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PHYSIOLOGICAL RESPONSES OF SOYBEAN AND
WHEAT TOWARDS NANOPARTICLE BASED
MICRONUTRIENTS FERTILIZATION
M.Sc. (Ag) Thesis
By
Milap Ram Sahu
DEPARTMENT OF PLANT PHYSIOLOGY,
AGRICULTURAL BIOCHEMISTRY, MEDICINAL AND
AROMATIC PLANTS COLLEGE OF AGRICULTURE, RAIPUR
FACULTY OF AGRICULTURE INDIRA GANDHI KRISHI VISHWAVIDYALAYA
RAIPUR (Chhattisgarh) 2016
PHYSIOLOGICAL RESPONSES OF SOYBEAN AND
WHEAT TOWARDS NANOPARTICLE BASED
MICRONUTRIENTS FERTILIZATION
Thesis
Submitted to the
Indira Gandhi Krishi Vishwavidyalaya, Raipur
By
Milap Ram Sahu
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
Master of Science
In
Plant Physiology
UN ID No. 20141520374 ID No. 120114156
JULY, 2016
iv
ACKNOWLEDGEMENT
The author of this manuscript praises the omniscient and almighty god and
his parents, who provided his this opportunity of submitting the present thesis for
award of M.Sc. (Ag.) Plant Physiology, Degree.
The word can never express indebtedness but I can take this opportunity to
express my deepest and heartfelt sense of gratitude to revered, chairman of
Advisory committee, Dr. A. Guhey, Head and Professor of Department, Plant
Physiology, Agril. Biochemistry and Medicinal and Aromatic Plants. College of
Agriculture, IGKV, Raipur (C.G.) for her guidance and inspiration to carry out the
present work. I am equally indebted and express my heartfelt sense of gratitude to
Co-chairman Dr. R. Elanchezhian, Principal Scientist, (Plant Physiology), ICAR-
Indian Institute of Soil Science, Bhopal (M.P.) for suggesting the problem,
providing necessary laboratory space and screen house facilities to carry out the
present work and for his healthy criticism in preparing the present manuscript of
this thesis to make this task a success. He has been a constant source of inspiration
and his love and affection to me will ever be remembered.
I wish to render my sincere thanks to the member of the thesis Advisory
Committee Dr. S.P. Tiwari (Asst. Professor) Department of Plant Physiology,
Agril. Biochemistry and Medicinal and Aromatic Plants, Dr. S.B. Verulkar (Head
and Professor) Department of Plant Molecular Biology and Biotechnology, and
Dr. G. Chandrakar (Professor) Department of Statistics, College of Agriculture,
IGKV, Raipur (C.G.) or their kind help and constant advisement.
I also feel great pleasure to express my heartfelt thanks to the Honorable
Vice Chancellor of IGKV, Raipur (C.G.) Dr. S.K. Patil, Dean faculty of
Agriculture, Dr. S.S. Rao, Director of Research Services, Dr. J.S. Urkurkar and
Director of Extension Services Dr. M.P. Thakur, Director of Instructions Dr. S.S.
Shaw, IGKV, Raipur for providing necessary facilities in carrying out this piece of
research work.
I take this opportunity to express sincere thanks to Dr. A. K. Patra,
Director ICAR-IISS, Bhopal who permitted me to work at IISS. I am also highly
grateful to Dr. A. K. Biswas, Head, Division of Soil Chemistry and fertility (ICAR-
IISS) for providing the required facilities for my research work.
I am deeply indebted to my teachers, Dr. A.K. Geda (Professor), Dr.
Pratiba Katiyar, (Professor), Dr. D. Khokhar (Scientist) and Mr. V.B.
Kuruwanshi (Asst. Professor) Department of Plant Physiology, Agril.
Biochemistry and Medicinal and Aromatic Plants, Raipur, for their encouragement
throughout the course of my studies.
I wish to record my grateful thanks to Dr. S. Shrivastava (Principal
Scientist), Dr. B. L. Lakaria (Principal Scientist), Dr. K. Ramesh (Principal
Scientist) Dr. P. Jha (Senior Scientist), Dr. I. Rashmi (Scientist), Dr. B. P. Meena
vi
TABLE OF CONTENTS
Chapter Title Page ACKNOWLEDGEMENT iv/v TABLE OF CONTENTS vi/viii LIST OF TABLES ix/x LIST OF FIGURES xi/xii LIST OF NOTATIONS Xiii LIST OF ABBREVIATIONS Xiv ABSTRACT xv/xvi
I INTRODUCTION 1-4 II REVIEW OF LITERATURE
2.1 Impact of Iron nanoparticle fertilizer on Plants 2.1.1 Morphological effect 2.1.2 Physiological and biochemical effect 2.2 Impact of Copper nanoparticle fertilizer on Plant
2.3.1 Morphological effect 2.3.2 Physiological and biochemical effect on Plants
2.3 Impact of Zink nanoparticle fertilizer 2.2.1 Morphological effect 2.2.2 Physiological and biochemical effect
2.4 Impact of Other metallic nanoparticle fertilizer on Plants 2.4.1 Morphological effect 2.4.2 Physiological and biochemical effect
5-18 5-8 5-7 7-8 9-14 9 10-14 10-14 10-13 13-14 15-18 15-16 16-18
III MATERIALS AND METHODS
3.1 Experimental site 3.2 Sand culture system setup 3.3 Climate 3.4 Experimental details
3.4.1 Design and layout 3.4.2 Collection of experimental data
3.5 Morphological parameters 3.5.1 Plant height 3.5.2 Root length 3.5.3 Plant biomass 3.5.4 Root biomass 3.5.5 Root volume
3.6 Growth parameters
19-29 19 19 19 22 22 22 22-23 22 22 23 23 23 23-24
vii
3.6.1 Leaf area 3.6.2 leaf area ratio 3.6.3 Specific leaf weight 3.6.4 Specific leaf area 3.6.5 Root shoot ratio
3.7 Physiological and biochemical parameters 3.7.1 Chlorophyll content estimation 3.7.2 Estimation of relative water content 3.7.3 Estimation of membrane stability 3.7.4 Proline content estimation 3.7.5 Assay of antioxidant enzyme
3.7.5.1 Super oxide dismutase (SOD) activity assay 3.7.5.2 Catalase (CAT) activity assay 3.7.5.3 Peroxidase (POX) activity assay
3.7.6 Estimation of total soluble protein 3.7.7 Estimation of total soluble sugar 3.7.8 Estimation of starch 3.7.9 Estimation of non-structural carbohydrate 3.7.10 Gas exchange parameter 3.7.11 SPAD Value 3.7.12 Grain Yield 3.7.13 Toxicity/ Deficiency symptom analysis
23 23 23 24 24 24-29 24-25 25 25 26 26-27 27 27 27 27 28 28 29 29 29 29 29
IV RESULTS AND DISCUSSION
4.1 Morphological parameters of Soybean 4.1.1 Plant height (cm) 4.1.2 Total root Length (cm) 4.1.3 Shoot dry weight (g) 4.1.4 Root dry weight (g) 4.1.5 Specific leaf area (cm²g-1) 4.1.6 Specific leaf weight (g cm-²) 4.1.7 Leaf area ratio 4.1.8 Leaf area (cm2) 4.1.9 Root volume (cm3) 4.1.10 Pod weight (g plant-1) 4.1.11 Grain weight (g plant-1) 4.1.12 Root shoot ratio
4.2 Biochemical parameters of Soybean 4.2.1 Chlorophyll content (mg g-1FW) 4.2.2 Membrane stability (%) 4.2.3 Relative water content (%) 4.2.4 Antioxidant enzyme 4.2.4.1 Super oxide dismutase (unit g-1)
30-114 30-47 30-31 31-32 32-34 34-36 37-38 38-39 39-40 41-43 44 45 45-46 46-47 47-63 47-50 51-52 52-53 52-57 53-55
viii
4.2.4.2 Catalase (unit H2O2 min-1 g-1) 4.2.4.3 Peroxidase (unit H2O2 min-1 g-1) 4.2.5 Proline content (μM g
-1) 4.2.6 Total soluble protein (mg g-1) 4.2.7 Total soluble sugar (%) 4.2.8 Non-structural carbohydrate (%)
4.3 Physiological parameters of Soybean 4.3.1 Photosynthesis rate (µM m-2 s-1) 4.3.2 Transpiration rate (mM m-2 s-1) 4.3.3 Stomatal conductance (µM m-2 s-1) 4.3.4. SPAD value 4.4 Morphological parameters of wheat
4.4.1 Plant height (cm) 4.4.2 Total root length (cm) 4.4.3 Shoot dry weight (g) 4.4.4 Root dry weight (g) 4.4.5 Specific leaf area (cm² g-1) 4.4.6 Specific leaf weight (g cm-²) 4.4.7 Leaf area (cm²) 4.4.8 Leaf area ratio 4.4.9 Number of tillers 4.4.10 Root volume ( cm3) 4.4.11 Panicle weight (g plant-1) 4.4.12 Grain weight (g plant-1) 4.4.13 Number of grain plant-1 4.4.14 Seed index (%) 4.4.15 Root shoot ratio
4.5 Biochemical parameters of wheat 4.5.1 Chlorophyll content (mg g-1FW) 4.5.2 Antioxidant enzyme activity 4.5.2.1 Super oxide dismutase (unit g-1) 4.5.2.2 Catalase (unit H2O2 min-1 g-1) 4.5.2.3 Peroxidase (unit H2O2 min-1 g-1) 4.5.3 Membrane stability (%) 4.5.4 Relative water content (%) 4.5.5 Proline content (μM proline g
-1) 4.5.6 Total soluble protein (mg g-1) 4.5.7 Total soluble sugar (%) 4.5.8 Non-structural carbohydrate (%)
4.6 Physiological parameters of wheat 4.6.1 Photosynthesis rate ( µM m-2 s-1) 4.6.2 Transpiration rate (mM m-2 s-1)
55-57 58 58-60 60-61 61-63 64 64-73 64 65-66 66-67 71-73 74-91 74 74-75 75-77 77-79 80-81 81-82 82 82-83 85 85-86 87 87 88 88 89 92-108 92-94 94-95 96-97 99-102 99-100 100-101 101-102 104 104-105 105-106 106-107 109-115 109 109-110
ix
4.6.3 Stomatal conductance (µM m-2 s-1) 4.6.4 SPAD value
110-111 113-114
V SUMMARY AND CONCLUSIONS
5.1 Summary 5.2 Conclusions 5.3 Suggestions for future research work
116-119 116-118 118 118-119
REFERENCES 120-128 APPENDICES
Appendix A Appendix B
129-130 129 130
RESUME 131
x
LIST OF TABLES
Table Title Page
3.1 Treament details 22 4.1 Effect of micronutrient NPs on plant height and total root length of
soybean 32
4.2 Effect of micronutrient NPs on shoot weight of soybean 34 4.3 Effect of micronutrient NPs on root dry weight of soybean 35 4.4 Effect of micronutrient NPs on specific leaf area and specific leaf
weight of soybean 39
4.5 Effect of micronutrient NPs on leaf area ratio and leaf area of soybean. 42 4.6 Effect of micronutrient NPs on root volume of soybean 45 4.7 Effect of micronutrient NPs on pod weight and grain weight of soybean 46 4.8 Effect of micronutrient NPs on root shoot ratio of soybean 47 4.9 Effect of micronutrient NPs on chlorophyll content of soybean 49 4.10 Effect of micronutrient NPs on membrane stability and relative water
content of soybean 52
4.11 Effect of micronutrient NPs on super oxide dismutase of soybean 53 4.12 Effect of micronutrient NPs on catalase and peroxidase of soybean 56 4.13 Effect of micronutrient NPs on proline and protein of soybean 60 4.14 Effect of micronutrient NPs on total soluble sugar and non-structural
carbohydrate of soybean 62
4.15 Effect of micronutrient NPs on photosynthesis rate of soybean 64 4.16 Effect of micronutrient NPs on transpiration rate of soybean 66 4.17 Effect of micronutrient NPs on stomatal conductance of soybean 67 4.18 Effect of micronutrient NPs on SPAD value of Soybean 72 4.19 Effect of micronutrient NPs on plant height and total root length of
wheat 75
4.20 Effect of micronutrient NPs on shoots and root dry weight of wheat 78 4.21 Effect of micronutrient NPs on specific leaf area and specific leaf
weight of wheat 82
4.22 Effect of micronutrient NPs on leaf area and leaf area ratio of wheat 83 4.23 Effect of micronutrient NPs number of tillers and root volume of wheat 86 4.24 Effect of micronutrient NPs on panicle weight and grain weight of
wheat 87
4.25 Effect of micronutrient NPs on number of grain and seed index of wheat
89
4.26 Effect of micronutrient NPs on root shoot ratio of wheat 90 4.27 Effect of micronutrient NPs on chlorophyll content of wheat 94 4.28 Effect of micronutrient NPs on membrane stability and relative water
content of wheat 97
4.29 Effect of micronutrient NPs on super oxide dismutase of wheat of 100
xi
wheat 4.30 Effect of micronutrient NPs on catalase and peroxidase of wheat 102 4.31 Effect of micronutrient NPs on proline and protein 105 4.32 Effect of micronutrient NPs on NPs on total soluble sugar and non-
structural carbohydrate of wheat 107
4.33 Effect of micronutrient NPs on Photosynthesis rate of wheat 109 4.34 Effect of micronutrient NPs on transpiration rate of wheat 110 4.35 Effect of micronutrient NPs on stomatal conductance of wheat 111 4.36 Effect of micronutrient NPs on SPAD value of wheat 114
xii
LIST OF FIGURE
Figure Title Page
3.1 A view of experimental set up of Soybean in sand culture 20 3.2 A view of experimental set up of Wheat in sand culture 20 3.3 Meteorological data during crop growth period. 21 4.1 Effect of micronutrient NPs on shoot weight of soybean 36 4.2 Effect of micronutrient NPs on root weight of soybean 36 4.3 Effect of micronutrient NPs on specific leaf area of soybean 40 4.4 Effect of micronutrient NPs on leaf area ratio of soybean. 43 4.5 Effect of micronutrient NPs on leaf area of soybean. 43 4.6 Effect of micronutrient NPs on chlorophyll content of soybean at 45
DAS 50
4.7 Effect of micronutrient NPs on chlorophyll content of soybean at 60 DAS
50
4.8 Effect of micronutrient NPs on super oxide dismutase of soybean 54 4.9 Effect of micronutrient NPs on catalase of soybean 57 4.10 Effect of micronutrient NPs on peroxidase of soybean 57 4.11 Effect of micronutrient NPs on TSS of soybean 63 4.12 Effect of micronutrient NPs on non-structural carbohydrate of soybean 63 4.13 Effect of micronutrient NPs on photosynthesis rate of soybean 68 4.14 Effect of micronutrient NPs on transpiration rate of soybean 69 4.15 Effect of micronutrient NPs on stomatal conductance of soybean 70 4.16 Effect of micronutrient NPs on SPAD value of Soybean 73 4.17 Effect of micronutrient NPs on plant height of wheat 76 4.18 Effect of micronutrient NPs on total root length of wheat 76 4.19 Effect of micronutrient NPs on shoot dry weight of wheat 79 4.20 Effect of micronutrient NPs on root dry weight of wheat 79 4.21 Effect of micronutrient NPs on leaf area of wheat 84 4.22 Effect of micronutrient NPs on leaf area ratio of wheat 84 4.23 Effect of micronutrient NPs on root shoot ratio of wheat 91 4.24 Effect of micronutrient NPs on chlorophyll content of wheat at 30 DAS 95 4.25 Effect of micronutrient NPs on chlorophyll content of wheat at 60 DAS 95 4.26 Effect of micronutrient NPs on membrane stability of wheat 98 4.27 Effect of micronutrient NPs on Relative water content of wheat 98 4.28 Effect of micronutrient NPs on Super oxide dismutase of wheat 103 4.29 Effect of micronutrient NPs on peroxidase of wheat 103 4.30 Effect of micronutrient NPs on TSS of wheat 108 4.31 Effect of micronutrient NPs on non-structural carbohydrate of wheat 108 4.32 Effect of micronutrient NPs on photosynthesis rate of wheat 112 4.33 Effect of micronutrient NPs on SPAD value of wheat 115
xiii
LIST OF NOTATIONS/SYMBOLS
Symbol/
notations
Detail
% Percent 0C Degree Celsius CD Critical difference cm Centimeter cm2 Square centimeter cm3 Cubic centimeter cm² g-1 Square centimeter per gram d-1 Per day g cm-2 Gram per square centimeter gm or g Gram ha-1 Per hectare Hrs Hours Kg Kilogram
m Meter m2 Square meter Mg Milligram mg g-1 Milligram per gram mg g-1 FW Milligram per gram fresh weight Ppm Part per million unit g-1 Unit per gram unit H2O2 min-1 Unit per H2O2 per minute μM g
-1 FW Micromole per gram fresh weight µM m-2 s-1 Micromole per meter per second mM m-2 s-1 Millimole per meter per second
xiv
LIST OF ABBREVIATIONS
Abbreviations Detail
CAT Catalase CHL Chlorophyll DAS Day after sowing DW Dry weight EC Electrical conductivity et al. And coworkers / and others i.e That is viz. Namely Fig. Figure FW Fresh weight ICAR Indian Council of Agriculture Research IISS Indian Institute of Soil Science IGKV Indira Gandhi Krishi Vishwavidyalaya LA Leaf area LAR Leaf area ratio MS Membrane stability NPs Nano particles NSC Non-structural carbohydrate NS None significant NSPs Nano scale particles POX Peroxidase RWC Relative water content SLA Specific leaf area SLW Specific leaf weight SOD Super oxide dismutase TSP Total soluble protein TSS Total soluble sugar
xvi
growth and gas exchange parameters of plants like Photosynthetic rate, stomatal
conductance and transpiration rate. In wheat, the nano-micronutrient fertilization of
plants with Fe NPs/ Cu NPs / Zn NPs had positively influenced most of the
morphological parameters (Plant height, total root length, shoot dry weight, root
dry weight, SLA, SLW, LA, LAR, root shoot ratio and grain yield) while reduced
concentration of Fe NPs/ Cu NPs and Zn NPs had positively influenced
biochemical metabolism (Chlorophyll content, SOD, POX, CAT, MS, RWC, TSS,
NSC) of plants. Gas exchange parameters were also positively influenced by NPs.
The above findings indicated that the effect of nanoparticles were crop or species
specific. Moreover, it is also envisaged that nanoparticle at reduced concentration
may be useful for the crop and they may act as catalyst for growth, metabolism and
yield of plants.
xviii
CHAPTER - I
INTRODUCTION
Soybean (Glycine max (L) Merr.) belongs to Fabaceae family and is an
annual crop. Due to having useful compounds such as unsaturated fatty acids,
protein, mineral salts and plant secondary metabolites such as isoflvin, soybean has
many important roles in human and animal nutrition. Achieving optimum quantity
and increasing quality of soybean seeds depend upon many factors among which,
weed control and plant nutrition have critical importance (Sedghi, 2007). Iron is
one of the essential elements for plant growth and plays an important role in the
photosynthetic reactions. Iron activates several enzymes and contributes in RNA
synthesis and improves the performance of photo systems (Malakouti and Tehrani,
2005). Soybean is sensitive to iron deficiency, but different genotypes are various
in efficiency of iron consumption. Application of iron in low–iron soils can
increase grain yield in soybean (Ghasemi et al, 2006). Iron compounds can use as
foliar on leaves and as seed coating (Debermann, 2006). Nanotechnology can
present solution to increasing the value of agricultural products and environmental
problems. With using of nano-particles and nano-powders, we can produce
controlled or delayed releasing fertilizers. Nano-particles have high reactivity
because of more specific surface area, more density of reactive areas, or increased
reactivity of these areas on the particle surfaces. These features simplify the
absorption of fertilizers and pesticides that produced in nano scale (Anonymous,
2009). Studies showed that the effect of nano-particles on plants can be beneficial
(seedling growth and development) or non-beneficial (to prevent root growth) (Zhu
et al,2008). This experiment was conducted to investigate the effects of nano-iron
oxide particles on soybean yield and agronomic traits.Nanoparticles (nano-scale
particles = NSPs) are atomic or molecular aggregates with at least one dimension
between 1 and 100nm (Ball 2002; Roco 2003), that can drastically modify their
physico-chemical properties compared to the bulk material (Nel et al. 2006). It is
worth noting that nanoparticles can be made from a variety of bulk materials and
that they can explicate their actions depending on both the chemical composition
1
and on the size and/or shape of the particles (Brunner et al. 2006). Depending on
the origin, a further distinction is made between three types of NSPs: natural, in-
cidental and engineered. Natural nanoparticles have existed from the beginning of
the earth‘ history and still occur in the environment (volcanic dust, lunar dust,
mineral composites, etc.). Incidental nanoparticles, also defined as waste or
anthropogenic particles, take place as the result of manmade industrial processes
(diesel exhaust, coal combustion, welding fumes, etc.). Currently as nanopaticles
are being widely used in consumer products, communication sector,
pharmaceutics, and energy sector (Zhao and Castranova 2011), these particles may
find their way into terrestrial environment where their fate and behavior are still
largely unknown. Nanofertilzers or nano-encapsulated nutrients might have
properties that are effective to crops, released the nutrients on-demand, controlled
release of chemicals fertilizers that regulate plant growth and enhanced target
activity (De Rosa et al. 2010; Nair et al. 2010). There have been publications on
effects of engineered nanomaterials on higher plants wherein both positive and
negative effects were reported (Monica and Cremonini 2009).
Wheat is the most important stable food crop for more than one third of the
world population and contributes more calories and proteins to the world diet than
any other cereal crops there is no doubt that the number of people who rely on
wheat for a substantial part of their diet amounts to several billions. Therefore, the
nutritional importance of wheat proteins should not be underestimated, particularly
in less developed countrieswhere bread, noodles and other products (e.g. bulgar,
couscous) may provide a substantial proportionof the diet. Wheat provides nearly
55% of carbohydrate and 20% of the food calories. It contains carbohydrate
78.10%, protein 14.70%, fat 2.10%, minerals 2.10% and considerrabble
proportions of vitamins (thiamine and Vitamin-B) and minerals (zinc, iron). Wheat
is also a good source of traces minerals like selenium and magnesium, nutrients
essential to good health. Wheat grain precisely known as caryopsis consists of the
pericarp or fruit and the true seed. In the endosperm of the seed, about72% of the
protein is stored, which forms 8-15% of total protein per grain weight. Wheat
grains are also rich in pantothenic acid, riboflavin and some minerals, sugars etc.
2
The barn, whichconsists of pericarp testa and aleurone, is also a dietary source for
fiber, potassium, phosphorus, magnesium, calcium, and niacin in small quantities.
Nanotechnology is a multidisciplinary field, as it combines the knowledge
from different disciplines: chemistry, physics, and biology amongst others
(Schmid, 2006; Schmid, 2010). Nanotechnology is the art and science of
manipulating matter at the atomic or molecular scale and holds the promise of
providing significant improvements in technologies for protecting the environment.
While many definitions for nanotechnology exist, the U.S. Environmental
Protection Agency (EPA) uses the definition developed by the National
Nanotechnology Initiative (NNI). According to National Nanotechnology Initiative
of the USA, nanotechnology is defined as: research and technology development at
the atomic, molecular, or macromolecular levels using a length scale of
approximately one to one hundred nm in any dimension; the creation and use of
structures, devices and systems that have novel properties and functions because of
their small size; and the ability to control or manipulate matter on an atomic scale
(USEPA, 2007). The technology has excellent prospects for exploitation across the
medical, pharmaceutical, biotechnology, engineering, manufacturing,
telecommunications and information technology markets.
Micronutrients (MNs) are important to world wild agriculture. Zinc (Zn),
iron (Fe), manganese (Mn) and copper (Cu) have become yield-limiting factors
and are partly responsible for low food nutrition. Although crops use low amounts
of MNs (<2.4 kg/ha), about half of the cultivated world‘s soils are deficient in
plant bioavailable MNs, due to their slow replenishment from the weathering of
soil minerals, soil cultivation for thousands of years and insufficient crop
fertilization. Relevant MN deficiencies occur more frequently in neutral to alkaline
soils, under anaerobic conditions and in arid or semi-arid regions. The MN use
efficiency (MUE) of most commercial fertilizers added to soils or foliage is 2.5%
to 5% of applied, due to their rapid stabilization by soil components, low leaf
penetration and low mobility in plants. In soil-plant systems, fertilizer-MNs
interact with macronutrients resulting in synergistic, antagonistic or neutral
response affecting yield and food quality. Some elements are directly involved in
plant metabolism (Arnon and Stout, 1939). However, this principle does not
3
account for the so-called beneficial elements, whose presence, while not required,
has clear positive effects on plant growth. Mineral elements those either stimulates
growth but are not essential, or that are essential only for certain plant species, or
under given conditions, are usually defined as beneficial elements.
Iron is one of the essential elements for plant growth and plays an
important role in the photosynthetic reactions. Iron activates several enzymes and
contributes in RNA synthesis and improves the performance of photosystems
(Malakouti and Tehrani, 2005). Iron deficiency in plants causes a reduction in
chlorophyll and the other anthocyanin contents. Its excess leads to ROS generation,
leading to the oxidative damage (Suh et al. 2002). Thus, Fe has a major role in
cellular redox reactions with 85% of its activity focused in plastids (Mengel and
Kirby 1987).
Zinc (Zn) is typically the second most abundant transition metal in
organisms after iron and the only metal represented in all six enzyme classes
(oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases) (Auld,
2001).
Copper is associated with enzymes involved in redox reactions being
reversibly oxidized from Cu+ to Cu
2+. An enzyme is plastocyanin, which is
involved in electron transfer during the light reaction of photosynthesis (Haehnel
1984).
Hence this study was undertaken to analyze the effect of nano sized
micronutrient fertilizers on the crop performance of soybean and wheat with the
following objectives.
Objectives of the investigation:
1. To study the response of different concentrations of nanoparticle on plant
growth, morphological and yield characteristics of soybean and wheat.
2. To study the response of different concentrations of nanoparticle on
physiological and biochemical characteristics of soybean and wheat.
4
CHAPTER - II
REVIEW OF LITERATURE
2.1 Impact of Iron nanoparticle fertilizer on plant
2.1.1 Morphological effect
Afshar et al. (2013) evaluated the impact of nano-iron on cowpea crop
under irrigation deficit and found significant increase in numbers of seed per pod.
It was observed that increasing nano-iron concentration increased number of seeds
per pod but decreased 1000 seed weight significantly in cowpea. Dhoke et al.
(2013) studied the effect of nano-FeO and nano-Zn Cu Fe oxide particles on the
growth of mung bean (Vigna radiata) seedling and found that the best performance
was observed for nano-Zn Cu Fe-Oxide followed by nano-FeO. The absorption of
nanoparticles by plant leaves was also detected by inductive coupled
plasma/atomic emission spectroscopy. Karimia et al. (2014) investigated the effect
of different concentrations of iron chelate nano fertilizer and Fe-EDDHA on
morphological characteristics and antioxidant enzymes activity of green gram and
observed that increasing nanoparticles concentration above 10 ppm reduced shoot
fresh weight, shoot dry weight and root weight. Nadi et al. (2013) studied the
effect of nano iron chelate fertilizer on grain yield, protein percent and chlorophyll
content of faba bean (Vicia faba L.) and found that the highest and lowest grain
yield belonged to nano-iron (6 g l-1
) and control, respectively.
Liu et al. (2005) reported that nano-Fe2O3 promoted the growth and
photosynthesis of peanut. Ngo et al. (2014) observed that the germination rates of
soybean seeds, treated with zerovalent Cu, Co and Fe were 65, 80 and 80%,
respectively, whereas 55% germination was observed in the control sample in the
field experiment. For all of the nanoscale metals studied, the number of nodules
increased 20–49% compared to the control sample, and the soybean crop yield
increased up to 16% in comparison with the control sample. Nano-iron oxide at the
concentration of 0.75 g l-1
increased leaves and pod dry weight of soybean and
5
thehighest grain yield was observed with 0.5 g l-1
nano-iron oxide that showed 48%
increase in grain yield in comparison with control (Sheykhbaglou et al. 2010).
Armin et al. (2014) carried out foliar application of nano Fe in wheat
(Triticum aestivum) at 2%, 4% and 6% and observed an increase of 12%, 22.09%
and 19.07% grain yield, respectively, over the control. Bakhtiari et al. (2015)
studied that effect of different concentration of Fe Nano-oxide solution at five
levels (0, 0.01%, 0.02%, 0.03% and 0.04%) of wheat. Higher spike weight, 1000
grain weight, biological yield, and grain yield were achieved in plants treated with
0.04% Fe nano-oxide concentration and the lowest values were achieved in the
control. Ghafari and Razmjoo (2013) reported that 2 g l-1
nano iron oxide has
increased harvest index, 1000-grain weight and yield of wheat. Many et al. (2013)
reported that iron nano composite, when sprayed in rice at two stages of nursery
and early earring led to increase in yield, 1000 grains weight and healthy grains
number in ear and decrease in hollow grains number in ear.
