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Novel effects of nanoparticulate delivery of zinc on growth, productivity and zinc bio-fortification in maize (Zea mays L.) Layam Venkata Subbaiah, Tollamadugu Naga Venkata Krishna Vara Prasad, Thimmavajjula Giridhara Krishna, Palagiri Sudhakar, Balam Ravindra Reddy, and Thalappil Pradeep J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00838 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on May 3, 2016

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Journal of Agricultural and Food Chemistry

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Novel effects of nanoparticulate delivery of zinc on growth, productivity and zinc biofortification in maize (Zea mays L.) Layam Venkata Subbaiaha, Tollamadugu Naga Venkata Krishna Vara Prasad*a, Thimmavajjula Giridhara Krishnaa, Palagiri Sudhakara, Balam Ravindra Reddyb and Thalappil Pradeepc a

Agri-Nanotechnology laboratory, Institute of Frontier Technologies, Regional Agricultural Research Station,

Acharya N .G. Ranga Agricultural University, Tirupati – 517 502, A.P., India b

S.V.Agricultural College, Acharya N. G. Ranga Agricultural University, Tirupati – 517 502, A.P., India

c

DST Unit on Nanoscience, Department of Chemistry, Indian Institute of Technology Madras, Chennai, T.N., India.

*Corresponding author: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

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In the present investigation, nanoscale zinc oxide particulates (ZnO-nanoparticulates) were prepared using

3

modified oxalate decomposition method. Prepared ZnO-nanoparticulates (mean size 25nm) were

4

characterized using techniques like, Transmission electron microscopy (TEM), Fourier transform infrared

5

spectroscopy

6

(50,100,200,400,600,800,1000,1500 and 2000 ppm) of ZnO-nanoparticulates were examined to reveal

7

their effects on maize crop on overall growth and translocation of zinc along with bulk ZnSO4 and control.

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Highest germination percentage (80%) and seedling vigor index (1923.20) were observed at 1500 ppm of

9

ZnO-nanoparticulates. The yield was 42% more compared to control and 15% higher compared to 2000

10

ppm of ZnSO4. Higher accumulation of zinc (35.96 ppm) in grains was recorded with application of 100

11

ppm followed by 400 ppm (31.05 ppm) of ZnO-nanoparticulates. These results indicate that ZnO-

12

nanoparticulates have significant effects on growth, yield and zinc content of maize grains which is an

13

important feature in terms of human health.

14

Key

words:

(FT-IR)

ZnO

and

zeta

nanoparticulates,

potential

Maize,

analyzer.

Bioavailability

15

16

17

18

19

20 2


Different

of

zinc,

concentrations

Biofortification

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1. Introduction

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Among the new innovations, nanotechnologies play an important role in modifying the agriculture

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and food production systems. Though, nanotechnologies have many applications in other sciences like,

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electronics, medicine and pharmaceuticals their impact is still at its infancy stage in terms of agricultural

25

applications. Nanoparticles (size 1-100 nm in at least one dimension) pose beneficial as well as inhibitory

26

effects on the biological systems. Reducing the size of the particles leads to the increase in specific surface

27

area of particles and as a consequence, the contact area of fertilizers with the plants will be increased

28

resulting in the higher nutrient uptake by the plants. Limited reports are available on the promotory effects

29

of nanoparticles on the agricultural crops1. Many studies have focused more on the effect or toxicity of

30

nanomaterials and experimentally proved that at low concentrations nanoparticles are able to enhance the

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physiological processes of plants2,3. The absorption, translocation and accumulation of nanoparticles reveal

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that they can penetrate through the seed coat and move through the conducting tissues in the plants.

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Magnetic nanoparticles (Fe2CO3) can able to penetrate, move and accumulate in Cucurbita maxima when

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they are grown in the aqueous culture medium whereas it is not observed in the case of Phaseolus

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limensis4. Nanoscale ZnO particles and AgNPs when they applied to maize and cabbage, the germination

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process is not inhibited in maize where as a concentration dependent germination inhibition has been

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observed in the cabbage when ZnO nanoparticles are used, illustrating the differential behavior of different

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plant species to the same nanoparticles5 and similar kind of results were observed in the case of radish,

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rape, ryegrass, lettuce, corn and cucumber6. To enhance the production and productivity of crops, the

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traditional fertilizers could be mould and make them to release the nutrients slowly, by coating a layer of

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nanomaterials, as and when the crops require the nutrients. In a study on the peanut, ZnO nanoparticles

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have shown positive effects on the seed germination and seedling vigor indices even at high concentrations 3



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(1000 ppm), whereas, at field scale studies the promotory effects of ZnO nanoparticles were evidenced at

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relatively low concentrations with the foliar application1. There are a few studies on the use of

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nanomaterials (nanoporous silica) as fertilizer components or enhancers to control the chemical as well as

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biological processes that are assumed to be taken place in the soil after fertilizer application7 and also the

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use of zinc-aluminium-layered nanocomposites for release of plant growth regulators in a controlled

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manner8. It has been reported that the application of slow-released fertilizer incorporated nano-carbon is

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beneficial in increasing the grain yield in rice crop as well as to save the nitrogenous fertilizers as it

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increases the nitrogen use efficiency of the crops9. Multi-walled carbon nanotubes (MWCNT) can able to

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penetrate the seed coat and hasten the germination process in tomato growth of mustard seeds10 and the

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growth, water and ion uptake by maize plants11. Foliar application of nano TiO2 has shown positive effects

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in the corn by increasing the crop yield12. β-cyclodextrin coated iron nanoparticles could able to penetrate

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through the biological membranes of maize plants and increase the chlorophyll pigments particularly

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chlorophyll-a up to 38% when compared to that of control13 and also stimulate the seedlings proliferation

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of maize compared to control in the early ontogenic stages of maize seedlings14.

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Maize (Zea mays L.) is a strategic cereal crop of the world and ranks third after wheat and rice in

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the world and India. In India, maize is cultivated in 8.67 million hectares and is having the productivity15 of

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2492 kg ha−1. Maize is one of the high yield potential crops among the cereals and yields double

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production when compared with other cereals16. In the recent past maize attracted the researchers being an

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unprecedented source of food for millions of people and livestock including poultry across the world thus

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having the most importance in the food security. But in the present scenario, the yield potential and nutrient

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content of maize is depleting due to several biotic and aboitic stresses including deficiencies of

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micronutrients in the soil, zinc (Zn) in particular, and maize is very sensitive to the zinc deficiency. 4


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Zinc (Zn) is an important transitional metal, abundantly available after iron and is the only metal

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present in all six classes of enzymes and act as functional component for several transcriptional factors1.

