Botanical Studies (2006) 47: 239-250.
*
Corresponding author: E-mail: ashrafm@fsd.paknet.com.
pk; Phone: 92-41-9200312; Fax: 92-41-9200764.
INTRODUCTION
Salt stress-induced imbalance in the hormonal levels
of plants is well known (Debez et al., 2001; Wang et al.,
2001). Abscisic acid (ABA) generally increases in re-
sponse to salinity, and auxins decline (Zhang and Zhang,
1994; Wang et al., 2001). Recent studies show that SA
is involved in the plant responses to salt and osmotic
stress by playing a role in the reactive oxygen species-
mediated damage caused by high salt and osmotic condi-
tions (Borsani et al., 2001) possibly through signalling
and gene regulation. Therefore, SA-diminished changes
in phytohormone levels are suggested to be responsible
for salt tolerance in some crop species (Shakirova et
al., 2003). Moreover, the inhibitory effect of salt stress
on plant growth involves an array of cellular processes,
and all of these may be regulated by an altered hormone
homeostasis under salt stress (Xiong and Zhu, 2002). For
example, elevations in endogenous ABA levels could act
as an internal inhibitor of shoot growth in some plants
(Lee et al., 1996). However, adequate levels of ABA could
regulate the export of organic substances from the leaf and
presumably participate in the regulation of the metabolism
of transportable assimilates (Kiseleva and Kaminskaya,
2002).
Polyamines (PAs), such as Spd and Spm and their
obligate precursor Put, are polybasic amines that are
implicated in many physiological processes in plants
(Smith, 1985; Tiburcio et al., 1993; Galston and Kaur-
Sawhney, 1995; Kumar et al., 1997). Because of their
polycationic nature at a physiological pH, PAs occur
PHYSIOLOGY
Does polyamine seed pretreatment modulate growth
and levels of some plant growth regulators in hexaploid
wheat (Triticum aestivum L.) plants under salt stress.
Muhammad IQBAL
1
, Muhammad ASHRAF
1,
*, Shafiq-Ur-REHMAN
2
, and Eui Shik RHA
3
1
Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan
2
Faculty of Biological Resources Science, College of Agriculture, Chonbuk National University, Chonju 561-756, Republic
of Korea
3
Life Resources Science, Plant Genetics & Breeding, Sunchon National University, Sunchon, 540-742, Republic of Korea
(Received July 12, 2005; Accepted December 19, 2005)
ABSTRACT.
The effect of seed presowing treatment with polyamines (2.5 mM putrescine, 5.0 mM
spermidine and 2.5 mM spermine) on growth and internal levels of different plant growth regulators in two
spring wheat (Triticum aestivum L.) cultivars MH-97 and Inqlab-91 was studied. The primed seeds of each
treatment and non-primed seeds were sown in a field in which NaCl salinity of 15 dS m
-1
was developed.
Although all three polyamines were effective in improving grain yield in both cultivars under saline
conditions, the effect of spermine (Spm) was more pronounced. All priming agents reduced leaf free abscisic
acid (ABA) levels in MH-97 as compared with untreated plants under saline conditions, but putrescine
(Put) priming caused a maximum increase in leaf ABA concentration in MH-97 under saline conditions. In
contrast, in Inqlab-91, Put proved to be very effective in increasing free indoleacetic acid (IAA) concentration
under saline conditions. Spermidine (Spd) and Spm were very effective in enhancing salicylic acid (SA)
concentration in both MH-97 and Inqlab-91 under saline conditions. Leaf free Put concentration was higher
in plants of MH-97 raised from seeds treated with Spd under saline conditions. In contrast, all priming agents
decreased leaf Put concentration compared with non-priming in Inqlab-91 under saline conditions. Plants
of both cultivars raised from seeds treated with Put had the maximum Spm levels under saline conditions.
Overall, the beneficial effects of priming agents (polyamines) were cultivar specific. Physiologically, the
beneficial effect of pre-sowing treatment with Spm on grain yield in both cultivars may be attributed to altered
hormonal balance.
Keywords: Hormone priming; Plant hormones; Pre-sowing treatment; Salinity; Salt tolerance.
pg_0002
240
Botanical Studies, Vol. 47, 2006
in plant cells not only as free forms, but also as bound
forms. The intracellular free PA pool depends on several
processes including a) PA synthesis, b) PA degradation,
c) PA conjugation, d) PA transport (Tiburcio et al., 1990;
Slocum, 1991; Bagni and Torrigiani, 1992; Tiburcio et al.,
1997) and e) interaction with other hormones like ABA
(Lee et al., 1997). Recent molecular genetic analyses have
shown that altered polyamine levels have profound effects
on plant growth and development (Fritze et al., 1995;
Kumar et al., 1996; Masgrau et al., 1997; Watson and
Malmberg, 1998). Polyamines, being cationic in nature,
can associate with anionic components of the membrane
such as phospholipids thereby stabilizing the bilayer
surface and retarding membrane deterioration (Roberts et
al., 1986; Galston and Kaur-Sawhney, 1987; Kramer and
Wang, 1989; Basra et al., 1994). It has been shown that
PAs in chloroplasts can covalently bound to chlorophyll-
protein complexes in thylakoids and Rubisco in the
stroma (Del Duca et al., 1994). These ionic interactions
are considered to increase stabilization of the subcellular
compounds and membranes under stress (Roberts et al.,
1986).