Bozorgi (2012) studied the effect of foliar spray of Ascophyllum nodosum
extract and nano iron chelate fertilizer on fruit yield and several attributes of
eggplant. The highest fruit yield was recorded from foliar spray of A. nodosum
extract @ 2 g l-1
with 37.89 ton ha-1
. Among nano iron chelate fertilizer treatments,
spray of 2 g l-1
recorded the maximum amount of fruit yield i.e. 37.11 ton ha-1
.
Amuamuha et al. (2012) evaluated the effect of iron on the flower and
essential oil yield of Pot Marigold (Calendula officinalis) and observed highest
yield of flower at first harvest (405.37 kg ha-1
) from nano iron foliar application at
stem initialized stage, and the lowest yield of flower (261.64 kg ha-1
) after second
harvest.
Moghadam et al. (2012) investigated the effect of different concentrations
of iron chelate Nano fertilizer (2 and 4 ppt) on growth and performance on
Spinach. Results showed that wet weight, dry weight and maximum leaf area index
were influenced by concentration of iron chelate Nano fertilizer. Use of 4 kg ha-1
Nano fertilizer caused 58 and 47% increase in wet weight and maximum leaf area
index, respectively compared to control.
Mohamadipoor et al. (2013) reported that nano iron fertilizer and FeSO4
treatments produced similar response in ornamental plants spathyphyllum to most
6
of the characteristics. He observed that use of nano iron fertilizer is superior
because of lower cost. Peyvandi et al. (2011) reported the positive effect of
spraying basil plants with iron nanofertilizer. Fe nanoparticles increased root
length, stem length, and shoot dry weight compared with the common iron
fertilizers. Salarpour et al. (2013) investigated the effect of Nano-iron chelates on
growth, peroxidase enzyme activity and oil essence of Cress (Lepidium sativum L.)
and observed 80% increase in plant height.
2.1.2 Physiological and Biochemical effect
Bakhtiari et al. (2015) studied the effect of different concentration of Fe
Nano-oxide solution (0, 0.01%, 0.02%, 0.03% and 0.04%) on wheat and observed
highest protein content in 0.04% Fe concentration. Ghafari and Razmjoo (2013)
reported that 2 g l-1
nano iron oxide has increased chlorophyll content, antioxidant
enzyme activities, protein and carbohydrate content of wheat.
Delfani et al. (2014) studied the effect of Iron (Fe) and magnesium (Mg) in
nano and common forms as foliar application on black-eyed pea. Iron had
significant effect on yield, leaf Fe content, stem Mg content, plasma membrane
stability, and chlorophyll content. The greatest effect was obtained by two
treatment combinations of 0.5 g l−1
common Fe + 0.5% nano-Mg and 0.5 g l−1
common Fe + 0.5 g l−1
common Mg. In general, almost all analyzed traits were
improved by foliar application of these two elements, probably as a result of more
efficient photosynthesis.
Karimia et al. (2014) compared iron chelated fertilizer and nano-iron
chelated fertilizer in different concentrations on some physiological and
biochemical responses of mung bean (Vigna radiata). The effects of nano-iron
chelated fertilizer and iron chelated fertilizer were determined on photosynthetic
pigments, leaf protein content and activity of antioxidant enzymes in the leaves.
Nadi et al. (2013) studied that the effect of nano iron chelate fertilizer on grain
yield, protein percent and chlorophyll content of Faba bean (Vicia faba L.) and
observed that increasing Nano-Iron concentration (6 g l-1
) had a positive and
significant effect on grain yield. Ngo et al. (2014) reported that the chlorophyll
index increased by 7–15% in soybean seeds treated with zerovalent Cu, Co and Fe.
7
Alidoust,D. and Isoda, A. (2013) et al study that was performed to
investigate the effect of 6-nm IONPs and citrate-coated IONPs (IONPs-Cit) on
photosynthetic characteristics and root elongation during germination of Glycine
max (L) Merr. Plant physiological performance was assessed after foliar and soil
IONPs fertilization. No adverse impacts at any growth stage of the soybeans were
observed after application of IONPs. The Fe2O3 nanoparticles produced a
significant positive effect on root elongation, particularly when compared to the
bulk counterpart (IOBKs) suspensions of concentrations greater than 500 mg L-1.
Furthermore, IONPs-Cit significantly enhanced photosynthetic parameters when
sprayed foliarly at the eight-trifoliate leaf stage (P\0.05). The increases in
photosynthetic rates following spraying were attributed to increases in stomatal
opening rather than increased CO2 uptake activity at the chloroplast level. We
observed more pronounced positive effects of IONPs via foliar application than by
soil treatment. This study concluded that IONPs coated with citric acid at IONPs to
citrate molar ratio of 1:3 can markedly improve the effectiveness of insoluble iron
oxide for Fe foliar fertilization.
Hokmabadi et al. (2006) reported that the iron nano fertilizers increased the
ratio of ferrous iron to ferric iron in chelate surface which resulted in increased
synthesis of chlorophyll of chrysanthemum. Salarpour et al. (2013) reported that
nano iron fertilizer has a positive effect on peroxidase enzyme activity, chlorophyll
content and oil essence of cress (Lepidium sativum L.).
Siva, G.V. and John Benita, L.F. et al, (2016) studied the Iron is an element
essential for plant growth and development. Nanoparticles like iron oxide
nanoparticles are being investigated as plant supplements for its promising targeted
delivery approach. The experiment is designed in a hydroponic system where
along with the Hoagland solution 100ppm concentration of iron oxide
nanoparticles are added to evaluate whether it gives beneficial results when
compared to EDTA chelated iron. The experimental data showed ginger roots
absorbed iron oxide nanoparticles also showed an increase with respect to protein
levels and iron content of rhizome. Iron oxide nanoparticles are an effective
supplement for chlorosis.
8
2.2 Impact of Copper nanoparticle fertilizer on Plant
2.2.1 Morphological effect
Adhikari et al. (2012) studied the effect of Cu oxide-nano particles (< 50
nm) on germination and growth of seeds of soybean and chickpea. In both the
crops, germination was not inhibited up to 2,000 ppm Cu (applied through CuO
NP), but the root growth was prevented above 500 ppm Cu. With increasing
concentration of NPs, the elongation of the roots was severely inhibited as
compared to that in control. In many cases root necrosis was occurred. Massive
adsorption of Cu oxide-nano particles into the root system was responsible for the
toxicity. A parallel experiment was also carried out to know the effect of copper
sulphate solution on seed germination, above 200 ppm Cu, it restricted the
germination of seeds, because of high salinity.
Hafeez et al. (2015) observed that Cu-NPs (10, 20, 30, 40 and 50 ppm)
significantly increased growth and yield of wheat as compared with control.
Among the graded concentration, 30 ppm Cu-NPs produced significantly higher
leaf area, number of spikes/pot, number of grains/spike, 100 grain weight and grain
yield. Hashemabadi et al. (2013) reported that 5 mg l-1
of copper nanoparticles
increased the vase life of cut flowers of chrysanthemum compared to control. Shah
and Belozerova (2009) reported that the seed germination in the presence of Cu
NPs showed an increase in shoot to root ratio compared to control lettuce plants.
Shobha (2014) reported that copper nanoparticles, which have shown positive and
negative impact on the micro-organism and the plants.
F. Yasmeen et al, (2015) reported that a reduction in germination
percentage on exposure to silver and copper nanoparticles while maximum
germination percentage was on application of iron nanoparticles. Similarly, while
root and shoot growth was also enhanced under iron nanoparticles application
while severereduction in root and shoot length was observed on exposure to copper
nanoparticles. So copper has inhibitory while iron has stimulatory effect on wheat
germination and growth.
2.2.2 Physiological and Biochemical effect
Hafeez et al. (2015) observed that 30 ppm Cu-NPs produced significantly
higher chlorophyll content in wheat. Nekrasova et al. (2011) reported the effect of
9
copper ions and copper oxide nanoparticle on lipid peroxidation rate, anti-oxidant
enzyme activities (superoxide dismutase, catalase and peroxidise) and
photosynthesis of Elodea densa planch. The results showed nanoparticles that are
accumulated by plants activated lipid peroxidation rate from 120 to 180% of the
control level by copper ions and nanoparticles, respectively. Catalase and
Superoxide dismutase activity increased by a factor of 1.5 to 2.0 when plants were
treated with NPs. Copper ions suppresses photosynthesis at a concentration of 0.5
mg l-1
, whereas nanoparticles produce such an effect only at 1.0 mg l-1
.
2.3 Impact of Zinc nanoparticle fertilizer on plant
2.3.1 Morphological effect
Asadzade et al. (2015) investigated the effect of foliar application of
conventional and nano-fertilizers (ZnO and SiO2) on yield, morphological and
physiological traits and harvest index of sunflowers. ZnO nano-fertilizer was found
to increase head diameter, seed yield, harvest index for seed in plant and seed in
head.
Avinash et al. (2010) observed increases in germination and growth rate in
the seeds of Cicer arietinum treated with nano-ZnO. Burman et al. (2013)
compared the effect of 1.5 and 10 ppm foliar spray of zinc oxide (ZnO)
nanoparticles on ten days old seedlings of chickpea (Cicer arietinum L var. HC-1)
with corresponding concentration of zinc sulphate and ZnO of normal size.
Maximum response with respect to shoot dry weight was observed in seedlings
treated with 1.5 ppm ZnO nanoparticles while at 10 ppm the nanoparticles exerted
adverse effects on root growth. However, overall biomass accumulation improved
in the ZnO nanoparticle treated seedlings. Mahajan et al. (2011) reported the
effects of ZnO nanoparticles on the growth of mung bean (Vigna radiata)
seedlings, the seeds of which were previously allowed to germinate in wet cotton
for 24 h in the dark, then the sprouted seeds were taken for further study of the
seedlings which grew in culture media containing nanoparticles. Maximum
seedling growth was observed with nano-ZnO concentration of 20 ppm and beyond
which the seedling growth was inhibited, which might be attributed to the toxic
level of nanoparticles.
10
Boonyanitipong et al. (2011) investigated the effect of zinc oxide
nanoparticles (nano-ZnO) on rice (Oryza sativa L.) root and found that there is no
reduction in the percent seed germination, however nano-ZnO is observed to have
detrimental effects on rice roots at early seedling stage. Nano-ZnO is found to stunt
root length and reduce number of roots. Ramesh et al. (2014) studied the positive
effects of bulk and nano-Titanium dioxide (TiO2) and Zinc oxide (ZnO) @ low
concentration on seed germination, shoot -root growth in Triticum aestivum
(Wheat).
Gokak and Taranath (2015) observed similar response in Zn and nano Zn
treated plants for seed germination and root – shoot elongation of Macrotyloma
uniflorum (Lam.) Verdc.
Laware et al. (2014) studied the effect of graded concentrations of zinc
oxide nanoparticles (ZnO NPs) along with sticker on onion crop and seed samples
obtained from NP treated plants along with control were tested for germination and
early seedling growth. The plants treated with ZnO NPs at the concentration of 20
and 30μg ml-1
showed better growth and flowered 12-14 days earlier than the
control. Treated plants also showed significantly higher values for seeded fruit per
umbel, seed weight per umbel and 1000 seed weight over control plants. These
result indicated that ZnO NPs can reduce flowering period by 12-14 days and even
produce healthy seeds. Rosa et al. (2013) reported that some nanoparticles (NPs)
affect seed germination; however, the biotransformation of metal NPs is still not
well understood. They investigated the NP toxicity on seed germination/root
elongation and the uptake of ZnO NPs and Zn2+
in alfalfa (Medicago sativa),
cucumber (Cucumis sativus), and tomato (Solanum lycopersicum) seedlings. Seeds
were treated with ZnO NPs at 0–1600 mg l–1
as well as 0–250 mg l–1
Zn2+
for
comparison purposes. Results showed that at 1600 mg l–1
ZnO NPs, germination in
cucumber increased by 10% and in alfalfa and tomato germination were reduced
by 40 and 20%, respectively. At 250 mg Zn2+
l–1
, only tomato germination was
reduced with respect to controls. The highest Zn content was of 4700 and 3500 mg
kg–1
dry weight for alfalfa seedlings germinated in 1600 mg l–1
ZnO NPs and 250
mg l–1
Zn2+
, respectively.
11
Singh et al. (2013) studied the effects of bulk and ZnO NPs on germination,
growth and biochemical parameters of cabbage, cauliflower and tomato vegetable
crops and observed that nano-zinc oxide particle enhanced germination and
seedling growth in all the three test crops.
Jayarambabu, N. Siva Kumari, B. Venkateswara Rao, K. and Prabhu, Y.T.
et al (2014) reported that ZnO nanoparticles application in different fields like seed
germination, sensors, biomedical, semiconductor etc. In present study, the different
concentration (0,20,40,60 and 100mg) of ZnO NPs are prepared in distilled water
and sonicator for 15 minutes are used for the treatment in Mungbean (Vigna
radiata L.) seeds to study the effect on bioavailability of seed germination and
observed early seedling growth and growth characteristics of Mungbean. The
experiment was carried out under greenhouse conditions.
Prasad et al. (2012) carried out an investigation to examine the effects of
nanoscale zinc oxide particles on plant growth and development on peanut. Peanut
seeds were separately treated with different concentrations of nanoscale zinc oxide
(ZnO) and chelated bulk zinc sulfate (ZnSO4) suspensions (a common zinc
supplement), respectively and the effect this treatment had on seed germination,
seedling vigor, plant growth, flowering, chlorophyll content, pod yield and root
growth were studied. Treatment of nanoscale ZnO (25 nm mean particle size) at
1000 ppm concentration promoted both seed germination and seedling vigor and in
turn showed early establishment in soil manifested by early flowering and higher
leaf chlorophyll content. These particles proved effective in increasing stem and
root growth. Pod yield per plant was 34% higher compared to chelated bulk
ZnSO4.
Sedghi et al. (2013) studied that the effect of nano zinc oxide on
germination parameters of soybeans seeds under drought stress conditions applied
by poly ethylene glycol (PEG). Results showed that the effect of different
concentrations of PEG and nano zinc oxide on germination rate and germination
percentage, root length, root fresh and dry weight, seed residual fresh and dry
weight were significant. Nano zinc oxide increased germination percentage and
rate over control.
12
Sunita et al. (2013) studied that the effects of zinc oxide (ZnO) engineered
nanoparticles (ENPs) on plant growth, and bioaccumulation in Brassica juncea.
The seed was germinated under hydroponic condition with a varying concentration
of ZnO ENPs (0, 200, 500, 1000, 1500 mg l-1
) for 96 h and significant decrease in
plant biomass was recorded.
2.3.2 Physiological and Biochemical effect
Burman et al. (2013) reported that foliar spray (1.5 and 10 ppm) of zinc
oxide (ZnO) nanoparticles has improved overall biomass accumulation in ten days
old seedlings of chickpea (Cicer arietinum L var. HC-1) in comparison to zinc
sulphate and ZnO of normal size. This response may be attributed to low reactive
oxygen species (ROS) levels which resulted in less lipid peroxidation as evident
from lower malondialdehyde (MDA) content. This was also associated with lower
activity of prominent antioxidant enzymes, superoxide dismutase (SOD), and
peroxidase in ZnO nanoparticle treated seedling when compared to control. The
study indicated importance in precise application of zinc, more so in deficient
system, where plant response varies with concentration and is important in
understanding the mechanism of action of specific nanomaterials.
Prasad et al. (2012) reported that seeds when treated with nanoscale ZnO
(25 nm mean particle size) at 1000 ppm concentration promoted chlorophyll
content of Peanut. Raliya and Tarafdar (2013) studied the effect of biologically
transformed ZnO nanoparticles on cluster bean (Cyamopsis tetragonoloba L.) to
enhance native phosphorous mobilizing enzymes and nano induced gum
production. ZnO nanoparticles were foliar sprayed at 10 ppm concentration on leaf
of 14-day-old cluster bean plants. A significant improvement in plant biomass
(27.1%), shoot length (31.5%), root length (66.3%), root area (73.5%), chlorophyll
content (276.2%), total soluble leaf protein (27.1%), rhizospheric microbial
population (11–14%), acid phosphatase (73.5%), alkaline phosphatase (48.7%),
and phytase (72.4%) activity in cluster bean rhizosphere was observed over control
in 6-week-old plants due to application of nano- ZnO. The gum content in cluster
bean seeds also improved by 7.5% after maturity which indicates that ZnO in nano
13
form may contribute more in industrial and medical applications besides
agricultural sector.
Ramesh et al. (2014) reported significant increases of chlorophyll and
protein content with low concentration nano-ZnO treated sample in wheat and no
changes were observed in bulk-ZnO and bulk and nano-TiO2 treated samples.
Singh et al. (2013) studied the effect of bulk and nano dimensional zinc
oxide particles (ZnO NPs) on germination, growth and biochemical parameters of
cabbage, cauliflower and tomato vegetable crops. They observed that bulk ZnO
particles was phytotoxic and adversely affected biochemical parameters of all the
test crops. On the contrary, nano-dimensional zinc oxide particle enhanced
germination, seedling growth, pigments, sugar and protein contents along with
increased activities of antioxidant enzymes in all the three test crops. Zinc oxide
NPs invariably increase pigments, protein and sugar contents and nitrate reductase
activities in cabbage (Brassica oleracea var. Capitata). It was observed in
cauliflower that higher concentration of ZnO NPs (9.0 µM) maintained the sugar
content at the level of control, while SOD and CAT to the level of bulk ZnO
treated seedlings. ZnO NPs induced activities of antioxidant enzymes viz. SOD,
CAT, APX and POD, however, were lower as compared to those treated with bulk
ZnO.
Sunita et al. (2013) studied that the effect of zinc oxide (ZnO) engineered
nanoparticles (ENPs) on antioxidative enzyme activity in Brassica juncea. The
seed was germinated under hydroponic culture with a varying concentration of
ZnO ENPs (0, 200, 500, 1000, 1500 mg l-1
) for 96 h. Increase in proline content
and lipid peroxidation upto a concentration of 1000 mg l-1
was observed. They
observed that ENPs caused a significant effect due to their accumulation along
with the generation of reactive oxygen species in plant tissues, thus signifying its
hazardous effect on B. juncea.
2.4 Impact of other metallic nanoparticle fertilizer on Plant
2.4.1 Morphological effect
Bao-shan et al. (2004) studied the effect of exogenous application of nano-
SiO2 on Changbai larch (Larix olgensis) seedlings and found that nano-SiO2
14
improved seedling growth and quality, including mean height, root collar diameter,
main root length, and the number of lateral roots of seedlings and also induced the
synthesis of chlorophyll. Under abiotic stress, nano-SiO2 augments seed
germination
Haghighi et al. (2012) in tomato and Siddiqui et al. (2014) in squash
reported that nano-SiO2 enhanced seed germination and stimulated the antioxidant
system under NaCl stress. Siddiqui and Al-Whaibi (2014) reported that the lower
concentrations of nano-SiO2 improved seed germination of tomato.
Liu et al. (2009) showed that 5 mg l-1
silver nano-particles reduced
bacterial colonies and extended the vase life of cut gerbera cv. ‗Ruikou‘. Lu et al.
(2002) studied the effect of nano-SiO2 and nano-TiO2 mixtures on soybean seeds
and found that the mixture increased the nitrate reductase in soybeans, increasing
their germination and growth.
Mazumdar (2014) reported that significant inhibition on shoot fresh weight
of V. radiata (p=0.008) and B. campestris (p=0.002) was observed at 1000 μg ml-1
silver nanoparticle solution after treatment period. V. radiata showed significant
retardation on dry weight of root at 1000 μg ml-1
of Ag+ ions solution after 12
th
day. The decrease on shoot dry weight with increase in nanoparticle and ion
concentration was also observed after 12th
day.
Najafi et al. (2014) studied the phytotoxic effects of Pb as Pb(NO3)2 and
silver nanoparticles on mung bean (Vigna radiata) planted on contaminated soil
was assessed in terms of growth, yield, at 120 ppm concentration. Yugandhar and
Savithramma (2013) observed the effect of calcium carbonate nanoparticles on
seed germination and seedling growth of Vigna mungo and showed that the bio-
synthesized calcium carbonate nanoparticles accelerate the seed germination and
seedling growth in V. mungo. Highest percentages of seed germination, seedling
vigor index, root and shoot length, fresh and dry weight and relative water content
was also observed in NP treated plants.
Savithramma et al. (2012) reported that biologically synthesized Silver NPs
improved the seed germination and seedling growth of Boswellia ovalifoliolata an
endemic, endangered and globally threatened medicinal tree species.
15
Suriyaprabha et al. (2012) studied plant responses to nano and bulk silica
treatments in terms of growth characteristics in maize crop. Growth characteristics
were much influenced with increasing concentration of Silica nanoparticles up to
15 kg ha-1
whereas at 20 kg ha-1
, no significant increments were noticed. Silica
accumulation in leaves was high at 10 and 15 kg ha-1
(0.57 and 0.82%)
concentrations of SNPs. Suriyaprabha et al. (2012) reported that nano-SiO2
increased seed germination by providing better nutrients availability to maize
seeds, and pH and conductivity to the growing medium.
Y.Farhat. et al. (2015) reported that a reduction in germination percentage
on exposure to silver and copper nanoparticles while maximum germination
percentage was on application of iron nanoparticles. Similarly, while root and
shoot growth was also enhanced under iron nanoparticles application while severe
reduction in root and shoot length was observed on exposure to copper
nanoparticles. So copper has inhibitory while iron has stimulatory effect on wheat
germination and growth.
Zheng et al. (2005) studied the effects of nano-TiO2 and non-nano-TiO2 on
the germination and growth of Spinacia oleracea by measuring the germination
rate and vigor indexes. An increase of these indexes was observed at 0.25-4%
nano-TiO2 treatments.
2.4.2 Physiological and Biochemical effect
Adhikari et al. (2013) observed good germination of seeds in the presence
of SiO2 nano particles which had showed no toxic effect on rice growth, whereas
root growth and elongation were arrested with Mo nano particles after 50 mg l-1
. In
many cases root necrosis was occurred. Massive adsorption of Mo nano particles
into the root system was responsible for the toxicity, which calls for more research
for recommending their safe use as biolabels in plants. The uptake of both the nano
particles was observed with rice seedlings. Application of silica nano particles
enhanced the root length, root volume and dry matter weight of shoot and root of
rice crop.
Agrawal and Rathore (2014) reported the positive morphological effects of
nano materials which include enhanced germination percentage and rate; length of
root and shoot, and their ratio; and vegetative biomass of seedlings along with
16
enhancement of physiological parameters like enhanced photosynthetic activity
and nitrogen metabolism in many crop plants. Amirnia et al. (2014) reported that
nanofertilizers improved saffron yield. In addition, it was also observed that Fe, P
and K nanofertilizers had positive effects on the saffron flowering.
Ashrafi et al. (2013) evaluated the effect of nanosilver application and
weed density in an integrated fertilization system on agronomic traits of soybean.
They found that coapplication of organic and nanosilver increased leaf chlorophyll
significantly. The highest grain P and K concentration was obtained from
nanosilver treated plants. Coapplication of compost, farmyard manure and
chemical fertilizer produced a higher amount of pods per plant, seed number per
pod and 100-seed mass. Nanosilver treated plants produced the highest grain yield.
Aslani et al. (2014) studied the influence of engineered nanomaterials
(carbon and metal/metal oxides based) on plant growth and observed that
engineered nanomaterials influences seed germination. It was found to affect the
shoot-to-root ratio and the growth of the seedlings. Hong et al. (2005) reported that
Nanoscale titanium dioxide (TiO2) promoted photosynthesis, and growth of
spinach.
Karthick and Chitrakala (2011) observed that chlorophyll a content was
significantly increased by Ag nanoparticles in green gram and sorghum. Najafi et
al. (2014) studied the Phytotoxic effects of Pb as Pb(NO3)2 and silver nanoparticles
on Mung bean (Vigna radiata) planted on contaminated soil in terms of
chlorophyll pigments, phenol and flavonoid content at 120 ppm concentration.
Mazumdar (2014) reported that exposure to 1000 μg ml-1
of Ag nanoparticles led
to significant retardation of total chlorophyll content in V. radiata (p=0.001) and B.
campestris (p=0.001) when compare to control after 12th
day of treatment. After
the treatment period no significant inhibition on chlorophyll ratio was observed
when exposed to both Ag nanoparticle and ion solutions.
Morteza et al. (2013) reported the effects of titanium dioxide spray on corn
(Zea mays L.). Results showed that effect of nano TiO2 was significant on
chlorophyll content (a and b), total chlorophyll (a + b), chlorophyll a/b, carotenoids
and anthocyanins. The maximum amount of pigment was recorded from the
treatment of nano TiO2 spray at the reproductive stage in comparison with control.
17
Suriyaprabha et al. (2012) studied the observed physiological changes including
expression of organic compounds such as proteins, chlorophyll, and phenols was
favorable to maize treated with nanosilica especially at 15 kg ha-1
compared with
bulk silica and control. Nanoscale silica regimes at 15 kg ha-1
have a positive
response on maize than bulk silica which helps to improve the sustainable farming
of maize crop as an alternative source of silica fertilizer. Qiang et al. (2008)
reported that compared to NPK chemical fertilizer, the application of
slow/controlled release fertilizer coated and felted by nanomaterials improved
grain yield with an insignificant increase in protein content and a decrease in
soluble sugar content in wheat. Rico et al. (2013) reported that rice grains from
nCeO2-treated plants had less Fe, S, prolamin, glutelin, lauric and valeric acids,
and starch. Moreover, the nCeO2 reduced all antioxidant values, except flavonoids
in grain. Medium- and low-amylose varieties accumulated more Ce in grains than
the high-amylose variety, but the grain quality of the medium-amylose variety
showed higher sensitivity to the nCeO2 treatment. These results indicated that
nCeO2 could compromise the quality of rice.
Zhu et al. (2008) reported that Cucurbita maxima grown in an aqueous
medium containing magnetic nanoparticles can absorb, move and accumulate the
particles in the plant tissues, whereas Phaseolus limensis is not able to absorb and
move particles. It indicated that different plants have different response to the same
nanoparticles.
18
CHAPTER - III
MATERIALS AND METHODS
A Laboratory experiment was conducted during kharif 2015 and Rabi 2015-
16 to study the physiological responses of soybean and wheat towards nano
particle based micronutrients fertilization. The details of materials used and the
experimental technique adopted during the course of investigation are described
below.
3.1 Experimental site
The laboratory experiment was conducted at Indian Institute of Soil
Science Bhopal at 230
18 N, 77024 E, with an altitude of 485 meter above the mean
sea level.
3.2 Sand culture system setup
Sand culture system was setup in china clay pots of 5 kg capacity having
size of 30 cm height and 14 cm diameter. Acid washed sand was washed with de-
ionized water continuously till it is devoid of macro and micro nutrients. Hoagland
nutrient solution was applied weekly to the sand culture system. The solution
composition is given in appendix B.
Description of crop: Soybean and Wheat
Soybean genotype – JS 355
Wheat genotype – HD 8729
3.3 Climate
The main research station of ICAR-Indian Institute of Soil Science Bhopal
is situated in Central Highlands (zone -10) of M.P state. The meteorological data
during crop season as recorded at the meteorological observatory, ICAR-Central
Institute of Agricultural Engineering, Bhopal situated near the research farm of
IISS Bhopal (Fig. 3.3 and appendix A).
19
Fig. 3.1 A view of experimental set up of soybean in sand culture system
Fig. 3.2 A view of experimental set up of wheat in sand culture system
20
Fig
. 3.3
Met
eoro
logic
al d
ata
duri
ng c
rop g
row
th p
erio
d
05
10
15
20
25
30
35
40
Tem
p. M
ax
Tem
p. M
in
0C
Tem
pra
ture
(0C
)
21
3.4 Experimental details
3.4.1 Design and layout
The experiment was laid out in completely randomized block design with
ten treatments and five replications. In each replication five plants were maintained
for growth and morphological analysis. The treatment details are Table 3.1.
Table: 3.1 Treatment details
(% given in parenthesis is either 100 or 50 % of corresponding nutrient in Hoagland solution)
3.4.2 Collection of experimental data
One plant was randomly selected from each container and was tagged for
recording various morphological observations at different stages.
3.5 Morphological parameters
3.5.1 Plant height
Plant height was recorded from base of the plant to the uppermost node of
main shoot of plant at 45 days after sowing (DAS) and 60 DAS and height was
expressed in cm.
3.5.2 Root length
Total root length of the five randomly selected plants was measured at 45
and 60 DAS in centimeters with the help of Graph paper ruled in millimeters scale.
