Evaluating Positive and Negative Effects of Seven Biogenic Metal-based Nanoparticles on Seed Germination and Seedling of Nano-primed Wheat and Flax Seeds

Seed germination is the rst and the most susceptible stage in plant’s growing phases, so could be considered as an index to evaluate the effect of newly developed materials like nanoparticles (NPs), providing useful information for researchers. In our experiments, germination tests have been carried out in Petri plates, containing wet lter paper and nano-primed seeds. We had biosynthesized seven nanoparticles in our previous researches, including calcinated and non-calcinated zinc oxide, zinc, magnesium oxide, silver, copper and iron nanoparticles. The effect of these biogenic nanoparticles and their counterpart metallic salts including zinc acetate, magnesium sulphate, silver nitrate, copper sulphate and iron (III) chloride was studied on two popularly grown plants, wheat and ax, in laboratory condition to obtain preliminary information for future eld experiments. Germination percentage, shoot length, root length, seedlings length, root-shoot ratio, seedling vigor index (SVI), shoot length stress tolerance index (SLSI) and root length stress tolerance index (RLSI) were calculated at 2 nd and 7 th days of the experiment. According to the results, the response of the plants to metal containing nanoparticles and metal salts mainly depend on type of the metal, plant species, concentration of the NP suspension or salt solution, condition of the exposure and the stage of growth. in These nanoparticles showed both promoting and inhibition effects on Moreover, the inuence of metal salts, which are used as the precursors of the NPs during their biosynthesis, is compared with their counterpart NPs at the same concentrations. To the best of our knowledge, this report is the rst to compare the positive and negative effects of these NPs on seedling parameters.


Introduction
The rapidly growing population and crop consumption cause a high demand of using fertilizers, which are critical for plant growth and improving the crop yield. In general, conventional mineral fertilizers are soluble salts, which easily dissolve in the soil media for plant uptake. However, a large portion of these soluble salts leach to the water resources, resulting in eutrophication and nutrient loss in fertilization. Solid forms of insoluble fertilizers also have been applied but the micronutrients are not so free to be easily bioavailable and easily transport to the water resources. Although, when the plants are in need, these solid minerals are less effective in supplying micronutrients in time due to their large size. To solve these problems, application of nanoparticles could be helpful hypothetically in both providing the micronutrients, minimizing the environmental contamination risks of soluble fertilizers and the problem of less bioavailability of solid fertilizers which is more environmentally benign (Singh et al. 2018;Liu et al. 2014). As the dimensions of materials reduce from a large size below 100 nm, signi cant changes in characteristics can occur mainly due to an increase in relative surface area (per unit mass) and then an enhanced chemical reactivity (Al-Hakkani 2020).
In recent years, a large number of reports have analyzed the in uence of various nanostructures on different crops especially on their early developmental stage, seed germination and seedling development. Seed germination, i.e., the emergence of the radicle and primary root's elongation, is considered as the most sensitive stage of a plant life cycle. Priming with nanoparticle (nano-priming) could lead to positive, negative, or no impacts on germination process, depending on NPs type, size, concentration, duration of the exposure, or the growing conditions. Exact outcomes of the seed priming also consider as a technique to control the hydrate content of the seeds, stimulating metabolic activities for germination (Salah et al. 2015;Szollosi et al. 2020). In priming with NPs, rst, the NPs have to penetrate the sclereids barrier of seed coat. Several studies revealed that NPs reach to the plant cells by crossing the intercellular spaces, bounding to a carrier protein, through aquaporin, ion channels, bounding to organic materials or endocytosis via making new pores. The NP-plant interaction may result in morphological and physiological changes, depending on characteristics of NPs (Singh et al. 2018;Aslani et al. 2014). It is also con rmed that some metal-based NPs can cross the seed coat and stimulate the embryonic differentiation through inducing the enzymes which interrupt seed dormancy. NPs may translocate to the other plant parts and cause various structural or functional changes in those parts (Alam et al. 2015). Previous reports also suggested different biochemical mechanisms for positive effect of nanomaterials upon NP exposure such as increased water uptake, remodeling of membrane lipids in seeds enhanced sugar metabolism and energy production, and the stimulated antioxidant defense (Szollosi et al. 2020). The impact of NPs on seed germination is related to their capability to enter the embryonic tissues through the seed coat. This capability is mostly related to the structure of seed coat and differs upon each plant species, physical and chemical characteristics of the ambience (Ko et al. 2017).
There is also another reason for evaluation of NP-plant interactions. Up to now, the amount of NPs existed in the environment is really lower than their toxic concentration. However, as NPs are widely commercialized, the potential biological impacts of NPs should be carefully assessed. As NPs have the potential to nd their pathway into the environment and plants continuously interact with soil, water and air, these NPs may penetrate and translocate into the plant (Yang et al. 2017;Rastogi et al. 2017). In this regard, there will be a need to study the impact of NPs on plants.
Metals generally affect seed germination process, biochemical and physiological pro les and plant growth. Essential metals such as Zn, Mg, Cu and Fe are crucial for living cells and their de ciency could lead to damages in cell wall and DNA. Nevertheless, the excessive amounts of these metals or presence of non-essential metals (e.g., Ag) could be toxic due to causing oxidative stress, stimulating loss of membrane integrity, and injuring to proteins and DNA in a phytotoxic manner (Rai et al. 2017).
The objective of this study encompasses assessment potential impact of seven metal-based nanoparticles, which we have biosynthesized, characterized and applied in our previous works (Bayat et al. 2021a;Bayat et al. 2021b;Bayat et al. 2019), on seedling and seedling growth of two popular plants of wheat and ax though a laboratory study. Calcinated zinc oxide (C-ZnO), non-calcinated zinc oxide (NC-ZnO), zinc (Zn), magnesium oxide (MgO), copper (Cu) and iron (Fe) include essential metals which are vital for plant growth. Ag NP is selected due to its extensive use in industry and it could reach to the plants by surface water. Essential and nonessential elements might be absorbed by plant and according to their concentration, may result in toxicity. The effect of NPs also studied in some earlier reports. These nanoparticles showed both promoting and inhibition effects on plants growth (Vanninia et al. 2014;Asanova et al. 2019;Gorczyca et al. 2018). Moreover, the in uence of metal salts, which are used as the precursors of the NPs during their biosynthesis, is compared with their counterpart NPs at the same concentrations. To the best of our knowledge, this report is the rst to compare the positive and negative effects of these NPs on seedling parameters.

