROS) accu-
mulation, and thus enhanced oxidative DNA damage
(Cassels and Curry 2001). The latter is typically associated
with the accumulation of 7,8-dihydro-8-oxoguanine (8-
oxo-dG), an oxidized form of guanine which is removed by
the OGG1 enzyme, a bifunctional DNA glycosylase/lyase
(Girard et al. 1997; Zielinska et al. 2011). OGG1 is a
component of the Base Excision Repair (BER) pathway
involved in the removal of oxidative base damage, alkyl-
ation, deamination, abasic (apurinic and/or apyrimidinic)
sites and single-strand breaks (Roldan-Arjona and Ariza
2009; Balestrazzi et al. 2011a). Plants own not only OGG1,
but also the FPG (FormamidoPyrimidine-DNA-Glycosy-
lase) enzyme involved in the removal of 8-oxo-dG and
FaPy lesions (Murphy and George 2005; Macovei et al.
2011a).
Although the multiple DNA repair pathways show high
fidelity, a fraction of damaged DNA can escape repair. This
has been demonstrated by Sanderson and Mosbaugh (1998)
who found an increased error frequency occurring during
the DNA synthesis step in BER. Another source of genetic
variability comes from the process of Translesion Synthesis
(TLS) in which specialised DNA polymerases assist the
replicative enzymes, ensuring the replication of lesion-
containing DNA templates. TLS is an error-prone mecha-
nism that generates mutations at relatively high frequency
(Pages and Fuchs 2002).
It has been hypothesised that mutations useful for
breeding purposes might arise from errors in the activity of
DNA repair enzymes, although this aspect of the plant
response to genotoxic injury is still poorly explored. For
this reason, a deeper knowledge on the molecular responses
induced by oxidative stress treatments under in vitro con-
ditions is expected to positively influence the output of
Mutation Breeding (Shu 2009).
Besides DNA repair, induction of antioxidant mecha-
nisms represents an essential step for plant defence
against the genotoxic effects of environmental stress (Gill
and Tuteja 2010; Balestrazzi et al. 2013). This response
includes metallothioneins (MTs), low molecular weight
cysteine-rich proteins with high binding affinity for bio-
logically relevant metal ions, such as Zn and Cu, and
cytotoxic metals. MTs, described as multifunctional pro-
teins, play a relevant role as ROS scavengers, providing
protection to the cellular components against oxidative
injury (Hassinen et al. 2010; Freisinger 2011). It has been
hypothesised that MTs might release metals during oxi-
dative stress, triggering a Zn-mediated antioxidant
response and that Zn would be released when ROS are
bound to the Cysteine residues of the MT protein, leading
to signalling cascades (Hassinen et al. 2010). The relevant
role played by MTs as ROS scavengers has been high-
lighted in plants, particularly in the nuclear compartment
(Balestrazzi et al. 2009) and it has been suggested that
MTs are part of the signalling pathway activated by nitric
oxide in plants (Balestrazzi et al. 2011b,c). Ascorbate
peroxidase (APX) is one of the key players of the enzy-
matic components of the cell antioxidant response.
Among the isozymes of ascorbate APX, the cytosolic
isoform is highly responsive to several environmental
changes while the corresponding genes are up-regulated
by H
2
O
2
accumulation (Ishikawa and Shigeoka 2008).
Up-regulation of the MtAPX gene encoding cytosolic
APX was observed in aerial parts of M. truncatula plants
grown in vitro and challenged with heavy metals and
osmotic stress, respectively (Macovei et al. 2010; Ba-
lestrazzi et al. 2010).
The activity of antioxidant and DNA repair enzymes
and/or the expression profiles of the related genes might be
used as indicators of the cell ability to withstand the oxi-
dative injury (Balestrazzi et al. 2010; Macovei et al. 2010,
2011a,b; Ventura et al. 2012), a genotype-dependent fea-
ture quite relevant in the context of Mutation Breeding.
This information might be exploited to improve mutation
breeding protocols that are currently used with ornamental
plants, such as P. 9hybrida (Shu 2009). The genus
Petunia includes not only elite varieties of commercial
value, cultivated in flower beds and pots (Angenent et al.
2005; Gerats and Vandenbussche 2005), but also model
genotypes that can be easily investigated for basic and
applied research purposes (McKim and Hay 2010). Muta-
tion-based techniques provide tools for increasing vari-
ability in ornamentals, as requested by consumers.
Aim of the present work was to assess the genotoxic
effects induced by in vitro culture on regenerating leaf
discs of an elite P. 9hybrida genotype, in terms of DNA
damage/repair and antioxidant response. This commer-
cially relevant P. 9hybrida genotype has been already
used for Mutation Breeding (Giovannini et al. 2012). DNA
damage/repair responses were also investigated using leaf
discs challenged with the genotoxic model agent hydrogen
peroxide (H
2
O
2
).
332 Acta Physiol Plant (2014) 36:331–341
123
Materials and methods
Plant material and H
2
O
2
treatments
The P. 9hybrida genotype Ariel
Ò
was kindly supplied by
Albani and Ruggieri Italina s.s.a. (Civitavecchia, Italy).
