Effect of Hydroxyapatite and Zinc Oxide Nanoparticles on the Germination of Some Seeds

The present study aims to investigate the effects of hydroxyapatite (HA-NPS) and zinc oxide (ZnO-NPs) Nanoparticles on seed germination of four plant species


INTRODUCTION
Nanotechnology has enormous potential uses and benefits. Nowadays tremendous research has been carried out to explore the positive impacts of Nanoparticles on plant growth and development while few studies reported their negative effect. Nanoparticles are atomic or molecular aggregates having size dimensions between 1 to 100 nm. They have diverse and unique physic-chemical properties as compared to other bulk materials (Nel et al., 2006). The Nanoparticles, with their ultra-small size, specific shape, geometric structure, and unique properties, may have the potential for increased toxicity (Arruda et al., 2015).
The present study aims to investigate the effects of hydroxyapatite (HA-NPS) and zinc oxide (ZnO-NPs) Nanoparticles on seed germination of four plant species; radish (Raphanus sativus), tomato (Solanum lycopersicum), wheat (Triticum aestivum L.) and cucumber (Cucumis sativus L.). Seeds were treated with DI-water (control) and nanoparticle suspensions (HA-NPS and ZnO-NPs) solutions, at six concentrations of 10, 20, 50, 100, 200 and 500 ppm. The treatment of HA-NPS showed an inhibition effect on all treated seed germination with significant dose dependence. The inhibition effect was related positively to HA-NPS concentration and was significant at the rate of 50 ppm in radishes and tomatoes, and at 100 ppm in cucumber and wheat seeds. In ZnO-NPs treatment, the inhibition effect was related positively to ZnO-NPs concentration and was significantly started from 20 ppm in radish, tomatoes, and cucumber. Meanwhile, wheat seeds weakly responded to ZnO-NPs inhibition showing a significant effect when applied at 200 ppm. The germination index data (combined seed germination and root elongation), indicated that the response of the tested seeds against Hydroxyapatite Nanoparticles was evident at the highest applied rate (500 ppm), and was as follows: Radish > Tomatoes > wheat > Cucumber. In ZnO-NPs treatment was Radish > Tomatoes > Cucumber > wheat. We can recommend the use of Hydroxyapatite and ZnO-NPs with previous seeds with low concentrations before planting.
Fertilizers are very important for plant growth and development. Most of the applied fertilizers are rendered unavailable to plants due to many factors, such as leaching, degradation by photolysis, hydrolysis, and decomposition (DeRose et al., 2010). Hence, it is necessary to minimize nutrient losses in fertilization and to increase crop yield through the exploitation of new applications with the help of Nanotechnology and Nanomaterials.
The presence of Nanoparticles on the root surface can change the surface chemistry of the roots and consequently affect the uptake of nutrients into the plant root (Mirzajani et al., 2013) thus; these have to be taken into consideration too.
Seed germination and seedling growth are being widely used to test the phytotoxicity of many chemical species such as fungicides which may be released into the environment because it is the crucial stage in plant growth and it is also an important phenomenon in agriculture because it is regarded as the thread of life of plants that ensure its survival (Iqbal et al., 2016).
Seed germination, shoot and root elongation measurements are quite rapid for use on acute phytotoxicity tests with several advantages: sensitivity, simplicity, low cost, and suitability for reactive chemicals and contaminated soil samples (Munzuroglu and Geckil, 2002). Therefore, this study aimed to compare the effect of different concentrations of selected Nanoparticles on seed germination of four plants when used as Nanofertilzers compared to untreated.

Nanoparticles:
Two types of Nanoparticles (hydroxyapatite and zinc oxide) were used in this study.

Preparation of Hydroxyapatite Nanoparticles:
Hydroxyapatite (HA-NPS) was synthesized from calcium hydroxide and orthophosphoric acid. Both chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxyapatite is prepared via the wet-chemical precipitation method as described by Bianco et al. (2007). In the present work, calcium ions react with phosphate ions based on a molar ratio of Ca/P = 1.67. Calcium hydroxide was dissolved in deionized water (Milli-Q, Millipore, USA) under vigorous stirring at 1000 rpm/min for 12 h at 25 °C. The prepared orthophosphoric acid solution was slowly added dropwise into the stirring suspension of calcium hydroxide in distilled water. The precipitated materials were allowed to settle overnight before filtration. The filtered precipitation was rinsed three times by using deionized water and then dried overnight in an oven at 100 °C for 2 h.

Physical, Chemical and Morphological Characterization:
The formation process of HA-NPS crystal was investigated by X-ray diffraction (XRD, X'pert Pro, PanAnalytical, Netherlands) in the 20 range 0° to 80° using CuKα1 radiation (λ=1.54056 Å). The morphology and size distribution of the synthesized HA-NPS powder was characterized with a transmission electron microscope (HR-TEM, Tecnia G20, FEI, Netherlands), operating at 80 kV. An aspect ratio can be defined by the ratio length/width of the HA-NPS Nanocrystals. All preparation procedures and characterization of calcium Nanoparticles were done at the Nanotechnology &Advanced Materials, Central Lab, ARC, Giza, Egypt.