Materials And Methods
The plant extraction method, synthesis and characterization of applied NPs are described in our previous works (Bayat et al. 2021a;Bayat et al. 2019). In summary, dried strawberry leaves boiled in distilled water, ltered and mixed with NPs precursors. NPs generated by reduction of 0.01M precursor salt's solutions under heating, continuous stirring and addition of the extract drop by drop. Produced NPs washed with distilled water after centrifuge and dried at room temperature. C-ZnO and MgO NPs calcinated in furnace at 500 o C for 4h. The biosynthesized NPs speci ed using different characterization techniques including UV-Vis Spectroscopy, XRD, FESEM, EDS, Photon Cross-Correlation Spectroscopy (PCCS) and FT-IR. Biosynthesized NPs, their counterpart precursors, their average sizes and shapes are listed in Table1.

Preparation of seeds
Seeds of wheat (Triticum aestivum L.) variety Firuza 40 and ax (Linum usitatissimum) variety Semi Lini were used in this experiment. Each treatment consisted 30 randomly selected seeds with three replications. Seeds were kept in dry place at room temperature prior to use.
(a) Preparation of wheat seeds: Viability of seeds checked visually and then by suspending in distilled water, discarding the seeds oating above were and selecting seeds settled at bottom of the water for further experiment. Seeds immersed in a 5% sodium hypochlorite solution and rinsed with distilled water after 10 minutes, for surface sterility of the seeds (USEPA 1996). Then seeds soaked in a prepared NPs suspensions or metal salt solution for 12 hours. A set of seeds was soaked in distilled water without providing any treatment as control.
(b) Preparation of ax seeds: Seeds were checked visually for removing damaged seeds from the samples. Then seeds soaked in NPs suspensions or metal salt solutions for 12 hours. A set of seeds was used without providing any treatment as a control and soaked in distilled water.
In vitro germination of seeds One piece of lter paper was placed into a Petri plate (10 cm in diameter), and for wetting the paper, 5 ml distilled water was added using a Pasteur pipette.
Then 30 nano-primed seeds were transferred onto each lter paper. The Petri plates incubated at room temperature for seven days.

