Phytotoxicity of Chitosan and SiO 2 Nanoparticles to Seed Germination of Wheat ( Triticum aestivum L . ) and Barley ( Hordeum vulgare L . ) Plants

Plants such as wheat and barley that are strategically important crops need to be considered to develop a comprehensive toxicity profile for nanoparticles (NPs). The present study was aimed to investigate the effects of chitosan and SiO2 NPs on wheat and barley plants. Two factorial experiments (seeds priming and direct exposure) were performed based on a completely randomized design in four replications. Results showed that the seeds priming with the NPs had not significant effect on germination parameters such as Germination Percentage (GP), Germination Rate (GR), Germination Value (GV), Mean Germination Time (MGT), Pick Value (PV) and Mean Daily Germination (MDG). In contrast, exposure of the seeds to the NPs had significant effects on these parameters. In both experiments, treatments had significant effects on shoot, seedling, root length, fresh and dry weight, as well as vigor indexes as compared to the control. In most traits, the best concentration of NPs was 30 ppm, whereas applications of the NPs with 90 ppm displayed adverse effects on majority of the studied traits. According to these results, selectivity in applications of NPs with suitable concentration and method is essential for different plant species.


Introduction
Nowadays, various researchers have studied the effects of nanoparticles (NPs) on plant germination and growth with the objective to promote its use for agricultural applications (Khot et al., 2012).NPs have a high surface to volume ratio that increases their reactivity and possible biochemical activity (Dubchak et al., 2010).The interaction mechanisms at the molecular level between NPs and biological systems are largely unknown (Barrena et al., 2009).Therefore, it is important to understand the course of plant germination and growth in relation to NPs.The reported data from various studies suggested that effect of NPs on seed germination was concentration dependent (Siddiqui et al., 2015).For example, application of 15 mg kg -1 of Fe/SiO2 NPs increased the shoot length of barley and maize; however, the concentration of 25 mg kg-1 had a negative effect on shoot length in barley (Najafi Disfani et al., 2016).Also ZnO NPs was reported to be one of the most toxic NPs that could terminate root growth of plants (cucumber, ryegrass, radish, corn, lettuce, and rape) (Lin and Xing, 2007).
On the other hand, SiO2 NPs are frequently used nanomaterials in a variety of technological applications such as packaging, industrial manufacturing, composite and ceramics materials, adsorption, drug delivery, bio sensing and catalytic applications (Martinez et al., 2010;Ghorbani et al., 2015).The great interest shown to SiO2 NPs is attributed to their low toxicity, high chemical and physical stability, and straightforward surface chemistry, which allows them to be combined or functionalized with a variety of functional species or molecules (Ghorbani et al., 2015).Silicon (Si) is the second most abundant element in soil, which accounts for approximately 32% of the total weight of soil (Abdel Latef and Tran, 2016).It plays an important role as a physic mechanical barrier, and is deposited on the walls of epidermis and vascular tissues of the stem, leaf sheath and hull in most plants especially monocots (Ma and Yamaji, 2006;Parven and Ashraf, 2010).Study on the influence of metal NPs (Si, Pd, Au, Cu) germination of lettuce seeds indicated that NPs (Pd, Au at low concentrations; Si, Cu at higher concentrations, and combination of Au and Cu) had a positive influence on seed germination, shoot to root ratio and growth of seedling (Shah and Belozerova, application of SiO (Glycine max) could increase nitrate reductase, enhance water and fertilizer absorbing capacity, stimulate antioxidant defense system and also hasten seed germination (2004) immersed the roots of changbai larch (Larix olgensis) seedlings in SiO clearly showed positive effects of SiO quality of the seedlings Chitosan is a line produced by de It has great potentials for being used in a wide range of industries such as pharmacology, medicine and agriculture (Bautista-Banos unique properties like biodegradability, antimicrobial activity and nontoxicity (Saharan et al., 2015).Such unique properties of the chitosan biopolymer can be further enhanced by using it in the form of NPs can instill differ physicochemical properties like surface area, size, cationic nature etc. Chitosan has promoted growth of various plants such as soybean sprouts (Lee (Kim et al., 2005) seeds resistance to certain diseases and improve their quality and ability to germinate (Reddy Banu (2016) stated that Ag promoted growth and biochemical variation capabilities of chickpea (Cicer arietinum with chitosan could accelerate their germination and improve their tolerance to stress conditions (Ruan and Xue, 2002).
Therefore, attention was given experimental design to study th SiO2 NPs with two consumption methods on germination of wheat (Triticum aestivum vulgare L.).

