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.”

Supervisory Committee Chairman: ___________________________ (Dr. Rahmatullah) Member: ___________________________ (Dr. Atta Muhammad Ranjha) Member: ___________________________ (Dr. Mumtaz Hussain)

DEDICATED TO

DR. MAQSOOD AHMAD GILL MY FATHER, MY MENTOR, MY INSPIRATION

ACKNOWLEDGEMENT

With the name of Allah, the Most Merciful and Beneficent

and His Holey and Beloved Prophet Hazrat Muhammad (May

Peace Be Upon Him), the guiding light for all mankind.

I appreciatively acknowledge the guidance and

supervision of Dr. Rahmatullah throughout my dissertation work

and write up of the manuscript. His critical reading of this

manuscript and valuable suggestions are highly appreciable. I

am also grateful for the suggestions and constructive criticism

offered by Dr. Atta Muhammad Ranhja and Dr. Mumtaz

Hussain in the write up of my manuscript.

I would also like to pay special thanks to Dr. Tariq Aziz,

Shamsa Kanwal, Haji Fazal Rasool, Sajid Ali and Akhtar Rasool.

In the end, I am forever indebted for the love and support

of my uncle Dr. Abdus Sattar Gill, my loving mother, my caring

sister and my adherent brother Muhammad Imran Maqsood.

Muhammad Aamer Maqsood

August, 2009

iv

ABSTRACT

Zinc (Zn) deficiency is a universal nutrient constraint of agricultural crops

produced on alkaline calcareous soils. Crop species and varieties differ

genetically in response to Zn applied to root medium. A series of solution and soil

culture experiments were conducted to assess the differential Zn utilization

efficiency of wheat genotypes having varying genetic attributes. In the first study,

twelve wheat genotypes were grown in solution culture at adequate and deficient

Zn levels. The plants were harvested twice after transplanting and Zn efficiency

traits, such as biomass accumulation, Zn uptake and Zn utilization efficiency

were identified. Based on the results, thus generated, the wheat genotypes

Sehar-06 and Vatan were categorized as Zn efficient and inefficient respectively

and selected for next studies. In second study, the wheat genotypes were grown

in plastic pots containing a Zn deficient soil. Plants were harvested at maturity.

The harvested plants were analyzed for uptake and distribution of Zn in wheat

straw and grain. Similar response of wheat genotypes to Zn stress was observed

in soil culture. Sehar-06 was selected as efficient and Vatan was selected and

inefficient genotype. In the third study selected wheat genotypes were grown in

solution culture to study Zn uptake, transport, and utilization efficiency. For this,

plants were harvested twice and shoot samples were separated in older and

younger leaves to estimate Zn translocations within plants. Wheat genotype

Sehar-06 efficiently translocated Zn from roots and older leaves to younger

leaves when subjected to Zn deficiency stress. The forth study involved growing

of these genotypes in solution culture with adequate and deficient Zn levels for

40 days to measure root exudates released in solution. Efficient wheat genotype

Sehar-06 released significantly higher amount of maleic acid under Zn deficient

conditions but no trend was observed in release of fumaric acid. It is hoped that

these findings will set forth useful information to categorize the wheat genotypes

under study into efficient and inefficient Zn utilizers. This in turn will be helpful for

researchers to plan their breeding experiments and to set genotype specific

recommendations for Zn deficient soils.

CONTENTS

No. TITLE PAGE

ACKNOWLEDGEMENT

LIST OF TABLES ii LIST OF FIGURES iii ABSTRACT iv CHAPTER 1 INTRODUCTION 1 CHAPTER 2 REVIEW OF LITERATURE 5 CHAPTER 3 EXPERIMENTATION

3.1 Differential growth response and zinc acquisition of wheat genotypes in hydroponics

16

3.1.1 Introduction 16

3.1.2 Materials and Methods 18 3.1.3 Results 23 3.1.4 Discussion 32

3.1.5 Conclusion 34

3.2 Differential growth response of wheat genotypes to applied zinc in soil culture

38

3.2.1 Introduction 38 3.2.2 Materials and Methods 39 3.2.3 Results 41 3.2.4 Discussion 44 3.2.5 Conclusion 46

3.3 Zinc translocation in wheat genotypes under zinc deficient environment

49

3.3.1 Introduction 49 3.3.2 Materials and Methods 50 3.3.3 Results 53 3.3.4 Discussion 60 3.3.5 Conclusion 64

3.4 Differences in organic acid extrusion by wheat genotypes under Zn deficiency

65

3.4.1 Introduction 65 3.4.2 Materials and Methods 66 3.4.3 Results 69 3.4.4 Discussion 75 3.4.5 Conclusion 77 CHAPTER 4 SUMMARY 80 LITERATURE CITED 83

CONTENTS

ii

LIST OF TABLES

No. TITLE PAGE

3.1.1 List of wheat genotypes 20

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.

STUDY I

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

STUDY I

21

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

shoot dry matter (SDM), root dry matter (RDM) (Table 3.1.2) and root shoot

ratio (RSR) of wheat genotypes (Table 3.1.2).

3.1.3.1 Total Dry Matter (TDM)

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

Total dry matter (g plant-1)

Shoot dry matter (g plant-1)

Root dry matter (g plant-1)

Root: shoot ratio

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)

ADEQ Zn (2 µM)

DEF Zn (0.2 µM)

Inqalab-91 3.66 3.29 3.15 2.66 0.51 0.63 0.16 0.24

Bhakar-2000 3.40 2.38 2.97 1.85 0.43 0.53 0.14 0.29

Pari-73 3.58 2.66 3.19 2.07 0.39 0.58 0.12 0.29

Yaqora 3.52 3.07 3.15 2.43 0.37 0.64 0.12 0.26

As-2002 3.77 3.22 3.29 2.42 0.48 0.80 0.15 0.32

Shafaq-06 4.04 2.99 3.51 2.45 0.53 0.53 0.15 0.22

Auqab-2000 3.92 3.75 3.42 2.95 0.50 0.79 0.15 0.27

Sehar-06 5.28 4.21 4.60 3.25 0.68 0.96 0.15 0.30

Dirk 2.13 1.35 1.95 1.14 0.17 0.20 0.09 0.19

Iqbal-2000 2.88 2.79 2.55 2.17 0.33 0.62 0.13 0.29

Vatan 1.81 1.70 1.48 1.08 0.33 0.62 0.22 0.57

SARC-1 4.68 3.29 4.01 2.30 0.67 0.99 0.16 0.43

Mean 3.56 2.89 3.11 2.23 0.45 0.66 0.145 0.306

LSD0.05 (Genotype x Zn Level)

1.95 1.63 0.46 0.17

STUDY I

25

hence these genotypes gained the lowest index scores of 1. All other

genotypes were medium in TDM production and their index score was 2.

3.1.3.2 Shoot Dry Matter (SDM)

Genotypes varied significantly (p<0.01) for shoot dry matter (SDM)

production (Table 3.1.2). Shoot dry matter of wheat genotypes ranged

between 1.08 g plant-1 in Vatan and 4.60 g plant-1 in Sehar-06. Sehar-06

produced the maximum SDM when grown either adequate or deficient Zn

application and minimum SDM was produced by Vatan and Dirk. Differences

in SDM among different crop genotypes with Zn application to rooting medium

had also been observed by other researchers (Cakmak et., 2001, Irshad et

al., 2004).

