7965 27953 2 PB PDF
7965 27953 2 PB PDF
7965 27953 2 PB PDF
RESEARCH ARTICLE
(on average, one-third of the world’s population, ranging fortification (Ahmad et al., 2012). Increasing the Zn
from 4 to 73 % in different countries), and related health content in food crops may be a good strategy to overcome
problems such as impairment in physical development, its deficiency in people in developing countries. Many
stunting in children, susceptibility to infectious diseases, researchers endeavoured to chalk out various strategies
increased morbidity and mortality, poor birth outcome of its better supplementation, which included; Zn
in women and poor immune system and brain function application to soil in the form of ZnSO4, Zn chelates,
(Hotz & Brown, 2004; Black et al., 2008; Cakmak, soil and foliar spray with different Zn compounds and
2008). seed priming (Singh & Abrol, 1985; Yilmaz et al., 1997;
Cakmak et al., 1998; Amar et al., 2000; Khattak et al.,
Alkaline soils with an apparently sufficient Zn level 2006; Khan et al., 2008; Maqsood et al., 2009). Realising
(>1 mg kg-1) may show reduced Zn availability to crops the importance of Zn in plant and human nutrition and
due to (a) Zn affinity towards adsorption/fixation on the the problems associated with its availability to plants in
clay adsorption sites, and (b) the high pH of soil (pH 8.0), alkaline soils, the present study was conducted to assess
which might have helped in the formation of unavailable the effect of various modes and levels of Zn application
forms of Zn as hydroxides. Zn has greater affinity for on wheat yield and protein content in order to evolve a
adsorption on clay and also it tends to make unavailable cost effective strategy to address Zn deficiency in cereals
zinc hydroxides due to increased pH (Khattak & Pulford, grown in alkaline soils.
1999). In such soils crops have responded positively to
Zn application by showing increased protein content in METHODS AND MATERIALS
grain as well as soil residual Zn concentration (Khattak
et al., 2006). A portion of additional Zn probably helps The present experiment was conducted in the greenhouse
in satisfying the adsorption sites and some of it might of the Institute of Biological and Life Sciences (IBLS),
be retained as available Zn to plants in solution form. University of Glasgow, UK during 2009 – 2010.
Besides fixation at adsorption sites and the formation of Wheat variety ‘Siran 2008’ obtained from the Institute
zinc hydroxide in alkaline conditions, low availability of of Biotechnology and Genetic Engineering (IBGE),
Zn is also attributed to a number of soil and environmental University of Agriculture, Peshawar, Pakistan was used
factors including low soil organic matter, calcareous for testing the effect of 0, 5, 10 and 15 kg Zn ha-1 (0.0,
nature of soil, water logging and arid climate (Morvedt 2.5, 5.0 and 7.5 mg Zn kg-1 soil in the form of ZnSO4)
et al., 1991; Tandon, 1995; Cakmak et al., 1998). as soil addition and 0, 0.5 and 1 % ZnSO4 solution
(0, 5 and 10 g ZnSO4 per litre of de-ionised water) as
Estimates suggest that 25 % of the world’s population foliar application on wheat yield and protein content
is at risk of Zn deficiency (Maret & Standstead, 2006). in alkaline soil. A total of 12 treatment combinations
Several approaches have been made to overcome (Table 1) arranged in RCB design with 4 replications
Zn deficiency in humans and these include Zn were applied in pots filled with 2 kg sandy loam soil of
supplementation, food diversification as well as food known physico-chemical characteristics (Table 2).
