EFFECT OF ZINC OXIDE NANOPARTICLES ON ......Effect of Zinc Oxide Nanoparticles on Germination, Growth and Yield of Maize (Zea mays L.) Name of student Major Guide Pankaj Kumar Tiwari

EFFECT OF ZINC OXIDE NANOPARTICLESON GERMINATION, GROWTH AND YIELD

OF MAIZE (Zea mays L.)

BYPANKAJ KUMAR TIWARI

M. Sc. (Agri.)

DEPARTMENT OF SOIL SCIENCE & AGRICULTURAL CHEMISTRYB. A. COLLEGE OF AGRICULTURE

ANAND AGRICULTURAL UNIVERSITYANAND - 388 110 (GUJARAT, INDIA)

2017

Reg. No. : 04-1600-2011/15

PAN

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EFFECT OF ZINC OXIDE NANOPARTICLESON GERMINATION, GROWTH AND YIELD

OF MAIZE (Zea mays L.)

ATHESIS

SUBMITTED TO THEANAND AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTSFOR THE AWARD OF THE DEGREE

OF

Doctor of Philosophy(AGRICULTURE)

IN

SOIL SCIENCE & AGRICULTURAL CHEMISTRY

BYPANKAJ KUMAR TIWARI

M. Sc. (Agri.)

DEPARTMENT OF SOIL SCIENCE & AGRICULTURAL CHEMISTRYB. A. COLLEGE OF AGRICULTURE

ANAND AGRICULTURAL UNIVERSITYANAND - 388 110 (GUJARAT, INDIA)

2017

Reg. No. : 04-1600-2011/15

Dedicated to My Beloved Parents,Wife

&Respected Guide

Dedicated to My Beloved Parents,Wife

&Respected Guide

ABSTRACT

Effect of Zinc Oxide Nanoparticles on Germination, Growthand Yield of Maize (Zea mays L.)

Name of student Major GuidePankaj Kumar Tiwari Dr. K. P. Patel

Department of Soil Science and Agricultural ChemistryB. A. College of Agriculture

Anand Agricultural University, Anand – 388 110

ABSTRACT

The present investigation was undertaken to investigate the effect of ZnO

nanoparticles (ZnO NPs) on seed germination, growth and yield of maize. The study

included four sequential experiments: (1) synthesis and characterization of ZnO NPs;

(2) effect of different concentrations of ZnO NPs on germination of maize seeds; (3)

effect of seed treatment with ZnO NPs on growth and yield of maize; and (4) effect of

foliar application of ZnO NPs on growth and yield of maize under microplot conditions.

In first experiment, ZnO NPs were synthesized, using oxalate decomposition

method and characterized by XRD, TEM, SEM, DLS, TGA and UV-vis spectroscopy

analysis. The instrumental analysis results clearly indicated that synthesized ZnO NPs

were of 65 nm particle size, nanorods, monodispersed, highly pure, and stable. The

particle size estimated by XRD and DLS were in good agreement with TEM, SEM and

UV-Vis spectroscopy results. Thermo-gravimetric analysis (TGA) results confirmed

the calcination temperature as more than 400 °C.

Synthesized ZnO NPs were tested for their efficacy for seed treatment of maize

in second experiment where in 3 levels each of ZnO NPs and bulk ZnO concentration

(500 ppm, 1000 ppm and 2000 ppm) along with ZnO slurry were repeated thrice in

completely randomized design (CRD). Seed germination test was carried out by paper

towel method of seed incubation for 9 days following standard protocol. Soaking time

for maize seeds with different Zn treatment was optimized at 2 hrs as there was no

significant difference from 4 hrs soaking with respect to seed vigour. Results revealed

that ZnO NPs at 1000 ppm concentration significantly increased seed germination, root

length and seedling vigour index over no Zn. However, higher concentration of ZnO

nanoparticles i.e. 2000 ppm reduced the root length and seed vigour.

Abstract

ii

Consequently, microplot study was conducted during Rabi and repeated during

summer seasons of the year 2015–2016 with 8 seed Zn treatments: no Zn; 500, 1000,

2000 ppm concentrations each of ZnO NPs and bulk ZnO, and ZnO slurry replicated

three times in CRD. Results of this experiment indicated that seed treatment with ZnO

NPs at 1000 ppm registered the highest grain, stover, and dry matter yield of maize.

Further, seed treatment with ZnO NPs either at 1000 and 2000 ppm recorded the highest

and statistically at par enhancement in grain, stover and root Zn concentrations. Zinc

uptake, partitioning and accumulation factor results corroborated the higher Zn

accumulation in grain. However, higher concentration of ZnO NPs caused detrimental

effect on germination and yield of maize. Important soil properties viz. pH, EC, OC (%)

and DTPA-extractable micronutrients contents were not affected significantly by any

of seed Zn treatments.

The effect of foliar application of three levels ZnO NPs (500, 1000, 2000 ppm)

along with corresponding concentrations of bulk ZnO and 0.5% ZnSO4 on maize was

investigated under microplot conditions for two consecutive seasons. Results suggested

that two foliar application of ZnO NPs to maize at 30 and 45 days of sowing proved to

be significantly superior in enhancing grain, stover and dry matter yield of maize, grain,

stover and root Zn concentration and uptake by maize, however, the results were at par

with 2000 ppm ZnO NPs. Like seed treatment experiment, ZnO NPs application did

not show any significant change in soil properties like pH, EC, OC (%) and DTPA-Zn.

The overall finding suggested that seed treatment with ZnO NPs at 1000 ppm

proved effective in increasing seed germination, seedling length, seedling vigour, plant

growth, grain, stover, dry matter yield, grain Zn concentration of maize. yield, stem and

root growth. If applied foliarly, ZnO NPs at 1000 ppm registered significantly enhanced

grain yield, Zn content and uptake by maize crop however, higher dose i.e. 2000 ppm

proved statistically at par. Thus, use of ZnO NPs at 1000 ppm was found beneficial in

increasing growth parameters and yield of maize over traditional application through

ZnSO4. However, the delivery mechanism may be improved upon to avoid health

hazards, if any due to the use of nanoparticles.

Dr. K. P. PatelPrincipal & DeanB. A. College of AgricultureAAU, Anand, Gujarat (India)

CERTIFICATE

This is to certify that the thesis entitled “Effect of Zinc Oxide

Nanoparticles on Germination, Growth and Yield of Maize ( Zea mays L.)”

submitted by Pankaj Kumar Tiwari (Reg. No. 04-1600-2011/15) in partial

fulfillment of the requirements for the award of the degree of

Doctor of Philosophy in the subject of Soil Science and Agricultural

Chemistry of B. A. College of Agriculture, Anand Agricultural University,

Anand is a record of bonafide research work carried out by him under my

personal guidance and supervision and the thesis has not previously formed the

basis for the award of any degree, diploma or other similar title.

Place: Anand (K. P. Patel)Date: .04.2017 Major Guide

ANAND AGRICULTURAL UNIVERSITYB. A. COLLEGE OF AGRICULTURE

ANAND – 388 110

DECLARATION

This is to declare that the whole of the research work reported in the thesis

entitled “Effect of Zinc Oxide Nanoparticles on Germination, Growth and

Yield of Maize (Zea mays L.)” for the partial fulfillment of the requirement for

the degree of Doctor of Philosophy (Agriculture) in the subject of Soil Science

and Agricultural Chemistry is the results of investigation done by the

undersigned under the direct guidance and supervision of Dr. K. P. Patel,

Principal & Dean, B. A. College of Agriculture, Anand Agricultural University,

Anand and no part of work has been submitted for any other degree so far.

Place : Anand (Pankaj Kumar Tiwari)

Date : .04.2017

COUNTER SIGNED BY

Dr. K. P. Patel

Principal & DeanB. A. College of Agriculture

Anand Agricultural UniversityAnand (Gujarat)

ACKNOWLEDGEMENT

This memorable occasion provides me a unique privilege to express my sincere

and deep sense of gratitude and respect to my major guide, Dr. K. P. Patel, Principal and

Dean, B. A. College of Agriculture, A.A.U, Anand for enlightening me the first glance of

research and allowing me to grow as a researcher. His endless support, untiring effort,

constructive criticism and persistent guidance have always given me courage

throughout the course of investigation and in the preparation of the manuscript. His

advice on both research as well as my career has been priceless.

I am deeply grateful to the members of my advisory committee, Dr. Y. M. Shukla,

Co-Guide Principal, College of Agriculture and Polytechnic in Agriculture, Anand

Agricultural University, Vaso, Dr. V. P. Ramani, Associate Research Scientist,

Micronutrient Research Project (ICAR), Dr. K. C. Patel, Associate Professor, Department

of Soil Science and Agricultural Chemistry, and Dr. P. R. Vaishnav, Professor & Head,

Department of Agricultural Statistics, B.A.C.A., A.A.U., Anand, for their technical

guidance, innovative ideas, valuable suggestions, encouragement and moral support

received throughout the course of investigation.

I would like to place my sincere gratitude to Dr. A. K. Shukla, Project

Coordinator (Micronutrients), ICAR-Indian Institute of Soil Science and Dr. V. R. Bhatt,

Professor and Head, Department of Soil Science and Agricultural Chemistry for their

ardent generosity to provide valuable suggestions and inspiration during my research

work.

I am extremely thankful to Dr. N. J. Jadav (Associate Professor) and

Er. Bhavin Ram (Assistant Professor) Technical Officers, Principal Office, B. A. College

of Agriculture, Anand Agricultural University, Anand for their co-operation throughout

my research work and pursuance to complete my research work expeditiously. I

gratefully acknowledge Dr. Dileep Kumar and Dr. G. J. Mistry, Assistant Research

Scientists, Micronutrient Research Project (ICAR), Anand Agricultural University, Anand

for their support and cooperation during my research work.

I place my sincere gratitude towards SSCSSN, Dharamsingh Desai Institute of

Technology (DDIT), Nadiad and Laboratory for Advanced Research in Polymeric

Materials (LARPM), CIPET, Bhubaneswar for the characterization of nanoparticles. I

am, personally obliged to Dr. Atindra Shukla, Professor (SSCSSN), DDIT, Nadiad and

Dr. Sunil Shah, Professor (SSCSSN), DDIT, Nadiad, for their ardent generosity to

provide valuable suggestions and technical guidance during the synthesis and

characterization of nanoparticles.

I avail this opportunity to express my profound thanks to Vijaybhai, Priteshbhai,

Vinukaka, Rameshbhai, Harishbhai, Baldev, Vikram, Mahipat, Suresh, Chandrakant,

Pritesh and all staff members of Micronutrient Project, (ICAR) for their kind cooperation

during the course of study and laboratory analysis works.

I shall always remain indebted to good-humoured friends Vimal, Hardik, Krunal,

Pratik, Gobinath, Shyam, Jugal, Dharmendra, Ramesh, Sangram, Sumit, Pradeep, Pawan,

Chandrakant Singh, Ravikiran, Arunbhai, Bhupendra, Nilesh, Punit, Yogesh, Ravi, Sagar,

Samar, Aniket, Altafbhai and Dhunilal for their unstinted cooperation, moral support

that rendered help during my stay in milk city Anand.

A part of my larger interest lies with my intimate colleagues and friends

Dr. N. K. Sinha, Dr. B. P. Meena, Dr. J. K. Thakur, Dr. A. O. Shirale, Siddique, Venny and

staff of AICRP on Micronutrients, ICAR-Indian Institute of Soil Science, Bhopal for their

keen interest in my career and extending their constant help and encouragement for

every arena of difficulty.

Words are quite inadequate to express my gratitude and indebtedness to my

family for their sacrifices, understanding and support. I particularly express my larger

debt and devotion to my father Shri. Adya Tiwari, mother Smt. Saraswati Devi, Brothers

Shri. Anil and Arun and Sisters Poonam, Sunita and Seema for the inspiration which

enabled me to complete this long cherished work.

My felicitousness overwhelms to express my deepest sense of reverence and

indebtedness to my treasured wife Dr. Priyanka Pandey Tiwari, my father-in law

Er. Shri. K. N. Pandey, brother-in laws Ashish and Shikhar and all members of my

extended family for their love, encouragement and prayers who sustained my spirit and

endeavour at every critical juncture of my educational career.

I gratefully acknowledge the Indian Council of Agricultural Research (ICAR)

and ICAR- Indian Institute of Soil Science, Bhopal for allowing me to complete my

Ph. D. study at A.A.U., Anand.

Place: Anand

Dated: April 15, 2017 (Pankaj Kumar Tiwari)

Chapter Title Page

No.

I. INTRODUCTION 1-8

II. REVIEW OF LITERATURE 10-38

2.1 Nanotechnology — Zinc Nutrition in Plants 10

2.2 Synthesis and Characterization of ZnO NPs 12

2.2.1 Top-down Methods 12

2.2.2 Bottom-up Methods 14

2.3 ZnO Nanoparticle — Seed Treatment 20

2.4 ZnO nanoparticle — Foliar, Nutrient Solution and

Soil Application27

2.4.1 Foliar Application 27

2.4.2 Nutrient Solution Application 32

2.4.3 Soil Application 34

III. MATERIALS AND METHODS 39-53

3.1 Laboratory studies 39

3.1.1 Synthesis and Characterization of ZnO

Nanoparticles39

3.1.1.1 Synthesis of ZnO Nanoparticles 39

3.1.1.2 Characterization of ZnO Nanoparticles 40

X-ray Diffraction Analysis (XRD) 41

Dynamic Light Scattering (DLS) 41

Scanning Electron Microscopic Analysis (SEM) 42

Transmission electron microscopy (TEM) 42

UV-vis Spectroscopic Analysis 43

Thermogravimetric Analysis (TGA) 43

3.1.1.3 ZnO NPs suspension preparation 43

3.1.2 Effect of seed treatment with ZnO NPs on

germination of maize seeds44

3.1.2.1 Seed 44

3.1.2.2 Seed treatment 44

CONTENTS

3.1.2.3 Seed germination 45

3.1.2.4 Optimization of seed soaking time 45

Shoot length (cm) 45

Root length (cm) 46

Germination percentage (%) 46

Seedling Vigour Index (SVI) 46

3.2 Microplot Studies 46

3.2.1 Experimental site 46

3.2.2 Climate and weather conditions 47

3.2.3 Physico-chemical properties of soil 47

3.2.4 Seed 48

3.2.5 Treatment Details 48

3.2.5.1 Effect of seed treatment with ZnO NPs on growth

and yield of maize48

3.2.5.2 Effect of foliar application of ZnO NPs on growth

and yield of maize49

3.2.6 Sowing, Fertilizers, Intercultural Operations and

Harvesting50

Sowing 50

Fertilizers and Manure 51

Irrigation, weeding and plant protection 51

Harvesting 51

3.2.7 Soil and Plant Samples Analysis 52

Soil Sampling and Analysis 52

Plant Sampling and Analysis 52

3.2.8 Computation of Nutrient uptake and Accumulation

Factor53

3.3 Statistical analysis 53

IV. RESULTS AND DISCUSSION 54-107

4.1 Synthesis and Characterization Of ZnO NPs 54

4.1.1 X-Ray Diffraction (XRD) 55

4.1.2 Scanning Electron Microscopy (SEM) 56

4.1.3 Transmission Electron Spectroscopy (TEM) 57

4.1.4 UV-vis Spectroscopy 58

4.1.5 Thermo-gravimetric Analysis (TGA) of Zinc Oxalate 58

4.1.6 Dynamic Light Scattering (DLS) 60

4.2 Effect of Seed Treatment with ZnO NPs on

Germination of Maize Seed63

4.2.1 Seed Germination 63

4.2.2 Root and Shoot Length of Seedlings 66

4.2.3 Seed Vigour Index 67

4.3 Effect of Seed Treatment with ZnO NPs on Growth

and Yield Of Maize70

4.3.1 Seed Germination (%) of Maize Seeds 70

4.3.2 Grain and Stover Yield 72

4.3.3 Zinc Concentration 77

4.3.4 Zinc Uptake 81

4.3.5 Zinc Uptake Partitioning and Bioaccumulation Factor 82

4.3.6 Soil Parameters after Harvest 85

Soil pH, EC and OC 86

DTPA-extractable micronutrients 88

4.4 Effect of Foliar Treatment with ZnO NPs on

Growth and Yield of Maize91

4.4.1 Grain and Stover Yield 91

4.4.2 Zinc Concentration 95

4.4.3 Zinc uptake 98

4.4.4 Zinc Uptake Partitioning and Bioaccumulation Factor 100

4.4.5 Soil Parameters after Harvest 102

Soil pH, EC and OC 102

DTPA-extractable micronutrients 105

V. SUMMARY AND CONCLUSION 108-116

References i-xxv

Table

No.Title

Page

No.

3.1 Physico-chemical properties of the soil used in microplot studies 48

4.1Effect of different Zn treatments and soaking time on seed germination

(%) of maize64

4.2Effect of different Zn treatments and soaking time on root and shoot

length (cm) of maize seedlings66

4.3Effect of different Zn treatments and soaking time on seed vigour

index of maize seedlings68

4.4Effect of different Zn seed treatments on germination (%) of maize

seeds71

4.5 Effect of different Zn seed treatments on grain yield of maize 73

4.6 Effect of different Zn seed treatments on stover yield of maize 74

4.7Effect of different Zn seed treatments on total dry matter yield of

maize75

4.8Effect of different Zn seed treatments on grain Zn concentration of

maize77

4.9Effect of different Zn seed treatments on stover Zn concentration of

maize79

4.10Effect of different Zn seed treatments on root Zn concentration of

maize79

4.11 Effect of different Zn seed treatments on soil pH after harvest of maize 86

4.12 Effect of different Zn seed treatments on soil EC after harvest of maize 87

4.13 Effect of different Zn seed treatments on soil OC after harvest of maize 87

4.14Effect of different Zn seed treatments on DTPA-extractable Zn and Fe

contents in soil after harvest of maize89

4.15Effect of different Zn seed treatments on DTPA-extractable Mn and

Cu contents in soil after harvest of maize90

4.16 Effect of different foliar Zn treatments on grain yield of maize 92

4.17 Effect of different foliar Zn treatments on stover yield of maize 93

LIST OF TABLES

4.18Effect of different foliar Zn treatments on total dry matter yield of

maize93

4.19Effect of different foliar Zn treatments on grain Zn concentration of

maize95

4.20Effect of different Zn foliar Zn treatments on stover Zn concentration

of maize96

4.21Effect of different foliar Zn treatments on root Zn concentration of

maize97

4.22Effect of different foliar Zn treatments on soil pH after harvest of

maize103

4.23Effect of different foliar Zn treatments on soil EC after harvest of

maize104

4.24Effect of different foliar Zn treatments on soil OC after harvest of

maize104

4.25Effect of different foliar Zn treatments on DTPA-extractable Zn and

Fe contents in soil after harvest of maize106

4.26Effect of different foliar Zn treatments on DTPA-extractable Mn and

Cu contents in soil after harvest of maize106

FigureNo.

TitleAfterpageNo.

3.1 Flow chart of synthesis of ZnO nanoparticles by oxalate

decomposition method

40

4.1 X-Ray diffraction pattern of ZnO Nanoparticles 55

4.2 SEM micrographs of ZnO NPs 56

4.3 TEM micrographs of ZnO NPs 57

4.4 UV-vis spectra of ZnO NPs 58

4.5 Thermogravimetric analysis of zinc oxalate molecule 59

4.6 Particle size distribution of the ZnO nanoparticles 61

4.7 Zeta potential of ZnO nanoparticles 62

4.8 Zn uptake by grain as influenced by different Zn seed treatments 81

4.9 Zn uptake by stover as influenced by different Zn seed

treatments81

4.10 Zn uptake by root as influenced by different Zn seed treatments 82

4.11 Zn uptake partitioning in different plant parts of maize as

influenced by different Zn seed treatments83

4.12 Zn bioaccumulation in maize plant as influenced by different Zn

seed treatments85

4.13 Zn uptake by grain as influenced by different foliar Zn

treatments98

4.14 Zn uptake by stover as influenced by different foliar Zn

treatments99

4.15 Zn uptake by root as influenced by different foliar Zn treatments 99

4.16 Zn uptake partitioning in different plant parts of maize as

influenced by different foliar Zn treatments101

4.17 Zn bioaccumulation in maize plant as influenced by different

foliar Zn treatments101

LIST OF FIGURES

PlateNo.

TitleAfter

Page No.

4.1Effect of different seed Zn treatments on germination ofmaize seeds (5th Day of incubation)

64

4.2Effect of different seed Zn treatments on germination ofmaize seeds (9th Day of incubation)

66

4.3Effect of different seed Zn treatments on seedling length andvigour of maize seeds

68

LIST OF PLATES

INTRODUCTION

I. INTRODUCTION

Maize (Zea mays L.), which is widely cultivated throughout the world and has

the highest production among all the cereals, is one of the most important cereal crops

of the world and contributes to food security in almost all the developing countries.

Maize, also known as “queen of cereals” is by far the largest component of global

coarse-grain trade and its importance lies in the fact that it is not only utilized for human

food and animal feed but at the same time it is also widely used for corn starch industry,

corn oil production, baby corns etc. The crop has tremendous genetic variability, which

enables it to thrive in tropical, subtropical and temperate climates. The worldwide

production of maize was more than 960 million MT in 2013-14 (FICCI, 2014).

In India, maize is emerging as third most important crop after rice and wheat

and it accounts for about 10% of total food grain production in the country. Though

maize is grown throughout the year in India but it is predominantly a kharif crop with

85% of the area under cultivation in the season. Maize production in India has increased

at a compound annual growth rate (CAGR) of 5.5% over the last ten years from 14

million MT in 2004-05 to 23 million MT in 2013-14. The area under maize cultivation

in the period has increased at a CAGR of 2.5% from 7.5 million ha in 2004-05 to 9.4

million ha in 2013-14 (FICCI, 2014).

Maize production is dominated by Andhra Pradesh and Karnataka along with

seven other states viz. Tamil Nadu, Rajasthan, Maharashtra, Bihar, Uttar Pradesh,

Madhya Pradesh and Gujarat and they account for about 85% of India’s maize

production and 80% of area under cultivation. In Gujarat, Mehsana, Banaskantha,

Rajkot and Kheda districts in the command areas of the Sabarmati and Mahi rivers are

the main producers contributing over 55% of the state’s production (Anonymous,

2011).

Introduction

2

Despite all the technological, varietal and mechanization interventions in maize

cultivation, its productivity in the country is half of the global average. The major

constraints for low productivity include: climatic variations resulting in drought or

excess water; increased pressure of diseases and pests; low adoption of single cross

hybrid; small farm holdings and limited resource availability; limited adoption of

improved production-protection technology; which deprives crop from proper

nutrients, especially micronutrients availability. Unsustainable intensification

accompanied by imbalanced soil nutrient management is one of the major causes of

declining productivity of crops and land degradation in the country (Shukla et al. 2016).

An increase in the productivity of a crop can be achieved either by increasing

the area under cultivation or by increasing the productivity per unit area. Since the area

is limited, yield level per unit area needs to be augmented to ensure food security of a

nation. Micronutrients play a significant role in plant growth and metabolic processes

associated with photosynthesis, chlorophyll formation, cell wall development,

respiration, water absorption, xylem permeability, resistance to plant diseases, enzyme

activities involved in the synthesis of primary and secondary metabolites along with

nitrogen fixation and reduction (Adhikary et. al., 2010; Vitti et al., 2014).

Micronutrient deficiencies in plants may lead to reduced yields and, in severe

cases, to plant death, also. Among the micronutrients, Zn deficiency is the most

detrimental to crop growth and yield of all the cereal crops including maize (Alloway,

2008; Marschner, 1995). Zinc, the 2nd most abundant transition metal in organisms after

Fe is generally absorbed as a divalent cation (Zn+2) by higher plants.

Zinc acts either as the metal component or as a functional, structural or a

regulatory co-factor of a large number of enzymes. For example, Zn is involved in a

number of fundamental functions in plant systems such as synthesis of indole-acetic

Introduction

3

acid (IAA), a phytohormone which dramatically regulates plant growth, protein

synthesis and function, detoxification of reactive oxygen species (ROS), chlorophyll

and carbohydrate synthesis, biosynthesis of cytochrome (a pigment that maintains the

plasma membrane integrity) and synthesis of leaf cuticle, reduces the uptake of heavy

metals such as cadmium (Cd) (Marschner, 1995; Buchanan et al., 2000; Cakmak,

2008a).

When facing Zn shortage, plants undergo a range of physiological and

molecular adjustment in order to maintain cellular homeostasis and to avoid abrupt

changes in the dynamic and complex process of development (Grusak, 2002).

Therefore, many physiological processes are adversely affected when plants are

exposed to Zn deficiency resulting in significant decrease in both productivity and

nutritional quality (Cakmak, 2008b). The most common symptoms of Zn deficiency in

maize include the development of whitish or yellowish stripes parallel to the midrib on

the young leaves and stunting appearances with shortened internodes. Necrotic spots

and reddish colour may develop on leaves at the advanced stage of Zn deficiency.

The deficiency of Zn in Indian as well as world soils is very well documented

constraint in crop production and since last couple of decades, it is considered to be the

4th most yield limiting nutrient after N, P, and K, respectively in India (Sillanpaa, 1990;

Katyal and Sharma, 1991; Singh, 2009; Shukla et al., 2014). Recent Indian studies also

report extensive deficiency of Zn in farms due to regular withdrawal of these nutrients

through crop uptake without sufficient replenishment (Shukla et al., 2014; Shukla et

al., 2015). Low plant-available Zn was reported for soils of various characteristics: high

and low pH, high and low organic matter, calcareous, sodic, sandy, wetland or ill-

drained, limed acid soils, etc. (Rehman et al., 2012). In cereal crops like maize, Zn

Introduction

4

deficiency is common in neutral to alkaline pH soils containing medium organic matter

and intensive cultivation without Zn replenishment.

Response to Zn application to maize has been reported from several countries

of the world (Alloway, 2008; Potarzycki and Grzebisz, 2009; Hossain et al., 2011) as

well as different states of our country (Singh and Behera, 2011; Rattan et al. 2008). Its

application significantly influenced all the yield attributes of maize viz. plant height,

cob length, test weight, number of grain per cob, shelling percentage and grain yield,

resulting in an average 10 to 20% increase in yield (Arya and Singh, 2000; Raskar et

al., 2012; Shukla et al., 2014).

Reviewing the data from several research centres in India, Rattan et al. (2008)

reported an average response to Zn application to the tune of 670 kg ha-1 in maize,

across the country. Based on several reviews, Shukla and Behera (2012) predicted that

additional production due to Zn fertilization could be about 17 Mt, which is 7.6 % of

the total cereal production in 2010-2011. Thus, adequate Zn fertilization can certainly

help in increasing cereal production in the country (Prasad et al., 2013).

Besides, enhancing grain yields of different crops, Zn supplementation is also

essential for maintaining optimum Zn content in human and animal (Singh, 2009,

Shukla et al., 2014). Maize, however, is very poor in protein and micronutrients

concentrations, especially Zn. Therefore, in countries like India where maize

consumption is very high, the incidence of micronutrient malnutrition particularly, Zn

is also very high. The enrichment of maize with high levels of Zn is a growing global

challenge in order to contribute to the well-being of human populations who rely on

maize for their nourishment.

Introduction

5

In Indian scenario, Zn deficiency is usually corrected by application of ZnSO4,

Zn chelates like Zn-EDTA, and ZnO which can be applied to crops via different

methods viz. seed treatment, soil application, foliar application etc. However, most of

these Zn fertilizers solubilize relatively slowly in soil, which in some cases may be too

slow to supply adequate amounts required for the vigorous plant growth (Rengel, 2002;

Rehman et al., 2012).

Even when soluble salts like ZnSO4 are used, soil equilibria result in conversion

of released Zn into less soluble forms, generally carbonates, oxides and various

hydroxides (Alloway, 2009). A number of studies evaluated the fertilizer effectiveness

of various synthetic and natural chelates as Zn carriers. Prasad and Sinha (1981) gave

the following order of relative efficiency with respect to yield and Zn uptake by maize

from a calcareous soil: Zn-DTPA > Zn-fulvate > Zn-EDTA > Zn-citrate > Zn-sulphate,

which reflects the stability of these compounds.

Besides soil application, foliar Zn supplementation has also proved to be vital

in alleviating Zn deficiency in crops, especially cereals like maize. However, low

penetration rates in thick leaves, rapid drying of spray solution, limited translocation

within the plant, and leaf damage are the problems of concern as most foliar applied Zn

carriers are not efficiently transported towards the roots (Marschner, 1995).

Concentrated liquid suspensions of ZnO are used for foliar application but their

performance is strongly determined by the size range specifications of the ZnO particles

present in the formulation. Leaf water repellency of adaxial or abaxial surface is also a

limiting factor, which can affect the Zn uptake through spray application processes

(Watanabe and Yamaguchi, 1991; Holder, 2007).

Introduction

6

Particle size of Zn fertilisers greatly influences their agronomic effectiveness.

Decreased particle size results in increased number of particles per unit weight of

applied Zn. Decreased particle size also increases the specific surface area of a fertilizer,

which increase the dissolution rate of fertilizers with low solubility in water such as

ZnO (Mortvedt, 1992). Granular ZnSO4 (1.4 to 2 mm) was somewhat less effective

than fine ZnSO4 (0.8 to 1.2 mm) whereas granular ZnO was completely ineffective.

Gradual increase in Zn uptake could be observed with decreasing granule size

and only the powder form could produce plants with Zn concentrations in the sufficient

range. Since granules of 1.5 mm size weigh less than that of 2.0 or 2.5 mm, smaller

granules were used for the same weight, resulting in a better distribution of Zn, and the

higher surface area of contact of Zn fertilizer resulted in better Zn uptake (Liscano et

al., 2000). It is evident that ample work has been done on the particle size of Zn carriers

with an aim to increase the efficiency of the fertilizers for better uptake and higher

yields.

In recent years, nanoscience and nanotechnology, which refer to the growing

knowledge base and technical framework for understanding and manipulating matter

on nanometer scales ranging from the atomic to the cellular, have been ascendant on

the world stage of science and technology (Bai, 2005). It is a fast-developing industry,

posing substantial impacts on economy, society and environment (Brumfiel, 2003;

Roco, 2005; Yang et al., 2006). Nanomaterials are increasingly being used for

commercial purposes such as fillers, opacifiers, catalysts, semiconductors, cosmetics,

microelectronics, and drug carriers (Biswas and Wu, 2005).

Nanoparticles (NPs) have always existed in our environment, from both natural

and anthropogenic sources. Nanoparticles in air were traditionally referred to as

ultrafine particles, while in soil and water they were colloids, with a slightly different

Introduction

7

size range (Klaine et al., 2008). Nanoparticles are atomic or molecular aggregates with

at least one dimension between 1 and 100 nm (Roco, 2003), that can drastically modify

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

Nanoparticles can be made from a variety of bulk materials and they can

explicate their actions depending on both the chemical composition, size and/or shape

of the particles (Monica and Cremonini, 2009). The field of Soil Science is gradually

emerging out as a frontier area of research in nanotechnology; because many natural

components of soil like soil colloids, clay fraction, microorganisms, nutrients etc. also

fall in the size range of nanoparticles.