Leaves were excised from 20-day-old plantlets grown
in vitro on MS0 medium (Murashige and Skoog 1962) and
cut into discs of 1 cm diameter. Leaf discs were transferred
to MS medium supplemented with benzyladenine and
naphthaleneacetic acid (regeneration medium; BA,
1mgl
-1
; NAA, 1 mg l
-1
; Duchefa Biochemicals, Haar-
lem, The Netherlands) and incubated in a climate chamber
at 22–24 °C with a 16-h light/8-h dark cycle photoperiod
and a photosynthetic photon flux of 65–70 lmol m
-2
s
-1
,
under a cool white fluorescence lamp. Leaf discs were
cultured with the abaxial side in contact with the medium
for 8 days. For oxidative stress treatments, P. 9hybrida
leaf discs maintained in vitro for 8 days as previously
described were incubated for 1 h in Petri dishes containing
increasing concentrations of hydrogen peroxide (0, 100,
150, 200 and 400 mM H
2
O
2
). For the evaluation of ROS
and DNA damage, treated and untreated leaf discs were
transferred to Petri dishes and collected at different time
points following reatments (0, 2, 4, 6 and 24 h). Tissues for
molecular analyses were collected and stored in liquid N
2
.
DNA diffusion test
The DNA diffusion assay (Singh 2000) was performed to
evaluate cell death (programmed cell death or PCD versus
necrosis). The analysis was carried out on untreated (CTRL)
and H
2
O
2
-treated leaf discs, at different time points fol-
lowing exposure to genotoxic agent. Nuclei were extracted
as described by Dona
`et al. (2013). Agarose precoated slides
were prepared by spreading 1 ml of 1 % agarose on each
slide and drying them at room temperature. Aliquots
(300 ll) of nuclei suspension were mixed with 200 llof
1 % Low Melting Point agarose (Sigma-Aldrich, Milan,
Italy) in phosphate-buffered saline (PBS) at 37 °C and
gently transferred onto slides. The gel was covered with a
cover glass, slides were cooled on ice for 1 min. Cover
glasses were removed and slides were immersed in lysing
solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris HCl
pH 7.5) for 20 min at room temperature. After lysis, slides
were washed twice in neutral solution TBE (89 mM Tris
Base, 89 mM Boric Acid, 2 mM EDTA, pH 8.3) for 5 min,
rinsed in 70 % ethanol (v/v) for 10 min at room tempera-
ture. Slides were air-dried and stored at room temperature,
stained with 20 llDAPI(4
0,6-diamidino-2-phenylindole,
Sigma-Aldrich, 1 lgml
-1
) (Menke et al. 2000) and cov-
ered with a cover slip for 10 min. One hundred nuclei were
analysed per slide. Cells undergoing PCD or necrosis were
distinguished from viable cells as indicated by Singh
(2003). In case of PCD, cell nuclei own an homogeneous
outline without any clear boundary due to nucleosomal-
sized DNA diffusing into the agarose. Necrotic cell nuclei
are bigger and poorly defined with a non-homogeneous halo
appearance. For each treatment, three replicated samples
were analysed in two independent experiments.
Single cell gel electrophoresis (SCGE)
Nuclei were extracted from P. 9hybrida leaf discs as
described by Dona
`et al. (2013). The suspension containing
the purified nuclei and a solution of 1 % low melting point
agarose (Sigma-Aldrich) in phosphate-buffered saline
(PBS) at 37 °C was mixed in equal volume. Two drops of
the resulting suspension were then pipetted onto agarose
precoated slides and solidified on ice. Slides were incubated
20 min at room temperature in high salt lysis buffer (2.5 M
NaCl, 100 mM Tris–HCl pH 7.5, 100 mM EDTA) to dis-
rupt the nuclear membrane. For alkaline SCGE, nuclei were
denatured in alkaline buffer (1 mM Na
2
EDTA, 300 mM
NaOH, pH [13) 20 min at 4 °C and then electrophoresed
in the same buffer for 30 min at 0.72 V cm
-1
in a cold
chamber. The FPG-SCGE assay was carried out by adding
the Escherichia coli enzyme Formamidopyrimidine-DNA
glycosylase (FPG, 8 U ml
-1
, New England BioLabs, Pero,
Italy) in reaction buffer (40 mM Hepes, 0.1 M KCl,
0.5 mM EDTA, 0.2 mg ml
-1
BSA) and by incubating
slides at 37 °C for 30 min. Control slides, obtained by
adding the reaction buffer without enzyme, were incubated
under the same conditions. After electrophoresis, slides
were washed in 0.4 M Tris–HCl pH 7.5 two times for
5 min, rinsed in 70 % ethanol (v/v) three times for 10 min
at 4 °C and dried overnight at room temperature. Subse-
quently, slides were stained with 20 ll DAPI (1 lgml
-1
,
Sigma-Aldrich) (Menke et al. 2000). For each slide, one
hundred nucleoids were scored using a fluorescence
microscope with an excitation filter of 340–380 nm and a
barrier filter of 400 nm. Nucleoids were classified and
results were expressed in arbitrary units (a.u.) according to
Collins (2004). For each treatment, three replicated samples
were analysed in two independent experiments.
