Cu-Chitosan Nanoparticle Mediated Sustainable Approach To

Jul 27, 2016 - Ram Chandra Choudhary,. † ... of Chemistry and Biochemistry, College of Basic Sciences and Humanities, Chaudhary Charan Singh Haryana...
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Cu-Chitosan Nanoparticles Mediated Sustainable Approach to Enhance Seedling Growth in Maize by Mobilizing Reserved Food Vinod Saharan, Kumaraswamy R.V., Ram Chandra Choudhary, Sarita Kumari, Ajay Pal, Ramesh Raliya, and Pratim Biswas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02239 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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Journal of Agricultural and Food Chemistry

Cu-Chitosan Nanoparticles Mediated Sustainable Approach to Enhance Seedling Growth in Maize by Mobilizing Reserved Food Vinod Saharan*†, R. V. Kumaraswamy†, Ram Chandra Choudhary†, Sarita Kumari†, Ajay Pal‡, Ramesh Raliya§ and Pratim Biswas§



Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan 313001, India ‡

Department of Chemistry and Biochemistry, College of Basic Sciences and Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana 125004, India

§

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, MO 63130, USA

*Corresponding author E-mail: [email protected] Phone: +91-9461180586; Fax: +91-294-2420447

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ABSTRACT: Food crop seedlings often have susceptibility towards various abiotic and biotic

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stresses. Therefore, in the present study, we studied the impact of Cu-chitosan nanoparticles

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(NPs) on physiological and biochemical changes during maize seedling growth. Higher values of

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percent germination, shoot, and root length, root number, seedling length, fresh and dry weight

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and seed vigor index were obtained at 0.04 to 0.12% concentrations of Cu-chitosan NPs as

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compared to water, CuSO4, and bulk chitosan treatments. Cu-chitosan NPs at the same

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concentrations induced the activities of α-amylase and protease enzymes and also increased the

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total protein content in germinating seeds. The increased activities of α-amylase and protease

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enzymes corroborated with decreased content of starch and protein, respectively in the

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germinating seeds. Cu-chitosan NPs at 0.16% and CuSO4 at 0.01% concentrations showed

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inhibitory effect on seedling growth. The observed results on seedling growth could be explained

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by the toxicity of excess Cu and growth promotory effect of Cu-chitosan NPs. Physiological and

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biochemical studies suggest that Cu-chitosan NPs enhance the seedling growth of maize by

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mobilizing the reserved food, primarily starch through the higher activity of α-amylase.

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Keywords: Chitosan. Cu-chitosan nanoparticles. Seedling growth.

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INTRODUCTION

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Application of nanomaterials in agriculture is the subject of intense research and development.

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The positive results of application of various nanomaterials in agriculture have encouraged the

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further utilization of this technology. The majority of the nanomaterials used in agriculture for

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plant growth and protection are metal based and have received a lot of attention and concern

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related to toxicity.1-8 More vigilance is, therefore, needed during application of highly active and

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non-degradable metal nanomaterials.9-11 The situation has galvanized the search for more

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efficient and eco-friendly alternatives. Biopolymer based nanomaterials having certain exclusive

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characteristics like biodegradability and biocompatibility could be utilized in agriculture.

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Chitosan, a chitin derived amino polysaccharide has been used in various fields including

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agriculture.12 Further, the benefits of nanotechnology innovations have been initiated to explore

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the synthesis of various chitosan-based nanoparticles.13-18 Earlier results of in-vitro and in-vivo

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studies have indicated a substantial effect of chitosan-based nanomaterials on plant growth and

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protection.16, 17, 19-21

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Chitosan has high affinity towards Cu as compared to other metals and with this

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distinctive ability, Cu-chitosan based nanomaterials have been synthesized and applied in various

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fields.13, 14, 16, 22, 23 We have already reported a reproducible method for the synthesis of stable

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and monodisperse Cu-chitosan NPs (mean size 374±8.2 nm) by an ionic gelation method.16 Our

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studies have resulted in a highly porous network of chitosan nanomaterial wherein Cu gets

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entrapped with 80% encapsulation efficacy.16 We have further explained the potential of Cu-

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chitosan NPs as an antifungal agent against various phytopathogenic fungi. Subsequently, we

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systematically studied the growth promotory effect of Cu-chitosan NPs on tomato seedlings by

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measuring germination, shoot-root length, and seed vigor index. Our findings unraveled that Cu-

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chitosan NPs had significant antifungal and seedling growth inducer activity.16, 20 Based on the

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studies, we predicted that Cu-chitosan NPs might enhance the activities of enzymes involved in

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the mobilization of stored food in the seed. Hence, further insight may only come through in-

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depth studies of physiological and biochemical responses of Cu-chitosan NPs on the plant. This

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could also lead to precise and safe application of Cu-chitosan NPs in the future as an antifungal

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and growth promotory agent.

