Jo urnal of Radiation Research
and Applied Sciences
J. Rad. Res. Appl. Sci., Vol. 2, No. 3, pp. 549-562 (2009)
Using of Hydrogel to Increase Maize Salt Tolerance
O. S. Hussein, A. F. Khafaga & N. Hamideldin
Natural Product Department,
National Centre for Radiation Research and Technology, Atomic Energy Authority,
P.O Box 29, Nasr City, Cairo, Egypt.
E-mail address: omyma_mah@yahoo.co.uk & n.hamideldin@yahoo.com
Received: 31/03/2009, Accepted: 01/07/2009.
ABSTRACT
Seeds of two cultivars (Giza 122 and 129) of Zea mays L. were sown in pots.
Pots were divided into two sets; soils of one mixed with hydrogel and the other
set considered as control. After germination, pots were irrigated by tap water or
by 4500 ppm NaCl solution.The results indicated that salt stress reduced growth
characters significantly. Addition of hydrogel to the soil improved growth
character especially in cultivar 129, hydrogel ameliorates the harmful effect of
salt on plant. In the two cultivars, proline contents increased under salt stress
but the presence of hydrogel reduced these contents significantly. Also, the
presence of hydrogel appeared to reduce phenol content significantly under salt
stress in cultivar (129) or insignificantly in cultivar (122).The appearance or
disappearance of protein bands and the alterations in peroxidase and esterase
pattern could be used as molecular marker for salt stress and hydrogel.
INTRODUCTION
The development of mankind has reached the point that new resources
need to be trapped in order to fill our basic needs for food, feed and freshwater.
It is foreseeable that freshwater resource will become limited, and that currently
used agricultural irrigation systems will steadily increase soil salinity in the near
future. To date, 30% of the farmland under irrigation cannot be used any more
for efficient crop farming due to salt accumulation. Mediterranean and
subtropical dry regions perform physiological, biochemical and molecular
genetically experiments with potential cash crop halophytes in order to identify
relevant parameters and to provide data for future breeding projects (1). Salt
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tolerance is complex genetically and physiologically. Tolerance to salinity stress
can be considered to contain three main components: Na+ exclusion, tolerance
to Na+ in the tissues and osmotic tolerance. To date, most experimental work on
salinity tolerance in cereals has focused on Na+ exclusion due in part to its ease
of measurement. It has become apparent, however, that Na+ exclusion is not the
sole mechanism for salinity tolerance in cereals, and research needs to expand
to study osmotic tolerance and tissue tolerance (2). Tolerance often shows the
characteristics of a multigenic trait, with quantitative trait loci (QTLs)
associated with tolerance. Physiologically salt tolerance is also complex, with
halophytes and less tolerant plants showing a wide range of adaptations.
Attempts to enhance tolerance have involved conventional breeding programs 3.
Salinity reduced the leaf area, dry matter weight, and plant height of maize
hybrid 704 (4). Polymeric soil conditioners were known since the 1950s (5).
However, their wide commercial application failed even though the scientific
basis for their use was quite well established. These polymers were developed
to improve the physical properties of soil in view of: increasing their waterholding capacity, increasing water use efficiency, enhancing soil permeability
and infiltration rates reducing irrigation frequency, reducing compaction
tendency, stopping erosion and water run-off and increasing plant performance
(especially in structure less soils in areas subject to drought). Polyacrylamide
(PAM) is one of the most widely employed soil conditioner. Anionic character is
imparted to polyacrylamide which is basically non ionic, either by
copolymerization with an unsaturated acid such as acrylic acid or by partial
hydrolysis of amide groups. More recently, polyelectrolyte such as
acrylamide/acrylate copolymers have attracted much attention as they have been
shown to be most effective in improving the physico-chemical properties of
soils. Polyacrylamide has also been used in combination with natural
polysaccharides for soil-conditioning purposes. The performance of the gel on
plant growth depends on the method of application. It was shown that spraying
the hydrogels as dry granules or mixing them with the entire root zone is not
effective(6). Better results seem to be obtained when the hydrogels are layered,
preferably a few inches below soil surface. Soluble and fusible polymer can be
transformed into a polymer gel by gamma-ray irradiation(7). This work aims to
use hydrogel for increasing maize tolerance to salt stress.