Interest is growing in the possible involvement of PAs
in the defense reactions of plants to various environmental
stresses (Flores, 1990; Kumar et al., 1997; Bouchereau et
al., 1999; Shen et al., 2000; Galston, 2001; He et al., 2002;
Maiale et al., 2004). Many reports have indicated that the
stress tolerance of plants is correlated with their capacity to
enhance the synthesis of PAs upon encountering the stress
(Evans and Malmberg, 1989; Chattopadhyay et al., 1997;
Bouchereau et al., 1999; Mo and Pau, 2002; Kasinathan
and Wingler, 2004; Kasukabe et al., 2004). Which of
the three PAs plays central roles in stress responses of
plants may depend on the plant species and the types
of stress (Kasukabe et al., 2004). In many, but not all,
cases, however, Spd is more closely associated with stress
tolerance of plants than Put and Spm (Bouchereau et al.,
1999; Li and Chen, 2000; Shen et al., 2000; He et al.,
2002; Martinez-Tellez et al., 2002; Maiale et al., 2004).
For example, salinity greatly enhanced the accumulation
of Spm and Spd associated with a decrease in Put content
in wheat cultivars (El-Shintinawy, 2000). Lefevre and
Lutts (2000) observed that an ionic rather than osmotic
component of salt stress is involved in the initial steps of
PAs accumulation in rice. Lin and Kao (1995) showed
contrasting results that increasing NaCl levels led to a
decrease in free putrescine levels, but an increase in Spd
levels in a salt-sensitive rice cultivar, cv. Taichung Native
1. In pea (Anderson and Martin, 1973) and barley (Smith,
1973), neither Put nor Spd accumulated when exposed to
increasing salinity. Salt stress increased the levels of Put
and decreased the concentration of Spm in wheat (Simon-
Sarkadi et al., 2002).
Even if PAs accumulate, this does not prove their
involvement in stress protection, especially as the role of
PAs may depend on their cellular localization and whether
they are free, bound to proteins, or conjugated to phenolic
acids (Bouchereau et al., 1999). However, a correlation
between stress tolerance and polyamine levels has been
demonstrated in a number of studies using a variety of
plant materials, but the physiological rationale for stress-
induced polyamine accumulation remains unknown. The
function of PAs is presumed to be protective, with a role
in scavenging free radicals being a favoured hypothesis
(Mansour, 2000). These reports indicate that the individual
PAs may have different roles during the response of plants
to salt stress. Seed priming with appropriate concentrations
of PAs has also been shown to be beneficial under salt
stress in some crops (Mansour et al., 2002; Mishra and
Sharma, 1994; Ali, 2000). Moreover, hydropriming is also
known to be effective under saline conditions (Twitchell,
1955; Zheng et al., 1998; Roy and Srivastava, 1999;
Junmin et al., 2000; Cassaro-Silva, 2002).
All these reports led us to hypothesize that pre-sowing
seed treatment with PAs could alleviate the adverse
effects of salt stress on the growth of spring wheat. Thus,
the primary objective of the present study was to assess
that up to what extent PAs applied as per-sowing seed
treatment could ameliorate the effect of salt stress on two
wheat cultivars differing in salt tolerance. In addition, it
was determined that how far pre-sowing seed treatment
with PAs can regulate the internal levels of PAs and other
plant growth regulators in two genetically diverse wheat
cultivars subjected to salt stress.
MATERIALS AND METHODS
The seeds of two moderately salt tolerant spring wheat
cultivars, MH-97 and Inqlab-91, were obtained from the
Wheat Section, Ayub Agricultural Research Institute,
Faisalabad, Pakistan. Solutions of 2.5 mM, 5.0 mM and
2.5 mM each of Put, Spd and Spm were used for seed
priming, respectively, following Mansour et al. (2002).
Distilled water was also used for priming and as a control.
Healthy wheat seeds (17 g for each treatment) were
primed separately in 100 mL of solutions of PAs as well
as in distilled water (DW) for 12 h at room temperature in
plastic cups (250 mL). After presoaking, the seeds were
surface dried on filter paper and then allowed to air-dry for
12 h at room temperature. The air-dried seeds were used
for field experiments.
A field experiment was conducted at the Botanic
Garden, University of Agriculture, Faisalabad (UAF)
during winter 2003-2004. Eight blocks having a length,
width and depth of 1097, 137 and 46 cm, respectively, and
lined with polythene sheets were filled with thoroughly
mixed sandy loam soil (pH = 7.56; electrical conductivity
of the saturation paste = 2.84 dS m
-1
; saturation percentage
= 25.5). In order to develop 15 dS m
-1
NaCl salinity,
the calculated amount of NaCl was dissolved in water
required for complete saturation of the soil in four blocks
(replicates). Thus, the soil was completely saturated with
salt solution (150 mM) so as to homogenize soil salinity.