Roots were placed on a shallow glass dish and graph paper was placed under the
dish. The roots were cut from the root-shoot joint and were positioned randomly
over the graph paper lines (representing a grid) with the help of forceps and needle
to avoid overlapping. The long branched roots were cut into smaller pieces
(Newman, 1966). The counts for intersection of roots (N) with vertical and
horizontal lines of 1 cm grid from the graph paper were recorded root length was
Treatment in sand culture system Elemental concentration of Fe, Cu and Zn
(salt / NP)
T1: 100% (Fe + Cu + Zn) =Normal salts 54 µM Fe + 0.5 µM Cu + 2 µM Zn =
Normal salts
T2: T1- Fe salt+ Fe NP (100%) 54 µM Fe NP + 0.5 µM Cu + 2 µM Zn
T3: T1- Fe salt + Fe NP (50%) 27 µM Fe NP + 0.5 µM Cu + 2 µM Zn
T4: 100% (Cu + Zn) salts + Fe salt (50%) 27 µM Fe salt + 0.5 µM Cu + 2 µM Zn
T5: T1- Cu salt + Cu NP (100%) 54 µM Fe + 0.5 µM Cu NP + 2 µM Zn
T6: T1- Cu salt + Cu NP (50%) 54 µM Fe + 0.25 µM Cu NP + 2 µM Zn
T7: 100% (Fe + Zn) salts + Cu salt (50%) 54 µM Fe + 0.25 µM Cu salt + 2 µM Zn
T8: T1- Zn salt + Zn NP (100%) 54 µM Fe + 0.5 µM Cu + 2 µM Zn NP
T9: T1- Zn salt + Zn NP (50%) 54 µM Fe + 0.5 µM Cu + 1 µM Zn NP
T10: 100% (Fe + Cu) salts + Zn salt (50%) 54 µM Fe + 0.5 µM Cu + 1µM Zn salt
22
computed using the modified version of Newman (1966) formula proposed by
Tennant (1975) as:
Root Length= 11/14* number of intersections (N) *grid unit.
3.5.3 Plant Biomass
The selected plant was removed from the plastic container. The whole plant
was divided into leaves, stem and roots and then weight of leaves and stem was
measured for fresh weight. The samples were dried in oven for 72 hrs at 65°C and
total dry weight (expressed in gram) of leaves and stem was recorded for dry
weight.
3.5.4 Root Biomass
The whole root was weighed immediately for fresh weight and dried in
oven for 72 hrs at 65°C and total dry weight (expressed in gram) of root was
recorded.
3.5.5 Root Volume
Root volume was determined by volume displacement method (Mishra and
Ahmed, 1987) using a measuring cylinder.
3.6 Growth parameters
3.6.1 Leaf area
Leaf area was measured by leaf area meter of LICOR make (Model 3100)
and expressed in cm2.
3.6.2 Leaf area ratio (LAR)
The leaf area ratio was worked out by the formula of Radford (1967) and
expressed as cm² g-1
.
Leaf area (cm2plant-
1)
LAR =
Total dry matter (g plant-1
)
3.6.3 Specific leaf weight (SLW)
The specific leaf weight (g cm-2
) indicates the leaf thickness and was
determined by the following formula.
Leaf dry weight (g)
SLW =
Leaf area (cm2)
23
3.6.4 Specific leaf area (SLA)
The inverse of specific leaf weight is the specific leaf area (cm² g-1
) and
was calculated as follows.
Leaf area (cm²)
SLA =
Leaf dry weight (g)
3.6.5 Root shoot ratio
The materials was put in paper bags and then put in an oven at 80oC for 24
hours. The samples were weighing by electronic balance (Sartorius Basic,
BA2105) and the average data on dry weights of root, leaf, and stem per plant was
worked out. Shoot dry weight per plant was obtained by adding leaf dry weight
with stem dry weight per plant (Amanullah et al., 2010; Amanullah & Shah, 2011).
The sum of the shoot and root dry weight was calculated as the total dry weight per
plant. Shoot to root ratio (S:R) at each growth stage was calculated using the
following formula:
Root dry weight (g plant-1
)
Root Shoot Ratio (S:R) =
Shoot dry weight (g plant-1
)
3.7 Physiological and biochemical parameters
3.7.1 Chlorophyll content estimation
The chlorophyll content was estimated at 30 and 60 DAP. Total
chlorophyll, chlorophyll a and chlorophyll b contents were determined by
following the method of Hiscox and Israelstom (1979). 500 mg of fresh leaf tissues
were cut into small pieces and incubated in 5.0 ml of DMSO (Dimethyl Sulfoxide)
at 50°C for 2.30 hours. At the end of incubation period the supernatant was
decanted and leaf tissues discarded. The absorbance was read at 645 and 663 nm in
UV-vis spectrophotometer (ELICO, 159). Total chlorophyll, chlorophyll a and
chlorophyll b content were calculated using the formula given by Arnon (1949)
and expressed in mg per gram fresh weight.
V
Chlorophyll 'a' = 12.7 (A663) - 2.69 (A645) X
1000 X W
24
V
Chlorophyll 'b' = 22.9 (A645) - 4.68 (A663) X
1000 X W
V
Total chlorophyll = 20.2 (A645) + 8.02 (A663) X
1000 X W
Where,
A = Absorbance at specific wave length (645, 663 nm)
V = Final volume of the chlorophyll extract (ml)
W = Fresh weight of the sample (g)
3.7.2 Estimation of relative water content
Relative water content is an important parameter to measure the water
content in plant tissues. It has taken two times during crop growth period in a fully
expanded upper most leaf and cut into small pieces, recorded their fresh weight.
After measuring the fresh weight, leaf pieces were incubated in fresh water for 3
hrs to allow them to gain full turgidity at room temperature. Removed water from
leaf segment and blotted with tissue paper to remove adhered water on leaf surface
and thereafter their turgid weight is recorded. Leaf pieces are dried to constant
weight at 600C in hot air oven and dry weight was calculated (Weatherly and
Slatyer, 1957).
Fresh weight – Dry weight
RWC (%) = X 100
Turgid weight – Dry weight
3.7.3 Estimation of membrane stability
Leaf tissue was cut, added 10 ml distilled water then placed for incubation
at 100C temperature for 24 hours. After incubation the sample was equilibrated for
1 hour at room temperature and the conductivity of medium was measured by
conductivity electrode. All samples were covered with wrap and autoclaved for 15
min 1210C. There after the sample was equilibrated for 1 hour at room temperature
and the conductivity of medium was measured by conductivity electrode.
(1-(T1/T2)
MS % = X 100
(1-C1/C2)
25
T1 - EC after 10oC temperature of sample
T2- EC after 121oC temperature of sample
C1 - EC after 10oC temperature of blank sample
C2- EC after 121oC temperature of blank sample
3.7.4 Proline content estimation
Extraction
Proline content was measured following the method of Bates et al. (1973).
0.5 gram of fresh plant sample (leaves) was taken and 10ml of 3% aqueous
sulphosalicylic acid added and ground in pestle and mortar, and then filtered
through What man No. 42 filter paper.
Assay
5 ml of aliquot from the colour filtrate was taken in a 30 ml test tube for
determination and then 2ml of glacial acetic acid and 2 ml acid ninhydrine mixture
solution (1.25g ninhydrine in 30 ml of acetic Acid +20 ml of 6M phosphoric acid)
was added into each tube. The reaction mixture was kept in boiling water for 1
hour to develop pink colour. Reaction was terminated by keeping in ice. Added 4
ml toluene and mixed thoroughly and aspirated toluene layer. The colour intensity
was measured by spectrophotometer at 520 nm wavelength after setting the
instruments to zero with blank. To determine the proline content, a standard curve
was made using pure proline. The content of proline was expressed in units of
μmol per gram fresh weight (μM g-1
FW).
3.7.5 Assay of antioxidant enzyme
Enzyme extraction
For enzyme assay leaf sample were collected from plant and kept in ice
box. 0.5gram of fresh leaf was homogenized in 3 ml of extraction buffer of 0.1 M
phosphate buffer (7.8) containing 0.5 mM EDTA in pre chilled mortar and pestle.
The homogenized tissue was centrifuged at 13,000g for 10 min at 4°C and the
collected supernatant was used for enzyme assay. Total process was carried at 4°C.
3.7.5.1 Super oxide dismutase (SOD) activity assay
SOD activity was assayed by following the method of Dhindsa et al.
(1981). Added 0.3 ml supernatant to reaction mixture containing 1.3μM
Riboflavin 63μM of NBT and 200mM of methionine. Tubes were covered with
26
aluminum foil to prevent light. Prepared blank without enzyme supernatant as
control. Exposed tubes to light in light box for 3 min. The colour intensity was
measured by spectrophotometer at 560 nm wavelength and SOD activity, in unit g-
1, was expressed as fold increase over Normal salt treated plants.
3.7.5.2 Catalase (CAT) activity assay
CAT activity was assayed by following the method given by Barber (1980).
The extract from SOD Assay was used for CAT assay. Added 1.5 ml phosphate
buffer, 1 ml H2O2 (0.005M) and 0.5 ml enzyme. Incubated at 200C for 1 min and
stopped reaction by 5ml 0.7 N H2SO4.Titrated reaction mixture against 0.01N
Kmo4 until a faint /light purple colour persists for at least 15 second. Prepared
blank by adding enzyme extract reaction mix without incubation and CAT was
expressed in unit H2O2 min-1
g-1
.
3.7.5.3 Peroxidase (POX) activity assay
POX activity was assayed by following the method of Summer and
Gjessing, (1943). The extract from SOD Assay was used for POX assay. Added
1ml O-dianisidine (0.01M in methanol), 0.5 ml H2O2 (0.02M), 1 ml phosphate
buffer, 2.4 ml distilled water and 0.2 ml enzyme and incubated at 300
C for 5 min.
The reaction was stopped by adding 1ml 2N H2SO4. Blank tube excluding H2O2
was prepared by adding 0.5 ml distilled water. The colour intensity was measured
by spectrophotometer at 430 nm wavelength and POX was expressed in unit H2O2
min-1
g-1
.
3.7.6 Estimation of total soluble protein
Extraction assay
For Protein assays leaf sample were collected and 0.5gram of fresh leaf was
homogenized in 3 ml extraction buffer of 0.1 M phosphate buffer (7.8) containing
0.5 mM EDTA in pre chilled mortar and pestle. The homogenized tissue was
centrifuged at 13,000g for 10 min at 40C and the collected supernatant was used for
enzyme assay. The extraction process was carried at 40C.
Total soluble protein assay
Total soluble protein was measured by following method given by
Bradford (1976). Used the extract for assay and added 0.2 ml sample, 4 ml 0.1%
protein reagent (100 mg Coomassie Brilliant Blue) and 0.8 ml distilled water. The
27
colour intensity was measured by spectrophotometer at 595nm wavelength.
Calculation of the total soluble protein content was done by creating a standard
curve using a standard bovine serum albumin (25 mg in 0.15 M NaCl and made up
to volume 25 ml stock and working stock was made by diluting 10 times) and was
expressed in mg per gram fresh weight (mg g-1
FW).
3.7.7 Estimation of total soluble sugar
Extraction
Total soluble sugar content was measured based on the Anthrone method
(Hodge and Hofreite, 1962 and Sadasivam and Manickam, 1992). 0.1 gram of
oven dry plant sample was taken, added 5 ml of 2.5 N HCl, hydrolyzed by keeping
in boiling water bath for 3 hours then cooled to room temperature. The reaction
mixture was neutralized by solid sodium carbonate and then volume made up to 50
ml. The mixture was centrifuged and collected the supernatant.
Assay
Taken supernatant 0.5 ml aliquots then added 4 ml of anthrone reagent
(dissolve 200 mg anthrone in 100 ml of ice cold 95% H2SO4). Heated for 8 min. in
boiling water bath, cooled rapidly and read the green to dark green colour. The
colour intensity was measured by spectrophotometer at 630nm wavelength.
Calculation of the total soluble sugar content was done by creating a standard
curve using a standard glucose (dissolve 100mg glucose in 100 ml in water and
working standard of 10 ml stock and made up to 100 ml) and was expressed as
percentage of dry weight.
3.7.8 Estimation of starch
Extraction
Starch was measured based on the Anthrone method (Hodge and Hofreite,
1962, Thayumanavan and Sadasivam, 1984 and Sadasivam and Manickam.1992).
0.1 Gram of oven dry plant sample was taken added 10 ml of 80% ethanol and then
centrifuged to remove sugar. Residue was dried over water both. Added 5 ml water
and 6.5 ml 52% perchloric acid to residue. Extract was centrifuged at 00C for 20
min. and collected supernatant and volume was made up to 50 ml.
28
Assay
Taken supernatant of 0.5 ml aliquots and then added 4 ml of anthrone
reagent (200 mg anthrone dissolved in 100 ml of ice cold 95% H2SO4). The
reaction mixture was kept for 8 min. in boiling water both, cooled rapidly and read
the green to dark green colour. The colour intensity was measured by
spectrophotometer at 630nm wavelength. Calculation of the starch was done by
creating a standard curve using a standard glucose as mentioned in TSS assay and
was expressed as percentage of dry weight.
3.7.9 Estimation of non-structural carbohydrate
Non-structural carbohydrate was calculated as sum of total soluble sugar
and starch and expressed as percentage of dry weight.
3.7.10 Gas exchange parameters
Gas exchange parameters viz. Photosynthesis rate (µM m-2
s-1
),
transpiration rate (mM m-2
s-1
) and stomatal conductance (µM m-2
s-1
) were
recorded in the morning (8 to 10 AM) in the experimental plant leaves using
Photosynthesis system (make: PP systems and model: CIRAS-2).
3.7.11 SPAD value
SPAD value was recorded in the evening (3 to 4 PM) in the experimental
plant leaves using SPAD-502Plus.
3.7.12 Grain yield
Grain yield was recorded from five plants and expressed in g plant-1
.
3.7.13 Toxicity/ Deficiency symptom analysis
Toxicity/ deficiency symptoms due to the application of micronutrients were
observed visually.
29
CHAPTER – IV
RESULTS AND DISCUSSION
4.1 Morphological parameters of Soybean
4.1.1 Plant height
Among all the treatments, highest plant height was found in plants treated
with Cu NP (0.5 µM) at both stage and lowest plant height was observed in plants
treated with Zn salt (1 µM) and Fe salt (27 µM) at 45 DAS and 60 DAS,
respectively (Table 4.1). Among Fe treatments, plant height was found higher in
plant treated with Normal salt and was followed by Fe NP (54 µM), Fe NP (27
µM) and Fe salt (27 µM) at 45 DAS. However at 60 DAS plants treated with Fe
NP (27 µM) were taller than Fe NP (54 µM) and Fe salt (27 µM). In the present
study there was not much increase in height of soybean plants treated with Fe NP
(27 µM) at 60 DAS. Fe nanoparticles were found to increase stem length of basil
plants (Peyvandi et al. 2011, Kumar 2015). Salarpour et al. (2013) observed 80%
increase in plant height of Lepidum sativum L treated with nano-iron chelates.
Among Cu Treated plants, higher plant height was obtained in Cu NP (0.5
µM) followed by Cu NP (0.25 µM), Normal salt and Cu salt (0.25µM) at 45DAS.
In second stage Cu NP (0.5 µM) followed by Normal salt, Cu NP (0.25 µM) and
Cu salt (0.25µM). The influence of Cu NPs on plant height is sparsely reported.
CuO NPs did not inhibit the seed germination upto 2000ppm but root growth was
inhibited at 500 ppm in soybean and chickpea (Adhikari et al. 2012). However,
improvement in growth of plants treated with Cu NPs were reported in wheat
(Hafeez et al. 2015), lettuce (Shah and Belozerova, 2009) and improvement in vase
life of chrysanthemum was also reported with Cu NPs (Hashemabadi et al. 2013).
In the present study it was observed that normal concentration of Cu NP (0.5 µM)
had positive influence on plant height of soybean while reduced concentration of
Cu NP (0.25 µM).
Among Zn treatments plants, higher plant height was noted in Normal salt
followed by Zn NP (1 µM), Zn NP (2 µM) and Zn salt at 45 DAS. But at 60 DAS,
30
maximum plant height was recorded in Zn NP (2µM) followed by Normal salt, Zn
NP (1 µM) and Zn salt (1µM). Foliar spray of 10 ppm ZnO nanoparticles on 14-
day-old cluster bean plants, significantly improved shoot length (Raliya and
Tarafdar 2013). Ramesh et al. (2014) also reported positive effects of nano-ZnO on
shoot-root growth of wheat. Nano-ZnO particle was found to enhance germination
and seedling growth of soybean (Sedghi et al. 2013) and vegetable crops like
cabbage, cauliflower and tomato (Singh et al. 2013). This was in conformity with
the present findings wherein Zn NP (2 µM) improved plant height of soybean
plants at 60 DAS. This shows that normal concentration of Zn NPs can positively
influence height of plants.
4.1.2 Total Root Length
Total root length was recorded in different stages at 45 DAS and 60 DAS
(Table 4.1). At 45 DAS, among Fe treatments, plant show higher root length in
plant treated with Fe NP (27 µM) followed by Fe NP (54µM), Fe salt (27µM) and
Normal salt. But at 60 DAS, higher root length was observed in plants treated with
Fe salt (27 µM) as compared to Fe NP (54 µM), Fe NP (27µM) and normal salt. Fe
nanoparticles were found to increase root length of basil plants (Peyvandi et al.
2011, Kumar 2015). In the present study there increase in root length was observed
at 60 DAS with Fe NP (27 µM) treatment, which may be attributed to the increased
branching of roots of soybean crop.
Among Cu treatments, higher total root length was observed in plants
treated with Cu salt (0.25 µM) followed by Cu NP (0.25 µM), normal salt and Cu
NP (0.5 µM) at 45 DAS. At 60 DAS, higher root length was recorded in plants
treated with Cu NP (0.25 µM) followed by Cu NP (0.5 µM), normal salt and Cu
salt (0.25 µM). The influence of Cu NPs on plant height is sparsely reported. CuO
NPs did not inhibit the seed germination upto 2000ppm but root growth was
inhibited at 500 ppm in soybean and chickpea (Adhikari et al. 2012). In the present
study it was observed that normal concentration of Cu NP (0.5 µM) had positive
influence on shoot part of soybean while reduced concentration of Cu NP (0.25
µM) had positively influenced root length at 45 DAS. This indicated that reduced
concentration of NP might have acted as catalyst for root growth and normal
concentration of NP for shoot growth.
Among Zn treated plants, higher total root length was noted in plant
treated with Zn salt (1µM) at both stages followed by Zn NP (2 µM) and normal
31
salt and Zn NP (1 µM) at 45 DAS, in the second stage Zn NP (1 µM) treated plants
produced longer roots than Zn NP (2 µM) and normal salt. Among all the ten
treatments, highest root length was obtained in plant treated with Fe NP (27 µM) at
45 DAS and lowest Cu salt (0.25 µM) 60 DAS. Ramesh et al. (2014) also reported
positive effects of nano-ZnO on shoot-root growth of wheat. However,
Boonyanitipong et al. (2011) observed negative effect of nano-ZnO on root length
of rice (Oryza sativa L.). Nano-ZnO particle was found to enhance germination
and seedling growth of soybean (Sedghi et al. 2013) and vegetable crops like
cabbage, cauliflower and tomato (Singh et al. 2013). This is conformity for root
length enhanced by reduced concentration of Zn treatments at both growth stages.
Table 4.1: Effect of micronutrient NPs on plant height and root length of Soybean
4.1.3 Shoot dry weight
Shoot dry weight differed significantly at 45 DAS, 60 DAS (Table 4.2 and
Fig 4.2). Among all the treatments highest Shoot dry weight was recorded in plants
treated with Zn NP (2µM) both stage at 45 DAS and 60 DAS similar to Fe NP (54
µM) at harvest and lowest shoot dry weight was recorded in Cu salt (0.25 µM) at
both stages 45 DAS and 60 DAS similar to Zn salt (1µM) at harvest stage. Among
Fe treatments, higher shoot weight was observed in plant treated with Fe NP (27
µM) better than Fe NP (54 µM) normal salt and Fe salt (27 µM) at 45 DAS.
However, at 60 DAS and harvest, plants treated with Fe NP (54µM) showed
enhanced performance as compared to normal salt, Fe NP (27 µM) and Fe salt
(27µM). Fe nanoparticles were found to increase shoot dry weight of basil plants
(Peyvandi et al. 2011, Kumar 2015). In the present study higher shoot dry weight
Treatment Plant height (cm) Total root length(cm)
45 DAS 60 DAS 45 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 56.0 64.0 14298.4 5834.5 T2: T1- Fe salt+ Fe NP (54 µM) 47.0 48.0 33858.6 7110.1 T3: T1- Fe salt + Fe NP (27 µM) 40.5 58.5 153789.5 8307.3 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 37.0 41.0 21655.2 12229.5 T5: T1- Cu salt + Cu NP (0.5 µM) 58.5 70.0 12115.7 7697.7 T6: T1- Cu salt + Cu NP (0.25 µM) 56.5 57.5 20827.2 13088.7 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 47.5 56.0 36981.7 1862.0 T8: T1- Zn salt + Zn NP (2 µM) 45.5 67.5 20545.8 5842.7 T9: T1- Zn salt + Zn NP (1 µM) 47.5 55.0 5865.0 19463.7 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 32.5 53.0 33079.5 27837.3 CD (5%) NS 10.20 37915.1 NS
32
was found in plants treated with normal concentration of Zn NP (2µM) at 45 DAS
and 30 DAS, respectively.
Among Cu NP treatments, higher shoot dry weight was recorded in plants
treated with Normal salt at 45 DAS followed by Cu NP (0.25µM), Cu NP (0.5 µM)
and Cu salt (0.25µM). At 60 DAS, Cu NP (0.5 µM) treated plants showed higher
shoot dry weight followed by normal salt, Cu NP (0.25 µM), Cu NP (0.5 µM) and
Cu salt but at harvest stage, higher shoot weight was recorded in plants treated with
normal salt as compared to Cu NP (0.25 µM), Cu NP (0.5 µM) and Cu salt
(0.25µM). However, improvement in growth of plants treated with Cu NPs were
reported in wheat (Hafeez et al. 2015), lettuce (Shah and Belozerova, 2009) and
improvement in vase life of chrysanthemum was also reported with Cu NPs
(Hashemabadi et al. 2013). In the present study it was observed that normal
concentration of Cu NP (0.5 µM) had positive influence on shoot dry weight of
soybean while reduced concentration of Cu NP (0.25 µM) had positively
influenced below ground part of plants at 45 DAS. This indicated that reduced
concentration of NP might have acted as catalyst for root growth and normal
concentration of NP for shoot growth.
Among Zn treatment higher shoot dry weight was observed in plants
treated with Zn NP (2µM), at 45 DAS, followed by Zn NP (1µM), normal salt and
Zn salt (1µM). At 60 DAS, Zn NP (2µM) treated plants recorded higher shoot dry
weight followed by normal salt, Zn NP (1µM) and Zn salt (1µM). However, at last
stage higher shoot dry weight was noted in plants treated with Normal salt
followed by Zn NP (1µM) and Zn NP (1µM) Zn salt (1µM). Foliar spray of 10
ppm ZnO nanoparticles on 14-day-old cluster bean plants, significantly improved
shoot length (Raliya and Tarafdar 2013). Ramesh et al. (2014) also reported
positive effects of nano-ZnO on shoot-root growth of wheat. Nano-ZnO particle
was found to enhance germination and seedling growth of soybean (Sedghi et al.
2013) and vegetable crops like cabbage, cauliflower and tomato (Singh et al.
2013). This was in conformity with the present findings wherein Zn NP (2 µM)
improved shoot dry weight of soybean plants at 60 DAS. This shows that normal
concentration of Zn NPs can positively influence shoot/ root growth of plants.
Table 4.2: Effect of micronutrient NPs on shoot weight of soybean
33
Treatment Shoot dry weight (g plant-1
)
45 DAS 60 DAS Harvest
T1: 100% (Fe + Cu + Zn) = Normal salts 3.66 5.10 10.11 T2: T1- Fe salt+ Fe NP (54 µM) 4.49 5.19 10.12 T3: T1- Fe salt + Fe NP (27 µM) 5.33 3.83 8.69 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 2.46 3.42 7.74 T5: T1- Cu salt + Cu NP (0.5 µM) 2.20 5.91 8.38 T6: T1- Cu salt + Cu NP (0.25 µM) 2.70 3.91 8.51 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.62 2.96 8.11 T8: T1- Zn salt + Zn NP (2 µM) 6.43 6.68 6.86 T9: T1- Zn salt + Zn NP (1 µM) 5.58 4.70 7.30 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 2.21 3.48 6.36 CD (5%) 0.635 1.0 NS
4.1.4 Root dry weight
Root dry weight differed at 45 DAS, 60 DAS and Harvest (Table 4.3Fig.
4.3). Out of all ten treatments the highest root dry weight was observed in plants
treated with Cu NP (0.25µM) at Harvest and lowest with Zn salt (1µM) at 45 DAS.
In the all Fe treated plants, higher root weight was recorded in plants treated with
Fe NP (27µM) in comparison to Fe NP (54µM), Fe salt (27µM) and normal salt at
45 DAS. In second growth stage higher root dry weight was recorded in plant
treated with Fe NP (27µM) followed by normal salt, Fe salt (27µM) and Fe NP
(54µM). At last stage Fe NP (27µM) treated plant showed higher root dry weight
in comparison to normal salt, Fe salt (27µM) and Fe NP (54µM). Karimia et al.
(2014) observed that increasing Fe nanoparticles concentration above 10 ppm
reduced root weight of green gram, indicating negative effect of Fe NP on crop
growth. However, in the present study there was increase root dry weight was
observed at 60 DAS with Fe NP (27 µM) treatment, which may be attributed to the
increased branching of roots of soybean crop.
Among Cu treatments, maximum root weight was obtained in plants treated
with Cu salt (0.25µM) followed by Cu NP (0.5µM), normal salt and Cu NP
(0.25µM) at first growth stage. In second growth stage higher root dry weight was
recorded in plants treated with Cu salt (0.25µM) followed by normal salt, Cu NP
(0.5µM) and Cu NP (0.25µM). At last stage Cu NP (0.25µM) treated plant showed
higher root dry weight in comparison to normal salt, Cu salt (27µM) and Cu NP
(0.5µM).The influence of Cu NPs on plant height is sparsely reported. CuO NPs
34
did not inhibit the seed germination upto 2000ppm but root growth was inhibited at
500 ppm in soybean and chickpea (Adhikari et al. 2012). In the present study it
was observed reduced concentration of Cu NP (0.25 µM) had positively influenced
root weight at 45 DAS. This indicated that reduced concentration of NP might
have acted as catalyst for root growth.
Among Zn treatments, higher root weight was noted in plants treated with
Zn NP (1µM) followed by Zn NP (2µM), normal salt and Zn salt (1µM) at 45
DAS. But in the second stage, Zn NP (2µM) treated plants was found maximum
root weight as compared to normal salt, Zn NP (1µM) and Zn salt (1µM). At
harvest, impact of Zn treatments was found higher in normal salt treated plant
followed by Zn (1µM), Zn salt and Zn NP (2µM). Ramesh et al. (2014) also
reported positive effects of nano-ZnO on shoot-root growth of wheat. However,
Boonyanitipong et al. (2011) observed negative effect of nano-ZnO on root length
of rice (Oryza sativa L.). Nano-ZnO particle was found to enhance germination
and seedling growth of soybean (Sedghi et al. 2013) and vegetable crops like
cabbage, cauliflower and tomato (Singh et al. 2013). This was in conformity with
the present findings wherein Zn NP (2 µM) improved root dry weight of soybean
plants at 60 DAS. This shows that normal concentration of Zn NPs can positively
influence height and shoot/ root growth of plants. However, Kumar 2015 has
reported not much improvement in root dry weight of maize plants with Zn NP
treatments.
Table 4.3: Effect of micronutrient NPs on root dry weight of soybean
Treatment Root dry weight (g plant-1
)
45DAS 60 DAS Harvest
T1: 100% (Fe + Cu + Zn) = Normal salts 0.365 0.700 0.92
T2: T1- Fe salt+ Fe NP (54 µM) 0.440 0.490 0.71
T3: T1- Fe salt + Fe NP (27 µM) 0.605 0.745 0.93
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.395 0.505 0.90
T5: T1- Cu salt + Cu NP (0.5 µM) 0.415 0.640 0.69
T6: T1- Cu salt + Cu NP (0.25 µM) 0.390 0.595 1.04
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.660 0.750 0.87
T8: T1- Zn salt + Zn NP (2 µM) 0.385 0.840 0.66
T9: T1- Zn salt + Zn NP (1 µM) 0.475 0.485 0.68
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.275 0.375 0.68
CD (5%) NS NS NS
35
Fig. 4.1: Effect of micronutrient NPs on shoot weight of soybean.
Fig. 4.2 Effect of micronutrient NPs on root dry weight of soybean
0
2
4
6
8
10
12
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 DAS HARVESTS
hoot
dry
Wt.
