American Journal of Plant Sciences, 2011, 2, 443-448
doi:10.4236/ajps.2011.23051 Published Online September 2011 (http://www.SciRP.org/journal/ajps)
443
Assessing Influence of Ozone in Tomato Seed
Dormancy Alleviation
N. Sudhakar1*, D. Nagendra-Prasad2, N. Mohan2, Bradford Hill3, M. Gunasekaran3, K. Murugesan2
1Department of Microbiology, Sengunthar Arts and Science College, Tiruchengode, India; 2Center for Advanced Studies in Botany,
University of Madras, Guindy Campus, Chennai, India; 3Department of Biology, Fisk University, Nashville, USA.
Received May 20th, 2011; revised June 29th, 2011; accepted July 18th, 2011.
ABSTRACT
The study was made on th e role of ozone (O3) gas treatmen t on seeds of Lycop ersicon esculent um cv. PKM 1 (tomato) to
release dormancy in advance. The experimental conditions followed a complete factorial design with 3 independent
factors, i.e. A faster start of germination in T2 treatment (98% - 99%) was observed than in other treatments (T1, T3 and
T4) compared to control seeds by measuring seedling growth rate on 5th day after treatment. Nevertheless a too long
and high concentration of O3 treatment seemed to be penalizing on germinatio n rate wherea s low O3 concen tration (T2)
for a moderate time interval (20 min) seemed to be most beneficial. The treated seeds were stored, checked 1, 3, 6
months later and found that, the seeds have retained their accelerated germination efficiency. In particular, 1T2 (1
month storage), 3T2 (3 month storage) had maximum germination rate among all the stored treated seeds, but 6T2 (6
month storage) didn’t show sustained germination acceleration efficiency. Therefore, it was found that when time pro-
longs, O3 treatment loss its effect steadily and the g ermination efficiency of all the treatments ar e more or less same at
six months after treatment. It is hypothesized that th e application of O3 acts as an important phenomenon in accelerat-
ing seed germination by breaking the dormancy in advance which is associated with reduced level of ABA in O3 treated
seeds.
Keywords: Abscissic Acid, Germination, Ozone , Tomato See d
1. Introduction
Germination is the process which leads to elongation of
the embryonic axis from a seed, allowing subsequent
seedling emergence. Seed dormancy is defined as the
failure of an intact viable seed to complete germination
under favorable conditions and is controlled by several
environmental factors, such as light, temperature and the
duration of seed storage (after ripening) [1,2]. Dormancy
may be related to the embryo itself or to its surrounding
structures i.e., seed coat which lead to distinguish several
classes of dormancy [3]. Dormancy and germination are
determined by the co-action of the growth potential of
the embryo and the restraints imposed by the tissues sur-
rounding it. It is known that, when used in the natural
state, seeds for agricultural production suffer from a cer-
tain number of deficiencies, giving rise in particular to a
small germination percentage, to growth defects, and to
pathology specific thereof.
Various studies have been done documenting the ex-
ogenous treatment of seeds to break seed dormancy.
Scarification is the weakening of seed coats by mechani-
cal abrasions or chemical treatments. Mechanically, the
abrasions are caused by machine threshing, or chemical
treatments. Chemically, the seeds are treated with che-
micals like potassium nitrate, thiourea, ethylene, chloro-
hydrine to make the seed coats soft and weak. In addition
the positive effects of oxidative stress on germination
have also been documented [4,5].
The biological effects of ozone (O3) on plants have
been studied for more than 50 years. Plants have evolved
a complex of defence response mechanisms to respond
various environmental stresses from morphological, bio-
chemical and physiological changes triggered by O3 [6-8].
Ozone is a strong oxidant and it is an effective disinfec-
tant because it has the unique ability to destroy toxic or
noxious industrial impurities and inactivates biological
(viral and bacterial) contaminants through the oxidation
of double bonds [9]. In seeds, reactive oxygen species
(ROS) production has been considered for a long time as
being very harmful, since the works dealing with ROS
were mainly focused on seed ageing or seed desiccation,
two stressful situations which often lead to oxidative

Assessing Influence of Ozone in Tomato Seed Dormancy Alleviation
444
stress [10]. Numerous recent works have nevertheless
brought new lines of evidence showing that the role of
ROS in seeds is not as unfavourable as it was considered
previously [11].
