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Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 11 (2017) pp. 3714-3724 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.611.435 Effect of Nanoparticles for Seed Quality Enhancement in Onion [Allium cepa (Linn) cv. CO (On)] 5 K. Anandaraj* and N. Natarajan Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore - 641 003, Tamil Nadu, India *Corresponding author ABSTRACT Keywords Onion, Seed Quality, Allium cepa, nano particle, Nano seed treatment, ZnO, Ag, CuO and TiO2 Nanoparticles, SEM, TEM, Particle Size Analyzer, Raman Spectroscopy. Article Info Accepted: 26 September 2017 Available Online: 10 November 2017 Zinc oxide (ZnO), Silver (Ag), Copper oxide (CuO) and Titanium oxide (TiO2) nanoparticles were synthesised using simple chemical route which were characterised using Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Particle Size Analyzer and Raman Spectroscopy. Size of Zinc oxide (ZnO), Silver (Ag) Copper oxide (CuO) and Titanium dioxide (TiO2) nanoparticles measured 16-50 nm, 50-100 nm, 60-150 nm and 100-120, respectively to conform the nano-size. Onion seeds when dry dressed with the synthesised nanoparticles each at 750, 1000, 1250 and 1500 mg kg -1, the dose of 1000 mg kg-1 outperformed in enhancing the germination (72%), shoot length (7.5 cm) root length (6.4) and thereby the vigour index (998) compared to control (60%, 6.0, 5.4 and 692) respectively. Introduction Onion (Allium cepa L.) belongs to the family Liliaceae and is one of the most important monocotyledonous and cool season vegetable crops in India. Amongst the onion producing countries in the World, India ranks second in area and production. Onion has been the largest item of export accounting to 76.2 per cent in the total export of vegetables from India. The unavailability of quality onion seed is greatly responsible for its lower yield. The seed quality parameters especially seed size and seed weight affect the final yield in onion production (Gamiely et al., 1991). Furthermore, high quality seed is considered as the critical input in onion on which all other inputs have to be managed for potential yield in onion. Onion is grown in an area of 1.01 m ha with a production of 16.8m tonnes keeping the productivity at 16.6 t ha-1.The prominent onion growing states are Maharashtra, Gujarat, Uttar Pradesh, Orissa, Karnataka, Tamil Nadu and Andhra Pradesh. Perambalur district in Tamil Nadu has the highest share of production (24.6%) followed by Trichy (14.2%), Coimbatore (13.7%) and Erode (10.8%) districts. In India onion seed is getting lost quickly due to the production of free radicals by lipid peroxidation during 3714 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 storage. As the current technologies available to prolong the vigour and viability of onion seed on a large scale are not satisfactorily alleviating the practical problem, an alternative simple and practicable seed treatment to control seed deterioration of onion is need of the hour. Several researchers reported that mid-term hydration-dehydration treatments performed better in improving germinability and seedling vigour after storage in soy bean (Basu 1994; Mandal et al., 2000) and okra (Kapri et al., 2003). Nanoparticles can be one of the ways to retain the vigour and viability during storage by preventing the losses due to biotic and abiotic stress. Lots of works have been done in biological system to address a wide range of field problems utilizing nanomaterials and nanodevices. (Natarajan and Sivasubramanian, 2008) elucidated various nanotechnological approaches especially in the field of agriculture including nano-polymer for seed hardening, nano-sensors, nano-barcodes and use of magnetic nanoparticles for aerial seeding. (Senthil kumar, 2011) and (Sridhar, 2012) further established the use of metal oxide nano-particles in improving germination up to 30 per cent in aged seeds of black gram and tomato respectively which could be probably due to the quenching of reactive oxygen species (ROS) generated during seed storage. Applications of nanotechnology in improving seed germination, emergence and growth of seedlings (Zhang et al., 2006), thwarting pest attack (Nair et al., 2010) and for early pathogen detection (Alocilja and Radke, 2003) are few of the multifarious beneficial interventions in the field of agriculture. Hence the