The other four blocks served as control (2.84 dS m
-1
).
After three weeks, when the moisture contents were
suitable for germination, the primed seeds (P-seeds) of
pg_0003
IQBAL et al. — Does polyamine-priming modulate growth under salt stress.
241
each treatment and non-primed seeds (NP-seeds) were
sown in rows keeping row to row spacing at 15 cm. All
pre-sowing seed treatments were randomized in each
block. The experiment was laid out in a split split plot
design.
At the boot stage, 12 plants from each treatment (three
plants replicate
-1
) were uprooted and washed. After being
dried with filter paper, roots were carefully removed, and
data for fresh weights of shoots were recorded. Samples
were dried in an oven at 65oC for two weeks, and shoot
dry weights were recorded. At maturity, grain yield plant
-1
(g) was recorded.
Determination of hormones (Auxins, Abscisic
acid) and Salicylic acid
Fresh leaves (3
rd
leaf from top) were harvested
from each treatment. Two grams of fresh leaves were
finely chopped in 30 mL of 80% cold (-70oC) aqueous
methanol (4:1, v/v) supplemented with 20 mg L
-1
butylated hydroxytoluene (BHT) and stored at -70oC
or -20oC. Fresh leaves already frozen and stored were
ground with a mortar and pestle using aqueous 80%
methanol supplemented with BHT (MeOH-BHT) as
extracting solvent. To check recoveries during extraction
and purification, 191.6 ng of IAA, 300 ng NAA, 287.4
ng salicylic acid (SA), and 191.6 ng abscisic acid
(ABA) were used as internal standards and were added
before homogenization in each sample and blank. The
homogenate was vortexed for 10 min and filtered with
suction through Whatman #42 paper (Whatman Shanghai,
China). The residues in the flask and on the filter were
rinsed thrice with 10-mL aliquots of extracting solvent
and twice with 100% MeOH. The extracts were combined
and mixed and then divided into two equal samples
(Kusaba et al., 1998). One sample was used for IAA
and ABA analyses and the other for SA analysis. The
extract was concentrated to an aqueous residue by rotary
flask evaporation (RFE) at 35oC. The sublimation losses
were reduced by adding phosphate buffer (pH 7). The
aqueous fraction was transferred to 50-mL polypropylene
centrifuge tubes. The flask, used for RFE, was rinsed with
10 mL of hexane to extract chlorophylls and lipids from
the aqueous residue. Similarly, the aqueous fraction was
partitioned three times against hexane after adjusting the
pH to 8. The pH of the aqueous residue was adjusted to 3
with HCl and again partitioned three times with hexane.
The hexane fractions were then discarded. The pH was
then adjusted to 2.8, and the samples were centrifuged
for 15 min at 13,000 g to remove any precipitate. The
supernatant fractions were decanted into clean centrifuge
tubes and partitioned thrice against 10-mL portions of cold
diethylether-BHT so as to extract any traces of hormones.
Each 10-mL portion of ether was partitioned, in turn,
against a 10-mL portion of 1 mM HCl. The same 10 mL of
1 mM HCl was used for all three 10-mL portions of ether.
All three ether fractions were combined and evaporated
by RFE to dryness in vacuo. The residue was immediately
dissolved into 500 μL of 80% ice-cold MeOH-BHT in a
1.5-mL Eppendorf microcentrifuge tube. These samples
were kept overnight at -70oC and then centrifuged (25,000
g) for 10 min at -10oC. (A lower temperature helped to
remove any residues easily.) Standards were also prepared
following the same procedure. The supernatant was
filtered and subjected to HPLC (high performance liquid
chromatography) analysis.
Analysis of IAA, SA, and ABA was performed by
HPLC system (Sykam GmbH, Kleinostheim, Germany),
equipped with S-1121 dual piston solvent delivery
system and S-3210 UV/VIS detector. Following Guinn
et al. (1986) with some modifications, the elution system
consisted of 100% methanol: 1% acetic acid (52:48 v/v)
as solvent, run isocratically with a flow rate of 1.10 mL
min
-1
at 40oC. Twenty microliters of filtered extracts (using
a Whatman nylon membrane filter of 0.45 μm pore size,
Whatman Int. Ltd., Maldstone, England) were injected
into a Hypersil ODS reverse-phase (C
18
) column (4.6 ×
250 mm, 5-
μ
m particle size: Thermo Hypersil GmbH,
Germany). Detection of different hormones was performed
at 280 nm by co-chromatography with authentic standards.
The peak areas were recorded and calculated by a
computer with SRI peak simple chromatography data
acquisition and integration software (SRI Instruments,
Torrance, California, USA). All values were corrected on
internal standards with known concentrations of different
hormones.
