physiological responses of soybean and wheat … · physiological responses of soybean and wheat...

PHYSIOLOGICAL RESPONSES OF SOYBEAN AND

WHEAT TOWARDS NANOPARTICLE BASED

MICRONUTRIENTS FERTILIZATION

M.Sc. (Ag) Thesis

By

Milap Ram Sahu

DEPARTMENT OF PLANT PHYSIOLOGY,

AGRICULTURAL BIOCHEMISTRY, MEDICINAL AND

AROMATIC PLANTS COLLEGE OF AGRICULTURE, RAIPUR

FACULTY OF AGRICULTURE INDIRA GANDHI KRISHI VISHWAVIDYALAYA

RAIPUR (Chhattisgarh) 2016

PHYSIOLOGICAL RESPONSES OF SOYBEAN AND

WHEAT TOWARDS NANOPARTICLE BASED

MICRONUTRIENTS FERTILIZATION

Thesis

Submitted to the

Indira Gandhi Krishi Vishwavidyalaya, Raipur

By

Milap Ram Sahu

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

Master of Science

In

Plant Physiology

UN ID No. 20141520374 ID No. 120114156

JULY, 2016

iv

ACKNOWLEDGEMENT

The author of this manuscript praises the omniscient and almighty god and

his parents, who provided his this opportunity of submitting the present thesis for

award of M.Sc. (Ag.) Plant Physiology, Degree.

The word can never express indebtedness but I can take this opportunity to

express my deepest and heartfelt sense of gratitude to revered, chairman of

Advisory committee, Dr. A. Guhey, Head and Professor of Department, Plant

Physiology, Agril. Biochemistry and Medicinal and Aromatic Plants. College of

Agriculture, IGKV, Raipur (C.G.) for her guidance and inspiration to carry out the

present work. I am equally indebted and express my heartfelt sense of gratitude to

Co-chairman Dr. R. Elanchezhian, Principal Scientist, (Plant Physiology), ICAR-

Indian Institute of Soil Science, Bhopal (M.P.) for suggesting the problem,

providing necessary laboratory space and screen house facilities to carry out the

present work and for his healthy criticism in preparing the present manuscript of

this thesis to make this task a success. He has been a constant source of inspiration

and his love and affection to me will ever be remembered.

I wish to render my sincere thanks to the member of the thesis Advisory

Committee Dr. S.P. Tiwari (Asst. Professor) Department of Plant Physiology,

Agril. Biochemistry and Medicinal and Aromatic Plants, Dr. S.B. Verulkar (Head

and Professor) Department of Plant Molecular Biology and Biotechnology, and

Dr. G. Chandrakar (Professor) Department of Statistics, College of Agriculture,

IGKV, Raipur (C.G.) or their kind help and constant advisement.

I also feel great pleasure to express my heartfelt thanks to the Honorable

Vice Chancellor of IGKV, Raipur (C.G.) Dr. S.K. Patil, Dean faculty of

Agriculture, Dr. S.S. Rao, Director of Research Services, Dr. J.S. Urkurkar and

Director of Extension Services Dr. M.P. Thakur, Director of Instructions Dr. S.S.

Shaw, IGKV, Raipur for providing necessary facilities in carrying out this piece of

research work.

I take this opportunity to express sincere thanks to Dr. A. K. Patra,

Director ICAR-IISS, Bhopal who permitted me to work at IISS. I am also highly

grateful to Dr. A. K. Biswas, Head, Division of Soil Chemistry and fertility (ICAR-

IISS) for providing the required facilities for my research work.

I am deeply indebted to my teachers, Dr. A.K. Geda (Professor), Dr.

Pratiba Katiyar, (Professor), Dr. D. Khokhar (Scientist) and Mr. V.B.

Kuruwanshi (Asst. Professor) Department of Plant Physiology, Agril.

Biochemistry and Medicinal and Aromatic Plants, Raipur, for their encouragement

throughout the course of my studies.

I wish to record my grateful thanks to Dr. S. Shrivastava (Principal

Scientist), Dr. B. L. Lakaria (Principal Scientist), Dr. K. Ramesh (Principal

Scientist) Dr. P. Jha (Senior Scientist), Dr. I. Rashmi (Scientist), Dr. B. P. Meena

vi

TABLE OF CONTENTS

Chapter Title Page ACKNOWLEDGEMENT iv/v TABLE OF CONTENTS vi/viii LIST OF TABLES ix/x LIST OF FIGURES xi/xii LIST OF NOTATIONS Xiii LIST OF ABBREVIATIONS Xiv ABSTRACT xv/xvi

I INTRODUCTION 1-4 II REVIEW OF LITERATURE

2.1 Impact of Iron nanoparticle fertilizer on Plants 2.1.1 Morphological effect 2.1.2 Physiological and biochemical effect 2.2 Impact of Copper nanoparticle fertilizer on Plant

2.3.1 Morphological effect 2.3.2 Physiological and biochemical effect on Plants

2.3 Impact of Zink nanoparticle fertilizer 2.2.1 Morphological effect 2.2.2 Physiological and biochemical effect

2.4 Impact of Other metallic nanoparticle fertilizer on Plants 2.4.1 Morphological effect 2.4.2 Physiological and biochemical effect

5-18 5-8 5-7 7-8 9-14 9 10-14 10-14 10-13 13-14 15-18 15-16 16-18

III MATERIALS AND METHODS

3.1 Experimental site 3.2 Sand culture system setup 3.3 Climate 3.4 Experimental details

3.4.1 Design and layout 3.4.2 Collection of experimental data

3.5 Morphological parameters 3.5.1 Plant height 3.5.2 Root length 3.5.3 Plant biomass 3.5.4 Root biomass 3.5.5 Root volume

3.6 Growth parameters

19-29 19 19 19 22 22 22 22-23 22 22 23 23 23 23-24

vii

3.6.1 Leaf area 3.6.2 leaf area ratio 3.6.3 Specific leaf weight 3.6.4 Specific leaf area 3.6.5 Root shoot ratio

3.7 Physiological and biochemical parameters 3.7.1 Chlorophyll content estimation 3.7.2 Estimation of relative water content 3.7.3 Estimation of membrane stability 3.7.4 Proline content estimation 3.7.5 Assay of antioxidant enzyme

3.7.5.1 Super oxide dismutase (SOD) activity assay 3.7.5.2 Catalase (CAT) activity assay 3.7.5.3 Peroxidase (POX) activity assay

3.7.6 Estimation of total soluble protein 3.7.7 Estimation of total soluble sugar 3.7.8 Estimation of starch 3.7.9 Estimation of non-structural carbohydrate 3.7.10 Gas exchange parameter 3.7.11 SPAD Value 3.7.12 Grain Yield 3.7.13 Toxicity/ Deficiency symptom analysis

23 23 23 24 24 24-29 24-25 25 25 26 26-27 27 27 27 27 28 28 29 29 29 29 29

IV RESULTS AND DISCUSSION

4.1 Morphological parameters of Soybean 4.1.1 Plant height (cm) 4.1.2 Total root Length (cm) 4.1.3 Shoot dry weight (g) 4.1.4 Root dry weight (g) 4.1.5 Specific leaf area (cm²g-1) 4.1.6 Specific leaf weight (g cm-²) 4.1.7 Leaf area ratio 4.1.8 Leaf area (cm2) 4.1.9 Root volume (cm3) 4.1.10 Pod weight (g plant-1) 4.1.11 Grain weight (g plant-1) 4.1.12 Root shoot ratio

4.2 Biochemical parameters of Soybean 4.2.1 Chlorophyll content (mg g-1FW) 4.2.2 Membrane stability (%) 4.2.3 Relative water content (%) 4.2.4 Antioxidant enzyme 4.2.4.1 Super oxide dismutase (unit g-1)

30-114 30-47 30-31 31-32 32-34 34-36 37-38 38-39 39-40 41-43 44 45 45-46 46-47 47-63 47-50 51-52 52-53 52-57 53-55

viii

4.2.4.2 Catalase (unit H2O2 min-1 g-1) 4.2.4.3 Peroxidase (unit H2O2 min-1 g-1) 4.2.5 Proline content (μM g

-1) 4.2.6 Total soluble protein (mg g-1) 4.2.7 Total soluble sugar (%) 4.2.8 Non-structural carbohydrate (%)

4.3 Physiological parameters of Soybean 4.3.1 Photosynthesis rate (µM m-2 s-1) 4.3.2 Transpiration rate (mM m-2 s-1) 4.3.3 Stomatal conductance (µM m-2 s-1) 4.3.4. SPAD value 4.4 Morphological parameters of wheat

4.4.1 Plant height (cm) 4.4.2 Total root length (cm) 4.4.3 Shoot dry weight (g) 4.4.4 Root dry weight (g) 4.4.5 Specific leaf area (cm² g-1) 4.4.6 Specific leaf weight (g cm-²) 4.4.7 Leaf area (cm²) 4.4.8 Leaf area ratio 4.4.9 Number of tillers 4.4.10 Root volume ( cm3) 4.4.11 Panicle weight (g plant-1) 4.4.12 Grain weight (g plant-1) 4.4.13 Number of grain plant-1 4.4.14 Seed index (%) 4.4.15 Root shoot ratio

4.5 Biochemical parameters of wheat 4.5.1 Chlorophyll content (mg g-1FW) 4.5.2 Antioxidant enzyme activity 4.5.2.1 Super oxide dismutase (unit g-1) 4.5.2.2 Catalase (unit H2O2 min-1 g-1) 4.5.2.3 Peroxidase (unit H2O2 min-1 g-1) 4.5.3 Membrane stability (%) 4.5.4 Relative water content (%) 4.5.5 Proline content (μM proline g

-1) 4.5.6 Total soluble protein (mg g-1) 4.5.7 Total soluble sugar (%) 4.5.8 Non-structural carbohydrate (%)

4.6 Physiological parameters of wheat 4.6.1 Photosynthesis rate ( µM m-2 s-1) 4.6.2 Transpiration rate (mM m-2 s-1)

55-57 58 58-60 60-61 61-63 64 64-73 64 65-66 66-67 71-73 74-91 74 74-75 75-77 77-79 80-81 81-82 82 82-83 85 85-86 87 87 88 88 89 92-108 92-94 94-95 96-97 99-102 99-100 100-101 101-102 104 104-105 105-106 106-107 109-115 109 109-110

ix

4.6.3 Stomatal conductance (µM m-2 s-1) 4.6.4 SPAD value

110-111 113-114

V SUMMARY AND CONCLUSIONS

5.1 Summary 5.2 Conclusions 5.3 Suggestions for future research work

116-119 116-118 118 118-119

REFERENCES 120-128 APPENDICES

Appendix A Appendix B

129-130 129 130

RESUME 131

x

LIST OF TABLES

Table Title Page

3.1 Treament details 22 4.1 Effect of micronutrient NPs on plant height and total root length of

soybean 32

4.2 Effect of micronutrient NPs on shoot weight of soybean 34 4.3 Effect of micronutrient NPs on root dry weight of soybean 35 4.4 Effect of micronutrient NPs on specific leaf area and specific leaf

weight of soybean 39

4.5 Effect of micronutrient NPs on leaf area ratio and leaf area of soybean. 42 4.6 Effect of micronutrient NPs on root volume of soybean 45 4.7 Effect of micronutrient NPs on pod weight and grain weight of soybean 46 4.8 Effect of micronutrient NPs on root shoot ratio of soybean 47 4.9 Effect of micronutrient NPs on chlorophyll content of soybean 49 4.10 Effect of micronutrient NPs on membrane stability and relative water

content of soybean 52

4.11 Effect of micronutrient NPs on super oxide dismutase of soybean 53 4.12 Effect of micronutrient NPs on catalase and peroxidase of soybean 56 4.13 Effect of micronutrient NPs on proline and protein of soybean 60 4.14 Effect of micronutrient NPs on total soluble sugar and non-structural

carbohydrate of soybean 62

4.15 Effect of micronutrient NPs on photosynthesis rate of soybean 64 4.16 Effect of micronutrient NPs on transpiration rate of soybean 66 4.17 Effect of micronutrient NPs on stomatal conductance of soybean 67 4.18 Effect of micronutrient NPs on SPAD value of Soybean 72 4.19 Effect of micronutrient NPs on plant height and total root length of

wheat 75

4.20 Effect of micronutrient NPs on shoots and root dry weight of wheat 78 4.21 Effect of micronutrient NPs on specific leaf area and specific leaf

weight of wheat 82

4.22 Effect of micronutrient NPs on leaf area and leaf area ratio of wheat 83 4.23 Effect of micronutrient NPs number of tillers and root volume of wheat 86 4.24 Effect of micronutrient NPs on panicle weight and grain weight of

wheat 87

4.25 Effect of micronutrient NPs on number of grain and seed index of wheat

89

4.26 Effect of micronutrient NPs on root shoot ratio of wheat 90 4.27 Effect of micronutrient NPs on chlorophyll content of wheat 94 4.28 Effect of micronutrient NPs on membrane stability and relative water

content of wheat 97

4.29 Effect of micronutrient NPs on super oxide dismutase of wheat of 100

xi

wheat 4.30 Effect of micronutrient NPs on catalase and peroxidase of wheat 102 4.31 Effect of micronutrient NPs on proline and protein 105 4.32 Effect of micronutrient NPs on NPs on total soluble sugar and non-

structural carbohydrate of wheat 107

4.33 Effect of micronutrient NPs on Photosynthesis rate of wheat 109 4.34 Effect of micronutrient NPs on transpiration rate of wheat 110 4.35 Effect of micronutrient NPs on stomatal conductance of wheat 111 4.36 Effect of micronutrient NPs on SPAD value of wheat 114

xii

LIST OF FIGURE

Figure Title Page

3.1 A view of experimental set up of Soybean in sand culture 20 3.2 A view of experimental set up of Wheat in sand culture 20 3.3 Meteorological data during crop growth period. 21 4.1 Effect of micronutrient NPs on shoot weight of soybean 36 4.2 Effect of micronutrient NPs on root weight of soybean 36 4.3 Effect of micronutrient NPs on specific leaf area of soybean 40 4.4 Effect of micronutrient NPs on leaf area ratio of soybean. 43 4.5 Effect of micronutrient NPs on leaf area of soybean. 43 4.6 Effect of micronutrient NPs on chlorophyll content of soybean at 45

DAS 50

4.7 Effect of micronutrient NPs on chlorophyll content of soybean at 60 DAS

50

4.8 Effect of micronutrient NPs on super oxide dismutase of soybean 54 4.9 Effect of micronutrient NPs on catalase of soybean 57 4.10 Effect of micronutrient NPs on peroxidase of soybean 57 4.11 Effect of micronutrient NPs on TSS of soybean 63 4.12 Effect of micronutrient NPs on non-structural carbohydrate of soybean 63 4.13 Effect of micronutrient NPs on photosynthesis rate of soybean 68 4.14 Effect of micronutrient NPs on transpiration rate of soybean 69 4.15 Effect of micronutrient NPs on stomatal conductance of soybean 70 4.16 Effect of micronutrient NPs on SPAD value of Soybean 73 4.17 Effect of micronutrient NPs on plant height of wheat 76 4.18 Effect of micronutrient NPs on total root length of wheat 76 4.19 Effect of micronutrient NPs on shoot dry weight of wheat 79 4.20 Effect of micronutrient NPs on root dry weight of wheat 79 4.21 Effect of micronutrient NPs on leaf area of wheat 84 4.22 Effect of micronutrient NPs on leaf area ratio of wheat 84 4.23 Effect of micronutrient NPs on root shoot ratio of wheat 91 4.24 Effect of micronutrient NPs on chlorophyll content of wheat at 30 DAS 95 4.25 Effect of micronutrient NPs on chlorophyll content of wheat at 60 DAS 95 4.26 Effect of micronutrient NPs on membrane stability of wheat 98 4.27 Effect of micronutrient NPs on Relative water content of wheat 98 4.28 Effect of micronutrient NPs on Super oxide dismutase of wheat 103 4.29 Effect of micronutrient NPs on peroxidase of wheat 103 4.30 Effect of micronutrient NPs on TSS of wheat 108 4.31 Effect of micronutrient NPs on non-structural carbohydrate of wheat 108 4.32 Effect of micronutrient NPs on photosynthesis rate of wheat 112 4.33 Effect of micronutrient NPs on SPAD value of wheat 115

xiii

LIST OF NOTATIONS/SYMBOLS

Symbol/

notations

Detail

% Percent 0C Degree Celsius CD Critical difference cm Centimeter cm2 Square centimeter cm3 Cubic centimeter cm² g-1 Square centimeter per gram d-1 Per day g cm-2 Gram per square centimeter gm or g Gram ha-1 Per hectare Hrs Hours Kg Kilogram

m Meter m2 Square meter Mg Milligram mg g-1 Milligram per gram mg g-1 FW Milligram per gram fresh weight Ppm Part per million unit g-1 Unit per gram unit H2O2 min-1 Unit per H2O2 per minute μM g

-1 FW Micromole per gram fresh weight µM m-2 s-1 Micromole per meter per second mM m-2 s-1 Millimole per meter per second

xiv

LIST OF ABBREVIATIONS

Abbreviations Detail

CAT Catalase CHL Chlorophyll DAS Day after sowing DW Dry weight EC Electrical conductivity et al. And coworkers / and others i.e That is viz. Namely Fig. Figure FW Fresh weight ICAR Indian Council of Agriculture Research IISS Indian Institute of Soil Science IGKV Indira Gandhi Krishi Vishwavidyalaya LA Leaf area LAR Leaf area ratio MS Membrane stability NPs Nano particles NSC Non-structural carbohydrate NS None significant NSPs Nano scale particles POX Peroxidase RWC Relative water content SLA Specific leaf area SLW Specific leaf weight SOD Super oxide dismutase TSP Total soluble protein TSS Total soluble sugar

xvi

growth and gas exchange parameters of plants like Photosynthetic rate, stomatal

conductance and transpiration rate. In wheat, the nano-micronutrient fertilization of

plants with Fe NPs/ Cu NPs / Zn NPs had positively influenced most of the

morphological parameters (Plant height, total root length, shoot dry weight, root

dry weight, SLA, SLW, LA, LAR, root shoot ratio and grain yield) while reduced

concentration of Fe NPs/ Cu NPs and Zn NPs had positively influenced

biochemical metabolism (Chlorophyll content, SOD, POX, CAT, MS, RWC, TSS,

NSC) of plants. Gas exchange parameters were also positively influenced by NPs.

The above findings indicated that the effect of nanoparticles were crop or species

specific. Moreover, it is also envisaged that nanoparticle at reduced concentration

may be useful for the crop and they may act as catalyst for growth, metabolism and

yield of plants.

CHAPTER - I

INTRODUCTION

Soybean (Glycine max (L) Merr.) belongs to Fabaceae family and is an

annual crop. Due to having useful compounds such as unsaturated fatty acids,

protein, mineral salts and plant secondary metabolites such as isoflvin, soybean has

many important roles in human and animal nutrition. Achieving optimum quantity

and increasing quality of soybean seeds depend upon many factors among which,

weed control and plant nutrition have critical importance (Sedghi, 2007). Iron is

one of the essential elements for plant growth and plays an important role in the

photosynthetic reactions. Iron activates several enzymes and contributes in RNA

synthesis and improves the performance of photo systems (Malakouti and Tehrani,

2005). Soybean is sensitive to iron deficiency, but different genotypes are various

in efficiency of iron consumption. Application of iron in low–iron soils can

increase grain yield in soybean (Ghasemi et al, 2006). Iron compounds can use as

foliar on leaves and as seed coating (Debermann, 2006). Nanotechnology can

present solution to increasing the value of agricultural products and environmental

problems. With using of nano-particles and nano-powders, we can produce

controlled or delayed releasing fertilizers. Nano-particles have high reactivity

because of more specific surface area, more density of reactive areas, or increased

reactivity of these areas on the particle surfaces. These features simplify the

absorption of fertilizers and pesticides that produced in nano scale (Anonymous,

2009). Studies showed that the effect of nano-particles on plants can be beneficial

(seedling growth and development) or non-beneficial (to prevent root growth) (Zhu

et al,2008). This experiment was conducted to investigate the effects of nano-iron

oxide particles on soybean yield and agronomic traits.Nanoparticles (nano-scale

particles = NSPs) are atomic or molecular aggregates with at least one dimension

between 1 and 100nm (Ball 2002; Roco 2003), that can drastically modify their

physico-chemical properties compared to the bulk material (Nel et al. 2006). It is

worth noting that nanoparticles can be made from a variety of bulk materials and

that they can explicate their actions depending on both the chemical composition

1

and on the size and/or shape of the particles (Brunner et al. 2006). Depending on

the origin, a further distinction is made between three types of NSPs: natural, in-

cidental and engineered. Natural nanoparticles have existed from the beginning of

the earth‘ history and still occur in the environment (volcanic dust, lunar dust,

mineral composites, etc.). Incidental nanoparticles, also defined as waste or

anthropogenic particles, take place as the result of manmade industrial processes

(diesel exhaust, coal combustion, welding fumes, etc.). Currently as nanopaticles

are being widely used in consumer products, communication sector,

pharmaceutics, and energy sector (Zhao and Castranova 2011), these particles may

find their way into terrestrial environment where their fate and behavior are still

largely unknown. Nanofertilzers or nano-encapsulated nutrients might have

properties that are effective to crops, released the nutrients on-demand, controlled

release of chemicals fertilizers that regulate plant growth and enhanced target

activity (De Rosa et al. 2010; Nair et al. 2010). There have been publications on

effects of engineered nanomaterials on higher plants wherein both positive and

negative effects were reported (Monica and Cremonini 2009).

Wheat is the most important stable food crop for more than one third of the

world population and contributes more calories and proteins to the world diet than

any other cereal crops there is no doubt that the number of people who rely on

wheat for a substantial part of their diet amounts to several billions. Therefore, the

nutritional importance of wheat proteins should not be underestimated, particularly

in less developed countrieswhere bread, noodles and other products (e.g. bulgar,

couscous) may provide a substantial proportionof the diet. Wheat provides nearly

55% of carbohydrate and 20% of the food calories. It contains carbohydrate

78.10%, protein 14.70%, fat 2.10%, minerals 2.10% and considerrabble

proportions of vitamins (thiamine and Vitamin-B) and minerals (zinc, iron). Wheat

is also a good source of traces minerals like selenium and magnesium, nutrients

essential to good health. Wheat grain precisely known as caryopsis consists of the

pericarp or fruit and the true seed. In the endosperm of the seed, about72% of the

protein is stored, which forms 8-15% of total protein per grain weight. Wheat

grains are also rich in pantothenic acid, riboflavin and some minerals, sugars etc.

2

The barn, whichconsists of pericarp testa and aleurone, is also a dietary source for

fiber, potassium, phosphorus, magnesium, calcium, and niacin in small quantities.

Nanotechnology is a multidisciplinary field, as it combines the knowledge

from different disciplines: chemistry, physics, and biology amongst others

(Schmid, 2006; Schmid, 2010). Nanotechnology is the art and science of

manipulating matter at the atomic or molecular scale and holds the promise of

providing significant improvements in technologies for protecting the environment.

While many definitions for nanotechnology exist, the U.S. Environmental

Protection Agency (EPA) uses the definition developed by the National

Nanotechnology Initiative (NNI). According to National Nanotechnology Initiative

of the USA, nanotechnology is defined as: research and technology development at

the atomic, molecular, or macromolecular levels using a length scale of

approximately one to one hundred nm in any dimension; the creation and use of

structures, devices and systems that have novel properties and functions because of

their small size; and the ability to control or manipulate matter on an atomic scale

(USEPA, 2007). The technology has excellent prospects for exploitation across the

medical, pharmaceutical, biotechnology, engineering, manufacturing,

telecommunications and information technology markets.

Micronutrients (MNs) are important to world wild agriculture. Zinc (Zn),

iron (Fe), manganese (Mn) and copper (Cu) have become yield-limiting factors

and are partly responsible for low food nutrition. Although crops use low amounts

of MNs (<2.4 kg/ha), about half of the cultivated world‘s soils are deficient in

plant bioavailable MNs, due to their slow replenishment from the weathering of

soil minerals, soil cultivation for thousands of years and insufficient crop

fertilization. Relevant MN deficiencies occur more frequently in neutral to alkaline

soils, under anaerobic conditions and in arid or semi-arid regions. The MN use

efficiency (MUE) of most commercial fertilizers added to soils or foliage is 2.5%

to 5% of applied, due to their rapid stabilization by soil components, low leaf

penetration and low mobility in plants. In soil-plant systems, fertilizer-MNs

interact with macronutrients resulting in synergistic, antagonistic or neutral

response affecting yield and food quality. Some elements are directly involved in

plant metabolism (Arnon and Stout, 1939). However, this principle does not

3

account for the so-called beneficial elements, whose presence, while not required,

has clear positive effects on plant growth. Mineral elements those either stimulates

growth but are not essential, or that are essential only for certain plant species, or

under given conditions, are usually defined as beneficial elements.

Iron is one of the essential elements for plant growth and plays an

important role in the photosynthetic reactions. Iron activates several enzymes and

contributes in RNA synthesis and improves the performance of photosystems

(Malakouti and Tehrani, 2005). Iron deficiency in plants causes a reduction in

chlorophyll and the other anthocyanin contents. Its excess leads to ROS generation,

leading to the oxidative damage (Suh et al. 2002). Thus, Fe has a major role in

cellular redox reactions with 85% of its activity focused in plastids (Mengel and

Kirby 1987).

Zinc (Zn) is typically the second most abundant transition metal in

organisms after iron and the only metal represented in all six enzyme classes

(oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases) (Auld,

2001).

Copper is associated with enzymes involved in redox reactions being

reversibly oxidized from Cu+ to Cu

2+. An enzyme is plastocyanin, which is

involved in electron transfer during the light reaction of photosynthesis (Haehnel

1984).

Hence this study was undertaken to analyze the effect of nano sized

micronutrient fertilizers on the crop performance of soybean and wheat with the

following objectives.

Objectives of the investigation:

1. To study the response of different concentrations of nanoparticle on plant

growth, morphological and yield characteristics of soybean and wheat.

2. To study the response of different concentrations of nanoparticle on

physiological and biochemical characteristics of soybean and wheat.

4

CHAPTER - II

REVIEW OF LITERATURE

2.1 Impact of Iron nanoparticle fertilizer on plant

2.1.1 Morphological effect

Afshar et al. (2013) evaluated the impact of nano-iron on cowpea crop

under irrigation deficit and found significant increase in numbers of seed per pod.

It was observed that increasing nano-iron concentration increased number of seeds

per pod but decreased 1000 seed weight significantly in cowpea. Dhoke et al.

(2013) studied the effect of nano-FeO and nano-Zn Cu Fe oxide particles on the

growth of mung bean (Vigna radiata) seedling and found that the best performance

was observed for nano-Zn Cu Fe-Oxide followed by nano-FeO. The absorption of

nanoparticles by plant leaves was also detected by inductive coupled

plasma/atomic emission spectroscopy. Karimia et al. (2014) investigated the effect

of different concentrations of iron chelate nano fertilizer and Fe-EDDHA on

morphological characteristics and antioxidant enzymes activity of green gram and

observed that increasing nanoparticles concentration above 10 ppm reduced shoot

fresh weight, shoot dry weight and root weight. Nadi et al. (2013) studied the

effect of nano iron chelate fertilizer on grain yield, protein percent and chlorophyll

content of faba bean (Vicia faba L.) and found that the highest and lowest grain

yield belonged to nano-iron (6 g l-1

) and control, respectively.

Liu et al. (2005) reported that nano-Fe2O3 promoted the growth and

photosynthesis of peanut. Ngo et al. (2014) observed that the germination rates of

soybean seeds, treated with zerovalent Cu, Co and Fe were 65, 80 and 80%,

respectively, whereas 55% germination was observed in the control sample in the

field experiment. For all of the nanoscale metals studied, the number of nodules

increased 20–49% compared to the control sample, and the soybean crop yield

increased up to 16% in comparison with the control sample. Nano-iron oxide at the

concentration of 0.75 g l-1

increased leaves and pod dry weight of soybean and

5

thehighest grain yield was observed with 0.5 g l-1

nano-iron oxide that showed 48%

increase in grain yield in comparison with control (Sheykhbaglou et al. 2010).