3.1.3.3 Root Dry Matter (RDM)

Genotypes differed significantly (p<0.01) for root dry matter (RDM)

production (Table 3.1.2). Root dry matter production ranged from 0.18 to 0.96

g plant-1. Similar to shoot, maximum RDM was observed by Sehar-06 when

grown under Zn deficient conditions. Minimum RDM was produced by Dirk.

3.1.3.4 Root:Shoot Ratio

Significant variations were observed in wheat genotypes for root:shoot

ratio (RSR) also (Table 3.1.2). Increase in root to shoot ratio was observed in

all wheat genotypes when grown in Zn deficient conditions compared to

STUDY I

26

adequate conditions. Root:shoot ratio ranged from 0.14 to 0.39. Maximum

RSR was exhibited by Vatan and minimum by Dirk.

3.1.3.5 Shoot Zn Concentration and Uptake

There were significant (p<0.01) main and interactive effects of

genotypes and Zn levels on shoot Zn concentration of wheat genotypes

(Table 3.1.3). Shoot Zn concentration was significantly lower in plants grown

under Zn deficient conditions than under Zn adequate conditions. Shoot Zn

concentration of wheat genotypes ranged between 18.71 µg g-1 in Shafaq-6

and 64.75 µg g-1 in Sehar-06. In plants grown under Zn deficient conditions Zn

concentration was lower than critical concentration (Reuter et al., 1997).

Significant genetic variability among wheat (Cakmak et al., 2002) and cotton

(Irshad et al., 2004) had been reported for shoot Zn concentration under

varying level of available Zn in root medium.

There were significant (p<0.01) main and interactive effects of

genotypes and Zn levels on shoot Zn uptake (Table 3.1.3). Zinc contents

were significantly (p<0.01) lower in plants grown in Zn deficient conditions.

Zinc uptake in shoots of wheat genotypes ranged from 24.59 µg plant-1 in

Vatan and 298.40 µg plant-1 in Sehar-06.

3.1.3.6 Root Zn Concentration and Uptake

Root Zn concentration varied significantly (p<0.01) among the

genotypes (Table 3.1.3). Zinc concentration in roots reduced significantly

STUDY I

27

Table 3.1.3 Shoot and root Zn concentration and uptake of wheat genotypes grown with adequate (ADEQ) and

deficient (DEF) Zn nutrient solution

Shoot Zn Conc. (µg g-1)

Root Zn Conc. (µg g-1)

Shoot Zn uptake (µg plant-1)

Root Zn uptake (µg plant-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)

ADEQ Zn (2 µM)

DEF Zn (0.2 µM)

Inqalab-91 55.64 19.51 48.04 14.70 175.80 52.15 24.63 9.26

Bhakar-2000 61.87 20.79 49.83 14.09 184.73 38.69 22.62 6.88

Pari-73 59.00 24.30 51.61 10.84 188.70 50.17 20.36 6.42

Yaqora 56.76 22.22 49.51 10.39 178.93 54.23 18.71 6.59

As-2002 55.80 26.86 57.51 12.15 186.30 64.50 28.67 9.35

Shafaq-06 63.31 18.71 57.13 11.79 222.48 44.85 30.50 6.28

Auqab-2000 54.04 19.83 48.31 12.15 185.81 58.58 24.43 9.61

Sehar-06 64.75 23.50 51.72 13.27 298.40 76.02 35.41 12.90

Dirk 51.16 25.26 41.57 7.53 100.50 29.72 7.24 1.56

Iqbal-2000 50.68 25.10 48.53 12.31 130.12 54.17 15.97 7.61

Vatan 55.64 22.86 82.12 9.35 81.96 24.59 26.73 5.90

SARC-1 61.07 25.10 36.62 10.55 242.28 57.97 26.48 10.10

Mean 57.47 22.83 51.87 11.59 181.33 50.46 23.48 7.70

LSD0.05 (Genotype x Zn Level)

13.23 15.50 98.56 20.20

STUDY I

28

(p<0.01) in plants grown in Zn deficient solution. Maximum Zn concentration

in roots was 82.12 µg g-1 in Vatan and minimum was 7.53 µg g-1 in Dirk. There

were also significant (p<0.01) effects of genotypes and Zn levels on root Zn

uptake (Table 3.1.4). Zinc uptake in roots of wheat genotypes ranged from

1.56 µg plant-1 to 35.41 µg plant-1.

3.1.3.7 Zinc Utilization Efficiency (ZnUE)

Data regarding shoot Zn use efficiency (ZnUE) of wheat genotypes is

presented in Table 3.1.4. Genotypes differed significantly (p<0.01) for Shoot

ZnUE. Interaction between Zn and genotypes was non-significant. Shoot Zn

use efficiency ranged between 26.93g2 DM mg-1 Zn to 141.63g2 DM mg-1 Zn

in plants. There were significant (p<0.01) main and interactive effects of

genotypes on root Zn use efficiency (ZnUE) of wheat (Table 3.1.4). It ranged

from 4.10 g2 DM mg-1 Zn in Vatan to 72.14 g2 DM mg-1 Zn in Sehar-06.

Differences among crop genotypes for Zn use efficiency had also been

reported by Irshad et al., (2004).

3.1.3.8 Relative Growth Rate

There were significant (p<0.01) main and interactive effects of

genotypes and Zn application on relative growth rate (RGR) of shoot of wheat

genotypes. Maximum relative growth rate (RGR) of shoot was 0.13 mg g-1

SDM day-1 in Sehar-06 and minimum was 0.07 mg g-1 SDM day-1 in Shafaq-

06 and Yaqora. There were significant (p<0.01) effects of genotypes and Zn

STUDY I

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Table 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

Shoot ZnUE (g2 SDW mg Zn-1)

Root ZnUE (g2 RDW mg Zn-1)

RGR shoot (mg g-1 SDM day-1)

RGR Root (mg g-1 RDM 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)

ADEQ Zn (2 µM)

DEF Zn (0.2 µM)

Inqalab-91 57.14 136.85 10.68 43.50 0.13 0.09 0.14 0.11

Bhakar-2000 47.95 90.08 8.49 45.64 0.11 0.08 0.10 0.11

Pari-73 54.26 88.10 7.62 54.34 0.11 0.09 0.10 0.14

Yaqora 55.92 110.08 7.54 62.83 0.11 0.07 0.11 0.11

As-2002 58.41 92.88 8.26 71.32 0.11 0.09 0.10 0.13

Shafaq-06 55.58 141.63 9.32 46.34 0.11 0.07 0.11 0.11

Auqab-2000 63.35 148.74 10.30 69.24 0.11 0.10 0.09 0.13

Sehar-06 71.42 140.39 13.19 72.14 0.13 0.09 0.13 0.13

Dirk 38.27 45.51 4.32 26.49 0.08 0.04 0.05 0.04

Iqbal-2000 50.44 87.55 6.92 53.24 0.10 0.10 0.07 0.13

Vatan 26.93 47.99 4.10 66.63 0.06 0.04 0.05 0.09

SARC-1 67.00 91.68 17.96 39.98 0.13 0.08 0.12 0.14

Mean 53.89 101.78 9.06 54.30 0.11 0.08 0.10 0.11

LSD0.05 (Genotype x Zn Level)

62.06 39.25 0.05 0.06

STUDY I

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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.

STUDY I

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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

STUDY I

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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

relative Zn contents. Zinc uptake significantly correlated (P<0.01) with shoot dry

matter and Zn utilization efficiency (Figure 3.1.3). This correlation shows the

importance of Zn uptake in explaining variation in Zn utilization efficiency. The

results obtained in this experiment are in agreement with the results obtained in

wheat (Cakmak et al., 1996) and chickpea (Khan et al., 2000).