Fig. 1. Images of SiO
Study on the influence of metal NPs (Si, Pd, Au, Cu) germination of lettuce seeds indicated that NPs (Pd, Au at low concentrations; Si, Cu at higher concentrations, and combination of Au and Cu) had a positive influence on seed germination, shoot to root ratio and growth of seedling (Shah and Belozerova, 2009).Lu application of SiO2 and TiO (Glycine max) could increase nitrate reductase, enhance water and fertilizer absorbing capacity, stimulate antioxidant defense system and also hasten seed and seedling growth.Also, Bao (2004) immersed the roots of changbai larch (Larix olgensis) seedlings in SiO2 clearly showed positive effects of SiO quality of the seedlings.
Chitosan is a linear β produced by de-acetylation of chitin (Radman It has great potentials for being used in a wide range of industries such as pharmacology, medicine and agriculture Banos et al., 2004 unique properties like biodegradability, antimicrobial activity and nontoxicity (Saharan et al., 2015).Such unique properties of the chitosan biopolymer can be further enhanced by using it in the form of NPs can instill different biological activities with altered physicochemical properties like surface area, size, cationic Chitosan has promoted growth of various plants such as soybean sprouts (Lee ., 2005).It was also report seeds resistance to certain diseases and improve their quality and ability to germinate (Reddy Banu (2016)  Study on the influence of metal NPs (Si, Pd, Au, Cu) germination of lettuce seeds indicated that NPs (Pd, Au at low concentrations; Si, Cu at higher concentrations, and combination of Au and Cu) had a positive influence on seed germination, shoot to root ratio and growth of seedling 2009).Lu et al. (2002) reported that and TiO2 NPs mixture on soybean (Glycine max) could increase nitrate reductase, enhance water and fertilizer absorbing capacity, stimulate antioxidant defense system and also hasten seed and seedling growth.Also, Bao (2004) immersed the roots of changbai larch (Larix 2 NPs for 6 hours.Their results clearly showed positive effects of SiO2 NPs on growth and ar β-(1, 4)-glucosamine polymer acetylation of chitin (Radman It has great potentials for being used in a wide range of industries such as pharmacology, medicine and agriculture . , 2004).Chitosan is known unique properties like biodegradability, antimicrobial activity and nontoxicity (Saharan et al., 2015).Such unique properties of the chitosan biopolymer can be further enhanced by using it in the form of NPs ent biological activities with altered physicochemical properties like surface area, size, cationic Chitosan has promoted growth of various plants such as soybean sprouts (Lee et al., 2005) and sweet basil It was also reported to increase wheat seeds resistance to certain diseases and improve their quality and ability to germinate (Reddy et al., 1999).Anusuya and Banu (2016) stated that Ag-Chitosan NPs significantly promoted growth and biochemical variation capabilities of L.).Similarly, rice seed coated with chitosan could accelerate their germination and improve their tolerance to stress conditions (Ruan and Xue, Therefore, attention was given experimental design to study the effects of chitosan and NPs with two consumption methods on germination Triticum aestivum L.) and barley (