Armin et al. (2014) carried out foliar application of nano Fe in wheat

(Triticum aestivum) at 2%, 4% and 6% and observed an increase of 12%, 22.09%

and 19.07% grain yield, respectively, over the control. Bakhtiari et al. (2015)

studied that effect of different concentration of Fe Nano-oxide solution at five

levels (0, 0.01%, 0.02%, 0.03% and 0.04%) of wheat. Higher spike weight, 1000

grain weight, biological yield, and grain yield were achieved in plants treated with

0.04% Fe nano-oxide concentration and the lowest values were achieved in the

control. Ghafari and Razmjoo (2013) reported that 2 g l-1

nano iron oxide has

increased harvest index, 1000-grain weight and yield of wheat. Many et al. (2013)

reported that iron nano composite, when sprayed in rice at two stages of nursery

and early earring led to increase in yield, 1000 grains weight and healthy grains

number in ear and decrease in hollow grains number in ear.

Bozorgi (2012) studied the effect of foliar spray of Ascophyllum nodosum

extract and nano iron chelate fertilizer on fruit yield and several attributes of

eggplant. The highest fruit yield was recorded from foliar spray of A. nodosum

extract @ 2 g l-1

with 37.89 ton ha-1

. Among nano iron chelate fertilizer treatments,

spray of 2 g l-1

recorded the maximum amount of fruit yield i.e. 37.11 ton ha-1

.

Amuamuha et al. (2012) evaluated the effect of iron on the flower and

essential oil yield of Pot Marigold (Calendula officinalis) and observed highest

yield of flower at first harvest (405.37 kg ha-1

) from nano iron foliar application at

stem initialized stage, and the lowest yield of flower (261.64 kg ha-1

) after second

harvest.

Moghadam et al. (2012) investigated the effect of different concentrations

of iron chelate Nano fertilizer (2 and 4 ppt) on growth and performance on

Spinach. Results showed that wet weight, dry weight and maximum leaf area index

were influenced by concentration of iron chelate Nano fertilizer. Use of 4 kg ha-1

Nano fertilizer caused 58 and 47% increase in wet weight and maximum leaf area

index, respectively compared to control.

Mohamadipoor et al. (2013) reported that nano iron fertilizer and FeSO4

treatments produced similar response in ornamental plants spathyphyllum to most

6

of the characteristics. He observed that use of nano iron fertilizer is superior

because of lower cost. Peyvandi et al. (2011) reported the positive effect of

spraying basil plants with iron nanofertilizer. Fe nanoparticles increased root

length, stem length, and shoot dry weight compared with the common iron

fertilizers. Salarpour et al. (2013) investigated the effect of Nano-iron chelates on

growth, peroxidase enzyme activity and oil essence of Cress (Lepidium sativum L.)

and observed 80% increase in plant height.

2.1.2 Physiological and Biochemical effect

Bakhtiari et al. (2015) studied the effect of different concentration of Fe

Nano-oxide solution (0, 0.01%, 0.02%, 0.03% and 0.04%) on wheat and observed

highest protein content in 0.04% Fe concentration. Ghafari and Razmjoo (2013)

reported that 2 g l-1

nano iron oxide has increased chlorophyll content, antioxidant

enzyme activities, protein and carbohydrate content of wheat.

Delfani et al. (2014) studied the effect of Iron (Fe) and magnesium (Mg) in

nano and common forms as foliar application on black-eyed pea. Iron had

significant effect on yield, leaf Fe content, stem Mg content, plasma membrane

stability, and chlorophyll content. The greatest effect was obtained by two

treatment combinations of 0.5 g l−1

common Fe + 0.5% nano-Mg and 0.5 g l−1

common Fe + 0.5 g l−1

common Mg. In general, almost all analyzed traits were

improved by foliar application of these two elements, probably as a result of more

efficient photosynthesis.

Karimia et al. (2014) compared iron chelated fertilizer and nano-iron

chelated fertilizer in different concentrations on some physiological and

biochemical responses of mung bean (Vigna radiata). The effects of nano-iron

chelated fertilizer and iron chelated fertilizer were determined on photosynthetic

pigments, leaf protein content and activity of antioxidant enzymes in the leaves.

Nadi et al. (2013) studied that the effect of nano iron chelate fertilizer on grain

yield, protein percent and chlorophyll content of Faba bean (Vicia faba L.) and

observed that increasing Nano-Iron concentration (6 g l-1

) had a positive and

significant effect on grain yield. Ngo et al. (2014) reported that the chlorophyll

index increased by 7–15% in soybean seeds treated with zerovalent Cu, Co and Fe.

7

Alidoust,D. and Isoda, A. (2013) et al study that was performed to

investigate the effect of 6-nm IONPs and citrate-coated IONPs (IONPs-Cit) on

photosynthetic characteristics and root elongation during germination of Glycine

max (L) Merr. Plant physiological performance was assessed after foliar and soil

IONPs fertilization. No adverse impacts at any growth stage of the soybeans were

observed after application of IONPs. The Fe2O3 nanoparticles produced a

significant positive effect on root elongation, particularly when compared to the

bulk counterpart (IOBKs) suspensions of concentrations greater than 500 mg L-1.

Furthermore, IONPs-Cit significantly enhanced photosynthetic parameters when

sprayed foliarly at the eight-trifoliate leaf stage (P\0.05). The increases in

photosynthetic rates following spraying were attributed to increases in stomatal

opening rather than increased CO2 uptake activity at the chloroplast level. We

observed more pronounced positive effects of IONPs via foliar application than by

soil treatment. This study concluded that IONPs coated with citric acid at IONPs to

citrate molar ratio of 1:3 can markedly improve the effectiveness of insoluble iron

oxide for Fe foliar fertilization.

Hokmabadi et al. (2006) reported that the iron nano fertilizers increased the

ratio of ferrous iron to ferric iron in chelate surface which resulted in increased

synthesis of chlorophyll of chrysanthemum. Salarpour et al. (2013) reported that

nano iron fertilizer has a positive effect on peroxidase enzyme activity, chlorophyll

content and oil essence of cress (Lepidium sativum L.).

Siva, G.V. and John Benita, L.F. et al, (2016) studied the Iron is an element

essential for plant growth and development. Nanoparticles like iron oxide

nanoparticles are being investigated as plant supplements for its promising targeted

delivery approach. The experiment is designed in a hydroponic system where

along with the Hoagland solution 100ppm concentration of iron oxide

nanoparticles are added to evaluate whether it gives beneficial results when

compared to EDTA chelated iron. The experimental data showed ginger roots

absorbed iron oxide nanoparticles also showed an increase with respect to protein

levels and iron content of rhizome. Iron oxide nanoparticles are an effective

supplement for chlorosis.

8

2.2 Impact of Copper nanoparticle fertilizer on Plant


Adhikari et al. (2012) studied the effect of Cu oxide-nano particles (< 50

nm) on germination and growth of seeds of soybean and chickpea. In both the

crops, germination was not inhibited up to 2,000 ppm Cu (applied through CuO

NP), but the root growth was prevented above 500 ppm Cu. With increasing

concentration of NPs, the elongation of the roots was severely inhibited as

compared to that in control. In many cases root necrosis was occurred. Massive

adsorption of Cu oxide-nano particles into the root system was responsible for the

toxicity. A parallel experiment was also carried out to know the effect of copper

sulphate solution on seed germination, above 200 ppm Cu, it restricted the

germination of seeds, because of high salinity.

Hafeez et al. (2015) observed that Cu-NPs (10, 20, 30, 40 and 50 ppm)

significantly increased growth and yield of wheat as compared with control.

Among the graded concentration, 30 ppm Cu-NPs produced significantly higher

leaf area, number of spikes/pot, number of grains/spike, 100 grain weight and grain

yield. Hashemabadi et al. (2013) reported that 5 mg l-1

of copper nanoparticles

increased the vase life of cut flowers of chrysanthemum compared to control. Shah

and Belozerova (2009) reported that the seed germination in the presence of Cu

NPs showed an increase in shoot to root ratio compared to control lettuce plants.

Shobha (2014) reported that copper nanoparticles, which have shown positive and

negative impact on the micro-organism and the plants.

F. Yasmeen et al, (2015) reported that a reduction in germination

percentage on exposure to silver and copper nanoparticles while maximum

germination percentage was on application of iron nanoparticles. Similarly, while

root and shoot growth was also enhanced under iron nanoparticles application

while severereduction in root and shoot length was observed on exposure to copper

nanoparticles. So copper has inhibitory while iron has stimulatory effect on wheat

germination and growth.


Hafeez et al. (2015) observed that 30 ppm Cu-NPs produced significantly

higher chlorophyll content in wheat. Nekrasova et al. (2011) reported the effect of

9

copper ions and copper oxide nanoparticle on lipid peroxidation rate, anti-oxidant

enzyme activities (superoxide dismutase, catalase and peroxidise) and

photosynthesis of Elodea densa planch. The results showed nanoparticles that are

accumulated by plants activated lipid peroxidation rate from 120 to 180% of the

control level by copper ions and nanoparticles, respectively. Catalase and

Superoxide dismutase activity increased by a factor of 1.5 to 2.0 when plants were

treated with NPs. Copper ions suppresses photosynthesis at a concentration of 0.5

mg l-1

, whereas nanoparticles produce such an effect only at 1.0 mg l-1

.

2.3 Impact of Zinc nanoparticle fertilizer on plant


Asadzade et al. (2015) investigated the effect of foliar application of

conventional and nano-fertilizers (ZnO and SiO2) on yield, morphological and

physiological traits and harvest index of sunflowers. ZnO nano-fertilizer was found

to increase head diameter, seed yield, harvest index for seed in plant and seed in

head.

Avinash et al. (2010) observed increases in germination and growth rate in

the seeds of Cicer arietinum treated with nano-ZnO. Burman et al. (2013)

compared the effect of 1.5 and 10 ppm foliar spray of zinc oxide (ZnO)

nanoparticles on ten days old seedlings of chickpea (Cicer arietinum L var. HC-1)

with corresponding concentration of zinc sulphate and ZnO of normal size.

Maximum response with respect to shoot dry weight was observed in seedlings

treated with 1.5 ppm ZnO nanoparticles while at 10 ppm the nanoparticles exerted

adverse effects on root growth. However, overall biomass accumulation improved

in the ZnO nanoparticle treated seedlings. Mahajan et al. (2011) reported the

effects of ZnO nanoparticles on the growth of mung bean (Vigna radiata)

seedlings, the seeds of which were previously allowed to germinate in wet cotton

for 24 h in the dark, then the sprouted seeds were taken for further study of the

seedlings which grew in culture media containing nanoparticles. Maximum

seedling growth was observed with nano-ZnO concentration of 20 ppm and beyond

which the seedling growth was inhibited, which might be attributed to the toxic

level of nanoparticles.

10

Boonyanitipong et al. (2011) investigated the effect of zinc oxide

nanoparticles (nano-ZnO) on rice (Oryza sativa L.) root and found that there is no

reduction in the percent seed germination, however nano-ZnO is observed to have

detrimental effects on rice roots at early seedling stage. Nano-ZnO is found to stunt

root length and reduce number of roots. Ramesh et al. (2014) studied the positive

effects of bulk and nano-Titanium dioxide (TiO2) and Zinc oxide (ZnO) @ low

concentration on seed germination, shoot -root growth in Triticum aestivum

(Wheat).

Gokak and Taranath (2015) observed similar response in Zn and nano Zn

treated plants for seed germination and root – shoot elongation of Macrotyloma

uniflorum (Lam.) Verdc.

Laware et al. (2014) studied the effect of graded concentrations of zinc

oxide nanoparticles (ZnO NPs) along with sticker on onion crop and seed samples

obtained from NP treated plants along with control were tested for germination and

early seedling growth. The plants treated with ZnO NPs at the concentration of 20

and 30μg ml-1

showed better growth and flowered 12-14 days earlier than the

control. Treated plants also showed significantly higher values for seeded fruit per

umbel, seed weight per umbel and 1000 seed weight over control plants. These

result indicated that ZnO NPs can reduce flowering period by 12-14 days and even

produce healthy seeds. Rosa et al. (2013) reported that some nanoparticles (NPs)

affect seed germination; however, the biotransformation of metal NPs is still not

well understood. They investigated the NP toxicity on seed germination/root

elongation and the uptake of ZnO NPs and Zn2+

in alfalfa (Medicago sativa),

cucumber (Cucumis sativus), and tomato (Solanum lycopersicum) seedlings. Seeds

were treated with ZnO NPs at 0–1600 mg l–1

as well as 0–250 mg l–1

Zn2+

for

comparison purposes. Results showed that at 1600 mg l–1

ZnO NPs, germination in

cucumber increased by 10% and in alfalfa and tomato germination were reduced

by 40 and 20%, respectively. At 250 mg Zn2+

l–1

, only tomato germination was

reduced with respect to controls. The highest Zn content was of 4700 and 3500 mg

kg–1

dry weight for alfalfa seedlings germinated in 1600 mg l–1

ZnO NPs and 250

mg l–1

Zn2+

, respectively.

11

Singh et al. (2013) studied the effects of bulk and ZnO NPs on germination,

growth and biochemical parameters of cabbage, cauliflower and tomato vegetable

crops and observed that nano-zinc oxide particle enhanced germination and

seedling growth in all the three test crops.

Jayarambabu, N. Siva Kumari, B. Venkateswara Rao, K. and Prabhu, Y.T.

et al (2014) reported that ZnO nanoparticles application in different fields like seed

germination, sensors, biomedical, semiconductor etc. In present study, the different

concentration (0,20,40,60 and 100mg) of ZnO NPs are prepared in distilled water

and sonicator for 15 minutes are used for the treatment in Mungbean (Vigna

radiata L.) seeds to study the effect on bioavailability of seed germination and

observed early seedling growth and growth characteristics of Mungbean. The

experiment was carried out under greenhouse conditions.

Prasad et al. (2012) carried out an investigation to examine the effects of

nanoscale zinc oxide particles on plant growth and development on peanut. Peanut

seeds were separately treated with different concentrations of nanoscale zinc oxide

(ZnO) and chelated bulk zinc sulfate (ZnSO4) suspensions (a common zinc

supplement), respectively and the effect this treatment had on seed germination,

seedling vigor, plant growth, flowering, chlorophyll content, pod yield and root

growth were studied. Treatment of nanoscale ZnO (25 nm mean particle size) at

1000 ppm concentration promoted both seed germination and seedling vigor and in

turn showed early establishment in soil manifested by early flowering and higher

leaf chlorophyll content. These particles proved effective in increasing stem and

root growth. Pod yield per plant was 34% higher compared to chelated bulk

ZnSO4.

Sedghi et al. (2013) studied that the effect of nano zinc oxide on

germination parameters of soybeans seeds under drought stress conditions applied

by poly ethylene glycol (PEG). Results showed that the effect of different

concentrations of PEG and nano zinc oxide on germination rate and germination

percentage, root length, root fresh and dry weight, seed residual fresh and dry

weight were significant. Nano zinc oxide increased germination percentage and

rate over control.

12

Sunita et al. (2013) studied that the effects of zinc oxide (ZnO) engineered

nanoparticles (ENPs) on plant growth, and bioaccumulation in Brassica juncea.

The seed was germinated under hydroponic condition with a varying concentration

of ZnO ENPs (0, 200, 500, 1000, 1500 mg l-1

) for 96 h and significant decrease in

plant biomass was recorded.


Burman et al. (2013) reported that foliar spray (1.5 and 10 ppm) of zinc

oxide (ZnO) nanoparticles has improved overall biomass accumulation in ten days

old seedlings of chickpea (Cicer arietinum L var. HC-1) in comparison to zinc

sulphate and ZnO of normal size. This response may be attributed to low reactive

oxygen species (ROS) levels which resulted in less lipid peroxidation as evident

from lower malondialdehyde (MDA) content. This was also associated with lower

activity of prominent antioxidant enzymes, superoxide dismutase (SOD), and

peroxidase in ZnO nanoparticle treated seedling when compared to control. The

study indicated importance in precise application of zinc, more so in deficient

system, where plant response varies with concentration and is important in

understanding the mechanism of action of specific nanomaterials.

Prasad et al. (2012) reported that seeds when treated with nanoscale ZnO

(25 nm mean particle size) at 1000 ppm concentration promoted chlorophyll

content of Peanut. Raliya and Tarafdar (2013) studied the effect of biologically

transformed ZnO nanoparticles on cluster bean (Cyamopsis tetragonoloba L.) to

enhance native phosphorous mobilizing enzymes and nano induced gum

production. ZnO nanoparticles were foliar sprayed at 10 ppm concentration on leaf

of 14-day-old cluster bean plants. A significant improvement in plant biomass

(27.1%), shoot length (31.5%), root length (66.3%), root area (73.5%), chlorophyll

content (276.2%), total soluble leaf protein (27.1%), rhizospheric microbial

population (11–14%), acid phosphatase (73.5%), alkaline phosphatase (48.7%),

and phytase (72.4%) activity in cluster bean rhizosphere was observed over control

in 6-week-old plants due to application of nano- ZnO. The gum content in cluster

bean seeds also improved by 7.5% after maturity which indicates that ZnO in nano

13

form may contribute more in industrial and medical applications besides

agricultural sector.

Ramesh et al. (2014) reported significant increases of chlorophyll and

protein content with low concentration nano-ZnO treated sample in wheat and no

changes were observed in bulk-ZnO and bulk and nano-TiO2 treated samples.

Singh et al. (2013) studied the effect of bulk and nano dimensional zinc

oxide particles (ZnO NPs) on germination, growth and biochemical parameters of

cabbage, cauliflower and tomato vegetable crops. They observed that bulk ZnO

particles was phytotoxic and adversely affected biochemical parameters of all the

test crops. On the contrary, nano-dimensional zinc oxide particle enhanced

germination, seedling growth, pigments, sugar and protein contents along with

increased activities of antioxidant enzymes in all the three test crops. Zinc oxide

NPs invariably increase pigments, protein and sugar contents and nitrate reductase

activities in cabbage (Brassica oleracea var. Capitata). It was observed in

cauliflower that higher concentration of ZnO NPs (9.0 µM) maintained the sugar

content at the level of control, while SOD and CAT to the level of bulk ZnO

treated seedlings. ZnO NPs induced activities of antioxidant enzymes viz. SOD,

CAT, APX and POD, however, were lower as compared to those treated with bulk

ZnO.

Sunita et al. (2013) studied that the effect of zinc oxide (ZnO) engineered

nanoparticles (ENPs) on antioxidative enzyme activity in Brassica juncea. The

seed was germinated under hydroponic culture with a varying concentration of

ZnO ENPs (0, 200, 500, 1000, 1500 mg l-1

) for 96 h. Increase in proline content

and lipid peroxidation upto a concentration of 1000 mg l-1

was observed. They

observed that ENPs caused a significant effect due to their accumulation along

with the generation of reactive oxygen species in plant tissues, thus signifying its

hazardous effect on B. juncea.

2.4 Impact of other metallic nanoparticle fertilizer on Plant


Bao-shan et al. (2004) studied the effect of exogenous application of nano-

SiO2 on Changbai larch (Larix olgensis) seedlings and found that nano-SiO2

14

improved seedling growth and quality, including mean height, root collar diameter,

main root length, and the number of lateral roots of seedlings and also induced the

synthesis of chlorophyll. Under abiotic stress, nano-SiO2 augments seed

germination

Haghighi et al. (2012) in tomato and Siddiqui et al. (2014) in squash

reported that nano-SiO2 enhanced seed germination and stimulated the antioxidant

system under NaCl stress. Siddiqui and Al-Whaibi (2014) reported that the lower

concentrations of nano-SiO2 improved seed germination of tomato.

Liu et al. (2009) showed that 5 mg l-1

silver nano-particles reduced

bacterial colonies and extended the vase life of cut gerbera cv. ‗Ruikou‘. Lu et al.

(2002) studied the effect of nano-SiO2 and nano-TiO2 mixtures on soybean seeds

and found that the mixture increased the nitrate reductase in soybeans, increasing

their germination and growth.

Mazumdar (2014) reported that significant inhibition on shoot fresh weight

of V. radiata (p=0.008) and B. campestris (p=0.002) was observed at 1000 μg ml-1

silver nanoparticle solution after treatment period. V. radiata showed significant

retardation on dry weight of root at 1000 μg ml-1

of Ag+ ions solution after 12

th

day. The decrease on shoot dry weight with increase in nanoparticle and ion

concentration was also observed after 12th

day.

Najafi et al. (2014) studied the phytotoxic effects of Pb as Pb(NO3)2 and

silver nanoparticles on mung bean (Vigna radiata) planted on contaminated soil

was assessed in terms of growth, yield, at 120 ppm concentration. Yugandhar and

Savithramma (2013) observed the effect of calcium carbonate nanoparticles on

seed germination and seedling growth of Vigna mungo and showed that the bio-

synthesized calcium carbonate nanoparticles accelerate the seed germination and

seedling growth in V. mungo. Highest percentages of seed germination, seedling

vigor index, root and shoot length, fresh and dry weight and relative water content

was also observed in NP treated plants.

Savithramma et al. (2012) reported that biologically synthesized Silver NPs

improved the seed germination and seedling growth of Boswellia ovalifoliolata an

endemic, endangered and globally threatened medicinal tree species.

15

Suriyaprabha et al. (2012) studied plant responses to nano and bulk silica

treatments in terms of growth characteristics in maize crop. Growth characteristics

were much influenced with increasing concentration of Silica nanoparticles up to

15 kg ha-1

whereas at 20 kg ha-1

, no significant increments were noticed. Silica

accumulation in leaves was high at 10 and 15 kg ha-1

(0.57 and 0.82%)

concentrations of SNPs. Suriyaprabha et al. (2012) reported that nano-SiO2

increased seed germination by providing better nutrients availability to maize

seeds, and pH and conductivity to the growing medium.

Y.Farhat. et al. (2015) reported that a reduction in germination percentage

on exposure to silver and copper nanoparticles while maximum germination

percentage was on application of iron nanoparticles. Similarly, while root and

shoot growth was also enhanced under iron nanoparticles application while severe

reduction in root and shoot length was observed on exposure to copper

nanoparticles. So copper has inhibitory while iron has stimulatory effect on wheat

germination and growth.

Zheng et al. (2005) studied the effects of nano-TiO2 and non-nano-TiO2 on

the germination and growth of Spinacia oleracea by measuring the germination

rate and vigor indexes. An increase of these indexes was observed at 0.25-4%

nano-TiO2 treatments.


Adhikari et al. (2013) observed good germination of seeds in the presence

of SiO2 nano particles which had showed no toxic effect on rice growth, whereas

root growth and elongation were arrested with Mo nano particles after 50 mg l-1

. In

many cases root necrosis was occurred. Massive adsorption of Mo nano particles

into the root system was responsible for the toxicity, which calls for more research

for recommending their safe use as biolabels in plants. The uptake of both the nano

particles was observed with rice seedlings. Application of silica nano particles

enhanced the root length, root volume and dry matter weight of shoot and root of

rice crop.

Agrawal and Rathore (2014) reported the positive morphological effects of

nano materials which include enhanced germination percentage and rate; length of

root and shoot, and their ratio; and vegetative biomass of seedlings along with

16

enhancement of physiological parameters like enhanced photosynthetic activity

and nitrogen metabolism in many crop plants. Amirnia et al. (2014) reported that

nanofertilizers improved saffron yield. In addition, it was also observed that Fe, P

and K nanofertilizers had positive effects on the saffron flowering.

Ashrafi et al. (2013) evaluated the effect of nanosilver application and

weed density in an integrated fertilization system on agronomic traits of soybean.

They found that coapplication of organic and nanosilver increased leaf chlorophyll

significantly. The highest grain P and K concentration was obtained from

nanosilver treated plants. Coapplication of compost, farmyard manure and

chemical fertilizer produced a higher amount of pods per plant, seed number per

pod and 100-seed mass. Nanosilver treated plants produced the highest grain yield.

Aslani et al. (2014) studied the influence of engineered nanomaterials

(carbon and metal/metal oxides based) on plant growth and observed that

engineered nanomaterials influences seed germination. It was found to affect the

shoot-to-root ratio and the growth of the seedlings. Hong et al. (2005) reported that

Nanoscale titanium dioxide (TiO2) promoted photosynthesis, and growth of

spinach.

Karthick and Chitrakala (2011) observed that chlorophyll a content was

significantly increased by Ag nanoparticles in green gram and sorghum. Najafi et

al. (2014) studied the Phytotoxic effects of Pb as Pb(NO3)2 and silver nanoparticles

on Mung bean (Vigna radiata) planted on contaminated soil in terms of

chlorophyll pigments, phenol and flavonoid content at 120 ppm concentration.

Mazumdar (2014) reported that exposure to 1000 μg ml-1

of Ag nanoparticles led

to significant retardation of total chlorophyll content in V. radiata (p=0.001) and B.

campestris (p=0.001) when compare to control after 12th

day of treatment. After

the treatment period no significant inhibition on chlorophyll ratio was observed

when exposed to both Ag nanoparticle and ion solutions.

Morteza et al. (2013) reported the effects of titanium dioxide spray on corn

(Zea mays L.). Results showed that effect of nano TiO2 was significant on

chlorophyll content (a and b), total chlorophyll (a + b), chlorophyll a/b, carotenoids

and anthocyanins. The maximum amount of pigment was recorded from the

treatment of nano TiO2 spray at the reproductive stage in comparison with control.

17

Suriyaprabha et al. (2012) studied the observed physiological changes including

expression of organic compounds such as proteins, chlorophyll, and phenols was

favorable to maize treated with nanosilica especially at 15 kg ha-1

compared with

bulk silica and control. Nanoscale silica regimes at 15 kg ha-1

have a positive

response on maize than bulk silica which helps to improve the sustainable farming

of maize crop as an alternative source of silica fertilizer. Qiang et al. (2008)

reported that compared to NPK chemical fertilizer, the application of

slow/controlled release fertilizer coated and felted by nanomaterials improved

grain yield with an insignificant increase in protein content and a decrease in

soluble sugar content in wheat. Rico et al. (2013) reported that rice grains from

nCeO2-treated plants had less Fe, S, prolamin, glutelin, lauric and valeric acids,

and starch. Moreover, the nCeO2 reduced all antioxidant values, except flavonoids

in grain. Medium- and low-amylose varieties accumulated more Ce in grains than

the high-amylose variety, but the grain quality of the medium-amylose variety

showed higher sensitivity to the nCeO2 treatment. These results indicated that

nCeO2 could compromise the quality of rice.

Zhu et al. (2008) reported that Cucurbita maxima grown in an aqueous

medium containing magnetic nanoparticles can absorb, move and accumulate the

particles in the plant tissues, whereas Phaseolus limensis is not able to absorb and

move particles. It indicated that different plants have different response to the same

nanoparticles.

18

CHAPTER - III

MATERIALS AND METHODS

A Laboratory experiment was conducted during kharif 2015 and Rabi 2015-

16 to study the physiological responses of soybean and wheat towards nano

particle based micronutrients fertilization. The details of materials used and the

experimental technique adopted during the course of investigation are described

below.

3.1 Experimental site

The laboratory experiment was conducted at Indian Institute of Soil

Science Bhopal at 230

18 N, 77024 E, with an altitude of 485 meter above the mean

sea level.

3.2 Sand culture system setup

Sand culture system was setup in china clay pots of 5 kg capacity having

size of 30 cm height and 14 cm diameter. Acid washed sand was washed with de-

ionized water continuously till it is devoid of macro and micro nutrients. Hoagland

nutrient solution was applied weekly to the sand culture system. The solution

composition is given in appendix B.

Description of crop: Soybean and Wheat

Soybean genotype – JS 355

Wheat genotype – HD 8729

3.3 Climate

The main research station of ICAR-Indian Institute of Soil Science Bhopal

is situated in Central Highlands (zone -10) of M.P state. The meteorological data

during crop season as recorded at the meteorological observatory, ICAR-Central

Institute of Agricultural Engineering, Bhopal situated near the research farm of

IISS Bhopal (Fig. 3.3 and appendix A).

