Comprehensive assessment of ZnO, P and TiO2 nanoparticles sustaining environment in response to seed germination, antioxidants activity, nutritional quality and yield of Spinach Beet (Beta vulgaris var. bengalensis)

Comprehensive assessment of ZnO, P and TiO2 nanoparticles sustaining environment in response to seed germination, antioxidants activity, nutritional quality and yield of Spinach Beet (Beta vulgaris var. bengalensis) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comprehensive assessment of ZnO, P and TiO 2 nanoparticles sustaining environment in response to seed germination, antioxidants activity, nutritional quality and yield of Spinach Beet ( Beta vulgaris var. bengalensis) BIMAL DAS, Rakesh Yonzone, Salil Saha, Dipak Kumar Murmu, Surajit Kundu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1315549/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A comprehensive investigation was carried out under laboratory and field by ZnO, P and TiO 2 NPs on seedling growth and antioxidant activity, nutrient accumulation, yield traits and biochemical response of spinach beet. Spinach beet variety All Green were treated with sixteen different concentrations nanoscale ZnO (5, 10, 15, 20, 25 ppm), P (10, 20, 30, 40, 50 ppm) and TiO 2 (10, 20, 30, 40, 50 ppm) including untreated control and each treatment has three replicates. TiO 2 NPs treatments found drastic positive impact on seed germination, root and shoot length, seed vigour index and antioxidant enzyme activity (CAT, PAD, SOD and GR). Application of ZnO, P and TiO 2 NPs improve morphological parameter and that of increase seed yield (gm plant -1 ) with 12.48% 13.01% and 10.93% respectively, compared to control. Study also revealed that zinc and iron content in leaf tissues increases by 50.76%, 7.71%, and 6.41% and 12.69%, 18.49% and 14.17%, respectively of ZnO, P and TiO 2 NPs NPs. Among the biochemical parameter maximum chlorophyll content obtained from 10 ppm TiO 2 , leaf protein and carotene from 50 ppm P, ascorbic acid from 40 ppm TiO 2 , crude fibre from 20 ppm ZnO and total phenol from 30 ppm TiO 2 . Our result clearly demonstrates that application of ZnO, P and TiO 2 NPs improve the antioxidant enzyme activity, yield and nutritional quality of spinach beet and these nanoparticles can be utilized with their effective doses which may reduce fertilizer cost and environmental pollution. Nanoparticles ZnO P TiO2 Spinach beet Germination Antioxidant Yield Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction To meet demand of food supply over increasing population, recently nanotechnology will occupied a prominent position in transforming agricultural sector and environment sustainability. The expansion of nano devices (<100nm size) could be use wide range of extensive applications for increasing seed germination and seedling growth, plant morho-physiological development and alleviation of stress environments. The unusual idiomatic properties of nanoparticles with high surface to volume ratio, better catalytic surface, high reactivity capacity, rapid chemical reaction and adsorb abundant water may result in different environmental behaviors than the bulk materials. The metal based NPs (ZnO, TiO 2 , Fe 2 O 3 , CuO) were capable to penetrate into the plant cell causing damage by interacting with sulfur and phosphorus containing compounds such as DNA and protein ( Amarendra and Krishna, 2010 ) and also optimizing plant metabolic processes at the early growth stages (Taran et al. 2014 ). The interaction between NPs and plant cell results changes of gene expression and connected biological pathways which finally changes the plant characteristics. The impact of NPs positive effect were documented in array of crop plants (Monica and Cremonini 2009 ) and their response dependent type of NPs use, concentration and species (Gao et al. 2006 ; Hong et al. 2005 ; Ma et al. 2010 ). Application of MWCNT (multiwalled carbon nanotubes) increases seed germination and seedling growth of different crop plants related to tomato (Khodakovskaya et al. 2010), soybean, corn, barley (Lahiani et al. 2013 ), maize, peanut and wheat (Srivastava et al. 2014; Joshi et al. 2018 ). Use of metal-based ZnO, TiO 2 and P NPs largely utilized in seed sciences and crop improvement, which exploit contradictory results on seed germination and seedling growth in tomato (Das et al. 2015 ). NPs application also acts as antioxidant enzyme activity in plants that protect from oxidative stress damage (Gupta et al. 2018 ; Jadczak et al. 2020 ). In addition to germination and seedling growth the nanoscale ZnO and TiO 2 NPs were reported to positive influences of crop growth and development with nutritional quality enhancement in many crop species including peanut (Prasad et al. 2012 ), soybean (Zhang et al. 2021 ), rice (Debnath et al. 2020 ), spinach (Yang et al. 2006 ) and carrot (Gholami et al. 2020 ). Application of TiO 2 NPs in spinach produced significant effects on growth, increases photosynthesis and nitrogen metabolism (Gao et al. 2006 ; Hong et al. 2005 ; Zheng et al. 2005 ) and induce redox reactions, chlorophyll formation and Rubisco activity ( Siddiqui et al. 2015 ; Hegde et al. 2016 ). NPs may enhanced nutrient use efficiency (Zulfiqar et al. 2019 ), nutrient content (Sabir et al. 2014 ) and improve yield and also decrease its accumulation in soil. Albeit reports of improvement on seedling growth, physiological attributes, yield and quality, information on the genetic mechanism underline these changes is meager. In viewpoint of the above facts the present study delineates ZnO, P and TiO 2 NPs laboratory observation of seed germination, seedling growth and antioxidant activity and field study of yield attributing and nutritional quality responsive traits in spinach beet (Figure 1 ). 2. Materials And Methods 2.1. Material used Titanium dioxide (TiO 2 ) NPs were purchased from Sigma-Aldrich Company, St. Louis, MO, USA with a purity of 99.5%, particle size of (<100 nm). Biosynthesized Zinc Oxide (ZnO) and Phosphorus (P) NPs were collected from Central Arid zone Research Institute (CAZRI), Jodhpur, Rajastan. Characterization of particles in stipulations of size (<100 nm), shape, structure and elemental proportion presented our previous report (Das et. al. 2015 ). 2.2. Seed treatment with ZnO, P and TiO 2 NPs The present study was conducted at research laboratory and Instructional farm of College of Agriculture (Extended Campus), Uttar Banga Krishi Viswavidyalaya, Majhian, West Bengal, India during 2018 and 2019. Healthy and uniform seeds of spinach beet variety All Green were selected and sterilized with 0.1% Mercuric Chloride solution for 1 min and rinsed properly with sterilized distilled water two to three times. Then seeds were treated with different concentrations of ZnO (0, 5, 10, 15, 20, 25 ppm), P (10, 20, 30, 40, 50 ppm) and TiO 2 (10, 20, 30, 40, 50 ppm) NPs in aerated solutions for 24 hrs at 25 ± 2 o C and these concentration were chosen based on our previous studies (Das et al. 2015 ). The untreated seeds with sterilized water considered as control. 2.3. Germination test and seedling growth Germination percentage of spinach beet seeds was estimated based on AOSA method (AOSA, 1990). In an incubator, three replicates of 50 seeds of each treatment were placed in 12 cm diameter petri plates, between the layers of moist Whatman-42 filter papers at 27°C and arranged in a complete randomized design (CRD). Germination percentage was counted based on emergence of seedling from embryo. The average root length, shoot length and their fresh seedlings weight recorded from 10 seedlings of each treatment at one week of age. Vigour Index (VI) was estimated as per following equation (Abdul Baki and Anderson, 1973 ): Vigour Index = (Shoot length average + Root length average)*Germination 2.4. Extraction and Antioxidant enzyme activity Catalase enzyme (CAT) activity was measured following the procedure of Aebi ( 1984 ) with some minor modifications. All steps in preparation of the enzyme extracts were carried out at 0 to 4°C. The ultra violet (UV) light absorbance of hydrogen peroxide solution can be measured between 230 and 250 nm. On decomposition of hydrogen peroxide by catalase, the absorption decreases with time. Fresh leaves (0.1 g) were homogenized in 5 mL of cooled phosphate buffer (50 mM pH 7.8) with a pinch of Poly Vinyl Pyrrolidone (PVP).The extracted leaves were centrifuged with 15000 rpm for 10 minutes and supernatant collected for enzyme estimation. Three ml of analysis mixture contained 50 µl distilled water whereas, control contained devoid of H 2 O 2 . The catalase activity was measured by reduce of H 2 O 2 absorbance on 240 nm and expressed as µmol H 2 O 2 decomposing/min/g. Peroxidase enzyme (POD) was estimated following the procedure of Castillo et al. ( 1994 ) at absorbance of 470 nm using a reaction mixture containing 12 mM hydrogen peroxide and 96 mM guaiacol in phosphate buffer (pH 7.0). The optical density increases due to oxidation which produce tetra-guaiacol and POD expressed as µmoles/cm/min/g fresh weigh. The Superoxide dismutase (SOD) was estimated following the procedure by Gong et al. ( 2005 ) by nitroblue tetrazolium (NBT) with an absorbance at 560 nm. Glutathione reductase (GR) was estimated following the procedure describe by Shanker et al. ( 2004 ). 2.5. Field study of NPs treated seeds and recording of yield attributing traits Beside seedling experiments All Green seeds with above sixteen different concentrations of NPs treatment including control also sown in field based on randomized block design (RBD) by maintaining spacing of 40cm x 20cm. The fertilizer applied in soil following recommended dose of NPK (75:40:40) and necessary cultural practices maintained for plant growth. The initial soil fertility status of experimental field was presented in Table 1 . One foliar exposure of above NPs treatment in each case was done at 30 days of sowing. During the entire growth period (150 days) of spinach beet plants morpho-phenotypic and yield attributes such as plant height (60 DAS), leaf number, leaf length (cm), leaf width (cm), stem thickness (mm), leaf area (cm 2 ), fresh leaf weight (gm) and seed yield plant −1 (gm) were recorded by selecting average of five plant in each treatment. The fresh weight (gm) of the leaves at was recorded after 50 days sowing. Table 1 Soil fertility status under field condition during crop growth stage Particular Values Status Texture class Sand 58% - Silt 27 - Clay 15 Sandy loam pH 5.54 Acidic Soil nutrients Organic carbon (%) 0.41 Medium Available N (Kg ha −1) 185.36 Medium Available P 2 O 5 (Kg ha −1) 12.07 Low Available K 2 O (Kg ha −1) 296.78 High Source: Regional Research Station (Old Alluvial Zone), UBKV, Majhian 2.6. Dry matter and Elemental Analysis For estimation of dry matter content each treated plants were taken out during maturity stage (60DAS), dried under shade and then placed in a hot air oven at 70 ± 5 o C for 48 hours to a constant weight, and weighed to record dry matter content (gm). The dried leaf were finely ground and digested for estimation of total NPK content. Nitrogen content from dried leaves was estimated by the micro-Kjeldahl method (Subbiah et al. 1956). For phosphorus and potassium content tri-acid mixture (HNO 3 :H 2 SO 4 :HClO 4 = 10:1:4) was used and determined by flame photometer (Jackson 1973 ). Iron and zinc contents each treated leaves extract digested in triple acid was estimated by employing the method given by Jackson ( 1973 ) by using atomic absorption spectroscopy (PinAAcle 990F; PerkinElmer, Singapore). 2.7. Estimation of Biochemical parameters Biochemical properties like total chlorophyll content from young leaf of spinach beet was estimated by spectrophotometric method (Davies 1976 ). Carotene content of spinach leaf was estimated by the standard procedure of AOAC ( 1984 ) using 80% of acetone. Protein content from the fresh leaves was estimated following the method of Lowry, et al., 1951 . Ascorbic acid content was determined by using 2-6, Dichlorophenyl Indophenol dye solution (Ranga 1979 ) and expressed as mg/100g of fresh weight. Total polyphenol concentrations of leaf material were determined using the Folin-Ciocalteu the method by Waterman and Mole ( 1994 ) as described by Mudau et al. ( 2006 ). The finely ground leaves samples were used crude fibre content by AOAC method (1970). 2.5 Statistical Analysis Each treatment was conducted with three replicates and the results were presented as mean±SE (standard error of the mean) and CD value. All the data were statistically analyzed by Fisher’s technique and Duncan’s Multiple Range Test (p ≤ 0.05). The analysis was carried out using the SPSS software 21 version and graphical representation done by OriginPro 21 software. 3. Result 3.1. Effect of ZnO, P and TiO 2 NPs for germination and seeding growth The use of ZnO, P and TiO 2 NPs treatments significantly affected germination and seedling growth of spinach beet. Improvement in germination percentage was found from all the ZnO, P and TiO 2 NPs treatments (Figure 2 A). Enhanced germination percentage over control was highlighted for almost all the treatments except 5 ppm of ZnO. Highest germination percentage was found in treatment with 30 ppm of TiO 2 , followed by 15 ppm of ZnO. The increment in germination percentage was observed to the tune of 10.92%, 13.66% and 17.01% in ZnO, P and TiO 2 respectively, as compare to control. The data regarding length of root and shoot at one week of age has been presented in the Figure 2 B. Most favourable effect on root and shoot length was highlighted by respective treatments of 40 ppm of TiO 2 . The data also depicts that the TiO 2 NPs provides maximum response of root and shoot length than other treatment which an increase of 28.14% and 22.34% respectively, over control. Similar to the data on fresh seedling weight the maximum was recorded with 50 ppm TiO 2 which was 36.46% higher than control, whereas, the least fresh seedling weight of shoot was associated with 20 ppm of P (Figure 2 C). Enhancement of vigor index was maximum by TiO 2 NPs at 40 ppm and 30 ppm. Over all vigor index increase with 25.16%, 26.92% and 30.39% by ZnO, P and TiO 2 NPs, respectively from the control. 