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Zinc affects several processes pertaining to plant life cycles. Many processes such as metabolism of

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saccharides, nucleic acids and lipids etc. are regulated by zinc and it plays an important role in the

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synthesis of proteins and carbohydrates17. Several research reports have established the essentiality and

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role of micronutrient-zinc on plant growth, development and yield

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the cultivating plants against biotic and abiotic stresses by involving in many physiological processes like,

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resistance against pathogens, drought or heat27,28. Zinc deficiency in the soils is an extensive problem

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globally and nearly 30% of the cultivated lands are deficient of zinc29. In countries where Zn deficiency is

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well documented as an important public health problem, cereal-based foods are predominant in daily food

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intake29. In India, nearly 50% of the soils are deficient in plant available zinc that are under cultivation of

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rice and wheat, that receives no or less Zn through fertilizers, particularly in rural areas, indicating the

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urgent need to enrich the cereal grains with zinc. Though the breeding methods are efficient in increasing

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the grain zinc content, it takes longer period to develop zinc efficient cultivars. Moreover, these cultivars

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could not perform well because of low soil zinc content. Therefore, the zinc biofortification of cereal

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grains, up to 2 or 3-fold, with zinc enriched fertilizers complements the breeding methods30. There are

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some reports that indicates across the globe over 30 billion people are suffering from the deficiencies of

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micronutrients in the food and to overcome this problem, a lot of research has to be done to develop new

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technologies to fortify the edible plants and grains with the deficient micronutrients 31 with ample emphasis

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on zinc.

18,26

. Zinc provides higher resistance to

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Therefore, herein, a field scale study and in vitro studies were conducted to evaluate the effects of

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nanoparticulate delivery of zinc through ZnO-nanoparticulates on the growth, productivity and zinc bio5



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fortification in maize. Further, the zinc content in the different parts of the plant was estimated to assess the

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translocation of zinc in the plants. It was also examined, whether or not the concentration of the

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nanoparticulate influences the translocation and bio-fortification of zinc in maize plant.

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2. Materials and methods

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2.1. Synthesis and characterization of ZnO-nanoparticulates

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Oxalate decomposition technique1 was used to prepare ZnO-nanoparticulates. Equimolar (0.2M)

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solutions of zinc acetate and oxalic acid were mixed to prepare zinc oxalate. The precipitate so formed as a

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result of mixing zinc acetate and oxalic acid was collected and thoroughly rinsed with double de-ionized

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water (DI-water) and allowed to dry in the air at ambient room temperature. Then the oxalate was made

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into fine powder form decomposed in the air by keeping it in a pre-heated muffle furnace for 45 minutes at

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500οC.

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The Nano ZnO particulates were characterized by High resolution transmission electron

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microscopy (HRTEM, JEOL 3010; Jeol Ltd, Peabody, MA, USA) for surface morphological studies,

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nanoparticle analyzer through dynamic light scattering technique for the measurement of hydrodynamic

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diameter (size) (Nanopartica SZ-100, Horiba), and FT-IR (BRUKER, TENSOR 27) to identify the

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functional groups present. The sample for TEM analysis was prepared by drop casting the nanoparticles

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suspension on the carbon coated Cu grids. The maize seeds after 3 hours of soaking in solutions of

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different concentrations (according to the treatments) were cut into thin slices and endospermic and

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embryonic regions were examined under scanning electron microscopy (SEM-EDAX, FEI Quanta 200) for

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the zinc uptake confirmation.

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2.2. Procurement of seeds 6


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Maize seeds (hybrid var.DHM-117) were procured from the Agricultural Research Station,

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Karimnagar, and were used without further modifications.

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2.3. In vitro studies

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2.3.1. Preparation of particle suspension and seed treatment

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The prepared ZnO-nanoparticulates were suspended in the de-ionized water directly and dispersed

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by ultrasonic vibration for 30 minutes. The aggregation of particles was avoided by stirring the suspensions

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with magnetic bars. The solutions of corresponding bulk ZnSO4 and respective treatments each of 100 ml

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were prepared. The control was maintained with double distilled de-ionized water. Five seeds of maize

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were placed in each 100 ml solutions/suspensions prepared for soaking for three hours. Three replications

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were maintained for each treatment.

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2.3.2. Germination and seedling vigor test

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Treated seeds were placed in to the Petri dishes (100 mm × 15 mm) provided with one piece of

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sterilized filter paper and 5 ml of corresponding solutions/suspensions were added to the Petri dishes,

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covered with the lid and placed in the incubator at 26±1οC for seven days. After seven days the Petri dishes

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were removed and data was recorded. Germination percentage was calculated based on the number seeds

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germinated among five seeds kept for germination. Seedling Vigor Index (SVI) was calculated as per the

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formula given by32. All the root and shoot lengths were measured by stretching the curved ones using a

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thread placed on a scale (0-30cm). Seedling Vigor Index = Germination % × (root length + shoot length)

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127 7



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2.3.3. Field experiment

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The field experiment was conducted at Regional Agricultural Research Station, Acharya N. G.

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Ranga Agricultural University, Tirupati (13°37′38.4″-N latitude and 79°22′26.6″- E longitude, with an

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altitude of 214 m above the mean sea level) during Kharif season, 2013-14. The experiment was laid out

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in sandy clay loam textured soil in RBD (Randomized Block Design) with three replications and with the

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plot size of 3 m×5 m. The initial soil parameters were pH = 6.42 (Neutral)33; E. C. = 0.132 dSm−1 33;

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organic carbon = 0.50 % (Low) 34; available nitrogen = 188.16 Kg ha−1(Low) 35; available P2O5 = 14.66 Kg

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ha−1 (36); available K2O = 564.4 Kg ha−1 (High)33; available zinc = 16.6 ppm 37 and total zinc content was

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21.3 ppm 38.

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2.3.4. Treatments

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Eleven treatments were considered in this study namely, Control-Distilled water (Foliar spray);

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Bulk-ZnSO4 of 2000 ppm (Foliar spray); T1, T2, T3, T4, T5, T6, T7, T8 and T9 corresponds to the foliar

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application of ZnO-nanoparticulates of 50 ppm, 100 ppm, 200 ppm, 400 ppm, 600 ppm, 800 ppm,1000

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ppm, 1500 ppm and 2000 ppm respectively. In all the treatments, recommended dose of fertilizers namely,

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Nitrogen – 100 kg ha-1(in the form of Urea), P – 60 kg ha-1(in the form of Single super phosphate), K – 50

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kg ha-1 (in the form of Murate of potash) were applied to the soil.