Quantitative real-time polymerase chain reaction
(QRT-PCR)
RNA extraction was carried out using the Aurum Total
RNA Fatty and Fibrous Tissue kit (Bio-Rad, Milan, Italy)
and cDNA synthesis was then performed with the iScript
cDNA Synthesis kit (Bio-Rad). QRT-PCR was carried out
using the SsoFast
TM
EvaGreenÒSupermix (Bio-Rad) and a
Rotor-Gene 6000 PCR apparatus (Corbett Robotics, Bris-
bane, Australia). To identify the most suitable reference
Acta Physiol Plant (2014) 36:331–341 333
123
genes for transcript normalisation, the geNorm software
(Kakar et al. 2008), which uses pair-wise comparison and
geometric averaging across biological samples to determine
gene expression stability, was chosen. Genes encoding actin
(DFCI Accession TC8485), b-tubulin (GenBank accession
AY396149) and ubiquitin (DFCI Accession TC8816) were
tested by QRT-PCR and the resulting expression profiles
were subjected to geNorm analysis and the b-tubulin gene,
found to be the most stable gene (data not shown), was used
for transcript normalisation (Brunner et al. 2004). To ana-
lyse the expression profiles of the PhOGG1 (GenBank
Accession EST881115 CV 292738), PhMT2 (TC4393
DFCI database) and PhAPX (TC2674 DFCI database)
genes, the oligonucleotide primers were designed using the
Real-Time PCR Primer Design program from GenScript
(Table 1). QRT-PCR conditions were as follows: denatur-
ation at 95 °C for 30 s, cycling at 95 °C (5 s), 60 °C (10 s),
72 °C (10 s). For each primer set, a no-template water was
used as negative control. The QRT-PCR results were
interpreted using the LinRegPCR computer software (Ra-
makers et al. 2003). For each set of PCR reactions, the
logarithm of the initial fluorescence (No) was calculated
based on the individual PCR efficiency.
In situ ROS detection and cell death analysis
In situ detection of hydrogen peroxide (H
2
O
2
) and super-
oxide radical (O
2-
) was carried out with 3,30-diam-
inobenzidine (DAB) and nitroblue tetrazolium (NBT)
(Sigma-Aldrich S.r.l.), as described by Thordal-Christensen
et al. (1997) and Fryer et al. (2002), respectively. The ana-
lysis was carried out in intact leaves excised from 20-day-old
plantlets grown in vitro and on leaf discs maintained in vitro
for 8 days, as previously described. Cell death was evaluated
using 0.025 % Evans blue (Sigma-Aldrich) (Yamamoto
et al. 2001). For each treatment, three leaf discs were ana-
lysed. Quantitation of dark areas was performed using a
Biostep GmbH apparatus with the Argus X1 3.3.0 software.
Statistical analysis
All experiments were performed in triplicates. Data were
subjected to Analysis of Variance (ANOVA) and the
statistical significance of mean differences was determined
by Tukey’s test, using SigmaStat program.
Results
In vitro culture enhances DNA damage
and the expression of antioxidant and DNA repair genes
in P. 9hybrida leaf discs
The elite genotype ArielÒwas previously characterised
for the high regeneration frequency and used for molec-
ular breeding applications (Giovannini et al. 2012). The
ROS enhancement in P. 9hybrida leaf discs cultured
in vitro for 8 days on regenerating medium was evaluated
by in situ detection of H
2
O
2
and O
2-
, using DAB and
NBT staining, respectively, in comparison with intact
leaves excised from plantlets grown in vitro. As shown in
Fig. 1a, DAB and NBT staining was absent from intact
leaves, indicating the presence of physiological ROS
levels, while Evans blue evidenced a reduced cell death
frequency. When leaf discs were cut and immediately
incubated with DAB and NBT, staining was observed only
at the wounded sites (data not shown). As for regenerating
leaf discs, the histochemical staining revealed ROS
accumulation in the external part of the explants, where
cell proliferation occurred (Fig. 1a, arrows). To better
characterise the genotoxic effects of in vitro culture,
alkaline SCGE was used to assess the total DNA damage
(SSBs ?DSBs) in both intact leaves and regenerating leaf
discs. As shown in Fig. 1b, the estimated level of DNA
damage was 21.0 ±3.0 a.u. in intact leaves while a sig-
nificant (P\0.001) increase in DNA damage (up to
83.0 ±7.0 a.u., 4.0-fold) was observed in regenerating
leaf discs.
QRT-PCR analysis revealed that the expression of the
PhOGG1 gene encoding 8-oxoguanine DNA glycosylase/
lyase, involved in removal of the oxidized base 8-oxo-dG,
was significantly (P\0.001) up-regulated (up to 2.0-fold)
in regenerating leaf discs, compared to intact leaves. A
similar response was observed for the PhMT2 and PhAPX
genes, encoding a type 2 metallothionein and the cytosolic
isoform of ascorbate peroxidase, respectively (Fig. 1c).
