Conjunctive and concentration dependent effects of nanoscale zinc and boron on the physiological, biochemical, nutrient uptake, and translocation processes in peanut (Arachis hypogaea L.)

Abstract In the present investigation, different concentrations (150, 200, 300, 400, and 500 mg/L) of nanoscale zinc oxide (ZnO) and boron (B) were applied through foliar spray (pot culture) to evaluate their effects on the growth and biochemical parameters of peanut. Nanoscale zinc oxide and nano boron were prepared using modified oxalate decomposition and encapsulation methods, respectively, and were characterized using techniques such as, X-ray diffraction (XRD), dynamic light scattering (DLS), ultraviolet–visible (UV–vis) spectroscopy, and transmission electron microscopy (TEM). The average size (37.2 nm, 53.6 nm) and zeta potential (−37.7 mV, −28.3 mV) of nano ZnO and nano boron were measured, respectively. Fourier transform infrared spectroscopic (FT-IR) studies revealed the functional groups that are present in the hydrosols. Highest germination percentage (98.7% and 96%), seedling vigor index (2359 and 1325) and total chlorophyll content (3.28 and 2.24 mg g−1) were recorded at 500 mg/L of nano zinc oxide and nano boron. Significantly higher peroxidase, catalase, superoxide dismutase (SOD) enzymatic activities, pod yield (39.76 g pot−1 and 38.14 g pot−1) and haulm yield (49.56 g pot−1 and 47.05 g pot−1) were also recorded with the application of nano ZnO and boron @ 500 mg/L. Translocation of nanoscale nutrients through stomata was clearly observed. Significant nutrient uptake with the foliar application of nanoscale nutrients has been recorded. The performance of groundnut (in terms of yield, quality and nutrient uptake) was significantly enhanced with the application of nanoscale nutrients and this point to achieving the nutrient bio-fortification with the desired levels for the betterment of human health.


Introduction
Agrinanotechnologies (nanotechnologies that are being used for agriculture and allied sectors) have been emerging as the technologies of the future to attain agricultural sustainability and global food security. Unique properties of nanoscale materials (size less than 99 nm in at least one easy uptake by plants. The nano boron and nano zinc may, therefore could, be useful to act as an alternative source of boron and zinc but their effective use in crops is not prominent. Only limited studies were documented on the promotory effects of nanoparticles on plants at relatively low concentrations. Therefore, a pot culture experiment was conducted to reveal the concentration-dependent effects of nanoscale zinc and boron on the growth, yield, and nutrient uptake in peanut.

Synthesis and characterization of nanoscale zinc oxide and boron
Synthesis of ZnO and boron nano particles Nanoscale ZnO was prepared by using the oxalate decomposition technique (Prasad et al. 2012). Equimolar (0.2 M) solutions of zinc acetate and oxalic acid were mixed to prepare zinc oxalate. The precipitate so formed as a result of mixing zinc acetate and oxalic acid was collected and thoroughly rinsed with double-deionized water (DI-water) and allowed to dry in the air at ambient room temperature. Then the oxalate was made into fine powder form and decomposed in the air by keeping it in a preheated muffle furnace for 45 min at 500 C. Nanoscale boron was prepared by using the encapsulation method. 15.2 g of borax and 0.1 g of nano zinc was dissolved in 100 mL of distilled water separately by continuous stirring on magnetic stirrer. Gum acacia was used as binding agent. The precipitate so formed as a result of mixing borax and nano zinc was collected and thoroughly rinsed with double-deionized water and allowed to dry in the air at ambient room temperature. Then the material was made into fine powder form and decomposed in the air by keeping it in a preheated muffle furnace for 45 min at 500 C.

Characterization of nanoparticles
Particle size and zeta potential analysis The aqueous suspension of the synthesized nanoparticles was filtered through a 0.22 lm syringe driven filter unit and the size and zeta potential of the distributed nanoparticles were measured by using the principle of dynamic light scattering technique made in a Nanopartica (HORIBA, SZ-100) compact scattering spectrometer.
Fourier transform infrared spectral analysis Fourier transformed infrared spectroscopic analysis was used to identify the organic functional groups that are present in the solution containing the nanoparticles. FT-IR spectrum was taken in the mid IR region of 400-4000 cm À1 . The dried sample was mixed with the potassium bromide crystal in a ratio of 1:200 and spectrum was recorded in transmission mode.
X-ray diffraction and determination of crystal structure X-ray diffraction analysis was performed on a Diffractometer (JEOL, and Model: JPX-8030) using CuKa radiation in the range of 40 kV and 20 A in order to identify the phases of the nanoscale particles. The built-in software (syn master 7935) program was used for the identification of XRD peaks corresponding to the Bragg's reflections.