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In the present study, foliar spray of 2000 ppm of bulk zinc sulphate was taken only as it was a

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recommendation to the farmers to all crops including maize to supplement the zinc nutrition to the standing

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crop. Therefore, the effects of all the concentrations of foliar applied ZnO-nanoparticulates were compared

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with the foliar application of 2000 ppm of bulk zinc sulphate. Further, the zinc content was calculated by

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considering 12% of zinc in zinc sulphate and 80% of zinc in zinc oxide. 8


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The treatments were imposed at two stages during tasseling at 48-58 Days after sowing (DAS) and

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milking stage of grains (100-105 DAS). The physiological parameters like, plant height (measured using

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the scale, 1-100cm), number of leaves, leaf area (measured using a leaf area meter (LICOR, Model LI

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3000) and expressed in cm2) and dry matter (measured using common balance) were recorded at 30 DAS,

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60 DAS and 90 DAS. At harvest, samples from each plot were collected to record the yield attributing

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characteristics like, number of cobs per plant, number of rows per cob, number of grains per row, cob

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height and test weight of grain. The post harvest analysis of leaf, cob and grain samples for zinc content

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was estimated using ICP-MS (Perkin Elmer, Nexion 300X) by feeding the digested samples. The digestion

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of samples was done by mixing 0.5 g of sample with 5 ml of HNO3 and 1 ml of H2O2 and 1ml of H2O in

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teflon tubes and digested in the microwave digester (Perkin Elmer, Multiwave 3000) and then feeding the

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digested liquid to the Instrument38 .

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2.4. Statistical Analysis

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The data recorded on various parameters of crop during the course of investigation was statistically

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analyzed following the analysis of variance for randomized block design39. Statistical significance was

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tested with ‘F’ test at 5 per cent level of probability and compared the treatmental means with critical

164

difference. The mean values were separated by Duncan's Multiple Range Test (DMRT).

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3. Results and discussion

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3.1. Size and surface morphological studies of ZnO-nanoparticulates

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ZnO-nanoparticulates were used in this study (mean size 25nm) as nanoparticulate systems for

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delivery of Zn in to plants. HRTEM micrograph (Fig.1) of ZnO-nanoparticulates appears to be slightly

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aggregated due to the absence of surface protecting ligands. The particles were crystalline in nature and the 9



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lattice of zinc oxide was clearly seen. The hydrodynamic diameter (size) of the ZnO-nanoparticulates was

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found to be 35.5 nm with a negative zeta potential of 39.6 mV (Fig. 2).

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Fourier Transform Infrared Spectroscopy (FT-IR) showed the presence of different functional

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groups on the surface of ZnO-nanoparticulates (Fig. 3). with the corresponding peaks formed at 3702.41

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cm-1(free alcohols), 2923.54 cm-1(alcohols, alkanes, carboxylic acids), 2856.19 cm-1(alcohols), 1838.87cm-

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1

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amides), 1396.96 cm-1(alkanes), 1161.58 cm-1(alcohols) and 1024.82 cm-1(alkanes).

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3.2. Influence of ZnO-nanoparticulates on maize seedlings (in vitro studies)

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3.2.1. Germination and seedling vigor index (SVI)

(aromatics), 1744.70 cm-1(ketones, amides), 1538.86 cm-1(ketones, amides), 1459.03 cm-1(nitrates,

The highest germination percentage (80%) (Fig.6), highest root length (11.47 cm), highest shoot

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length (12.57 cm) were recorded with the treatment 1500 ppm of ZnO-nanoparticulates (T8)

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significantly differed from the control (40%, 39%, 35%) and 2000 ppm of ZnSO4 (20%, 60%, 55%)

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respectively (Fig.7). Significantly highest seedling vigor index (1923.2) was recorded when the grains were

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treated with ZnO-nanoparticulates of 1500 ppm (T8) compared to all other treatments. The SEM images of

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treated seeds were evidenced (Fig.6, Fig.7) the zinc rich portions of endospermic and embryonic regions

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along with starch granules. It is clear from the SEM micrographs that the endospermic and embryonic

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regions of the seeds treated with 100 ppm and 400 ppm of ZnO-nanoparticulates were relatively enriched

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with the zinc content and which were correlated with the enhanced yield and grain zinc content of maize

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discussed hereafter.

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3.2.2. Physiological parameters of maize as influenced by ZnO-nanoparticulates

10


and were

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Plant height of maize plants was measured at different growth stages during the crop period at 30,

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60 and 90 DAS and presented in Table-I. The plant height at 30 DAS was not differed significantly

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because the treatments were not applied at this stage. However, there was a significant difference in the

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plant height of maize plants at 60 DAS. Among the treatments, application of 400 ppm of ZnO-

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nanoparticulates had shown a significant increase in plant height (167.50 cm) (25% increment over control

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and 11% increment over bulk ZnSO4 of 2000 ppm) and at 90 DAS similar trend has been observed with the

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same concentration 400 ppm of ZnO-nanoparticulates (T4) (187.56 cm, 19% increment over control and

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8% increment over bulk ZnSO4 of 2000 ppm).

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There was a significant difference in the leaf area at 60 DAS (Table-I) and the maximum leaf area

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(4533.42 cm2) was recorded with application of 400 ppm ZnO-nanoparticulates (64% increment over

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control and 31% increment over bulk ZnSO4 of 2000 ppm). The other treatments in which notable leaf area

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recorded were 100 ppm of ZnO-nanoparticulates (4246.25 cm2) and 200 ppm of ZnO-nanoparticulates

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(4110.22 cm2). At 90 DAS, application of 400 ppm of ZnO-nanoparticulates had shown the maximum leaf

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area (5715.10 cm2) - significantly superior over control (52%) and bulk ZnSO4 of 2000 ppm (19%)

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respectively (Table-I).

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At 30 DAS there was no significant difference in the dry weight of maize plants was observed as

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the treatments were not imposed, whereas, at 60DAS significant dry weight (105.56 g plant-1) was

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recorded with the application of 400 ppm of ZnO-nanoparticulates (56% increment over control and 13%

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increment over bulk ZnSO4 of 2000 ppm) and at 90 DAS 145.30 g plant-1 dry weight (Table-I) was

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recorded with the application of 400 ppm of ZnO-nanoparticulates (34% increment over control and 16%

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increment over bulk ZnSO4 of 2000 ppm). Higher chlorophyll index was recorded (44.467) with the

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application of 400 ppm of ZnO-nanoparticulates and was significantly higher than the control (23% 11



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increment over control and 16% increment over bulk ZnSO4 of 2000 ppm). The treatment of maize seeds

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with the nanosilica powder enhanced the water uptake by seeds and thereby increasing the activity of

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enzymes in the seeds resulting in the high germination percentage 40. Earlier it has been reported that the

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ZnO-nanoparticulates at high concentrations also increased the germination percentage of peanut1.