3.1.5 Conclusion

Genotypes differed significantly for their biomass production and Zn

contents. Significant interaction among genotypes and Zn application for biomass

production and Zn contents indicated large genetic variations which can be

exploited to select more Zn efficient wheat genotypes. Sehar-06 and Auqab-2000

were efficient in dry matter yield, and Zn contents both in shoot and root.

However, further studies both in solution as well as soil culture experiments are

needed to identify mechanisms responsible for these contrasting differences

among genotypes.

STUDY I

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Figure 3.1.1 Dry matter partitioning among shoot and root of wheat genotypes grown with adequate (+) and

deficient (-) Zn nutrient solution

0

1

2

3

4

5

6

Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn- Zn+ Zn-

Inqalab-91 Bhakar-2000

Pari-73 Yaqora As-2002 Shafaq-06 Auqab-2000

Sehar-06 Dirk Iqbal-2000 Vatan SARC-1

Wheat Genotypes

Dry

ma

tte

r y

ield

(g

)Root Shoot

STUDY I

36

Figure 3.1.2 Categorization of wheat genotypes according to Zn efficiency

Inqalab-91

Bhakar-2000Pari-73

Yaqora

As-2002

Shafaq-06Auqab-2000

Sehar-06

Dirk

Iqbal-2000

Vatan (v-87092)

SARC-1

0

20

40

60

80

100

120

140

160

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

SDM (g)

Zn

UE

(g2 S

DW

mg

Zn-1

)

NE-LSW

ME-LSW

HE-LSW

NE-HSW

ME-HSW

HE-HSW

NE-MSW

ME-MSW

HE-MSW

STUDY I

37

Figure 3.1.2 Correlation of various Zn related physiological parameters

CHAPTER 3, STUDY II

Differential growth response of wheat genotypes to applied zinc in soil culture.

3.2.1 Introduction

Wheat is an important staple food crop of the entire world as well as Pakistan.

But the average yield of wheat is low due to many factors. Nutrient deficiency is

one of the important factors. The universal deficiency of nitrogen and phosphorus

is followed by Zn deficiency. Almost 50% of the world soils used for cereal

production are Zn deficient (Gibbson, 2006). As a result, approximately 2 billion

people suffer from Zn deficiency all over the world. Bhutta et al. (1999) have

reported Zn deficiency in Pakistan in children less than five years of age and

women of reproductive age. Low soil Zn is attributed to a number of soil and

environmental factors including low soil organic matter, high soil pH,

calcareousness, water logging and arid climate. (Cakmak, 1998; Tandon, 1995;

Mortvedt, 1991). Several approaches have been investigated to overcome the

common problem of Zn deficiency. These include Zn supplementation, food

diversification as well as food fortification. Increasing the Zn content of food crops

can be a good strategy to overcome its deficiency in people of developing

countries. The Zn content of crops can be increased via Zn fertilization but the

resource poor farmers are unable to bear the relatively high cost of Zn fertilizers.

STUDY II

39

Like many other crop species, wheat genotypes possess great variation in their

Zn acquisition and utilization (Oikeh et al., 2003; Banziger & Long, 2000). Most

studies emphasize on the effect of Zn application on vegetative growth and grain

production and not on the Zn status in grain. The objective of the present study

was to compare the response of twelve indigenous wheat genotypes to Zn

deficiency on a calcareous soil and to estimate the Zn status in grain.

3.2.2 Materials and Methods

3.2.2.1 Soil

Bulk surface soil sample (0-15 cm) was collected for Pindorian series (Udic

Haplustalf). The sample was air-dried and ground to pass through a 2 mm sieve.

A portion of the prepared soil sample was analyzed for various physico-chemical

properties. Soil had pH 7.36 which was measured in 1: 1 soil: water suspension

by calomel glass electrode assembly by using a Beckman pH meter. Soil texture

determined by hydrometer method was sandy loam (Gee & Bauder, 1986).

Organic matter content in the soil sample was 1.66 % according to Walkley-Black

method (Nelson & Sommer, 1982). Free lime was 1.5% which was estimated by

acid dissolution (Allison & Moodie, 1965). Plant available Zn in the soil was 0.75

mg kg-1 which was extracted with 0.005 M DTPA (Lindsay & Norvell, 1978) and

determined by atomic absorption spectrophotometer.

3.2.2.2 Pot Study

Five kilograms of thoroughly mixed soil was filled each in 72 polyethylene lined

plastic pots. Thirty mg N kg-1 soil as urea, 45 mg P kg-1 soil as monoammonium

phosphate (MAP) and 30 mg K kg-1 soil as K2SO4 was applied uniformly to all

STUDY II

40

pots in solution form. Two rates of Zn (0, 6 mg Zn kg-1 soil) were applied as

ZnSO4.7H2O in solution form. Before planting, soil in all the pots was moistened

with distilled water, dried and thoroughly mixed for equilibration. The pots were

arranged according to a completely randomized design in the green house (Steel

& Torrie, 1980). During the experimental period, the average temperatures in the

green house were 20± 5 ˚C at different times of the day and 12± 3 ˚C during the

night. Light intensity varied from 300 to 1400 µmol photon m-2 s-1 and relative

humidity varied from 35 % (midday) to 85 % (midnight). Eight seeds of each of

the twelve wheat genotypes were sown per pot. After germination two plants per

pot were allowed to grow. Distilled water was used to maintain moisture contents

at field capacity in all the pots during the experimental period. Two weeks after

sowing, a second dose of 30 mg N kg-1 soil as urea and 15 mg P kg-1 soil as

MAP were applied uniformly to all pots in solution form. Plant were harvested at

maturity, washed with distilled water and blotted dried with tissue paper. The

grains were separated from the straw. The straw samples were air-dried and

then oven-dried at 70 ˚C to a constant weight for dry matter yield in a forced air

oven. The straw and grain samples were then finely ground with a Wiley mill

fitted with a stainless steel chamber and blades. A portion of finely ground plant

and grain samples was digested in a diacid (HNO3:HClO4) mixture. The Zn

concentration in the digest was estimated by atomic absorption

spectrophotometer. Zinc stress factor (ZnSF) was calculated following Irshad et

al., 2004.

Znic Stress Factor = Shoot Dry Wt. at Adq Zn – Shoot Dry Wt at Def Zn X 100 SDW at Adq Zn

STUDY II

41

The data obtained for straw yield, grain yield, Zn concentration and its uptake by

wheat plants were statistically analyzed using Microsoft Excel and MSTAT-C

computer software (Russell & Eisensmith, 1983).

3.2.3 Results

The wheat genotypes produced significantly (P<0.01) different grain yield. The

sandy loam soil had marginally available DTPA extractable Zn up to 0.75 mg kg-

1. The application of Zn therefore had a significant (P<0.05) effect on grain yield

(Table 1). Maximum grain yield was produced by Sehar-06 and minimum by

Iqbal-2000 by Zn application and control treatment. Overall there was a 9.5%

increase in grain production when Zn was applied. There was a significant effect

(P<0.05) of zinc application on straw production of wheat genotypes over control

(Table 1). Under adequate Zn supply maximum straw was produced by Sehar-06

(15.83g) and minimum was produced by Iqbal-2000 (11.23).

There was a significant effect (P<0.05) of wheat genotypes and Zn application on

the grain to straw ratio. Grain to straw ratio ranged between 1.4 to 0.78. Yaqora,

Sehar-06 and Auqab-2000 had a higher grain to straw ratio compared to other

genotypes.