NPs (right) and chitosan NPs (left) by Field Emission
Behboudi F et al / Study on the influence of metal NPs (Si, Pd, Au, Cu) germination of lettuce seeds indicated that NPs (Pd, Au at low concentrations; Si, Cu at higher concentrations, and combination of Au and Cu) had a positive influence on seed germination, shoot to root ratio and growth of seedling .( 2002) reported that NPs mixture on soybean (Glycine max) could increase nitrate reductase, enhance water and fertilizer absorbing capacity, stimulate antioxidant defense system and also hasten seed and seedling growth.Also, Bao-shan (2004) immersed the roots of changbai larch (Larix NPs for 6 hours.Their results NPs on growth and glucosamine polymer acetylation of chitin (Radman et al., 2003).It has great potentials for being used in a wide range of industries such as pharmacology, medicine and agriculture Chitosan is known for its unique properties like biodegradability, antimicrobial activity and nontoxicity (Saharan et al., 2015).Such unique properties of the chitosan biopolymer can be further enhanced by using it in the form of NPs, as in this form it ent biological activities with altered physicochemical properties like surface area, size, cationic Chitosan has promoted growth of various plants ., 2005) and sweet basil ed to increase wheat seeds resistance to certain diseases and improve their quality ., 1999).Anusuya and Chitosan NPs significantly promoted growth and biochemical variation capabilities of Similarly, rice seed coated with chitosan could accelerate their germination and improve their tolerance to stress conditions (Ruan and Xue, Therefore, attention was given to appropriate e effects of chitosan and NPs with two consumption methods on germination L.) and barley (Hordeum NPs (right) and chitosan NPs (left) by Field Emission

Materials
Two factorial experiments were carried out to assess the effects of SiO germinations in a completely randomized design with four replications.The experimental factors included the NPs with concentrations 0 (control, no NPs), 30, 60 and 90 ppm as well as plant species (wheat and barley).T experiment was performed in a germinator with an average temperature of 21 ± 1 °C for 16/8 hours (day/night) in laboratory of the College of Agriculture, Tarbiat Modares University, Tehran, Iran (May, 2016).

Description of materials NPs
Two types of N primary particles size chitosan NPs (Fig. 1) were determined through Field Emission

Plant materials
Seeds of wheat ( barley ( materials for the present investigation.The seeds were purchased from the Plant, Breeding and Seed Institute of Karaj.

SiO
The SiO2 NPs (5 g) were suspended dir water (1,000 mL) and dispersed by ultrasonic vibration (100 W, 40 KHz) for 30 min.Small magnetic bars were placed in the suspensions for stirring before use, to avoid aggregation of the particles (Adhikari suspension for preparing different doses of SiO2 NPs suspensions (0, 30, 60 and 90 ppm).

Chitosan NPs
The chitosan NPs (5 g) was solubilized in deionized water (1,000 mL) with acetic acid (10 mL) under constan stirring chitosan NPs.Then, the Study on the influence of metal NPs (Si, Pd, Au, Cu) on germination of lettuce seeds indicated that NPs (Pd, Au at low concentrations; Si, Cu at higher concentrations, and combination of Au and Cu) had a positive influence on seed germination, shoot to root ratio and growth of seedling .( 2002) reported that NPs mixture on soybean (Glycine max) could increase nitrate reductase, enhance water and fertilizer absorbing capacity, stimulate antioxidant defense system and also hasten seed shan et al. (2004) immersed the roots of changbai larch (Larix NPs for 6 hours.Their results NPs on growth and glucosamine polymer ., 2003).It has great potentials for being used in a wide range of industries such as pharmacology, medicine and agriculture for its unique properties like biodegradability, antimicrobial activity and nontoxicity (Saharan et al., 2015).Such unique properties of the chitosan biopolymer can be further as in this form it ent biological activities with altered physicochemical properties like surface area, size, cationic Chitosan has promoted growth of various plants ., 2005) and sweet basil ed to increase wheat seeds resistance to certain diseases and improve their quality ., 1999).Anusuya and Chitosan NPs significantly promoted growth and biochemical variation capabilities of Similarly, rice seed coated with chitosan could accelerate their germination and improve their tolerance to stress conditions (Ruan and Xue, to appropriate e effects of chitosan and NPs with two consumption methods on germination Hordeum

Materials and Methods
Two factorial experiments were carried out to assess the effects of SiO2 and chitosan NPs on wheat and barley germinations in a completely randomized design with four replications.The experimental factors included the NPs with concentrations 0 (control, no NPs), 30, 60 and 90 ppm as well as plant species (wheat and barley).T experiment was performed in a germinator with an average temperature of 21 ± 1 °C for 16/8 hours (day/night) in laboratory of the College of Agriculture, Tarbiat Modares University, Tehran, Iran (May, 2016).