Determination of polyamines
Fresh leaves (3
rd
leaf from top) were harvested from
plants of each treatment. Two grams of fresh leaves were
finely chopped in 20 mL of cold 5% aqueous perchloric
acid and were stored at -70oC.
For polyamine extraction and HPLC analysis, the
benzoylation method was performed as described
previously (Flores and Galston, 1982) with some
modifications. Leaf tissue (2 g fresh weight) was
homogenized in 20 mL of cold 5% perchloric acid
with a mortar and a pestle containing 200 nmol of 1,
6-hexanediamine as an internal standard. The homogenates
were incubated at 4oC for 30 min and then centrifuged
at 13,000 g for 20 min. Aliquots of 1 mL of supernatant
were added to 1 mL of 4 M NaOH with 10
μ
L of benzoyl
chloride. The mixture was vigorously vortexed for 30 s
and then incubated for 30 min at room temperature. The
reaction was terminated by adding 2 mL of saturated
NaCl. The benzoyl-polyamines were extracted with 3 mL
of cold diethyl ether for 30 min. Then the samples were
centrifuged at 2,500 g for 5 min, and the ether phase was
collected and dried under N
2
flow. The residues were
redissolved in 500
μ
L of methanol in a 1.5-mL Eppendorf
microcentrifuge tube. Polyamine standards (Sigma, USA)
were prepared similarly to plant samples. These samples
were filtered and stored at -20oC for HPLC analysis.
The polyamine contents were analyzed by HPLC
(Sykam GmbH, Kleinostheim, Germany) equipped
pg_0004
242
Botanical Studies, Vol. 47, 2006
in the grain yield of both cultivars. An exception was in
the plants of Inqlab-91, raised from seeds treated with Put
under saline conditions.
Saline substrate significantly altered the leaf free IAA
contents of both wheat cultivars (P . 0.001; Table 3).
Cultivars also differed significantly from one another.
However, different priming agents altered leaf IAA
concentrations differently in both cultivars. For example,
distilled water proved to be the priming agent most
effective at increasing free IAA concentration in MH-97
under saline conditions. In contrast, in Inqlab-91, Put,
and Spd proved to be very effective at increasing the free
IAA concentration under saline conditions and non-saline
control, respectively (Table 4). Overall, the increase in
free IAA concentration in MH-97 due to different priming
agents was higher than that in Inqlab-91 under both saline
and non-saline conditions.
Leaf free ABA concentrations of both cultivars
changed significantly under salt stress. The effects of
priming agents differed significantly in altering leaf ABA
concentration of both cultivars (P . 0.001; Table 3).
Except hydropriming, all priming agents reduced leaf free
ABA levels in MH-97 as compared with untreated plants
under saline conditions. However, Put priming increased
leaf ABA concentrations the most in MH-97 under saline
conditions.
Salt stress significantly changed the leaf free SA
concentrations of both cultivars (P . 0.001; Table 3).
Priming agents also showed different effects on free SA
concentration in leaves of both cultivars (P . 0.001). The
pattern of increase or decrease in leaf SA concentration in
both cultivars due to salt stress was not consistent (Table
4). However, Spd was a very effective priming agent
in enhancing SA concentration in MH-97 under saline
conditions. In contrast, pre-treatment with Spm slightly
with S-1121 dual piston solvent delivery system and
S-3210 UV/VIS detector. The elution system consisted of
methanol: water (65:35 v/v) as solvent, run isocratically
with a flow rate of 0.70 mL min
-1
at 50oC. Twenty
microliters of benzoylated extracts were injected into a
Hypersil ODS reverse-phase (C
18
) column (4.6 × 250 mm,
5-
μ
m particle size: Thermo Hypersil GmbH, Germany)
and detected at 254 nm. The peak areas were recorded
and calculated by a computer with SRI peak simple
chromatography data acquisition and integration software
(SRI Instruments, Torrance, California, USA).
Data analysis
Analysis of variance (ANOVA) of data for all attributes
was computed using a COSTAT computer package
(CoHort Software, 2003, Monterey, California). The
comparisons of means were done by COSTAT computer
package, using Duncan’s New Multiple Range (DMR)
test.
RESULTS
Saline growth medium caused a significant reduction
in fresh weights of shoots of plants of both cultivars (P
. 0.001; Table 1). Plants derived from seeds treated with
different priming agents did not improve shoot fresh
weight in either cultivar.
Salt stress imposed in the growth medium caused a
significant reduction in the grain yield of both cultivars
(P . 0.01; Table 1). However, different priming agents
caused a significant increase in grain yield (P . 0.001). Of
the priming agents used, Spm was found very effective
in enhancing the grain yield of both cultivars under salt
stress (Table 2). Overall, in comparison with non-priming,
all priming treatments caused a significant improvement
Table 1. Mean squares from analysis of variance of data for shoot fresh mass and grain yield per plant of two cultivars of hexaploid
wheat raised from seeds primed with different polyamines.