Table 1 Sequences of oligonucleotide primers utilised in QRT-PCR
Gene Forward primer Reverse primer Efficiency
a
PhOGG1 50-ACAATGAGCCATTCCCTGAT-3050-ATGGCCAGTGATCTTTGCT-301.65
PhMT2 50-GATGCGGGATGTACCTTGAC-3050-CAGCCATGTCCTCCTTCTGT-301.75
PhAPX 50-ACTATTGGAGCCCATCAAGG-3050-AGGTGGCTCTGTCTTGTCCT-301.78
PhTUB 50-ACATTCAACGTTCCGGCTAT-3050-ACACCATCACCAGAGTCCAA-301.85
a
Efficiency of the primer pair in QRT-PCR
334 Acta Physiol Plant (2014) 36:331–341
123
Since both metallothionein and ascorbate peroxidase play
active roles in ROS scavenging, it is evident that the P. 9
hybrida regenerating leaf discs cultivated in vitro for
8 days were exposed to oxidative stress conditions with the
consequent activation of antioxidant functions and DNA
repair mechanisms.
To assess the DNA repair ability of P. 9hybrida
regenerating leaf discs, treatments with hydrogen peroxide
(H
2
O
2
) as an oxidative stress agent were carried out. The
use of increasing H
2
O
2
doses allowed to estimate the range
of oxidative DNA damage in which cells of the elite
genotype can actively diplay their response to genotoxic
stress.
Dose- and time-dependent fluctuations of PCD/necrosis
events in response to H
2
O
2
The DNA diffusion test was used to assess the occurrence
of cell death in P. 9hybrida leaf discs exposed to oxi-
dative stress for 1 h. Increasing H
2
O
2
doses (0, 100, 150,
200 and 400 mM) were tested. The analysis was carried out
on untreated (CTRL) and H
2
O
2
-treated leaf discs, at dif-
ferent time points (0, 2, 4, 6 and 24 h) following exposure
to genotoxic agent.
The percentage of viable nuclei and the percentage of
nuclei with cell death morphologies typical of programmed
cell death (PCD) and necrosis, respectively, were esti-
mated. Viable nuclei were small and compact (Fig. 2a). In
the case of PCD, nuclei showed an undefined outline
without any clear boundary, due to nucleosomal sized
DNA diffusing into the agarose (Fig. 2b) while necrotic
nuclei appeared bigger and non-homogeneous (Fig. 2c).
Results of the DNA diffusion test are reported in Fig. 2d.
Statistical significance of differences was determined using
Tuckey’s test (P\0.001). The percentage of viable nuclei
in the untreated control (CTRL) was within the range
94–96 % throughout the tested time points. In the absence
of oxidative stress, the PCD events were barely detectable
while necrosis accounted for 4–5 % of the total cells. In the
explants exposed to 100 mM H
2
O
2
, the viable nuclei
accounted for 88.0 ±2.0 and 87 ±1.0 % of the total
population analysed, respectively, at 6 and 24 h following
treatments. In the same samples, PCD morphology was
detected in a small fraction (4.5 ±0.5 %) only at 6 h while
necrosis occurred at significantly higher frequency
(7.5 ±2.5 %, 6 h; 13.5 ±1.5 %, 24 h).
As expected, increased H
2
O
2
concentrations severely
affected cell viability. The 150 mM dose resulted in a
significant reduction of viable nuclei (78.5 ±4.5 % at 6 h
and 57.5 ±3.5 % at 24 h) (Fig. 2d). PCD morphology was
detected in 14.5 ±2.5 % of the total cells, 6 h following
treatments but the value dropped dramatically
(2.5 ±0.5 %) at 24 h. By contrast, the estimated per-
centage of necrotic nuclei at 6 h was 7.0 ±2.0 % and
subsequently there was a significant enhancement
(40.0 ±3.0 %) at 24 h (Fig. 2d). In explants treated with
200 mM H
2
O
2
, cell viability was further reduced
(62.5 ±2.5 % at 6 h and 46.5 ±3.5 % at 24 h). Under
this conditions, PCD frequency increased up to
28.5 ±1.0 % at 6 h and subsequently dropped to
9.0 ±0.0 % at 24 h. Necrotic nuclei accounted for
9.5 ±1.5 % of the total cells at 6 h with a significant
increase (44.5 ±3.5 %) at 24 h. The cytotoxic effects of
the highest H
2
O
2
dose (400 mM) resulted in a percentage
of viable nuclei that dropped from 75.5 ±3.5%(6h)to
9.0 ±2.0 % (24 h). PCD morphology was 2.0 ±1.0 % at
6 h and then increased up to 40.5 ±1.0 % at 24 h.
Necrosis was the predominant cell death event, with an
estimated percentage of 22.5 ±2.5 % (6 h) and
86.0 ±0.0 % (24 h). For all the above described treat-
ments, the estimated PCD:necrosis ratio was calculated. At
6 h the PCD:necrosis ratio was found to increase with the
increasing H
2
O
2
doses (100, 150, 200 mM), reaching the
highest value in response to the 200 mM dose.