Description of materials NPs
Two types of N primary particles size chitosan NPs (Fig. 1) were determined through Field Emission-Scanning Electron Microscope (FE

Plant materials
Seeds of wheat ( barley (Hordeum vulgare materials for the present investigation.The seeds were purchased from the Plant, Breeding and Seed Institute of Karaj.

SiO 2 NPs suspension
The SiO2 NPs (5 g) were suspended dir water (1,000 mL) and dispersed by ultrasonic vibration (100 W, 40 KHz) for 30 min.Small magnetic bars were placed in the suspensions for stirring before use, to avoid aggregation of the particles (Adhikari suspension of SiO for preparing different doses of SiO2 NPs suspensions (0, 30, 60 and 90 ppm).

Chitosan NPs suspension
The chitosan NPs (5 g) was solubilized in deionized water (1,000 mL) with acetic acid (10 mL) under constan stirring (80-90 ℃) for 2 h until complete dissolution of the chitosan NPs.Then, the NPs (right) and chitosan NPs (left) by Field Emission-Scanning Electron Microscope (FE 249 ethods Two factorial experiments were carried out to assess the and chitosan NPs on wheat and barley germinations in a completely randomized design with four replications.The experimental factors included the NPs with concentrations 0 (control, no NPs), 30, 60 and 90 ppm as well as plant species (wheat and barley).T experiment was performed in a germinator with an average temperature of 21 ± 1 °C for 16/8 hours (day/night) in laboratory of the College of Agriculture, Tarbiat Modares University, Tehran, Iran (May, 2016).

Description of materials
Two types of NPs were used in the primary particles sizes were > 80 nm.The size of SiO chitosan NPs (Fig. 1) were determined through Field Scanning Electron Microscope (FE

