EFFICIENCY OF ZINC UTILIZATION IN WHEAT GENOTYPES A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY By Muhammad Aamer Maqsood M.Sc. (Hons.) Agriculture INSTITUTE OF SOIL & ENVIRONMENTAL SCIENCES FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE FAISALABAD 2009
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EFFICIENCY OF ZINC UTILIZATION IN WHEAT GENOTYPES
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY
By
Muhammad Aamer Maqsood M.Sc. (Hons.) Agriculture
INSTITUTE OF SOIL & ENVIRONMENTAL SCIENCES FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE FAISALABAD
2009
The Controller of Examinations,
University of Agriculture,
Faisalabad
“We, the supervisory committee, certify that the contents and format
of the thesis submitted by Mr. Muhammad Aamer Maqsood (Reg. No.
2000-ag-1138) have been found satisfactory and recommend that it be
processed for evaluation by External Examiner(s) for the award of degree.”
3.1.2 Table 3.1.2 Total, shoot and root dry matter and root: shoot ratio of wheat genotypes grown with adequate (ADEQ) and deficient (DEF) Zn nutrient solution
24
3.1.3 Table 3.1.3 Shoot and root Zn concentration and uptake of wheat genotypes grown with adequate (ADEQ) and deficient (DEF) Zn nutrient solution
27
3.1.4 Shoot and root Zn utilization efficiency (ZnUE) and Relative growth rate (RGR) of shoot and root of wheat genotypes grown with adequate (ADEQ) and deficient (DEF) Zn nutrient solution
29
3.1.5 Specific absorption rate (SAR), specific utilization rate (SUR) and zinc transport rate (ZnTR) of wheat genotypes grown with adequate (ADEQ) and deficient (DEF) Zn nutrient solution
31
3.2.1 Grain and straw yield and grain/straw ratio of twelve wheat genotypes grown at adequate and deficient zinc levels
42
3.2.2 Zinc concentration and zinc uptake in straw and grain of twelve wheat genotypes grown at adequate and deficient zinc levels.
43
3.3.1 Biomass production of wheat genotypes 56
3.3.2 Zinc concentration of wheat genotypes 56
3.3.3 Zinc uptake of wheat genotypes 58
3.3.4 Zinc utilization efficiency of wheat genotypes 58
3.4.1 Shoot dry weight of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
71
3.4.2 Root dry weight of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
71
3.4.3 Shoot Zn concentration of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
71
3.4.4 Root Zn concentration of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
72
3.4.5 Shoot Zn uptake of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
72
3.4.6 Root Zn uptake of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
72
3.4.7 Maleic acid released (µM 2h-1 g-1) by wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
74
3.4.8 Fumaric acid released (µM 2h-1 g-1) by wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels
74
CONTENTS
iii
LIST OF FIGURES
No. TITLE PAGE
3.1.1 Dry matter partitioning among shoot and root of wheat genotypes grown with adequate (+) and deficient (-) Zn nutrient solution
35
3.1.2 Categorization of wheat genotypes according to Zn efficiency 36
3.1.3 Correlation of various Zn related physiological parameters 37
3.2.1 Percent increase over control by Zn application in straw and grain yield of twelve wheat genotypes
45
3.2.2 Correlation between shoot dry matter and Zn use efficiency of wheat genotypes
47
3.2.3 Relative reduction in shoot dry matter (ZnSF) in twelve wheat genotypes due to Zn deficiency
48
3.3.1 Relative growth of young and old leaves of Sehar-06 and Vatan by Zn application
61
3.3.2 Proportion of total Zn contents in young leaves of wheat at two harvests
62
3.3.3 Proportion of total Zn contents in old leaves of wheat at two harvest
62
3.3.4 Zn utilization efficiency in young and old leaves and roots of Sehar-06
63
3.3.5 Zn utilization efficiency in young and old leaves and roots of Vatan
63
3.4.1 Maleic Acid exudation by Sehar-6 under in relation to Zn supply
78
3.4.2 Maleic Acid exudation by Vatan under in relation to Zn supply
78
3.4.3 Fumaric Acid exudation by Sehar-6 under in relation to Zn supply
79
3.4.4 Fumaric Acid exudation by Vatan under in relation to Zn supply
79
CHAPTER 1
INTRODUCTION
Wheat is an important cereal crop grown in the world including Pakistan. It
is an important part of the daily diet of every Pakistani. Unfortunately our region
still lacks sufficiency in wheat production due to limitations such as over
cultivated soils, lack of good quality water, impure and insufficient seed and poor
management practices. The average yield of wheat in Pakistan during 2006 was
2.61 Mg ha-1(Government of Pakistan, 2006). This is well below the average yield
in developed countries. A quantity of 0.815 million tons of wheat were imported to
meet the shortfall. Repetitive cropping caused the mining of essential nutrients
from our soils after the advent of high yielding varieties during the green
revolution era. More than 50% of our soils are Aridisols with low micronutrient
availability (Gibson, 2006). According to Rahmatullah et al., (1988), the soils
present in Pakistan are young and most of the micronutrients especially Zn are
present in the un-weathered fraction. This may be one of the reasons that we are
still unable to realize the potential yield of our cereal crops.
Zinc deficiency is one of the most wide spread nutrient deficiencies in the
world (Imtiaz et al., 2006). About 50% of the world’s soils used for growing
cereals are Zn deficient (Graham and Welch, 1996). Statistics show that more
INTRODUCTION
2
nitrogen and phosphorus is applied, while little or no attention is paid to
micronutrients especially zinc (NFDC, 2000). Losses in yield in many Zn deficient
soils have major economic impact on the farmers’ income as a result of low yield.