3.2 Effect of ZnO, P and TiO 2 Nanoparticles for antioxidant enzymatic activity NPs treatments significantly influence the antioxidant enzymatic response during seedling growth of spinach beet. The data indicate highly significant (p ≤ 0.05) differences of antioxidant enzyme activity (CAT, POD, SOD and GR) under seedling stage of spinach beet (1 week old seedling) in response to ZnO, P and TiO 2 NPs application (Figure 3 A, 3 B). The catalase (CAT) activity increased statistically (p ≤ 0.05) with the treatments (Figure 3 A). The maximum expression of catalase activity was recorded when treated with TiO 2 at 40 ppm followed by 20 ppm and 30 ppm. The average cumulative effect of catalase activity increased from 21.79%, 8.08% and 43.78% respectively, by ZnO, P and TiO 2 NPs respectively, over the control. The maximum peroxidase enzyme activity was recorded in 20 ppm TiO 2 and an average increment by ZnO, P and TiO 2 NPs treatments found with 17.31%, 6.15% and 34.05% respectively, as compared to control plants (Figure 3 A). During seedling growth, the SOD activity tended to produce considerably at different NPs treatment (Figure 3 B). The SOD enhancement occurred by ZnO, P and TiO 2 NPs treatments was 22.59%, 22.02% and 45.22% respectively, from control. On the other hand the expression of GR activity in spinach beet leaves was found maximum with TiO 2 NPs at 30 ppm (49.22%). Overall enhancement of the GR activity was noticed 18.41%, 10.68% and 44.50% by ZnO, P and TiO 2 NPs treatments (Figure 3 B). 3.3 Effect of ZnO, P and TiO 2 NPs on morphological and yield traits of spinach beet Application of ZnO, P and TiO 2 NPs treatments of spinach beet had profound effect on plant morphological traits (Figure 4 ). At maturity stage of spinach beet (80 DAS), the maximum plant height was found in TiO 2 NPs treatments and increased by 35.27% at 30 ppm (Table 2 ). Shortest plants were observed under untreated control condition. The positive effect of plant height with statistically at per treatments were 40 ppm and 50 ppm TiO 2 NPs, 20 ppm and 30 ppm P NPs, 10 ppm and 15 ppm ZnO NPs. Other characteristics noticed maximum response with the application of TiO 2 NPs at 30 ppm of leaf number; at 40 ppm of leaf length, leaf width and leaf area; at 10 ppm of stem thickness. For ZnO NPs treatments the promising effect found at higher concentration of 25 ppm for plant height, number of leaf plant −1 and leaf area whereas lower concentration beneficial for stem thickness. In case of P NPs treatment the higher concentration found satisfactory results almost all the morphological characteristics. In situation to yield characteristics of spinach beet leaf weight and seed yield showed significant (p <0.05) impact with by application of ZnO, P and TiO 2 NPs treatments (Table 2 ). Significantly enhancement of fresh leaf weight (gm plant −1 ) exhibited with 25 ppm of ZnO, 40 ppm P and 20 ppm of TiO 2 with an increase of 17.16%, 18.33% and 16.41% respectively, in assessment to control. Whereas, seed yield (gm plant −1 ) an average increase by ZnO, P and TiO 2 NPs occurred with 12.48% 13.01% and 10.93% respectively, compared to control (Table 2 ). Table 2 Effect of ZnO, P and TiO 2 NPs plant morphological and yield traits of spinach beet Treatment (ppm) Plant height (cm) Number of leaf plant −1 Leaf length (cm) Leaf width (cm) Stem thickness (mm ) Leaf area (cm 2 ) Fresh leaf weight (gm) Seed yield plant −1 (gm) Control 79.81 ±1.77h 13.81 ±1.01g 14.57±1.17e 10.35±0.52f 11.07 ± 0.28g 193.12 ±3.62k 71.45 ±0.75k 12.45 ±0.30h ZnO-5 80.35 ±0.10h 14.72 ±1.05efg 17.43 ±0.43abcd 12.46 ±0.28def 12.04 ±0.41fg 198.44 ±2.95jk 74.89 ±0.35j 14.46 ±0.26bcde ZnO-10 85.40 ±2.84fgh 17.67 ±1.12bcd 19.16 ±0.59a 12.05 ±0.91ef 15.93 ±0.87ab 212.31 ±3.24hi 78.36 ±0.27hi 13.13±0.12fgh ZnO-15 84.80 ±2.78fgh 16.07 ±0.80defg 17.15 ±0.65abcd 11.53 ±1.24f 14.09 ±0.51cd 227.86 ±5.73efg 75.99 ±0.75 ij 15.35 ±0.37ab ZnO-20 89.43 ±2.38ef 16.48 ±0.85def 17.75 ±1.12abcd 11.87 ±0.91ef 12.52±0.40efg 225.90 ±5.13fg 84.80 ±0.33bcd 13.89 ±0.32def ZnO-25 92.88 ±1.61de 20.87 ±1.51a 17.75 ±0.49abcd 11.95 ±0.77ef 15.72 ±0.50ab 239.81 ±2.70cd 86.24 ±1.09 ab 16.08 ±0.34a Avg. of ZnO 86.57 17.16 17.85 11.97 14.06 220.69 80.06 86.57 P-10 82.50 ±1.81gh 15.67 ±0.54defg 18.29 ±1.05ab 15.67 ±1.27abc 13.61 ±0.67cde 219.47 ±3.34gh 80.13 ±1.18 gh 14.59 ±0.43bcd P-20 98.15 ±1.21d 17.98 ±0.67bcd 18.31 ±0.67ab 17.98 ±0.30a 12.79 ±0.61def 207.12 ±2.98ij 83.98 ±0.94bcde 14.42 ±0.34bcde P-30 96.17 ±1.57d 14.73 ±0.55fg 15.61 ±0.93de 14.74 ±0.79bcd 12.77 ±0.05def 229.80 ±3.20ef 87.49 ±0.38a 14.37 ±0.25bcde P-40 93.05 ±2.55de 16.82 ±0.75de 17.62 ±0.70abc 16.82 ±1.01ab 12.92 ±0.63def 248.70 ±3.50bc 77.75 ±2.22hi 12.86 ±0.31gh P-50 87.55 ±1.17efg 14.10 ±0.94fg 18.91 ±0.53a 14.11 ±0.74cde 13.63 ±0.57cde 259.21 ±1.79a 83.46 ±0.38cdef 15.32 ±0.77ab Avg. of P 91.49 15.86 17.75 15.86 13.14 231.80 82.56 91.49 TiO 2 -10 117.07 ±1.76b 17.09 ±0.66cde 19.11 ±1.33a 11.70 ±0.66f 16.67 ±0.21a 241.88 ±3.10cd 81.15 ±0.59fg 13.07 ±0.35fgh TiO 2 -20 120.84 ±1.67ab 19.33 ±0.58abc 15.47 ±0.60de 10.72 ±1.01f 13.44 ±0.48cdef 236.86 ±2.58de 82.29 ±0.23efg 13.91 ±0.20def TiO 2 -30 123.29 ±1.30a 19.77 ±0.73ab 15.89 ±1.04cde 10.79 ±0.70f 13.09 ±0.38def 254.77 ±2.89ab 82.54 ±0.25defg 13.56 ±0.26efg TiO 2 -40 111.00 ±1.12c 17.38 ±0.49cde 18.48 ±1.18ab 12.43 ±0.41f 13.92 ±0.83cde 234.24 ±2.75def 85.48 ±0.36abc 14.25 ±0.18cde TiO 2 -50 110.18 ±2.62c 19.29 ±0.76abc 16.26 ±0.60bcde 11.29 ±0.53f 14.71 ±0.28bc 258.60 ±2.62a 84.39 ±0.20bcde 15.10 ±0.12abc Avg. of TiO 2 116.48 18.57 17.04 11.39 14.37 244.66 83.17 116.48 *Mean value± SE. Each value represents the mean of three replication of each treatment. Different letter of mean value are significantly different at P<0.05 according to the Duncan’s multiple range test (DMRT) and the same letters indicate no significant difference P<0.05. 3.2.2 Effect of ZnO, P and TiO 2 NPs on dry matter production and nutrients accumulation in spinach beet Effect of NPs on dry matter and nutrients accumulation were presented in Table 3 . Our study revealed that maximum dry matter was observed by ZnO NPs at 25 ppm (36.55 g), P NPs at 20 ppm (35.66 g) and TiO 2 NPs at 40 ppm (34.35g) that which 22.74%, 20.81% and 17.76% enhancement monitored as compared to control. The nutrient uptake in spinach beet leaves exposed by NPs, indicating their differential response with the treatments. Among the analyzed elements, significant difference in total N, P, K, Zn and Fe contents was found in leaves exposed to NPs. Our results depicted that improvement of major and minor elements in the leaves with an average increase 15.60%, 9.39% and 12.01% of N, 21.62%, 52.44% and 16.00% of P, 10.13%, 14.11%, and 11.75% of K, 50.76%, 7.71%, and 6.41% of Zn and 12.69%, 18.49% and 14.17% of Fe by ZnO, P, and TiO 2 NPs respectively, over the control plants (Table 3 ). Table 3 Effect of ZnO, P and TiO 2 NPs on dry matter production and nutrients accumulation in spinach beet Treatment (ppm) Dry matter (gm plant −1 ) N (%) P (%) K (%) Zn (ppm) Iron (ppm) Control (0 ppm) 28.24 ± 1.14f 0.608 ± 0.02e 0.775 ± 0.05d 3.56 ± 0. 29e 40.26 ± 1.64e 212.36 ± 2.40g ZnO-5 29.26 ± 2.60ef 0.636 ± 0.02e 0.955 ± 0.05cd 3.69 ± 0.25de 63.56 ± 2.22c 232.55 ± 2.20f ZnO-10 30.24 ± 1.12cdef 0.672 ± 0.06cde 0.966 ± 0.05cd 3.88 ± 0.05bcde 70.69 ± 0.99b 236.56 ± 5.17ef ZnO-15 29.65 ± 2.35def 0.756 ± 0.02abc 0.942 ± 0.05cd 4.12 ± 0.12abcd 88.66 ± 1.76a 254.36 ± 4.86bc ZnO-20 33.56 ± 0.91abc 0.779 ± 0.02a 1.020 ± 0.09c 4.13 ± 0.13abcd 92.21 ± 3.00a 241.37 ± 4.26def ZnO-25 36.56 ± 1.24a 0.759 ± 0.05ab 1.061 ±0.02c 3.99 ± 0.02bcde 93.66 ± 1.28a 251.33 ± 2.70cd Avg. of ZnO 31.86 0.72 0.99 3.96 81.76 243.23 P-10 34.66 ± 0.92bcde 0.656 ± 0.03de 1.454 ± 0.11b 3.89 ± 0.04bcde 42.34 ± 1.75de 244.12 ± 2.20de P-20 35.66 ± 1.11ab 0.689 ± 0.03bcde 1.560 ± 0.18ab 3.97 ± 0.10bcde 44.22 ±1.19de 245.36 ± 3.02cde P-30 28.66 ± 0.52f 0.671 ± 0.03cde 1.661 ± 0.09ab 4.23 ± 0.12ab 41.66 ± 2.17de 262.56 ± 2.49b P-40 34.66 ± 0.92ab 0.685 ± 0.03bcde 1.713 ± 0.12a 4.12 ± 0.16abcd 46.25 ± 1.76d 274.38 ± 4.17a P-50 33.55 ± 0.94abc 0.654 ± 0.04de 1.760 ± 0.02a 4.53 ± 0.18a 43.66 ± 1.76de 276.22 ± 5.47a Avg. of P 33.02 0.67 1.63 4.15 43.62 260.53 TiO 2 -10 33.25 ± 0.58abcd 0.668 ± 0.03de 0.911 ± 0.05cd 4.12 ± 0.24abcd 42.34 ± 2.18de 232.25 ± 2.83f TiO 2 -20 33.22 ± 0.97abcd 0.724 ± 0.01abcd 0.923 ± 0.06cd 3.87 ± 0.13bcde 41.34 ± 0.35de 233.67 ± 1.21f TiO 2 -30 32.67 ± 0.88bcde 0.723 ± 0.01abcd 0.922 ± 0.02cd 4.16 ± 0.20abc 44.22 ± 2.25de 254.36 ± 2.65bc TiO 2 -40 34.35 ± 1.2ab 0.653 ± 0.02de 0.932 ± 0.01cd 4.33 ± 0.16ab 45.61 ± 2.26de 263.12 ± 2.69b TiO 2 -50 33.46 ± 0.96abc 0.687 ± 0.01bcde 0.925 ± 0.03cd 3.70 ± 0.15cde 41.58 ± 1.71de 254.68 ± 4.56bc Avg. of TiO 2 33.39 0.69 0.92 4.04 43.02 247.61 *Mean value± SE. Each value represents the mean of three replication of each treatment. Different letter of mean value are significantly different at P<0.05 according to the Duncan’s multiple range test (DMRT) and the same letters indicate no significant difference P<0.05. 3.2.2 Effect of ZnO, P and TiO 2 NPs on biochemical responses in spinach beet The application of different NPs treatments increase total chlorophyll content in leaves where, TiO 2 NPs at 10 ppm proved to be best and increased by 17.18%, over the control (Table 4 ). All though successful improvement also detected from 10 ppm and 50 ppm of TiO 2 which were statistically at per. A progressive increase of total chlorophyll content by ZnO, P, and TiO 2 treatments with 30.17%, 28.24%, and 40.69% respectively, compared to control. Table 4 Effect of ZnO, P and TiO 2 NPs on biochemical response in spinach beet Treatment (ppm) Total Chlorophyll (mg g-1 FW) Protein (g/100g) Carotene (mg/100g) Ascorbic acid (mg/100g) Total phenol (mg/100g D.W) Crude fibre (%) Control (0 ppm) 2.11 ± 0.11f 3.01 ± 0.22d 9.26 ± 0.61d 93.55 ± 2.54h 608.37 ± 4.51h 7.16 ± 0.15d ZnO-5 2.52 ± 0.30ef 3.31 ± 0.10cd 10.26 ± 1.11cd 115.26 ± 2.69g 725.34 ± 4.84g 8.66 ± 0.09abc ZnO-10 3.15 ±0.15bcd 3.57 ± 0.46bcd 12.36 ± 0.59abc 118.78 ± 3.19g 736.56 ± 1.90g 9.55 ± 0.44ab ZnO-15 3.39 ± 0.10bc 3.49 ± 0.27bcd 11.38 ± 0.91abcd 144.66 ± 3.30e 825.45 ± 9.23cd 9.58 ± 0.47ab ZnO-20 3.02 ± 0.10cd 4.08 ± 0.05ab 12.33 ± 1.08abc 135.44 ± 2.20f 837.55 ± 2.23ab 9.65 ± 0.83a ZnO-25 3.03 ± 0.08cd 3.95 ± 0.24abc 10.36 ± 1.04bcd 138.34 ± 1.19ef 822.66 ± 4.07d 8.56 ± 0.31abc Avg. of ZnO 3.02 3.68 11.34 130.50 789.51 9.20 P-10 3.13 ± 0.09bcd 3.48 ± 0.16bcd 12.56 ± 1.20abc 136.66 ± 2.45f 765.64 ± 10.49e 8.69 ± 0.46abc P-20 3.24 ± 0.13bc 3.92 ± 0.17abc 12.36 ± 0.98abc 144.68 ± 2.15e 789.56 ± 4.01e 9.12 ± 0.57abc P-30 2.46 ± 0.14 ef 3.86 ± 0.15abc 12.78 ± 0.58ab 166.57 ± 1.61c 796.22 ± 4.65e 8.68 ± 0.65abc P-40 3.13 ± 0.17bcd 4.25 ± 0.05a 12.88 ± 0.19a 175.66 ± 5.42b 801.67 ± 3.34e 8.79 ± 0.54abc P-50 2.74 ± 0.22de 4.25 ± 0.20a 12.36 ± 0.49abc 167.67 ± 1.54c 796.66 ± 2.42e 9.00 ± 0.26abc Avg. of P 2.94 3.95 12.59 158.25 789.95 8.86 TiO 2 -10 4.05 ± 0.13a 3.91 ± 0.34abc 11.66 ± 0.90abcd 156.65 ± 2.79d 842.33 ± 2.89 b 8.22 ± 0.46cd TiO 2 -20 3.08 ± 0.11cd 3.96 ± 0.08ab 12.58 ± 0.72abc 179.66 ± 2.86b 848.66 ± 3.30ab 8.34 ± 0.17bcd TiO 2 -30 3.55 ± 0.19b 4.03 ± 0.31ab 12.67 ± 0.81abc 189.66 ± 3.20a 857.12 ± 4.63a 8.55 ± 0.36abc TiO 2 -40 3.08 ± 0.11cd 4.09 ± 0.11ab 10.26 ± 1.03cd 193.24 ± 2.36a 844.21 ± 3.71ab 8.11 ± 0.41cd TiO 2 -50 4.02 ± 0.13a 4.09 ± 0.21ab 9.22 ± 0.82d 179.23 ± 1.94b 837.67 ± 3.83ab 7.88 ± 0.31cd Avg. of TiO 2 3.56 4.02 11.28 179.69 846.00 8.22 *Mean value± SE. Each value represents the mean of three replication of each treatment. Different letter of mean value are significantly different at P<0.05 according to the Duncan’s multiple range test (DMRT) and the same letters indicate no significant difference P<0.05. The perusal of data in Table 4 revealed that use of NPs treatment significantly influences the leaf protein content of spinach beet. Protein content recorded maximum 4.25 g that of 40 ppm and 50 ppm concentration of P, where as plants under control had produced 3.15g only. ZnO, P, and TiO 2 NPs treatments protein content increased by 11.57%, 21.21%, and 21.09% respectively, over the control. Carotene content among the treatments varied considerable with the treatment. It was revealed that moderate concentration of NPs was found to be more effective for carotene content in the leaves. The application of NPs at higher concentration shows the decreasing tends of carotene. Total Carotene content of ZnO, P, and TiO 2 NPs treatments increased by 18.30%, 26.43%, and 17.85% respectively, compared to control. On the other hand ascorbic acid content on leaves also significantly influenced by NPs treatment (Table 4 ). The maximum of ascorbic acid content was recorded from 40 ppm TiO 2 , which was also statistically at par with 40 ppm TiO 2 . Total ascorbic acid content increased by 28.31%, 40.88%, and 48.03% of ZnO, P, and TiO 2 treatments respectively, as compared to control. The maximum total phenol was recorded from 30 ppm of TiO 2 NPs. An average effect of ZnO, P, and TiO 2 treatments on total phenol content increased by 22.94%, 22.99%, and 28.09% respectively, compared to control. In response to crude fibre content maximum was found when were treated with 20 ppm of ZnO NPs, whereas an enhancement of 22.21%, 19.18%, and 12.92% occurred by ZnO, P, and TiO 2 treatments respectively, compared to control. 4. Discussion 4.1. Germination and seeding development Seed germination considered as an index of plant growth which required adequate water absorption that responses to yield characteristic. Our study exhibited that seed germination in spinach beet significantly enhanced by TiO 2 NPs than ZnO and P NPs and the result fully agreement with Hajra et al (2017). TiO 2 NPs found better germination percentage over ZnO NPs and P NPs may be due to production of reactive anions which accumulate more water absorption and boost the oxygen rate for uptake during germination ( Khot et al. 2012 ). In viewpoint of Zheng et al. ( 2007 ) suggested that due to small particle size of TiO 2 NPs can easily penetrate into the seed coat of spinach and enhanced activity during growth. Zheng et al. ( 2005 ) in spinach and Feizi et al. ( 2012 ) in wheat observed accelerated germination in proper concentration of TiO 2 treatment. The study revealed that seed priming and presoaking techniques using different NPs can significantly improve spinach beet performance by increasing seedling growth and vigour, although response varied with different NPs and concentrations. In general, treatments with lower to moderate doses of TiO 2 NPs than ZnO and P NPs showed significant positive response in most of the seedling characters. Previously, Haghighi et al. (2014) opined that higher doses of TiO 2 NPs (up to 200 ppm) in tomato and onion be capable of enhanced root and shoot lengths. Seedling growth enhances by TiO 2 NPs treatment possibly due to it regulates the activity of nitrogen-metabolizing enzyme which helps to convert nitrogen for chlorophyll accumulation (Yang et al. 2006 ; Mishra et al. 2014 ). The enhancement of seedling weight and vigor index by ZnO, P and TiO 2 NPs also described our earlier report in tomato (Das et al. 2015 ) and TiO 2 NPs in rice (Debnath et al. 2020 ). 