Zinc Supported Multiwalled Carbon Nanotube Nanocomposite; A

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Biological and Medical Applications of Materials and Interfaces

Zinc Supported Multiwalled Carbon Nanotube Nanocomposite; A Synergism to Micronutrient Release and A Smart Distributor to Promote the Growth of Onion Seeds in Arid Conditions. Vinay Kumar, Divya Sachdev, Renu Pasricha, Priyanka H Maheshwari, and Neetu Kumra Taneja ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b13464 • Publication Date (Web): 04 Oct 2018 Downloaded from http://pubs.acs.org on October 5, 2018

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ACS Applied Materials & Interfaces

Zinc Supported Multiwalled Carbon Nanotube Nanocomposite; A Synergism to Micronutrient Release and A Smart Distributor to Promote the Growth of Onion Seeds in Arid Conditions. Vinay Kumar1, Divya Sachdev1**, Renu Pasricha2*, Priyanka H. Maheshwari3, Neetu Kumra Taneja1, 1

**National Institute of Food Technology Entrepreneurship and Management-Sonepat, Haryana, India 2*

New York University-Abu Dhabi

3

National Physical Laboratory, CSIR-New Delhi, India.

** Corresponding Author- Divya Sachdev * Co-Corresponding author- Renu Pasricha Email: [email protected], [email protected]

Keywords:

Multiwalled

Carbon nanotubes,

Onion seeds,

Epifluorescence,

Raman

spectroscopy, Water flux, Scanning Transmission Electron Microscopy

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Abstract In the current scenario, nanotechnological applications in the agriculture sector showing potential impacts on the improvement of plant growth in terms of protection and safety are at a very nascent stage. The present study deals with the synergistic role of zinc (Zn) and multiwalled carbon nanotubes (MWCNTs) synthesized as ZnO/MWCNTs nanocomposite, a prospective applicant to modulate the micronutrient supply and enhance the growth of onion seeds thereby replacing harmful, unsafe chemical fertilizers. To the best of our knowledge, this is the first report wherein MWCNTs have been envisaged as a micronutrient distributor and a nutrient stabilizer enhancing the growth of onion plant under arid conditions. The growth trend of onion seeds was evaluated in an aqueous medium with varied concentrations of (i) MWCNTs (ii) Zinc oxide nanoparticles (ZnONPs) and (iii) ZnO/MWCNTs nanocomposites. ZnO/MWCNTs nanocomposites with 15 µg/ml of concentration displayed the best seedling growth with maximum number of cells in telophase. Significant growth trend with increased concentration of ZnO/MWCNTs displayed no negative impact on plant growth in contrast to the use of MWCNTs. The synergistic role of Zn nanoparticles and MWCNTs in ZnO/MWCNTs nanocomposites on the rate of germination was explained via mechanism supported by Scanning Transmission Electron Microscopy (STEM). 1. Introduction New advances in the nanosized engineered materials have demonstrated great potentials in the field of agriculture science and plant biology1. As a part of this technological innovations carbon nanotubes (CNTs) with its distinct morphological characters, pore size and surface properties have been demonstrated to have distinct properties which can be intensively employed for nanobiotechnological application2. In the recent past we have noticed that with the advent of nanotechnology in agriculture, more investigations are focussed on studying the detrimental impacts of different nanomaterials on plant ecology and environment rather than its affirmative 2 ACS Paragon Plus Environment

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implications

3-5

. Especially increased investigations on examining the impact of CNTs on the

germination of varied plants have been allured because of their ability to penetrate plant cells6. It has been shown that the interactions of plant with CNTs have displayed activation of physiological processes in plants that may either lead to the enhancement or retardation of growth in a plant7,8. There are reports of carbon nano-onions derived from biowaste that stimulates metallic micronutrients in gram plants9. Recent investigations of dispersible functionalized MWCNTs on tomato plant have displayed its character in triggering the water channel protein and consequently enhancing the growth10. In a similar study, MWCNTs were evaluated and shown to affect the plant phenotype and the composition of soil microbiota11. Eventually both MWCNTs and single walled carbon nano tubes (SWCNTs) have shown to affect the growth of some important plants such as tomato10-12, corn13, barley, soyabean

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, rice15, mustard

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, wheat, maize zucchini, cucumber,

ryegrass5, 12, 17. The demand of the hour is to exploit the nanotechnology for boosting the crop production and its quality while capitalizing the associated economic benefits

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. The agricultural production is

experiencing a plateau nowadays, which will adversely affect the growth rate of countries at large, hence employing diminutive quantities of these nanomaterials can work wonders to impart high levels of efficiency in the field of agriculture19. The size of nanomaterials renders them high fraction of surface atoms with high surface energy and reduced imperfections as compared to their bulk counterparts.20. In this context efficacy of zinc (Zn), an important micronutrient involved in a wide range of physiological processes in plants21 such as in preserving structural orientation of macromolecules while keeping the ion transport systems intact22, acting as a prosthetic component of enzymes within cells23 and upholding the important role in production of plant growth hormone21, 24 ; all this can be improved by supplying it in nano form while ensuring and increasing the bioavailability of the zinc. Although the most