present investigation was made to study the effect of ZnO, Ag, CuO and TiO2 nanopartilcle on the vigour and viability of onion seed. Materials and Methods The first experiment synthesis of nanoparticles and characterization was carried out at Department of Nano Science and Technology and the second experiment study of seed quality enhancement was carried at Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore -03, during the year of 2012–13. The chemicals used for synthesis of nanoparticles viz., Zinc nitrate (Zn (NO3)2.4H2O), AgNO3, Trisodium citrate, copper nitrate trihydrate,TiO2 pellets, NaOH and Ethanol were purchased from THE I.L.E. Co. Pvt. Ltd., Coimbatore, Tamil Nadu. Synthesis of ZnO, Ag, CuO and TiO2 Nanoparticles Zinc oxide nanoparticles ZnO NPs were synthesized by adding 0.45 M aqueous solution of zinc nitrate (Zn(NO3)2.4H2O) and 0.9 M aqueous solution of sodium hydroxide (NaOH) in distilled water taken in two separate 250 ml glass beakers. The Zn(NO3)2 solution (100 ml) transferred to a burette was added drop wise (slowly for 40 min.) to the 100 ml of NaOH contained in the beaker placed over a magnetic stirrer with hot plate set at 55oC with high-speed stirring. The beaker after adding 100 ml Zn(NO3)2 was removed from the hot plate, sealed with aluminium foil and kept undisturbed for 2h for precipitation and settlement. The precipitated ZnO NPs were washed with millipore water followed by ethanol and then vacuum dried at 60oC (Moghaddam et al., 2009). Nanoparticles such synthesized were transferred to air tight screw cap vial (10 ml) and stored at ambient temperature for further investigations. 3715 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 Silver nanoparticles The Ag NPs were prepared by using chemical reduction method according to the description outlined by (Lee and Meisel, 2005). Fifty milliliter of AgNO3 0.005 M taken in a beaker was boiled on a magnetic stirrer with hot plate. To this solution, 5ml of 1% trisodium citrate was added drop by drop from 10 ml measuring cylinder with vigorous mixing on the stirrer until pale yellow colour appeared. Then the beaker was removed and kept at ambient temperature where the chemical reaction occurred would have been 4Ag+ + C6H5O7Na3 + 2H2O → 4Ag0 + C6H5O7H3 + 3Na+ + H+ + O2↑ Copper oxide Nanoparticles CuO NPs were synthesised using copper nitrate trihydrate (CuN2O6.3H20, SigmaAldrich), and sodium hydroxide anhydrous pellets (NaOH, Carlo erba) in the presence of polyvinyl alcohol (PVA, Sigma Aldrich) as starting precursor (Wongpisutpaisan et al., 2011). Sodium hydroxide was dissolved in deionized water and thus obtained solution (0.5M, 50 ml) was added drop wise to an aqueous CuN2O6.3H20 solution (0.1 M, 50 ml) for 30 min. Sonication of the solution was performed using Sonics Model VCX 1500 until complete precipitation. Finally, precipitated powder was calcined at 6000C for 2 h to obtain the nanoparticles. decanted with deionized water several times and dried at 60o C for 24 h to obtain the nanoparticles (Arami et al., 2007). Characterization nanoparticles of synthesized Characterization of the synthesized nanoparticles was performed by using Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Particle Size Analyzer and Raman Spectroscopy. Scanning Electron Microscope (SEM) FEI QUANTA 250 was used to characterize the size and morphology of the nanoparticles. Sample of test nanoparticles (0.5 to 1.0 mg) was dusted on one side of the double sided adhesive carbon conducting tape, and then mounted on the 8mm diameter aluminum stub. Sample surface were observed at different magnification and the images were recorded. Transmission Electron Microscope (TEM) FEI TECHNAI SPRIT make was used to analyze the sample. Dilute suspensions of NPs (0.50 mg) in pure ethanol (15 ml) were prepared by ultrasonication. A drop of the suspension placed on 300-mesh lacy carbon coated copper grid upon drying, was examined and the images were recorded at different magnification. Titatium oxide nanoparticles Particle size analyzer TiO2 NPs were synthesized by dissolving 0.5 g TiO2 pellets in 30 ml of NaOH solution (10 M) under vigorous stirring at room temperature for 2 h. Thus obtained yellow solution was irradiated in an ultra sonicator (Soncis, VCX 1500, 20 kHz and 350 W) for 2h in ambient temperature. The resultant precipitate was then centrifuged, washed and The particle size analyzer was