Plant materials
Seeds of wheat (Triticum aestivum Hordeum vulgare cv.'Reyhan') were used as plant materials for the present investigation.The seeds were purchased from the Plant, Breeding and Seed Institute of suspension The SiO2 NPs (5 g) were suspended dir water (1,000 mL) and dispersed by ultrasonic vibration (100 W, 40 KHz) for 30 min.Small magnetic bars were placed in the suspensions for stirring before use, to avoid aggregation of the particles (Adhikari of SiO2 NPs was diluted with deionized water for preparing different doses of SiO2 NPs suspensions (0, 30, 60 and 90 ppm).
suspension The chitosan NPs (5 g) was solubilized in deionized water (1,000 mL) with acetic acid (10 mL) under constan ) for 2 h until complete dissolution of the chitosan NPs.Then, the suspension was alkalized to pH 6.5 ng Electron Microscope (FE Two factorial experiments were carried out to assess the and chitosan NPs on wheat and barley germinations in a completely randomized design with four replications.The experimental factors included the NPs with concentrations 0 (control, no NPs), 30, 60 and 90 ppm as well as plant species (wheat and barley).T experiment was performed in a germinator with an average temperature of 21 ± 1 °C for 16/8 hours (day/night) in laboratory of the College of Agriculture, Tarbiat Modares University, Tehran, Iran (May, 2016).e used in the experiment.Average were > 80 nm.The size of SiO chitosan NPs (Fig. 1) were determined through Field Scanning Electron Microscope (FE-SEM).
Triticum aestivum cv.'Pishtaz') and cv.'Reyhan') were used as plant materials for the present investigation.The seeds were purchased from the Plant, Breeding and Seed Institute of The SiO2 NPs (5 g) were suspended directly in distilled water (1,000 mL) and dispersed by ultrasonic vibration (100 W, 40 KHz) for 30 min.Small magnetic bars were placed in the suspensions for stirring before use, to avoid aggregation of the particles (Adhikari et al NPs was diluted with deionized water for preparing different doses of SiO2 NPs suspensions (0, The chitosan NPs (5 g) was solubilized in deionized water (1,000 mL) with acetic acid (10 mL) under constan ) for 2 h until complete dissolution of the uspension was alkalized to pH 6.5 ng Electron Microscope (FE-SEM) Two factorial experiments were carried out to assess the and chitosan NPs on wheat and barley germinations in a completely randomized design with four replications.The experimental factors included the NPs with concentrations 0 (control, no NPs), 30, 60 and 90 ppm as well as plant species (wheat and barley).The experiment was performed in a germinator with an average temperature of 21 ± 1 °C for 16/8 hours (day/night) in laboratory of the College of Agriculture, Tarbiat Modares experiment.Average were > 80 nm.The size of SiO2 and chitosan NPs (Fig. 1) were determined through Field SEM).
cv. 'Pishtaz') and cv.'Reyhan') were used as plant materials for the present investigation.The seeds were purchased from the Plant, Breeding and Seed Institute of ectly in distilled water (1,000 mL) and dispersed by ultrasonic vibration (100 W, 40 KHz) for 30 min.Small magnetic bars were placed in the suspensions for stirring before use, to avoid et al., 2013).The NPs was diluted with deionized water for preparing different doses of SiO2 NPs suspensions (0, The chitosan NPs (5 g) was solubilized in deionized water (1,000 mL) with acetic acid (10 mL) under constan ) for 2 h until complete dissolution of the uspension was alkalized to pH 6.5 243 Two factorial experiments were carried out to assess the and chitosan NPs on wheat and barley germinations in a completely randomized design with four replications.The experimental factors included the NPs with concentrations 0 (control, no NPs), 30, 60 and 90 he experiment was performed in a germinator with an average temperature of 21 ± 1 °C for 16/8 hours (day/night) in laboratory of the College of Agriculture, Tarbiat Modares experiment.Average and chitosan NPs (Fig. 1) were determined through Field cv.'Pishtaz') and cv.'Reyhan') were used as plant materials for the present investigation.The seeds were purchased from the Plant, Breeding and Seed Institute of ectly in distilled water (1,000 mL) and dispersed by ultrasonic vibration (100 W, 40 KHz) for 30 min.Small magnetic bars were placed in the suspensions for stirring before use, to avoid ., 2013).The NPs was diluted with deionized water for preparing different doses of SiO2 NPs suspensions (0, The chitosan NPs (5 g) was solubilized in deionized water (1,000 mL) with acetic acid (10 mL) under constant ) for 2 h until complete dissolution of the uspension was alkalized to pH 6.5 with 1 N NaOH (Li et al., 2008).Further, the suspension of chitosan NPs was diluted with deionized water for preparing different doses of chitosan NPs suspensions (0, 30, 60 and 90 ppm).

Seeds treatment
Plants culture system and test procedures for two experiments have been adapted from U.S. EPA and ISTA guidelines (USEPA, 1996;ISTA, 2009).In the first experiment, the seeds of wheat and barley were immersed in a 5% sodium hypochlorite solution for 10 min to ensure surface sterility.Then they were soaked in distilled water, SiO2 and chitosan NPs suspension for about 12 hours at 25 ℃, after being rinsed three times with distilled water.Treated wheat and barley seeds were shade-dried for 1 hour.Then the 25 seeds were placed in a Petri dish (100 mm × 15 mm) with one piece of sterilized filter paper (Whatman No. 1) and 5 mL of distilled water was added.Watering was done for all Petri dish.
For the second experiment, the seeds of wheat and barley were immersed in a 5% sodium hypochlorite solution for 10 min to ensure surface sterility.Then seeds after being rinsed three times with distilled water were shade-dried for 1 hour.The 25 seeds were placed in a Petri dish (100 mm x 15 mm) with one piece of sterilized filter paper (Whatman No. 1) and 5 mL of distilled water, SiO2 and chitosan NPs suspension were added to them.Finally, petri dishes for two experiments were covered and placed in a germinator at 21 ± 1 ℃ for 16/8 hours (day/night) for fourteen days.