(gpla
nt-1
)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 DAS HARVEST
Root
dry
wt.
(g
pla
nt-1
)
36
4.1.5 Specific leaf area
The information on specific leaf area (SLA) as influenced by various
micronutrient treatments during different growth periods are furnished in Table
4.4. Out of all treatments, highest SLA was noted in plant treated with Cu NP
(0.5µM) at 45 DAS. Lowest SLA was found in plant treated with Cu salt (0.25µM)
at 60 DAS. Among Fe treated plant higher SLA found was observed in plants
treated with Fe NP (54µM) followed by Fe NP (27µM), Fe salt (27µM) and
normal salt at 45 DAS. At 60 DAS, higher SLA was noted in plants treated with Fe
NP (27µM) than Fe NP (54µM), normal salt and Fe salt (27µM). There were not
many reports on the impact of nanoparticles on leaf growth parameter. It was
reported that nano-iron fertilizer caused 58 and 47% increase in fresh weight of
spinach (Moghadam et al. 2012). This was in conformity with the present findings
wherein soybean plants treated with reduced concentration i.e. Fe NP (27 µM)
exhibited higher SLA at 60 DAS. At 45 DAS, normal concentration of Fe NP (54
µM) treated plants showed higher SLA. This may be due to the growth promoting
effect of reduced concentration of Fe NP (27 µM) on growth characteristics of
soybean in comparison to Fe salts.
Among Cu treatments, higher SLA was observed in plants treated with Cu
NP (0.5µM) followed by Cu NP (0.25µM), normal salt and Cu salt (0.25µM) at 45
DAS. But at 60 DAS, normal salt treated plants showed higher SLA followed by
Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Enhanced leaf area of
wheat treated with 30 ppm of Cu NP was reported by Hafeez et al. (2015). In the
present study, higher SLA was reported from soybean plants treated with Cu NP @
0.5 µM at 45 DAS, which is much lower concentration than the 30 ppm used by
Hafeez et al. 2015.
Among the Zn treatment, higher SLA was observed in plant treated with
Zn (1 µM) as compared to Zn NP (2 µM), Zn salt (1µM) and normal salt at 45
DAS. However, at 60 DAS Zn NP (2µM) treated plants exhibited higher SLA than
normal salt, Zn NP (1µM) and Zn salt (1µM). In the present study, Zn NP treated
soybean plant has advantage over other treated plants for leaf growth
characteristics like SLA. Similarly, Avinash et al. (2010) observed increases in
37
germination and growth rate in the seeds of Cicer arietinum treated with nano-
ZnO.
4.1.6 Specific leaf weight
Specific leaf weight (SLW) as influenced by the application of
micronutrients based NPs at different growth stage is presented in Table 4.4. No
significant differences were observed plants at 45 DAS and at 60 DAS. Among Fe
treatment plant, higher SLW was recorded in plant treated with Normal salt
followed by Fe NP (27 µM) and Fe NP (54 µM). At 60 DAS higher SLW was fond
in plants treated with Fe NP (54 µM) followed by normal salt, Fe NP (27 µM) and
Fe salt (27 µM). There were not many reports on the impact of nanoparticles on
leaf growth parameter. It was reported that nano-iron fertilizer caused 58 and 47%
increase in fresh weight of spinach (Moghadam et al. 2012). This was in
conformity with the present findings wherein soybean plants treated with reduced
concentration i.e. Fe NP (27 µM) exhibited higher SLW at 60 DAS. At 45 DAS,
normal concentration of Fe NP (54 µM) treated plants showed higher SLW. This
may be due to the growth promoting effect of reduced concentration of Fe NP (27
µM) on growth characteristics of soybean in comparison to Fe salts.
Among the Cu treatments, Cu salt (0.25 µM) treated plants recorded
highest SLW at 45 DAS in comparison to normal salt, Cu NP (0.5 µM) and Cu NP
(0.25µM). In Second growth stage normal salt treated plants showed higher SLW
followed by Cu NP (0.5 µM) and Cu NP and Cu salt (0.25µM). Enhanced leaf
weight of wheat treated with 30 ppm of Cu NP was reported by Hafeez et al.
(2015). In the present study, higher SLW was reported from soybean plants treated
with Cu NP @ 0.5 µM at 45 DAS, which is much lower concentration than the 30
ppm used by Hafeez et al. 2015.
Among Zn treatments, higher SLW was noted in plants treated with normal
salt in comparison to Zn NP (1 µM), Zn NP (2 µM) and Zn salt (1 µM) at 45 DAS.
But at second growth stage, Normal salt treated plants recorded higher SLW
followed by Zn salt (1 µM) and Zn NP (2µM). Among all the ten treatments
highest SLW was obtained in plants treated with normal salt, Fe NP (54µM) at 60
DAS and lowest SLW noted in plant treated with Zn NP (2µM) at 60 DAS. In the
38
present study, Zn NP treated soybean plant has advantage over other treated plants
for leaf growth characteristics SLW. Similarly, Avinash et al. (2010) observed
increases in germination and growth rate in the seeds of Cicer arietinum treated
with nano-ZnO.
Table 4.4: Effect of micronutrient NPs on SLA and SLW of Soybean
Treatment Specific leaf area
(cm²g-1
)
Specific leaf weight
(g-1
cm-²)
45 DAS 60 DAS 45 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 222.20 187.25 0.0070 0.0090
T2: T1- Fe salt+ Fe NP (54 µM) 338.75 195.75 0.0000 0.0090
T3: T1- Fe salt + Fe NP (27 µM) 241.70 370.65 0.0030 0.0070
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 236.75 169.45 0.0030 0.0030
T5: T1- Cu salt + Cu NP (0.5 µM) 386.80 152.45 0.0060 0.0060
T6: T1- Cu salt + Cu NP (0.25 µM) 325.70 166.00 0.0010 0.0020
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 289.60 109.40 0.0090 0.0020
T8: T1- Zn salt + Zn NP (2 µM) 341.90 206.45 0.0020 0.0000
T9: T1- Zn salt + Zn NP (1 µM) 349.80 177.65 0.0030 0.0010
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 253.85 167.95 0.0010 0.0060
CD (5%) NS 97.582 NS NS
4.1.7 Leaf area ratio
It is evident form Table 4.5, highest leaf area ratio (LAR) was observed in
plants treated with Zn NP (1 µM) at 45 DAS and Fe NP (27µM) at 60 DAS and
lowest LAR was noted in plants treated with Zn salt (1 µM) at both stages. The
lower concentration of Fe NP (27µM) performed better as compared to higher
concentration of Fe NP (54µM), normal salt Fe salt (27µM) at both stages. There
were not many reports on the impact of nanoparticles on leaf growth parameter. It
was reported that nano-iron fertilizer caused 58 and 47% increase in leaf area index
of spinach (Moghadam et al. 2012). This was in conformity with the present
findings wherein soybean plants treated with reduced concentration i.e. Fe NP (27
µM) exhibited higher LAR at 60 DAS. This may be due to the growth promoting
effect of reduced concentration of Fe NP (27 µM) on growth characteristics of
soybean in comparison to Fe salts.
Among Cu treatments, higher LAR was observed in those plants treated
with Cu NP (0.5µM) followed by Cu NP (0.25µM), Cu salt (0.25µM) and normal
salt at 45 DAS but in second growth stage higher LAR was obtained in plants
39
Fig
4.3
Eff
ect
of
mic
ronu
trie
nt
NP
s on s
pec
ific
lea
f ar
ea o
f so
ybea
n
0
50
10
0
15
0
20
0
25
0
30
0
35
0
40
0
45
0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
45
DA
S6
0 D
AS
Specificleaf area (cm²g-1)
40
treated with Cu NP (0.25µM) as compared normal salt, Cu NP (0.5µM) Cu salt
(0.25µM). Enhanced leaf area ratio of wheat treated with 30 ppm of Cu NP was
reported by Hafeez et al. (2015). In the present study, LAR was reported from
soybean plants treated with Cu NP @ 0.5 µM at 45 DAS, which is much lower
concentration than the 30 ppm used by Hafeez et al. 2015. At 60 DAS, LAR was
found to higher with reduced concentration of Cu NP. This again reiterates that Cu
nanoparticle at lower concentration may act as catalyst for growth promotion.
Similar observations were also recorded in maize (Kumar 2015; Elanchezhian et al
2015).
Among Zn NP treatments, higher LAR was found in plant treated with Zn
NP (1 µM) followed by Normal salt, Zn NP (2µM) and Zn salt (1µM) at 45 DAS
and at second growth stage, higher LAR was obtained in plant treated with normal
salt followed by Zn NP (1µM), Zn NP (2µM) and Zn salt (1µM). In the present
study, Zn NP treated soybean plant has advantage over other treated plants for leaf
growth characteristics like LAR. Similarly, Avinash et al. (2010) observed
increases in germination and growth rate in the seeds of Cicer arietinum treated
with nano-ZnO.
4.1.8 Leaf area
The leaf area presented in Table 4.5 indicated significant differences
between the treatments at the both growth stages. Among all ten treatments,
highest leaf area was obtained in plants treated with Zn NP (2µM) and Fe NP
(27µM) at 45 DAS and 60 DAS, respectively and lowest Leaf area was observed in
plants treated with Cu salt (0.25 µM) and Zn salt (1 µM) at 45 DAS and 60 DAS,
respectively. Plants treated with Fe showed higher leaf area in plants treated with
lower concentration of Fe NP (27 µM) when compared to the high concentration of
Fe NP (54µM) followed by normal salt at both stage. There were not many reports
on the impact of nanoparticles on leaf growth parameter. It was reported that nano-
iron fertilizer caused 58 and 47% increase in leaf area index of spinach
(Moghadam et al. 2012). This was in conformity with the present findings wherein
soybean plants treated with reduced concentration i.e. Fe NP (27 µM) exhibited
higher leaf area and LAR at 60 DAS. This may be due to the growth promoting
41
effect of reduced concentration of Fe NP (27 µM) on growth characteristics of
soybean in comparison to Fe salts.
Among Cu treatments, normal salt treated plant was performing better than
Cu NP (0.5µM), Cu NP (0.25µM) Cu salt (0.25µM) but at 60 DAS, higher LA was
recorded in plants treated with Cu NP (0.5µM) than normal salt, Cu NP (0.25µM)
and Cu salt (0.25µM). Enhanced leaf area of wheat treated with 30 ppm of Cu NP
was reported by Hafeez et al. (2015). In the present study, LAR was reported from
soybean plants treated with Cu NP @ 0.5 µM at 45 DAS, which is much lower
concentration than the 30 ppm used by Hafeez et al. 2015. At 60 DAS, LA was
found to higher with normal concentration of Cu NP while LAR was found to
higher with reduced concentration of Cu NP. This again reiterates that Cu
nanoparticle at lower concentration may act as catalyst for growth promotion.
Similar observations were also recorded in maize (Kumar 2015; Elanchezhian et al
2015).
Among Zn treated plants higher leaf area was found in plants treated with
Zn NP (2µM) as compared to Zn NP (1µM), normal salt and Zn salt (1µM) but at
second stage Zn NP (2µM) treated plants noted higher LA followed by normal salt,
Zn NP (1µM) and Zn salt (1µM). In the present study, Zn NP treated soybean plant
has advantage over other treated plants for leaf growth characteristics like leaf
area. Similarly, Avinash et al. (2010) observed increases in germination and
growth rate in the seeds of Cicer arietinum treated with nano-ZnO.
Table 4.5: Effect of micronutrient NPs on LAR and LA of soybean
Treatment Leaf area ratio(cm² g-1) Leaf area(cm²)
45 DAS 60 DAS 45 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 138.66 109.31 517.60 537.2
T2: T1- Fe salt+ Fe NP (54 µM) 130.83 116.53 585.85 604.2
T3: T1- Fe salt + Fe NP (27 µM) 152.70 190.01 599.75 726.3
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 112.71 102.24 373.30 347.5
T5: T1- Cu salt + Cu NP (0.5 µM) 175.39 93.50 389.90 554.5
T6: T1- Cu salt + Cu NP (0.25 µM) 163.13 135.72 447.70 530.5
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 138.94 64.59 214.55 255.8
T8: T1- Zn salt + Zn NP (2 µM) 123.06 98.16 786.10 653.7
T9: T1- Zn salt + Zn NP (1 µM) 216.76 105.43 620.95 365.3
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 111.26 52.17 476.55 248.0
CD (5%) NS 35.71 272.64 132.70
42
Fig 4.4: Effect of micronutrient NPs on leaf area ratio of soybean.
Fig 4.5: Effect of micronutrient NPs on leaf area of soybean.
0
50
100
150
200
250
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 DAS
Lea
f ar
ea r
atio
(cm
² g
-1)
0
100
200
300
400
500
600
700
800
900
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 DAS
Lea
f ar
ea (
cm²
)
43
4.1.9 Root volume
Among Fe treatments, higher root volume was observed in plants treated
with Fe NP (27µM) as compared to Fe NP (54µM), Fe salt (27µM) and normal salt
at 45 DAS (Table 4.6). AT 60 DAS, plants were performing better when treated
with Fe salt (27µM) followed by Fe NP (27µM), Fe NP (54µM) and normal salt.
There were many reports on the impact of nanoparticles on root volume for
conformity that nano particles could enhance and maintain the growth of maize
plant. The plant parameters like root length and root volume were all improved due
to application of nano-particle (Adhikari, T. et al.2015). In the present study
soybean plants treated with reduced concentration i.e. Fe NP (27 µM) exhibited
higher root volume at 60 DAS.
Among Cu treated plants, Root volume obtained was higher in plant
treated with Cu NP (0.25 µM) as compared to Cu salt (0.25µM), Cu NP (0.5µM)
and normal salt at both stages. CuO NPs did not inhibit the seed germination upto
2000ppm but root growth was inhibited at 500 ppm in soybean and chickpea
(Adhikari et al. 2012). However, improvement in growth of plants treated with Cu
NPs were reported in wheat (Hafeez et al. 2015), lettuce (Shah and Belozerova,
2009) and improvement in vase life of chrysanthemum was also reported with Cu
NPs (Hashemabadi et al. 2013). In the present study it was observed that normal
concentration of Cu NP (0.5 µM) had positive influence on above ground part as
well as shoot of soybean while reduced concentration of Cu NP (0.25 µM) had
positively influenced root volume at 45 DAS. This indicated that reduced
concentration of NP might have acted as catalyst for root growth and normal
concentration of NP for shoot growth.
In the case of Zn treated plants, higher root volume was observed in plant
treated with Zn salt (1µM) as compared to Zn NP (2µM), normal salt, Zn NP
(1µM) at 45 DAS but at 60 DAS, higher root volume was noted in Zn (1µM)
followed by Zn salt (1µM), Zn NP (2µM) and normal salt. Among all the ten
treatments, highest root volume was recorded in plants treated with Fe NP (27µM)
at 45 DAS and lowest root volume was recorded in plant treated Normal salt at 60
DAS.
44
Table 4.6: Effect of micronutrient NPs on root volume of soybean
Treatment Root Volume (cc)
45 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 4.0
2.0
T2: T1- Fe salt+ Fe NP (54 µM) 4.5
3.0
T3: T1- Fe salt + Fe NP (27 µM) 12.0
4.0
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 4.5
4.5
T5: T1- Cu salt + Cu NP (0.5 µM) 4.0
3.5
T6: T1- Cu salt + Cu NP (0.25 µM) 4.5
5.5
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 4.5
3.0
T8: T1- Zn salt + Zn NP (2 µM) 4.0
3.0
T9: T1- Zn salt + Zn NP (1 µM) 3.5
5.0
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 8.5
4.0
CD (5%) NS
NS
4.1.10 Pod weight
The Pod weight was observed after harvest furnished in Table 4.7. Out of
all the ten treatments, highest pod weight was observed in plants treated with Fe
NP (54µM) and lowest Pod weight was noted in Cu salt (0.25µM). Among Fe
treatments, highest pod weight was recorded in plants treated with Fe NP (54µM)
followed by normal salt, Fe NP (27µM) and Fe salt (27µM). Among Cu treatment,
higher Pod weight was recorded in plants treated with normal salt followed by Cu
NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM). In the case of Zn treatments,
Zn NP (1µM) performed better than normal salt, Zn NP (2µM) and Zn salt (1µM).
4.1.11 Grain weight
The Grain weight after harvest was furnished in Table 4.7. Highest and
lowest Grain weight was noted in Zn NP (2µM) and Cu salt (0.25µM) treated
plants, respectively. Among Fe treatment, higher Grain weight was recorded in
plants treated with Fe NP (54µM) in comparison of Fe NP (27µM) followed by
normal and Fe salt (27µM). In the case of Cu treatments, Cu NP (0.5µM) was
performed better than normal salt, Cu NP (0.25µM) and Cu salt (0.25µM). Among
Zn treatment, highest Grain weight was recorded in plants treated with Zn NP
(2µM) followed by Zn NP (1µM), normal salt and Zn salt (1µM).
Increases in grain yield and straw yield of rice with Zn application was
reported (Maharana et al., 1993). In present study NP treated soybean plants, the
pod weight and grain yield was higher with normal concentration of Fe NP (54
45
µM), Cu NP (0.5 µM) and Zn NP (2 µM). This indicated that normal concentration
of NP might have sustained the crop growth for effective grain yield.
Table 4.7: Effect of micronutrient NPs on Pod weight and Grain weight of
Soybean
Treatment Pod weight
(g plant-1
)
Grain weight
(g plant-1
)
Harvest Harvest
T1: 100% (Fe + Cu + Zn) = Normal salts 1.99 0.57
T2: T1- Fe salt+ Fe NP (54 µM) 2.68 0.94
T3: T1- Fe salt + Fe NP (27 µM) 1.94 0.74
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 1.90 0.48
T5: T1- Cu salt + Cu NP (0.5 µM) 1.48 0.57
T6: T1- Cu salt + Cu NP (0.25 µM) 1.39 0.48
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.26 0.45
T8: T1- Zn salt + Zn NP (2 µM) 1.69 1.03
T9: T1- Zn salt + Zn NP (1 µM) 2.09 0.77
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 1.33 0.46
CD (5%) NS NS
4.1.12 Root Shoot Ratio
Root shoot ratio was influence by various treatments at different stages
(Table 4.8). Among all the treatments, highest Root Shoot ratio was found in plants
treated with Cu NP (0.25 µM) at 45 DAS and lowest in plants treated with Zn salt
(27 µM) at 45 DAS. Among Fe treated plants, higher root shoot ratio was obtained
in lower concentration of Fe NP (27 µM) as compared to normal concentration of
Fe NP (54 µM) followed by normal salt and Fe salt (27 µM) at both stages. In the
case of Cu treatments, maximum root shoot ratio was obtained in plants treated
with Cu NP (0.25 µM) better than Cu NP (0.5 µM) and Cu salt (0.25 µM) and
Normal salt at 45 DAS, but in second stage, Cu NP (0.5 µM) treated plants noted
higher root shoot ratio in comparison to Cu NP 0.25µM), Normal salt and Cu salt
(0.25µM). Among Zn treatments, higher root shoot ratio was recorded in plant
treated with Zn NP (2µM) followed by Normal salt, Zn NP (1µM) and Zn salt
(1µM) at 45 DAS. But it was changed in second growth stage, maximum root
shoot ratio was observed in plants treated with Normal salt followed by Zn NP
(2µM), Zn NP (1µM) and Zn salt (1µM).
46
In soybean, root/shoot dry weight were found to be higher with normal
concentration of Zn NP (2 µM). In the present set of investigation, the response in
wheat was similar to soybean and maize crop (Kumar 2015).
Table 4.8: Effect of micronutrient NPs on root shoot ratio of soybean
Treatment Root Shoot Ratio
45 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 0.105 0.145
T2: T1- Fe salt+ Fe NP (54 µM) 0.114 0.145
T3: T1- Fe salt + Fe NP (27 µM) 0.159 0.195
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.098 0.095
T5: T1- Cu salt + Cu NP (0.5 µM) 0.193 0.255
T6: T1- Cu salt + Cu NP (0.25 µM) 0.385 0.155
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.155 0.110
T8: T1- Zn salt + Zn NP (2 µM) 0.125 0.125
T9: T1- Zn salt + Zn NP (1 µM) 0.085 0.105
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.055 0.103
CD (5%) NS NS
4.2 Biochemical parameters of Soybean
4.2.1 Chlorophyll content
It is evident from Table 4.9 and Fig 4.6 and 4.7 that the chlorophyll ‗a‘
content did not differ significantly between treatments at both stages. Among all
the treatments highest chlorophyll content ‗a‘ was found in plants treated with Cu
NP (0.5µM) and Zn NP (2 µM) at 45 DAS, respectively but at 60 DAS, highest
chlorophyll content ‗a‘ was recorded in plants treated with Cu NP (0.25 µM).
Lowest chlorophyll contents ‗a‘ was obtained in plants treated with Fe salt (27µM)
at both stages. Among Fe treated plants at 45 DAS, chlorophyll content ‗a‘ was
found to be higher in plants treated with Fe NP (27 µM) followed by NP (54µM),
normal salt and Fe salt (27 µM) at 45 DAS. At 60 DAS, normal salt treated plants
performance was better than Fe NP (54µM), Fe NP (27µM) and Fe salt (27 µM).
Among Cu Treated plants higher Chlorophyll content ‗a‘ was found higher in
plants treated with Cu NP (0.5 µM) as compare to Cu NP (0.25µM), Cu salt
(0.25µM) and Normal salt at 45 DAS, but in second stage, Cu NP (0.25µM)
treated plants, performed best as compared to Normal salt, Cu NP (0.5µM) and Cu
salt (0.25µM). In the case of Zn treatments, higher chlorophyll content ‗a‘ was
observed in plants treated with Zn NP (2µM) followed by Zn NP (1µM), Zn salt
47
(1µM) and Normal salt at 45 DAS. But in second growth stage, Normal salt, Zn
NP (2µM), Zn NP (1µM) treated plants was performing better than Zn salt (1µM)
respectively.
Significantly higher chlorophyll content ‗b‘ was observed in leaves at both
the growth stages of plants treated with Normal salt at 45 DAS and Fe NP (27 µM)
at 60 DAS (Table 4.9and Fig. 4.6 and 4.7). Among Fe treated plants significant
differences were observed in chlorophyll content ‗b‘ of plants treated with Normal
salt followed by Fe NP (27 µM), Fe NP (54 µM) and Fe salt (27 µM) at 45 DAS.
In second growth stage, Fe NP (27 µM) treated plants exhibited significantly
higher chlorophyll content ‗b‘ in comparison to Fe NP (54 µM), normal salt and Fe
salt (27 µM) treated plants. Among Cu treated plants higher chlorophyll content
‗b‘ was recorded in plants treated with Normal salt followed by Cu NP (0.25 µM),
Cu NP (0.5µM) Cu salt (0.25 µM) plants at first stage. At 60 DAS, maximum
chlorophyll content ‗b‘ was observed in plants treated with Normal salt as
compared to Cu NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM). Among Zn
treated plants, chlorophyll ‗b‘ content was found be significantly higher in plants
treated with Normal salt better than Zn NP (1 µM), Zn NP (2 µM) and Zn salt (1
µM) treated plants at both stages. Among all the treatments, lowest chlorophyll
content ‗b‘ was found in Fe salt (27 µM) at 45 DAS and Zn salt (1 µM) at 60 DAS.
Significant differences in total chlorophyll content were observed at 45
DAS and at 60 DAS (Table 4.9, Fig. 4.6 and 4.7). Among all the treatments,
highest total chlorophyll content was found significantly higher in plants treated
with Fe NP (27 µM) and lowest in plants treated with Zn salt (1 µM) at 45 DAS.
Among Fe treated plants significant differences were observed in plants treated
with Fe NP (27 µM) as compared to Fe NP (54 µM), Normal salt and Fe salt (27
µM) at 45 DAS, but at 60 DAS, Fe NP (27 µM) treated plants showed higher
chlorophyll content in comparison to Normal salt, Fe NP (54 µM) and Fe salt (27
µM). At 45 DAS, in Cu treatments, total chlorophyll content was found
significantly higher in plants treated with Normal salt followed by Cu NP (0.5
µM), Cu NP (0.25 µM) and Cu salt (0.25µM). However, at 60 DAS significant
differences were observed in plants treated with Normal salt as compared to Cu NP
(0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Among Zn treated plants higher
48
total chlorophyll Content was recorded in plants treated with Normal salt when
compared to Zn NP (1µM), Zn NP (2 µM) and Zn salt (1 µM) treated plants at
both growth stages.
Among Fe treated soybean plants, chlorophyll a, total chlorophyll content
was found to be high in plants treated with Fe NP (27 µM). This was in conformity
to the results obtained in maize crop for the trait of membrane stability
(Elanchezhian et al 2015; Kumar 2015). This indicated that the reduced
concentration of Fe NP may be inducing biochemical changes for chlorophyll
content. These results were in conformity with the findings of Delfani et al. (2014)
wherein nano-iron either alone or in combination with nano-magnesium had
significant effect on chlorophyll content. In the present study, Ghafari and
Razmjoo, (2013) also observed similar results of increased chlorophyll content. In
soybean, higher chlorophyll a content was observed in plants treated with Cu NP
(0.5 µM). This was in conformity with the earlier findings of Hafeez et al. (2015).
Zinc oxide (ZnO) nanoparticles resulted in chlorophyll content (Burman et al.
2013). Similar results were obtained with present set of experiment with soybean
wherein increased chlorophyll content was observed with Zn NP (2 µM). Similar
results were also obtained for enhanced chlorophyll content of peanut (Prasad et al.
2012), chlorophyll content of cluster bean (Raliya and Tarafdar 2013) and wheat
(Ramesh et al. 2014).
Table 4.9: Effect of micronutrient NPs on chlorophyll content of soybean
Treatment Chlorophyll Content (mg g-1
FW)
CHL a CHL b Total
DAS
45 60 45 60 45 60
T1: 100% (Fe + Cu + Zn) = Normal salts 0.28 0.30 0.49 0.33 0.63 0.76
T2: T1- Fe salt+ Fe NP (54 µM) 0.28 0.29 0.40 0.41 0.70 0.68
T3: T1- Fe salt + Fe NP (27 µM) 0.29 0.29 0.48 0.46 0.74 0.77
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.27 0.26 0.15 0.30 0.56 0.42
T5: T1- Cu salt + Cu NP (0.5 µM) 0.31 0.30 0.41 0.27 0.57 0.71
T6: T1- Cu salt + Cu NP (0.25 µM) 0.29 0.31 0.47 0.22 0.52 0.76
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.29 0.30 0.40 0.19 0.50 0.69
T8: T1- Zn salt + Zn NP (2 µM) 0.31 0.30 0.40 0.17 0.46 0.71
T9: T1- Zn salt + Zn NP (1 µM) 0.30 0.30 0.41 0.24 0.54 0.71
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.29 0.28 0.39 0.11 0.39 0.68
CD (5%) NS NS 0.11 0.07 0.09 0.11
49
Fig 4.6: Effect of micronutrient NPs on chlorophyll content of soybean at 45 DAS
Fig 4.7: Effect of micronutrient NPs on chlorophyll content of soybean at 60 DAS
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
Chlorophyll a Chlorophyll b Total
Ch
loro
ph
yll
Co
nte
nt
(mg g
-1)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
Chlorophyll a chlorophyll b Total
Ch
loro
phyll
Conte
nt
(mg g
-1)
50
4.2.2 Membrane stability
Among all the treatments, highest membrane stability (MS) was observed
in plants treated with Cu NP (0.5µM) and lowest in plants treated with Cu salt
(0.5µM) (Table 4.10). Among Fe treatments, significantly higher MS was observed
in plants treated with Fe NP (27 µM) as compared to Fe NP (54µM), Normal salt
and Fe salt (27µM). Among Cu treatments, maximum MS was found in plants
treated with Cu NP (0.5 µM) in comparison to Normal salt, Cu NP (0.25µM) and
Cu salt (0.25µM). Among Zn treatments, higher MS was obtained in plants treated
with Normal salt when compared to Zn NP (2µM), Zn NP (1µM) and Zn salt
(1µM).
4.2.3 Relative water content
Relative Water Content (RWC) was found to be significantly different
among all the treatments (Table 4.10). Highest RWC was found in plants treated
with Fe NP (27 µM) and lowest RWC was found in plants treated with Cu salt
(0.25 µM). Among Fe treatments plants, maximum RWC was obtained with lower
concentration of Fe NP (27 µM) as compared to higher concentration of Normal
salt, Fe NP (54µM) and Fe salt (27 µM). Among Cu treatments, higher RWC was
recorded in plants treated with Normal salt followed by Cu NP (0.25 µM), Cu NP
(0.5 µM) and Cu salt (0.25 µM). In the case of Zn treatments, Normal salt treated
plants was performing better than Zn NP (1 µM), Zn NP (2µM) and Zn salt (1
µM).