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Maize is one of the important food crops of the world population. Along with other crops,

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maize seedlings have susceptibility towards various abiotic and biotic stress factors. Therefore,

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the early vigor of seedlings is also pivotal for yield improvement. Application of Cu-chitosan

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NPs in maize to understand the physiological and biochemical responses is therefore very

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crucial. In the present study, Cu-chitosan NPs synthesized with well controlled characteristics

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were evaluated for Cu content in treated seeds, seedling growth, starch and protein content, α-

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amylase and protease activity in germinating maize seeds.

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MATERIALS AND METHODS

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The experimental plan and methodology are summarized in Table 1 and the following sections

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are outlined here.

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Materials. Chitosan (low molecular weight and 80% N-deacetylation) was procured

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from Sigma-Aldrich, St. Louis, USA. Sodium tripolyphosphate (TPP) was supplied by SRL,

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Mumbai, India. PVDF (polyvinylidene difluoride) syringe filters (pore size 0.22 µm) and

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chemicals for enzyme analysis including dinitrosalicylic acid, anthrone reagent, folin and

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ciocalteus phenol were procured from HiMedia, Mumbai, India. All the chemicals and

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reagents were used as received. The seeds of maize cultivar Surya local were obtained from

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the Department of Plant Breeding and Genetics, Rajasthan College of Agriculture, Maharana

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Pratap University of Agriculture and Technology, Udaipur, India.

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Preparation of Cu–chitosan NPs. Cu-chitosan NPs were prepared following the methods

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developed in our laboratory based on the ionic gelation of chitosan with TPP anions.16

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Synthesized NPs

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Scattering (DLS), Fourier Transform Infrared, Transmission Electron Microscopy, Scanning

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Electron Microscopy and Energy Dispersive X-ray Spectroscopy and double beam Atomic

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Absorption Spectrophotometry. The characteristic details of synthesized NPs were same as we

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reported in our earlier report.16

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Seedling bioassay. Maize cultivar Surya local was used to study the efficacy of NPs on seedling

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growth using standard methods with slight modifications.24 Briefly, maize seeds were surface

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sterilized by immersing in 10% sodium hypochlorite solution for 10 min and then rinsed thrice

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with deionised water. The seeds were treated for 4 h with deionized water (control), bulk

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chitosan (0.01%), CuSO4 (0.01%) and Cu-chitosan NPs at different concentrations (0.01, 0.04,

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0.08, 0.012 and 0.16 % w/v). The treated seeds were placed in Petri plates (90 × 15 mm,

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HiMedia, Mumbai, India), having moistening filter paper with 5.0 ml of deionized water. Each

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treatment was performed in triplicates with 10 seeds in each plate. Sealed Petri plates were

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maintained at 28 ± 2ºC in a dark growth room. Deionized water was applied daily to the Petri

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plates to maintain required moisture level. Percent germination was recorded when seed showed

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at least 1.5-2.0 mm shoot. After 10 days of growth, percent germination, shoot-root length, and

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root number, seedling length, fresh and dry weight were measured. Seed vigor index (SVI) was

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calculated by the formula described elsewhere.25

were characterized for physicochemical analyses using

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Seed vigor index = (Germination %) × (Seedling Length)

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Copper content. To estimate Cu content, treated seeds were oven dried for ~96 h at 75ºC till

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constant dry weight was achieved. The dried seed samples were ground to a fine powder and

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digested with 65% HNO3. Cu content in digested samples was measured using atomic absorption

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spectrophotometer (AAS 4141 model, Electronics Corporation of India Ltd., India) by following

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a method described elsewhere.26

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Measurement of enzyme activity

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Alpha (α) amylase assay. α-amylase at different growth stages (0, 1, 3, 5, 7 and 9 days of

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germination) was extracted from germinating seeds by homogenizing in sodium acetate buffer

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(100 mM, pH 4.7) at 4ºC. To initiate the enzyme reaction, diluted enzyme extract (1 ml) was

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mixed with 100 mM sodium acetate buffer (pH 4.7) and 1 ml of starch solution (1% w/v) and

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incubated at 37ºC for 15 min. The reaction was stopped by adding 2 ml 3, 5-Dinitrosalicylic acid

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(DNS) followed by incubation at 100ºC for 5 min in the pre-heated oven. Before cooling the

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reaction mixture, 1 ml of potassium tartrate (40% w/v) was added, and mixed by quick

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vortexing. The absorbance of the enzyme activity was recorded at 560 nm. The enzyme activity

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was expressed as µ mole/min/g dry weight basis which corresponded to µ mole of glucose

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equivalent released per minute under the assay conditions.27

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Protease assay. At various germination stages (0, 1, 3, 5, 7 and 9 days of germination), protease

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was extracted from germinating seed tissues by homogenizing in 100 mM phosphate buffer at

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4ºC. The enzyme reaction was started by adding casein (1% w/v), a substrate for protease assay.