19

http://www.ciae.nic.in/



Fig. 3.1 A view of experimental set up of soybean in sand culture system

Fig. 3.2 A view of experimental set up of wheat in sand culture system

20

Fig

. 3.3

Met

eoro

logic

al d

ata

duri

ng c

rop g

row

th p

erio

d

05

10

15

20

25

30

35

40

Tem

p. M

ax

Tem

p. M

in

0C

Tem

pra

ture

(0C

)

21

3.4 Experimental details

3.4.1 Design and layout

The experiment was laid out in completely randomized block design with

ten treatments and five replications. In each replication five plants were maintained

for growth and morphological analysis. The treatment details are Table 3.1.

Table: 3.1 Treatment details

(% given in parenthesis is either 100 or 50 % of corresponding nutrient in Hoagland solution)

3.4.2 Collection of experimental data

One plant was randomly selected from each container and was tagged for

recording various morphological observations at different stages.

3.5 Morphological parameters

3.5.1 Plant height

Plant height was recorded from base of the plant to the uppermost node of

main shoot of plant at 45 days after sowing (DAS) and 60 DAS and height was

expressed in cm.

3.5.2 Root length

Total root length of the five randomly selected plants was measured at 45

and 60 DAS in centimeters with the help of Graph paper ruled in millimeters scale.

Roots were placed on a shallow glass dish and graph paper was placed under the

dish. The roots were cut from the root-shoot joint and were positioned randomly

over the graph paper lines (representing a grid) with the help of forceps and needle

to avoid overlapping. The long branched roots were cut into smaller pieces

(Newman, 1966). The counts for intersection of roots (N) with vertical and

horizontal lines of 1 cm grid from the graph paper were recorded root length was

Treatment in sand culture system Elemental concentration of Fe, Cu and Zn

(salt / NP)

T1: 100% (Fe + Cu + Zn) =Normal salts 54 µM Fe + 0.5 µM Cu + 2 µM Zn =

Normal salts

T2: T1- Fe salt+ Fe NP (100%) 54 µM Fe NP + 0.5 µM Cu + 2 µM Zn

T3: T1- Fe salt + Fe NP (50%) 27 µM Fe NP + 0.5 µM Cu + 2 µM Zn

T4: 100% (Cu + Zn) salts + Fe salt (50%) 27 µM Fe salt + 0.5 µM Cu + 2 µM Zn

T5: T1- Cu salt + Cu NP (100%) 54 µM Fe + 0.5 µM Cu NP + 2 µM Zn

T6: T1- Cu salt + Cu NP (50%) 54 µM Fe + 0.25 µM Cu NP + 2 µM Zn

T7: 100% (Fe + Zn) salts + Cu salt (50%) 54 µM Fe + 0.25 µM Cu salt + 2 µM Zn

T8: T1- Zn salt + Zn NP (100%) 54 µM Fe + 0.5 µM Cu + 2 µM Zn NP

T9: T1- Zn salt + Zn NP (50%) 54 µM Fe + 0.5 µM Cu + 1 µM Zn NP

T10: 100% (Fe + Cu) salts + Zn salt (50%) 54 µM Fe + 0.5 µM Cu + 1µM Zn salt

22

computed using the modified version of Newman (1966) formula proposed by

Tennant (1975) as:

Root Length= 11/14* number of intersections (N) *grid unit.

3.5.3 Plant Biomass

The selected plant was removed from the plastic container. The whole plant

was divided into leaves, stem and roots and then weight of leaves and stem was

measured for fresh weight. The samples were dried in oven for 72 hrs at 65°C and

total dry weight (expressed in gram) of leaves and stem was recorded for dry

weight.

3.5.4 Root Biomass

The whole root was weighed immediately for fresh weight and dried in

oven for 72 hrs at 65°C and total dry weight (expressed in gram) of root was

recorded.

3.5.5 Root Volume

Root volume was determined by volume displacement method (Mishra and

Ahmed, 1987) using a measuring cylinder.

3.6 Growth parameters

3.6.1 Leaf area

Leaf area was measured by leaf area meter of LICOR make (Model 3100)

and expressed in cm2.

3.6.2 Leaf area ratio (LAR)

The leaf area ratio was worked out by the formula of Radford (1967) and

expressed as cm² g-1

.

Leaf area (cm2plant-

1)

LAR =

Total dry matter (g plant-1

)

3.6.3 Specific leaf weight (SLW)

The specific leaf weight (g cm-2

) indicates the leaf thickness and was

determined by the following formula.

Leaf dry weight (g)

SLW =

Leaf area (cm2)

23

3.6.4 Specific leaf area (SLA)

The inverse of specific leaf weight is the specific leaf area (cm² g-1

) and

was calculated as follows.

Leaf area (cm²)

SLA =

Leaf dry weight (g)

3.6.5 Root shoot ratio

The materials was put in paper bags and then put in an oven at 80oC for 24

hours. The samples were weighing by electronic balance (Sartorius Basic,

BA2105) and the average data on dry weights of root, leaf, and stem per plant was

worked out. Shoot dry weight per plant was obtained by adding leaf dry weight

with stem dry weight per plant (Amanullah et al., 2010; Amanullah & Shah, 2011).

The sum of the shoot and root dry weight was calculated as the total dry weight per

plant. Shoot to root ratio (S:R) at each growth stage was calculated using the

following formula:

Root dry weight (g plant-1

)

Root Shoot Ratio (S:R) =

Shoot dry weight (g plant-1

)

3.7 Physiological and biochemical parameters

3.7.1 Chlorophyll content estimation

The chlorophyll content was estimated at 30 and 60 DAP. Total

chlorophyll, chlorophyll a and chlorophyll b contents were determined by

following the method of Hiscox and Israelstom (1979). 500 mg of fresh leaf tissues

were cut into small pieces and incubated in 5.0 ml of DMSO (Dimethyl Sulfoxide)

at 50°C for 2.30 hours. At the end of incubation period the supernatant was

decanted and leaf tissues discarded. The absorbance was read at 645 and 663 nm in

UV-vis spectrophotometer (ELICO, 159). Total chlorophyll, chlorophyll a and

chlorophyll b content were calculated using the formula given by Arnon (1949)

and expressed in mg per gram fresh weight.

V

Chlorophyll 'a' = 12.7 (A663) - 2.69 (A645) X

1000 X W

24

V

Chlorophyll 'b' = 22.9 (A645) - 4.68 (A663) X

1000 X W

V

Total chlorophyll = 20.2 (A645) + 8.02 (A663) X

1000 X W

Where,

A = Absorbance at specific wave length (645, 663 nm)

V = Final volume of the chlorophyll extract (ml)

W = Fresh weight of the sample (g)

3.7.2 Estimation of relative water content

Relative water content is an important parameter to measure the water

content in plant tissues. It has taken two times during crop growth period in a fully

expanded upper most leaf and cut into small pieces, recorded their fresh weight.

After measuring the fresh weight, leaf pieces were incubated in fresh water for 3

hrs to allow them to gain full turgidity at room temperature. Removed water from

leaf segment and blotted with tissue paper to remove adhered water on leaf surface

and thereafter their turgid weight is recorded. Leaf pieces are dried to constant

weight at 600C in hot air oven and dry weight was calculated (Weatherly and

Slatyer, 1957).

Fresh weight – Dry weight

RWC (%) = X 100

Turgid weight – Dry weight

3.7.3 Estimation of membrane stability

Leaf tissue was cut, added 10 ml distilled water then placed for incubation

at 100C temperature for 24 hours. After incubation the sample was equilibrated for

1 hour at room temperature and the conductivity of medium was measured by

conductivity electrode. All samples were covered with wrap and autoclaved for 15

min 1210C. There after the sample was equilibrated for 1 hour at room temperature

and the conductivity of medium was measured by conductivity electrode.

(1-(T1/T2)

MS % = X 100

(1-C1/C2)

25

T1 - EC after 10oC temperature of sample

T2- EC after 121oC temperature of sample

C1 - EC after 10oC temperature of blank sample

C2- EC after 121oC temperature of blank sample

3.7.4 Proline content estimation

Extraction

Proline content was measured following the method of Bates et al. (1973).

0.5 gram of fresh plant sample (leaves) was taken and 10ml of 3% aqueous

sulphosalicylic acid added and ground in pestle and mortar, and then filtered

through What man No. 42 filter paper.

Assay

5 ml of aliquot from the colour filtrate was taken in a 30 ml test tube for

determination and then 2ml of glacial acetic acid and 2 ml acid ninhydrine mixture

solution (1.25g ninhydrine in 30 ml of acetic Acid +20 ml of 6M phosphoric acid)

was added into each tube. The reaction mixture was kept in boiling water for 1

hour to develop pink colour. Reaction was terminated by keeping in ice. Added 4

ml toluene and mixed thoroughly and aspirated toluene layer. The colour intensity

was measured by spectrophotometer at 520 nm wavelength after setting the

instruments to zero with blank. To determine the proline content, a standard curve

was made using pure proline. The content of proline was expressed in units of

μmol per gram fresh weight (μM g-1

FW).

3.7.5 Assay of antioxidant enzyme

Enzyme extraction

For enzyme assay leaf sample were collected from plant and kept in ice

box. 0.5gram of fresh leaf was homogenized in 3 ml of extraction buffer of 0.1 M

phosphate buffer (7.8) containing 0.5 mM EDTA in pre chilled mortar and pestle.

The homogenized tissue was centrifuged at 13,000g for 10 min at 4°C and the

collected supernatant was used for enzyme assay. Total process was carried at 4°C.

3.7.5.1 Super oxide dismutase (SOD) activity assay

SOD activity was assayed by following the method of Dhindsa et al.

(1981). Added 0.3 ml supernatant to reaction mixture containing 1.3μM

Riboflavin 63μM of NBT and 200mM of methionine. Tubes were covered with

26

aluminum foil to prevent light. Prepared blank without enzyme supernatant as

control. Exposed tubes to light in light box for 3 min. The colour intensity was

measured by spectrophotometer at 560 nm wavelength and SOD activity, in unit g-

1, was expressed as fold increase over Normal salt treated plants.

3.7.5.2 Catalase (CAT) activity assay

CAT activity was assayed by following the method given by Barber (1980).

The extract from SOD Assay was used for CAT assay. Added 1.5 ml phosphate

buffer, 1 ml H2O2 (0.005M) and 0.5 ml enzyme. Incubated at 200C for 1 min and

stopped reaction by 5ml 0.7 N H2SO4.Titrated reaction mixture against 0.01N

Kmo4 until a faint /light purple colour persists for at least 15 second. Prepared

blank by adding enzyme extract reaction mix without incubation and CAT was

expressed in unit H2O2 min-1

g-1

.

3.7.5.3 Peroxidase (POX) activity assay

POX activity was assayed by following the method of Summer and

Gjessing, (1943). The extract from SOD Assay was used for POX assay. Added

1ml O-dianisidine (0.01M in methanol), 0.5 ml H2O2 (0.02M), 1 ml phosphate

buffer, 2.4 ml distilled water and 0.2 ml enzyme and incubated at 300

C for 5 min.

The reaction was stopped by adding 1ml 2N H2SO4. Blank tube excluding H2O2

was prepared by adding 0.5 ml distilled water. The colour intensity was measured

by spectrophotometer at 430 nm wavelength and POX was expressed in unit H2O2

min-1

g-1

.

3.7.6 Estimation of total soluble protein

Extraction assay

For Protein assays leaf sample were collected and 0.5gram of fresh leaf was

homogenized in 3 ml extraction buffer of 0.1 M phosphate buffer (7.8) containing

0.5 mM EDTA in pre chilled mortar and pestle. The homogenized tissue was

centrifuged at 13,000g for 10 min at 40C and the collected supernatant was used for

enzyme assay. The extraction process was carried at 40C.

Total soluble protein assay

Total soluble protein was measured by following method given by

Bradford (1976). Used the extract for assay and added 0.2 ml sample, 4 ml 0.1%

protein reagent (100 mg Coomassie Brilliant Blue) and 0.8 ml distilled water. The

27

colour intensity was measured by spectrophotometer at 595nm wavelength.

Calculation of the total soluble protein content was done by creating a standard

curve using a standard bovine serum albumin (25 mg in 0.15 M NaCl and made up

to volume 25 ml stock and working stock was made by diluting 10 times) and was

expressed in mg per gram fresh weight (mg g-1

FW).

3.7.7 Estimation of total soluble sugar

Extraction

Total soluble sugar content was measured based on the Anthrone method

(Hodge and Hofreite, 1962 and Sadasivam and Manickam, 1992). 0.1 gram of

oven dry plant sample was taken, added 5 ml of 2.5 N HCl, hydrolyzed by keeping

in boiling water bath for 3 hours then cooled to room temperature. The reaction

mixture was neutralized by solid sodium carbonate and then volume made up to 50

ml. The mixture was centrifuged and collected the supernatant.

Assay

Taken supernatant 0.5 ml aliquots then added 4 ml of anthrone reagent

(dissolve 200 mg anthrone in 100 ml of ice cold 95% H2SO4). Heated for 8 min. in

boiling water bath, cooled rapidly and read the green to dark green colour. The

colour intensity was measured by spectrophotometer at 630nm wavelength.

Calculation of the total soluble sugar content was done by creating a standard

curve using a standard glucose (dissolve 100mg glucose in 100 ml in water and

working standard of 10 ml stock and made up to 100 ml) and was expressed as

percentage of dry weight.

3.7.8 Estimation of starch

Extraction

Starch was measured based on the Anthrone method (Hodge and Hofreite,

1962, Thayumanavan and Sadasivam, 1984 and Sadasivam and Manickam.1992).

0.1 Gram of oven dry plant sample was taken added 10 ml of 80% ethanol and then

centrifuged to remove sugar. Residue was dried over water both. Added 5 ml water

and 6.5 ml 52% perchloric acid to residue. Extract was centrifuged at 00C for 20

min. and collected supernatant and volume was made up to 50 ml.

28

Assay

Taken supernatant of 0.5 ml aliquots and then added 4 ml of anthrone

reagent (200 mg anthrone dissolved in 100 ml of ice cold 95% H2SO4). The

reaction mixture was kept for 8 min. in boiling water both, cooled rapidly and read

the green to dark green colour. The colour intensity was measured by

spectrophotometer at 630nm wavelength. Calculation of the starch was done by

creating a standard curve using a standard glucose as mentioned in TSS assay and

was expressed as percentage of dry weight.

3.7.9 Estimation of non-structural carbohydrate

Non-structural carbohydrate was calculated as sum of total soluble sugar

and starch and expressed as percentage of dry weight.

3.7.10 Gas exchange parameters

Gas exchange parameters viz. Photosynthesis rate (µM m-2

s-1

),

transpiration rate (mM m-2

s-1

) and stomatal conductance (µM m-2

s-1

) were

recorded in the morning (8 to 10 AM) in the experimental plant leaves using

Photosynthesis system (make: PP systems and model: CIRAS-2).

3.7.11 SPAD value

SPAD value was recorded in the evening (3 to 4 PM) in the experimental

plant leaves using SPAD-502Plus.

3.7.12 Grain yield

Grain yield was recorded from five plants and expressed in g plant-1

.

3.7.13 Toxicity/ Deficiency symptom analysis

Toxicity/ deficiency symptoms due to the application of micronutrients were

observed visually.

29

CHAPTER – IV

RESULTS AND DISCUSSION

4.1 Morphological parameters of Soybean

4.1.1 Plant height

Among all the treatments, highest plant height was found in plants treated

with Cu NP (0.5 µM) at both stage and lowest plant height was observed in plants

treated with Zn salt (1 µM) and Fe salt (27 µM) at 45 DAS and 60 DAS,

respectively (Table 4.1). Among Fe treatments, plant height was found higher in

plant treated with Normal salt and was followed by Fe NP (54 µM), Fe NP (27

µM) and Fe salt (27 µM) at 45 DAS. However at 60 DAS plants treated with Fe

NP (27 µM) were taller than Fe NP (54 µM) and Fe salt (27 µM). In the present

study there was not much increase in height of soybean plants treated with Fe NP

(27 µM) at 60 DAS. Fe nanoparticles were found to increase stem length of basil

plants (Peyvandi et al. 2011, Kumar 2015). Salarpour et al. (2013) observed 80%

increase in plant height of Lepidum sativum L treated with nano-iron chelates.

Among Cu Treated plants, higher plant height was obtained in Cu NP (0.5

µM) followed by Cu NP (0.25 µM), Normal salt and Cu salt (0.25µM) at 45DAS.

In second stage Cu NP (0.5 µM) followed by Normal salt, Cu NP (0.25 µM) and

Cu salt (0.25µM). The influence of Cu NPs on plant height is sparsely reported.

CuO NPs did not inhibit the seed germination upto 2000ppm but root growth was

inhibited at 500 ppm in soybean and chickpea (Adhikari et al. 2012). However,

improvement in growth of plants treated with Cu NPs were reported in wheat

(Hafeez et al. 2015), lettuce (Shah and Belozerova, 2009) and improvement in vase

life of chrysanthemum was also reported with Cu NPs (Hashemabadi et al. 2013).

In the present study it was observed that normal concentration of Cu NP (0.5 µM)

had positive influence on plant height of soybean while reduced concentration of

Cu NP (0.25 µM).

Among Zn treatments plants, higher plant height was noted in Normal salt

followed by Zn NP (1 µM), Zn NP (2 µM) and Zn salt at 45 DAS. But at 60 DAS,

30

maximum plant height was recorded in Zn NP (2µM) followed by Normal salt, Zn

NP (1 µM) and Zn salt (1µM). Foliar spray of 10 ppm ZnO nanoparticles on 14-

day-old cluster bean plants, significantly improved shoot length (Raliya and

Tarafdar 2013). Ramesh et al. (2014) also reported positive effects of nano-ZnO on

shoot-root growth of wheat. Nano-ZnO particle was found to enhance germination

and seedling growth of soybean (Sedghi et al. 2013) and vegetable crops like

cabbage, cauliflower and tomato (Singh et al. 2013). This was in conformity with

the present findings wherein Zn NP (2 µM) improved plant height of soybean

plants at 60 DAS. This shows that normal concentration of Zn NPs can positively

influence height of plants.

4.1.2 Total Root Length

Total root length was recorded in different stages at 45 DAS and 60 DAS

(Table 4.1). At 45 DAS, among Fe treatments, plant show higher root length in

plant treated with Fe NP (27 µM) followed by Fe NP (54µM), Fe salt (27µM) and

Normal salt. But at 60 DAS, higher root length was observed in plants treated with

Fe salt (27 µM) as compared to Fe NP (54 µM), Fe NP (27µM) and normal salt. Fe

nanoparticles were found to increase root length of basil plants (Peyvandi et al.

2011, Kumar 2015). In the present study there increase in root length was observed

at 60 DAS with Fe NP (27 µM) treatment, which may be attributed to the increased

branching of roots of soybean crop.

Among Cu treatments, higher total root length was observed in plants

treated with Cu salt (0.25 µM) followed by Cu NP (0.25 µM), normal salt and Cu

NP (0.5 µM) at 45 DAS. At 60 DAS, higher root length was recorded in plants

treated with Cu NP (0.25 µM) followed by Cu NP (0.5 µM), normal salt and Cu

salt (0.25 µM). The influence of Cu NPs on plant height is sparsely reported. CuO

NPs did not inhibit the seed germination upto 2000ppm but root growth was

inhibited at 500 ppm in soybean and chickpea (Adhikari et al. 2012). In the present

study it was observed that normal concentration of Cu NP (0.5 µM) had positive

influence on shoot part of soybean while reduced concentration of Cu NP (0.25

µM) had positively influenced root length at 45 DAS. This indicated that reduced

concentration of NP might have acted as catalyst for root growth and normal

concentration of NP for shoot growth.

Among Zn treated plants, higher total root length was noted in plant

treated with Zn salt (1µM) at both stages followed by Zn NP (2 µM) and normal

31

salt and Zn NP (1 µM) at 45 DAS, in the second stage Zn NP (1 µM) treated plants

produced longer roots than Zn NP (2 µM) and normal salt. Among all the ten

treatments, highest root length was obtained in plant treated with Fe NP (27 µM) at

45 DAS and lowest Cu salt (0.25 µM) 60 DAS. Ramesh et al. (2014) also reported

positive effects of nano-ZnO on shoot-root growth of wheat. However,

Boonyanitipong et al. (2011) observed negative effect of nano-ZnO on root length

of rice (Oryza sativa L.). Nano-ZnO particle was found to enhance germination


cabbage, cauliflower and tomato (Singh et al. 2013). This is conformity for root

length enhanced by reduced concentration of Zn treatments at both growth stages.

Table 4.1: Effect of micronutrient NPs on plant height and root length of Soybean

4.1.3 Shoot dry weight

Shoot dry weight differed significantly at 45 DAS, 60 DAS (Table 4.2 and

Fig 4.2). Among all the treatments highest Shoot dry weight was recorded in plants

treated with Zn NP (2µM) both stage at 45 DAS and 60 DAS similar to Fe NP (54

µM) at harvest and lowest shoot dry weight was recorded in Cu salt (0.25 µM) at

both stages 45 DAS and 60 DAS similar to Zn salt (1µM) at harvest stage. Among

Fe treatments, higher shoot weight was observed in plant treated with Fe NP (27

µM) better than Fe NP (54 µM) normal salt and Fe salt (27 µM) at 45 DAS.

However, at 60 DAS and harvest, plants treated with Fe NP (54µM) showed

enhanced performance as compared to normal salt, Fe NP (27 µM) and Fe salt

(27µM). Fe nanoparticles were found to increase shoot dry weight of basil plants

(Peyvandi et al. 2011, Kumar 2015). In the present study higher shoot dry weight

Treatment Plant height (cm) Total root length(cm)

45 DAS 60 DAS 45 DAS 60 DAS

T1: 100% (Fe + Cu + Zn) = Normal salts 56.0 64.0 14298.4 5834.5 T2: T1- Fe salt+ Fe NP (54 µM) 47.0 48.0 33858.6 7110.1 T3: T1- Fe salt + Fe NP (27 µM) 40.5 58.5 153789.5 8307.3 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 37.0 41.0 21655.2 12229.5 T5: T1- Cu salt + Cu NP (0.5 µM) 58.5 70.0 12115.7 7697.7 T6: T1- Cu salt + Cu NP (0.25 µM) 56.5 57.5 20827.2 13088.7 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 47.5 56.0 36981.7 1862.0 T8: T1- Zn salt + Zn NP (2 µM) 45.5 67.5 20545.8 5842.7 T9: T1- Zn salt + Zn NP (1 µM) 47.5 55.0 5865.0 19463.7 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 32.5 53.0 33079.5 27837.3 CD (5%) NS 10.20 37915.1 NS

32

was found in plants treated with normal concentration of Zn NP (2µM) at 45 DAS

and 30 DAS, respectively.

Among Cu NP treatments, higher shoot dry weight was recorded in plants

treated with Normal salt at 45 DAS followed by Cu NP (0.25µM), Cu NP (0.5 µM)

and Cu salt (0.25µM). At 60 DAS, Cu NP (0.5 µM) treated plants showed higher

shoot dry weight followed by normal salt, Cu NP (0.25 µM), Cu NP (0.5 µM) and

Cu salt but at harvest stage, higher shoot weight was recorded in plants treated with

normal salt as compared to Cu NP (0.25 µM), Cu NP (0.5 µM) and Cu salt

(0.25µM). However, improvement in growth of plants treated with Cu NPs were

reported in wheat (Hafeez et al. 2015), lettuce (Shah and Belozerova, 2009) and

improvement in vase life of chrysanthemum was also reported with Cu NPs

(Hashemabadi et al. 2013). In the present study it was observed that normal

concentration of Cu NP (0.5 µM) had positive influence on shoot dry weight of

soybean while reduced concentration of Cu NP (0.25 µM) had positively

influenced below ground part of plants at 45 DAS. This indicated that reduced



Among Zn treatment higher shoot dry weight was observed in plants

treated with Zn NP (2µM), at 45 DAS, followed by Zn NP (1µM), normal salt and

Zn salt (1µM). At 60 DAS, Zn NP (2µM) treated plants recorded higher shoot dry

weight followed by normal salt, Zn NP (1µM) and Zn salt (1µM). However, at last

stage higher shoot dry weight was noted in plants treated with Normal salt

followed by Zn NP (1µM) and Zn NP (1µM) Zn salt (1µM). Foliar spray of 10

ppm ZnO nanoparticles on 14-day-old cluster bean plants, significantly improved

shoot length (Raliya and Tarafdar 2013). Ramesh et al. (2014) also reported

positive effects of nano-ZnO on shoot-root growth of wheat. Nano-ZnO particle

was found to enhance germination and seedling growth of soybean (Sedghi et al.

2013) and vegetable crops like cabbage, cauliflower and tomato (Singh et al.

2013). This was in conformity with the present findings wherein Zn NP (2 µM)

improved shoot dry weight of soybean plants at 60 DAS. This shows that normal

concentration of Zn NPs can positively influence shoot/ root growth of plants.

Table 4.2: Effect of micronutrient NPs on shoot weight of soybean

33

Treatment Shoot dry weight (g plant-1

)

45 DAS 60 DAS Harvest

T1: 100% (Fe + Cu + Zn) = Normal salts 3.66 5.10 10.11 T2: T1- Fe salt+ Fe NP (54 µM) 4.49 5.19 10.12 T3: T1- Fe salt + Fe NP (27 µM) 5.33 3.83 8.69 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 2.46 3.42 7.74 T5: T1- Cu salt + Cu NP (0.5 µM) 2.20 5.91 8.38 T6: T1- Cu salt + Cu NP (0.25 µM) 2.70 3.91 8.51 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.62 2.96 8.11 T8: T1- Zn salt + Zn NP (2 µM) 6.43 6.68 6.86 T9: T1- Zn salt + Zn NP (1 µM) 5.58 4.70 7.30 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 2.21 3.48 6.36 CD (5%) 0.635 1.0 NS

4.1.4 Root dry weight

Root dry weight differed at 45 DAS, 60 DAS and Harvest (Table 4.3Fig.

4.3). Out of all ten treatments the highest root dry weight was observed in plants

treated with Cu NP (0.25µM) at Harvest and lowest with Zn salt (1µM) at 45 DAS.

In the all Fe treated plants, higher root weight was recorded in plants treated with

Fe NP (27µM) in comparison to Fe NP (54µM), Fe salt (27µM) and normal salt at

45 DAS. In second growth stage higher root dry weight was recorded in plant

treated with Fe NP (27µM) followed by normal salt, Fe salt (27µM) and Fe NP

(54µM). At last stage Fe NP (27µM) treated plant showed higher root dry weight

in comparison to normal salt, Fe salt (27µM) and Fe NP (54µM). Karimia et al.

(2014) observed that increasing Fe nanoparticles concentration above 10 ppm

reduced root weight of green gram, indicating negative effect of Fe NP on crop

growth. However, in the present study there was increase root dry weight was

observed at 60 DAS with Fe NP (27 µM) treatment, which may be attributed to the

increased branching of roots of soybean crop.

Among Cu treatments, maximum root weight was obtained in plants treated

with Cu salt (0.25µM) followed by Cu NP (0.5µM), normal salt and Cu NP

(0.25µM) at first growth stage. In second growth stage higher root dry weight was

recorded in plants treated with Cu salt (0.25µM) followed by normal salt, Cu NP

(0.5µM) and Cu NP (0.25µM). At last stage Cu NP (0.25µM) treated plant showed

higher root dry weight in comparison to normal salt, Cu salt (27µM) and Cu NP

(0.5µM).The influence of Cu NPs on plant height is sparsely reported. CuO NPs

34

did not inhibit the seed germination upto 2000ppm but root growth was inhibited at

500 ppm in soybean and chickpea (Adhikari et al. 2012). In the present study it

was observed reduced concentration of Cu NP (0.25 µM) had positively influenced

root weight at 45 DAS. This indicated that reduced concentration of NP might

have acted as catalyst for root growth.

Among Zn treatments, higher root weight was noted in plants treated with

Zn NP (1µM) followed by Zn NP (2µM), normal salt and Zn salt (1µM) at 45

DAS. But in the second stage, Zn NP (2µM) treated plants was found maximum

root weight as compared to normal salt, Zn NP (1µM) and Zn salt (1µM). At

harvest, impact of Zn treatments was found higher in normal salt treated plant

followed by Zn (1µM), Zn salt and Zn NP (2µM). Ramesh et al. (2014) also

reported positive effects of nano-ZnO on shoot-root growth of wheat. However,

Boonyanitipong et al. (2011) observed negative effect of nano-ZnO on root length

of rice (Oryza sativa L.). Nano-ZnO particle was found to enhance germination


cabbage, cauliflower and tomato (Singh et al. 2013). This was in conformity with

the present findings wherein Zn NP (2 µM) improved root dry weight of soybean

plants at 60 DAS. This shows that normal concentration of Zn NPs can positively

influence height and shoot/ root growth of plants. However, Kumar 2015 has

reported not much improvement in root dry weight of maize plants with Zn NP

treatments.