Among Fe treated soybean plants, membrane stability and RWC was found
to be high in plants treated with Fe NP (27 µM). This was in conformity to the
results obtained in maize crop for the trait of membrane stability (Elanchezhian et
al 2015; Kumar 2015). In soybean, MS was observed in plants treated with Cu NP
(0.5 µM). This was in conformity with the earlier findings of Hafeez et al. (2015).
51
Table 4.10: Effect of micronutrient NPs on MS and RWC of soybean at 60 DAS
4.2.4 Antioxidant enzyme
4.2.4.1 Super oxide dismutase enzyme activity
Super oxide dismutase (SOD) was found to be significantly different in
treatments (Table 4.11 and Fig. 4.9). Highest SOD enzyme activity was recorded
in plants treated Zn salt (1 µM) at 60 DAS and lowest in plants treated with Cu NP
(0.25 µM) at 45 DAS. Among Fe treated plants, higher SOD activity was observed
in plants treated with Fe NP (54 µM) when compared to Fe NP (27 µM), Fe salt
(27 µM) and Normal salt at the first stage but in second growth stage, higher SOD
enzyme activity was observed in plants treated with Fe NP (54µM) followed by Fe
salt (27 µM), Fe NP (27 µM) and Normal salt. In the present study, SOD activity
was also found to be more in soybean plants treated with Fe NP (54 µM) indicating
the involvement of Fe NP in antioxidant stress response. Ghafari and Razmjoo,
(2013) also observed similar results of increased antioxidant enzyme activities of
wheat. This again reiterates the species specificity of different nanoparticles.
Among Cu treated plants maximum SOD activity was recorded in plants
treated with Cu NP (0.5µM) better than Cu salt (0.25 µM), Cu NP (0.25µM) and
Normal salt at both stages. In soybean, SOD was observed in plants treated with
Cu NP (0.5 µM). This was in conformity with the earlier findings of Hafeez et al.
(2015).
Treatment MS% RWC%
T1: 100% (Fe + Cu + Zn) = Normal salts 46.89 58.75
T2: T1- Fe salt+ Fe NP (54 µM) 47.38 52.80
T3: T1- Fe salt + Fe NP (27 µM) 53.24 59.30
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 40.74 47.60
T5: T1- Cu salt + Cu NP (0.5 µM) 54.84 47.95
T6: T1- Cu salt + Cu NP (0.25 µM) 45.51 50.00
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 33.82 41.25
T8: T1- Zn salt + Zn NP (2 µM) 42.67 50.45
T9: T1- Zn salt + Zn NP (1 µM) 36.84 51.55
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 36.73 47.40
CD (5%) NS 8.40
52
In the case of Zn treatments, maximum SOD enzyme activity was obtained
in plants treated with Zn NP (2µM) than Zn salt (1µM), Zn NP (1µM) and Normal
salt at 45 DAS. But in second stage it was changed and higher enzyme activity was
recorded in plants treated with Zn salt (1µM) followed by Zn NP (1µM), Zn NP
(2µM) and Normal salt. Zinc oxide (ZnO) nanoparticles resulted in lesser lipid
peroxidation and also associated with lower activity of prominent antioxidant
enzymes, superoxide dismutase (SOD) (Burman et al. 2013). Similar results were
obtained with present set of experiment with soybean wherein increased SOD was
observed with Zn NP (2 µM).
Table 4.11: Effect of micronutrient NPs on super oxide dismutase enzyme activity
of soybean
4.2.4.2 Catalase enzyme activity
Catalase (CAT) activity was found to be significantly different among
various treatments (Table 4.12). Out of all ten treatments, highest catalase activity
was recorded in plants treated with Cu NP (0.25µM) and Fe NP (54 µM) at 45
DAS and 60 DAS, respectively. Among Fe NP treated plant higher catalase
activity was observed in plants treated with Fe salt (27µM) when compared to Fe
NP (54 µM), Fe NP (27 µM) and Normal salt at 45 DAS but at 60 DAS, maximum
CAT activity was noted in plants treated with Fe NP (54 µM) as compared to
Normal salt, Fe NP (27µM) and Fe salt (27µM). Higher CAT was observed with
10 ppm of nano-iron chelate (Karimia et al. 2014). This again reiterates the species
specificity of different nanoparticles.
Treatment SOD (unit g-1
)
45 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 0.00 0.00
T2: T1- Fe salt+ Fe NP (54 µM) 20.97 22.94
T3: T1- Fe salt + Fe NP (27 µM) 14.44 17.45
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 11.27 21.17
T5: T1- Cu salt + Cu NP (0.5 µM) 19.38 18.21
T6: T1- Cu salt + Cu NP (0.25 µM) 8.89 17.03
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 11.32 17.88
T8: T1- Zn salt + Zn NP (2 µM) 14.69 15.37
T9: T1- Zn salt + Zn NP (1 µM) 9.07 27.67
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 11.37 30.35
CD (5%) 9.74 11.81
53
Fig
. 4.8
: E
ffec
t of
mic
ron
utr
ient
NP
s on s
uper
ox
ide
dis
muta
se e
nzym
e ac
tivit
y o
f so
ybea
n
-505
10
15
20
25
30
35
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
45
DA
S6
0 D
AS
SOD(Unit g-1)
54
Maximum catalase activity was recorded in plants treated with Cu NP (0.25
µM) followed by Cu NP (0.5 µM), Normal salt and Cu salt (0.25µM) at 45 DAS,
in the case of 60 DAS, higher CAT enzyme activity was observed in plants treated
with Cu NP (0.25 µM) better than Normal salt, Cu salt (0.25 µM) and Cu NP
(0.5µM). The enhanced antioxidant enzyme CAT activity with Cu NP treatment as
observed in the present set of experiment with soybean was corroborated with the
findings of Nekrasova et al. (2011).
Among Zn NP treated plants higher catalase activity was recorded in plants
treated with Zn salt (1µM) when compared to Zn NP (2 µM), Zn NP (1 µM) and
Normal salt at 45. After 15 days interval, CAT enzyme activity was recorded
higher in plants treated with Zn NP (2µM) followed by Normal salt, Zn salt (1 µM)
and Zn NP (1 µM).
4.2.4.3 Peroxidase enzyme activity
Significant differences among various treatments in terms of Peroxidase
(POX) activity were observed (Table 4.12). Among all the treatments, higher
Peroxidase enzyme activity was recorded in plants treated with Normal at both
stages and lowest POX enzyme activity was recorded in plants treated with Cu salt
(0.25µM) at 60 DAS. Among Fe treated plants, higher Peroxidase activity was
observed in plants treated with Normal salt when compared to Fe NP (54 µM) Fe,
Fe NP (27 µM) and salt (27 µM) at both stages. In the present study, POX activity
was also found to be more in soybean plants treated with Fe NP (54 µM) indicating
the involvement of Fe NP in antioxidant stress response. Ghafari and Razmjoo,
(2013) also observed similar results of increased antioxidant enzyme activities of
wheat. Higher POX was observed with 10 ppm of nano-iron chelate (Karimia et al.
2014). This again reiterates the species specificity of different nanoparticles.
Among Cu treated plants maximum Peroxidase activity was recorded in
plants treated with Normal salt, better than Cu NP (0.5 µM) followed by Cu NP
(0.25µM) and Cu salt (0.25 µM) at 45 DAS, but in the second growth stage,
Normal salt performed better than Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt
(0.25µM). The enhanced antioxidant enzyme peroxidase activity with Cu NP
55
treatment as observed in the present set of experiment with soybean was
corroborated with the findings of Nekrasova et al. (2011).
Among Zn treated plants higher POX was obtained in plants treated with
Normal salt followed by Zn NP (2µM), Zn NP (1 µM) and Zn salt (1µM) at both
growth stages. Zinc oxide (ZnO) nanoparticles resulted in lesser lipid peroxidation
and also associated with lower activity of prominent antioxidant enzymes,
peroxidase (Burman et al. 2013). Similar results were obtained with present set of
experiment with soybean wherein increased SOD was observed with Zn NP (2
µM).
Table 4.12: Effect of micronutrient NPs on catalase enzyme activity and
peroxidase enzyme activity of soybean
Treatment CAT (unit H2O2
min-1
g-1
)
POX (unit H2O2
min-1
g-1
)
45
DAS
60
DAS
45
DAS
60
DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 15.30 21.20 140.24 122.79 T2: T1- Fe salt+ Fe NP (54 µM) 17.60 24.80 112.61 103.56 T3: T1- Fe salt + Fe NP (27 µM) 17.20 19.20 100.68 87.95 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 20.40 17.60 72.27 69.01 T5: T1- Cu salt + Cu NP (0.5 µM) 15.30 21.60 122.81 108.15 T6: T1- Cu salt + Cu NP (0.25 µM) 25.65 17.60 85.89 122.66 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 15.10 20.80 75.79 61.87 T8: T1- Zn salt + Zn NP (2 µM) 17.70 24.80 79.21 109.09 T9: T1- Zn salt + Zn NP (1 µM) 17.20 17.60 74.90 98.49 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 25.40 18.40 74.05 83.44 CD (5%) 6.32 3.76 26.31 37.25
56
Fig 4.9: Effect of micronutrient NPs on catalase enzyme activity of soybean.
Fig. 4.10: Effect of micronutrient NPs on peroxidase enzyme activity of soybean
0
5
10
15
20
25
30
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 ADSC
AT
(u
nit
H2O
2m
in-1
g-1
)
0
20
40
60
80
100
120
140
160
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 ADS
PO
X (
un
it H
2O
2m
in-1
g-1
)
57
4.2.5 Proline content
Proline content was found to be significantly different in plants at 45 DAS
and 60 DAS, respectively (Table 4.13 and Fig. 4.12). Among Fe treated plants,
proline content was significantly higher in plants treated with Fe NP (54µM), when
compared to Fe NP (27 µM), Normal salt and Fe salt (27 µM) at first growth stage
but at 60 DAS, Fe NP (27 µM) was performed best as compared to Fe NP (54µM),
Normal salt and Fe salt (27 µM).The proline content was found to be high in plants
treated with Fe NP (54 µM). These results were in conformity with the findings of
Delfani et al. (2014) wherein nano-iron either alone or in combination with nano-
magnesium had significant effect on proline content.
Among Cu treated plants higher proline content was recorded in plants
treated with Cu NP (0.5 µM) followed by Cu NP (0.25 µM), Cu salt (0.25 µM) and
normal salt at 45 DAS. In the case of 60 DAS, higher proline content was recorded
in plants treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Normal salt
and Cu salt (0.25µM). In soybean, higher proline was observed in plants treated
with Cu NP (0.5 µM). This was in conformity with the earlier findings of Hafeez et
al. (2015). The enhanced proline content with Cu NP treatment as observed in the
present set of experiment with soybean was corroborated with the findings of
Nekrasova et al. (2011).
Higher proline content was observed in plants treated with Zn NP (1 µM)
as compared to Zn NP (2µM), Normal salt and Zn salt (1µM) at 45 DAS. At 60
DAS, maximum proline content was recorded in plants treated with Zn NP (2µM)
which is better than Normal salt, Zn NP (1µM) and Zn salt (1µM). Among all the
treatments, highest proline content was observed in plants treated with Fe NP (27
µM) at 60 DAS and lowest proline content was observed in plants treated with Zn
salt (27 µM) at 45 DAS. Some results were obtained for protein content of cluster
bean (Raliya and Tarafdar 2013) and wheat (Ramesh et al. 2014). However,
proline was found to be higher with Zn NP (1 µM) treated plants. Similar increase
in proline content with ZnO NPs was also reported by Sunita et al. (2013) in
Brassica juncea.
58
4.2.6 Total soluble protein
Total soluble protein (TSP) content was found similar during the growth
period (Table 4.13). Protein content was observed to be not significant among all
NP treated plants at 45 DAS and 60 DAS. Among all the treatments, higher
magnitude of TSP content was observed in plants treated with Zn NP (2 µM) at 60
DAS, and lower TSP content was recorded in plants treated with Zn salt (1µM) at
45 DAS. Among Fe treatments, higher TSP content was noted in plants treated
with Fe NP (54 µM) as compared to Normal salt, Fe NP (27µM) and Fe salt (27
µM) at 45 DAS. However, at 60 DAS, Fe NP (54µM) treated plants performed
better than Fe NP (27µM), Normal salt and Fe salt (27µM). Total soluble protein
content was found to be high in plants treated with Fe NP (54 µM). These results
were in conformity with the findings of Delfani et al. (2014) wherein nano-iron
either alone or in combination with nano-magnesium had significant effect on seed
protein content.
Among Cu treated plants higher protein content was observed in plants
treated with Cu NP (0.25 µM) followed by Normal salt, Cu NP (0.5µM) and Cu
salt (0.25µM) at 45 DAS, but in second stage maximum protein content was
recorded in plants treated with Cu NP (0.5 µM) as compared to Cu NP (0.25µM),
Normal salt and Cu salt (0.25µM). In soybean, higher TSP was observed with Cu
NP (0.25 µM). This was in conformity with the earlier findings of Hafeez et al.
(2015). The enhanced protein content with Cu NP treatment as observed in the
present set of experiment with soybean was corroborated with the findings of
Nekrasova et al. (2011).
Among Zn treated plants, higher protein content was found in plants treated
with lower concentration of Zn NP (1 µM), better than Normal salt, Zn NP (2 µM)
and Zn salt (1 µM) at 45 DAS. But in the case of 60 DAS, higher protein content
was found in plants treated with higher concentration of Zn NP (2 µM) in
comparison to lower concentration of Zn NP (1µM) followed by Zn salt (1µM) and
Normal salt. Some results were obtained for protein content of cluster bean (Raliya
and Tarafdar 2013) and wheat (Ramesh et al. 2014). However, TSP was found to
be higher with Zn NP (1 µM) treated plants. Similar increase in protein content
with ZnO NPs was also reported by Sunita et al. (2013) in Brassica juncea.
59
Table 4.13: Effect of micronutrient NPs on proline and protein of soybean
Treatment Proline (μM g-1
) TSP (mg g-1
)
45 DAS 60 DAS 45 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 0.02 0.05 1.13 1.30 T2: T1- Fe salt+ Fe NP (54 µM) 0.10 0.09 1.15 1.45 T3: T1- Fe salt + Fe NP (27 µM) 0.07 0.28 1.12 1.44 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.02 0.03 1.10 1.28 T5: T1- Cu salt + Cu NP (0.5 µM) 0.10 0.20 1.12 1.41 T6: T1- Cu salt + Cu NP (0.25 µM) 0.06 0.25 1.14 1.38 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.03 0.03 1.12 1.25 T8: T1- Zn salt + Zn NP (2 µM) 0.03 0.24 1.11 1.50 T9: T1- Zn salt + Zn NP (1 µM) 0.04 0.02 1.14 1.40 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.02 0.02 1.08 1.34 CD (5%) 0.02 0.14 NS NS
4.2.7 Total soluble sugar
Total soluble sugar was found to be different at all the growth stages with
the various treatments (Table 4.14 and Fig.4.13). Out of all ten treatments, highest
TSS content was observed in plants treated with Cu NP (0.25 µM) at 45 DAS. At
60 DAS, maximum TSS was observed in plants treated with Normal salts. Among
Fe treated plants, higher TSS content was found in plants treated with Fe NP (27
µM) as compared to plants treated with Fe NP (54 µM), Normal salt and Fe salt
(27µM) at 45 DAS but at 60 DAS, Normal salt was obtained higher TSS better
than Fe NP (27µM), Fe NP (54 µM) and Fe salt (27µM). Among Fe treated
soybean plants, TSS was found to be high in plants treated with Fe NP (27 µM).
This was in conformity to the results obtained in maize crop for the trait of
membrane stability (Elanchezhian et al 2015; Kumar 2015). This indicated that the
reduced concentration of Fe NP may be inducing biochemical changes for soluble
carbohydrate. Ghafari and Razmjoo, (2013) also observed similar results of
increased carbohydrate content of wheat.
Maximum TSS content was observed in plants treated with Cu NP (0.25
µM) at 45 DAS among Cu treated plants as compared to Cu (0.5 µM) followed by
Cu salt (0.25 µM) and Normal salt. However, at 60 DAS, maximum TSS was
recorded in plants treated with Normal salt when compared to Cu NP (0.5 µM), Cu
NP (0.25 µM) and Cu salt (0.25 µM). In soybean, higher TSS was observed with
Cu NP (0.25 µM). This was in conformity with the earlier findings of Hafeez et al.
60
(2015). However, there was not much report impact of NPs on carbohydrate status
of plants.
Among Zn treatments, higher TSS content was found in plants treated with
Zn NP (1µM) followed by Zn NP (2µM), Normal salt and Zn salt (1µM) at 45
DAS. But in case of 60 DAS, maximum TSS was observed in plants treated with
Normal salt as compared to Zn NP (1µM), Zn NP (2µM) and Zn salt (1µM).
However lowest TSS content was recorded in plants treated with Fe salt (27 µM) at
both the stages. The TSS content was found to be higher with Zn NP (1 µM)
treated plants was also reported by Sunita et al. (2013) in Brassica juncea. Singh et
al. (2013) reported that sugar content of Zn NP treated plants were similar to
control, in the present study also similar results were obtained at 60 DAS.
4.2.8 Non-structural carbohydrate
Non-structural carbohydrate (NSC) content was found to be significantly
higher at both the growth stages with various treatments (Table 4.14 and Fig 4.14).
Among all the treatments, maximum NSC content was recorded in plants treated
with Cu NP (0.25 µM) and Fe NP (27µM) at 45 and 60 DAS, respectively. Among
Fe treated plants higher NSC was observed in plants treated with Fe NP (27 µM)
when compared to all Fe treatments like Fe NP (54 µM), Normal salt and Fe salt
(27 µM) at 45 DAS and at 60 DAS, Fe NP (27 µM) treated plants exhibited higher
NSC as compared to Normal salt, Fe NP (54µM) and Fe salt (27 µM). Among Fe
treated soybean plants, NSC was found to be high in plants treated with Fe NP (27
µM). This was in conformity to the results obtained in maize crop for the trait of
membrane stability (Elanchezhian et al 2015; Kumar 2015). This indicated that the
reduced concentration of Fe NP may be inducing biochemical changes for soluble
carbohydrate. Ghafari and Razmjoo, (2013) also observed similar results of
increased carbohydrate content of wheat.
Maximum NSC was found in Cu NP (0.25µM) treated plants at 45 DAS
among Cu treatments, followed by Cu NP (0.5µM), Cu salt (0.25µM) and Normal
salt. But in second growth stage, Cu NP (0.25 µM) showed higher NSC as
compared to Cu NP (0.5µM), Normal salt and Cu salt (0.25µM). In soybean,
higher NSC was observed with Cu NP (0.25 µM). This was in conformity with the
61
earlier findings of Hafeez et al. (2015). However, there was not much report
impact of NPs on carbohydrate status of plants.
Among Zn treated plants, higher NSC content was found in plants treated
with Zn NP (1 µM) at first growth stage followed by Zn NP (2µM), Normal salt
and Zn salt (1µM) at 45 DAS. But in the second growth stage higher NSC was
recorded in plants treated with Zn NP (2µM) as compare to Zn NP (1µM), Normal
salt and Zn salt. However lowest NSC content was recorded in plants treated with
Zn salt (1 µM) at 45 DAS and Fe salt (27 µM) at 60 DAS. The NSC content was
found to be higher with Zn NP (1 µM) treated plants was also reported by Sunita et
al. (2013) in Brassica juncea. Singh et al. (2013) reported that sugar content of Zn
NP treated plants were similar to control, in the present study also similar results
were obtained at 60 DAS.
Table 4.14: Effect of micronutrient NPs on TSS and NSC of soybean
Treatment TSS (%) NSC (%)
45
DAS
60
DAS
45
DAS
60
DAS
T1: 100% (Fe + Cu + Zn) =Normal salts 2.65 3.92 3.19 3.43 T2: T1- Fe salt+ Fe NP (54 µM) 3.01 3.50 3.95 3.25 T3: T1- Fe salt + Fe NP (27 µM) 3.21 3.75 3.98 4.31 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 1.94 2.48 2.49 2.59 T5: T1- Cu salt + Cu NP (0.5 µM) 3.48 3.72 4.26 3.83 T6: T1- Cu salt + Cu NP (0.25 µM) 3.64 3.68 4.40 4.05 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 3.04 3.16 3.59 3.37 T8: T1- Zn salt + Zn NP (2 µM) 3.26 3.76 3.27 4.00 T9: T1- Zn salt + Zn NP (1 µM) 3.43 3.77 3.43 3.70 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 2.26 2.62 2.27 3.12 CD (5%) 0.84 0.34 0.83 0.42
62
Fig 4.11: Effect of micronutrient NPs on TSS of soybean
Fig 4.12: Effect of micronutrient NPs on non-structural carbohydrate of soybean
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 DAS
Tota
lS
olu
ble
Sugar
(%
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 DAS
Non
stru
ctura
l ca
rbohydra
te (
%)
63
4.3 Physiological parameters of Soybean
4.3.1 Photosynthesis rate
The Photosynthesis rate presented in Table 4.15 and Fig. 4.15 indicated
significant differences between the treatments at the all growth stages. Out of all
treatments, highest and lowest Photosynthesis rate was obtained in plants treated
with Normal salt at 50 DAS and 70 DAS. Among Fe treated plants, higher
Photosynthesis rate was noted in plant treated with Fe NP (54 µM) followed by Fe
NP (27 µM), normal salt and Fe salt (27µM) at 20 DAS. At 35 DAS and 50 DAS,
higher photosynthesis rate was noted in plants treated with Normal salt followed
by Fe NP (54µM), Fe NP (27µM) and Fe salt (27µM). But at last stage higher
photosynthesis rate was recorded in plants treated with Fe NP (54µM) as compared
to Fe NP (27µM) and normal salt. Among Cu treatments, photosynthesis rate was
found to be significantly higher in plant treated with normal followed by Cu NP
(0.25 µM), Cu NP (0.5 µM) and Cu salt (0.25 µM) at first stages. At 35 DAS,
among Cu treatments, higher photosynthesis rate was recorded in plant treated with
Cu NP (0.25µM) followed by normal salt, Cu NP (0.25µM) and Cu salt (0.25µM).
At 50 DAS, plants showed higher photosynthesis rate with normal salt in
comparison to Cu NP (0.5 µM), Cu NP (0.25 µM) and cu salt (0.25 µM). In the
case of Zn Treatments, higher photosynthesis rate was observed in plants treated
with Zn NP (1µM) in comparison to normal salt, Zn NP (2 µM) Zn salt (1µM) at
20 DAS. Among Zn treated plants, higher photosynthesis rate was noted in plants
treated with Normal salt as compared to Zn NP (2µM), Zn NP (1µM), and Zn salt
(1µM) at 35 DAS and 50 DAS. At last growth stage (70 DAS) there was no
significant impact of Zn treatments.
Table 4.15: Effect of micronutrient NPs on photosynthesis rate of soybean
Treatment Photosynthesis rate (µM m-2
s-1
)
20 DAS 35DAS 50 DAS 70DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 4.75 8.75 13.30 0.35
T2: T1- Fe salt+ Fe NP (54 µM) 9.45 7.30 11.70 5.25
T3: T1- Fe salt + Fe NP (27 µM) 7.45 4.95 10.90 2.65
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 3.40 3.40 7.60 -0.95
T5: T1- Cu salt + Cu NP (0.5 µM) 4.60 7.60 6.95 1.30
T6: T1- Cu salt + Cu NP (0.25 µM) 4.70 11.00 6.50 0.60
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 4.40 3.80 2.80 -0.05
64
T8: T1- Zn salt + Zn NP (2 µM) 3.80 7.10 4.60 1.40
T9: T1- Zn salt + Zn NP (1 µM) 5.35 6.05 2.85 0.00
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 3.50 4.70 2.80 -1.20
CD (5%) 3.114 4.023 6.785 1.539
4.3.2 Transpiration rate
The Transpiration rate furnished in Table 4.16 and Fig 4.16 indicated
significant differences between the treatments at the all growth stages. Among all
the treatments, highest transpiration rate was noted in plants treated with Normal
salt at 50 DAS and lowest Transpiration rate was obtained in plants treated with Fe
salt (27 µM) at last growth stage. Among Fe treatment plants, higher Transpiration
rate was recrded in plant treated with Fe NP (27 µM) followed by Fe NP (54 µM),
normal salt and Fe salt (27µM) at20 DAS. At 35 DAS and 50 DAS, plant showed
higher transpiration rate in plants treated with normal salt as compared to Fe NP
(54 µM), Fe NP (27µM) and Fe salt (27µM). But at last growth stage, maximum
transpiration was recorded in plants treated with Fe NP (54 µM) followed by
normal salt, Fe NP (27 µM) and Fe salt (27µM). Among Cu treatments,
transpiration rate was found to be significantly higher in plant treated with higher
concentration of Cu NP (0.5µM) as compared to Cu NP (0.25 µM), normal salt and
Cu salt (0.25µM) at 20 DAS. At 35 DAS higher transpiration rate was recorded in
plant treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Cu salt (0.25µM)
and normal salt. In the case of 50 DAS, maximum transpiration rate was noted in
plants treated with normal salt in comparison to Cu NP (0.5µM), Cu NP (0.25µM)
and Cu salt (0.25µM), but in last stage Cu NP (0.5µM) performed better followed
by normal salt, Cu NP (0.25µM) and Cu salt (0.25µM). Among Zn Treatments,
higher Transpiration rate was observed in plant treated with Zn NP (2µM) when
compared with other Zn treated plants at 20 DAS. At last growth stage, higher
transpiration was observed in Zn NP (2µM) treated plants followed by Zn NP
(1µM), normal salt and Zn salt (1µM).
65
Table 4.16: Effect of micronutrient NPs on transpiration rate of soybean
Treatment Transpiration rate (mM m-2
s-1
)
20 DAS 35DAS 50 DAS 70 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 3.40 3.50 5.35 2.40
T2: T1- Fe salt+ Fe NP (54 µM) 3.40 3.30 4.45 2.90
T3: T1- Fe salt + Fe NP (27 µM) 3.55 3.20 4.30 2.35
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 3.00 2.70 3.35 1.55
T5: T1- Cu salt + Cu NP (0.5 µM) 3.50 3.55 3.15 2.70
T6: T1- Cu salt + Cu NP (0.25 µM) 3.50 4.25 3.70 2.25
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 3.35 3.50 2.85 2.15
T8: T1- Zn salt + Zn NP (2 µM) 3.55 4.10 2.80 2.60
T9: T1- Zn salt + Zn NP (1 µM) 3.50 4.15 3.10 2.55
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 3.45 3.95 2.55 2.35
CD (5%) NS 0.42 0.84 0.39
4.3.3 Stomatal conductance
Stomatal conductance was found significantly different between treatments
at all growth stages (Table 4.17 and Fig. 4.17). Among Fe treatment plants, higher
Stomatal conductance was recorded in plant treated with Fe NP (27 µM) followed
by normal salt, Fe NP (27 µM) and Fe salt (27µM) at 20 DAS and 35 DAS. At 50
DAS, higher stomatal conductance was observed in plants treated with Fe (57µM)
in comparison to normal salt, Fe NP (27µM) and Fe salt (27µM) but in the last
stage, maximum stomatal conductance was noted in Fe NP (27µM) followed by
normal salt, Fe NP (54µM) and Fe salt (2µM). Among Cu treatments, Stomatal
conductance was found significantly higher in plant treated with Cu NP (0.25µM)
as compared to Cu NP (0.5 µM), normal salt and Cu salt (0.25µM) at first stages.
At 35 DAS Cu treatments plants, higher Stomatal conductance was recorded in
plant treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Cu salt (0.25µM)
and normal salt. In next stage (50 DAS), higher stomatal conductance was noted in
plants treated with normal salt in comparison to Cu NP (0.5µM), Cu NP (0.25µM)
and Cu salt (0.25µM. But at the last stage good impact of Cu treatments was
shown in Cu NP (0.25µM) in comparison to normal salt, Cu NP (0.5µM) and Cu
salt (0.25µM). Among Zn treatments, higher Stomatal conductance was observed
in plant treated with Zn NP (1µM) when compared with the all Zn treated plants
like Zn NP (2 µM) Zn salt (1µM) and normal salt at first two growth stages viz. 20
DAS and 35 DAS. But at 50 DAS Zn treated plants, higher Stomatal conductance
was noted in plants treated with normal salt followed by Zn (2µM), Zn NP (1µM)
66
and Zn salt (1µM). In the case of last growth stage, maximum stomatal
conductance was noted in plants treated with Zn (2µM) as compare to normal salt,
Zn NP (1µM) and Zn salt (1µM). Among all the ten treatments, highest Stomatal
conductance was obtained in plants treated with Cu (0.25µM) at 35 DAS and
lowest is Fe salt (27 µM) at last growth stages.
Gas exchange parameters viz photosynthetic rate, stomatial conductance
and transpiration rate were positively influenced by the NPs. Photosynthetic rate
was enhanced by Fe NP (54µM) and Cu NP (0.25µM) treatments in soybean.