Measurements
Number of germinated seeds was noted daily for 7 days.Seeds were considered germinated when the radicle showed at least 2 mm in length (ISTA, 2009).On the 14th day, from each Petri dish, 10 seedlings were randomly sampled and morphological parameters including root, shoot and seedling length were measured.Subsequently, they were oven-dried at 70 °C overnight to estimate their dry weight with a sensitive scale (Gubbins et al., 2011).Also, the germination parameters were used according to the following formula: MGT (mean germination time) was calculated based on equation 1 (Matthews and Khajeh-Hosseini, 2007): MGT= (Σ( × ))/Σ Eq. 1 Where F is the number of seeds newly germinated at the time of X, and X is the number of days from sowing.
GR was determined based on equation 2 (Maguire, 1982): Where a, b, c… n are numbers of germinated seeds after 1, 2, 3… N days from the start of imbibition.
Seedling vigor index were computed based on equations 3 and 4 (Vashisth and Nagarajan, 2010): Vigor index I = Germination% × Seedling length (cm) Eq. 3 Vigor index II = Germination % × Seedling weight (g) Eq. 4 Evaluations of MDG, PV and GV were calculated by the following equation 5 (Hartmann et al., 1990): MDG = Germination % / total experiment days Eq. 5 PV = Maximum germinated seed number at one day / day number GV= PV × MDG GP was calculated by the following equation (Hosseini et al., 2013): GP= ∑ ×100 Eq. 6 Where Σn is the number of seeds germinated until the last day of experiments, and N is the total number of seeds.

Statistical analysis
Data were statistically analyzed using two way analysis of variance (SAS Institute, 9.1.3).The significance of differences among treatment means were compared by Duncan's multiple range tests at p < 0.05.

Characterization of chitosan and SiO 2 NPs suspension
Specific surface area of SiO2 NPs was > 80 m2 g-1, average primary particle size was > 80 nm and purity was > 99.5%.The pH of SiO2 NPs suspension was 6.8.Also, specific surface area of chitosan NPs was > 80 m2 g-1, average primary particle size was > 80 nm and purity was >99%.The pH of chitosan NPs suspension was 6.8.

First experiment Germination parameters
Analysis of variance showed that wheat and barley seeds priming with chitosan and SiO2 NPs had not significant effect on GP, GR, GV, PV, MDG and MGT of the seeds (Table 1).Different concentration of SiO2 or chitosan NPs led to 97-100% seeds germination of wheat and barley.Also, analysis of variance showed that main effects and two-way interaction between plant species and NPs had significant effect on vigor index I (Table 1).Pre-treatment of the barley seeds with 60 ppm chitosan NPs demonstrated the lowest value of vigor index I (1,134.24cm), while the highest this value (1,642.33cm) was encountered in 30 ppm chitosan NPs for wheat seeds (Table 2).Wheat seeds priming with 90 ppm of both NPs adversely affected vigor index I as compared to the control (NPs = 0 ppm), but had not significant effect on barley seeds.Using of 30 ppm of both NPs in barley seeds significantly increased vigor index I as compared to the control.Also, analysis of variance showed that plant species significantly affected the seedling vigor index II (Table 1), among which barley showed the maximum value (Fig. 2).

Seedling, shoot and root lengths
Results in Table 3 showed that shoot, root and seedling elongation of wheat and barley were significantly affected by main effects, and tow-way interaction.Wheat seeds priming with 30 ppm chitosan NPs led to relatively highe increase in seedling and root length (Table 2).In contrast, wheat seeds priming with SiO2 NPs significantly decreased seedling and root length.The highest shoot length (9.77 cm) was observed at barley seeds priming with 30 ppm chitosan NPs.The lowest shoot and seedling length was obtained in 60 ppm chitosan NPs in barley seeds.Wheat seeds priming with 30 ppm chitosan NPs was led to the highest root length (8.20 cm).