Table 1: Details of Zn treatments as soli application and foliar spray applied to wheat crop during experiment
December 2015 Journal of the National Science Foundation of Sri Lanka 43(4)
Soli and foliar Zn effect on yield and protein content in wheat 305
Initially, seed grains were soaked in distilled water on background electrolyte. The solution in the tubes was
a double layered towel paper pad tray and kept in the then analysed by the ICP (OES).
growth chamber under 18 ‒ 22 oC, 9 hrs light/15 hrs dark,
60 ‒ 70 % humidity and light 150 µMole m-2 S-1. After Grain protein
germination, 6 healthy seedlings were transferred to each
pot and kept in a glass house under day and night lengths To assess the wheat grain protein content, 0.5 g of
of 16 and 8 hrs, respectively with mercury reflector wheat flour samples were taken in triplicate to which,
lighting of 400 watts/base E40. 0.25 mL protein buffer was added, mixed by Vortex
Mixer, and then centrifuged for 15 min at 13 K rpm.
Fertiliser at recommended levels i.e. N:P2O5:K2O at The supernatant was removed for spectroscopic analysis
the rate of 120:65:110 kg ha-1 (60:32.5:55 mg kg-1 soil) at E-280 nm by ultraviolet spectrophotometer in
was added to each treatment in solution form in two mg/mL i.e. approximately equal to 1 optical density (OD).
splits. Zn as soil treatment or foliar spray was applied in The absorbance was multiplied by the dilution factor and
required amount in the form of ZnSO4. To assess the yield the protein percentage was calculated as follow;
trend with Zn application at lower doses of NPK, the same
layout was repeated with half the recommended NPK % protein = absorbance reading × dilution × 100
levels (N:P2O5:K2O at the rate of 60:32.5:55 kg ha-1). 1000
Water was provided through plastic trays beneath the
pots to avoid nutrient leaching. At boot stage, two The effect of Zn on the composition of proteins
in wheat grain was studied using sodium dodecyl
healthy plants were cut at the base for analysis of ion
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
retention by plants. At maturity, spikes were removed
for grain yield and 1000 grain weight determination,
while the remaining plants were harvested to determine RESULTS AND DISCUSSION
the total biomass yield.
Soil samples were analysed before sowing for various
Soil analysis physico-chemical characters. The results (Table 2) revealed
that the soil texture was a sandy loam, free from excess salinity
The composite soil sample was subjected to various and alkaline in reaction (pH > 7.0). The soil was moderate in
physical and chemical analyses. Existing standard organic matter content (OM > 2 %), and sufficient in macro
methods were followed for the determination of soil (N, P, K, S, Ca and Mg) and micronutrients (Fe, Zn, Mn, Cu
pH (McClean, 1982) and electrical conductivity (EC) and B contents) (Table 2).
(Rhoades, 1996) using 1:5 soil and water suspension.
The texture and organic matter content were determined Table 2: Physico-chemical characters of the
by Bouyoucous method (Bouyoucous, 1962) and Black experimental soil before sowing
procedures (1965), respectively.
Property Mean values
The nutrient ions were determined by Induced
Textural class Sandy loam
Coupled Plasma-Optical Emission Spectrometer (ICP- pH (1:5) 8.52
OEC; Perkin Elmer Optima 4300 DV). A 10 g soil sample EC (1:5) (dS m-1) 0.398
was taken in triplicate in 250 mL Erlenmeyer flask to Organic matter (%) 2.34
which 20 mL of ammonium bicarbonate di-ethylene N (%) 0.041
triamine penta acetic acid (AB-DTPA) solution was P (mg kg-1) 34.44
added. Flasks were shaken on a reciprocating shaker K (mg kg-1) 235.35
at 180 rpm per min for 15 min. The suspension was Ca (mg kg-1) 197.2
filtered through a filter paper No. 42 and 5 mL of the Mg (mg kg-1) 33.38
AB-DTPA extract from each sample was transferred S (mg kg-1) 76.6
into 15 mL falcon tubes to which 0.5 mL concentrated Zn (mg kg-1) 1.95
HCl was added and mixed on a rotary shaker for 15 Cu (mg kg-1) 1.79
Mn (mg kg-1) 138.8
min to drive off CO2. After this, 4.5 mL of distilled
Fe (mg kg-1) 216.4
water was added to each tube in order to bring the pH
B (mg kg-1) 0.618
of the sample to be in accordance with the pH of the
Journal of the National Science Foundation of Sri Lanka 43(4) December 2015
306 Sabir Gul Khattak et al.
Application of Zn, both as soil and foliar treatments, to imbalanced use of fertilisers and micronutrient
significantly (p < 0.05) increased the wheat grain deficiencies, specially of zinc and boron. Qayyum et al.
