EFFECT OF ZINC OXIDE NANOPARTICLES ON ......Effect of Zinc Oxide Nanoparticles on Germination,...
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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
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
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.
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
Review of Literature
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
Review of Literature
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).
Review of Literature
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.
Review of Literature
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
Review of Literature
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
Review of Literature
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.
Review of Literature
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
Review of Literature
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),
Review of Literature
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
Review of Literature
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
Review of Literature
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
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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).
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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.
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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
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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/
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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.
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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
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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
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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
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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.
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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 nano- particles. 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.
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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
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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
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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
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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-
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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
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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
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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.
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
Materials and Methods
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
Materials and Methods
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).
Materials and Methods
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).
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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
T3 : ZnONPs suspension at 1000 ppm
T4 : ZnONPs suspension at 2000 ppm
T5 : Bulk ZnO suspension at 500 ppm
T6 : Bulk ZnO suspension at 1000 ppm
T7 : Bulk ZnO suspension at 2000 ppm
T8 : Seed treatment with ZnO slurry @10 mL bulk ZnO /kg seeds
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
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
T3 : Seed treatment with ZnONPs suspension at 1000 ppm
T4 : Seed treatment with ZnONPs suspension at 2000 ppm
T5 : Seed treatment with Bulk ZnO suspension at 500 ppm
T6 : Seed treatment with Bulk ZnO suspension at 1000 ppm
T7 : Seed treatment with Bulk ZnO suspension at 2000 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
Materials and Methods
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
T3 : Foliar spray of ZnONPs suspension at 1000 ppm
T4 : Foliar spray of ZnONPs suspension at 2000 ppm
T5 : Foliar spray of Bulk ZnO suspension at 500 ppm
T6 : Foliar spray of Bulk ZnO suspension at 1000 ppm
T7 : Foliar spray of Bulk ZnO suspension at 2000 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
Materials and Methods
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
Materials and Methods
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)
Materials and Methods
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.
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
Results and Discussion
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).
Results and Discussion
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.
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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.
Results and Discussion
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.
Results and Discussion
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)
Results and Discussion
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
Results and Discussion
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
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 4.2: Effect of different seed Zn treatments on germination ofmaize seeds (9th Day of incubation)
Results and Discussion
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
Results and Discussion
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
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 4.3: Effect of different seed Zn treatments on seedling length andvigour of maize seeds
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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.
Results and Discussion
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
T1: No Zn (Control) 313.7 287.7 300.7T2: ZnO NPs at 500 ppm 350.3 316.7 333.5T3: ZnO NPs at 1000 ppm 407.0 377.4 392.2T4: ZnO NPs at 2000 ppm 377.4 329.3 353.4T5: Bulk ZnO at 500 ppm 324.3 311.2 317.8T6: Bulk ZnO at 1000 ppm 376.0 349.0 362.5T7: Bulk ZnO at 2000 ppm 365.1 341.4 353.3T8: ZnO slurry @10 mL kg -1 seed 337.3 327.3 332.3Mean 356.4 330.0 343.2
S. Em. (±)Zn 6.54 5.03 5.94Season - - 2.06Zn x Season - - 5.83
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
Results and Discussion
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
T1: No Zn (Control) 577.1 627.3 602.2T2: ZnO NPs at 500 ppm 691.0 789.8 740.4T3: ZnO NPs at 1000 ppm 813.5 914.0 863.7T4: ZnO NPs at 2000 ppm 737.0 812.5 774.8T5: Bulk ZnO at 500 ppm 683.6 758.1 720.8T6: Bulk ZnO at 1000 ppm 761.3 845.7 803.5T7: Bulk ZnO at 2000 ppm 758.3 818.7 788.5T8: ZnO slurry @10 mL kg -1 seed 720.3 778.0 749.1Mean 717.8 793.0 755.4
S. Em. (±)Zn 14.17 12.20 9.86Season - - 4.67Zn x Season - - 13.22
C. D. (p=0.05)Zn 42.49 36.56 28.34Season - - 13.50Zn x Season - - NS
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.
Results and Discussion
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
S. Em. (±)Zn 15.86 15.02 11.17Season - - 5.46Zn x Season - - 15.45
C. D. (p=0.05)Zn 44.56 45.03 32.08Season - - 16.00Zn x Season - - NS
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
Results and Discussion
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).