MATERIALS AND METHODS
Seeds of two varieties (Giza 122 and Giza 129) of Zea mays L. were kindly
O. S. HUSSEIN et al. / J. Rad. Res. Appl. Sci., Vol. 2, No. 3 (2009)
551
obtained from, the Agricultural Research Center, Ministry of Agriculture, Giza,
Egypt. Seeds were sown in pots (50 cm) filled with (2:1) "washed" sand and
clay soil (v/v). Pots were divided into two sets; soils of one set mixed with 10%
hydrogel, which layered few inches below soil surface. Hydrogels
(polyacrylamide/Na-acrylate) prepared by radiation, obtained from hydrogel lab
in National Center for Radiation Research and Technology, Nasr City, Cairo,
Egypt. Seeds were sown in sand/clay soil without gel and considered as control,
ten replicates were carried out. After germination, two irrigation regimes were
used. One of which, was irrigated with tap water. The other, subjected to saltstress, using sodium chloride solution (4500 ppm), irrigation was carried out
weekly. Plants were collected after 50 days from sowing. The studied characters
were: Growth criteria, contents of proline, contents of phenol and molecular
genetic markers (protein patterns and two isozymes).
Growth Parameters
length of shoot (cm), stem diameter (cm), number of leaves, leaf area
(cm ), fresh and dry weight (g) in the end of the experiment. The data were
subjected to the standard analysis of variance procedure. The differences
between the means were compared by Duncan's multiple range tests(8).
2
Proline content
Proline was determined using the method of Bates et.al. (9).
Phenol content
Total phenol contents were determined according to the method described
by Malik and Singh (10).
Protein electrophoresis
Sodium dodycil sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
was performed according to the method previously described (11), as
modified12).Fully expanded leaf samples (at a constant node from the top) were
taken from each cultivar under normal and salt stress conditions, in the absence
or presence of hydrogel.
Isoezyme electrophoresis
Native-polyacrylamide gel electrophoresis (Native-PAGE) was conducted
to identify isozyme variations among studied cultivars as stated before (13). Fully
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552
expanded leaf samples (at a constant node from the top) were taken from each
cultivar under normal and salt stress conditions, in the absence or presence of
hydrogel, were used separately for isozymes extraction.
RESULTS
Growth characters
Using sodium chloride solution in irrigation decreased measured growth
criteria significantly. Meanwhile mixing hydrogel with soil improve growth
character in both cultivars used as shown in Table 1. On the other hand (Fig. 1)
ascertain that polyacrylamide/Na-acrylate ameliorate the harmful effect of salts,
as appeared in picture (1 & 5). While in Fig. 1 (3 & 7) salinity stress conditions
resulted in weak growth of plant and leaf tips become burned. Also, It was
noticed that hydrogel improve growth characters in water irrigation treatment as
compared with its corresponding control. It was observed that cultivar Giza 129
was more tolerate to salinity than cultivar Giza 122 as declare in Table 1 and
Fig. 1.
Table 1. Vegetative characters for two maize cultivars (Giza 122 and129), sown
in the presence or absence of hydrogel and irrigated by H2 O or
NaCl solution.
Growth criterea
Shoot
length
(Cm)
Stem
diameter
(Cm)2
C122+NaCl
40.14C
0.84 A
6.8c
C122+H2O
53.76B
0.90A
8.2AB
111.9BC 16.74AB
2.5BC
C129+NaCl
39.46C
O.78 A
8.0AB
53.14D
6. 32C
1.5CD
C129+H2O
64.1A
1.38 A
8.8A
170.8A
20.44 A
4.54A
C122+NaCl
35.8C
0.74 A
7.6BC
44.6D
4.5C
1.78BCD
C122+H2O
52.8 .B
0.84A
8.0AB
99.3BC
13.94B
2.94B
C129+NaCl
35.1C
0.66 A
6.8c
51.08D
3.96C
1.12D
C129+H2O
58.32AB
1.04A
8.6AB
157.65A 19.82A
4.12AB
Treatments
Fresh
Number Leaf area
weight
2
of leaves (Cm)
(g)
77.14.3CD
5.6C
Dry
weight
(g)
1.48CD
Gel present
Gel absent
Different letters indicate significant variation.