ROS derivate from the reduction of oxygen which
gives rise to superoxide (2
O), hydrogen peroxide (H2O2),
hydroxyl radical (HO) and singlet oxygen (1O2]. ROS
been playing a role in cellular signaling, and these com-
pounds could facilitate the shift from a dormant to a
nondormant status in seeds [10]. Indeed, it is known that
ROS can accumulate during seed storage in the dry state,
as previously mentioned [12-14]. There was a clear cut
relationship between seed dormancy alleviation and ac-
cumulation of ROS and peroxidation products in cells of
embryonic axes, thus suggesting that ROS might play a
role of signal in dormancy alleviation [15,16]. Several
reviews have described the signalling roles of ROS in
plants and their role in plant growth and development is
well documented.

Therefore, aim of the present study is to examine the
influence of O3 gas generation in eliciting ROS on to-
mato seeds to release dormancy in advance and the
grounds for acceleration of germination will be deliber-
ated.
2. Materials and Methods
2.1. Ozone Generation
Ozone gas was generated by passing dry oxygen gas
through a corona discharge type O3 generator (V can
Network model M221), passed through a concentration
equalization tank, and bubbled into the deionized water
with a glass diffuser. The O3 gas output was estimated
with an O3 analyzer kit (BMT 961).
2.2. Seed Material
Lycopersicon esculentum cv. PKM1 (tomato) seeds iso-
lated from ripe fruits were used for the study. All the
seeds used for the study were incubated in 1% HCl for 1
h to remove the remnants of the mucilaginous locular
tissue. Subsequently, the seeds were rinsed with tap wa-
ter, dried at room temperature, and stored in closed plas-
tic container in a refrigerator at 7˚C until use.
2.3. Seed Treatments
In order to investigate the effects of O3 on germination,
the seeds were sterilized in the laboratory using Sodium
Hypochlorite of 7% for 1 hr and washed three times with
sterile distilled water. Tomato seeds were humidified in a
plastic flask and distilled water was added, until all the
seeds were get completely immersed. For each treatment
fifty seeds were used. Seeds were introduced in the reac-
tor and treated with different O3 concentration, T1 = 0.001
grams per gram (g O3 g
–1) of seeds, T2 = 0.01 g O3 g –1
seeds, T3 = 0.1 g O3 g
–1seeds and T4 = 1 g O3 g –1seeds
were performed in the sterilized-diffusers for a various
time duration of 10, 20 and 30 mins respectively. While
for the control (C) tomato seeds, charcoal filtered air
(Cl-F) were passed. After treatment, seed were removed
from the reactor, dried at room temperature, and stored in
closed plastic container in a refrigerator at 7˚C until use.
2.4. Germination Test
The germination tests were performed using treated seeds,
by giving four different periods of rest time before ger-
mination test, i.e. 1) Germination test performed imme-
diately after treatment (referred to as T1, T2, T3 and T4
seeds); 2) One month after treatment (referred to as 1T1,
1T2, 1T3 and 1T4 seeds); 3) Three months after treatment
(referred to as 3T1, 3T2, 3T3 and 3T4 seeds), 4) Six
months after treatment (referred to as 6T1, 6T2, 6T3 and
6T4 seeds). All the seeds including control seeds were
sown in Petri dishes on one layer of filter paper (What-
mann No.1) moistened with 1.5 mL of 0.25 mg·L–1
thiomersal to prevent fungal growth, without growth
regulators or osmotic material. The Petri dishes were
sealed with Parafilm to ensure closed-system models. All
the plates were placed in closed plastic boxes and incu-
bated at 26˚C ± 1˚C in the dark unless otherwise men-
tioned. In experiments with intact seeds, visible radicle
protrusion was used as the criterion for germination
unless mentioned otherwise [17]. These experimental
conditions followed a complete factorial design with 3
independent factors, i.e. O3 concentration, treatment du-
ration, and rest time before germination.
2.5. ABA Determinations
Quantitative determinations of endogenous ABA were
performed according to techniques described by [18]
using Gas chromatograph equipped with an electron
capture detector. Recovery was determined with the use
of ("4C) ABA that was added to the first extraction me-
dium. It was shown to be at least 95%.