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Similarly, exposure of maize seeds to higher concentration 2000 ppm of ZnO-nanoparticulates (T9) had not

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shown any inhibition on seed germination whereas, a complete germination inhibition was observed in the

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seeds treated with ZnSO45. In the present study, it was observed that all the physiological parameters like,

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plant height, leaf area, number of leaves and dry matter were recorded high (Table-I) with the application

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of 400 ppm of ZnO-nanoparticulates (T4) and significantly differed with the control and all other

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treatments. On the other hand, non-significant differences were observed at 30DAS before the application

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of ZnO-nanoparticulates. The results clearly indicate the influence of the ZnO-nanoparticulates on the

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growth and physiological parameters of maize.

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3.2.3. Yield and yield attributes of maize as influenced by ZnO nanoparticulates

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The highest grain yield of 3298 kg ha-1 was recorded with the application of ZnO-

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nanoparticulates of 400 ppm (T4) which was significantly higher (42%) compared to control (1884 kg ha-

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1

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application of 100 ppm of ZnO-nanoparticulates (3182 kg ha-1) and 200 ppm of ZnO-nanoparticulates

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(3120 kg ha-1) and followed by 50 ppm of ZnO-nanoparticulates (Table-II). The number of cobs per plant

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recorded (one) were same in all the treatments (Table-II). The other cobs that were produced by the plants

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were not considered as they were very small and without matured grains. The highest cob length of 16.40

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cm was recorded with 400 ppm of ZnO-nanoparticulates (T4) and was significantly superior to control

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(18%) and bulk ZnSO4 of 2000 ppm (10%) (Table-II). Application of ZnO-nanoparticulates of 400 ppm

) and 15% higher compared to 2000 ppm of bulk ZnSO4 (2787 kg ha-1). The next best treatments were

12


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(T4) resulted in producing more number of rows (15) per cob which was significantly higher than control

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(11%) and bulk ZnSO4 of 2000 ppm (8%) (Table-II). More number of grains per row (38.5) was produced

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with the application of 400 ppm of ZnO-nanoparticulates (T4) and was significantly differed from control

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(36%) and bulk ZnSO4 of 2000 ppm (27%). However, application of ZnO-nanoparticulates of 100 ppm and

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200 ppm recorded notable grains per row 34.9 and 33.2 respectively. The highest test weight (weight of

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100 grains) of 35.2 g was recorded (Table-II) with the application of ZnO-nanoparticulates of 400 ppm

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(T4) and which was significantly higher than control and bulk ZnSO4 of 2000 ppm (16% over control and

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11% increment over bulk).

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Several researchers have noticed the effect of different nanoscale materials in increasing the yield

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of different crops. Use of iron oxide nanoparticles as a source of iron nutrition could able to increase the

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yield of soybean crop by increasing the pod dry weight41. The nano-carbon particles when applied along

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with the nitrogenous fertilizers have increased the yield of the rice crop as well as the nitrogen use

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efficiency of rice42. Yield of soybean had significantly increased over control due to the efficacy of the

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nano silver particles in preventing the undesirable action of ethylene on the flowering43. Iron nanoparticles

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had shown the promising results in increasing the yield of wheat

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reports indicating the positive effects of TiO2 nanoparticles in increasing the yield of wheat when

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compared to that of control and bulk TiO2 even under water deficit conditions

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amount of chlorophyll content in the leaves of maize crop thereby increasing the photosynthetic efficiency

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of plants resulting in the high yields12. Similarly, in the present study, significantly higher grain yield (3298

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kg ha-1) of maize was recorded with the application of 400 ppm of ZnO-nanoparticulates (T4) followed by

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(T2)100 ppm (3182 kg ha-1) and (T3)200 ppm (3120 kg ha-1) in contrast to the application of bulk ZnSO4

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of 2000 ppm (2787 kg ha-1).

44

13


and Phaseolus vulgaris45. There were

46

or by enhancing the


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3.3. Post harvest zinc concentration in plant parts and soil as influenced by ZnO-nanoparticulates

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The total zinc content in the post harvest samples was estimated using ICP-MS. It was observed

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that the translocation of the zinc content was dependent on the concentration of the ZnO- nanoparticulates

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and different parts of the maize plant were enriched with zinc at different concentrations. The highest zinc

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concentration of 22.0 ppm (Table-III) was recorded in leaf at 2000 ppm of ZnO-nanoparticulates (T9)

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which is significantly higher than that of control (53%) and bulk zinc sulphate of 2000 ppm (50%).

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Application of 2000 ppm of ZnO-nanoparticulates (T9) also resulted in higher zinc accumulation (Table-

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III) in maize cobs (47.6 ppm) compared to control (21.4 ppm) and 2000 ppm of bulk zinc sulphate (31.3

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ppm).

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The highest grain zinc content of 35.96 ppm (Table-III) was recorded with the application of

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100 ppm of ZnO-nanoparticulates (T2) and was significantly higher over control (37%) and bulk zinc

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sulphate of 2000 ppm (29%). The more accumulation of zinc in the grains of maize at 100 ppm of ZnO-

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nanoparticulates (T2) may be attributed to the enhanced translocation of zinc into the grains when

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compared to other treatments. It has been reported47 that the sucrose loading into the wheat grain had not

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affected with the higher concentrations of Zn and Mn, whereas, the Zn and Mn concentrations were

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limited due to membrane saturation. Labeling the ears with isotopes (Zn65 and Mn54) at various stages of

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grain development had shown more translocation of Zn and Mn to the endosperm and the embryo as the

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grains of wheat mature47. Likewise, in our study, it has been found that at lower concentration (T2-100

274

ppm) of ZnO- nanoparticulates the translocation of Zn into grains is significantly maximum (35.96 ppm)

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and decreased with increased concentration (Table-III) and recorded minimum with T9-2000 ppm (25.34

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ppm). Seed treatment studies also evidenced the higher content of zinc in endospermic region with the

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application of 400 ppm of ZnO- nanoparticulates. 14


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Saturation of membrane transporters leads to the limited loading of Zn to the phloem and the xylem

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and phloem interactions may play an important role in the regulation of Zn transport to the maturing grains.