O. S. HUSSEIN et al. / J. Rad. Res. Appl. Sci., Vol. 2, No. 3 (2009)
Giza 122
553
Giza 129
NaCl+ Gel
H2O+Gel
NaCl+Gel
H2O+Gel
NaCl
H2O
NaCl
H2O
Fig. 1. Plants of two maize cultivar (Giza 122 and 129), sown in the presence or absence
of hydrogel and irrigated by H2O or NaCl solution.
Proline content
Proline concentration was increased under salt stress but the presence of
hydrogel reduced its concentration significantly in the two cultivars (Table 2). The
increasing in proline levels at high salinity concentration might be one of the earliest
metabolic responses triggered in the translocation pathway that links the perception of
many environmental stresses to the elicitation of physiological responses at the
cellular level(14).
Total Phenol content
Phenol concentration increased under salt stress (Table 2) but the presence of
hydrogel reduced it under salt stress significantly in cultivar (129) but insignificantly
in cultivar (122).
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554
Table 2. Proline and total phenol contentes of two maize cultivar (Giza 122 or
129) sown in the presence or absence of hydrogel and irrigated by
H2 O or NaCl solution.
Chemical analysis
Proline
Total Phenol
100.27E
113.36D
155.3C
163.54B
102.7E
72.12F
248.38A
125.35D
316AB
313.3B
302C
312.7B
322AB
315.3B
317.3AB
304.7C
Treatments
Gel present
Gel absent
NaCl+ C122
C122+H2O
C129+NaCl
C129+H2O
C122+NaCl
C122+H2O
C129+NaCl
C129+H2O
Different letters indicate significant variation.
Protein profile
SDS- PAGE patterns of water-soluble protein fraction for the two cultivars of
maize (Fig. 2 and Table 3), exhibited a maximum number of 35 bands which were not
necessarily present in all samples but were different in density and intensity. The
protein patterns of the cultivar 122 differ from that of cultivar 129 in the presence or
absence of hydrogel and under control or salt stress condition.
Fig. 2. SDS-PAGE profiles for leaf protein (water soluble fraction) of two maize
cultivar (Giza 122and 129), sown in the presence or absence of hydrogel
and irrigated by H 2O or NaCl solution. 1= C122+NaCl, 2=C122+H2O,
3=C129+NaCl, 4= C129+H2O in presence of hydrogel and 5=C122+ NaCl,
6=C122+H2O, 7=129+NaCl, 8=C129+H2O in absence of hydrogel,
M=marker.
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555
Table 3. Band number and presence or absence records of water soluble Protein SDSPAGE of two maize cultivar (Giza 122 and 129), sown in the presence or
absence of hydrogel and irrigated byH2O or NaCl solution.
Band No.
Marker
1
2
3
4
5
6
7
8
1
115.55
+
2
114.738
+
+
+
3
113.931
+
+
+
4
105.92
+
+
+
+
5
104.43
+
+
+
6
96.409
+
+
7
94.612
+
+
+
+
8
90.69
+
+
+
+
+
9
86.727
+
+
10
85.110
+
+
+
11
84.115
+
+
+
12
73.389
+
+
+
+
+
+
+
+
13
65.708
+
14
65.246
+
+
+
15
64.788
+
+
16
62.102
+
+
+
+
+
+
17
58.008
+
+
+
+
18
55.47
+
+
+
+
19
51.815
+
20
49.901
+
+
+
21
48.741
+
+
+
+
22
46.941
+
23
45.207
+
+
+
24
42.526
+
25
40.955
+
+
+
+
+
26
35.733
+
27
32.144
+
+
+
+
28
32.397
+
+
+
29
28.916
+
+
+
30
25.890
+
+
+
+
+
+
+
31
20.415
+
32
18.49
+
+
+
+
+
+
33
17.195
+
+
+
+
34
16.061
+
+
+
+
35
14.516
+
1= C122+ NaCl, 2= C122+H2O, 3= C129+NaCl, 4= C129+H2O in presence of
hydrogel and 5= C122+ NaCl, 6 =C122+H2O, 7= C129+NaCl, 8=C129+H2O in
absent of hydrogel, M=marker.
