2.

REVIEW OF LITERATURE

Global population is increasing drastically and threatening the food and nutritional
security. With the stagnant and limited availability of arable land and water resources
achieving food security is a major challenge in 21st century. According to predictions, to meet
the demand of food for the increasing population at a reducing cultivable land the food
production must be doubled in next two decades. Therefore, increasing food grain production
and nutritional security is essential.

Human beings and animals depend on plant products for their primary nutritional
requirement and if the plants fail to provide adequate nutritional quantities then it leads to
nutritional insecurity in population. Excessive application of major nutrients especially
Nitrogen, Phosphorous and Potassium fertilizers to the high yielding varieties lead to the
occurrence of micronutrient deficiency in the soils of many developing countries. Zinc and
iron deficiency is reported as the major ones worldwide compared to other nutrients (Chen
and Barak, 1982). Millions of hectares of cereal growing areas are suffering with zinc
deficiency leading to reduction in yield and quality of food crops (Graham et al., 1992).
Estimates reveal that about 50 percent of soils used for cereal production in the world have
low levels of plant available Zn (Graham and Welch, 1996). Results of 298 soil samples
collected world wide from different countries suggested that Zn deficiency is the major
problem found in maximum number of soils (Sillanpaa and Vlek, 1985). In India, 2.52 lakhs
surface soil samples analysis revealed that 49 percent of soils are majorly deficient in
micronutrient especially Zn. For reclamation of sodic soils, zinc is recommended in package
of practices (Singh, 2010).

In India, Nitrogen, phosphorus, and potassium are the three major nutrients for crop
growth and zinc is considered as the fourth most important nutrients limiting crop yield and is
required for many basic physiological processes of plants viz chlorophyll synthesis, pollen
formation, fertilization and germination (Kaya and Higgs, 2002; Pandey, et al., 2006;
Cakmak, 2008). Among the micronutrients, zinc and manganese provide the susceptibility to
plants under abiotic stresses especially drought and other biotic stresses (Khan, et al., 2003).

Zn is a basic micronutrient for both plants and people and around three billion
individuals are at present micronutrient malnourished. Zinc deficiency is known to cause a
few dangerous health problems in people. In this view, bio fortification has been utilized in


the vast majority of the plant species and the objective of bio fortification is to create plants
that have a higher content of bio available supplements in their consumable parts and is a
dependable answer for Zn insufficiency issue.

2.1 Zinc in soil and its deficiency: A global scenario

The basic/critical soil levels for occurrence of zinc deficiency are between 0.6 ppm
and 2 mg zinc kg–1 relying upon the strategy for extraction utilized. Calcareous soils (pH> 7)
with moderate to high organic content (>1.5 % natural C) are probably going to be Zn
insufficient because of high HCO3– in the soil solution.

Low Zn availability in neutral and calcareous soil is one of the widespread nutritional
problem in crop plants. Zn deficiency is observed especially in cereals globally and
particularly in Turkey, Australia, China and India. Rice and wheat cereal crops suffer severely
due to zinc deficiency. Eighty percent grain yield reduction along with low levels of zinc in
grains has been observed (Cakmak, 1998). This has serious implication for human health in
countries where consumption of cereal-based diets predominates. Further, plants grown on
zinc-deficient soils tend to accumulate heavy metals, which again is a potential human health
hazard (Hart et al., 1998).

In India, there is a zinc deficiency in 10 M ha of region and 85 percent of rice-wheat

cropping takes place in the Indo gangetic plain, which has calcareous soils with elevated pH
and therefore low availability of Zn. Improving crop production in these areas by applying
zinc to soil is a vital need to feed the country, however, its not economical or eco friendly in
long run because only 20 percent of the zinc applied is available for plant uptake and rest is
adsorbed and becomes immobile and toxic in soil and crop growth. Plant genotypes with
greater resistance to zinc deficiency need to be selected and/ or bred both in terms of greater
grain output and greater grain zinc content. This strategy is plausible given the large
genotypic variations in zinc sensitivity among plant species especially when its availability to
the roots is restricted (Rengel and Graham, 1995 a, b).

2.2 Relative Sensitivity of Crops to Zinc Deficiency

Zinc deficiency is known to affect a wide range of crops in many parts of the world,
genotypic differences makes the crops more susceptible to deficiency than others. According
to the reports of ILZRO, 1975 (International Lead Zinc Research Organisation), the relative
 

sensitivity of crops to zinc deficiency is classified as highly sensitive, medium sensitive and
low sensitive.

Crops such as maize, rice, sorghum and beans are extremely susceptible to zinc
deficiency and wheat is less sensitive. Even though wheat crop is less sensitive to zinc
deficiency compared to other crops but still it is affected in majority of cultivated areas.

2.3 Zn is an essential micronutrient in human

Zn and iron are very important elements from human nutritional point of view and
adult human body contains nearly 2g Zn. Zinc is present evenly in the human body as a
component of thousands of Zn-binding proteins or Zn metal-proteins and as nucleic acids. So,
unlike iron zinc is not easily detectable and giving diagnosis is challenging (Hambidge, et al.,
1986).