Evans blue staining, used to assess the occurrence of
H
2
O
2
-induced cell death in P. 9hybrida leaf discs,
revealed a dose-dependent pattern of cell death (data not
Fig. 1 a ROS detection and evaluation of cell death were carried out
using intact leaves excised from 20-day-old P. 9hybrida plantlets
grown in vitro and leaf discs (1 cm diameter) cultured in vitro for
8 days, respectively. H
2
O
2
and O
2-
accumulation was evidenced by
histochemical staining with DAB and NBT, while Evans Blue
staining was used to reveal cell death. bThe level of total DNA
damage (SSBs ?DSBs) in intact leaves and regenerating leaf discs
was estimated by alkaline SCGE and expressed as arbitrary units (a.
u.). cThe expression profiles of PhOGG1,PhMT2 and PhAPX and
genes were evaluated by QRT-PCR in intact leaves and regenerating
leaf discs
Acta Physiol Plant (2014) 36:331–341 335
123
shown). As for regeneration ability, only the P. 9hybrida
leaf discs exposed to 100 mM H
2
O
2
were able to regen-
erate shoots, most of them with abnormal morphology. No
shoot regeneration was observed with the 150, 200 and
400 mM H
2
O
2
doses. The untreated control resulted in the
expected regeneration frequency and normal shoot pro-
duction (data not shown).
Dose-dependent accumulation/repair of oxidative DNA
damage in H
2
O
2
-treated P. 9hybrida cells
Leaf discs treated with increasing H
2
O
2
concentration for
1 h and untreated controls were analysed by FPG-SCGE
to assess the level of oxidative DNA damage at different
time points following treatments. Assays were carried
out with the enzyme Formamidopyrimidine-DNA glyco-
sylase (FPG) that acts on 8-oxo-dG and ring-opened
purine derivatives (formamidopyrimidine), cleaving the
damaged sites. Incubation of P. 9hybrida nucleoids
with the reaction buffer without enzyme (B-E) high-
lighted the total DNA strand breaks (SSBs ?DSBs)
while incubation with the FPG enzyme (B ?E) revealed
the FPG-sensitive sites (Table 2). For each sampling
time (0, 2, 4, 6 and 24 h), the number of a.u. detected in
the FPG-treated sample (B ?E), compared to control
(B-E), measured the extent of oxidative DNA damage
[(B ?E)–(B-E)] (Table 2). Statistical significance of
differences was determined using Tuckey’s test
(P\0.001).
As shown in Table 2, slight but significant fluctuations
in the level of oxidative DNA damage were observed in the
untreated leaf discs (CTRL) during the tested time points.
Soon after exposure to the genotoxic compound (T
0
), there
was a significant increase in the amount of oxidative DNA
damage that positively correlated with H
2
O
2
concentration.
At T
0
, the estimated values observed in samples treated
with 100, 150, 200 and 400 mM H
2
O
2
were 77.0, 110.5,
161.0 and 311.0 a.u. (Table 2, oxidative damage). Thus,
1 h of incubation with the genotoxic agent was sufficient to
induce DNA damage. However, at the subsequent time
points (2, 4, 6 and 24 h,) the level of oxidative DNA
damage was significantly reduced possibly as a result of
DNA repair. With 100 mM H
2
O
2
, the amount of oxidative
DNA damage decreased progressively (from 34.0 a.u. at
2 h to 8.5 a.u. at 24 h) as well as with 150 mM H
2
O
2
(from
71.5 a.u. at 2 h to 35.5 a.u. at 24 h). Similarly, oxidative
DNA lesions were reduced in samples exposed to 200 mM
H
2
O
2
at 2 h (116.0 a.u.), 4 h (86.5 a.u.), 6 h (66.5 a.u.) and
24 h (35.5 a.u.) (Table 2, oxidative damage). Finally, no
significant changes in the level of oxidative DNA damage
occurred in response to 400 mM H
2
O
2
throughout the
0–24 h period.
Based on the results obtained with FPG-SCGE, the
percentage of oxidized bases was calculated for each H
2
O
2
5 µm
D
A B C
viable PCD necrosis
CTRL 100 150 200 400 CTRL 100 150 200 400 CTRL 100 150 200 400
H2O2(mM)
0 h 6 h 24 h
H2O2(mM) H2O2(mM)
Nuclear morphology (%)
0
50
100
Fig. 2 Evaluation of PCD/
necrosis events by DNA
diffusion assay. The analysis
was carried out on untreated
(CTRL) and H
2
O
2
-treated leaf
discs, at different time points
following exposure to genotoxic
agent. Morphologies of nuclei
extracted from viable (a), PCD
(b) and cNecrotic cells are
shown. dResults from DNA
diffusion assay carried out at 0,
6 and 24 h following treatments.
Two hundred cells were scored
for each sample. Values are
expressed as mean ±SD of
three replicates from two
independent experiments.