HR-TEM (high-resolution transmission electron microscopy) analysis
For TEM analysis, the samples were prepared by drop casting the nanoparticles suspension on the carbon coated Cu grids and allowed samples to dry in air. The nanoparticles surface morphology and size were studied by using transmission electron microscopy [JEOL (JEM-1010)] with an accelerating voltage of 80 kV.
SEM (scanning electron microscopy) and energy dispersive X-ray spectroscopy analysis (EDAX) Groundnut leaves treated with nano ZnO at 500 mg/Land nano boron 500 mg/L were collected and upper, middle and lower portions of the leaf were examined under scanning electron microscopy (SEM-EDAX, FEI Quanta 200) for the uptake and translocation of nano ZnO and nano boron. EDS was carried out for determination of the elemental composition and purity of the sample by atom %. Samples were prepared on carbon-coated copper grids and kept under vacuum desiccation for 3 h before loading them onto a specimen holder. Elemental analysis on single particles was carried out using SEM-EDAX, FEI Quanta 200.
In vitro study Invitro study was conducted at nanotechnology laboratory, Regional Agricultural Research Station, Tirupati to know the effect of different concentrations of nano ZnO and boron on groundnut seed germination, seedling vigor index in comparison with bulk zinc sulfate (ZnSO 4 ) and boron.

Preparation of particle suspension
The prepared nanoparticulates of ZnO and boron were suspended in the deionized water directly and dispersed by ultrasonic vibration (100 W, 40 KHz) for 30 min. The aggregation of particles was avoided by stirring the suspensions with magnetic bars. Bulk ZnSO 4 , borax, nanoscale ZnO and boron suspensions were prepared at the concentrations of 150, 200, 300, 400, and 500 mg/L and control (distilled water) were maintained and replicated thrice in a completely randomized design. Groundnut seeds of variety Dharani were procured from Regional Agricultural Research Station, Tirupati, Acharya N. G. Ranga Agricultural University, Andhra Pradesh, India. Seeds of uniform size were selected randomly and placed in petri dishes (100 mm Â 15 mm) provided with one piece of sterilized filter paper. Daily 5 mL of solution was added to respective petri dishes to maintain the moisture for seed germination and kept at room temperature (28 ± 2 C) for 14 days.
Germination and seedling vigor test Germination was calculated based on the number of seeds germinated and produced marginal seedlings in each petri dishand expressed as germination percentage. Root and shoot lengths of seedling were measured at 14th day after start of the experiment and expressed in cm. Seedling vigor index (SVI) was calculated as per the formula given in ref Abdul-Baki and Anderson (1973).

Estimation of chlorophyll content in leaves
Chlorophyll content in leaves was estimated colorimetrically by dimethyl sulphoxide (DMSO) method as described by Hiscox and Stam (1979) on 14th day after start of the experiment. 0.1 g of fresh leaf sample was soaked in 10 mL of Dimethyl sulphoxide for 24 h under dark condition. The optical densities of the supernatant liquid were recorded at 645 nm and 663 nm using UV 2450 visible spectrophotometer. The chlorophyll content was calculated by using the following formulae (Arnon 1949 where, D is the optical density, V is the final volume of DMSO, W is the fresh weight of sample taken, and total chlorophyll is the chlorophyll a þ chlorophyll b.

Pot culture experiment
The pot culture experiment was conducted at the Regional Agricultural Research Station, Acharya N. G. Ranga Agricultural University, Tirupati during rabi, 2018 to study the effect of nano ZnO and boron on growth and yield of groundnut. The experiment was laid out (Variety Dharani) in completely randomized design with twenty-one treatments and replicated thrice. The top 30 cm soil of experimental field was collected and filled in plastic pots often kg capacity. Proper care was taken to use similar soil in all the pots to minimize soil heterogeneity effects. Randomly selected groundnut seeds were treated with mancozeb @ 3 g per kg seed against seed borne diseases and they were dibbled @ 6 pot À1 filled with equal quantity of soil and watered to field capacity. After establishing, three seedlings were retained. The treatment details are: T 1 : Nano ZnO @ 150 mg/L, T 2 : Nano ZnO @ 200 mg/L, T 3 : Nano ZnO @ 300 mg/L, T 4 : Nano ZnO @ 400 mg/L, T 5 : Nano ZnO @ 500 mg/L, T 6 : Nano boron @ 150 mg/L, T 7 : Nano boron@ 200 mg/L, T 8 : Nano boron@ 300 mg/L, T 9 : Nano boron @ 400 mg/L, T 10 : Nano boron @ 500 mg/L, T 11 : ZnSO 4 @ 150 mg/L, T 12 : ZnSO 4 @ 200 mg/L, T 13 : ZnSO 4 @ 300 mg/L, T 14 : ZnSO 4 @ 400 mg/L, T 15 : ZnSO 4 @ 500 mg/L, T 16 : Boron @ 150 mg/L, T 17 : Boron @ 200 mg/L, T 18 : Boron @ 300 mg/L, T 19 : Boron @ 400 mg/L, T 20 : Boron @ 500 mg/L, and T 21 : control. The treatments were imposed at 45 days after sowing (DAS) as foliar spray. Boron was applied in the form of borax. The pots were kept clean and weed free throughout the crop growth. Proper agronomic and plant protection management was done to all the treated plants for their maximum growth expression.
The growth parameters, such as plant height (measured from the base of the plant to the tip of the growing point using the scale 1-100 cm) and dry matter production were recorded at peg formation, pod development stages and harvest(sampled plants without roots were shade dried followed by oven dried at 65 C till a constant weight was obtained and the dry weight of these samples were recorded using a common balance and expressed in g pot À1 ).The SPAD (soil plant analytical development) chlorophyll meter readings (SCMR) were taken for third leaf from the terminal bud of main axis (with four leaflets) at peg formation, pod development and harvest stages by handheld portable Minolta SCMR meter (SPAD-502 Minolta, Tokyo, Japan) and instant readings were furnished directly. In recording SCMR, care was taken to ensure the SPAD meter sensor to cover the leaf lamina properly. Biochemical parameters, that is, peroixdase activity, Catalase activity, Superoxide dismutase activity were analysed at peg formation stage of groundnut.