4.2. Antioxidant enzymatic activity Spinach beet one of the important vegetables contains high antioxidant capacity and it protects the plant cells from oxidative damage during stress. Our results depicted that the enzymatic activities of CAT, POD, SOD, and GR responses significantly by application of ZnO, P and TiO 2 NPs treatment during seedling stage and varies upon changes of concentration (Figure 3 A, 3 B). Higher concentration of Phosphorus and ZnO NPs the enzymatic activity were increasing trends which suggested that these elemental particles actively utilized various metabolic processes as well as protein synthesis also decrease H 2 O 2 levels and involve oxidative stress response in spinach beet. On the other hand higher concentration of TiO 2 a decreasing trend of CAT and GR activity was noticed, which might affect the failure of plant defense system. The highest activity of peroxidase was noticed by ZnO NPs at 15 ppm and positive effect also agreements with previous study (Faizan et al. 2017). Pretreatments of seeds by ZnO nanoparticles can improve germination and seedlings as well as antioxidant enzymes (García-López et al. 2018 ). The observed increase in GR activity suggests that glutathione is also involved in spinach plants, either directly to oxidative stress response or indirectly via the production of phytochelatins (Potters et al. 2002 ). The antioxidant activity effective towards dose-dependent with lower doses of ZnO NPs increases the catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) but decreases at extremely high concentration (200 ppm) in rapeseed (Sohail et al. 2020 ), in potato (Raigond et al. 2017 ) and in wheat and maize (Srivastav et al. 2021 ; Wang et al. ( 2019 ). Additionally, Khater ( 2015 ) reported that TiO 2 increased the production of antioxidant content in coriander. Additionally, Çatav et al. ( 2020 ) showed that higher doses of Cd severely increased GR, POD and SOD in wheat. 4.3. Morphological and yield traits of spinach beet Our finding clearly depicted positive response plant growth and yield responses in nanoparticles treated plants as compared to control plants. Lower concentration of ZnO showed optimum improvement for the characters plant height, leaf length, leaf width, number of leaves and exposure to extremely high concentration showed optimum improvement for the characters stem thickness and leaf area (Table 2 ). Similarly the economic product fresh leaf weight showed optimum response at moderately high concentration. With high concentration of P NPs were found to produce effective improvement for majority of the characters except leaf length and number of leaves, where comparatively lower concentration had given the effective result. Extremely higher concentration of TiO 2 NPs treatments produced optimum improvement in economic product fresh leaf weight along with its component like plant height, number of leaves, stem thickness. Our present study both laboratory and field evaluation found a positive impact on spinach beet growth by NPs and the suggested mechanisms explain by increased nutrient adsorption and active transport of nutrients to the roots, activate enzyme for nutrient accumulation in leaf, enhanced synthesis of starch which ultimately increased the carbohydrate production and photosynthesis (Song et al. 2018). These mechanisms could be a contributing factor for increase average leaf yield found in spinach beet. In addition, the small size and high surface ratio with photo catalysis property of NPs which can interact with cellular system that contribute to overall effect of plant phenotypic changes and yield. Interestingly, Elizabath et al. ( 2017 ) reported a combination of ZnO and FeO NPs at 50ppm found good response of plant height, leaves plant −1 , petiole length, leaf area (cm 2 ) and yield of carrot. Recently Zhang et al. ( 2021 ) reported ZnO NPs application obtained higher yield of rice with more panicle number (4.83–13.14%), spikelets panicle −1 (4.81–10.69%), test weight (3.82–6.62%) and filled grain rate (0.28–2.36%). Kheyri et al. ( 2019 ) suggest that Zn and Si NPs applications in rice enhance grain yield upto 9.5, 9.2, and 6.9%, respectively, which reduce fertilizer costs as well as environmental pollution. Tarafder et al. (2014) reported grain yield in pear millet at crop maturity was improved by 37.7% due to application of zinc nanofertilizer. On the other hand, Khater, ( 2015 ) described TiO 2 with low concentration had enhances the plant height, branches and fruit yield in coriander. Recently, Waani et al. ( 2021 ) suggested TiO 2 with dose dependant action both positive and negative effect on plant growth and yield of rice. Earlier report also suggested that CeO and iron oxide NPs increased wheat and soybean yield by 36.6% and 48%, respectively (Yang et al. 2006 ; Sheykhbaglou et al. 2010 ). 4.4. Effect of NPs nutrient accumulation of spinach beet The application of NPs found to be beneficial in increasing dry matter of plant as compared control. Better production of dry matter by NPs treatments suggested that plant accumulate higher nutrient supply in respective plant parts in particularly leaf and produce larger size. Our study revealed that by ZnO NPs with higher concentration maximum accumulation of nitrogen content found in spinach beet leaf and this might have accelerated the activity of several enzymes which in turn results better nitrogen content. On the other hand the phosphorus accumulation in treated leaves significantly increased by P NPs (Table 3 ). This result also supported by application of TiO 2 NPs that enhanced acid phosphatase activity and increase phosphorus nutrients (Raliya and Tarafdar 2014 ). In another report, it has been suggested that use of 500 mg kg −1 TiO 2 NPs increased the P content in shoots (138.9%) in rice (Zahra et al. 2017 ). The maximum potassium content of observed in 50 ppm of P NPs. Our investigation also suggested that application of ZnO NPs significantly increased the zinc and iron content in spinach beet. The enrichment of zinc and iron content in spinach beet leaves might be due to availability and increased uptake of ZnO NPs by plants. Du et al. ( 2019 ) recommended that to overcome the problem of Zn deficiency in plants Zn NPs will be best responsive slow release fertilizer. Recently, Zhang et al. ( 2021 ) reported that ZnO NPs significantly increased Zn content in polished rice which could be remarkable achievement under biofortified nutrition rich rice grain. The application of TiO 2 NPs at higher concentration (500 mg kg −1 ) significantly many fold changes of Fe, Zn, Cu and P accumulation occurred in rice (Arshad et al. 2021 ). This contrasts with results by Hruby et al. ( 2002 ), who found that Avena sativa L. showed increased Mg and Fe uptake with increased Ti exposure at 2–18 mg L −1 . Additionally, Li et al. ( 2021 ) reported hydroxyapatite NPs were promising beneficial response for nutrient uptake of Cu, Zn, Fe, Mn, and B in soybean tissues. With the application of Fe 3 O 4 NPs tremendous nutrient accumulation of P, K, Mn, Ca and Fe content recorded in different tissues of common bean (Souza et al. 2019 ). 4.5. Biochemical trait responses in spinach beet The experimental results indicate that TiO 2 and ZnO NPs with moderate to higher concentration could significantly improve spinach beet growth and accelerate maximum chlorophyll synthesis. Surprisingly, Morteza et al. ( 2013 ) described utilizing of nano TiO 2 at 0.03% concentration could increase chlorophyll content in corn. In contrast to this, opposite response was found by ZnO NPs plants which accumulate more chlorophyll and carotene content than TiO 2 NPs treated plants (Hajra et al. 2017). Spinach beet being a leafy vegetable contains good source of vitamin C, protein, fibre, calcium and iron. In the current study, maximum protein content in spinach beet was observed at 50 ppm of P NPs (Table 4 ) suggesting that acceleration of nutrients particularly nitrogen which enhances protein content in spinach beet leaves. Tarafder et al. (2014) found significant enhancement in chlorophyll content (24.4%), leaf protein (38.7%) and plant dry biomass (12.5%) in Pennisetum americanum by use of Zn NPs. Following nano TiO 2 treatments also accumulate more photosynthetic activity, enhanced mRNA expression and protein content in spinach (Gao et al. 2006 ). Our results showed that chlorophyll and carotene content were increased by increasing the doses of TiO 2 NPs and this reports are agreement with recent study in carrot (Gholami et al. 2020 ). Moreover, Sohail et al. ( 2020 ) reported that application of Zn NPs drastically improved protein content, total soluble sugar, proline, total flavonoid, and phenolic content in rapeseed. Whereas, Ghafari et al. (2013) suggested that nano-iron oxide (2 g L −1 ) increases chlorophyll, grain protein and iron contents in wheat. Conclusion In this study a comprehensive assessment of ZnO, P and TiO 2 NPs was achieved to target for improvement of seed germination antioxidant activity, nutritional quality and yield of spinach beet. NPs treatments showed the capability to increase seedling growth and many fold volume of antioxidant enzyme in spinach beet. Variable response to different concentrations of nanoparticles was noticed changes of plant phenotypic character in spinach beet. Higher concentration of P and moderate to high concentration of ZnO and TiO 2 NPs improve the plant nutrient (Zn and Fe content) content and quality traits as well as economic yield of spinach beet. Development of nanotechnology paved the way or increase productivity of spinach beet but at the same time it has raised concern for its adverse effect on biological system, food safety and environment system. It needs further study to get answer of question as to what extent molecular and genetic factors may mediate plant response to nano exposure with mitigation of adverse effect on plant development and environment. Declarations Acknowledgments The authors are grateful to Dr. J. C. Tarafdar, Central Arid Zone Research Institute (CAZRI), Jodhpur, Rajasthan for providing nanoparticles. Authors’ Contributions: (1) Conceptualization: Bimal Das, Surajit Kundu. (2) Methodology: Bimal Das, Dipak Kumar Murmu, Rakesh Yonzone. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1315549","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":85309841,"identity":"d3f75642-45ed-48d9-8858-4898a1f60ab9","order_by":0,"name":"BIMAL DAS","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACA2Yg8YABSLI3H//xAchhYydGSwJIC8+xBMkZIC3MhLQwwLRI5BhI84B4hLSYs3MnfkhgsJaXn5GWYGzza5s8HzMD44ePObi1WDbzbpZIYEg33HDm8YHk3L7bhm3MDMySM7fhcdhh3g1ALYcZN7CnJRzO7bnNCNTCxsyLX8vmH0At9vMbcgybLXtu2xOjZRvIlsSGEznGzAw/bicS1AL0yzaLBIP05A1njqUx9jbcTm5jZmzG6xdz/rObb3yosLad3958jOHHn9sgxsEPH/FogToPSjO2gckGQuqRwR9SFI+CUTAKRsFIAQDvsVEKIwXhkwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7865-6120","institution":"North Bengal Agricultural University: Uttar Banga Krishi Viswavidyalaya","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"BIMAL","middleName":"","lastName":"DAS","suffix":""},{"id":85309842,"identity":"63eefab9-6a0d-46d5-8e95-4345c7b9cc48","order_by":1,"name":"Rakesh Yonzone","email":"","orcid":"","institution":"Uttar Banga Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rakesh","middleName":"","lastName":"Yonzone","suffix":""},{"id":85309843,"identity":"05865430-73d5-4bd9-a361-1190edc3cb9b","order_by":2,"name":"Salil Saha","email":"","orcid":"","institution":"Uttar Banga Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Salil","middleName":"","lastName":"Saha","suffix":""},{"id":85309844,"identity":"f5caa74d-a480-48ac-b1aa-c3c86e004be4","order_by":3,"name":"Dipak Kumar Murmu","email":"","orcid":"","institution":"Uttar Banga Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dipak","middleName":"Kumar","lastName":"Murmu","suffix":""},{"id":85309845,"identity":"a464e07a-f6a5-461a-a84c-3c0317f5c3fa","order_by":4,"name":"Surajit Kundu","email":"","orcid":"","institution":"Uttar Banga Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Surajit","middleName":"","lastName":"Kundu","suffix":""}],"badges":[],"createdAt":"2022-01-31 18:22:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1315549/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1315549/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18569820,"identity":"c2d2d660-990c-4db3-b9b2-c1961e70e36f","added_by":"auto","created_at":"2022-02-24 14:59:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129284,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs under laboratory and field study of Spinach beet variety All Green\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1315549/v1/e20b6875cd085b2051f67499.png"},{"id":18569825,"identity":"d41301ec-16fd-4c22-9912-cf9ce6bfc274","added_by":"auto","created_at":"2022-02-24 14:59:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113082,"visible":true,"origin":"","legend":"\u003cp\u003eA. Effect of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs treatments for germination percentage of Spinach beet\u003c/p\u003e\u003cp\u003eB. Effect of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs treatments for root length and shoot length of Spinach beet\u003c/p\u003e\u003cp\u003eC. Effect of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs treatments for fresh seedling weight and vigour index of Spinach beet\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1315549/v1/ecc81f9bfb5c991b56f945fc.png"},{"id":18569823,"identity":"c46ed44c-4cb8-4875-8f53-efffffe02587","added_by":"auto","created_at":"2022-02-24 14:59:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78139,"visible":true,"origin":"","legend":"\u003cp\u003eA. Effect of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs treatments for Catalase (CAT) and Peroxidase (POD) enzyme activity of Spinach beet at one week of seedling\u003c/p\u003e\u003cp\u003eB. Effect of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs treatments for Superoxide dismutase (SOD) and Glutathione reductase (GR) enzyme activity of Spinach beet at one week of seedling\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1315549/v1/af355eb957b0d63e245f3c07.png"},{"id":18570123,"identity":"2c39c949-a817-4a1d-b46e-2ff618ad7a99","added_by":"auto","created_at":"2022-02-24 15:02:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":632117,"visible":true,"origin":"","legend":"\u003cp\u003eZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs treatments responses under field evaluation of Spinach beet variety All Green\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1315549/v1/3dfa881d894101ce63d39f85.png"},{"id":19166815,"identity":"4842dd08-b007-4f35-aac9-2b6d390d2514","added_by":"auto","created_at":"2022-03-13 07:50:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1594152,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1315549/v1/b541c5ec-aacf-4add-bc42-8a04c969b940.