Statistical significance of
differences was determined
using Student’s ttest (P\0.05)
336 Acta Physiol Plant (2014) 36:331–341
123
dose and tested time point and then reported in graphics
(Fig. 3). This allowed to represent the dynamics of DNA
repair occurring in P. 9hybrida cells treated with H
2
O
2
.
As shown in Fig. 3a, the oxidized bases accounted for
49.36 % of the total DNA damage observed soon after
treatment with 100 mM H
2
O
2
while a descrease occurred
at 2 h (30.09 %), 4 h (14.29 %), and 6 h (16.49 %). At the
end of the tested period (24 h), the level of oxidized bases
was 9.39 % of the total DNA damage. This reduction is
indicative of the activation of repair mechanisms respon-
sible for the removal of oxidative DNA damage. As for
treatments with 150 mM H
2
O
2
(Fig. 3b), the amount of
oxidized bases at 0 h was 57.55 % with a subsequent
decrease (40.89 % at 6 h) that was indicative of impair-
ment in DNA repair. At the end of the tested period (24 h),
the level of oxidized bases was 31.14 % of the total DNA
damage. In cells challenged with 200 mM H
2
O
2
(Fig. 3c),
the estimated amount of oxidized bases at 0 h was 64.92 %
while there was a reduction at the subsequent time points
(58.59 %, 2 h; 51.49 %, 4 h; 45.24 %, 6 h). At the end of
the tested period (24 h), the level of oxidized bases was
Table 2 Level of oxidative DNA damage estimated by FPG-SCE analysis of Petunia 9hybrida leaf discs treated with increasing H
2
O
2
concentrations
Time after treatment (h)
Treatment T
0h
T
2h
T
4h
T
6h
T
24h
(B-E)
a
(B ?E)
b
Oxidative
damage
c
(B-E) (B ?E) Oxidative
damage
(B-E) (B ?E) Oxidative
damage
(B-E) (B ?E) Oxidative
damage
(B-E) (B ?E) Oxidative
damage
CTRL 66.0 ±3.0 92.0 ±3.0 26.0 86.0 ±4.0 122.0 ±2.0 36.0 72.0 ±3.0 110.0 ±1.0 38.0 77.0 ±5.0 106.0 ±5.0 29.0 83.0 ±4.0 108.0 ±4.0 25.0
100 mM
H
2
O
2
79.0 ±1.0 156.0 ±5.0 77.0 79.0 ±5.0 113.0 ±3.0 34.0 78.0 ±2.0 91.0 ±5.0 13.0 78.5 ±0.5 94.0 ±2.0 15.5 82.0 ±2.0 90.5 ±4.0 8.5
150 mM
H
2
O
2
81.5 ±1.5 192.0 ±4.0 110.5 78.5 ±2.5 150.0 ±4.0 71.5 75.5 ±2.5 134.0 ±3.0 58.5 73.0 ±2.0 123.5 ±3.0 50.5 78.5 ±1.5 114.0 ±4.0 35.5
200 mM
H
2
O
2
87.0 ±3.0 248.0 ±3.0 161.0 82.0 ±2.0 198.0 ±4.0 116.0 81.5 ±7.5 168.0 ±2.0 86.5 80.5 ±2.5 147.0 ±5.0 66.5 89.5 ±2.5 125.0 ±5.0 35.5
400 mM
H
2
O
2
89.0 ±4.0 400.0 ±0.0 311.0 82.5 ±3.5 398.0 ±1.0 315.5 86.5 ±1.5 397.5 ±3.0 311.0 82.5 ±1.5 396.0 ±1.0 313.5 83.0 ±1.0 386.0 ±8.0 303.0
a
(B-E), sample incubated only with buffer
b
(B ?E), sample incubated with buffer and enzyme FPG
c
Oxidative damage =(B ?E)-(B-E)
100 mM H2O2
0
50
100
DNA damage (%)
150 mM H2O2
400 mM H2O2
Strand breaks
Oxidized bases
AB
Time after
treatment (h)
0 2 4 6 24
DNA damage (%)
100
50
0
Time after
treatment (h)
0 2 4 6 24
200 mM H2O2
C
DNA damage (%)
100
50
0
Time after
treatment (h)
0 2 4 6 24
D
100
50
0
Time after
treatment (h)
0 2 4 6 24
CTRL
E
100
50
0
DNA damage (%)
Time after
treatment (h)
0 2 4 6 24
DNA damage (%)
Fig. 3 Repair dynamics highlighted by changes in the percentage of
oxidized bases in P. 9hybrida regenerating leaf discs exposed to
increasing H
2
O
2
concentrations
Acta Physiol Plant (2014) 36:331–341 337
123
28.40 % of the total DNA damage. The highest level of
oxidized bases was induced by the 400 mM H
2
O
2
dose at
0 h (77.75 %) and only slight changes were subsequently
observed, indicating lack of DNA repair activity (Fig. 3d).
Finally, in the untreated cells the percentage of basal oxi-
dized DNA was in the range 20–26 % (Fig. 3e).