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The reaction mixture was incubated at 37º C for 20 min. of the enzyme reaction was terminated

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by adding trichloroacetic acid (5% v/v), cause precepetation of protein. The resulted precipitate

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was removed by centrifugation at 10000 rpm for 15 min. and the supernatant was allowed to

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react with alkaline copper reagent at room temperature for 10 min. To the reaction mixture, Folin

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Ciocalteau Reagent (FCR) was added and incubated in dark at room temperature for 30 min. The

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absorbance was measured at 620 nm after the development of blue color using a UV-visible

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spectrophotometer. The enzyme activity was expressed as µ mole/min/g dry weight basis which

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corresponded to µ mole of tyrosine equivalent released per minute under the assay conditions.28

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Starch and protein estimation. The starch content in the germinating seeds was determined

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using anthrone reagent.29 The seeds were homogenized in ethanol (85% v/v) and boiled for 10

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min. The samples were centrifuged at 5000 rpm for 10 min, and the supernatant was removed.

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One additional extraction in 10 ml of hot ethanol (80% v/v) was also carried out. The pooled

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supernatant was allowed to react with 0.2% anthrone reagent. The absorbance was measured at

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630 nm and starch content was expressed as mg/g dry weight basis. The protein content of

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germinating tissue was determined according to the Lowry method28 using bovine serum

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albumin as standard and the results were expressed as mg/g dry weight.

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Statistical analysis. Statistical analyses of the data were performed with JMP software version

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12 using the Turkey-Kramer HSD test to determine significant differences among treatment

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groups at p = 0.05. Each experiment was repeated twice, and each treatment consisted of three

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replicates.

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RESULTS AND DISCUSSION

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Cu-chitosan NPs. Well characterized Cu-chitosan NPs, as reported in our previous research

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article16 were used to study its effect on physiological and biochemical response in seedling

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growth. The mean physical diameter of the NPs was 150 ± 12.4 nm and mean hydrodynamic

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diameter was 374.3 ± 8.2 nm along with zeta potential + 22.6 mV. The striking feature which

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could play an important role in the growth aspect of the plants is the Cu component of NPs

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which is entrapped in the chitosan network. The present study was conducted to compare the

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effect of bulk chitosan, CuSO4 and Cu-chitosan NPs on growth aspects of maize seedlings.

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Cu content in seed. The aim of this experiment was to quantify Cu content in differentially

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treated seeds and make a further comparison with the seedling growth. Laboratory synthesized

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Cu-chitosan NPs, water (control), bulk chitosan and CuSO4 were used separately to treat maize

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seeds for 4h. The treated seeds were dried and used for estimation of Cu content by AAS.

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Increasing amount (0.01, 0.04, 0.08, 0.12 and 0.16%) of Cu-chitosan NPs showed increasing

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contents (0.012, 0.022, 0.028, 0.035 and 0.041 mg/g) of Cu into the treated seeds (Table 2).

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Maximum 0.041 mg/g Cu content was recorded in 0.16% Cu-chitosan NPs treated seeds. CuSO4

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(0.01%) treated seeds had 0.027 mg/g dw Cu whereas in water and bulk chitosan treatment, the

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almost same content of Cu was found (Table 2).

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Effect of NPs on seedling growth. To study the effect of various NPs on seedling growth seeds

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treated for 4h were grown for 10 days and data were recorded for % seed germination, root-shoot

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length, root number, seedling length, fresh and dry weight and seed vigor index (SVI). The

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statistical analysis showed that Cu-chitosan NPs exert a statistically significant difference in all

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parameters used for seedling growth measurement (Figure 1). Significantly higher values of

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shoot length, root lengths, root number, seedling length, fresh weight, and SVI were recorded in

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0.04, 0.08 and 0.12% Cu-chitosan NPs treated seeds (Figure 2 b - f and h). However, %

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germination and dry weight were not statistically different among the treatments but

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considerably higher in NPs treated seeds (Fig 2a and g). Effect of bulk chitosan on various

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growth parameters was higher as compared to control and CuSO4 except % germination and

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SVI. However, bulk chitosan has a significantly lower growth promotion effect than Cu-chitosan

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NPs. But, Cu-chitosan NPs at 0.16% concentration was found to be strong growth inhibitory for

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germinating seedlings followed by CuSO4 treatment (Figure 2). Most dramatic effect of 0.16%

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Cu-chitosan NPs was on root number and length which was significantly lower as compared to

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all other treatments (Figure 2c and d). From these experiments, it can be concluded that 0.16%

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Cu-chitosan NPs exert seedling growth inhibitory effect while its lower concentrations showed a

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growth promotion. The observed results on seedling growth were also correlated with the Cu

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content of treated seeds. In general, higher content of Cu in seeds showed growth inhibitory

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effect on seedlings (Figure 2).

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Effect of NPs on starch and protein content in germinating seeds. To determine the starch

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and protein mobilization pattern during germination of maize seed, seed tissues were collected

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on 0, 1, 3, 5, 7 and 9 days of seedlings for starch and protein estimation. From zero to first day, a

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slower and similar pattern of starch mobilization was observed in all treatments. From third day

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onward, starch started to mobilize rapidly and maximum mobilization was observed in Cu-

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chitosan NPs treated seeds. In control, bulk chitosan and CuSO4 treated seeds, starch

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mobilization was slower and its content remained higher on all days as compared to Cu-chitosan

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NPs treated seeds (Figure 3). Among Cu-chitosan NPs, 0.01, 0.04, 0.08 and 0.12%, induced

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maximum mobilization of starch over the germination period. After nine days, starch could not

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be estimated in Cu-chitosan NPs treated seeds because the seed material got almost exhausted

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(Figure 3a). Total protein content was also measured in germinating seeds which increased from

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the first day and reached a maximum at the third day in all the treatments. After the third day,

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protein content started to decline (Figure 3b). From these experiments, we can observe that seeds

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treated with Cu-chitosan NPs showed higher starch mobilization and have enhanced de nova

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synthesis of proteins.