Zinc oxide nanoparticles (nano-ZnO) is a commonly used metal oxide

engineered nano particles. In addition, nano scale ZnO is one of the five Zn compounds

that are currently listed as GRAS (Generally Recognized As Safe) by USFDA (United

States Food and Drug Administration). It usually appears as a white powder and is

sparingly soluble in water. Zinc oxide is used in a range of applications such as

sunscreens and other personal care products, electrodes and bio-sensors, photo-

catalysis and solar cells (Kumar and Chen, 2008). Owing to increasing use in consumer

products, it is likely that through both deliberate application and accidental release,

engineered NPs will find their way into aquatic, terrestrial and atmospheric

environments.

Zinc oxide nanoparticles (ZnONPs) with small size and large surface area are

expected to be the ideal candidates for use as a Zn fertilizer in plants (Adhikari et al.,

2015). There is a huge scope of research on biological effects of nanoparticles on higher

plants. Several studies are concerned with the synthesis of nanomaterials using

biological routes. Most of these studies are focused on the potential toxicity of

engineered NPs to plants and both positive and negative or inconsequential effects have

Introduction

8

been reported. However, majority of the reports available in the literature indicate

phytotoxicity of engineered NPs (Lin and Xing, 2007; Ma et al., 2010; Rico et al., 2011;

Wang et al., 2012; Zhang et al., 2012). Limited studies have been reported on the

promotory effects of metal nanoparticles on plants in low concentrations.

To address the issues relating to increase fertilizer use efficiency of Zn,

development of new agricultural technologies is crucial in meeting the ecological needs

and achieving the anticipated food demands of the growing population in the near

future. In this context, nanotechnology in soil science has to be introduced, which is

likely to bring a sea-change in the production of fertilizers, thereby expected to improve

agricultural production and productivity. Moreover, in order to understand the possible

benefits of applying nanotechnology to agriculture, the first step should be to analyze

penetration and transport of nanoparticles in plants. Against this backdrop, a sequential

study was taken up to investigate the promotory and/ or inhibitory effects of various

concentrations of ZnO nanoparticles (ZnONPs) on growth, development and yield of

maize (Zea mays L.) with following objectives:

1. To synthesize zinc oxide nanoparticles (ZnONPs) and characterize for size and

morphological characteristics.

2. To study the efficacy of ZnONPs seed treatment in maize at different levels and

its effect on seed germination and to estimate the optimum soaking time.

3. To investigate the effect of different levels of ZnONPs seed treatment on growth

and yield of maize.

4. To study the effect of foliar application of ZnONPs at different levels on growth

and yield of maize.

REVIEW OF LITERATURE

II. REVIEW OF LITERATURE

Nanotechnology is a multidisciplinary and rapidly growing field in the area of

science and technology which involves the manufacture, processing and application of

nanometer scale assemblies of atoms and molecules. Nanomaterials are generally

defined as materials with at least one dimension less than 100 nm (Powers et al., 2006).

Due to their extremely small size and greater surface activity, they possess unique

physical and chemical characteristics which deviate vastly from those of individual

atoms or molecules and also the same material at bulk scale. Therefore, their reactivity

enables them to have novel applications in different sectors (Banfield and Zhang, 2001).

As there have been very few studies on the fate of nano-scale materials in

terrestrial environments, it is necessary to conduct research on the solution,

transformation, diffusion, mobility and availability of these materials in these complex

systems including soil. Accordingly, focus of this thesis was to develop a better

understanding of the reactions of ZnO nanoparticles (ZnO NPs) in the soil-plant system

in order to evaluate the possibilities of its use as a source of Zn to improve crop yield

and Zn contents in maize. Keeping all these facts in view, the available literatures

related to present investigation have been reviewed under the following subheads:

2.1. Nanotechnology — Zinc Nutrition in Plants

2.2. Synthesis and Characterization of ZnO Nanoparticles

2.3 ZnO nanoparticles — Seed Treatment

2.3 ZnO nanoparticles — Foliar, Nutrient Solution and Soil Application

2.1 Nanotechnology — Zinc Nutrition in Plants

Nanoparticles have smaller particle sizes, higher specific surface area and an

increased proportion of reactive surface atoms as compared to bulk particles

Review of Literature

10

(Wigginton et al., 2007). These unique properties have led to their wide range of

application in the fields of energy, electronics, medicines and environmental

remediation as well in material science and nanotechnology-based industries (Baruah

and Dutta, 2009; Rickerby and Morrison, 2007; Aitken et al. 2006; Liu, 2006; Luther,

2004).

Since engineered nanoparticles do not occur naturally in the environment so,

they are intentionally synthesized for specific applications. The USEPA (2005) has

grouped manufactured nanomaterials into four types: (i) carbon (C)-based

nanoparticles that are composed entirely of C; (ii) metal-based materials such as nano-

Zn, nano-Al, and nano-scale metal oxides like TiO2, ZnO, Fe2O3, Al2O3; (iii)

dendrimers; which are nano-sized polymers built from branched units capable of being

tailored to perform specific chemical functions; and (iv) composites, which combine

nanoparticles with other nanoparticles or with bulk materials.

The use of nanoparticles in agriculture is a promising area which could

potentially improve prevailing crop management techniques in long term prospective.

Use of nano-capsulated pesticides have been successfully applied to release chemicals

in controlled and specifically targeted manner which provides a safer and easier control

system for pests (Beddington, 2010; Nair et al. 2010). In near future, nanotechnology

tools may provide smart devices that are capable of soil monitoring which will enable

early remedial actions and synchronization of delivering agricultural inputs, especially

chemicals, precisely according to plant needs (Dewick et al. 2004; DeRosa et al. 2010;

Nair et al. 2010).

One potential application of nanotechnology in soil science is to address issues

related to micronutrients deficiency, which is one of the major problem in agricultural

productivity. Zinc deficiency is the most extensive micronutrient problem in Indian


11

soils as almost 40.0% of the soils are deficient in Zn availability (Shukla et al. 2016).

The solubility and particle size of Zn source in the conventional Zn fertilizers are among

the main parameters that determine their effectiveness (Mortvedt, 1992).

Application of nanotechnological tools in Zn fertilizer formulations may

improve their performance in enhancing crop yields. Since the dissolution kinetics of

particles depends on surface area, it is expected that rate and extent of dissolution is

greater for nanoparticles compared to that of micron sized particles and/ or bulk

materials (Borm et al., 2006). Mortvedt (1992) also cited that fine ZnSO4 particles

(<0.15 mm in diameter) were more effective than larger ZnSO4 particles (1.4-2.0 mm

and 0.8-1.2 mm in diameter). Additionally, when smaller Zn particles are used, the

number of Zn particles per unit of applied Zn to soil would increase. Owing to all these

characteristics, nano Zn formulations are expected to enhance dissolution rate of Zn

sources, especially in Zn sources with lower solubility such as ZnO.

In addition, ZnO nanoparticles is the most common Zn nanomaterial which is

being used as UV protector (e.g. in personal care products, coatings and paints),

biosensors, electronics, and rubber manufacture (Brayner et al., 2010; Kool et al.,

2011). The wide range of industrial applications for ZnO nanoparticles can predict

future increase in the production volume of these nanoparticles by developing

economical synthesis methods and reducing the manufacture costs. Hence, economical

application of ZnO nanoparticles as Zn fertilizers can turn out to be practical in large

scale globally.

Moreover, nanotechnology may assist fertilizer industry by designing Zn

fertilizers which could release Zn on demand and therefore preventing the interactions

of Zn in soil with soil compartments, water and microorganisms which reduce

availability of Zn for crops (DeRosa et al., 2010). However, it is important to consider


12

that different properties of soils (pH, ionic strength, organic matter, solid phases etc.)

may strongly affect the fate of nanoparticles in the soil. Therefore, the behaviour of

nanoparticles can deviate from the ones theoretically expected.

So far, research on the transformation of ZnO nanoparticles in soil-plant system

is in infancy stage. Hence, it is critical to develop understanding on the fate and

behaviour of ZnO NPs in soils and their possible influence on the Zn uptake by plants

in Zn deficient area. A proactive understanding of the environmental impact and fate of

nanotechnology-based products is needed to ensure safe and sustainable use of

nanoparticles in agriculture and better management of their associated risks (Thomas

et al. 2011; Bernhardt et al. 2010; Klaine et al. 2008; Rickerby and Morrison, 2007;

Weisner et al., 2006). Thus, this present study presents the use of in-house synthesized

ZnO nanoparticles and its effect on the maize yield.

2.2 Synthesis and Characterization of ZnO Nanoparticles

There are wide variety of methods that are used to synthesize ZnO NPs, but the

fundamental approaches in nanoparticle fabrication can be categorized into two groups:

top-down and bottom-up methods. However, hybrid techniques using both of these

methods are also under exploration.

2.2.1 Top-down Methods

Top-down methods reduce macroscopic particles to nano-size scale by different

physical methods like high energy ball milling, mechano-chemical processing, etching,

electro-explosion, sonication, sputtering or laser-ablation (Luther, 2004). However,

these methods usually are not suitable for generating uniformly shaped nanoparticles

(Schmid, 2001).


13

It has been shown that there is an equilibrium limit to the size of particle that

could be achieved by mechanical grinding, such as ball milling, and this could be as

high as 300 nm (Yokoyama and Huang, 2005). Wet grinding, using fine ceramic beads

below 30 µm in diameter was shown to be highly effective. Where materials are highly

crystalline, the size after milling could be as low as 1 to 10 nm (Klaine et al., 2008).

Metal oxanes are examples of a top-down procedure in which a mineral is cut into

smaller parts by an organic acid in aqueous solution (Weisner et al. 2006). The

nanoparticles produced may or may not have properties different from those of the bulk

material from which they were developed (USEPA, 2005; Zhang, 2003).

Shen et al. (2006) reported controlled mechano-chemical synthesis of ZnO NPs

in presence of oxalic acid and zinc acetate. The initial reactant mixture of zinc acetate

and oxalic acid was milled from 30 minutes to 4 hours and thermally treated at 450 °C

for 30 minutes. The ZnO NPs thus formed were quite uniform with size range of 20 -

40 nm. Similarly, Sun et al. (2006) also reported one step rapid synthesis of ZnO NPs

using zinc acetate, CTAB and NaOH at room temperature. In this typical method, zinc

acetate dihydrate, CTAB and NaOH were mixed (molar ratio 1:0.4:3) and ground

together in an agate mortar for 50 min at room temperature. ZnO NPs of 10-30 nm

diameters can be synthesized conveniently with this method.

However, these preparation methods are generally complicated and expensive,

especially when organo-metallic precursors, catalysts and complex process controls are

involved. Another major drawback of top-down approach is surface structure

imperfection and significant crystallographic damage to the particles. Also, the

possibility of achieving the nano-sized particles is relatively less than the bottom up

approach. However, this approach leads to the production of nano-material in bulk.


14

Regardless of the defects produced by top down approach, they continue to play an

important role in the synthesis of nano structures.

2.2.2 Bottom-up Methods

Bottom up approach refers to the build-up of a material from the bottom: atom

by atom, molecule by molecule or cluster by cluster. In other words, it represents

constructing nanomaterials from basic building blocks such as atoms or molecules

(Tavakoli et al. 2007) and usually include aggregation of atoms or molecules in solution

or in the gas to form particles with distinctive size, shape and structure (Schmid, 2001).

Colloidal dispersion is also a good example of bottom up approach in the synthesis of

nano particles.

The bottom up approach involves two fundamental methods of synthesis viz.

chemical and biological synthesis (green synthesis). Chemical synthesis involves a

direct chemical synthesis route that yields particles in the nano size range; while a

biological synthesis route comprises a plant/ plant extracts or microorganism.

Biological synthesis considered as safe alternatives to chemical methods, hence it is

also known as green synthesis. On the contrary, these methods involve extensive

process of maintaining cell cultures, intracellular synthesis and multiple purification

steps and hence, are somewhat complex than chemical synthesis methods.

Chemical synthesis of nanoparticles involves variety of methods such as

hydrothermal, sol-gel, thermal decomposition, spray pyrolysis, chemical vapour

deposition, and laser ablation. Among these techniques, the hydrothermal method has

been considered to be the most attractive due to its robust and reliable control on the

shape and size of the nanoparticles without requiring the expensive and complex

equipment. Broadly, synthesis of metal nanoparticles using bottom-up methods can be


15

achieved through approaches identified as gas phase or chemical phase synthesis

(Schmid, 2001).

The chemical routes for synthesis of nanoparticles are based on the reduction of

positively charged metal atoms by chemical reductants or decomposition of

organometallic precursors with extra energy to form atoms followed by aggregation of

atoms (Tavakoli et al. 2007). Molecular hydrogen, citrate, alcohol, borohydrides,

hydroxylamine hydrochloride, formaldehyde, carbon monoxide, and many other

reducing agents have been used as chemical reductants (Schmid, 2001).

Moreover, the energy required for decomposition of metal precursors can be

supplied through thermal energy, electricity, photoenergy (ultraviolet and visible light)

or sonochemical energy (Tavakoli et al. 2007). In addition, many other methods for

synthesizing ZnO nanoparticles have been published, such as hydrothermal synthesis,

the micro-emulsion hydrothermal process, chemical vapour deposition (CVD) and a

catalyst-free CVD method (Jiang et al. 2005; Sun et al. 2003; Wu and Liu, 2002a; Wu

and Liu, 2002b).

In a study reported by Ni et al. (2005), ZnO nanorods with the mean size of 50

nm × 250 nm were successfully synthesized via a hydrothermal synthesis route in the

presence of cetyl trimethyl ammonium bromide (CTAB) at a reaction temperature of

120 °C for 5 hours in the presence of ZnCl2 and KOH as precursors. The resultant ZnO

NPs was characterized using X-ray Diffractometer (XRD), Transmission Electron

Microscopy (TEM) and selected area electron diffraction (SAED).

In another investigation, Aneesh et al. (2007) presented the synthesis of stable,

OH free ZnO NPs via hydrothermal route at variable growth temperature and

concentration of the precursors. The formation of ZnO nanoparticles were confirmed


16

by XRD, TEM, and SAED studies. The average particle size was found to be about 7-

24 nm. Diffuse reflectance spectroscopy (DRS) results showed that the band gap of

ZnO NPs is blue shifted with decrease in particle size.

Ni et al. (2008) also reported synthesis of ZnO NPs by hydrothermal method

where ZnO NPs with an average particle size of 20-30 nm were readily synthesized

through the reaction between zinc acetate and oxalic acid under hydrothermal

conditions. Similarly, Chen et al. (2008) also demonstrated that ZnO NPs of uniform

size (20-25 nm) can be synthesized by hydrothermal method using zinc nitrate and urea.

Liu et al. (2007) followed sol-gel route using zinc acetate and NaOH for

synthesis of ZnO NPs with an average diameter of about 20 nm. Tang et al. (2008) also

reported uniform synthesis of ZnO NPs using urea, zinc nitrate and sodium dodecyl

sulfonate (anionic surfactant) to block the growth of ZnO with size range of 20-25 nm.

Similarly, Zak et al. (2011) successfully demonstrated protocol for synthesis of ZnO

NPs (average size: 33 nm) by sol-gel route; wherein the precursor molecules were zinc

acetate and Tri-ethanol amine (TEA) to control the growth of ZnO.

Saleem et al. (2012) prepared nano-crystalline ZnO thin films by multi-step sol-

gel method using spin coating technique in which zinc acetate dihydrate, 2-

methoxyethanol and mono ethanolamine were used as a starting material, solvent and

stabilizer, respectively. According to XRD results, the as-deposited films exhibited a

hexagonal wurtzite structure with (002) preferential orientation after annealing at 400˚C

in air ambiance for 1 hour. The XRD pattern consists of a single (002) peak which

occurred due to ZnO crystals and grows along the c-axis. The grain size and thickness

of the films are estimated to be 16 nm and 266 nm. SEM micrograph of ZnO thin film

showed that the small grains made a smooth and transparent surface.


17

In a study Jurablu et al (2015), reported the synthesis of ZnO nano-powders via

sol-gel method from an ethanol solution of ZnSO4.7H2O in the presence of diethylene

glycol surfactant. Detailed structural and microstructural investigations were carried

out using XRD, HRTEM, FE-SEM, Fourier transform infrared spectroscopy (FTIR)

and UV-Vis spectrophotometer. XRD pattern showed that the zinc oxide nanoparticles

exhibited hexagonal wurtzite structure. The average particle size of ZnO was achieved

around 28 nm as estimated by XRD technique and direct HRTEM observation. The

surface morphological studies from SEM and TEM depicted spherical particles with

formation of clusters.

Hasnidawani et al. (2016) synthesized ZnO NPs via sol gel method using zinc

acetate dehydrate (Zn(CH3COO)2.2H2O) as a precursor and ethanol as solvent, while,

NaOH and distilled water were used as medium. Result of EDX characterization

showed that the ZnO NPs has good purity with (Zn: 55.38% and O2: 44.62%). While,

XRD result spectrum displayed mainly O2 and Zn peaks, which indicate the crystallinity

in nature as exhibited. The FESEM micrographs shows that synthesized ZnO have a

rod-like structure. The obtained ZnO NPs are homogenous and consistent in size which

corresponds to the XRD result that exhibit good crystallinity. Through this method ZnO

NPs were successfully synthesized in nano-size range within 81.28 nm to 84.98 nm.

A vital step in production of ZnO NPs, independent of the method used, is

stabilizing their growth and dispersion. Surface ligands such as organic polymers (poly

vinyl pyrrolidine, poly vinyl alcohol or poly methyl ether) or surfactants provide

stabilizing agents that control nanoparticle growth and solubility, prevent aggregation

and limit surface oxidation of nanoparticles (Lin and Samia, 2006). The difficult

component in chemical synthesis route is controlling the size and shape precisely. In

chemical synthesis, the size and shape of nanoparticle is controlled and modified by


18

capping agents like different biomolecules and surfactants such as CTAB, TEA,

Chitosan, Cyclodextrin etc.

In another study, Yang et al. (2004) reported the synthesis of ZnO NPs by

thermal decomposition using zinc acetate and capping agents like β-Cyclodextrin (β-

CD), amylose and poly ethylene oxide (PEO). However, β-cyclodextrin was proved to

be the most favourable for preparation of uniform ZnO NPs with mean size of 18-20

nm range.

Sridevi and Rajendran (2009) used low temperature CTAB assisted

hydrothermal method for the synthesis of ZnO NPs (range: 25 nm) where the size is

controlled by CTAB molecule. While, Prasad et al. (2012) reported synthesis of ZnO

NPs of uniform size of 25 nm diameter by oxalate decomposition method

(hydrothermal method) where capping agent was oxalate molecule.

In a fast and efficient combustion method for synthesis of ZnO nano powder,

Asgari and Rashedi (2015) obtained ZnO crystallite size of 21 nm (calculated based on

XRD data). Particle Analyzer supported the XRD calculations of crystallite size while

SEM picture showed that particles were arranged on one another. However, they were

of the opinion that synthesis of ZnO nano particles is still in its infancy and more

research needs to be focused on the mechanism of nanoparticle formation which may

lead to fine tuning of the process ultimately leading to the synthesis of nanoparticles

with a strict control over the size and shape parameters.

Narendhran et al. (2016) reported the successful synthesis of ZnO NPs via

biological as well as chemical methods. Synthesized nanoparticles were confirmed with

Ultra Violet-visible spectroscopy (UV-vis), Fourier transform infrared spectrometer

(FTIR), Energy dispersive X-ray spectrometer (EDX), X-ray diffractometer (XRD),


19

Field Emission Scanning Electron Microscopy (FE-SEM) and High-Resolution

Transmission Electron Microscopy (HR-TEM).

Large-scale and inexpensive synthesis of ZnO NPs can also be performed using

a simple mixing technique of precipitation. As demonstrated by Sadraei (2016), ZnSO4

and NH4OH can be used as precipitating agent in aqueous solutions. Alkali solution

(25% NH4OH) was slowly dropped into the mother solution of 0.2 M ZnSO4 at

controlled temperature of about 50-60 ºC before drying at 60 ºC for 8 hours in oven.

Characterization done through SEM images, EDX and XRD pattern indicated that the

prepared ZnO NPs have uniform structure (average of particle size about 30 nm) and

high purity.

Askarinejad et al. (2011) described synthesis of ZnO NPs through a simple

sonochemical method involving direct transformation of Zn(OAc)2 and NaOH as

precursors to create the ZnO NPs without high temperature calcination. The SEM

analysis showed that ZnO NPs had an average diameter of 20- 50 nm which varied by

different factors.

Keeping in view the above studies, the hydrothermal approach was considered

for the synthesis of ZnO NPs and Oxalate decomposition method was chosen for

present study (details in Materials and Methods chapter).

2.3 ZnO Nanoparticle — Seed Treatment

Significantly important role of Zn nutrition in seed germination, seedling

emergence, initial crop stand establishment and ultimately crop growth and yield is very

well documented in scientific literature. Yilmaz et al. (1998) noticed that wheat plants

emerging from seeds with low Zn have poor seedling vigour and field establishment on

Zn-deficient soils. Similarly, Rengel and Graham (1995) reported from pot culture


20

experiments on wheat plants that increasing seed Zn content from 0.25 to 0.70 μg per

seed significantly improved root and shoot growth under Zn deficiency.

These results highlighted the involvement of Zn in physiological processes

during early seedling development, possibly in protein synthesis, cell elongation

membrane function and resistance to abiotic stresses (Cakmak, 2000). In addition,

higher seed Zn contents may better resist invasion of soil-borne pathogens during

germination and seedling development thus ensuring good crop stands (Marschner,

1995) and ultimately better yield. Hence, it may be concluded that high Zn content in

seed could act as a starter fertilizer and improve root and shoot growth of the plants.

As far as mode of application of Zn fertilizers is concerned, Zn can be applied

to the soil, foliar sprayed or added as seed treatments. Although the required amounts

of Zn can be supplied by any of these methods, soil and foliar sprays have been more

effective in yield improvement and grain enrichment. Though soil application is the

most common method in providing required Zn to plants (Mortvedt and Gilkkes, 1993)

but the effectiveness of Zn fertilizers in providing required Zn in deficient soils mainly

depends on the solubility of the Zn source in soil (Amrani et al., 1999; Mortvedt, 1968).

Likewise, foliar application is employed at later growth stages when crop stands

are already established. Besides this, the high cost of Zn application has restricted wider

adaption of these two methods, particularly by resource-poor farmers (Johnson et al.,

2005). Hence, seed treatment is a better option from an economical perspective as less

micronutrient is needed, it is easy to apply and seedling growth is improved (Singh et

al., 2003).

Seed treatment with Zn, which has potential to meet its crop requirements, can

be performed either by soaking in Zn containing solution of a specific concentration for


21

a specific duration (seed priming) or by seed coating (Farooq et al. 2012, 2009). In seed

priming, seeds are partially hydrated to allow metabolic events to occur without actual

germination, and then re-dried (near to their original weight) to permit routine handling.

Zinc carriers are used as osmotica and such seeds germinate faster than non-primed

seeds (Singh, 2007). Primed seeds usually have better and more synchronized

germination (Farooq et al., 2009) owing simply to less imbibition time (Brocklehurst

and Dearman, 2008) and build-up of germination-enhancing metabolites (Basra et al.,

2005).

Various Zn compounds which vary considerably in Zn content, chemical state,

effectiveness for crops and associated cost have been used as Zn fertilizers. Four main

sources for Zn fertilizers include inorganic compounds, synthetic chelates, natural

organic, and inorganic complexes (Mortvedt and Gilkkes, 1993). Although chelated Zn

sources are more agronomically effective (more response per unit of applied

micronutrient), inorganic sources of Zn like zinc sulphate are more economical to apply

and mainly are preferred to chelated ones in large scale application (Takkar and Walker,

1993).

Inorganic sources of Zn such as zinc sulphate (ZnSO4.H2O or ZnSO4.7H2O) and

zinc oxides (ZnO) are the most commonly used Zn fertilizers to correct Zn deficiency

(Mortvedt, 1992). Recently, with the advent of Zn and ZnO NPs, several experiments

on impact of seed treatment with these nanomaterials have been initiated. The

accessible scientific studies, reported that the synthesized metal oxide nanoparticles

including ZnO NPs have both positive and negative consequences on the plant growth

that depends on the different size and other parameters of engineered nanoparticles

(Arif et al. 2016; Siddiqui et al. 2015; Sekhon 2014; Sabir et al. 2014). So, an attempt


22

has been made in this section to review the outcomes emanated from different studies,

with special emphasis on ZnO NPs.

Although ZnSO4 is more soluble than ZnO, the experiment conducted by

Giordano and Mortvedt (1973) showed that ZnO was more effective in providing rice

plants with adequate Zn and resulted in higher dry matter production than ZnSO4. It is

also to be noticed that immediate dissolution of ZnSO4 after application in soil may

result in a sharp increase in the Zn concentration of soil solution followed by a rapid

decline. In contrast to ZnSO4 which is known to fall off quickly, ZnO dissolve more

slowly and retains sufficient level of Zn in the soil solution for longer period of time

(Pandey et al., 2010; Mortvedt, 1985).

Seed priming with Zn can improve crop emergence, stand establishment, and

subsequent growth and yield of different crops. For example, results of 7 field trials

indicated that seed priming in maize in 1% ZnSO4 solution (for 16 h) substantially

improved crop growth, grain yield and grain Zn content (Harris et al., 2007). Ozturk et

al. (2006) found that Zn in newly developed radicles and coleoptiles of wheat during

seed germination was much higher. Likewise, in another study, Yilmaz et al. 1998

demonstrated that seed priming with Zn was more effective in increasing grain yield

and grain Zn concentration of wheat grown on Zn-deficient soils.

Similarly, Slaton et al. (2001) reported that treating seeds with ZnO greatly

increased rice grain yield while, Ajouri et al. (2004) reported that seed priming with Zn

was very effective in improving seed germination and seedling development in barley.

Seed priming with Zn improved germination, seedling development, and yield and

related traits in common bean also (Kaya et al., 2007).


23

In an investigation by Adhikari et al. (2016a) with ZnO NPs coated and

uncoated seeds of maize revealed better germination percentage (93-100%) due to ZnO

coating as compared to uncoated seeds (80%). Pot culture experiment conducted with

coated seeds also revealed that the crop growth with ZnO coated seeds were similar to

that observed with soluble Zn treatment applied as ZnSO4 at 2.5 ppm Zn. They also

anticipated that seed coating with ZnO NPs did not exert any osmotic potential at the

time of germination of the seed, thus, the total requirement of Zn of the maize can be

loaded with the seed effectively through ZnO NPs.

The results of a study conducted by Yang et al. (2015) showed that seed

germination of maize and rice was not affected by ZnO NPs at lower concentration.

However, at the higher concentration of 2000 mg L-1, the root elongation was

significantly inhibited by ZnO NPs (50.45% for maize and 66.75% for rice). Further,

they opined that the phytotoxic effects of ZnO NPs (25 to 2000 mg L-1) were

concentration dependent, and were not caused by the corresponding Zn2+.

Boonyanitipong et al. (2011) indicated that application of ZnO NPs (10-1000

mgL-1) led to 100 % germination of rice seeds showing that ZnO NPs did not adversely

affect seed germination. Little increase in root length and number of roots were

observed at low concentration i.e. 10 mg L-1, however, at higher concentration toxicity

of ZnO NPs to rice roots was apparent from root length and number of roots.

No marked negative effect on germination and root elongation in radish,

rapeseed, ryegrass, lettuce, maize and cucumber was observed with application of nano-

Zn and ZnO NPs (Lin and Xing, 2007). However, suspensions of higher concentrations

i.e. 2000 mg L-1 nano-Zn or ZnO NPs nearly terminated root elongation of the tested

plant species. These findings clearly indicated that at low concentrations either Zn or

ZnO NPs may be beneficial in enhancing crop performances.


24

In soybean, similar results were obtained by Sedghi et al. (2013) wherein

germination parameters improved significantly besides enhancing drought tolerance. In

another experiment, Zn speciation in soybean seeds germinated in a petri dish system

with 0, 500, 1000, 2000 and 4000 mg Zn L-1 as ZnO NPs showed that the ZnO NPs

were not present in the root. Synchrotron X-ray absorption spectroscopy results showed

that at the 4000 mg L-1 spike rate, Zn coordinated in the same manner as Zn nitrate or

Zn acetate and no ZnO was present in the root. Nevertheless, application of ZnO NPs

slightly increased seed germination of soybean plants up to 1000 mg Zn L-1 in the

solution culture, however, the uptake of Zn was reduced by increasing the spike rate of

ZnO NPs above 1000 mg L-1 (Lopez-Moreno et al., 2010a).

When groundnut seeds were dry dressed with ZnO NPs of 35-45 nm size

synthesised using template-free aqueous solution and characterized through SEM, TEM

and XRD, it outperformed in enhancing germination (75%), shoot length (20.97 cm)

root length (17.98) and thereby the vigour index (2949) compared to control (55%,

16.92, 15.21 and 1759), respectively (Shyla and Natarajan, 2014). Prasad et al. (2012)

also observed that treating groundnut seeds with nanoscale ZnO particles with a

concentration of 1000 ppm caused significant increment in germination, shoot length,

root length and vigour index of groundnut seeds over other concentrations of the same

material and varying concentrations of another material (chelated zinc sulphate) tested.

Pandey et al. (2010) observed a positive response with ZnO NPs seed

germination and root growth of Cicer arietinum seeds. The effect of these ZnO NPs on

the reactivity of phytohormones, especially indole acetic acid (IAA) involved in the

phytostimulatory actions was also observed. Due to oxygen vacancies, the oxygen

deficient, i.e. zinc-rich ZnO NPs increased the level of IAA in roots (sprouts), which in


25

turn indicate the increase in the growth rate of plants as Zn is an essential nutrient for

plants.

The results of a study conducted by Jayarambabu et al. (2014) on effect of ZnO

NPs on mungbean seeds (Vigna radiata L.) revealed significant improvement in

germination, root length and shoot length at lower concentration of ZnO NPs however,

at higher concentration the growth started retarding. In tomato, also, Panwar et al.

(2012) reported no toxic effect of ZnO NPs up to 250 mg L-1 on seed germination.

However, the root and shoot growth of the seedlings were higher when exposed to lower

concentration i.e. 100 mg ZnO NPs L-1. Laware and Raskar (2014) reported that onion

seeds treated with ZnO NPs at the concentration of 20 and 30 μg ml-1 showed better

growth and flowered 12-14 days earlier than the control. Treated plants showed

significantly higher values for seeded fruit per umbel, seed weight per umbel and 1000

seed weight as well.