Similar results were obtained by Alidoust and Isoda (2013) with foliar spray of Fe
NPs in soybean. In addition to Fe and Cu being important element in
photosynthetic reaction pathways, it is envisaged that they may be stimulating
photosynthetic electron transport, which might enhance the photosynthetic rate.
Moreover, transpiration rate and stomatal conductance was found to higher with
reduced concentration of NP in both the crops which indicated that the NPs may
positively regulate stomatal opening and closure.
Table 4.17: Effect of micronutrient NPs on stomatal conductance of soybean
Treatment stomatal conductance (µM m-2
s-1
)
20
DAS
35
DAS
50
DAS
70
DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 147.5 124.5 147.5 68.5
T2: T1- Fe salt+ Fe NP (54 µM) 143.5 114.5 154.5 65.5
T3: T1- Fe salt + Fe NP (27 µM) 154.0 144.5 112.0 77.0
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 120.0 97.5 111.0 32.5
T5: T1- Cu salt + Cu NP (0.5 µM) 150.5 143.0 94.0 61.5
T6: T1- Cu salt + Cu NP (0.25 µM) 151.5 176.5 78.5 72.0
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 141.5 130.5 73.5 58.5
T8: T1- Zn salt + Zn NP (2 µM) 152.0 159.0 70.0 69.5
T9: T1- Zn salt + Zn NP (1 µM) 159.5 162.0 68.5 64.5
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 151.5 153.5 59.0 60.0
CD (5%) 16.5 20.1 47.8 16.4
67
Fig
4.1
3:
Eff
ect
of
mic
ronutr
ient
NP
s on p
hoto
syn
thes
is r
ate
of
soybea
n.
-4-202468
10
12
14
16
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
20
DA
S3
5 D
AS
50
DA
S7
0 D
AS
Photosinthesisrate (µM m-2s-1)
68
Fig
4.1
4:
Eff
ect
of
mic
ronutr
ient
NP
s on t
ransp
irat
ion r
ate
of
soybea
n
0123456
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
20
DA
S3
5 D
AS
50
DA
S7
0 D
AS
Transpiration rate (mMm-2s-1)
69
Fig
. 4.1
5:
Eff
ect
of
mic
ronutr
ient
NP
s on s
tom
atal
conduct
ance
of
soybea
n
0
20
40
60
80
10
0
12
0
14
0
16
0
18
0
20
0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
20
DA
S3
5 D
AS
50
DA
S7
0 D
AS
Stomatal conductance (µM m-2s-1)
70
4.3.4 SPAD Value
SPAD value was found significantly difference between treatments
at all growth stages show in table no. 4.18 and fig. 4.18. Among all treatments,
highest SPAD value was obtain in plants treated with Cu NP (0.25µM) at 35 DAS
and lowest SPAD value was obtained in plants treated with Cu salt (0.25 µM) at 70
DAS. Among Fe treatments, higher SPAD value was obtained in plant treated with
Normal salt as compared to Fe NP (27µM), Fe NP (54µM), and Fe salt (27µM) at
20 DAS. But at 35 DAS and 50 DAS, higher SPAD value was recorded in plants
treated with Fe NP (27µM) followed by Fe NP (27µM), Normal salt and Fe salt
(27µM). At last stage maximum SPAD value was noted in plants treated with Fe
NP (54µM) when compared to Fe NP (27µM), Normal salt and Fe salt (27µM).
This was in conformity to the results obtained in maize crop for the trait of
membrane stability (Elanchezhian et al 2015; Kumar 2015). Among Fe treated
soybean plants, chlorophyll content was found to be high in plants treated with Fe
NP (27 µM). This indicated that the reduced concentration of Fe NP may be
inducing biochemical changes for chlorophyll content.
Among Cu treatment plants, higher SPAD Value was obtained in plant
treated with Cu NP (0.25 µM) in comparison of Normal salt, Cu NP (0.25µM) and
Cu salt (0.25µM) at 20 DAS. Plants were show maximum SPAD value in plants
treated with Cu NP (0.25µM) followed by Cu NP (0.5µM), Normal salt and Cu salt
(0.25µM) at both stages 35 DAS and 50 DAS. At last stage, Cu NP (0.25µM) was
show better performance in comparison Normal salt, Cu NP (0.5µM) and Cu salt
(0.25µM). In soybean, higher chlorophyll a content was observed in plants treated
with Cu NP (0.25 µM). This was in conformity with the earlier findings of Hafeez
et al. (2015).
Among Zn Treatments, higher SPAD value was noted in plant treated with
Zn NP (2µM) when compared with the plants treated with Normal salt, Zn NP
(1µM) and Zn salt (1µM) at both stages 20 DAS and 35 DAS. But at 50 DAS Zn
treated plants; higher SPAD value was noted in plants treated with Zn (2µM) as
compared to Zn NP (1µM), Normal salt and Zn salt (1µM). At last stage,
maximum SPAD value was noted in plants treated with Zn NP (1µM) followed by
Zn NP (2µM), Normal salt and Zn salt (1µM).Among Zn treatments, obtained
71
increased chlorophyll content was observed with Zn NP (2 µM). Zinc oxide (ZnO)
nanoparticles resulted in chlorophyll content (Burman et al. 2013).
Table 4.18: Effect of micronutrient NPs on SPAD value of soybean
Treatment SPAD Value
20 DAS 35DAS 50 DAS 70 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 31.91 42.55 29.60 22.15 T2: T1- Fe salt+ Fe NP (54 µM) 29.15 42.85 34.95 31.35 T3: T1- Fe salt + Fe NP (27 µM) 29.85 45.95 39.25 28.55 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 27.10 42.10 27.70 15.70 T5: T1- Cu salt + Cu NP (0.5 µM) 27.40 42.60 30.15 17.75 T6: T1- Cu salt + Cu NP (0.25 µM) 32.00 46.80 31.70 23.90 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 27.25 41.65 24.71 15.25 T8: T1- Zn salt + Zn NP (2 µM) 35.00 43.25 38.90 37.15 T9: T1- Zn salt + Zn NP (1 µM) 31.00 41.95 31.65 42.05 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 28.90 40.90 18.65 21.50 CD (5%) 4.12 2.69 4.42 10.29
72
Fig
. 4.1
6:
Eff
ect
of
mic
ronutr
ient
NP
s on S
PA
D v
alue
of
So
ybea
n
05
10
15
20
25
30
35
40
45
50
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
20
DA
S3
5D
AS
50
DA
S7
0 D
AS
SPAD Value
73
4.4 Morphological parameters of wheat
4.4.1 Plant height
Among all the treatments, plant height was found to be significantly higher
in plants treated with Cu NP (0.5 µM) and lower in plants treated with Fe salt (27
µM) at 60 DAS (Table 4.19). Among Fe treatments, plant height was found to be
similar in Fe NP (54 µM) and Normal salt when compared to Fe salt (27 µM) at 60
DAS. Among Cu treated plants, higher plant height was found in Cu NP (0.5 µM)
followed by normal salt, Cu NP (0.25µM) and Cu salt (0.25µM) treated plant at 60
DAS. Plants treated with normal salt exhibited higher plant height when compared
to plants treated with Zn NP (1 µM), Zn NP (2 µM) Zn salt (1 µM) at 60 DAS.
The positive effects of nano materials on morphology of crop plants have
been reported by several workers (Agrawal and Rathore 2014; Elanchezhian et al.
2014; Elanchezhian et al. 2015). There were selected reports on effect of Fe
nanoparticles on plant morphology (Elanchezhian et al. 2015, Kumar 2015). Fe
nanoparticles were found to increase stem length of basil plants (Peyvandi et al.
2011, Kumar 2015). Salarpour et al. (2013) observed 80% increase in plant height
of Lepidum sativum L treated with nano-iron chelates. In the present study,
increase in height of wheat plants treated with Cu NP (0.5µM) at panicle initiation
stage.
4.4.2 Total Root Length
Total root length was found to be significant at all stages (Table 4.19 and
Fig. 4.20). Among Fe treatments, higher root length was obtained with Fe NP
(54µM) as compared to Fe NP (27µM), normal salt and Fe salt (27µM) at first
growth stage but at second stage, normal salt treated performed better with Fe NP
(27µM), Fe NP (54µM) and Fe salt (27µM). Fe nanoparticles were found to
increase root length of basil plants (Peyvandi et al. 2011, Kumar 2015). In the
present study there increase in root length was observed with normal Fe NP
treatment, which may be attributed to the increased branching of roots of wheat
crop.
Among Cu treated plants, plant treated with normal salt exhibited higher
length followed by Cu salt (0.25µM), Cu NP (0.5µM) and Cu NP (0.25µM) at 30
DAS. However, with Cu treatments, normal salt treated plant obtained higher root
length than Cu NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM) at 60 DAS.
74
Among Zn treated plants maximum root length was observed in plants
treated with Zn NP (1µM) as compared to Zn NP (2µM), normal salt and Zn salt
(1µM) at 30 DAS. But in 60 DAS, higher root length was noted in plants treated
with Zn salt (1µM) in comparison to normal salt, Zn NP (1µM) Zn NP (2µM).
Among all the treatments, highest total root length was observed in plants treated
with Zn salt (1µM) at 60 DAS and lowest root length was noted in plants treated
with Cu NP (0.25µM) at 30 DAS. In wheat, higher root length were observed with
Zn NP (1 µM) treatments. In the present set of investigation, the response in wheat
was similar to soybean and maize crop (Kumar 2015).
Table 4.19: Effect of micronutrient NPs on plant height and root length of wheat
4.4.3 Shoot dry weight
Shoot dry weight of wheat differed between all treatments significantly at 30 DAS
and 60 DAS (Table 4.20 and Fig. 4.21). Among all the treatments, significantly
highest shoot dry weight was recorded in plants treated with Zn NP (2 µM).
Among Fe treatments, significantly higher shoot dry weight was obtained in plants
treated with Fe NP (54µM) followed by Fe NP (27 µM), normal salt and Fe salt
(27 µM) at 30 DAS. But at second stage, higher shoot dry weight was obtained in
plants treated with Fe NP (27µM) as compared to normal salt, Fe NP (54µM) and
Fe salt (27µM). Fe nanoparticles were found to increase shoot dry weight of basil
plants (Peyvandi et al. 2011, Kumar 2015). However, Karimia et al. (2014)
observed that increasing Fe nanoparticle s concentration above 10 ppm reduced
shoot fresh weight, shoot dry weight of green gram, indicating negative effect of
Treatment Plant
height(cm)
Total root length (cm)
60 DAS 30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 65.5 53414.15 108586.70
T2: T1- Fe salt+ Fe NP (54 µM) 65.5 90206.71 51991.34
T3: T1- Fe salt + Fe NP (27 µM) 56.0 54023.31 71747.04
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 34.0 58782.90 57234.35
T5: T1- Cu salt + Cu NP (0.5 µM) 66.0 17085.53 42064.23
T6: T1- Cu salt + Cu NP (0.25 µM) 51.5 9627.24 22780.70
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 46.0 47227.17 22346.60
T8: T1- Zn salt + Zn NP (2 µM) 63.0 70612.72 24978.31
T9: T1- Zn salt + Zn NP (1 µM) 65.0 75127.64 39642.50
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 41.5 22817.21 130143.98
CD (5%) 16.8 32245.9 35380.4
75
Fig 4.17: Effect of micronutrient NPs on plant height of wheat at 60 DAS.
Fig 4.18: Effect of micronutrient NPs on total root length of wheat.
0
10
20
30
40
50
60
70
80
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
60 DASP
lant
heg
ht
(cm
)
0
20000
40000
60000
80000
100000
120000
140000
160000
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DAS
Tota
lro
ot
rength
(cm
)
76
Fe NP on crop growth. In the present study there higher shoot dry weight was
obtained in plants treated with Fe NP (54µM).
Among the plants treated with Cu, higher shoot dry weight was recorded in
plants treated with Cu NP (0.25µM) in comparison to normal salt, Cu NP (0.5 µM)
and Cu salt (0.25µM) at first growth stage. In the second stage higher shoot dry
weight was recorded in plants treated with normal salt followed by Cu NP
(0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Improvement in growth of plants
treated with Cu NPs were reported in wheat (Hafeez et al. 2015), lettuce (Shah and
Belozerova, 2009) and improvement in vase life of chrysanthemum was also
reported with Cu NPs (Hashemabadi et al. 2013). In the present study it was
observed that normal concentration of Cu NP (0.25 µM) had positive influence on
shoot dry weight of wheat at 30 DAS, while normal concentration of Cu NP
(0.5µM) had positively influenced shoot weight at 60 DAS.
Among Zn treated plants, maximum shoot dry weight was obtained in
plants treated with Zn NP (2µM) when compared to all Zn treated plants at 30
DAS. But at 60 DAS, higher shoot dry weight was observed in plants treated with
Zn NP (2µM) followed by normal salt, Zn NP (1µM) and Zn salt (1µM). Lowest
shoot dry weight was noted in plants treated with Cu salt (0.25µM). Ramesh et al.
(2014) reported positive effects of nano-ZnO on shoot-root growth of wheat.
However, Boonyanitipong et al. (2011) observed negative effect of nano-ZnO on
root length of rice (Oryza sativa L.). Nano-ZnO particle was found to enhance
germination and seedling growth of soybean (Sedghi et al. 2013) and vegetable
crops like cabbage, cauliflower and tomato (Singh et al. 2013). This was in
conformity with the present findings wherein Zn NP (2 µM) improved shoot dry
weight of wheat plants at both stages. This shows that normal concentration of Zn
NPs can positively influence shoot growth of plants.
4.4.4 Root dry weight
Root dry weight differed significantly at 30 DAS and at 60 DAS (Table
4.20). Among the Fe treatments, plants treated with high concentration Fe NP
(54µM) exhibited significantly higher root dry weight as compared to Fe NP
(27µM), normal salt and Fe salt (27µM) at 30 DAS. But in the second growth stage
77
higher root dry weight was obtained in plant treated with Fe NP (27µM) as
compared to normal salt, Fe NP (54µM) Fe salt (27µM).
Among Cu treated plants root dry weight was found significantly higher in
plant treated with normal salt followed by Cu salt (0.25µM), Cu NP (0.25µM) and
Cu NP (0.5µM) at 30 DAS. But at 60 DAS, best performance of plants was
observed with Cu NP (0.5µM) as compared to Cu NP (0.25µM), normal salt and
Cu salt (0.25µM). In wheat, maximum root dry weight was obtained with Cu NP
(0.5 µM) treatment at 60 DAS. The effective concentration of Cu NP in wheat (0.5
µM) was similar to the effective concentration in soybean.
Among Zn treatments, higher root weight was recorded in plants treated
with Zn NP (2µM) than Zn NP (1µM), normal salt and Zn salt (1µM) at 30 DAS.
But at 60 DAS, maximum root weight was noted in plants treated with Zn salt
(1µM) as compared to Zn NP (2µM), normal salt and Zn NP (1µM). Among all
treatments, highest root dry weight was obtained in plants treated with Fe NP
(54µM) at 60 DAS and lowest in plants treated with Cu NP (0.5 µM) at 30 DAS.
Higher root dry weight was found to be higher with normal concentration of Zn NP
(2 µM). In the present set of investigation, the response in wheat was similar to
soybean and maize crop (Kumar 2015).
Table 4.20: Effect of micronutrient NPs on shoots and root dry weight of wheat
Treatment Shoot dry weight
(g plant-1
)
Root dry weight
(g plant-1
)
30 DAS 60 DAS 30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 0.90 3.13 0.39 0.55
T2: T1- Fe salt+ Fe NP (54 µM) 1.52 2.79 0.81 0.53
T3: T1- Fe salt + Fe NP (27 µM) 1.06 3.34 0.58 0.77
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.88 2.59 0.36 0.37
T5: T1- Cu salt + Cu NP (0.5 µM) 0.54 1.79 0.18 0.66
T6: T1- Cu salt + Cu NP (0.25 µM) 0.93 2.40 0.23 0.56
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.46 1.09 0.39 0.44
T8: T1- Zn salt + Zn NP (2 µM) 1.80 3.35 0.65 0.74
T9: T1- Zn salt + Zn NP (1 µM) 1.17 2.66 0.57 0.44
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.51 1.90 0.25 0.77
CD (5%) 0.424 0.670 0.310 0.189
78
Fig 4.19: Effect of micronutrient NPs on shoot dry weight of wheat.
Fig 4.20: Effect of micronutrient NPs on root dry weight of wheat.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DASS
hoot
dry
wt.
(g p
lant-1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DAS
Root
dry
wei
ght
(g p
lant-1
)
79
4.4.5 Specific leaf area
The information on specific leaf area (SLA) as influenced by various
treatments during different growth periods are furnished in Table 4.21. Highest
SLA was observed in plants treated with Cu NP (0.5 µM) at both the stages and
lowest SLA was recorded in plants treated with Zn salt (1µM) at 30 DAS. Among
Fe treated plants maximum SLA was observed in plant treated with Fe NP (27 µM)
followed by Fe NP (54 µM), normal salt and Fe salt (27µM) at 30 DAS. At 60
DAS plants treated with Fe NP (54 µM) showed higher SLA as compared to Fe NP
(27µM), normal salt and Fe salt (27µM). There were not many reports on the
impact of nanoparticles on leaf growth parameter. It was reported that nano-iron
fertilizer caused 58 and 47% increase in fresh weight and leaf area index of
spinach (Moghadam et al. 2012). This was in conformity with the present findings
wherein wheat plants treated with reduced concentration i.e. Fe NP (27 µM)
exhibited SLA at 30 DAS. At 60 DAS, normal concentration of Fe NP (54 µM)
treated plants showed higher SLA. This may be due to the growth promoting effect
of reduced concentration of Fe NP (27 µM) on growth characteristics of wheat in
comparison to Fe salts.
In the case of Cu treatments, higher SLA was noted in plant treated with
Cu NP (0.5µM) as compared to normal salt, Cu NP (0.25µM) and Cu salt
(0.25µM). But in the case of second growth stage, higher SLA was noted in plants
treated with Cu NP (0.5µM) followed by Cu NP (0.25µM), Cu salt (0.25µM) and
normal salt. The effective concentration of Cu NP in wheat (0.5 µM) was similar to
the effective concentration in soybean. Leaf growth characteristics like SLA wheat
plant at 30 DAS were found to be higher with normal concentration of Cu NP (0. 5
µM).
Among Zn treated plants higher SLA was recorded in plant treated with Zn
NP (1 µM) as compared to Zn NP (2µM), normal salt and Zn salt (1µM) at first
sages but at 60 DAS, higher SLA was recorded in plants treated with Zn NP (1µM)
followed by normal salt, Zn NP (2µM) Zn salt (1µM). The SLA was found to be
higher with normal concentration of Zn NP (1µM). In the present set of
investigation, the response in wheat was similar to soybean and maize crop (Kumar
2015).
80
4.4.6 Specific leaf weight
There were significant differences in SLW among various treatments at 30
DAS and at 60 DAS (Table 4.21). Out of all ten treatments, highest SLW was
found in plant treated with Cu NP (0.25 µM) at 60 DAS and lowest SLW was
noted in plants treated with Fe salt (27µM) at 30 DAS. Among Fe treatments
plants, SLW was found significantly higher in plant treated with normal salt as
compared to Fe NP (54µM), Fe NP (27 µM) and Fe salt (27µM) at 30 DAS.
However, at 60 DAS, higher SLW was found in plant treated with Fe (27µM) as
compared to Fe NP (54µM) normal salt and Fe salt (27µM). There were not many
reports on the impact of nanoparticles on leaf growth parameter. It was reported
that nano-iron fertilizer caused 58 and 47% increase in fresh weight and leaf area
index of spinach (Moghadam et al. 2012). This was in conformity with the present
findings wherein wheat plants treated with reduced concentration i.e. Fe NP (27
µM) exhibited SLW at 30 DAS. At 60 DAS, normal concentration of Fe NP (54
µM) treated plants showed higher SLW. This may be due to the growth promoting
effect of reduced concentration of Fe NP (27 µM) on growth characteristics of
wheat in comparison to Fe salts.
Among Cu treatments, Cu NP (0.5 µM) and Cu NP (0.25 µM) treated
plants exhibited similar SLW at 30 DAS and found better than Cu salt (0.25 µM)
and normal salt. At 60 DAS, maximum SLW was observed in plants treated with
Cu NP (0.25 µM) followed by normal salt, Cu NP (0.5µM) and Cu salt (0.25 µM).
The effective concentration of Cu NP in wheat (0.5 µM) was similar to the
effective concentration in soybean. Leaf growth characteristics like SLW wheat
plant at 30 DAS were found to be higher with normal concentration of Cu NP (0. 5
µM).
Among Zn treatments, highest SLW was recorded in plant treated with Zn
NP (2 µM) as compared to Zn NP (1µM), Zn salt (1 µM) and normal salt at 30
DAS but it was decreased at 60 DAS and maximum SLW was noted in Zn NP
(1µM) followed by normal salt, Zn NP (2 µM) and Zn salt (1 µM). The SLW were
found to be higher with normal concentration of Zn NP (1µM). In the present set of
investigation, the response in wheat was similar to soybean and maize crop (Kumar
2015).
81
Table 4.21: Effect of micronutrient NPs on SLA and SLW of wheat
Treatment SLA (cm2g
-1) SLW (g cm
-2)
30 DAS 60 DAS 30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 132.55 90.55 0.006 0.006
T2: T1- Fe salt+ Fe NP (54 µM) 158.15 223.90 0.003 0.009
T3: T1- Fe salt + Fe NP (27 µM) 174.15 104.25 0.001 0.011
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 110.75 86.80 0.001 0.001
T5: T1- Cu salt + Cu NP (0.5 µM) 256.55 424.75 0.009 0.003
T6: T1- Cu salt + Cu NP (0.25 µM) 119.00 247.60 0.009 0.017
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 101.25 94.55 0.006 0.001
T8: T1- Zn salt + Zn NP (2 µM) 127.60 220.60 0.015 0.005
T9: T1- Zn salt + Zn NP (1 µM) 168.95 313.65 0.009 0.007
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 54.65 152.70 0.009 0.001
CD (5%) 79.28 100.21 0.006 0.006
4.4.7 Leaf area
Significant differences between the treatments for leaf area were observed
at both the growth stages (Table 4.22). Among all the treatments, highest leaf area
was obtained in the plants treated with Zn NP (2µM) at 60 DAS, and lowest leaf
area was noted in Zn salt (1µM) treated plants at 30 DAS. Among Fe treated plants
maximum leaf area was recorded in Plants treated with Fe NP (54 µM) followed
by Fe NP (27µM), normal salt and Fe salt (27 µM) at both stage. Significant
differences were observed among Cu treated plants, higher LA was observed in
plants treated with Cu NP (0.5 µM) followed by normal salt, Cu NP (0.25µM) and
Cu salt (0.25µM) at first stage, but in second stage maximum LA was obtained in
plants treated with Cu NP (0.5 µM) in comparison to Cu NP (0.25 µM), normal
salt and Cu salt (0.25 µM). Among Zn treatments, higher leaf area was observed in
plants treated with Zn NP (1µM) as compared to Zn NP (2µM), normal salt Zn salt
(1µM) at 30 DAS. At 60 DAS, maximum LA was noted in plants treated with Zn
NP (2µM) than Zn NP (1µM), Zn salt (1 µM) and normal salt.
4.4.8 Leaf area ratio
It is evident form Table 4.22 that out of all ten treatments, highest leaf area
ratio (LAR) was noted in plant treated with Cu NP (0.5µM) at 30 DAS and Cu NP
(0.25µM) at 60 DAS and lowest LAR was recorded in plants treated with Fe salt
(27 µM) at 60 DAS. Among Fe treated plants, LAR was higher in plants treated
with Fe NP (27 µM) as compared to Fe NP (54µM) and Fe salt (27µM), normal
salt and Fe salt (27 µM) at 30 DAS but at 60 DAS, it was decreased and higher
82
LAR was found in plants treated with Fe NP (54µM) followed by Fe NP (27µM),
normal salt and Fe salt (27µM). Among Cu treatments, maximum LAR was
observed in plants treated with Cu NP (0.5µM) than Cu NP (0.25µM) normal salt,
and Cu salt (0.25µM). At 60 DAS, higher LAR was obtained with Cu NP
(0.25µM) in comparison to Cu NP (0.5µM), normal salt and Cu salt (0.25µM).
Among Zn treated plants higher LAR was observed in plant treated with lower
concentration of Zn NP (1µM) as compared to normal salt, Zn NP (2µM) and Zn
salt (1µM) at 30 DAS. But in the case of 60 DAS, maximum LAR was observed in
plants treated with Zn NP (2µM) followed by Zn NP (1µM), Zn salt (1µM) and
normal salt.
There was significant positive influence of Fe NP treatment (54 µM) on
morphology of wheat plant including leaf growth characteristics like leaf area and
LAR 60 DAS. This was in conformity to the findings in soybean plant, where there
was increase in growth parameters with the Fe NP treatments. The effective
concentration of Cu NP in wheat (0.5 µM) was similar to the effective
concentration in soybean. Leaf growth characteristics like Leaf area and LAR of
wheat plant at 30 DAS were found to be higher with normal concentration of Cu
NP (0.5 µM). In wheat, higher LA and LAR were observed with Zn NP (1 µM)
treatments. In the present set of investigation, the response in wheat was similar to
soybean and maize crop (Kumar 2015).
Table 4.22: Effect of micronutrient NPs on LA and LAR of wheat
Treatment Leaf Area (cm2) LAR (cm² g
-1)
30 DAS 60 DAS 30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 76.25 69.64 87.19 25.15
T2: T1- Fe salt+ Fe NP (54 µM) 143.85 155.08 94.77 59.82
T3: T1- Fe salt + Fe NP (27 µM) 123.04 88.04 126.08 27.29
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 73.30 57.46 86.27 18.51
T5: T1- Cu salt + Cu NP (0.5 µM) 77.31 139.94 149.90 79.86
T6: T1- Cu salt + Cu NP (0.25 µM) 72.00 92.18 89.98 86.74
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 41.47 56.25 77.21 23.48
T8: T1- Zn salt + Zn NP (2 µM) 85.97 192.99 77.14 57.61
T9: T1- Zn salt + Zn NP (1 µM) 137.40 143.77 119.13 54.85
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 39.08 103.50 48.31 54.16
CD (5%) 20.82 20.57 NS 23.24
83
Fig 4.21: Effect of micronutrient NPs on leaf area of wheat.
Fig 4.22: Effect of micronutrient NPs on leaf area ratio of wheat.
0
50
100
150
200
250
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DASL
eaf
area
( c
m2)
0
20
40
60
80
100
120
140
160
180
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DAS
Lea
far
ea r
atio
(cm
² g
-1)
84
4.4.9 Number of Tillers
It is evident form Table 4.23 that Number of Tillers was highest in Zn NP
(2µM) treated plant at both stages and lowest in Cu salt (0.25µM) at 30 DAS.
Among Fe treated plants, Number of tiller obtained was higher in plants treated
with Fe NP (54 µM or 27µM) followed by normal salt and Fe salt (27µM) at 30
DAS, but at 60 DAS plants treated with Fe NP (27 µM) showed better
performance as compared to normal salt, Fe NP (54 µM) and Fe salt (27µM).
Among Cu treated plants higher no. of tillers was observed in plant treated with Cu
NP (0.25µM) as compared to Cu NP (0.5µM), normal salt and Cu salt (0.25µM) at
30 DAS, but at 60 DAS, and maximum tillers was noted in plants treated with Cu
NP (0.5µM) in comparison to normal salt, Cu NP (0.25µM) and Cu salt (0.25µM).
Among Zn treatments, best performance was noted in plants treated with Zn NP
(2µM) at 30 DAS followed by Zn NP (1µM), Zn salt (1µM) and normal salt at 30
DAS, but in the case of 60 DAS, maximum number of tiller was recorded in plants
treated with Zn NP (2µM) followed by normal salt, Zn NP (1µM) and Zn salt
(1µM). The plant parameters like number of tillers were all improved due to
application of nano-particle (Adhikari, T. et al.2015). In the present study wheat
plants treated with normal concentration i.e. Fe NP (54 µM) exhibited higher tillers
at 30 DAS.
4.4.10 Root volume
Among Fe treatments, higher root volume was obtained in plants
treated with Fe NP (54µM) as compared to Fe NP (27µM), normal salt Fe salt
(27µM) at 30 DAS, but at 60 DAS, higher root volume was recorded in plants
treated with normal salt as compared to all Fe treatment (Table 4.23). There were
many reports on the impact of nanoparticles on root volume for conformity that
nano particles could enhance and maintain the growth of maize plant. The plant
parameters like root length and root volume were all improved due to application
of nano-particle (Adhikari, T. et al.2015). In the present study wheat plants treated
with normal concentration i.e. Fe NP (54 µM) exhibited higher root volume at 30
DAS.