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Effect of Cu-chitosan NPs on α-amylase and protease activity in germinating seeds. The

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aim of this experiment was to correlate the mobilization of starch and protein with α-amylase and

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protease activity. α-amylase activity was measured at 0, 1, 3, 5, 7 and 9 days of germinating

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seeds (Figure 4a). At zero day, the negligible activity of α-amylase was observed in all

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treatments. The activity increased from 1st to 5th day and declined rapidly in proceeding days in

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all treatments. On 5th day α-amylase activity was maximum (2.32, 2.93, 2.14, 1.96 and 0.59

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µmol/min/g dw) in Cu-chitosan NPs treatments (0.01, 0.04, 0.08, 0.12 and 0.16%) as compared

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to all other the treatment groups. Among the treatments, lower α-amylase activity was recorded

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in 0.16% Cu-chitosan NPs and CuSO4 treatments. Bulk chitosan induced comparatively higher

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α-amylase activity as compared to control, CuSO4 and 0.16% Cu-chitosan NPs but considerably

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lower to other concentrations of NPs (Figure 4a). Similar to α-amylase activity, protease activity

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was also effected by Cu-chitosan NPs in the same manner (Figure 4b). Protease activity was

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maximum at a 3rd day in all treatments of Cu-chitosan NPs except 0.16%. In 0.16% Cu-chitosan

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NPs, lowest protease activity was recorded followed by CuSO4 treatment. After the 3rd day, the

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enzyme activity got fall-off in all the treatments (Figure 4b). These data suggest that Cu-chitosan

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NPs significantly induced starch and protein mobilization by enhancing the α-amylase and

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protease activity in germinating seeds. As starch comprises about 80% of reserve food in maize

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seed, it is, therefore, the main source of energy for germinating seeds.

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In the present study, we have deciphered the physiological and biochemical effects of Cu-

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chitosan NPs on seedling growth of maize. We ascertain that seed priming by Cu-chitosan NPs

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significantly induces the seedling growth as compared to other treatment (Figs. 1 and 2). Further,

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we observed that the elevated seedling growth is due to rapid mobilization of starch and protein

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in germination process due to higher activity of α-amylase and protease in Cu-chitosan NPs

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treatments (Figs. 3 and 4). Our study also revealed that Cu-chitosan NPs significantly enhanced

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the total protein content in the germinating seed which may be attributed to de nova synthesis of

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proteins (Figure 3b). Few studies have been conducted on the applications of chitosan NPs in

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enhancing the plant growth and protection with astonishing results.17,

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studies we have established that in addition to antifungal activity, Cu-chitosan NPs can stimulate

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the seedling growth in tomato16. But, further reports on the physiological and biochemical

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response of seedlings are unavailable.

20, 21

. In our previous

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Germination assay is a fundamental procedure to determine growth and toxicity of metals

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and NPs on plants.9, 11 In the seed germination process, embryonic cells become metabolically

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active through complex biochemical changes like protein biosynthesis including various

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enzymes of the glycolytic pathway and carbon metabolism such as α-amylase.30 For vigor and

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rapid seedling growth, the reserved food should be efficiently mobilized to the embryonic mass.

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Chitosan and Cu, which are the core components of NPs could enhance the seedling growth by

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inducing the higher activity of α-amylase and protease enzymes (Figs. 2 and 4). The two facts

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could explain the physiological and biochemical responses of NPs in our experiments. First, in a

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recent study, it has been found that chitosan biopolymer entails up-regulation of a number of

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genes related to carbohydrate metabolism in the plant15 and thus application of chitosan/or its

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NPs could persuade the growth of plants through up-regulation of various enzymes. Second, the

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role of Cu is well established in plants as a micronutrient, acts as a key structural and catalytic

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component of various enzymes of electron transfer and redox reactions and is imperative for

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growth.31, 32 Thus, Cu component of NPs may contribute in accelerating the metabolic processes

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in germinating seed. Alongside, it is interesting to reiterate that bulk chitosan (0.01%) treatment

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did not show any significant effect on seedling growth, α-amylase and protease activity and total

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protein content as compared to NPs treatment (Figs. 2, 3 and 4).

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We foresee that Cu-chitosan NPs has a weighty effect on plant cells as they easily pass

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into the seeds along with encapsulated Cu and participate strongly in the metabolism of

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germinating seeds. Whereas, large sized polymeric bulk chitosan with low surface area cannot

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readily pass into the seeds and is not as effective as chitosan NPs towards plant cells (Figure 5).