Source of variation
Degrees of freedom
Shoot fresh mass
Grain yield plant
-1
Salinity (S)
1
3721.1***
26.90**
Main plot error
3
14.09
0.276
Cultivars (C)
1
6.045ns
0.121ns
C x S
1
0.559ns
0.319*
Subplot error
6
16.72
0.039
Treatments (T)
4
31.74*
0.578***
T x S
4
10.961ns
0.061**
T x C
4
25.44*
0.137***
T x C x S
4
26.86*
0.129***
Error
48
9.469
0.016
*, **, *** = significant at 0.05, 0.01, and 0.001 levels, respectively.
ns = non-significant.
pg_0005
IQBAL et al. — Does polyamine-priming modulate growth under salt stress.
243
Table 2. Shoot fresh mass and grain yield per plant of two cultivars of hexaploid wheat at 2.84 dS m
-1
(control) or 15 dS m
-1
NaCl
(salt) when the seeds were primed with solutions of different polyamines. Values in parentheses are SE of means (based on four
replicates).
Pre-sowing seed
treatments
MH-97
Inqlab-91
2.84 dS m
-1
15 dS m
-1
2.84 dS m
-1
15 dS m
-1
Shoot fresh biomass (g plant
-1
)
Put (2.5 mM)
27.72 (1.56)
14.51 (2.94)
29.79 (0.89)
18.39 (0.87)
Spd (5.0 mM)
27.29 (1.87)
14.97 (2.21)
29.05 (0.77)
15.79 (0.81)
Spm (2.5 mM)
31.23 (0.53)
14.84 (2.61)
31.13 (0.82)
14.87 (0.76)
Distilled water
29.82 (0.25)
15.98 (1.71)
24.66 (0.80)
16.43 (0.56)
Untreated
27.14 (0.47)
15.28 (0.89)
28.95 (0.89)
14.64 (0.73)
LSD 5%
4.375
Grain yield (g dry weight plant
-1
)
Put (2.5 mM)
3.55 (0.144)
2.66 (0.059)
3.75 (0.150)
2.24 (0.094)
Spd (5.0 mM)
3.98 (0.112)
2.93 (0.103)
3.75 (0.083)
2.50 (0.017)
Spm (2.5 mM)
3.69 (0.108)
3.05 (0.074)
3.84 (0.078)
2.56 (0.021)
Distilled water
3.60 (0.044)
2.25 (0.137)
3.58 (0.025)
2.52 (0.029)
Untreated
3.43 (0.059)
2.12 (0.150)
3.57 (0.094)
2.25 (0.049)
LSD 5%
0.178
Table 3. Mean squares from analysis of variance of data for IAA, ABA, and SA and polyamine concentrations of two cultivars of
hexaploid wheat raised from seeds primed with different polyamines.
Source of variation Degrees of freedom
IAA
ABA
SA
Salinity (S)
1
792614.2***
100509.1***
8461635.1***
Main plot error
3
421.2
548.2
9414.9
Cultivars (C)
1
19438.5*
50605.4***
12246589.0***
C x S
1
73564.6**
94511.6***
5254614.9***
Subplot error
6
2821.9
12.7
39618.8
Treatments (T)
4
52291.8***
11815.3***
5132013.4***
T x S
4
78157.3***
9782.8***
1312861.3***
T x C
4
76262.8***
19010.7***
2249342.3***
T x C x S
4
87476.9***
23998.0***
1849103.4***
Error
48
1327.4
649.8
40559.5
Putrescine
Spermidine
Spermine
Salinity (S)
1
4450.3**
385946.6***
3081.3ns
Main plot error
3
46.8
118.4
574.9
Cultivars (C)
1
133.6ns
3027.8ns
188.2ns
C x S
1
705.4**
26786.5*
2513.1**
Subplot error
6
38.3
2609.3
96.6
Treatments (T)
4
635.7***
25959.7***
2191.4***
T x S
4
902.5***
31620.2***
2628.6***
T x C
4
1238.4***
5230.5ns
1547.7***
T x C x S
4
809.4***
8347.2ns
549.6ns
Error
48
19.8
3251.5
271.3
*, **, *** = significant at 0.05, 0.01, and 0.001 levels, respectively; ns = non-significant.
IAA, indoleacetic acid; ABA, abscisic acid; SA, salicylic acid.
pg_0006
244
Botanical Studies, Vol. 47, 2006
increased leaf SA concentration in Inqlab-91 under saline
conditions.
Salt stress of the growth medium significantly affected
leaf Put concentration of both cultivars, but the cultivars
also did not differ significantly (Table 3). However, the
priming agents differed significantly in altering the Put
levels in both wheat cultivars (P . 0.01). Leaf free Put
concentration was higher in plants of MH-97 raised from
seeds treated with Spd under saline conditions (Table
5). Of all priming agents, Put was the most effective at
increasing leaf Put concentration in Inqlab-91 under saline
conditions. In contrast, the remaining priming agents
decreased leaf Put content compared with non-priming in
Inqlab-91 under saline conditions.