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The xylem discontinuity was reported in the case of wheat48 whereas, in rice, there is no xylem

281

discontinuity49, 50, which facilitates the continuous transport of Zn into the grain through vascular bundles

282

during grain filling. An increased Zn uptake by the rice plants resulted in enrichment of vegetative parts

283

with Zn rather than reproductive parts. But at lower Zn supply levels, the grains seem to be enriched with

284

Zn, due to net Zn remobilization from the leaves to the grain during grain filling51. Similar trend was

285

observed in our study (Table-III) with higher concentration of ZnO-nanoparticulates (T7-1000 ppm, T8-

286

1500 ppm and T9-2000 ppm) elevated Zn content was recorded both in leaf (16.64, 17.47, 22.04 ppm

287

respectively) and cob (36.56, 40.64, 47.64 ppm respectively) and at lower concentration (100 ppm) grain

288

enrichment with zinc (35.96 ppm).

289

The zinc concentration in the soils was estimated and more content of zinc content of 24.3 ppm

290

(Table-III) of soil was found in the treatment with ZnO-nanoparticulates of 1000 ppm (T7) indicating that

291

the translocation of zinc from the leaves to the soil through the plant body system and accumulating in the

292

soils. The soil zinc content had shown high significant difference among control and bulk treatments. The

293

high content of zinc in the soils at high concentration of ZnO-nanoparticulates reveals that the plants were

294

unable to use such high amount of zinc nutrient when supplied to the plant through foliar spraying and the

295

extra amount of zinc was translocated to the soil through the stem and roots.

296

Zn deficiency is one of the most critical micronutrient deficiency problems in human health. Zinc

297

deficiency is widespread among children and represents a major cause of child death in the world30.

298

Increasing evidence is available from field trails showing that soil and/ or foliar application of Zn fertilizers

299

improves grain Zn concentration up to 2 to 3 folds. Nicotinamine (NA) is a chelator of metal cations and it 15



Page 16 of 35

16

300

is biosynthesized from S-adenosyl methionine via NA synthase (NAS). All higher plants synthesize and

301

utilize NA for the internal transport of Zn and other metals. Reports suggest that NA plays an essential role

302

in Zn translocation to seeds, including Fe

303

seeds of rice plants 34 and wheat 52. Endosperm-specific ferritin expression also contributes to the increased

304

Zn concentration in rice seeds. The germination studies (in vitro) with the delivery of zinc through different

305

concentrations of the ZnO-nanoparticulates clearly indicate the novel effects of nanoparticulate

306

concentration. The same was reflected in field scale experiments (in vivo) as the translocation and

307

localization of zinc content was greatly influenced by the concentration of the ZnO-nanoparticulates.

308

Second, nanoparticulate concentration dependent localization of the zinc content leads to the production of

309

designer agricultural produce with the desired nutrient levels. This is entirely novel and observed when

310

nanoscale materials were applied on agricultural crops as nutrients. As the nanoscale is a quantum

311

confinement with the increased surface to volume ratio, we assume that delivery of zinc through ZnO-

312

nanoparticulates can be effective in adequate and timely supply of this micronutrient to the crop plant.

313

Further, reports evidenced1, 43, 44 that nanoparticles could be translocate to all the parts of the plant system

314

with the increased time of retention. Thus, the content in the nanoparticulate could be available to the plant

315

for the extended periods of time and consequently stimulate and participate in the plant biochemical

316

processes (Fig-8).

(52)

. Higher NA production increases the Zn concentration in

317

Yield attributes like, cob length, number of grain rows per cob, number of grains per row and test

318

weight followed the similar trend as that of yield. These results indicate that at lower concentration of

319

ZnO-nanoparticulates, the delivery of Zn content is adequate to maintain the required nutrient flux in the

320

plant system thereby promoting the translocation of Zn to the various parts of the plant as and when

321

needed. 16


Page 17 of 35


17

322

ZnO-nanoparticulates used in this study are specific kind and aimed to agricultural applications

323

and the results cannot be extrapolated to any other crop or nanomaterials. This novel technology such as

324

application of ZnO-nanoparticulates on the crops through foliar method had established the fact that the

325

bio-availability of Zn can be enhanced by active uptake, translocation and maximum accumulation of Zn in

326

the grains (Fig-8).

327

Application of nanomaterials in agriculture as nutrients is promising. Further, site specific

328

translocation of a particular nutrient in plant system appears to be bright with the application of nanoscale

329

materials which leads to the production of designer agricultural produce with the required levels of

330

nutrients through fortification. In the present investigation, it was clear that the nanoparticulate delivery of

331

Zn through ZnO-nanoparticulates could be effective and was beneficial in order to enhance the growth,

332

yield and yield attributes of maize crop. In the controlled conditions (in vitro) even at the high

333

concentrations of ZnO-nanoparticulates showed promotory effects, whereas, at field scale conditions, even

334

at the low concentrations of ZnO-nanoparticulates increase in the yield and uptake of zinc was recorded.

335

Therefore, it is evident that nanoparticulate delivery of nutrients in to the plant system can be effective and

336

micronutrient deficiencies could be corrected using nanoscale materials through agronomic bio-

337

fortification which could reflect in human health. Abbreviations ZnO -

Zinc oxide

DAS -

Days after sowing

HRTEM -

High resolution transmission electron microscope

ppm -

Parts per million

17



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18

SVI -

Seedling vigor index

ICP-MS -

Inductively coupled plasma mass spectroscopy

FT-IR -

Fourier transform infrared spectroscopy

nm -

Nanometer

NA -

Nicotinamine

NAS -

Nicotinamine synthase

SEM -

Scanning electron microscopy

Acknowledgements The authors are thankful to Acharya N G Ranga Agricultural University for providing research facilities at Institute of Frontier Technology, Regional Agricultural Research Station, Tirupati to carry out this research programme. LVS is thankful to Acharya N G Ranga agricultural University for providing financial assistance to carry out this research programme. LVS is thankful to Dr.K.Murali Krishna, Senior Scientist, Agricultural Research Station, Karimnagar, Telangana state for sparing the maize seed for experimentation. LVS is thankful to Dr.S.Adam whose inputs are instrumental in writing the manuscript. References 1. Prasad, T.N.V.K.V.;Sudhakar, P.; Sreenivasulu,Y.; Latha, P.; Munaswamy, V.; Raja Reddy, K.; Sreeprasad, T.S.; Sajanlal P.R.; Pradeep, T. Effect of nanoscale zinc oxide particles on the germination, growth and yield of peanut. J. Plant Nut. 2012, 35, 905-927. 2. Ghodake, G.; Seo, Y.D.; Park, D.; Lee, D.S. Phytotoxicity of carbon nanotubes assessed by Brassica juncea and Phaseolus mungo. J. Nanoelectron. Optoelectron. 2010, 5, 157-160. 3. Khodakovskaya, M.; Dervishi, E.; Mahmood, M.; Xu, Y.; Li, Z.; Watanabe, F.; Biris, A. S. Carbon nanotubes are able to penetrate plant seed coat and dramatically affect seed germination and plant growth. ACS Nano. 2009, 3, 3221-3227. 18