Several approaches such as addition of inorganic fertilizers, organic matter
addition and synthetic chelates have been employed from time to time to cope
with Zn deficiency (Alvarez and Rico 2003). But external addition of Zn to soil is
not always the best strategy due to economic and agronomic factors (Graham
and Rengel, 1993). Added Zn in our soils quickly forms insoluble compounds due
high pH and CaCO3 content, which are not easily available to plants (Malik et al.,
1988)
Wheat is classified as a moderately sensitive species to Zn deficiency
(Clark, 1990). It is also severely affected by Zn deficiency in Pakistan, India and
other regions with soils having low Zn supply capacities. Symptoms of Zn
deficiency in wheat appear on the middle aged leaves, which show color from
healthy green to a muddy grey green, followed by small necrotic spots, that
gradually extent to the margins (Snowball and Robson, 1983). Zinc deficiency
results in the shortening of internodes causing stunted growth and delay in
flowering and maturity (Grundon, 1987). The deficiency of Zn in plants not only
effects the plant growth but also has a pronounced effect on human and animal
health. According to Black, (2003) about two billion people are deficient in Zn. In
Pakistan mostly children under the age of five and women of reproductive age
suffer from some degree of Zn deficiency (Bhutta et al., 1999). As mentioned
above the addition of Zn to soil may alleviate the problem of Zn deficiency in
INTRODUCTION
3
cereals and ultimately humans but it is not a sound option in developing
countries. On the other hand the area under wheat can not be increased beyond
a certain limit due to a limitation of productive land and good quality irrigation
water. The only option is to produce high yielding varieties that have high nutrient
use efficiency.
Wheat shows pronounced genotypic variation in tolerance to Zn deficiency
(Imtiaz et al., 2006). Zinc absorption by plants is a dynamic and complex process
which depends on ion concentrations at the root surface, root absorption capacity
and plant demand (Fageria et al., 2002). Because the concentration of Zn in soil
is very low, supply by mass flow is less important than diffusion and root
interception. Initial Zn uptake by plant roots is very rapid due to binding within the
root cell wall, and is followed by slower linear phase of transport across the
plasma membrane. Very few studies have investigated the mechanism of Zn
uptake into plant roots. Zinc transport in the plant is a metabolically controlled
process (Kochian, 1991), but still very few studies have investigated the
mechanism of Zn uptake into the plant. Plant roots are also known to exude
organic acids under nutrient deficient conditions in order to improve their chances
of nutrient acquisition (Hoffland et al., 1989).
Little work has been done on the categorization of wheat genotypes and
cultivars for their Zn use efficiency, which may be important for two reasons i.e.,
selecting low Zn requiring genotypes for resource poor farmers in Zn deficient
areas and evaluating Zn-responsive cultivars where Zn addition is not a
problem. Work in this direction is direly needed not only for categorizing the
INTRODUCTION
4
genetic potential but also for providing database to breeders for their future
ventures.
The present research encompasses a series of solution and soil culture
studies to achieve the following objectives
1. Study differential growth and Zn acquisition of wheat genotypes
2. Understand Zn remobilization within plants under induced Zn
deficiency
3. Assess the nature and amount of organic acids extrusion under Zn
deficiency and their role in Zn acquisition by wheat genotypes
CHAPTER 2
REVIEW OF LITERATURE
Zinc (Zn) is an essential micronutrient, required for the growth and normal
development of higher plants (Kochian, 1993; Marschner, 1995). Research in
recent years has shown that Zn plays important roles in a number of different
processes of plant physiology and biochemistry such as enzyme activity,
oxidative stress tolerance, root stress resistance, and plasma membrane function
(Kochian, 1993; Cakmak, 2000). Zinc deficiency is a global problem (Takkar and
Walker, 1993). In the recent past the problem of Zn deficiency in various crops
have increased because of intensive cultivation of high yielding varieties which
remove more Zn from soil.
2.1 Global distribution of Zn deficiency
Zinc deficiency is one of the most widespread nutritional disorders
observed for sustained crop production on alkaline calcareous soils (Kauser et
al., 1976). This Zn deficiency in soil is associated with high soil pH, low organic
matter, clay content and leaching (Takkar and Walker, 1993). According to Hewit,
(1963) and Loneragan and Webb, (1993) the anatoginstic relationship of Zn with
elements such as P and Cu may also lead to Zn deficiency in plants. Zn
deficiency has been reported in almost every country except Malta and Belgium
(Sillanpaa, 1982). Pakistan, Iran, Turkey, India, Syria, Lebonon, Mexico, Italy,
REVIEW OF LITERATURE
6
Tanzania and Thaialand are among the countries with the lowest levels of
reported available Zn (Sillanpaa, 1982). Western Australia has the largest tract
of low Zn soils in the world, occupying 8 million ha in the south-west of the state
where most of the agricultural production is based (Welch et al., 1991). In many
countries Zn deficiency has been induced by the use of lime to increase the soil
pH.
2.1.1 Occurrence of Zn deficiency in Pakistan
According to Rashid et al., (1994) Zn deficiency is the most widespread
micronutrient disorder on alkaline calcareous soils of Pakistan. This is a widely
recognized micronutrient deficiency in rice growing areas where not only is N and
P fertilizer is applied but also ZnSO4 on a regular basis (Malik et al., 1988).
Flooded soil conditions are responsible for Zn and Cu deficiency in rice (Malik et
al., 1988). Zinc deficiency has also been reported in crops such as maize
(Rashid et al., 1976), rape seed and mustard (Rashid et al., 1994) and citrus
orchards (Haq et al., 1995). The soils of Pakistan are generally light to medium
textured and have high pH, low organic matter content and are deficient in N and
P. Zinc along with other micronutrients such as Cu, Fe and Mn form insoluble
compounds that are not easily available to plants. The indiscriminate use of
ground water, high in carbonates and the introduction of high yielding crop
varieties and intensive cropping have further aggravated the situation (Tahir,
1981). The low Zn content of crops is transferred to humans as well. Bhutta et
REVIEW OF LITERATURE
7
al., (1999) have reported Zn deficiency in children less than the age of five and in
women of reproductive age.
2.2 Soil zinc
Zinc availability to plants from the soil depends upon several factors
including the concentration of Zn in soil solution, the presence of Zn in various
soil fractions, the interaction of Zn with other soil micronutrients and
macronutrients (Hewitt, 1963; Carroll, 1967; Shuman, 1985). Total Zn
concentration in soils mainly depends upon the parent material (Graham, 1953;
Sillanpaa, 1982). In igneous rocks, silicate containing minerals contain highest
concentration of Zn. In basalt rocks, magnetite is the most important Zn
containing mineral. In sedimentary rocks, highest Zn concentration is found in
clay sediments and shales while sandstone and limestone have lower
concentration of Zn (Pendias and Pendias, 1984). The total Zn concentration in
soil ranges from 10 to 300 mg Zn kg-1 soil with an average of 50 mg Zn kg-1 soil
(Mengel and Kirkby, 1978). The highest Zn concentrations are found in alluvial
soils while lower are found in sandy soils.