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eComprehensive assessment of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003enanoparticles sustaining environment in response to seed germination, antioxidants activity, nutritional quality and yield of Spinach Beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e var. bengalensis)\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTo meet demand of food supply over increasing population, recently nanotechnology will occupied a prominent position in transforming agricultural sector and environment sustainability. The expansion of nano devices (\u0026lt;100nm size) could be use wide range of extensive applications for increasing seed germination and seedling growth, plant morho-physiological development and alleviation of stress environments. The unusual idiomatic properties of nanoparticles with high surface to volume ratio, better catalytic surface, high reactivity capacity, rapid chemical reaction and adsorb abundant water may result in different environmental behaviors than the bulk materials. The metal based NPs (ZnO, TiO\u003csub\u003e2\u003c/sub\u003e, Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, CuO) were capable to penetrate into the plant cell causing damage by interacting with sulfur and phosphorus containing compounds such as DNA and protein \u003cb\u003e(\u003c/b\u003eAmarendra and Krishna, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and also optimizing plant metabolic processes at the early growth stages (Taran et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The interaction between NPs and plant cell results changes of gene expression and connected biological pathways which finally changes the plant characteristics. The impact of NPs positive effect were documented in array of crop plants (Monica and Cremonini \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and their response dependent type of NPs use, concentration and species (Gao et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hong et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Application of MWCNT (multiwalled carbon nanotubes) increases seed germination and seedling growth of different crop plants related to tomato (Khodakovskaya et al. 2010), soybean, corn, barley (Lahiani et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), maize, peanut and wheat (Srivastava et al. 2014; Joshi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Use of metal-based ZnO, TiO\u003csub\u003e2\u003c/sub\u003e and P NPs largely utilized in seed sciences and crop improvement, which exploit contradictory results on seed germination and seedling growth in tomato (Das et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). NPs application also acts as antioxidant enzyme activity in plants that protect from oxidative stress damage (Gupta et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jadczak et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition to germination and seedling growth the nanoscale ZnO and TiO\u003csub\u003e2\u003c/sub\u003e NPs were reported to positive influences of crop growth and development with nutritional quality enhancement in many crop species including peanut (Prasad et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), soybean (Zhang et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), rice (Debnath et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), spinach (Yang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and carrot (Gholami et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Application of TiO\u003csub\u003e2\u003c/sub\u003e NPs in spinach produced significant effects on growth, increases photosynthesis and nitrogen metabolism (Gao et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hong et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and induce redox reactions, chlorophyll formation and Rubisco activity \u003cb\u003e(\u003c/b\u003eSiddiqui et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hegde et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). NPs may enhanced nutrient use efficiency (Zulfiqar et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), nutrient content (Sabir et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and improve yield and also decrease its accumulation in soil. Albeit reports of improvement on seedling growth, physiological attributes, yield and quality, information on the genetic mechanism underline these changes is meager. In viewpoint of the above facts the present study delineates ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs laboratory observation of seed germination, seedling growth and antioxidant activity and field study of yield attributing and nutritional quality responsive traits in spinach beet (Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Material used\u003c/h2\u003e\n \u003cp\u003eTitanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e) NPs were purchased from Sigma-Aldrich Company, St. Louis, MO, USA with a purity of 99.5%, particle size of (\u0026lt;100 nm). Biosynthesized Zinc Oxide (ZnO) and Phosphorus (P) NPs were collected from Central Arid zone Research Institute (CAZRI), Jodhpur, Rajastan. Characterization of particles in stipulations of size (\u0026lt;100 nm), shape, structure and elemental proportion presented our previous report (Das et. al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Seed treatment with ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs\u003c/h2\u003e\n \u003cp\u003eThe present study was conducted at research laboratory and Instructional farm of College of Agriculture (Extended Campus), Uttar Banga Krishi Viswavidyalaya, Majhian, West Bengal, India during 2018 and 2019. Healthy and uniform seeds of spinach beet variety All Green were selected and sterilized with 0.1% Mercuric Chloride solution for 1 min and rinsed properly with sterilized distilled water two to three times. Then seeds were treated with different concentrations of ZnO (0, 5, 10, 15, 20, 25 ppm), P (10, 20, 30, 40, 50 ppm) and TiO\u003csub\u003e2\u003c/sub\u003e (10, 20, 30, 40, 50 ppm) NPs in aerated solutions for 24 hrs at 25 \u0026plusmn; 2\u003csup\u003eo\u003c/sup\u003eC and these concentration were chosen based on our previous studies (Das et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The untreated seeds with sterilized water considered as control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. Germination test and seedling growth\u003c/h2\u003e\n \u003cp\u003eGermination percentage of spinach beet seeds was estimated based on AOSA method (AOSA, 1990). In an incubator, three replicates of 50 seeds of each treatment were placed in 12 cm diameter petri plates, between the layers of moist Whatman-42 filter papers at 27\u0026deg;C and arranged in a complete randomized design (CRD). Germination percentage was counted based on emergence of seedling from embryo. The average root length, shoot length and their fresh seedlings weight recorded from 10 seedlings of each treatment at one week of age. Vigour Index (VI) was estimated as per following equation (Abdul Baki and Anderson, \u003cspan class=\"CitationRef\"\u003e1973\u003c/span\u003e):\u003c/p\u003e\n \u003cp\u003eVigour Index = (Shoot length average + Root length average)*Germination\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Extraction and Antioxidant enzyme activity\u003c/h2\u003e\n \u003cp\u003eCatalase enzyme (CAT) activity was measured following the procedure of Aebi (\u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e) with some minor modifications. All steps in preparation of the enzyme extracts were carried out at 0 to 4\u0026deg;C. The ultra violet (UV) light absorbance of hydrogen peroxide solution can be measured between 230 and 250 nm. On decomposition of hydrogen peroxide by catalase, the absorption decreases with time. Fresh leaves (0.1 g) were homogenized in 5 mL of cooled phosphate buffer (50 mM pH 7.8) with a pinch of Poly Vinyl Pyrrolidone (PVP).The extracted leaves were centrifuged with 15000 rpm for 10 minutes and supernatant collected for enzyme estimation. Three ml of analysis mixture contained 50 \u0026micro;l distilled water whereas, control contained devoid of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The catalase activity was measured by reduce of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e absorbance on 240 nm and expressed as \u0026micro;mol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposing/min/g.\u003c/p\u003e\n \u003cp\u003ePeroxidase enzyme (POD) was estimated following the procedure of Castillo et al. (\u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e) at absorbance of 470 nm using a reaction mixture containing 12 mM hydrogen peroxide and 96 mM guaiacol in phosphate buffer (pH 7.0). The optical density increases due to oxidation which produce tetra-guaiacol and POD expressed as \u0026micro;moles/cm/min/g fresh weigh. The Superoxide dismutase (SOD) was estimated following the procedure by Gong et al. (\u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e) by nitroblue tetrazolium (NBT) with an absorbance at 560 nm. Glutathione reductase (GR) was estimated following the procedure describe by Shanker et al. (\u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Field study of NPs treated seeds and recording of yield attributing traits\u003c/h2\u003e\n \u003cp\u003eBeside seedling experiments All Green seeds with above sixteen different concentrations of NPs treatment including control also sown in field based on randomized block design (RBD) by maintaining spacing of 40cm x 20cm. The fertilizer applied in soil following recommended dose of NPK (75:40:40) and necessary cultural practices maintained for plant growth. The initial soil fertility status of experimental field was presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. One foliar exposure of above NPs treatment in each case was done at 30 days of sowing. During the entire growth period (150 days) of spinach beet plants morpho-phenotypic and yield attributes such as plant height (60 DAS), leaf number, leaf length (cm), leaf width (cm), stem thickness (mm), leaf area (cm\u003csup\u003e2\u003c/sup\u003e), fresh leaf weight (gm) and seed yield plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e (gm) were recorded by selecting average of five plant in each treatment. The fresh weight (gm) of the leaves at was recorded after 50 days sowing.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSoil fertility status under field condition during crop growth stage\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParticular\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eValues\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eStatus\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTexture class\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSilt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSandy loam\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcidic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSoil nutrients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrganic carbon (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvailable N (Kg ha\u003csup\u003e\u0026minus;1)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e185.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvailable P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e (Kg ha\u003csup\u003e\u0026minus;1)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvailable K\u003csub\u003e2\u003c/sub\u003eO (Kg ha\u003csup\u003e\u0026minus;1)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e296.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003eSource: Regional Research Station (Old Alluvial Zone), UBKV, Majhian\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6. Dry matter and Elemental Analysis\u003c/h2\u003e\n \u003cp\u003eFor estimation of dry matter content each treated plants were taken out during maturity stage (60DAS), dried under shade and then placed in a hot air oven at 70 \u0026plusmn; 5\u003csup\u003eo\u003c/sup\u003eC for 48 hours to a constant weight, and weighed to record dry matter content (gm). The dried leaf were finely ground and digested for estimation of total NPK content. Nitrogen content from dried leaves was estimated by the micro-Kjeldahl method (Subbiah et al. 1956). For phosphorus and potassium content tri-acid mixture (HNO\u003csub\u003e3\u003c/sub\u003e:H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e:HClO\u003csub\u003e4\u003c/sub\u003e = 10:1:4) was used and determined by flame photometer (Jackson \u003cspan class=\"CitationRef\"\u003e1973\u003c/span\u003e). Iron and zinc contents each treated leaves extract digested in triple acid was estimated by employing the method given by Jackson (\u003cspan class=\"CitationRef\"\u003e1973\u003c/span\u003e) by using atomic absorption spectroscopy (PinAAcle 990F; PerkinElmer, Singapore).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7. Estimation of Biochemical parameters\u003c/h2\u003e\n \u003cp\u003eBiochemical properties like total chlorophyll content from young leaf of spinach beet was estimated by spectrophotometric method (Davies \u003cspan class=\"CitationRef\"\u003e1976\u003c/span\u003e). Carotene content of spinach leaf was estimated by the standard procedure of AOAC (\u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e) using 80% of acetone. Protein content from the fresh leaves was estimated following the method of Lowry, et al., \u003cspan class=\"CitationRef\"\u003e1951\u003c/span\u003e. Ascorbic acid content was determined by using 2-6, Dichlorophenyl Indophenol dye solution (Ranga \u003cspan class=\"CitationRef\"\u003e1979\u003c/span\u003e) and expressed as mg/100g of fresh weight. Total polyphenol concentrations of leaf material were determined using the Folin-Ciocalteu the method by Waterman and Mole (\u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e) as described by Mudau et al. (\u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). The finely ground leaves samples were used crude fibre content by AOAC method (1970).