Zn is involved in signal transduction, gene expression, apoptosis (Chesters, 1978) and

structure (biological membranes, enzymes as numerous central roles in DNA and RNA
metabolism) and function (transcription factors, hormonal receptor sites) of proteins (Donald,
2000). Zn metal enzymes and Zn-dependent enzymes are involved in nucleic acid metabolism
and cellular proliferation, differentiation and growth. Zn plays major role in maintaining
human immune system catalytic or structural component in various body enzymes. Optimum
dietary intake is 15 mg Zn per day and Zn deficiency in humans leads to weakness in body
muscles, skin problems hair and memory loss. Zn deficiency in man leads to infertility and
insufficient intake of Zn during pregnancy leads to stunted brain development of the foetus.
According to FAO/WHO, (2002) the recommended daily allowance (RDA) of male is 11 mg,
female is 9 mg and pregnant and lactating women 13 to 14 mg. Infants from 7 months to 3
years need 3 mg, 4 to 8 years need 5 mg and children from 9 to 13 years need 8 mg of Zn
daily (Hotz and Brown, 2004). The foods rich in Zn are nuts like roasted peanuts, almonds,
walnuts, oats and dairy products like yogurt, cheese and milk, beef, pork and chicken
(Cakmak, 2002).

2.4 Zinc is an essential micronutrient in plant

Zinc is an essential plant micronutrient which is involved in many physiological

functions and its inadequate supply will reduce crop yields. The essentiality of Zn in plants
was first shown in maize, and subsequently in barley and dwarf sunflower (Sommer and
 

Lipman, 1926). Zinc deficiency leads to decrease in the synthesis of carbohydrate,

cytochrome, nucleotide, auxin and chlorophyll. Under stress conditions Zn deficiency leads to
membrane damage and susceptibility to abiotic stresses especially heat stress. Further, Zn-
containing compounds are also hindered like alcohol dehydrogenase, carbonic anhydrase,
Cu/Zn superoxide dismutase, alkaline phosphatase, phospholipase, carboxypeptidase, and
RNA polymerase. Depending on the zinc level, zinc deficiency status of plants can be
classified as follows: less than 10 mg kg–1 – definite zinc deficiency; between 10 and 15 mg
kg–1 – likely to be zinc deficient; between 15 and 20 mg kg–1 – likely to be zinc-sufficient;
more than 20 mg kg–1 – Zn-sufficient (Hotz and Brown, 2004).

Zn is a co-factor of important antioxidant enzymes like super oxide dismutase (SOD)

and peroxidase. Studies of Misra and Srivastava, 2010 in Geranium plant clearly indicates that
many physiological processes like chlorophyll content; net photosynthetic rate, fresh weight
and dry weight were found normal under optimum zinc supply in Hoagland nutrient solution.
Whereas, both toxicity and deficiency have negative effects on the plant. Zn is also found
essential in the synthesis of tryptophan, a precursor of indole acetic acid (IAA). Zn-deficient
leads to reduction in IAA synthesis or enhanced degradation of IAA and the disorder is called
“little leaf disease" (Cakmak et al., 1989). Zn deficiency symptoms are identified by
characteristically small misshapen fruit, often dieback of twigs, mottled leaves and highly
reduced leaf size (Spiegel-Roy and Goldschmidt, 2008).

Carbonic anhydrase (CA) is another important Zn requiring enzyme which catalyzes

the inter conversion of HCO3 and CO2 in C4 photosynthetic pathway. Among wheat zinc
efficient and inefficient genotypes studied, zinc efficient genotypes showed higher expression
and high activity of CA and Cu/Zn SOD (Hacisalihoglu and Kochian, 2003). An investigation
done in algae revealed that when zinc is supplied in optimum concentration the algal cell
specific growth rate and cell density increased two-fold while extracellular CA activity
increased 10.5 times higher than that in control (Karim et al., 2011).

2.5 Nanotechnology and its application in agriculture

The increasing world population has led to increases in food production. To increase
food production it is necessary to use the different technologies in agriculture.
Nanotechnology appears to have an encouraging potential and alternative technology to make
more supportable future of agriculture. Nanotechnology has been described as relating to
 

materials, systems and processes which operate at a scale of 100 nanometres or less (Mousavi
and Rezai 2011, Srilatha 2011, Ditta 2012). Use of nano particles as fertilizer has a countless
opportunity to increase crop yield and minimize environmental hazard. Nanotechnology
shows potential in the field of interdisciplinary research. Application of nanotechnology
include improvement of agricultural production using bio conjugated NPs (encapsulation),
animal breeding , transfer of DNA in plants for the growth of insect pest resistant varieties,
nano formulations of agrochemicals for applying pesticides and fertilizers for crop
improvement, nanosensors/nanobiosensors in crop protection for the identification of diseases
and residues of agrochemicals, plant disease diagnostics, animal health, poultry production
and postharvest management. The effect of NPs on plants varies from species to species and
plant to plant (Farooqui et al., 2016).

In recent days, nanofertilizers are emerging as an alternative to conventional fertilizers

and predicting to improve nutrient use efficiency and minimize cost of production. Nano
particles/nutrients will be released slowly throughout crop growth period and so available to
the plants throughout its lifecycle and avoid waste by leaching. Fertilizer particles are
sometimes coated / cemented with nano sized membranes helps for slow and steady release of
inside nutrients and further helps in regulation of releasing rate. A nano-composite consists of
nitrogen, phosphorous, potassium and important micronutrients (zinc and iron), mannose and
amino acids that increase the uptake and utilization of nutrients by grain crops (Tulasi et al.,
2015).

Nanotechnology is a versatile field and has found applications in pharmaceuticals,

medical, industrial and agriculture fields of science. Nanotechnology has the potential to
revolutionize the agriculture with new tools to enhancing the ability of plants to absorb
nutrients and can interact with bio molecules directly inside the living cell. Employment of
nano particles in agriculture is a present ambition for improving crop growth, yield and
control of plant diseases. Particles of size below 100 nm fall in the group of nano particle and
research in the agricultural crops is increasing in the present scenario (Savithramma et al.,
2012).