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legitimate source of uptake of Zn for controlled dispersion in nano form is ZnO (alone or in stabilized form)25, which will ultimately convert to Zn2+ in different pH environment of plant26. Zn uptake varies among the plant species although the accurate reasons of specific location of nutrient uptake by plant species are yet unexplored. Raskar et al.27revealed the positive effect of ZnO nanoparticles (ZnO-NPs) on seed germination. While the ZnO nanoparticles (ZnO-NPs) employed by Rasker et al. have shown to be linked up in alleviating the germination of plant that ultimately leads to higher crop production, however the germination trend decreases at higher concentrations. Likewise, reduced root growth was observed in some plants like corn and ryegrass with increased ZnO-NPs concentrations8,28. Onion (Allium cepa L.) of family Amaryllidaceae is one such crop wherein Zn plays an important role in plant growth and yield. Yet there are reports indicating the retarded growth in onions due to phytotoxicity of aggregated ZnO-NPs29. Observations of certain degree of phytotoxicity, especially at higher concentration can be an output of high interactions within the nanoparticles that leads to the agglomeration 30and therefore refrain the actual bioavailability of these micronutrients via treatment of plant by nanoparticles. In this view to prevent agglomeration of metal nanoparticles, CNTs have been procured as a smart vehicle to deliver the desired micronutrient to seeds slowly and steadily during germination. Therefore for the first time the synergistic effects of Zn supported on MWCNTs have been employed for the germination of onion seeds. Herein MWCNTs served the dual purpose, on one hand it stabilizes the Zn nanoparticles (not allowing to aggregate) while on the other hand penetrates the cell in delivering the Zn ions (a, micronutrient) in a controlled manner. Since the Zn uptake and translocations in a plant system are profoundly dependent on their availability in the growth media, therefore a controlled release of micronutrient Zn will be significant for enhancing the onion seed growth. This endeavours our goal in keeping Zn 4 ACS Paragon Plus Environment

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nanoparticles dispersed while nourishing the plant effectively with minimum quantity of Zn and increasing the utilization of less Eco-toxic micronutrient. 2. Experimental 2.1 Synthesis of Zn supported MWCNTs nanocomposites Commercially available Nanocyl 7000 multiwalled carbon nanotubes (MWCNTs) with diameter in the range of 20-30 nm and aspect ratio >1000 were used as such. These were further employed as a support for the synthesis of Zn nanoparticles. To prepare the same, MWCNTs were dispersed in ethanol by ultra-sonication, the dispersed MWCNTs were placed in three neck round bottom flask and kept for refluxing at ~80 °C with continuous magnetic stirring. 0.5 mM solution of anhydrous zinc chloride (ZnCl2) prepared in ethanol was added such that the ratio of MWCNTs: ZnCl2 is 9: 1 by weight. A simultaneous reduction of the ZnCl2 was done via sodium borohydride (NaBH4) during refluxing for an hour. The solution was filtered followed by washing (with fresh Ethanol) and dried in oven to obtain Zn supported MWCNTs nanocomposites. It is to be mentioned here that these nanocomposites were hereafter named as (ZnO/MWCNTs) for the reason given in results and discussions section (3.1). 2.2 Synthesis of bare Zinc Oxide (ZnO) nanoparticles ZnO nanoparticles were obtained on simultaneous addition of 0.1mM solution of zinc acetate and 0.05mM sodium hydroxide solution in a beaker containing water with continuous stirring. Once the addition is over, the white precipitate is filtered and washed thoroughly and dried for further use. The solid white powder was named as ZnO-NPs. 2.3 Material characterization details Thermal stability and ZnO-NPs loading in samples was estimated by Thermo Gravimetric Analysis (TGA). The measurements were carried out on TGA/DSC 1600 by Mettler Toledo at a constant



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heating rate of 10 °C/min in air. The X-ray Diffraction (XRD) was performed on Rikagu powder Xray diffractometer model: XRG 2KW using Cu Kα radiation. The mean crystallite size and lattice parameters were calculated from line broadening and d-spacing using Debye Scherrer formula. The purity of the prepared samples was enumerated from Raman analysis. Raman spectroscopy was carried out using Renishaw InVia Reflex Micro Raman Spectrometer equipped with CCD detector at room temperature and in air. Green laser (excitation line 514 nm) was used to excite the samples. Five scans per sample was recorded wherein the samples were exposed to the laser power of 25mW for 10 sec. The MWCNTs samples were imaged under Transmission Electron Microscope (TEM)31using Tecnai G2 F30 S-Twin instrument operating at an accelerating voltage of 300 kV, having a point resolution of 0.2 nm and a lattice resolution of 0.14 nm. The samples were dispersed uniformly in iso-propyl alcohol

32

and the solution was drop casted on the carbon coated copper

grid and allowed to dry. The size and distribution of Zn nanoparticles supported on MWCNTs as ZnO/MWCNTs were determined by High Resolution Transmission Electron Microscope (HRTEM). 2.4 Preparations of Seeds: The onion seeds were purchased from a local market from Satguru Seeds Pvt. Ltd., Haryana India of truthful label (variety DR-301). Prior to using of seeds in the experiment, the physical purity of the seeds was tested and established, by placing the seeds in water for few minutes, the seeds floating on water were discarded and rest of them were used for experiment. The experimental set up consisting of petri-plates, distilled water and filter papers were properly sterilized in autoclave. 2.5 Seed Treatment and Germination: Seeds were surface sterilized by treating with (50%, (v/v)) sodium hypochlorite solution for ten minutes and rinsed once with distilled water. They were kept in a dark room before use. The


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sterilized seeds were then transferred to the varied concentrations of solutions (mentioned below) of the MWCNTs, ZnO-NPs and ZnO/MWCNTs. 2.6 Preparation of MWCNTs stock solution and germination of seeds Stock solution of MWCNTs (70µg/ml) only, ZnO-NPs (72µg/ml) only and ZnO/MWCNTs (84µg/ml) were uniformly dispersed in water by ultrasonic probe sonicator (Cole-parmer (Model08895-06). MWCNTs and ZnO/MWCNTs of different concentrations (viz. 2µg/ml, 5µg/ml, 10µg/ml, 15 µg/ml, 20 µg/ml, and 40 µg/ml) were prepared from stock solutions while ZnO-NPs solution with only 20 µg/ml concentrations was prepared. The sterilized onion seeds were soaked in the above solutions for 20 hours and also in distilled water so as to serve as control. After the seed treatment, the seeds were washed carefully with distilled water. The set up used (i.e filter paper, petri-plates) for germination of seeds were all sterilized with each set (at room temperature of 22 ±1°C) containing twenty five seeds in three replications for individual concentrations of nanoparticles and for different time period intervals. 2.7 Determining the Germinated Seed Characterization The total number of onion seeds germinated were counted every day (the seed was considered to be germinated only if the length of the radical was 2 mm length). Germination of seed was considered till 12 days and at the end of the 10th day, the potential of seed germination was assessed in the terms of Germination Percentage (GP), where; GP= number of germinated seeds per day/ total number of seeds x 100 At the end of the 10th day growth, length of radical and plumule were noted. The length of root and shoot was taken with the help of a thread and scale. Furthermore, in order to get the moisture content of seedling, fresh weight (FW) and dry weight (DW)20 (seedling dried at 60ºC) were also weighed for the moisture content in seeds.