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eEach treatment was conducted with three replicates and the results were presented as mean\u0026plusmn;SE (standard error of the mean) and CD value. All the data were statistically analyzed by Fisher\u0026rsquo;s technique and Duncan\u0026rsquo;s Multiple Range Test (p \u0026le; 0.05). The analysis was carried out using the SPSS software 21 version and graphical representation done by OriginPro 21 software.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1. Effect of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs for germination and seeding growth\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe use of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments significantly affected germination and seedling growth of spinach beet. Improvement in germination percentage was found from all the ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments (Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Enhanced germination percentage over control was highlighted for almost all the treatments except 5 ppm of ZnO. Highest germination percentage was found in treatment with 30 ppm of TiO\u003csub\u003e2\u003c/sub\u003e, followed by 15 ppm of ZnO. The increment in germination percentage was observed to the tune of 10.92%, 13.66% and 17.01% in ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e respectively, as compare to control. The data regarding length of root and shoot at one week of age has been presented in the Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB. Most favourable effect on root and shoot length was highlighted by respective treatments of 40 ppm of TiO\u003csub\u003e2\u003c/sub\u003e. The data also depicts that the TiO\u003csub\u003e2\u003c/sub\u003e NPs provides maximum response of root and shoot length than other treatment which an increase of 28.14% and 22.34% respectively, over control. Similar to the data on fresh seedling weight the maximum was recorded with 50 ppm TiO\u003csub\u003e2\u003c/sub\u003e which was 36.46% higher than control, whereas, the least fresh seedling weight of shoot was associated with 20 ppm of P (Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Enhancement of vigor index was maximum by TiO\u003csub\u003e2\u003c/sub\u003e NPs at 40 ppm and 30 ppm. Over all vigor index increase with 25.16%, 26.92% and 30.39% by ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs, respectively from the control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.2 Effect of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e Nanoparticles for antioxidant enzymatic activity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNPs treatments significantly influence the antioxidant enzymatic response during seedling growth of spinach beet. The data indicate highly significant (p \u0026le; 0.05) differences of antioxidant enzyme activity (CAT, POD, SOD and GR) under seedling stage of spinach beet (1 week old seedling) in response to ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs application (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The catalase (CAT) activity increased statistically (p \u0026le; 0.05) with the treatments (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The maximum expression of catalase activity was recorded when treated with TiO\u003csub\u003e2\u003c/sub\u003e at 40 ppm followed by 20 ppm and 30 ppm. The average cumulative effect of catalase activity increased from 21.79%, 8.08% and 43.78% respectively, by ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs respectively, over the control. The maximum peroxidase enzyme activity was recorded in 20 ppm TiO\u003csub\u003e2\u003c/sub\u003e and an average increment by ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments found with 17.31%, 6.15% and 34.05% respectively, as compared to control plants (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). During seedling growth, the SOD activity tended to produce considerably at different NPs treatment (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The SOD enhancement occurred by ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments was 22.59%, 22.02% and 45.22% respectively, from control. On the other hand the expression of GR activity in spinach beet leaves was found maximum with TiO\u003csub\u003e2\u003c/sub\u003e NPs at 30 ppm (49.22%). Overall enhancement of the GR activity was noticed 18.41%, 10.68% and 44.50% by ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 Effect of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs on morphological and yield traits of spinach beet\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eApplication of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments of spinach beet had profound effect on plant morphological traits (Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). At maturity stage of spinach beet (80 DAS), the maximum plant height was found in TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments and increased by 35.27% at 30 ppm (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Shortest plants were observed under untreated control condition. The positive effect of plant height with statistically at per treatments were 40 ppm and 50 ppm TiO\u003csub\u003e2\u003c/sub\u003e NPs, 20 ppm and 30 ppm P NPs, 10 ppm and 15 ppm ZnO NPs. Other characteristics noticed maximum response with the application of TiO\u003csub\u003e2\u003c/sub\u003e NPs at 30 ppm of leaf number; at 40 ppm of leaf length, leaf width and leaf area; at 10 ppm of stem thickness. For ZnO NPs treatments the promising effect found at higher concentration of 25 ppm for plant height, number of leaf plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e and leaf area whereas lower concentration beneficial for stem thickness. In case of P NPs treatment the higher concentration found satisfactory results almost all the morphological characteristics. In situation to yield characteristics of spinach beet leaf weight and seed yield showed significant (p \u0026lt;0.05) impact with by application of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Significantly enhancement of fresh leaf weight (gm plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e) exhibited with 25 ppm of ZnO, 40 ppm P and 20 ppm of TiO\u003csub\u003e2\u003c/sub\u003e with an increase of 17.16%, 18.33% and 16.41% respectively, in assessment to control. Whereas, seed yield (gm plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e) an average increase by ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs occurred with 12.48% 13.01% and 10.93% respectively, compared to control (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs plant morphological and yield traits of spinach beet\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlant height (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of leaf plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLeaf length (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLeaf width (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStem thickness (mm )\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLeaf area (cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFresh leaf weight (gm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSeed yield plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e (gm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.81 \u0026plusmn;1.77h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.81 \u0026plusmn;1.01g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.57\u0026plusmn;1.17e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.35\u0026plusmn;0.52f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.07 \u0026plusmn; 0.28g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e193.12 \u0026plusmn;3.62k\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.45 \u0026plusmn;0.75k\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.45 \u0026plusmn;0.30h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.35 \u0026plusmn;0.10h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.72 \u0026plusmn;1.05efg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.43 \u0026plusmn;0.43abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.46 \u0026plusmn;0.28def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.04 \u0026plusmn;0.41fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198.44 \u0026plusmn;2.95jk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.89 \u0026plusmn;0.35j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.46 \u0026plusmn;0.26bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.40 \u0026plusmn;2.84fgh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.67 \u0026plusmn;1.12bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.16 \u0026plusmn;0.59a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.05 \u0026plusmn;0.91ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.93 \u0026plusmn;0.87ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e212.31 \u0026plusmn;3.24hi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.36 \u0026plusmn;0.27hi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.13\u0026plusmn;0.12fgh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.80 \u0026plusmn;2.78fgh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.07 \u0026plusmn;0.80defg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.15 \u0026plusmn;0.65abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.53 \u0026plusmn;1.24f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.09 \u0026plusmn;0.51cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e227.86 \u0026plusmn;5.73efg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.99 \u0026plusmn;0.75 ij\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.35 \u0026plusmn;0.37ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.43 \u0026plusmn;2.38ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.48 \u0026plusmn;0.85def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.75 \u0026plusmn;1.12abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.87 \u0026plusmn;0.91ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.52\u0026plusmn;0.40efg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e225.90 \u0026plusmn;5.13fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.80 \u0026plusmn;0.33bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.89 \u0026plusmn;0.32def\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.88 \u0026plusmn;1.61de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.87 \u0026plusmn;1.51a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.75 \u0026plusmn;0.49abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.95 \u0026plusmn;0.77ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.72 \u0026plusmn;0.50ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e239.81 \u0026plusmn;2.70cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.24 \u0026plusmn;1.09 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.08 \u0026plusmn;0.34a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of ZnO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e220.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.50 \u0026plusmn;1.81gh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.67 \u0026plusmn;0.54defg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.29 \u0026plusmn;1.05ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.67 \u0026plusmn;1.27abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.61 \u0026plusmn;0.67cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e219.47 \u0026plusmn;3.34gh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.13 \u0026plusmn;1.18 gh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.59 \u0026plusmn;0.43bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.15 \u0026plusmn;1.21d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.98 \u0026plusmn;0.67bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.31 \u0026plusmn;0.67ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.98 \u0026plusmn;0.30a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.79 \u0026plusmn;0.61def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e207.12 \u0026plusmn;2.98ij\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.98 \u0026plusmn;0.94bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.42 \u0026plusmn;0.34bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.17 \u0026plusmn;1.57d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.73 \u0026plusmn;0.55fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.61 \u0026plusmn;0.93de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.74 \u0026plusmn;0.79bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.77 \u0026plusmn;0.05def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e229.80 \u0026plusmn;3.20ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.49 \u0026plusmn;0.38a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.37 \u0026plusmn;0.25bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.05 \u0026plusmn;2.55de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.82 \u0026plusmn;0.75de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.62 \u0026plusmn;0.70abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.82 \u0026plusmn;1.01ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.92 \u0026plusmn;0.63def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e248.70 \u0026plusmn;3.50bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.75 \u0026plusmn;2.22hi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.86 \u0026plusmn;0.31gh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.55 \u0026plusmn;1.17efg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.10 \u0026plusmn;0.94fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.91 \u0026plusmn;0.53a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.11 \u0026plusmn;0.74cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.63 \u0026plusmn;0.57cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e259.21 \u0026plusmn;1.79a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.46 \u0026plusmn;0.38cdef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.32 \u0026plusmn;0.77ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e231.