Preparation of zinc oxide Nanoparticles:
Zinc oxide was synthesized by the precipitation method. In a typical procedure, 14.38 g of zinc sulfate heptahydrate was dissolved in 50 ml of deionized water (Milli-Q, Millipore, USA) and then, 4 g of sodium hydroxide in 50 ml of deionized water was added dropwise under magnetic stirring. After the addition was completed, the stirring was continued for 30 min. The precipitates were filtered and washed using pure water several times. Then the precipitates were dried at 60 • C for 24 h and dried at 400 • C for 2 h. The crystalline and phase structure of the synthesized ZnO NPs was studied by an X-ray diffractometer (XRD, X'Pert Pro, PanAlytical, Netherlands). The morphology and size were determined by transmission electron microscopy ((TEM, Tecnai G20, FEI, Netherlands).

Preparation of HA-NPS and ZnO NPs Suspensions:
Weight of 1 g from HA-NPS and ZnO-NPs were suspended individually in 1 liter of deionized water (DI-water) and dispersed by ultrasonic vibration (100 W, 40 kHz) for 30 min to prepare 1000 ppm stock solution for each. The stock solutions were diluted by DIwater to make serial concentrations of10, 20, 50, 100, 200 and 500 ppm. Small magnetic bars were placed in the prepared solution for stirring before use to avoid aggregation of the particles. Germination Assay: Germination assay was carried out according to Yang and Watts (2005). Seeds were immersed in sodium hypochlorite (10%) solution for 10 min to ensure surface sterility, then rinsed three times with DI water. After rinsing, seeds were prepared for assay by soaking in DI water for control treatment and in the suspension of HA-NPS or ZnO-NPs for 2 h., (Kikui et al., 2005). One piece of filter paper was put into a Petri dish (10 mm) and 10 soaked seeds were transferred onto the filter paper and 5 ml of DI-water or test Nanoparticle suspension was added/dish with three replicates for each treatment/concentration. Petri dishes were covered and placed in an incubator for 7 days in the dark at 28±2ºC. After the incubation period, root and shoot length, germination percentage, relative elongation %, relative germination rate and germination index were recorded. The results were compared with the untreated (control) for each seed type.
The relative elongation %, relative germination rate and germination index were calculated as follows: Relative germination rate = Seeds germinated in test sample × 100 Seeds germinated in the control Relative root elongation = Mean root length in test sample × 100 Mean root length in control Germination Index = Relative germination rate × Relative root elongation 100 Statistical Analysis: The statistical analysis was done by using SPSS statistical software (Landau and Everitt, 2004). The results were presented as mean ± SD (standard deviation). Each of the experimental values was compared with the corresponding control. Statistical significance was accepted when the probability of the result assuming the null hypothesis (p) is less than 0.05.

Transmission Electron Microscopy (TEM)
The TEM image of the HA-NPS Nanoparticles is shown in Figure (2). The needlelike morphology of the apatite particles were less than 10-16 nm and a length was 30-55 nm was clearly observed.

XRD Analysis
The XRD pattern of the synthesized ZnO-NPs s is shown in Figure (3 (100)

Characterization of Synthesized Zinc Oxide Nanoparticles 1. TEM Analysis
High-Resolution Transmission Electron Microscopic (HR-TEM) studies were carried out to find out the exact particle size of synthesized ZnO-NPs. TEM images as illustrated in Figure (4) ZnO-NPs which having a particle size in the range of 20-32 nm with nearly spherical shaped particles.

Effects of HA-NPS and ZnO-NPs Suspensions on Seed Germination 1. Effects of HA-NPS
Data in Table (1a) and (1b) showed that HA-NPS suspension decreased the root and shoot length of all treated seeds compared with the control. The results showed an inhibition effect on all treated seed germination with a clear significant dose-dependent. The inhibition effect was related positively to HA-NPS concentration and was significant at the rate of 50 ppm in radishes and tomatoes, and at 100 ppm in cucumber and wheat seeds. The concentration of 500 ppm showed the highest inhibition effect compared to control and significantly decreased root and shoot length of all tested seeds being 4.32, 5.31, 3.02 and5.32 cm for root and 5.25, 3.08, 1.27 and 5.01 cm for the shoot of radish, tomatoes, cucumber and wheat seeds, respectively.
The data revealed that the relative % root or shoot length at the concentration of 500 ppm of HA-NPS recorded 48.16 or 87.06 % for radish, 78.32 or 68.29 % for tomatoes, 84.59 or 61.95% for cucumber, 81.60 or 77.78% for wheat, respectively. The germination index data (combined seed germination and root elongation), indicated that the sensitivity of the tested seeds against Hydroxyapatite Nanoparticles at the highest applied rate (500 ppm) was as follows: Radish > Tomatoes > wheat > Cucumber Values are means of three replicates of each parameter ± standard deviation. Means within each column followed by the same letter are not significant at p > 0.05. * Relative germination rate compared with control **Germination Index = (Relative germination rate × Relative root length)/100 Values are means of three replicates of each parameter ± standard deviation. Means within each column followed by the same letter are not significant at p > 0.05. * Relative germination rate compared with control **Germination Index = (Relative germination rate × Relative root length)/100

