Effect of iron nanoparticles on germination and biometric parameters of fiber flax seedlings

. The article investigates the effect of iron nanoparticles introduced into the composition of a liquid-phase biological agent (LPB) on germination and biometric parameters, such as the average length and average weight of one seedling, fiber flax. Iron nanoparticles were obtained by green synthesis using a 0.5 mol/L FeCl3∙6H2O solution as a precursor and green tea extract as a reducing agent. The finished nanoparticles were introduced into LPB in a volume ratio of 1:50 to obtain the LPB-Fe biological agent. Both biological agents were tested during the germination of fiber flax seeds. Two ways of using biological agents were studied - seed watering and inoculation, while in both cases the concentration of biological agents varied from 0.05 to 1.0%. Water was used as a control. As a result, it was found that in the case of flax seeds, watering is more effective than inoculation. Thus, when irrigating seeds, the maximum germination rate of 99% was obtained using 0.4% LPB-Fe, and the maximum average length of one seedling was 14.4 ± 0.8 cm at a LPB-Fe concentration of 0.1%. Whereas, when seeds were inoculated in LPB-Fe, the maximum germination rate of 95% and seedling length of 13.9 ± 0.6 were obtained at a concentration of 0.2%. When irrigating with LPB, the highest germination rate of 96% was obtained at a biological agent concentration of 1.0%, and the maximum average length of one seedling of 14.5 ± 0.7 cm was obtained at a biological agent concentration of 0.6%. At the same time, none of the biological agents affected the average weight of one seedling.


Introduction
Iron and iron oxide nanoparticles are very often used in catalysis for hydrogenation, reduction, and oxidation of alcohols and for many other reactions, as well as in biomedicine for drug delivery, magnetic resonance imaging, hyperthermia, cell labeling, and protein separation [1].In addition, due to their physicochemical properties, iron nanoparticles are also widely used in agriculture as antimicrobial agents, plant growth stimulants, for targeted delivery of other nutrients, and to eliminate the effects of pesticides and heavy metals [2].Since iron (Fe) is an essential nutrient that plants require in small amounts to maintain proper growth and development, its application is mandatory to optimize crop yields [3].
For example, R. Sheykhbaglou et al. [4] found that foliar spraying of soybean plants with nano iron oxide at a concentration of 0.75 g/l led to an increase in protein content up to 34% and lipids up to 25% compared with the control, in which their content reached 28% and 20%, respectively.In addition, iron oxide nanoparticles with a concentration of 0.75 g/l affected the mineral composition of seeds.Thus, the content of iron, magnesium, phosphorus and calcium increased by 1.7, 3.3, 4.0 and 3.7 mg/g, respectively, compared with the control.In addition, when plants were treated with Fe2O3 nanoparticles at a concentration of 0.75 g/l, the content of chlorophyll in soybean seeds increased, which could positively affect the antioxidant role of soybean oil, providing a beneficial effect from the point of view of food science and technology [4].It was shown in [5] that after treatment with nanoparticles at a concentration of 500 mg iron/kg of soil, the biomass of plants of the Talya clover (Arabidopsis thaliana) increased by 38% due to increased photosynthesis, which was confirmed by the gas exchange system, the ratio of carbon isotopes, and analysis of the chlorophyll content.In addition, iron uptake by the plant was increased by roots and leaves.Accumulation of carbohydrates such as glucose, sucrose, and starch was increased by increased photosynthesis, and photosynthesis-related inorganic nutrients such as phosphorus, manganese, and zinc maintain homeostasis in line with increased iron intake.These data indicated that iron nanoparticles have additional or alternative benefits as a nanofertilizer and CO2 uptake stimulator in plants [5].
The "green" synthesis of nanoparticles is one of the most promising ways to obtain iron nanoparticles for agricultural needs, since it is environmentally friendly and does not require large expenditures on equipment and reagents.Along with other plant extracts, green tea extract (Camellia sinensis) has been successfully used for the synthesis of iron nanoparticles, since it contains a large amount of catechin, a polyphenolic antioxidant compound found in plant leaves as a secondary metabolite [6].For example, K.S.V. Gottimukkala et al. [7] obtained iron nanoparticles with an average diameter of 128 nm by mixing green tea extract and 0.01 M iron(III) chloride solution in a 1:1 volume ratio.According to the authors, epigallocatechin gallate, which reduces Fe 3+ to Fe 0 , takes an active part in the formation of iron nanoparticles, due to its standard potential of 0.58 V, since the standard potential of iron is 0.036 V.In this case, when an iron precursor is added to tea, the gallate complex is first formed epigallkatechin with iron when the -OH bond is broken, and then a partial bond is broken and electrons are transferred to reduce metal ions to nanoparticles [7].
Currently, microbial preparations or biological products are an alternative to traditional inorganic and organic fertilizers, as they contain a large number of symbiotic, associative and rhizospheric microorganisms (1 ml or 1 g of the preparation contains up to 5 billion bacterial cells), which compete with the native microflora, healing the root-inhabited soil layer and shifting the balance of organomineral compounds towards enrichment with available forms of nitrogen and phosphorus [8].Such preparations can increase the length and mass of shoots and roots of plants, positively influence various biochemical processes occurring in the soil and plants, and, as a result, increase crop yields [9].However, the effect of the impact of the drugs used does not always meet the desired.For example, the effectiveness of such drugs can be significantly reduced when plants are massively infected with pests or diseases, as well as when temperature and humidity change.In this regard, it is relevant to develop a new approach to the creation of preparations (biologically active agents) for crop production of a complex composition, containing several active principles, which can primarily lead to an increase in their effectiveness.
As such an approach, in this work it is proposed to use a combination of the microbial biological agent LPB developed earlier [10] with iron nanoparticles obtained using green tea extract.The introduction of iron nanoparticles, obtained by an environmentally safe , 010 (2023) BIO Web of Conferences CIBTA-II-2023 https://doi.org/10.1051/bioconf/2023710105252 71 method of green synthesis, into this biological product at the ripening stage, as a result of the synergistic effect, will enhance the effectiveness of the microflora acting in it.