3. Results and Discussion
Seed dormancy and germination are very complex pheno-
mena which involve tightly controlled signalling path-
ways and molecular regulations. Seed germination is
sensitive to both endogenous plant growth regulators and
environmental factors. Although ROS have been widely
considered as detrimental to seeds, recent advances in
plant physiology signalling pathways has lead to recon-
sider their role [19]. ROS accumulation can therefore be
also beneficial for seed germination and seedling growth
by regulating cellular growth, ensuring a protection against
pathogens or controlling the cell redox status. ROS

Assessing Influence of Ozone in Tomato Seed Dormancy Alleviation
445
probably also act as a positive signal in seed dormancy
release. They interact with abscisic acid and gibberellins
transduction pathway and are likely to control numerous
transcription factors and properties of specific protein
through their carbonylation [19].
Reports have shown that the transition from a quies-
cent seed to a metabolically active organism is associated
with ROS generation, suggesting that it is a widespread
phenomenon. Production of hydrogen peroxide has been
demonstrated at the early imbibition period of tomato
seeds [20]. Nitric oxide, hydroxyl radicals and superox-
ide radicals also accumulate during the germination of
seeds of various species [21,22]. However, the external
sources of ROS production and application methods are
poorly documented. Therefore we made an attempt to
demonstrate the influence of O3 in generation of ROS in
accelerating germination in tomato seeds.
When different concentrations of O3 (T1, T2, T3 and T4)
passed to tomato seeds and immediately tested for the
germination efficiency of treatments, although germina-
tion was noticed in all treatments within 5 days, on the
whole maximum frequency of germination of tomato
seeds were observed in the 20 min O3 treatment than 10
and 30 min treatment. Therefore, hereafter results are
discussed only about the 20 min O3 treatment, unless
otherwise mentioned. Treated tomato seeds resulted in a
faster germination rate vs. control samples. This early
germination start led to a larger number of germinated
seeds with longer roots at 5th day. A faster start of ger-
mination was observed in 0T1, 0T2 and 0T3 ozone treated
samples, whereas in 0T4 doesn’t. The germination of
tomato seeds after treatments (before storage) were 0T1 =
86%, 0T2 = 97% and 0T3 = 78%, 0T4 = 44% but for con-
trol (0C) 72 percentage germination was obtained (Table
1). Comparing to Control seeds, germination % is rela-
tively higher in 0T2 = 26%, followed by 0T1 = 16% and
0T3 = 8%. But, 0T4 treatment shows 40% reduced germi-
nation than the control seeds. Nevertheless, too long an
O3 treatment seemed to be unfavorable for seed growth,
whereas a short one seemed to be most beneficial. The
O3 treatment oxidizes the seeds and therefore, germina-
tion efficiency of the seeds decreased as the concentra-
tion (0T4 = 40%) and treatment duration (30 min) of O3
increased (Table 1). Tests performed on seeds of tomato,
representative of major existing families, have shown
that O3 treatment without an additional agent serves to
significantly improve the germination rate of the treated
seeds.
Table 1. Effect of ozone treatments on germination of mature tomato seeds based on 3 independent factors, i.e. Ozone con-
centration, treatment duration, and rest time befor e ger mination.
Germination frequency (%)
Treated seeds rest time before germination testO3 Treatment duration of seeds
S.No
Duration O3 Treatments 10 min 20 min 30 min
0C 72 ± 02c 72 ± 03 d 73 ± 02 c
0T1 78 ± 02b 86 ± 02 b 81 ± 02 ab
0T2 86 ± 03a 97 ± 03 a 84 ± 03 a
0T3 64 ± 02d 78 ± 03 c 54 ± 02 d
1 Immediate
0T4 53 ± 02e 44 ± 01e 32 ± 04e
1C 71 ± 02bc 71 ± 03d 72 ± 02bc
1 T1 74 ± 03b 83 ± 02b 74 ± 02b
1T2 84 ± 03a 93 ± 03a 82 ± 02a
1T3 61 ± 02d 75 ± 02c 57 ± 0d
2 1 month
1T4 56 ± 02e 48 ± 04e 36 ± 03e
3C 70 ± 02b 69 ± 03c 69 ± 02ab
3 T1 68 ± 03bc 78 ± 03b 69 ± 02ab
3T2 76 ± 03a 89 ± 03a 71 ± 02a
3T3 57 ± 03de 69 ± 04c 62 ± 03c
3. 3 month
3T4 58 ± 03d 52 ± 02d 39 ± 03d
6C 66 ± 03ab 66 ± 02bc 68 ± 03a
6 T1 61 ± 02b 68 ± 03b 65 ± 02ab
6T2 69 ± 03a 73 ± 02a 64 ± 03b
6T3 46 ± 03e 62 ± 02c 54 ± 04c
4. 6 month
6T4 53 ± 04d 50 ± 03d 43 ± 03d
Each value is the mean of three experiments with ten replications each (n = 3). Statistically the means of the three experiments were not significantly different
(P < 0.05). Means in the same column of respective S.No. with different letters are significantly different at P < 0.05 in accordance with Fisher’s least signifi-
cant difference test. T1= 0.001 g O3 g
–1 seeds, T2 = 0.01 g O3 g –1 seeds, T3 = 0.1 g O3 g –1 seeds and T4 = 1 g O3 g –1 seeds were performed in the steril-
ized-diffusers for a time duration of 10, 20 and 30 min. While for the control (C) tomato seeds, charcoal filtered air (Cl-F) were passed. Seeds were given dif-
ferent rest time i.e. 1, 3, 6 months before germination. Treatments are referred to different categories i.e. 1 month (1C, 1T1, 1T2, 1T3, 1T4), 3 month (3C, 3T1,
3T2, 3T3, 3T4) and 6 month (6C, 6T1, 6T2, 6T3, 6T4) respectively.