Besides, ZnO NPs there are other metal oxide nanoparticles like Fe, Ti, Al etc.

are also being evaluated for their potential application in agriculture especially nutrient

management. For example, Racuciu and Creanga (2007) investigated the influence of

magnetic nanoparticles on the growth of maize in early growth stages. The results

revealed that small concentrations of aqueous ferrofluid solution (a source of Fe), added

in culture medium had a stimulating effect on the growth of the plantlets while the

enhanced concentration of aqueous ferrofluid solution induced an inhibitory effect.

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

germination and growth of naturally aged seeds of spinach by measuring the

germination rate and vigour indices. An increase of these indices was observed at 0.25-

4.00% with nano-TiO2 treatments. During the growth stage the plant dry weight was

increased as the chlorophyll formation, the ribulose bisphosphate carboxylase/


26

oxygenase activity and the photosynthetic rate increased. These results showed that the

physiological effects were related to the nanometer-size particles.

Similarly, Hong et al. (2005) reported that the nano TiO2 treatments induced an

increase in Hill reaction and activity of chloroplasts in spinach, which accelerated FeCy

reduction and oxygen evolution. Moreover, non-cyclic photophosphorylation activity

was higher than cyclic photophosphorylation activity. The explanation of these effects,

on the opinion of the authors, could be that the nano-TiO2 might enter the chloroplast

and its oxidation-reduction reactions might accelerate electron transport and oxygen

evolution. Gao et al. (2006) also reported 2.67 times increase in Rubisco carboxylase

activity in nano-anatase TiO2 treated Spinacia oleracea seeds over control.

The results of the above-mentioned studies indicated that seed treatment with

ZnO NPs at lower concentration resulted in improvement in seedling emergence and

stand establishment, yield, and grain Zn enrichment in different crops. However, it may

be toxic at high levels with effective concentrations (EC50 - substrate Zn concentration

resulting in 50% biomass reduction) varying from 43 to 996 mg Zn L-1 within various

plant species (Paschke et al., 2006). Many studies showed that excess of Zn2+ reduced

the germination in a variety of plant seeds, and was also inhibitory to growth of their

roots, stems and leaves. The general symptom of Zn2+ phytotoxicity is a retardation of

growth, with the plants being stunted (El-Ghamery et al., 2003).

Keeping these findings in view, it would be wise to optimize ZnO NPs seed

priming treatment protocol of maize in the laboratory and test the seeds in soil for

germination prior to priming the whole batch.


27

2.4 ZnO nanoparticle — Foliar, Nutrient Solution, and Soil Application

2.4.1 Foliar Application

The initial step in determining the effect of nanoparticles on plant growth and

possible benefits of applying nanoparticles in agriculture would be to understand the

uptake mechanisms, translocation and transformation of nanoparticles following

application to soil-plant system. Although the majority of studies on plant uptake and

negative or positive effects of nanoparticles have been conducted on seed germination

and root elongation in culture media (Peralta- Videa et al., 2011). The results provide

evidence that plant uptake of nanoparticles and response to the exposed nanoparticles

are primarily dependent on the physicochemical properties of nanoparticles (e.g.

composition, shape and size) and plant type (Ma et al., 2010).

In plant uptake processes, solutes translocated by diffusion or mass flow to the

external surface of plant roots, are taken up by movement across the cell wall and water-

filled intercellular spaces of the root cortex (Marschner, 1995). The main barrier against

passive solute movement in the apoplast is the Casparian strip in the endodermis, the

innermost layer of cells of the cortex. To date, studies on plant uptake of nanoparticles

from soil have suggested that the possible interactions of nanoparticles with higher

plants are adsorption onto the root surfaces, incorporation onto the cell walls and uptake

into the cells (Ma et al., 2010; Nair et al., 2010; Nowack and Bucheli, 2007).

Dissolution of ZnO NPs in soils and uptake of dissolved ions is also a critical pathway

which may affect plant growth.

The permeability of the cuticle to water and to lipophilic organic molecules

increases with mobility (distribution coefficient) and solubility (partition coefficient)

of ZnO NPs within the transport-limiting barrier of the cuticles. Ions being highly water

soluble might face some hindrance in penetrating the lipophilic cuticle. This may act as


28

a limiting factor in the case of chelated ZnSO4. But custom-made ZnO NPs, which is

having less hydrophilicity and being more dispersible in lypophilic substances

compared to the ions, can penetrate through the leaf surface compared to ZnSO4 (Da

Silva et al., 2006). The bioavailability of the nanoparticles because of its size and lower

water solubility (which inhibit rapid falling off compared to ionic supplements) can also

be higher compared to chelated ZnSO4.

Application of foliar sprays implies that the nutrients applied will be absorbed

and exported from the point of application (leaf) to the point of utilization. Thus, in

foliage applications, nutrients need to first travel through the leaf cuticle (Monreal et

al., 2016). For Zn applied either in chelated or in sulphate salt form, an extensive

nutrient fixation by cuticle may occur at the point of application (Ferrandon and

Chamel, 1988). In an experiment, foliar absorption of Zn was lower from chelates than

from the inorganic salt, but the translocation within the plant was greater when chelated

forms were applied (Rengel et al. 1999).

Given that the pore diameter of cell walls of plants is generally in the range of

3.5-3.8 nm for root hairs, only nanoparticles or aggregates with diameters less than the

cell wall pore-diameter can enter the cell wall of undamaged cells (Dietz and Herth,

2011). However, formation of new and large size pores, which allows internalization

of nanoparticles through cell walls has also been reported (Ma et al., 2010; Navarro et

al., 2008). Further internalization is possible by endocytosis which provides a cavity

structure around the nanoparticles by the plasma membrane (Nair et al., 2010) or it can

enter xylem via cortex and apoplastic bypass (Dietz and Herth, 2011).

Eichert et al. (2008) demonstrated that the mechanism of foliar uptake pathway

for aqueous solutes and water-suspended nanoparticles in Allium porrum and Vicia faba

and observed that the stomatal pathway differs fundamentally from the cuticular foliar


29

uptake pathway. However, the uptake and translocation mechanism of foliarly applied

ZnO NPs is yet to be fairly understood.

In some experiments, it has been observed that ZnO NPs significantly

influenced the growth, yield, and Zn content of maize grains (Subbaiah et al. 2016).

Analogous results were obtained by Adhikari et al. (2015) on maize plant where in

results of solution culture study showed that the application of ZnO NPs at relatively

lower level enhanced the growth of maize plant as compared to conventional Zn

fertilizer i.e. ZnSO4.

On the other side, Cu being an essential micronutrient, Cu NPs positively

influence growth of maize plant by assimilating into the metabolic routes of plant and

regulating the enzymatic activities. Apart from that, Mn and Fe nanoparticles delivery

have been reported to display positive impacts on the seed germination and were also

enhanced agronomic productivity (Adhikari et al., 2016b).

Farnia and Omidi (2015) also reported positive increase in grain row per cob,

number of grain per cob and grain yield of maize due to application of nano Zn

fertilizer. Afshar et al. (2014) studied the foliar application of different amount of ZnO

nanoparticles and bulk ZnO on arable irrigated wheat plant and results revealed that

foliar spray of nano ZnO with 60 g ha-1 was superior.

Ten days old seedlings of chickpea were foliar sprayed with 1.5 or 10 ppm

aqueous solution of ZnO NPs in an experiment by Burman et al. (2013). Results

indicated that maximum promotery response with respect to shoot dry weight was

observed in seedlings treated with 1.5 ppm ZnO NPs while at 10 ppm, they exerted

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

the ZnO NPs treated seedlings. The study indicated importance in precise application


30

of Zn, more so in deficient system, where plant response varies with concentration and

is important in understanding the mechanism of action of specific nanomaterials.

Raliya and Tarafdar (2013) demonstrated dramatic increase in biomass of

cluster bean when leaves were sprayed with ZnO NPs compared to bulk ZnO. Increased

accumulation of Zn from ZnO NPs in different crop species was also observed by

Watson et al. (2015).

The results from an experiment by Prasad et al. (2012) revealed that the

response of groundnut to lower dose of nanoscale ZnO was highly significant. In

general, foliar application of nanoscale ZnO at 2 g 15 L−1 significantly increased pod

yield and shelling per cent and other biometric parameters. In addition, the post-harvest

leaf and kernel samples analysis revealed a significant increment in Zn content in leaves

(42%, 29%) and kernels (42%, 36.6%) when supplied with nanoscale ZnO compared

to chelated ZnSO4 (in two consecutive Rabi seasons, respectively). Similarly, nanoscale

nutrients at high concentrations are detrimental just as the bulk nutrients.

In spinach, foliar spray of ZnO NPs at the concentration of 500 and 1000 ppm

exhibited increased leaf length, width, surface area and colour of leaf samples when

compared to no Zn leaf samples. Further, ZnO NPs treated plants showed higher values

of protein and dietary fibre content suggesting that the ZnO NPs sprayed spinach is

more nutritious to vegetarian diet (Kisan et al., 2015).

Davarpanah et al. (2016) also demonstrated the same with his findings on

exogenous foliar application of Zn and B nanofertilizers to the pomegranate (Punica

granatum cv. Ardestani). As reported, their application increased the yield and quality

of pomegranate fruit and also improved the nutrient availability to the tree.


31

Ghafari and Razmjoo (2013) studied the effect of foliar application of nano-iron

oxide (2 and 4 g L-1), iron chelate (4 and 8 g L-1) and iron sulphate (4 and 8 g L-1) on

grain yield, yield components and foliar chlorophyll and carotenoid content, peroxidase

(POX), catalase (CAT) and ascorbate peroxidase (APX) activities of bread wheat. The

results suggested that Fe fertilization increased antioxidant enzyme activities and

chlorophyll content, yield components and the grain quality of wheat, however,

application of 2 g L-1 nano iron oxide was more effective than other sources.

Armin et al. (2014) evaluated the effect of concentration and time of foliar

application of nano-Fe on yield and yield components of wheat. The results revealed

that foliar application of nano-Fe at tillering + stem elongation and at tillering had

9.17% and 5.19% more grain yield, respectively compared to foliar application of Fe at

stem elongation. Foliar application of nano- Fe at 2%, 4% and 6% produced an increase

of 12%, 22.09% and 19.07% grain yield, respectively, over the control.

In an effort to study the effect of different concentrations of nano-iron oxide

(0.25, 0.50, 0.75 and 1.0 g L-1) in soybean, Sheykhbaglou et al. (2010) observed that

spraying of nano-iron oxide at the concentration of 0.75 g L-1 increased leaf + pod dry

weight and pod dry weight. However, the highest grain yield was observed with using

0.5 g L-1 nano-iron oxide that showed 48% increase in grain yield in comparison with

control. Other measured traits were not affected by the iron nanoparticles. Similar

promotory effect of nanoscale SiO2 and TiO2 on germination was reported in soybean

(Lu et al., 2002), in which authors noticed increased nitrate reductase enzyme activity

and enhanced antioxidant system.


32

2.4.2 Nutrient Solution Application

Lv et al. (2015) conducted an integrated study through microscopic and

spectroscopic techniques to comparatively investigate the uptake of ZnO NPs and Zn2+

ions by maize in order to further elucidate plant uptake pathways of ZnO NPs. The

results demonstrated that the majority of Zn taken up was derived from Zn2+ released

from ZnO NPs, and Zn accumulated in the form of Zn phosphate. ZnO NPs were

observed mainly in the epidermis, a small fraction of ZnO NPs was present in the cortex

and root tip cells, and some further entered the vascular system through the sites of the

primary root-lateral root junction. However, no ZnO nanoparticle was observed to

translocate to shoots, possibly due to the dissolution and transformation of ZnO NPs

inside the plants.

In a plant agar method based study, Mahajan et al. (2011) noticed that presence

of ZnO NPs in the nutrient media affected the growth of mung and chickpea seedlings

at different concentrations. The maximum effect was found at 20 ppm for mung and 1

ppm for gram seedlings however, beyond this concentration, the growth was inhibited.

They also noticed that the effective growth at certain optimum concentration and

inhibited growth beyond this concentration may be attributed to the accumulation and

uptake of nano-ZnO particle by the roots, which varied with exposure concentrations

of ZnO NPs.

Experiments on cell internalization and upward translocation of ZnO NPs by

ryegrass have also been conducted in hydroponic culture (Lin and Xing, 2008). Electron

microscopy images confirmed that ZnO NPs concentration in the rhizosphere, adhered

to the root surface, damaged the epidermal and cortical cells upon intake and increased

Zn concentration in roots 3.6 times more than soluble Zn source when 1000 mg L-1 Zn

were added to the hydroponic solution. However, translocation of Zn from roots to


33

shoots for ZnO NPs remained very low, much lower than that for Zn2+. The reported

phytotoxicity may be due to high rates of Zn (1000 mg L-1) applied to the solution

culture. Moreover, it can be assumed that the observed toxic effects would be less in

soil systems due to partitioning to the soil solid phase.

In an attempt to investigate comparative effects of ZnO NPs, ZnO bulk, and

Zn2+ ions on rapeseed after long-term exposure to a wide range of concentrations, Kouhi

et al. (2015a) found that the inhibitory effects of treatments were in the order Zn2+ >>

ZnO bulk > ZnO NPs. Results further indicated that the toxicity of ZnO NPs on

rapeseed was lower than toxicity of Zn2+ or ZnO bulk. Since NPs tend to aggregate in

aqueous medium or absorb on solid surface due to its higher surface energy, they could

not show significant toxicity on the plants. In high concentrations, NP toxicity may be

due in part to the toxic effects of Zn2+ ion dissolution, probably induced by root

exudates or due to the physical interaction of ZnO particles with roots, for instance

particle aggregation on the root surface, and induction of structural and functional

disorders Kouhi et al. (2015b).

The effect of ZnO NPs on the root growth of garlic (Allium sativum L.) was

investigated by Shaymurat et al. (2012). It was noticed that ZnO NPs caused a

concentration-dependent inhibition of root length. When treated with 50 mg L-1 ZnO

NPs for 24h, the root growth of garlic was completely blocked. The 50% inhibitory

concentration (IC50) was estimated to be 15 mg L-1. However, 0, 0.5, 1, 1.5 and 2 mg

L-1 of dissolved Zn2+ ions (equivalent to the concentrations in the supernatants of ZnO

NPs suspensions after centrifugations, respectively) did not show any toxicity to the

growth of the root tips of garlic.

Different plants have different response to the same nanoparticles as Zhu et al.

(2008) showed that Cucurbita maxima growing in an aqueous medium containing


34

magnetite nanoparticles can absorb, move and accumulate the particles in the plant

tissues, on the contrary Phaseolus limensis is not able to absorb and move particles. Liu

et al. (2005) also observed that the application of nano-iron oxide significantly affects

peanut and caused increase in growth and photosynthesis. Nano-iron oxide compared

to other treatments such as organic materials and iron citrate facilitated the

photosynthate and iron transferring to the leaves of peanut.

Lee et al. (2008) examined bioavailability of Cu nanoparticles to the plants

Phaseolus radiatus and Triticum aestivum, employing plant agar test as growth

substrate for homogeneous exposure of nanoparticles. The growth rates of both plants

were inhibited and as result of exposure to nanoparticles and the seedling lengths of

tested species were negatively related to the exposure concentration of nanoparticles.

Bioaccumulation is concentration dependent and the contents of nanoparticles in plant

tissues increased with increasing nanoparticles concentration in growth media.

2.4.3 Soil Application

As outlined above, the majority of studies investigating the effect of

nanoparticles on plants have been conducted in vitro, in petri dishes or in hydroponic

culture media. Interactions of NPs with soil surfaces, effects of soil on NP dissolution

and the mode of uptake of elements by roots in soil will all markedly affect the

outcomes from NP dosing experiments. There is therefore a need to study the uptake,

translocation and biotransformation of NPs in natural soil environments. Potential

dissolution of metal-based nanoparticles in soil or dissolution of nanoparticles within

plant root cells may also affect plant growth by production of dissolved species.

Liu et al. (2015) confirmed a high Zn bioavailability in ZnO NPs-spiked soil to

maize as they observed significant positive correlations with ZnO NPs dose, indicating


35

the Zn in plants is at least partly from ZnO NPs. Further, compared with bulk ZnSO4,

ZnO NPs produced similar plant Zn uptake or even higher shoot Zn concentration,

indicating that the bioavailability of Zn released from ZnO NPs is similar to or higher

than that from ZnSO4. Another evidence was that soil DTPA-extractable Zn

concentrations correlated significantly with ZnO NPs dose and Zn concentrations in

plants, indicating ZnO NPs indeed released Zn2+ or other exchangeable forms into soil.

They also cautioned that at low doses, ZnO NPs may serve as a Zn fertilizer and supply

Zn2+ for plant growth, but at high doses, they will be toxic because they release excess

amount exceeding plant requirement.

Watson et al. (2015) observed that phytotoxicity of ZnO NPs to young wheat

seedlings was dependent on the soil properties: phytotoxicity was observed in acid but

not alkaline soils. However, although the extent of solubility of Zn from the NPs was a

100-fold less in the alkaline than the acid soil, an increased uptake of Zn into the shoots

from the NPs occurred in the calcareous alkaline soil. These findings indicate that use

of NPs such as ZnO NPs as a fertilizer or a pesticide would have to be tuned to the soil

being treated to avoid phytotoxic effects yet retain beneficial Zn uptake.

In the study carried out in a clay loamy soil, Du et al. (2011) investigated the

effect of applying ZnO NPs on growth of wheat plants. The results indicated that Zn

concentration of wheat tissue increased as a result of application of ZnO NPs to soil,

however, no ZnO NPs were observed in primary root. Therefore, uptake of ZnO NPs

may not be responsible for Zn accumulation in the plants and more likely it was

dissolution of ZnO NPs during the 2 months’ incubation period which increased soil

Zn availability.

As demonstrated by Priester et al. (2012), Zn substantially moved aboveground

in soybean from nano-ZnO treated soils as Zn concentrations increased in a dose-


36

dependent fashion in the stem, leaf, and soybean pod tissues. When compared with no

Zn, high nano-ZnO treatment registered 6 times more Zn in the stem, 4 times more in

the leaf, and nearly 3 times more in the soybean pod. Such Zn concentrations in various

plant tissues were similar for equivalently dosed soybean (on a Zn mass basis) grown

with Zn salts (Shute and Macfie, 2006). They also suggested that nano-ZnO must have

been highly bioavailable in this study soil, as Zn also substantially bioaccumulated in

nano-ZnO-treated plants in a previous hydroponic study (Lopez-Moreno et al. 2010b).

In a recent experiment conducted by Kim et al. (2011), application of 2000 mg

kg-1 Zn NPs and ZnO NPs compared to soluble Zn sources in a natural soil did not

affect biomass production and Zn concentration in cucumber plant tissue. However, Zn

concentration in soils treated with nanoparticles were significantly higher than control

plants and soil treated with soluble Zn. This may indicate that retention of nanoparticles

in natural soil can effectively reduce plant toxicity of nanoparticles.

In a bid to compare silica nanoparticles against conventional bulk silica,

Suriyaprabha et al. (2012) observed that nanoscale silica regimes at 15 kg ha-1 has a

positive response of maize than bulk silica which help to improve the sustainable

farming of maize crop as an alternative source of silica fertilizer. The observed

physiological changes showed that the expression of organic compounds such as

proteins, chlorophyll, and phenols favoured to maize treated with nano-silica,

especially at 15 kg ha-1 when compared with bulk silica.

Yang and Watts (2005) investigated the effect of Al oxide nanoparticles on root

elongation of maize, cucumber, soybean, cabbage and carrot and reported that root

elongation can be inhibited in the presence of uncoated Al oxide. This effect on root

elongation was reduced effectively by coating the Al oxide nanoparticles with

phenanthrene which indicates relevance of surface modifications in reduction of


37

phytotoxicity. Doshi et al. (2008) also investigated effect of Al nanoparticles on kidney

beans and ryegrass and reported that up to 10,000 mg kg-1, it did not significantly affect

the Al concentration in kidney beans whereas the Al concentration in ryegrass almost

doubled.

In a nutshell, different plants may also behave differently to addition of the same

nanoparticles (Nair et al., 2010) and also their response may be dependent on the

growth stage. In accordance with most of the experimental reports, other metal

nanoparticles and their oxides Ag NPs (Hordeum vulgare L.) (Gruyer et al., 2013), Au

NPs (Brassica juncea) (Arora et al., 2012), Ti NPs (wheat) (Jaberzadeh et al., 2013),

Fe3O4NPs (iron oxide) (wheat) (Bakhtiari et al., 2015) positively responds to the crop

productivity.

On the contrary to these results mentioned above, ZnO NPs have also been

reported to exhibit phytotoxicity in maize and cucumber (Zhang et al., 2015). Similarly,

Da Costa and Sharma (2016) propounded exogenous application of CuO NPs in rice

that resulted in decrease in germination rate, growth parameters and biomass.

Simultaneously, Liu et al. (2016) documented that CuO and ZnO NPs display toxicity

on the germination of lettuce seeds. Previous studies also reported phytotoxicity of

metal oxide NPs, high concentrations of NPs, from 1000 to 4000 mg L-1 (Rao and

Shekhawat, 2014; Pokhrel and Dubey, 2013; Mazumdar and Ahmed, 2011; Lopez-

Moreno et al. 2010b; Lee et al. 2010; Stampoulis et al. 2009; Lin and Xing, 2007).

However, the outcomes from many of the discrete studies of Zn-plant

interactions suggest the potential use of commercial nanoparticles of ZnO as Zn sources

in crops produced in Zn-deficient soils. Reports indicated that NPs of ZnO (<100 nm)

used with a variety of crops such as cucumber (Zhao et al. 2013); peanuts (Prasad et al.

2012); cabbage, cauliflower, and tomato (Singh et al., 2013); and common chickpea


38

(Pandey et al. 2010) increased biomass, yield, and nutrient accumulation. Recently, a

review by Liu and Lal (2015) indicated that synthetic NPs have a great potential as

fertilizers (including micronutrients) for increasing crop production and reduce adverse

environmental impacts by excess nutrients from conventional fertilizer sources.

MATERIALS AND METHODS

III. MATERIALS AND METHODS

In order to attain the objectives, four sequential experiments were undertaken

in Laboratory as well as microplots. In first experiment, ZnO nanoparticles (ZnO NPs)

were synthesized by Oxalate Decomposition Method and characterized for different

properties. While, their comparative effect at different concentrations on germination

of maize seeds were evaluated in second experiment. In another third and fourth

experiments, the effects of seed treatment with ZnO NPs and foliar application of ZnO

NPs, respectively were studied in separate microplot studies. The details of the

materials used, experimental methods followed and techniques adopted during the

course of investigation are furnished below.

3.1 Laboratory Studies

Two successive experiments were conducted in Soil and Seed Laboratories of

Anand Agricultural University, Anand. The first experiment comprised of synthesis

ZnO NPs by Oxalate Decomposition Method and subsequently characterization. In next

laboratory study, different concentrations of nano-sized as well as bulk ZnO were

compared for their efficacy in maize seed germination.

3.1.1 Synthesis and characterization of ZnO nanoparticles

3.1.1.1 Synthesis of ZnO nanoparticles

Zinc oxide (ZnO) nanoparticles were prepared via oxalate decomposition

technique (hydrothermal method) in the Laboratory of Micronutrient Project (ICAR),

Anand Agricultural University, Anand. As demonstrated by Prasad et al. (2012), first

the zinc oxalate was prepared through mixing equimolar (0.2 M) solution of zinc acetate

and oxalic acid. The resultant precipitate was collected and rinsed extensively with

deionized water (DI-water) and dried in air to obtain zinc oxalate. Subsequently, the

Materials and Methods

40

zinc oxalate was ground and allowed to decompose in air for 45 min inside pre-heated

furnace at a temperature of 500 °C. The step-wise method of synthesis of ZnO NPs is

presented in Fig. 3.1.

Fig. 3.1: Flow chart of synthesis of ZnO nanoparticles by oxalate decomposition

method

3.1.1.2 Characterization of ZnO nanoparticles

After the preparation of ZnO NPs, different characterization techniques were

used to investigate their structure and optical properties. The nanoparticles were

characterized at three different institutions viz. (1) Dharamsinh Desai Institute of

Technology (DDIT), Nadiad (Gujarat); (2) Sophisticated Instrumentation Centre for


41

Applied Research and Testing (SICART), Anand (Gujarat) and (3) Laboratory for

Advanced Research in Polymeric Materials (LARPM), Bhubaneswar (Odisha). The

equipment, materials and methods used for the characterization of synthesized ZnO

NPs are described below.

X-ray diffraction analysis (XRD)

The crystal-phase structure and the crystallite size of the ZnO NPs were

determined using X-ray diffractometer (Philips X’Pert MPD (Japan)) using

monochromatic CuKa1 radiation of wavelength k = 1.5418Å from a fixed source

operated at 40kV and 30 mA in the 2θ scan range of 20–80°. The ZnO NP crystallite

size was calculated using the Scherrer equation (Equation 1):

= / [Eq. 1]

Where, k is the Scherer constant (k=0.89), λ is the X-ray wavelength, β is full width of

the peak at half maximum (FWHM) intensity (in radians) and θ is the Bragg’s

diffraction angle.

Dynamic light scattering (DLS)

Particles size and particle size distribution was confirmed by DLS (Dynamic

Light Scattering) or Zeta Potential. DLS is a laser diffraction method with a multiple

scattering technique which was used to determine the particle size distribution of the

powder. It was based on Mie-scattering theory. In order to find out the particles size

distribution, the ZnO powder was dispersed in water by horn type ultrasonic processor

[Vibronics, model: VPLP1]. Then experiment was carried out in computer controlled

particle size analyzer [ZETA Sizers Nanoseries (Malvern Instruments Nano ZS) to find


42

out the particles size distribution and stability of synthesized nanoparticles in aqueous

media.

The zeta potential of the nanoparticles was also evaluated by analyzing 0.1 g of

ZnO NPs in 10 ml of water (or additives solutions) using the Zetasizer Nano ZS

(Malvern Instruments Ltd., GB). Before zeta potential measurements all samples were

sonicated for 5 minutes. Zetasizer Nano ZS uses Laser Doppler Velocimetry to

determine electrophoretic mobility. The zeta potential was obtained from the

electrophoretic mobility by the Smoluchowski equation.

Scanning Electron Microscopic Analysis (SEM)

The coarse and fine microstructures and the morphology of all the ZnO NPs

were depicted by using SEM; EVO MA 15 Germany, scanning electron microscope

(SEM). The SEM micrographs were used to analyze the surface morphology of the

sample, and the topographic filature of the sample, in order to examine the diameter,

length, shape and density of the ZnO nanoparticles. For the analysis, the ZnO

nanoparticles were sonicated with distilled water, small drop of this sample was

placed on glass slide and allowed to dry. A thin layer of gold was coated to make the

samples conductive. SEM was operated at a vacuum of the order of 10-5 torr. The

accelerating voltage of the microscope was kept in the range 10-20 kV.

Transmission electron microscopy (TEM)

The average particle size and the size distribution of the ZnO NPs were further

investigated using Transmission Electron Microscope (JEOL 1200EX, Japan).


43

UV-VIS Spectroscopic Analysis

ZnO nanoparticles were characterized by UV (Perkin-Elmer UV-VIS

spectrophotometer, Lambda-19) to know the kinetic behaviour of ZnO nanoparticles.

The scanning range for the samples was 200-800 nm at a scan speed of 480 nm/min.

Base line correction of the spectrophotometer was carried out by using a blank

reference.

Thermogravimetric Analysis (TGA)

Thermogravimetric analysis of zinc oxalate was carried out in order to observe

characteristic weight loss with temperature during formation of ZnO nanoparticle. The

analysis was carried out using Q50 (M/s TA instrument, USA) at scan rate of 100C/min.

3.1.1.3 ZnO nanoparticle suspension preparation

Synthesized ZnONPs at different concentrations were suspended directly in de-

ionized water and dispersed ultrasonic vibration (100 W, 40 KHz) for 30 minutes at

Department of Microbiology, B. A. College of Agriculture, Anand. Small magnetic bar

was placed in the suspension for stirring through Ultrasonicator (Make: Enertech India

Pvt. Ltd.) to avoid aggregation of the particles. Several suspensions of concentration

range up to maximum possible limit were tried for uniform particle dispersion, stability

and clear suspension (trial and error method). Based on the results, 3 concentrations

each of bulk ZnO and nano ZnO (500, 1000, 2000 mg L-1) were selected for their

evaluation in maize seed germination and growth study. The pH of all the prepared

suspensions was found to be neutral (6.70 to 7.00).


44

3.1.2 Effect of seed treatment with ZnONPs on germination of maize seeds

3.1.2.1 Seed

The seeds of maize (Zea mays L.) variety GM – 6 (Gujarat Maize – 6), procured

from Main Maize Research Station, Godhra, Anand Agricultural University (Anand)

were used in the present study. This cultivar is an early, drought escaping and white

flint grained composite variety recommended for Gujarat, Rajasthan and Madhya

Pradesh states of India. Uniform seeds were screened and used freshly for the

experiment to minimize errors in seed germination and seedling vigour study.

3.1.2.2 Seed treatment

In order to evaluate the effect of different concentration of ZnO NPs along with

corresponding levels of bulk ZnO and recommended dose of seed treatment seeds were

soaked-in for 2 and 4 hrs. The experiment was statistically designed under Factorial

Completely Randomised Design (FCRD) with soaking time as one factor while Zn

levels as another with 3 repetitions.

Factor 1: Soaking Time (2 levels — S2: 2 hours and S4: 4 hours)

Factor 2: Zinc levels (8 levels)

T1 : Pure water (Control)

T2 : ZnONPs suspension at 500 ppm



T5 : Bulk ZnO suspension at 500 ppm



T8 : Seed treatment with ZnO slurry @10 mL bulk ZnO /kg seeds


45

Seeds (200 Nos.) were soaked in sufficient volume of each of the Zn level

suspension, separately for 2.0 hrs and 4.0 hrs in two distinct sets and shade dried to near

original moisture content of the seed. Thus, treated seeds were stored in air-tight plastic

bags for later use in seed germination study.

3.1.2.3 Seed germination

The germination study was carried out in the Laboratory of Department of Seed

Science and Technology, B. A. College of Agriculture, Anand Agricultural University

(Anand). Maize seeds treated with Zn were germinated through Paper Towel Method

following standard procedure laid down by ISTA. Two brown corrugated paper towel

sheets for each set were moistened (not dripping wet just wet). One wet paper towel

was placed in shallow plastic container and seeds (50 nos. for each set) were placed

evenly on it and another wet paper towel was used to cover the seeds. Then both the

sheets were rolled in and wrapped in a butter paper and loosely gripped through rubber

bands. After proper labelling, rolled-in towels with seeds were placed vertically on

racks in a Seed Incubator at prescribed temperature (20-30 0C) and aeration. Seeds were

monitored daily till the germination was complete and re-moistened, if needed.