Measurement of physiological indexes
Germination percentages, shoot length, root length, seedlings length, root-shoot ratio, seedling vigor index (SVI), shoot length stress tolerance index (SLSI) and root length stress tolerance index (RLSI) were calculated at 2 nd and 7 th days. Means and standard deviations were derived from measurements on three replicates for each treatment and controls. A seed was considered germinated after the emergence of radicles or plumules from the seed coat (Ahmed et al., 2019). The length of roots and shoots were measured using a ruler with centimeter and millimeter scale ( g. 1).
(a) Shoot and root length: At 2 nd and 7 th days of the experiment, 10 seedlings from petri plate randomly selected to measure shoot and root lengths (Rawat et al. 2018).
(b) Seedling length: is considered as the sum of shoot length and root length of a seed (Rawat et al. 2018).
(c) Germination percentage: The germination percentage was calculated based on the total number of germinated seeds at the day of the experiment.
Germination percentage calculated using the following equation (Raskar et al. 2013): Germination percentage (%) = (average number of germinated seeds/total number of seeds) × 100 (d) Root/Shoot Ratio: The root to shoot ratio for each seedling was calculated as follows (Raskar et al. 2013): Root/Shoot Ratio = average root length/ average shoot length SLSI (%) =average shoot length of treated seedlings/average shoot length of control seedlings ×100 (g) Root length stress tolerance index (RLSI) calculated as follow (Ahmed et al. 2019;Raskar et al. 2013): RLSI (%) =average root length of treated seedlings/average root length of control seedlings ×100

Statistical analysis
The obtained data statistically analyzed using Microsoft Excel software (version 2019), SAS and MSTAT-C statistical programs. One-way analysis of variance (ANOVA) applied for performing statistical analysis and p-value <0.05 considered as signi cant. Mean comparison performed by Least Signi cant Different (LSD) test. Means and standard deviations obtained from measurements on three replicates for control and each treatment.

Results
In the present research, we carried out experiments to compare the effect of our synthesized biogenic NPs (C-ZnO, NC-ZnO, Zn, MgO, Ag, Cu and Fe) and their counterpart metallic salts (Zn(CH 3 COO) 2 , MgSO 4 , AgNO 3 , CuSO 4 and FeCl 3 ) on germination and seedling growth of wheat and ax by considering their effect on different parameters including germination percentage, shoot length, root length, seedlings length, root/shoot ratio, seedling vigor index (SVI), shoot length stress tolerance index (SLSI) and root length stress tolerance index (RLSI). Three different concentrations of 50, 100 and 150ppm were used for seed priming. These concentrations were selected considering previous reports (Gorczyca et al. 2018;Younes et al. 2020). Wheat and ax plantlets were grown In vitro and all the observations were recorded up to 7 days. Results are tabulated in Tables 2 to 9. We observed that plant growth parameters of wheat and ax, varied considerably among the plants and also different priming solutions with various concentrations. Moreover, both effects of "stimulation" and "phytotoxicity" and in some cases no signi cant effect of NPs and their counterpart salts was observed on germination and seedling growth. The p-values less than 0.05 indicate there is a signi cant difference within the results.

Effect of biogenic NPs and their counterpart salts on physiological characteristics of wheat seedling
Tables 2-5 summarize the effect of priming with biogenic NPs and their counterpart metal salts (precursors) on seed germination parameters of wheat on 2 nd and 7 th day.

Germination percentage
The data showed that exposure to different concentrations of biogenic NPs resulted in an increase in germination percentage (G%) of all the treatments over the control at 2 nd day ( Table 2). The maximum G% is related to the seeds primed with Zn NP at concentration of 150 and then 100ppm i.e., 98 and 96% respectively. Vise a versa, a decrease in G% occurred in samples treated with metal salts, comparing to the control, except the sample primed with 50ppm of zinc acetate in which G% was similar to the control (Table 4).
At 7 th day, an increase was observed in most of the samples and the highest G% was related to the concentrations of 50 and 150ppm of Zn NPs and also 50ppm of Cu NPs treated seeds (Table 3). Interestingly, for the samples treated with metal salts, the G% values are similar or close to the control except to the seeds primed with iron chloride, which showed a concentration dependent increase (Table 5). Among all of the applied NPs, Zn NPs found to be more effective in developing the seed germination of wheat seeds.