Results and Discussion
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
T1: No Zn (Control) 18.08 20.00 19.04T2: ZnO NPs at 500 ppm 23.58 27.42 25.50T3: ZnO NPs at 1000 ppm 30.17 34.50 32.33T4: ZnO NPs at 2000 ppm 27.33 31.50 29.42T5: Bulk ZnO at 500 ppm 21.33 24.08 22.71T6: Bulk ZnO at 1000 ppm 27.00 29.17 28.08T7: Bulk ZnO at 2000 ppm 25.17 28.83 27.00T8: ZnO slurry @10 mL kg -1 seed 23.92 25.92 24.92Mean 24.57 27.68 26.13
S. Em. (±)Zn 0.67 0.68 0.48Season - - 0.24Zn x Season - - 0.68
C. D. (p=0.05)Zn 2.02 2.05 1.38Season - - 0.69Zn x Season - - NS
C. V. (%) 4.75 4.28 4.50
Results and Discussion
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.
Results and Discussion
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
T1: No Zn (Control) 37.42 44.08 40.75T2: ZnO NPs at 500 ppm 46.33 48.42 47.38T3: ZnO NPs at 1000 ppm 56.33 60.58 58.46T4: ZnO NPs at 2000 ppm 52.08 56.17 54.13T5: Bulk ZnO at 500 ppm 44.67 47.50 46.08T6: Bulk ZnO at 1000 ppm 51.67 59.67 55.67T7: Bulk ZnO at 2000 ppm 49.33 57.92 53.63T8: ZnO slurry @10 mL kg -1 seed 45.67 51.58 48.63Mean 47.94 53.24 50.59
S. Em. (±)Zn 1.22 1.38 0.92Season - - 0.46Zn x Season - - 1.30
C. D. (p=0.05)Zn 3.65 4.14 2.65Season - - 1.33Zn x Season - - NS
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
T1: No Zn (Control) 42.67 47.33 45.00T2: ZnO NPs at 500 ppm 50.50 53.00 51.75T3: ZnO NPs at 1000 ppm 58.33 64.00 61.17T4: ZnO NPs at 2000 ppm 54.67 59.75 57.21T5: Bulk ZnO at 500 ppm 49.67 51.83 50.75T6: Bulk ZnO at 1000 ppm 54.67 58.33 56.50T7: Bulk ZnO at 2000 ppm 54.92 60.08 57.50T8: ZnO slurry @10 mL kg -1 seed 52.50 54.75 53.63Mean 52.24 56.14 54.19
S. Em. (±)Zn 0.83 1.05 0.67Season - - 0.33Zn x Season - - 0.95
C. D. (p=0.05)Zn 2.50 3.14 1.93Season - - 0.96Zn x Season - - NS
C. V. (%) 2.76 3.24 3.03
Results and Discussion
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.
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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.
Results and Discussion
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
Results and Discussion
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
T1: No Zn (Control) 8.33 8.19 8.26T2: ZnO NPs at 500 ppm 8.35 8.21 8.28T3: ZnO NPs at 1000 ppm 8.35 8.18 8.26T4: ZnO NPs at 2000 ppm 8.36 8.17 8.27T5: Bulk ZnO at 500 ppm 8.34 8.17 8.25T6: Bulk ZnO at 1000 ppm 8.35 8.20 8.27T7: Bulk ZnO at 2000 ppm 8.33 8.17 8.25T8: ZnO slurry @10 mL kg -1 seed 8.33 8.16 8.25Mean 8.34 8.18 8.26
S. Em. (±)Zn 0.01 0.02 0.01Season - - 0.01Zn x Season - - 0.02
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.