Effect of ZnO-NPs Root and Shoot Length
Data presented in Table (2a) and (2b), revealed that ZnO-NPs showed a similar pattern to HA-NPS. ZnO-NPs inhibited root growth and decreased the root length of all treated seeds compared with the untreated. The inhibition effect was related positively to ZnO-NPs concentration and was significantly started from 20 ppm in radish, tomatoes, and cucumber. Wheat seeds were more tolerant effect to ZnO-NPs and were significant when applied at 200 ppm. At 500 ppm showed the highest inhibition effect compared to the control and significantly decreased roots and shoot length of all tested seeds being 1.13, 1.01, 1.02, and 4.1 cm for root and 4.02, 1.61, 1.25 and 5.01 cm for the shoot of radish, tomatoes, cucumber and wheat seeds, respectively.

Relative Root and Shoot Length
The data in Table (2a) and (2b) revealed that the relative % of root or shoot length was correlated negatively with increasing ZnO-NPs applied rate and showed the minimum relative % at the concentration of 500 ppm and recorded 12.60 or 66.67% for radish, 14.90 or 35.70% for tomatoes, 28.57 or 60.98% for cucumber and 62.88 or 94.00% for wheat seeds, respectively. According to the germination index data (combined seed germination and root elongation), the sensitivity of the tested seeds against Zinc oxide Nanoparticles at the highest applied rate (500 ppm), was as follows: Radish > Tomatoes > Cucumber > wheat Values are means of three replicates of each parameter ± standard deviation. Means within each column followed by the same letter are not significant at p > 0.05. * Relative germination rate compared with control **Germination Index = (Relative germination rate × Relative root length)/100 Values are means of three replicates of each parameter ± standard deviation. Means within each column followed by the same letter are not significant at p > 0.05. * Relative germination rate compared with control **Germination Index = (Relative germination rate × Relative root length)/100 Accordingly, it concluded that cucumber and wheat seeds showed the lowest significant response against HA-NPS and ZnO-NPs Nanoparticles treatment compared to radish and tomatoes seed, which might depend on the Nanoparticles type or the plant species (Ngo et al., 2014 andDewez et al., 2005). Figure 5, indicated that (1) ZnO-NPs showed more inhibition effect than HA-NPS on seed germination index except for wheat which showed a converse result. The inhibition effect of HA-NPs on seeds germination/growth may be due to that HA-NPs can reduce the plant uptake of water and element to plant, (Li et al., 2016).
The treatment with HA-NPs with high concentrations (1,000 and 2,000µg/ml) could cause some negative effects on seeds germination/growth as reported by Bala et al. (2014). Also, ZnO-NPs caused a concentration-dependent inhibition of the root length of garlic (Allium sativum L.) when treated with 50 mg L−1 , for 24 h where the root growth was completely blocked, which could be due to the total percentage of abnormal cells increased with the increase of ZnO-NPs concentration and the prolonging of treatment time, (Hossain et al., 2016). The results of ZnO-NPs are in agreement with Shaymurat et al. (2012), who indicated that ZnO-NPs have been shown to induce oxidative stress in soybean (G. max) seedlings at a concentration of 500 mg L −1 . Plant growth, the rigidity of roots, and root cell viability were markedly affected by ZnO-NPs stress. Oxidation-reduction cascade-related genes, such as GDSL motif lipase 5, SKU5 similar 4, galactose oxidase, and quinone reductase were down-regulated in ZnO-NPs treatment.
Nanoparticles are taken up by plant roots and transported to the above-ground parts of the plant through the vascular system, depending on the composition, shape, size of the Nanoparticle, and anatomy of the plant. Some Nanoparticles remain adhered to the plant roots. Toxicity of Nanoparticles may be attributed to two different actions: (1) a chemical toxicity based on the chemical composition, e.g., the release of toxic ions; and (2) stress or stimuli caused by the surface, size and/or shape of the particles (Lin, 2007).
ZnO NPs in the concentration range from 50 to 1600 mg/L are used to stimulate the in vitro germination process of Allium cepa L. and 'Sochaczewska' seeds without negative effects on the further growth and development of seedlings (Tymoszuk and Wojnarowicz, 2020). Nano fertilizers improve crops by enhancing seed germination, shoot and root growth, chlorophyll contents, photosynthesis, abiotic stress tolerance and increasing finally crop yield and quality (Kumar and Bera, 2021).
The low value of nano-scale fertilizers as a tool for reducing the rate of fertilizer input in agriculture, while still maintaining equivalent or even increased yields, compared to bulk-scale fertilizers (Sharma et al., 2022).

Conclusion
The results of experiments vary depending on NPs' type, shape, concentration, and plant genotype. That is relevant for agricultural and horticultural practices related to the stimulation of seed germination as well as plant micropropagation.