2.5.1 Nano particles

Nano particles (NPs,) are atomic or molecular aggregates with the dimensions
between 1 and 100 nm (Ball, 2002: Roco, 2003), that can drastically modify their physico-
 

chemical properties compared to the bulk material (Nel et al., 2006). Nano particles are made
from its bulk particles and are unique because of modification of their physical and chemical
properties especially shape and size (Brunnert, et al., 2006).

Depending on the origin, NPs are classified as natural, incidental and engineered.
Engineered NPs are grouped into four types viz Carbon based single walled carbon nanotube
(SWCNT) and multi walled carbon nanotubes (MWCNT). Metal based materials like
quantum dots, nanogold, nanozinc, nanoaluminum and nanoscales metal oxides like TiO2,
ZnO and Al2O3. Dendrimers which are nano-sized polymers built from branched units,
capable of being tailored to perform specific chemical function. In present days, engineered
nanomaterials have gained much higher attention because of its positive application in the
sectors like pharmaceutics, medical, textiles, cosmetics, transportation, energy and
agriculture. In agriculture, the application has to increase keeping the safety issues into
considerations (Roco, 2003).

2.5.2 Chemical and physical properties of zinc oxide nano particles

Zinc oxide (ZnO) powders are accessible as powders and dispersions and have
antibacterial, anti-corrosive, antifungal and UV filtering characteristics. Zinc is an aspect of
Block D, Period 4 element while Oxygen is an aspect of Block P, Period 2. The chemical
symbol is ZnO with chemical composition of zinc and oxygen with atomic mass and atomic
number of 80.34 percent and 19.6 percent respectively. The physical properties like Density
and Molecular mass of ZnO is 5600 kg m-3 and 81.40 g mol-1 respectively (AZo nano, 2013).

2.5.3 Conventional fertilizers versus nano fertilizers

Conventional fertilizers are supplied to the crop by soil application or by foliar spray
and important factor that decides the application methods is the final concentration which
reaches the plant. Because of unknown and undesirable fate of zinc after application such as
leaching, drift, runoff, evaporation, soil humidity hydrolysis and photolytic and microbial
degradation, very less concentration actually reaches the plant’s grain.

About 40-70 per cent of nitrogen, 80-90 per cent of phosphorous and 50-90 per cent
potassium content of applied fertilizers have been estimated to be lost to the atmosphere and
unable to reach the plant which causes sustainable and economic losses (Trenkel 1997;
Ombodi and Saigusa, 2000). Because of these problems farmer goes for repeated use of
  

fertilizer which adversely affects the inherent nutrient balance of the soil and leading to
destroy of soil microflora and natural ecosystem (Tilman et al., 2002). Hence, it is very
important to optimize the use of chemical fertilization and to minimize the application and to
reduce the risk of environmental pollution. Hence, there is a need to use the favourable
nutrient and method of application which can improve crop yield keeping the soil structure in
good shape and maintaining ecological balance (Miransari, 2011).

Advantages of nano based formulations against conventional bulk fertilizers (Cui et al.,
2010)

Sl.
Properties Nano formulations Conventional bulk fertilizers
No.

1 Solubility and High solubility and dispersion and Fixed in soil and low bio
dispersion reduce soil absorption and fixation available to plants
and enhances bioavailability

2 Nutrient Higher fertilizer uptake ratio Not available to roots and low
uptake efficiency
efficiency

3 Controlled Release rate (timely release) and Un controlled excess release

release and release pattern of nutrients is leads to toxicity. Available to
utilization precisely controlled by plants at the time of application
encapsulation. Available when and later converted into insoluble
required by the plant salt and fixed in the soil

4 Loss of Low leaching/leakage High losses due to leaching, run

fertilizer off and drift.

Nanotechnology is a science of using nano scale materials as fertilizers and controlled

release vectors to build so called intelligent fertilizers as emerging facility to enhance nutrient
use efficiency and reduce environmental costs (Chinnamuthu and Boopati, 2009). A nano-
fertilizer refers to a product which is either directly applied as emulsions or encapsulated
using protective polymer film inside with nano material (DeRosa et al.,2010). Delivery of
macro and micro nutrient and plant protection chemicals is an important application of
nanotechnology in agriculture. These advantages of nano particles are due to their high
surface area to volume ratio, high mobility, high solubility, specific targeting due to small size
and low toxicity (Sasson et al., 2007). A clear picture of advantages and difference between
nano and bulk or conventional fertilizers from agriculture point of view is clearly mentioned
in the above table by Cui and Co worker, (2010).
  

2.6 Application of Zinc oxide nano particles in agriculture

Researchers have both positive and negative opinions about impact of engineered nano
particles (ENPs) on plants growth and development. Based on their findings, they reported
that the effect is mainly dependent on the composition, concentration, size, physical and
chemical properties of ENPs as well as plant species (Ma et al., 2010). Zn oxide (ZnO) nano
fertilizers are preferred over bulk sized particles because small particle size results in
increased number of particles and also increasing the surface area and enhancing the
dissolution rate of fertilizers with low solubility in water. (Mortvedt et al., 1972, Liscano et
al., 2000).

In a study by Allen and Terman (1966) granular Zn sulphate of size 1.4 to 2 mm was
less effective than fine ZnSO4 of size 0.8 to 1.2 mm whereas granular ZnO was completely
ineffective. So this concludes that size is very important to increase thee efficacy of fertilizer
for better uptake and higher yields. Pure alumina nano-particles of size 13 nm (without any
modification) reduced the root elongation and growth potential of various plants viz., maize,
soybean, carrot, cucumber and Brassica oleracea (Yang and Watts, 2005).