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Raman spectroscopy of plant roots was carried out using Renishaw InVia Reflex Micro Raman Spectrometer equipped with CCD detector at room temperature and in air. Red laser (excitation line 784 nm) was used to excite the onion root samples. Five scans per sample was recorded wherein the samples were exposed to the laser power of 25mW for 10 sec. Raman spectra facilitated the evaluation of carbon present during the growth in ZnO/MWCNTs and control. Moreover Scanning Electron Microscope (SEM) analysis of sectioned roots was carried out to observe the presence of ZnO/MWCNTs in the rootsby using a model ZEISS-EVO MA10. Epi-fluoroscence microscopyof the vertically sectioned roots placed on the slide was done on NIKON INTENSILIGHT, C-HGFI with the camera employed DS-Fi2. The fluorescence in the roots were observed with FITC, (mercury lamp) with excitation at 465-495 nm, dichoric beam splitter (DM) at 505nm and barrier filter with emission at 515-555 nm. In order to study the mitotic cell division in the onion root tip, individual roots of each sample of approximately 1cm length were cut and transferred into a plastic vial. The roots were incubated with 1.5ml of 1N hydrochloric acid (HCl) for 15min, which allowed the cells to completely stabilize and gets fixed. The solution in the vial was heated at 60ºC for few more minutes before final washing of the root tip with distilled water. For staining, the root tip was rinsed thrice with distilled water and incubated with acetocarmine solution for 15 min. The root tip was removed from the stain solution and was again rinsed 2-3 times with water before microscopic analysis on NIKON instrument under bright field mode. 2.8 Transmission Electron Microscopy (TEM) of onion roots Sample Preparation: Onion roots were analysed by (TEM) by placing in petri dishes and covering with a fixing solution containing; 2% paraformaldehyde, 2.5% glutaraldehyde, 1.5mM calcium chloride (CaCl2) and 1.5mM Magnesium chloride(MgCl2) in 0.05 M PIPES buffer to maintain pH 6.9 (calcium and magnesium ions insertion reduce the cellular components and also enhance the


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retention of phospholipids)31. These roots were then cut into approximately 1mm X 1mm with a sharp razor blade while submerged and further fixed. Further the root pieces were washed three times for 20 min each in 0.05M PIPES buffer containing 1.5mM CaCl2 and MgCl2 and placed at 4ºC in the same solution overnight. Samples were rinsed one more time in the buffer solution and then briefly post fixed at room temperature for 20 min in 1% osmium tetroxide, 0.8% potassium ferricyanide, 1.5 mM CaCl2, and 1.5 mM MgCl2 in 0.05 M PIPES buffer (pH 6.9). After fixing, tissues were restored to 4 °C by rinsing in cold distilled water three times for 20 min each and dehydrating in an ascending ethanol series starting from 10 to 70% ethanol (EtOH), with each time 10% increments for 20 min each. Tissues were then stained in 1% uranyl acetate and 70% EtOH for 1.5 h at 4 °C, followed by two 5 min rinses in 70% EtOH, with the temperature brought back to room temperature during the second rinse. Dehydration was continued by washing tissues once in 85% and 95% EtOH and twice in 100% EtOH, (total time taken for each stepis 15 min). Finally, two washes were done in propylene oxide for 10 min each preceded the embedment of material into (Spurr’s resin). Thin sections were cut from the embedded samples using RMC ultramicrotome equipped with a diamond knife. Sections were mounted oncopper slot grids. To understand the effects of activation of ZnO/MWCNTs on root elongation the TEM studies were simultaneously carried out on control (without ZnO/MWCNTs)and treated onion roots (ZnO/MWCNTs). Scanning Transmission Electron Microscopy/ Energy-dispersive X-ray analysis (STEM-EDAX) HRTEM for the ultrathin sections of chemically fixed onion seedwas carried out using a Talos F200X Scanning/Transmission Electron Microscope with a lattice-fringe resolution of 0.14 nm at an accelerating voltage of 200 kV equipped with CETA 16M camera. High resolution images of periodic structures were analysed using TIA software. Chemical mapping was carried out in STEM-EDAX mode wherein the Energy-Dispersive X-ray Analysis (EDAX) was carried out using 9 ACS Paragon Plus Environment


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a super-X EDS detector which has a superior sensitivity with resolution of 136eV@Mn-Ka for 10kcps at zero degree sample tilt. The detector provides quick data even for low intensity EDS signals as it is the sum of 4 detectors reducing data collection time for elemental mapping to minutes from hours. The STEM-EDAX data was analysed using Velox analytical software.