3.1.2.4 Optimization of seed soaking time

After 8 days of incubation, seeds were taken out to register observations on

various seed quality parameters viz., shoot length, root length and seed germination

(%). Later on, seed vigour index was computed and results were compared for statistical

difference between two soaking durations.

Shoot length (cm)

Shoot length of the germinated maize seeds was recorded in centimeter with the


46

help of measuring scale following random representative sampling. Then, at the end

average shoot length was calculated for each treatment.

Root length (cm)

Root length of the germinated seeds used for shoot length measurement were

recorded in centimeter with the help of scale. Then, at the end mean root length was

computed out for each treatment.

Germination percentage (%)

Germination percentage was calculated by taking the ratio of number of seeds

sown (50 nos. for each set) to the number of seeds germinated in a paper towel roll at

the end of 8 days and expressed as percentage.

Seedling Vigour Index (SVI)

Seedling Vigour Index (SVI) was computed by using the formula described by

Baki and Anderson (1973).

Seed Vigour Index = Germination% × (root length + shoot length)

3.2 Microplot Studies

The synthesized ZnO NPs were evaluated for their suitability for seed treatment

and foliar application in maize and their effect on growth and yield of maize in two

separate experiments under microplot conditions.

3.2.1 Experimental site

The experiments were conducted in Microplots laid down at Model Laboratory

of Micronutrient Project (ICAR), Anand Agricultural University, Anand (Gujarat)

during Rabi (2015-2016) season and repeated in ensuing summer (2016) season. The


47

permanent brick-cemented microplot each of net size of 1.35 X 0.90 m2 filled with soil

were used for the studies.

3.2.2 Climate and weather conditions

Geographically, Anand is situated at 220° 35’ N latitude, 720° 55’ E longitude

with an elevation of 45.1 m above the mean sea level. The climate of this region is semi-

arid and sub-tropical. Monsoon commences by the 3rd week of June and retreats by

middle of September with an average rainfall of 864 - 870 mm received entirely during

south west monsoon. In general, rainfall is adequate in this region but partial failure of

rain once in 3-4 years is very common. July and August are the months of heavy

precipitation and there is no rainfall in winter and summer in almost all part of Gujarat.

Except some sporadic showers in Rabi season, winter is severe and sets in the month

of November and continues till the end of January. Summer is hot and dry, spread over

for the months of April-May.

3.2.3 Physico-chemical properties of soil

The soil used in the experiment was representative of the soils of the middle

Gujarat region and is locally known as “Goradu” soil. The texture of the soil is loamy

sand, very deep and moisture retentive belongs to the soil order Inceptisols (Typic

Ustochrepts). It responds well to manuring and is suitable to variety of crop of tropical

region. After survey, Zn deficient bulk soil was collected from village Vadod, which is

5 km from Anand Agricultural University, Anand. The soil was analyzed for different

physical and chemical properties including DTPA-extractable micronutrients status.

The results of chemical analysis, presented in Table 3.1 which indicated that with

respect to DTPA-extractable- Zn, the soil of the experimental field was found deficient

in its availability as required for the investigation.


48

3.2.4 Seed

The seeds of maize (Zea mays L.) variety GM – 6 (Gujarat Maize – 6), procured

from Main Maize Research Station, Godhra, Anand Agricultural University (Anand)

were used in the microplot studies.

Table 3.1: Physico-chemical properties of the soil used in microplot studies

S. No. Properties Initial valuePhysical Properties1. Mechanical analysis (International pipette method) (0-15 cm)

Clay (%) 9.8Silt (%) 7.2Fine sand (%) 82.3Coarse sand (%) 0.3

2. Texture Loamy sandChemical Properties3. pH (1:2.5) Potentiometric (Jackson, 1973) 8.044. EC (1:2.5) (dSm-1) Conductometric (Jackson, 1973) 0.185. Organic carbon (%)

Walkley and Black method (Jackson, 1973)0.41

6. Available N (kg ha-1)Alkaline permanganate method (Subbaiah and Asija, 1956)

160.8

7. Available P2O5 (0.5 M NaHCO3 extractable-P) (kg ha-1)Olsen’s method (Jackson, 1973) 76.3

8. Available K2O (NH4OAc extractable-K) (kg ha-1)Flame photometric method (Jackson, 1973)

235.7

9. Micronutrients (0.005 M DTPA-extractable) (mg kg-1)Lindsay and Norvell (1978)

Zn 0.29Fe 3.66

Mn 3.23Cu 0.46

3.2.5 Treatment details

3.2.5.1 Effect of seed treatment with ZnONPs on growth and yield of maize

Two experiments in microplots (Gross size: 1 x 1.5 m2) were conducted during

Rabi and Summer seasons of the year 2015-2016 at Model Laboratory, Micronutrient


49

Research Project (ICAR), Anand Agricultural University, Anand to carry out study on

“Effect of ZnO nanoparticles seed treatment on germination, growth and yield of

maize”. The treatments (8) and experimental details are furnished below.

Treatments : 8

Treatment details :

T1 : Control

T2 : Seed treatment with ZnONPs suspension at 500 ppm



T5 : Seed treatment with Bulk ZnO suspension at 500 ppm



T8 : Seed treatment with ZnO slurry@10 mL ZnO (30% Zn)/kg seeds

No. of repetitions : 3

No. of microplots : 24 (8 X 3)

Experimental design : Completely Randomised Design (CRD)

Based on the results of laboratory study, seeds were soaked-in for 2 hrs and

dried to near original moisture content of the seed. Treated seeds were stored in air-

tight plastic bags and used in the study.

3.2.5.2 Effect of foliar application of ZnONPs on growth and yield of maize

Similarly, another two experiments in parallel microplots were conducted each

during Rabi and Summer seasons of the year 2015-2016 at Model Laboratory,

Micronutrient Research Project (ICAR), Anand Agricultural University, Anand to carry

out study on “Effect of foliar application of ZnO nanoparticles on growth and yield of


50

maize”. The treatments (8) and experimental details are furnished below.

Treatments : 8

Treatment details :

T1 : Control

T2 : Foliar spray of ZnONPs suspension at 500 ppm



T5 : Foliar spray of Bulk ZnO suspension at 500 ppm



T8 : 0.5% foliar spray of ZnSO4

Schedule of foliar spray : 30 and 45 days after sowing (DAS)

No. of repetitions : 3

No. of microplots : 24 (8 X 3)

Experimental design : Completely Randomised Design (CRD)

The foliar application of designed treatments was done at 30 and 45 DAS (Days

after sowing). The ZnO (nano as well as bulk) were sonicated through Ultrasonicator

(100 W, 40 KHz) appropriately for 30 minutes before spraying.

3.2.6 Sowing, Fertilizers, Intercultural Operations and Harvesting

Sowing

The seeds (treated for one experiment and untreated seeds for another) were

planted into microplots (1.5 x 1 m2), and thinning was done to maintain 14 plants after

germination per microplot in two rows with a spacing of 45 cm x 60 cm. The


51

observations like germination percentage were taken during crop growth and the maize

plants were allowed to grow upto maturity.

Fertilizers and Manure

The recommended dose of N: P2O5: K2O (100:50:0) kg ha-1) was given to maize

crop in each season. Urea was used as N source, while di-ammonium phosphate (DAP)

as P source. Full dose of P and 50% of N in each plot in the form of di-ammonium

phosphate and urea, respectively were applied in furrows before sowing. Remaining

50% nitrogen was applied in the form of urea in two equal splits at 30 and 45 DAS in

both season. Organic manure in the form of vermicompost, procured from Department

of Agronomy, B. A. College of Agriculture, Anand was applied uniformly in all the

microplots before sowing in summer season at the rate of 2 kg per microplot.

Irrigation, weeding and plant protection

Maize crop being very sensitive to moisture stress; moisture availability to the

crop throughout the growth period was maintained. According to evapotranspiration

rate, water was supplemented by irrigation. The manual weeding was performed at

regular interval of 10-15 days to remove the weeds from the microplots. Dimethoate

30% EC was applied along with water (10 ml per 10 lit) as and when required to control

infestation of sucking pests. Chlorpyriphos was applied along with irrigation water as

and when required to control the infestation of termite.

Harvesting

At maturity (120 DAS), plants were uprooted gently along with the whole soil

mass. The harvested plants with whole root were washed thoroughly with tap water and

then with deionized water. Finally, the plants were kept in laboratory shed for drying


52

and used for further analysis. Roots were separated and used for recording the

parameters. Similarly, mature, filled and unfilled cobs were dried and dry weight per

plant was recorded. After complete drying of cobs, grain was separated manually and

weighed to record total dry matter. Different plant parts were separately dried and kept

in air-tight polythene bags for chemical analysis.

3.2.7 Soil and Plant samples analysis

Soil sampling and Analysis

Representative soil samples from 0-15 cm depth were collected from each plot

after harvest of maize. Then soil samples were air-dried and ground to pass through 2

mm sieve. The samples were labelled and stored in polythene bags for further analysis.

The processed soil samples were analyzed for important soil properties viz. pH,

EC (1:2.5; soil: water ratio), organic carbon, and DTPA (Diethylene Triamine Penta

Acetic acid)-extractable micronutrients (Fe, Zn, Mn and Cu) using standard methods as

shown in Table 3.1.

Plant sampling and analysis

The sample of different plant parts (root, stem, leaf, grain) were washed with

dilute 0.01 N HCl, single, and double demineralized water in a sequence and air dried.

Then samples were oven- dried in brown paper bags at 70 0C till constant weight in a

hot air oven and preserved for further analysis. Dried plant samples (root, stem, leaf,

grain and shell) were ground in a stainless steel mixer to avoid contamination of

micronutrients. The processed samples were labeled and preserved in air tight

polyethylene bags for chemical analysis.

Dried plant samples were wet digested in di-acid mixture (HNO3: HClO4 – 4:1)


53

and volume was made with double distilled water (Jackson, 1973). The extract was

filtered through Whatman filter paper No. 42 and extract was used for analysis of

micronutrients (Fe, Mn, Zn and Cu) on Atomic Absorption Spectrophotometer (AAS

Model: PE 3110).

3.2.8 Computation of nutrient uptake and Accumulation Factor

Nutrient uptake was calculated by using yield (expressed in g microplot-1) and

nutrient content (expressed in mg kg-1) data through following formula.

Nutrient uptake of Zn(mg plot-1)

= Nutrient content (mg kg-1) X Yield (g plot-1)

1000

In order to compute the bioaccumulation/ accumulation factor, following

formula, suggested by Nirmalkumar et al. (2009) was used.

Accumulation/Bioaccumulation Factor

=Mean plant (Root+Straw+Leaves) concentration (µg g-1)

Mean soil available concentration (µg g-1)

3.3 Statistical analysis

The statistical analysis of the data generated during the course of investigation

was carried out as per the method suggested by Steel and Torrie (1982). The value of

‘F’ was worked out and compared with value of ‘F’ at 5% level of significance. The

values of standard error (mean) (S. Em. ±), Critical difference (C. D.) and coefficient

of variation (C. V. %) were also calculated and appropriately used for interpretation of

data, which are presented in respective tables.

RESULTS & DISCUSSION

IV. RESULTS AND DISCUSSION

The results obtained from the sequential studies under present investigation

entitled “Effect of Zinc Oxide nanoparticles on germination, growth and yield of maize

(Zea mays L.)” conducted during rabi and summer seasons of 2015-16 in the

Laboratory as well as microplots are presented in this chapter. Data pertaining to the

behaviour of various Zinc Oxide nanoparticles (ZnO NPs) treatments on seed

germination, crop growth, yield and nutrient uptake by maize and their significance on

soil properties were subjected to statistical analysis in order to test their significance.

An endeavour has been made to discuss the findings of the present investigation for

precise interpretation in view of the available results are furnished under following sub-

headings:

4.1. Synthesis and characterization of ZnO NPs

4.2. Effect of seed treatment with ZnO NPs on germination of maize seeds

4.3. Effect of seed treatment with ZnO NPs on growth and yield of maize

4.4. Effect of foliar application of ZnO NPs on growth and yield of maize

4.1. Synthesis and characterization of ZnO NPs

As mentioned in the preceding chapter, after thorough review of available

literature, hydrothermal method (oxalate decomposition) was selected for the synthesis

of ZnO NPs. Typical ZnO NPs with an average particles size of about 65 nm, computed

using Scherrer equation were prepared by mixing equimolar (0.2 M) solutions of zinc

acetate and oxalic acid, which immediately formed bulky precipitates of zinc oxalate.

The resultant precipitates were collected, washed extensively with deionized water and

dried in air. The final precipitates of zinc oxalate were then calcinated in a furnace for

Results and Discussion

55

4 hours at 500 °C. The synthesized ZnO NPs were characterized by XRD, DLS, TEM,

SEM, TGA and UV-Vis Spectroscopy and the results are discussed in ensuing section.

4.1.1. X-Ray Diffraction (XRD)

The crystallite size and purity of synthesized ZnO NPs were determined by

XRD. From the XRD pattern of ZnO NPs, presented in Fig. 4.1, it was noticed that all

the peaks matched well with the standard wurtzite structure corresponding to JCPDS

Card No. 36-1451 (Morkoc and Ozgur, 2009). Peaks at diffraction angles (2θ) of 31o,

34o, 35o, 47o, 56o, 62o, 67o, 68o and 69o correspond to the reflection from (100), (002),

(101), (102), (110), (103), (200), (112) and (201) crystal planes of the hexagonal

wurtzite ZnO structure (Yang et al., 2004).

Fig. 4.1: X-Ray diffraction pattern of ZnO Nanoparticles

Further, the mean size of the ZnO NPs was estimated using Debye-Sherrer

equation, mentioned in preceding chapter, was found to be 65 nm. Additionally, no

traces of peak corresponding to impurity could be noticed which confirmed the high


56

purity level of ZnO NPs. Moreover, all the diffraction peaks of the product show sharp

peak intensities, indicating good crystalline nature of obtained nanoparticles.

4.1.2 Scanning Electron Microscopy (SEM)

A scanning electron microscope (SEM) can produce very high resolution

images of a sample surface, revealing details about less than 1 to 5 nm in size. Due to

the very narrow electron beam, SEM micrographs have a large depth of field yielding

a characteristic three-dimensional appearance useful for understanding the surface

structure of a sample. Figure 4.2 showed the sub-microscopic images i.e. micrographs

of ZnO NPs with magnification of 500X and 1.74 KX, respectively.

Fig. 4.2: SEM micrographs of ZnO NPs (left: 500X and right: 1.74 KX)

These micrographs clearly indicated that the aggregates of ZnO NPs and the

size of these aggregates was nearly similar. Further, images showed large

agglomerates or clusters of ZnO NPs. Surface of these aggregates were rough in

nature that may be attributed to the nanorods of ZnO. A number of researchers have

reported similar magnification images and showed homogeneous shape and size for

ZnO NPs (Zak et al. 2011).


57

4.1.3 Transmission Electron Spectroscopy (TEM)

The morphology of the synthesized ZnO NPs was characterized by transmission

electron microscopy (TEM) using an accelerating voltage of 200 kV, having a

resolution of ~ 1 Å. For this analysis, the ZnO NPs sample were dispersed in TDW

through a probe sonicator; a drop of the same was placed onto a carbon coated copper

grid and dried at room temperature. It is evident from the TEM micrographs, shown in

Fig. 4.3 that ZnO NPs are rod shaped with a diameter of 60-65 nm and length of 135-

138 nm.

Fig. 4.3: TEM micrographs of ZnO NPs

These TEM images confirmed the formation of ZnO NPs and substantiated the

approximate rod-shape of the ZnO NPs. Additionally, rod like structure is considered

to be the best nanostructure as compared to others one-dimensional nanostructures (viz.


58

nanorods, nanowires, and nanotubes) owing to decreased grain boundaries, surface

defects, disorders, and discontinuous interfaces that facilitate more efficient carrier

transport ability (Morkoc and Ozgur, 2009; Moezzi, et al., 2012).

4.1.4 UV-vis Spectroscopy

A perusal of UV-vis absorption spectra of the ZnO NP, depicted in Fig. 4.4

indicated formation of ZnO NPs, which was confirmed by the presence of excitonic

absorption at 262 nm. Moreover, very sharp absorption peak of ZnO was also noticed,

indicating the monodispersed nature of the nanoparticle distribution as mentioned by

several researchers (Ng et al., 2003; Sharma et al., 2003). The monodispersed nature of

particle distribution was also confirmed by SEM analysis.

Fig. 4.4: UV-vis spectra of ZnO NPs

4.1.5 Thermo-gravimetric Analysis (TGA) of Zinc Oxalate

Thermo-gravimetric analysis (TGA) of zinc oxalate was carried out to observe

characteristic weight loss with temperature during synthesis of ZnO NPs and the results


59

are presented in Fig. 4.5. To synthesize ZnO NPs, zinc oxalate was calcined at a

temperature range of 500 C. From the analysis, it was noticed that the weight loss in

zinc oxalate took place in three steps at 65 0C, 167 0C and 406 0C.

Fig. 4.5: Thermogravimetric analysis of zinc oxalate molecule

The degradation peaks at 65 0C represents the evaporation of water in the form

of moisture and ethanol from the test sample. Further, peak at 167 0C indicated the

decomposition of oxalate molecule owing to weight loss due to acetic acid and crystal

water in oxalic acid molecule. The major weight loss peak observed at 406 C exhibited

the complete decomposition of oxalate molecule that in turn leads to the release of

carbon monoxide (CO) and carbon dioxide (CO2) molecule from the decomposition of

oxalic acid. This final weight loss peak revealed the successful formation of ZnO

nanoparticles as explained by Shen et al. (2006).

In addition, there was no weight loss beyond 406 °C, owing to the complete

decomposition of zinc oxalate. Hence, it was confirmed that 406 °C was the optimum


60

calcination temperature. This was in accordance with the reaction conditions employed

to synthesize ZnO NPs, wherein a temperature of 5000C (~900C higher to optimum

decomposition temperature) was chosen for final decomposition in order to ensure the

complete decomposition of zinc oxalate precursor. In accordance with the results

obtained by Chung et al. (2015), schematic representation of the reaction occurred

during synthesis of ZnO NPs are presented below.

4.1.6 Dynamic Light Scattering (DLS)

Particle size distribution of the ZnO NPs synthesized via hydrothermal method,

was evaluated at its various concentrations (500, 1000 and 2000 ppm) of suspension

prepared in deionized water. The graphs representing particle size distribution of ZnO

NPs, presented in Fig. 4.6, indicated that the particle size distribution of ZnO NPs

varied significantly with suspension concentration. Figure 4.6 (a), (b) and (c) revealed

that the 500 ppm, 1000 ppm and 2000 ppm suspensions of ZnO NPs showed a particle

size distribution in the range of 60-70 nm, 110-140 nm and 160-180 nm, respectively.

The lower particle size and narrow range of distribution was obtained in 500

ppm suspension, wherein, the larger concentration of particle was found to be of 75-87

nm. The particle size obtained for 500 ppm suspension was in accordance with the

results obtained from TEM analysis, wherein the diameter of the ZnO NPs were found


61

to be in the range of 60-65 nm. It can be considered as a good result because the particle

size of synthesized ZnO is below than 100 nm (Hasnidawani et al., 2016).

Fig. 4.6: Particle size distribution of the ZnO nanoparticles (a) 500 ppm, (b) 1000ppm and (c) 2000 ppm


62

Furthermore, the magnitude of zeta potential is an indicator of the repulsive

forces between particles and therefore it can provide a good estimation of the

suspension stability (Hunter, 1981). The larger zeta potential values represent lower

degree of aggregation that leads to higher degree of stability of nanoparticles and

smaller z-averaged hydrodynamic diameter. At lower zeta values, the nanoparticles

flocculate early and the stability in nano-suspension reduces.

The common dividing line between unstable and stable suspensions is taken as

+30 or -30 mV; particles having zeta potentials beyond these limits are generally

considered as stable (Zak et al., 2011). The zeta potential values of ZnO NPs are

presented in Fig 4.7. From the analysis, the zeta potential value was found to be (-29.8

mV), revealing the better stability of synthesized ZnO NPs in aqueous suspension.

Fig. 4.7: Zeta potential of ZnO nanoparticles (500 ppm)

Hence, from results of above mentioned analysis it was observed that the

method employed for ZnO NPs synthesis i.e. Oxalate Decomposition Method is an

efficient method of ZnO NPs synthesis. Thus, synthesized ZnO NPs could be efficiently

utilised in soil-plant studies.


63

4.2. Effect of Seed Treatment with ZnO NPs on Germination of Maize Seed

In order to evaluate the efficacy of different ZnO oxide treatments (including

nanoparticles as well as bulk) on germination of maize, seeds were incubated under in

vitro conditions. The seeds were soaked in different concentrations of ZnO NPs and

bulk ZnO for 2 hours and 4 hours, separately. Fifty maize seeds for each treatment were

incubated on a wet uniform substrate i.e. rolled paper towel at an optimum temperature

(20-30 0C) with at least 8 hours/ day cool white florescent light in a seed incubator for

9 days. On 5th day of incubation, first germination count was recorded wherein the

number of normal seedlings, abnormal seedlings and ungerminated seed were counted.

Similarly, after the prescribed period of incubation i.e. 9th day, the seedlings were

examined and germination as well as seedling length were estimated.

4.2.1 Seed Germination

Germination is normally known as a physiological process beginning with water

imbibition by seeds and culminating in the emergence of the radicles and plumules.

Seed germination test is known to be most widely used phytotoxicity test as it is a direct

exposure method. Therefore, in order to assess the efficacy of ZnO nanoparticles on

maize seeds, germination test was carried out. The data pertaining to the effect of ZnO

NPs and corresponding bulk ZnO upto concentrations of 2000 mg L-1 on seed

germination (%) of maize after 5th day and 9th day of incubation was estimated and

presented in Table 4.1.


64

Table 4.1: Effect of different Zn treatments and soaking time on seed germination

(%) of maize

Treatment Germination (%)(5th days)

Germination (%)(9th day)

2 hours 4 hours 2 hours 4 hoursT1: No Zn (Control) 40.7 51.3 80.0 79.7T2: ZnONPs at 500 ppm 50.3 61.3 92.0 92.3T3: ZnONPs at 1000 ppm 60.7 70.7 98.3 97.7T4: ZnONPs at 2000 ppm 56.7 64.7 95.7 94.0T5: Bulk ZnO at 500 ppm 46.0 63.3 94.7 93.3T6: Bulk ZnO at 1000 ppm 57.3 68.0 95.3 93.0T7: Bulk ZnO at 2000 ppm 55.3 62.0 93.0 94.0T8: ZnO slurry @10 mL kg -1 seed 46.7 54.0 92.3 92.3Mean 51.7 61.9 92.7 92.0

S. Em. (±)S 0.42 0.39Zn 0.84 0.79S x Zn 1.18 1.11

C. D. (p=0.05)S 1.20 NSZn 2.41 2.26S x Zn 3.40 NS

C. V. (%) 3.60 2.09

A perusal of data revealed that there was significant increase in germination (%)

in maize seeds following treatment with ZnO NPs. The results further revealed that

after 5th day of incubation, the difference in germination (%) between the two soaking

time was significant wherein the seeds soaked-in for 4 hours (61.9%) recorded higher

seed germination than that of 2 hours of soaking (51.7%). However, the difference

could not be observed at final count i.e. 9th day of incubation as difference in

germination (%) between both the soaking times was non-significant. After completion

of incubation period, seed germination (%) varied from 79.7% in control to 98.3% in

treatment receiving ZnONPs at 1000 ppm.

Overall, the seed germination increased significantly in all the Zn treatments

over control. Significant difference during early germination stage can be attributed to

increased mobilization of phyto-metabolites within the seeds during soaking with water

No Zn (Control) ZnO NPs at 500 ppm

ZnO NPs at 1000 ppm ZnO NPs at 2000 ppm

Bulk ZnO at 500 ppm Bulk ZnO at 1000 ppm

Bulk ZnO at 2000 ppm ZnO Slurry

Plate 4.1: Effect of different seed Zn treatments on germination ofmaize seeds (5th Day of incubation)


65

loaded with ZnO. However, the slow pace of germination in seeds soaked-in for 2 hours

made it up at the completion of incubation. It has been reported by several workers that

seed treatment with Zn induces a range of biochemical changes in the seed, required to

start the germination process, such as breaking of dormancy, hydrolysis or

metabolization of inhibitors, imbibition and enzyme activation (Ajouri et al. 2004;

Harris et al. 2007, Samad et al. 2014).

Among the treatments, maximum seed germination (%) at 5th day of incubation

was recorded significantly maximum in the treatment receiving ZnONPs at 1000 ppm

while the minimum seed emergence was observed in treatment where no Zn was

applied and soaked-in for 2 hours. Similarly, in the treatment which received bulk ZnO

NP at 1000 ppm also early seed germination was prominent. Similar trend was also

observed among the ZnO treatments where maize seeds were soaked-in for 4 hrs.

Overall, all Zn treatments, resulted in an addition of 12-13% in maize seed

germination post-incubation under in vitro conditions. At the end of incubation also,

ZnO NPs application at 1000 ppm resulted in the highest increase in germination of

maize seed over no Zn control. Almost 98% of maize seeds germinated successfully

when ZnO NPs was applied at 1000 ppm. Though, a dose lower and greater than 1000

ppm, also caused significant increase in seed germination over control however, at

higher dose i.e. 2000 ppm there was significant decrease over 1000 ppm ZnO NPs.

Similar findings were reported by Prasad et al. (2012) in groundnut wherein the

results also showed that nanoscale ZnO at lower concentration promoted seed

germination. Promotory effect of Zn in increasing seed germination was also witnessed

when seeds treated with recommended dose of ZnO registered significant increase in

seed germination (%). Zinc has a number of fundamental functions in plant systems


66

such as synthesis of indole acetic acid (IAA), a phyto-hormone which dramatically

regulates plant growth (Cakmak, 2000).

4.2.2 Root and Shoot Length

The root length (cm) and shoot length (cm) were measured by steel tape after

completion of incubation period. Seedling length gives an idea about the ability of seeds

to germinate and establish in given media. Greater root and shoot length of the seedlings

indicated the higher rate of seed germination. The results on difference in root shoot

length of the maize seedlings are presented in Table 4.2. Perusal of data indicated that

root length of maize seedlings was not significantly affected by soaking times.

Similarly, shoot length of maize seedlings were indifferent to the duration of seed

soaking i.e. 2 hrs and 4 hrs.

Table 4.2: Effect of different Zn treatments and soaking time on root and shoot

length (cm) of maize seedlings

Treatment Root Length (cm) Shoot Length (cm)2 hours 4 hours 2 hours 4 hours

T1: No Zn (Control) 4.59 4.55 1.26 1.25T2: ZnONPs at 500 ppm 6.31 6.25 1.71 1.73T3: ZnONPs at 1000 ppm 6.82 6.75 1.94 1.91T4: ZnONPs at 2000 ppm 5.83 5.83 1.57 1.55T5: Bulk ZnO at 500 ppm 5.87 5.85 1.42 1.39T6: Bulk ZnO at 1000 ppm 6.61 6.52 1.78 1.78T7: Bulk ZnO at 2000 ppm 6.12 6.13 1.53 1.52T8: ZnO slurry @10 mL kg -1 seed 6.02 6.01 1.40 1.41Mean 6.02 5.98 1.58 1.57

S. Em. (±)S 0.02 0.01Zn 0.04 0.02S x Zn 0.06 0.03

C. D. (p=0.05)S NS NSZn 0.13 0.06S x Zn NS NS

C. V. (%) 1.82 3.40





Plate 4.2: Effect of different seed Zn treatments on germination ofmaize seeds (9th Day of incubation)


67

Among different Zn treatments, ZnONPs at 1000 ppm registered the highest

growth of root as well as shoot of maize seedlings at both the soaking durations. It is

noteworthy, that increase in ZnO concentration level from 1000 to 2000 ppm caused

significant reduction in root as well as shoot length indicating that higher rates of ZnO

NPs may be detrimental to seed germination and growth. As in case with seed

germination, Zn supplied through recommended dose of ZnO also registered significant

increase in seedling length over control. Such increase could be ascribed to higher

precursor activity of Zn, especially, ZnO NPs in auxin production (Kobayashi and

Mizutani, 1970).

4.2.3 Seed Vigour Index

Seed vigour index or germination vigour index is calculated by determining the

seedling length (root+shoot) and germination (%) of the same seed lot. The computed

results, presented in Table 4.3 showed that maize seeds responded variably towards

various concentrations of both bulk ZnO and nano ZnO.

Since, there was no significant difference in germination (%), root length (cm)

and shoot length (cm), the seed vigour index of maize seedlings also did not show any

significant difference with respect to change in soaking time. So, it is evident from the

results that both of the soaking time i.e. 2 hrs and 4 hrs gave identical results with

respect to seed vigour index.

In general, all the ZnO treatments were found significantly superior over no Zn

control. Among different Zn treatments, ZnO NPs at 1000 ppm recorded the highest

seed vigour index of germinated maize seed at both the soaking times. Though, different

levels of bulk ZnO also enhanced seed vigour index of maize seedlings but the

magnitude of increase was less than their corresponding nano levels. Further, it was


68

also observed that vigour index increased up to 1000 ppm of ZnO application however,

at higher dose i.e. 2000 ppm the vigour of seedlings declined. Overall, bulk ZnO also

showed significant positive effect on seedling of growth.

Table 4.3: Effect of different Zn treatments and soaking time on seed vigour index

of maize seedlings

Treatment Seed Vigour Index2 hours 4 hours

T1: No Zn (Control) 468.3 461.4T2: ZnONPs at 500 ppm 737.4 736.2T3: ZnONPs at 1000 ppm 861.4 845.4T4: ZnONPs at 2000 ppm 707.9 693.0T5: Bulk ZnO at 500 ppm 690.0 675.6T6: Bulk ZnO at 1000 ppm 800.0 772.3T7: Bulk ZnO at 2000 ppm 711.8 718.2T8: ZnO slurry @10 mL kg -1 seed 684.1 685.0Mean 708 698

S. Em. (±)S 3.33Zn 6.66S x Zn 9.41

C. D. (p=0.05)S NSZn 19.17S x Zn NS

C. V. (%) 2.32

Plants emerging from seed with low Zn concentration have poor seedling

vigour and field establishment on Zn-deficient soils (Yilmaz et al., 1998). Rengel and

Graham (1995) reported that increasing seed Zn content significantly improved root

and shoot growth of wheat under Zn deficiency. They opined that high Zn concentration

in seed could act as a starter fertilizer.