Table 4.3: Effect of micronutrient NPs on root dry weight of soybean

Treatment Root dry weight (g plant-1

)

45DAS 60 DAS Harvest

T1: 100% (Fe + Cu + Zn) = Normal salts 0.365 0.700 0.92

T2: T1- Fe salt+ Fe NP (54 µM) 0.440 0.490 0.71

T3: T1- Fe salt + Fe NP (27 µM) 0.605 0.745 0.93

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.395 0.505 0.90

T5: T1- Cu salt + Cu NP (0.5 µM) 0.415 0.640 0.69

T6: T1- Cu salt + Cu NP (0.25 µM) 0.390 0.595 1.04

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.660 0.750 0.87

T8: T1- Zn salt + Zn NP (2 µM) 0.385 0.840 0.66

T9: T1- Zn salt + Zn NP (1 µM) 0.475 0.485 0.68

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.275 0.375 0.68

CD (5%) NS NS NS

35

Fig. 4.1: Effect of micronutrient NPs on shoot weight of soybean.

Fig. 4.2 Effect of micronutrient NPs on root dry weight of soybean

0

2

4

6

8

10

12

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 DAS HARVESTS

hoot

dry

Wt.

(gpla

nt-1

)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 DAS HARVEST

Root

dry

wt.

(g

pla

nt-1

)

36

4.1.5 Specific leaf area

The information on specific leaf area (SLA) as influenced by various

micronutrient treatments during different growth periods are furnished in Table

4.4. Out of all treatments, highest SLA was noted in plant treated with Cu NP

(0.5µM) at 45 DAS. Lowest SLA was found in plant treated with Cu salt (0.25µM)

at 60 DAS. Among Fe treated plant higher SLA found was observed in plants

treated with Fe NP (54µM) followed by Fe NP (27µM), Fe salt (27µM) and

normal salt at 45 DAS. At 60 DAS, higher SLA was noted in plants treated with Fe

NP (27µM) than Fe NP (54µM), normal salt and Fe salt (27µM). There were not

many reports on the impact of nanoparticles on leaf growth parameter. It was

reported that nano-iron fertilizer caused 58 and 47% increase in fresh weight of

spinach (Moghadam et al. 2012). This was in conformity with the present findings

wherein soybean plants treated with reduced concentration i.e. Fe NP (27 µM)

exhibited higher SLA at 60 DAS. At 45 DAS, normal concentration of Fe NP (54

µM) treated plants showed higher SLA. This may be due to the growth promoting

effect of reduced concentration of Fe NP (27 µM) on growth characteristics of

soybean in comparison to Fe salts.

Among Cu treatments, higher SLA was observed in plants treated with Cu

NP (0.5µM) followed by Cu NP (0.25µM), normal salt and Cu salt (0.25µM) at 45

DAS. But at 60 DAS, normal salt treated plants showed higher SLA followed by

Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Enhanced leaf area of

wheat treated with 30 ppm of Cu NP was reported by Hafeez et al. (2015). In the

present study, higher SLA was reported from soybean plants treated with Cu NP @

0.5 µM at 45 DAS, which is much lower concentration than the 30 ppm used by

Hafeez et al. 2015.

Among the Zn treatment, higher SLA was observed in plant treated with

Zn (1 µM) as compared to Zn NP (2 µM), Zn salt (1µM) and normal salt at 45

DAS. However, at 60 DAS Zn NP (2µM) treated plants exhibited higher SLA than

normal salt, Zn NP (1µM) and Zn salt (1µM). In the present study, Zn NP treated

soybean plant has advantage over other treated plants for leaf growth

characteristics like SLA. Similarly, Avinash et al. (2010) observed increases in

37

germination and growth rate in the seeds of Cicer arietinum treated with nano-

ZnO.

4.1.6 Specific leaf weight

Specific leaf weight (SLW) as influenced by the application of

micronutrients based NPs at different growth stage is presented in Table 4.4. No

significant differences were observed plants at 45 DAS and at 60 DAS. Among Fe

treatment plant, higher SLW was recorded in plant treated with Normal salt

followed by Fe NP (27 µM) and Fe NP (54 µM). At 60 DAS higher SLW was fond

in plants treated with Fe NP (54 µM) followed by normal salt, Fe NP (27 µM) and

Fe salt (27 µM). There were not many reports on the impact of nanoparticles on

leaf growth parameter. It was reported that nano-iron fertilizer caused 58 and 47%

increase in fresh weight of spinach (Moghadam et al. 2012). This was in

conformity with the present findings wherein soybean plants treated with reduced

concentration i.e. Fe NP (27 µM) exhibited higher SLW at 60 DAS. At 45 DAS,

normal concentration of Fe NP (54 µM) treated plants showed higher SLW. This

may be due to the growth promoting effect of reduced concentration of Fe NP (27

µM) on growth characteristics of soybean in comparison to Fe salts.

Among the Cu treatments, Cu salt (0.25 µM) treated plants recorded

highest SLW at 45 DAS in comparison to normal salt, Cu NP (0.5 µM) and Cu NP

(0.25µM). In Second growth stage normal salt treated plants showed higher SLW

followed by Cu NP (0.5 µM) and Cu NP and Cu salt (0.25µM). Enhanced leaf

weight of wheat treated with 30 ppm of Cu NP was reported by Hafeez et al.

(2015). In the present study, higher SLW was reported from soybean plants treated

with Cu NP @ 0.5 µM at 45 DAS, which is much lower concentration than the 30

ppm used by Hafeez et al. 2015.

Among Zn treatments, higher SLW was noted in plants treated with normal

salt in comparison to Zn NP (1 µM), Zn NP (2 µM) and Zn salt (1 µM) at 45 DAS.

But at second growth stage, Normal salt treated plants recorded higher SLW

followed by Zn salt (1 µM) and Zn NP (2µM). Among all the ten treatments

highest SLW was obtained in plants treated with normal salt, Fe NP (54µM) at 60

DAS and lowest SLW noted in plant treated with Zn NP (2µM) at 60 DAS. In the

38

present study, Zn NP treated soybean plant has advantage over other treated plants

for leaf growth characteristics SLW. Similarly, Avinash et al. (2010) observed

increases in germination and growth rate in the seeds of Cicer arietinum treated

with nano-ZnO.

Table 4.4: Effect of micronutrient NPs on SLA and SLW of Soybean

Treatment Specific leaf area

(cm²g-1

)

Specific leaf weight

(g-1

cm-²)


T1: 100% (Fe + Cu + Zn) = Normal salts 222.20 187.25 0.0070 0.0090

T2: T1- Fe salt+ Fe NP (54 µM) 338.75 195.75 0.0000 0.0090

T3: T1- Fe salt + Fe NP (27 µM) 241.70 370.65 0.0030 0.0070

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 236.75 169.45 0.0030 0.0030

T5: T1- Cu salt + Cu NP (0.5 µM) 386.80 152.45 0.0060 0.0060

T6: T1- Cu salt + Cu NP (0.25 µM) 325.70 166.00 0.0010 0.0020

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 289.60 109.40 0.0090 0.0020

T8: T1- Zn salt + Zn NP (2 µM) 341.90 206.45 0.0020 0.0000

T9: T1- Zn salt + Zn NP (1 µM) 349.80 177.65 0.0030 0.0010

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 253.85 167.95 0.0010 0.0060

CD (5%) NS 97.582 NS NS

4.1.7 Leaf area ratio

It is evident form Table 4.5, highest leaf area ratio (LAR) was observed in

plants treated with Zn NP (1 µM) at 45 DAS and Fe NP (27µM) at 60 DAS and

lowest LAR was noted in plants treated with Zn salt (1 µM) at both stages. The

lower concentration of Fe NP (27µM) performed better as compared to higher

concentration of Fe NP (54µM), normal salt Fe salt (27µM) at both stages. There

were not many reports on the impact of nanoparticles on leaf growth parameter. It

was reported that nano-iron fertilizer caused 58 and 47% increase in leaf area index

of spinach (Moghadam et al. 2012). This was in conformity with the present

findings wherein soybean plants treated with reduced concentration i.e. Fe NP (27

µM) exhibited higher LAR at 60 DAS. This may be due to the growth promoting



Among Cu treatments, higher LAR was observed in those plants treated

with Cu NP (0.5µM) followed by Cu NP (0.25µM), Cu salt (0.25µM) and normal

salt at 45 DAS but in second growth stage higher LAR was obtained in plants

39

Fig

4.3

Eff

ect

of

mic

ronu

trie

nt

NP

s on s

pec

ific

lea

f ar

ea o

f so

ybea

n

0

50

10

0

15

0

20

0

25

0

30

0

35

0

40

0

45

0

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

45

DA

S6

0 D

AS

Specificleaf area (cm²g-1)

40

treated with Cu NP (0.25µM) as compared normal salt, Cu NP (0.5µM) Cu salt

(0.25µM). Enhanced leaf area ratio of wheat treated with 30 ppm of Cu NP was

reported by Hafeez et al. (2015). In the present study, LAR was reported from

soybean plants treated with Cu NP @ 0.5 µM at 45 DAS, which is much lower

concentration than the 30 ppm used by Hafeez et al. 2015. At 60 DAS, LAR was

found to higher with reduced concentration of Cu NP. This again reiterates that Cu

nanoparticle at lower concentration may act as catalyst for growth promotion.

Similar observations were also recorded in maize (Kumar 2015; Elanchezhian et al

2015).

Among Zn NP treatments, higher LAR was found in plant treated with Zn

NP (1 µM) followed by Normal salt, Zn NP (2µM) and Zn salt (1µM) at 45 DAS

and at second growth stage, higher LAR was obtained in plant treated with normal

salt followed by Zn NP (1µM), Zn NP (2µM) and Zn salt (1µM). In the present

study, Zn NP treated soybean plant has advantage over other treated plants for leaf

growth characteristics like LAR. Similarly, Avinash et al. (2010) observed

increases in germination and growth rate in the seeds of Cicer arietinum treated

with nano-ZnO.

4.1.8 Leaf area

The leaf area presented in Table 4.5 indicated significant differences

between the treatments at the both growth stages. Among all ten treatments,

highest leaf area was obtained in plants treated with Zn NP (2µM) and Fe NP

(27µM) at 45 DAS and 60 DAS, respectively and lowest Leaf area was observed in

plants treated with Cu salt (0.25 µM) and Zn salt (1 µM) at 45 DAS and 60 DAS,

respectively. Plants treated with Fe showed higher leaf area in plants treated with

lower concentration of Fe NP (27 µM) when compared to the high concentration of

Fe NP (54µM) followed by normal salt at both stage. There were not many reports

on the impact of nanoparticles on leaf growth parameter. It was reported that nano-

iron fertilizer caused 58 and 47% increase in leaf area index of spinach

(Moghadam et al. 2012). This was in conformity with the present findings wherein

soybean plants treated with reduced concentration i.e. Fe NP (27 µM) exhibited

higher leaf area and LAR at 60 DAS. This may be due to the growth promoting

41



Among Cu treatments, normal salt treated plant was performing better than

Cu NP (0.5µM), Cu NP (0.25µM) Cu salt (0.25µM) but at 60 DAS, higher LA was

recorded in plants treated with Cu NP (0.5µM) than normal salt, Cu NP (0.25µM)

and Cu salt (0.25µM). Enhanced leaf area of wheat treated with 30 ppm of Cu NP

was reported by Hafeez et al. (2015). In the present study, LAR was reported from

soybean plants treated with Cu NP @ 0.5 µM at 45 DAS, which is much lower

concentration than the 30 ppm used by Hafeez et al. 2015. At 60 DAS, LA was

found to higher with normal concentration of Cu NP while LAR was found to

higher with reduced concentration of Cu NP. This again reiterates that Cu

nanoparticle at lower concentration may act as catalyst for growth promotion.

Similar observations were also recorded in maize (Kumar 2015; Elanchezhian et al

2015).

Among Zn treated plants higher leaf area was found in plants treated with

Zn NP (2µM) as compared to Zn NP (1µM), normal salt and Zn salt (1µM) but at

second stage Zn NP (2µM) treated plants noted higher LA followed by normal salt,

Zn NP (1µM) and Zn salt (1µM). In the present study, Zn NP treated soybean plant

has advantage over other treated plants for leaf growth characteristics like leaf

area. Similarly, Avinash et al. (2010) observed increases in germination and

growth rate in the seeds of Cicer arietinum treated with nano-ZnO.

Table 4.5: Effect of micronutrient NPs on LAR and LA of soybean

Treatment Leaf area ratio(cm² g-1) Leaf area(cm²)



T2: T1- Fe salt+ Fe NP (54 µM) 130.83 116.53 585.85 604.2

T3: T1- Fe salt + Fe NP (27 µM) 152.70 190.01 599.75 726.3


T5: T1- Cu salt + Cu NP (0.5 µM) 175.39 93.50 389.90 554.5

T6: T1- Cu salt + Cu NP (0.25 µM) 163.13 135.72 447.70 530.5


T8: T1- Zn salt + Zn NP (2 µM) 123.06 98.16 786.10 653.7

T9: T1- Zn salt + Zn NP (1 µM) 216.76 105.43 620.95 365.3


CD (5%) NS 35.71 272.64 132.70

42

Fig 4.4: Effect of micronutrient NPs on leaf area ratio of soybean.

Fig 4.5: Effect of micronutrient NPs on leaf area of soybean.

0

50

100

150

200

250

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 DAS

Lea

f ar

ea r

atio

(cm

² g

-1)

0

100

200

300

400

500

600

700

800

900

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 DAS

Lea

f ar

ea (

cm²

)

43

4.1.9 Root volume

Among Fe treatments, higher root volume was observed in plants treated

with Fe NP (27µM) as compared to Fe NP (54µM), Fe salt (27µM) and normal salt

at 45 DAS (Table 4.6). AT 60 DAS, plants were performing better when treated

with Fe salt (27µM) followed by Fe NP (27µM), Fe NP (54µM) and normal salt.

There were many reports on the impact of nanoparticles on root volume for

conformity that nano particles could enhance and maintain the growth of maize

plant. The plant parameters like root length and root volume were all improved due

to application of nano-particle (Adhikari, T. et al.2015). In the present study

soybean plants treated with reduced concentration i.e. Fe NP (27 µM) exhibited

higher root volume at 60 DAS.

Among Cu treated plants, Root volume obtained was higher in plant

treated with Cu NP (0.25 µM) as compared to Cu salt (0.25µM), Cu NP (0.5µM)

and normal salt at both stages. CuO NPs did not inhibit the seed germination upto

2000ppm but root growth was inhibited at 500 ppm in soybean and chickpea

(Adhikari et al. 2012). However, improvement in growth of plants treated with Cu

NPs were reported in wheat (Hafeez et al. 2015), lettuce (Shah and Belozerova,

2009) and improvement in vase life of chrysanthemum was also reported with Cu

NPs (Hashemabadi et al. 2013). In the present study it was observed that normal

concentration of Cu NP (0.5 µM) had positive influence on above ground part as

well as shoot of soybean while reduced concentration of Cu NP (0.25 µM) had

positively influenced root volume at 45 DAS. This indicated that reduced



In the case of Zn treated plants, higher root volume was observed in plant

treated with Zn salt (1µM) as compared to Zn NP (2µM), normal salt, Zn NP

(1µM) at 45 DAS but at 60 DAS, higher root volume was noted in Zn (1µM)

followed by Zn salt (1µM), Zn NP (2µM) and normal salt. Among all the ten

treatments, highest root volume was recorded in plants treated with Fe NP (27µM)

at 45 DAS and lowest root volume was recorded in plant treated Normal salt at 60

DAS.

44

Table 4.6: Effect of micronutrient NPs on root volume of soybean

Treatment Root Volume (cc)

45 DAS 60 DAS

T1: 100% (Fe + Cu + Zn) = Normal salts 4.0

2.0

T2: T1- Fe salt+ Fe NP (54 µM) 4.5

3.0

T3: T1- Fe salt + Fe NP (27 µM) 12.0

4.0

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 4.5

4.5

T5: T1- Cu salt + Cu NP (0.5 µM) 4.0

3.5

T6: T1- Cu salt + Cu NP (0.25 µM) 4.5

5.5

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 4.5

3.0

T8: T1- Zn salt + Zn NP (2 µM) 4.0

3.0


5.0

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 8.5

4.0

CD (5%) NS

NS

4.1.10 Pod weight

The Pod weight was observed after harvest furnished in Table 4.7. Out of

all the ten treatments, highest pod weight was observed in plants treated with Fe

NP (54µM) and lowest Pod weight was noted in Cu salt (0.25µM). Among Fe

treatments, highest pod weight was recorded in plants treated with Fe NP (54µM)

followed by normal salt, Fe NP (27µM) and Fe salt (27µM). Among Cu treatment,

higher Pod weight was recorded in plants treated with normal salt followed by Cu

NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM). In the case of Zn treatments,

Zn NP (1µM) performed better than normal salt, Zn NP (2µM) and Zn salt (1µM).

4.1.11 Grain weight

The Grain weight after harvest was furnished in Table 4.7. Highest and

lowest Grain weight was noted in Zn NP (2µM) and Cu salt (0.25µM) treated

plants, respectively. Among Fe treatment, higher Grain weight was recorded in

plants treated with Fe NP (54µM) in comparison of Fe NP (27µM) followed by

normal and Fe salt (27µM). In the case of Cu treatments, Cu NP (0.5µM) was

performed better than normal salt, Cu NP (0.25µM) and Cu salt (0.25µM). Among

Zn treatment, highest Grain weight was recorded in plants treated with Zn NP

(2µM) followed by Zn NP (1µM), normal salt and Zn salt (1µM).

Increases in grain yield and straw yield of rice with Zn application was

reported (Maharana et al., 1993). In present study NP treated soybean plants, the

pod weight and grain yield was higher with normal concentration of Fe NP (54

45

µM), Cu NP (0.5 µM) and Zn NP (2 µM). This indicated that normal concentration

of NP might have sustained the crop growth for effective grain yield.

Table 4.7: Effect of micronutrient NPs on Pod weight and Grain weight of

Soybean

Treatment Pod weight

(g plant-1

)

Grain weight

(g plant-1

)

Harvest Harvest

T1: 100% (Fe + Cu + Zn) = Normal salts 1.99 0.57

T2: T1- Fe salt+ Fe NP (54 µM) 2.68 0.94

T3: T1- Fe salt + Fe NP (27 µM) 1.94 0.74

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 1.90 0.48

T5: T1- Cu salt + Cu NP (0.5 µM) 1.48 0.57

T6: T1- Cu salt + Cu NP (0.25 µM) 1.39 0.48

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.26 0.45

T8: T1- Zn salt + Zn NP (2 µM) 1.69 1.03

T9: T1- Zn salt + Zn NP (1 µM) 2.09 0.77

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 1.33 0.46

CD (5%) NS NS

4.1.12 Root Shoot Ratio

Root shoot ratio was influence by various treatments at different stages

(Table 4.8). Among all the treatments, highest Root Shoot ratio was found in plants

treated with Cu NP (0.25 µM) at 45 DAS and lowest in plants treated with Zn salt

(27 µM) at 45 DAS. Among Fe treated plants, higher root shoot ratio was obtained

in lower concentration of Fe NP (27 µM) as compared to normal concentration of

Fe NP (54 µM) followed by normal salt and Fe salt (27 µM) at both stages. In the

case of Cu treatments, maximum root shoot ratio was obtained in plants treated

with Cu NP (0.25 µM) better than Cu NP (0.5 µM) and Cu salt (0.25 µM) and

Normal salt at 45 DAS, but in second stage, Cu NP (0.5 µM) treated plants noted

higher root shoot ratio in comparison to Cu NP 0.25µM), Normal salt and Cu salt

(0.25µM). Among Zn treatments, higher root shoot ratio was recorded in plant

treated with Zn NP (2µM) followed by Normal salt, Zn NP (1µM) and Zn salt

(1µM) at 45 DAS. But it was changed in second growth stage, maximum root

shoot ratio was observed in plants treated with Normal salt followed by Zn NP

(2µM), Zn NP (1µM) and Zn salt (1µM).

46

In soybean, root/shoot dry weight were found to be higher with normal

concentration of Zn NP (2 µM). In the present set of investigation, the response in

wheat was similar to soybean and maize crop (Kumar 2015).

Table 4.8: Effect of micronutrient NPs on root shoot ratio of soybean

Treatment Root Shoot Ratio

45 DAS 60 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 0.114 0.145

T3: T1- Fe salt + Fe NP (27 µM) 0.159 0.195


T5: T1- Cu salt + Cu NP (0.5 µM) 0.193 0.255

T6: T1- Cu salt + Cu NP (0.25 µM) 0.385 0.155


T8: T1- Zn salt + Zn NP (2 µM) 0.125 0.125

T9: T1- Zn salt + Zn NP (1 µM) 0.085 0.105


CD (5%) NS NS

4.2 Biochemical parameters of Soybean

4.2.1 Chlorophyll content

It is evident from Table 4.9 and Fig 4.6 and 4.7 that the chlorophyll ‗a‘

content did not differ significantly between treatments at both stages. Among all

the treatments highest chlorophyll content ‗a‘ was found in plants treated with Cu

NP (0.5µM) and Zn NP (2 µM) at 45 DAS, respectively but at 60 DAS, highest

chlorophyll content ‗a‘ was recorded in plants treated with Cu NP (0.25 µM).

Lowest chlorophyll contents ‗a‘ was obtained in plants treated with Fe salt (27µM)

at both stages. Among Fe treated plants at 45 DAS, chlorophyll content ‗a‘ was

found to be higher in plants treated with Fe NP (27 µM) followed by NP (54µM),

normal salt and Fe salt (27 µM) at 45 DAS. At 60 DAS, normal salt treated plants

performance was better than Fe NP (54µM), Fe NP (27µM) and Fe salt (27 µM).

Among Cu Treated plants higher Chlorophyll content ‗a‘ was found higher in

plants treated with Cu NP (0.5 µM) as compare to Cu NP (0.25µM), Cu salt

(0.25µM) and Normal salt at 45 DAS, but in second stage, Cu NP (0.25µM)

treated plants, performed best as compared to Normal salt, Cu NP (0.5µM) and Cu

salt (0.25µM). In the case of Zn treatments, higher chlorophyll content ‗a‘ was

observed in plants treated with Zn NP (2µM) followed by Zn NP (1µM), Zn salt

47

(1µM) and Normal salt at 45 DAS. But in second growth stage, Normal salt, Zn

NP (2µM), Zn NP (1µM) treated plants was performing better than Zn salt (1µM)

respectively.

Significantly higher chlorophyll content ‗b‘ was observed in leaves at both

the growth stages of plants treated with Normal salt at 45 DAS and Fe NP (27 µM)

at 60 DAS (Table 4.9and Fig. 4.6 and 4.7). Among Fe treated plants significant

differences were observed in chlorophyll content ‗b‘ of plants treated with Normal

salt followed by Fe NP (27 µM), Fe NP (54 µM) and Fe salt (27 µM) at 45 DAS.

In second growth stage, Fe NP (27 µM) treated plants exhibited significantly

higher chlorophyll content ‗b‘ in comparison to Fe NP (54 µM), normal salt and Fe

salt (27 µM) treated plants. Among Cu treated plants higher chlorophyll content

‗b‘ was recorded in plants treated with Normal salt followed by Cu NP (0.25 µM),

Cu NP (0.5µM) Cu salt (0.25 µM) plants at first stage. At 60 DAS, maximum

chlorophyll content ‗b‘ was observed in plants treated with Normal salt as

compared to Cu NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM). Among Zn

treated plants, chlorophyll ‗b‘ content was found be significantly higher in plants

treated with Normal salt better than Zn NP (1 µM), Zn NP (2 µM) and Zn salt (1

µM) treated plants at both stages. Among all the treatments, lowest chlorophyll

content ‗b‘ was found in Fe salt (27 µM) at 45 DAS and Zn salt (1 µM) at 60 DAS.

Significant differences in total chlorophyll content were observed at 45

DAS and at 60 DAS (Table 4.9, Fig. 4.6 and 4.7). Among all the treatments,

highest total chlorophyll content was found significantly higher in plants treated

with Fe NP (27 µM) and lowest in plants treated with Zn salt (1 µM) at 45 DAS.

Among Fe treated plants significant differences were observed in plants treated

with Fe NP (27 µM) as compared to Fe NP (54 µM), Normal salt and Fe salt (27

µM) at 45 DAS, but at 60 DAS, Fe NP (27 µM) treated plants showed higher

chlorophyll content in comparison to Normal salt, Fe NP (54 µM) and Fe salt (27

µM). At 45 DAS, in Cu treatments, total chlorophyll content was found

significantly higher in plants treated with Normal salt followed by Cu NP (0.5

µM), Cu NP (0.25 µM) and Cu salt (0.25µM). However, at 60 DAS significant

differences were observed in plants treated with Normal salt as compared to Cu NP

(0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Among Zn treated plants higher

48

total chlorophyll Content was recorded in plants treated with Normal salt when

compared to Zn NP (1µM), Zn NP (2 µM) and Zn salt (1 µM) treated plants at

both growth stages.

Among Fe treated soybean plants, chlorophyll a, total chlorophyll content

was found to be high in plants treated with Fe NP (27 µM). This was in conformity

to the results obtained in maize crop for the trait of membrane stability

(Elanchezhian et al 2015; Kumar 2015). This indicated that the reduced

concentration of Fe NP may be inducing biochemical changes for chlorophyll

content. These results were in conformity with the findings of Delfani et al. (2014)

wherein nano-iron either alone or in combination with nano-magnesium had

significant effect on chlorophyll content. In the present study, Ghafari and

Razmjoo, (2013) also observed similar results of increased chlorophyll content. In

soybean, higher chlorophyll a content was observed in plants treated with Cu NP

(0.5 µM). This was in conformity with the earlier findings of Hafeez et al. (2015).

Zinc oxide (ZnO) nanoparticles resulted in chlorophyll content (Burman et al.

2013). Similar results were obtained with present set of experiment with soybean

wherein increased chlorophyll content was observed with Zn NP (2 µM). Similar

results were also obtained for enhanced chlorophyll content of peanut (Prasad et al.

2012), chlorophyll content of cluster bean (Raliya and Tarafdar 2013) and wheat

(Ramesh et al. 2014).

Table 4.9: Effect of micronutrient NPs on chlorophyll content of soybean

Treatment Chlorophyll Content (mg g-1

FW)

CHL a CHL b Total

DAS

45 60 45 60 45 60

T1: 100% (Fe + Cu + Zn) = Normal salts 0.28 0.30 0.49 0.33 0.63 0.76

T2: T1- Fe salt+ Fe NP (54 µM) 0.28 0.29 0.40 0.41 0.70 0.68

T3: T1- Fe salt + Fe NP (27 µM) 0.29 0.29 0.48 0.46 0.74 0.77

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.27 0.26 0.15 0.30 0.56 0.42

T5: T1- Cu salt + Cu NP (0.5 µM) 0.31 0.30 0.41 0.27 0.57 0.71

T6: T1- Cu salt + Cu NP (0.25 µM) 0.29 0.31 0.47 0.22 0.52 0.76

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.29 0.30 0.40 0.19 0.50 0.69

T8: T1- Zn salt + Zn NP (2 µM) 0.31 0.30 0.40 0.17 0.46 0.71

T9: T1- Zn salt + Zn NP (1 µM) 0.30 0.30 0.41 0.24 0.54 0.71

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.29 0.28 0.39 0.11 0.39 0.68

CD (5%) NS NS 0.11 0.07 0.09 0.11

49

Fig 4.6: Effect of micronutrient NPs on chlorophyll content of soybean at 45 DAS

Fig 4.7: Effect of micronutrient NPs on chlorophyll content of soybean at 60 DAS

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Chlorophyll a Chlorophyll b Total

Ch

loro

ph

yll

Co

nte

nt

(mg g

-1)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Chlorophyll a chlorophyll b Total

Ch

loro

phyll

Conte

nt

(mg g

-1)

50

4.2.2 Membrane stability

Among all the treatments, highest membrane stability (MS) was observed

in plants treated with Cu NP (0.5µM) and lowest in plants treated with Cu salt

(0.5µM) (Table 4.10). Among Fe treatments, significantly higher MS was observed

in plants treated with Fe NP (27 µM) as compared to Fe NP (54µM), Normal salt

and Fe salt (27µM). Among Cu treatments, maximum MS was found in plants

treated with Cu NP (0.5 µM) in comparison to Normal salt, Cu NP (0.25µM) and

Cu salt (0.25µM). Among Zn treatments, higher MS was obtained in plants treated

with Normal salt when compared to Zn NP (2µM), Zn NP (1µM) and Zn salt

(1µM).

