Influence of Exogenous Application of Hydrogen Peroxide on Root and Seedling Growth on Wheat (Triticum aestivum L.)

Abstract

Effect of exogenous application of hydrogen peroxide (90 mM H2O2) was studied on initial roots and seedling growth in wheat. Fresh weight was significantly higher (p

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2004/06–2–366–369
http://www.ijab.org
Influence of Exogenous Application of Hydrogen Peroxide on
Root and Seedling Growth on Wheat (Triticum aestivum L.)
AMJAD HAMEED
1
, SHAFQAT FAROOQ, NAYYER IQBAL AND RUBINA ARSHAD
Plant Molecular Breeding Group, Mutation Breeding Division, Nuclear Institute for Agriculture and Biology (NIAB),
Faisalabad–Pakistan
1
Corresponding author’s Email: amjad46pk@yahoo.com
ABSTRACT
Effect of exogenous application of hydrogen peroxide (90 mM H
2
O
2
) was studied on initial roots and seedling growth in
wheat. Fresh weight was significantly higher (p<0.05) in stressed seedlings (124% of the control) on 8th day. Mean weight
gain per day from 5th to 8th day was significantly (p<0.01) higher in stressed roots and whole seedlings. Number of roots was
higher in stressed seedlings on all days with a significant increase (p<0.01) on 8
th
day. Root length was significantly shorter
(p<0.01) in stressed seedlings on all days indicating suppression of cell elongation probably by premature initiation of
secondary wall formation. Maximum stress in terms of reduced fresh weight and length of roots (p<0.001) observed on 5
th
day, suggested that exogenous application of H
2
O
2
for improvement of root growth or to control root diseases should be made
after 5
th
day. The study provided evidence that exogenous application of H
2
O
2
provided more vigorous root system in wheat.
Key Words. Abiotic stress; Hydrogen peroxide; Oxidative stress; Root growth; Reactive oxygen species
INTRODUCTION
The imposition of biotic and abiotic stress conditions
such as drought and salinity are known to raise
concentrations of reactive oxygen species (ROS) such as
hydrogen peroxide, super oxide and hydroxyl ions, resulting
in oxidative damage at the cellular level (Zhang et al.,
2001). Exogenous hydrogen peroxide (H
2
O
2
) signals the
induction of defense responses in plants against pathogen
attack (Levine et al., 1994; Alvarez et al., 1998) abiotic
(Prasad et al., 1994; VanCamp et al., 1998) and oxidative
stresses (Morita et al., 1999).
Root growth of plants growing under different type of
stresses can be modulated by exogenous application of
hydrogen peroxide. For example low concentrations of
hydrogen peroxide when applied exogenously inhibit root
development in alpine larch (Shearer, 1961). An increase
concentration of H
2
O
2
in rice roots cells, preceded root
growth reduction caused by abscisic acid (ABA) (Lin &
Kao, 2001). Contrary to this, low doses of hydrogen
peroxide can increase in mass and length of roots
(Narimanov & Korystov, 1997). In addition to this
hydrogen peroxide has also been reported to stimulate
germination of seeds and growth of sprouts (Narimanov &
Korystov, 1997). Hydrogen peroxide can also be used for
surface sterilization and disinfestations of pine (Barnett,
1976; James & Genz, 1981) and lettuce seeds (Pernezny et
al., 2001) to reduce root and leaf diseases caused by
different soil born bacteria and fungi. However, some
problems with seedling toxicity and reduced seed
germination have also been reported (Edwards &
Sutherland, 1979; James & Genz, 1981; Pernezny et al.,
2001) that warns its cautious application as seed disinfectant
The present study was, therefore, designed to
characterize the influence of exogenous application of
hydrogen peroxide on growth and development of wheat
roots and the intact etiolated seedlings of various ages. The
ultimate aim of study was to find out seedling age suitable
for hydrogen peroxide induced wheat root growth
enhancement and reducing soil and water borne diseases of
roots. It is anticipated that any beneficial role of hydrogen
peroxide on wheat root growth and physiology would be of
major agricultural significance especially under water
stressed environment and salinity.