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Isozymes electrophoresis
Peroxidase Isozymes
A maximum number of five bands were shown in zymogram and diagram of
peroxidase isozymes for the two cultivars of maize in Fig. 3. Cultivar122 exerts no
change in the number of bands when compared between treatment used and the
control. There were changes in intensity of bands 3, 4 and 5 when comparing bands
of salt stress and the control in the presence or absence of hydrogel. In cultivar129,
the salt with hydrogel treatment (lane 3) and the salt treatment (lane 7) showed the
same bands.In the control with hydrogel treatment (lane 4) and the control treatment
(lane 8) showed that the band number four appeared in the first conditions. While, the
salt with hydrogel treatment (lane 3) and the control with hydrogel treatment (lane 4)
showed that the band number five was absent in the control conditions in the presence
or absence of hydrogel, while it was appeared in the salt conditions in the presence or
absence of hydrogel, so it can be used as a negative molecular marker for salt stress.
Fig. 3. Peroxidase and Esterase isozyme of two maize cultivar (Giza 122 and 129)
sown in the
presence or absence of hydrogel and irrigated by
H2O or NaCl solution. 1=C122+NaCl, 2=C122+H2O, 3=C129+NaCl,
4= C129+H2O in presence of hydrogel and 5= C122+ NaCl, 6 =C122+H2O,
7= C129+NaCl, 8=C129+H2O in absent of hydrogel, M=marker.
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557
Esterase Isozymes
A maximum number of four bands were shown in diagram and zymograms
of esterase isozymes for the two cultivars of maize (Fig.3). In cultivar122, the
hydrogel induced changes in the number and intensity of bands (lane 1 and 2) as
compared by control (lane 6), the band number two was disappeared in the
presence of hydrogel. In cultivar129, the band number in the presence of
hydrogel under salt irrigation (lane 3) gain the same pattern as the control under
water irrigation (lane8), but the intensity of bands was different.
The salt with hydrogel treatment (lane 3) caused the new appearance of
bands 3 and 4, while the control with hydrogel treatment (lane 4) showed the
disappearance of these two bands. So, these bands can be considered as positive
markers for the salt stress. Also, the salt treatment (lane 7) and the control
treatment (lane 8) showed that the bands number 3 was present in the control
conditions but it was absent in the salt conditions. So, it can be considered a
negative marker for salt stress.
DISCUSSION
The results obtained in concern to vegetative characters are in agreement
with Hussein (15), who found that salinity stress conditions reduced plant growth
and yield. This reduction is in consistent with the fact that salinity induces
accumulation of certain ions and deficiency of others and in the mean time
lowers the external water potential below that in the cell. Hydrogel reduced the
effect of salt stress on growth character of plant which sown in the soil mixed
with it but insignificantly.The addition of Polyacrylamide/sodium alginate
(PAAm/Na-alginate) copolymer in small quantities to sandy soil increased its
ability to retain water(16). Researchers(6, 17) have reported that the use of
hydrogels increased the amount of available moisture in the root zone, thus
implying longer intervals between irrigations. It must be pointed out that the
polymers do not reduce the amount of water used by plants. The water-holding
capacity depends on the texture of the soil, the type of hydrogel and particle size
(powder or granules), the salinity of the soil solution and the presence of ions.
Cross-linked polyacrylamides hold up to 400 times their weight in water and
release 95% of the water retained within the granule to growing plants.
Hydrogels help in reduceing water stress of plants resulting in increased growth
and plant performance (18-20).