used to determine the particle size and the distribution pattern of synthesized ZnO, Ag, CuO and TiO2 nanoparticles. The particle size distribution (PSD) of a powder indicates a list of values or a mathematical function that defines the relative amount of particles 3716 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 present, sorted according to size. In the present study, HORIBA nanoparticle size analyser SZ 100 was used. Accurately, 0.5 mg of sample was dispersed in 10 ml pure water through ultrasonication and the measurements were taken. Raman spectroscopy Raman spectroscopy is a spectroscopic technique based on inelastic scattering of monochromatic light, usually from a laser source. Inelastic scattering means that the frequency of photons in monochromatic light changes upon interaction with the sample. Photons of the laser light are absorbed by the sample and then reemitted. Frequency of the reemitted photons can be shifted either up or down in comparison to the original monochromatic frequency which is called the Raman Effect. This shift provides information about vibrational, rotational and other low frequency transitions happening in the molecules. Raman spectroscopy can be used to study solid, liquid and gaseous samples. Raman spectrum is a spectral “fingerprint”. If number of different compounds is present in a mixture, the resulting Raman spectra will be a superposition of the spectrum of each of the components. The relative intensities of the peaks can be used to give quantitative information on the composition of mixture of known compounds. The Raman spectrum was measured for the synthesized nanoparticles using Raman spectrum Model- R- 3000- QE. The powdered, dried NPs kept in a polythene bag were spread to an extent of 1 cm2 and Raman probe was placed on the sample packets without exposing the sample directly to the probe (Fig. 2). Seed treatment Fresh seeds of onion (CO 5) obtained from the Department of Vegetable Crops, Horticultural College and Research Institute, Coimbatore were dry dressed with each of the synthesized nanoparticles viz., ZnO, Ag, CuO and Tio2 @ 750, 1000, 1250, and 1500 mg kg-1 in screw capped glass bottles at room temperature. The glass bottles containing seeds and nanoparticles were manually shaken gently for 3 min., 5 times in a span of 3h. Seeds shaken without nanoparticles served as control. After dry dressing with the nanoparticles, the seeds were packed in cloth bag and stored under ambient condition (25 ± 30C temperature and 95 ± 3% RH). Seed samples were drawn at monthly intervals up to six months and evaluated for the following seed quality parameters. viz., germination percentage, shoot length, root length, and vigour index. Germination test in quadruplicate using 100 seeds each with four replicates of 25 seeds was carried out in paper medium. The test conditions of 25 ± 2 0C and 95 ± 3 per cent RH were maintained in the germination room. At the end of 14 days, the number of normal seedlings was counted and the mean was expressed as percentage (ISTA, 2005). Root length of all the normal seedlings from the germination test was measured from collar region to the root tip and the mean was expressed in centimetre. Shoot length of all the normal seedlings from the germination test was measured from collar region to the shoot apex and the mean was expressed in centimetre. Vigour index was computed by adopting the method suggested by (Abdul-Baki and Anderson, 1973) and expressed as whole number. Vigour index = Germination percentage × Seedling length in cm. 3717 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 Results and Discussion Characterization of nanoparticles (ZnO, Ag, CuO and TiO2) The surface morphology of Zinc Oxide (ZnO), Silver (Ag), Copper Oxide (CuO), and Titanium Oxide (TiO2) nanoparticles were examined under SEM, TEM, Particle Size Analyzer and Raman Spectroscopy. The morphology of different nanoparticles observed are presented below. The particle size analyzer was used to analyze the size of the particle using laser scattering principle for estimating the average particle size and distribution pattern for synthesized ZnO, Ag, CuO, and TiO2 nanoparticles. The particle size distribution of ZnO, Ag, CuO and TiO2 was found to be 16, 53.7 nm, 183 nm and 387 nm respectively (Fig. 1). Raman spectroscopy was employed to identify the chemical composition and to confirm the four