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4. Zhu, H.; Han, J.; Xiao, J.Q.; Jin, Y. Uptake, translocation and accumulation of manufactured iron oxide nanoparticles by pumpkin plants. J. Environ. Monit. 2008,10,713-717. 5. Pokhrel; Lok, R.; Brajesh, Dubey. Evaluation of developmental responses of two crop plants exposed to silver and zinc oxide nanoparticles. Sci. Total Environ. 2013, 452, 321-332. 6. Lin, D.; Xing, B. Phytotoxicity of nanoparticles: Inhibition of seed germination and root growth. Environ. Poll. 2007, 150,243-250. 7. Hossain, K.Z.; Monreal, C.M.; Sayari, A. Adsorption of urease on PE-MCM-41 and its catalytic effect on hydrolysis of urea. Colloid Surface B. 2008, 62, 42–50. 8. Hussein, M. Z. B.; Zainal, Z.; Yahaya, A. H.; Wong vui foo, D. Controlled release of a plant growth regulator, alpha-naphthalene acetate from the lamella of Zn-Al-layered double hydroxide nanocomposite. J. Control Rel. 2002, 82, 417-427. 9. Wu, M.Y. Effects of incorporation of nano-carbon into slow-released fertilizer on rice yield and nitrogen loss in surface water of paddy soil. Adv. J. Food Sci. Technol.2013, 5, 398-403. 10. Mondal, A.; Basu, R.; Das, S.; Nandy, P. Beneficial role of carbon nanotubes on mustard plant growth: an agricultural prospect. J. Nanopart. Res.2011, 13, 4519-4528. 11. Tiwari, D.K.; Dasgupta-Schubert, N.; Cendejas, L.V.; Villegas, J.; Montoya, L.C.; Garcia, S.B. Interfacing carbon nanotubes (CNT) with plants: enhancement of growth, water and ionic nutrient uptake in maize (Zea mays) and implications for nanoagriculture. Appl. Nanosci. 2014, 4, 577-591. 12. Morteza, E.; Moaveni, P.; Farahani, H.A.; Kiyani, M. Study of photosynthetic pigments changes of maize (Zea mays L.) under nano TiO2 spraying at various growth stages. Springer Plus. 2013, 2, 247. 19



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13. Racuciu, M. Iron oxide nanoparticles with β-cyclodextrin polluted of Zea mays plantlets. Nanotech. Dev.2012, 2, 31-35. 14. Suriyaprabha, R.; Karunakaran, G.; Yuvakkumar, R.; Prabu, P.; Rajendran, V.; Kannan, N. Growth and physiological responses of maize (Zea mays L.) to porous silica nanoparticles in soil. J. Nanopart. Res. 2012, 14, 1-14. 15. Ministry of Agriculture, India. Report from State of Indian Agriculture 2011-12, Agricultural Production and Programmes. 2012, 101. 16. Tollenar, M.; Alee, E. Yield potential, yield stability and stress tolerance in maize. Field Crop Res. 2002, 75, 161-169. 17. Sajedi, N.A.; Ardakani, M.R.; Naderi, A.; Madani, H.; Boojar, M.M.A. Response of maize to nutrients foliar application under water deficit stress conditions. Am. J. Agric. Biol. Sci. 2009, 4, 242-248. 18. Camp, A.; Fudge, B.R. Zinc as a nutrient in plant growth. Soil Sci. 1945, 60, 157-164. 19. Chapman, H.D. Zinc. In: Diagnostic Criteria for Plant and Soils, Chapman, H. D. Eds.; University of California: Riverside, CA. 1966, 6484-499. 20. Viets, F.G. Zinc Deficiency in the Soil-Plant System. In: Zn Metabolism, Prasad, A. S., Eds.; Springfield: IL, Thomas, 1966, 90-128. 21. Anderson, W.B. Zinc in soils and plant nutrition. Adv. Agron. 1972, 24,147-186. 22. Mengel, L.; Kirkby, E.A. Principles of Plant Nutrition. Basel, Switzerland: International Potash Institute, 1978. 23. Marschner, H. Zinc Uptake from Soils. In: Zinc in Soils and Plants, Robson, A. D., Eds.; Kluwer Academic Publishers: Dordrecht, the Netherlands, 1993, 59-77. 20


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24. Brown, P. H; Cakmak, I; Zheng, Q. Forms and Function of Zinc in Plants. In: Zinc in Soil and Plants. Robson, A. D., Eds.; Kluwer Academic Publishers: Dordrecht, the Netherlands, 1993, 93-106. 25. Fageria, N.K.; Baligar, V.C.; Clark, R.B. Micronutrients in crop production. Adv. Agron. 2012,77,189-272. 26. Grzebisz, W.; Wronska, M.; Diatta, J.B.; Dullin, P. Effect of zinc foliar application at an early stage of maize growth on patterns of nutrients and dry matter accumulation by the canopy. I: Zinc uptake patterns and its redistribution among maize organs. J. Elemen. 1999, 13, 17-28. 27. Grusak, M.A.; Pearson, J.N.; Marentes, E. The physiology of micronutrient homeostasis in field crops. Field Crop Res. 1999, 60, 41-56. 28. Marschner, H. Mineral Nutrition in Higher Plants. Academic Press: 1986, 300-312. 29. Cakmak, I.; Yilmaz, A.; Kalayci, M.; Ekiz, H.; Torun, B.; Ereno, B.; Braun, H.J. Zinc deficiency as a critical nutritional problem in wheat production in Central Anatola. Plant and Soil. 1996, 180, 172-195. 30. Cakmak, I. Enrichment of fertilizers with zinc: An excellent investment for humanity and crop production in India. J. Trace Elem. Med. Biol. 2009, 23,281-289. 31. Graham, R.D.; Welch, R.M.; Bouis, H.E. Addressing micronutrient malnutrition through enhancing the nutritional quality of staple foods: Principles, perspectives and knowledge gaps. Adv. Agron. 2001,70,77-142. 32. Abdul-Baki, A.A.; Anderson, J.D. Vigor determination in soybean seed by multiple criteria. Crop Sci.1973, 13, 630-633. 33. Jackson, M.L. Soil chemical analysis. Prentice hall of India Pvt Ltd. New Delhi.1973, 134-204. 21