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We assume that bulk chitosan might have developed a film on the seed surface which had

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prevented water uptake by the seed. The reduced water uptake has subsequently affected the seed

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germination and seed vigor index. Earlier results also concluded that chitosan NPs as compared

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to bulk chitosan has better growth enhancing activity in plants as it can easily cross the cell

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membrane and endows higher activity.10, 16, 19 However, a high concentration of Cu in plant cell

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becomes toxic and induces metabolic disturbances.22, 33

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In the AAS study, we estimated Cu content in the treated seeds and correlated it with

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seedling growth characters and α-amylase and a protease activity. We observed that the pattern

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of the seedling growth was correlated with the Cu content. As Cu content increases in the seeds,

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the seedling growth abates and goes to a minimum at high Cu content (Table 2 and Figure 2). In

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aerial parts of the plant, the upper critical concentration of Cu has been found from 15-30 mg/g

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in various plant species.33. Seeds treated with 0.01, 0.04, 0.08, 0.12 and 0.16% Cu-chitosan NPs

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showed 0.012; 0.022; 0.028; 0.035 and 0.041 mg Cu content per g of seeds, respectively (Table

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2). The acute toxicity on seedling growth and enzymes activities at 0.16% of Cu-chitosan NPs

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could be explained by the higher Cu content (0.041 mg/g) in germinating seeds (Table 2 and

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Figure 2). Cu-chitosan NPs (0.01 to 0.12% ) showed higher vigor, seedling growth, enhanced

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amylase and protease activity in germinating seeds compared to all other treatments (Figs. 2 and

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4). With respect to Cu toxicity in seedlings, the Cu content found in 0.08 and 0.12% NPs treated

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seed was 0.028 and 0.035 mg/g which was higher than the seeds treated with CuSO4 (Table 2).

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CuSO4 (0.01%) showed the more inhibitory effect on seedling growth, α-amylase and protease

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activity as compared to treatment with 0.08 and 0.12% Cu-chitosan NPs (Figs. 2 and 4).

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To understand the response of CuSO4 on seedling growth, we must understand the

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physicochemical properties of Cu-chitosan NPs. Cu-chitosan NPs exhibits porous network

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wherein Cu is entrapped in the pores.16 The porous network of chitosan NPs slowly releases Cu

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from the nanostructures.13, 16 Therefore, we assume that after entering into seeds the Cu-chitosan

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NPs releases Cu slowly and steadily, and the exposure of Cu to cellular system is squat. Thus,

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chitosan entrapped Cu, and its slow release imposes a mild effect on the cell. Contrarily, in

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CuSO4 treatment, a sudden and rapid exposure of Cu to cellular system exerts growth inhibitory

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effect on seedlings (Figure 5). These results are in line with the previous results where PVP

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entrapped Ag exhibited less toxicity to plants as compared to AgNO3.34

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Cu-chitosan NPs at 0.12% concentration caused a little decline in growth but not as

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cumbersome as it was at 0.16% concentration (Figure 1). Growth inhibitory effect of 0.16% Cu-

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chitosan NPs could be because of excess Cu that has been reported to decrease the level35 and

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activity of α-amylase36, and other metabolic enzymes in germinating seeds thereby inhibiting

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seedling growth.22,

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compared to other growth parameters (Figure 2). It appears that Cu was much more cytotoxic to

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the root cells at higher concentration. In addition, roots have been found to have more affinity

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towards Cu and as a result increase in lipid peroxidation and a striking decrease of the K content

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has been reported.37 In the present study, the higher seedling growth of maize under Cu-chitosan

35, 36

Root number and length were drastically decreased at 0.16% NPs as

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NPs treatment may be due to the involvement of chitosan and Cu in metabolic complex reactions

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during germination which ultimately provides higher energy to growing seedlings.

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In summary, the present study shows that Cu-chitosan NPs can significantly enhance

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maize seedling growth by up-regulating the enzymes responsible for mobilization of stored food.

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It can also be concluded that micronutrient like Cu could be used in plants as Cu-chitosan NPs

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for seed treatments for higher and rapid growth of seedlings. These findings will open the

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possibility of using other micronutrients in the form of metal chitosan NPs to enhance seedling

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growth. Our earlier and present study reveals that Cu-chitosan NPs shows promising antifungal

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activity in plants. Therefore, we conclude that seed priming with Cu- chitosan NPs is beneficial

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for seedling growth and may also help in disease protection especially for seed born and soil

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born disease. Further investigations in this line are under progress.

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Acknowledgments. The authors are indebted to the financial support from Rashtriya Kristi

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Vikas Yojna (RKVY), Government of Rajasthan, India. The authors gratefully acknowledge the

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PhD grants, IF140505 from DST and DBT/2015RCOA-2012/372 DBT, Government of India to

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R.V. Kumaraswamy and Ram Chandra Choudhary. Authors are thankful to the Nano Research

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Facility, Washington University in St. Louis for providing assistance in nanoparticle synthesis

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and characterization.