A pot test was carried out to study the impacts of time and concentration of application
of nano zinc sulfide (ZnS) on chlorophyll levels of sunflower (Helianthus annuus L) by Singh
et al (2017) during rabi season 2014-15. Foliar sprayed nano-ZnS 400 ppm at 35 DAS
recorded higher chlorophyll content contrast with different treatments which were on par with
500 ppm nano-ZnS sprayed at 35 DAS.

Lin and Xing (2007) treated the seeds of alfalfa (Medicago sativa), corn (Zea mays),
cucumber (Cucumis sativus) and tomato (Lycopersicon esculentum) with nano zinc oxide with
−1
concentrations ranging from 0 to 4000 mg L . Uptake of nano has reduced significantly
germination (30 percent each) of corn, tomato and cucumber germination of 20 percent at
−1
.
2000 mg L However, the root growth of cucumber and corn were significantly promoted at
−1
2000 mg L except alfalfa and tomato where it was reduced. This differed response of
different crops is evident.

Burmana et al. (2013) studied the effect of nano ZnO on chickpea seedlings by foliar
spray of 1.5 ppm and plants showed an improvement in seedling growth, biomass
accumulation that may be due to the production of lower reactive oxygen species evident by
the lower levels of melondialdehyde content. In Soybean (Glycine max) zinc oxide nano
  

particles showed root elongation at 500 ppm concentration but as the concentration increases
there is a reduction in root growth (Lopez-Moreno et al., 2010b). Zhao et al (2014) studied
the effect of ZnO and CeO2 nanoparticles (400 ppm) on Cucumis sativus and as a result both
nano particles improved fruit quality and starch content and altered carbohydrate pattern.

Avinash et al (2010) conducted an experiment in Bengal gram (Cicer arietinum) to

study the effect of nano ZnO (20-30 nm) on the reactivity of phytohormones, especially phyto
stimulatory actions of indole acetic acid (IAA) on plant growth. Zn-rich Nano ZnO increased
the level of IAA in roots, which in turn increase the growth rate of plants.

Mahajan et al (2011) studied the effect of nano ZnO particles on the growth of
seedlings of mung bean (Vigna radiate) and Bengal gram (Cicer arietinum) by conducting
experiment in agar media. Various concentrations were supplied in the agar media, and its
entry into the root and shoot was examined by SEM and is evident by the adsorption of NPs
on the root surface. Absorption of nano particles by seedlings root was also detected by
inductive coupled plasma/atomic emission spectroscopy (ICP-AES). At optimum
concentration seedlings showed good growth over control and above that there is retardation
in growth.

Boonyanitipong et al (2011) conducted experiment in rice (Oryza sativa L.) to know

the effect of zinc oxide nano particles and titanium dioxide nano particles on roots. The result
says that there is no reduction in the percent seed germination with both nanoparticles.
However, at the early seedling stage, nano ZnO is noted to have harmful impacts on rice
roots. It is discovered that nano ZnO inhibits growth of root length and reduces the amount of
roots formation, while nano TiO2 does not affect the length of the root. This research showed
that direct exposure to particular kinds of nano particles creates substantial phytotoxicity,
highlighting the need for environmental friendly disposal of particle wastes.

Prasad et al (2012) reported that application of nano scale ZnO (25 nm) at 1000 ppm
concentration in peanut promoted seed germination, seedling vigour, early flowering and
higher chlorophyll content in leaf. Pod yield per plant was 34 percent higher compared to
chelated bulk ZnSO4. Foliar application of nano ZnO at 15 time’s lower dose recorded 26.3
percent and 29.5 percent higher pod yield compared to the chelated ZnSO4.

Hydroponically grown Mung bean (Vigna radiata) seedlings were supplied with
optimum concentrations of nano zinc oxide, nano iron oxide and nano zinc+copper+iron
 

oxide in suspension form. Seedlings showed good growth over control in all the three
treatments. The best performance was observed for nano Zinc + Copper +iron Oxide followed
by nano iron oxide and nano zinc oxide. Inductive couple plasma atomic emission
spectroscopy (ICP-OES) showed the absorption of nano particles by plant leaves. (Shailesh et
al.,2013).

Singh et al. (2013) studied the effects of bulk and nano zinc oxide particles on
germination, growth and biochemical parameters of cabbage, cauliflower and tomato crops. In
all three crops, bulk zinc oxide particles caused adverse phytotoxic effects on germination,
seedling growth and biochemical parameters, pigment, sugar and protein contents but zinc
oxide nano particles had promotory effect. The activities of antioxidant enzymes (SOD, CAT,
APX and POD) in zinc oxide NPs treated seedlings were lower as compared to those treated
with bulk ZnO. The order of sensitivity of the crops to ZnO and nano ZnO was cauliflower
followed by tomato and then by cabbage. Nano ZnO appeared to mitigate the phytotoxic
effects unlike bulk ZnO.

Salama (2012) studied the effects of silver nano particles on plant growth parameters
such as shoot and root length, leaf area, chlorophyll content, carbohydrate and protein content
of economically important pulses like common bean (Phaseolus vulgaris L.) and cereals like
corn (Zea mays L.). The results showed that lower concentrations of silver nano particles had
a stimulating effect on the growth of the plants, whereas under higher concentrations induced
an inhibitory effect. Increased concentration of silver nano particles up to 60 ppm has led to
an increase in shoot and root length, leaf surface area, chlorophyll content, carbohydrate and
protein contents.