Materials and methods
Obtaining an extract and biosynthesis of iron nanoparticles.The synthesis of iron nanoparticles was carried out using an extract of commercially available green tea brand "Princess Java, Traditional", produced by "NEP" LLC, which contains a large amount of polyphenols capable of reducing metal ions [11].The tea extract was prepared as follows: distilled water (2 liters) was brought to a boil, tea (100 g) was poured and continued to boil for 30 minutes.After that, the obtained extract was cooled, the tea leaves were filtered first through gauze, and then through a paper filter using a vacuum pump.
The synthesis of iron nanoparticles was performed using a FeCl3•6H2O solution with a concentration of 0.5 mol/l as a precursor.The solution was mixed with green tea in a 1:1 volume ratio and heated in a boiling water bath for 20 minutes.The formation of nanoparticles in this case was evidenced by the change in the color of the solution from yellow-green to black, associated with the excitation of surface plasmon resonance [12].
Obtaining biological means LPB-Fe.Green tea containing iron or copper nanoparticles was mixed with a ready-made liquid-phase biological agent (LPB) to enhance its multifunctional properties in a volume ratio of 1:50.
Laboratory study of the obtained biological agent on flax seeds.In order to study the effect of the obtained biological product LPB-Fe, as well as pure LPB on the plant organism, a model experiment was carried out on flax seeds of the Tverskoy variety.In the experiment, seeds of the fourth reproduction were used, category II in terms of varietal purity, phytopathological evaluation of seeds was not carried out, their quality was determined by carefully examining them for infection with diseases, followed by calibration by size and weight.Before direct use, flax seeds were disinfected with a 1% solution of potassium permanganate for 5 min.
Flax seeds were germinated according to GOST 12038-84 for seven days in glass Petri dishes on filter paper at a temperature of 22±1°C in the dark.In the experiment, two methods of using biological agents were used: watering and inoculation.In the first case, seeds were put into Petri dishes on dry filter paper, and then they were watered once with 5 ml of ready-made biological products.During inoculation, the seeds were pre-soaked before sowing for 3 hours in solutions of biological agents, and then they were laid out on filter paper pre-moistened with distilled water.The concentration of drugs varied from 0.05 to 1.0%.In each variant, 4 repetitions were provided (35 seeds in each).Evaluation of the effectiveness of biological agents was carried out by determining the germination of seeds, as well as determining the average length and average weight of one seedling.Distilled water was used as a control.
Statistical processing of the results was carried out using the program Microsoft Office Excel 2007.The data in the tables are presented as the mean value of four repetitions ± confidence interval.

Results
When irrigating flax seeds with the LPB biological agent (figure 1), the maximum seed germination (96%) was obtained at a biological agent concentration of 1%.At the same time, in this case, there was no dependence of seed germination on the concentration of the biological agent.In the case of using LPB-Fe for irrigation, a dependence of seed germination on concentration was observed.At the same time, the maximum germination , 010 (2023) BIO Web of Conferences CIBTA-II-2023 https://doi.org/10.1051/bioconf/2023710105252 71 rate of 99% was obtained at a biological product concentration of 0.4%, while at concentrations of 0.05 -0.2%, the germination of seeds in this variant was almost equal to the values in the control (87 -88%).An increase in the concentration of LPB-Fe led to a decrease in seed germination, which was equal to 96% when using a 1% agent, as well as in the case of pure LPB.