Rahmatullah et al., (1988) concluded from a series of experiments that a
significant proportion of total Zn was associated with the silt fraction in Pakistan
soils. Presumably the soils in Pakistan are relatively less weathered and
therefore, about one third of the total Zn was recovered from the coarser fraction,
silt. This is contrary to other studies that reported clay as the principal reservoir of
total Zn in soils (Iyengar et al., 1981; White, 1957).
REVIEW OF LITERATURE
8
2.2.1 Behavior of Zn in soils
The different chemical forms of Zn found in soils exhibit different levels of
reactivity, solubility and availability to plants. Sequential extraction procedures
are applied in soils to partition metal into different fractions. The bioavailability of
metals in soils is related to these chemical fractions and not to total metal content
(Almendros et al., 2008). According to Viets (1962) Zn is present in five different
soil pools which include, soil solution, soil exchange sites, complexed with
organic matter, co-precipitated with oxides and hydroxides of Fe and Al and held
in primary and secondary minerals. The availability of Zn to plants decreases in
these successive pools. Plants absorb Zn from the soil solution, but the labile Zn
held on solid soil surface is also made available via desorption when solution Zn
is low (Lindsay, 1981). Takkar and Sidhu (1977) also proposed that sorption and
desorption reactions in the soil control the concentration of Zn in soil solution and
its availability to plants. These reactions are controlled by various soil properties
such as soil texture, pH, cation exchange capacity (CEC), organic matter content
and iron and aluminum oxides (Shuman, 1975).
The availability of all micronutrient except Mo increases as the pH of the
soil decreases. According to Lindsay, (1981) the availability of Zn decreases 100-
fold for every unit increase in soil pH. The availability of Zn decreases with
increase in pH above 6.0 due to increase adsorption by Fe and Al oxides and
hydroxides and by reaction with cobonates and bi-carobnates in alkaline
calcareous soils. Clark and Graham, (1968) have reported that alkaline soils
require more Zn compared to acidic soils for maximum plant growth. In alkaline
REVIEW OF LITERATURE
9
calcareous soil there is the precipitation of Zn(OH)2, ZnCO3 and calcium zincate
compounds (Clark and Graham, 1968) or there may be the adsorption of Zn by
various carbonates (Udo et al., 1970). In addition to this, high solution Ca inhibits
the Zn uptake and may also be a factor responsible for reduced Zn availability in
alkaline calcerous soils (Chaudhry and Lonaragan, 1972).
The availability of Zn to plants has also reported to be influenced by soil
organic matter (Shuman, 1975; Barrow, 1993). Soil organic matter plays an
important role in both bonding and sorption of Zn (Pendias and Pendias, 1984).
Zinc deficiency has been reported in peat soils or in soil where high amounts of
organic matter have been added. Zinc may be bound to organic compounds that
make Zn unavailable for plant uptake (Lindsay, 1972). However the effect of
organic compounds on Zn uptake by plants has been shown to be inconsistent,
as not only decrease but increase in Zn uptake have been reported (Bell et al.,
1991). Humic compounds are the most stable organic compounds. Humic
compound can be further divided in to humic acid and fulvic acid (Stevenson,
1991). These organic compounds have a great affinity for Zn and other
micronutrients. Humic acid is more soluble under alkaline conditions whereas
fulvic acid is soluble in both alkaline and acidic conditions (Stevenson, 1991).
2.3 Improving crop yields under Zn deficient conditions
In order to get higher yield under Zn deficient conditions, either of the
following strategies are employed. First, by improving the soil conditions by
REVIEW OF LITERATURE
10
organic or inorganic soil amendments. Second, by tailoring the crops to fit the soil
conditions.
2.3.1 Use of soil amendments
The application of inorganic fertilizers is a direct and simple method to
cope with Zn deficiency. There are various methods of Zn fertilizer application
including, direct soil application, foliar application, coating of Zn fertilizer on seed
and dipping of young seedlings in Zn solution (Slaton et al., 2001). Poshtmasari
et al., (2008) studied the response of common bean to different levels and
methods of Zn application. They found that foliar application of 40 mg Zn kg-1
soil resulted in highest Zn content in leaves and seed. Khan et al., (2008)
conducted an experiment to evaluate the best level of Zn for wheat crop on
alkaline calcareous soil of Pakistan and reported the use of 5 kg ha-1 ZnSO4 gave
the highest marginal rate of return. The effectiveness of Zn fertilizer depends
upon its water solubility and whether it is in granular or powder form (Mortvedt,
1991). According to Mortved, (1991) ZnO in granular form is ineffective because
of the decrease in surface area and its low solubility in water. Various other
fertilizers have been reported to contain significant amount of Zn (Tiller, 1983).
Single super phosphate contains 400-700 mg kg-1 whereas diammonium
phosphate contains 70 mg kg-1 Zn (Brennen, 1986). Depending upon the rate
and frequency of fertilizer use, the Zn present in these fertilizers as an impurity
can supply sufficient amount of Zn under deficient soil Zn availability. Srivastava
REVIEW OF LITERATURE
11
and Sethi, (1981) reported that the application of manures to soil can also
alleviate Zn deficiency.
2.3.2 Tailoring crops to tolerate soil Zn deficiency
Nutrient efficiency is defined as the ability of a genotype to grow and
produce maximum yield in a soil too deficient in Zn for a standard genotype
(Graham 1984). Zinc efficiency (ZE) has been attributed to the efficiency of
acquisition of Zn under low zinc availability conditions (Salama and Fouly 2008).
Gupta et al., (1994) classified the genotypes that show the highest percent
response to added Zn and decrease in growth without Zn as susceptible to Zn
deficiency and vice versa. It was further suggested that the responsive cultivars
absorb naturally available Zn. However, non responsive cultivars may utilize
naturally available Zn more effectively (Gupta et al. 1994). The cultivation of Zn
efficient crop genotypes is considered a better approach on soils with low
available Zn (Cakmak et al., 1999), especially in areas where Zn fertilizers are
expensive and not always effective in overcoming Zn deficiency (Genc and
Donald, 2004).