different nanoparticles synthesized by observing the peaks. The peaks were observed at 308, 908, 1152 and 1280 cm-1 for CuO while at 528, 871, 945 and 1411 cm-1 for Ag, 276, 637, 1327 and 1458 cm-1 for TiO2 and 366, 723, 1066 and 1219 cm-1 for ZnO nanoparticle confirming the respective chemical compounds (Fig. 2). Seed germination and seedling vigour Nanoparticles of ZnO, Ag, CuO and TiO2 when treated in different concentrations viz., 750, 1000, 1250 and 1500 mg kg-1 had significantly outperformed control in terms of germination, shoot length, root length and vigour index. Significant differences were also observed between the nanoparticles and doses. Nano seed treatment improved the germination of aged onion seeds variably towards the treatment at different concentrations. Fig.1 Particle analyzer average size and intestity distribution of ZnO nanoparticles Peak No 1 2 3 Total S.P. Area Ratio 1.00 ----1.00 Mean 16.1nm --- nm --- nm 16.1 nm 3718 S.D 0.7 nm --- nm --- nm 0.7 nm Mode 16.0 nm --- nm --- nm 16.0 nm Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 Fig.2 Raman spectra of (a) Zno, (b)Ag, (c)CuO and (d) TiO2 nanoparticles (a) (b) (c) (d) Plate.1 SEM images of (a) Zno, (b) silver, (c) CuO and (d) TiO nanoparticles 2 (a) (b) 3719 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 (c) (d) Plate.2 TEM images of (a) Zno, (b) silver, (c) CuO and (d) TiO2 nanoparticles (a) (b) (c) (d) 3720 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 Table.1 Effect of nanoparticles on germination % of stored (6 months old) seeds of onion (CO 5) Treatments (mg /kg-1) 750 1000 1250 1500 Mean Control ZnO 64(53.13) 72(58.05) 70(56.79) 66(54.33) 68(55.55) 60(50.76) T 0.84 1.66** SEd CD Ag 65(53.73) 69(56.18) 67(54.94) 66(54.33) 67(54.94) Germination % CuO 60(50.76) 66(54.33) 66(54.33) 61(51.35) 63(52.53) D 0.75 1.48** TD 1.68 NS TiO2 60(50.76) 65(53.73) 60(50.76) 62(51.94) 62(51.94) Mean 62(51.94) 68(55.55) 66(54.33) 64(53.13) 65(53.73) Table.2 Effect of nanoparticles on shoot length (cm) of stored (6 months old) seeds of onion (CO 5) Treatments (mg /kg-1) 750 1000 1250 1500 Mean Control ZnO 7.2 7.5 7.3 7.4 7.3 6.0 T 0.09 0.17** SEd CD Ag 6.7 7.5 7.4 7.5 7.3 Shoot length (cm) CuO 6.5 6.4 6.7 6.5 6.5 D 0.08 NS TD 0.18 NS TiO2 6.5 6.7 6.7 6.4 6.6 Mean 6.725 7.025 7.025 6.95 6.925 Table.3 Effect of nanoparticles on root length (cm) of stored (6 months old) seeds of onion (CO 5) Treatments (mg /kg-1) ZnO Ag 6.2 6.4 6.4 6.3 6.3 5.4 750 1000 1250 1500 Mean Control T SEd CD 5.6 6.3 6.2 5.8 6.0 D 0.07 0.15** Root length (cm) CuO TiO2 5.2 5.3 5.3 5.4 5.5 6.4 5.6 6.3 5.4 5.9 TD 0.06 0.13** 0.15 0.30** 3721 Mean 5.575 5.85 6.125 6 5.9 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 Table.4 Effect of nanoparticles on vigour index of stored (6 months old) seeds of onion (CO 5) Treatments (mg /kg-1) 750 1000 1250 1500 Mean Control SEd CD ZnO 858 998 954 909 929 692 T 11.50 22.64** Ag 796 958 918 887 890 Vigour index CuO 704 775 798 735 753 D 10.28 20.25** TD 23.00 NS TiO2 707 795 779 791 768 Mean 766.25 881.5 862.25 830.5 835 Characterization of Nanoparticles (ZnO, Ag, CuO and TiO2) Nanoparticles ZnO Ag CuO TiO2 Morphological Descriptions SEM Lanceolated nanoscaled rods measuring 50-80 nm diameter; appeared to be radiating from a central core (Plate 1a) Appeared as a bundle of spheres measuring for 400-450 nm (Plate 1b) Uniform spherical to oval sized particle measuring 60 – 150 nm (Plate 1c) Irregular spherical shaped peanut like particle with an average diameter of 120 nm (Plate 1d) The values recorded for control was 60 per cent. Among the nanoparticles treatment, seeds treated with ZnO NPs @ 1000 mg kg-1 had the highest germination of 72 per cent which was followed by Ag NPs @ 1000 mg kg-1 (69%). Control recorded the lowest germination (60%) (Table 1). Among the dosages, seeds treated @1000 (72%) found to register maximum germination than other dosages. Interaction among the NPs and dosage revealed that ZnONPs @ 1000 mg kg1and Ag NPs @ 1000 mg kg-1 recorded in the maximum germination of 72 percent while the minimum in the seed treated with TiO2 at the 750 mg kg-1. The beneficial effect of the ZnO NPs in improving the germination TEM Rod shaped fused at centre to form a radiating structure as observed in SEM (Plate 2a) Spherical in shape with a size ranging from 50 -100 nm (Plate 2b) Uniform crystalline particles measuring 80-140 nm (Plate 2c) Rutile