Figure 1. Germination of flax seeds when irrigated with biological agents of various concentrations.
In addition to seed germination, the average length and average weight of one seedling were also determined.The results are presented in table 1.When watering LPB-Fe, the maximum length of one seedling of 14.4 ± 0.8 cm was obtained at a concentration of 0.1%, and was 3 cm more than in the control variant.Almost the same maximum seedling length (14.5 ± 0.7) was also obtained using LPB at a concentration of 0.6%.At the same time, in the case of using LPB-Fe, the average length of one seedling gradually decreases with increasing concentration of the biological agent.
The maximum average weight of one seedling, 62.3 ± 3.6 mg, was obtained when using LPB-Fe at a concentration of 0.1%, while when irrigating with pure LPB, the largest mass was 58.4 ± 0.4 mg at a biological agent concentration of 0.2% .At the same time, it is important to note that when using both biological agents, there was no dependence of the average weight of one seedling on their concentration.When seeds are inoculated in the biological agents of LPB and LPB-Fe, the dependence of germination on their concentration is observed (figure 2).So the maximum germination of 94% and 95% was obtained with 0.8% LPB and 0.2% LPB-Fe, while in the control variant the germination was 88%.

Figure 2. Germination of flax seeds after inoculation in biological agents of various concentrations.
Seed inoculation in LPB-Fe had no effect on the average length and average weight of one seedling.So the length of one seedling in this case varied in the range of 12-13 cm, and the average weight from 54 to 59 mg, which was practically equal to the values of the average weight (58.1 ± 0.2 mg) and average length (12.2 ± 0.3) in the control variant.
When using pure LPB, the average length of one seedling varied in the range of 13.0-14.5cm, while the maximum length of 14.4 ± 0.8 cm was obtained at a biological agent concentration of 1%.The highest average weight of one seedling 62.4 ± 0.4 mg was obtained using 0.4% LPB, while the use of a biological product of lower concentration led to a decrease in the weight of the seedling to 52-57 mg.

Discussion
When seeds were irrigated with biological means of LPB and LPB-Fe, the maximum germination rate of 99% was obtained when using LPB-Fe at a concentration of 0.4% (figure 1).This germination was 12 and 6% higher than the values obtained in the control variant and LPB of the same concentration, respectively.In general, it should be noted that when irrigating LPB-Fe with a concentration of more than 0.4%, the germination of seeds is greater than irrigating with pure LPB.
Iron nanoparticles in the LPB-Fe biological product had a positive effect on the average length of one seedling (table 1).In all experimental variants, the average length of one seedling was 1.2-1.3times higher than in the control.At the same time, when using LPB-Fe, to obtain the maximum length of one seedling (14.4 ± 0.8 cm), a concentration of 0.1% of the biological agent was sufficient, while when using pure LPB, the same length of the seedling (14.5 ± 0.7 cm) was obtained at a much higher concentration -0.6%.Thus, with the combined use of LPB and iron nanoparticles, a synergistic effect was observed due to their interaction, expressed in an increase in germination and average length of one seedling, compared with the control at a lower concentration of the biological agent.However, it is also worth noting that the average length of one seedling gradually decreases with an increase in the concentration of LPB-Fe (table 1).This is probably due to the fact that at high concentrations of the biological agent, the concentration of iron also increases, which in this case inhibits plant growth.
The inoculation of flax seeds in biological agents significantly affected only the germination of seeds, the maximum values of which were 94% when using 0.8% LPB and 95% when soaking in 0.2% LPB-Fe (figure 2).At the same time, the germination of seeds during inoculation in LPB-Fe was 4-9% less compared to the germination during irrigation with the same biological agent.In addition, inoculation in LPB-Fe did not affect the average length and average weight of one seedling (Table 2).This is probably due to the fact that during inoculation, iron nanoparticles penetrate the seeds to a greater extent, thereby inhibiting not only their germination, but also further growth.Thus, from the point of view of the studied parameters, seed watering is preferable to inoculation.

Conclusion
The addition of biogenic iron nanoparticles to LPB made it possible to obtain a new biological agent of LPB-Fe, which has properties that stimulate plant growth.A laboratory experiment showed that the maximum germination of 99% was obtained by watering flax seeds with 0.4% LPB-Fe.Whereas when using pure LPB for irrigation, the highest germination rate was 96% at a biological product concentration of 1.0%.In addition, the addition of iron nanoparticles also affected the average length of one seedling.Thus, the maximum average length of one seedling of 14.4 ± 0.8 cm was obtained using 0.1% LPB-Fe, while using pure LPB the same average length of one seedling was obtained at a concentration of 0.8%.In the case of seed inoculation in biological agents, the maximum germination rate of 95% and the average length of one seedling 13.9 ± 0.6 were obtained using 0.2% LPB-Fe.Thus, from the point of view of the studied parameters, seed watering is preferable to inoculation.

Table 2 .
Average length and average weight of one seedling during seed inoculation with experimental biological agents.