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The increases in concentrations of phenols and proline under salt stress
condition are agreed with many searchers. Many studies suggest that proline is a
protective agent of enzyme and membrane (21). Moreover, Maiti et al., (22)
demonstrated that proline increased in all barley genotypes with the increase in
salt stress. They added also that proline is a good parameter to evaluate the
effect of salinity. Concerning this Hamada (23) revealed that salinity and water
deficit induces accumulation of proline in seedlings. It has been concluded that
total free amino acids, free proline and protein increased with increasing salinity
concentration (p> 0.01) particularly in the presence of hydrogel polymer
(p>0.05) (24). It has been observed that saline (NaCl) stress in barley seedlings
causes an increase in total phenolic compounds, flavonoids and enhancement of
peroxidase and indoleacetic acid oxidase activities and consequent decrease in
growth rate (25). Salt stress induced changes in the content of phenolic and
flavonoids compounds, as well as pungency levels (26).
The appearance or disappearance of protein bands, peroxidase and esterase
isozymes under salt stress are agreed with many authors (27). Increase in the
concentration or synthesis of new proteins were critical for plant adaptation to
unfavorable condition (28) .Also,changes in the concentration, disappearance or
appearance of new protein bands under salt stress were determined (29).
Peroxidase isozymes activities increased in plant tissues as a defensive response
to salt stress-dependent formation of H2 O2 and of superoxides (30).There were
many puplications could use the appearance or disappearance of bands in
peroxidase isozyme electrophoresis as molecular marker for salt stress in maize