yield and 1000 grain weight while the effect on wheat (1987) have also reported a yield loss of 27.8 % in maize
biological yield was not significant. The treatment that when Zn was omitted from the treatments involving NPK
received 5 kg ha-1 ZnSO4 as soil treatment + 1.0 % ZnSO4 and micronutrients.
as foliar spray recorded 55 % higher grain yield while that
with 15 kg ha-1 ZnSO4 as soil treatment + 1.0 % ZnSO4 A positive correlation (R2 = 0.39) was observed
as foliar spray recorded 48 % higher grain yield over the between the Zn uptake by the plant at boot stage and
control. However, the grain yield in both these treatments total grain yield (Figure 1). Takkar and Randhawa (1978)
was not significantly different (Table 3). These results have reported a similar positive correlation between the
are in line with those reported by Alloway (2008) who Zn concentration and grain yield and suggested that a
states that Zn deficiency in wheat reduces grain yield. Zn concentration below 30 mg Zn kg-1 plant dry matter
Kausar et al., (2001) have reported that despite a sharp would result in yields not attaining their maximum
increase in fertiliser use in Pakistan, the corresponding potential for the site, and the value of 20 mg Zn kg-1 is
increase in yield was only 15 %, which was ascribed the lower critical concentration. Previous studies (Viets,
December 2015 Journal of the National Science Foundation of Sri Lanka 43(4)
Soli and foliar Zn effect on yield and protein content in wheat 307
1966; Rashid & Fox, 1992) have suggested the critical value Different modes and levels of Zn demonstrated variable
of 15 mg Zn kg-1 as a general value for the interpretation of effects on 1000 grain weight, and both increase and
grain analyses, and more recent work has shown a value of decrease in 1000 grain weight was observed with respect
10 mg Zn kg-1 (Brennan et al., 1993). The treatment of such to the control due to Zn application (Table 3). The highest
deficiencies with Zn fertilisers or foliar sprays can increase 1000 grain weight (50.6 g) was recorded in the treatment
receiving 15 kg ZnSO4 as soil and 0.5 % ZnSO4 solution
the yield and also improve the plant’s resistance to ‘foot rot’
as foliar spray. 1000 grain weight was statistically
fungus (F. graminarum) (Gooding & Davies, 1997). different among the levels of Zn applied as soil addition
with the best results shown by 15 kg ZnSO4 ha-1 (48.3 g),
The grain and biomass yields were plotted (Figure 2) while on average over the levels of soil application,
to observe their trends with the increasing levels of Zn different concentrations of foliar application did not show
application. The line for grain yield in the graph showed any significant change in 1000 grain weight. Previous
a significant response (R2 = 0.51) to increasing levels investigations (Khattak et al., 2006) have reported an
of Zn, while the biomass was independent (R2 = 0.06) increase in the grain yield of maize and wheat with Zn
of increasing levels of Zn. The biomass yield was not addition at the rate of 5 kg ha-1 or 2 times as foliar spray
affected by 50 % Zn treatments, while the remaining of 0.5 % ZnSO4, and this treatment showed an economical
50 % increased positively. This is in conformity with value greater than all the other concentrations added to
maize. Yilmaz (1997) has shown increased wheat grain
the results obtained by Rengel and Graham (1996), who
yield by 260 % and 124 %, with Zn application as soil
reported that the relative production of root and shoot dry
addition and foliar application, respectively.