Results and Discussion
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
T1: No Zn (Control) 0.20 0.18 0.19T2: ZnO NPs at 500 ppm 0.21 0.19 0.20T3: ZnO NPs at 1000 ppm 0.22 0.18 0.20T4: ZnO NPs at 2000 ppm 0.22 0.19 0.21T5: Bulk ZnO at 500 ppm 0.20 0.19 0.20T6: Bulk ZnO at 1000 ppm 0.21 0.19 0.20T7: Bulk ZnO at 2000 ppm 0.22 0.19 0.21T8: ZnO slurry @10 mL kg -1 seed 0.21 0.19 0.20Mean 0.21 0.19 0.20
S. Em. (±)Zn 0.01 0.01 0.00Season - - 0.00Zn x Season - - 0.01
C. D. (p=0.05)Zn NS NS NSSeason - - 0.01Zn x Season - - NS
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
T1: No Zn (Control) 0.31 0.41 0.36T2: ZnO NPs at 500 ppm 0.33 0.44 0.39T3: ZnO NPs at 1000 ppm 0.34 0.42 0.38T4: ZnO NPs at 2000 ppm 0.34 0.43 0.39T5: Bulk ZnO at 500 ppm 0.34 0.44 0.39T6: Bulk ZnO at 1000 ppm 0.34 0.44 0.39T7: Bulk ZnO at 2000 ppm 0.33 0.44 0.39T8: ZnO slurry @10 mL kg -1 seed 0.34 0.42 0.38Mean 0.33 0.43 0.38
S. Em. (±)Zn 0.01 0.01 0.01Season - - 0.00Zn x Season - - 0.01
C. D. (p=0.05)Zn NS NS NSSeason - - 0.01Zn x Season - - NS
C. V. (%) 4.41 4.32 4.39
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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
S. Em. (±)Zn 0.05 0.05 0.04 0.01 0.01 0.01Season - - 0.02 - - 0.00Zn x Season - - 0.05 - - 0.01
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),
Results and Discussion
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
Results and Discussion
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
S. Em. (±)Zn 7.18 5.90 6.91Season - - 2.32Zn x Season - - 6.57
C. D. (p=0.05)Zn 21.51 17.68 23.13Season - - 6.70Zn x Season - - NS
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.
Results and Discussion
93
Table 4.17: Effect of different foliar Zn treatments on stover yield of maize
Treatment Stover Yield (g plot-1)Rabi Summer Pooled
T1: No Zn (Control) 668.8 763.3 716.1T2: ZnO NPs at 500 ppm 834.0 943.4 888.7T3: ZnO NPs at 1000 ppm 965.9 1057.3 1011.6T4: ZnO NPs at 2000 ppm 951.5 1060.8 1006.2T5: Bulk ZnO at 500 ppm 732.0 821.8 776.9T6: Bulk ZnO at 1000 ppm 747.7 845.3 796.5T7: Bulk ZnO at 2000 ppm 756.0 857.3 806.7T8: 0.5% ZnSO4 856.0 983.5 919.8Mean 814.0 916.6 865.3
S. Em. (±)Zn 16.69 18.15 12.12Season - - 6.16Zn x Season - - 17.44
C. D. (p=0.05)Zn 50.04 54.42 34.81Season - - 17.80Zn x Season - - NS
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
S. Em. (±)Zn 16.84 22.56 13.78Season - - 7.04Zn x Season - - 19.91
C. D. (p=0.05)Zn 50.48 67.65 39.60Season - - 20.30Zn x Season - - NS
C. V. (%) 2.35 3.02 2.72
Results and Discussion
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.
Results and Discussion
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
T1: No Zn (Control) 20.42 24.08 22.25T2: ZnO NPs at 500 ppm 28.67 36.00 32.33T3: ZnO NPs at 1000 ppm 34.25 41.17 37.71T4: ZnO NPs at 2000 ppm 33.92 41.50 37.71T5: Bulk ZnO at 500 ppm 20.92 25.83 23.38T6: Bulk ZnO at 1000 ppm 21.08 27.33 24.21T7: Bulk ZnO at 2000 ppm 22.25 27.42 24.83T8: 0.5% ZnSO4 27.00 34.58 30.79Mean 26.06 32.24 29.15
S. Em. (±)Zn 0.66 1.15 0.71Season - - 0.33Zn x Season - - 0.93
C. D. (p=0.05)Zn 1.96 3.44 2.04Season - - 0.95Zn x Season - - NS
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.
Results and Discussion
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
T1: No Zn (Control) 43.67 48.25 45.96T2: ZnO NPs at 500 ppm 55.00 59.83 57.42T3: ZnO NPs at 1000 ppm 64.25 70.42 67.33T4: ZnO NPs at 2000 ppm 65.17 68.75 66.96T5: Bulk ZnO at 500 ppm 46.00 50.92 48.46T6: Bulk ZnO at 1000 ppm 48.83 52.58 50.71T7: Bulk ZnO at 2000 ppm 47.67 51.08 49.38T8: 0.5% ZnSO4 54.33 61.92 58.13Mean 53.11 57.97 55.54
S. Em. (±)Zn 1.62 1.70 1.17Season - - 0.59Zn x Season - - 1.66
C. D. (p=0.05)Zn 4.84 5.10 3.35Season - - 1.69Zn x Season - - NS
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
Results and Discussion
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
T1: No Zn (Control) 49.33 52.00 50.67T2: ZnO NPs at 500 ppm 61.83 62.67 62.25T3: ZnO NPs at 1000 ppm 70.17 73.42 71.79T4: ZnO NPs at 2000 ppm 67.67 75.17 71.42T5: Bulk ZnO at 500 ppm 50.33 54.17 52.25T6: Bulk ZnO at 1000 ppm 52.00 55.00 53.50T7: Bulk ZnO at 2000 ppm 52.83 56.33 54.58T8: 0.5% ZnSO4 60.08 62.00 61.04Mean 58.03 61.34 59.69
S. Em. (±)Zn 1.57 1.63 1.17Season - - 0.57Zn x Season - - 1.60
C. D. (p=0.05)Zn 4.72 4.88 3.35Season - - 1.63Zn x Season - - NS
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.