MATERIALS AND METHODS
Uniform sized seeds (44.05 ± 3.07 mg) of wheat strain
1076 (Triticum aestivum L.) were germinated in darkness
for 24 h at 25 ± 1°C on wet filter paper in petridishes.
Germinated seeds were then covered with a lid to minimize
the evaporation, and growth was continued for 24 h in
darkness at 25 ± 1°C. To apply hydrogen peroxide
treatment, water as the medium was changed with 90 mM
hydrogen peroxide (Merck, Germany), and the growth of
the seedlings was continued at 25 ± 1°C for another 6 days
in darkness. Experiment was repeated thrice and for each
replication at least 12 seedlings were used. The reagent
solution was changed once a day for freshly prepared
solutions to insure exogenous exposure of the seedlings to a
uniform level of hydrogen peroxide, which is a pre-
requisite, to study detailed effect of oxidative stress imposed
by H
2
O
2
on initial root growth of wheat.
Measurement of growth. Age of seedling was estimated in

H
2
O
2
APPLICATION IN WHEAT / Int. J. Agri. Biol., Vol. 6, No. 2, 2004
367
days starting from the beginning of the seed soaking time.
Fresh weights of seedlings (starting from 3 to 8 days of age)
were obtained immediately after taking out of petre plates.
At least 36 treated and control seedlings were studied every
day starting from 3
rd
to 8
th
day. After measuring total
seedling fresh weight roots were separated and fresh weight
of roots were taken. Root length (cm) was measured by
spreading the roots on a scale calibrated in centimeters.
Total number of roots for each seedling was also recorded
each day to measure the rate of new root emergence.
Changes in root weight, length and number were kept
determined continuously at different days in both stressed
and control seedlings.
Statistical analysis. All experiments were repeated three
times, every time with three replications (12 seedlings per
replication). Similar results and identical trends were
obtained each time. The data being presented here is for one
experiment replicated three times with 12 seedlings per
replication for each day. The descriptive statistics including
mean, standard deviation, median and sample variance were
applied to analyze and organize the data. The significance of
differences between means (for stressed & control
seedlings) for different parameters was measured using
Student’s t-Test (two tailed) assuming unequal variances at
0.01 and where applicable at 0.05 significance level (add
reference).
RESULTS
Fresh weight of seedlings. Fresh weight of both control and
stressed seedlings increased up to 4
th
day (Fig. 1) and then
decreased. The age (day) at which seedling fresh weight was
minimum differed in control and stressed seedlings. It was
minimum (55.3mg) on 5
th
day in stressed seedlings and
(117.8 mg) on 6
th
day in control seedlings. After 5
th
and 6
th
day there was a steady increase in fresh weight in both
stressed and control seedlings respectively. Mean weight
gain/day from 5
th
to 8
th
day in stressed seedlings (31.4
mg/day) was significantly higher (p<0.01) compared to that
measured from 6
th
to 8
th
day in control (26.1 mg/day).
Statistically, fresh weight of stressed seedlings (Fig. 1) was
significantly lower (p<0.01) than control on 3
rd
to 7
th
day
however the difference became non significant (p>0.01) on
8
th
day where percent fresh weight of stressed seedlings was
87.9 % of control. Maximum and highly significant
difference (P<0.001) in fresh weight of stressed and control
seedlings were observed on 5
th
day, here stressed seedlings
fresh weight was just 44.404 % as that of control.
Fresh weight of roots. Fresh weight of roots in control and
stressed seedlings increased slightly up to 4
th
day (Fig. 2)
and then decreased significantly to a minimum of 5.7±1.8
mg on 5
th
day that was 44.1 % of control. Fresh weight of
roots growing under stress was significantly (p<0.01) low
(Fig. 2) compared to control on 3
rd
to 7
th
(p<0.05) day with
the lowest (P<0.01) on 5
th
day. On 8
th
day however, mean
fresh weight of roots in stressed seedlings increased
significantly (24% of control). After 5
th
day there was a
steady increase in fresh weight of roots in both stressed and
control seedlings. Mean weight gain per day from 5
th
to 8
th
day was significantly higher (p<0.01) in stressed seedlings
(6.9 mg/day) compared to control (2.5/day).