Assessing Influence of Ozone in Tomato Seed Dormancy Alleviation
446
In order to check, whether O3 treated stored seeds re-
tain the accelerated germination efficiency, it was
checked and found that, all the T2 seeds, i.e. 1T2, 3T2 and
6T2 had maximum germination rate compared to other
stored seeds (T1, T3, T4 seeds). Among the T2 stored
seeds, 1T2 shows 24% and 3T2 shows 22% enhanced
germination compared to control but 6T2 didn’t show su-
stained germination acceleration efficiency i.e. O3 treated
seeds have more or less same or vaguely (negligibly)
higher in germination frequency (9%) compared to con-
trol seeds. We infer that the seeds loss its temperament
when time prolongs and posses more or less normal ger-
mination efficiency as that of control. The seeds of T3
and T4, O3 treated for 30 min duration were found to be
injured and when time prolongs the seeds recovered from
the injury (1T3 = 21%, 3T3 = 10%; 1T4 = 50%, 3T4 =
43%, and 6T4 = 37%) and show enhanced germination
(6T3 = 21%) compared to initial stage. Therefore it was
found that when time prolongs, O3 treatment loss its ef-
fect steadily and the germination efficiency of all the
treatments are neither more or less same nor meagrely
higher at six months after treatment. The data from our
present study show that inducing dormancy could be
break up by O3 treatment to the seeds resulted in an oxi-
dative stress, which allows the seeds to germinate. It now
appears more and more clear that ROS would play a key
signalling role in the achievement of major events of
seed life, such as germination or dormancy release.
In developing or germinating seeds, the active mito-
chondria are probably one of the major sources of ROS,
generating superoxide, and subsequently H2O2 [23]. In
seeds of some species, such as tobacco, tomato, pepper
or Arabidopsis, germination is constraint by the micro-
pylar endosperm, which covers the radicle tip [24]. Ger-
mination can proceed if the mechanical resistance im-
posed by the endosperm decreases to such a level that
radicle can protrude through the weakened tissues. The
GA3/ABA ratio is the most important hormone factor,
which promote germination in seeds and led to improved
germination performance [25]. This endosperm weaken-
ing is under the regulation of abscissic acid (ABA) and
gibberellic acid (GA) and several hydrolases have been
suspected to contribute to cell-wall loosening [26]. There
is an increasing bundle of evidence suggesting that ROS
would play a key role in this phenomenon and it has been
proposed that they would be involved in cell wall loos-
ening in growing tissues. Therefore, in order to examine
the cause of O3 treatments on seed germination, ABA
contents were analyzed (Table 2). Among the treatments,
T2 treatment significantly reduced (5 fold) the ABA level
in seeds compared to other treatments T1 (1 fold), T3 (2
fold) and T4 (below 1 fold). The results revealed that the
ABA levels were drastically reduced compared to ma-
tured control seeds (Table 2).