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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.
Results and Discussion
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
Results and Discussion
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
Results and Discussion
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
T1: No Zn (Control) 8.32 8.19 8.25T2: ZnO NPs at 500 ppm 8.30 8.20 8.25T3: ZnO NPs at 1000 ppm 8.29 8.21 8.25T4: ZnO NPs at 2000 ppm 8.33 8.22 8.28T5: Bulk ZnO at 500 ppm 8.32 8.18 8.25T6: Bulk ZnO at 1000 ppm 8.31 8.20 8.25T7: Bulk ZnO at 2000 ppm 8.34 8.18 8.26T8: 0.5% ZnSO4 8.29 8.22 8.25Mean 8.31 8.20 8.26
S. Em. (±)Zn 0.14 0.05 0.07Season - - 0.04Zn x Season - - 0.11
C. D. (p=0.05)Zn NS NS NSSeason - - 0.11Zn x Season - - NS
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.
Results and Discussion
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
T1: No Zn (Control) 0.22 0.19 0.20T2: ZnO NPs at 500 ppm 0.23 0.21 0.22T3: ZnO NPs at 1000 ppm 0.24 0.19 0.21T4: ZnO NPs at 2000 ppm 0.23 0.20 0.22T5: Bulk ZnO at 500 ppm 0.24 0.20 0.22T6: Bulk ZnO at 1000 ppm 0.24 0.20 0.22T7: Bulk ZnO at 2000 ppm 0.24 0.19 0.22T8: 0.5% ZnSO4 0.24 0.21 0.23Mean 0.23 0.20 0.22
S. Em. (±)Zn 0.01 0.01 0.01Season - - 0.00Zn x Season - - 0.01
C. D. (p=0.05)Zn NS NS NSSeason - - 0.01Zn x Season - - NS
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
T1: No Zn (Control) 0.36 0.42 0.39T2: ZnO NPs at 500 ppm 0.39 0.44 0.42T3: ZnO NPs at 1000 ppm 0.38 0.46 0.42T4: ZnO NPs at 2000 ppm 0.39 0.45 0.42T5: Bulk ZnO at 500 ppm 0.38 0.44 0.41T6: Bulk ZnO at 1000 ppm 0.37 0.46 0.42T7: Bulk ZnO at 2000 ppm 0.40 0.44 0.42T8: 0.5% ZnSO4 0.39 0.45 0.42Mean 0.38 0.44 0.41
S. Em. (±)Zn 0.01 0.01 0.01Season - - 0.00Zn x Season - - 0.01
C. D. (p=0.05)Zn NS NS NSSeason - - 0.01Zn x Season - - NS
C. V. (%) 4.99 3.54 4.23
Results and Discussion
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.
Results and Discussion
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
S. Em. (±)Zn 0.01 0.01 0.01 0.09 0.08 0.06Season - - 0.00 - - 0.03Zn x Season - - 0.01 - - 0.08
C. D. (p=0.05)Zn NS NS NS NS NS NSSeason - - 0.01 - - 0.08Zn x Season - - NS - - NS
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
S. Em. (±)Zn 0.08 0.07 0.05 0.01 0.01 0.01Season - - 0.03 - - 0.00Zn x Season - - 0.08 - - 0.01
C. D. (p=0.05)Zn NS NS NS NS NS NSSeason - - 0.08 - - 0.01Zn x Season - - NS - - NS
C. V. (%) 4.07 3.32 3.68 4.50 4.29 4.39
Results and Discussion
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).
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%
Summary and Conclusion
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
Summary and Conclusion
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.
Summary and Conclusion
112
5.3.3 Zinc Concentration
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.
5.3.6 Soil Parameters after Harvest
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
Summary and Conclusion
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.
5.4.2 Zinc Concentration
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.
Summary and Conclusion
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.
5.4.5 Soil Parameters after Harvest
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.
Summary and Conclusion
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
Summary and Conclusion
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.
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