Among Cu treated plants higher Root volume was observed in plant treated
with normal salt in comparison to Cu NP (0.5µM), Cu salt (0.25µM) and Cu NP
85
(0.25µM) at 30 DAS. But at second stage, normal salt treated plants performed
much better as compared to Cu NP (0.25µM), Cu salt (0.25µM) and Cu NP
(0.25µM). CuO NPs did not inhibit the seed germination upto 2000ppm but root
growth was inhibited at 500 ppm in soybean and chickpea (Adhikari et al. 2012).
However, improvement in growth of plants treated with Cu NPs were reported in
wheat (Hafeez et al. 2015), lettuce (Shah and Belozerova, 2009) and improvement
in vase life of chrysanthemum was also reported with Cu NPs (Hashemabadi et al.
2013). In the present study it was observed that normal concentration of Cu (0.5
µM) had positive influence on above ground part as well as shoot of soybean while
reduced concentration of Cu NP (0.25 µM) had positively influenced root volume
at both stages.
Among all Zn treatments, maximum root volume was observed in plants
treated with Zn NP (2µM) followed by Zn NP (1µM), normal salt and Zn salt
(1µM) at 30 DAS. But at second growth stage, maximum root volume was noted in
Zn salt (1µM) treated plants followed by Zn NP (2µM), Normal salt and Zn NP
(1µM). Among all the treatments, highest in plants treated with Fe NP (54µM)
similar to Zn NP (2µM) at 30 DAS and lowest is Cu NP (0.25µM) at both stages.
In wheat, higher root volume was found with normal concentration of Zn NP (2
µM). In the present set of investigation, the response in wheat was similar to
soybean and maize crop (Kumar 2015).
Table 4.23: Effect of micronutrient NPs on no. of tillers and root volume of wheat
Treatment Number of Tillers
(Plant-1
)
Root volume
(cm3 Plant
-1)
30 DAS 60 DAS 30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 3.0 3.5 3.5 3.5
T2: T1- Fe salt+ Fe NP (54 µM) 4.5 3.0 6.0 3.0
T3: T1- Fe salt + Fe NP (27 µM) 4.5 3.5 4.0 2.5
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 2.5 2.5 2.5 2.5
T5: T1- Cu salt + Cu NP (0.5 µM) 3.0 3.5 2.0 3.0
T6: T1- Cu salt + Cu NP (0.25 µM) 4.0 3.0 1.5 1.0
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 2.0 2.5 2.5 1.8
T8: T1- Zn salt + Zn NP (2 µM) 5.5 4.0 6.0 3.5
T9: T1- Zn salt + Zn NP (1 µM) 4.5 3.0 5.0 2.0
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 4.0 2.5 2.0 4.0
CD (5%) NS NS 1.73 NS
86
4.4.11 Panicle weight
Among all the treatments, highest panicle weight were recorded in plants
treated with Zn NP (2µM) and lowest in Cu salt (0.25µM) treated plants at harvest
(Table 4.24). In the case of Fe treatments, Fe NP (54µM) treated plants performed
better than Fe NP (27µM), normal salt and Fe salt (27µM). Among Cu treatments,
higher Panicle weight was recorded in plants treated with Cu NP (0.25µM) in
comparison to normal salt, Cu NP (0.5µM) and Cu salt (0.25µM). Among Zn
Treated plants, maximum panicle weight was noted in plants treated with Zn NP
(2µM) followed by Zn NP (1µM), Normal salt and Zn salt (1µM).
4.1.12 Grain weight
Out of all the ten treatments, highest grain weight was recorded in plants
treated with Zn NP (1µM) and lowest in Cu salt (0.25µM) treated plants (Table
4.24). Among Fe treatment, higher grain weight was recorded in plants treated with
Fe NP (27µM) in comparison to normal salt, Fe NP (54µM) and Fe salt (27µM). In
the case of Cu treatments, Cu NP (0.25µM) performed better than normal salt, Cu
NP (0.5µM) and Cu salt (0.25µM). Among Zn treatments, maximum grain weight
was obtained in plants treated with Zn NP (1µM) followed by Zn NP (2µM), Zn
salt (1µM) and normal salt.
Table 4.24: Effect of micronutrient NPs on panicle weight and grain weight of
wheat
Treatment
Panicle weight
(g plant-1
)
Grain weight
(g plant-1
)
Harvest Harvest
T1: 100% (Fe + Cu + Zn) = Normal salts 2.9 1.78
T2: T1- Fe salt+ Fe NP (54 µM) 3.7 1.38
T3: T1- Fe salt + Fe NP (27 µM) 3.3 2.50
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 1.9 1.70
T5: T1- Cu salt + Cu NP (0.5 µM) 2.1 1.52
T6: T1- Cu salt + Cu NP (0.25 µM) 3.8 2.56
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.3 0.93
T8: T1- Zn salt + Zn NP (2 µM) 3.9 2.41
T9: T1- Zn salt + Zn NP (1 µM) 3.7 2.97
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 2.7 2.15
CD (5%) NS NS
87
4.4.13 Number of Grain
Among the Fe treatments, Fe NP (27µM) treated plants performed better
than Fe NP (54µM), normal salt and Fe salt (27µM) ( Table 4.25). Among Cu
treatments, maximum number of grain was recorded in plants treated with Cu NP
(0.5µM) in comparison to Cu NP (0.25µM), normal salt and Cu salts (0.25µM). In
the case of Zn treatments, higher number of grain was recorded in plants treated
with Zn NP (1µM) followed by Zn (2µM), Zn salt (1µM) and normal salt. Among
all the treatment, highest number of grain was recorded in plants treated with Zn
NP (1µM) and lowest in plant treated with Cu salt (0.25µM).
4.4.14 Seed index (100 seed weight)
Among the all treatments, highest 100 seed Grain weight (Table 4.26) was
recorded in plants treated with Zn NP (1µM) and lowest in Cu salt (0.25µM).
Among Fe treatments, higher grain weight was recorded in plants treated with Fe
NP (54µM) in comparison to Fe NP (27µM), normal salt, Fe salt (27µM). In the
case of Cu treatments, Cu NP (0.25µM) performed better than Cu NP (0.5µM),
normal salt and Cu salt (0.25µM). Among Zn treatments, maximum Seed Index
was be noted in plants treated with Zn NP (1µM) followed by Zn NP (2µM), Zn
salt (1µM) and normal salt.
In case of wheat plants, the number of tillers and panicle weight was found
to higher with normal concentration of Fe NP (54 µM) and Zn NP (2 µM) while
grain weight and number of grains were found to be higher with Fe NP (27 µM) as
well as Zn NP (1 µM). However, wheat plants exhibited higher tillers, panicle
weight, grain weight and 100 seed weight with reduced concentration of Cu NP
(0.25 µM).
The above findings indicated that nanoparticle at reduced concentration
may be useful and they may act as catalyst for growth of plants. Moreover, it is
also envisaged that the effect of nanoparticles was crop or species specific.
88
Table 4.25: Effect of micronutrient NPs on number of grain and seed index of
wheat
Treatment Number of Grain
(plant-1
)
100 Seed Weight
(g plant-1
)
Harvest Harvest
T1: 100% (Fe + Cu + Zn) = Normal salts 62.8 1.91
T2: T1- Fe salt+ Fe NP (54 µM) 72.3 2.90
T3: T1- Fe salt + Fe NP (27 µM) 75.0 2.16
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 60.0 1.86
T5: T1- Cu salt + Cu NP (0.5 µM) 71.5 2.48
T6: T1- Cu salt + Cu NP (0.25 µM) 65.0 2.71
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 40.3 1.85
T8: T1- Zn salt + Zn NP (2 µM) 84.8 3.14
T9: T1- Zn salt + Zn NP (1 µM) 86.3 4.40
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 76.5 2.49
CD (5%) NS NS
4.4.15 Root Shoot Ratio
Among all the treatments, highest Root Shoot ratio was found in plants
treated with Cu NP (0.25 µM) at 30 DAS and lowest in plants treated with Fe salt
(27 µM) at 60 DAS (Table 4.26). Among Fe treated plants, higher root shoot ratio
was obtained with Fe NP (27 µM) as compared to Fe NP (54 µM) followed by
normal salt and Fe salt (27 µM) at both stages. In the case of Cu treatments,
maximum root shoot ratio was obtained in plants treated with Cu NP (0.25 µM)
than Normal salt, Cu NP (0.5 µM) and Cu salt (0.25 µM) at 30 DAS, but in second
stage, Cu NP (0.25 µM) treated plants exhibited higher root shoot ratio in
comparison to Cu NP 0.5µM), Cu salt (0.25µM) and normal salt. Among Zn
treatments, higher root shoot ratio was recorded in plant treated with Zn NP (1µM)
followed by Zn NP (2µM), normal salt and Zn salt (1µM) at 30 DAS. But at
second growth stage, maximum root shoot ratio was observed in plants treated with
Zn NP (1µM) followed by Zn NP (2µM), normal salt and Zn salt (1µM).
In wheat, root / shoot dry weight were found to be higher with
normal concentration of Zn NP (2 µM). In the present set of investigation, the
response in wheat was similar to soybean and maize crop (Kumar 2015).
89
Table 4.26: Effect of micronutrient NPs on root shoot ratio of wheat
Treatment Root Shoot Ratio
30DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 0.47
0.18
T2: T1- Fe salt+ Fe NP (54 µM) 0.54
0.20
T3: T1- Fe salt + Fe NP (27 µM) 0.60
0.24
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.44
0.14
T5: T1- Cu salt + Cu NP (0.5 µM) 0.34
0.38
T6: T1- Cu salt + Cu NP (0.25 µM) 0.84
0.40
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.25
0.24
T8: T1- Zn salt + Zn NP (2 µM) 0.48
0.22
T9: T1- Zn salt + Zn NP (1 µM) 0.50
0.41
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.36
0.17
CD (5%) NS
0.073
90
Fig
4.2
3:
Eff
ect
of
mic
ronutr
ient
NP
s on
root
shoot
rati
o o
f w
hea
t
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
45
DA
S6
0 D
AS
Root Shoot ratio
91
4.5 Biochemical parameters of Wheat
4.5.1 Chlorophyll content
It is evident from Table 4.27 and Fig 4.28 and 4.29 that the chlorophyll ‗a‘
content did not differ significantly between treatments at both stages. Among all
the treatments higher magnitude of chlorophyll content ‗a‘ was found in plants
treated with Normal salt and Cu NP (0.5µM) at 30 DAS and 60 DAS, respectively
and lower chlorophyll content ‗a‘ was obtained in plants treated with Fe salt
(27µM) at both stages.
Significant chlorophyll ‗b‘ content was observed at both the growth stages
of plants treated with Normal salt at 30 DAS and Fe NP (27 µM) at 60 DAS (Table
4.27 and Fig. 4.28 and 4.29). Among Fe treated plants significant differences were
observed for chlorophyll ‗b‘ in plants treated with Normal salt followed by Fe NP
(27 µM), Fe NP 54 µM) and Fe salt (27 µM) at 30 DAS. In second growth stage,
Fe NP (27 µM) treatments exhibited significantly higher chlorophyll ‗b‘ in
comparison to Normal salt, Fe NP (54 µM) and Fe salt (27 µM). Among Cu treated
plants higher chlorophyll ‗b‘ was recorded in plants treated with Cu NP (0.25µM)
followed by Cu NP (0.5 µM), Normal salt and Cu salt (0.25 µM) at first stage. At
60 DAS, maximum chlorophyll ‗b‘ was observed in plants treated with Normal salt
as compared to Cu NP (0.5µM), Cu NP (0.25µM) and Cu salt (0.25µM). Among
Zn treated plants, chlorophyll ‗b‘ was found be significantly higher in plants
treated with Zn NP (2 µM) than Normal salt, Zn NP (1µM) and Zn salt (1 µM)
treated plants at 30 DAS, however at 60 DAS, maximum chlorophyll b was
recorded in plants treated with Normal salt when compared to Zn NP (2 µM), Zn
NP (1 µM) and Zn salt (1 µM). Among all the treatments, lowest chlorophyll
content ‗b‘ was found in Cu salt (0.25µM) at 30 DAS and Fe salt (27 µM) at 60
DAS.
Significant differences in total chlorophyll content were observed at 30
DAS and at 60 DAS (Table 4.27, Fig. 4.28 and 4.29). Among all the treatments,
highest total chlorophyll content was observed in plants treated with Normal salt at
60 DAS and lowest in plants treated with Fe salt (27µM) at 60 DAS. Among Fe
treated plants significant differences were observed in plants treated with Fe NP
(27 µM) as compared to Normal salt, Fe NP (54 µM) and Fe salt (27 µM) at 30
92
DAS, but at 60 DAS, higher chlorophyll content was observed in Normal salt in
comparison to Fe NP (27 µM), Fe NP (54 µM) and Fe salt (27 µM). At 30 DAS
higher total chlorophyll content was found in plants treated with Cu NP (0.5 µM)
among Cu treatments, followed by Normal salt, Cu NP (0.25 µM) and Cu salt
(0.25µM). However, at 60 DA significant differences were observed in plants
treated with Normal salt as compared to Cu NP (0.5µM), Cu NP (0.25µM), and Cu
salt. Among Zn treated plants higher total chlorophyll Content was recorded in
plants treated with Zn NP (2 µM) when compared to Normal salt, Zn NP (1 µM)
and Zn salt (1 µM) treated plants at both growth stages.
The present study on influence of Fe NP on biochemical parameters of
wheat revealed that there was significant improvement in chlorophyll b and total
chlorophyll content with the application of Fe NP (27 µM). In general, the
biochemical parameters were found to be higher with Fe NP treatments. The
response towards Fe NP in wheat with respect to chlorophyll content was similar to
maize (Elanchezhian et al 2015; Kumar 2015). Biochemical parameters viz. chl b,
total chl, was found to be higher with treatment of Cu NP. However, chl b, were
found to be higher with reduced concentration of Cu NP (0.25 µM). Similar results
were also reported earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al.
2011; Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a
catalyst for improvement in biochemical metabolism of plants. Among Zn
treatments, the chl b and total chl were found to be high with Zn NP treatments
either 2 µM or 1 µM. Similar results were observed in the present set of
investigation with soybean crop as mentioned above with Zn NP as well as by
Singh et al. (2013) and Kumar 2015 with maize crop.
93
Table 4.27: Effect of micronutrient NPs on chlorophyll content of wheat
Treatment Chlorophyll content (mg g-1
)
CHL a CHL b Total
DAS
30 60 30 60 30 60
T1: 100% (Fe + Cu + Zn) = Normal salts 0.45 0.60 0.61 0.68 1.21 1.28
T2: T1- Fe salt+ Fe NP (54 µM) 0.31 0.59 0.56 0.40 1.14 1.01
T3: T1- Fe salt + Fe NP (27 µM) 0.32 0.60 0.64 0.46 1.24 1.08
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.31 0.58 0.53 0.31 1.11 0.94
T5: T1- Cu salt + Cu NP (0.5 µM) 0.31 0.61 0.61 0.49 1.22 1.10
T6: T1- Cu salt + Cu NP (0.25 µM) 0.31 0.61 0.63 0.45 1.20 1.07
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.31 0.58 0.46 0.35 1.07 0.98
T8: T1- Zn salt + Zn NP (2 µM) 0.31 0.60 0.62 0.44 1.21 1.06
T9: T1- Zn salt + Zn NP (1 µM) 0.32 0.61 0.58 0.44 1.19 1.06
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.31 0.60 0.58 0.36 1.18 0.98
CD (5%) NS NS 0.72 0.12 0.05 0.12
4.5.2 Membrane stability
Among all the treatments, highest membrane stability (MS) was observed
in plants treated with Fe NP (54µM) and Fe NP (27µM) at 30 DAS and 60 DAS,
respectively and lowest in plants treated with Fe salt (27µM) and Zn salt (1µM) at
30 DAS and 60 DAS (Table 4.28 and Fig 4.30). Among Fe treatments,
significantly higher MS was observed in plants treated with Fe NP (54 µM) as
compared to Fe NP (27µM), Normal salt and Fe salt (27µM) at 30 DAS, but in
second stage higher MS was found in plants treated with Fe NP (27µM) followed
by Fe NP (54µM), Normal salt and Fe salt (27µM). Among Fe treated soybean
plants, membrane stability was found to be high in plants treated with Fe NP (54
µM). This was in conformity to the results obtained in maize crop for the trait of
membrane stability (Elanchezhian et al 2015; Kumar 2015). This indicated that the
normal concentration of Fe NP may be inducing biochemical changes for
membrane stability.
94
Fig 4.24: Effect of micronutrient NPs on chlorophyll content of wheat at 30 DAS
Fig 4.25: Effect of micronutrient NPs on chlorophyll content of wheat at 60 DAS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
Chlorophyll a Chlorophyll b TotalC
hlo
rop
hyll
Co
nte
nt
(mg g
-1)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
Chlorophyll a chlorophyll b Total
Ch
loro
ph
yll
Conte
nt
(mg g
-1)
95
Maximum MS was found in plants treated with Cu NP (0.25 µM) among
Cu treatments in comparison to Normal salt, Cu NP (0.25µM) and Cu salt
(0.25µM) at first growth stage. At 60 DAS, higher MS % was obtained in plants
treated with Cu NP (0.5µM) when compared to Normal salt, Cu NP (0.25µM) and
Cu salt (0.25µM). The MS were found to be higher with treatment of Cu NP.
However, MS were found to be higher with reduced concentration of Cu NP (0.25
µM). Similar results were also reported earlier as mentioned above (Hafeez et al.
2015; Nekrasova et al. 2011; Elanchezhian et al 2015). The reduced dose of Cu NP
might be used as a catalyst for improvement in biochemical metabolism of plants.
Among Zn treatments, higher MS was observed in plants treated with
Normal salt as compared to Zn NP (2µM), Zn NP (1µM) and Zn salt (1µM).
However at 60 DAS, Normal salt treated plants performed better than Zn NP
(1µM), Zn NP (2µM) and Zn salt (1µM)
4.5.3 Relative water content
Out of all ten treatments, highest RWC was found in plants treated with
Normal salt and Fe NP (27 µM) at 30 DAS and 60 DAS, respectively (Table 4.28).
Among Fe treatments plants, maximum RWC was obtain in plants treated with
Normal salt as compared to Fe NP (54 µM) and Fe NP (27 µM) and Fe salt (27
µM). But in second growth stage, higher MS was recorded in plants treated with Fe
NP (54µM) as compared to Fe NP (27 µM), Normal salt and Fe salt (27 µM).
Among Fe treated soybean plants, RWC was found to be high in plants treated
with Fe NP (54 µM). This was in conformity to the results obtained in maize crop
for the trait of membrane stability (Elanchezhian et al 2015; Kumar 2015). This
indicated that the normal concentration of Fe NP may be inducing biochemical
changes for water relations.
Among Cu treatments, higher RWC was recorded in plants treated with
Normal salt followed by Cu NP (0.5 µM), Cu NP (0.25 µM) and Cu salt (0.25
µM). At 60 DAS, maximum RWC was noted in plants treated with Cu NP (0.5
µM) followed by Normal salt, Cu NP (0.25µM) and Cu salt (0.25µM). The RWC
was found to be higher with treatment of Cu NP. Similar results were also reported
earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011;
96
Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a catalyst
for improvement in biochemical metabolism of plants.
Among Zn treatments, Normal salt treated plants performed better than Zn
NP (2µM), Zn NP (1µM) and Zn salt (1 µM) at 30 DAS. However, at 60 DAS,
higher RWC was observed in plants treated with Zn NP (1µM) as compared to Zn
NP (2µM), Normal salt and Zn salt (1µM). Among all treatments lowest RWC was
found in plants treated with Cu salt (0.25 µM) at 30 DAS.
Table 4.28: Effect of micronutrient NPs on MS and RWC of wheat
Treatment MS %
RWC %
30
DAS
60 DAS 30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 76.40 75.11 67.05 52.72 T2: T1- Fe salt+ Fe NP (54 µM) 81.07 91.42 66.05 58.59 T3: T1- Fe salt + Fe NP (27 µM) 77.88 98.2 63.77 53.73 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 52.76 74.34 48.34 50.88 T5: T1- Cu salt + Cu NP (0.5 µM) 72.39 77.93 57.68 53.73 T6: T1- Cu salt + Cu NP (0.25 µM) 77.35 75.07 47.66 49.55 T7: 100% (Fe + Zn) salts + Cu salt (0.25
µM) 65.66 69.04 43.44 47.79 T8: T1- Zn salt + Zn NP (2 µM) 69.66 66.53 57.71 57.52 T9: T1- Zn salt + Zn NP (1 µM) 68.71 70.84 50.81 58.19 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 66.08 62.04 49.03 45.98 CD (5%) 12.71 10.90 9.16 4.18
97
Fig 4.26: Effect of micronutrient NPs on membrane stability of wheat
Fig 4.27: Effect of micronutrient NPs on Relative water content of wheat
0
20
40
60
80
100
120
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DAS
Mem
bra
ne
Sta
bil
ity (
%)
0
10
20
30
40
50
60
70
80
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DAS
Rel
ativ
e W
ater
Conte
nt
(%)
98
4.5.4 Antioxidant enzyme
4.5.4.1 Super oxide dismutase enzyme activity
Highest SOD enzyme activity was recorded in plants treated Zn NP (1 µM)
at 60 DAS and lowest in plants treated with Cu NP (0.25 µM) at 60 DAS (Table
4.29 and Fig. 4.32). Among Fe treated plant higher SOD activity was observed in
plants treated with Fe NP (27 µM) when compared to Fe NP (57 µM), Fe salt (27
µM) and Normal salt at the first stage but in second growth stage, higher SOD
enzyme activity was observed in plants treated with Fe salt (27µM) followed by Fe
NP (27 µM), Fe NP (54 µM) and Normal salt. The present study on influence of Fe
NP on biochemical parameters of wheat revealed that there was significant
improvement in activity of SOD with the application of Fe NP (27 µM). In general,
the biochemical parameters were found to be higher with Fe NP treatments. The
response towards Fe NP in wheat with respect to antioxidant enzymes was similar
to maize (Elanchezhian et al 2015; Kumar 2015).
Among Cu treated plants maximum SOD activity was recorded in plants
treated with Cu NP (0.5µM) than Cu salt (0.25 µM), Cu NP (0.25µM) and Normal
salt at first stages. At second growth stage, maximum SOD enzyme activity was
noted in plants treated with Cu NP (0.25µM) followed by Cu salt (0.25µM) and Cu
NP (0.5µM) and normal salt. The antioxidant enzyme like SOD activity was found
to be higher with treatment of Cu NP. Similar results were also reported earlier as
mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al
2015). The reduced dose of Cu NP might be used as a catalyst for improvement in
biochemical metabolism of plants.
In the case of Zn treatments, maximum SOD enzyme activity was obtained
in plants treated with Zn NP (1µM) than Zn NP (2µM), Zn salt (1µM) and Normal
salt at 30 DAS. But in second stage it was changed and higher enzyme activity was
recorded in plants treated with by Zn NP (1µM) followed by Zn salt (1µM), Zn NP
(2µM) and Normal salt. The higher SOD activity was found with Zn NP treatments
either 2 µM or 1 µM. Similar results were observed in the present set of
investigation with soybean crop as mentioned above with Zn NP as well as by
Singh et al. (2013) and Kumar 2015 with maize crop.
99
Table 4.29: Effect of micronutrient NPs on Super oxide dismutase enzyme activity
of wheat
4.5.4.2 Catalase enzyme activity
There was not much change in Catalase (CAT) activity among various
treatments (Table 4.30). Out of all ten treatments, highest catalase activity was
recorded in plants treated with Fe NP (54 µM) and Fe NP (27 µM) at 30 DAS and
60 DAS respectively. Lowest catalase activity was noted in plants treated with Cu
NP (0.5 µM) at 60 DAS. Among Fe NP treated plants higher catalase activity was
observed in plants treated with Fe NP (54 µM) followed by Fe NP (27µM),
Normal salt and Fe salt (27µM) at 30 DAS. But in 60 DAS, maximum CAT
activity was noted in plants treated with Fe NP (27 µM) as compared to Fe NP
(54µM), Normal salt and Fe salt (27µM). The present study on influence of Fe NP
on biochemical parameters of wheat revealed that there was significant
improvement enhanced activity of CAT with the application of Fe NP (27 µM).
In general, the biochemical parameters were found to be higher with Fe NP
treatments. The response towards Fe NP in wheat with respect to antioxidant
enzymes was similar to maize (Elanchezhian et al 2015; Kumar 2015).
Maximum catalase activity, among Cu treatments, was recorded in plants
treated with Cu NP (0.25 µM), followed by Cu NP (0.5 µM), Normal salt and Cu
salt (0.25µM) at 30 DAS; and at 60 DAS, higher CAT enzyme activity was
observed in plants treated with Cu NP (0.25 µM) than Normal salt, Cu salt (0.25
µM) and Cu NP (0.5µM). The CAT activity was found to be higher with treatment
of Cu NP. However, CAT was found to be higher with reduced concentration of
Treatment SOD (unit g-1
)
30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 0.00 0.00
T2: T1- Fe salt+ Fe NP (54 µM) 14.92 5.77
T3: T1- Fe salt + Fe NP (27 µM) 15.69 7.18
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 15.36 15.06
T5: T1- Cu salt + Cu NP (0.5 µM) 12.15 7.18
T6: T1- Cu salt + Cu NP (0.25 µM) 9.29 18.24
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 11.72 11.65
T8: T1- Zn salt + Zn NP (2 µM) 10.72 9.85
T9: T1- Zn salt + Zn NP (1 µM) 13.26 18.71
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 9.39 10.00
CD (5%) 5.67 9.35
100
Cu NP (0.25 µM). Similar results were also reported earlier as mentioned above
(Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al 2015). The reduced
dose of Cu NP might be used as a catalyst for improvement in biochemical
metabolism of plants.
Among Zn NP treated plants higher catalase activity was recorded in
plants treated with Zn NP (1µM) when compared to Normal Salt Zn NP (2µM) and
Zn salt (1 µM) at 30. At 60 DAS, CAT enzyme activity was recorded higher in
plants treated with Zn NP (2µM) followed by Zn salt (1 µM), Normal salt and Zn
NP (1 µM). Higher CAT activity was found to be high with Zn NP treatments
either 2 µM or 1 µM. Similar results were observed in the present set of
investigation with soybean crop as mentioned above with Zn NP as well as by
Singh et al. (2013) and Kumar 2015 with maize crop.
4.5.4.3 Peroxidase enzyme activity
Among all the treatments, highest Peroxidase enzyme activity was recorded
in plants treated with Normal at 30 DAS and lowest POX enzyme activity was
recorded in plants treated with Fe salt (27µM) at 60 DAS (Table 4.30). Among Fe
treated plants, higher POX activity was observed in plants treated with Normal salt
when compared to Fe NP (54 µM) Fe, Fe NP (27 µM) and salt (27 µM) at first
stages but at second stage higher POX enzyme activity was recorded in plants
treated with Fe NP (27µM) when compared to Fe NP (54 µM), Normal salt and Fe
salt (27µM). The present study on influence of Fe NP on biochemical parameters
of wheat revealed that there was significant improvement enhanced activity of
POD with the application of Fe NP (27 µM). In general, the biochemical
parameters were found to be higher with Fe NP treatments. The response towards
Fe NP in wheat with respect to antioxidant enzymes was similar to maize
(Elanchezhian et al 2015; Kumar 2015).
Among Cu treated plants, maximum POX activity was recorded in plants
treated with Normal salt than Cu NP (0.25 µM) followed by Cu NP (0.5µM) and
Cu salt (0.25 µM) at 30 DAS, but in the second growth stage, Cu salt (0.25 µM)
performed better than Normal salt, Cu NP (0.5µM), and Cu NP (0.25µM). The
POX activity was found to be higher with treatment of Cu NP. Similar results were
101
also reported earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al.
2011; Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a
catalyst for improvement in biochemical metabolism of plants.
Among Zn treated plants higher POX was obtained in plants treated with
Normal salt followed by Zn NP (2µM), Zn NP (1 µM) and Zn salt (1µM) at both
growth stages. Higher POX activity was found to be high with Zn NP treatments
either 2 µM or 1 µM. Similar results were observed in the present set of
investigation with soybean crop as mentioned above with Zn NP as well as by
Singh et al. (2013) and Kumar 2015 with maize crop.