Plant species have been observed to differ in Zn requirement (Shukla and
Raj, 1974; Graham and Rengel, 1993; Cakmak et al., 1996). Legumes have
higher Zn concentration than cereals when grown on the same soil (Gladstone
and Loneragan, 1967). Within cereals there is a large variation in susceptibility to
Zn deficiency. Cakmak et al., (1996) reported the susceptibility of cereal to Zn
deficiency decreased in the following order durum wheat>oat>bread
REVIEW OF LITERATURE
12
wheat>barley>triticale>rye. Differences in the reduction in growth due to Zn
deficiency have been observed in genotypes of various plant species (Rengel,
2001). Genotypes of wheat (Graham, et al., 1992; Cakmak et al., 1994),
chickpea (Khan et al., 2000), durum wheat (Cakmak et al., 2001) and rapeseed
(Grewal et al., 1997) differ in their ability to tolerate Zn deficiency.
Ssemakula et al., (2008) conducted an experiment on cassava plants and
found significant effect of genotype (G) and environment (E) on the root Zn
concentration. Genc et al., (2002) found that the effect of Zn deficiency on
appearance of deficiency symptoms and of Zn fertilization on increase in growth
were more pronounced on an Zn inefficient genotype compared to a Zn efficient
genotype of barley. Ambler and Brown (1969) showed that the Zn concentration
in plant tissue of navy bean were greater in an efficient genotypes than an
inefficient genotype. Up to 25% higher Zn concentration has been observed in
the seed of an efficient genotype of navy bean than inefficient genotype grown on
the same soil (Moraghan and Grafton, 1999). Moraghan and Grafton (1999)
suggested the selection of genotypes with higher seed Zn concentration because
they can be better source of Zn for human nutrition. Graham et al., (1992)
observed contrasting result where Zn efficient wheat cultivar had lower Zn
concentration in grain than an inefficient cultivar. According to Behl et al., (2003)
screening and further breeding of efficient wheat genotypes can be an
economical strategy. Also, it may be possible to combine the high seed Zn trait
with seedling vigor, nutritional quality and high yield.
REVIEW OF LITERATURE
13
2.4 Mechanisms of Zn efficiency
The mechanisms that are responsible for the variation in Zn efficiency
within plant species are not fully clear. However, according to Dong et al., (1995)
efficient genotypes are characterized by higher Zn acquisition form soil by
modifying their root morphology in the form of longer and thinner roots. In some
plant species increase in Zn translocation from roots to shoots has been found
under Zn deficiency (Reuter et al., 1980). Modification in soil rhizosphere pH, root
exudation is also related to Zn efficiency in plants (Rengel 1999).
2.4.1 Zinc translocation
Zinc translocation in plants is dependent on the level of Zn supply, N
status of the plant and the demand for Zn within the plant. Riceman and Jones
(1958) found that Zn accumulation in reproductive organ of subterranean clover
was related to the decrease in total Zn in leaves and petioles. The depletion of
Zn in roots and stems is due to the remobilization and re-translocation of Zn to
the developing leaves and grain. However, Zn is variably mobile within the
plants, hence the distribution and redistribution patterns are complex
(Longnecker and Robson, 1993). Zinc concentration in plants varies with the
levels of Zn supply, growth stage, plant parts and also leaf position. In Zn-
deficient plants, the Zn concentration in leaves usually ranges between 10 and
15 mg Zn kg-1 and in healthy leaves, the Zn concentration ranges from 15 to 100
mg Zn kg-1 (Longnecker and Robson, 1993). At the reproductive stage, the Zn
concentration tends to be higher than at vegetative growth stage with low to
REVIEW OF LITERATURE
14
adequate Zn supply. At adequate Zn supply there is higher Zn concentration in
growing plant parts than in mature leaves. Reuter (1980) reported that maximum
Zn concentration was in the youngest folded leaves (YFL) of subterranean clover
and followed by youngest open leaves (YOL). In Zn-deficient plants, the Zn
concentration in the leaf blades generally is higher than in the petioles, but with
increasing Zn supply, the differences in Zn concentration between blades and
petioles disappeared (Riceman and Jones, 1958).
Reuter (1980) has reported that Zn accumulation in roots of subterranean
clover was greater in plants grown with high Zn supply than in those with low Zn
supply. when soybean plants were grown with an adequate Zn supply, the Zn
concentration in the leaves was 92 mg Zn kg-1 and that in the roots was 35 mg
Zn kg-1, but with toxic Zn supply, the Zn concentration in the leaves was 133 mg
Zn kg-1 and in the roots was 1335 mg Zn kg-1 (White et al., 2002). Cogliatti et
al., (1991) investigated the Zn distribution between roots and shoots in wheat
plants by using 65Zn–labelled nutrient solution, and found that Zn accumulated in
the wheat roots for the first 2 hours but after 4 hours, Zn in roots decreased,
indicating that Zn had been remobilized and transported to the shoot.
2.4.2 Excretion of organic acids and chelating agents
Plant roots exude organic acids especially under nutrient deficiency
(Grinsted et al., 1982). Under phosphorus deficiency, increased excretion of
organic acids has been reported in white lupin (Gardner et al., 1983). Dinkelaker
et al. (1989) reported that the roots of lupin excreted large amounts of citric acids
REVIEW OF LITERATURE
15
that caused rhizosphere pH decline, that could have mobilized Zn in the
rhizosphere soils and increased the availability of Zn. Degryse et al., (2008)
conducted a resin buffered solution culture experiments to study the effect of Zn
and Cu deficiency on root exudation by dicotyledonous plants. They found than
the Cu and Zn concentrations in the nutrient solution increased with time, except
in plant-free controls, indicating that the plant roots released organic ligands that
mobilized Cu and Zn from the resin. Hoffland et al. (2006) also reported a
significant increase in exudation of low-molecular weight organic acids by rice
under Zn-deficient conditions.
Plant roots can also excrete chelating compounds under nutrient
deficiency. Under Fe deficiency, grasses excrete phytosiderophores that have an
important role in acquisition of iron (Römheld and Marschner, 1990). These
compounds also form stable chelates with Cu, Zn and to lesser extent Mn, and
may mobilize these nutrients, especially Fe and Zn from calcareous soils, and
enhance uptake by plants (Marschner, 1993). Zhang et al. (1989) reported that
root-induced changes in the rhizosphere soil of barley and wheat occur in
response to Zn deficiency. Cakmak et al., (1996) found that phytosiderophore
release was more pronounced for Zn-efficient than for Zn-inefficient genotypes of
wheat.