3.2.3.1 Zinc Concentration and Uptake

There was a significant (P<0.05) main and interactive effect of different wheat

genotypes and Zn application on concentration and uptake of Zn in wheat grain

and straw (Table 2). Zinc concentration in wheat straw ranged from 29.80 to

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Table 3.2.1. Grain yield, straw yield and grain/straw ratio of twelve wheat genotypes grown at adequate and deficiency zinc levels.

Grain Yield (g) Straw Yield (g) Grain/Straw Ratio Wheat Genotypes -Zn

(Control) +Zn

@ 6 µg g-1 -Zn

(Control) +Zn

@ 6 µg g-1 -Zn

(Control) +Zn

@ 6 µg g-1 Inqalab-91 14.06 14.42 12.53 15.01 1.12 0.96 Bhakar-2000 14.14 12.73 12.64 13.03 1.12 0.98 Pari-73 11.72 12.92 11.03 12.91 1.06 1.00 Yaqora 14.65 15.24 10.43 12.09 1.40 1.26 As-2002 10.29 10.84 13.65 13.85 0.75 0.78 Shafaq-06 11.73 14.28 13.01 13.46 0.90 1.06 Auqab-2000 13.47 13.80 12.86 13.45 1.05 1.03 Sehar-06 14.38 15.35 14.01 15.83 1.03 0.97 Dirk 10.84 11.24 11.61 13.24 0.93 0.85 Iqbal-2000 8.26 8.73 10.38 11.23 0.80 0.78 Vatan 10.31 10.83 12.19 12.64 0.85 0.86 SARC-1 10.91 12.49 13.96 15.20 0.78 0.82 Mean 12.06 12.74 12.36 13.50 0.98 0.95 LSD0.05 (Gentype x Zn Level) 2.85 1.36 .078

STUDY II

43

Table 3.2.2. Zinc concentration and zinc uptake in straw and grain of twelve wheat genotypes grown at adequate and deficiency zinc levels.

Zn Concentration in

Straw (µg g-1) Zn Concentration in

Grain (µg g-1) Total Zn Uptake in Straw (µg plant-1)

Total Zn Uptake in Grain (µg plant-1)

Genotype -Zn

(Control) +Zn

@ 6 µg g-1 -Zn

(Control) +Zn

@ 6 µg g-1 -Zn

(Control) +Zn

@ 6 µg g-1 -Zn

(Control) +Zn

@ 6 µg g-1 Inqalab-91 29.80 35.58 52.41 60.58 373.66 534.72 735.52 865.99 Bhakar-2000 41.87 44.76 54.29 59.90 433.63 583.30 761.26 753.61 Pari-73 34.73 39.32 40.00 55.82 381.57 515.12 472.63 719.91 Yaqora 33.71 40.85 51.05 59.05 351.24 497.37 746.45 899.25 As-2002 33.64 42.72 38.64 51.73 456.56 591.44 390.23 562.58 Shafaq-06 36.77 43.76 34.90 68.23 481.05 592.43 409.83 975.23 Auqab-2000 32.35 34.05 52.76 54.80 414.89 459.21 710.53 750.24 Sehar-06 34.19 51.22 54.12 57.35 585.89 809.46 778.94 883.74 Dirk 36.77 39.49 55.65 69.93 433.77 519.47 591.04 787.52 Iqbal-2000 37.96 40.68 45.61 61.60 398.17 455.36 372.82 538.43 Vatan 39.15 45.27 51.39 51.90 477.61 572.87 531.97 542.89 SARC-1 32.58 40.78 48.16 56.16 454.72 620.58 519.53 703.45 Mean 35.29 41.54 48.25 58.92 436.90 562.61 585.06 748.57 LSD0.05

(Gentype x Zn Level)

5.57 2.85 75.45 112.19

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51.22 µg g-1. An 18.1% increase in straw Zn concentration was observed by Zn

application. Both under control and adequate Zn supply Zn uptake was maximum

in Sehar-6 and minimum in Iqbal-2000. There was a significant (P<0.05) main

and interactive effect of wheat genotypes and Zn application on the grain Zn

concentration and uptake (Table 2). Zinc concentration in wheat grain ranged

from 34.9 to 69.93 µg g-1. Maximum grain Zn uptake was observed by Shafaq-06

under adequate Zn supply and by Sehar-06 under deficient Zn supply.

3.2.4 Discussion

The genotypes exhibited wide variation for straw as well as grain biomass

production. Aycicek & Yildirim, 2006 also reported differences in grain yield and

yield components in different wheat genotypes. Cakmak et al., 1998 and Hoffland

et al., 2006 reported that by Zn addition to the root medium, straw production in

different crops increases. They correlated genotypic variation of crop plants to

their tolerance to low Zn availability in soil. The genotypes Shafaq-06, Sehar-06

and SARC-1 showed an overall increase in grain production by Zn application

over control compared to increase in straw production (Figure 1). This attribute is

an important parameter in improving nutrient content of the edible parts of plants.

Total Zn uptake both in grain and straw ranged from 975.23 to 351.24 µg plant-1.

Wheat grains are mostly used fro human consumption. A 48.3 to 62.2 % of Zn

was stored in grains.

Zinc contents in straw and grain is an important parameter indicating the relative

acquisition efficiencies of these genotypes from a Zn deficient soil. Efficient

genotypes such as Sehar-06, Shafaq-06 and Inqalab-91 accumulated more Zn

STUDY II

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Figure 1. Percent increase (%) over control by Zn application in straw and grain yield of twelve wheat genotypes-15

-10

-5

0

5

10

15

20

25

Inqalab-91

Bhakar-2000

Pari-73

Yaqora

As-2002

Shafaq-06

Auqab-2000

Sehar-06

Dirk

Iqbal-2000

Vatan (v-87092)

SARC-1

Genotypes

Per

cen

t In

crea

se (

%)

straw

Grain

STUDY II

46

even under deficient soil Zn conditions. Zinc use efficiency is the amount of dry

matter produced per unit of Zn absorbed (Siddiqui & Glass, 1981). Present

results also indicated significant differences in wheat genotypes for Zn utilization

efficiency. Zinc use efficiency was positively correlated with dry matter production

(Fig 2). Differences in Zn use efficiency have been reported in various crops such

as wheat, rye, barley and oats (Cakmak et al., 1998) and cotton (Shukla & Raj,

1987).

Relative reduction in shoot dry matter or Zn stress factor (ZnSF) is a useful

parameter in assessing relative tolerance of crops genotypes to Zn deficiency

(Babikar, 1986; Yaseen, et al., 2000; Irshad et al., 2004). Significant differences

for ZnSF (Fig. 3) were exhibited among wheat genotypes. The maximum relative

reduction in SDM (%) due to Zn deficiency was exhibited by Inqalab-91 (16.5 %)

while As-2002 and Bhakar-2000 showed negligible reduction in SDM.

3.2.5 Conclusion

Useful genetic variations existed among wheat genotypes for Zn acquisition and

its utilization to produce biomass. Cultivars efficient in Zn acquisition and

utilization (Sehar-06, Shafaq-06 and Inqalab-91) showed more growth than

inefficient cultivars. Efficient cultivars also translocated more Zn from the straw to

the grains. Nevertheless verification of the results is needed under field

conditions.