4.2.3 Relative water content

Relative Water Content (RWC) was found to be significantly different

among all the treatments (Table 4.10). Highest RWC was found in plants treated

with Fe NP (27 µM) and lowest RWC was found in plants treated with Cu salt

(0.25 µM). Among Fe treatments plants, maximum RWC was obtained with lower

concentration of Fe NP (27 µM) as compared to higher concentration of Normal

salt, Fe NP (54µM) and Fe salt (27 µM). Among Cu treatments, higher RWC was

recorded in plants treated with Normal salt followed by Cu NP (0.25 µM), Cu NP

(0.5 µM) and Cu salt (0.25 µM). In the case of Zn treatments, Normal salt treated

plants was performing better than Zn NP (1 µM), Zn NP (2µM) and Zn salt (1

µM).

Among Fe treated soybean plants, membrane stability and RWC was found

to be high in plants treated with Fe NP (27 µM). This was in conformity to the

results obtained in maize crop for the trait of membrane stability (Elanchezhian et

al 2015; Kumar 2015). In soybean, MS was observed in plants treated with Cu NP

(0.5 µM). This was in conformity with the earlier findings of Hafeez et al. (2015).

51

Table 4.10: Effect of micronutrient NPs on MS and RWC of soybean at 60 DAS

4.2.4 Antioxidant enzyme

4.2.4.1 Super oxide dismutase enzyme activity

Super oxide dismutase (SOD) was found to be significantly different in

treatments (Table 4.11 and Fig. 4.9). Highest SOD enzyme activity was recorded

in plants treated Zn salt (1 µM) at 60 DAS and lowest in plants treated with Cu NP

(0.25 µM) at 45 DAS. Among Fe treated plants, higher SOD activity was observed

in plants treated with Fe NP (54 µM) when compared to Fe NP (27 µM), Fe salt

(27 µM) and Normal salt at the first stage but in second growth stage, higher SOD

enzyme activity was observed in plants treated with Fe NP (54µM) followed by Fe

salt (27 µM), Fe NP (27 µM) and Normal salt. In the present study, SOD activity

was also found to be more in soybean plants treated with Fe NP (54 µM) indicating

the involvement of Fe NP in antioxidant stress response. Ghafari and Razmjoo,

(2013) also observed similar results of increased antioxidant enzyme activities of

wheat. This again reiterates the species specificity of different nanoparticles.

Among Cu treated plants maximum SOD activity was recorded in plants

treated with Cu NP (0.5µM) better than Cu salt (0.25 µM), Cu NP (0.25µM) and

Normal salt at both stages. In soybean, SOD was observed in plants treated with

Cu NP (0.5 µM). This was in conformity with the earlier findings of Hafeez et al.

(2015).

Treatment MS% RWC%


T2: T1- Fe salt+ Fe NP (54 µM) 47.38 52.80

T3: T1- Fe salt + Fe NP (27 µM) 53.24 59.30


T5: T1- Cu salt + Cu NP (0.5 µM) 54.84 47.95

T6: T1- Cu salt + Cu NP (0.25 µM) 45.51 50.00


T8: T1- Zn salt + Zn NP (2 µM) 42.67 50.45

T9: T1- Zn salt + Zn NP (1 µM) 36.84 51.55


CD (5%) NS 8.40

52

In the case of Zn treatments, maximum SOD enzyme activity was obtained

in plants treated with Zn NP (2µM) than Zn salt (1µM), Zn NP (1µM) and Normal

salt at 45 DAS. But in second stage it was changed and higher enzyme activity was

recorded in plants treated with Zn salt (1µM) followed by Zn NP (1µM), Zn NP

(2µM) and Normal salt. Zinc oxide (ZnO) nanoparticles resulted in lesser lipid

peroxidation and also associated with lower activity of prominent antioxidant

enzymes, superoxide dismutase (SOD) (Burman et al. 2013). Similar results were

obtained with present set of experiment with soybean wherein increased SOD was

observed with Zn NP (2 µM).

Table 4.11: Effect of micronutrient NPs on super oxide dismutase enzyme activity

of soybean

4.2.4.2 Catalase enzyme activity

Catalase (CAT) activity was found to be significantly different among

various treatments (Table 4.12). Out of all ten treatments, highest catalase activity

was recorded in plants treated with Cu NP (0.25µM) and Fe NP (54 µM) at 45

DAS and 60 DAS, respectively. Among Fe NP treated plant higher catalase

activity was observed in plants treated with Fe salt (27µM) when compared to Fe

NP (54 µM), Fe NP (27 µM) and Normal salt at 45 DAS but at 60 DAS, maximum

CAT activity was noted in plants treated with Fe NP (54 µM) as compared to

Normal salt, Fe NP (27µM) and Fe salt (27µM). Higher CAT was observed with

10 ppm of nano-iron chelate (Karimia et al. 2014). This again reiterates the species

specificity of different nanoparticles.

Treatment SOD (unit g-1

)

45 DAS 60 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 20.97 22.94

T3: T1- Fe salt + Fe NP (27 µM) 14.44 17.45


T5: T1- Cu salt + Cu NP (0.5 µM) 19.38 18.21

T6: T1- Cu salt + Cu NP (0.25 µM) 8.89 17.03


T8: T1- Zn salt + Zn NP (2 µM) 14.69 15.37

T9: T1- Zn salt + Zn NP (1 µM) 9.07 27.67


CD (5%) 9.74 11.81

53

Fig

. 4.8

: E

ffec

t of

mic

ron

utr

ient

NP

s on s

uper

ox

ide

dis

muta

se e

nzym

e ac

tivit

y o

f so

ybea

n

-505

10

15

20

25

30

35

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

45

DA

S6

0 D

AS

SOD(Unit g-1)

54

Maximum catalase activity was recorded in plants treated with Cu NP (0.25

µM) followed by Cu NP (0.5 µM), Normal salt and Cu salt (0.25µM) at 45 DAS,

in the case of 60 DAS, higher CAT enzyme activity was observed in plants treated

with Cu NP (0.25 µM) better than Normal salt, Cu salt (0.25 µM) and Cu NP

(0.5µM). The enhanced antioxidant enzyme CAT activity with Cu NP treatment as

observed in the present set of experiment with soybean was corroborated with the

findings of Nekrasova et al. (2011).

Among Zn NP treated plants higher catalase activity was recorded in plants

treated with Zn salt (1µM) when compared to Zn NP (2 µM), Zn NP (1 µM) and

Normal salt at 45. After 15 days interval, CAT enzyme activity was recorded

higher in plants treated with Zn NP (2µM) followed by Normal salt, Zn salt (1 µM)

and Zn NP (1 µM).

4.2.4.3 Peroxidase enzyme activity

Significant differences among various treatments in terms of Peroxidase

(POX) activity were observed (Table 4.12). Among all the treatments, higher

Peroxidase enzyme activity was recorded in plants treated with Normal at both

stages and lowest POX enzyme activity was recorded in plants treated with Cu salt

(0.25µM) at 60 DAS. Among Fe treated plants, higher Peroxidase activity was

observed in plants treated with Normal salt when compared to Fe NP (54 µM) Fe,

Fe NP (27 µM) and salt (27 µM) at both stages. In the present study, POX activity

was also found to be more in soybean plants treated with Fe NP (54 µM) indicating

the involvement of Fe NP in antioxidant stress response. Ghafari and Razmjoo,

(2013) also observed similar results of increased antioxidant enzyme activities of

wheat. Higher POX was observed with 10 ppm of nano-iron chelate (Karimia et al.

2014). This again reiterates the species specificity of different nanoparticles.

Among Cu treated plants maximum Peroxidase activity was recorded in

plants treated with Normal salt, better than Cu NP (0.5 µM) followed by Cu NP

(0.25µM) and Cu salt (0.25 µM) at 45 DAS, but in the second growth stage,

Normal salt performed better than Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt

(0.25µM). The enhanced antioxidant enzyme peroxidase activity with Cu NP

55

treatment as observed in the present set of experiment with soybean was

corroborated with the findings of Nekrasova et al. (2011).

Among Zn treated plants higher POX was obtained in plants treated with

Normal salt followed by Zn NP (2µM), Zn NP (1 µM) and Zn salt (1µM) at both

growth stages. Zinc oxide (ZnO) nanoparticles resulted in lesser lipid peroxidation

and also associated with lower activity of prominent antioxidant enzymes,

peroxidase (Burman et al. 2013). Similar results were obtained with present set of

experiment with soybean wherein increased SOD was observed with Zn NP (2

µM).

Table 4.12: Effect of micronutrient NPs on catalase enzyme activity and

peroxidase enzyme activity of soybean

Treatment CAT (unit H2O2

min-1

g-1

)

POX (unit H2O2

min-1

g-1

)

45

DAS

60

DAS

45

DAS

60

DAS

T1: 100% (Fe + Cu + Zn) = Normal salts 15.30 21.20 140.24 122.79 T2: T1- Fe salt+ Fe NP (54 µM) 17.60 24.80 112.61 103.56 T3: T1- Fe salt + Fe NP (27 µM) 17.20 19.20 100.68 87.95 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 20.40 17.60 72.27 69.01 T5: T1- Cu salt + Cu NP (0.5 µM) 15.30 21.60 122.81 108.15 T6: T1- Cu salt + Cu NP (0.25 µM) 25.65 17.60 85.89 122.66 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 15.10 20.80 75.79 61.87 T8: T1- Zn salt + Zn NP (2 µM) 17.70 24.80 79.21 109.09 T9: T1- Zn salt + Zn NP (1 µM) 17.20 17.60 74.90 98.49 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 25.40 18.40 74.05 83.44 CD (5%) 6.32 3.76 26.31 37.25

56

Fig 4.9: Effect of micronutrient NPs on catalase enzyme activity of soybean.

Fig. 4.10: Effect of micronutrient NPs on peroxidase enzyme activity of soybean

0

5

10

15

20

25

30

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 ADSC

AT

(u

nit

H2O

2m

in-1

g-1

)

0

20

40

60

80

100

120

140

160

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 ADS

PO

X (

un

it H

2O

2m

in-1

g-1

)

57

4.2.5 Proline content

Proline content was found to be significantly different in plants at 45 DAS

and 60 DAS, respectively (Table 4.13 and Fig. 4.12). Among Fe treated plants,

proline content was significantly higher in plants treated with Fe NP (54µM), when

compared to Fe NP (27 µM), Normal salt and Fe salt (27 µM) at first growth stage

but at 60 DAS, Fe NP (27 µM) was performed best as compared to Fe NP (54µM),

Normal salt and Fe salt (27 µM).The proline content was found to be high in plants

treated with Fe NP (54 µM). These results were in conformity with the findings of

Delfani et al. (2014) wherein nano-iron either alone or in combination with nano-

magnesium had significant effect on proline content.

Among Cu treated plants higher proline content was recorded in plants

treated with Cu NP (0.5 µM) followed by Cu NP (0.25 µM), Cu salt (0.25 µM) and

normal salt at 45 DAS. In the case of 60 DAS, higher proline content was recorded

in plants treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Normal salt

and Cu salt (0.25µM). In soybean, higher proline was observed in plants treated

with Cu NP (0.5 µM). This was in conformity with the earlier findings of Hafeez et

al. (2015). The enhanced proline content with Cu NP treatment as observed in the

present set of experiment with soybean was corroborated with the findings of

Nekrasova et al. (2011).

Higher proline content was observed in plants treated with Zn NP (1 µM)

as compared to Zn NP (2µM), Normal salt and Zn salt (1µM) at 45 DAS. At 60

DAS, maximum proline content was recorded in plants treated with Zn NP (2µM)

which is better than Normal salt, Zn NP (1µM) and Zn salt (1µM). Among all the

treatments, highest proline content was observed in plants treated with Fe NP (27

µM) at 60 DAS and lowest proline content was observed in plants treated with Zn

salt (27 µM) at 45 DAS. Some results were obtained for protein content of cluster

bean (Raliya and Tarafdar 2013) and wheat (Ramesh et al. 2014). However,

proline was found to be higher with Zn NP (1 µM) treated plants. Similar increase

in proline content with ZnO NPs was also reported by Sunita et al. (2013) in

Brassica juncea.

58

4.2.6 Total soluble protein

Total soluble protein (TSP) content was found similar during the growth

period (Table 4.13). Protein content was observed to be not significant among all

NP treated plants at 45 DAS and 60 DAS. Among all the treatments, higher

magnitude of TSP content was observed in plants treated with Zn NP (2 µM) at 60

DAS, and lower TSP content was recorded in plants treated with Zn salt (1µM) at

45 DAS. Among Fe treatments, higher TSP content was noted in plants treated

with Fe NP (54 µM) as compared to Normal salt, Fe NP (27µM) and Fe salt (27

µM) at 45 DAS. However, at 60 DAS, Fe NP (54µM) treated plants performed

better than Fe NP (27µM), Normal salt and Fe salt (27µM). Total soluble protein

content was found to be high in plants treated with Fe NP (54 µM). These results

were in conformity with the findings of Delfani et al. (2014) wherein nano-iron

either alone or in combination with nano-magnesium had significant effect on seed

protein content.

Among Cu treated plants higher protein content was observed in plants

treated with Cu NP (0.25 µM) followed by Normal salt, Cu NP (0.5µM) and Cu

salt (0.25µM) at 45 DAS, but in second stage maximum protein content was

recorded in plants treated with Cu NP (0.5 µM) as compared to Cu NP (0.25µM),

Normal salt and Cu salt (0.25µM). In soybean, higher TSP was observed with Cu

NP (0.25 µM). This was in conformity with the earlier findings of Hafeez et al.

(2015). The enhanced protein content with Cu NP treatment as observed in the

present set of experiment with soybean was corroborated with the findings of

Nekrasova et al. (2011).

Among Zn treated plants, higher protein content was found in plants treated

with lower concentration of Zn NP (1 µM), better than Normal salt, Zn NP (2 µM)

and Zn salt (1 µM) at 45 DAS. But in the case of 60 DAS, higher protein content

was found in plants treated with higher concentration of Zn NP (2 µM) in

comparison to lower concentration of Zn NP (1µM) followed by Zn salt (1µM) and

Normal salt. Some results were obtained for protein content of cluster bean (Raliya

and Tarafdar 2013) and wheat (Ramesh et al. 2014). However, TSP was found to

be higher with Zn NP (1 µM) treated plants. Similar increase in protein content

with ZnO NPs was also reported by Sunita et al. (2013) in Brassica juncea.

59

Table 4.13: Effect of micronutrient NPs on proline and protein of soybean

Treatment Proline (μM g-1

) TSP (mg g-1

)


T1: 100% (Fe + Cu + Zn) = Normal salts 0.02 0.05 1.13 1.30 T2: T1- Fe salt+ Fe NP (54 µM) 0.10 0.09 1.15 1.45 T3: T1- Fe salt + Fe NP (27 µM) 0.07 0.28 1.12 1.44 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.02 0.03 1.10 1.28 T5: T1- Cu salt + Cu NP (0.5 µM) 0.10 0.20 1.12 1.41 T6: T1- Cu salt + Cu NP (0.25 µM) 0.06 0.25 1.14 1.38 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.03 0.03 1.12 1.25 T8: T1- Zn salt + Zn NP (2 µM) 0.03 0.24 1.11 1.50 T9: T1- Zn salt + Zn NP (1 µM) 0.04 0.02 1.14 1.40 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.02 0.02 1.08 1.34 CD (5%) 0.02 0.14 NS NS

4.2.7 Total soluble sugar

Total soluble sugar was found to be different at all the growth stages with

the various treatments (Table 4.14 and Fig.4.13). Out of all ten treatments, highest

TSS content was observed in plants treated with Cu NP (0.25 µM) at 45 DAS. At

60 DAS, maximum TSS was observed in plants treated with Normal salts. Among

Fe treated plants, higher TSS content was found in plants treated with Fe NP (27

µM) as compared to plants treated with Fe NP (54 µM), Normal salt and Fe salt

(27µM) at 45 DAS but at 60 DAS, Normal salt was obtained higher TSS better

than Fe NP (27µM), Fe NP (54 µM) and Fe salt (27µM). Among Fe treated

soybean plants, TSS was found to be high in plants treated with Fe NP (27 µM).

This was in conformity to the results obtained in maize crop for the trait of

membrane stability (Elanchezhian et al 2015; Kumar 2015). This indicated that the

reduced concentration of Fe NP may be inducing biochemical changes for soluble

carbohydrate. Ghafari and Razmjoo, (2013) also observed similar results of

increased carbohydrate content of wheat.

Maximum TSS content was observed in plants treated with Cu NP (0.25

µM) at 45 DAS among Cu treated plants as compared to Cu (0.5 µM) followed by

Cu salt (0.25 µM) and Normal salt. However, at 60 DAS, maximum TSS was

recorded in plants treated with Normal salt when compared to Cu NP (0.5 µM), Cu

NP (0.25 µM) and Cu salt (0.25 µM). In soybean, higher TSS was observed with

Cu NP (0.25 µM). This was in conformity with the earlier findings of Hafeez et al.

60

(2015). However, there was not much report impact of NPs on carbohydrate status

of plants.

Among Zn treatments, higher TSS content was found in plants treated with

Zn NP (1µM) followed by Zn NP (2µM), Normal salt and Zn salt (1µM) at 45

DAS. But in case of 60 DAS, maximum TSS was observed in plants treated with

Normal salt as compared to Zn NP (1µM), Zn NP (2µM) and Zn salt (1µM).

However lowest TSS content was recorded in plants treated with Fe salt (27 µM) at

both the stages. The TSS content was found to be higher with Zn NP (1 µM)

treated plants was also reported by Sunita et al. (2013) in Brassica juncea. Singh et

al. (2013) reported that sugar content of Zn NP treated plants were similar to

control, in the present study also similar results were obtained at 60 DAS.

4.2.8 Non-structural carbohydrate

Non-structural carbohydrate (NSC) content was found to be significantly

higher at both the growth stages with various treatments (Table 4.14 and Fig 4.14).

Among all the treatments, maximum NSC content was recorded in plants treated

with Cu NP (0.25 µM) and Fe NP (27µM) at 45 and 60 DAS, respectively. Among

Fe treated plants higher NSC was observed in plants treated with Fe NP (27 µM)

when compared to all Fe treatments like Fe NP (54 µM), Normal salt and Fe salt

(27 µM) at 45 DAS and at 60 DAS, Fe NP (27 µM) treated plants exhibited higher

NSC as compared to Normal salt, Fe NP (54µM) and Fe salt (27 µM). Among Fe

treated soybean plants, NSC was found to be high in plants treated with Fe NP (27

µM). This was in conformity to the results obtained in maize crop for the trait of


reduced concentration of Fe NP may be inducing biochemical changes for soluble

carbohydrate. Ghafari and Razmjoo, (2013) also observed similar results of

increased carbohydrate content of wheat.

Maximum NSC was found in Cu NP (0.25µM) treated plants at 45 DAS

among Cu treatments, followed by Cu NP (0.5µM), Cu salt (0.25µM) and Normal

salt. But in second growth stage, Cu NP (0.25 µM) showed higher NSC as

compared to Cu NP (0.5µM), Normal salt and Cu salt (0.25µM). In soybean,

higher NSC was observed with Cu NP (0.25 µM). This was in conformity with the

61

earlier findings of Hafeez et al. (2015). However, there was not much report

impact of NPs on carbohydrate status of plants.

Among Zn treated plants, higher NSC content was found in plants treated

with Zn NP (1 µM) at first growth stage followed by Zn NP (2µM), Normal salt

and Zn salt (1µM) at 45 DAS. But in the second growth stage higher NSC was

recorded in plants treated with Zn NP (2µM) as compare to Zn NP (1µM), Normal

salt and Zn salt. However lowest NSC content was recorded in plants treated with

Zn salt (1 µM) at 45 DAS and Fe salt (27 µM) at 60 DAS. The NSC content was

found to be higher with Zn NP (1 µM) treated plants was also reported by Sunita et

al. (2013) in Brassica juncea. Singh et al. (2013) reported that sugar content of Zn

NP treated plants were similar to control, in the present study also similar results

were obtained at 60 DAS.

Table 4.14: Effect of micronutrient NPs on TSS and NSC of soybean

Treatment TSS (%) NSC (%)

45

DAS

60

DAS

45

DAS

60

DAS

T1: 100% (Fe + Cu + Zn) =Normal salts 2.65 3.92 3.19 3.43 T2: T1- Fe salt+ Fe NP (54 µM) 3.01 3.50 3.95 3.25 T3: T1- Fe salt + Fe NP (27 µM) 3.21 3.75 3.98 4.31 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 1.94 2.48 2.49 2.59 T5: T1- Cu salt + Cu NP (0.5 µM) 3.48 3.72 4.26 3.83 T6: T1- Cu salt + Cu NP (0.25 µM) 3.64 3.68 4.40 4.05 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 3.04 3.16 3.59 3.37 T8: T1- Zn salt + Zn NP (2 µM) 3.26 3.76 3.27 4.00 T9: T1- Zn salt + Zn NP (1 µM) 3.43 3.77 3.43 3.70 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 2.26 2.62 2.27 3.12 CD (5%) 0.84 0.34 0.83 0.42

62

Fig 4.11: Effect of micronutrient NPs on TSS of soybean

Fig 4.12: Effect of micronutrient NPs on non-structural carbohydrate of soybean

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 DAS

Tota

lS

olu

ble

Sugar

(%

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 DAS

Non

stru

ctura

l ca

rbohydra

te (

%)

63

4.3 Physiological parameters of Soybean

4.3.1 Photosynthesis rate

The Photosynthesis rate presented in Table 4.15 and Fig. 4.15 indicated

significant differences between the treatments at the all growth stages. Out of all

treatments, highest and lowest Photosynthesis rate was obtained in plants treated

with Normal salt at 50 DAS and 70 DAS. Among Fe treated plants, higher

Photosynthesis rate was noted in plant treated with Fe NP (54 µM) followed by Fe

NP (27 µM), normal salt and Fe salt (27µM) at 20 DAS. At 35 DAS and 50 DAS,

higher photosynthesis rate was noted in plants treated with Normal salt followed

by Fe NP (54µM), Fe NP (27µM) and Fe salt (27µM). But at last stage higher

photosynthesis rate was recorded in plants treated with Fe NP (54µM) as compared

to Fe NP (27µM) and normal salt. Among Cu treatments, photosynthesis rate was

found to be significantly higher in plant treated with normal followed by Cu NP

(0.25 µM), Cu NP (0.5 µM) and Cu salt (0.25 µM) at first stages. At 35 DAS,

among Cu treatments, higher photosynthesis rate was recorded in plant treated with

Cu NP (0.25µM) followed by normal salt, Cu NP (0.25µM) and Cu salt (0.25µM).

At 50 DAS, plants showed higher photosynthesis rate with normal salt in

comparison to Cu NP (0.5 µM), Cu NP (0.25 µM) and cu salt (0.25 µM). In the

case of Zn Treatments, higher photosynthesis rate was observed in plants treated

with Zn NP (1µM) in comparison to normal salt, Zn NP (2 µM) Zn salt (1µM) at

20 DAS. Among Zn treated plants, higher photosynthesis rate was noted in plants

treated with Normal salt as compared to Zn NP (2µM), Zn NP (1µM), and Zn salt

(1µM) at 35 DAS and 50 DAS. At last growth stage (70 DAS) there was no

significant impact of Zn treatments.

Table 4.15: Effect of micronutrient NPs on photosynthesis rate of soybean

Treatment Photosynthesis rate (µM m-2

s-1

)

20 DAS 35DAS 50 DAS 70DAS


T2: T1- Fe salt+ Fe NP (54 µM) 9.45 7.30 11.70 5.25

T3: T1- Fe salt + Fe NP (27 µM) 7.45 4.95 10.90 2.65

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 3.40 3.40 7.60 -0.95

T5: T1- Cu salt + Cu NP (0.5 µM) 4.60 7.60 6.95 1.30

T6: T1- Cu salt + Cu NP (0.25 µM) 4.70 11.00 6.50 0.60

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 4.40 3.80 2.80 -0.05

64

T8: T1- Zn salt + Zn NP (2 µM) 3.80 7.10 4.60 1.40

T9: T1- Zn salt + Zn NP (1 µM) 5.35 6.05 2.85 0.00

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 3.50 4.70 2.80 -1.20

CD (5%) 3.114 4.023 6.785 1.539

4.3.2 Transpiration rate

The Transpiration rate furnished in Table 4.16 and Fig 4.16 indicated

significant differences between the treatments at the all growth stages. Among all

the treatments, highest transpiration rate was noted in plants treated with Normal

salt at 50 DAS and lowest Transpiration rate was obtained in plants treated with Fe

salt (27 µM) at last growth stage. Among Fe treatment plants, higher Transpiration

rate was recrded in plant treated with Fe NP (27 µM) followed by Fe NP (54 µM),

normal salt and Fe salt (27µM) at20 DAS. At 35 DAS and 50 DAS, plant showed

higher transpiration rate in plants treated with normal salt as compared to Fe NP

(54 µM), Fe NP (27µM) and Fe salt (27µM). But at last growth stage, maximum

transpiration was recorded in plants treated with Fe NP (54 µM) followed by

normal salt, Fe NP (27 µM) and Fe salt (27µM). Among Cu treatments,

transpiration rate was found to be significantly higher in plant treated with higher

concentration of Cu NP (0.5µM) as compared to Cu NP (0.25 µM), normal salt and

Cu salt (0.25µM) at 20 DAS. At 35 DAS higher transpiration rate was recorded in

plant treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Cu salt (0.25µM)

and normal salt. In the case of 50 DAS, maximum transpiration rate was noted in

plants treated with normal salt in comparison to Cu NP (0.5µM), Cu NP (0.25µM)

and Cu salt (0.25µM), but in last stage Cu NP (0.5µM) performed better followed

by normal salt, Cu NP (0.25µM) and Cu salt (0.25µM). Among Zn Treatments,

higher Transpiration rate was observed in plant treated with Zn NP (2µM) when

compared with other Zn treated plants at 20 DAS. At last growth stage, higher

transpiration was observed in Zn NP (2µM) treated plants followed by Zn NP

(1µM), normal salt and Zn salt (1µM).

65

Table 4.16: Effect of micronutrient NPs on transpiration rate of soybean

Treatment Transpiration rate (mM m-2

s-1

)

20 DAS 35DAS 50 DAS 70 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 3.40 3.30 4.45 2.90

T3: T1- Fe salt + Fe NP (27 µM) 3.55 3.20 4.30 2.35


T5: T1- Cu salt + Cu NP (0.5 µM) 3.50 3.55 3.15 2.70

T6: T1- Cu salt + Cu NP (0.25 µM) 3.50 4.25 3.70 2.25


T8: T1- Zn salt + Zn NP (2 µM) 3.55 4.10 2.80 2.60

T9: T1- Zn salt + Zn NP (1 µM) 3.50 4.15 3.10 2.55


CD (5%) NS 0.42 0.84 0.39

4.3.3 Stomatal conductance

Stomatal conductance was found significantly different between treatments

at all growth stages (Table 4.17 and Fig. 4.17). Among Fe treatment plants, higher

Stomatal conductance was recorded in plant treated with Fe NP (27 µM) followed

by normal salt, Fe NP (27 µM) and Fe salt (27µM) at 20 DAS and 35 DAS. At 50

DAS, higher stomatal conductance was observed in plants treated with Fe (57µM)

in comparison to normal salt, Fe NP (27µM) and Fe salt (27µM) but in the last

stage, maximum stomatal conductance was noted in Fe NP (27µM) followed by

normal salt, Fe NP (54µM) and Fe salt (2µM). Among Cu treatments, Stomatal

conductance was found significantly higher in plant treated with Cu NP (0.25µM)

as compared to Cu NP (0.5 µM), normal salt and Cu salt (0.25µM) at first stages.