The FPG-SCGE analysis allowed to assess the genotoxic
effects of the model substrate H
2
O
2
, highlighting the dose-
dependent accumulation of oxidative injury as well as the
most effective repair response.
Response of PhAPX,PhOGG1 and PhMT2 genes
The expression profiles of PhAPX,PhOGG1 and PhMT2
genes were evaluated in P. 9hybrida leaf discs exposed
to increasing H
2
O
2
concentration and results from QRT-
PCR experiments are shown in Fig. 4. Statistical signifi-
cance of differences was determined using Tuckey’s test
(P\0.001).
When the lowest H
2
O
2
concentration (100 mM) was
used, the PhAPX gene was significantly up-regulated,
reaching the highest transcript amount (2.0-fold, compared
to CTRL) at 2 h while at the end of the tested period
(24 h), the level of APX mRNA was similar to that of the
untreated control. Similarly, the amount of PhOGG1
transcript was significantly enhanced (up to 1.5-fold) at 0, 2
and 6 h, compared to untreated control. The accumulation
of PhMT2 mRNA progressively increased from 0 to 6 h
with the highest level (2.5-fold) recorded at 6 h (Fig. 4a).
A significant decrease in PhOGG1 and PhMT2 transcripts
occurred at 24 h. All the tested genes were further up-
regulated with 150 mM H
2
O
2
as shown in Fig. 3b. The
PhAPX gene expression was enhanced (up to 12.0-fold)
compared to the untreated control in the time period 2–24 h
while the amount of PhOGG1 mRNA increased (up to 8.0-
fold) throughout the tested period (0–24 h), compared to
control. Effects were observed on the PhMT2 gene that was
up-regulated in the 0–6 h period (up to 15.0-fold) and then
the highest transcript accumulation (up to 35.0-fold) was
recorded at 24 h (Fig. 4b). When 200 mM H
2
O
2
was
provided to P. 9hybrida leaf discs, the PhAPX transcript
was downregulated at 0 and 2 h, and subsequently up-
regulated at 4 and 24 h (up to 4.0-fold) compared to control
(Fig. 4c). Fluctuations in the amount of PhOGG1 mRNA
were observed in the 0–24 h period. The PhMT2 gene was
up-regulated, reaching the maximum transcript levels at
24 h (3.7-fold compared to control).
Discussion
The search for novel commercial traits resulting from the
enhancement of natural genetic variability is a primary goal
for ornamental breeders (Cassels and Curry 2001; Shu
2009). In the present work, P. 9hybrida leaf discs
maintained for 8 days on regeneration medium were used
to assess the effects of oxidative stress induced by in vitro
culture, in terms of ROS accumulation and genotoxicity.
The investigation, carried out by comparing the response of
intact leaves excised from P. 9hybrida plantlets grown
in vitro and the regenerating leaf discs, demonstrated that
both ROS accumulation and DNA damage were enhanced
in the explants cultured in vitro. On the other hand, up-
regulation of genes involved in DNA repair and antioxidant
defence was observed in the explants cultured in vitro, thus
A
100 mM H2O2
CTRL 0 2 4 6 24
Time after treatment (h)
150 mM H2O2
CTRL 0 2 4 6 24
Time after treatment (h)
B
200 mM H2O2
CTRL 0 2 4 6 24
C
0
2
1
3
4
5
0
10
20
30
0
2
4
6
Relative expression
Relative expressionRelative expression
Time after treatment (h)
PhAPX
PhOGG1
PhMT2
Fig. 4 Expression profiles of PhOGG1 (a), PhMT2 (b) and PhAPX
(c) genes were evaluated by QRT-PCR in regenerating P. 9hybrida
leaf discs exposed to increasing H
2
O
2
concentrations and untreated
control (CTRL). Expression was monitored at different time points (0,
2, 4, 6 and 24 h) following treatments. Values are expressed as
mean ±SD of three independent experiments
338 Acta Physiol Plant (2014) 36:331–341
123
confirming that the molecular responses to oxidative stress
were activated. It should be hypothesised that the observed
enhancement in DNA repair and antioxidant functions
represents a sort of adaptive response, a complex process
by which organisms generally respond to long-term envi-
ronmental stresses through permanent genetic/epigenetic
changes (Dimova et al. 2008).