Shoot length and SLSI
At 2 nd day of the experiment, the best results of shoot elongation were related to the seeds treated with NC-ZnO NPs among all of the used NPs (Table 2) and also the maximum SLSI caused by 50 and 100ppm of NC-ZnO and 50ppm of Fe NPs treatments (about 115 to 120%). In the case of priming with salts (Table  4), the shoot elongations and SLSI values were less than the control, except 100ppm zinc acetate which was close to the control. Ag NPs induced a signi cant decrease in shoot length and SLSI, both in 2 nd and 7 th day. According to the Tables 3 and 5, the maximum shoot lengths and SLSI at 7 th day are related to the concentrations of 50ppm of Zn and Fe, and also 50 and 150ppm of Cu NPs (108 to 112%). In salt primed samples, just priming with 100ppm of FeCl 3 had a positive effect on SLSI (125%) and no other signi cant increase was observed. There were also decrease of shoot length in CuSO 4 primed samples.

Root length and RLSI
According to the Tables 2&4, at the 2 nd day of the test, the most signi cant promoting effect on root length and RLSI is related to the soaking with NC-ZnO NPs. Root lengths of the Zn NPs treated seeds were similar to the control and the other NP treatments had an inverse effect on root elongation. Considering the results of the priming with salts, the increase in root lengths and RLSI observed in zinc acetate treated seeds, also in 150ppm of MgSO 4 and AgNO 3 priming and also in 50ppm of CuSO 4 treatments. For seeds treated with FeCl 3 , 50 ppm MgSO 4 and 100ppm CuSO 4 , a signi cant decline was indicated.
In 7 th day (Table 3&5), there were a signi cant improvement in root length for all of the NP-primed seeds, except Ag NP-primed samples. NC-ZnO had the best effect on root length development and Ag NP showed a dose dependent inhibition effect on root lengths. For metal salt priming cases, the best results in root elongation and RLSI were related to the 100 and 150ppm of FeCl 3 and then 100 and 150ppm of zinc acetate priming. For the other samples there were a decrease in root length, mainly in CuSO 4 primed seeds.

Seedling length
In 2 nd day (Tables 2&4), for Ag primed seeds, there were a remarkable decrease in seedling length. Soaking with 100&150ppm NC-ZnO and 50&150ppm Zn NPs exhibited an increase in seedling development. Additionally, zinc acetate was the most effective salt in improving seedling length and CuSO 4 was the most toxic one. For other samples the results were similar to the control or just a little lower than the control.
In 7 th day (Tables 3&5), for all NP treatments we observed seedling length improvement in comparison with the control, except Ag NP treated seeds. The maximum effect obtained with 150ppm C-ZnO, 100ppm NC-ZnO and 150ppm MgO NPs (about 25% more than the control).
For metal salt primed seeds, application of CuSO 4 , AgNO 3 and MgSO 4 led to a notable inhibition of seedling growth respectively. FeCl 3 treatments, had the best improving effect in a dose dependent manner.
Root/soot ratio (R/S) Under speci c conditions, higher proportion of roots can help plants to compete more e ciently for water uptake and soil resources, while a higher proportion of shoots can help plants to collect more light energy (Allaby, 2006). In 2 nd day (Table 2&4), all of the samples had the R/S of more than 1, indicating root lengths longer than shoot lengths and the values were similar to the control or less than that, except the Ag NP treatments. In Ag NP primed seeds, a great improvement in R/S observed mainly at the concentration of 100ppm which was about 1.5 times more than its respective control and the minimum was related to the 50ppm Fe NP which was 0.3 times lower than the control. For metal salt treatments, AgNO 3 showed the results close to the Ag NP treatments, zinc acetate had the most effect in R/S values and CuSO 4 had a dose dependent decrease. Overall, the R/S in salt treated seeds was more than NP treated ones.
In 7 th day, R/S ratios of the NP treated seeds are higher than the control, except 150ppm Ag treated one and the maximum was for 150ppm C-ZnO NP treatments (Table 3). For salt priming, the amounts are less than NP treated seeds and just 100&150ppm zinc acetate and 110&150ppm FeCl 3 root to shoot ratios were higher than the control (Table 5).