At 35 DAS Cu treatments plants, higher Stomatal conductance was recorded in

plant treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Cu salt (0.25µM)

and normal salt. In next stage (50 DAS), higher stomatal conductance was noted in

plants treated with normal salt in comparison to Cu NP (0.5µM), Cu NP (0.25µM)

and Cu salt (0.25µM. But at the last stage good impact of Cu treatments was

shown in Cu NP (0.25µM) in comparison to normal salt, Cu NP (0.5µM) and Cu

salt (0.25µM). Among Zn treatments, higher Stomatal conductance was observed

in plant treated with Zn NP (1µM) when compared with the all Zn treated plants

like Zn NP (2 µM) Zn salt (1µM) and normal salt at first two growth stages viz. 20

DAS and 35 DAS. But at 50 DAS Zn treated plants, higher Stomatal conductance

was noted in plants treated with normal salt followed by Zn (2µM), Zn NP (1µM)

66

and Zn salt (1µM). In the case of last growth stage, maximum stomatal

conductance was noted in plants treated with Zn (2µM) as compare to normal salt,

Zn NP (1µM) and Zn salt (1µM). Among all the ten treatments, highest Stomatal

conductance was obtained in plants treated with Cu (0.25µM) at 35 DAS and

lowest is Fe salt (27 µM) at last growth stages.

Gas exchange parameters viz photosynthetic rate, stomatial conductance

and transpiration rate were positively influenced by the NPs. Photosynthetic rate

was enhanced by Fe NP (54µM) and Cu NP (0.25µM) treatments in soybean.

Similar results were obtained by Alidoust and Isoda (2013) with foliar spray of Fe

NPs in soybean. In addition to Fe and Cu being important element in

photosynthetic reaction pathways, it is envisaged that they may be stimulating

photosynthetic electron transport, which might enhance the photosynthetic rate.

Moreover, transpiration rate and stomatal conductance was found to higher with

reduced concentration of NP in both the crops which indicated that the NPs may

positively regulate stomatal opening and closure.

Table 4.17: Effect of micronutrient NPs on stomatal conductance of soybean

Treatment stomatal conductance (µM m-2

s-1

)

20

DAS

35

DAS

50

DAS

70

DAS


T2: T1- Fe salt+ Fe NP (54 µM) 143.5 114.5 154.5 65.5

T3: T1- Fe salt + Fe NP (27 µM) 154.0 144.5 112.0 77.0


T5: T1- Cu salt + Cu NP (0.5 µM) 150.5 143.0 94.0 61.5

T6: T1- Cu salt + Cu NP (0.25 µM) 151.5 176.5 78.5 72.0


T8: T1- Zn salt + Zn NP (2 µM) 152.0 159.0 70.0 69.5

T9: T1- Zn salt + Zn NP (1 µM) 159.5 162.0 68.5 64.5


CD (5%) 16.5 20.1 47.8 16.4

67

Fig

4.1

3:

Eff

ect

of

mic

ronutr

ient

NP

s on p

hoto

syn

thes

is r

ate

of

soybea

n.

-4-202468

10

12

14

16

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

20

DA

S3

5 D

AS

50

DA

S7

0 D

AS

Photosinthesisrate (µM m-2s-1)

68

Fig

4.1

4:

Eff

ect

of

mic

ronutr

ient

NP

s on t

ransp

irat

ion r

ate

of

soybea

n

0123456

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

20

DA

S3

5 D

AS

50

DA

S7

0 D

AS

Transpiration rate (mMm-2s-1)

69

Fig

. 4.1

5:

Eff

ect

of

mic

ronutr

ient

NP

s on s

tom

atal

conduct

ance

of

soybea

n

0

20

40

60

80

10

0

12

0

14

0

16

0

18

0

20

0

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

20

DA

S3

5 D

AS

50

DA

S7

0 D

AS

Stomatal conductance (µM m-2s-1)

70

4.3.4 SPAD Value

SPAD value was found significantly difference between treatments

at all growth stages show in table no. 4.18 and fig. 4.18. Among all treatments,

highest SPAD value was obtain in plants treated with Cu NP (0.25µM) at 35 DAS

and lowest SPAD value was obtained in plants treated with Cu salt (0.25 µM) at 70

DAS. Among Fe treatments, higher SPAD value was obtained in plant treated with

Normal salt as compared to Fe NP (27µM), Fe NP (54µM), and Fe salt (27µM) at

20 DAS. But at 35 DAS and 50 DAS, higher SPAD value was recorded in plants

treated with Fe NP (27µM) followed by Fe NP (27µM), Normal salt and Fe salt

(27µM). At last stage maximum SPAD value was noted in plants treated with Fe

NP (54µM) when compared to Fe NP (27µM), Normal salt and Fe salt (27µM).

This was in conformity to the results obtained in maize crop for the trait of

membrane stability (Elanchezhian et al 2015; Kumar 2015). Among Fe treated

soybean plants, chlorophyll content was found to be high in plants treated with Fe

NP (27 µM). This indicated that the reduced concentration of Fe NP may be

inducing biochemical changes for chlorophyll content.

Among Cu treatment plants, higher SPAD Value was obtained in plant

treated with Cu NP (0.25 µM) in comparison of Normal salt, Cu NP (0.25µM) and

Cu salt (0.25µM) at 20 DAS. Plants were show maximum SPAD value in plants

treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Normal salt and Cu salt

(0.25µM) at both stages 35 DAS and 50 DAS. At last stage, Cu NP (0.25µM) was

show better performance in comparison Normal salt, Cu NP (0.5µM) and Cu salt

(0.25µM). In soybean, higher chlorophyll a content was observed in plants treated

with Cu NP (0.25 µM). This was in conformity with the earlier findings of Hafeez

et al. (2015).

Among Zn Treatments, higher SPAD value was noted in plant treated with

Zn NP (2µM) when compared with the plants treated with Normal salt, Zn NP

(1µM) and Zn salt (1µM) at both stages 20 DAS and 35 DAS. But at 50 DAS Zn

treated plants; higher SPAD value was noted in plants treated with Zn (2µM) as

compared to Zn NP (1µM), Normal salt and Zn salt (1µM). At last stage,

maximum SPAD value was noted in plants treated with Zn NP (1µM) followed by

Zn NP (2µM), Normal salt and Zn salt (1µM).Among Zn treatments, obtained

71

increased chlorophyll content was observed with Zn NP (2 µM). Zinc oxide (ZnO)

nanoparticles resulted in chlorophyll content (Burman et al. 2013).

Table 4.18: Effect of micronutrient NPs on SPAD value of soybean

Treatment SPAD Value

20 DAS 35DAS 50 DAS 70 DAS

T1: 100% (Fe + Cu + Zn) = Normal salts 31.91 42.55 29.60 22.15 T2: T1- Fe salt+ Fe NP (54 µM) 29.15 42.85 34.95 31.35 T3: T1- Fe salt + Fe NP (27 µM) 29.85 45.95 39.25 28.55 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 27.10 42.10 27.70 15.70 T5: T1- Cu salt + Cu NP (0.5 µM) 27.40 42.60 30.15 17.75 T6: T1- Cu salt + Cu NP (0.25 µM) 32.00 46.80 31.70 23.90 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 27.25 41.65 24.71 15.25 T8: T1- Zn salt + Zn NP (2 µM) 35.00 43.25 38.90 37.15 T9: T1- Zn salt + Zn NP (1 µM) 31.00 41.95 31.65 42.05 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 28.90 40.90 18.65 21.50 CD (5%) 4.12 2.69 4.42 10.29

72

Fig

. 4.1

6:

Eff

ect

of

mic

ronutr

ient

NP

s on S

PA

D v

alue

of

So

ybea

n

05

10

15

20

25

30

35

40

45

50

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

20

DA

S3

5D

AS

50

DA

S7

0 D

AS

SPAD Value

73

4.4 Morphological parameters of wheat

4.4.1 Plant height

Among all the treatments, plant height was found to be significantly higher

in plants treated with Cu NP (0.5 µM) and lower in plants treated with Fe salt (27

µM) at 60 DAS (Table 4.19). Among Fe treatments, plant height was found to be

similar in Fe NP (54 µM) and Normal salt when compared to Fe salt (27 µM) at 60

DAS. Among Cu treated plants, higher plant height was found in Cu NP (0.5 µM)

followed by normal salt, Cu NP (0.25µM) and Cu salt (0.25µM) treated plant at 60

DAS. Plants treated with normal salt exhibited higher plant height when compared

to plants treated with Zn NP (1 µM), Zn NP (2 µM) Zn salt (1 µM) at 60 DAS.

The positive effects of nano materials on morphology of crop plants have

been reported by several workers (Agrawal and Rathore 2014; Elanchezhian et al.

2014; Elanchezhian et al. 2015). There were selected reports on effect of Fe

nanoparticles on plant morphology (Elanchezhian et al. 2015, Kumar 2015). Fe

nanoparticles were found to increase stem length of basil plants (Peyvandi et al.

2011, Kumar 2015). Salarpour et al. (2013) observed 80% increase in plant height

of Lepidum sativum L treated with nano-iron chelates. In the present study,

increase in height of wheat plants treated with Cu NP (0.5µM) at panicle initiation

stage.

4.4.2 Total Root Length

Total root length was found to be significant at all stages (Table 4.19 and

Fig. 4.20). Among Fe treatments, higher root length was obtained with Fe NP

(54µM) as compared to Fe NP (27µM), normal salt and Fe salt (27µM) at first

growth stage but at second stage, normal salt treated performed better with Fe NP

(27µM), Fe NP (54µM) and Fe salt (27µM). Fe nanoparticles were found to

increase root length of basil plants (Peyvandi et al. 2011, Kumar 2015). In the

present study there increase in root length was observed with normal Fe NP

treatment, which may be attributed to the increased branching of roots of wheat

crop.

Among Cu treated plants, plant treated with normal salt exhibited higher

length followed by Cu salt (0.25µM), Cu NP (0.5µM) and Cu NP (0.25µM) at 30

DAS. However, with Cu treatments, normal salt treated plant obtained higher root

length than Cu NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM) at 60 DAS.

74

Among Zn treated plants maximum root length was observed in plants

treated with Zn NP (1µM) as compared to Zn NP (2µM), normal salt and Zn salt

(1µM) at 30 DAS. But in 60 DAS, higher root length was noted in plants treated

with Zn salt (1µM) in comparison to normal salt, Zn NP (1µM) Zn NP (2µM).

Among all the treatments, highest total root length was observed in plants treated

with Zn salt (1µM) at 60 DAS and lowest root length was noted in plants treated

with Cu NP (0.25µM) at 30 DAS. In wheat, higher root length were observed with

Zn NP (1 µM) treatments. In the present set of investigation, the response in wheat

was similar to soybean and maize crop (Kumar 2015).

Table 4.19: Effect of micronutrient NPs on plant height and root length of wheat

4.4.3 Shoot dry weight

Shoot dry weight of wheat differed between all treatments significantly at 30 DAS

and 60 DAS (Table 4.20 and Fig. 4.21). Among all the treatments, significantly

highest shoot dry weight was recorded in plants treated with Zn NP (2 µM).

Among Fe treatments, significantly higher shoot dry weight was obtained in plants

treated with Fe NP (54µM) followed by Fe NP (27 µM), normal salt and Fe salt

(27 µM) at 30 DAS. But at second stage, higher shoot dry weight was obtained in

plants treated with Fe NP (27µM) as compared to normal salt, Fe NP (54µM) and

Fe salt (27µM). Fe nanoparticles were found to increase shoot dry weight of basil

plants (Peyvandi et al. 2011, Kumar 2015). However, Karimia et al. (2014)

observed that increasing Fe nanoparticle s concentration above 10 ppm reduced

shoot fresh weight, shoot dry weight of green gram, indicating negative effect of

Treatment Plant

height(cm)

Total root length (cm)

60 DAS 30 DAS 60 DAS

T1: 100% (Fe + Cu + Zn) = Normal salts 65.5 53414.15 108586.70

T2: T1- Fe salt+ Fe NP (54 µM) 65.5 90206.71 51991.34

T3: T1- Fe salt + Fe NP (27 µM) 56.0 54023.31 71747.04

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 34.0 58782.90 57234.35

T5: T1- Cu salt + Cu NP (0.5 µM) 66.0 17085.53 42064.23

T6: T1- Cu salt + Cu NP (0.25 µM) 51.5 9627.24 22780.70

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 46.0 47227.17 22346.60

T8: T1- Zn salt + Zn NP (2 µM) 63.0 70612.72 24978.31

T9: T1- Zn salt + Zn NP (1 µM) 65.0 75127.64 39642.50

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 41.5 22817.21 130143.98

CD (5%) 16.8 32245.9 35380.4

75

Fig 4.17: Effect of micronutrient NPs on plant height of wheat at 60 DAS.

Fig 4.18: Effect of micronutrient NPs on total root length of wheat.

0

10

20

30

40

50

60

70

80

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

60 DASP

lant

heg

ht

(cm

)

0

20000

40000

60000

80000

100000

120000

140000

160000

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DAS

Tota

lro

ot

rength

(cm

)

76

Fe NP on crop growth. In the present study there higher shoot dry weight was

obtained in plants treated with Fe NP (54µM).

Among the plants treated with Cu, higher shoot dry weight was recorded in

plants treated with Cu NP (0.25µM) in comparison to normal salt, Cu NP (0.5 µM)

and Cu salt (0.25µM) at first growth stage. In the second stage higher shoot dry

weight was recorded in plants treated with normal salt followed by Cu NP

(0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Improvement in growth of plants

treated with Cu NPs were reported in wheat (Hafeez et al. 2015), lettuce (Shah and

Belozerova, 2009) and improvement in vase life of chrysanthemum was also

reported with Cu NPs (Hashemabadi et al. 2013). In the present study it was

observed that normal concentration of Cu NP (0.25 µM) had positive influence on

shoot dry weight of wheat at 30 DAS, while normal concentration of Cu NP

(0.5µM) had positively influenced shoot weight at 60 DAS.

Among Zn treated plants, maximum shoot dry weight was obtained in

plants treated with Zn NP (2µM) when compared to all Zn treated plants at 30

DAS. But at 60 DAS, higher shoot dry weight was observed in plants treated with

Zn NP (2µM) followed by normal salt, Zn NP (1µM) and Zn salt (1µM). Lowest

shoot dry weight was noted in plants treated with Cu salt (0.25µM). Ramesh et al.

(2014) reported positive effects of nano-ZnO on shoot-root growth of wheat.

However, Boonyanitipong et al. (2011) observed negative effect of nano-ZnO on

root length of rice (Oryza sativa L.). Nano-ZnO particle was found to enhance

germination and seedling growth of soybean (Sedghi et al. 2013) and vegetable

crops like cabbage, cauliflower and tomato (Singh et al. 2013). This was in

conformity with the present findings wherein Zn NP (2 µM) improved shoot dry

weight of wheat plants at both stages. This shows that normal concentration of Zn

NPs can positively influence shoot growth of plants.

4.4.4 Root dry weight

Root dry weight differed significantly at 30 DAS and at 60 DAS (Table

4.20). Among the Fe treatments, plants treated with high concentration Fe NP

(54µM) exhibited significantly higher root dry weight as compared to Fe NP

(27µM), normal salt and Fe salt (27µM) at 30 DAS. But in the second growth stage

77

higher root dry weight was obtained in plant treated with Fe NP (27µM) as

compared to normal salt, Fe NP (54µM) Fe salt (27µM).

Among Cu treated plants root dry weight was found significantly higher in

plant treated with normal salt followed by Cu salt (0.25µM), Cu NP (0.25µM) and

Cu NP (0.5µM) at 30 DAS. But at 60 DAS, best performance of plants was

observed with Cu NP (0.5µM) as compared to Cu NP (0.25µM), normal salt and

Cu salt (0.25µM). In wheat, maximum root dry weight was obtained with Cu NP

(0.5 µM) treatment at 60 DAS. The effective concentration of Cu NP in wheat (0.5

µM) was similar to the effective concentration in soybean.

Among Zn treatments, higher root weight was recorded in plants treated

with Zn NP (2µM) than Zn NP (1µM), normal salt and Zn salt (1µM) at 30 DAS.

But at 60 DAS, maximum root weight was noted in plants treated with Zn salt

(1µM) as compared to Zn NP (2µM), normal salt and Zn NP (1µM). Among all

treatments, highest root dry weight was obtained in plants treated with Fe NP

(54µM) at 60 DAS and lowest in plants treated with Cu NP (0.5 µM) at 30 DAS.

Higher root dry weight was found to be higher with normal concentration of Zn NP

(2 µM). In the present set of investigation, the response in wheat was similar to

soybean and maize crop (Kumar 2015).

Table 4.20: Effect of micronutrient NPs on shoots and root dry weight of wheat

Treatment Shoot dry weight

(g plant-1

)

Root dry weight

(g plant-1

)



T2: T1- Fe salt+ Fe NP (54 µM) 1.52 2.79 0.81 0.53

T3: T1- Fe salt + Fe NP (27 µM) 1.06 3.34 0.58 0.77


T5: T1- Cu salt + Cu NP (0.5 µM) 0.54 1.79 0.18 0.66

T6: T1- Cu salt + Cu NP (0.25 µM) 0.93 2.40 0.23 0.56


T8: T1- Zn salt + Zn NP (2 µM) 1.80 3.35 0.65 0.74

T9: T1- Zn salt + Zn NP (1 µM) 1.17 2.66 0.57 0.44


CD (5%) 0.424 0.670 0.310 0.189

78

Fig 4.19: Effect of micronutrient NPs on shoot dry weight of wheat.

Fig 4.20: Effect of micronutrient NPs on root dry weight of wheat.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DASS

hoot

dry

wt.

(g p

lant-1

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DAS

Root

dry

wei

ght

(g p

lant-1

)

79

4.4.5 Specific leaf area

The information on specific leaf area (SLA) as influenced by various

treatments during different growth periods are furnished in Table 4.21. Highest

SLA was observed in plants treated with Cu NP (0.5 µM) at both the stages and

lowest SLA was recorded in plants treated with Zn salt (1µM) at 30 DAS. Among

Fe treated plants maximum SLA was observed in plant treated with Fe NP (27 µM)

followed by Fe NP (54 µM), normal salt and Fe salt (27µM) at 30 DAS. At 60

DAS plants treated with Fe NP (54 µM) showed higher SLA as compared to Fe NP

(27µM), normal salt and Fe salt (27µM). There were not many reports on the

impact of nanoparticles on leaf growth parameter. It was reported that nano-iron

fertilizer caused 58 and 47% increase in fresh weight and leaf area index of

spinach (Moghadam et al. 2012). This was in conformity with the present findings

wherein wheat plants treated with reduced concentration i.e. Fe NP (27 µM)

exhibited SLA at 30 DAS. At 60 DAS, normal concentration of Fe NP (54 µM)

treated plants showed higher SLA. This may be due to the growth promoting effect

of reduced concentration of Fe NP (27 µM) on growth characteristics of wheat in

comparison to Fe salts.

In the case of Cu treatments, higher SLA was noted in plant treated with

Cu NP (0.5µM) as compared to normal salt, Cu NP (0.25µM) and Cu salt

(0.25µM). But in the case of second growth stage, higher SLA was noted in plants

treated with Cu NP (0.5µM) followed by Cu NP (0.25µM), Cu salt (0.25µM) and

normal salt. The effective concentration of Cu NP in wheat (0.5 µM) was similar to

the effective concentration in soybean. Leaf growth characteristics like SLA wheat

plant at 30 DAS were found to be higher with normal concentration of Cu NP (0. 5

µM).

Among Zn treated plants higher SLA was recorded in plant treated with Zn

NP (1 µM) as compared to Zn NP (2µM), normal salt and Zn salt (1µM) at first

sages but at 60 DAS, higher SLA was recorded in plants treated with Zn NP (1µM)

followed by normal salt, Zn NP (2µM) Zn salt (1µM). The SLA was found to be

higher with normal concentration of Zn NP (1µM). In the present set of

investigation, the response in wheat was similar to soybean and maize crop (Kumar

2015).

80

4.4.6 Specific leaf weight

There were significant differences in SLW among various treatments at 30

DAS and at 60 DAS (Table 4.21). Out of all ten treatments, highest SLW was

found in plant treated with Cu NP (0.25 µM) at 60 DAS and lowest SLW was

noted in plants treated with Fe salt (27µM) at 30 DAS. Among Fe treatments

plants, SLW was found significantly higher in plant treated with normal salt as

compared to Fe NP (54µM), Fe NP (27 µM) and Fe salt (27µM) at 30 DAS.

However, at 60 DAS, higher SLW was found in plant treated with Fe (27µM) as

compared to Fe NP (54µM) normal salt and Fe salt (27µM). There were not many

reports on the impact of nanoparticles on leaf growth parameter. It was reported

that nano-iron fertilizer caused 58 and 47% increase in fresh weight and leaf area

index of spinach (Moghadam et al. 2012). This was in conformity with the present

findings wherein wheat plants treated with reduced concentration i.e. Fe NP (27

µM) exhibited SLW at 30 DAS. At 60 DAS, normal concentration of Fe NP (54

µM) treated plants showed higher SLW. This may be due to the growth promoting


wheat in comparison to Fe salts.

Among Cu treatments, Cu NP (0.5 µM) and Cu NP (0.25 µM) treated

plants exhibited similar SLW at 30 DAS and found better than Cu salt (0.25 µM)

and normal salt. At 60 DAS, maximum SLW was observed in plants treated with

Cu NP (0.25 µM) followed by normal salt, Cu NP (0.5µM) and Cu salt (0.25 µM).

The effective concentration of Cu NP in wheat (0.5 µM) was similar to the

effective concentration in soybean. Leaf growth characteristics like SLW wheat

plant at 30 DAS were found to be higher with normal concentration of Cu NP (0. 5

µM).

Among Zn treatments, highest SLW was recorded in plant treated with Zn

NP (2 µM) as compared to Zn NP (1µM), Zn salt (1 µM) and normal salt at 30

DAS but it was decreased at 60 DAS and maximum SLW was noted in Zn NP

(1µM) followed by normal salt, Zn NP (2 µM) and Zn salt (1 µM). The SLW were

found to be higher with normal concentration of Zn NP (1µM). In the present set of

investigation, the response in wheat was similar to soybean and maize crop (Kumar

2015).

81

Table 4.21: Effect of micronutrient NPs on SLA and SLW of wheat

Treatment SLA (cm2g

-1) SLW (g cm

-2)



T2: T1- Fe salt+ Fe NP (54 µM) 158.15 223.90 0.003 0.009

T3: T1- Fe salt + Fe NP (27 µM) 174.15 104.25 0.001 0.011


T5: T1- Cu salt + Cu NP (0.5 µM) 256.55 424.75 0.009 0.003

T6: T1- Cu salt + Cu NP (0.25 µM) 119.00 247.60 0.009 0.017


T8: T1- Zn salt + Zn NP (2 µM) 127.60 220.60 0.015 0.005

T9: T1- Zn salt + Zn NP (1 µM) 168.95 313.65 0.009 0.007


CD (5%) 79.28 100.21 0.006 0.006

4.4.7 Leaf area

Significant differences between the treatments for leaf area were observed

at both the growth stages (Table 4.22). Among all the treatments, highest leaf area

was obtained in the plants treated with Zn NP (2µM) at 60 DAS, and lowest leaf

area was noted in Zn salt (1µM) treated plants at 30 DAS. Among Fe treated plants

maximum leaf area was recorded in Plants treated with Fe NP (54 µM) followed

by Fe NP (27µM), normal salt and Fe salt (27 µM) at both stage. Significant

differences were observed among Cu treated plants, higher LA was observed in

plants treated with Cu NP (0.5 µM) followed by normal salt, Cu NP (0.25µM) and

Cu salt (0.25µM) at first stage, but in second stage maximum LA was obtained in

plants treated with Cu NP (0.5 µM) in comparison to Cu NP (0.25 µM), normal

salt and Cu salt (0.25 µM). Among Zn treatments, higher leaf area was observed in

plants treated with Zn NP (1µM) as compared to Zn NP (2µM), normal salt Zn salt

(1µM) at 30 DAS. At 60 DAS, maximum LA was noted in plants treated with Zn

NP (2µM) than Zn NP (1µM), Zn salt (1 µM) and normal salt.

4.4.8 Leaf area ratio

It is evident form Table 4.22 that out of all ten treatments, highest leaf area

ratio (LAR) was noted in plant treated with Cu NP (0.5µM) at 30 DAS and Cu NP

(0.25µM) at 60 DAS and lowest LAR was recorded in plants treated with Fe salt

(27 µM) at 60 DAS. Among Fe treated plants, LAR was higher in plants treated

with Fe NP (27 µM) as compared to Fe NP (54µM) and Fe salt (27µM), normal

salt and Fe salt (27 µM) at 30 DAS but at 60 DAS, it was decreased and higher

82

LAR was found in plants treated with Fe NP (54µM) followed by Fe NP (27µM),

normal salt and Fe salt (27µM). Among Cu treatments, maximum LAR was

observed in plants treated with Cu NP (0.5µM) than Cu NP (0.25µM) normal salt,

and Cu salt (0.25µM). At 60 DAS, higher LAR was obtained with Cu NP

(0.25µM) in comparison to Cu NP (0.5µM), normal salt and Cu salt (0.25µM).

Among Zn treated plants higher LAR was observed in plant treated with lower

concentration of Zn NP (1µM) as compared to normal salt, Zn NP (2µM) and Zn

salt (1µM) at 30 DAS. But in the case of 60 DAS, maximum LAR was observed in

plants treated with Zn NP (2µM) followed by Zn NP (1µM), Zn salt (1µM) and

normal salt.

There was significant positive influence of Fe NP treatment (54 µM) on

morphology of wheat plant including leaf growth characteristics like leaf area and

LAR 60 DAS. This was in conformity to the findings in soybean plant, where there

was increase in growth parameters with the Fe NP treatments. The effective

concentration of Cu NP in wheat (0.5 µM) was similar to the effective

concentration in soybean. Leaf growth characteristics like Leaf area and LAR of

wheat plant at 30 DAS were found to be higher with normal concentration of Cu

NP (0.5 µM). In wheat, higher LA and LAR were observed with Zn NP (1 µM)

treatments. In the present set of investigation, the response in wheat was similar to


Table 4.22: Effect of micronutrient NPs on LA and LAR of wheat

Treatment Leaf Area (cm2) LAR (cm² g

-1)



T2: T1- Fe salt+ Fe NP (54 µM) 143.85 155.08 94.77 59.82

T3: T1- Fe salt + Fe NP (27 µM) 123.04 88.04 126.08 27.29


T5: T1- Cu salt + Cu NP (0.5 µM) 77.31 139.94 149.90 79.86

T6: T1- Cu salt + Cu NP (0.25 µM) 72.00 92.18 89.98 86.74


T8: T1- Zn salt + Zn NP (2 µM) 85.97 192.99 77.14 57.61

T9: T1- Zn salt + Zn NP (1 µM) 137.40 143.77 119.13 54.85


CD (5%) 20.82 20.57 NS 23.24

83

Fig 4.21: Effect of micronutrient NPs on leaf area of wheat.

Fig 4.22: Effect of micronutrient NPs on leaf area ratio of wheat.

0

50

100

150

200

250

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DASL

eaf

area

( c

m2)

0

20

40

60

80

100

120

140

160

180

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DAS

Lea

far

ea r

atio

(cm

² g

-1)

84

4.4.9 Number of Tillers

It is evident form Table 4.23 that Number of Tillers was highest in Zn NP

(2µM) treated plant at both stages and lowest in Cu salt (0.25µM) at 30 DAS.

Among Fe treated plants, Number of tiller obtained was higher in plants treated

with Fe NP (54 µM or 27µM) followed by normal salt and Fe salt (27µM) at 30

DAS, but at 60 DAS plants treated with Fe NP (27 µM) showed better

performance as compared to normal salt, Fe NP (54 µM) and Fe salt (27µM).