3. Results and Discussions 3.1 Characterization of MWCNTs, ZnO/MWCNTs and ZnO-NPs 3.1.1 XRD The XRD curves for (a) ZnO-NPs (b) MWCNTs and (c) ZnO/MWCNTs is shown in Fig. 1 while (Supporting Information,S-1) gives a detailed account of the lattice parameters, i.e. miller indices, FWHM, and crystallite size for the given diffraction angle of the samples. The diffraction peak at

ߠ =25.7º corrosponds to graphitic crystallographic planes (002)33 of MWCNTs which is intact during the preparation of ZnO/MWCNTs nanocomposites. The X-ray diffraction spectra (Fig. 1 (A)) for ZnO-NPs shows characterstic peaks at 2ߠ value 31.85, 34.68 and 36.42 which is respectively indexed to (100), (002) and (101) hexagonal planes of ZnO (JCPDS card no. 89-0510). Further it was observed that the XRD pattern (Fig 1(A)) for ZnO/MWCNTs , shows peaks at 2ߠ value 31.96, 34.61 and 36.42 that are similar to ZnO-NPs. However the significant variation in 2ߠ peaks in case of ZnO/MWCNTs corresponds to the adherence of ZnO nanoparticle ssupported by MWCNTs surface. There is an increase in the line broadening of the diffraction peaks of ZnO/MWCNTs as compared to that of the ZnO-NPs indicating a substantial decrease in the average crystallite size of ZnO-NPs on MWCNTs computed from PXRD data (Supporting Information,S-1) ~ 3.11 nm (also observed from HRTEM in (Fig.1(E) inset)) as compared to 8.54 nm for bare ZnO-NPs. It is important to mention here that MWCNTs is an appropriate matrix that defies the agglomeration of ZnO-NPs,


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while providing the surface for supporting ZnO-NPs34 . In order to keep the surface of MWCNTs unchanged, the methodologies adopted for depositing Zn nanoparticles on MWCNTs surface and synthesis of (bare) ZnO-NPs varied, though the outcome for both the procedures attributed to the formation of ZnO-NPs as observed from

PXRD25,35-36. Well-dispersed ZnO-NPs results in

increased reactivity even at low concentrations which in turn acts as defects and refrains the ordering of MWCNTs structure. 3.1.2. Raman Spectroscopy Fig. 1(B) shows the Raman spectra of MWCNT and ZnO/MWCNTs nanocomposites. Three bands: D (defect/ disorder induced) band, G (graphite like) band, and G΄ (second order harmonic to D) band were identified. A small peak of D+G band is also present, which is due to defect in CNTs 37. The intensities of the bands were determined by the area under the spectral curve. The intensity of the G-band 11was used as a reference in determining the relative intensities of the D band (ID) and G΄ band (IG΄)38. The relative intensity of D band (ratio ID/IG) for MWCNTs sample is nearly 1.15, which indicates a large number of defects39. The high value of ID/IG is probably because of the commercial MWCNTs (Nanocyl 7000 grade) procured and used as such without any purification. Moreover the surface morphology as depicted by TEM images (Fig. 1 (D&E)), are quite uneven, wavy and the graphitic planes are disoriented with respect to the nanotube axis. The TGA curve (Fig. 1(C)) of MWCNTs also confirms defects in the pure carbon structure (Nanocyl 7000 grade) that shows the presence of 8.7% of residue. The incorporation of ZnO-NPs (Fig.1(E)) on the tubes, further results in generating stresses in the hexagonal carbon layer that is reflected by the increase in the relative intensity of the D band in Raman (Fig.1(B)).The relative intensity of G΄ band (indicating interlayer coupling ) decreases very slightly in ZnO/MWCNTs nanocomposite as compared to MWCNTs, which indicates that ZnONPs are attached and have penetrated on walls of the nanotubes

40

. This probably affects the

coupling of only a few surface layers leaving the inner/core of MWCNTs walls unchanged.



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3.1.3. TGA The TGA and DTG curve of the MWCNTs and ZnO/ MWCNTs nanocomposites is shown in Fig 1(C). the residual content in the MWCNTs is nearly 8.7 %. This is probably due to the presence of some impurities like metal oxides (used as catalyst during the synthesis of MWCNTs). For ZnO/MWCNTs the residual content was nearly 18.8%, indicating the incorporation of nearly 10 % ZnO in the nanocomposite. The thermal decomposition of MWCNTs takes place around 610 °C as also reported elsewhere41. The thermal stability of the ZnO/MWCNTs reduces as compared to MWCNTs. This is because, (i) the ZnO-NPs act as defects and catalyze the thermal decomposition process in TGA; and (ii) there is a probability that defects are also introduced in the nanotube structure during the deposition of ZnO-NPs process, which in turn reduces its thermal stability41-43. 3.2 Germination of onion seeds Germination of seeds is an important process that requires water, oxygen and suitable temperature. Uptake of water follows a triphasic pattern in which- Phase I encompasses seed imbibitions that commences with the rapid uptake of water, due to the high potential difference between water and dry seed. Phase II is a lag phase wherein there is sparse water uptake, while the seed exhibits an increase in biomass due to an enhanced metabolic activity post water uptake and utilization of the stored reserves (proteins, fats and lipids) for germination. Phase III exhibits a radical protrusion wherein the water imbibed growing seeds start differentiating into root and shoot system. In order to see the impact of ZnO/MWCNTs for effect on water uptake and the controlled release of Zn on the onion seed germination, the triphasic pattern of germination was investigated (Fig.2). 3.2.1 Water Uptake and seed germination of Onions: The germination profile (popularly referred as priming) of the seeds imbibed in water alone (control) and in various nanoparticle solutions [MWCNTs, ZnO/MWCNTs, and ZnO-NPs 12 ACS Paragon Plus Environment