Root length. Mean root length increased significantly but
only in control seedlings between 3
rd
and 4
th
day. After that
a significant (p< 0.01) shrinkage was observed on 5
th
day in
Fig. 1. Mean fresh weight of control (hatched
columns) and stressed (white columns) seedlings on
various days
0
50
100
150
200
345678
Seedling age (days)
Fresh weight (mg
)
Fig. 2. Mean fresh weight of roots in control (hatched
columns) and stressed (white columns) seedlings on
various days
0
10
20
30
40
345678
Seedling age (days)
Fresh weight (mg
)
Fig. 3. Comparison of seedling age and root length
in control (hatched columns) and stressed (white
columns) seedlings
0
1
2
3
4
5
6
7
345678
Seedling age (days)
Mean root length (cm)

HAMEED et al. / Int. J. Agri. Biol., Vol. 6, No. 2, 2004
368
both stressed and control seedlings (Fig. 3), which resulted
into a decrease in root length.
After 5
th
day, an increase in mean root length was
observed in control seedlings while in stressed seedlings no
considerable change occurred between 6
th
and 7
th
day. Mean
increase in root length/day from 5
th
to 8
th
day was almost
similar in control (0.39 cm/day) and stressed seedlings (0.37
cm/day in). In general mean root length was significantly
lower (p<0.01) in stressed seedlings compared to control
(Fig. 3) on all days.
Primary root length (cm) was significantly lower
(p<0.01) in stressed seedlings on all days with highly
significant (P<0.001) difference on 5
th
, 7
th
and 8
th
day (Fig.
4). Shrinkage in primary root length was observed from 3
rd
to 5
th
day in control as well as stressed seedlings with
maximum shrinkage observed on 5
th
day. However the rate
of shrinkage in maximum root length per day from 3
rd
to 5
th
day was almost similar stressed and control. After 5
th
day
primary root length started increased gradually in control
and stressed seedlings with a mean increase of 1.357cm/day
in control and 1.053 cm/day in stressed seedlings. Root
elongation process was more rapid in control (R
2
0.497) then
in stressed (R
2
0.172) seedlings.
Root number. Number of roots in control and stressed
seedlings increased with increasing age (Fig. 5) but rate of
increase was more rapid in stressed seedlings (R
2
0.873)
compared to that in control (R
2
0.717). Contrary to other
parameters such as root weight and length, root number was
higher in stressed seedlings on all days in general and on 7
th
and 8
th
day old seedlings in particular. However difference
in root number of stressed and control seedlings became
significant (P<0.01) on 8
th
day) (123.077% of control).
DISCUSSION
The influence of oxidative stress induced by hydrogen
peroxide on growth and development of roots in wheat
during early ontogenesis has not yet been studied
nevertheless it has been documented (Anonymous, 2002)
recently that oxidative stress produce at cellar level by low
dose irradiation of seeds can stimulates seed germination
and other stages of plant development. For example,
oxidative stress induced by ionizing radiation and hydrogen
peroxide can stimulates growth of sprouts and roots in
barley, wheat, pea, maize and melon (Anonymous, 2002).
Irradiation of seeds can also stimulate growth of roots in
terms of increase in mass and length (Narimanov &
Korystov, 1997). In present study, it was observed
significantly higher weight gain per day in stressed
seedlings (Fig. 1) and roots (Fig. 2) compared to that in
control after 5
th
day. This indicated that oxidative stress
induced by H
2
O
2
could accelerate seedling and specifically
root growth in wheat. The process of weight gain started one
day earlier in stressed seedlings (Fig. 1) and 24% more fresh
weight of stressed roots on 8
th
day (Fig. 2) provides not only
the further evidences for growth enhancing effect of H
2
O
2
but at the same time it also
signify the importance of
exogenous application of H
2
O
2
for wheat root growth
enhancement and prevention from diseases (Barnett, 1976;
James & Genz 1981; Pernezny et al., 2001). This also
suggest that exogenous application of H
2
O
2
should be made
after 5
th
day of seedling age especially in wheat, thus
bypassing the period in which H
2
O
2
treatment may have
growth suppressive effect.