Different priming agents altered leaf free Spd
concentration under both saline and non-saline conditions,
but cultivars also did not differ significantly. Similarly, the
difference among priming agents in altering the Spd levels
of plants of both cultivars was also insignificant (Tables 3
and 5).
Salt stress imposed in the growth medium had no
significant effect on leaf spermine (Spm) concentration in
either cultivar, and neither cultivar differed significantly
from the other. However, priming agents differed
significantly in altering free Spm concentration in both
cultivars (Tables 3 and 5). For example, plants of both
cultivars raised from seeds treated with Put had the
maximum Spm levels under saline conditions.
DISCUSSION
It should now be evident that growth regulator balance
can be changed at high salinities, and this effect can
be partially alleviated with application of exogenous
growth promoter substances (Gadallah, 1999; Khan
et al., 2000; Debez et al., 2001). In the present study,
priming with Put alleviated the adverse effects of salt
stress on hormonal balance in the plants of Inqlab-91.
For example, put pre-treatment considerably increased
leaf ABA and IAA concentrations in Inqlab-91 under
saline conditions. In contrast, in MH-97, it decreased free
ABA levels compared with the untreated plants under
saline conditions. The increased ABA levels can regulate
the pathways of photoassimilate utilization in leaves by
partitioning carbon flows either to the synthesis of high
molecular weight compounds (cellulose, hemicellulose,
and proteins), used for cell growth in leaves, or to
Table 4. IAA, ABA and SA concentrations (ng g
-1
fresh weight) of two cultivars of hexaploid wheat at 2.84 dS m
-1
(control) or 15
dS m
-1
NaCl (salt) when the seeds were primed with solutions of different polyamines. Values in parentheses are SE of means (based
on four replicates).
Pre-sowing seed
treatments
MH-97
Inqlab-91
2.84 dS m
-1
15 dS m
-1
2.84 dS m
-1
15 dS m
-1
Free IAA
Put (2.5 mM)
209.2 (2.51)
164.1 (26.54)
121.9 (4.74)
182.9 (10.37)
Spd (5.0 mM)
85.4 (0.11)
125.1 (13.13)
633.2 (12.19)
94.8 (6.97)
Spm (2.5 mM)
231.2 (4.74)
119.6 (15.43)
273.7 (8.45)
112.9 (6.70)
Distilled water
421.8 (18.83)
180.9 (18.79)
561.4 (49.02)
88.2 (1.05)
Untreated
472.5 (19.29)
138.1 (35.93)
288.9 (21.48)
101.5 (1.74)
LSD 5%
51.79
Free ABA
Put (2.5 mM)
183.6 (16.82)
231.5 (12.80)
59.5 (19.78)
187.1 (7.59)
Spd (5.0 mM)
31.5 (0.88)
178.5 (2.38)
173.8 (1.34)
110.7 (0.37)
Spm (2.5 mM)
116.9 (9.91)
137.9 (9.40)
145.2 (18.85)
122.6 (10.60)
Distilled water
119.3 (0.90)
329.4 (13.94)
93.3 (3.47)
102.9 (5.91)
Untreated
96.5 (9.53)
368.5 (21.15)
168.1 (19.50)
127.3 (5.56)
LSD 5%
36.24
Free SA
Put (2.5 mM)
1588.4 (10.4)
1634.6 (31.0)
1524.9 (117.9)
1572.1 (34.7)
Spd (5.0 mM)
1851.5 (74.3)
2851.5 (39.7)
3929.5 (204.7)
1959.6 (62.2)
Spm (2.5 mM)
2434.3 (51.6)
1668.8 (128.2)
4744.4 (90.9)
2574.5 (71.2)
Distilled water
2331.5 (63.2)
1147.2 (81.0)
2332.6 (207.6)
1659.5 (57.7)
Untreated
919.6 (90.1)
1133.8 (108.9)
3069.3 (61.5)
2019.9 (63.1)
LSD 5%
286.3
pg_0007
IQBAL et al. — Does polyamine-priming modulate growth under salt stress.
245
the synthesis of transportable forms of carbohydrates
(Kiseleva and Kaminskaya, 2002). Similarly, auxins
play a critical role in the regulation of plant growth and
development, including cell division and differentiation
(Thimann, 1977; Dietz et al., 1990; Normanly, 1997;
Davies, 1995; Pospi.ilova, 2003). Thus, the improvement
in growth only in Inqlab-91 due to priming with Put seems
to have been due to increased leaf ABA and IAA levels
under salt stress.