Zhao et al. (2013) reported that nano particles (NPs) can interact with naturally
occurring inorganic and organic substances in soils and these substances may change their
transport behaviour in soil and plants. Results indicated that nano ZnO coexisting with Zn
dissolved species were released continuously to the soil solution to replenish the Zn ions or
-1
nano ZnO scavenged by roots. Nano ZnO at concentrations of 400 and 800 mg kg in absence
of alginate (component of soil organic matter) significantly reduced the root and shoot
biomass production. However, plants treated with these NP concentrations along with alginate
recorded significantly higher Zn in tissues with no reduction in biomass production. Alginate
significantly reduced the activity of stress enzymes i.e. catalase and peroxidase which are
responsible for damage in the defence system.
 

Sedghi et al. (2013) reported that significantly higher germination percentage, root
length, root fresh and dry weight in soybean crop were noticed when seeds were treated with
different concentrations of poly ethylene glycol (PEG) and nano zinc oxide under drought
stress conditions. Application of nano zinc oxide followed by stress occurrence caused
decrease in seed residual fresh and dry weight.

Cluster bean (Cyamopsis tetragonoloba) showed a relative improvement in plant

biomass, shoot and root growth, root area, chlorophyll and protein synthesis in plant when
treated with zinc oxide nano particles. When studied the microbial population, acid
phosphatase, alkaline phosphatase and phytase activity in rhizosphere was also improved in
the zinc oxide treated soil (Raliya and Tarafdar 2013)

The effect of NPs on germination varies from plants to plants and it depends on
concentrations of NPs. Different concentrations of nano ZnO were tried in cucumber, alfalfa
and tomato crop where seed germination was enhanced only in cucumber crop (Rosa et al.,
2013).

Afshar et al. (2014) studied the effects of different spraying concentrations of nano
zinc oxide and zinc oxide on wheat crop. Results revealed that nano-compounds are quickly
absorbed by plants in large amounts and hence it can be toxic on plants and plant products
when it applied at higher concentration.

Jayarambabu et al. (2014) used different concentrations (0, 20, 40, 60 and 100 mg l-1)
of nano ZnO for seed treatment in mungbean (Vigna radiata L.) and studied seed
germination. Nano particles of metal oxides with 20 mg l-1 recorded the lowest germination
per cent. But due to their quick movement into the plant it showed good shoot and root
growth compared to other concentrations and control.

Laware and Raskar (2014) conducted experiment to study the effect of nano ZnO on
growth, flowering and seed productivity of onion. The results indicated that the plants treated
-1
with nano ZnO at the concentrations of 20 and 30 ȝg ml showed improved growth and
flowered 12-14 days earlier than the control. Further, the treated plants showed significantly
higher seeds per umbel, seed weight per umbel and 1000 seed weight over control plants.
These results indicate that nano ZnO can reduce flowering period in onion by 12-14 days and
even produce healthy seeds.
 

Vinoth Kumar and Udayasoorian (2014) investigated the toxic potential of three
different metal oxide nano particles viz., zinc oxide (ZnO), aluminum oxide (Al O ) and
2 3

titanium oxide (TiO ) by using maize as test plant for germination study. A significant toxic
2

effect was observed on growth parameters like germination percentage, vigour index, shoot
and root length of maize. Among the tested concentrations of three different metal oxide
-1
particles, the concentration of 2000 mg L displayed highest toxic effect followed by 1000
-1
mg L . A significant toxic effect of metal oxide nano particles was observed on growth
parameters of maize and it was in the order of: ZnO followed by TiO and then by Al O .
2 2 3

Hesham et al., 2016b carried out the experiment to discover the function of zinc oxide
nano particles in stress resistance, five tomato cultivars with various salt stress resistance were
-1 -1
subjected to various treatments of NaCl (0, 3 and 6 g L ) and nano ZnO (0,15 and 30 mg L ).
-1
Treatments with NaCl at both 3 and 6 g L reduced the mRNA levels of superoxide dismutase
(SOD) and glutathione peroxidase (GPX) levels in all cultivars while plants treated with nano
ZnO with NaCl, demonstrated improvement in the mRNA expression levels. This
demonstrated that nano ZnO had a positive response on plant physiological processes under
salt stress. Higher expression of mRNA was obtained in the salt resistant cultivars.

Davoody et al. (2013) conducted a study to confirm the role of nano ZnO in foxtail
millet water stress condition. Water stress declined peduncle length, stomatal conductivity,
germination rate and grain yield. Impact of ZnO application on relative water content (RWC)
at the pre-anthesis stage was significant. The highest RWC before flowering were recorded in
the ZnO nano particles treatment (250 ppm). The insignificant impact of bulk and ZnO nano
particles medicines can be ascribed to a low zinc requirement in millet. Probably, nutrient
imbalance in the soil may also have contributed to the prevention of any positive effects of
zinc consumption on plant growth parameters.

Zhao et al. (2015) studied the modification in the physiological and agronomic
characteristics of corn plants when exposed to engineered nano particles (nano cesium oxide
or nano zinc Oxide) at 400 and 800 mg kg-1. Results showed that nano cesium oxide at both
concentrations did not impact gas exchange in leaves at all the growth stages, while nano zinc
oxide at 800 mg kg-1 reduced net photosynthesis (12 per cent), stomatal conductance (15
percent), chlorophyll content (10 percent) at 20 days after treatment and yield was reduced by
40 percent.
  