Significant reduction in root length under stressed
condition (Fig. 3 & 4) indicates suppression of root
elongation process i.e. cell elongation and division. In the
present study however, reduction in root length may have
occurred due to inhibition of cell elongation process alone
and not due to cell division, as there was a significant
increase in root number and weight, which is possible only
if active cell division in taking place. Hydrogen peroxide
therefore enhances cell division either as primary or
secondary effect to counter balance the inhibition process of
cell elongation. As reported earlier (Potikha et al., 1999),
exogenous application of H
2
O
2
prematurely promoted the
secondary wall formation; therefore inhibition of root cell
elongation may be a consequence of premature secondary
wall formation in root cells thus blocking the further cell
Fig. 4. Comparison of seedlin
g
a
g
e and mean maximum
(primary) root length in control (hatched columns) and
stressed (white columns) seedlings
For control
y = 0.591x + 5.4023
R
2
= 0.497
For stressed
y = 0.2354x + 2.6296
R
2
= 0.1717
0
2
4
6
8
10
12
345678
Seedling age (days)
Mean max root length (cm)..
Fi
g
. 5. Comparison between seedlin
g
a
g
e and root no in
control (hatched columns) and stressed (white columns)
seedlings
For control
y = 0.2678x + 3.6985
R
2
= 0.7169
For stressed
y = 0.4068x + 3.7476
R
2
= 0.8723
0
1
2
3
4
5
6
7
8
345678
Seedling age (days)
Number of roots

H
2
O
2
APPLICATION IN WHEAT / Int. J. Agri. Biol., Vol. 6, No. 2, 2004
369
elongation.
Oxidative stress induce by irradiation of seeds with 20
cGy, has been reported (Narimanov & Korystov, 1997) to
significantly increase the number of lateral roots as
compared to control melon seedlings. In the present study
we have also observed higher number of roots (Fig. 5) in
stressed seedlings on all days with a significant increase on
8
th
day, which increases root weight of stressed seedlings up
to 24% on the same day. Thus H
2
O
2
induced oxidative stress
appeared to have the ability of enhancing the process of new
root emergence, which can be attributed to plant defense
response to abolish the effect of stress (reduction in root
length & weight) thereby, helping the plant to establish
properly for attaining proper growth under stress condition.
Moreover a significantly low root length (Fig. 3 & 4) and
high root number (Fig. 5) in stressed seedlings indicates that
primary root growth is halted while secondary root
emergence and growth is promoted under H
2
O
2
induced
oxidative stress. This observation can be supported
indirectly by the study of Ren et al. (2000), who observed
an increased level of H
2
O
2
in wheat root cells, which
enhanced the root growth under drought stress. Exogenous
application of H
2
O
2
to wheat seedlings can thus result in to a
significant increase in fresh weight and number of root by
promoting new roots emergence that may provide more
vigorous root system to wheat plant. Furthermore, it has
been reported very recently in a field trial that early
vigorous root growth was major factor for higher nitrogen
up take in wheat (Liao et al., 2004). More vigorous root
growth by exogenous hydrogen peroxide consequently will
cause higher nitrogen uptake ensuing better growth and
yield of wheat plant.
Collectively root growth enhancement by exogenous
application of H
2
O
2
may be of tremendous agricultural
importance especially in water deficient and saline area
where crops cannot survive due to poorly developed root
system hence well-developed root system can improve
seedling survival rate. In addition vigorous root growth by
applying exogenous hydrogen peroxide can be used to
increase nitrogen uptake resulting in better growth and
yield.
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(Received 06 January 2004; Accepted 10 February 2004)