Since ZnO is insoluble in water, the particles of bulk and nano ZnO remain

suspended in water. When maize seeds were soaked-in the suspension, the particles of

bulk and nano ZnO adhered to the seed surface, however, the size range differs

significantly from microparticles (bulk ZnO) to nanoparticles (ZnO NPs). Bulk ZnO





Plate 4.3: Effect of different seed Zn treatments on seedling length andvigour of maize seeds


69

have size range of the order of 1 x 10-6 m, whereas ZnO NPs possess size of the order

of 1x10-9 m. Since the size of nanoparticles is so small, the number of particle per unit

surface area increases as compared to bulk ZnO (macroparticles). Zhou et al. (2011)

reported that ZnO NPs with high specific surface and surface reactivity cannot only be

easily adsorbed on physical surface, but also react with biological proteins and even

absorbed into the cell. Lin and Xing (2008) also pronounced that ZnO NPs were

primarily adsorbed onto the cell surface and then their uptake followed.

As far as detrimental effect of ZnO NPs on maize seedlings is concerned, higher

dose i.e. 2000 ppm showed decline in seedling length as well as seed vigour index.

Since roots are the first target tissues affected with high specific surface area of ZnO

NPs, beneficial or toxic symptoms seem to appear more in roots rather than in shoots

(Sresty and Rao, 1999). Such inhibitory effect of ZnO nanoparticles at higher dose has

also been reported by Lin and Xing (2007) on radish, rape and ryegrass.

Seed qualities (seedling length and vigour) have profound influence on the

establishment and the yield of crops. Healthy plant with well-developed root system

can more effectively mobilize limiting nutrients from the soil and can better withstand

adverse conditions (e.g. dry spells). Vigorous early seedling growth has been shown to

be associated with higher yield (Harris et al., 1999). The vigour of seeds can be

improved by seed treatment, which enhances the speed and uniformity of germination

(Heydecker et al., 1975).

In nutshell, most of the physiological and biochemical processes that precede

the germination are triggered by seed treatment with Zn and persist following the

redesiccation of the seeds. Thus upon seeding, treated seeds can rapidly imbibe and


70

revive the seed metabolism, resulting in a higher germination rate and a reduction in

the inherent physiological heterogeneity in germination.

4.3. Effect of Seed Treatment with ZnO NPs on Growth and Yield of Maize

In order to investigate the effect of seed treatment with ZnO NPs on growth and

yield of maize, two consecutive Microplot studies were conducted during the rabi and

summer seasons of the year 2015-16. Data pertaining to the behaviour of various ZnO

treatments on germination, yield, Zn concentration and uptake by different plant parts

of maize, Zn partitioning and bioaccumulation as well as important soil properties were

recorded and subjected to statistical analysis. The detailed discussion of different results

obtained in the present study is given under appropriate subheadings.

4.3.1 Seed Germination (%)

Results obtained on seed germination (%) of maize as influenced by different

Zn seed treatments are presented in Table 4.4. Overall, the data specified that seed

treatment with Zn induced significant increase in seed germination irrespective of

sources and level of ZnO. A perusal of data indicated that seed germination of maize,

which varied from 83.7 to 98.0% in rabi, 83.0 to 97.0% in summer was significantly

affected by different Zn seed treatments.

Among the Zn treatments, ZnO NPs application at 1000 ppm registered

maximum seed germination which was significantly higher than all other Zn treatments

including bulk ZnO, in both seasons as well pooled analysis. Application of lower dose

of ZnO NPs i.e. 500 ppm also resulted in significant increase in seed germination over

control however, it was at par with all three doses of bulk ZnO and standard dose of

ZnO slurry during both seasons. However, seed germination was significantly

hampered by increasing the level of ZnO NPs to 2000 ppm across the seasons and


71

pooled results. Results clearly indicated that seed treatment with ZnO NPs at 1000 ppm

treatments was superior over its lower and higher doses as well as it corresponding bulk

concentrations.

Table 4.4: Effect of different Zn seed treatments on germination (%) of maize

seeds

Treatment Germination (%)Rabi Summer Pooled

T1: No Zn (Control) 83.7 83.0 83.3T2: ZnO NPs at 500 ppm 93.7 92.3 93.0T3: ZnO NPs at 1000 ppm 98.0 97.0 97.5T4: ZnO NPs at 2000 ppm 91.0 90.7 90.8T5: Bulk ZnO at 500 ppm 91.7 91.0 91.3T6: Bulk ZnO at 1000 ppm 94.0 92.3 93.2T7: Bulk ZnO at 2000 ppm 93.7 93.0 93.3T8: ZnO slurry @10 mL kg -1 seed 94.3 93.3 93.8Mean 92.5 91.6 92.0

S. Em. (±)Zn 1.18 1.06 0.77Season - - 0.40Zn x Season - - 1.13

C. D. (p=0.05)Zn 3.55 3.20 2.20Season - - NSZn x Season - - NS

C. V. (%) 2.22 2.02 2.12

The beneficial effect of Zn on seed germination in maize has been explained in

detail in preceding section. However, the probable reason for the enhanced seed

germination due to ZnO NPs over its bulk form might be due to the nano size of

particles which allow them to penetrate through seed coat easily and hence, provide

better absorption and utilization of these particles by seeds (Korishettar et al., 2016).

The positive effect of the these NPs in improving the germination could also be

ascribed to higher precursor activity of ZnO NPs in production of essential

biomolecules vis-a-vis essential nutrients required for maize growth. Zinc is also an

important component of various enzymes which are responsible for driving many


72

metabolic reactions. Further, ZnO NPs is also expected to induce oxidation-reduction

reactions via the superoxide-ion-radical during germination, resulting the quenching of

free radicals in the germinating seeds. Similar findings have also reported positive

impact of ZnO NPs in different crops (Pandey et al., 2010; Panwar et al., 2012, Prasad

et al., 2012; Shailesh et al., 2013; Laware and Raskar, 2014; Shyla and Natarajan,

2014). Adhikari et al. (2016a) also reported that maize seeds coated with ZnO NPs

enhanced seed germination significantly over no Zn.

In the present investigation, it was noticed that, ZnO NPs at higher

concentration i.e. 2000 ppm decreased seed germination. The probable reason for

decreased germination at higher concentration could be the increased absorption and

accumulation of these ZnO NPs both in extracellular space and within the cells resulted

in reduction in cell division, cell elongation and inhibition of the hydrolytic enzymes

involved in food mobilization during the process of seed germination. Similar results

were noticed by several workers who observed that ZnO NPs at higher concentration

had inhibitory effect on growth and development in in different crops including maize

(Lee et al., 2010; Prasad et al., 2012; Yang et al. 2015).

The overall experimental results indicated that ZnO NPs upto 1000 ppm level

promoted the seed germination which may potentially result in increase in seedling

growth, dry matter production and ultimately economic yield as suggested by Avinash

et al. (2010).

4.3.2 Grain and Stover Yield

The data pertaining to the effect of seed treatment with ZnO nanoparticles on

grain and stover yield as well as total dry matter production rabi, summer and on pooled

basis are presented as under.


73

An appraisal of data, presented in Table 4.5 revealed that application of Zn in

the form of either nano or bulk ZnO through seed treatment caused significant increase

in grain yield of maize over no Zn control during both the crop seasons.

Table 4.5: Effect of different Zn seed treatments on grain yield of maize

Treatment Grain Yield (g plot-1)Rabi Summer Pooled



C. D. (p=0.05)Zn 19.61 15.07 19.86Season - - 5.90Zn x Season - - NS

C. V. (%) 3.18 2.64 2.94

Among different nano Zn treatments, ZnO NPs at 1000 ppm registered the

highest grain yield (407.0 g pot-1 in rabi and 377.4 g plot-1 in summer) which was

significantly superior to 500 ppm and 2000 ppm of ZnO NPs as well as corresponding

level of bulk ZnO. Further, seed treatment of bulk ZnO at 1000 ppm also resulted in

significant increase in grain yield however, its magnitude was lower than ZnO NPs.

Recommended rate of ZnO slurry also resulted in significant increase in grain yield

over no Zn.

Interestingly, the lowest level of ZnO NPs i.e. 500 ppm was much better in

enhancing the yield over its corresponding bulk level. It is worth mentioning here that

higher dose of ZnO NPs i.e. 2000 ppm caused significant reduction in grain yield of


74

maize. Overall, grain yield of maize was significantly greater during rabi than summer

season.

The perusal of data presented in Table 4.6 indicated that on the contrary to the

results of grain yield, stover yield of maize in rabi (717.8 mg plot-1) was significantly

lower than the same in summer (793.0 mg plot-1). However, the effect of Zn application

through seed treatment was significantly positive in all the Zn treatments over no Zn

control.

Table 4.6: Effect of different Zn seed treatments on stover yield of maize

Treatment Stover Yield (g plot-1)Rabi Summer Pooled




C. V. (%) 3.42 2.66 3.03

The data further revealed that seed treatment with ZnO NPs at 1000 ppm

resulted in significantly the highest stover yield in rabi (813.5 mg plot-1), summer

(914.0 mg plot-1) as well as pooled analysis (863.7 mg plot-1) over other Zn treatments.

Dose lower than this also resulted in significantly higher stover yield over control

however, higher level of ZnO NPs caused significant decline in stover yield which was

reflected in corresponding grain yield.


75

As far as bulk ZnO is concerned, all three levels resulted in significant increase

in stover yield of maize however, stover yield at its 1000 ppm level was at par with

2000 ppm in both seasons as well pooled results. However, the magnitude of increase

was less than that of corresponding ZnO NPs levels. Moreover, seed treatment with

ZnO slurry also resulted in significant enhancement in stover yield (Table 4.6).

Table 4.7: Effect of different Zn seed treatments on total dry matter yield of maize

Treatment Dry Matter Yield (g plot-1)Rabi Summer Pooled

T1: No Zn (Control) 891 915 903T2: ZnO NPs at 500 ppm 1041 1106 1074T3: ZnO NPs at 1000 ppm 1220 1291 1256T4: ZnO NPs at 2000 ppm 1114 1142 1128T5: Bulk ZnO at 500 ppm 1008 1069 1039T6: Bulk ZnO at 1000 ppm 1137 1195 1166T7: Bulk ZnO at 2000 ppm 1123 1160 1142T8: ZnO slurry @10 mL kg -1 seed 1058 1105 1081Mean 1074 1123 1099



C. V. (%) 2.56 2.32 2.44

Similar trends and variations were also observed in case of total dry matter yield

of maize as reflected from the data furnished in Table 4.7. Seed treatment with ZnO

NPs at 1000 ppm registered the highest total dry matter yield of maize in rabi, summer

and pooled results.

Forgoing results indicated that the seed treatment with Zn produced higher

grain, stover and total dry matter yield in maize which suggested that being an

essential nutrient Zn plays a vital role in plant growth and development. Zinc also

plays as an activator of enzymes in plants and is directly involved in the biosynthesis


76

of auxin, which produces more cells and dry matter that in turn will be stored in seeds

as sink. Significantly important role of Zn nutrition in seed germination, seedling

emergence, initial crop stand establishment and ultimately crop growth and yield is very

well documented in scientific literature (Pandey et al., 2010; Boonyanitipong et al.,

2011; Prasad et al., 2012; Sedghi et al., 2013; Jayarambabu et al., 2014; Yang et al.,

2015; Adhikari et al., 2016a).

Yilmaz et al. (1998) noticed that wheat plants emerging from seeds with low

Zn have poor seedling vigour and field establishment on Zn-deficient soils. Similarly,

Rengel and Graham (1995) reported from pot culture experiments on wheat plants that

increasing seed Zn content from 0.25 to 0.70 μg per seed significantly improved root

and shoot growth under Zn deficiency. In addition, the result also suggested that seed

treatment with Zn has potential to meet its crop requirements as noticed by Farooq et

al. (2009, 2012) also.

Further, trends in enhancement in seed germination by different Zn treatments

(refer section 4.2) had significant bearings on grain and stover yield of maize.

Furthermore, increase in grain and stover yield of maize by seed treatment with ZnO

NPs may be due to small size and large effective surface area of nanoparticles. Due

to these unique properties, ZnO NPs could have easily be adhered to the cell surface

and later on quickly dissolved in rhizosphere leading to better uptake of Zn (Lopez-

Moreno et al., 2010a). However, at higher concentration of ZnO NPs, grain yield

decreased, these results were in accordance with reports on radish, rape, ryegrass,

corn and lettuce etc. (El-Ghamery et al., 2003; Paschke et al., 2006; Lin and Xing,

2007).


77

Significant increase in stover yield of maize during summer season over rabi

might be attributed to uniform application of organic manure i.e. vermicompost

before sowing in summer season. However, it could not enhance the respective grain

yield and probable reason for low grain yield during summer can be ascribed to more

favourable climatic conditions for maize seed setting during rabi months.

4.3.3 Zinc Concentration

The scrutiny of data given in Table 4.8 revealed that application of Zn through

seed treatment with different forms of ZnO resulted in significant escalation in grain

Zn concentration of maize in both seasons of experiments as well as pooled results.

On average, seed treatment with Zn resulted in 37, 40 and 39% increase in grain Zn

concentration during rabi, summer and pooled analysis, respectively over no Zn

control.

Table 4.8: Effect of different Zn seed treatments on grain Zn concentration of

maize

Treatment Grain Zn concentration (mg kg-1)Rabi Summer Pooled




C. V. (%) 4.75 4.28 4.50


78

Among nano ZnO treatments, seed treatments with ZnO NPs at 1000 ppm

resulted in 68.9% increase on pooled analysis which was significantly greater than its

immediate lower level and all other Zn treatments. However, there was no significant

difference between the effects of seed treatment with 1000 ppm and 2000 ppm ZnO

NPs as both were gave statistically at par results.

Seed treatment with bulk ZnO at 1000 ppm and 2000 ppm also registered

significant enhancement of grain Zn concentration of maize however, the magnitude

was slightly less as compared to corresponding ZnO NPs. Application of ZnO slurry at

recommended dose was also effective in enhancing Zn content in maize grain. Despite

registering low grain yield during summer, grain Zn concentration of maize was

significantly greater during summer when compared to rabi season (Table 4.8). All the

treatment performed uniformly during both the experimental season as Zn x Season

effect was recorded as non-significant.

A glance at data pertaining to influence of seed treatment with ZnO on stover

and root Zn concentration, given in Table 4.9 and 4.10 indicated that Zn contents in

stover and root of maize were significantly enhanced by Zn application during both

seasons as well as pooled results.

The magnitude of increase in Zn content of maize stover by three ZnO NPs

levels i.e. 500, 1000 and 2000 was 16, 43, and 38%, respectively in pooled analysis.

The results further indicated that seed treatment with ZnO NPs was maximum at 1000

ppm application level which was at par with 2000 ppm level. More or less similar

results were also recorded under the treatments receiving corresponding bulk ZnO.


79

Table 4.9: Effect of different Zn seed treatments on stover Zn concentration ofmaize

Treatment Stover Zn concentration (mg kg-1)Rabi Summer Pooled




C. V. (%) 4.40 4.49 4.46

Table 4.10: Effect of different Zn seed treatments on root Zn concentration ofmaize

Treatment Root Zn concentration (mg kg-1)Rabi Summer Pooled




C. V. (%) 2.76 3.24 3.03


80

Likewise, root Zn concentration of maize was also enhanced significantly by

seed treatment with Zn (Table 4.10). Further, results indicated that ZnO NPs at 1000

ppm resulted in significantly the highest increase in Zn concentration in roots of maize

in both season and consequently in pooled analysis. More or less, the trend of increase

was similar to that of grain and stover Zn concentrations.

As comprehended in preceding results on Zn content, significant increase in

Zn concentration in various parts of the plant due to Zn application have been reported

by several contemporary workers in cereal crops including maize (Rengel et al., 1999;

Sharma and Bapat, 2000; Haslett et al. 2001; Varshney et al., 2008; Dhaliwal et al.,

2009; Dhaliwal et al. 2010; Prasad et al. 2012).

Du et al. (2011) suggested that nano ZnO and bulk ZnO have higher solubility

in soil and no ZnO NPs were observed in wheat primary roots grown in soil with ZnO

NPs. However, the total Zn content of wheat tissues increased compared to control

indicating that ZnO NPs dissolved in the root rhizosphere and thereby enhancing the

uptake of Zn+2 ions. Similarly, Lopez-Moreno et al. (2010a), Priester et al. (2012) and

Sedghi et al. (2013) observed similar results in soybean plants.

These studies also opined that ZnO NPs significantly affected the root Zn

content as compared to control as roots accumulate higher Zn at higher dose of ZnO

NPs. Similar results were also obtained by Pandey et al. (2010) in gram, Prasad et al.

(2012) in groundnut, Boonyanitipong et al. (2011) in rice, Jayarambabu et al. (2014)

in mungbean, and Laware and Raskar (2014) in onion wherein ZnO NPs resulted in

positive increase in Zn concentration in different plant parts. It is therefore, clear from

the results that due to greater dissolution in the rhizosphere, the ZnO NPs induced

better content and uptake as compared to bulk counter parts.


81

4.3.4 Zinc Uptake

The data pertaining to Zn uptake by maize grain, stover and root are depicted

graphically in Fig. 4.8, 4.9 and 4.10, respectively. An appraisal of data illustrated in

figures revealed that the highest Zn uptake in all three plant parts was registered under

the treatment receiving 1000 ppm ZnO NPs. Notably, at higher concentration i.e. 2000

ppm ZnO NPs Zn uptake by different maize plant parts decreased. The data further

suggested maximum of Zn uptake was by stover part while grain uptake was

minimum.

Fig. 4.8: Zn uptake by grain as influenced by different Zn seed treatments

Fig. 4.9: Zn uptake by stover as influenced by different Zn seed treatments

0.0

5.0

10.0

15.0

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

BulkZnO 500

BulkZnO1000

BulkZnO2000

ZnOSlurry

Gra

in Z

n up

take

(mg

pot-1

) Rabi Summer Pooled

0.0

20.0

40.0

60.0

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

Bulk ZnO500

Bulk ZnO1000

Bulk ZnO2000

ZnOSlurry

Stov

er Z

n up

take

(mg

pot-1

)

Rabi Summer Pooled


82

Fig. 4.10: Zn uptake by root as influenced by different Zn seed treatments

As mentioned in preceding section, Zn plays an activator role in several

enzymes and is directly involved in biosynthesis of growth substance auxin which

produce more plant cells and dry matter that is in turn stored in the seeds as a sink.

Slaton et al. (2001) reported that treating seeds with ZnO greatly increased rice grain

yield and uptake while, Ajouri et al. (2004) reported that seed priming with Zn was

very effective in improving seed germination and seedling development in barley. Seed

priming with Zn improved germination, seedling development, and yield and related

traits in common bean also (Kaya et al., 2007).The translocation of Zn is desirable for

Zn dense seed which seems to be more appropriate with the seed treatment of ZnO

NPs.

4.3.5 Zinc Uptake Partitioning and Bioaccumulation Factor

The results on partitioning of Zn uptake by different plant parts of maize viz.

grain, stover and root as influenced by different Zn seed treatments are depicted

graphically in Fig 4.11. An overview of the results indicated that total Zn uptake by

maize plant was increased by twofold following seed treatment with ZnO NPs at 1000

0.0

10.0

20.0

30.0

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

Bulk ZnO500

Bulk ZnO1000

Bulk ZnO2000

ZnOSlurry

Roo

t Zn

upta

ke (

mg

pot-1

) Rabi Summer


83

ppm over no Zn control. In general, stover retained relatively greater amount of Zn than

the root and grain of maize following Zn applied through seed treatment. Root Zn

uptake by maize ranged from 28-30% of total uptake; the minimum root uptake was

observed under no Zn treatment which was significantly lower than rest of the Zn

treatments. Zinc uptake by maize stover varied from 57-58% of total Zn uptake while

the same by grain was recorded between 13 to 15% of total Zn uptake. In general, Zn

uptake by grain was relatively higher when seeds were treated with Zn through ZnO

NPs.

Fig. 4.11: Zn uptake partitioning in different plant parts of maize as influencedby different Zn seed treatments (pooled basis)

Plants possess a number of transport mechanisms to control the acquisition,

partitioning and the deposition of the micronutrients metals like Zn. This control is

important because the plants must obtain adequate levels of these micronutrients for

both vegetative and reproductive tissues and these control processes vary temporally

and spatially within a given plant. Much of Zn found in the roots is thought to be in

soluble fraction, incorporated into enzymes and low-molecular-weight organic

compounds. The cell walls also contain a large proportion of the Zn found in roots and

0%

20%

40%

60%

80%

100%

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

BulkZnO 500

BulkZnO 1000

BulkZnO 2000

ZnOSlurry

% o

f T

otal

Zn

Upt

ake

Root Stover Grain


84

this provides a reservoir for uptake if the Zn supply becomes limiting. Zinc is probably

sequestered in vacuoles of root cells as phytate, and vacuoles of leaves with

endogenous organic acids.

Owing to greater biomass accumulation, Zn uptake by stover as a consequence

of seed treatment with ZnO NPs was increased drastically in comparison to its uptake

by root. This signified higher absorption of Zn by foliage of maize but its further

translocation to grain was not in the same proportion; even though increase in Zn uptake

by grain was noticed. However, percentage of total uptake by grain was decreased as

its greater uptake by straw and root was noted. An attempt to understand transfer

coefficient of Zn as influenced by various Zn treatments is made in the ensuing section.

Review of available literature also suggested that significant increase in total Zn

uptake by ZnO NPs was observed due to increase in Zn availability within the plant

system as observed in maize by Kar et al. (2007) and in sorghum by Krishnasamy

(1996). Similar results were also observed by Chaube et al. (2007) and Sharma et al.

(1986) also. However, the uptake of Zn was reduced by increasing the application rate

of ZnO NPs above 1000 mg L-1 which is supported by the findings of Lopez-Moreno

et al., 2010a.

In order to understand the possible mechanism of Zn accumulation and mobility

within the plant system as influenced by different ZnO seed treatments,

Bioaccumulation Factor or Accumulation Factor was computed and presented in Fig.

4.12. A scrutiny of data indicated that application of ZnO NPs at 1000 ppm resulted in

the highest 37.3% increase in accumulation of Zn by maize plant. Further, Zn

bioaccumulation was significantly higher in the treatment receiving 2000 ppm of ZnO

NPs which was substantially greater than the same under 500 ppm ZnO NPs.


85

Interestingly, Zn applied to seeds through bulk ZnO also caused marked increase in Zn

accumulation by maize plant.

Fig. 4.12: Zn bioaccumulation in maize plant as influenced by different Zn seed

treatments

Apparently, the first point of micronutrients entry into the plant is the root

system, and thus up-regulating the necessary ion acquisition processes bring more of

that micronutrient into the plant. In contrast to Fe, Zn is mobilized from old wheat

leaves, especially flag leaf, into developing grains to a considerable extent. Rengel et

al. (1999) reported that Zn was remobilized from leaves of wheat and that a greater

percentage of Zn was remobilized from leaves in plant with a deficient Zn supply.

4.3.6 Soil Parameters after Harvest

The important soil properties viz. pH, EC, OC (%), and DTPA- extractable

micronutrients contents of the experimental microplots were determined at the end of

the experiment i.e. after harvest of maize crop in both the seasons. Results of soil

analysis pertaining to different soil properties are presented under this section.

0.0

60.0

120.0

180.0

Control ZnONPs 500

ZnONPs1000

ZnONPs2000

BulkZnO500

BulkZnO1000

BulkZnO2000

ZnOSlurry

Zn

Bio

accu

mul

atio

n F

acto

r


86

Soil pH, EC and OC

The set of data obtained after the estimation of soil pH (1:2.5), presented in

Table 4.11 indicated that seed treatment with either of Zn sources at any given level did

not cause any significant change in soil reaction. The soil pH of experimental site which

ranged from 8.33 to 8.36 in rabi and 8.16 to 8.21 in summer was slightly alkaline in

nature. However, soil pH in rabi season was significantly higher than the same in

summer season (Table 4.11).

Table 4.11: Effect of different Zn seed treatments on soil pH after harvest of maize

Treatment Soil pHRabi Summer Pooled



C. D. (p=0.05)Zn NS NS NSSeason - - 0.02Zn x Season - - NS

C. V. (%) 0.30 0.41 0.36

Similarly, the results on soil EC, presented in Table 4.12 clearly suggested that

the change in soil EC was found non-significant due to seed treatment with ZnO

nanoparticles and bulk particles in both, rabi (0.20-0.21 dSm-1) and summer (0.18-0.19

dSm-1) seasons. However, pooled analysis of results indicated that soil EC decreased

significantly during summer in comparison to rabi season.


87

Table 4.12: Effect of different Zn seed treatments on soil EC after harvest of maize

Treatment Soil EC (dSm-1)Rabi Summer Pooled




C. V. (%) 5.47 6.09 5.76

Table 4.13: Effect of different Zn seed treatments on soil OC after harvest of maize

Treatment Soil OC (%)Rabi Summer Pooled




C. V. (%) 4.41 4.32 4.39


88

Likewise, organic carbon content of soil, which varied between 0.31-0.34% in

rabi and 0.41-44 in summer did not change significantly due to seed treatment with

ZnO NPs and bulk ZnO particles in both rabi and summer seasons (Table 4.13).

Nevertheless, the effect of season on soil OC (%) was significant as organic carbon

content in summer season (0.43%) was greater than that of in rabi season (0.33%).

The results mentioned above clearly indicated that changes in soil pH (1:2.5),

EC (1:2.5) and OC (%) were non-significant due to application of bulk as well as nano

ZnO through seed treatment. However, effect of season was observed significantly as

pH and EC of the soil decreased while in summer season experiments. It is noteworthy

to mention here that uniform application of organic manure (vermicompost) done

before sowing in summer season might have bearing on decrease in pH and EC of the

soil. Further, application of organic manure might have resulted in significant increase

in organic carbon content of soil in summer season as well.

The favourable influence of organic manures on soil pH and EC through their

effect on soil physical, chemical and biological properties of soil have been very well

documented by several workers (Watson et al., 2002; Eigenberg et al., 2002; Dikinya

and Mufwanzala, 2010; Azeez and Van Averbeke, 2012). Several studies have also

reported that the addition of organic residues increases the soil OC level initially

however, with the course of time it decreases in soil up to a certain period (Manivannan

et al., 2009; Gulser et al., 2010). Further secretion of root exudates and other

biochemical reactions taking place in rhizosphere during the cropping period can also

be ascribed to these slight changes in these soil properties (Roy and Kashem, 2014).

DTPA-extractable micronutrients

An examination of results, presented in Table 4.14 indicated that different Zn

seed treatments including nano sized ZnO did not induce any significant change in


89

DTPA- extractable Zn content of soil after harvest of maize in both the season as well

as pooled analysis. Conversely, effect of season on availability of Zn in soil was

prominent as DTPA- extractable Zn content in rabi was significantly lower than

summer season.

Table 4.14: Effect of different Zn seed treatments on DTPA-extractable Zn and

Fe content in soil after harvest of maize

Treatment DTPA-extractable Zn(mg kg-1)

DTPA-extractable Fe(mg kg-1)

Rabi Summer Pooled Rabi Summer PooledT1: No Zn (Control) 0.26 0.36 0.31 3.74 4.65 4.19T2: ZnO NPs at 500 ppm 0.29 0.35 0.32 3.82 4.69 4.25T3: ZnO NPs at 1000 ppm 0.28 0.38 0.33 3.90 4.72 4.31T4: ZnO NPs at 2000 ppm 0.28 0.41 0.34 3.89 4.73 4.31T5: Bulk ZnO at 500 ppm 0.28 0.39 0.34 3.82 4.53 4.18T6: Bulk ZnO at 1000 ppm 0.28 0.42 0.35 3.85 4.76 4.30T7: Bulk ZnO at 2000 ppm 0.26 0.40 0.33 3.90 4.83 4.37T8: ZnO slurry @10 mL kg -1 seed 0.30 0.42 0.36 3.98 4.76 4.37Mean 0.28 0.39 0.33 3.86 4.71 4.28

S. Em. (±)Zn 0.01 0.02 0.01 0.10 0.06 0.06Season - - 0.01 - - 0.03Zn x Season - - 0.02 - - 0.08

C. D. (p=0.05)Zn NS NS NS NS NS NSSeason - - 0.02 - - 0.08Zn x Season - - NS - - NS

C. V. (%) 6.71 8.32 7.92 4.43 2.12 3.27

Similar to DTPA- extractable Zn content in soil, available Fe content in soil also

did not show any significant change due to different Zn seed treatments. Likewise, in

the succeeding season (summer) significantly positive effect on DTPA-extractable Fe

content across the treatments was noticed (Table 4.14).

The perusal of results depicted in Table 4.15 indicated that similar to Zn and Fe,

DTPA-extractable Mn and Cu in soil did not change due to application of ZnO either

nano or bulk irrespective of their dose. However, in pooled results, the availability of


90

these micronutrients were increased significantly in summer season when compared

with rabi.

Table 4.15: Effect of different Zn seed treatments on DTPA-extractable Mn and

Cu content in soil after harvest of maize

Treatment DTPA-extractable Mn(mg kg-1)

DTPA-extractable Cu(mg kg-1)

Rabi Summer Pooled Rabi Summer PooledT1: No Zn (Control) 3.10 3.67 3.39 0.39 0.51 0.45T2: ZnO NPs at 500 ppm 3.12 3.87 3.50 0.40 0.51 0.45T3: ZnO NPs at 1000 ppm 3.13 3.67 3.40 0.39 0.50 0.44T4: ZnO NPs at 2000 ppm 3.10 3.77 3.44 0.38 0.52 0.45T5: Bulk ZnO at 500 ppm 3.11 3.81 3.46 0.40 0.50 0.45T6: Bulk ZnO at 1000 ppm 3.15 3.85 3.50 0.38 0.51 0.45T7: Bulk ZnO at 2000 ppm 3.21 3.83 3.52 0.42 0.49 0.46T8: ZnO slurry @10 mL kg -1 seed 3.18 3.76 3.47 0.40 0.49 0.45Mean 3.14 3.78 3.46 0.39 0.50 0.45


C. D.(p=0.05)

Zn NS NS NS NS NS NSSeason - - 0.05 - - 0.01Zn x Season - - NS - - NS

C. V. (%) 2.74 2.37 2.54 4.97 4.89 4.96

Higher availability of micronutrients during summer season wherein

vermicompost was applied may be due to mineralization of their organically bound

forms in the organic manure and formation of organic chelates of higher stability which

decreased their susceptibility to adsorption, fixation and precipitation resulting in their

enhanced availability in soil (Kher, 1993).

Organic sources like vermicompost might have also contributed to its enhanced

availability in soil. Hodgson (1963) found that the addition of organic matter to soil

encouraged microorganisms, which under certain conditions aided in the liberation of

trace elements. The results are in conformation to the findings of Singh et al. (1999),


91

Sudhir et al. (2002), Kumar and Yadav (2005), Behera and Singh (2009), Kumar and

Singh (2010), and Shambhavi (2011).

As far as fate of ZnO NPs in soil is concerned, Wang et al. (2010) reported that

ZnO NPs and bulk particles have higher solubility in soil environment. Similar findings

were reported by Du et al. (2011) that ZnO NPs were no longer retained in the soil for

longer period of time and dissolved in the soil, leaving no significant change in soil

chemical properties at the end of crop growth period.