STUDY II

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Figure 2. Correlation between shoot dry matter and Zn use efficiency of wheat genotypes

R2 = 0.51

10

10.5

11

11.5

12

12.5

13

13.5

14

14.5

250 270 290 310 330 350 370 390 410 430 450

Shoot Zn use efficiency

Sh

oo

t d

ry m

atte

r (g

/pla

nt)

STUDY II

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Figure 3. Relative reduction in shoot dry matter (ZnSF%) in tewlve wheat genotpes due to Zn deficiency

0

2

4

6

8

10

12

14

16

18

Inqalab-91Pari-7

3Yaqora Dirk

Sehar-06

SARC-1

Iqbal-2000

Auqab-2000

Vatan (v-87092)

Shafaq-06

Bhakar-2000

As-2002

Genotype

Zn

Str

ess

Fac

tor

(%)

CHAPTER 3, STUDY III

Zinc translocation in wheat genotypes under zinc deficient environment

3.3.1 Introduction

Zinc deficiency in cereal crops such as wheat, is a well documented cause

of reduction in agricultural production in almost every part of the world

(Marschner, 1995). Due to agronomic and economic factors, fertilization is not

always the only option to cope with Zn deficiency (Graham and Rengel, 1993).

Cultivation of efficient Crop species/genotypes may also be an alternative

approach to combat the situation. Genotypes of crops have great variation in Zn

utilization efficiency, as reported for wheat (Cakmak et al., 2001) and rice (Sakal

et al., 1989).

Zinc is relatively immobile in soils. It undergoes numerous physical,

chemical and biological changes in soil which render it unavailable for plant

uptake. But inside plants foliar applied Zn is translocated to both upper and lower

parts of the plant (Haslett et al. 2001). Not much is known about transport of Zn

from roots to leaves and from leaves to other plant organs in case of Zn stress.

On mechanism that might contribute towards improved Zn utilization efficiency in

crops is enhanced translocation of Zn from root to shoot meristems and its

translocation from older to growing parts of the plant under Zn deficiency. The

superior ability of genotypes for Zn translocation from root to shoot and its

STUDY III

50

utilization under deficient Zn supply was shown to play a role in improved Zn

efficiency in wheat genotypes (Cakmak et al., 1996). According to Hajiboland et

al (2001), Zn deficiency tolerance of a Zn-efficient rice genotype is linked to its

ability to re-translocate zinc from older to growing parts of the shoot. Among

dicots, Zn-efficient chickpea genotypes transported more than 70% of the total

absorbed zinc to the shoot compared with inefficient genotypes (Khan et al.,

2000).

We can use such variations to exploit, select and produce more Zn

efficient crop genotypes. Significant differences were present among wheat

genotypes for Zn acquisition and utilization in our previous solution culture and

soil culture experiments. Not much is known about the reasons for these

contrasting differences. It is assumed that differences in Zn absorption,

translocation and remobilization might be the possible adaptive mechanism

contributing towards differences in Zn efficiency. The current study was planned

to evaluate Zn re-mobilization in selected wheat genotypes.

3.3.2 Materials and Methods

3.3.2.1 Plant Growth

The nursery was grown in pre-washed sand. Sand, washed with 0.05 N

hydrochloric acid (HCl), was subsequently washed with tap water and distilled

water until it was acid free. It was then added to iron trays (9X18 inch) lined with

STUDY III

51

polyethylene sheets. Seeds of two selected genotypes (Sehar-06 and Vatan)

were sown in these trays. During seed germination distilled water was used for

irrigation. Eight Seedlings of each genotype were transplanted seven days after

germination, in thermopal sheets floating on half strength Johnson’s nutrient

solution (1000 ml) contained in plastic cups separately. Aquarium pumps were

used for continuously aerating solution during the growth period.

3.3.2.2 Composition of Nutrient Solution

Nutrient solution contained 6 mM N, 2 mM P, 3 mM K, 2 mM Ca, 1 mM

Mg, 2 mM S, 1µM Zn, 100 µM Fe 10 µM B, 1 µM Mn, 0.5 µM Cu, 0.1 µM Mo and

50 µM Cl, The salts used were; KH2PO4, Ca(NO3)2, K2SO4, MgSO4, KCl, H3BO3,

MnSO4, CuSO4, (NH4)6 Mo7O4 F(III)-EDTA and ZnSO4.7H2O. The pH of nutrient

solution was maintained at 6.0 ± 0.5 with 1N NaOH or 1N HCl.

3.3.2.3 First Harvest

Four replicates of both genotypes were harvested after 30 days of

transplanting. These plants were separated into shoots and roots and washed

with distilled water. Young leaves were then separated from shoots (including

mature leaves) and stored in paper bags. Top 3 leaves were considered as

young leave and the rest of shoot and leaves were called mature leaves. The

plant samples were air dried in a wire house for two days. Air dried samples were

then dried at 75°C in a forced air-driven oven (EYELA oven, WFO-600ND) for 48

h. Oven dry weight of the samples was recorded using top loading balance.

STUDY III

52

3.3.2.4 Change of Nutrient Solution

After harvesting of four replicates, zinc deficiency was induced by

replacing the existing nutrient solution with Zn-free nutrient solution.

3.3.2.5 Second Harvest

The remaining four replicates of each genotype were allowed to grow

further for another 10 days under Zn deficiency to study the remobilization of

absorbed Zn. After 10 days the plants were harvested and separated into shoots

and roots. Samples were washed with distilled water and blotted dry with tissue

papers. Young leaves were separated from shoot samples and stored separately

in paper bags. After air drying, the samples were dried at 75°C in a forced air-

driven oven (EYELA windy oven, WFO-600ND) for 48 hours and the dry weight

was recorded.

3.3.2.6 Zinc Concentration

Samples from both harvests were fine ground in a Wiley Mill to pass

through a 1 mm sieve. Uniform samples of various tissues (young leaves, stem

and roots) from both harvests were digested in a di-acid mixture (3:1) of nitric

and perchloric acid (Miller, 1998). The digest was analyzed for Zn on atomic

absorption spectrophotometer (Perkin Elmer Analyst-100). Zn uptake (content)

was calculated by multiplying the Zn concentration with dry weight of tissue in

both the harvests.

STUDY III

53

3.3.2.7 Zinc Utilization Efficiency (ZnUE)

Zinc utilization efficiency (ZnUE) of various wheat genotypes was

calculated according to following formula (Siddiqui and Glass, 1981)

1

ZnUE (g2 SDM mg-1 Zn) = Zn conc. (mg g-1) x SDW (g)

Where as SDW is the shoot dry weight (g) and Zn conc. is concentration of Zn in shoot.

3.3.2.8 Statistical Analysis

The data collected at each harvest was statistically analyzed separately

using computer software ‘MSTAT-C’ (Russell and Eisensmith, 1983). Completely

randomized design was employed for analysis of variance. ). Least significant

difference (LSD) test was used to separate the treatment means (Steel and

Torrie, 1980).

3.3.3 Results

STUDY III

54

3.3.3.1 Biomass Production

Data regarding dry weights of shoots and roots of wheat genotypes is

presented in Table 3.3.1. Genotypes differed significantly (p<0.01) for young

leaves, mature leaves and root dry matter production at both harvests. Biomass

of both genotypes was more at second harvest than biomass produced at first

harvest. More growth was observed in Sehar-06 than Vatan. Dry weight of young

leaves was more in Sehar-06 and of mature leaves was more in Vatan. Several

researchers had reported significant differences among genotypes of several

crop species for biomass production grown at varying levels of Zn supply

(Erenoglu et al., 2002; Hajiboland et al., 2000).