Peroxidase activity
Peroxidase activity was estimated by following the procedure of Angelini, Manes, and Federico (1990). One gram of fresh leaf sample was grounded to fine powder with liquid nitrogen and macerated with 3 mL of 0.1 M sodium phosphate buffer and centrifuged at 8000 rpm at 4 C for 15 min. The supernatant was collected and 0.1 mL of this supernatant was added to test tube containing 3 mL of sodium phosphate buffer (0.1 M), 50 mL of 20 mM guaiacol solution and 30 ml of 1% hydrogen peroxide solution. Blank was maintained without the enzyme extract. Initial and final absorbances were recorded 436 nm in a time difference of one minute using UV 2450 visible spectrophotometer. The peroxidase activity was expressed in min À1 g À1 using the following formula Peroxidaseactivity ¼ change in OD value=min=gram of leaf sample:

Catalase activity
The Catalase activity was assayed by following the procedure of Aebi (1974). 1.0 g of fresh leaf tissue was grounded to fine powder with liquid nitrogen and macerated with 2.5 mL of 0.05 M Sodium phosphate buffer (pH-7.2) and one mL of 1% poly vinyl pyrrolidine and centrifuged at 10,000 rpm at 4 C for 15 min. From the supernatant 50 ml of enzyme extract was added to test tube containing 2 mL of 0.05 M sodium phosphate buffer (pH-7.0) and 950 ml of 1% hydrogen peroxide solution. Besides this, blank was also run without the enzyme extract. Initial and final absorbances were recorded 240 nm using UV 2450 visible spectrophotometer. The catalase activity was expressed in units min À1 g À1 using the following formula Catalaseactivity ¼ change in OD value=min=gram of leaf sample:

Super oxide dismutase activity
The superoxide dismutase activity was estimated according to Sadasivam and Manickam (2013). One gram of fresh leaf tissue was grounded to fine powder with liquid nitrogen and macerated with 10 mL of 50 mM potassium phosphate buffer (pH-7.8) and centrifuged at 10,000 rpm at 4 C for 10 min. From the supernatant 50 mL of enzyme extract was added to test tube containing 0.6 mL of 50 mM potassium phosphate buffer (pH 7.8), 0.39 mL of 100 mM methionine, 0.0006 mL of 10 mM riboflavin, 0.06 mL of 5 mM ethylenediamine tetraacetic acid (EDTA), 0.3 mL of 750 mM nitroso blue tetrazolium (NBT) and make up to 3 mL with distilled water. Along with the sample test tubes, blank (without enzyme extract and NBT) and reference (without enzyme extract) were also maintained. All tubes were kept under fluorescent light for 15 min and absorbance was recorded at 560 nm by UV-vis spectrophotometer. Calculated the percent inhibition and the 50% inhibition of the reaction between riboflavin and NBT in the presence of methionine was taken as one unit of SOD activity and the enzyme activity was expressed as OD min À1 g À1 of protein.
SODactivity ¼ change in OD value=min=gram of leaf sample: At harvest, samples from each pot were collected to record the yield and yield-attributing characteristics, such as the number of pods per plant, filled pods plant À1 and test weight (100 kernel weight, g). Quality parameters (oil and protein content, %) were measured by using Infratech 1241 Grain analyzer in which 100 grams of kernels were kept inside the nuclear magnetic resonance (NMR) instrument and it display the results in percentage. To analyse the nutrient contents, samples were collected at peg formation, pod development and harvest stages of the crop. Nitrogen (micro Kjeldahl distillation method), phosphorus (spectrophotometrically at 420 nm by vanadomolybdate phosphoric acid yellow color method), potassium (from di-acid extract by using flame photometer), micronutrients, that is, iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) by using atomic absorption spectroscopy (AAS) from di-acid extract) and boron (curcumin method) contents were estimated as per the standard procedures.
From the chemical analytical data, uptake of macronutrients and micronutrients at different stages of the crop was calculated as shown below.

Statistical analysis
The data recorded on various parameters of the crop during the course of investigation was statistically analyzed following the analysis of variance for completely randomized design (Panse and Sukhatme 1985). The comparison between treatment means were carried out using Duncan's multiple range test (DMRT) in Statistical package for the social sciences (SPSS) software.

Results
Nanoscale ZnO and boron applied through foliar spray with different concentrations (both independent and conjunctive) resulted in interesting effects on the growth, biochemical processes, and nutrient uptake in peanut compared to their bulk counter parts. Nutrient translocation through stomata has been observed in peanut which may be extended to reveal the mechanistic aspects of translocation and mobility of nanoscale nutrients in plant system, peanut in particular.