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Ghormade, V.; Deshpande, M. V.; Paknikar, K. M. Perspectives for nano-biotechnology enabled protection and nutrition of plants. Biotechnology Advances 2011, 29, 792-803. Khot, L. R.; Sankaran, S.; Maja, J. M.; Ehsani, R.; Schuster, E. W. Applications of nanomaterials in agricultural production and crop protection: A review. Crop Protection 2012, 35, 64-70. Jayaseelan, C.; Ramkumar, R.; Rahuman, A. A.; Perumal, P. Green synthesis of gold nanoparticles using seed aqueous extract of Abelmoschus esculentus and its antifungal activity. Industrial Crops and Products 2013, 45, 423-429. Raliya, R.; Nair, R.; Chavalmane, S.; Wang, W.-N.; Biswas, P. Mechanistic evaluation of translocation and physiological impact of titanium dioxide and zinc oxide nanoparticles on the tomato (Solanum lycopersicum L.) plant. Metallomics 2015, 7, 1584-1594. Raliya, R.; Tarafdar, J.; Singh, S.; Gautam, R.; Choudhary, K.; Maurino, V. G.; Saharan, V. MgO nanoparticles biosynthesis and its effect on chlorophyll contents in the leaves of clusterbean (Cyamopsis tetragonoloba L.). Advanced Science, Engineering and Medicine 2014, 6, 538-545. Raliya, R.; Tarafdar, J. C.; Biswas, P. Enhancing the mobilization of native phosphorous in mung bean rhizosphere using ZnO nanoparticles synthesized by soil fungi. Journal of Agricultural and Food Chemistry 2016, 64, 3111-3118. Anderson, C. W. N.; Bhatti, S. M.; Gardea-Torresdey, J.; Parsons, J. In Vivo Effect of Copper and Silver on Synthesis of Gold Nanoparticles inside Living Plants. ACS Sustainable Chemistry & Engineering 2013, 1, 640-648. Hernandez-Viezcas, J. A.; Castillo-Michel, H.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Interactions between CeO2 nanoparticles and the desert plant mesquite: a spectroscopy approach. ACS Sustainable Chemistry & Engineering 2016, 4, 1187-1192. Feizi, H.; Moghaddam, P. R.; Shahtahmassebi, N.; Fotovat, A. Impact of bulk and nanosized titanium dioxide (TiO2) on wheat seed germination and seedling growth. Biological trace element research 2012, 146, 101-106. Shukla, S. K.; Mishra, A. K.; Arotiba, O. A.; Mamba, B. B. Chitosan-based nanomaterials: A state-of-the-art review. International Journal of Biological Macromolecules 2013, 59, 4658. Thuesombat, P.; Hannongbua, S.; Akasit, S.; Chadchawan, S. Effect of silver nanoparticles on rice (Oryza sativa L. cv. KDML 105) seed germination and seedling growth. Ecotoxicology and Environmental Safety 2014, 104, 302-309. Dzung, N. A.; Khanh, V. T. P.; Dzung, T. T. Research on impact of chitosan oligomers on biophysical characteristics, growth, development and drought resistance of coffee. Carbohydrate Polymers 2011, 84, 751-755. Brunel, F.; El Gueddari, N. E.; Moerschbacher, B. M. Complexation of copper (II) with chitosan nanogels: Toward control of microbial growth. Carbohydrate Polymers 2013, 92, 1348-1356. Jaiswal, M.; Chauhan, D.; Sankararamakrishnan, N. Copper chitosan nanocomposite: synthesis, characterization, and application in removal of organophosphorous pesticide from agricultural runoff. Environmental Science and Pollution Research 2012, 19, 2055-2062.