CHAPTER 3, STUDY I
Differential growth response and zinc acquisition of wheat genotypes in hydroponics
3.1.1 Introduction
Zinc deficiency is a common micronutrient deficiency in cereals such as
wheat grown in arid and semi arid regions of the world (Takkar and walker,
1993). In Pakistan the major reasons for Zn deficiency in soils are high pH,
clay content, CaCO3 and low organic matter along with high
evapotranspiration rate (Khattak and Perveen, 1985). According to
Rahmatullah et al., (1988) a significant amount of Zn is present in soil matrix
but only a small fraction of it is bio-available. Zinc status of a plant can be
improved by applying organic and inorganic fertilizers. But there are some
constraints in application of fertilizers. One being the increasing cost of Zn
based fertilizers and second is that when Zn fertilizer is applied to the soil it
undergoes a number of chemical reactions which reduce its availability to
plants (Rahmatullah et al., 1988).
The other option to combat Zn deficiency is tailoring plants to suit the
soil conditions. Tailoring plants here refers to improvement of nutrient use
efficiency. Scientists such as Graham et al., (1992) and Graham and Rengel,
(1992) have identified crop genotypes that are able to grow and give higher
STUDY I
17
yield in soils too deficient in Zn for standard genotypes. Thus exploitation of
the plants’ genetic capacity for efficient nutrient uptake and utilization can
prove to be a promising tool to cope with nutrient deficiency stress (Irshad et
al., 2004).
In screening of crop genotypes in hydroponic conditions we are unable
to replicate all soil related factors important in Zn uptake; however, it is a
quick and effective method for evaluating Zn deficiency tolerance (Trostle et
al., 2001). But Zn is an ever-present contaminant in the laboratory. It’s also a
micronutrient, making it quite difficult to impose Zn deficiency using regular
nutrient solution techniques (Brown, 1986). The introduction of chelartor
buffered nutrient solution (Parker et al., 1995) is a major advancement in
studying micronutrient activities which can be consistently maintained in near
plant roots, thus mimicking the situation in a soil (Yang et al., 1994).
According to Epstien (1972) hydroponics culture satisfies many requirements
of screening for breeding plant genotypes to tolerate Zn deficiency by
providing a homogenous growth medium that can be easily maintained and
controlled. Keeping in view the above, a screening experiment was conducted
in DTPA buffered nutrient solution to select Zn efficient and responsive wheat
genotypes.
STUDY I
18
3.1.2 Materials and Methods
The experiment was conducted in a rain protected wire house under
natural conditions. The temperature of wire house varied from a minimum of
7°C to a maximum of 22°C with a mean value of 12°C.
3.1.2.1 Plant Culture
Seeds of twelve wheat genotypes (Table 3.1) were collected from Ayub
Agriculture Research Institute (AARI). The seeds were sown in polyethylene
lined iron trays containing washed river-bed sand. Optimum moisture for
germination was maintained using distilled water. Uniform seedlings were
transplanted, one week after germination in foam-plugged holes of thermopal
sheets floating on continuously aerated 50L half strength modified Johnson’s
nutrient solution (Johnson et al., 1957) in polyethylene lined two iron tubs.
The solution contained 6 mM N, 2mM P, 3 mM K, 2 mM Ca, 1mM Mg, 2 mM
S, 50 µM Cl, 25 µM B, 2 µM Mn, 1 µM Cu, 0.05 µM Mo and 50 µM Fe. Two
Zn levels i.e., adequate (2 µM) with ZnSO4.2H2O and deficient (0.2 µM) were
maintained in nutrient solution. There were 6 tubs (3 tubs per treatment) and
24 plants in each tub. Zinc deficient level in 3 tubs was induced by addition of
50µM DTPA with additional concentration of Fe, Cu and Mn. Hydrogen ion
activity (pH) of nutrient solution in tubs was monitored daily and adjusted daily
at 6.0 ± 0.5 with 1N NaOH or 1N HCl.
STUDY I
19
3.1.2.2 Harvesting
Plants were harvested 20 and 32 days after transplanting. They were
washed in distilled water, blotted dry and separated into shoots and roots
before air drying for 2 d. The samples were then oven dried at 75°C in a
forced air driven oven for 48 h to record dry matter yield (g plant-1).
3.1.2.3 Zinc Concentration
Samples of dried shoots and roots were ground in a mechanical grinder
(MF 10 IKA, Werke, Germany) to pass through a 1 mm sieve. Ground
samples were then mixed uniformly. A 0.5 g portion of plant sample was
digested in a di-acid mixture of nitric acid and perchloric acid (3:1) at 150°C
(Miller, 1998). Zinc concentration in shoot and root digest was estimated
using atomic absorption spectrophotometer (Perkin Elmer Analyst-100). Zinc
contents (mg Zn plant-1) were calculated in shoots and roots by multiplying Zn
concentration in the respective tissue with dry matter and on whole plant
basis by adding up shoot and root Zn contents.
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20
Table 3.1.1 List of Wheat Genotypes (Triticum aestivum)
3.1.2.4 Relative Growth Rate
Relative growth rate (RGR) of shoot and root (mg g-1 day-1) was
calculated according to Hunt, (1978) as given below:
ln W2 – ln W1
RGR =
rT
S. No. Genotype
1 Inqalab-91
2 Bhakar-2000
3 Pari-73
4 Yaqora
5 As-2002
6 Shafaq-06
7 Auqab-2000
8 Sehar-06
9 Dirk
10 Iqbal-2000
11 Vatan
12 SARC-1
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Whereas W1 and W2 are the shoot or root dry weight (g) at harvest time T1
and T2 (day), respectively and rT is the time interval (days) between two
harvests.
3.1.2.5 Specific Absorption Rate of Zn (mg Zn mg-1 RDM day-1)
Specific absorption rate (SAR) of Zn in wheat genotypes was
calculated according to Hunt (1978) as given below:
SAR = Zn2 – Zn1 x RGR (root) R2 – R1
Where, Zn1 and Zn2 are total Zn uptake at harvest 1 and 2, respectively. R1
and R2 are root dry weight (g) at first and second harvest, respectively and
RGR (root) is the relative growth rate of root.