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34. Walkley, A.; Black, C.A. An estimation of dertjareff method for determining soil organic matter and proposed modification of chromic acid titration method. Soil sci.1934, 37, 29-37. 35. Subbaiah, B.V.; Asija, G.L. A rapid procedure for determination of available nitrogen in soils. Curr. Sci. 1956, 25,259-260. 36. Olsen, S.R.; Cole, C.V.; Watanabe, F.S.; Dean, L.A. Estimation of available phosphorous in soils by extraction with sodium bicarbonate. U. S. Department of Agriculture, 1954, Circular number: 939. Banderis, A.D.; Barter, D.H.; Anderson, K. Agricultural and Advisor. 37. Lindsay, W.L.; Norvell, W. A. Development of a DTPA Soil test for zinc iron, manganese and Copper. Soil Sci. Soc. Amer. J. 1978, 42,421-428. 38. Wu, S.; Fen, X.; Wittmeier, A. Microwave digestion of plant and grain reference materials in nitric acid or a mixture of nitric acid and hydrogen peroxide for the determination of multi elements by Inductively Coupled plasma-mass spectrometry. J. Analytic. Atomic Spec.1977, 12, 797-806. 39. Panse, V.G.; Sukhatme, P.V. Statistical methods for agricultural workers, 4th ed., ICAR, New Delhi, 1985, 347. 40. Yuvakkumar, R.; Elango, V.; Rajendran, V.; Kannan, N.S.; Prabu, P. Influence of nanosilica powder on the growth of maize crop (Zea mays L.). Int. J. Green Nanotech. 2011, 3,180-190. 41. Sheykhbaglou, R.; Sedghi, M.; Shishevan, M.T.; Sharifi, R.S. Effects of nano-iron oxide particles on agronomic traits of soybean. Notulae Scientia Biologicae. 2010, 2, 112-113. 42. Fan, L.; Wang, Y.; Shao, X.; Geng, Y.; Wang, Z.; Ma, Y.; Liu, J. Effects of combined nitrogen fertilizer and nano-carbon application on yield and nitrogen use of rice grown on saline-alkali soil. J. Food Agri. Envi. 2012, 10, 558-562. 22


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43. Ashrafi, M.; Sarajuoghi, M.; Mohammadi, K.; Zarei, S. Effect of nanosilver application on agronomic traits of soybean in relation to different fertilizers and weed density in field conditions. Envi. Exp. Biol. 2013, 11, 53-58. 44. Ghafari, H.; Razmjoo, J. Effect of foliar application of nano-iron oxidase, iron chelate and iron sulphate rates on yield and quality of wheat. Int. J. Agro. Plant Prod. 2013, 4, 2997-3003. 45. Jahanara, F.; Sadeghi, S.M.; Ashouri, M. Effect of nano-iron (Fe) fertilization and Rhizobium leguminosarum on the qualitative and quantitative traits of Phaseolus vulgaris genotypes. Int. J. Agri. Crop Sci. 2013, 5, 572-578. 46. Jaberzadeh, A.; Moaveni, P.; Moghadam, H.R.T.; Zahedi, H. Influence of bulk and nanoparticles titanium foliar application on some agronomic traits, seed gluten and starch contents of wheat subjected to water deficit stress. Notulae Botanicae Horti Agrobo. 2013, 41, 201-207. 47. Pearson, J.N.; Rengel, Z.; Jenner, C.F.; Graham, R.D. Dynamics of zinc and manganese movement in developing wheat grains. Aus. J. Plant Physiol. 1998, 25, 139-144. 48. Zee, S.Y.; O’Brien, T.P. A special type of tracheary element associated with ‘xylem discontinuity’ in the floral axis of wheat. Aus. J. Biol. Sci. 1970, 23,783-791. 49. Zee, S.Y. Vascular tissue and transfer cell distribution in the rice spikelet. Aus. J. Biol. Sci. 2011, 3,180-190. 50. Krishnan, S.; Dayanandan, P. Structural and histo-chemical studies on grain filling in the caryopsis of rice (Oryza sativa L.). J. Biosci. 2003, 28,455-469.

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51. Jiang, W.; Jiang, W.; Struik, P.C.; Van Keulen, H.; Zhao, M.; Jin, L.N.; Stomph, T.J. Does increased zinc uptake enhance grain zinc mass concentration in rice. Ann. App. Biol. 2008, 153, 135-147. 52. Masuda, H.; Ishimaru, Y.; Aung, M.S.; Kobayashi, T.; Kakei, Y.; Takahashi, M.; Higuchi, K.; Nakanishi, H.; Nishizawa, N.K. Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition. Scientific Reports 2. 2012,543.

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Table. I. Effect of nanoparticulate delivery of zinc through zinc oxide nanoparticles on plant growth and physiological traits of maize

Leaf number

Dry weight (g plant−1)

Plant height (cm)

Leaf area (cm2)

Leaf number


Plant height (cm)

Leaf area (cm2)

Leaf number


90 DAS

Leaf area (cm2)

60 DAS

Plant height (cm)