At first harvest, dry matter production of young leaves was 0.25 g plant-1 in

Sehar-06 and 0.30g plant-1 in Vatan. At second harvest, it was 0.46 g plant-1 in

Sehar-6 and 0.41 g plant-1 in Vatan. Dry matter production of mature leaves at

the time of first harvest was 0.37 g plant-1 in Sehar-06 and 0.54 g plant-1 in Vatan.

At second harvest, dry matter of mature leaves was 0.41 g plant-1 in Sehar-06

and 0.72 g plant-1 in Vatan.

Genotypes differed significantly (p<0.01) for root dry weight (RDW) at both

harvests (Table 3.3.1). At first harvest, RDW it was 0.07 g plant-1 in Seahar-6 and

0.14 g plant-1 in Vatan. At second harvest, RDW was 0.17 g plant-1 in Sehar-06

and 0.27 g plant-1 in Vatan.

STUDY III

55

3.3.3.2 Zinc Concentration

Genotypes differed significantly (p<0.01) for their Zn concentration in

young and mature leaves; and roots in plants at both harvests (Table 3.3.2).

Variations among genotypes for Zn concentration in young leaves were more

pronounced when plants were grown under induced Zn deficiency. Zinc

concentration in young leaves significantly reduced in both genotypes when their

plants were grown in Zn free solution for 10 days.

STUDY III

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Table 3.3.1. Biomass production of wheat genotypes (Values are means of four replicates + SE)

Biomass production g plant -1

Young leaves Mature Leaves Roots Genotypes

30 D 40 D 30 D 40 D 30 D 40 D

Sehar-06 0.250 +.01 0.458 +.01 0.374 +.01 0.411 +.02 0.071 +.01 0.173 +.01

Vatan 0.303 +.01 0.418 +.01 0.544 +.01 0.716 +.01 0.140 +.01 0.275 +.01

ANALYSIS OF VARIANCE

F Value 27.3 5.99 114 216 2085 48.6

Probability .002 .050 .000 .000 .000 .000

Table 3.3.2. Zinc concentration of wheat genotypes

(Values are means of four replicates + SE)

Zinc Concentration µg g -1

Young leaves Mature Leaves Roots Genotypes

30 D 40 D 30 D 40 D 30 D 40 D

Sehar-06 29.35 +.78 28.14 +.76 40.54 +.49 29.27 +.77 68.22 +1.16 24.57 +.38

Vatan 25.47 +.40 23.81 +.61 35.85 +.55 26.07 +.38 58.15 +.36 35.15 +.26

Analysis of Variance

F Value 19.7 19.6 40.8 13.9 68.9 534

Probability .004 .004 .000 .009 .000 .000

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57

Zinc concentration in mature leaves of wheat genotypes was lower at

second harvest than Zn concentration at first harvest. Zinc concentration in

mature leaves was 40.54 µg g -1 in Sehar-06 and 35.85 µg g -1 in Vatan at first

harvest and it was 29.27 µg g -1 in Sehar-06 and 26.07 µg g -1 in Vatan at second

harvest.

Zinc concentration was reduced significantly in roots at second harvest as

plants were grown with Zn free solution (Table 3.3.2). It was 68.22µg g -1 in Sehar-

06 and 58.15µg g -1 in Vatan at first harvest. In plants grown in Zn free solution,

root Zn concentration was 24.57µg g -1 in Sehar-06 and 35.15µg g -1 in Vatan.

Genetic differences in plants for their tissue Zn concentration had been reported

in several crop species (Cakmak et., 2001, Irshad et al., 2004, Gao et al.,

2006).

3.3.3.3 Zinc Uptake

Zinc deficiency in root medium significantly (p<0.01) influenced partitioning

of Zn in plant tissues. Genotypes did not differ significantly for Zn uptake in

young leaves at first harvest (Table 3.3.3). Zinc uptake in young leaves of plants

at second were significantly (p<0.01) more than those at first harvest. Zinc

uptake in young leaves ranged from 7.32 µg plant-1in Sehar-06 and 7.86 µg plant-1

STUDY III

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Table 3.3.3. Zinc uptake of wheat genotypes (Values are means of four replicates + SE)

Zinc Uptake µg plant -1

Young leaves Mature Leaves Roots Genotypes

30 D 40 D 30 D 40 D 30 D 40 D

Sehar-06 7.32 +.31 13.00 +.38 14.82 +.53 12.00 +.19 4.94 +.19 4.42 +.28

Vatan 7.86 +.22 10.28 +.40 19.67 +.34 18.66 +.45 8.11 +.09 9.21 +.29

Analysis of Variance

F Value 3.39 24.2 60.1 187 227 141

Probability .115 .003 .000 .000 .000 .000

Table 3.3.4. Zinc utilization efficiency of wheat genotypes

(Values are means of four replicates + SE)

Zinc utilization efficiency g2 DM mg -1 Zn

Young leaves Mature Leaves Roots Genotypes

30 D 40 D 30 D 40 D 30 D 40 D

Sehar-06 7.42 +.49 17.01 +.19 9.23 +.24 13.97 +.41 1.10 +.06 6.73 +.19

Vatan 11.96 +.25 15.86 +.30 14.90 +.30 27.47 +.59 2.40 +.02 8.19 +.21

Analysis of Variance

F Value 67.5 10.5 217 351 448 25.9

Probability .000 .018 .000 .000 .000 .002

STUDY III

59

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

STUDY III

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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

STUDY III

63

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

STUDY III

64

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

66

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%.

STUDY IV

67

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

K2SO4, 1mM MgSO4, 50 µM KCl, 10 µM H3BO3, 1 µM MnSO4, 0.5 µM CuSO4,

0.02 µM (NH4)6 Mo7O4 and 100 µM F(III)-EDTA.

3.4.2.3 Zinc Levels

There were two zinc (Zn) levels in nutrient solution (viz 2 µM as adequate

and 0.2µM as deficient Zn) using zinc sulphate (ZnSO4.7H2O). The pH of nutrient

solution was maintained at 6.0 ± 0.5 with 1N NaOH or 1N HCl. It was monitored

regularly and complete solution in each tub was changed after every 4 days.

3.4.2.4 Collection of Root Exudates

Plants were grown for 30 days. After 14, 21 and 28 days, the roots of

intact plants were carefully washed with distilled water and submerged for 120

minutes in 500 ml aerated distilled water contained in plastic bottles. After 120

minutes of incubation, the solution was measured and it was stored at – 20 ºC for

further analysis of root exudates (Fang et al., 2008).

STUDY IV

68

3.4.2.5 Harvesting of Plants

After 30 days, the plants were harvested. The plants were washed with

distilled water and blotted dry with tissue papers to record their fresh weights.

Plants samples were then oven dried at 72º C in a forced air oven for 48 h. Dry

weights of both shoots and roots were recorded using top loading balance.

3.4.2.6 Concentration and Uptake of Zn

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 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 uptake (µg Zn plant-1) were

calculated in shoots and roots by multiplying Zn concentration in the respective

tissue with its dry matter.

3.4.2.7 Identification and Quantification of Organic Acids

The stored solution was vacuum evaporated to dryness by means of a

rotary evaporator. The residue was re-dissolved in 20 ml of deionized water and

stored at -20ºC. Before measuring organic acids the solution was passed through

a C18-E column (S201-13, Strata, Torrance, CA, USA). The solution (20 µL) was

analyzed by reverse phase HPLC in the ion suppression mode. Separation was

STUDY IV

69

conducted on a 250X4 nm reversed phase column (Dionex, Germany) equipped

with a 205X4 nm hypersil ODS guard column (Dionex, Germany). Sample

solution was injected onto the column, and 18 mM KH2PO4 adjusted to pH 2.1

with H3PO4 was used for isocratic elution, with a flow rate of 0.5 ml min-1 at 28 ºC

and UV detection at 215 nm. Identification of organic acids was performed by

comparing retention times and absorption spectra with those of known standards

(Fang et al., 2008).