4.4. Effect of Foliar Treatment with ZnO NPs on Growth and Yield of Maize

Microplot studies were carried out to evaluate the effect of foliar treatment with

ZnO (nano as well bulk) and conventional ZnSO4 on growth and yield of maize for two

seasons and results were pooled analyzed. The detail results after statistical analysis is

discussed as under.

4.4.1. Grain and Stover Yield

The data pertaining to the effect of foliar application of ZnO NPs on grain yield,

stover yield and total dry matter for rabi, summer and on pooled basis were recorded at

the termination of experiments and statistically analysed. An appraisal of data, given in

Table 4.16 revealed that the response of maize to two foliar application of ZnO NPs at

1000 ppm was found superior over control and ZnO NPs at 500 ppm in both the seasons

as well as on pooled basis, however, it was with at par 2000 ppm of ZnO NPs. Results

further indicated that bulk ZnO was inferior in supplying Zn through foliar application

in maize whereas two sprays of conventional 0.5% ZnSO4 was significantly better.

Furthermore, overall grain yield during summer season was significantly lower

than in rabi however, the performance trend of different foliar treatment remained


92

constant over the seasons. The results clearly suggested that foliar application of ZnO

at 1000 ppm has potential to meet the Zn requirement and enhancing maize grain yield.

Table 4.16: Effect of different foliar Zn treatments on grain yield of maize

Treatment Grain Yield (g plot-1)Rabi Summer Pooled

T1: No Zn (Control) 369.3 331.7 350.5T2: ZnO NPs at 500 ppm 452.0 391.7 421.8T3: ZnO NPs at 1000 ppm 488.3 428.0 458.2T4: ZnO NPs at 2000 ppm 492.0 422.3 457.2T5: Bulk ZnO at 500 ppm 382.0 336.7 359.3T6: Bulk ZnO at 1000 ppm 382.3 344.7 363.5T7: Bulk ZnO at 2000 ppm 387.3 354.7 371.0T8: 0.5% ZnSO4 457.7 397.0 427.3Mean 426.4 375.8 401.1



C. V. (%) 2.91 2.71 2.84

In case of stover yield of maize as influenced by different foliar Zn treatments

also, ZnO NPs at 1000 ppm registered significantly the highest value (965.9, 1057.3,

1011.6 mg plot-1 in rabi, summer and pooled results, respectively) which was at par

with the stover yield obtained under ZnO NPs at 2000 ppm (Table 4.17). Effect of

different foliar Zn treatments on total dry matter yield of maize was also in the line

of the results obtained for grain and stover yields (Table 4.18).

The forgoing results on yield indicated that foliar application of ZnO NPs was

much superior to bulk ZnO in enhancing yield of maize. It is also worthy to mention

here that owing to application of vermicompost before sowing of summer crop

resulted in significantly higher stover and biomass yield; however, it could not

translate in to enhanced grain yield due to relatively less favourable environmental

for pollination and seed setting in maize.


93

Table 4.17: Effect of different foliar Zn treatments on stover yield of maize

Treatment Stover Yield (g plot-1)Rabi Summer Pooled




C. V. (%) 3.55 3.43 3.49

Table 4.18: Effect of different foliar Zn treatments on total dry matter yield of

maize

Treatment Dry Matter Yield (g plot-1)Rabi Summer Pooled

T1: No Zn (Control) 1038 1095 1067T2: ZnO NPs at 500 ppm 1286 1335 1311T3: ZnO NPs at 1000 ppm 1454 1485 1470T4: ZnO NPs at 2000 ppm 1443 1483 1463T5: Bulk ZnO at 500 ppm 1114 1159 1136T6: Bulk ZnO at 1000 ppm 1130 1190 1160T7: Bulk ZnO at 2000 ppm 1143 1212 1178T8: 0.5% ZnSO4 1314 1381 1347Mean 1240 1292 1266



C. V. (%) 2.35 3.02 2.72


94

Positive effect of Zn on grain yield on Zn deficient soil is one of the most widely

documented facts across the world (Patel, 2011; Behera et al., 2015). However, impact

of foliar application of ZnO NPs on crop growth and yield is not yet properly explored.

Application of foliar sprays implies that the nutrients applied will be absorbed and

exported from the point of application (leaf) to the point of utilization. Thus, in foliage

applications, nutrients need to first travel through the leaf cuticle (Monreal et al., 2016).

Since the pore diameter of cell walls of root hairs of plants is in the range of

3.5-3.8 nm, only nanoparticles or aggregates with diameters less than the cell wall pore-

diameter can enter the cell wall of undamaged cells (Dietz and Herth, 2011). Moreover,

custom-made ZnO NPs, which is having less hydrophilicity and being more dispersible

in lypophilic substances compared to the ions, can penetrate through the leaf surface

compared to ZnSO4 (Da Silva et al., 2006). The bioavailability of the nanoparticles

because of its size and lower water solubility (which inhibit rapid falling off compared

to ionic supplements) can also be higher compared to chelated ZnSO4.

Fittingly, in some experiments, it has been observed that ZnO NPs significantly

influenced the growth, yield, and Zn content of maize grains (Subbaiah et al., 2016).

Analogous results were obtained by Adhikari et al. (2015) on maize plant where in

results of solution culture study showed that the application of ZnO NPs at relatively

lower level enhanced the growth of maize plant as compared to conventional Zn

fertilizer i.e. ZnSO4.

Likewise, Farnia and Omidi (2015) also reported positive increase in grain yield

of maize due to application of nano Zn fertilizer. The results from experiments by

Prasad et al. (2012) in groundnut, Kisan et al. (2015) in spinach, and Davarpanah et al.

(2016) in pomegranate also suggested that application of ZnO NPs increased the crop

yields.


95

4.4.2. Zinc Concentration

The scrutiny of data, presented in Table 4.19 revealed that ZnO NPs

application through foliar spray induced significant increase in grain Zn concentration

of maize over no Zn control.

Table 4.19: Effect of different foliar Zn treatments on grain Zn concentration of

maize

Treatment Grain Zn concentration (mg kg-1)Rabi Summer Pooled




C. V. (%) 4.35 6.16 5.55

Among different concentration levels of ZnO NPs, 1000 ppm caused

significantly the highest enhancement in Zn content of maize grain, however, it was

statistically at par with 2000 ppm ZnO NP. Quantitatively, foliar supplementation of

ZnO NPs at 1000 ppm resulted in 68, 71 and 69% increase in grain Zn concentration

over respective control in rabi, summer and pooled results, respectively. The

quantum of increase was almost similar at higher dose of ZnO NPs i.e. 2000 ppm

however, at 500 ppm level the increase was significantly low.


96

It is evident from the results that foliar application of bulk ZnO was much

inferior to ZnO NPs with respect to Zn fortification in grain maize. However, standard

foliar Zn supplementation through ZnSO4 proved significantly better than bulk ZnO

levels but its performance was at par with results at ZnO NPs at 500 ppm level (Table

4.19).

Significantly higher grain Zn concentration was recorded during summer

season which could be ascribed to enhanced Zn availability to plant in summer. In

addition, low impact of metal dilution effect due to low grain yield might also be

responsible for greater Zn accumulation in grain.

Table 4.20: Effect of different Zn foliar Zn treatments on stover Zn concentration

of maize

Treatment Stover Zn concentration (mg kg-1)Rabi Summer Pooled




C. V. (%) 5.27 5.08 5.17

Similar to the results obtained in case of grain Zn concentration, Zn contents

in stover and root of maize were also influenced significantly by foliar treatment of

ZnO as well as ZnSO4 (Table 4.20 and 4.21). Application of ZnO NPs at 1000 ppm


97

concentration to foliage registered the highest Zn contents in stover and root however,

the results were at par with ZnO NPs at 2000 ppm. As witnessed in case of grain Zn,

bulk ZnO at all three levels were proved inferior to their corresponding ZnO NPs level.

In general, Zn content in stover and root during summer were significantly greater than

those recorded in rabi.

Table 4.21: Effect of different foliar Zn treatments on root Zn concentration of

maize

Treatment Root Zn concentration (mg kg-1)Rabi Summer Pooled




C. V. (%) 4.70 4.60 4.65

As evident from the results obtained by Subbaiah et al. (2016) and Adhikari et

al. (2015), ZnO NPs significantly influenced Zn content of different plant parts of maize

including grain. Further, they also opined that application of ZnO NPs at lower level

enhanced the Zn content in maize grain as compared to conventional Zn fertilizer i.e.

ZnSO4. Analogous results were also reported by Prasad et al. (2012), wherein the post-

harvest leaf and kernel samples analysis revealed a significant increment in Zn content

in leaves and kernels of groundnut when supplied with ZnO NPs compared to ZnSO4.


98

Inferiority of ZnO for foliar Zn supplementation can be ascribed to comparatively larger

size, lower surface area as well low solubility in water (Takkar and Walker, 1993).

4.4.3. Zinc Uptake

The results on Zn uptake by grain, stover and root as influenced by different

foliar Zn treatments were computed-out for each season and pooled basis which are

depicted graphically here (Fig. 4.17).

Fig. 4.13: Zn uptake by grain as influenced by different foliar Zn treatments

A summation of results, presented in Fig 4.3 indicated that two foliar

application of ZnO NPs either at 1000 or 2000 ppm resulted in more than two fold

increase in grain Zn uptake (17.17 and 17.12 mg plot-1, respectively) over no Zn

control (7.76 mg plot-1) in pooled results. Foliar supplementation with lower dose of

ZnO NPs i.e. 500 ppm and 0.5% ZnSO4 performed equally in enhancing grain Zn

uptake. Further, bulk ZnO sprays at all three levels caused no substantial increase in

Zn uptake by grain during both seasons as well as pooled results.

0

5

10

15

20

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

Bulk ZnO500

Bulk ZnO1000

Bulk ZnO2000

0.5%ZnSO4

Gra

in Z

n up

take

(m

g pl

ot-1

) Rabi Summer Pooled


99

Fig. 4.14: Zn uptake by stover as influenced by different foliar Zn treatments

Similar to grain Zn uptake, ZnO NPs at 1000 and 2000 ppm level were equally

good in improving the Zn uptake by stover as well as root. Almost two times increase

in stover and root Zn uptake was registered by these ZnO NPs treatments (Fig. 4.14 and

4.15).

Fig. 4.15: Zn uptake by root as influenced by different foliar Zn treatments

As Zn uptake is dependent on yield of particular plant part and Zn concentration

in respective parts, it also follows the same trend. The forgoing results were in

0

20

40

60

80

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

Bulk ZnO500

Bulk ZnO1000

Bulk ZnO2000

0.5%ZnSO4

Stov

er Z

n up

take

(m

g pl

ot-1

)

Rabi Summer Pooled

0

10

20

30

40

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

Bulk ZnO500

Bulk ZnO1000

Bulk ZnO2000

0.5%ZnSO4

Roo

t Z

n up

take

(m

g pl

ot-1

)

Rabi Summer Pooled


100

corroboration with the findings of Prasad et al. (2012), Subbaiah et al. (2016), Adhikari

et al. (2016a). Similar results were also obtained by Eichert et al. (2008) who

demonstrated the mechanism of foliar uptake pathway for aqueous solutes and water-

suspended nanoparticles in Allium porrum and Vicia faba. They also observed that the

stomatal pathway differs fundamentally from the cuticular foliar uptake pathway.

However, the uptake and translocation mechanism of foliarly applied ZnO NPs is yet

to be fairly understood.

4.4.4. Zinc Uptake Partitioning and Bioaccumulation Factor

Micronutrients, especially Zn differ widely in their distribution within plants

and their ability to be remobilized from certain organs or tissue for transport to

developing seeds. In either case, the nature of the Zn storage pool and the capacity for

phloem loading of Zn dictate its mobility. The trafficking of Zn from the phloem to its

deposition in the cereal grains constitutes the last phase in a long series of events from

uptake in the roots until storage in the grain. In order to understand this variation in

distribution within plants, the graphical presentation of the partitioning of Zn as

influenced by various foliar Zn treatments is shown in Fig. 4.16.

As evident from the following graph, on average Zn accumulation by root,

stover and grain was in the ratio of 2:4:1, respectively. However, foliar application of

Zn through ZnO NPs caused greater accumulation in grain as compared to ZnO bulk.

About 13-15% of Zn was accumulated in grain in the treatments receiving ZnO NPs.

Moreover, more Zn from root was re-mobilized to upper plant parts resulting in higher

grain Zn concentration.


101

Fig. 4.16: Zn uptake partitioning in different plant parts of maize as influenced

by different foliar Zn treatments (pooled basis)

Data pertaining to accumulation factor of Zn as affected by various foliar Zn

treatments including ZnO NPs is presented in Fig. 4.17.

Fig. 4.17: Zn bioaccumulation in maize plant as influenced by different foliar Zn

treatments (pooled basis)

0%

20%

40%

60%

80%

100%

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

Bulk ZnO500

Bulk ZnO1000

Bulk ZnO2000

0.5%ZnSO4

% o

f T

otal

Zn

Upt

ake

Root Stover Grain

0

30

60

90

120

150

180

Control ZnO NPs500

ZnO NPs1000

ZnO NPs2000

BulkZnO 500

BulkZnO1000

BulkZnO2000

0.5%ZnSO4

Zn

Bio

accu

mul

atio

n F

acto

r


102

A summarized perusal of data indicated that accumulation of Zn by plant parts

increased significantly in treatments receiving foliar applied ZnO NPs over no Zn.

Among ZnO NPs treatments, 1000 and 2000 ppm levels enhanced plant accumulation

of Zn by one and half times. The enhanced availability in Zn to plants has significant

bearing on Zn bioaccumulation by maize plant during summer.

From the present results, it appeared that increased Zn availability to plant may

not necessarily increase its content in the grain. However, under treatments involving

ZnO NPs, significant increase in accumulation factor was registered which might have

resulted in increased grain Zn concentration. The summary of above results on

partitioning and accumulation factor suggested that accumulation of Zn in grain was

increased with its foliar application through ZnO NPs and ZnSO4. Similar explanation

was also put forth by Moretti et al. (2014).

4.4.5. Soil Parameters after Harvest

After harvest of maize in each season, soil samples were collected from all the

microplots and analyzed for important soil properties viz. pH, EC, OC (%) and DTPA-

extractable micronutrients. The effect of different foliar Zn treatments were statistically

analysed for both the seasons individually and on pooled basis and results are provided

in this section.

Soil pH, EC and OC

Generally, the soil reaction of experimental site was slightly alkaline in nature

as pH of soil varied from 8.29 to 8.34 during rabi season while, from 8.18 to 8.22 during

summer (Table 4.22). Perusal of data indicated that different foliar treatments of Zn

could not induce any significant change in soil pH in both the seasons as well as pooled

results. However, pooled analysis showed that soil pH during summer (8.20) was


103

significantly lower than in the rabi season (8.31). Effect of Season x Zn on pH was

found non-significant.

Table 4.22: Effect of different foliar Zn treatments on soil pH after harvest of

maize

Treatment Soil pHRabi Summer Pooled




C. V. (%) 2.99 1.03 2.24

The results on soil EC, presented in Table 4.23 clearly suggested that foliar

application of ZnO NPs and ZnO bulk did not have any significant effect on soil EC

during both the experimental seasons and pooled results. Nevertheless, in rabi season

soil EC which varied from 0.22 to 0.24 dSm-1, with a mean value of 0.23 dSm-1 was

significantly greater than that of summer (mean 0.20 dSm-1). Statistically, Zn x Season

effect was also non-significant.

Similar to pH and EC, soil organic carbon content of soil was not altered

significantly by various foliar Zn treatments including nano as well as bulk ZnO (Table

4.24) in both seasons and pooled results.


104

Table 4.23: Effect of different foliar Zn treatments on soil EC after harvest of

maize

Treatment Soil EC (dSm-1)Rabi Summer Pooled




C. V. (%) 5.77 5.05 5.50

Table 4.24: Effect of different foliar Zn treatments on soil OC after harvest of

maize

Treatment Soil OC (%)Rabi Summer Pooled




C. V. (%) 4.99 3.54 4.23


105

In general, soil organic content was quite low and categorized as the soil having

low fertility with respect to organic carbon. Nonetheless, effect of season was seen as

soil OC (%) increased in summer season by 0.05 unit over rabi.

The above described positive trends in pH, EC and OC (%) between the seasons

may be accredited to uniform addition of vermicompost during summer maize. Several

workers reported decrease in pH and EC of soil due to the incorporation of

vermicompost and other similar organics to different crops (Hangarge et al., 2004;

Rajshree et al., 2005; Ghuman and Sur, 2006; Vijayashankar et al., 2007; Gathala et al.

2007). This decrease in pH and EC with application of vermicompost might be

attributed to the release of organic acids as a result of decomposition due to added

organics (Raghuwanshi et al., 1998). In addition, these changes might also be due to

better root growth and more plant residue left across all the microplots (Pawar et al.,

1987).

DTPA-extractable micronutrients

The content of DTPA-extractable Zn ranged from 0.35-0.38 mg kg-1 in rabi and

0.40-0.44 mg kg-1 in summer, respectively; while DTPA-Fe varied from 3.83-3.93 mg

kg-1 in rabi and 4.32-4.55 mg kg-1 in summer, respectively (Table 4.25).

A scrutiny of data indicated that DTPA-extractable Zn and Fe contents were

also not affected by any of the foliar Zn treatments during both the seasons as well as

in pooled analysis. However, DTPA- Zn and Fe contents were increased at the end of

summer season in comparison to rabi. Similar to the trends observed in changes of

DTPA-extractable Zn and Fe, DTPA-extractable Mn and Cu contents in soil were also

not affected by different foliar Zn treatments (Table 4.26). At the end of summer

experiment, their contents were enhanced across the treatments.


106

Table 4.25: Effect of different foliar Zn treatments on DTPA-extractable Zn andFe content in soil after harvest of maize

Treatment DTPA-extractable Zn(mg kg-1)

DTPA-extractable Fe(mg kg-1)

Rabi Summer Pooled Rabi Summer PooledT1: No Zn (Control) 0.35 0.41 0.38 3.83 4.32 4.08T2: ZnO NPs at 500 ppm 0.37 0.42 0.40 3.89 4.31 4.10T3: ZnO NPs at 1000 ppm 0.38 0.43 0.41 3.86 4.32 4.09T4: ZnO NPs at 2000 ppm 0.35 0.40 0.38 3.88 4.39 4.14T5: Bulk ZnO at 500 ppm 0.36 0.42 0.39 3.85 4.38 4.12T6: Bulk ZnO at 1000 ppm 0.37 0.44 0.41 3.88 4.35 4.11T7: Bulk ZnO at 2000 ppm 0.37 0.43 0.40 3.93 4.40 4.17T8: 0.5% ZnSO4 0.36 0.42 0.39 3.86 4.55 4.20Mean 0.36 0.42 0.39 3.87 4.38 4.12



C. V. (%) 4.98 3.64 4.27 3.98 3.07 3.50

Table 4.26: Effect of different foliar Zn treatments on DTPA-extractable Mn andCu contents in soil after harvest of maize

Treatment DTPA-extractable Mn(mg kg-1)

DTPA-extractable Cu(mg kg-1)

Rabi Summer Pooled Rabi Summer PooledT1: No Zn (Control) 3.36 3.70 3.53 0.45 0.51 0.48T2: ZnO NPs at 500 ppm 3.40 3.73 3.57 0.47 0.55 0.51T3: ZnO NPs at 1000 ppm 3.31 3.73 3.52 0.45 0.54 0.49T4: ZnO NPs at 2000 ppm 3.37 3.70 3.53 0.46 0.53 0.49T5: Bulk ZnO at 500 ppm 3.39 3.75 3.57 0.47 0.54 0.50T6: Bulk ZnO at 1000 ppm 3.42 3.78 3.60 0.49 0.52 0.50T7: Bulk ZnO at 2000 ppm 3.44 3.77 3.61 0.45 0.54 0.49T8: 0.5% ZnSO4 3.31 3.74 3.53 0.46 0.52 0.49Mean 3.37 3.74 3.56 0.46 0.53 0.50



C. V. (%) 4.07 3.32 3.68 4.50 4.29 4.39


107

The above specified increase in DTPA-micronutrients status of soil in summer

season following application of vermicompost might be due to direct addition of

micronutrients to soil and release of chelating agents which might have prevented

micronutrients from precipitation, oxidation and leaching (Datt et al., 2003). In

addition, the increase in micronutrient cations might be a result of transformation of

sound phase to soluble metal complexes i.e. DTPA-extractable form as reported by

Ismail et al. (2001) and Arbad et al. (2008).

Further, Zn forms relatively stable chelates with organic ligand, which decrease

its susceptibility to adsorption, fixation and precipitation. The incorporation of organic

manures might have resulted in the formation of such organic chelates of higher

stability (Jagtap et al., 2007; Singh et al., 2009). While, The increase in DTPA-

extractable Fe might be due to intensified microbial and chemical reduction of Fe3+ and

also formation of stable complexes with organic ligands which might have decreased

fixation or precipitation reaction in soil resulting in its greater availability in soil.

Likewise, increase in DTPA-extractable Cu and Mn contents in soil with organic

manures might be due to mineralization and release of native forms of these nutrients

(Harikrishna et al., 2002).

SUMMARY AND CONCLUSION

V. SUMMARY AND CONCLUSION

Despite all the technological, varietal, and mechanization interventions in maize

cultivation, its productivity in India is almost half of the global average. One of the

major constraints for low productivity is unsustainable intensification accompanied by

imbalanced soil nutrient management which deprives crop from proper nutrients,

especially Zn availability. However, use efficiency of sources of Zn supplementation

such as, sulphates, oxides and chelated Zn fertilizer hardly crosses 5%. As opined by

several researchers, decreased in particle size of Zn fertilizers is expected to increase

the dissolution rate of Zn fertilizers with low water solubility.

Nanoparticles with small size and large effective surface area fall in the

transition zone between individual molecules and corresponding bulk materials which

generate both positive and negative effects on plants growth and yield. Zinc oxide

nanoparticles, which is one of the most commonly used engineered metal oxide nano

particles is expected to be the ideal material for use as a Zn fertilizer in plants. Against

this milieu, a sequential study was taken up to investigate the effect of various

concentrations of ZnO nanoparticles (ZnONPs) on growth, development and yield of

maize. Salient results of the study are summarized as under.

5.1. Synthesis and Characterization of ZnO NPs

From the XRD pattern of ZnO NPs, it was noticed that all the peaks matched

well with the standard crystal planes of the hexagonal wurtzite structure corresponding

to JCPDS Card No. 36-1451 with high purity level. Further, the mean size of the ZnO

NPs was estimated using Debye-Sherrer equation, was estimated as 65 nm. The SEM

micrographs clearly indicated that the aggregates of ZnO NPs and the size of these

Summary and Conclusion

109

aggregates was nearly similar. Surface of these aggregates were rough in nature that

may be attributed to the nanorods of ZnO.

The TEM images confirmed the formation of ZnO NPs and substantiated the

approximate rod-shape of the ZnO NPs, which is considered to be the best

nanostructure as compared to others one-dimensional nanostructures. Formation of

ZnO NPs, was further confirmed by the presence of excitonic absorption at 262 nm in

UV-vis absorption spectra which further indicated the monodispersed nature of the

nanoparticle distribution.

From results of TGA analysis of zinc oxalate, calcination temperature was

computed as 406 °C which was in accordance with the reaction conditions employed to

synthesize ZnO NPs. Particle size distribution analysis through DLS showed a particle

size distribution in the range of 60-70 nm in 500 ppm ZnO NPs suspension. From the

analysis, the zeta potential value was found to be (-29.8 mV), revealing the better

stability of synthesized ZnO nanoparticles in aqueous suspension.

5.2. Effect of Seed Treatment with ZnO NPs on Germination of Maize Seeds

5.2.1. Seed Germination (%)

Significant increase in germination in maize seeds was noticed after 5th day and

9th day of incubation following treatment with ZnO NPs. After 5th day of incubation,

the difference in germination (%) between the two soaking time was significant wherein

the seeds soaked-in for 4 hours recorded higher seed germination than that of 2 hours

of soaking. However, the difference could not be observed at final count i.e. 9th day of

incubation as difference in germination (%) between both the soaking times was non-

significant. Among different Zn treatments, ZnO NPs application at 1000 ppm resulted

in the highest increase in germination of maize seed over no Zn control. Almost 98%


110

of maize seeds germinated successfully when ZnO NPs was applied at 1000 ppm.

However, bulk ZnO applied at 1000 ppm was statistically at par. Interestingly, a dose

lower and greater than 1000 ppm, also caused significant increase in seed germination

over control however, at higher dose i.e. 2000 ppm, there was significant decrease over

1000 ppm level.

5.2.2. Root and Shoot Length of Seedlings

Two soaking durations i.e. 2 hrs and 4 hrs did not show any significant

difference in root and shoot lengths of maize seedlings. However, seed treatment with

Zn through either of sources caused significant increase in seedling length over no Zn

control. Among different Zn treatments, ZnO NPs at 1000 ppm registered the highest

growth of root as well as shoot of maize seedlings at both the soaking durations. As in

case with seed germination, Zn supplied through recommended dose of ZnO also

registered significant increase in seedling length over control. It is noteworthy, that

increase in ZnO concentration level from 1000 to 2000 ppm caused significant

reduction in root as well as shoot length indicating that higher rates of ZnO either

through nano or bulk material may be detrimental to seed germination and growth.

5.2.3. Seed Vigour Index

Since, there was no significant difference in germination, root and shoot lengths,

the seed vigour index of maize seedlings also did not show any significant difference

with respect to change in soaking time. In general, all the ZnO treatments were found

significantly superior over no Zn control. Among different Zn treatments, ZnO NPs at

1000 ppm recorded the highest seed vigour index of germinated maize seed at both the

soaking times. Though, different levels of bulk ZnO also enhanced seed vigour index

of maize seedlings but the magnitude of increase was less than their corresponding nano


111

levels. As far as toxic or detrimental effect of ZnO NPs on maize seedlings are

concerned, the higher dose i.e. 2000 ppm showed decline in seedling length as well as

seed vigour index.

5.3. Effect of seed treatment with ZnO NPs on growth and yield of maize

5.3.1 Seed Germination (%)

Among the Zn treatments, ZnO NPs application at 1000 ppm registered

maximum seed germination in maize which was significantly higher than all other Zn

treatments including bulk ZnO in both seasons as well pooled analysis. Application of

lower dose of ZnO NPs i.e. 500 ppm also resulted in significant increase in seed

germination over control however, it was at par with all three doses of bulk ZnO and

standard dose of ZnO slurry during both seasons. Moreover, seed germination was

significantly hampered by increasing the level of ZnO NPs to 2000 ppm across the

seasons and pooled results.

5.3.2 Grain and Stover Yield

In general, application of Zn in the form of either nano or bulk ZnO through

seed treatment caused significant increase in grain, stover and total dry matter yield of

maize over no Zn control during both the crop seasons. Among different ZnO NPs

treatments, ZnO NPs at 1000 ppm registered significantly the highest grain, stover and

total dry matter yield during both the seasons. Interestingly, the lowest level of ZnO

NPs i.e. 500 ppm was much better in enhancing the yield over its corresponding bulk

level. It is worth mentioning here that higher dose of ZnO NPs i.e. 2000 ppm caused

significant reduction in grain yield of maize. Overall, grain, stover and total dry matter

yield of maize was significantly greater during rabi than summer season.


112


Application of Zn through seed treatment with different forms of ZnO resulted

in significant escalation in grain, stover and root Zn concentration of maize in both

seasons of experiments as well as pooled results. On average, seed treatment with

ZnO NPs resulted in 37, 40 and 39% increase in grain Zn concentration during Rabi,

summer and pooled analysis, respectively over no Zn control.

5.3.4 Zinc Uptake

The highest Zn uptake in all three plant parts i.e. grain, stover and root was

registered under the treatment receiving 1000 ppm ZnO NPs through seed treatment.

Notably, at higher concentration i.e. 2000 ppm ZnO NPs Zn uptake by different maize

plant parts decreased.

5.3.5 Zinc Uptake Partitioning and Bioaccumulation Factor

Total Zn uptake by maize plant increased by two-fold following seed treatment

with ZnO NPs at 1000 ppm over no Zn control. In general, stover retained relatively

greater quantity of total Zn uptake than the root as well as grain of maize following Zn

applied through seed treatment. Application of ZnO NPs at 1000 ppm also resulted in

the highest (37.3%) increase in accumulation of Zn by maize plant.


The important soil properties viz. pH, EC, OC (%), and DTPA- extractable

micronutrients contents of the experimental microplots, determined at the end of the

experiment i.e. after harvest of maize crop in both the seasons did not show any

significant change due to various Zn seed treatments. However, soil pH and EC of the

experimental site in summer was slightly lower than in Rabi season while OC (%), and


113

DTPA- extractable micronutrients contents increased significantly in summer possibly

as a consequence of vermicompost application.

5.4. Effect of Foliar Application of ZnO NPs on Growth and Yield of Maize

5.4.1 Grain, Stover and Dry Matter Yield

The response of maize to two foliar application of ZnO NPs at 1000 ppm was

found superior in enhancing grain, stover and total dry matter yield of maize over no

Zn control as well ZnO NPs at 500 ppm in both the seasons as well as on pooled basis,

however, it was at par 2000 ppm of ZnO NPs. Results further indicated that bulk ZnO

was inferior in supplying Zn through foliar application in maize whereas two sprays of

conventional 0.5% ZnSO4 was significantly better. Furthermore, overall grain yield of

maize during summer season was significantly lower than in rabi however, the

performance trend of different foliar treatment remained constant over the seasons.


Zinc oxide nanoparticles application through foliar spray induced significant

increase in grain, straw and root Zn concentration of maize over no Zn control.

Among different concentration levels of ZnO NPs, 1000 ppm resulted in significantly

the highest enhancement in Zn contents in different plant parts of maize, however, it

was statistically at par with 2000 ppm ZnO NP. Further, foliar application of bulk

ZnO was noticed to be much inferior to ZnO NPs in Zn fortification in grain of maize.

However, standard foliar Zn supplementation through ZnSO4 proved significantly

better than bulk ZnO levels but its performance was poorer than ZnO NPs even at 500

ppm level.


114

5.4.3 Zinc Uptake

Zinc uptake by grain, stover and root was influenced significantly by different

foliar Zn treatments during both the seasons and pooled basis. Among treatments, two

foliar application of ZnO NPs either at 1000 or 2000 ppm resulted in more than two

fold increase in grain, stover and root Zn uptake over no Zn control. Foliar

supplementation with lower dose of ZnO NPs i.e. 500 ppm, 0.5% ZnSO4 performed

equally in enhancing Zn uptake by plant parts. However, bulk ZnO sprays at all three

levels caused no substantial increase in Zn uptake by grain during both seasons as

well as pooled results.