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(dispersed individually in water)] were compared for their rate of germination and phase analysis over a period of 20 h (Fig.2). The seeds soaked in nanoparticle solutions exhibited an enhanced germination profile (MWCNTs > ZnO-NPs > Control > ZnO/MWCNTs) while ZnO/MWCNTs exhibited marginally delayed seed growth during initial 20h (corresponding to Phase I of seed germination). This enhanced water uptake during Phase I of germination in case of the nanoparticle solutions is likely to be due to an altered water potential that occurs upon addition of nanoparticles in water. Emergence of radical from cotyledons is not only an indication of viability of seed germination but also an extension to the influence of these nanoparticles on the growth of onion seeds while crossing its seed coat6, 16. 3.2.2Controlled release of micronutrient Zn during onion seed germination: In order to study the impact of MWCNTs for the controlled release of Zn, we investigated the growth of onion seeds in the presence of different experimental solutions containing ZnO-NPs (in triplicate, along with the control) at a final concentration of 20 µg/ml. The seeds were monitored for the appearance of roots (which indicated the initiation of germination) on daily basis for 12 days as per protocols described by Raskar et.al27. Onion seeds treated in different sets of MWCNTs at varying concentration range (from 2 to 40µg/ml each) exhibited a steady growth pattern wherein the seed germination commence from the second day and continued till the 10th day. The Germination percentage (GP) in the presence of MWCNTs reached almost 50% within merely three days (Fig.2(a)). Maximum GP for MWCNTs was attained at a concentration of 15µg/ml with no further appreciation with time or concentration. Therefore, the same concentration (i.e. 15µg/ml) was chosen for further studies At higher concentration of MWCNTs, the GP of seeds declined at later time points hence eliminated for further studies (Fig. 2 (a)). Concurrently, the germination of seeds soaked in varied sets of ZnO/MWCNTs solution (ranging from 2 to 40µg/ml) were also monitored, wherein a 40% GP was achieved in nearly eight days



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under similar conditions and tested concentrations. The ZnO-NPs alone also exhibited a pattern similar to ZnO/MWCNTs attaining 50% GP by day 11. Fig.2(c) shows the comparative variations in GP of onion seeds present in ZnO, MWCNT, ZnO/MWCNTs and control. Whilst an accelerated growth of seeds occurred in MWCNTs and control solutions (in terms of GP) in comparison to ZnO/MWCNTs and ZnO-Nps, it is pertinent to observe that ZnO/MWCNTs supported growth of onion seeds at lower concentrations (15µg/ml). When the concentration of ZnO/MWCNTs was increased (with 40µg/ml), the GP increased to almost twice the initial (Fig. 2(b)). It is important to mention here that presence of Zn affects the capacity for water uptake and transport in a plant system44-46. Moreover there is a possibility that the water intake required by the seedlings grown in the presence of the ZnO/MWCNT is reduced thereby implicating that ZnO/MWCNTs could support seed germination at low water levels. 3.2.3Onion seed germination in arid conditions Investigating the decrease of GP in ZnO/MWCNTs, (shown in Fig.2) wherein seedlings grown in each case were watered every alternate day, there isprobably an excessive translocation of water occuring in the case of ZnO/MWCNTs treated seeds (due to reduced water requirement), that hindered the passage of atmospheric air inside the seed during the initial phase of the growth resulting in a decreased GP. To verify the above hypothesis we conducted an experiment in which equal number of onion seeds imbibed initially with solutions of MWCNTs (15µg/ml), ZnO-NPs (20µg/ml) alone and ZnO/MWCNTs (15µg/ml), and control (water) were placed in appropriately labelled petriplates (in sets of three: S1, S2, S3) and kept under laboratory conditions. Each set was watered differently: S1 was watered after every 4th day, S2 was watered after 6th day and S3 was watered after 8th days (Fig. 3). The image in Fig. 3 shows the effect of watering patterns on the GP. Surprisingly we observed that the GP in case of ZnO/MWCNTs (as shown in Fig. 3) accelerated when the seeds


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were watered on 6th day and 8th day unlike the control and MWCNT sets where the GP declined while in ZnO-NPs it remained almost same. It can therefore be concluded that s supports a reduced water demand for germination of seeds and exclusively establish its application and usage in supporting growth of onion seeds under arid conditions. This is one of the exclusive results of our work which will initiate and boost the use of ZnO/MWCNTs nanocomposite for seed germination of onions and possibly other crop plants as well. We continued to monitor the growth pattern of seeds exposed to nanoparticles (MWCNTs, ZnONPs and ZnO/MWCNTs) with varied concentrations for next 10 days. Fig.4 (each with the plant images) showed the germination response of onion seedlings in terms of root, shoot length and root-shoot ratios for each individual concentration of MWCNTs nanomaterial. Morphology of the developed seedlings after 10 days were analysed and it was observed that seeds exposed to 15µg/ml each containing MWCNTs and ZnO/MWCNTs, showed maximum impact on the root length (onion seedlings shown in Fig.4(A) and 5(A)). Seedlings in MWCNTs with (15µg/ml) concentration showed almost equivalent root length (1.51cm in Fig. 4(B)), ZnO/MWCNTs (15µg/ml) and ZnO-NPs showed root elongation (2.83 cm and 2.31 cm resp.) as compared to the control (1.44cm) (Fig. 5 (B)). It is important to mention here that with increasing concentration of ZnO/MWCNTs, the root length almost remained the same however in case of MWCNTs alone, a decreasing trend of the root length with rising concentration was observed. Another important observation in case of MWCNTs was bulb formations (Fig.4(C)) between the root and shoot region; this may be due to the accumulation of MWCNTs thus resulting in growth retardation. Similar trend of increased shoot length was observed in presence of ZnO/MWCNTs and MWCNTs. 3.2.4 Transportation of ZnO/MWCNTs in onion seedlings Previously reported growth trends of in-vitro studies of onion seedlings in presence of SWCNT (Single wall Carbon nanotube) and fSWCNT (functionalized) by Canas et.al. observed no



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retardation which is in contrast to our results.12 This may be due to the basic structural difference between SWCNTs and MWCNTs; wherein the co-axial graphitic cylinders present in MWCNTs affects the porosity network and ultimately alters the water flux required by the onion seedlings47. Onion seed being porous and semi permeable in nature (pore size ~ 100nm) allows MWCNTs to enter easily and being hydrophobic in nature slips to endocarp-the bilipid layer, however once it enters the seed the diffusive flux of water molecules inside the plant changes. It is very well known fact that flow of water will be frictionless if the MWCNTs are vertically aligned however change of density of CNTs (from SWCNTs to MWCNTs) leads to change in porous network and ultimately the water flux17. Although the hydrophobic MWCNTs pass through the bilipid layer but the theoretical calculations have shown that the lengthy MWCNTs have the tendency to accumulate in parellel