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15. Chandra, S.; Chakraborty, N.; Dasgupta, A.; Sarkar, J.; Panda, K.; Acharya, K. Chitosan nanoparticles: A positive modulator of innate immune responses in plants. Scientific Reports 2015, 5. 16. Saharan, V.; Sharma, G.; Yadav, M.; Choudhary, M. K.; Sharma, S.; Pal, A.; Raliya, R.; Biswas, P. Synthesis and in vitro antifungal efficacy of Cu–chitosan nanoparticles against pathogenic fungi of tomato. International Journal of Biological Macromolecules 2015, 75, 346-353. 17. Xing, K.; Shen, X.; Zhu, X.; Ju, X.; Miao, X.; Tian, J.; Feng, Z.; Peng, X.; Jiang, J.; Qin, S. Synthesis and in vitro antifungal efficacy of oleoyl-chitosan nanoparticles against plant pathogenic fungi. International Journal of Biological Macromolecules 2016, 82, 830-836. 18. Tan, C.; Xie, J.; Zhang, X.; Cai, J.; Xia, S. Polysaccharide-based nanoparticles by chitosan and gum arabic polyelectrolyte complexation as carriers for curcumin. Food Hydrocolloids 2016, 57, 236-245. 19. Van, S. N.; Minh, H. D.; Anh, D. N. Study on chitosan nanoparticles on biophysical characteristics and growth of Robusta coffee in green house. Biocatalysis and Agricultural Biotechnology 2013, 2, 289-294. 20. Saharan, V.; Mehrotra, A.; Khatik, R.; Rawal, P.; Sharma, S.; Pal, A. Synthesis of chitosan based nanoparticles and their in vitro evaluation against phytopathogenic fungi. International Journal of Biological Macromolecules 2013, 62, 677-683. 21. Manikandan, A.; Sathiyabama, M. Preparation of Chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International Journal of Biological Macromolecules 2016, 84, 58-61. 22. Lou, L.-q.; Shen, Z.-g.; Li, X.-d. The copper tolerance mechanisms of Elsholtzia haichowensis, a plant from copper-enriched soils. Environmental and Experimental Botany 2004, 51, 111-120. 23. Saharan, V.; Khatik, R.; Kumari, M.; Raliya, R.; Nallamuthu, I.; Pal, A. Nano-materials for plant protection with special reference to Nano-chitosan. International Conference on Advances in Biotechnology (BioTech). Proceedings 2014, 23. 24. Association, I. S. T. In International rules for seed testing: rules l996, 1996; International Seed Testing Association: 1996. 25. Abdul-Baki, A. A.; Anderson, J. D. Vigor determination in soybean seed by multiple criteria. Crop Science 1973, 13, 630-633. 26. Smiri, M.; Chaoui, A.; El Ferjani, E. Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium. Journal of plant physiology 2009, 166, 259-269. 27. Bernfeld, P.; Colowick, S.; Kaplan, N. Methods in enzymology. by SP Colowick and NO Kaplan, Academic Press Inc., New York 1955, 149. 28. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 1951, 193, 265-275. 29. Yemm, E.; Willis, A. The estimation of carbohydrates in plant extracts by anthrone. Biochemical Journal 1954, 57, 508. 30. Zhang, H.; Lian, C.; Shen, Z. Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa. Annals of Botany 2009, 103, 923-930. 31. Ahmad, P. Plant Metal Interaction: Emerging Remediation Techniques. Elsevier: 2015. 32. Rajasekaran, P.; Santra, S. Hydrothermally treated chitosan hydrogel loaded with copper and zinc particles as a potential micronutrient-based antimicrobial feed additive. Frontiers in Veterinary Science 2015, 2, doi: 10.3389/fvets.2015.00062.

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33. Adrees, M.; Ali, S.; Rizwan, M.; Ibrahim, M.; Abbas, F.; Farid, M.; Zia-ur-Rehman, M.; Irshad, M. K.; Bharwana, S. A. The effect of excess copper on growth and physiology of important food crops: a review. Environmental Science and Pollution Research 2015, 22, 8148-8162. 34. Yasur, J.; Rani, P. U. Environmental effects of nanosilver: impact on castor seed germination, seedling growth, and plant physiology. Environmental Science and Pollution Research 2013, 20, 8636-8648. 35. Ahsan, N.; Lee, D.-G.; Lee, S.-H.; Kang, K. Y.; Lee, J. J.; Kim, P. J.; Yoon, H.-S.; Kim, J.S.; Lee, B.-H. Excess copper induced physiological and proteomic changes in germinating rice seeds. Chemosphere 2007, 67, 1182-1193. 36. Singh, D.; Nath, K.; Sharma, Y. K. Response of wheat seed germination and seedling growth under copper stress. Journal of Environmental Biology 2007, 28, 409. 37. Adhikari, T.; Sarkar, D.; Mashayekhi, H.; Xing, B. Growth and enzymatic activity of maize (zea mays l.) plant: solution culture test for copper dioxide nanoparticles. Journal of Plant Nutrition 2015, 99-115.

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List of Figures

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Figure 1. Effect of Cu-chitosan NPs on seed germination and seedling growth of the maize.

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Figure 2. Effect of Cu-chitosan NPs on (a) % germination; (b) shoot and (c) root length; (d) root

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number; (e) seedling length; (f) fresh weight; (g) dry weight and (h) shoot vigor index

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of maize seedling. Data were recorded after 10 days. Each value is mean of triplicate,

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and each experiment consisted of 10 seedlings. The same letter in the graph of each

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treatment is not significantly different at p=0.05 as determined by Tukey-Kramer

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HSD, Control with water. BCH (Bulk chitosan, 0.01%) dissolved in 0.1% acetic acid.

398

CuSO4 (0.01%). Cu content was measured in 4h treated seed.

399 400

401 402

403 404

Figure 3. Effect of Cu-chitosan NPs on (a) starch and (b) total protein content in germinating seeds of maize. Each value is mean of triplicate Figure 4. Effect of Cu-chitosan NPs on (a) α-amylase and (b) protease activity in germinating seeds of maize. Each value is mean of triplicate Figure 5. A hypothetical presentation of the effect of CuSO4, bulk chitosan and Cu-chitosan NPs on seedling growth of maize

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Figures

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Figure 1.

407 408 409

Control

BCH

CuSO4

0.01 % 0.04 % 0.08 % 0.12 % 0.16% I-------------------------------- Cu-chitosan NPs ---------------------------

----I

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Figure 2.