Among Cu treated plants higher no. of tillers was observed in plant treated with Cu

NP (0.25µM) as compared to Cu NP (0.5µM), normal salt and Cu salt (0.25µM) at

30 DAS, but at 60 DAS, and maximum tillers was noted in plants treated with Cu

NP (0.5µM) in comparison to normal salt, Cu NP (0.25µM) and Cu salt (0.25µM).

Among Zn treatments, best performance was noted in plants treated with Zn NP

(2µM) at 30 DAS followed by Zn NP (1µM), Zn salt (1µM) and normal salt at 30

DAS, but in the case of 60 DAS, maximum number of tiller was recorded in plants

treated with Zn NP (2µM) followed by normal salt, Zn NP (1µM) and Zn salt

(1µM). The plant parameters like number of tillers were all improved due to

application of nano-particle (Adhikari, T. et al.2015). In the present study wheat

plants treated with normal concentration i.e. Fe NP (54 µM) exhibited higher tillers

at 30 DAS.

4.4.10 Root volume

Among Fe treatments, higher root volume was obtained in plants

treated with Fe NP (54µM) as compared to Fe NP (27µM), normal salt Fe salt

(27µM) at 30 DAS, but at 60 DAS, higher root volume was recorded in plants

treated with normal salt as compared to all Fe treatment (Table 4.23). There were

many reports on the impact of nanoparticles on root volume for conformity that

nano particles could enhance and maintain the growth of maize plant. The plant

parameters like root length and root volume were all improved due to application

of nano-particle (Adhikari, T. et al.2015). In the present study wheat plants treated

with normal concentration i.e. Fe NP (54 µM) exhibited higher root volume at 30

DAS.

Among Cu treated plants higher Root volume was observed in plant treated

with normal salt in comparison to Cu NP (0.5µM), Cu salt (0.25µM) and Cu NP

85

(0.25µM) at 30 DAS. But at second stage, normal salt treated plants performed

much better as compared to Cu NP (0.25µM), Cu salt (0.25µM) and Cu NP

(0.25µM). CuO NPs did not inhibit the seed germination upto 2000ppm but root

growth was inhibited at 500 ppm in soybean and chickpea (Adhikari et al. 2012).

However, improvement in growth of plants treated with Cu NPs were reported in

wheat (Hafeez et al. 2015), lettuce (Shah and Belozerova, 2009) and improvement

in vase life of chrysanthemum was also reported with Cu NPs (Hashemabadi et al.

2013). In the present study it was observed that normal concentration of Cu (0.5

µM) had positive influence on above ground part as well as shoot of soybean while

reduced concentration of Cu NP (0.25 µM) had positively influenced root volume

at both stages.

Among all Zn treatments, maximum root volume was observed in plants

treated with Zn NP (2µM) followed by Zn NP (1µM), normal salt and Zn salt

(1µM) at 30 DAS. But at second growth stage, maximum root volume was noted in

Zn salt (1µM) treated plants followed by Zn NP (2µM), Normal salt and Zn NP

(1µM). Among all the treatments, highest in plants treated with Fe NP (54µM)

similar to Zn NP (2µM) at 30 DAS and lowest is Cu NP (0.25µM) at both stages.

In wheat, higher root volume was found with normal concentration of Zn NP (2

µM). In the present set of investigation, the response in wheat was similar to


Table 4.23: Effect of micronutrient NPs on no. of tillers and root volume of wheat

Treatment Number of Tillers

(Plant-1

)

Root volume

(cm3 Plant

-1)



T2: T1- Fe salt+ Fe NP (54 µM) 4.5 3.0 6.0 3.0

T3: T1- Fe salt + Fe NP (27 µM) 4.5 3.5 4.0 2.5


T5: T1- Cu salt + Cu NP (0.5 µM) 3.0 3.5 2.0 3.0

T6: T1- Cu salt + Cu NP (0.25 µM) 4.0 3.0 1.5 1.0


T8: T1- Zn salt + Zn NP (2 µM) 5.5 4.0 6.0 3.5

T9: T1- Zn salt + Zn NP (1 µM) 4.5 3.0 5.0 2.0


CD (5%) NS NS 1.73 NS

86

4.4.11 Panicle weight

Among all the treatments, highest panicle weight were recorded in plants

treated with Zn NP (2µM) and lowest in Cu salt (0.25µM) treated plants at harvest

(Table 4.24). In the case of Fe treatments, Fe NP (54µM) treated plants performed

better than Fe NP (27µM), normal salt and Fe salt (27µM). Among Cu treatments,

higher Panicle weight was recorded in plants treated with Cu NP (0.25µM) in

comparison to normal salt, Cu NP (0.5µM) and Cu salt (0.25µM). Among Zn

Treated plants, maximum panicle weight was noted in plants treated with Zn NP

(2µM) followed by Zn NP (1µM), Normal salt and Zn salt (1µM).

4.1.12 Grain weight

Out of all the ten treatments, highest grain weight was recorded in plants

treated with Zn NP (1µM) and lowest in Cu salt (0.25µM) treated plants (Table

4.24). Among Fe treatment, higher grain weight was recorded in plants treated with

Fe NP (27µM) in comparison to normal salt, Fe NP (54µM) and Fe salt (27µM). In

the case of Cu treatments, Cu NP (0.25µM) performed better than normal salt, Cu

NP (0.5µM) and Cu salt (0.25µM). Among Zn treatments, maximum grain weight

was obtained in plants treated with Zn NP (1µM) followed by Zn NP (2µM), Zn

salt (1µM) and normal salt.

Table 4.24: Effect of micronutrient NPs on panicle weight and grain weight of

wheat

Treatment

Panicle weight

(g plant-1

)

Grain weight

(g plant-1

)

Harvest Harvest


T2: T1- Fe salt+ Fe NP (54 µM) 3.7 1.38

T3: T1- Fe salt + Fe NP (27 µM) 3.3 2.50


T5: T1- Cu salt + Cu NP (0.5 µM) 2.1 1.52

T6: T1- Cu salt + Cu NP (0.25 µM) 3.8 2.56


T8: T1- Zn salt + Zn NP (2 µM) 3.9 2.41

T9: T1- Zn salt + Zn NP (1 µM) 3.7 2.97


CD (5%) NS NS

87

4.4.13 Number of Grain

Among the Fe treatments, Fe NP (27µM) treated plants performed better

than Fe NP (54µM), normal salt and Fe salt (27µM) ( Table 4.25). Among Cu

treatments, maximum number of grain was recorded in plants treated with Cu NP

(0.5µM) in comparison to Cu NP (0.25µM), normal salt and Cu salts (0.25µM). In

the case of Zn treatments, higher number of grain was recorded in plants treated

with Zn NP (1µM) followed by Zn (2µM), Zn salt (1µM) and normal salt. Among

all the treatment, highest number of grain was recorded in plants treated with Zn

NP (1µM) and lowest in plant treated with Cu salt (0.25µM).

4.4.14 Seed index (100 seed weight)

Among the all treatments, highest 100 seed Grain weight (Table 4.26) was

recorded in plants treated with Zn NP (1µM) and lowest in Cu salt (0.25µM).

Among Fe treatments, higher grain weight was recorded in plants treated with Fe

NP (54µM) in comparison to Fe NP (27µM), normal salt, Fe salt (27µM). In the

case of Cu treatments, Cu NP (0.25µM) performed better than Cu NP (0.5µM),

normal salt and Cu salt (0.25µM). Among Zn treatments, maximum Seed Index

was be noted in plants treated with Zn NP (1µM) followed by Zn NP (2µM), Zn

salt (1µM) and normal salt.

In case of wheat plants, the number of tillers and panicle weight was found

to higher with normal concentration of Fe NP (54 µM) and Zn NP (2 µM) while

grain weight and number of grains were found to be higher with Fe NP (27 µM) as

well as Zn NP (1 µM). However, wheat plants exhibited higher tillers, panicle

weight, grain weight and 100 seed weight with reduced concentration of Cu NP

(0.25 µM).

The above findings indicated that nanoparticle at reduced concentration

may be useful and they may act as catalyst for growth of plants. Moreover, it is

also envisaged that the effect of nanoparticles was crop or species specific.

88

Table 4.25: Effect of micronutrient NPs on number of grain and seed index of

wheat

Treatment Number of Grain

(plant-1

)

100 Seed Weight

(g plant-1

)

Harvest Harvest


T2: T1- Fe salt+ Fe NP (54 µM) 72.3 2.90

T3: T1- Fe salt + Fe NP (27 µM) 75.0 2.16


T5: T1- Cu salt + Cu NP (0.5 µM) 71.5 2.48

T6: T1- Cu salt + Cu NP (0.25 µM) 65.0 2.71


T8: T1- Zn salt + Zn NP (2 µM) 84.8 3.14

T9: T1- Zn salt + Zn NP (1 µM) 86.3 4.40


CD (5%) NS NS

4.4.15 Root Shoot Ratio

Among all the treatments, highest Root Shoot ratio was found in plants

treated with Cu NP (0.25 µM) at 30 DAS and lowest in plants treated with Fe salt

(27 µM) at 60 DAS (Table 4.26). Among Fe treated plants, higher root shoot ratio

was obtained with Fe NP (27 µM) as compared to Fe NP (54 µM) followed by

normal salt and Fe salt (27 µM) at both stages. In the case of Cu treatments,

maximum root shoot ratio was obtained in plants treated with Cu NP (0.25 µM)

than Normal salt, Cu NP (0.5 µM) and Cu salt (0.25 µM) at 30 DAS, but in second

stage, Cu NP (0.25 µM) treated plants exhibited higher root shoot ratio in

comparison to Cu NP 0.5µM), Cu salt (0.25µM) and normal salt. Among Zn

treatments, higher root shoot ratio was recorded in plant treated with Zn NP (1µM)

followed by Zn NP (2µM), normal salt and Zn salt (1µM) at 30 DAS. But at

second growth stage, maximum root shoot ratio was observed in plants treated with

Zn NP (1µM) followed by Zn NP (2µM), normal salt and Zn salt (1µM).

In wheat, root / shoot dry weight were found to be higher with

normal concentration of Zn NP (2 µM). In the present set of investigation, the

response in wheat was similar to soybean and maize crop (Kumar 2015).

89

Table 4.26: Effect of micronutrient NPs on root shoot ratio of wheat

Treatment Root Shoot Ratio

30DAS 60 DAS

T1: 100% (Fe + Cu + Zn) = Normal salts 0.47

0.18

T2: T1- Fe salt+ Fe NP (54 µM) 0.54

0.20

T3: T1- Fe salt + Fe NP (27 µM) 0.60

0.24

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.44

0.14

T5: T1- Cu salt + Cu NP (0.5 µM) 0.34

0.38

T6: T1- Cu salt + Cu NP (0.25 µM) 0.84

0.40

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.25

0.24


0.22


0.41

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.36

0.17

CD (5%) NS

0.073

90

Fig

4.2

3:

Eff

ect

of

mic

ronutr

ient

NP

s on

root

shoot

rati

o o

f w

hea

t

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

45

DA

S6

0 D

AS

Root Shoot ratio

91

4.5 Biochemical parameters of Wheat

4.5.1 Chlorophyll content

It is evident from Table 4.27 and Fig 4.28 and 4.29 that the chlorophyll ‗a‘

content did not differ significantly between treatments at both stages. Among all

the treatments higher magnitude of chlorophyll content ‗a‘ was found in plants

treated with Normal salt and Cu NP (0.5µM) at 30 DAS and 60 DAS, respectively

and lower chlorophyll content ‗a‘ was obtained in plants treated with Fe salt

(27µM) at both stages.

Significant chlorophyll ‗b‘ content was observed at both the growth stages

of plants treated with Normal salt at 30 DAS and Fe NP (27 µM) at 60 DAS (Table

4.27 and Fig. 4.28 and 4.29). Among Fe treated plants significant differences were

observed for chlorophyll ‗b‘ in plants treated with Normal salt followed by Fe NP

(27 µM), Fe NP 54 µM) and Fe salt (27 µM) at 30 DAS. In second growth stage,

Fe NP (27 µM) treatments exhibited significantly higher chlorophyll ‗b‘ in

comparison to Normal salt, Fe NP (54 µM) and Fe salt (27 µM). Among Cu treated

plants higher chlorophyll ‗b‘ was recorded in plants treated with Cu NP (0.25µM)

followed by Cu NP (0.5 µM), Normal salt and Cu salt (0.25 µM) at first stage. At

60 DAS, maximum chlorophyll ‗b‘ was observed in plants treated with Normal salt

as compared to Cu NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM). Among

Zn treated plants, chlorophyll ‗b‘ was found be significantly higher in plants

treated with Zn NP (2 µM) than Normal salt, Zn NP (1µM) and Zn salt (1 µM)

treated plants at 30 DAS, however at 60 DAS, maximum chlorophyll b was

recorded in plants treated with Normal salt when compared to Zn NP (2 µM), Zn

NP (1 µM) and Zn salt (1 µM). Among all the treatments, lowest chlorophyll

content ‗b‘ was found in Cu salt (0.25µM) at 30 DAS and Fe salt (27 µM) at 60

DAS.

Significant differences in total chlorophyll content were observed at 30

DAS and at 60 DAS (Table 4.27, Fig. 4.28 and 4.29). Among all the treatments,

highest total chlorophyll content was observed in plants treated with Normal salt at

60 DAS and lowest in plants treated with Fe salt (27µM) at 60 DAS. Among Fe

treated plants significant differences were observed in plants treated with Fe NP

(27 µM) as compared to Normal salt, Fe NP (54 µM) and Fe salt (27 µM) at 30

92

DAS, but at 60 DAS, higher chlorophyll content was observed in Normal salt in

comparison to Fe NP (27 µM), Fe NP (54 µM) and Fe salt (27 µM). At 30 DAS

higher total chlorophyll content was found in plants treated with Cu NP (0.5 µM)

among Cu treatments, followed by Normal salt, Cu NP (0.25 µM) and Cu salt

(0.25µM). However, at 60 DA significant differences were observed in plants

treated with Normal salt as compared to Cu NP (0.5µM), Cu NP (0.25µM), and Cu

salt. Among Zn treated plants higher total chlorophyll Content was recorded in

plants treated with Zn NP (2 µM) when compared to Normal salt, Zn NP (1 µM)

and Zn salt (1 µM) treated plants at both growth stages.

The present study on influence of Fe NP on biochemical parameters of

wheat revealed that there was significant improvement in chlorophyll b and total

chlorophyll content with the application of Fe NP (27 µM). In general, the

biochemical parameters were found to be higher with Fe NP treatments. The

response towards Fe NP in wheat with respect to chlorophyll content was similar to

maize (Elanchezhian et al 2015; Kumar 2015). Biochemical parameters viz. chl b,

total chl, was found to be higher with treatment of Cu NP. However, chl b, were

found to be higher with reduced concentration of Cu NP (0.25 µM). Similar results

were also reported earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al.

2011; Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a

catalyst for improvement in biochemical metabolism of plants. Among Zn

treatments, the chl b and total chl were found to be high with Zn NP treatments

either 2 µM or 1 µM. Similar results were observed in the present set of

investigation with soybean crop as mentioned above with Zn NP as well as by

Singh et al. (2013) and Kumar 2015 with maize crop.

93

Table 4.27: Effect of micronutrient NPs on chlorophyll content of wheat

Treatment Chlorophyll content (mg g-1

)

CHL a CHL b Total

DAS

30 60 30 60 30 60

T1: 100% (Fe + Cu + Zn) = Normal salts 0.45 0.60 0.61 0.68 1.21 1.28

T2: T1- Fe salt+ Fe NP (54 µM) 0.31 0.59 0.56 0.40 1.14 1.01

T3: T1- Fe salt + Fe NP (27 µM) 0.32 0.60 0.64 0.46 1.24 1.08

T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.31 0.58 0.53 0.31 1.11 0.94

T5: T1- Cu salt + Cu NP (0.5 µM) 0.31 0.61 0.61 0.49 1.22 1.10

T6: T1- Cu salt + Cu NP (0.25 µM) 0.31 0.61 0.63 0.45 1.20 1.07

T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.31 0.58 0.46 0.35 1.07 0.98

T8: T1- Zn salt + Zn NP (2 µM) 0.31 0.60 0.62 0.44 1.21 1.06

T9: T1- Zn salt + Zn NP (1 µM) 0.32 0.61 0.58 0.44 1.19 1.06

T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.31 0.60 0.58 0.36 1.18 0.98

CD (5%) NS NS 0.72 0.12 0.05 0.12

4.5.2 Membrane stability

Among all the treatments, highest membrane stability (MS) was observed

in plants treated with Fe NP (54µM) and Fe NP (27µM) at 30 DAS and 60 DAS,

respectively and lowest in plants treated with Fe salt (27µM) and Zn salt (1µM) at

30 DAS and 60 DAS (Table 4.28 and Fig 4.30). Among Fe treatments,

significantly higher MS was observed in plants treated with Fe NP (54 µM) as

compared to Fe NP (27µM), Normal salt and Fe salt (27µM) at 30 DAS, but in

second stage higher MS was found in plants treated with Fe NP (27µM) followed

by Fe NP (54µM), Normal salt and Fe salt (27µM). Among Fe treated soybean

plants, membrane stability was found to be high in plants treated with Fe NP (54

µM). This was in conformity to the results obtained in maize crop for the trait of


normal concentration of Fe NP may be inducing biochemical changes for

membrane stability.

94

Fig 4.24: Effect of micronutrient NPs on chlorophyll content of wheat at 30 DAS

Fig 4.25: Effect of micronutrient NPs on chlorophyll content of wheat at 60 DAS

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Chlorophyll a Chlorophyll b TotalC

hlo

rop

hyll

Co

nte

nt

(mg g

-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Chlorophyll a chlorophyll b Total

Ch

loro

ph

yll

Conte

nt

(mg g

-1)

95

Maximum MS was found in plants treated with Cu NP (0.25 µM) among

Cu treatments in comparison to Normal salt, Cu NP (0.25µM) and Cu salt

(0.25µM) at first growth stage. At 60 DAS, higher MS % was obtained in plants

treated with Cu NP (0.5µM) when compared to Normal salt, Cu NP (0.25µM) and

Cu salt (0.25µM). The MS were found to be higher with treatment of Cu NP.

However, MS were found to be higher with reduced concentration of Cu NP (0.25

µM). Similar results were also reported earlier as mentioned above (Hafeez et al.

2015; Nekrasova et al. 2011; Elanchezhian et al 2015). The reduced dose of Cu NP

might be used as a catalyst for improvement in biochemical metabolism of plants.

Among Zn treatments, higher MS was observed in plants treated with

Normal salt as compared to Zn NP (2µM), Zn NP (1µM) and Zn salt (1µM).

However at 60 DAS, Normal salt treated plants performed better than Zn NP

(1µM), Zn NP (2µM) and Zn salt (1µM)

4.5.3 Relative water content

Out of all ten treatments, highest RWC was found in plants treated with

Normal salt and Fe NP (27 µM) at 30 DAS and 60 DAS, respectively (Table 4.28).

Among Fe treatments plants, maximum RWC was obtain in plants treated with

Normal salt as compared to Fe NP (54 µM) and Fe NP (27 µM) and Fe salt (27

µM). But in second growth stage, higher MS was recorded in plants treated with Fe

NP (54µM) as compared to Fe NP (27 µM), Normal salt and Fe salt (27 µM).

Among Fe treated soybean plants, RWC was found to be high in plants treated

with Fe NP (54 µM). This was in conformity to the results obtained in maize crop

for the trait of membrane stability (Elanchezhian et al 2015; Kumar 2015). This

indicated that the normal concentration of Fe NP may be inducing biochemical

changes for water relations.

Among Cu treatments, higher RWC was recorded in plants treated with

Normal salt followed by Cu NP (0.5 µM), Cu NP (0.25 µM) and Cu salt (0.25

µM). At 60 DAS, maximum RWC was noted in plants treated with Cu NP (0.5

µM) followed by Normal salt, Cu NP (0.25µM) and Cu salt (0.25µM). The RWC

was found to be higher with treatment of Cu NP. Similar results were also reported

earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011;

96

Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a catalyst

for improvement in biochemical metabolism of plants.

Among Zn treatments, Normal salt treated plants performed better than Zn

NP (2µM), Zn NP (1µM) and Zn salt (1 µM) at 30 DAS. However, at 60 DAS,

higher RWC was observed in plants treated with Zn NP (1µM) as compared to Zn

NP (2µM), Normal salt and Zn salt (1µM). Among all treatments lowest RWC was

found in plants treated with Cu salt (0.25 µM) at 30 DAS.

Table 4.28: Effect of micronutrient NPs on MS and RWC of wheat

Treatment MS %

RWC %

30

DAS

60 DAS 30 DAS 60 DAS

T1: 100% (Fe + Cu + Zn) = Normal salts 76.40 75.11 67.05 52.72 T2: T1- Fe salt+ Fe NP (54 µM) 81.07 91.42 66.05 58.59 T3: T1- Fe salt + Fe NP (27 µM) 77.88 98.2 63.77 53.73 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 52.76 74.34 48.34 50.88 T5: T1- Cu salt + Cu NP (0.5 µM) 72.39 77.93 57.68 53.73 T6: T1- Cu salt + Cu NP (0.25 µM) 77.35 75.07 47.66 49.55 T7: 100% (Fe + Zn) salts + Cu salt (0.25

µM) 65.66 69.04 43.44 47.79 T8: T1- Zn salt + Zn NP (2 µM) 69.66 66.53 57.71 57.52 T9: T1- Zn salt + Zn NP (1 µM) 68.71 70.84 50.81 58.19 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 66.08 62.04 49.03 45.98 CD (5%) 12.71 10.90 9.16 4.18

97

Fig 4.26: Effect of micronutrient NPs on membrane stability of wheat

Fig 4.27: Effect of micronutrient NPs on Relative water content of wheat

0

20

40

60

80

100

120

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DAS

Mem

bra

ne

Sta

bil

ity (

%)

0

10

20

30

40

50

60

70

80

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DAS

Rel

ativ

e W

ater

Conte

nt

(%)

98

4.5.4 Antioxidant enzyme

4.5.4.1 Super oxide dismutase enzyme activity

Highest SOD enzyme activity was recorded in plants treated Zn NP (1 µM)

at 60 DAS and lowest in plants treated with Cu NP (0.25 µM) at 60 DAS (Table

4.29 and Fig. 4.32). Among Fe treated plant higher SOD activity was observed in

plants treated with Fe NP (27 µM) when compared to Fe NP (57 µM), Fe salt (27

µM) and Normal salt at the first stage but in second growth stage, higher SOD

enzyme activity was observed in plants treated with Fe salt (27µM) followed by Fe

NP (27 µM), Fe NP (54 µM) and Normal salt. The present study on influence of Fe

NP on biochemical parameters of wheat revealed that there was significant

improvement in activity of SOD with the application of Fe NP (27 µM). In general,

the biochemical parameters were found to be higher with Fe NP treatments. The

response towards Fe NP in wheat with respect to antioxidant enzymes was similar

to maize (Elanchezhian et al 2015; Kumar 2015).

Among Cu treated plants maximum SOD activity was recorded in plants

treated with Cu NP (0.5µM) than Cu salt (0.25 µM), Cu NP (0.25µM) and Normal

salt at first stages. At second growth stage, maximum SOD enzyme activity was

noted in plants treated with Cu NP (0.25µM) followed by Cu salt (0.25µM) and Cu

NP (0.5µM) and normal salt. The antioxidant enzyme like SOD activity was found

to be higher with treatment of Cu NP. Similar results were also reported earlier as

mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al

2015). The reduced dose of Cu NP might be used as a catalyst for improvement in

biochemical metabolism of plants.

In the case of Zn treatments, maximum SOD enzyme activity was obtained

in plants treated with Zn NP (1µM) than Zn NP (2µM), Zn salt (1µM) and Normal

salt at 30 DAS. But in second stage it was changed and higher enzyme activity was

recorded in plants treated with by Zn NP (1µM) followed by Zn salt (1µM), Zn NP

(2µM) and Normal salt. The higher SOD activity was found with Zn NP treatments




99

Table 4.29: Effect of micronutrient NPs on Super oxide dismutase enzyme activity

of wheat

4.5.4.2 Catalase enzyme activity

There was not much change in Catalase (CAT) activity among various

treatments (Table 4.30). Out of all ten treatments, highest catalase activity was

recorded in plants treated with Fe NP (54 µM) and Fe NP (27 µM) at 30 DAS and

60 DAS respectively. Lowest catalase activity was noted in plants treated with Cu

NP (0.5 µM) at 60 DAS. Among Fe NP treated plants higher catalase activity was

observed in plants treated with Fe NP (54 µM) followed by Fe NP (27µM),

Normal salt and Fe salt (27µM) at 30 DAS. But in 60 DAS, maximum CAT

activity was noted in plants treated with Fe NP (27 µM) as compared to Fe NP

(54µM), Normal salt and Fe salt (27µM). The present study on influence of Fe NP

on biochemical parameters of wheat revealed that there was significant

improvement enhanced activity of CAT with the application of Fe NP (27 µM).

In general, the biochemical parameters were found to be higher with Fe NP

treatments. The response towards Fe NP in wheat with respect to antioxidant

enzymes was similar to maize (Elanchezhian et al 2015; Kumar 2015).

Maximum catalase activity, among Cu treatments, was recorded in plants

treated with Cu NP (0.25 µM), followed by Cu NP (0.5 µM), Normal salt and Cu

salt (0.25µM) at 30 DAS; and at 60 DAS, higher CAT enzyme activity was

observed in plants treated with Cu NP (0.25 µM) than Normal salt, Cu salt (0.25

µM) and Cu NP (0.5µM). The CAT activity was found to be higher with treatment

of Cu NP. However, CAT was found to be higher with reduced concentration of

Treatment SOD (unit g-1

)

30 DAS 60 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 14.92 5.77

T3: T1- Fe salt + Fe NP (27 µM) 15.69 7.18


T5: T1- Cu salt + Cu NP (0.5 µM) 12.15 7.18

T6: T1- Cu salt + Cu NP (0.25 µM) 9.29 18.24


T8: T1- Zn salt + Zn NP (2 µM) 10.72 9.85

T9: T1- Zn salt + Zn NP (1 µM) 13.26 18.71


CD (5%) 5.67 9.35

100

Cu NP (0.25 µM). Similar results were also reported earlier as mentioned above

(Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al 2015). The reduced

dose of Cu NP might be used as a catalyst for improvement in biochemical

metabolism of plants.

Among Zn NP treated plants higher catalase activity was recorded in

plants treated with Zn NP (1µM) when compared to Normal Salt Zn NP (2µM) and

Zn salt (1 µM) at 30. At 60 DAS, CAT enzyme activity was recorded higher in

plants treated with Zn NP (2µM) followed by Zn salt (1 µM), Normal salt and Zn

NP (1 µM). Higher CAT activity was found to be high with Zn NP treatments




4.5.4.3 Peroxidase enzyme activity

Among all the treatments, highest Peroxidase enzyme activity was recorded

in plants treated with Normal at 30 DAS and lowest POX enzyme activity was

recorded in plants treated with Fe salt (27µM) at 60 DAS (Table 4.30). Among Fe

treated plants, higher POX activity was observed in plants treated with Normal salt

when compared to Fe NP (54 µM) Fe, Fe NP (27 µM) and salt (27 µM) at first

stages but at second stage higher POX enzyme activity was recorded in plants

treated with Fe NP (27µM) when compared to Fe NP (54 µM), Normal salt and Fe

salt (27µM). The present study on influence of Fe NP on biochemical parameters

of wheat revealed that there was significant improvement enhanced activity of

POD with the application of Fe NP (27 µM). In general, the biochemical

parameters were found to be higher with Fe NP treatments. The response towards

Fe NP in wheat with respect to antioxidant enzymes was similar to maize

(Elanchezhian et al 2015; Kumar 2015).

Among Cu treated plants, maximum POX activity was recorded in plants

treated with Normal salt than Cu NP (0.25 µM) followed by Cu NP (0.5µM) and

Cu salt (0.25 µM) at 30 DAS, but in the second growth stage, Cu salt (0.25 µM)

performed better than Normal salt, Cu NP (0.5µM), and Cu NP (0.25µM). The

POX activity was found to be higher with treatment of Cu NP. Similar results were

101

also reported earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al.

2011; Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a

catalyst for improvement in biochemical metabolism of plants.