Generally, salinity caused a marked increase in Put
levels in both cultivars. Both arginine decarboxylase
(ADC) and ornithine decarboxylase (ODC) are responsible
for salt (NaCl) induced increase in Put biosynthesis
(Friedman et al., 1989; Lee and Chen, 1998). However,
plants show diverse responses to high saline conditions
with respect to PA biosynthesis: some systems show
increased PA levels while others show smaller increases or
even decreases (Krishnamurthy and Bhagwat, 1989; Lin
and Kao, 1995; Lee and Chen, 1998). In the present study,
in comparison with plants raised from untreated seeds,
pre-treatment with Put increased endogenous free Put
levels only in Inqlab-91 under saline conditions. Serrano
et al. (1997, 1998) have hypothesized that the increase in
PAs levels in response to stress is immediate, indicating
it is a consequence of stress rather than a protective
mechanism. After a period of time, even if the stress
situation continues, PA levels decrease and resemble those
from non-stressed plants. However, many contrasting
reports have suggested the change in PAs under salt
stressed conditions to be a protective mechanism (Roberts
et al., 1986; Kramer and Wang, 1989; Basra et al., 1994;
Basra et al., 1997; Kasukabe et al., 2004).
Pre-sowing seed treatment with Spd also improved
the grain yield of both cultivars under saline conditions.
However, the salinity induced decrease in leaf free IAA
was not alleviated by pre-sowing treatment with Spd
in either cultivar. It also caused a decrease in leaf ABA
levels and an increase in SA levels in both cultivars except
Inqlab-91, in which pre-sowing treatment with Spd did not
ameliorate salt-induced reduction in leaf SA levels under
saline conditions. SA is an endogenous growth regulator
which controls plant growth and development (Schettel
and Balke, 1983), photosynthesis and transpiration rates
(Pancheva et al., 1996), and ion uptake and transport
(Harper and Balke, 1981; Uzunova and Popova, 2000).
SA is also known to reverse salt stress-induced decreases
Table 5. Polyamine concentrations (μg g
-1
fresh weight) of two cultivars of hexaploid wheat at 2.84 dS m
-1
(control) or 15 dS m
-1
NaCl (salt) when the seeds were primed with solutions of different polyamines. Values in parentheses are SE of means (based on
four replicates).
Pre-sowing seed
treatments
MH-97
Inqlab-91
2.84 dS m
-1
15 dS m
-1
2.84 dS m
-1
15 dS m
-1
Putrescine
Put (2.5 mM)
8.66 (0.35)
22.37 (0.92)
10.52 (1.14)
24.24 (6.77)
Spd (5.0 mM)
28.16 (1.32)
35.71 (3.69)
15.17 (2.07)
17.99 (0.36)
Spm (2.5 mM)
15.88 (0.82)
23.87 (4.36)
8.04 (0.30)
15.14 (0.28)
Distilled water
10.72 (0.85)
16.48 (0.66)
13.40 (1.51)
21.60 (4.09)
Untreated
9.18 (0.20)
19.06 (0.50)
8.70 (1.44)
81.14 (2.77)
LSD 5%
6.32
Spermidine
Put (2.5 mM)
43.9 (15.5)
308.9 (24.8)
58.5 (4.35)
355.1 (80.1)
Spd (5.0 mM)
108.1 (0.37)
228.3 (27.7)
61.6 (17.6)
182.7 (18.7)
Spm (2.5 mM)
67.9 (3.91)
303.2 (66.5)
141.7 (24.6)
169.5 (71.6)
Distilled water
79.1 (11.3)
145.6 (18.9)
134.4 (12.6)
136.2 (15.2)
Untreated
151.5 (0.46)
342.2 (5.00)
175.7 (6.59)
240.1 (27.9)
LSD 5%
(S x V x T) = ns
Spermine
Put (2.5 mM)
6.31 (1.35)
44.47 (8.51)
4.19 (0.06)
25.42 (5.81)
Spd (5.0 mM)
6.80 (0.58)
12.53 (0.23)
2.80 (0.13)
5.89 (1.28)
Spm (2.5 mM)
6.54 (0.21)
17.73 (3.33)
8.72 (0.28)
9.13 (0.32)
Distilled water
5.63 (0.21)
7.40 (0.39)
14.46 (1.96)
6.38 (1.01)
Untreated
6.86 (0.38)
18.13 (0.36)
20.19 (6.48)
26.06 (2.74)
LSD 5%
(S x V x T) = ns
pg_0008
246
Botanical Studies, Vol. 47, 2006
in total and reducing sugar contents by providing a pool
of compatible osmolytes in the presence of sodium (Tari
et al., 2002). In the present study, the greater improvement
in grain yield in MH-97 could be attributed to increased
accumulation of SA under saline conditions. Furthermore,
the pattern of accumulation of PAs was not consistent in
both cultivars due to pre-sowing treatment with Spd under
saline conditions. For example, Spd proved to be the most
effective of all priming agents in accumulating leaf free
Put levels in MH-97 while it failed to cause accumulation
of leaf Put contents in Inqlab-91 under saline conditions.
Pre-sowing seed treatment with Spd did not affect leaf
free Spd and Spm levels in either cultivar under saline
conditions. This contravenes the findings reported in
some earlier studies, e.g., salt-tolerant rice cultivars
(Krishnamurthy and Bhagwat, 1989), Limonium sp.