Ambily et al. (2017) noticed higher plant height, number of leaves plant-1, petiole
length plant-1, and leaf area, root diameter, root length, root yield/treatment and root yield
hectare-1 in the treatment combination of nano ZnO at 100ppm + FeO NPs at 50ppm. Nano
particles interact with plants causing many morphological and physiological changes,
depending on the properties of NPs. Efficacy of NPs is determined by their chemical
composition, size, surface covering, reactivity, and most importantly the dose at which they
are effective (Khodakovskaya et al., 2012). Nano ZnO supplemented with MS media
promoted somatic embryogenesis, shooting, regeneration of plantlets, and also induced
proline synthesis, activity of superoxide dismutase, catalase, and peroxidase thereby
improving tolerance to biotic stress (Helaly et al., 2014)

Priyanka and Venkatachalam (2016) investigated the impact of ZnO NP on Gossypium

hirsutum. ZnO NP presentation for 21 days significantly improved development (125 percent)
and biomass (132 percent) in the treated plants contrasted with control. Curiously,
photosynthetic pigments specially chl a (134 percent), chl b (132 percent), carotenoids (160
percent), and TSS (165 percent) increased altogether, however the level malondialdehyde
(MDA) content (74 percent) reduced in the nano ZnO supplied plants contrasted with the
control. The outcomes demonstrated that there were huge increments in superoxide dismutase
(SOD, 267 percent) and peroxidase (POX, 175 percent) contents, while reduction in catalase
(CAT, 83 percent) action was recorded in the NPs treated plants contrasted with the control.
ZnO NP treatment did not indicate clearly the presence or absence of DNA in the banding
pattern of RAPD study.

The Spinach (45-50 days) plants sprayed with nano ZnO at the concentration of 500
and 1000 ppm showed the increased leaf length and width and colour of leaf samples when
compared to control. Similarly treated plants with nano ZnO at the concentration of 500 and
1000 ppm showed increased levels of protein and dietary fibre content in comparison to
control leaf samples. The study suggested that the nano-zinc oxide sprayed spinach is more
nutritious to vegetarian diet by providing, protein, fibre and required amount of vegetarian fat
to diet (Kisan et al., 2015).

An exposure of rice cultivar KMJ-6-1-2 to Zn NP (at 0, 25, 50, 75, 100 & 150 mg L-1)
caused significant changes in root and shoot length, mass (fresh & dry mass) and RWC in
rice. Growing rice seedlings treated with Zn NP at 25 & 50 mg L-1 showed better growth
 


which possibly protected plants from ROS damage by improving levels of antioxidant
enzyme activities (Upadhyaya et al., 2015).

2.7 Use of zinc oxide nano particles in hydroponic studies

Growing of plants without using soil in nutrient solution is called Hydroponics (Resh.,
2001). Even though it has many advantages like nutrient can be supplied in optimum
controlled condition and chances of pest and disease attack is less, but still the technology is
not extensively adopted on a large scale. Hydroponics is very useful in studying the nutrient
uptake and its interactions with other available nutrients inside the solution, at the site of
uptake (rhizosphere) and inside the plant system (translocation site) (Chaoui et al., 1997).

Rice varieties JP-05, Swat-I, IRRI-6 and Basmati-2000 were germinated and grown
hydroponically. The effect of the Zn dosages (0, 1 and 2ȝM) on the plant height, crude
protein, chlorophyll a and b, zinc and iron contents of plant were evaluated. IRRI-6 and
Basmati-2000 attained greater height relative to JP-05 and Swat-I at 1ȝM of Zn dosage
whereas crude proteins were maximum in JP-05 and Basmathi 2000 at 1ȝM Zn dosage and
Swat-1 and IRRI-6 at 2ȝM Zn dosage. A similar pattern for the leaf chlorophyll a and b was
also observed. Basmati 2000 and JP-5 had got maximum amount of chlorophyll a and b at 1
ȝM Zn dosage while Swat-1 showed higher contents of chlorophyll at 2 ȝM Zn dosage. The
Zn contents of the plants increased upto 1ȝM Zn dosage, however, the Fe contents was
observed to decrease. This signified the competitive behaviour of Fe with Zn. The Zn dosage
1 ȝM was found potentially effective in obtaining maximum plant height, crude protein, and
chlorophyll a and b for the rice plants (Humaira et al., 2015).

Samreen et al., (2013) conducted an hydroponic experiment (0, 1 and 2 ȝM of zinc

concentrations) to study the effect of zinc on protein, chlorophyll content, and interaction with
other minerals in the solution and in the plant using four varieties of mungbeans (Ramazan,
Swat mung I, NM92 and KMI). After two months plant showed higher plant growth,
chlorophyll contents, crude proteins and Zn contents in treatments with higher zinc
application. P levels have decreased as zinc concentration increased from 1 ȝM to 2 ȝM
indicating the antagonistic effect of P/Zn in solution preventing the entry to the roots.
Potassium, Sodium, and manganese content not much altered, where as copper and
Magnesium content increased and iron content is reduced with increase in zinc level. Zinc
  

concentration upto 1 ȝM was found to be optimum and above that is considered as non
supportive for crop growth and other nutrients absorption. .

Raliya et al. (2015) conducted a study in tomato crop with the objective to compare
the impact of TiO2 and ZnO nano particles at the concentrations of 0 to 1000 mg kg-1 on the
translocation and accumulation of nano particles and also its impact on plant growth. The
results indicated that there is a critical level of nano particles upto which the growth was
promoted and beyond that there is no improvement and concluded that aerosol mediated
application is better than soil application.