Treatments*

30 DAS

Control

48.8

981.5

5.1

12.3

124.4g

1598.5h

11.4i

46.3g

151.0g

2704.5g

12.5i

95.6h

Bulk

50.2

1274.5

5.6

20.6

147.4e

3090.6e

12.2ef

91.1d

171.2de

4610.0e

13.0fg

122.3f

T-1

54.9

993.5

5.6

24.0

155.3c

3727.2c

12.5d

96.6c

178.2b

4941.2d

13.5d

134.7c

T-2

68.8

894.0

6.0

17.3

162.5b

4246.2b

12.8b

101.5b

180.0b

5692.1b

14.0b

138.6b

T-3

50.6

1188.0

5.3

18.6

156.1c

4110.2b

12.6c

98.8bc

179.6b

5277.6c

13.7c

135.4c

T-4

39.6

1078.4

5.8

22.0

167.5a

4533.4a

13.0a

105.5a

187.5a

5715.1a

14.6a

145.3a

T-5

53.4

991.5

5.8

14.6

150.3d

3387.9d

12.5d

92.2d

173.7c

4936.5d

13.2e

131.0d

T-6

81.2

1161.7

5.8

21.0

149.2de

3683.2c

12.3e

91.9d

171.0e

4698.1 e

13.0g

122.2f

T-7

46.2

797.2

6.1

23.6

147.0e

3080.8e

12.1f

90.4d

173.4cd

4594.0e

13.1 f

124.6e

T-8

50.2

934.4

5.0

16.0

146.7e

2246.3g

11.8h

81.3e

161.1f

4334.0f

12.8h

114.9f

T-9

59.1

934.8

5.7

15.3

133.9f

2583.4f

11.9g

55.7f

160.8f

4165.8f

12.8h

113.6g

S.Em±

22.0

132.2

0.3

3.4

0.8

64.5

0.03

1.3

0.75

59.8

0.03

1.0

N.S

N.S

N.S

N.S

2.5

191.8

0.1

4.0

2.2

177.7

0.10

2.9

C. D. (P=0.05)

The data represents the mean of three replications. In each column, values with same alphabet are not significantly different at 5% probability level. Treatments: Control-Distilled water; Bulk-ZnSO4of 2000 ppm (Foliar spray); T1-nano ZnO of 50 ppm (Foliar spray); T2-nano ZnO of 100 ppm (Foliar spray); T3-nano ZnO of 200 ppm (Foliar spray); T4- nano ZnO of400 ppm (Foliar spray); T5-nano ZnO of 600 ppm (Foliar spray); T6-nano ZnO of 800 ppm (Foliar spray); T7- nano ZnO of 1000 ppm (Foliar spray); T8- nano ZnO of 1500 ppm (Foliar spray); T9- nano ZnO of 2000 ppm (Foliar spray). *Soil application of Nitrogen (N) – 100 kg ha-1(in the form of Urea), P – 60 kg ha-1(in the form of Single super phosphate), K – 50 kg ha-1 (in the form of Murate of potash) was done for all the treatments.

25



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26

Table. II. Effect of nanoparticulate delivery of zinc through zinc oxide nanoparticles on the yield and yield attributes of maize

Treatments*

Yield (kg ha−1)

Control-Distilled water (Foliar spray)

Yield attributing characteristics

1884f

Cobs Per plant 1

Cob length (cm) 13.3h

No. of rows/cob 13.3f

No. of grains/row 24.5d

Test weight (g) 29.6g

Bulk-ZnSO4@2000 ppm (Foliar spray)

2787d

1

14.8d

13.8de

28.2c

31.3e

T1-nano ZnO@50 ppm (Foliar spray)

2951c

1

15.3c

14.1c

29.5c

33.4c


3182b

1

15.8b

14.7ab

34.9ab

34.4b


3120b

1

15.6b

14.4b

33.2b

33.4c

T4- nano ZnO@400 ppm(Foliar spray)

3298a

1

16.4a

14.9a

38.5a

35.2a


2938c

1

15.2c

14.4bc

31.7bc

32.8d


2880c

1

14.2e

14.2c

29.4c

30.9f

T7- nano ZnO@1000 ppm (Foliar spray)

2747d

1

14.1ef

14.1cd

28.9c

30.7f


2780d

1

14.0f

13.5e

27.9c

30.8f


2644e

1

13.6g

13.3ef

26.8cd

30.7f

S.Em±

24.0

---

0.1

0.2

1.3

0.1

C. D. (P=0.05)

71.3

N.S.

0.2

0.5

4.0

0.4

The data represents the mean of three replications. In each column, values with same alphabet are not

significantly different at 5% probability level. * Soil application of Nitrogen (N) – 100 kg ha-1(in the form of Urea), P – 60 kg ha-1(in the form of Single

super phosphate), K – 50 kg ha-1 (in the form of Murate of potash) was done for all the treatments.

26


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27

Table.III. Evidence on concentration dependent translocation of zinc content through zinc oxide nanoparticulates to leaves, cobs and grains of maize and soil (after harvest) estimated using Inductively coupled plasma mass spectrometry (ICP-MS) Zinc content in mg kg−1 of dry matter

Treatments* Control-Distilled water (Foliar spray)

Leaf

Cob

e

g

10.3

21.4

Grain 22.7

g

Soil 21.3f

Bulk-ZnSO4@2000 ppm (Foliar spray)

12.1de

31.3e

25.6e

21.9de


13.4d

35.8cd

31.0b

22.2cd


13.8d

29.3ef

36.0a

21.5ef


11.7e

25.7fg

24.0f

21.7e

T4- nano ZnO@400 ppm(Foliar spray)

12.2d

38.1b

31.0b

20.4g


14.4 cd

26.0f

26.2d

23.4b


13.4d

31.4de

27.4c

23.2b


16.6bc

36.5bc

26.6d

24.3a


17.5b

40.6b

25.7e

22.5c


22.0a

47.6a

25.3e

23.0b

S.Em±

0.76

1.5

0.13

0.16

C. D. (P=0.05)

2.25

4.44

0.38

0.49

The data represents the mean of three replications. In each column, values with same alphabet are not significantly different at 5% probability level * Soil application of Nitrogen (N) – 100 kg ha-1(in the form of Urea), P – 60 kg ha-1(in the form of Single

super phosphate), K –50 kg ha-1 (in the form of Murate of potash) was done for all the treatments.

27



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28

Figures

Fig.1. (A)High resolution transmission electron microscopic (HRTEM) image of ZnO-nanparticulates at 100nm scale and a highly focused image of single ZnO-nanoparticulate(inset) at 5nm; (B) enlarged image of ZnO nanoparticulate at 20 nm showing clearly the structure of ZnO particulates.

28


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29

Fig.2. (A) Micrograph representing zeta potential of ZnO-nanoparticulates (-39.6mV); (B) Particle size distribution pattern of ZnO-nanoparticlulates (35.5 nm)

29



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Fig.3. Fourier transform infrared spectroscopic micrograph cleary showing the various functional groups present on the surface of ZnO-nanoparticulates

Fig. 4. Effect of different concentrations of ZnO-nanoparticulates along with bulk zinc sulphate on the germination of maize seeds. (In each column, values with same alphabet are not significantly different at 5% probability level) 30


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31

Fig. 5. Effect of different concentrations of ZnO-nanoparticulates and bulk zinc sulphate on the root and shoot growth of maize seedlings. (In each column, values with same alphabet are not significantly different at 5% probability level)

31



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Fig.6. SEM images (50µmscale) showing endospermic region enriched with zinc of maize grains of (A) Control (B)50 ppm (C),(D) and (E) ZnO-nanoparticulates at 100 ppm, 400 ppm and 1500 ppm respectively.

32


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33

Fig.7. SEM images (50µm scale) showing embryonic region enriched with zinc of maize grains of (A) Control (B)50 ppm (C),(D) and (E) ZnO-nanoparticulates at 100 ppm, 400 ppm and 1500 ppm respectively.

33



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Fig. 8. Schematic representation of nanoparticulate delivery, translocation and bio-fortification of Zn in different parts of maize plant when supplied through foliar application.

34


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