Table 4.30: Effect of micronutrient NPs on catalase enzyme activity and
peroxidase enzyme activity of wheat
Treatment CAT (unit H2O2-1
min-1
g-1
)
POX (unit H2O2-1
min-1
g-1
)
30
DAS
60
DAS
30
DAS
60
DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 67.2 9.6 139.90 59.82
T2: T1- Fe salt+ Fe NP (54 µM) 72.0 12.8 123.68 60.34
T3: T1- Fe salt + Fe NP (27 µM) 72.0 14.4 122.66 80.23
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 60.8 9.6 124.19 48.78
T5: T1- Cu salt + Cu NP (0.5 µM) 68.8 8.0 123.32 59.16
T6: T1- Cu salt + Cu NP (0.25 µM) 70.4 11.2 123.55 58.09
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 54.4 9.6 122.79 63.65
T8: T1- Zn salt + Zn NP (2 µM) 65.6 14.4 111.57 104.53
T9: T1- Zn salt + Zn NP (1 µM) 70.4 8.0 111.90 79.05
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 64.0 12.8 105.42 52.25
CD (5%) NS NS 14.39 26.96
102
Fig. 4.28: Effect of micronutrient NPs on Super oxide dismutase enzyme activity
of wheat
Fig. 4.29: Effect of micronutrient NPs on peroxidase enzyme activity of wheat
-5
0
5
10
15
20
25
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 DASS
OD
(Un
it g
m-1
)
0
20
40
60
80
100
120
140
160
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
45 DAS 60 ADS
PO
X (
un
it H
2O
2m
in-1
g-1
)
103
4.5.5 Proline content
Proline content was found to be significantly different among treatments at
30 and 60 DAS, respectively (Table 4.31 and Fig. 4.34). Among Fe treated plants,
Proline content was significantly higher in plants treated with Fe NP (27 µM),
when compared to Fe NP (54 µM), Normal salt and Fe salt (27 µM) at first growth
stage but at 60 DAS, plants treated with Fe NP (54 µM) and Normal salt performed
best as compared to Fe NP (27µM) and Fe salt (27 µM). Among Cu treated plants
higher Proline content was recorded in plants treated with Normal salt followed by
Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM) at 30 DAS. At 60 DAS,
higher Proline content was recorded in plants treated with Normal salt followed by
Cu NP (0.25µM), Cu NP (0.5µM) and Cu salt (0.25µM). Among Zn treated plants
higher Proline content was observed in plants treated with Normal salt as
compared to Zn NP (2µM), Zn NP (1 µM) and Zn salt (1µM) at both growth
stages. Among all the treatments, highest Proline content was observed in plants
treated with Fe NP (27 µM) at 60 DAS and lowest Proline content was observed in
plants treated with Cu salt (27 µM) at 30 DAS.
The present study on influence of Fe NP on biochemical parameters of
wheat revealed that there was significant improvement in proline with the
application of Fe NP (27 µM). In general, the biochemical parameters were found
to be higher with Fe NP treatments. The response towards Fe NP in wheat with
respect to proline content was similar to maize (Elanchezhian et al 2015; Kumar
2015).
4.5.6 Total soluble protein
Total soluble protein (TSP) content was found similar during the growth
period (Table 4.31). Protein content was observed to be not significant among all
NPs treated plants at 30 DAS and 60 DAS. Among all the treatments, higher
magnitude of TSP content was observed in plants treated with Fe NP (54µM) at 30
DAS, and lowest TSP content was recorded in plants treated with Fe salt (27µM)
at 60 DAS.
Among Fe treatments, higher protein content was found with normal
concentration of Fe NP (54µM) similar results was observed of increased protein
and of wheat Ghafari and Razmjoo, (2013).
104
Table 4.31: Effect of micronutrient NPs on proline and protein of wheat
Treatment Proline (μM g-1
) TSP (mg g-1
)
30 DAS 60 DAS 30 DAS 60DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 0.008 0.025 1.695 1.705
T2: T1- Fe salt+ Fe NP (54 µM) 0.012 0.025 1.775 1.645
T3: T1- Fe salt + Fe NP (27 µM) 0.015 0.020 1.735 1.645
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 0.005 0.015 1.609 1.546
T5: T1- Cu salt + Cu NP (0.5 µM) 0.005 0.015 1.735 1.705
T6: T1- Cu salt + Cu NP (0.25 µM) 0.006 0.021 1.751 1.695
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 0.004 0.015 1.702 1.678
T8: T1- Zn salt + Zn NP (2 µM) 0.006 0.025 1.734 1.645
T9: T1- Zn salt + Zn NP (1 µM) 0.006 0.019 1.725 1.700
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 0.005 0.018 1.725 1.625
CD (5%) 0.009 0.000 NS NS
4.5.7 Total soluble sugar
Out of all ten treatments, highest TSS content was observed in plants
treated with Fe NP (54 µM) at both stages and lowest TSS contents was noted in
plants treated with Zn salt (1 µM) at 30 DAS and Cu salt (0.25 µM) at 60 DAS
(Table 4.32 and Fig.4.35). Among Fe treated Plants, higher TSS content was found
in plants treated with Fe NP (54 µM) as compared to plants treated with Fe NP (27
µM), Normal salt and Fe salt (27µM) at 30 DAS. However, at 60 DAS, Fe NP
(54µM) treated plants exhibited higher TSS than Normal salt, Fe NP (27 µM) and
Fe salt (27µM). The response towards Fe NP (27 µM) in wheat with respect to
TSS content was found higher. Similar to the reports of enhanced carbohydrate
content with Fe NP in wheat (Ghafari and Razmjoo, 2013), we could also find
significant improvement of TSS in comparison to normal salt treatment.
Maximum TSS content was observed in plants treated with Cu NP (0.25
µM) at 30 DAS among Cu treated plants as compared to Cu (0.5 µM) followed by
Cu salt (0.25 µM) and Normal salt. However, at 60 DAS, maximum TSS was
recorded in plants treated with Normal salt when compared to Cu NP (0.5 µM), Cu
NP (0.25 µM) and Cu salt (0.25 µM). In wheat, biochemical parameters viz. TSS
was found to be higher with treatment of Cu NP. However, TSS was found to be
higher with reduced concentration of Cu NP (0.25 µM). Similar results were also
reported earlier as mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011;
105
Elanchezhian et al 2015). The reduced dose of Cu NP might be used as a catalyst
for improvement in biochemical metabolism of plants.
Among Zn treatments, higher TSS content was found in plants treated with
Zn NP (1 µM) followed by Zn NP (2µM), Normal salt and Zn salt (1µM) at 30
DAS. But at 60 DAS, maximum TSS was observed in plants treated with Normal
salt as compared to, Zn NP (1µM), Zn NP (2µM) and Zn salt (1µM). Among Zn
treatments, TSS was found to be high with Zn NP treatments either 2 µM or 1 µM.
Similar results were observed in the present set of investigation with soybean crop
as mentioned above with Zn NP as well as by Singh et al. (2013) and Kumar 2015
with maize crop
4.5.8 Non-structural carbohydrate
Among all the treatments, maximum NSC content was recorded in plants
treated with Fe NP (27 µM) and normal salt at 30 and 60 DAS, respectively (Table
4.32 and Fig 4.36). Among Fe treated plants higher NSC was observed in plants
treated with Fe NP (27 µM) when compared to all Fe treatments at 30 DAS; and at
60 DAS, Normal salt treated plants showed higher NSC. The response towards Fe
NP (27 µM) in wheat with respect to NSC content was found higher. Similar to the
reports of enhanced carbohydrate content with Fe NP in wheat (Ghafari and
Razmjoo, 2013), we could also find significant improvement of TSS and NSC in
comparison to normal salt treatment.
Maximum NSC was found in Cu NP (0.25µM) treated plants at 30 DAS,
among Cu treatments. But at second growth stage, plants treated with Normal salt
showed higher NSC as compared to Cu NP (0.25 µM), Cu NP (0.5µM) and Cu salt
(0.25µM). In wheat, biochemical parameters like NSC were found to be higher
with treatment of Cu NP. However, NSC were found to be higher with reduced
concentration of Cu NP (0.25 µM). Similar results were also reported earlier as
mentioned above (Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al
2015). The reduced dose of Cu NP might be used as a catalyst for improvement in
biochemical metabolism of plants.
Among Zn treated plants, higher NSC content was found in plants treated
with Zn NP (1 µM) at first growth stage followed by Normal salt, Zn NP (2µM)
106
and Zn salt (1µM) at 30 DAS. But in the second growth stage higher NSC was
recorded in plants treated with Normal salt as compared to Zn NP (1µM), Zn NP
(2µM) and Zn salt (1µM). However lowest NSC was recorded in plants treated
with Zn salt (1 µM) at 30 DAS and Cu salt (27 µM) at 60 DAS. Among Zn
treatments, NSC was found to be high with Zn NP treatments either 2 µM or 1 µM.
Similar results were observed in the present set of investigation with soybean crop
as mentioned above with Zn NP as well as by Singh et al. (2013) and Kumar 2015
with maize crop.
Table 4.32: Effect of micronutrient NPs on TSS and NSC of wheat
Treatment TSS (%) NSC (%)
30 DAS 60 DAS 30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) =Normal salts 3.225 7.555 4.81 5.21 T2: T1- Fe salt+ Fe NP (54 µM) 4.214 8.225 5.90 5.02 T3: T1- Fe salt + Fe NP (27 µM) 3.945 6.329 6.29 4.93 T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 2.865 5.902 4.37 3.86 T5: T1- Cu salt + Cu NP (0.5 µM) 2.195 6.075 4.49 4.30 T6: T1- Cu salt + Cu NP (0.25 µM) 3.699 6.469 5.94 4.39 T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.965 4.045 4.19 3.10 T8: T1- Zn salt + Zn NP (2 µM) 2.485 5.695 4.22 4.34 T9: T1- Zn salt + Zn NP (1 µM) 3.269 7.184 5.40 5.16 T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 1.635 5.555 3.97 4.22 CD (5%) 1.104 0.531 1.22 0.279
107
Fig 4.30: Effect of micronutrient NPs on TSS of wheat
Fig 4.31: Effect of micronutrient NPs on non-structural carbohydrate of wheat
0.0
1.0
2.0
3.0
4.0
5.0
6.0
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
30 DAS 60 DAST
ota
lS
olu
ble
Su
gar
(%
)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
T1 T2 T3 T4 T5 T6 T7 T8 T9 T1030 DAS 60 DAS
No
nS
tru
ctu
ral C
arb
ohydra
te (
%)
108
4.6 Physiological parameters of Wheat
4.6.1 Photosynthesis rate
The Photosynthesis rate furnished in Table 4.33 indicated significant
differences among the treatments at the all growth stages. Out of ten treatments,
highest Photosynthesis rate was obtained in plants treated with Zn NP (2µM) at 60
DAS and lowest in plants treated with Zn salt (1µM) at both stages. Among Fe
treatment plants, higher Photosynthesis rate was noted in plant treated with Normal
salt followed by Fe NP (27 µM), Fe NP (54 µM) and Fe salt (27µM) at both
stages. Among Cu treatments, photosynthesis rate was found significantly higher
in plant treated with Normal salt in comparison to Cu NP (0.5µM), Cu NP
(0.25µM) and Cu salt (0.25 µM) at both stages. In the case of Zn Treatments,
higher photosynthesis rate was observed in plant treated with Normal salt when
compared to plants treated with Zn NP (2µM) Zn NP (1µM) and Zn salt (1 µM) at
first stages. At 60 DAS, maximum photosynthesis rate was noted in plant treated
with Zn NP (2µM) followed by Zn (µM), normal salt and Zn salt (µM).
Table 4.33: Effect of micronutrient NPs on Photosynthesis rate of wheat
Treatment Photosynthesis rate (µM m-2
s-1
)
30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 6.25 6.65
T2: T1- Fe salt+ Fe NP (54 µM) 5.35 5.15
T3: T1- Fe salt + Fe NP (27 µM) 5.45 6.20
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 4.75 3.90
T5: T1- Cu salt + Cu NP (0.5 µM) 4.15 6.45
T6: T1- Cu salt + Cu NP (0.25 µM) 4.00 5.20
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 3.30 4.95
T8: T1- Zn salt + Zn NP (2 µM) 4.10 10.35
T9: T1- Zn salt + Zn NP (1 µM) 3.75 8.7
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 2.65 3.75
CD (5%) NS 2.76
4.6.2 Transpiration rate
Among all the treatments, highest transpiration rate was noted in plants
treated with Zn NP (2µM) and lowest in plants treated with Zn salt (1µM) at 60
DAS (Table 4.34). Among Fe treatment, higher Transpiration rate was noted in
plant treated with Fe NP (54 µM) followed by Fe NP (27 µM), normal salt and Fe
salt (27µM) at both stages.Among Cu treatments, Transpiration rate was found
109
significantly higher in plants treated with higher concentration of normal salt and
Cu NP (0.5µM) as compared to lower concentration of Cu NP (0.25 µM), Cu salt
(0.5µM) at 30 DAS. But at second stage, among Cu treatments, higher
Transpiration rate was recorded in plant treated with Cu NP (0.25µM) and Normal
salt followed by Cu NP (0.5µM) and Cu salt (0.25µM. Among Zn treated plants,
higher Transpiration was observed in plants treated with Norma salt when
compared to all Zn treated plants like Zn NP (2µM), Zn NP (1µM) and Zn salt
(1µM) at 30 DAS, but at 60 DAS, maximum Transpiration rate was recorded in
plants treated with Zn NP (2µM) in comparison to Zn NP (1µM), normal salt and
Zn salt (1µM).
Table 4.34: Effect of micronutrient NPs on transpiration rate of wheat
Treatment Transpiration rate (mM m-2
s-1
)
30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 1.9 2.5
T2: T1- Fe salt+ Fe NP (54 µM) 2.5 2.8
T3: T1- Fe salt + Fe NP (27 µM) 2.0 2.8
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 1.6 2.1
T5: T1- Cu salt + Cu NP (0.5 µM) 1.6 2.4
T6: T1- Cu salt + Cu NP (0.25 µM) 1.3 2.5
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 1.3 1.8
T8: T1- Zn salt + Zn NP (2 µM) 1.7 2.9
T9: T1- Zn salt + Zn NP (1 µM) 1.6 2.8
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 1.3 1.5
CD (5%) NS NS
4.6.3 Stomatal conductance
Stomatal conductance was found significantly different between treatments
at all growth stages (Table. 4.35). Among Fe treatments, higher stomatal
conductance was obtained in plant treated with Fe NP (27µM) as compared to Fe
NP (54µM), normal salt and Fe salt (27µM) at both stages. Among Cu treatment
plants, higher Stomatal conductance was obtained in plant treated with higher
concentration of Cu NP (0.5 µM) in comparison to Normal salt, Cu NP (0.25µM)
and Cu salt (0.25µM) at 30 DAS. But at 60 DAS, plants exhibited maximum
Stomatal conductance with Normal salt followed by Cu NP (0.25µM), Cu NP
(0.5µM) and Cu salt (0.25µM). Among Zn Treatments, higher Stomatal
conductance was noted in plant treated with normal salt when compared to Zn NP
(1µM), Zn NP (2 µM) and Zn salt (1µM) at 30 DAS. But at 60 DAS, among Zn
treated plants, higher Stomatal conductance was noted in plants treated with Zn
110
(2µM) as compared to Normal salt, Zn NP (1µM) and Zn salt (1µM). Among all
treatments, highest stomatal conductance was obtained in plants treated with Fe NP
(27µM) at both stages and lowest Stomatal conductance was obtained in plants
treated with Zn salt (1 µM) at both stages.
Alidoust and Isoda (2013) was reported that foliar spray of Fe NPs in
soybean Gas exchange parameters viz photosynthetic rate, stomatial conductance
and transpiration rate were positively influenced by the NPs. In present study,
photosynthetic rate was enhanced by Fe NP (54µM) and Cu NP (0.25µM)
treatments in wheat. In addition to Fe and Cu being important element in
photosynthetic reaction pathways, it is envisaged that they may be stimulating
photosynthetic electron transport, which might enhance the photosynthetic rate.
Moreover, transpiration rate and stomatal conductance was found to higher with
reduced concentration of NP in both the crops which indicated that the NPs may
positively regulate stomatal opening and closure.
Table 4.35: Effect of micronutrient NPs on stomatal conductance of wheat
Treatment Stomatal Conductance
(µM m-2
s-1
)
30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 71.5 106.5
T2: T1- Fe salt+ Fe NP (54 µM) 79.0 109.5
T3: T1- Fe salt + Fe NP (27 µM) 102.5 117.0
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 59.0 81.5
T5: T1- Cu salt + Cu NP (0.5 µM) 78.5 88.5
T6: T1- Cu salt + Cu NP (0.25 µM) 44.0 91.5
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 43.5 60.5
T8: T1- Zn salt + Zn NP (2 µM) 44.0 108.5
T9: T1- Zn salt + Zn NP (1 µM) 55.0 102.5
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 42.0 50.5
CD (5%) NS NS
111
Fig
4.3
2:
Eff
ect
of
mic
ronutr
ient
NP
s on p
hoto
syn
thes
is r
ate
of
wh
eat
02468
10
12
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
30
DA
S6
0 D
AS
Photosinthesisrate (µM m-2s-1)
112
4.6.4 SPAD value
SPAD value was found significantly different between treatments at
all growth stages (Table. 4.36 and fig. 4.38). Among all treatments, highest SPAD
value was obtained in plants treated with Zn NP (2µM) and lowest SPAD value
was obtained in plants treated with Zn salt (1 µM) at 60 DAS. Among Fe
treatments, higher SPAD value was obtained in plant treated with Normal salt as
compared to Fe NP (27µM), Fe NP (54µM), and Fe salt (27µM) at first stages. But
in second stage, higher SPAD value was recorded in plants treated with Fe NP
(54µM) followed by Normal salt, Fe NP (27µM) and Fe salt (27µM). In general,
the biochemical parameters were found to be higher with Fe NP treatments. The
response towards Fe NP in wheat with respect to chlorophyll content was similar to
maize (Elanchezhian et al 2015; Kumar 2015).The present study on influence of Fe
NP on biochemical parameters of wheat revealed that there was significant
improvement in chlorophyll content with the application of Fe NP (54 µM).
Among Cu treatment plants, higher SPAD Value was obtained in plant
treated with Normal salt in comparison to Cu NP (0.5 µM), Cu NP (0.25µM) and
Cu salt (0.25µM) at 30 DAS. But at 60 DAS, plants showed maximum SPAD
value in plants treated with Normal salt followed by Cu NP (0.25µM), Cu NP
(0.5µM) and Cu salt (0.25µM). Among Cu treatments, chlorophyll was positively
influenced by Cu NPs Similar results were also reported earlier as mentioned
above (Hafeez et al. 2015; Nekrasova et al. 2011; Elanchezhian et al 2015).
Among Zn Treatments, higher SPAD value was noted in plant treated with
Normal salt when compared with the plants treated with Zn NP (1µM), Zn NP (2
µM) and Zn salt (1µM) at 30 DAS. But at last growth stage, among Zn treated
plants; higher SPAD value was noted in plants treated with Zn (2µM) as compared
to Normal salt, Zn NP (1µM) and Zn salt (1µM). Among Zn treatments,
chlorophyll content was found to be high with Zn NP treatments 2µM. Similar
results were observed in the present set of investigation with soybean crop as
mentioned above with Zn NP as well as by Singh et al. (2013) and Kumar 2015
with maize crop.
113
Table 4.36: Effect of micronutrient NPs on SPAD value of wheat
Treatment SPAD Value
30 DAS 60 DAS
T1: 100% (Fe + Cu + Zn) = Normal salts 48.05 48.10
T2: T1- Fe salt+ Fe NP (54 µM) 43.70 48.30
T3: T1- Fe salt + Fe NP (27 µM) 44.40 46.55
T4: 100% (Cu + Zn) salts + Fe salt (27 µM) 42.85 44.20
T5: T1- Cu salt + Cu NP (0.5 µM) 40.45 44.00
T6: T1- Cu salt + Cu NP (0.25 µM) 40.15 46.25
T7: 100% (Fe + Zn) salts + Cu salt (0.25 µM) 39.00 41.05
T8: T1- Zn salt + Zn NP (2 µM) 41.25 49.75
T9: T1- Zn salt + Zn NP (1 µM) 41.30 43.05
T10: 100% (Fe + Cu) salts + Zn salt (1 µM) 40.60 38.35
CD (5%) 4.28 5.61
114
Fig
. 4.3
3:
Eff
ect
of
mic
ronutr
ient
NP
s on S
PA
D v
alue
of
whea
t.
0
10
20
30
40
50
60
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0
30
DA
S6
0 D
AS
SPAD Value
115
CHAPTER –V
SUMMARY AND CONCLUSIONS
5.1 SUMMARY
The investigation entitled ―Physiological responses of soybean and wheat
crops towards nanoparticle based micronutrients fertilization‖ was carried out at
ICAR-Indian Institute of Soil Science, Bhopal (M.P.) during – 2015-16. The major
objective of this study was to study the effect of different concentrations of
nanoparticles viz. Fe, Cu and Zn on plant morphological, physiological and
biochemical characteristics of soybean and wheat. The material for study
comprised of soybean and wheat were grown in CRD (Completely Randomized
Block Design) with five replications. Research work has been carried out in the
experimental screen house of ICAR-IISS Bhopal.
1. All the nano-micronutrients positively influenced the growth and yield
traits of soyeban and wheat plants for most of the parameters studied.
2. Plant height of Soybean and wheat was enhanced by nano- micronutrient
fertilization of Cu NP (0.5 µM). However, Zn NP (2µM) also promoted
plant height in soybean.
3. Root length of soybean was found to be maximum with Fe NP (27 µM).
Cu NP (0.5 µM) treated plants also exhibited maximum root length in
wheat.
4. Leaf growth characteristics of soybean viz. leaf area and LAR were found
to be promoted by Zn NP (2µM) and Fe NP (27µM). SLA and SLW were
found to be higher with Fe NP (27µM) and Cu NP (0.5µM). In wheat, leaf
area was found to be higher in Fe NP (54µM) and Zn NP (2µM), but LAR,
SLA and SLW were found to be positively influenced by Cu NPs.
5. Plant biomass of soybean and wheat was enhanced by Zn NP (2µM) and Fe
116
NP (54 µM) treatments.
6. Grain yield was positively affected by increased concentration of Zn NP
(2µM) and Fe NP (54µM) in soybean. However, in the case of wheat,
maximum grain yield was found with reduced concentration of NPs viz. Zn
NP (1µM), Fe NP (27µM) and Cu NP (0.25 µM).
7. Membrane stability was found to be higher with Cu NP (0.5 µM) and Fe
NP (27µM) treatments in soybean. However, in case of wheat greater MS
and RWC was obtained with Fe NP (54µM). While RWC was positively
influenced by Fe NP (27 µM) in Soybean.
8. Proline accumulation was found to be increased with Fe NP (54 µM and
27µM) treatments in soybean and wheat. Total soluble protein content in
soybean and wheat was higher in increased concentration of micronutrient
NPs viz. Fe NP (54 µM) and Zn NP (2 µM). However, TSP was found
positively influenced by reduced concentration of Cu NP (0.25 µM) in both
crops.
9. Anti-oxidant enzyme - SOD was found to be higher with micronutrient
fertilization of Fe NP (54µM) in soybean and reduced concentration of NPs
viz. Fe NP (27 µM), Zn (1 µM) and Cu NP (0.25 µM) in wheat. CAT
activity was enhanced by Cu NP (0.25µM) and Fe NP (54 µM) in soybean
and Fe NP (54 µM) in wheat crop. POX activity was improved by Zn NP
(2µM) in wheat.
10. Total Chlorophyll and chl b content was positively influenced by reduced
concentration of Fe NP (27 µM) in both the crop. While chl a content was
enhanced by Cu NP (0.5 and 0.25µM) in soybean and wheat. SPAD value
recorded also corroborated that reduced concentration of Fe NP (27 µM)
positively influenced greenness of the leaf.
11. Total soluble sugars and non-structural carbohydrate content was found to
be higher with Cu NP (0.25µM) and Fe NP (27µM) treatments in soybean.
However, TSS and NSC were positively influenced by Fe NP (54 µM) and
Fe NP (27 µM), respectively, in wheat crop.
117
12. Photosynthesis rate was enhanced by Fe NP (54µM) and Cu NP (0.25µM)
in soybean. But in the case wheat photosynthesis rate was enhanced in Zn
NP (2µM) treatments. The transpiration rate was found to be higher with
NP treatments viz. Fe NP (27µM and 54 µM), Zn NP (2µM) and Cu NP
(0.25µM) in both the crops. The stomatal conductance was positively
enhanced by reduced concentration of NPs viz. Zn NP (1µM), Cu NP
(0.25µM) and Fe NP (27µM) in soybean. In the wheat crop, maximum
stomatal conductance was obtained in reduced concentration of Fe NP
(27µM) and Cu NP (0.25µM).
5.2 CONCLUSIONS
1. In Soybean, the nano-micronutrient fertilization of plants with normal
concentration of NPs has positively influenced the shoot growth and
biochemical metabolism of plants. However, reduced concentration of
NPs had positively influenced root growth and gas exchange parameters
of plants.
2. In wheat, the nano-micronutrient fertilization of plants with Fe NPs/ Cu
NPs / Zn NPs had positively influenced most of the morphological
parameters while reduced concentration of Fe NPs/ Cu NPs and Zn NPs
had positively influenced biochemical metabolism of plants. Gas
exchange parameters were also positively influenced by NPs.
3. The above findings indicated that the effect of nanoparticles were crop
or species specific. Moreover, it is also envisaged that nanoparticle at
reduced concentration may be useful for the crop and they may act as
catalyst for growth, metabolism and yield of plants.
5.3 SUGGESTIONS FOR FUTURE RESEARCH WORK
The present investigation on nano sized micro-nutrient fertilization
revealed that impact of NPs were crop specific, which may be due to the different
size, shape of NPs which on entering the plant system may influence the growth
and metabolism differentially. It is observed that reduced concentration of NPs
affected positively the traits associated with growth and metabolism.
118
The concentration of NPs also should be optimized and standardized for
further exploitation in the manipulation of growth, yield potential and quality of
the crops.
There should be study on the impact of different shape, size of NPs on plant
system and integration on NPs in plant system to understand the basic mechanism
of NP uptake and utilization in the plants.
There should be more study on transport of NP inside plant system and
their movement to animal system for their impact on metabolism of animal system
to ascertain their toxicity for health concern of animal or human system
119
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APPENDICES
Appendix A
Table: Metrological data during crop period 2015-16
Month Temperature Maximum Temperature Minimum
Jul-15 30.7 25.2
Aug-15 29.3 24.6
Sep-15 32.5 24.8
Oct-15 34.0 23.3
Nov-15 31.1 18.3
Dec-15 26.6 11.8
Jan-16 26.7 11.2
Feb-16 29.0 13.8
Mar-16 35.4 20.4
Source - Central Institute of Agricultural Engineering, Bhopal (M.P.)
129
Ap
pen
dix
B
Tab
le:
Com
posi
tion o
f a
modif
ied H
oag
land n
utr
ient
solu
tion f
or
gro
win
g p
lants
Co
mp
ou
nd
M
ole
cu
lar
weig
ht
(g m
ol–
1)
Co
nce
ntr
ati
on
of
sto
ck s
olu
tio
n
(mM
)
Co
nce
ntr
ati
on
of
sto
ck s
olu
tion
(g l
–1)
Volu
me o
f st
ock
solu
tio
n p
er l
iter
of
fin
al s
olu
tio
n
(ml)
Ele
men
t
Fin
al
co
ncen
trat
ion
Ele
men
t (μ
M)
(pp
m)
Ma
cro
nu
trie
nts
KN
O3
10
1.1
0
1,0
00
101.1
0
6.0
N
1
6,0
00
2
24
Ca(N
O3)2
4H
2O
2
36
.16
1,0
00
236.1
6
4.0
K
6
,00
0
23
5
NH
4H
2 P
O4
11
5.0
8
1,0
00
115.0
8
2.0
C
a
4,0
00
1
60
MgS
O4·7
H2
24
6.4
8
1,0
00
246.4
8
1.0
P
2
,00
0
62
S
1,0
00
3
2
Mg
1
,00
0
24
Mic
ron
utr
ien
ts
KC
l 7
4.5
5
25
1.8
64
Cl
50
1.7
7
H3B
O3
Mn
SO
4.H
2O
61
.83
16
9.0
1
12
.5
1.0
0.7
73
0.1
69
B
Mn
25
2.0
0.2
7
0.1
1
Zn
SO
4.7
H2O
2
87
.54
1
.0
0.2
88
2.0
Z
n
2.0
0
.13
Cu
SO
4.5
H2O
2
49
.68
0
.25
0.0
62
Cu
0
.5
0.0
3
H2M
oO
4
(85%
Mo
O3)
16
1.9
7
0.2
5
0.0
40
Mo
0.5
0.0
5
NaF
eD
TP
A
(10%
)
46
8.2
0
64
30
.0
1.0
Fe
53
.7
3.0
0
Fe
3O
4 N
ano
2
31
.53
6
4
14
.81
1
.0
Fe
53
.7
3.0
0
Cu
O N
an
o
79
.55
0
.25
0.0
19
8
2.0
C
u
0.5
0
.03
Zn
O N
an
o
81
.39
1
.0
0.0
81
39
2
.0
Zn
2
.0
0.1
3
130
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