SVI
For evaluating the effect of metal NPs or metal salts on seedling growth, the seedling vigor index (SVI) could be used as a phytotoxicity index (Zhao et al. 2016). In 2 nd day, Ag NP priming resulted in minimum values of SVI. NC-ZnO and Zn NPs had values higher than the control and in salt treated seeds SVIs were less than NP treated seeds. The minimum amounts are due to the CuSO 4 and FeCl 3 treatments. In 7 th day, for NP treatments, all SVI amounts were considerably more than the control. The maximum SVI observed in 150ppm of C-ZnO, 50&150ppm of Zn NP and 150ppm of MgO NP primed samples. For salt treatments, 100&150ppm of zinc acetate and 150ppm of FeCl 3 solutions had the maximums SVIs. Besides, CuSO 4 and AgNO 3 showed the most inhibition effect.

Effect of biogenic NPs and their counterpart salts on physiological characteristics of ax seedling
Tables 6-9 summarize the effect of priming with biogenic NPs and their counterpart metal salts (precursors) on seed germination parameters of ax on 2 nd and 7 th day.

Germination percentage
Considering the results of the Tables 6&8, in early stages of seedling, the maximum seed germination percentage (90%) was related to the ax seeds soaked with 100ppm C-ZnO NPs and all of the other treatments showed G% similar or less than the control. The minimum G% was related to the seeds soaked with 100ppm of Cu NPs. Among metal salt, zinc acetate had improving effect on G% and except 100ppm of CuSO 4 and FeCl 3 primed seeds, no increase in G% was observed with respect to the control. At 7 th day of the experiment (Tables 7&9), just 150ppm of C-ZnO NPs suspension had improving effect over the control and all of the other NP treatments showed inhibition effect. Among salts, G% of the 150ppm of MgSO 4 treated seeds was close to the control and the others were less than the control. 150ppm of AgNO 3 had such a severe toxic effect so no gemination was observed and there are not any data reported in following for this case.

Shoot length and SLSI
According to the Tables 6&8, at 2 nd day of the test, all of the NP treatments showed enhancement in shoot length over the control. The most effective NP was NC-ZnO and the less effective one was Ag. The most effective salts in improving shoot length were MgSO 4 and then zinc acetate. 50ppm of AgNO 3 was also very effective in this regard and 100ppm concentration of AgNO 3 and CuSO 4 had the worst effect on shoot elongation. Tables 7&9 record the results of the 7 th day of the experiment which show that the most effective NP in shoot length development of ax seeds was Ag.
MgSO 4 and FeCl 3 were the most effective in shoot length development and 100ppm AgNO 3 was the most toxic one, completely inhibiting the germination of seeds.

Root length and RLSI
Flax seeds responded differently toward the treatment at various concentrations of NPs. At 2 nd day (Table 6&8), Zn NPs had the best effect on root growth. Ag and Fe NPs had the most inhibition effect. MgSO 4 was the most effective salt in root length development and CuSO 4 was the most toxic one. At 7 th day of the experiment, a signi cant increase in root length observed in most of the treatments, especially in Zn NPs treated samples and the root lengths were about 2 to 3 times higher than the control which is considered as a great positive effect. Cu 100ppm besides 50 and 150ppm concentrations of Fe NPs showed a notable toxic effect on root length parameter. Zinc acetate and MgSO 4 solutions induced a signi cant root length increase of about twofold over the control. 100 and 150ppm of CuSO 4 priming inhibited the root growth to the lengths about 27% of the control.

Seedling length
Like root and shoot length, the best results of seedling length is related to Zn NPs treated samples. After 48h (Tables 6&8), Ag and Fe had seedling lengths less than the control. For AgNO 3 primed samples, 50ppm concentration resulted in maximum seedling length and 100ppm resulted in no seedling. Moreover, MgSO 4 showed the best increasing effect in seedling length. After the rst week (Tables 7&9), in the case of nano-primed seeds, except Cu and Fe primed samples which had seedling lengths less than the control, the other treatments had seedling lengths more than the control. Zinc acetate and MgSO 4 were most effective in increase of seedling length and the minimum was related to the 100ppm AgNO 3 treatments.