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Germinatin % 100

a

Germination %

ab b

80

Cu content a

a ab

ab

b

60 40 20 0 Control BCH

CuS04

0.01

0.04

0.08

0.12

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/g dw)

(a)

0.16

412 413

Shoot length (cm)

9 8 7 6 5 4 3 2 1 0

Cu content

a

abc

ab

ab

abc abc bc c

Control BCH

CuS04

0.01

0.04

0.08

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0.16

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/g dw)

Shoot length

(b)

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(c)

Root length

Cu content

Root length (cm)

14 a

12 10 8 6

b

b

bc

b

b

b

4

c

2 0 Control

BCH

CuS04

0.01

0.04

0.08

0.12

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/g dw)

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0.16

415

Number of roots

6

a

Number of roots

5

ab

4 3

Cu content

bc

ab

ab

abc bc

2 c

1 0 Control

BCH

CuS04

0.01

0.04

0.08

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0.16

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/g dw)

(d)

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Seedling length

Cu content

Seedling length (cm)

25

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

a 20 b 15

cd

bcd

10

d

c

bcd

e

5 0 Control BCH CuSO4

0.01

0.04

0.08

0.12

Cu content (mg/g dw)

(e)

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0.16

417

Fresh weight 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00

Cu content a

a

a

Control BCH

a

a

a

a

a

Copper

0.01

0.04

0.08

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0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/gm dw)

Fresh weight (g)

(f)

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(g)

Dry weight

Cu content

0.25

Dry weight (g)

0.20

a

a

a a

a

a

a a

0.15 0.10 0.05 0.00 Control BCH Copper

0.01

0.04

0.08

0.12

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/g dw)

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419

Shoot vigour index 2000 1800 1600 1400 1200 1000 800 600 400 200 0

Cu content 0.045

a

0.04

ab

0.035

bc cd

d

0.03

cd

0.025

d

0.02 0.015

e

0.01 0.005 0

Control BCH CuS04

0.01

0.04

0.08

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0.16

Cu content (mg/g dw)

Shoot vigour index

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Figure 3. Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

(a)

Bulk chitosan (0.01%) Cu-chitosan NPs (0.01%) Cu-chitosan NPs (0.08%) Cu-chitosan NPs (0.16%)

Starch (mg/g dw)

1000 800 600 400 200 0 0

1

424

Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

(b)

Total protein (mg/g dw)

3 5 Time (days)

7

9

Bulk chitosan (0.01%) Cu-chitosan NPs (0.01%) Cu-chitosan NPs (0.08%) Cu-chitosan NPs (0.16%)

4 3 2 1 0 0

425

1

3 5 Time (days)

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Figure 4. Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

Activity (µmole/min/g dw)

(a)

3 2.5 2 1.5 1 0.5 0 0

1

3

5

7

9

Time (days)

428

Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

Activity (µmole/min/g dw)

(b)

Bulk chitosan (0.01%) Cu-chitosan NPs (0.01%) Cu-chitosan NPs (0.08%) Cu-chitosan NPs (0.16%)

8 6 4 2 0 0

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Bulk chitosan (0.01%) Cu-chitosan NPs (0.01%) Cu-chitosan NPs (0.08%) Cu-chitosan NPs (0.16%)

1

3

5

Time (days)

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Figure 5.

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Tables

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Table 1 Experimental outlines

437 Experiment Laboratory synthesis of Cu-chitosan NPs

Analysis/method

Notes

Ionic gelation approach following the method of Saharan et al. (2015)

Cu–chitosan NPs were synthesized

Seedling bioassay

Blotter test method

To determine the efficacy of NPs on seedling growth

Cu content in seed

AAS (Atomic Absorption Spectrophotometer) DNS reagents method

To determine Cu content in seeds

α-Amylase enzyme assay

To estimate α-amylase activity

Protease assay

Folin Ciocalteau reagent (FCR) method

To estimate protease activity

Starch estimation

Anthrone reagent

To estimate the mobilization pattern of starch during germination

Statistical analysis

JMP software version 10 using Turkey-Kramer HSD test

438 439 440 441 442 443

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Table 2 Cu content in seeds treated for 4h with various treatments Treatment (%)

Cu content (mg/g)

Control (water)

0.012± 0.0016d

BCH (0.01)

0.014±0.0014d

CuSO4 (0.01)

0.027±0.0017c

Cu-Chitosan NPs

445 446 447

0.01

0.011±0.0008d

0.04

0.021±0.0013c

0.08

0.027±0.0012bc

0.12

0.034±0.0017ab

0.16

0.041±0.0012ab

Each value is mean of triplicate. The same letter in a column of each treatment is not significantly different at p=0.05 as determined by Tukey-Kramer HSD, Control with water. BCH (Bulk chitosan, 0.01%) dissolved in 0.1% acetic acid. CuSO4 (0.01%).

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TOC Figure: Cu – Chitosan nanoparticles increase the activity of α – amylase and protease that

452

enhances the mobilization of reserve food of the seed, resulting in an increased germination rate.

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