matter at deficient compared to sufficient Zn supply for
two wheat genotypes tested had no difference. Khattak The present study reveals that Zn with higher levels of
et al., (2006) have concluded from their experiment on NPK performed well (R2 = 0.47) and almost an increasing
maize tested with various application levels of Zn that trend in the grain yield was observed. With lower doses
there was no statistically significant difference among of NPK (half NPK levels to that of the recommended
the treatments regarding fresh biomass yield; anyhow dose), application of Zn at lower levels both as soil and
they observed 8 ‒ 11 % increase in biomass yield in foliar spray recorded poor performance as compared to
various treatments. half NPK only treatment (Figure 3). This behaviour of
Figure 2: Effect of various modes and levels of Zn on grain vs Figure 3: Effect of various modes and levels of Zn application on
biomass yield in the presence of full dose of NPK grain yield in the presence of full and half doses of NPK
Zn could be attributed to its ineffectivity at lower levels potential as in the case of full dose of NPK. Yet, the R2
at the highly alkaline calcareous conditions in the soil value (0.55) showed that Zn increment, irrespective of
under study (Table 1), its reaction with clay minerals its mode of application, improved the overall yield of the
(Abat et al., 2012), or might be due to some negative crop better in the case of half NPK dose as compared
interaction with P (Brown, 2008). With higher doses of to full NPK dose (Figure 3). Increased phosphorus
Zn, although the yield improved, due to lack of major supply enhances the symptoms of Zn deficiency (Brown,
nutrients the total yield could not attain its maximum 2008), yet, nitrogen appears to affect the Zn status of
Journal of the National Science Foundation of Sri Lanka 43(4) December 2015
308 Sabir Gul Khattak et al.
crops by promoting plant growth and by changing the modes. These findings are supported by the findings
pH of the root environment (Alloway, 2008). In many of Yilmaz et al. (1997) who concluded that soil and
soils, nitrogen is the chief factor limiting the growth foliar Zn increased the grain yield of wheat by 260 and
and yield and therefore in the case of full dose of NPK, 124 %, respectively in Zn deficient calcareous soils. The
improvements in yield have been found through positive interaction plot (Figure 4) shows that increasing levels of
interactions between nitrogen and Zn fertilisers. soil application of Zn has an obvious increasing effect on
grain yield.
Further analysis of the data using minitab statistical
package to find the significance of Zn as soil, foliar, The value cost ratio (VCR) for 5 kg ha-1 ZnSO4 as
and soil and foliar treatments revealed that Zn as soil soil treatment + 1.0 % ZnSO4 as foliar spray (10.23) was
treatment had a significant (p = 0.031) effect on the grain three times higher over the VCR for 15 kg ha-1 ZnSO4
yield and nearly significant (p = 0.084) effect of foliar as soil treatment + 1.0 % ZnSO4 as foliar spray (3.46),
spray on grain yield, but no interaction effect of the two confirming the superiority of the former over the latter
Note: Wheat grain rate = Pakistan rupees (Pak Rs) 24 kg-1 and ZnSO4 = Pakistan rupees (Pak Rs) 60 kg-1
December 2015 Journal of the National Science Foundation of Sri Lanka 43(4)
Soli and foliar Zn effect on yield and protein content in wheat 309
in terms of effectiveness (Table 4). Although VCR for concentration of foliar spray. The bars (Figure 5) clearly
0.5 % ZnSO4 (VCR:12) and 1.0 % ZnSO4 (VCR:11) indicate an initial gradual increase in protein with low Zn
as foliar application was the highest among the list of concentration levels, but with medium Zn concentration
treatments (Table 4), yet, taking into account the yield levels it tends to decrease irrespective of its application
per unit area of the treatments, the performance of 0.5 % method. With higher application of Zn at 15 kg as soil
ZnSO4 (1626 kg ha-1) and 1.0 % ZnSO4 (1752 kg ha-1) as treatment and 1.0 % ZnSO4 as foliar spray, it shows an
foliar application only was not satisfactory as compared increase in the protein content. The regression coefficient
to 5 kg ha-1 ZnSO4 as soil + 1.0 % ZnSO4 as indicated an overall increase of 21 % in grain protein
foliar (2294 kg ha-1) and 15 kg ha-1 ZnSO4 as soil + 1.0 % content with increasing Zn level applied either as foliar
ZnSO4 as foliar (2132 kg ha-1). Thus in a country like application or as soil application (Figure 5).