5.4.4 Zinc Uptake Partitioning and Accumulation Factor

On an average, Zn accumulation by root, stover and grain was found in the ratio

of 2:4:1, respectively. However, foliar application of Zn through ZnO NPs caused

greater accumulation in grain as compared to bulk ZnO. Moreover, greater Zn from

root was re-mobilized to upper plant parts resulting in higher grain Zn concentration.

A summarized perusal of data indicated that accumulation factor of Zn by different

plant parts of maize was also increased significantly in treatments receiving foliar

applied ZnO NPs over no Zn. Among ZnO NPs treatments, 1000 and 2000 ppm levels

enhanced plant bioaccumulation of Zn by one and half time. The enhanced availability

in Zn to plants has significant bearing on Zn bioaccumulation by maize plant during

summer.


Results clearly indicated that different foliar treatments of ZnO (both nano and

bulk) could not induce any significant change in soil pH, soil EC, organic carbon

content and DTPA-micronutrients in both the seasons as well as in pooled results.


115

However, pooled analysis showed that pH and EC decreased while organic carbon

content and DTPA-micronutrients contents increased during summer over rabi season

because of uniform application of vermicompost.

CONCLUSIONS

The results obtained in the present investigation are discussed earlier and

summarized above; the salient findings from the same are concluded as under.

1 Zinc Oxide Nanoparticle (ZnO NPs) of mean size 65 nm was synthesized

successfully through Oxalate Decomposition Method and characterized through

XRD, SEM, TEM, UV-vis spectroscopy. The nanorods of size range 60-65 nm of

monodispersed nature with a zeta potential of -29.8 mV (stable range) was formed

with the highest purity. TGA results confirmed the calcination temperature as

more than 400 °C.

2 Seed treatment with ZnO NPs at 1000 ppm resulted in significantly maximum seed

germination in maize over no Zn application. Healthy and most vigorous seedling

with the largest seedling length was observed under the treatment receiving 1000

ppm of ZnO NPs. However, ZnO NPs at higher concentration i.e. 2000 ppm was

detrimental to seedling growth in comparison to lower dose. Seed soaking with

ZnO NPs for 2 hrs was equally effective to 4 hrs as seed germination, seedling

length and seed vigour index of maize.

3 Under microplot conditions, seed treatment with ZnO NPs at 1000 ppm registered

the highest grain, stover and dry matter yield of maize. Further, seed treatment

with ZnO NPs either at 1000 and 2000 ppm recorded the highest and statistically

at par enhancement in grain, stover and root Zn concentrations. Zinc uptake,

partitioning and accumulation factor results corroborated the higher Zn


116

accumulation in grain. However, higher concentration of ZnO NPs caused

detrimental consequences on germination and yield of maize. Important soil

properties viz. pH, EC, OC (%) and DTPA-extractable micronutrients contents

were not influenced significantly by any of seed Zn treatments.

4 Two foliar application of ZnO NPs to maize at 30 and 45 days of sowing was

found to be significantly superior in enhancing grain, stover and dry matter yield

of maize however, the results were at par with 2000 ppm ZnO NPs. In addition,

1000 or 2000 ppm of ZnO NPs applied foliarly enhanced grain, stover and root Zn

concentration of maize and Zn uptake which was confirmed through Zn

partitioning in different plant parts and Zn accumulation factor. ZnO NPs

application did not show any significant change in soil properties like pH, EC, OC

(%) and DTPA-Zn were unaffected.

The findings of the present study suggest that Zn could be delivered into maize

seeds through ZnO NPs which improved the germination, root growth, plant growth,

and grain yield. Zinc concentration and uptake by maize grain could also be enhanced

further by foliar application of ZnO NPs as compared to seed treatment. The results

pointed towards the usage of nanoparticles as a fertilizer, especially in maize. Further,

the results emphasize that the nanoscale nutrients can be supplied to the crops either

through seed treatment or foliar application at a much lowered dose to get desired

results. However, the delivery mechanism may be improve upon to avoid health

hazards, if any due to the use of nanoparticles.

REFERENCES

REFERENCES

Adhikari, T., Kundu, S. and Subba Rao, A. (2016a). Zinc delivery to plants through

seed coating with nano-zinc oxide particles. Journal of Plant Nutrition, 39(1):

136-146.

Adhikari, T., Kundu, S., Biswas, A. K., Tarafdar, J. C. and Subba Rao, A. (2015).

Characterization of zinc oxide nano particles and their effect on growth of maize

(Zea mays L.) plant. Journal of Plant Nutrition, 38: 1505-1515.

Adhikari, T., Sarkar, D., Mashayekhi, H. and Xing, B. (2016b). Growth and enzymatic

activity of maize (Zea mays L.) plant: Solution culture test for copper dioxide

nano particles. Journal of Plant Nutrition, 39(1): 99-115.

Adhikary, B.H., Shrestha, J. and Baral, B.R. (2010). Effects of micronutrients on

growth and productivity of maize in acidic soil. International Research Journal

of Applied and Basic Sciences, 1(1): 8-15.

Afshar, I., Akbar, R. and Minoo, S. (2014). Comparison of the effects of spraying

different amounts of nano ZnO and bulk ZnO on wheat. International Journal

of Plant, Animal and Environmental Sciences, 4: 688-693.

Aitken, R. J., Chaudhry, M. Q., Boxall, A. B. and Hull, M. (2006). Manufacture and

use of nanomaterials: current status in the UK and global trends. Occupational

Medicine (London), 56(5): 300-306.

Ajouri, A., Asgedom, H. and Becker, M. (2004). Seed priming enhances germination

and seedling growth of barley under conditions of P and Zn deficiency. Journal

of Plant Nutrition and Soil Science, 167: 630-636.

Alloway, B. J. (2008). Zinc in soils and crop nutrition. 2nd edition. Brussels, Belgium:

International Zinc Association; and Paris, France: International Fertilizer

Industry Association. pp. 135.

Alloway, B. J. (2009). Soil factors associated with zinc deficiency in crops and humans.

Environmental Geochemistry & Health, 31: 537-548.

Amrani, M., Westfall, D.G. and Peterson, G.A. (1999). Influence of water solubility of

granular zinc fertilizers on plant uptake and growth. Journal of Plant Nutrition,

22: 1815- 1827.

References

ii

Aneesh, P. M., Vanaja, K. A. and Jayaraj, M. K. (2007). Synthesis of ZnO nanoparticles

by hydrothermal method. Nanophotonic Materials, 6639(5): 277-278.

Anonymous (2011). District-wise area, production and yield of important food & non-

food crops in Gujarat state, Directorate of Agriculture Gujarat State, Krishi

Bhavan, Gandhinagar.

Arbad, B. K., Ismail, S., Shinde, D. N. and Pardeshi, R. G. (2008). Effect of integrated

nutrient management practices on soil properties and yield in sweet sorghum

(Sorghum biocolor L. Moench) in Vertisols. An Asian Journal of Soil Science,

3(2): 329-332.

Arif, N., Yadav, V., Singh, S., Singh, S., Mishra, R. K., Sharma, S., Dubey, N. K.,

Tripathi, D. K. and Chauhan, D. K. (2016). Current trends of engineered

nanoparticles (ENPs) in sustainable agriculture: an overview. Journal of

Environmental and Analytical Toxicology, 6(5): 397-401.

Armin, M., Akbari, S. and Mashhadi S. (2014). Effect of time and concentration of

nano-Fe foliar application on yield and yield components of wheat.

International Journal of Biosciences, 4(9): 69-75.

Arora, S., Sharma, P., Kumar, S., Nayan, R., Khanna, P. K. and Zaidi, M. G. H. (2012).

Gold nanoparticle induced enhancement in growth and seed yield of Brassica

juncea. Plant Growth Regulation, 66(3): 303-310.

Arya, K. C. and Singh, S. N. (2000). Effect of different levels of phosphorus and zinc

on yield and nutrient uptake of maize (Zea mays L.) with and without irrigation.

Indian Journal of Agronomy, 45(4): 717-721.

Asgari, F. and Rashedi, F. (2015). Synthesis of ZnO nanoparticles. Indian Journal of

Fundamental and Applied Life Sciences, 5(S3): 809-811.

Askarinejad, A., Alavi, M. A. and Morsali A. (2011). Sonochemically assisted synthesis

of ZnO nanoparticles: a novel direct method. Iranian Journal of Chemistry and

Chemical Engineering, 30(3): 75-81.

Avinash, C., Pandey, S., Sanjay, S. and Yadav, S. (2010). Application of ZnO

nanoparticles in influencing the growth rate of Cicer arietinum. Journal of

Experimental Nanoscience, 5(6): 488-497.

References

iii

Azeez, J. O. and Van Averbeke, W. (2012). Dynamics of soil pH and electrical

conductivity with the application of three animal manures. Communications in

Soil Science and Plant Analysis, 43: 865-874.

Bai, C. L. (2005). Ascent of nanoscience in China. Science, 309: 61-63.

Bakhtiari, M., Moaveni, P. and Sani, B. (2015). The effect of iron nanoparticles

spraying time and concentration on wheat. Biological Forum, 7: 679-683.

Baki, A., and Anderson, J. D. (1973). Relationship between decarboxylation of

glutamic acid and vigour in soybean seed. Crop Science, 13:222-226.

Banfield, J. and Zhang, H. (2001). Nanoparticles in the environment. Reviews in

Mineralogy and Geochemistry, 44(1): 1-58.

Baruah, S. and Dutta, J. (2009). Nanotechnology applications in pollution sensing and

degradation in agriculture: a review. Environmental Chemistry Letters, 7: 191-

204.

Basra, S.M.A., Farooq, M., Tabassum, R. and Ahmad, N. (2005). Physiological and

biochemical aspects of seed vigor enhancement treatments in fine rice (Oryza

sativa L.). Seed Science and Technology, 33: 623-628.

Beddington, J. (2010). Food security: contributions from science to a new and greener

revolution. Philosophical Transactions of the Royal Society B: Biological

Sciences, 365(1537): 61-71.

Behera, S. K, Shukla, A. K., Singh, M. V., Wanjari, R. H. and Singh, P. (2015). Yield

and zinc, copper, manganese and iron concentration in maize (Zea mays L.)

Grown on Vertisols as influenced by zinc application from various zinc

fertilizers. Journal of Plant Nutrition, 38: 1544-1557.

Behera, S. K. and Singh, D. (2009). Effect of 31 years of continuous cropping and

fertilizer use on soil properties and uptake of micronutrients by Maize (Zea

mays)-wheat (Triticum aestivum) system. Indian Journal of Agricultural

Sciences, 79: 264-267.

Bernhardt, E. B., Colman, B. P., Hochella, M. F., Cardinale, B. J., Nisbet, R. M.,

Richardson, C. J. and Yin, L. (2010). An ecological perspective on nanomaterial

impacts in the environment. Journal of Environmental Quality, 39(6): 1954-65.

References

iv

Biswas, P. and Wu, C. Y. (2005). Critical review: nanoparticles and the environment.

Journal of the Air & Waste Management Association, 55: 708-746.

Boonyanitipong, P., Kositsup, B., Kumar, P., Baruah, B. and Dutta, J. (2011). Toxicity

of ZnO and TiO2 nanoparticles on germinating rice seed Oryza Sativa L.

International Journal of Bioscience, Biochemistry and Bioinformatics, 1(4):

282-285.

Borm, P., Klaessig, F.C., Landry, T.D., Moudgil, B., Pauluhn, J., Thomas, K., Trottier,

R. and Wood, S. (2006). Research strategies for safety evaluation of

nanomaterials, Part V: Role of dissolution in biological fate and effects of

nanoscale particles. Toxicological Sciences, 90: 23-32.

Brayner, R., Dahoumane, S.A., Yepremian, C., Djediat, C., Meyer, M., Coute, A., and

Fievet, F. (2010). ZnO nanoparticles: synthesis, characterization, and

ecotoxicological studies. Langmuir, 26: 6522-6528.

Brocklehurst, P. A. and Dearman, J. (2008). Interaction between seed priming

treatments and nine seed lots of carrot, celery and onion. II. Seedling emergence

and plant growth. Annals of Applied Biology, 102: 583-593.

Brumfiel, G. (2003). Nanotechnology: a little knowledge. Nature, 424: 246-248.

Buchanan, B. B., Gruissem, W. and Jones, R. L. (2000). Biochemistry and molecular

biology of plants. 1st Ed. American Society of Plant Physiologists, Rockville.

pp. 4-6.

Burman, U., Saini, M. and Kumar, P. (2013). Effect of zinc oxide nanoparticles on

growth and antioxidant system of chickpea seedlings. Toxicological &

Environmental Chemistry, 95(4): 605-612

Cakmak, I. (2000). Role of zinc in protecting plant cells from reactive oxygen species.

New Phytologist, 146: 185-205.

Cakmak, I. (2008a). Possible roles of zinc in protecting plant cells from damage by

reactive oxygen species. New Phytologist, 146(2): 185-205.

Cakmak, I. (2008b). Enrichment of cereal grains with zinc: agronomic or genetic

biofortification? Plant Soil, 302: 1-17.

Chaube, A. K., Ruhella, R., Chakraborty, R., Gangwar, M. S., Srivastava, P. C. and

Singh, S. K. (2007). Management of zinc fertilizer under pearl millet-wheat

References

v

cropping system in a Typic Ustipsamment. Journal of the Indian Society of Soil

Science, 55(2): 196-202.

Chen, L. A., McCrate, J. M. and Lee, J. C. (2008). Preparation of ZnO nanoparticles

and nanorods using hydrothermal method. Nanotechnology, 22: 1080-1098.

Chung, Y.T., Ba-Abbad, M. M., Mohammad, A. W., Hairom, N. H. H. and Benamor,

A. (2015). Synthesis of minimal-size ZnO nanoparticles through sol-gel

method: Taguchi design optimization, Materials and Design, 87: 780-787.

Da Costa, M. V. J. and Sharma, P. K. (2016). Effect of copper oxide nanoparticles on

growth, morphology, photosynthesis, and antioxidant response in Oryza sativa.

Photosynthetica, 54: 110-119.

Da Silva, L. C., Oliva, M. A., Azevedo, A. A. and De Araujo, M. J. (2006). Response

of resting plant species to pollution from an iron pelletization factory. Water,

Air and Soil Pollution, 175: 241-256.

Datt, N., Sharma, R. P. and Sharma, G. D. (2003). Effect of supplementary use of

farmyard manure along with chemical fertilizers on productivity and nutrient

uptake by vegetable pea and build-up of soil fertility in Lahaul valley of

Himachal Pradesh. The Indian Journal of Agricultural Sciences, 73(5): 266-

268.

Davarpanah, S., Tehranifar, A., Davarynejad, G., Abadia, J. and Khorasani, R. (2016).

Effects of foliar applications of zinc and boron nano-fertilizers on pomegranate

(Punica granatum cv. Ardestani) fruit yield and quality. Scientia Horticulturae,

210: 57-64.

DeRosa, M. C., Monreal, C., Schnitzer, M., Walsh, R. and Sultan, Y. (2010).

Nanotechnology in fertilizers. Nature Nanotechnology, 5: 91-91.

Dewick, P., Green, K. and Miozzo, M. (2004). Technological Change, Industry

Structure and the Environment. Futures, 36: 267-293.

Dhaliwal, S.S., Sadana, U.S., Khurana, M.P.S., Dhadli, H.S. and Manchanda, J.S.

(2010). Enrichment of rice grains with zinc and iron through ferti-fortification.

Indian Journal of Fertilizers, 6(7): 28-35.

Dhaliwal, S.S., Sadana, U.S., Manchanda, J.S. and Dhadli, H.S. (2009). Biofortification

of wheat grains with zinc and iron in Typic Ustochrept soils of Punjab. Indian

Journal of Fertilizers, 5(11): 13-16 & 19-20.

References

vi

Dietz, K. J. and Herth, S. (2011). Plant nanotoxicology. Trends in Plant Sciences, 16:

582-589.

Dikinya, O. and Mufwanzala, N. (2010). Chicken manure-enhanced soil fertility and

productivity: effects of application rates. Journal of Soil Science and

Environmental Management, 1: 46-54.

Doshi, R., Braida, W., Christodoulatos, C., Wazne, M. and O'Connor, G. (2008). Nano-

aluminium: Transport through sand columns and environmental effects on

plants and soil communities. Environmental Research, 106: 296-303.

Du, W. C., Sun, Y. Y., Ji, R., Zhu, J. G., Wu, J. C. and Guo, H. Y. (2011). TiO2 and

ZnO nanoparticles negatively affect wheat growth and soil enzyme activities in

agricultural soil. Journal of Environmental Monitoring, 13: 822-828.

Eichert, T., Kurtz, A., Steiner, U. and Goldbach, H. E. (2008). Size exclusion limits and

lateral heterogeneity of the stomatal foliar uptake pathway for aqueous solutes

and water suspended nanoparticles. Physiologia Plantarum, 134: 151-160.

Eigenberg, R. A., Doran, J. W., Nienaber, J. A., Ferguson, R. B. and Woodbury, B. L.

(2002). Electrical conductivity monitoring of soil condition and available N

with animal manure and cover crop. Agriculture, Ecosystems & Environment,

88: 183-193.

El-Ghamery, A. A., El-Kholy, M. A. and Abou El-Yousser, M. A. (2003). Evaluation

of cytological effects of Zn2+ in relation to germination and root growth of

Nigella sativa L. and Triticum aestivum L. Mutation Research, 537: 29-41.

Farnia, A. and Omidi, M. M. (2015). Effect of nano-zinc chelate and nano-biofertilizer

on yield and yield components of maize (Zea mays L.), under water stress

condition. Indian Journal of Natural Sciences, 5(29): 4614-4624.

Farooq, M., Basra, S. M. A., Wahid, A., Khaliq, A. and Kobayashi, N. (2009). Rice

seed invigoration. In: E. Lichtfouse (Ed.). Sustainable Agriculture Reviews,

Springer, the Netherlands. pp. 137-175.

Farooq, M., Wahid, A. and Siddique, K. H. M. (2012). Micronutrient application

through seed treatments - a review. Journal of Soil Science and Plant Nutrition,

12(1): 125-142.

References

vii

Ferrandon, M. and Chamel, A. R. (1988). Cuticular retention, foliar absorption and

translocation of Fe, Mn and Zn supplied in organic and inorganic form. Journal

of Plant Nutrition, 11: 247-263.

FICCI (2014). Maize Report: Maize in India — Global Maize Scenario, India Maize

Summit- 2014, pp: 7-13.

Gao, F., Hong, F., Liu, C., Zheng, L., Su, M., Wu, X., Yang, F., Wu, C. and Yang P.

(2006). Mechanism of nano-anatase TiO2 on promoting photosynthetic carbon

reaction of spinach. Biological Trace Element Research, 111: 239-253.

Gathala, M. K., Kanthaliya, P. C., Verma, A., Chauhan, M.S. (2007). Effect of

integrated nutrient management on soil properties and humus fractions in the

long term fertilizer experiments. Journal of the Indian Society of Soil Science,

53(3): 360-363.

Ghafari, H. and Razmjoo, J. (2013). Effect of foliar application of nano iron oxide, iron

chelate and iron sulphate rates on yield and quality of wheat. International

Journal of Agronomy and Plant Production, 4(11): 2997-3003.

Ghuman, B. S. and Sur, H. S. (2006). Effect of manuring on soil properties and yield

of rainfed wheat. Journal of the Indian Society of Soil Science, 54(1): 6-11.

Giordano, P. M., and Mortvedt, J. J. (1973). Zinc source and method of application for

rice. Agronomy Journal, 65: 51-53.

Grusak, M. L. (2002). Enhancing mineral content in plant food product. Journal of the

American College of Nutrition, 21: 178-183.

Gruyer, N., Dorais, M., Bastien, C., Dassylva, N. and Triffault-Bouchet, G. (2013).

Interaction between silver nanoparticles and plant growth. In: Proceedings of

International Symposium on New Technologies for Environment Control.

Energy-Saving and Crop Production in Greenhouse and Plant Factory, 1037:

795-800.

Gulser, C., Demir, Z. and Serkan, I.C. (2010). Changes in some soil properties at

different incubation periods after tobacco waste application. Journal of

Environmental Biology, 31: 671-674.

Hangarge, D. S., Raut, R. S., Gaikawad, G. K., Adsul, P. B. and Dixit, R. S. (2004).

Influence of vermicompost and other organics on fertility and productivity of

soil under chilli-spinach cropping system. Soil and Crops, 14(1): 181-186.

References

viii

Harikrishna, B. L., Channal, H. T., Hebsur, N. S., Dharmatti, P. R. and Sarangamath,

P. A. (2002). Integrated nutrient management (INM) on availability of

nutrients, uptake and yield of tomato. Karnataka Journal of Agricultural

Sciences, 15(2): 275-278.

Harris, D., Joshi, A., Khan, A. P., Gothkar, P. and Sodhi, S. P. (1999). On-farm seed

priming in semiarid agriculture: development and evaluation in maize, rice and

chickpea in India using participatory methods. Experimental Agriculture, 35:

15-29.

Harris, D., Rashid, A., Miraj, G., Arif, M. and Shah, H. (2007). On-farm seed priming

with zinc sulphate solution - A cost-effective way to increase the maize yields

of resource-poor farmers. Field Crops Research, 10: 119-127.

Haslett, B. S., Reid, R. J. and Rengel, Z. (2001). Zinc mobility in wheat: uptake and

distribution of zinc applied to leaves or roots. Annals of Botany, 87: 379-386.

Hasnidawani, J. N., Azlina, H. N., Norita, H., Bonnia, N. N., Ratim, S. and Ali, E. S.

(2016). Synthesis of ZnO nanostructures using sol-gel method. Procedia

Chemistry, 19: 211-216.

Heydecker, W., Higgins, J. and Turner, Y.J. (1975). Invigoration of seed. Seed Science

and Technology, 3: 881-888.

Hodgson, J. F. (1963). Chemistry of micronutrient elements in soils. Advances in

Agronomy, 15: 119-150.

Holder, C. D. (2007). Leaf water repellency of species in Guatemala and Colorado

(USA) and its significance to forest hydrology studies. Journal of Hydrology,

336: 147-154.

Hong, F., Zhou, J., Liu, C., Yang, F., Wu, C., Zheng, L. and Yang P. (2005). Effects of

Nano-TiO2 on photochemical reaction of chloroplasts of Spinach. Biological

Trace Element Research, 105: 269-279.

Hossain, M. A., Jahiruddin, M. and Khatun, F. (2011). Response of maize varieties to

zinc fertilization. Bangladesh Journal of Agricultural Research, 36: 437-447.

Hunter, R. J. (1981). Zeta Potential in Colloid Science: Principles and Applications.

Academic Press; London.

References

ix

Ismail, S., Adsul, P. B., Shinde, G. G. and Deshmukh, A. S. (2001). Impact of FYM

and fertilizer nitrogen on yield and soil properties of sorghum grown on

Vertisols. International Sorghum and Millets Newsletter, 42: 29-31.

Jaberzadeh, A., Moaveni, P., Moghadam, H. R. T. and Zahedi, H. (2013). Influence of

bulk and nanoparticles titanium foliar application on some agronomic traits,

seed gluten and starch contents of wheat subjected to water deficit stress.

Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 41(1): 201-207.

Jackson, M. L. (1973). Soil Chemical Analysis. Prentice Hall of India Pvt. Ltd., New

Delhi.

Jagtap, P. B., Patil J. D., Nimbalkar, C. A. and Kadlag, A. D. (2007). Influence of

integrated nutrient management on soil properties and release of nutrients in a

saline-sodic soil. Journal of the Indian Society of Soil Science, 55(2): 147-156.

Jayarambabu, M., Sivakumari, B., Rao, K. V. and Prabhu, Y. T. (2014). Germination

and growth characteristics of mungbean seeds affected by synthesized ZnO

nanoparticles. International Journal of Current Engineering and Technology,

4: 3411-3416.

Jiang, C., Zhang, W., Zou, G., Yu, W. and Qian, Y. (2005). Precursor-induced

hydrothermal synthesis of flowerlike cupped-end microrod bundles of ZnO.

The Journal of Physical Chemistry-B, 109(4): 1361-1363.

Johnson, S. E., Lauren, J. G., Welch, R. M. and Duxbury, J. M. (2005). A comparison

of the effects of micronutrient seed priming and soil fertilization on the mineral

nutrition of chickpea (Cicer arietinum), lentil (Lens culinaris), rice (Oryza

sativa) and wheat (Triticum aestivum) in Nepal. Experimental Agriculture, 41:

427-448.

Jurablu, S., Farahmandjou, M. and Firoozabadi, T. P. (2015). Sol-gel synthesis of zinc

oxide (ZnO) nanoparticles: study of structural and optical properties. Journal

of Sciences, Islamic Republic of Iran, 26(3): 281-285.

Kar, D., Ghosh, D. and Srivastava, P.C. (2007). Efficacy evaluation of different zinc

organo-complexes in supplying zinc to maize (Zea mays L.) plant. Journal of

the Indian Society of Soil Science, 55(1): 67-72.

References

x

Katyal, J. C. and Sharma, B. D. (1991). DTPA-extractable and total Zn, Cu, Mn and Fe

in Indian soils and their association with some soil properties. Geoderma, 49:

165-179.

Kaya, M., Atak, M., Khawar, K.M., Ciftci, C.Y. and Ozcan, S. (2007). Effect of pre-

sowing seed treatment with zinc and foliar spray of humic acids on yield of

common bean (Phaseolus vulgaris L.). International Journal of Agriculture and

Biology, 7: 875-878.

Kher, D. (1993). Effect of continuous liming, manuring and cropping on DTPA-

extractable micronutrients in an Alfisol. Journal of the Indian Society of Soil

Science, 41: 366-367.

Kim, S., Kim, J. and Lee, I. (2011). Effects of Zn and ZnO nanoparticles and Zn2+ on

soil enzyme activity and bioaccumulation of Zn in Cucumis sativus. Journal of

Chemical Ecology, 27: 49-55.

Kisan, B., Shruthi, H., Sharanagouda, H., Revanappa, S. B. and Pramod, N. K. (2015).

Effect of nano-zinc oxide on the leaf physical and nutritional quality of spinach.

Agrotechnology, 5: 132-134.

Klaine, S. J., Alvarez, P. J. J., Batley, G. E., Fernandes, T. F., Handy, R. D., Lyon, D.

Y., Mahendra, S., McLaughlin, M.J. and Lead, J.R. (2008). Nanomaterials in

the environment: behaviour, fate, bioavailability and effects. Environmental

Toxicology and Chemistry, 27(9): 1825-1851.

Kobayashi, Y., and Mizutani, S. (1970). Studies on the wilting treatment of corn plant:

The influence of the artificial auxin control in nodes on the behavior of rooting.

Proceedings of the Crop Science Society of Japan 39: 213-220.

Kool, P. L., Ortiz, M. D. and van Gestel, C. A. M. (2011). Chronic toxicity of ZnO

nanoparticles, non-nano ZnO and ZnCl2 to Folsomia candida (Collembola) in

relation to bioavailability in soil. Environmental Pollution, 159: 2713-2719.

Korishettar, P., Vasudevan, S. N., Shakuntala, N. M., Doddagoudar, S. R., Hiregoudar,

S. and Kisan, B. (2016). Seed polymer coating with Zn and Fe nanoparticles:

An innovative seed quality enhancement technique in pigeon pea. Journal of

Applied and Natural Science, 8(1): 445-450.

Kouhi, S. M. M., Lahouti, M., Ganjeali, A. and Entezari. M. H. (2015a). Comparative

Effects of ZnO nanoparticles, ZnO bulk particles, and Zn2+ on Brassica napus

References

xi

after long-term exposure: changes in growth, biochemical compounds,

antioxidant enzyme activities, and Zn bioaccumulation. Water, Air and Soil

Pollution, 226: 364-375.

Kouhi, S. M. M., Lahouti, M., Ganjeali, A. and Entezari. M. H. (2015b). Long-term

exposure of rapeseed (Brassica napus L.) to ZnO nanoparticles: anatomical and

ultrastructural responses. Environmental Science and Pollution Research, 22:

10733-10743.

Krishnasamy, R. (1996). Effect of zinc chelates on dry matter production and uptake of

micronutrients by sorghum. Madras Agricultural Journal, 3: 387-388.

Kumar, A. and Yadav, D. S. (2005). Influence of continuous cropping and fertilization

on nutrient availability and productivity of an alluvial soil. Journal of the Indian

Society of Soil Science, 53: 194-198.

Kumar, S. A. and Chen, S. M. (2008). Nanostructured zinc oxide particles in chemically

modified electrodes for biosensor applications. Analytical Letters, 41(2): 141 -

158.

Kumar, V. and Singh, A. P. (2010). Long-term effect of green manuring and farmyard

manure on yield and soil fertility status in rice-wheat cropping system. Journal

of the Indian Society of Soil Science, 58: 409-412.

Laware, S. L. and Raskar, S. (2014). Influence of zinc oxide nanoparticles on growth,

flowering, and seed productivity in onion. International Journal of Current

Microbiology and Applied Sciences, 3(7): 874-881.

Lee W. M., An, Y. J., Yoon, H. and Kwbon, H. S. (2008). Toxicity and bioavailability

of copper nanoparticles to the terrestrial plants mung bean (Phaseolus radiatus)

and wheat (Triticum aestivum): plant agar test for water-insoluble

nanoparticles. Environmental Toxicology and Chemistry, 27: 1915-1921.

Lee, C. W., Mahendra, S., Zodrow, K., Li, D., Tsai, Y. C. and Braam, J. (2010).

Developmental phytotoxicity of metal oxide nanoparticles to Arabidopsis

thaliana. Environmental Toxicology and Chemistry, 29: 669-675.

Lin D. and Xing B. (2007). Phytotoxicity of nanoparticles: inhibition of seed

germination and root growth. Environmental Pollution, 150: 243-250.

Lin, D., and Xing, B. (2008). Root uptake and phytotoxicity of ZnO nanoparticles.

Environmental Science & Technology, 42: 5580-5585.

References

xii

Lin, X. M. and Samia, A. C. S. (2006). Synthesis, assembly and physical properties of

magnetic nanoparticles. Journal of Magnetism and Magnetic Materials, 305:

100-109.

Lindsay, W.L. and Norvell, W.A. (1978). Development of a DTPA soil test for zinc,

iron, manganese and copper. Soil Science Society of America Journal, 42(3):

421-428.

Liscano, J. F., Wilson, C. E., Norman, Jr. R. J. and Slaton, N. A. (2000). Zinc

availability to rice from seven granular fertilizers. AAES Research Bulletin, 963:

1-31.

Liu, R. and Lal, R. (2015). Potentials of engineered nanoparticles as fertilizers for

increasing agronomic productions. Science of the Total Environment, 514:131-

139.

Liu, R., Zhang, H. and Lal, R. (2016). Effects of stabilized nanoparticles of copper,

zinc, manganese, and iron oxides in low concentrations on lettuce (Lactuca

sativa) seed germination: nanotoxicants or nanonutrients? Water, Air, & Soil

Pollution, 227: 1-14.