32, 48

which modulates disordered flow of water and as a consequence retardation in root

length is observed. While in contrast to the MWCNTs alone; the optimal transport of water in case of ZnO/MWCNTs is achieved by ordered transport of water (shown mechanistically in Scheme1) which is due to the interactions of water molecule with the supported Zn. Water molecule clings at the edges of the pore consisting of Zn atoms via Hydrogen bonding that create charges and builds up a field while promoting the entry of water in anorganized manner (Scheme 1); this in turn raises the capillary action in a seedling. These implications were found to be true since seedlings grown in presence of ZnO/MWCNTs displayed results that indicated maximum moisture content. Apart from this germinated onion seedlings exhibited significant increase in the vegetative fresh mass, each for MWCNTs and ZnO/MWCNTs with different concentrations ranging from 2µg/ml to 40µg/ml. The biomass trend (Supporting Information,S-4) for the (fresh weight, FW) of ZnO/MWCNTs displayed maximum accumulation of mass within ten days compared to the MWCNTs, ZnO-NPs alone and control. Furthermore, the difference (W1-W2) of FW (W1) and dry weight DW (W2) gives an indication of the moisture content present in the seedlings. Substantial amount of moisture was absorbed by seedlings that were exposed to ZnO/MWCNTs as compared to other mediums


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(Supporting Information,S-4). Particularly, with increasing concentration to 40µg/ml, the absorption capacity increases indicating a better growth in presence of Zn nanoparticles supported on MWCNTs that are working synergistically. 3.3 Detection and uptake of MWCNTs in onion seedlings The penetration of MWCNTs, ZnO/MWCNTs in onion seedlings was probed by Raman analysis, SEM and TEM imaging and Epifluorescence microscopy. 3.3.1 Raman analysis and SEM imaging Fig.9 (A1) shows the Raman spectra of the seedlings grown in different media (i.e. (a) the control, (b) ZnO-NPs, (c) MWCNTs and (d) ZnO/MWCNTs nanocomposites. A broad hump observed in samples (a) and (b) is probably due to the presence of non-graphitic carbon primarily present in all the biological materials. The incorporation of CNTs in the seeds is marked by the increase in the peak intensity (curve (c)) which is observed at nearly

1430 cm-1 , which decreases on Zn

inclusion (curve (d)) as expected11, 49. There is a shift in the peak position (plausibly due to the merging of the D and G peaks) along with an increase in the line broadening of the root samples as compared to that obtained for the original nanomaterial (Fig. 6(A1)). This is probably the result of interference of the graphitic carbon of MWCNTs with the native one. SEM imaging was also conducted to observe the uptake of ZnO/MWCNTs within the onion seedling, for this the root surface was cross-sectioned and analysed. MWCNTs penetrated the pores of the onion seeds (Fig. 6 (B1a&b)) and showed characteristic enhancement in the elongation of the roots. The exposed end of the root surface shows the presence of the nanotubes alone and in bundles aligned on the root surface (Fig. 6 (B1(a&b)). 3.3.2 Epifluoroscent Microscopy and TEM Imaging



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During the synthesis protocol, MWCNTs as well as ZnO-NPs attached to the outer wall of the MWCNTshave the tendency to aggregate while forming clusters (as shown in Fig 1(E)). However the original properties of nanomaterials (here ZnO/MWCNTs) can change, once they embed inside the biological test systems andare exposed (cluster to single fragments). These potential changes in nanomaterialsleading to change in size, shape, or state of aggregation is due to the uptake and bioaccumulation of nanomaterials via plants and most crucially governed by many environmental, physiochemical aspects and the nature of interaction with cell organelles50-51. Henceforth in order to observe these ZnO/MWCNTs inside the cells, TEM of onion stem was performed (Fig. 7) wherein we found that the ZnO/MWCNTs are present in sub-cellular organelles. Our observations corroborated the prior findings 6, 52as we noticed that ZnO/MWCNTs directly penetrated the cells as they were normally observed in the cytoplasm. Moreover, the images depict that ZnO/MWCNTs of different lengths and sizesare dispersed randomly inside the cells which are shown in Fig 7 (B and B1)53. The tissues of the fully grown onion seedlings on penetration of ZnO/MWCNTs were observed under the epifluoroscent microscope, since chlorophyll acts as a fluorophore and emit blue-green fluorescent images under UV radiations (lambda=465-495nm). The images for the seedlings grown in different media MWCNTs, ZnO/MWCNTs, and control were observed and recorded.

These images (Fig. 8) clearly depict that the MWCNTs have

penetrated inside the seed and translocated further to the root and shoot portion of the seedling. Fig. 8 ((B) and (C)) shows images of the onion vascular system with arrowsindicating penetration of carbon nanotubes, since these seedlings were grown in MWCNTs and ZnO/MWCNTs medium; while the seedlings grown in control medium shows none. It is important to mention here that the image (Fig. 8(C)) in presence of ZnO/MWCNTs are different from MWCNTs since fluorescent spots were observed here which may be due to the presence of nano sized Zn known to be fluorescent in nature54. The results were further complimented with Scanning Transmission Electron Microscopy (STEM) wherein the electron transparent sections were imaged initially using