Pakistan, which faces food deficit due to increased
population, the growers must adopt the technology In calcareous soils, Zn precipitates in unavailable
that could increase their production per unit area with forms for plants, and its uptake and transition to the shoot
acceptable margin of profit. Thus, this study suggested is inhibited by high concentrations of bicarbonate (Dogar
the best level of Zn as 5 kg ha-1 as soil treatment + 1.0 % & Van Haj, 1980). However, Zn deficiency in the plants
ZnSO4 as foliar application as a better option to improve grown in calcareous soils can be recovered fairly readily
the grain yield on alkaline soil (Table 4).
Journal of the National Science Foundation of Sri Lanka 43(4) December 2015
310 Sabir Gul Khattak et al.
by application of inorganic Zn salts such as ZnSO4 to in rice grains after Zn fertilisation of plants growing on
the soil (Nayyar & Takkar, 1980), and such application calcareous soil (Singh, 1991).
from external sources linearly increased the grain yield
and protein content of two wheat genotypes (Morshedi No major differences were observed in protein profiles
of the samples isolated from different Zn treatments
& Farahbakhsh, 2010). Szakal (1989) and Fecenko and
(Figure 6) despite the increase or decrease in protein
Lozek (1998) have reported similar findings. Khattak content in various treatments. Alloway (2008) reported
et al., (2006) have reported a maximum increase in that in general the amount of protein in the grains of
protein content in the grains of maize obtained from Zn deficient plants is greatly reduced, but the protein
the treatment supplied with 0.5 % ZnSO4 as foliar composition remains almost unchanged. In Zn deficient
spray at the stage when the crop was only 20 ‒ 25 days bean leaves, the concentration of free amino acids was
old. Several researchers have reported significant seed 6.5 times greater than in the control but these decreased,
protein and dry matter yield response to Zn fertilisation and the protein content increased after the administration
(Biswas et al., 1977; Tandon, 1992; Zaidan et al., 2010.; of Zn for 48 or 72 hours. The mechanism by which Zn
deficiency affects protein synthesis is considered to
Keram, 2014). Zn is required for the synthesis of auxin
be due to a reduction in RNA and the deformation and
[a growth regulating compound indole acetic acid (IAA)] reduction of ribosome. The importance of Zn in protein
(Brown et al., 1993). Tryptophan is the most likely synthesis suggests that a relatively high Zn concentration
precursor for the biosynthesis of IAA and Zn is required is required by meristematic tissues where cell division
for the synthesis of tryptophan (Marschner, 1995). This as well as the synthesis of nucleic acid and protein is
includes an observed increase in the tryptophan content actively taking place (Brown et al., 1993).
Figure 6: Effect of various modes of treatment and levels of Zn on composition of protein in wheat grain
Legends: Coomassie-blue strained SDS-PAGE gel of wheat grain proteins isolated from the 12
Zn treatments. Molecular weight standards are shown on the left.
Lane 1, S0F0: L2, S0F1: L3, S0F2: L4, S1F0: L5, S1F1: L6, S1F2: L7, S2F0: L8, S2F1: L9,
S2F2: L10, S3F0: L11, S3 F1: L12, S3F2
S = soil Zn application at 0, 1 (5 kg ha-1), 2 (10 kg ha-1), 3 (15 kg ha-1)
F = foliar ZnSO4 application at 0,1 (0.5 %), and 2 (1 %).
December 2015 Journal of the National Science Foundation of Sri Lanka 43(4)
Soli and foliar Zn effect on yield and protein content in wheat 311
302: 1 ‒ 17.
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December 2015 Journal of the National Science Foundation of Sri Lanka 43(4)