UV-vis spectral analysis
The characteristic UV-vis absorbance of nanoscale zinc oxide and boron was depicted in Figure  S1. It is clear from Figure S1 that nanoscale zinc oxide recorded an absorbance at 230 nm, whereas, nanoboron recorded at 290 nm.

Particle size and zeta potential analysis
The nano ZnO and nano boron were analyzed using the dynamic light scattering technique for the size measurements ( Figure 1) and the hydrodynamic diameter of nano ZnO was found to be 37.2 nm and recorded a relatively higher negative zeta potential of À37.7 mV. Nano boron was found to be 53.6 nm and recorded a relatively higher negative zeta potential of À28.3 mV clearly indicates that the particles were highly stable.
High-resolution transmission electron microscopy HR-TEM image (100 nm bar scale) of nano ZnO and nano boron ( Figure 2) shows nanoparticulates appear to be slightly aggregated due to the absence of protecting ligands on the surface. The lattice of individual particles is clearly seen and particles were spherical in shape and crystalline in nature.

Fourier transform infrared spectral analysis
The FT-IR micrograph representing the different functional groups that are present in the hydrosols of nanoscale zinc oxide and nanoscale boron was shown in Figure 3A, B. The band present at 1636 could be attributed to C ¼ C Alkenyl stretch, 1050 to C-O Primary alcohol stretch, 3357 to O-H Hydroxy group H-bonded OH stretch (broad), and the bands at 2989, 2885, and 2821 could be attributed to Methyl C-H stretch. Similar kind of stretches has been observed in both the hydrosols and which may arise due to their synthesis protocol.

X-ray diffraction analysis
The X-ray diffraction pattern of nanoscale particles was presented in Figure 3C, D. XRD patterns of nano ZnO particles showed the peaks corresponding to Bragg's diffraction signals from the crystal planes (100)   indexed as hexagonal wurtzite phase ((JCPDS No. 01-089-0510) of ZnO (Rajeswari et al. 2009). XRD patterns of nano boron showed the peaks corresponding to Bragg's diffraction signals from the crystal planes (111), (002), (200), (220), (311), (022), and (113) could be indexed to the tetragonal structure of nano boron (JCPDS No. 00-006-0634). Further it also confirms that the synthesized nanopowder was free of impurities as it does not contain any characteristics XRD peaks other than ZnO and boron, respectively.
Energy dispersive X-ray spectroscopy analysis The energy dispersive spectra of the sample obtained from EDS attached with SEM. EDS analysis the confirmed the elemental presence of boron ( Figure 4) Nanoparticles delivery, absorption, and uptake by the plant In general, nutrients are delivered to plants in three ways: seed treatment, soil application and foliar spray. Seed treatment or soil application of the fertilizer is performed on the basis of nutrient deficiency in the soil, whereas foliar application is performed on the basis of nutrient deficiency symptoms exhibited by plants. The major significance of foliar applications is that they need low exposure dose, potentially multiple application times and timing of the application based on whether to avoid loss of nutrients. Comparative investigations of nanoparticle delivery to plants by spraying on the leaves versus soil application indicate that foliar application has considerable advantages for nanoscale nutrient uptake (Raliya et al. 2015;Alidoust and Isoda 2013). The uptake of nanoparticles by plants is affected by nature of the nanoparticle, plant physiology and the interaction of the nanoparticles with the environment. Properties of the nanofertilizer are particularly important for foliar application, and particle size seems to be one of the major restrictions for penetration into plant tissues whereby size exclusion may limit uptake via the stomatal pathway . It is a pre-requisite that foliar applied nanoparticles must cross the cuticle, the primary barrier of the plant cell following the lipophilic or the hydrophilic pathway. The lipophilic one involves diffusion through cuticular waxes, whereas, the hydrophilic pathway is accomplished through polar aqueous pores presented in the cuticle and/or stomata . Because the diameter of cuticular pores has been estimated around 2 nm , the stomatal pathway appears as the most likely route for nanoparticle penetration, with a size exclusion limit above 10 nm ). In the present investigation, it is clear from the SEM micrographs (Figures 4 and 5) that boron and zinc nanoparticles entering the leaf through the stomata by following the hydrophilic pathway. After foliar spray nanoparticles moving toward stomata for their uptake. After entering the stomata, nanoparticles are subsequently translocated by passing through the phloem and reaching the roots of groundnut plants by following the vascular system À phloem transport pathways. Wang, Tarafdar, and Biswas (2013) studied the uptake and transport of nanoparticles inside the watermelon plants.
Once the nanoparticles entered into the plant, there are two ways for them to transport through tissues: the apoplast and the symplast pathway (Schwab et al. 2016). The schematic diagram representing the translocation of nanoscale nutrients applied through foliar spray was shown in Figure 6.
The way nanoparticles move inside plants is really important, because it can give indications about what parts of the plant they can reach, and where they might end and accumulate. In case the nanoparticles are transported mainly through the xylem, they will likely move mainly from root to shoot and leaves. On contrary, if the nanoparticles moving along the phloem will likely accumulate in plant organs acting as sink, such as grains and fruits. However, in this study, applied nano ZnO and nano boron moved through phloem and enhanced the yield and zinc, boron content of groundnut pods discussed hereafter.
Effect of nano ZnO and boron on groundnut seed germination, seedling vigor index Germination of the groundnut seeds responded variably toward the treatment at various concentrations of bulk ZnSO 4 , boron, nano ZnO, and nano boron ( Figure S2). All the nano scale ZnO and boron concentrations recorded significantly higher values compared to bulk ZnSO 4 , boron and control. Among the different concentrations of nano ZnO and nano boron, 500 mg/L recorded significantly highest germination percentage (98.7% and 96%), root length (11.1 cm and 6.4 cm), shoot length (12.8 cm and 7.4 cm) and seedling vigor index (2359 and 1325), respectively,  over bulk ZnSO 4 , boron and control followed by 400 mg/L (Table 1). There is a gradual increase in the above said parameters with the increasing concentrations of nano scale ZnO and boron. Significant higher germination and seedling vigor index of groundnut seeds were recorded with 1000 mg/L nanoscale ZnO (Prasad et al. 2012); and seed priming with nano boron@ 300 mg/L (Hanumanthappa, Sushmitha, and Gnanesh 2019). These results are in accordance with the findings of Deepa et al. (2015) with 500 mg/L nano calcium oxide (CaO) in peanut. Irrespective of concentrations, nano ZnO and nano boron increased leaf chlorophyll content compared to bulk ZnSO 4 , boron and control. Nano ZnO at 500 mg/L (T 5 ) recorded the maximum chlorophyll a (1.94 mg g À1 ), chlorophyll b (1.34 mg g À1 ) and total chlorophyll contents (3.28 mg g À1 ) and was significantly differed from the control and bulk ZnSO4. Among the different concentrations of nano boron, 500 mg/L recorded significantly highest chlorophyll content compared to control and bulk boron ( Figure S3).