245
Seedling, shoot and root weight Seedling, shoot and root fresh weight, as well as shoot dry weight, were significantly affected by two-way interaction between plant species and NPs (Table 3).Also, plant species had a significant effect on seedling dry weight.Treatments had not significant effect on root dry weight of barley and wheat plants.Use of both NPs in 90 ppm greatly decreased shoot, root and seedling fresh weight of both plants as compared to the control (Table 2).Whereas, use of both NPs in low concentration had not significant effect on fresh weight and shoot dry weight as compared to the control.The lowest shoot dry weight was observed in wheat seeds priming with 90 ppm SiO2 NPs.Also, seedling dry weight in barley was more than wheat (Fig. 3).

Second experiment Germination parameters
Analysis of variance showed that the main effects and two-way interaction between plant species, and NPs significantly affected all measured traits (Table 4).Results in Table 5 showed that usage 90 ppm SiO2 NPs in barley seeds significantly decreased GP, GR, MDG and seed vigor index II as compared to the control.Also, exposure of barley seeds to relatively high concentration of either SiO2 or chitosan NPs significantly led to diminished GV, PV and vigor index I.In contrast, application of chitosan NPs with low concentration in wheat seeds led to the height GV (46.96),PV (1.93) and vigor index I (1,877.87cm).In general, low concentration of both NPs had not significant effects on most studied traits.On the other hand, as a general rule, lower MGT represents a faster germination speed.Exposure of barley seeds to 90 ppm SiO2 NPs demonstrated the highest MGT (2.50 day).
Seedling, shoot and root length Analysis of variance showed that two-way interaction between plant species and NPs was significant for seedling, shoot and root length (Table 6).Barley seeds exposed to both NPs exhibited the sharp decline in length compared to the control (Table 7).The lowest seedling, shoot and root length was observed when barley seeds treated with 90 ppm chitosan NPs.Low concentration of both NPs led to increasing wheat seedling length.
Seedling, shoot and root weight Analysis of variance showed that two-way interaction between plant species and NPs was significant for seedling, shoot and root weight of barley and wheat plants (Table 6).Using both NPs in wheat seeds had not significant effect on seedling, shoot and root fresh weight as well as seedling and root dry weight (Table 7).Whereas high concentration of both NPs significantly decreased dry and fresh weight of barley seeds as compared to the control.The lowest seedling,