Effects of different nano-devices (0, 0.35, 0.70, 2.80 cm2 L-1) on growth, physiological
characteristics and quality of Lettuce in hydroponics were studied in this experiment. The
results showed that yield, root volume, and concentration of antioxidant enzymes viz
Superoxide dismutase, Peroxidase, Catalase activity, root activity and nitrate reductase
activity of lettuce increased with the use of nano-devices in nutrient solution, in which higher
amount results in better effects. The best treatment was 2.80 cm2 L-1 nano-devices in nutrient
solution, in which yield, root volume, nitrate reductase activity, content of soluble sugar in
lettuce were increased by 14.57 percent, 20.26 percent, 177.01 percent, 223.44 percent,
115.67 percent, respectively. Activities of antioxidant enzymes increased more than 10percent
compared with control. While treatment A (0.35 cm2 L-1) had the highest flavonoid and
soluble phenolic contents among the four treatments, which increased 34.76 percent, 37.08
percent, respectively, compared with control. In conclusion, nano-devices could increase
yields and improve quality of hydroponic lettuce (Li et al., 2013).

Ghodake et al. (2011) investigated the phytotoxicity of zinc and cobalt oxide nano
particles in the roots of onion bulbs (Allium cepa) as an indicator vegetable plant. Onion bulb
roots were treated with zinc and cobalt oxide nano particle dispersions at concentration of 5,
10 and 20 ȝg ml−1. Bulbs showed reduction in root growth with increase in the concentration
of both nano particles when compared with control. The study indicates that nano particle has
phytotoxic effect on plant root growth at higher concentrations.

Sagardoy et al. (2009) studied the effect of high zinc concentration on growth and
interaction with other essential nutrients in hydroponically grown sugar beet (Beta vulgaris
L.). Zinc Sulphate concentrations of 50, 200 and 300µM showed decreased root and shoot
fresh and dry mass and increased root » shoot ratios, when compared to control conditions (1.2
  

µm Zn). Plants showed short lateral roots, inward rolling of leaf edges and damaged brownish
root. At 50 and 100 µM of zinc treatments plants showed iron deficiency symptoms but at 300
µM there was a decrease in photosynthetic activity but did not have leaf iron deficiency
symptoms. At higher concentrations of zinc there is a decrease in the N, Mg, K and Mn
concentration, but an increase in P and Ca concentrations were observed only in shoots. Leaf
zinc content was high at higher concentration but at very high concentration there was a
reduction in the uptake of zinc by leaves. Sugar beet can become a good model for study of
zinc homeostasis.

2.8 Uptake of ZnO nano particles

The uptake, translocation and accumulation of zinc oxide nano particles (nano ZnO) in
food crops are not clearly known. In ryegrass, scanning electron microscopy (SEM) studies
revealed the adsorption and aggregation of the zinc oxide nano particles to the root surface.
The high amplification TEM pictures of the ryegrass root cross-segments additionally
demonstrated the nearness of particles in the apoplast, cytoplasm and cores of the endodermal
cells and the vascular chamber, attempted to be nano ZnO (Lin and Xing, 2008).

Lopez et al. (2010 b) examined the uptake and aggregation of 8 nm ZnO by soybean
(Glycine max) seedlings. The soybean seeds are treated with nano ZnO in the range of 500–
4000 mg L−1 and the Zn uptake by the seedlings was significantly higher at 500 mg L−1,
perhaps because at this concentration the NPs have lesser aggregation and at 1000-4000 mg
L−1 there is an increase in the agglomeration. These scientists performed XAS examination of
the ZnO NP treated samples and results indicated Zn2+ inside the plant tissues but the
spectrum resembled more than one of Zn acetate and nitrate than nano ZnO. In spite of the
fact that nano ZnO are required to be a hotspot for Zn2+ found inside tissues, the examination
neglects to feature whether the Zn2+ was contributed by the biotransformation of the nano
ZnO on/in roots. It might be speculated that root exudates ionized the nano ZnO on the root
surface, as no hint of nano ZnO were appeared by the XAS spectra.

The impact of zinc oxide NPs (10 nm) on biological system and its accumulation is
studies in velvet mesquite (Prosopis juliflora velutina) The seedlings are grown on 500 to
4000 mg L-1 of ZnO for 15 days and then the zinc concentration in the tissues was 2101 mg
kg-1 in root, 1135 mg kg-1 in stem and 628 mg/kg in leaves and at 2000 mg nano ZnO L-1. The
XRF image results suggest that zinc accumulation in the root is because of absorption but not
  

by adsorption. Biochemical estimations showed the stress enzymes catalase in root, stem and
leaves and peroxidase in stem and leaves are increased by ZnO uptake. Study of the XRF
images regarding localization of nano ZnO and bulk ZnO are complementary to each other
(Viezcas et al., 2011).

Mechanism of foliar uptake pathway for aqueous solutes and water suspended nano
particles in Allium porrum and Vicia faba was observed that stomatal pathway differs
fundamentally from the cuticular foliar uptake pathway and the translocation mechanism of
foliarly applied nano ZnO is not yet clearly understood. (Eichert et al., 2008).

A pot experiment was conducted at UAS, Dharwad during 2016 to study the response
of sunflower to three different nano ZnS foliar application and the results indicated an
improvement in the growth, yield and physiological parameters at foliar spray of 400 ppm of
concentration (Betageri, 2017).

2.9 Toxicity of engineered nano particals in the edible plants

Boonyanitipong et al. (2011) studied the phytotoxic nature of Zn oxide nano particles
(nano-ZnO) on rice (Oryza sativa L.). Results of parameters like seed germination percentage,
root length and number of roots showed that nano ZnO is observed to have detrimental effects
on rice roots at early seedling stage. Nano ZnO is found to show stunted root growth and
reduce number of roots. The study shows that direct exposure to specific types of NPs leads to
phytotoxicity and emphasis is needed for ecological responsible disposal of nano particle
wastes.