Effect of nanoscale and bulk nutrients on groundnut crop (pot culture experiment)
Physiological parameters of groundnut Physiological and biochemical parameters of the groundnut were influenced by application of nanoscale and bulk nutrients ZnO and boron. Plant height had a continuous increase till the maturity of crop. Increase in plant height was observed in all nano applied treatments. Among the different concentrations of nano ZnO and nano boron, the maximum plant height (8.62, 18.64, and 19.80 cm) was observed with nano ZnO @ 500 mg/L (T 5 ) compared to bulk ZnO and control whereas nano boron at 500 mg/L had shown a significant increase in plant height (167.50 cm) over bulk boron at peg formation, pod development and harvest stages of the groundnut, respectively (S-I). This increase in height was due to extended internodal length and could be ascribed to role of boron and zinc in improving the meristematic activity, cell division, and cell elongation. The zinc deficiency symptoms were directly visible in the plant height as Zn deficiency reduces internodal distance in plants and hence reduces the overall plant height. Similarly, chlorophyll content (SPAD readings) significantly increased with 500 mg/L nano ZnO (32.1, 40.5, and 30.8) and nano boron (30.9, 39.4, and 29.6) compared to control and the respective bulk ZnSO 4 concentrations at different stages of the groundnut (Figure 7). Gradual increase in plant height and chlorophyll content of leaves was observed with increasing the concentrations of nano scale and bulk nutrients over control. The amount of chlorophyll in the reproductive stage was even more than vegetative stage which decreased as growth progressed. Biochemical parameters of groundnut Among the nano ZnO and nano boron treatments, 500 mg/L showed highest catalase activity (1.526 and 1.516 g À1 fresh weight min À1 , respectively) compared with bulk ZnSO 4 , boron and control ( Figure 8). Catalase is naturally occurring plant enzyme catalyzes the decomposition of H 2 O 2 to water and oxygen and thus protects the cell from oxidative damage by H 2 O 2 and OH À (Bandopadhyay, Das, and Bannerjee 1999). 500 mg/L nano ZnO (0.697 g À1 fresh weight min À1 ) and nano boron (0.571 g À1 fresh weight min À1 ) showed highest SOD activity over bulk ZnSO 4 , boron and boron. SOD is naturally occurring potent antioxidant enzyme for quenching superoxide free radicals generated at stress conditions. Application of 500 mg/L of nano ZnO and nano boron recorded higher SOD activity compared to other treatments attains prominence in using this treatment specially under abiotic stress conditions. Highest peroxidise activity was recorded in T 5 (0.511 g À1 fresh weight min À1 ) and T 10 (0.502 g À1 fresh weight min À1 ) among the various concentrations of nano ZnO and nano boron over bulk ZnSO 4 , boron and boron.
Yield and yield attributes of groundnut Economic yield is expressed as a function of factors that contribute to yield, which are known as yield attributes. Among the different concentrations of nanoscale nutrients, nano ZnO and nano boron @ 500 mg/L recorded highest no of pods per plant (15.46 and 13.91, respectively), filled pods per plant (15.14 and 13.51) and test weight (38.01 and 36.31) compared to bulk ZnSO 4 , boron and control (Table 2). Nano ZnO and nano boron @ 500 mg/L recorded highest pod (39.76 and 38.14 g pot À1 , respectively) and haulm yield (49.56 and 47.05 g pot À1 , respectively) of groundnut compared to other concentrations of nanoscale nutrients, bulk ZnSO 4 , boron and control (Figure 9).