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e117.07 \u0026plusmn;1.76b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.09 \u0026plusmn;0.66cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.11 \u0026plusmn;1.33a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.70 \u0026plusmn;0.66f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.67 \u0026plusmn;0.21a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e241.88 \u0026plusmn;3.10cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.15 \u0026plusmn;0.59fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.07 \u0026plusmn;0.35fgh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120.84 \u0026plusmn;1.67ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.33 \u0026plusmn;0.58abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.47 \u0026plusmn;0.60de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.72 \u0026plusmn;1.01f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.44 \u0026plusmn;0.48cdef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e236.86 \u0026plusmn;2.58de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.29 \u0026plusmn;0.23efg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.91 \u0026plusmn;0.20def\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e123.29 \u0026plusmn;1.30a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.77 \u0026plusmn;0.73ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.89 \u0026plusmn;1.04cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.79 \u0026plusmn;0.70f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.09 \u0026plusmn;0.38def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e254.77 \u0026plusmn;2.89ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.54 \u0026plusmn;0.25defg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.56 \u0026plusmn;0.26efg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111.00 \u0026plusmn;1.12c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.38 \u0026plusmn;0.49cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.48 \u0026plusmn;1.18ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.43 \u0026plusmn;0.41f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.92 \u0026plusmn;0.83cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e234.24 \u0026plusmn;2.75def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.48 \u0026plusmn;0.36abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.25 \u0026plusmn;0.18cde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.18 \u0026plusmn;2.62c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.29 \u0026plusmn;0.76abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.26 \u0026plusmn;0.60bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.29 \u0026plusmn;0.53f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.71 \u0026plusmn;0.28bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e258.60 \u0026plusmn;2.62a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.39 \u0026plusmn;0.20bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.10 \u0026plusmn;0.12abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of TiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e244.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003e*Mean value\u0026plusmn; SE. Each value represents the mean of three replication of each treatment. Different letter of mean value are significantly different at P\u0026lt;0.05 according to the Duncan\u0026rsquo;s multiple range test (DMRT) and the same letters indicate no significant difference P\u0026lt;0.05.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003e3.2.2 Effect of ZnO, P and TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003eNPs on dry matter production and nutrients accumulation in spinach beet\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eEffect of NPs on dry matter and nutrients accumulation were presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Our study revealed that maximum dry matter was observed by ZnO NPs at 25 ppm (36.55 g), P NPs at 20 ppm (35.66 g) and TiO\u003csub\u003e2\u003c/sub\u003e NPs at 40 ppm (34.35g) that which 22.74%, 20.81% and 17.76% enhancement monitored as compared to control. The nutrient uptake in spinach beet leaves exposed by NPs, indicating their differential response with the treatments. Among the analyzed elements, significant difference in total N, P, K, Zn and Fe contents was found in leaves exposed to NPs. Our results depicted that improvement of major and minor elements in the leaves with an average increase 15.60%, 9.39% and 12.01% of N, 21.62%, 52.44% and 16.00% of P, 10.13%, 14.11%, and 11.75% of K, 50.76%, 7.71%, and 6.41% of Zn and 12.69%, 18.49% and 14.17% of Fe by ZnO, P, and TiO\u003csub\u003e2\u003c/sub\u003e NPs respectively, over the control plants (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs on dry matter production and nutrients accumulation in spinach beet\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDry matter (gm plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e )\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eK (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZn (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIron (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl (0 ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.24 \u0026plusmn; 1.14f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.608 \u0026plusmn; 0.02e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.775 \u0026plusmn; 0.05d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.56 \u0026plusmn; 0. 29e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.26 \u0026plusmn; 1.64e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e212.36 \u0026plusmn; 2.40g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.26 \u0026plusmn; 2.60ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.636 \u0026plusmn; 0.02e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.955 \u0026plusmn; 0.05cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.69 \u0026plusmn; 0.25de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.56 \u0026plusmn; 2.22c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232.55 \u0026plusmn; 2.20f\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.24 \u0026plusmn; 1.12cdef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.672 \u0026plusmn; 0.06cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.966 \u0026plusmn; 0.05cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.88 \u0026plusmn; 0.05bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.69 \u0026plusmn; 0.99b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e236.56 \u0026plusmn; 5.17ef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.65 \u0026plusmn; 2.35def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.756 \u0026plusmn; 0.02abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.942 \u0026plusmn; 0.05cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.12 \u0026plusmn; 0.12abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.66 \u0026plusmn; 1.76a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e254.36 \u0026plusmn; 4.86bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.56 \u0026plusmn; 0.91abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.779 \u0026plusmn; 0.02a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.020 \u0026plusmn; 0.09c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.13 \u0026plusmn; 0.13abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.21 \u0026plusmn; 3.00a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e241.37 \u0026plusmn; 4.26def\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.56 \u0026plusmn; 1.24a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.759 \u0026plusmn; 0.05ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.061 \u0026plusmn;0.02c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.99 \u0026plusmn; 0.02bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.66 \u0026plusmn; 1.28a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e251.33 \u0026plusmn; 2.70cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of ZnO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e243.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.66 \u0026plusmn; 0.92bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.656 \u0026plusmn; 0.03de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.454 \u0026plusmn; 0.11b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.89 \u0026plusmn; 0.04bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.34 \u0026plusmn; 1.75de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e244.12 \u0026plusmn; 2.20de\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.66 \u0026plusmn; 1.11ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.689 \u0026plusmn; 0.03bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.560 \u0026plusmn; 0.18ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.97 \u0026plusmn; 0.10bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.22 \u0026plusmn;1.19de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e245.36 \u0026plusmn; 3.02cde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.66 \u0026plusmn; 0.52f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.671 \u0026plusmn; 0.03cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.661 \u0026plusmn; 0.09ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.23 \u0026plusmn; 0.12ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.66 \u0026plusmn; 2.17de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e262.56 \u0026plusmn; 2.49b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.66 \u0026plusmn; 0.92ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.685 \u0026plusmn; 0.03bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.713 \u0026plusmn; 0.12a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.12 \u0026plusmn; 0.16abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.25 \u0026plusmn; 1.76d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e274.38 \u0026plusmn; 4.17a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.55 \u0026plusmn; 0.94abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.654 \u0026plusmn; 0.04de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.760 \u0026plusmn; 0.02a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.53 \u0026plusmn; 0.18a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.66 \u0026plusmn; 1.76de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e276.22 \u0026plusmn; 5.47a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e260.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.25 \u0026plusmn; 0.58abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.668 \u0026plusmn; 0.03de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.911 \u0026plusmn; 0.05cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.12 \u0026plusmn; 0.24abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.34 \u0026plusmn; 2.18de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232.25 \u0026plusmn; 2.83f\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.22 \u0026plusmn; 0.97abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.724 \u0026plusmn; 0.01abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.923 \u0026plusmn; 0.06cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.87 \u0026plusmn; 0.13bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.34 \u0026plusmn; 0.35de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e233.67 \u0026plusmn; 1.21f\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.67 \u0026plusmn; 0.88bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.723 \u0026plusmn; 0.01abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.922 \u0026plusmn; 0.02cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.16 \u0026plusmn; 0.20abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.22 \u0026plusmn; 2.25de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e254.36 \u0026plusmn; 2.65bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.35 \u0026plusmn; 1.2ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.653 \u0026plusmn; 0.02de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.932 \u0026plusmn; 0.01cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.33 \u0026plusmn; 0.16ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.61 \u0026plusmn; 2.26de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e263.12 \u0026plusmn; 2.69b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.46 \u0026plusmn; 0.96abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.687 \u0026plusmn; 0.01bcde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.925 \u0026plusmn; 0.03cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.70 \u0026plusmn; 0.15cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.58 \u0026plusmn; 1.71de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e254.68 \u0026plusmn; 4.56bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of TiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e247.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e*Mean value\u0026plusmn; SE. Each value represents the mean of three replication of each treatment. Different letter of mean value are significantly different at P\u0026lt;0.05 according to the Duncan\u0026rsquo;s multiple range test (DMRT) and the same letters indicate no significant difference P\u0026lt;0.05.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec15\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.2.2 Effect of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs on biochemical responses in spinach beet\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe application of different NPs treatments increase total chlorophyll content in leaves where, TiO\u003csub\u003e2\u003c/sub\u003e NPs at 10 ppm proved to be best and increased by 17.18%, over the control (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). All though successful improvement also detected from 10 ppm and 50 ppm of TiO\u003csub\u003e2\u003c/sub\u003e which were statistically at per. A progressive increase of total chlorophyll content by ZnO, P, and TiO\u003csub\u003e2\u003c/sub\u003e treatments with 30.17%, 28.24%, and 40.69% respectively, compared to control.