3.1.2.6 Specific Utilization Rate
Specific utilization rate (SUR) of Zn (mg DW mg-1 Zn day-1) for wheat
genotypes was calculated (Hunt, 1978) as given below:
SUR = TDW2 – TDW1 X lnZn2 - lnZn1
T2 – T1 Zn2 - Zn1
Where TDW is total dry weight, Zn1 and Zn2 are total Zn uptake at harvest 1
and 2, respectively
STUDY I
22
3.1.2.7 Zinc Transport Rate (mg Zn g-1 SDW day-1)
Zinc transport rate, which is the rate of Zn transport relative to shoot
dry weight of wheat genotypes, was calculated according to Pitman, (1972) as
given below:
ZnTR = Zn2 – Zn1 x RGR (shoot) S2 – S1
Where Zn1 and Zn2 are the zinc uptake in Shoot, and S1 and S2 are SDM at
first and second harvest, respectively and RGR (shoot) is the relative growth
rate of shoot.
3.1.2.8 Zinc Utilization Efficiency
Zinc utilization efficiency (g2 SDM mg-1 Zn) was calculated by the
following formula (Siddiqui and Glass, 1981):
1 Zinc Utilization Efficiency =
Shoot Zn concentration X Dry matter
3.1.2.9 Index Score Calculation
Wheat genotypes were grouped into three classes on the basis of
genotypic mean (µ) and standard deviation (SD) for seven parameters. The
genotypes were assigned as low if their mean were< µ-SD, medium if their
mean is between µ-SD to µ+SD and high if cultivar mean were > µ+SD.
STUDY I
23
These classes were assigned the numerical value as index score for each
parameter as 1 to low, 2 to medium and 3 to high (Gill et al., 2004).
3.1.2.10 Statistical Analysis
The data were subjected to statistical treatments using computer
software “MS-Excel” and “MSTAT-C” (Russell and Eisensmith, 1983).
Completely randomized factorial design was employed for analysis of
variance (ANOVA). Least significant difference (LSD) test was used to
separate the treatment means (Steel and Torrie, 1980).
3.1.3 Results
There were significant (p<0.05) effects of genotypes and Zn levels on
Genotypes varied significantly (p<0.01) for their total dry matter (TDM)
(Table. 3.1.2). Total dry matter of genotypes ranged from 1.35g plant-1 in Dirk
to 5.29 g plant-1 in Sehar-06. On the basis of genotypic means, Sehar-06,
Shafaq-6 and SARC produced > 4.01 g plant-1 TDM and gained maximum
index score of 3. Total dry matter of Dirk and Vatan was < 2.51 g plant-1,
STUDY I
24
Table 3.1.2 Total, shoot and root dry matter and root: shoot ratio of wheat genotypes grown with adequate (ADEQ) and deficient (DEF) Zn nutrient solution
application on relative growth rate (RGR) of root of Wheat (Table 3.1.4).
Interaction between Zn and genotypes was non-significant. It ranged from
0.05 mg g-1 RDM day-1 in Vatan to .14 mg g-1 RDM day-1 in SARC-1.
3.1.3.9 Specific Absorption Rate (SAR)
Data concerning Specific absorption rate (SAR) of Zn by wheat
genotypes is presented in Table 3.1.5. Genotypes differed significantly
(p<0.01) for SAR. Interaction between Zn and genotypes was also significant.
Specific absorption rate varied between 3.56 µg Zn g-1 RDM day-1 to 66.15 µg
Zn g-1 RDM day-1in plants.
3.1.3.10 Specific Utilization Rate (SUR)
There were significant (p<0.01) effects of wheat genotypes on Specific
utilization rate (SUR) of Zn (Table 3.1.5). However, interaction between Zn and
genotypes was non-significant for SUR. It ranged from 1.10 mg DW mg-1 Zn
day-1 in Vatan to 4.13 mg DW mg-1 Zn day-1 in Auqab-2000.
3.1.3.11 Zinc Transport Rate (ZnTR)
Data regarding Zinc Transport Rate (ZnTR) of wheat genotypes is
presented in Table 3.1.5. Genotypes differed significantly (p<0.01) for ZnTR.
Interaction between Zn and genotypes was also significant (p<0.01). Zinc
Transport Rate ranged between 0.40µg Zn g-1 SDM day-1 to 9.05 µg Zn g-1
SDM day-1 in plants.
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Table 3.1.5 Specific absorption rate (SAR), specific utilization rate (SUR) and zinc transport rate (ZnTR) of wheat genotypes grown with adequate (ADEQ) and deficient (DEF) Zn nutrient solution
SAR (µg Zn g-1 RDM day-1)
SUR (mg DW mg-1 Zn day-1)
ZnTR (µg Zn g-1 SDM day-1)
Genotypes
ADEQ Zn (2 µM)
DEF Zn (0.2 µM)
ADEQ Zn (2 µM)
DEF Zn (0.2 µM)
ADEQ Zn (2 µM)
DEF Zn (0.2 µM)
Inqalab-91 54.63 6.49 2.74 3.87 7.71 1.31
Bhakar-2000 54.37 5.87 2.10 3.47 7.55 1.25
Pari-73 59.36 8.99 2.22 3.77 7.38 1.85
Yaqora 59.97 5.65 2.18 3.08 6.71 1.19
As-2002 47.97 8.35 2.15 3.47 6.80 2.07
Shafaq-06 56.86 4.11 1.98 3.37 7.49 0.84
Auqab-2000 43.07 6.30 2.23 4.13 6.41 1.45
Sehar-06 66.15 8.38 2.32 3.72 9.05 1.89
Dirk 36.15 6.81 1.55 1.22 4.35 0.70
Iqbal-2000 38.95 9.58 2.06 3.94 5.50 2.31
Vatan 22.51 0.56 1.10 1.59 4.11 0.40
SARC-1 53.87 5.39 2.62 3.40 8.56 1.55
Mean 49.49 6.37 2.10 3.25 6.80 1.40
LSD0.05 (Genotype x Zn Level)
18.89 1.79 2.45
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3.1.4 Discussion
Zinc deficiency is one of the most widespread micronutrient deficiencies in
cereals (Behl et al., 2003). Selection and breeding for increased Zn efficiency is a
promising strategy to sustain crop productivity in low input and environmental
friendly agriculture systems (Cakmak et al., 1999). Genotypes that are more
efficient in Zn acquisition from deficient conditions are generally considered
better adaptable to Zn deficiency in soils. Sufficient genetic variability exists
among several crop species for Zn acquisition and utilization under low Zn
environments (Irshad et al., 2004, Cakmak et al., 2001).