Root/Shoot ratio
In early stages of ax seedling, the shoot grew with a higher speed in comparison with root. Considering the results of the Tables 6&8, at 2 nd day, all of the R/S values were less than the control in NP and salt primed samples. At 7 th day, except Fe NPs and 100-150ppm Cu NPs primed seeds, the other samples had R/S more than the control. 100 and 150ppm of CuSO 4 (like Cu NP) and 100ppm of FeCl 3 treated seeds had R/S less than the control.

SVI
At 2 nd day of the experiment, maximum SVI was related to the 150ppm C-ZnO and 50ppm of Zn NPs due to their high seedling length values and the minimum was related to the 100ppm of Cu NPs primed samples. At 7 th day, Fe NPs primed samples had the minimum SVIs due to their minimum seedling lengths. MgSO 4 primed samples had the maximum SVIs and 50ppm zinc acetate and 100ppm AgNO 3 had the minimum SVIs. Effect of biogenic NPs and their counterpart salts on physiological characteristics of wheat seedling As seed germination is the rst step to start a successful crop improvement, it could be considered as an index to assay the enhancive or inhibitive effect of newly developed agrochemicals such as nanomaterials (Ahmed et al., 2019).
In present study, the seeds were exposed to the NPs or salts only for 12 hours but the priming effect was observed up to several days. In this regard, it could be suggested that the NPs or metal ions are absorbed on the surface of the seeds and gradually release to show their effect during a period of seven days. Also, the reason of difference between response of nano-primed seeds with salt-primed ones may be illustrated as the result of gradually release of ions from NPs by sub-toxic levels rather than the exposure to a large number of ions in the case of priming with metal salts which may cause stress in the germination process (Szollosi et al. 2020).
Previous ndings have reported that the zinc nanoparticulate priming were more effective than zinc salt in enhancing the seedling growth. For instance, it is found that Zn NP treated wheat seeds surpassed elemental Zn values over ZnSO 4 , indicating that NPs are more e cient at delivering Zn to plant tissues than ZnSO 4 , which suggests it is done during a particle-speci c mechanism (Baddar et al. 2018). Similar studies have shown that accumulation of Zn from NP treatment was more than predicted values upon dissolved Zn concentration (Ahmed et al. 2019). However, Zn is an essential metal for plant growth, it may be a phytotoxic metal when exceeds the tolerance limit depending on the plant species or plant's studied part (Zaeem et al. 2020).
Previous studies also reported similar results to our ndings, on seedling growth of ZnO treated wheat seeds (Rawat et al. 2018;Awasthi et al. 2017). In the study conducted by Ahmed et al., C-ZnO NPs with very high concentrations of 0.05, 0.5, 2, 5 mg/ml were applied on four different seeds such as radish, cucumber, tomato and alfalfa to study the toxicity effect of NPs on seeds. They reported that C-ZnO exhibited no obvious toxic effect on germination, root and shoot growth of these seeds (Ahmed et al. 2019). Similarly, in our study C-ZnO and NC-ZnO NP treatment improved the seedling growth. Several mechanisms could be found in the literature illustrating various effects of NPs on plant parts and cell reactions. For example, the effect of NPs on speci c enzymatic reactions and different enzymes such as amylase could elucidate NPs effect on seed germination. It is not clear at this point whether NPs toxicity is stimulated by particles or the dissolved ions (Ko et al. 2017). The effect of NPs may also be due to the interaction of NPs with some parts of the plants such as cell wall or membrane components. The size of the NPs is consistent with structure of the plant cell wall to enter the cell, at the point that the accumulation of reactive oxygen species (ROS) can be started (Zaeem et al., 2020). ROS can in uence the permeability of the cells as it is interfered with the plasma membrane. Consequently, more NPs can result in intense stress after reaching the cells and stimulating the formation of stress-induced secondary metabolites (Zaeem et al. 2020).
In another study, Ag NPs toxicity on rockcress seeds was shown to be dependent on the size and concentration (Szollosi et al. 2020). Ag NPs with size of 80nm were only deteriorative at higher concentrations and those of 20 and 40 nm resulted in severe root growth inhibition. The researchers supposed that Ag NPs apoplastically transported through the root tissues (Szollosi et al. 2020). The inhibitory effect of Ag NPs on the germination index was also seen in the case of cucumber (Szollosi et al. 2020). Similar results are reported by Vanninia et al. (2014) as 10 ppm concentration of Ag NPs in uenced the seedling growth of wheat seeds adversely. They also reported induction of morphological modi cations in root tip cells by Ag NPs. According to the microscopy of the treated seeds roots, Ag NPs did not enter the root cells and located in the outer cells of the root cup. It was suggested by TEM analysis, that the toxicity effect of Ag NPs is resulted from release of Ag + ions from Ag NPs (Vanninia et al., 2014). Abbasi Khalaki et al. (2016) reported an enhancement in seedling growth of thymus kotschyanus seeds treated with 20 and 60% concentrations of Ag NPs.
The results reported by Zakharova et al. (2019) is comparable with our obtained results, in which wheat seeds were soaked in the presence of CuO NPs. They also reported that exposure of wheat seeds to 10ppm CuO NPs showed a 14.5% improvement in germination and a twofold increase in root and shoot length in comparison with control. At higher concentrations of CuO NPs, both stimulation and toxic effects were observed (decline in root length) (Zakharova et al. 2019).