The P. 9hybrida leaf explants were then challenged
with increasing H
2
O
2
concentrations to assess the geno-
toxic effects of the resulting oxidative stress and the cell
ability to withstand the oxidative injury. When cells are
subjected to a range of stress levels, the rate of PCD
increases until a critical threshold is reached and, above
that threshold, an increase in the rate of necrosis is
observed (Reape and McCabe 2010; Hogg et al. 2011). The
response of P. 9hybrida leaf discs to different degrees of
oxidative stress was assessed by monitoring the nuclear
morphology with the DNA diffusion assay. This versatile
technique, widely used with animal cells, was tested for the
first time in plants by Gichner et al. (2005) in tobacco
seedlings subjected to different stress treatments such as
H
2
O
2
, alkylating agents, and heat shock. The nuclear DNA
of apoptotic cells has abundant alkali-labile sites and under
alkaline conditions small DNA fragments are released and
diffuse in agarose, giving rise to a circular gradient with a
dense central zone surrounded by a lighter external halo
(Singh 2005). In case of necrosis, the nuclear DNA has an
heterogeneous appearance, with irregular borders and these
hallmarks can be easily scored. When considering the PCD
response of P. 9hybrida cells, it is evident that the dose
range 150–200 mM H
2
O
2
defines the threshold required to
induce PCD while at the highest tested dose (400 mM
H
2
O
2
) cells loose their ability to control the incoming
damage. The cell death profiles obtained by monitoring the
nuclear morphology in P. 9hybrida leaf discs were
associated with a dose-dependent loss of cell viability and
regeneration capacity.
As for the genotoxic effects of H
2
O
2
, the FPG-SCGE
analysis highlighted both the dose-dependent accumulation
of oxidative DNA damage in P. 9hybrida cells and the
repair of oxidative lesions occurring during recovery.
According to the FPG-SCGE analysis, cells were highly
efficient in removing the oxidized bases when treated with
the lowest H
2
O
2
dose. At 2 h, almost 50 % of the oxidized
lesions were removed and the level of DNA damage was
further lowered until 24 h, when approximately 80–85 %
of the damage disappeared. By contrast, the repair response
was slower in cells exposed to the 150 and 200 mM doses.
The repair dynamics resulting from FPG-SCGE analysis
clearly showed that DNA repair mechanisms, activated
soon after treatment, were able to remove oxidative lesions
within 24 h with a dose-dependent efficiency. Although
H
2
O
2
has been used with different plant systems to
investigate the cellular mechanisms underlying the
response to oxidative stress, only a few reports are cur-
rently available describing in detail the related DNA
damage profiles expressed as FPG-sensitive sites (Stavreva
and Gichner 2002). FPG-SCGE has been recently used
(Dona
`, persopnal communication) to monitor the repair
response of P. 9hybrida leaf explants exposed to gamma
rays under different total dose and dose rate conditions. In
this case, the amount of FPG-sensitive sites was progres-
sively reduced within the 6 h following irradiation.
Although additional investigation is required to better
describe from a quantitative and qualitative point of view
the mutagenic effects exerted by the prolonged in vitro
culture of P. 9hybrida leaf discs, some indications have
been provided by RAPD analysis carried out in a parallel
study (Dona
`et al. manuscript in preparation). The simi-
larity index calculated comparing the RAPD profiles
obtained with leaf discs excised from the plant and with
regenerating leaf discs cultured in vitro for one week was
consistent with the idea that prolonged in vitro culture
causes genetic variability.
In animal cells, the expression profiles of the OGG1
gene are frequently used to monitor the DNA damage
responses under oxidative stress conditions in combination
with FPG-SCGE (Wessels et al. 2011). For this reason, the
PhOGG1 gene was used as molecular indicator of the DNA
repair response in P. 9hybrida cells treated with H
2
O
2
.
The observed up-regulation, induced by 100 and 150 mM
H
2
O
2
, confirmed the involvement of the BER pathway,
differently from that observed in the case of gamma rays
treated P. 9hybrida explants in which no fluctuations of
the PhOGG1 mRNA were detected (Dona
`et al. 2013). The
expression profiles evidenced by QRT-PCR might be used
as molecular indicators of the response to oxidative injury
in H
2
O
2
-treated P. 9hybrida cells.
In conclusion, novel informations have been obtained,
concerning the genotoxic effects of prolonged in vitro
culture in P. 9hybrida tissues with high regeneration
potential. The reliability of FPG-SCGE for the quantitative
analysis of the dose-dependent genotoxic effects induced
by the model agent hydrogen peroxide has been demon-
strated. We believe that the acquired knowledge will pro-
vide the bases for the optimisation of Mutation Breeding
protocols designed on a genotype-scale.
Author contribution L. V., A. M. and M. D. developed
the SGE and DNA diffusion assay on Petunia 9hybrida
explants. L. V. and S. P. carried out SCGE and DNA dif-
fusion assay. A. Buttafava provided the expertise in
radiobiology and radiation chemistry for assessing irradi-
ation conditions. A. G. developed the in vitro culture
protocol for the elite genotype and contributed to the
design of in vitro experiments. A. Balestrazzi, D. C., A.
Acta Physiol Plant (2014) 36:331–341 339
123
Buttafava and A. G. were involved in the design of the
experimental work and paper writing. L. V. was supported
by a Fellowship from the Italian Ministry of Agriculture-
MiP.A.F. (‘MUTAFLOR’ project).
Acknowledgments Authors would like to thank Dr. Raffaele
Langhella and Albani Vincenzo e Ruggeri Italina s.s.a., via Font-
anatetta 158, 00053 Civitavecchia (RO) for supplying the P. 9
hybrida genotype. This research was supported by the ‘MUTAFLOR’
project from the Italian Ministry of Agriculture-MiP.A.F.
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