and wheat (31, 32). The enhancement of the esterase isozyme bands in shoots
pattern of maize genotype (Zea mays L) single cross 124 was grown in water
culture in presence or absence of 150 mM NaCl for 15 days (33).
CONCLUSION
Hydrogel reduced the effect of salt stress on growth character of plant and
decrease proline and total phenol contents. The appearance or disappearance of
bands in protein, peroxidase and esterase isozymes can be used as molecular
markers for salt stress and hydrogel.
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‫ﻣﺠﻠﺔ اﻟﺒﺤﻮث اﻹﺷﻌﺎﻋﯿﺔ‬
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‫ﻣﺠﻠﺪ ‪ 2‬ﻋﺪد ‪ 3‬ص ص ‪(2009) 562 – 549‬‬
‫اﺳﺘﺨﺪام اﻟﮭﯿﺪروﭼﯿﻞ ﻟﺰﯾﺎدة ﺗﺤﻤﻞ اﻟﺬرة ﻟﻠﻤﻠﻮﺣﺔ‬
‫أﻣﯿﻤﮫ ﺳـﯿﺪ ﺣﺴـﯿﻦ ‪ ,‬أﺣﻤﺪ ﻓﺎﯾﺰ ﺧﻔﺎﺟﺔ و ﻧﮭـﻠﺔ ﺣﻤـﯿﺪاﻟﺪﯾﻦ‬
‫ﻗﺴﻢ اﻟﻤﻨﺘﺠﺎت اﻟﻄﺒﯿﻌﯿﺔ‪ ،‬اﻟﻤﺮﻛﺰ اﻟﻘﻮﻣﻲ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﯿﺎ اﻹﺷﻌﺎع‪،‬ھﯿﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﯾﺔ‪ ،‬اﻟﻘﺎھﺮة‪ ،‬ﻣﺼﺮ‪.‬‬
‫زرﻋﺖ ﺑﺬور ﺻﻨﻔﯿﻦ ﻣﻦ اﻟﺬرة ) ﺟﯿﺰة ‪ (129 ، 122‬ﻓﻲ أﺻﺺ ﺑﻼﺳﺘﯿﻜﯿﮫ‪ .‬وﻗﺪ ﻗﺴﻤﺖ إﻟﻰ‬
‫ﻗﺴﻤﯿﻦ ‪ ،‬وﺿﻊ ﻓﻲ أﺣﺪھﻤﺎ اﻟﺘﺮﺑﺔ ﻣﺨﺘﻠﻄﺔ ﻣﻊ اﻟﮭﯿﺪروﭼﯿﻞ واﻷﺧﺮى ﺑﺪون ھﯿﺪروﭼﯿﻞ ﻛﻨﺘﺮول‬
‫ﻟﻠﻤﻘﺎرﻧﺔ‪ .‬ﺑﻌﺪ اﻹﻧﺒﺎت ﺗﻢ اﻟﺮي ﺑﻤﺎء اﻟﺼﻨﺒﻮر أو ﺑﻤﺤﻠﻮل ‪ 4500‬ﺟﺰء ﻓﻲ اﻟﻤﻠﯿﻮن ﻣﻦ ﻛﻠﻮرﯾﺪ‬
‫اﻟﺼﻮدﯾﻮم‪ .‬ﺗﺸﯿﺮ اﻟﻨﺘﺎﺋﺞ إﻟﻰ اﻧﺨﻔﺎض اﻟﻨﻤﻮ وﺧﺼﺎﺋﺼﮫ إﻟﻰ ﺣﺪ ﻛﺒﯿﺮ ﻓﻲ اﻷﺻﺺ اﻟﻤﺮوﯾﺔ‬
‫ﺑﺎﻟﻤﺤﻠﻮل اﻟﻤﻠﺤﻲ‪ .‬وﻗﺪ أدى إﺿﺎﻓﺔ اﻟﮭﯿﺪروﭼﯿﻞ ﻟﻠﺘﺮﺑﺔ إﻟﻰ ﺗﺨﻔﯿﻒ اﻟﺘﺄﺛﯿﺮ اﻟﻀﺎر ﻟﻠﻤﻠﺢ ﻋﻠﻰ ﻧﻤﻮ‬
‫اﻟﻨﺒﺎﺗﺎت اﻟﺘﻲ زرﻋﺖ ﻓﻲ اﻟﺘﺮﺑﺔ اﻟﻤﺨﺘﻠﻄﺔ ﻓﻲ ﻛﻼ ﻣﻦ اﻟﺼﻨﻔﯿﻦ اﻟﻤﺴﺘﺨﺪﻣﯿﻦ وﺧﺎﺻﺔ ﻓﻲ اﻟﺼﻨﻒ‬
‫ﺟﯿﺰة ‪ .125‬أﻣﺎ اﻟﺒﺮوﻟﯿﻦ ﻓﻘﺪ ازداد ﻋﻨﺪ اﻟﺮي ﺑﻤﺤﻠﻮل اﻟﻤﻠﺢ ﻓﻲ ﻛﻼ ﻣﻦ اﻟﺼﻨﻔﯿﻦ اﻟﻤﺰروﻋﯿﻦ‬
‫ﻓﻲ اﻟﺘﺮﺑﺔ ﺑﺪون ھﯿﺪروﭼﯿﻞ )ﻛﻨﺘﺮول( ‪ ،‬وﻟﻜﻦ وﺟﻮد اﻟﮭﯿﺪروﭼﯿﻞ ﻋﻤﻞ ﻋﻠﻰ ﺧﻔﺾ اﻟﺒﺮوﻟﯿﻦ‬
‫ﺑﺸﻜﻞ ﻣﻠﺤﻮظ ‪ ،‬وﻋﻤﻞ أﯾﻀﺎ ﻋﻠﻰ ﺧﻔﺾ ﺗﺮﻛﯿﺰ اﻟﻔﯿﻨﻮﻻت اﻟﻜﻠﯿﺔ ﻓﻲ اﻟﻤﻌﺎﻣﻼت اﻟﻤﺮوﯾﺔ ﺑﺎﻟﻤﻠﺢ‬
‫ﺧﺎﺻﺔ اﻟﺼﻨﻒ ) ‪ .(129‬وﯾﻤﻜﻦ اﺳﺘﺨﺪام ﻇﮭﻮر اﻟﺤﺰم أو اﺧﺘﻔﺎؤھﺎ أﺛﻨﺎء اﻟﺘﻔﺮﯾﺪ اﻟﻜﮭﺮﺑﻲ ﻟﻠﺒﺮوﺗﯿﻦ‬
‫أو ﻣﺸﺎﺑﮭﺎت إﻧﺰﯾﻤﻰ اﻟﺒﺮوﻛﺴﯿﺪﯾﺰ واﻹﺳﺘﯿﺮﯾﺰ ﻛﺪﻻﺋﻞ ﺟﺰﯾﺌﯿﮫ ﻟﻠﻤﻠﻮﺣﺔ أو اﻟﮭﯿﺪروﭼﯿﻞ‪.‬‬