The effect of wheat seed treatment with Cu NPs on germination and seedling vigor index has been studied by Yasmeen et al. under laboratory conditions (Yasmeen et al. 2015). Germination percentage, root and shoot lengths were calculated and the results indicated exposure of wheat seeds to Cu NPs lead to a decline in germination percentage and severe reduction of root and shoot length. Therefore, Cu NPs adversely affect germination and growth of wheat seeds (Yasmeen et al. 2015). This substantial decrease in the plantlet growth is consistent with previous wheat eld studies, where the application of excessive NPs resulted in reduced plantlet length and distorted plantlet physiology (Du et al. 2011 Effect of biogenic NPs and their counterpart salts on physiological characteristics of ax seedling The special structure of ax seed's coat was the reason we chose this plant for our experiments. The envelope or testa of the ax seed contain about 15% of mucilage, that mainly contains distinct types of arabinoxylans and water-soluble hydrocolloid/polysaccharides, which contribute to its gel qualities by forming large aggregates in solution (Mehtre et al. 2017). It is suggested that nano-priming of the ax seeds and also the NPs behavior may be affected by mucilage of the seed coat due to the thick chemical environment in which NPs were trapped. Therefore, according to the obtained results, it could be considered as one the reasons that we observed different results of ax seeds seedling in comparison with wheat seeds.
There are a few works studying the in uence of NPs on seedling parameters of ax seeds. In support of our results, the effect of different metal and metal oxide NPs have been presented. As an example, it was reported that the application of biosynthesized MgO NPs enhanced the seed germination and growth parameters of peanut seeds as compared with control. The authors by using physicochemical methods including UV and SEM analyses, indicated that the MgO NPs penetrates into the seed coat, support water uptake inside the seeds, and then affect seed germination and growth rate mechanism (Jhansi et al. 2017).
In contrast of the results obtained from our research, Gorczyca et al. noticed that the 100ppm of Ag NP treatments applied to ax seeds had a limited effect on the germination and early development of the seedlings in comparison with the control. The response of the ax seeds to the NPs was reported as an increase of chlorophyll content (Gorczyca et al. 2018). Zaeem et al. (2020) investigated the effect of green synthesized C-ZnO NPs at concentrations of 0, 1, 10, 100, 500 and 1000ppm on growth of ax seeds. All the treated ax seeds had root development of different lengths, ranging from 2.62cm (for 1000 ppm ZnO NPs) to 7.08cm (for 10ppm ZnO NPs) with 3.85cm for the control. These results indicate e ciency of different concentrations of ZnO NPs in seed germination. At ZnO NPs concentration of above 10ppm, the higher the concentration of NPs, the lower the root length. The increased sensitivity of radicle to NPs is due to the large surface area of the NPs. They suggested that the observed inhibitory effect on seed germination may be because of the very small size of NPs and the dissolution power of ZnO to Zn 2+ ions (Zaeem et al. 2020).
To the best of our knowledge, there was not any report on positive or negative effect of Cu NPs on ax seedling to be compare with our ndings. In previously reported researches, despite our ndings, stimulating effects of Fe NPs on seedling of different species have been described, for example in rocket (Ko et al. 2017), rice (Hao et al. 2016) and soybean (Ngo et al. 2014). Clearly, different results obtained from ax seed priming in comparison to the wheat seeds in germination and seedling growth parameters.
A comparison between the effect of biogenic NPs and their counterpart salts on physiological characteristics of wheat and ax seedlings At 7 th day of the experiment, in a comparison between the effect of studied NPs in applied concentrations, the most effective one in shoot and root development was Zn NP and the less effective NPs were Ag for wheat and Fe for ax seeds (Fig. 2). It shows Zn NPs are not toxic at the applied concentrations and even show stimulating effect on both wheat and ax seeds. Vice versa the applied concentrations of Ag NPs are toxic for wheat but stimulating for ax. As all of the experiment conditions are the same for all of the samples, it could be concluded these differences are related to the seed species. Among the tested metal slats, zinc acetate had the most stimulating effect and CuSO 4 was the most toxic one for both ax, and wheat. Nano forms of metals and metal oxides, have been reported to signi cantly improve root or shoot elongation and seed germination of wheat in comparison with bulk materials (Feizi et al. 2012). This kind of growth development mainly depends on the concentration of NP, duration of nano-priming, growth medium and species of plant (Ahmed et al. 2019).
Among measured parameters, root length is more sensitive than shoot length. Between wheat and ax roots, the ax root length was more sensitive against NP and salt treatments and wheat shoot length was the less sensitive parameter. All over, ax seeds were more sensitive to the treatments compared to the wheat seeds. Although the factors which impact the root and shoot elongation following NP exposure are not clear yet, it could be suggested that the polymeric network of ax seeds mucilage trap NPs or metal ions and then the accessibility of them for ax seeds differs from wheat seeds in the period of our experiment (Ahmed et al. 2019).
Figure 3 provides an image for better comparison between germination percentage variations with changes in concentrations of used NPs and salts. In majority of the cases wheat seeds had more G% than ax seeds. 150ppmof AgNO 3 solution had such a toxic effect on ax seeds that no germination was observed in this treatment. Similarly, metal-based NPs have been reported to show dual impacts on plants growth, such as seed germination. Positive effects of metal-based NPs treatments were displayed in different plants (Szollosi et al. 2020). Seed germination of soybean seeds enhanced by nano-priming with Co, Fe and Cu NPs (Mehtre et al. 2017), also similar ndings were reported in the case of some Solanaceae crops after treatment with ZnO and TiO 2 NPs (Younes et al. 2020). The obtained results were comparable with those reported by Feizi et al. (Feizi et al., 2012), in which seed treatment with TiO 2 NPs at low concentrations (1-2ppm) resulted in an improve in germination of wheat seeds and also seedling elongation compared to untreated wheat, but no signi cant effect at concentration of 100ppm was observed (Feizi et al., 2012).