(Bouchereau et al., 1999) and Bromus spp. (Santa-Cruz et
al., 1997). It may be possible that high levels of binding of
Put and Spd to proteins in actively dividing young tissues
and lower levels in mature non-dividing tissues might
have resulted in lower leaf free Put levels in Inqlab-91 and
non-significant levels of Spd and Spm in both cultivars
under saline conditions. Such accumulation and non-
accumulation of Spd and Spm has already been reported
in tissues of Chrysanthemum (Aribaud et al., 1995).
Spm was the most effective of all the polyamines
for seed priming because it maximized improvement in
grain yield in both cultivars under both saline and non-
saline conditions. Priming of seeds of Inqlab-91 with Spm
proved to be effective in reducing the drastic effect of salt
stress on plant hormonal balance. For example, salinity-
induced reduction in SA was alleviated by Spm priming
of Inqlab-91 under saline conditions. However, leaf free
ABA and SA levels in MH-97 were not affected by pre-
soaking with Spm under saline conditions. Thus, the
increase in grain yield is probably due to the increased rate
of translocation of photosynthates from leaves to grains
caused by pre-sowing Spm treatment. Such increases in
grain yield following hormone pre-treatments has already
been found in wheat (Lovell, 1971; Dewdeny and Mcwha,
1978; Ray and Choudhuri, 1981; Aldesuquy and Ibrahim,
2001).
Hydro-priming did not show a pronounced effect
on shoot growth or grain yield in either cultivar under
saline conditions, except for Inqlab-91, which showed
improved grain yield. Failure of hydro-priming to
improve germination and growth has already been
reported in wheat (Chaudhri and Wiebe, 1968) and
Kentucky bluegrass (Poa pratensis) (Pill and Necker,
2001). However, some reports do exhibit the considerable
effectiveness of hydro-priming on germination and
later growth in different plant species under both saline
and non-saline conditions, e.g., wheat (Kamboh et al.,
2000), and Acacia tortilis (Rehman et al., 1998). Hydro-
priming also failed to show a consistent effect on hormone
homeostasis in both cultivars. For example, it increased
leaf free IAA in MH-97, but decreased it in Inqlab-91,
under saline conditions. Moreover, the decrease or
increase in leaf free ABA and SA due to hydropriming was
not pronounced in either cultivar under saline conditions.
In conclusion, of the different priming agents used
in this study, Spm was most effective at alleviating the
adverse effect of salt stress on grain yield in both wheat
cultivars. Although all other priming agents were also
effective in inducing salt tolerance in wheat cultivars, the
beneficial effects of these priming agents were cultivar
specific. Physiologically, the beneficial effect of pre-
sowing treatment with Spm on grain yield in both cultivars
may be attributed to altered hormonal balance.
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Botanical Studies, Vol. 47, 2006
以多元胺預先處理種子能否½節發芽後處於鹽逆境六倍體小
麥之生長及若干植物生長½節物½之含量高低?
Muhammad IQBAL, Muhammad ASHRAF, Shafiq-Ur-REHMAN, and Eui Shik
RHA
1
Department of Botany, University of Agriculture Faisalabad 38040, Pakistan
2
Faculty of Biological Resources Science, College of Agriculture
Chonbuk National University, Chonju 561-756, Republic of Korea
3
Life Resources Science, Plant Genetics & Breeding, Sunchon National University
Sunchon, 540-742, Republic of Korea
  種子發芽前,分別以多元胺 (2.5 mM putrescine, 5.0 mM spermidine 及 2.5 mM spermine) 預先處理
對之後幼苗之生長及內在生長½節物½之量的影響以兩種春小麥 (Triticum aestivum L.) 品系 MH-97 及
Inqlab-91 測試。實驗組及對照組之種子均種在鹽度為 15 dS m
-1
之實驗田。雖然對兩種供試小麥之½
粒產量受鹽害之減低情形,三種多元胺均可改善;spermine 之效果最好。三種多元胺均對 MH-97 葉內
自由型離層酸 (ABA) 之減少 (實驗組比對對照組) 均有所改善,但以 putrescine 之效果最佳。相對地,
putrescine 在品系 Inqlab-91 內則最有效地增加葉內自由型 indoleacetic acid, spermidine 及 spermine 則在
兩種供試品系處於鹽逆境下有效地增強葉中之水楊酸 (salicylic acid) 含量。以 spermidine 預處理之種
子,之後在鹽逆境下葉中自由型之 putrescine 則以 MH-97 較對照組為高。相反地,在品系 Inqlab-91 所
有三種多元胺之種子處理均導致之後發芽幼苗在鹽逆境下均比對照組有較低之葉中自由型 putrescine,
兩種品系之種子經 putrescine 預處理後,之後發芽之植物體在鹽逆境下均有最大量之葉中自由型
spermidine。整體而言,多元胺之種子預處理其好處依品種而異。自生理觀點而言,種子以 spermine 預
處理之所以在鹽逆境下能改善兩種供試品種之½粒產量,可能歸因於植物激素的平衡之改變。
關鍵詞:植物生長½節物½預處理;植物生長½節物½;發芽前之預處理;鹽度;高鹽耐受性。