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\" id=\"Tab4\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs on biochemical response in spinach beet\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment (ppm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Chlorophyll (mg g-1 FW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein (g/100g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCarotene (mg/100g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAscorbic acid (mg/100g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal phenol (mg/100g D.W)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCrude fibre (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl (0 ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.11 \u0026plusmn; 0.11f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.01 \u0026plusmn; 0.22d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.26 \u0026plusmn; 0.61d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.55 \u0026plusmn; 2.54h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e608.37 \u0026plusmn; 4.51h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.16 \u0026plusmn; 0.15d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.52 \u0026plusmn; 0.30ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.31 \u0026plusmn; 0.10cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.26 \u0026plusmn; 1.11cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115.26 \u0026plusmn; 2.69g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e725.34 \u0026plusmn; 4.84g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.66 \u0026plusmn; 0.09abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.15 \u0026plusmn;0.15bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.57 \u0026plusmn; 0.46bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.36 \u0026plusmn; 0.59abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118.78 \u0026plusmn; 3.19g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e736.56 \u0026plusmn; 1.90g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.55 \u0026plusmn; 0.44ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.39 \u0026plusmn; 0.10bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.49 \u0026plusmn; 0.27bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.38 \u0026plusmn; 0.91abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e144.66 \u0026plusmn; 3.30e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e825.45 \u0026plusmn; 9.23cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.58 \u0026plusmn; 0.47ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.02 \u0026plusmn; 0.10cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.08 \u0026plusmn; 0.05ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.33 \u0026plusmn; 1.08abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135.44 \u0026plusmn; 2.20f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e837.55 \u0026plusmn; 2.23ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.65 \u0026plusmn; 0.83a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnO-25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.03 \u0026plusmn; 0.08cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.95 \u0026plusmn; 0.24abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.36 \u0026plusmn; 1.04bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e138.34 \u0026plusmn; 1.19ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e822.66 \u0026plusmn; 4.07d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.56 \u0026plusmn; 0.31abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of ZnO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e130.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e789.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.13 \u0026plusmn; 0.09bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.48 \u0026plusmn; 0.16bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.56 \u0026plusmn; 1.20abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e136.66 \u0026plusmn; 2.45f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e765.64 \u0026plusmn; 10.49e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.69 \u0026plusmn; 0.46abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.24 \u0026plusmn; 0.13bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.92 \u0026plusmn; 0.17abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.36 \u0026plusmn; 0.98abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e144.68 \u0026plusmn; 2.15e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e789.56 \u0026plusmn; 4.01e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.12 \u0026plusmn; 0.57abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.46 \u0026plusmn; 0.14 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.86 \u0026plusmn; 0.15abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.78 \u0026plusmn; 0.58ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e166.57 \u0026plusmn; 1.61c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e796.22 \u0026plusmn; 4.65e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.68 \u0026plusmn; 0.65abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.13 \u0026plusmn; 0.17bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.25 \u0026plusmn; 0.05a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.88 \u0026plusmn; 0.19a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175.66 \u0026plusmn; 5.42b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e801.67 \u0026plusmn; 3.34e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.79 \u0026plusmn; 0.54abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.74 \u0026plusmn; 0.22de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.25 \u0026plusmn; 0.20a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.36 \u0026plusmn; 0.49abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e167.67 \u0026plusmn; 1.54c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e796.66 \u0026plusmn; 2.42e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.00 \u0026plusmn; 0.26abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e158.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e789.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.05 \u0026plusmn; 0.13a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.91 \u0026plusmn; 0.34abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.66 \u0026plusmn; 0.90abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156.65 \u0026plusmn; 2.79d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e842.33 \u0026plusmn; 2.89 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.22 \u0026plusmn; 0.46cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.08 \u0026plusmn; 0.11cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.96 \u0026plusmn; 0.08ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.58 \u0026plusmn; 0.72abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e179.66 \u0026plusmn; 2.86b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e848.66 \u0026plusmn; 3.30ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.34 \u0026plusmn; 0.17bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.55 \u0026plusmn; 0.19b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.03 \u0026plusmn; 0.31ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.67 \u0026plusmn; 0.81abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e189.66 \u0026plusmn; 3.20a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e857.12 \u0026plusmn; 4.63a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.55 \u0026plusmn; 0.36abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.08 \u0026plusmn; 0.11cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.09 \u0026plusmn; 0.11ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.26 \u0026plusmn; 1.03cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e193.24 \u0026plusmn; 2.36a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e844.21 \u0026plusmn; 3.71ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.11 \u0026plusmn; 0.41cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.02 \u0026plusmn; 0.13a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.09 \u0026plusmn; 0.21ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.22 \u0026plusmn; 0.82d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e179.23 \u0026plusmn; 1.94b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e837.67 \u0026plusmn; 3.83ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.88 \u0026plusmn; 0.31cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAvg. of TiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e179.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e846.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e*Mean value\u0026plusmn; SE. Each value represents the mean of three replication of each treatment. Different letter of mean value are significantly different at P\u0026lt;0.05 according to the Duncan\u0026rsquo;s multiple range test (DMRT) and the same letters indicate no significant difference P\u0026lt;0.05.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe perusal of data in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e revealed that use of NPs treatment significantly influences the leaf protein content of spinach beet. Protein content recorded maximum 4.25 g that of 40 ppm and 50 ppm concentration of P, where as plants under control had produced 3.15g only. ZnO, P, and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments protein content increased by 11.57%, 21.21%, and 21.09% respectively, over the control. Carotene content among the treatments varied considerable with the treatment. It was revealed that moderate concentration of NPs was found to be more effective for carotene content in the leaves. The application of NPs at higher concentration shows the decreasing tends of carotene. Total Carotene content of ZnO, P, and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments increased by 18.30%, 26.43%, and 17.85% respectively, compared to control. On the other hand ascorbic acid content on leaves also significantly influenced by NPs treatment (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The maximum of ascorbic acid content was recorded from 40 ppm TiO\u003csub\u003e2\u003c/sub\u003e, which was also statistically at par with 40 ppm TiO\u003csub\u003e2\u003c/sub\u003e. Total ascorbic acid content increased by 28.31%, 40.88%, and 48.03% of ZnO, P, and TiO\u003csub\u003e2\u003c/sub\u003e treatments respectively, as compared to control. The maximum total phenol was recorded from 30 ppm of TiO\u003csub\u003e2\u003c/sub\u003e NPs. An average effect of ZnO, P, and TiO\u003csub\u003e2\u003c/sub\u003e treatments on total phenol content increased by 22.94%, 22.99%, and 28.09% respectively, compared to control. In response to crude fibre content maximum was found when were treated with 20 ppm of ZnO NPs, whereas an enhancement of 22.21%, 19.18%, and 12.92% occurred by ZnO, P, and TiO\u003csub\u003e2\u003c/sub\u003e treatments respectively, compared to control.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e4.1. Germination and seeding development\u003c/h2\u003e\n \u003cp\u003eSeed germination considered as an index of plant growth which required adequate water absorption that responses to yield characteristic. Our study exhibited that seed germination in spinach beet significantly enhanced by TiO\u003csub\u003e2\u003c/sub\u003e NPs than ZnO and P NPs and the result fully agreement with Hajra et al (2017). TiO\u003csub\u003e2\u003c/sub\u003e NPs found better germination percentage over ZnO NPs and P NPs may be due to production of reactive anions which accumulate more water absorption and boost the oxygen rate for uptake during germination \u003cstrong\u003e(\u003c/strong\u003eKhot et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). In viewpoint of Zheng et al. (\u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e) suggested that due to small particle size of TiO\u003csub\u003e2\u003c/sub\u003e NPs can easily penetrate into the seed coat of spinach and enhanced activity during growth. Zheng et al. (\u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e) in spinach and Feizi et al. (\u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e) in wheat observed accelerated germination in proper concentration of TiO\u003csub\u003e2\u003c/sub\u003e treatment. The study revealed that seed priming and presoaking techniques using different NPs can significantly improve spinach beet performance by increasing seedling growth and vigour, although response varied with different NPs and concentrations. In general, treatments with lower to moderate doses of TiO\u003csub\u003e2\u003c/sub\u003e NPs than ZnO and P NPs showed significant positive response in most of the seedling characters. Previously, Haghighi et al. (2014) opined that higher doses of TiO\u003csub\u003e2\u003c/sub\u003e NPs (up to 200 ppm) in tomato and onion be capable of enhanced root and shoot lengths. Seedling growth enhances by TiO\u003csub\u003e2\u003c/sub\u003e NPs treatment possibly due to it regulates the activity of nitrogen-metabolizing enzyme which helps to convert nitrogen for chlorophyll accumulation (Yang et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mishra et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). The enhancement of seedling weight and vigor index by ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs also described our earlier report in tomato (Das et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) and TiO\u003csub\u003e2\u003c/sub\u003e NPs in rice (Debnath et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003e4.2. Antioxidant enzymatic activity\u003c/h2\u003e\n \u003cp\u003eSpinach beet one of the important vegetables contains high antioxidant capacity and it protects the plant cells from oxidative damage during stress. Our results depicted that the enzymatic activities of CAT, POD, SOD, and GR responses significantly by application of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs treatment during seedling stage and varies upon changes of concentration (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Higher concentration of Phosphorus and ZnO NPs the enzymatic activity were increasing trends which suggested that these elemental particles actively utilized various metabolic processes as well as protein synthesis also decrease H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels and involve oxidative stress response in spinach beet. On the other hand higher concentration of TiO\u003csub\u003e2\u003c/sub\u003e a decreasing trend of CAT and GR activity was noticed, which might affect the failure of plant defense system. The highest activity of peroxidase was noticed by ZnO NPs at 15 ppm and positive effect also agreements with previous study (Faizan et al. 2017). Pretreatments of seeds by ZnO nanoparticles can improve germination and seedlings as well as antioxidant enzymes (Garc\u0026iacute;a-L\u0026oacute;pez et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The observed increase in GR activity suggests that glutathione is also involved in spinach plants, either directly to oxidative stress response or indirectly via the production of phytochelatins (Potters et al. \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e). The antioxidant activity effective towards dose-dependent with lower doses of ZnO NPs increases the catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) but decreases at extremely high concentration (200 ppm) in rapeseed (Sohail et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), in potato (Raigond et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) and in wheat and maize (Srivastav et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, Khater (\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported that TiO\u003csub\u003e2\u003c/sub\u003e increased the production of antioxidant content in coriander. Additionally, \u0026Ccedil;atav et al. (\u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) showed that higher doses of Cd severely increased GR, POD and SOD in wheat.