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HR-TEM along with the versatile advanced analytical microscopy, STEM coupled with Elemental X-ray spectral mapping techniques (EDAX) was done to assess the localization of ZnO-NPs on MWCNTs in the onion stem. The STEM mode along with EDAX resulted in obtaining high contrast images as shown in Fig. 9 (C and D). The contrast obtained in the STEM images was derived from the interactions of electron beam with ZnO-NPs and other heavier molecular weight elements that emit backscattered electrons. Fig 9 (A) shows the low resolution image of the whole section and we could clearly observe the distribution of MWCNTs. In higher resolution (Fig 9 (B) we also observed MWCNTs in the cell along with lot of high contrast particles. These were due to the presence of all the heavy metal (osmium and uranyl acetate) we used during the sample preparation. On further zooming in, Fig 9 (C) we could obtain the presence of ZnO-NPs on the clustered MWCNTs. This was confirmed by the EDAX (Fig 9(D)) analysis on the MWCNTs inside the section. The accumulation of ZnO-NPs onto the MWCNTs surface suggests that the particles are impregnated to the surface at a fairly good concentration. Elemental X-ray spectral mapping (EDAX) of ZnO-NPs was used to confirm location of NPs. Integration of characteristic peaks of ZnO-NPs centered at 1.012 and 8.637 keV in the EDX spectra (Fig 9, (D)) indicated the detectable limits of ZnO-NPs present on MWCNTs in onion section. It is to be mentioned here that due to the resolution limit of HR-TEM pertaining to the sample preparation of biological specimen by chemical route and the associated staining agents employed, it was not possible to resolve the nanosized ZnO-NPs on MWCNTs inside the cell. 3.4 Zinc as a mode of action for enhanced growth The synergism between MWCNTs and Zn in ZnO/MWCNTs ultimately led to a better growth of onion seeds. The enhanced growth of root and shoot length, germination percentage in presence of less water and higher biomass indicates favourable plant growth in presence of ZnO/MWCNTs medium. In contrast, seeds grown in presence of MWCNTs alone exhibits retarded root, shoot length and biomass as compared to the control medium. The significant difference in both the 19 ACS Paragon Plus Environment


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medium reveals that Zn (a micronutrient) slowly released from ZnO/MWCNTs is essential for the growth of onion and thence an experiment was designed to investigate this. In order to establish the role of Zn in the growth of onion seeds, succinic acid a ligand was chosen and incorporated in the following set of onion seeds. Initially 30 sterilized onion seeds were soaked for 10 hours in solutions of MWCNTs (15µg/ml), ZnO/MWCNTs (15µg/ml, 10µg/ml), and control (water only) separately. Once the seeds were removed from the solutions they were subsequently dipped in 0.001 molar solution of succinic acid for 10 hours. At the end ofthe ligand treatment, the seeds were washed carefully with distilled water and placed on filter paper inside thesterilized petriplates and allowed to germinate at room temperature of 24±1°C. Each set of seeds were under observation for ten days and to our surprise we found that the germination percentage of seeds in case of ZnO/MWCNTs with different concentrations was nil for 5 days

as shown in (Supporting

Information, S-2 (As showed 10% growth rate after 6 days and 30 % after ten days (Supporting Information, S-2 (B)). The seeds while in the other two sets MWCNTs and succinic acid showed the usual trend of germination rate of nearly 80% and 83% respectively as in the normal case (control) without succinic acid (Supporting Information, S-2(B)). This kind of behaviour is very much expected with ZnO/MWCNTs, since the reduced form of Zn is present on the surface of MWCNTs which is more lipophilic and can form a tetrahedral complex55with the succinic acid (as shown in Supporting Information, S-3) and become more hydrophilic, this in turn prevents its penetration inside the lipid region of the seedand abrogates the provision of contribution towards an essential nutrient to the plant which thereby leads to a poor plant growth56. Subsequently the mitotic cell division of onion root tip (Fig. 10) observed under an optical microscope (resolution~10x) for the seeds imbibed by different nutrients (here MWCNTs, ZnONPs, ZnO/MWCNTs) was also investigated. The mitotic cell divisions observed (Fig.10) under an optical set area for different seeds exposed (repeated twice), confirms the enhanced growth of onion 20 ACS Paragon Plus Environment

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radical in presence of ZnO/MWCNTs. The (Supporting Information, S-5) clearly indicates that the mitotic cell divisions for ZnO/MWCNTs showed maximum number of Telophase that indicated the enhanced growth rateas compared to seeds imbibed with other ion sources showed maximum number of metaphase and anaphase. This further indicates that rapid mitotic cell divisions57takes place in case of onion seeds treated with ZnO/MWCNTs.

4. Conclusion In the present scenario, MWCNTs and micronutrient, Zn supported MWCNTs nanocomposites have shown immense applications in diverse fields. Apart from increasing implications of nanotechnology in electronics, energy and structural applications, it is now heading towards the field of agriculture, plant biology and horticulture. The present study depicts the innovative technique of employing, MWCNTs as a smart vehicle for the nutrient delivery of Zn (acting as a nanofertilizer), to onion seeds. Although Zn plays an essential role in the metabolic activities of onion, especially inregulating the crop yield and the quality of produce but the excess of Zn also promotes negative interferences with other nutrients and enzyme activities. Therefore adequate amount of Zn supply to the plant is an important factor in raising the crop. In this context, Zn was incorporated to MWCNTs in the reduced form and solutions with different percentages i.e. 2µg/ml, 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, 40 µg/ml were prepared andemployed for the growth of onion seeds. We observed that the 15 µg/ml of ZnO/MWCNTs showed highest germination response at a laboratory scale, in terms of root and shoot length. Moreover our findings also demonstrated that while employing ZnO/MWCNTs for onion seeds, less water is required for enhancing the germination percentage which has been explained mechanistically. Finally, the paper also depicts explicitly the importance of Zn in ZnO/MWCNTs in terms of increased rate of cell divisions i.e. maximum numbers of telophase via mitotic cell divisions were seen in ZnO/MWCNTs. While in an another experiment declined or no growth of onion seedlings was



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observed when a Zn binding ligand like succinic acid was added, further illustrating the importance of Zn in the growth of onion seeds. The finding supported by characterization analysis corroborates the synergistic role of Zn and MWCNTs in the enhanced growth of onion seeds; in fact this performance in future will certainly foster nano-based fertilizers as nutrient uplifters in the plant germination and yield in arid conditions.