Liu, W. T. (2006). Nanoparticles and their biological and environmental applications.

The Journal of Bioscience and Bioengineering, 102(1): 1-7.

Liu, X. M., Zhang, F. D., Zhang, S. Q., He, X. S., Fang, R., Feng, Z. and Wang, Y.

(2005). Effects of nano-ferric oxide on the growth and nutrients absorption of

peanut. Plant Nutrition and Fertilizer Science, 11: 14-18.

Liu, X., Wang, F., Shi, Z., Tong, R. and Shi, X. (2015). Bioavailability of Zn in ZnO

nanoparticle-spiked soil and the implications to maize plants. The Journal of

Nanoparticle Research, 17: 175-185.

Liu, Y., Zhou, J., Larbot, A. and Persin, M. (2007). Preparation and Characterization of

Nano-Zinc Oxide. Journal of Materials Processing Technology, 189(1-3): 379-

383.

Lopez-Moreno, M. L., de la Rosa, G., Hernandez -Viezcas, J. A., Castillo-Michel, H.,

Botez, C. E., Peralta-Videa, J. R. and Gardea-Torresdey, J. L. (2010a).

Evidence of the differential biotransformation and genotoxicity of ZnO and

CeO2 nanoparticles on soybean (Glycine max) plants. Environmental Science &

Technology, 44: 7315-7320.

References

xiii

Lopez-Moreno, M. L., de la Rosa, G., Hernandez-Viezcas, J. A., Peralta-Videa, J. R.

and Gardea-Torresdey, J. L. (2010b). X-ray absorption spectroscopy (XAS)

corroboration of the uptake and storage of CeO2 nanoparticles and assessment

of their differential toxicity in four edible plant species. Journal of Agricultural

and Food Chemistry, 58: 3689-3693.

Lu, C.M., Zhang, C.Y., Wen, J.Q., Wu, G.R. and Tao, M.X. (2002). Research of the

effect of nanometer materials on germination and growth enhancement of

Glycine max and its mechanism. Soya Bean Science, 21: 168-172.

Luther, W. (2004). Industrial Application of Nanomaterials — Chances and risks, VDI

Technologiezentrum GmbH, Dusseldorf, Germany.

Lv, J., Zhang, S., Luo, L., Zhang, J., Yang, K. and Christie, P. (2015). Accumulation,

speciation and uptake pathway of ZnO nanoparticles in maize. Environmental

Science: Nano, 2: 68-77.

Ma, X., Geiser-Lee, J., Deng, Y. and Kolmakov, A. (2010). Interactions between

engineered nanoparticles (ENPs) and plants: Phytotoxicity, uptake and

accumulation. Science of the Total Environment, 408: 3053-3061.

Mahajan, P., Dhoke, S. K. and Khanna, A. S. (2011). Effect of nano-ZnO particle

suspension on growth of mung (Vigna radiata) and gram (Cicer arietinum)

seedlings using plant agar method. Journal of Nanotechnology, 2011: 1-7.

Manivannan, S., Balamurugan, M., Parthasarathi, K., Gunasekaran, G. and

Ranganathan, L. S. (2009). Effect of vermicompost on soil fertility and crop

productivity-beans (Phaseolus vulgaris). Journal of Environmental Biology,

30: 275-281.

Marschner, H. (1995). Mineral Nutrition of Higher Plants (2nd Ed.) Academic Press,

London. pp. 889.

Mazumdar, H. and Ahmed, G. U. (2011). Synthesis of silver nanoparticles and adverse

effect on seed germination in Oryza sativa, Vigna radiata and Brassica

campestris. International Journal of Advanced Biotechnology Research, 2(4):

404-413.

Moezzi, A., McDonagh, A. M. and Cortie, M. B. (2012). Zinc oxide particles:

Synthesis, properties and applications. Chemical Engineering Journal, 185-

186: 1-22.

References

xiv

Monica, R. C. and Cremonini, R. (2009). Nanoparticles and higher plants. Caryologia,

62(2): 161-165.

Monreal, C. M., DeRosa, M., Mallubhotla, S. C., Bindraban, P. S. and Dimkpa, C.

(2016). Nanotechnologies for increasing the crop use efficiency of fertilizer-

micronutrients, Biology and Fertility of Soils, 52: 423-437.

Moretti, D., Biebinger, R., Bruins, M. J., Hoeft, B. and Kraemer, K. (2014).

Bioavailability of iron, zinc, folic acid, and vitamin A from fortified maize.

Annals of the New York Academy of Sciences, 1312: 54–65.

Morkoc, H. and Ozgur, U. (2009). General Properties of ZnO. In: Zinc Oxide:

Fundamentals, Materials and Device Technology. Wiley-VCH. pp. 1-12.

Mortvedt, J. J. (1968). Crop response to applied zinc in ammoniated phosphate

fertilizers. Journal of Agricultural and Food Chemistry, 16: 241-245.

Mortvedt, J. J. (1985). Micronutrient fertilizers and fertilization practices. Nutrient

Cycling in Agroecosystems, 7: 221-235.

Mortvedt, J. J. (1992). Crop response to level of water -soluble zinc in granular zinc

fertilizers. Fertilizer Research, 33: 249-255.

Mortvedt, J. J., and Gilkkes, R. J. (1993). Zinc Fertilizers, In A. D. Robson, ed. Zinc

in Soils and Plants. Kluwer Academic Publishers, Dordrecht; Boston.

Nair, R., Varghese, S. H., Nair, B. G., Maekawa, T., Yoshida, Y. and Kumar, D.S.

(2010). Nanoparticulate material delivery to plants. Plant Science, 179(3): 154-

163.

Narendhran, S., Rajiv, P., and Sivaraj, R. (2016). Influence of zinc oxide nanoparticles

on growth of Sesamum indicum L. in zinc deficient soil. International Journal

of Pharmacy and Pharmaceutical Sciences, 8(3): 365-371.

Navarro, E., Baun, A., Behra, R., Hartmann, N. B., Filser, J., Miao, A. J., Quigg, A.,

Santschi, P.H. and Sigg, L. (2008). Environmental behaviour and ecotoxicity of

engineered nanoparticles to algae, plants, and fungi. Ecotoxicology, 17: 372-

386.

Nel, A., Xia, T., Madler, L. and Li, N. (2006). Toxic potential of materials at the nano

level. Science, 311: 622-627.

References

xv

Ng, H. T., Chen, B., Li, J., Han, J., Meyyappan, M., Wu, J., Li, S. X. and Haller, E. E.

(2003). Optical properties of single-crystalline ZnO nanowires on m-sapphire.

Applied Physics Letters, 82(13): 2023-2003.

Ni, Y., Wei, X. and Hong, J. (2008). Hydrothermal preparation of ZnO nanoparticles.

Materials Science, 16(4): 523-536.

Ni, Y., Wei, X., Hong, J. and Ye, Y. (2005). Hydrothermal preparation and optical

properties of ZnO nanorods. Materials Science and Engineering B, 121: 42-47.

Nirmalkumar, J. I., Soni, H., Kumar, R. N. and Bhatt, I. (2009). Hyperaccumulation

and mobility of heavy metals in vegetable crops in India. The Journal of

Agriculture and Environment, 10: 29-38.

Nowack, B. and Bucheli, T. D. (2007). Occurrence, behaviour and effects of

nanoparticles in the environment. Environmental Pollution, 150: 5-22.

Ozturk, L., Yazici, M. A., Yucel, C., Torun, A., Cekic, C., Bagci, A., Ozkan, H., Braun,

H.J., Sayers, Z. and Cakmak, I. (2006). Concentration and localization of zinc

during seed development and germination in wheat. Physiologia Plantarum,

128: 144-152.

Pandey, A. C., Sanjay, S. S. and Yadav, R. S. (2010). Application of ZnO nanoparticles

in influencing the growth rate of Cicer arietinum. Journal of Experimental

Nanoscience, 5(6): 488-497.

Panwar, J., Jain, N., Bhargaya, A., Akhatar, S. A., and Yun, Y. S. (2012). Positive effect

of ZnO nanoparticles on Tomato plants. Conference paper: 3rd International

Conference on Environmental Research and Technology (ICERT), University

of Sains, Pinang, Malaysia.

Paschke, M. W., Perry, L. G. and Redente, E. F. (2006). Zinc toxicity thresholds for

reclamation forb species. Water, Air & Soil Pollution, 170: 317-330.

Patel, K. P. (2011). Crop response to zinc-cereal crops. Indian Journal of Fertilisers,

7(10): 84-100.

Pawar, V. C., Joshi, A. C. and Umrani, N. K. (1987). Response of kharif sorghum to

nitrogen, phosphorus and potassium. Sorghum Newsletter, 30-46.

References

xvi

Peralta-Videa, J. R., Zhao, L. J., Lopez-Moreno, M. L., de la Rosa, G., Hong, J. and

Gardea-Torresdey, J. L. (2011). Nanomaterials and the environment: A review

for the biennium 2008-2010. Journal of Hazardous Materials, 186: 1-15.

Pokhrel, L. R. and Dubey, B. (2013). Evaluation of developmental responses of two

crop plants exposed to silver and Zn oxide nanoparticles. Science of the Total

Environment, 452-453: 321-332.

Potarzycki, J. and Grzebisz, W. (2009). Effect of zinc foliar application on grain yield

of maize and its yielding components. Plant Soil and Environment, 55: 519-

527.

Powers, K.W., Brown, S. C., Krishna, V. B., Wasdo, S. C., Moudgil, B. M. and Roberts,

S. M. (2006). Research strategies for safety evaluation of nanomaterials. Part

VI. Characterization of nanoscale particles for toxicological evaluation.

Toxicological Sciences, 90: 296-303.

Prasad, B. and Sinha, M. K. (1981). The relative efficiency of zinc carriers on growth

and zinc nutrition of corn. Plant and Soil, 62: 45-52.

Prasad, R., Shivay, Y. S. and Kumar, D. (2013). Zinc fertilization of cereals for

increased production and alleviation of zinc malnutrition in India. Agricultural

Research 2(2): 111-118.

Prasad, T. N. V. K. V., Sudhakar, P., Sreenivasulu, Y., Latha, P., Munaswamy, V., Raja

Reddy, K., Sreeprasad, T. S., Sajanlal, P. R. and Pradeep, T. (2012). Effect of

nanoscale zinc oxide particles on the germination, growth and yield of peanut.

Journal of Plant Nutrition, 35: 905-927.

Priester, J. H., Ge, Y., Mielke, R. E., Horst, A. M., Moritz, S. C., Espinosa, K., Gelb, J.

Walker, S. L., Nisbet, R. M., An, Y., Schimel, J. P. Palmer, R. G., Hernandez-

Viezcas, J. A., Zhao, L., Gardea-Torresdey, J. L. and Holden, P. A. (2012).

Soybean susceptibility to manufactured nanomaterials with evidence for food

quality and soil fertility interruption. Proceedings of the National Academy of

Sciences of the United States of America (PNAS), 109(37): 14734-14735.

Racuciu, M. and Creanga, D. E. (2007). TMA-OH coated magnetic nanoparticles

internalized in vegetal tissues. Romanian Journal of Physics, 52: 395-395.

References

xvii

Raghuwanshi, R. S., Pawar, K. B. and Patil, J. D. (1998). Effect of biofertilizer and N

levels on yield and nitrogen economy in pearl millet under dryland condition.

The Madras Agricultural Journal, 184(11/12): 556-558.

Rajshree, M., Wandile, Maya, M., Raul, Swati, V., Washimakar, and Bharti, S.B.

(2005). Residual effect of long-term application of N, P, Zn and FYM on soil

properties of Vertisols, yield, protein and oil content of soybean. Journals of

Soils and Crops, 15(1): 155-159.

Raliya, R. and Tarafdar, J. C. (2013). ZnO nanoparticle biosynthesis and its effect on

phosphorous-mobilizing enzyme secretion and gum contents in cluster bean

(Cyamopsis tetragonoloba L.). Agricultural Research, 2: 48-57.

Rao, S. and Shekhawat, G. S. (2014). Toxicity of ZnO engineered nanoparticles and

evaluation of their effect on growth, metabolism and tissue specific

accumulation in Brassica juncea. Journal of Environmental Chemical

Engineering, 2: 105-114.

Raskar, S. S., Sonani, V. V. and Shelke, A. V. (2012). Effects of different levels of

nitrogen, phosphorus and zinc on yield and yield attributes of maize (Zea mays

L.). Advance Research Journal of Crop Improvement, 3(2): 126-128.

Rattan, R. K., Datta, S. P. and Katyal, J. C. (2008). Micronutrient management-research

achievements and future challenges. Indian Journal of Fertilisers, 4(12): 93-

118.

Rehman, H., Aziz, T., Farooq, M., Wakeel, A. and Rengel, Z. (2012). Zinc nutrition in

rice production systems: a review. Plant and Soil, 361: 203-226.

Rengel, Z. (2002). Agronomic approaches to increasing zinc concentration in staple

food crops. In: I. Cakmak, R.M. Welch (ed.) Impacts of Agriculture on Human

Health and Nutrition. UNESCO, EOLSS Publishers, Oxford, UK.

Rengel, Z. and Graham, R. D. (1995). Importance of seed zinc content for wheat growth

on zinc-deficient soil-I. Vegetative growth. Plant and Soil, 173: 259-266.

Rengel, Z., Batten, G. D. and Crowley, D. E. (1999). Agronomic approaches for

improving the micronutrient density in edible portions of field crops. Field

Crop Research, 60: 27-40.

References

xviii

Rickerby, D. G. and Morrison, M. (2007). Nanotechnology and the environment: A

European perspective. Science and Technology of Advanced Materials, 8: 19-

24.

Rico, C. M., Majumdar, S., Duarte-Gardea, M., Peralta-Videa, J. R. and Gardea-

Torresdey, J. L. (2011). Interaction of nanoparticles with edible plants and their

possible implications in the food chain. Journal of Agricultural and Food

Chemistry, 59: 3485-3498.

Roco, M. C. (2003). Broader societal issue of nanotechnology. Journal of Nanoparticle

Research, 5: 181-189.

Roco, M. C. (2005). Environmentally responsible development of nanotechnology.

Environmental Science and Technology, 39: 106A-112A.

Roy, S. and Kashem, M. A. (2014). Effects of organic manures in changes of some soil

properties at different incubation periods. Open Journal of Soil Science, 4: 81-

86.

Sabir, S., Arshad, M. and Chaudhari, S. K. (2014). Zinc oxide nanoparticles for

revolutionizing agriculture: synthesis and applications. The Scientific World

Journal, 2014: 1-8.

Sadraei, R. (2016). A Simple Method for Preparation of Nano-sized ZnO. Research and

Reviews Journal of Chemistry, 5(2): 45- 49.

Saleem, M., Fang, L., Wakeel, A., Rashad, M. and Kong, C. Y. (2012). Simple

preparation and characterization of nano-crystalline zinc oxide thin films by

sol-gel method on glass substrate. World Journal of Condensed Matter Physics,

2: 10-15.

Samad, A., Khan, M. J., Shah, Z. and Jan, M. T. (2014). Determination of optimal

duration and concentration of zinc and phosphorus for priming wheat seed.

Sarhad Journal of Agriculture, 30(1): 27-34.

Schmid, G. (2001). Metals, In: Nanoscale Materials in Chemistry, Ed: Kenneth J.

Klabunde, Wiley-Interscience, New York. p. 15-59.

Sedghi, M., Hadi, M., and Toluie, S. G. (2013). Effect of nano zinc oxide on the

germination parameters of soybean seeds under drought stress. Annals of West

University of Timişoara, ser. Biology, XVI (2): 73-78.

References

xix

Sekhon, B. S. (2014). Nanotechnology in agri-food production: an overview.

Nanotechnology, science and applications, 7: 31-53.

Shailesh, K.D., Pramod, M., Rajashree, K. and Anand, K. (2013). Effect of

nanoparticles suspensions on the growth of mung (Vigna radiata) seedlings by

foliar spray method. Nanotechnology Development, 3(1): 1-5.

Shambhavi, S. (2011). Dynamics of micronutrient cations in soil-plant system as

influenced by long-term application of chemical fertilizers and amendments in

an acid Alfisol. Ph. D. Thesis, Department of Soil Science, CSK HPKVV,

Palampur, India, p 222.

Sharma, B. L. and Bapat, P. N. (2000). Levels of micronutrient cations in various plant

parts of wheat as influenced by zinc and phosphorus application. Journal of the

Indian Society of Soil Science, 48(1):130-134.

Sharma, B. L., Rathore, G. S., Dubey, S. B., Khamparia, R. S. and Sinha, S. B. (1986).

Response of rice to zinc and evaluation of some soil test methods for zinc.

Journal of the Indian Society of Soil Science, 34(1): 106-110.

Sharma, P., Sreenivas, K. and Rao, K. V. (2003). Analysis of ultraviolet

photoconductivity in ZnO films prepared by unbalanced magnetron sputtering.

Journal of Applied Physics, 93(7): 3963-3970.

Shaymurat, T., Gu, J., Xu, C., Yang, Z., Zhao, Q., Liu, Y. and Liu, Y. (2012).

Phytotoxic and genotoxic effects of ZnO nanoparticles on garlic (Allium

sativum L.): a morphological study. Nanotoxicology, 6(3): 241-248.

Shen, L., Bao, N., Yanagisawa, K., Domen, K., Gupta, A. and Grimes, C.A. (2006).

Direct synthesis of ZnO nanoparticles by a solution-free mechanochemical

reaction. Nanotechnology, 17(20):5117-5123.

Sheykhbaglou, R., Sedghi, M., Shishevan, T. and Seyed Sharifi, R. (2010). Effects of

nano-iron oxide particles on agronomic traits of soybean. Notulae Scientia

Biologicae, 2(2): 112-113.

Shukla, A. K. and Behera, S. K. (2012). Micronutrient fertilizers for higher

productivity. Indian Journal of Fertilisers, 8(4): 100-117.

Shukla, A. K., Behera, S. K., Lenka, N. K., Tiwari, P. K., Chandra Prakash, Malik, R.

S., Sinha, N. K., Singh, V. K., Patra, A. K. and Chaudhary, S. K. (2016). Spatial

References

xx

variability of soil micronutrients in the intensively cultivated Trans-Gangetic

Plains of India. Soil & Tillage Research, 163: 282-289.

Shukla, A. K., Malik, R. S., Tiwari, P. K., Prakash, C., Behera, S. K., Yadav, H. and

Narwal, R.P. (2015). Status of micronutrient deficiencies in soils of Haryana:

impact on crop productivity and human health. Indian Journal of Fertilisers,

11: 16-27.

Shukla, A. K., Tiwari, P. K. and Prakash, C. (2014). Micronutrients deficiencies vis-a-

vis food and nutritional security of India. Indian Journal of Fertilisers, 10: 94-

112.

Shute, T. and Macfie, S. M. (2006). Cadmium and zinc accumulation in soybean: A

threat to food safety? Science of the Total Environment, 371: 63-73.

Shyla, K. K. and Natarajan, N. (2014). Customizing zinc oxide, silver and titanium

dioxide nanoparticles for enhancing groundnut seed quality. Indian Journal of

Science and Technology, 7(9): 1376-1381.

Siddiqui, M. H., Al-Whaibi, M. H., Firoz, M. and Al-Khaishany, M. Y. (2015). Role of

nanoparticles in plants. In: Nanotechnology and Plant Sciences. Springer

International Publishing, pp: 19-35.

Sillanpaa, M. (1990). Micronutrient assessment at the country level: an international

study. FAO Soils Bulletin 63. FAO/Finnish International Development Agency,

Rome, Italy.

Singh, B., Natesan, S. K.A., Singh, B.K. and Usha, K. (2003). Improving zinc

efficiency of cereals under zinc deficiency. Current Science, 88: 36-44.

Singh, M. V. (2007). Efficiency of seed treatment for ameliorating zinc deficiency in

crops. In: Zinc Crops 2007, Improving Crop Production and Human Health,

Istanbul, Turkey.

Singh, M. V. (2009). Micronutrient nutritional problems in soils of India and

improvement for human and animal’s health. Indian Journal of Fertilisers, 5(4):

11-26.

Singh, M. V. and Behera, S. K. (2011). All India Coordinated Research Project of

Micro- and Secondary Nutrients and Pollutant Elements in Soils and Plants - A

profile. Research Bulletin No. 10. Bhopal, India: Indian Institute of Soil

Science.

References

xxi

Singh, N. B., Amist, N., Yadav, K., Singh, D., Pandey, J. K. and Singh, S. C. (2013).

Zinc oxide nanoparticles as fertilizer for the germination, growth and

metabolism of vegetable crops. Journal of Nanoengineering and

Nanomanufacturing, 3: 353-364.

Singh, N. P., Sachan, R. S., Pandey, P. C. and Bisht, P. S. (1999). Effect of decade long-

term fertilizers and manure application on soil fertility and productivity of rice-

wheat system in a Mollisol. Journal of the Indian Society of Soil Science, 47:

72-80.

Slaton, N. A., Wilson-Jr, C. E., Ntamatungiro, S., Norman, R. J. and Boothe, D. L.

(2001). Evaluation of zinc seed treatments for rice. Agronomy Journal, 93: 152-

157.

Sresty T. V. S. and Rao, K. V. M. (1999). Ultrastrucutural aeration in response to zinc

and nickel stress in root cells of pigeon pea. Environmental Experimental

Botany, 41: 3-13.

Sridevi, D. and Rajendran, K. V. (2009). Preparation of ZnO Nanoparticles and

nanorods by using CTAB assisted hydrothermal method. International Journal

of Nanotechnology and Applications, 3: 43-48.

Stampoulis, D., Sinha, S. K. and White, J. C. (2009). Assay dependent phytotoxicity of

nanoparticles to plants. Environmental Science & Technology, 43: 9473-9479.

Steel, R. G. and Torrie, J. H. (1982). Principles and procedures of statistics. McGraw

Hill Book Company, New Delhi-110 001.

Subbaiah, B. V. and Asija, G. L. (1956). A rapid procedure for the estimation of

available nitrogen in Soil. Current Science, 25(8): 259-260.

Subbaiah, L. V., Prasad, T. N. V. K. V., Krishna, T. G., Sudhakar, P., Reddy, B. R. and

Pradeep, T. (2016). Novel effects of nanoparticulate delivery of zinc on growth,

productivity, and zinc biofortification in maize (Zea mays L.). Journal of

Agricultural and Food Chemistry, 64(19): 3778-3788.

Sudhir, K., Srikanth, K. and Jayaprakash, S. M. (2002). Sustained crop production

under long term fertilizer use in red soil in India. 17th WCSS, 14-21 August

2002, Thailand. 108: 1- 10.

References

xxii

Sun, X. M., Chen, X., Deng, Z. X. and Li, Y. D. (2003). A CTAB-assisted

hydrothermal orientation growth of ZnO nanorods. Materials Chemistry and

Physics, 78(1): 99-104.

Sun, Z., Liu, L., Zhang, L. and Jia, D. (2006). Rapid synthesis of ZnO nano-rods by

one-step, room-temperature, solid-state reaction and their gas-sensing

properties. Nanotechnology, 17: 2266-2270.

Suriyaprabha, R., Karunakaran, G., Yuvakkumar, R., Prabu, P., Rajendran, V. and

Kannan, N. (2012). Growth and physiological responses of maize (Zea mays

L.) to porous silica nanoparticles in soil. The Journal of Nanoparticle Research,

14:1294-1307.

Takkar, P. N., and Walker, C. D. (1993). The distribution and correction of zinc

deficiency, In A. D. Robson, ed. Zinc in Soils and Plants, Vol. 55. Kluwer

Academic Publishers, Dordrecht; Boston.

Tang, E., Tian, B., Zheng, E., Fu, C. and Cheng, G. (2008). Preparation of ZnO

nanoparticles via uniform precipitation method. Chemical Engineering

Communication, 195: 479-491.

Tavakoli, A., Sohrabi, M. and Kargari, A. (2007). A review of methods for synthesis

of nanostructured metals with emphasis on iron compounds. Chemical Papers,

61: 151- 170.

Thomas, C., George, S., Horst, A., Ji, Z., Miller, R. and Peralta-Videa, J. R. (2011).

Nanomaterials in the environment: From materials to high- throughput

screening to organisms. American Chemical Society (ACS)-Nano, 5: 13-20.

USEPA (2005). Nanotechnology white paper -external review draft [Online]. Available

by U. S. Environmental Protection Agency http://www.epa.gov/osa/pdfs/EPA_

nanotechnology_ white_paper_external_review_draft_12-02-2005.pdf.

Varshney, P., Singh, S.K. and Srivastava, P.C. (2008). Frequency and rates of zinc

application under hybrid rice-wheat sequence in a Mollisol of Uttarakhand.

Journal of the Indian Society of Soil Science, 56(1): 92-98.

Vijayashankar, B. M., Mastan Reddy, C., Mastan Reddy, A., Subramanyam, D. and

Balaguravaiah, D. (2007). Effect of integrated use of organic and inorganic

fertilizers on soil properties and yield of sugarcane. Journal of the Indian

Society of Soil Science, 55(2): 161-166.

References

xxiii

Vitti, A., Nuzzaci, M., Scopa, A., Tataranni, G., Tamburrino, I. and Sofo, A. (2014).

Hormonal response and root architecture in Arabidopsis thaliana subjected to

heavy metals. International Journal of Plant Biology, 5: 5226-5232.

Wang, Z. Y., Xie, X. Y., Zhao, J., Liu, X. Y., Feng, W. Q., White, J.C. and Xing, B. S.

(2012). Xylem-and phloem-based transport of CuO nanoparticles in maize (Zea

mays L.). Environmental Science and Technology, 46: 4434-4441.

Watanabe, T. and Yamaguchi, I. (1991). Evaluation of wettability of plant leaf surfaces.

Journal of Pesticide Science, 16: 491-498.

Watson, C. A., Atkinson, D., Gosling, P., Jackson, L. R. and Rayns, F. W. (2002).

Managing soil fertility in organic fanning systems. Soil Use and Management,

18: 239-247.

Watson, J. L., Fang, T., Dimkpa, C. O, Britt, D. W., McLean, J. E., Jacobson, A. and

Anderson, A.J. (2015). The phytotoxicity of ZnO nanoparticles on wheat varies

with soil properties. Biometals, 28(1): 101-112.

Weisner, M. R., Lowry, G.V., Alvarez, P., Dionysiou, and Biswas, P. (2006). Assessing

the risks of manufactured nanomaterials. Environmental Science and

Technology, 40(14): 4336-4345.

Wigginton, N. S., Haus, K. L. and Hochella Jr. M. F. (2007). Aquatic environmental

nanoparticles. Journal of Environmental Monitoring, 9: 1306 -1316.

Wu, J. J. and Liu, S. C. (2002a). Low-temperature growth of well-aligned ZnO

nanorods by chemical vapor deposition. Advanced Materials, 14(3): 215-218.

Wu, J. J. and Liu, S. C. (2002b). Catalyst-Free Growth and Characterization of ZnO

Nanorods. The Journal of Physical Chemistry-B, 106(37): 9546-9551.

Yang, K., Wang, X. L., Zhu, L. Z. and Xing, B. S. (2006). Competitive sorption of

pyrene, phenanthrene, and naphthalene on multiwalled carbon nanotubes.

Environmental Science and Technology, 40: 5804-5810.

Yang, L. and Watts, D. J. (2005). Particle surface characteristics may play an important

role in phytotoxicity of alumina nanoparticles. Toxicology Letters, 158: 122-

132.

References

xxiv

Yang, Y., Chen, H., Zhao, B. and Bao, X. (2004). Size control of ZnO nanoparticles

via thermal decomposition of zinc acetate coated on organic additives. Journal

of Crystal Growth, 263(1-4): 447-453.

Yang, Z., Chen, J., Dou, R., Gao, X., Mao, C. and Wang, L. (2015). Assessment of the

phytotoxicity of metal oxide nanoparticles on two crop plants, maize (Zea mays

L.) and rice (Oryza sativa L.). International Journal of Environmental Research

and Public Health, 12: 15100-15109.

Yilmaz, A., Ekiz, H., Gultekin, I., Torun, B., Barut, H., Karanlik, S. and Cakmak, I.

(1998). Effect of seed zinc content on grain yield and zinc concentration of

wheat growth in zinc-deficient calcareous soils. Journal of Plant Nutrition, 21:

2257-2264.

Yokoyama, T. and Huang, C. C. (2005). Nanoparticle technology in the production of

functional materials. Kona Journal, 23: 7-17.

Zak, A. K., Razali, R., Majid, W. and Darroundi, M. (2011). Synthesis and

characterization of a narrow size distribution of ZnO nanoparticles.

International Journal of Nanomedicine, 6: 1399-1403.

Zhang, P., Ma, Y. H., Zhang, Z. Y., He, X., Guo, Z., Tai, R. Z., Ding, Y. Y., Zhao, Y.

L. and Chai, Z. F. (2012). Comparative toxicity of nanoparticulate/bulk Yb2O3

and YbCl3 to cucumber (Cucumis sativus). Environmental Science and

Technology, 46: 1834-1841.

Zhang, R., Zhang, H., Tu, C., Hu, X., Li, L., Luo, Y. and Christie, P. (2015).

Phytotoxicity of ZnO nanoparticles and the released Zn (II) ion to corn (Zea

mays L.) and cucumber (Cucumis sativus L.) during germination.

Environmental Science and Pollution Research, 22(14): 11109- 11117.

Zhang, S. G. (2003). Fabrication of novel biomaterials through molecular self-

assembly. Nature Biotechnology, 21: 1171-1178.

Zhao, L., Sun, Y., Hernandez-Viezcas, J. A., Servin, A. D., Hong, J., Niu, G., Peralta-

Videa, J. R., Duarte-Gardea, M. and Gardea-Torresdey, J. L. (2013). Influence

of CeO2 and ZnO nanoparticles on cucumber physiological markers and

bioaccumulation of Ce and Zn: a life cycle study. Journal of Agricultural and

Food Chemistry, 61: 11945-11951.

References

xxv

Zheng, L., Hong, F., Lu, S. and Liu, C. (2005). Effect of nano-TiO2 on strength of

naturally aged seeds and growth of spinach. Biological Trace Element

Research, 104: 83-91.

Zhou, D., Jin, S., Li, L., Wang, Y. and Weng, N. (2011). Quantifying the adsorption

and uptake of CuO nanoparticles by wheat root based on chemical extractions.

Journal of Environmental Sciences, 23(11): 1852-1857.

Zhu, H., Han J., Xiao J. Q. and Jin, Y. (2008). Uptake, translocation and accumulation

of manufactured iron oxide nanoparticles by pumpkin plants. Journal of

Environmental Monitoring, 10: 713-717.

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EFFECT OF ZINC OXIDE NANOPARTICLES ON ......Effect of Zinc Oxide Nanoparticles on Germination, Growth and Yield of Maize (Zea mays L.) Name of student Major Guide Pankaj Kumar Tiwari