Quality parameters of groundnut
Oil and protein content of groundnut was increased in all the nano nutrients applied treatments compared to control but there was no significant difference between the treatments (Table 2). Among the different concentrations of nanoscale nutrients, nano ZnO and nano boron @ 500 mg/L recorded highest oil content (48.5% and 48.4%) and protein content (26.8% and 26.6%) compared to bulk ZnSO 4 , boron and control, respectively (Table 2).There was an increasing trend in qualitative characteristics like percentage of oil and protein content of groundnut with the increase in the level of nano ZnO and nano boron from 150 to 500 mg/L. Nutrient content and uptake of groundnut Macronutrient like nitrogen (N), phosphorus (P) and potassium (K) contents were significantly affected (S-II). Micronutrient (Fe, Mn and Cu) contents of groundnut were increased in all the treatments compared to control but the increase was numerical and there was no significant difference between the treatments (S-III). Nano ZnO and nano boron @ 500 mg/L showed highest macronutrient (N and K) and micronutrient (Fe, Mn, and Cu) content at peg formation, pod development, pod and haulm of groundnut at harvest stages, respectively among the different concentrations of nanoscale nutrients, compared to bulk ZnSO 4 , boron and control. Nitrogen,  potassium, and micronutrient (Fe, Mn, and Cu) content in groundnut plant were generally higher at pod development and further, there was decline in the concentration toward maturity and it was higher in pods as compared to haulms whereas in case of micronutrient (Fe, Mn, and Cu) contents it was higher in haulm than in pod. The decreasing trend of concentration with advancement in age of crop might be attributed to dilution effect, as a result of increased biomass production. That decrease in content in plants at harvest indicated withdrawal of nutrient from source for pod development and filling. There was slight increase in Fe content with application of Zn might be due to their similar nutrient uptake and transporting system. It was observed that the potassium content was high in haulm compared to pod and it was due to less mobilization of nutrient from haulm to pod. Among the different concentrations of nanoscale nutrients, nano boron @ 500 mg/L recorded highest phosphorus content (0.8%, 0.97%, 0.91%, and 0.73%)compared to bulk ZnSO 4 , boron and control at peg formation, pod development, pod and haulm of groundnut at harvest stages, respectively. The decrease in phosphorus content in zinc applied treatments was attributed due to the antagonistic effect of zinc and phosphorus. Phosphorus content was decreased from peg formation to pod development and it might be due to the dilution effect of nutrients. Zinc and boron contents of groundnut were significantly increased in all the treatments compared to control (Table 3). Among the different concentrations of nanoscale nutrients, nano ZnO (34.73,47.91,35.73,and 44.31 mg kg À1 ) recorded highest zinc content compared to bulk ZnSO 4 , boron and control at peg formation, pod development, pod and haulm of groundnut at harvest stages, respectively. Zinc concentration decreased toward maturity of crop thus at harvest, concentration of Zn in haulm ranged from 31.02 to 44.31 mg kg À1 , while zinc content in pod ranged from 22.19 to 35.73 mg kg À1 . The decrease in the concentration of zinc in source is due to transfer of this nutrient element to the sink (kernel). These findings are in accordance with those reported by Chahal and Ahluwalia (1977). Among the different concentrations of nanoscale nutrients, nano boron (28.0, 43.21, 30.6, and 40.6 mg kg À1 ) recorded highest boron content compared to bulk ZnSO 4 , boron and control at peg formation, pod development, pod and haulm of groundnut at harvest stages, respectively. Boron content in groundnut plant was generally higher at pod development and further, there was decline in the concentration toward maturity and it was higher in haulm than in pod. The decreasing trend of concentration with advancement in age of crop might be attributed to dilution effect, as a result of increased biomass production. That decrease in content in plants at harvest indicated withdrawal of nutrient from source for pod development and filling.
Application of nano ZnO, nano boron, and bulk ZnSO 4 and boron to the groundnut crop altered the nutrient uptake to a noticeable extent (Tables 4-6). Nano ZnO and nano boron @ 500 mg/L showed highest macronutrient (N and K) and micronutrient (Fe, Mn, and Cu) uptake at peg formation, pod development, pod and haulm of groundnut at harvest stages, respectively, among the different concentrations of nanoscale nutrients, compared to bulk ZnSO 4 , boron and control whereas highest phosphorus (177.28, 397.35, 347.07, and 343.48 mg pot À1 ) uptake was observed with nano boron @ 500 mg/L.
Zinc and boron uptake of groundnut were significantly increased in all the treatments compared to control. Among the different concentrations of nanoscale nutrients, nano ZnO (894.51, 2214ZnO (894.51, .34, 1420ZnO (894.51, .53, and 2196.04 mg pot À1 ) recorded highest zinc uptake compared to bulk ZnSO 4 , boron and control at peg formation, pod development, pod and haulm of groundnut at harvest stages, respectively. Nano boron @ 500 mg/L recorded highest boron uptake (621.15, 1907.45, 1166.79, and 1910.01 mg pot À1 ) among the various concentrations of nanoscale nutrients compared to bulk ZnSO 4 , boron and control at peg formation, pod development, pod and haulm of groundnut at harvest stages, respectively. The increase in boron uptake might be due to increased availability of the nutrient.