In another study the harmful impact of Zn oxide nano particles (Nano ZnO) on the
root development, root apical meristem mitosis and mitotic distortions of garlic (Allium
sativum L.) were explored. Nano ZnO caused inhibition of root length. When treated with 50
mg L-1 Nano ZnO for 24 h, the root development of garlic was totally blocked. The 50 percent
inhibitory concentration was assessed to be 15 mg L-1. The mitosis was likewise reduced in a
concentration and time dependent manner. Nano ZnO additionally induced several kinds of
mitotic deviations (Talgarshaymurat et al., 2011)

Burmana et al. (2013) studied the effect of nano ZnO, bulk ZnSO4 and bulk ZnO on
10 days old seedlings of chickpea (Cicer arietinum L var. HC-1) by foliar sprayed with 1.5 or
10 ppm of liquid solution. Concentrations of 1.5 ppm of nano ZnO showed maximum shoot
  

dry weight, while 10 ppm treated seedlings exerted adverse effects on root growth. However,
overall biomass accumulation improved in the ZnO nano particle treated seedlings and may
be because of reduction of ROS levels which resulted in less lipid peroxidation estimated by
MDA formation. Even the antioxidant enzymes, superoxide dismutase (SOD) and peroxidase
were less in ZnO particle treated seedling compared to control. The study indicates the need
for application of optimum concentration and understanding the mechanism of action of
specific nano materials.

As demonstrated by Priester et al. (2012), Zn substantially moved above ground in

soybean from nano-ZnO treated soils as Zn concentrations increased in a dose dependent
fashion in the stem, leaf, and soybean pod tissues. When compared with no Zn, high nano-
ZnO treatment registered 6 times more Zn in the stem, 4 times more in the leaf, and nearly 3
times more in the soybean pod. Such Zn concentrations in various plant tissues were similar
for equivalently dosed soybean (on a Zn mass basis) grown with Zn salts (Shute and Macfie,
2006). They also suggested that nano-ZnO must have been highly bio available in this study
soil, as Zn also substantially bio accumulated in nano-ZnO-treated plants in a previous
hydroponic study (Lopez-Moreno et al., 2010b).

Hashemi et al. (2016) synthesised nano ZnO by leaf concentrate of Aloe vera and their
impacts were assessed on development and physiological parameters of soybean. Properties
of zinc oxide nano particles were detected by UV–Vis spectroscopy, SEM, XRD and dynamic
light dissipating. Zinc oxide nano particles were confirmed by the absorption maxima at the
wavelength of 360 nm. SEM pictures revealed that nano ZnO were oval shaped. Examinations
of nano ZnO on soybean have demonstrated quicker germination and improved development
of plants. Treatment of zinc oxide nano particles indicated increment in lipid peroxidation and
hydrogen peroxide substance.

Nano ZnO concentration extending from 500 to 1500 mg L-1 adversely influences the
Brassica nigra seed germination and seedling development and furthermore lead to an
expansion in the antioxidative activities and non-enzymatic. While, culturing the stem
explants of Brassica on Murashige and Skoog (MS) medium at lower concentration of nano
ZnO (1–20 mg L-1) resulted in white slim roots with thick root hairs. At 10 mg L-1 nano ZnO,
shoots rise is additionally observed. The calli/roots demonstrated 79 percent DPPH (2,2-
diphenyl-1-picryl hydrazyl) radical scavenging activity at 10 mg L-1. The total antioxidant and
  

reducing power potential also significantly influenced in presence of nano ZnO (Zafar et al.,
2016).

Yang et al. (2015) examined the phytotoxicity of seven metal oxide nano particles
(NPs) titanium dioxide (nTiO2), silicon dioxide (nSiO2), cerium dioxide (nCeO2), magnetite
(nFe3O4), alluminum oxide (nAl2O3), zinc oxide (nZnO) and copper oxide (nCuO) was
evaluated on two agriculturally important crop plants (maize and rice). The outcomes
demonstrated that seed germination was not influenced by any of the seven metal oxide NPs.
But, at the concentration of 2000 mg L-1, the root expansion was altogether repressed by
nCuO (95.73 percent for maize and 97.28 percent for rice), nZnO (50.45 percent for maize
and 66.75 percent for rice). Unexpectedly, minor phytotoxicity of nAl2O3 was just seen in
maize, and no conspicuous lethal impacts were found in the other four metal oxide NPs. By
further investigation we found that the phytotoxic impacts of nZnO, nAl2O3 and nCuO (25 to
2000 mg L-1) were concentration dependent, and were not brought about by the relating Cu2+,
Zn2+ and Al3+ particles (0.11 mg L-1, 1.27 mg L-1 and 0.74 mg L-1 separately). Moreover, nano
ZnO (<50 nm) indicated more prominent toxicity than ZnO microparticles (MPs) (<5 nm) to
root expansion of both maize and rice.

By and large, this examination gave important data to the use of built NPs. Phytotoxic
effects and accumulation of zinc oxide nano particles were studied in aquatic plant Hydrilla
verticillata and Phragmites australis in Hoagland nutrient solution culture. The dissolution
percentage of nano ZnO Hoagland nutrient solution was inversely related to the concentration
of applied nano ZnO so the lower concentration also may cause toxic effects. Plant Growth,
chlorophyll content, antioxidant enzymes and zinc content were adversely affected at lower
concentration and also at concentration above 1000 mg L-1 in both the plants (Song and Lee,
2016).

With the advancement of nano technology there are many emerging new ways to
address the food and nutritional security problems in ever growing population by maintaining
or enhancing the agriculture productivity in various crops. Application of nano particles at
different concentrations and at different times and different methods would definitely alter or
bring about the changes in physiology of plants and can greatly modify the source sink
relationships. The need for addressing the toxic effect of nano particles besides considering
their potential advantage is highly emphasized.