Conclusion
Suggesting that using green synthesized nano-size minerals for seed treatment could be helpful for seedling growth improvement. Present study provides new information on possible positive or toxic effects of seed priming with NPs on germination percentages, shoot length, root length, seedlings length, root/shoot ratio, seedling vigor index (SVI), shoot length stress tolerance index (SLSI) and root length stress tolerance index (RLSI) which were calculated at 2 nd and 7 th days on two popular early growth plants, wheat and ax. Plant's dual responses varied among NPs type and correlated to the tested concentrations. According to the obtained results, the response of the tested plants to a certain NP was different between ax and wheat. Moreover, it differed between applied concentrations of the NPs. For example, Ag NPs showed a signi cant positive effect on root and shoot elongation of ax seedlings but there were a dose dependent decrease of root and shoot elongation of wheat seedlings over their respective controls. Another important result is that ax seed was more sensitive to priming with metal salts and NPs in comparison with wheat seed. Furthermore, the in uence of these treatments was investigated in earlier stages of the growth, in 2 nd day of the experiment, in comparison with 7 th day of the experiment. Among the studied NPs, Zn and Ag NPs exhibited the best biological effects on growth and development of wheat and ax respectively. The effect of the nanoparticle's counterpart metal salts on seedling parameters also studied for comparison with nanoparticulate ones. Over all, nanoparticle treatments were more effective than metal salt treatments in root and shoot development. The basic mechanisms need to be investigated in future investigations.  Dose response effect of NPs and their correspondent metal salts on shoot and root stress tolerance index (SLSI and RLSI, respectively) of wheat and ax seeds at 7th day of the experiment.