Differential growth response and Zn acquisition efficiency by twelve wheat
genotypes was evaluated in a solution culture experiment. They varied
significantly (p<0.01) for their total biomass, shoot dry matter, root dry matter and
root: shoot ratio.
Biomass production by plants under Zn deficient conditions as compared
to adequate Zn supply conditions indicates relative tolerance of crop genotypes
against Zn deficiency in the growth medium (Graham et al., 1992). Relative
biomass production in Sehar-06 grown under Zn deficient conditions was 80% of
its maximum SDM production grown with adequate Zn supply. Genotypes
showing higher relative biomass production such as Sehar-06, Iqbal-2000 and
Inqalab-91 can be cultivated on soils with low Zn availability. However, absolute
dry matter yield should also be considered before selecting genotypes for
cultivation on low Zn soils. Genotypes were classified on the basis of their SDM
production and Zn uptake in Zn deficient conditions (Gill et al., 2004). On these
STUDY I
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base, the genotypes were grouped into 5 categories viz i) low dry matter and low
Zn utilization efficiency (LDM-NE), ii) medium dry matter with medium Zn
utilization efficiency (MDM-ME), iii) medium dry matter with high Zn utilization
efficiency (MDM-HE) and iv) high dry matter with high Zn utilization efficiency
(HDM-HE), iv) high dry matter with medium Zn utilization efficiency (HDM-ME)
(Fig. 3.1.3). Genotypes with HDM-HE were Sehar-06 and Auqab-2000. Both of
these genotypes were efficient in Zn acquisition and its utilization for biomass
production under low Zn availability (Table 3.1.2 & 3.1.3) hence these can be
selected for soils with wide range of Zn concentrations.
Genotypes of group LDM-NE (Vatan and Dirk) were least efficient in both
biomass and Zn uptake. Zinc efficiency traits such as RDM and Zn use efficiency
of genotypes in this group were lower than from genotypes falling in group HDM-
HE. Genotypes Inqalab-91 was medium in ZnUE but efficient in biomass
production indicating its efficiency in Zn utilization. Shafaq-06 was medium in
biomass production but accumulated more Zn, hence was less efficient in Zn
utilization. All other genotypes were medium in biomass as well as Zn utilization
efficiency (Figure 3.1.2).
Increased root growth at the expense of shoot is one of the possible
mechanisms to cope Zn deficiency in soil (Marschner et al., 1986). Effect of Zn
deficiency was more pronounced on SDM than on RDM. This resulted in an
increase in root:shoot ratio of all the genotypes (Figure 3.1.1) when grown in Zn
deficient nutrient solution. Since Zn is an immobile nutrient in soil and its
movement is diffusion dependent, hence, increased root:shoot ratio equips plants
STUDY I
34
with more root surface area for Zn absorption and exploration of root medium
(Marschner et al., 1986).
Genotypes showing less decrease in Zn contents of shoots and roots
when grown under low Zn conditions were considered to be tolerant. Relative Zn
content of plants grown with adequate Zn compared to deficient Zn varied
significantly among genotypes. Sehar-06 was the most efficient in accumulating
in Vatan at first harvest. When plants were grown with Zn free nutrient solution, it
ranged from 14.82 mg/pot in Sehar-06 to 19.67 mg/pot in Vatan.
Genotypes differed significantly for Zn uptake in their mature leaves when
grown with adequate Zn supply in root medium at first harvest (Table 3.3.3). Zinc
uptake in mature leaves at second harvest were lower in Sehar-06 than Zn
uptake in mature leaves at first harvest but opposite in case of Vatan. Zinc
uptake in mature leaves at first harvest was 13.0 µg plant-1 in Sehar-06 and 10.28
µg plant-1 in Vatan . Zinc uptake in mature leaves at second harvest, was 12.0 µg
plant-1 in Sehar-06 and 18.66 µg plant-1 in Vatan. Genotypes differed significantly
for root Zn uptake at first harvest (Table 3.3.3). Root Zn uptake at second harvest
were more in Vatan at both harvest.
3.3.3.4 Zinc Utilization Efficiency
Genotypes differed significantly (p<0.01) for Zinc utilization efficiency
(ZnUE) of young leaves (Table 3.3.4) during 30 days of growth with adequate Zn
supply. ZnUE of young leaves was 7.42 g2 SDW mg-1 Zn in Sehar-06 and 11.96
g2 SDW mg-1 Zn in Vatan grown with adequate Zn. Zinc utilization efficiency
significantly increased at second harvest solution and ranged in young leaves
from 17.01 g2 SDW mg-1 Zn in Sehar-06 to 15.86 g2 SDW mg-1 Zn in Vatan.
Zinc utilization efficiency (ZnUE) of mature leaves also differed
significantly (p<0.01) among genotypes at both harvests (Table 3.3.4). It
increased significantly due to induced Zn deficiency at 2nd harvest, irrespective of
genotypes. Zinc use efficiency was 9.23 g2 SDW mg-1 Zn in Sehar-06 and 14.90
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g2 SDW mg-1 Zn in Vatan at first harvest. At second harvest, ZnUE was 13.97 g2
SDW mg-1 Zn in Sehar-06 and 27.47g2 SDW mg-1 Zn in Vatan. Increase in ZnUE
of young leaves was more than that of mature leaves at second harvest in Sehar-
06 but it decreased at in case of Vatan.
3.3.4 Discussion
A number of adaptive mechanisms have been proposed in higher plants to
cope with Zn deficiency in the root medium. Among physiological adaptations,
decreased growth rate and remobilization of internal Zn from relatively inactive
sites to metabolically active sites are important (Erenoglu et al., 2002). The
present experiment was conducted to study Zn remobilization within different
plant organs under Zn deficiency in selected wheat genotypes differing in Zn
acquisition. The genotypes were grown for 30 days at adequate Zn (2 µM) and
four replications were harvested. The rest of the plants were grown further for 10
days in Zn free nutrient solution.