Among Zn treated plants higher POX was obtained in plants treated with

Normal salt followed by Zn NP (2µM), Zn NP (1 µM) and Zn salt (1µM) at both

growth stages. Higher POX activity was found to be high with Zn NP treatments




Table 4.30: Effect of micronutrient NPs on catalase enzyme activity and

peroxidase enzyme activity of wheat

Treatment CAT (unit H2O2-1

min-1

g-1

)

POX (unit H2O2-1

min-1

g-1

)

30

DAS

60

DAS

30

DAS

60

DAS


T2: T1- Fe salt+ Fe NP (54 µM) 72.0 12.8 123.68 60.34

T3: T1- Fe salt + Fe NP (27 µM) 72.0 14.4 122.66 80.23


T5: T1- Cu salt + Cu NP (0.5 µM) 68.8 8.0 123.32 59.16

T6: T1- Cu salt + Cu NP (0.25 µM) 70.4 11.2 123.55 58.09


T8: T1- Zn salt + Zn NP (2 µM) 65.6 14.4 111.57 104.53

T9: T1- Zn salt + Zn NP (1 µM) 70.4 8.0 111.90 79.05


CD (5%) NS NS 14.39 26.96

102

Fig. 4.28: Effect of micronutrient NPs on Super oxide dismutase enzyme activity

of wheat

Fig. 4.29: Effect of micronutrient NPs on peroxidase enzyme activity of wheat

-5

0

5

10

15

20

25

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 DASS

OD

(Un

it g

m-1

)

0

20

40

60

80

100

120

140

160

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

45 DAS 60 ADS

PO

X (

un

it H

2O

2m

in-1

g-1

)

103

4.5.5 Proline content

Proline content was found to be significantly different among treatments at

30 and 60 DAS, respectively (Table 4.31 and Fig. 4.34). Among Fe treated plants,

Proline content was significantly higher in plants treated with Fe NP (27 µM),

when compared to Fe NP (54 µM), Normal salt and Fe salt (27 µM) at first growth

stage but at 60 DAS, plants treated with Fe NP (54 µM) and Normal salt performed

best as compared to Fe NP (27µM) and Fe salt (27 µM). Among Cu treated plants

higher Proline content was recorded in plants treated with Normal salt followed by

Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM) at 30 DAS. At 60 DAS,

higher Proline content was recorded in plants treated with Normal salt followed by

Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Among Zn treated plants

higher Proline content was observed in plants treated with Normal salt as

compared to Zn NP (2µM), Zn NP (1 µM) and Zn salt (1µM) at both growth

stages. Among all the treatments, highest Proline content was observed in plants

treated with Fe NP (27 µM) at 60 DAS and lowest Proline content was observed in

plants treated with Cu salt (27 µM) at 30 DAS.

The present study on influence of Fe NP on biochemical parameters of

wheat revealed that there was significant improvement in proline with the

application of Fe NP (27 µM). In general, the biochemical parameters were found

to be higher with Fe NP treatments. The response towards Fe NP in wheat with

respect to proline content was similar to maize (Elanchezhian et al 2015; Kumar

2015).

4.5.6 Total soluble protein

Total soluble protein (TSP) content was found similar during the growth

period (Table 4.31). Protein content was observed to be not significant among all

NPs treated plants at 30 DAS and 60 DAS. Among all the treatments, higher

magnitude of TSP content was observed in plants treated with Fe NP (54µM) at 30

DAS, and lowest TSP content was recorded in plants treated with Fe salt (27µM)

at 60 DAS.

Among Fe treatments, higher protein content was found with normal

concentration of Fe NP (54µM) similar results was observed of increased protein

and of wheat Ghafari and Razmjoo, (2013).

104

Table 4.31: Effect of micronutrient NPs on proline and protein of wheat

Treatment Proline (μM g-1

) TSP (mg g-1

)

30 DAS 60 DAS 30 DAS 60DAS


T2: T1- Fe salt+ Fe NP (54 µM) 0.012 0.025 1.775 1.645

T3: T1- Fe salt + Fe NP (27 µM) 0.015 0.020 1.735 1.645


T5: T1- Cu salt + Cu NP (0.5 µM) 0.005 0.015 1.735 1.705

T6: T1- Cu salt + Cu NP (0.25 µM) 0.006 0.021 1.751 1.695


T8: T1- Zn salt + Zn NP (2 µM) 0.006 0.025 1.734 1.645

T9: T1- Zn salt + Zn NP (1 µM) 0.006 0.019 1.725 1.700


CD (5%) 0.009 0.000 NS NS

4.5.7 Total soluble sugar

Out of all ten treatments, highest TSS content was observed in plants

treated with Fe NP (54 µM) at both stages and lowest TSS contents was noted in

plants treated with Zn salt (1 µM) at 30 DAS and Cu salt (0.25 µM) at 60 DAS

(Table 4.32 and Fig.4.35). Among Fe treated Plants, higher TSS content was found

in plants treated with Fe NP (54 µM) as compared to plants treated with Fe NP (27

µM), Normal salt and Fe salt (27µM) at 30 DAS. However, at 60 DAS, Fe NP

(54µM) treated plants exhibited higher TSS than Normal salt, Fe NP (27 µM) and

Fe salt (27µM). The response towards Fe NP (27 µM) in wheat with respect to

TSS content was found higher. Similar to the reports of enhanced carbohydrate

content with Fe NP in wheat (Ghafari and Razmjoo, 2013), we could also find

significant improvement of TSS in comparison to normal salt treatment.

Maximum TSS content was observed in plants treated with Cu NP (0.25

µM) at 30 DAS among Cu treated plants as compared to Cu (0.5 µM) followed by

Cu salt (0.25 µM) and Normal salt. However, at 60 DAS, maximum TSS was

recorded in plants treated with Normal salt when compared to Cu NP (0.5 µM), Cu

NP (0.25 µM) and Cu salt (0.25 µM). In wheat, biochemical parameters viz. TSS

was found to be higher with treatment of Cu NP. However, TSS was found to be

higher with reduced concentration of Cu NP (0.25 µM). Similar results were also

reported earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011;

105

Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a catalyst

for improvement in biochemical metabolism of plants.

Among Zn treatments, higher TSS content was found in plants treated with

Zn NP (1 µM) followed by Zn NP (2µM), Normal salt and Zn salt (1µM) at 30

DAS. But at 60 DAS, maximum TSS was observed in plants treated with Normal

salt as compared to, Zn NP (1µM), Zn NP (2µM) and Zn salt (1µM). Among Zn

treatments, TSS was found to be high with Zn NP treatments either 2 µM or 1 µM.

Similar results were observed in the present set of investigation with soybean crop

as mentioned above with Zn NP as well as by Singh et al. (2013) and Kumar 2015

with maize crop

4.5.8 Non-structural carbohydrate

Among all the treatments, maximum NSC content was recorded in plants

treated with Fe NP (27 µM) and normal salt at 30 and 60 DAS, respectively (Table

4.32 and Fig 4.36). Among Fe treated plants higher NSC was observed in plants

treated with Fe NP (27 µM) when compared to all Fe treatments at 30 DAS; and at

60 DAS, Normal salt treated plants showed higher NSC. The response towards Fe

NP (27 µM) in wheat with respect to NSC content was found higher. Similar to the

reports of enhanced carbohydrate content with Fe NP in wheat (Ghafari and

Razmjoo, 2013), we could also find significant improvement of TSS and NSC in

comparison to normal salt treatment.

Maximum NSC was found in Cu NP (0.25µM) treated plants at 30 DAS,

among Cu treatments. But at second growth stage, plants treated with Normal salt

showed higher NSC as compared to Cu NP (0.25 µM), Cu NP (0.5µM) and Cu salt

(0.25µM). In wheat, biochemical parameters like NSC were found to be higher

with treatment of Cu NP. However, NSC were found to be higher with reduced

concentration of Cu NP (0.25 µM). Similar results were also reported earlier as

mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al

2015). The reduced dose of Cu NP might be used as a catalyst for improvement in

biochemical metabolism of plants.

Among Zn treated plants, higher NSC content was found in plants treated

with Zn NP (1 µM) at first growth stage followed by Normal salt, Zn NP (2µM)

106

and Zn salt (1µM) at 30 DAS. But in the second growth stage higher NSC was

recorded in plants treated with Normal salt as compared to Zn NP (1µM), Zn NP

(2µM) and Zn salt (1µM). However lowest NSC was recorded in plants treated

with Zn salt (1 µM) at 30 DAS and Cu salt (27 µM) at 60 DAS. Among Zn

treatments, NSC was found to be high with Zn NP treatments either 2 µM or 1 µM.

Similar results were observed in the present set of investigation with soybean crop

as mentioned above with Zn NP as well as by Singh et al. (2013) and Kumar 2015

with maize crop.

Table 4.32: Effect of micronutrient NPs on TSS and NSC of wheat

Treatment TSS (%) NSC (%)


T1: 100% (Fe + Cu + Zn) =Normal salts 3.225 7.555 4.81 5.21 T2: T1- Fe salt+ Fe NP (54 µM) 4.214 8.225 5.90 5.02 T3: T1- Fe salt + Fe NP (27 µM) 3.945 6.329 6.29 4.93 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 2.865 5.902 4.37 3.86 T5: T1- Cu salt + Cu NP (0.5 µM) 2.195 6.075 4.49 4.30 T6: T1- Cu salt + Cu NP (0.25 µM) 3.699 6.469 5.94 4.39 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.965 4.045 4.19 3.10 T8: T1- Zn salt + Zn NP (2 µM) 2.485 5.695 4.22 4.34 T9: T1- Zn salt + Zn NP (1 µM) 3.269 7.184 5.40 5.16 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 1.635 5.555 3.97 4.22 CD (5%) 1.104 0.531 1.22 0.279

107

Fig 4.30: Effect of micronutrient NPs on TSS of wheat

Fig 4.31: Effect of micronutrient NPs on non-structural carbohydrate of wheat

0.0

1.0

2.0

3.0

4.0

5.0

6.0

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

30 DAS 60 DAST

ota

lS

olu

ble

Su

gar

(%

)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

T1 T2 T3 T4 T5 T6 T7 T8 T9 T1030 DAS 60 DAS

No

nS

tru

ctu

ral C

arb

ohydra

te (

%)

108

4.6 Physiological parameters of Wheat

4.6.1 Photosynthesis rate

The Photosynthesis rate furnished in Table 4.33 indicated significant

differences among the treatments at the all growth stages. Out of ten treatments,

highest Photosynthesis rate was obtained in plants treated with Zn NP (2µM) at 60

DAS and lowest in plants treated with Zn salt (1µM) at both stages. Among Fe

treatment plants, higher Photosynthesis rate was noted in plant treated with Normal

salt followed by Fe NP (27 µM), Fe NP (54 µM) and Fe salt (27µM) at both

stages. Among Cu treatments, photosynthesis rate was found significantly higher

in plant treated with Normal salt in comparison to Cu NP (0.5µM), Cu NP

(0.25µM) and Cu salt (0.25 µM) at both stages. In the case of Zn Treatments,

higher photosynthesis rate was observed in plant treated with Normal salt when

compared to plants treated with Zn NP (2µM) Zn NP (1µM) and Zn salt (1 µM) at

first stages. At 60 DAS, maximum photosynthesis rate was noted in plant treated

with Zn NP (2µM) followed by Zn (µM), normal salt and Zn salt (µM).

Table 4.33: Effect of micronutrient NPs on Photosynthesis rate of wheat

Treatment Photosynthesis rate (µM m-2

s-1

)

30 DAS 60 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 5.35 5.15

T3: T1- Fe salt + Fe NP (27 µM) 5.45 6.20


T5: T1- Cu salt + Cu NP (0.5 µM) 4.15 6.45

T6: T1- Cu salt + Cu NP (0.25 µM) 4.00 5.20


T8: T1- Zn salt + Zn NP (2 µM) 4.10 10.35

T9: T1- Zn salt + Zn NP (1 µM) 3.75 8.7


CD (5%) NS 2.76

4.6.2 Transpiration rate

Among all the treatments, highest transpiration rate was noted in plants

treated with Zn NP (2µM) and lowest in plants treated with Zn salt (1µM) at 60

DAS (Table 4.34). Among Fe treatment, higher Transpiration rate was noted in

plant treated with Fe NP (54 µM) followed by Fe NP (27 µM), normal salt and Fe

salt (27µM) at both stages.Among Cu treatments, Transpiration rate was found

109

significantly higher in plants treated with higher concentration of normal salt and

Cu NP (0.5µM) as compared to lower concentration of Cu NP (0.25 µM), Cu salt

(0.5µM) at 30 DAS. But at second stage, among Cu treatments, higher

Transpiration rate was recorded in plant treated with Cu NP (0.25µM) and Normal

salt followed by Cu NP (0.5µM) and Cu salt (0.25µM. Among Zn treated plants,

higher Transpiration was observed in plants treated with Norma salt when

compared to all Zn treated plants like Zn NP (2µM), Zn NP (1µM) and Zn salt

(1µM) at 30 DAS, but at 60 DAS, maximum Transpiration rate was recorded in

plants treated with Zn NP (2µM) in comparison to Zn NP (1µM), normal salt and

Zn salt (1µM).

Table 4.34: Effect of micronutrient NPs on transpiration rate of wheat

Treatment Transpiration rate (mM m-2

s-1

)

30 DAS 60 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 2.5 2.8

T3: T1- Fe salt + Fe NP (27 µM) 2.0 2.8


T5: T1- Cu salt + Cu NP (0.5 µM) 1.6 2.4

T6: T1- Cu salt + Cu NP (0.25 µM) 1.3 2.5


T8: T1- Zn salt + Zn NP (2 µM) 1.7 2.9

T9: T1- Zn salt + Zn NP (1 µM) 1.6 2.8


CD (5%) NS NS

4.6.3 Stomatal conductance

Stomatal conductance was found significantly different between treatments

at all growth stages (Table. 4.35). Among Fe treatments, higher stomatal

conductance was obtained in plant treated with Fe NP (27µM) as compared to Fe

NP (54µM), normal salt and Fe salt (27µM) at both stages. Among Cu treatment

plants, higher Stomatal conductance was obtained in plant treated with higher

concentration of Cu NP (0.5 µM) in comparison to Normal salt, Cu NP (0.25µM)

and Cu salt (0.25µM) at 30 DAS. But at 60 DAS, plants exhibited maximum

Stomatal conductance with Normal salt followed by Cu NP (0.25µM), Cu NP

(0.5µM) and Cu salt (0.25µM). Among Zn Treatments, higher Stomatal

conductance was noted in plant treated with normal salt when compared to Zn NP

(1µM), Zn NP (2 µM) and Zn salt (1µM) at 30 DAS. But at 60 DAS, among Zn

treated plants, higher Stomatal conductance was noted in plants treated with Zn

110

(2µM) as compared to Normal salt, Zn NP (1µM) and Zn salt (1µM). Among all

treatments, highest stomatal conductance was obtained in plants treated with Fe NP

(27µM) at both stages and lowest Stomatal conductance was obtained in plants

treated with Zn salt (1 µM) at both stages.

Alidoust and Isoda (2013) was reported that foliar spray of Fe NPs in

soybean Gas exchange parameters viz photosynthetic rate, stomatial conductance

and transpiration rate were positively influenced by the NPs. In present study,

photosynthetic rate was enhanced by Fe NP (54µM) and Cu NP (0.25µM)

treatments in wheat. In addition to Fe and Cu being important element in

photosynthetic reaction pathways, it is envisaged that they may be stimulating

photosynthetic electron transport, which might enhance the photosynthetic rate.

Moreover, transpiration rate and stomatal conductance was found to higher with

reduced concentration of NP in both the crops which indicated that the NPs may

positively regulate stomatal opening and closure.

Table 4.35: Effect of micronutrient NPs on stomatal conductance of wheat

Treatment Stomatal Conductance

(µM m-2

s-1

)

30 DAS 60 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 79.0 109.5

T3: T1- Fe salt + Fe NP (27 µM) 102.5 117.0


T5: T1- Cu salt + Cu NP (0.5 µM) 78.5 88.5

T6: T1- Cu salt + Cu NP (0.25 µM) 44.0 91.5


T8: T1- Zn salt + Zn NP (2 µM) 44.0 108.5

T9: T1- Zn salt + Zn NP (1 µM) 55.0 102.5


CD (5%) NS NS

111

Fig

4.3

2:

Eff

ect

of

mic

ronutr

ient

NP

s on p

hoto

syn

thes

is r

ate

of

wh

eat

02468

10

12

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

30

DA

S6

0 D

AS

Photosinthesisrate (µM m-2s-1)

112

4.6.4 SPAD value

SPAD value was found significantly different between treatments at

all growth stages (Table. 4.36 and fig. 4.38). Among all treatments, highest SPAD

value was obtained in plants treated with Zn NP (2µM) and lowest SPAD value

was obtained in plants treated with Zn salt (1 µM) at 60 DAS. Among Fe

treatments, higher SPAD value was obtained in plant treated with Normal salt as

compared to Fe NP (27µM), Fe NP (54µM), and Fe salt (27µM) at first stages. But

in second stage, higher SPAD value was recorded in plants treated with Fe NP

(54µM) followed by Normal salt, Fe NP (27µM) and Fe salt (27µM). In general,

the biochemical parameters were found to be higher with Fe NP treatments. The

response towards Fe NP in wheat with respect to chlorophyll content was similar to

maize (Elanchezhian et al 2015; Kumar 2015).The present study on influence of Fe

NP on biochemical parameters of wheat revealed that there was significant

improvement in chlorophyll content with the application of Fe NP (54 µM).

Among Cu treatment plants, higher SPAD Value was obtained in plant

treated with Normal salt in comparison to Cu NP (0.5 µM), Cu NP (0.25µM) and

Cu salt (0.25µM) at 30 DAS. But at 60 DAS, plants showed maximum SPAD

value in plants treated with Normal salt followed by Cu NP (0.25µM), Cu NP

(0.5µM) and Cu salt (0.25µM). Among Cu treatments, chlorophyll was positively

influenced by Cu NPs Similar results were also reported earlier as mentioned

above (Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al 2015).

Among Zn Treatments, higher SPAD value was noted in plant treated with

Normal salt when compared with the plants treated with Zn NP (1µM), Zn NP (2

µM) and Zn salt (1µM) at 30 DAS. But at last growth stage, among Zn treated

plants; higher SPAD value was noted in plants treated with Zn (2µM) as compared

to Normal salt, Zn NP (1µM) and Zn salt (1µM). Among Zn treatments,

chlorophyll content was found to be high with Zn NP treatments 2µM. Similar

results were observed in the present set of investigation with soybean crop as

mentioned above with Zn NP as well as by Singh et al. (2013) and Kumar 2015

with maize crop.

113

Table 4.36: Effect of micronutrient NPs on SPAD value of wheat

Treatment SPAD Value

30 DAS 60 DAS


T2: T1- Fe salt+ Fe NP (54 µM) 43.70 48.30

T3: T1- Fe salt + Fe NP (27 µM) 44.40 46.55


T5: T1- Cu salt + Cu NP (0.5 µM) 40.45 44.00

T6: T1- Cu salt + Cu NP (0.25 µM) 40.15 46.25


T8: T1- Zn salt + Zn NP (2 µM) 41.25 49.75

T9: T1- Zn salt + Zn NP (1 µM) 41.30 43.05


CD (5%) 4.28 5.61

114

Fig

. 4.3

3:

Eff

ect

of

mic

ronutr

ient

NP

s on S

PA

D v

alue

of

whea

t.

0

10

20

30

40

50

60

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0

30

DA

S6

0 D

AS

SPAD Value

115

CHAPTER –V

SUMMARY AND CONCLUSIONS

5.1 SUMMARY

The investigation entitled ―Physiological responses of soybean and wheat

crops towards nanoparticle based micronutrients fertilization‖ was carried out at

ICAR-Indian Institute of Soil Science, Bhopal (M.P.) during – 2015-16. The major

objective of this study was to study the effect of different concentrations of

nanoparticles viz. Fe, Cu and Zn on plant morphological, physiological and

biochemical characteristics of soybean and wheat. The material for study

comprised of soybean and wheat were grown in CRD (Completely Randomized

Block Design) with five replications. Research work has been carried out in the

experimental screen house of ICAR-IISS Bhopal.

1. All the nano-micronutrients positively influenced the growth and yield

traits of soyeban and wheat plants for most of the parameters studied.

2. Plant height of Soybean and wheat was enhanced by nano- micronutrient

fertilization of Cu NP (0.5 µM). However, Zn NP (2µM) also promoted

plant height in soybean.

3. Root length of soybean was found to be maximum with Fe NP (27 µM).

Cu NP (0.5 µM) treated plants also exhibited maximum root length in

wheat.

4. Leaf growth characteristics of soybean viz. leaf area and LAR were found

to be promoted by Zn NP (2µM) and Fe NP (27µM). SLA and SLW were

found to be higher with Fe NP (27µM) and Cu NP (0.5µM). In wheat, leaf

area was found to be higher in Fe NP (54µM) and Zn NP (2µM), but LAR,

SLA and SLW were found to be positively influenced by Cu NPs.

5. Plant biomass of soybean and wheat was enhanced by Zn NP (2µM) and Fe

116

NP (54 µM) treatments.

6. Grain yield was positively affected by increased concentration of Zn NP

(2µM) and Fe NP (54µM) in soybean. However, in the case of wheat,

maximum grain yield was found with reduced concentration of NPs viz. Zn

NP (1µM), Fe NP (27µM) and Cu NP (0.25 µM).

7. Membrane stability was found to be higher with Cu NP (0.5 µM) and Fe

NP (27µM) treatments in soybean. However, in case of wheat greater MS

and RWC was obtained with Fe NP (54µM). While RWC was positively

influenced by Fe NP (27 µM) in Soybean.

8. Proline accumulation was found to be increased with Fe NP (54 µM and

27µM) treatments in soybean and wheat. Total soluble protein content in

soybean and wheat was higher in increased concentration of micronutrient

NPs viz. Fe NP (54 µM) and Zn NP (2 µM). However, TSP was found

positively influenced by reduced concentration of Cu NP (0.25 µM) in both

crops.

9. Anti-oxidant enzyme - SOD was found to be higher with micronutrient

fertilization of Fe NP (54µM) in soybean and reduced concentration of NPs

viz. Fe NP (27 µM), Zn (1 µM) and Cu NP (0.25 µM) in wheat. CAT

activity was enhanced by Cu NP (0.25µM) and Fe NP (54 µM) in soybean

and Fe NP (54 µM) in wheat crop. POX activity was improved by Zn NP

(2µM) in wheat.

10. Total Chlorophyll and chl b content was positively influenced by reduced

concentration of Fe NP (27 µM) in both the crop. While chl a content was

enhanced by Cu NP (0.5 and 0.25µM) in soybean and wheat. SPAD value

recorded also corroborated that reduced concentration of Fe NP (27 µM)

positively influenced greenness of the leaf.

11. Total soluble sugars and non-structural carbohydrate content was found to

be higher with Cu NP (0.25µM) and Fe NP (27µM) treatments in soybean.

However, TSS and NSC were positively influenced by Fe NP (54 µM) and

Fe NP (27 µM), respectively, in wheat crop.

117

12. Photosynthesis rate was enhanced by Fe NP (54µM) and Cu NP (0.25µM)

in soybean. But in the case wheat photosynthesis rate was enhanced in Zn

NP (2µM) treatments. The transpiration rate was found to be higher with

NP treatments viz. Fe NP (27µM and 54 µM), Zn NP (2µM) and Cu NP

(0.25µM) in both the crops. The stomatal conductance was positively

enhanced by reduced concentration of NPs viz. Zn NP (1µM), Cu NP

(0.25µM) and Fe NP (27µM) in soybean. In the wheat crop, maximum

stomatal conductance was obtained in reduced concentration of Fe NP

(27µM) and Cu NP (0.25µM).

5.2 CONCLUSIONS

1. In Soybean, the nano-micronutrient fertilization of plants with normal

concentration of NPs has positively influenced the shoot growth and

biochemical metabolism of plants. However, reduced concentration of

NPs had positively influenced root growth and gas exchange parameters

of plants.

2. In wheat, the nano-micronutrient fertilization of plants with Fe NPs/ Cu

NPs / Zn NPs had positively influenced most of the morphological

parameters while reduced concentration of Fe NPs/ Cu NPs and Zn NPs

had positively influenced biochemical metabolism of plants. Gas

exchange parameters were also positively influenced by NPs.

3. The above findings indicated that the effect of nanoparticles were crop

or species specific. Moreover, it is also envisaged that nanoparticle at

reduced concentration may be useful for the crop and they may act as

catalyst for growth, metabolism and yield of plants.

5.3 SUGGESTIONS FOR FUTURE RESEARCH WORK

The present investigation on nano sized micro-nutrient fertilization

revealed that impact of NPs were crop specific, which may be due to the different

size, shape of NPs which on entering the plant system may influence the growth

and metabolism differentially. It is observed that reduced concentration of NPs

affected positively the traits associated with growth and metabolism.

118

The concentration of NPs also should be optimized and standardized for

further exploitation in the manipulation of growth, yield potential and quality of

the crops.

There should be study on the impact of different shape, size of NPs on plant

system and integration on NPs in plant system to understand the basic mechanism

of NP uptake and utilization in the plants.

There should be more study on transport of NP inside plant system and

their movement to animal system for their impact on metabolism of animal system

to ascertain their toxicity for health concern of animal or human system

119

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APPENDICES

Appendix A

Table: Metrological data during crop period 2015-16

Month Temperature Maximum Temperature Minimum

Jul-15 30.7 25.2

Aug-15 29.3 24.6

Sep-15 32.5 24.8

Oct-15 34.0 23.3

Nov-15 31.1 18.3

Dec-15 26.6 11.8

Jan-16 26.7 11.2

Feb-16 29.0 13.8

Mar-16 35.4 20.4

Source - Central Institute of Agricultural Engineering, Bhopal (M.P.)

129


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nts

KN

O3

10

1.1

0

1,0

00

101.1

0

6.0

N

1

6,0

00

2

24

Ca(N

O3)2

4H

2O

2

36

.16

1,0

00

236.1

6

4.0

K

6

,00

0

23

5

NH

4H

2 P

O4

11

5.0

8

1,0

00

115.0

8

2.0

C

a

4,0

00

1

60

MgS

O4·7

H2

24

6.4

8

1,0

00

246.4

8

1.0

P

2

,00

0

62

S

1,0

00

3

2

Mg

1

,00

0

24

Mic

ron

utr

ien

ts

KC

l 7

4.5

5

25

1.8

64

Cl

50

1.7

7

H3B

O3

Mn

SO

4.H

2O

61

.83

16

9.0

1

12

.5

1.0

0.7

73

0.1

69

B

Mn

25

2.0

0.2

7

0.1

1

Zn

SO

4.7

H2O

2

87

.54

1

.0

0.2

88

2.0

Z

n

2.0

0

.13

Cu

SO

4.5

H2O

2

49

.68

0

.25

0.0

62

Cu

0

.5

0.0

3

H2M

oO

4

(85%

Mo

O3)

16

1.9

7

0.2

5

0.0

40

Mo

0.5

0.0

5

NaF

eD

TP

A

(10%

)

46

8.2

0

64

30

.0

1.0

Fe

53

.7

3.0

0

Fe

3O

4 N

ano

2

31

.53

6

4

14

.81

1

.0

Fe

53

.7

3.0

0

Cu

O N

an

o

79

.55

0

.25

0.0

19

8

2.0

C

u

0.5

0

.03

Zn

O N

an

o

81

.39

1

.0

0.0

81

39

2

.0

Zn

2

.0

0.1

3

130

ISSN : 0972-5210 Plant Archives

R.N.I. No. UPENG/2001/5119

(International Journal of Plant Research) Dr. R.S. Yadav, Principal, Ch. Charan Singh P.G. College, Heonra (Saifai), Etawah – (U.P.)

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I thankfully acknowledge the receipt of your manuscript. You are requested to refer the para(s)

ticked (✔) below :

✔ 1. I am pleased to inform you that your manuscript entitled “Morphological and biochemical

responses of soybean (Glycine max) plants fertilized with nano-iron (Fe3O4) micronutrient” by Milap Ram

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Your paper will appear in Vol 16 No.2 October 2016.

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