Discussion
Type, concentration and consumption methods of NPs play an important role in the behavior, in reactivity and in toxicity of plants.Seed germination and root elongation is widely used as acute phytotoxicity test with several advantages like sensitivity, simplicity, low cost and suitability for unstable chemicals or samples (Wang et al., 2005).In seeds priming with the NPs, there were no toxicity effect on seeds germination parameters.This could be due to the incompatibility between the priming time and permeability of the NPs through seed coat, which could have prevented easy entrance of the latter in the seed coats.Also, in seeds priming, vigor indexes were affected by NPs, that is due to NPs effect on length and weight of seedling.Direct exposure of the seeds to low concentration of NPs (30 ppm) increased some germination parameters that may be due to the absorption and utilization of NPs by seeds (Suriyaprabha et al., 2012).In contrast, in high concentration of NPs (90 ppm), possibly NPs did not fully penetrate the seed coat and endosperm and thus had limited effects on the embryos.Therefore, NPs might find the seed coat and endosperm to be effective barriers, especially when they are agglomerated (Duke and Kakefuda, 1981).These results are supported by similar data of Adhikari et al. (2013), Azimi et al. (2014), Kananont et al. (2010) and Shao et al. (2005).
Furthermore, the toxic effects of NPs in high concentrations were more pronounced in seedling, shoot and root length, especially in wheat seeds priming with SiO2 NPs, as well as direct exposure of barley seeds to the NPs (Table 8).In contrast, seeds priming with 30 ppm NPs (except wheat seeds priming with SiO2 NPs) gave the highest seedling and root length of the corresponding plants, as well as direct seed exposure of wheat seeds to 30 ppm NPs (Table 8).Different morphological effects depend on the nanomaterial type, size, concentration, surface properties, consumption methods, chemistry, crystallinity, agglomeration state and plant species (Casals et al., 2008;Rico et al., 2011).Also, root elongation of plant species would have a dose dependent response (Sresty and Rao, 1999).Therefore, radicles, after penetrating the seed coatings could contact the NPs directly.The presence of NPs in large quantities on the root surface could alter the surface chemistry of the root and clog the root openings and both hydraulic and nutrient uptake in roots is inhibited.Therefore, plant growth is negatively affected because of NPs.Siddiqui and Al-Whaibi (2014) found that SiO2 NPs inhibiting root elongation of tomato at high concentration, whereas, in middle concentration of NPs (8 g.L -1 ) increased root elongation as compared to the control.Also, seeds priming with chitosan solution (0, 0.01%, 0.05%, 0.1%, 0.2% and 0.5%) had not significant effect on shoot length of ajowan (Carum copticum), but increased root length as compared to the control (Mahdavi and Rahimi, 2013).
On the other hand, using of 90 ppm NPs in both experiments decreased seedling, shoot and root weight especially in wheat seeds priming, as well as direct exposure of barley seeds to NPs (Table 8).A decrease in biomass beyond such concentrations suggested toxic effects of NPs.It might be probable that increasing the concentration of both NPs could have induced aggregation of particles and resulted in clogging of root pores that interrupted water uptake et al., 2009).Saharan et al. (2016) found that Cu-chitosan NPs at low concentration had positive effect on fresh and dry weight of maize, whereas in high concentration showed inhibitory effect on seedling growth.In the case of tomato seeds, Haghighi et al. (2012) found that high concentration of nano-silicon (2 mM) decreased dry weight of tomato compared to the control and low concentration.Moreover, chickpea seeds treated with chitosan NPs showed positive morphological effects such as enhanced percentage germination, seed vigor index and vegetative biomass of seedlings (Sathiyabama and Parthasarathy, 2016).

Conclusions
The effect of NPs on plants is complex and many factors can affect plant growth and germination.In the current study, both positive and negative effects of NPs were observed for wheat and barley plants respectively.Using SiO2 and chitosan NPs at low dosage could promote seed germination and growth parameters of both crops, while these parameters decreased following an exposure to high concentrations of both NPs.Therefore, the results obtained in the two experiments suggest that the micro nutrients, SiO2 and chitosan, can be delivered into seeds of wheat and barley through NPs in suitable concentration and methods.
stated that Ag promoted growth and biochemical variation capabilities of Cicer arietinum with chitosan could accelerate their germination and improve their tolerance to stress conditions (Ruan and Xue, Therefore, attention was given experimental design to study th NPs with two consumption methods on germination Triticum aestivum Images of SiO 2 NPs (right) and chitosan NPs (left) by Field EmissionBehboudi F

Fig. 2 .
Fig. 2. Effect of plant species on vigor index II.Means by the uncommon letter are significantly different according to Duncan tests (p < 0.05)

Fig. 3 .
Fig. 3. Effect of plant species on seedling dry weight.Means by the uncommon letter are significantly different according to Duncan tests (p < 0.05)

Table 1 .
Analysis of variance for some related germination parameters of barley and wheat

Table 2 .
Effects of SiO2 and chitosan NPs on wheat and barley growth parameters Barley VI, L, F.W, D.W show vigor index, length, fresh weight, dry weight, respectively.Means by the uncommon letter in each row and column are significantly different according to Duncan tests (p<0.05)

Table 3 .
Analysis of variance for some growth parameters of barley and wheat .W, D.W show vigor index, length, fresh weight, dry weight, respectively.ns Non-significant, ** and * significant at the 1% , 5% probability levels, respectively

Table 4 .
Analysis of variance for some germination parameters of barley and wheat

Table 5 .
Means comparison the influence of application SiO2 and chitosan NPs on germination parameters of wheat and barley

Table 7 .
Means comparison the effect of SiO2 and chitosan NPs on growth parameters of wheat and barley