Acknowledgments DS is grateful to Akshay Bisht for preparing samples for TEM. We are also thankful to the anonymous reviewers for their valuable comments to improve this manuscript.

Supporting Information PXRD indexing and Crystallite sizes of ZnO-NPs, MWCNTs and ZnO/MWCNTs. Images depicting role of Zn for the growth of onion seeds and its disappearance by complexing it with succinic acid (S.A), Zn-S.A complex formation. Biomass and moisture content , mitotic division and varied phases in onion seedlings imbibed with ZnO-NPs, MWCNTs and ZnO/MWCNTs. References (1) Aouada, F. A.; de Moura, M. R. Nanotechnology Applied in Agriculture: Controlled Release of Agrochemicals. In Nanotechnologies in Food and Agriculture; Springer: 2015; pp 103-118. (2) Fortina, P.; Kricka, L. J.; Surrey, S.; Grodzinski, P. Nanobiotechnology: The Promise and Reality of New Approaches to Molecular Recognition. Trends Biotechnol. 2005, 23 (4), 168-173. (3) Kokini, J. L.; Yao, Y. Advances in Nanotechnology as They Pertain to Food and Agriculture: Benefits and Risks. Annu. Rev.Food.Sci.and Tech. 2016, 7 (1). (4) Servin, A. D.; White, J. C. Nanotechnology in Agriculture: Next Steps for Understanding Engineered Nanoparticle Exposure and Risk. NanoImpact 2016. (5) De La Torre-Roche, R.; Hawthorne, J.; Deng, Y.; Xing, B.; Cai, W.; Newman, L. A.; Wang, Q.; Ma, X.; Hamdi, H.; White, J. C. Multiwalled Carbon Nanotubes and C60 Fullerenes Differentially Impact the Accumulation of Weathered Pesticides in Four Agricultural Plants. Environ. Sci. Technol. 2013, 47 (21), 12539-12547. 22 ACS Paragon Plus Environment

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Figures, Tables and Schemes.



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Fig. 1 Evaluating MWCNTs, ZnO-NPs, ZnO/MWCNTs structural profiles via (A) Powder Xray diffraction (B) Raman Spectroscopy (C) Thermal gravimetric (TGA) analysis (D) Image showing TEM of typical MWCNT structure encompassed with layers (E) HR-TEM of ZnO/MWCNTs;Inset depicting the particle size distribution of the ZnO-NPs on MWCNTs


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Fig. 2 Germination percentages (GP) for seeds imbibed with (a) MWCNTs (b) ZnO/MWCNTs&ZnO-NPs and (c) overall performance at an optimum concentration.The images of seedlings (in petri-plates) with varied GP for (i) 15µg/ml MWCNT (ii) ZnO –NPs (iii) Control (iv) 15µg/ml ZnO/MWCNTs after 3rd day



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Fig. 3 GP of onion seeds imbibed for 20h with MWCNT (15 µg/ml), ZnO-NPs (20 µg/ml), ZnO/MWCNTs (15 µg/ml) and control when it is watered (S1) every 4th day; (S2) every 6th day;and (S3) watered every 8th day. The GP of ZnO/MWCNTs increased in the S3 group. Graphical representation of GP as a function of number of days for the sets/groups (S1, S2, S3).


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Fig. 4 (A) Image depicting the growth characteristics of onion seeds imbibed with MWCNTs with different concentrations (5µg/ml,10 µg/ml,15 µg/ml, 20µg/ml) (B) Effect of MWCNTs on the root and shoot elongation of onion seedlings with decreasing trend from control. (C) Images showing the bulb formation on the rear (root) end of the seedling.

Fig. 5 (A) Image depicting the growth characteristics of onion seeds imbibed with ZnO/MWCNTs of varied concentration (2µg/ml. 5µg/ml,10 µg/ml , 15 µg/ml , 20µg/ml, 40 µg/ml) (B) Effect of ZnO/MWCNTs on the root, and shoot elongation of onion seedlings with increasing trend from control. 31 ACS Paragon Plus Environment


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Scheme 1 Mechanistic depiction of the passage of water through (A) MWCNTs (B) ZnO/MWCNTs


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Fig. 6. (A1) Raman spectra of the onion roots as (a) control, (b)ZnO-NPs, (c) MWCNTs and (d) ZnO/MWCNTs (B1) SEM micrographs showing CNTs in the cross section of onion roots.



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Fig. 7 TEM images of onion roots (A, A1, A2 and A3) under control conditions with resolution from 10µm to 2 µm and to 0.2 µm respectively. B and B1 images of onion specimen incubated with ZnO/MWCNTs. View of MWCNTs (as thin white fringes) in the cytoplasm in B and B1 of the cell structure of onion are clearly visible on zooming the image.


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Fig. 8Auto fluorescence of cross-section of onion roots observed under epifluorescence microscope (filter exc- 465-495 nm and filter em- 515-555nm) (A) under control conditions (B) incubated with MWCNT only (C) incubated with ZnO/MWCNTs.



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Fig. 9 (A) and (B), HR-STEM image of MWCNT nanocompositesin onion section at different magnifications complimented with high contrast images of ZnO/MWCNTs observed by STEM in (C) for ZnO/MWCNTs. (The circled image shows the MWCNT nanocomposites in onion section) verified with EDAX in image (D) depicting the presence of Zinc on MWCNTs.


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a

b

c

d

Fig.10 Mitotic division of onion root tip under the microscope at resolution (10x) incubated under (a) control conditions (b) MWCNTs (c) ZnO-NPs (d) ZnO/MWCNTs, Inset showing resolution at (40x).



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“For Table of Contents Only”

Caption : ZnO/MWCNTs promotes the efficient growth of onion seeds in arid conditions.


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“For Table of Contents Only”

ACS Paragon Plus Environment

Zinc Supported Multiwalled Carbon Nanotube Nanocomposite; A

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