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003e4.3. Morphological and yield traits of spinach beet\u003c/h2\u003e\n \u003cp\u003eOur finding clearly depicted positive response plant growth and yield responses in nanoparticles treated plants as compared to control plants. Lower concentration of ZnO showed optimum improvement for the characters plant height, leaf length, leaf width, number of leaves and exposure to extremely high concentration showed optimum improvement for the characters stem thickness and leaf area (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly the economic product fresh leaf weight showed optimum response at moderately high concentration. With high concentration of P NPs were found to produce effective improvement for majority of the characters except leaf length and number of leaves, where comparatively lower concentration had given the effective result. Extremely higher concentration of TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments produced optimum improvement in economic product fresh leaf weight along with its component like plant height, number of leaves, stem thickness. Our present study both laboratory and field evaluation found a positive impact on spinach beet growth by NPs and the suggested mechanisms explain by increased nutrient adsorption and active transport of nutrients to the roots, activate enzyme for nutrient accumulation in leaf, enhanced synthesis of starch which ultimately increased the carbohydrate production and photosynthesis (Song et al. 2018). These mechanisms could be a contributing factor for increase average leaf yield found in spinach beet. In addition, the small size and high surface ratio with photo catalysis property of NPs which can interact with cellular system that contribute to overall effect of plant phenotypic changes and yield. Interestingly, Elizabath et al. (\u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported a combination of ZnO and FeO NPs at 50ppm found good response of plant height, leaves plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e, petiole length, leaf area (cm\u003csup\u003e2\u003c/sup\u003e) and yield of carrot. Recently Zhang et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported ZnO NPs application obtained higher yield of rice with more panicle number (4.83\u0026ndash;13.14%), spikelets panicle\u003csup\u003e\u0026minus;1\u003c/sup\u003e (4.81\u0026ndash;10.69%), test weight (3.82\u0026ndash;6.62%) and filled grain rate (0.28\u0026ndash;2.36%). Kheyri et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) suggest that Zn and Si NPs applications in rice enhance grain yield upto 9.5, 9.2, and 6.9%, respectively, which reduce fertilizer costs as well as environmental pollution. Tarafder et al. (2014) reported grain yield in pear millet at crop maturity was improved by 37.7% due to application of zinc nanofertilizer. On the other hand, Khater, (\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) described TiO\u003csub\u003e2\u003c/sub\u003e with low concentration had enhances the plant height, branches and fruit yield in coriander. Recently, Waani et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) suggested TiO\u003csub\u003e2\u003c/sub\u003e with dose dependant action both positive and negative effect on plant growth and yield of rice. Earlier report also suggested that CeO and iron oxide NPs increased wheat and soybean yield by 36.6% and 48%, respectively (Yang et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sheykhbaglou et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003e4.4. Effect of NPs nutrient accumulation of spinach beet\u003c/h2\u003e\n \u003cp\u003eThe application of NPs found to be beneficial in increasing dry matter of plant as compared control. Better production of dry matter by NPs treatments suggested that plant accumulate higher nutrient supply in respective plant parts in particularly leaf and produce larger size. Our study revealed that by ZnO NPs with higher concentration maximum accumulation of nitrogen content found in spinach beet leaf and this might have accelerated the activity of several enzymes which in turn results better nitrogen content. On the other hand the phosphorus accumulation in treated leaves significantly increased by P NPs (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). This result also supported by application of TiO\u003csub\u003e2\u003c/sub\u003e NPs that enhanced acid phosphatase activity and increase phosphorus nutrients (Raliya and Tarafdar \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). In another report, it has been suggested that use of 500 mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e TiO\u003csub\u003e2\u003c/sub\u003e NPs increased the P content in shoots (138.9%) in rice (Zahra et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The maximum potassium content of observed in 50 ppm of P NPs. Our investigation also suggested that application of ZnO NPs significantly increased the zinc and iron content in spinach beet. The enrichment of zinc and iron content in spinach beet leaves might be due to availability and increased uptake of ZnO NPs by plants. Du et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) recommended that to overcome the problem of Zn deficiency in plants Zn NPs will be best responsive slow release fertilizer. Recently, Zhang et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that ZnO NPs significantly increased Zn content in polished rice which could be remarkable achievement under biofortified nutrition rich rice grain. The application of TiO\u003csub\u003e2\u003c/sub\u003e NPs at higher concentration (500 mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e) significantly many fold changes of Fe, Zn, Cu and P accumulation occurred in rice (Arshad et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). This contrasts with results by Hruby et al. (\u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e), who found that \u003cem\u003eAvena sativa\u003c/em\u003e L. showed increased Mg and Fe uptake with increased Ti exposure at 2\u0026ndash;18 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Additionally, Li et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported hydroxyapatite NPs were promising beneficial response for nutrient uptake of Cu, Zn, Fe, Mn, and B in soybean tissues. With the application of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs tremendous nutrient accumulation of P, K, Mn, Ca and Fe content recorded in different tissues of common bean (Souza et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec21\"\u003e\n \u003ch2\u003e4.5. Biochemical trait responses in spinach beet\u003c/h2\u003e\n \u003cp\u003eThe experimental results indicate that TiO\u003csub\u003e2\u003c/sub\u003e and ZnO NPs with moderate to higher concentration could significantly improve spinach beet growth and accelerate maximum chlorophyll synthesis. Surprisingly, Morteza et al. (\u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) described utilizing of nano TiO\u003csub\u003e2\u003c/sub\u003e at 0.03% concentration could increase chlorophyll content in corn. In contrast to this, opposite response was found by ZnO NPs plants which accumulate more chlorophyll and carotene content than TiO\u003csub\u003e2\u003c/sub\u003e NPs treated plants (Hajra et al. 2017).\u003c/p\u003e\n \u003cp\u003eSpinach beet being a leafy vegetable contains good source of vitamin C, protein, fibre, calcium and iron. In the current study, maximum protein content in spinach beet was observed at 50 ppm of P NPs (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) suggesting that acceleration of nutrients particularly nitrogen which enhances protein content in spinach beet leaves. Tarafder et al. (2014) found significant enhancement in chlorophyll content (24.4%), leaf protein (38.7%) and plant dry biomass (12.5%) in \u003cem\u003ePennisetum americanum\u003c/em\u003e by use of Zn NPs. Following nano TiO\u003csub\u003e2\u003c/sub\u003e treatments also accumulate more photosynthetic activity, enhanced mRNA expression and protein content in spinach (Gao et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Our results showed that chlorophyll and carotene content were increased by increasing the doses of TiO\u003csub\u003e2\u003c/sub\u003e NPs and this reports are agreement with recent study in carrot (Gholami et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, Sohail et al. (\u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that application of Zn NPs drastically improved protein content, total soluble sugar, proline, total flavonoid, and phenolic content in rapeseed. Whereas, Ghafari et al. (2013) suggested that nano-iron oxide (2 g L\u003csup\u003e\u0026minus;1\u003c/sup\u003e) increases chlorophyll, grain protein and iron contents in wheat.\u003c/p\u003e"},{"header":"Conclusion ","content":"\u003cp\u003eIn this study a comprehensive assessment of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs was achieved to target for improvement of seed germination antioxidant activity, nutritional quality and yield of spinach beet. NPs treatments showed the capability to increase seedling growth and many fold volume of antioxidant enzyme in spinach beet. Variable response to different concentrations of nanoparticles was noticed changes of plant phenotypic character in spinach beet. Higher concentration of P and moderate to high concentration of ZnO and TiO\u003csub\u003e2\u003c/sub\u003e NPs improve the plant nutrient (Zn and Fe content) content and quality traits as well as economic yield of spinach beet. Development of nanotechnology paved the way or increase productivity of spinach beet but at the same time it has raised concern for its adverse effect on biological system, food safety and environment system. It needs further study to get answer of question as to what extent molecular and genetic factors may mediate plant response to nano exposure with mitigation of adverse effect on plant development and environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments \u003c/strong\u003eThe authors are grateful to Dr. J. C. Tarafdar, Central Arid Zone Research Institute (CAZRI), Jodhpur, Rajasthan for providing nanoparticles.\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; \u003c/strong\u003e\u003cstrong\u003eContributions:\u003c/strong\u003e (1) Conceptualization: Bimal Das, Surajit Kundu. (2) Methodology: Bimal Das, Dipak Kumar Murmu, Rakesh Yonzone. (3) Data recording: Bimal Das, Dipak Kumar Murmu, Rakesh Yonzone, Surajit Kundu (4) Statistical analysis: Salil Saha. (5) Writing\u0026mdash;original draft preparation: Bimal Das, Rakesh Yonzone, Surajit Kundu. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive funds from any organization\u003cstrong\u003e \u003c/strong\u003eduring this work.\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data recorded during study were presented in this article. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdul Baki A, Anderson JD (1973) Vigor determination in Soybean seed by multiple criteria. 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Plant Science 289:11027\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanoparticles, ZnO, P, TiO2, Spinach beet, Germination, Antioxidant, Yield","lastPublishedDoi":"10.21203/rs.3.rs-1315549/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1315549/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA comprehensive investigation was carried out under laboratory and field by ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs on seedling growth and antioxidant activity, nutrient accumulation, yield traits and biochemical response of spinach beet. Spinach beet variety All Green were treated with sixteen different concentrations nanoscale ZnO (5, 10, 15, 20, 25 ppm), P (10, 20, 30, 40, 50 ppm) and TiO\u003csub\u003e2\u003c/sub\u003e (10, 20, 30, 40, 50 ppm) including untreated control and each treatment has three replicates. TiO\u003csub\u003e2\u003c/sub\u003e NPs treatments found drastic positive impact on seed germination, root and shoot length, seed vigour index and antioxidant enzyme activity (CAT, PAD, SOD and GR). Application of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs improve morphological parameter and that of increase seed yield (gm plant\u003csup\u003e-1\u003c/sup\u003e) with 12.48% 13.01% and 10.93% respectively, compared to control. Study also revealed that zinc and iron content in leaf tissues increases by 50.76%, 7.71%, and 6.41% and 12.69%, 18.49% and 14.17%, respectively of ZnO, P and TiO\u003csub\u003e2\u003c/sub\u003e NPs NPs. Among the biochemical parameter maximum chlorophyll content obtained from 10 ppm TiO\u003csub\u003e2\u003c/sub\u003e, leaf protein and carotene from 50 ppm P, ascorbic acid from 40 ppm TiO\u003csub\u003e2\u003c/sub\u003e, crude fibre from 20 ppm ZnO and total phenol from 30 ppm TiO\u003csub\u003e2\u003c/sub\u003e. Our result clearly demonstrates that application of ZnO, P and TiO\u003csub\u003e2 \u003c/sub\u003eNPs improve the antioxidant enzyme activity, yield and nutritional quality of spinach beet and these nanoparticles can be utilized with their effective doses which may reduce fertilizer cost and environmental pollution.\u003c/p\u003e","manuscriptTitle":"Comprehensive assessment of ZnO, P and TiO2 nanoparticles sustaining environment in response to seed germination, antioxidants activity, nutritional quality and yield of Spinach Beet (Beta vulgaris var. bengalensis)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-24 14:59:10","doi":"10.21203/rs.3.rs-1315549/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6344722d-65f9-4c3d-8df7-3a44c91921ae","owner":[],"postedDate":"February 24th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-03-13T07:50:48+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-24 14:59:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1315549","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1315549","identity":"rs-1315549","version":["v1"]},"buildId":"i_I_jhV-QviBjPLXJga9V","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}