nano particle, primarily tetragonal in shape with an average size of 100 nm (Plate 2d). could be ascribed to higher precursor activity of nanoscale zinc in auxin production. Apart from this, zinc is one of the essential nutrients required for plant growth. It is an important component of various enzymes that are responsible for driving many metabolic reactions in all crops. Zinc oxide NPs are reported to also exhibit positive effect on the reactivity of phytohormones especially Indole Acetic Acid (IAA) facilitating in the phytostimulatory actions. Zinc-rich ZnO NPs could increase the level of IAA in roots (sprouts), which in turn can increase growth rate of seedlings. Enhanced physiological parameters could be attributed and quenching of free radicals by nanoparticles which could 3722 Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 3714-3724 entered through cracks present seed coat, reached into free radicals resulting in enhanced seed vigour. Nanoparticle treated germinated seeds exhibited higher root and shoot length than control. ZnO NPs treated seeds induced maximum shoot length (7.5 cm) compared to control (6.0 cm) after six months of storage. Among the different nano particle treatments, seeds treated with ZnO and Ag NPs @ 1000 mg kg-1 produced the lengthiest shoot length (7.5 and 7.5 cm) than control (6.0cm) (Table 2). Nanoparticles treatment did not influence the root length of seedlings immediately after treatment. After six months of storage, nanoparticles treated seeds had higher root length (6.4cm) compared to control (5.4 cm). Among the different nanoparticle treatments, seed treated with ZnO NPs @ 1000 and Tio2 @ 1250 mg kg-1 produced the lengthiest root (6.4 cm) than control (5.4 cm) (Table 3). Such promoting effect of nanoscale SiO2 and TiO2 on germination was reported in soya bean, in which authors noticed increased nitrate reductase enzyme activity and enhanced antioxidant system. Similar results were observed by (Zheng et al., 2005) when Spinacia oleracea seeds were treated with nanoscale TiO2 particles. The results revealed the promotory effect of ZnO nanoparticles at optimum concentrations and inhibitory effect at high concentrations on root and shoot growth. An increase of the shoot/root ratio compared to that of the control was reported by (Shah and Belozerova, 2009) while analyzing the influence of metal nanoparticles on germination of Lactuca seeds. Significant variation was observed for vigour index due to nano seed treatment, their dosages and their interactions (Table 4). Among the nano seed treatments, seeds treated with ZnO NPs resulted in maximum vigour index (998) than other treatments and the control (692). Interaction between nano seed treatment and its dosages revealed that highest vigour Index was observed in seeds treated with ZnO @ 1000 mg kg-1 (998) which was followed by Ag @ 1000 mgkg-1 (958). The beneficial effect of nanoparticle in improving the seed quality may be attributed that nano particles would induce oxidationreduction reactions via the superoxide ion radical during germination, resulting the quenching of free radicals in the germinating seeds. In turn, oxygen produced in such process could also be used for respiration, which would further promote germination. The experiments carried out by (Senthil kumar, 2011) revealed that black gram seeds treated with ZnO nano rods and ZVI NPs enhanced the physiological and biochemical properties resulting in improved vigour and viability of aged seeds. The reason attributed was the donation of electrons by the nano particles in scavenging the free radicals in the aged seeds. Nanoparticles tested in the investigation were supportive in enhancing the germination and seedling vigour of the onion seeds which are supposed to be highly prone for deterioration in storage. Application of nanoparticles especially ZnO @ 1000mg kg-1 seed improved germination and related physiological parameters. However, the findings are to be verified under large scale field condition before recommending to farmer for adoption. References Abdul-Baki, A.A., Anderson, J. D., 1973. Vigour deterioration of soybean seeds by multiple criteria. Crop Sci., 13:630–633. Alocilja, E. C., and S. M. Radke., 2003. Market analysis of biosensors for food safety. Biosensors and Bielectronics., 18: 841846. Arami, H., M. Mazloumi., R. Khalifehzadeh., and S. K. Sadmezhaad., 2007. 3723