Discussion
The present in vitro study revealed that germination and seedling vigor index of groundnut was influenced with application of various concentrations of bulk ZnSO 4 , boron, nano ZnO, and nano boron. The beneficial effect of the nano ZnO in improving the germination could be ascribed to higher precursor activity of zinc in auxin production (Kobayashi and Mizutani 1970).As zinc is involved in many vital physiological processes in early stage of radical and coleoptile development during seed germination (Ozturk et al. 2006), its addition in the petriplates, in optimum range, further increased the germination rate as compared to control. Increase in root and shoot related parameters may be attributed to the fact that Zn is involved in synthesis of protein, cell division and cell enlargement or cell elongation, meristematic growth of radical and plumule (Cakmak 2000). Enhanced physiological performance due to nano particles treatment could be attributed to the quenching of free radicals in the germinating seeds by inducing oxidation-reduction reactions via the superoxide ion radical during germination. Smaller size of the nanoparticles would have easily entered through the cracks present on the outer seed surface, reacted with free radicals resulting in reducing damage to the biological system and in turn, oxygen produced in such process could also be used for respiration, which would further promote germination, enhanced seed vigor and viability (Zhang et al. 2006). Efficacy of 500 mg/L of nano ZnO and nano boron nutrients might be due to effective absorption and accumulation of applied nanoparticles accelerates the seed germination and seedling growth.
Gradual increase in plant height and chlorophyll content of leaves was observed with increasing the concentrations of nano scale and bulk nutrients over control. In fact, nano zinc can improve structure of chlorophyll and better capture of sunlight, can facilitate manufacture of pigments and transformation of light energy to active electron and chemical activity and increases photosynthetic efficiency, stimulates rubisco activase and also increases photosynthesis . Prasad et al. (2012) observed higher chlorophyll content of groundnut with 1000 mg/L nano ZnO and suggested that higher leaf chlorophyll content might be due to complementary effect of other inherent nutrients like magnesium, iron and sulfur. Similar findings have been reported by with spraying of nano zinc sulfide 400 mg/L at 35 DAS in sunflower (Singh and Kumar 2017); nano ZnO @ 400 mg/L in maize (Subbaiah et al. 2016) and nano Fe 2 O 3 increased plant height and chlorophyll content of peanut . These results further establish advantage of spraying of spraying of nanoscale ZnO and boron @ 500 mg/L for sustained chlorophyll activity till harvest in terms of higher leaf area index (LAI), leaf angle distribution (LAD) and total chlorophyll. Increase in catalase, peroxidase and SOD activities in this study suggests that nanoscale ZnO and boron may significantly alter antioxidant metabolism in groundnut and protected plants from reactive oxygen species (ROS) damage by improving levels of antioxidant enzyme activities. Such increase in catalase, peroxidase, and SOD activities in both leaf and root tissues of Vigna radiate has been reported under salinity stress conditions (Panda 2001). These results are in conformity with the findings of Upadhyaya et al. (2017) who reported that application of Zn nanoparticles increased catalase, peroxidise and SOD enzyme activities of rice. Possible roles of zinc in protecting plant cells form damage by reactive oxygen species and its effect on plant metabolism has also been well reviewed (Cakmak 2000;Broadley et al. 2007). However, little information about the effect of nanoscale zinc and boron induced biochemical changes in groundnut is available. Spraying nano ZnO and nano boron @ 500 mg/L proved superior in activating peroxidase, catalase, and SOD activities in groundnut crop. As groundnut crop is mostly grown under rainfed condition, significance of this treatment can be explored.  Increased pod and kernel yield with application of ZnO and boron nanoparticles suggested that due to the small size and large effective surface area, nanoparticles might have penetrated through the leaf surface and translocated to the other parts of the plants and thereby lead to better uptake of zinc and boron. Zinc that plays very essential role in plant metabolism by affecting the activities of hydrogenase and carbonic anhydrase. Plant enzymes activated by Zn are involved in carbohydrate metabolism, maintenance of the rectitude of cellular membranes, protein composition and regulation of auxin synthesis and pollen formation. On the other hand, boron micronutrient plays important role in cell wall, cell division, sugar transport, flowering and fruiting and plant hormone regulation that lead to improve yield production (Devlin and Witham 1983). Boron plays an important role in retaining flowering and fruit setting in pulses. Application of boron also favored better root growth and nitrogen assimilation with higher nodulation which in turn resulted in better growth and development of sink size (number of pods plant À1 , pod, and kernel yield). The results also showed that yield and all the yield attributing characters measured during this study increased with increasing levels of nano boron and bulk boron. The beneficial effects of boron and zinc on yield of groundnut have been reported by Nasef, Nadia, and Badran (2006), Vishwakarma, Brajendra, and Pathak (2008), and Elayaraja and Singaravel (2014). Nanoparticles induced increased activity of chloroplast (Hong et al. 2005), rubisco ), antioxidant enzyme system (Nekrasova et al. 2011), and nitrate reductase (Lu et al. 2002) might be the possible underlying mechanism responsible for enhanced growth and yield. Significant increase in number of pods plant À1 , number of filled pods plant À1 , pod yield and shelling percent was noticed in groundnut with the foliar application of nanoscale ZnO at 2 g 15 L À1 (Prasad et al. 2012) and nano CaO @ 500 