3.4.3 Results

Selected wheat genotypes were grown at two levels of Zn for 30 days,

Differential exudation of organic anions by roots in response to Zn deficiency was

evaluated in solution culture.

3.4.3.1 Biomass Production

Shoot dry weight (SDW) production was significantly (p<0.01) higher in

wheat genotypes grown with adequate Zn than those grown with deficient Zn

supply in the root medium (Table 3.4.1). In plants grown under deficient Zn level,

Sehar-06 accumulated 0.25 g plant-1 and Vatan 0.22 g plant-1. With adequate Zn

supply, it was 0.71 g plant-1 in Sehar-06 and 0.68 g plant-1 in Vatan.

Genotypes did not differ significantly for their root dry weight (RDW) when

grown under adequate as well as under deficient Zn supply in root medium

(Table 3.4.2). Root dry weight was 0.09 g plant- 1 in Sehar-06 to 0.12 g plant-1 in

STUDY IV

70

Vatan when grown under deficient Zn supply. In plants grown under adequate Zn

level, RDW was 0.10 g plant-1 in Sehar-06 to 0.12 g plant-1 in Vatan. Shoot dry

weight was influenced by Zn supply more than RDW.

3.4.3.2 Zinc Concentration

There were significant (p<0.05) main effects of genotypes and Zn levels

on shoot Zn concentration (Table 3.4.3). Zinc concentration in shoots of wheat

plants grown at adequate Zn supply was significantly (p<0.01) higher than

deficient Zn supply in growth medium. Zinc concentration in shoots of both

genotypes was lower than its critical limit of Zn sufficiency in wheat i.e.25 mg Kg-1

(Reuter et al., 1997). Plants grown under Zn deficient conditions exhibited clear

Zn deficiency symptoms.

Shoot Zn concentration was higher in Sehar-06 than Vatan in deficient as

well as adequate level of Zn supply. Variations in shoot Zn concentration among

genotypes of different crop species have been reported by various researchers

(Irshad et al., 2004; Cakmak et al., 1998).

STUDY IV

71

Table 3.4.1 Shoot dry weight of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels

(Values are means of four replicates)

Table 3.4.2 Root dry weight of wheat genotypes grown with deficient (0.2

µM) and adequate (2 µM) Zn levels (Values are means of four replicates)

Table 3.4.3 Shoot Zn concentration of wheat genotypes grown with

deficient (0.2 µM) and adequate (2 µM) Zn levels (Values are means of four replicates)

Shoot dry weight (g plant-1) Genotypes

Deficient Zn Adequate Zn

Sehar-06 0.25 0.71

Vatan 0.22 0.68 LSD0.05 (Genotype x Zn Level) 0.02

Root dry weight (g plant-1) Genotypes

Deficient Zn Adequate Zn

Sehar-06 0.09 0.10

Vatan 0.12 0.12 LSD0.05 (Genotype x Zn Level) 1.53

Shoot Zn concentration (µg Zn g-1 SDW) Genotypes

Deficient Zn Adequate Zn

Sehar-06 25.50 34.95

Vatan 19.99 30.66 LSD0.05 (Genotype x Zn Level) 2.08

STUDY IV

72

Table 3.4.4 Root Zn concentration of wheat genotypes grown with deficient (0.2 µM) and adequate (2 µM) Zn levels

(Values are means of four replicates)

Table 3.4.5 Shoot Zn uptake of wheat genotypes grown with deficient (0.2

µM) and adequate (2 µM) Zn levels (Values are means of four replicates)

Table 3.4.6 Root Zn uptake of wheat genotypes grown with deficient (0.2

µM) and adequate (2 µM) Zn levels (Values are means of four replicates)

Root Zn concentration (µg Zn g-1 RDW) Genotypes

Deficient Zn Adequate Zn

Sehar-06 26.17 68.22

Vatan 25.82 58.15 LSD0.05 (Genotype x Zn Level) 3.00

Shoot Zn contents (µg Zn plant-1) Genotypes

Deficient Zn Adequate Zn

Sehar-06 6.38 11.07

Vatan 4.46 13.77 LSD0.05 (Genotype x Zn Level) 0.89

Root Zn contents (µg Zn plant-1) Genotypes

Deficient Zn Adequate Zn

Sehar-06 2.45 4.94

Vatan 2.99 8.11 LSD0.05 (Genotype x Zn Level) 0.47

STUDY IV

73

Root Zn concentration in two wheat genotypes was not different at

deficient level of Zn supply in the root medium. At deficient Zn supply genotypes

did not differ for root Zn concentration (Table 3.4.4). However, genotypes varied

significantly for root Zn concentration at adequate Zn supply. Root Zn

concentration was significantly higher in plants grown at adequate Zn supply than

those grown under Zn deficient conditions. Root Zn concentration was 26.17 µg

g-1 in Sehar-06 and 25.82 µg g-1 in Vatan at deficient level of Zn supply while it

ranged between 68.22 µg g-1 in Sehar-06 and 58.15 µg g-1 in Vatan at adequate

Zn supply in root medium.

3.4.3.3 Zinc Uptake

There was significant (p<0.01) main effect of Zn levels on Shoot Zn uptake

(Table 3.4.5. Zinc deficiency significantly (p<0.01) reduced root Zn uptake in both

genotypes (Table 3.5.6). More root Zn uptake was observed in Vatan compared

to Sehar-06. In plants grown with adequate Zn supply, root Zn uptake was 4.94

µg plant-1 in Sehar-06 to 8.11 µg plant-1 in Vatan.

3.4.3.4 Organic Acids in Root Exudates

The genotypes varied significantly (p<0.05) both release of organic acids

by plants when grown with adequate as well as deficient Zn supply in root

medium. Maleic acid and fumaric acid were the major organic acids exuded by

the wheat genotypes. Minor quantities of malic acid, citric acid, oxalic acid and

acetic acid were also detected in root exudates.

STUDY IV

74

Table 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 (Values are means of 4 replicates)

Sehar-06 Vatan Sampling period (Days) Deficient Zn Adequate Zn Deficient Zn Adequate Zn

14 2.59 3.24 4.09 2.67

21 4.57 3.46 3.96 3.30

28 6.18 2.89 3.85 3.11

Mean 4.45 3.20 3.96 3.03 LSD0.05 (Genotype x Zn Level) 0.81

Table 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 (Values are means of 4 replicates)

Sehar-06 Vatan Sampling period (Days) Deficient Zn Adequate Zn Deficient Zn Adequate Zn

14 1.75 2.40 2.15 2.11

21 1.45 2.05 2.79 2.63

28 2.26 1.63 2.50 2.76

Mean 1.82 2.03 2.48 2.50 LSD0.05 (Genotype x Zn Level) 0.93

Amount of Maleic acid released by wheat genotypes were higher in plants

grown under Zn deficiency (Table 3.4.7). Maximum Maleic acid was released by

Sehar-06 and minimum was released by Vatan when grown under adequate Zn

supply.

STUDY IV

75

The was no significant effect of Zn levels on the release of fumaric acid

(Table 3.4.8). Fumaric acid observed in Vatan was 2.48 µM 2h-1 g-1 DM and in

Sehar-06 was 1.82 µM 2h-1 g-1 DM when grown at deficient Zn supply. In plants

grown under adequate Zn supply, maximum amount of fumaric acid release was

2.50µM 2h-1 g-1 DM in Vatan and minimum was 2.03 µM 2h-1 g-1 DM in Sehar-06.