Biomass differed significantly among genotypes at both harvests (figure
3.3.1). Zinc deficiency did not significantly affect Zn contents in young leaves. As
Zn was withdrawn from solution during next 10 days, plants were unable to take
more Zn from solution, so the plants re-translocated Zn from inactive (mature
leaves) to metabolically active sites (young leaves) for sustaining growth
(Hajiboland et al., 2000). In Sehar-06 the proportion of total Zn was 27% in young
leaves and 55%
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Figure 3.3.1 Relative growth of young and old leaves of Sehar-06 and Vatan by Zn application
in mature leaves, whereas in Vatan it’s proportion was 22% in young leaves and
55% in mature leaves after 30 days of growth in Zn adequate nutrient solution.
Proportion of total Zn retained in young leaves increased from 44% in Sehar-06
and just 27% in Vatan during 10 days of growth in Zn free environment (Fig.
3.3.2). We found a significant impact of Zn remobilization/translocation on
differences in growth of these genotypes.
Root Zn contents were also lower at second harvest in Sehar-06 but a
slight increase in root Zn uptake was observed in Vatnan. Variations among
these genotypes in distributing Zn within plant tissues depict the efficiency of
genotypes in biomass accumulation. Sehar-06 (efficient in Zn acquisition and
utilization) produced higher biomass at both harvests; it also had higher
mobilization of Zn towards young leaves. This suggests that
0
5
10
15
20
25
30
35
40
45
50
Sehar Vatan
Genotype
% in
crea
se in
bio
mas
s
Young LeavesOld Leaves
Sehar-06 Vatan
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Figure 3.3.2 Proportion of total Zn contents in young leaves of wheat at two harvests Figure 3.3.3 Proportion of total Zn contents in old leaves of wheat at two harvests
0
5
10
15
20
25
30
35
40
45
50
sehar vatan
Genotypes
Pro
po
rtio
n o
f T
ota
l Zn
in y
ou
ng
lea
ve
s
(%)
30d40d
0
10
20
30
40
50
60
sehar vatan
Genotypes
Pro
po
rtio
n o
f to
tal Z
n in
old
lea
ves
(%)
30d40d
Sehar-06
Sehar-06
Vatan
Vatan
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Figure 3.3.4 Zn utilization efficiency in young and old leaves and roots of Sehar-06 Figure 3.3.5 Zn utilization efficiency in young and old leaves and roots of Vatan
0
2
4
6
8
10
12
14
16
18
20
30D 40D
Harvest Time
Zn
uti
lizat
ion
Eff
icie
ncy
(g
2 S
DW
mg
Zn
-1)
Young Leaves
Old Leaves
Root
0
5
10
15
20
25
30
30D 40D
Harvet Period
Zn
uti
lizat
ion
Eff
icie
ncy
(g
2 SD
W m
g Z
n-1
)
Young LeavesOld LeavesRoot
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mobilization of Zn within plant body is an adaptive mechanism in wheat and it
relates to differences in Zn efficiency of wheat genotypes.
3.5.5 Conclusion
Genotypes behaved differently for their growth and Zn mobilization when
grown under Zn deficiency at later stage of growth. Sehar-06 remobilized more
absorbed Zn from mature leaves to young leaves when Zn deficiency was
induced in root medium as indicated by increased Zn contents in young leaves at
2nd harvest. The variations in growth rate and Zn remobilization might explain
differences in growth of these genotypes.
CHAPTER 3, STUDY IV
Differences in organic acid extrusion by wheat genotypes under Zn deficiency
3.4.1 Introduction
Soils with low levels of phyto-available Zn are present in different climatic
regions all over the world (Takkar and Walker, 1993). The total Zn content in
soils range from 50 to 300 mg kg-1 soil. But due to soil constraints such as high
pH, low organic matter content and high carbonate content a major portion of this
is not phyto-available (Alloway, 2004). Zinc application to the soil is a simple
strategy but it is not always the best option due to certain agronomic, economic
and environmental constraints (Graham and Rengel, 1993). The alternate
strategy is to select and breed crop genotypes that can tolerate low Zn
availability (Neue et al., 1998). The superior ability of some crop genotypes to
tolerate low Zn availability over others is sill not clearly understood (Hacisalihoglu
and Kochain, 2003). These tolerant genotypes may have low Zn requirement,
better Zn translocation ability from root to shoot or they may have better ability to
solublize immobile Zn form soil. Kirk and Bajita, (1995) have also suggested that
the processes occurring in the rhizosphere may significantly effect the
bioavailability of Zn. Plants can modify the rhizosphere to increase the acquisition
of nutrients, especially for diffusion dependent ions such as Fe, Zn and P
(Marschner, 1995). In crops such as wheat, oats, rice, sorghum and maize,
STUDY IV
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differences in response to Zn deficiency might be related to different capacities of
the plant species and their genotypes to release Zn mobilizing organic acids.
It has been suggested that organic acids can increase soil Zn availability
by two means. First, they are released with protons as counter ions (Jones,
1998). Thus reducing rhizosphere pH and increasing Zn availability. Second, the
organic acids act as chelating agents for micronutrients, such as Fe, Mn and Zn.
It has been shown that organic acids such as malate and citrate can release Fe
from goethite and ferrihydrite (Jones et al., 1996).
In my previous studies, significant variations in Zn acquisition and
utilization among wheat genotypes were evident. But little is known whether
different wheat genotypes differ in amount and nature of organic acids released
from roots under Zn deficiency. The present experiment was conducted to
evaluate differences in root exudation under Zn deficiency of selected wheat
genotypes differing in Zn acquisition.
3.4.2 Materials and Methods
3.4.2.1 Growth Conditions
The experiment was conducted in a growth chamber under controlled
conditions at the Institute of Soil and Environmental Sciences. In the growth
chamber, fluorescent lamps provided a light intensity of approximately 400 µmol
m-2S-1 with 16 hour day and 8 hour night period at 22ºC and 15ºC respectively.
Relative humidity was 60%.
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3.4.2.2 Plant Growth and Nutrient Solution
Seeds of both selected genotypes (Sehar-06 and Vatan) were sown in a
plastic tray containing quartz sand. Four seven days old uniform seedlings of
both genotypes were transferred to plastic tubs containing 5 L of ½ strength
Johnsons’s nutrient solution. After 4th day of transplanting, the concentration of
the nutrient solution was increased to full strength. The full strength nutrient
solution had the following composition; 1 mM KH2PO4, 2 mM Ca(NO3)2, 1.5 mM