mg/L (Deepa et al. 2015). Similar kind of results has been reported by Naseeruddin et al. (2018) in sweet sorghum with the application of nanoscale zinc oxide, calcium oxide and magnesium oxide. There was an increasing trend in qualitative characteristics like percentage of oil and protein content of groundnut with the increase in the level of nano ZnO and nano boron from 150 to 500 mg/L. The channelization of photosynthates during reproductive stage might have been influenced by zinc by the way of its involvement in electron transfer and activation of various enzymes which in turn can directly or indirectly affect the synthesis of carbohydrates and protein. Increase in oil content was noticed with the spraying of nano boron @ 300 mg/L at 30 DAS (Hanumanthappa, Sushmitha, and Gnanesh 2019); and application of 0.2% nano boron either seed priming or spray to capitulum at ray floret opening stage along with the RDF gives the higher oil content in sunflower (Kavitha et al. 2018). Chitdeshwari and Poongothai (2003) observed that the positive role of boron in quality improvement through its involvement in the synthesis of protein and amino acids further increased the pod yield of groundnut.
Boron and zinc application promoted the absorption of nitrogen by groundnut and thus, helped in increasing plant growth and development. Such increase could be attributed to the synergistic effect between nitrogen and zinc which might be due to increase enzymatic activity by zinc application. These results are in line with findings of Kumar and Salakinkop (2017) who stated that zinc fertilization increased the potassium concentration in plant. The study also showed that application of boron fertilizer increased the phosphorus content of groundnut and this might be due to the beneficial effect of boron on metabolic processes and growth which in turn reflected positively on the chemical content of groundnut seeds. It was observed that the phosphorus content in pod was higher than that of haulm. This might be due to the translocation of large proportions of phosphorus from other parts of the plant to the kernel (Hussaini et al. 2008). It was reported that the increase in Zn concentrations in plants with nanoparticles application might be due to higher penetration of nanoparticles into plant cells. The positive effect of foliar applied zinc and boron to sustain proper leaf nutrition and improving photosynthetic capacity is well-established (Gosavi et al. 2017). The post-harvest leaf and kernel samples analysis revealed a significant increment in zinc content in leaves (42% and 29%) and kernels (42% and 36.6%) when supplied with nano scale ZnO compared to chelated ZnSO 4 (Prasad et al. 2012). Munir et al. (2018) reported that seed priming of ZnO NPs significantly increased concentration of Zn in the roots, shoot and grains of wheat than the control which confirmed that these particles could be used as a source of Zn aiming to reduce Zn deficiency in plants. These results are in accordance with the findings of El-Metwally, Basha, and El-Aziz (2018).
Foliar application of nanoscale and bulk nutrients to the groundnut crop altered the nutrient uptake to a noticeable extent. Nanofertilizers are easily absorbed by the epidermis of leaves translocated to stems which facilitated the uptake of active molecules and enhanced growth and productivity of wheat (Abdel-Aziz, Mohammed, and Aya 2018). Nano fertilizer have large surface area and particle size less than the pore size of leaves of the plant which can increase penetration into the plant tissues from applied surface and improve uptake and nutrient use efficiency and uptake of the nutrients (Dimkpa et al. 2015;Qureshi, Singh, and Dwivedi 2018). The increase in N and K uptake could be attributed to synergistic effect between N and Zn and due to the positive interaction of K and Zn, respectively. Increased P uptake of groundnut with the application of boron might be due to the effect of boron on plasma lemma permeability, causing an increase in P absorption (Patel and Golakiya 1986). Hosseini et al. (2007) found enhancement in the uptake of phosphorus with application of boron.
Zinc application enhanced the uptake of zinc and this increase was due to higher concentration of zinc in plant parts (Tripathy, Patra, and Samui 1999). An increase in uptake of zinc due to nanoscale nutrients was also reported by Prasad et al. (2012) in groundnut; Subbaiah et al. (2016) in maize with application of 100 mg/L ZnO nanoparticles. Similar favorable effect of boron on B uptake was reported by Shamsuddoha et al. (2011) in mung bean crop. The inherent small size and the associated large surface area of nano scale boron and ZnO fertilizer may increase the uptake. The mobility of the nanoparticles is known to be very high and through the phloem transport ensures the nutrient to reach all parts of the plant there by affecting the enzyme reactions, increased dry matter production which led to increased nutrient uptake. All these factors may be responsible to give higher yields for nano scale ZnO compared to bulk ZnSO 4 and boron.

Summary
Application of nanoscale materials as nutrients in agriculture is promising. Interestingly, transportation and translocation within the plant system and bioavailability of nutrients at specific sites were observed to be enhanced with the foliar application of nanoscale nutrients compared to their bulk counter parts. In the present investigation, it was evident from the in vitro studies that there was a significant increase in seed germination percentage, followed by seedling vigor with the application of various concentrations of nanoscale nutrients. Further, it is evident that boron and zinc nanoparticles entering the leaf through the stomata by following the hydrophilic pathway. The performance of groundnut (in terms of yield, quality and nutrient uptake) was significantly improved with the application of nanoscale nutrients compared to their bulk counter parts and this points to achieving the goal of nutrient bio-fortification with the desired levels for the betterment of human health.