There are also antagonistic effects of ABA and ethyl-
ene on dormancy and germination. Stimulation of ethyl-
ene synthesis by environmental stresses, such as O3, UV
irradiation, and wounding, involve generation of reactive
oxygen species [27]. Some responses of plant tissue to
O3 fumigation may be due to active production of ethyl-
ene by the injured tissue [28]. Tomato, tobacco, and bean
plants treated with O3 had increased rates of ethylene
production [29]. Ethylene may promote germination by
interfering with the ABA action on seed dormancy
and/or maintenance of dormancy [30]. In some species,
such as sunflower, ethylene can break seed dormancy
[31]. Several components of the ethylene signal trans-
duction have been identified and their signaling pathway
has been characterized [32]. Ethylene signal transduction
mutant studies highlight the interaction between ABA
and ethylene signaling suggesting that ethylene sup-
presses dormancy by inhibiting ABA action [32]. The
results from our study also confirm that the O3 treatment
suppresses the ABA level significantly which may be
due to the association of ethylene.
The elicitation of GA3 at mild concentration of O3 was
observed [33], whereas, no statistically significant dif-
ferences were found in the GA3 levels between the con-
trols and the needles of trees which were treated with
increased levels of O3. Similarly, from the results of our
study it is anticipated that the generation of mild concen-
tration of O3 (T1 and T2) on the seeds could elicit higher
level of GA3 rather than the other treatments (T3 and T4)
(Table 1).
On the one hand, there exists evidence suggesting that
hydrogen peroxide alleviates seed dormancy. It was al-
ready reported that H2O2 reverse the inhibitory effect of
ABA upon endosperm rupture, underlining the cross talk
between these two compounds [34]. Antioxidants which
Table 2. Effect of ozone treatments on ABA content of ma-
ture tomato seeds.
S.No Treatments ABA content ng·seed–1
1.
2.
3.
4.
5.
C
T1
T2
T3
T4
1.8 ± 0.2
0.8 ± 0.1
0.3 ± 0.0
0.6 ± 0.0
0.9 ± 0.0
The seeds were extracted at harvest (nondried) and each value in the data
represent the mean of three experiments with ten replications each (n = 3).
T1 = 0.001 g O3 g–1 seeds, T2 = 0.01 g O3 g–1 seeds, T3 = 0.1 g O3 g–1 seeds
and T4 = 1 g O3 g
–1 seeds were performed in the sterilized-diffusers for a
time duration of 20 mins. While for the control (C) tomato seeds, charcoal
filtered air (Cl-F) were passed.

Assessing Influence of Ozone in Tomato Seed Dormancy Alleviation447
act as ROS scavenger in seed biology play a very impor-
tant role in the growth processes occurring at early em-
bryogenesis during seed development, participate in the
mechanisms underlying radicle protrusion during seed
germination and seed aging [35,36]. Exogenous H2O2
stimulates the germination of dormant seeds of barley,
Fontaine et al. 1994; rice, [37] apple [38] and tomato
[20]. Treatment of dormant barley seeds with hydrogen
peroxide results in a decrease in endogenous ABA level
[39] and alleviation of apple embryo dormancy by cya-
nide induces a simultaneous increase in H2O2 level and
decrease in ABA content [38]. H2O2 promoted seed ger-
mination in a dose‑dependent manner as did respiratory
inhibitors, indicating that H2O2 itself possibly promotes
seed germination rather than O2 [40]. Therefore, in the
present study, it is predicted that H2O2 activity in tomato
endosperm would have greatly enhanced when the tissue
was rupture, or wounded by the generation of O3 which
results in the radicle penetration through the micropylar
endosperm.
In general, the reaction of O3 on the surface of seeds
takes place by way of a gas-solid type interchange, fol-
lowed by diffusion within the structure. Among the pos-
sible interplay between ROS and plant hormones, the
relationship between H2O2 and ABA appears as the most
probable. Oxidative stress accelerates the accumulation
of H2O2 in seeds. The hypothesis shown postulates that
ROS play a central role with hormones and particularly
ABA, in dormancy release and germination completion.
After treatment there is an accumulation of ROS by non-
enzymatic ROS production. This accumulation might
reduce ABA level and/or block ABA signalling, stimu-
late GA and ethylene signalling, modify redox status and
downstream events and alter protein function through
oxidative modifications.
The present work constitutes the basis for the future
invention that it is possible to improve the germination
properties and/or the growth of seedlings by treatment
with O3, preferably produced from vector gases, without
calling on an additional oxidizing agent, and with the
treatment being relatively short in duration, ease in han-
dling at very low cost. In general it is authenticated that,
the person skilled in the art will find it relatively easy to
determine the optimum conditions for implementation of
the process.
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