3.4.4 Discussion

Two genotypes were selected from previous experiments for their genetic

differences for Zn acquisition and utilization. Genotypes were grown for 30 days

in nutrient solution containing adequate (2 µM) or deficient (0.2 µM) Zn levels

The present study reports differences in nature and amount of organic acids

exuded in response to Zn deficiency by wheat genotypes..

These genotypes varied significantly (p<0.01) for biomass production and

Zn acquisition grown at deficient as well as adequate Zn supply. Maximum

growth was observed in Sehar-6 and minimum in Vatan at both levels of Zn

supply in the root medium. Effect of Zn deficiency was more pronounced on

shoot than on root growth.

Zinc is mainly absorbed as Zn2+ in plants, but in Zn deficient soils Zn is

predominantly present as insoluble Zn oxides or carbonates which cannot be

utilized by plants as such . Therefore the ability of a specific crop genotype to

tolerate Zn deficiency might involve process such as rhizosphere acidification

(Godo and Reisenauer, 1994). Organic acid release in rhizosphere is related to

increased Zn acquisition as reported by earlier researchers (Hoffland et al., 2006;

Rengel and Romheld, 2000). Nature and amount of organic acids released by

STUDY IV

76

wheat genotypes under Zn deficiency differed significantly (p<0.01). Total

amount of maleic acids released was higher in plants grown under deficient Zn

level (Figure 3.4.1 and 3.4.2). The Genotype efficient in biomass production and

Zn acquisition released more organic acids indicating the importance of root

exudates in Zn acquisition. Maleic acid and fumaric acid were the major organic

acids found in root exudates of wheat genotypes grown either with adequate or

deficient level of Zn in nutrient solution. Amount of maleic acid released was

more in Sehar-06 grown under Zn deficiency (Figure 3.4.1). But under adequate

Zn supply the difference in maleic acid release between the two genotypes was

not significant. This indicates that the response of efficient wheat genotype is an

active reaction. Fang et al., (2008) found similar response of wheat to Mn

deficiency. The active release of maleic acid from the roots of Zn efficient wheat

genotypes results in rhizosphere acidification, which could improve Zn

bioavailability to wheat plants in Zn deficient soils. No significant trend was found

in release of fumaric acid in relation to Zn deficiency in either genotype (Figure

3.4.3 and 3.4.4). Increased exudation of organic acids in the rhizosphere by

some crop species is well documented under Zn deficiency (Rengel and

Romheld, 2000; Cakmak et al., 1994; Cakmak and . Marschner, 1988).

STUDY IV

77

3.4.5 Conclusions

The wheat genotypes were significantly (p<0.01) different in biomass

accumulation when grown at adequate or deficient Zn supply. Various strategies

adopted by the genotypes included reduced shoot growth, increased root growth,

and increased extrusion of organic acids especially maleic acid in higher

quantity. Significant genetic variability existed among both wheat genotypes in

adoption or expression of these strategies; hence they differ in growth response

to applied Zn. Field verification of these results is warranted before employing

them for breeding and selection program for improving yield in low Zn soils.

STUDY IV

78

Figure 3.4.1 Maleic Acid exudation by Sehar-6 under in relation to Zn supply

Figure 3.4.2 Maleic Acid exudation by Vatan under in relation to Zn supply

0

1

2

3

4

5

6

7

14 21 28

Sampling Period (Days)

Mal

eic

Ac

id e

xud

atio

n r

ate

(uM

2h

-1g

-1 R

DM

) Def

Adq

2

2.5

3

3.5

4

4.5

5

14 21 28

Samling Period (Days)

Mal

iec

Aci

d E

xud

atio

n r

ate

(uM

2h

-1g

-1 R

DM

)

DefAdq

STUDY IV

79

Figure 3.4.3 Fumaric Acid exudation by Sehar-6 under in relation to Zn supply

Figure 3.4.4 Fumaric Acid exudation by Vatan under in relation to Zn supply

0.75

1.25

1.75

2.25

2.75

3.25

14 21 28

Sampling period (Days)

Fu

mar

ic a

cid

exu

dat

ion

ra

te(u

M 2

h-1

g-1

RD

M)

DefAdq

1

1.5

2

2.5

3

3.5

14 21 28

Sampling Period (Days)

Fu

mar

ic A

cid

Exu

dat

ion

Rat

e (u

M 2

h-1

g-1

RD

M)

Def

Adq

CHAPTER 4

SUMMARY

Wheat is the primary staple food crop of Pakistan. But our soils are

alkaline calcareous on which most of our crops commonly suffer from zinc

(Zn) deficiency. Application of Zn fertilizer is problematic both under intensive

and extensive agriculture systems because of cost and environmental

concerns. An alternative approach is to select or breed crops with greater

ability to yield in low Zn soils and enhanced responsiveness to applied Zn.

Categorization of genotypes for Zn acquisition and use would not only be

helpful for sustaining wheat production in the country but would also provide

necessary database for breeders working on increasing Zn efficiency in

crops.

The experiments were planned with following main objectives

· Study differential growth and Zn acquisition of wheat genotypes in

order to:

i. Select low Zn requiring wheat cultivars for resource poor farmers

and environmental friendly agriculture

ii. Evaluate Zn responsive cultivars for areas where Zn addition is

not a problem

· Understand Zn remobilization within plants under induced Zn

deficiency

SUMMARY

81

· Assess the nature and amount of organic acids extrusion under Zn

deficiency and their role in Zn acquisition by wheat genotypes

A series of solution and soil culture experiments were conducted

during 2006-2008 to achieve these objectives. In the first experiment, 12

wheat genotypes were grown at adequate and deficient Zn levels in solution

culture. Differential growth and Zn contents among genotypes were the base

for further experiments. These genotypes were then grown on a Zn deficient

soil. Genotypes exhibited significant growth differences and Zn uptake in both

of these experiments. Two contrasting genotypes were further evaluated in

solution culture for Zn translocation and remobilization under induced Zn

deficiency. In an other solution culture experiment, these genotypes were

grown for 30 days in Zn adequate as well as in Zn deficient nutrient solution

to study root exudates. The main Salient findings from these studies are:

1. Genotypes showed significant differences for biomass production

when grown either with or without Zn in growth medium. Genotypes

with higher Zn use efficiency (such as Sehar-06, Auqab-2000, Inqalab-

91) were efficient in producing shoot biomass.

2. In all solution and soil culture experiments, Zn deficiency caused a

significant decrease in biomass production and plant Zn contents

irrespective of genotypes.

3. Both the screened genotypes (Sehar-06 and Vatan) showed

translocation and remobilization of absorbed Zn under induced Zn

SUMMARY

82

deficiency. However, Sehar-06 remobilized Zn more efficiently, which

might explain variations in Zn efficiency.

4. Under Zn deficiency in solution, genotype Sehar-06 increased

exudation of maleic acid

It is concluded that significant variation existed among the indigenous

wheat genotypes for Zn acquisition and utilization. Exploitation of this

variation to select and breed wheat genotypes such a Sehar-06 is a promising

approach to improve the Zn status in wheat grain. Wheat being the principle

diet of the masses can then serve to alleviate Zn deficiency in humans.

This research warrants further studies on local wheat germplasm.

Compounds such as Phytate in wheat grain are highly related to Zn

bioavailability therefore detailed analysis of wheat grain for form of Zn storage

and it availability to humans is required.

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