Effects of Pre-sowing Seed Treatment on the Growth Rate of Seedlings and the Activity of the Excretory System of the Wheat Root in Aquatic Culture

The qualitative and quantitative composition of the rhizosphere is known to be the result of the metabolic activity of the root and it regulates the functional activity of the root. Therefore, it works as autoregulation in the root activity. The study of the system of autoregulation is actually not only for the physiology of plants, but also for modern biotechnologies. The contents of proteins, amino acids and total carbohydrates were determined during the formation of the rhizosphere of the wheat’s roots from 1 to 3 day of growth in aquatic culture. In addition to that, the role of microorganisms in the formation of the root microenvironment with different models of pre-sowing seed treatment was investigated. It was found that, the pre-sowing seed treatment affects the qualitative and quantitative composition of root exudates. The pre-sowing treatment of seeds with potassium permanganate increased the protein content in the composition of exudates in the 1st day of seedling about 75%, but it decreased in the 2nd day about 40% if compared with the control variant. The pre-sowing treatment of seeds with ethyl alcohol and sodium hypochlorite reduced the protein content of exudates in the 2nd day of seedlings about 45% and did not affect this indicator in 1 and 3rd day of seedlings in comparison with the control variant. The pre-sowing treatment with potassium permanganate insignificantly affected the mount of microorganisms, while the pre-sowing treatment with alcohol and sodium hypochlorite reduced the amount of microorganisms in the root exudates two times if compared with the control variant.

As it is known, from the first hours of growth of wheat sprouts, as well as other plants, the rhizosphere forms (Dennis et al. 2010;Mendes et al. 2014;Chaparro et al. 2014). The system of formation and functioning of the root rhizosphere has a great interest for the development of root biotechnologies. This can be explained by several important points: firstly, the root exudates are a unique natural complex of biologically active compounds that can be used in pharmacy, biotechnology and agro biotechnology; Secondly, the system of "root-rhizosphere" is a unique natural model for studying excretory mechanisms; thirdly, the study of this system will make it possible to understand the mechanisms of allelopathy Dmitrović et al. 2015;Bouhaouel et al. 2015;Cai et al. 2012).
At the same time, the stages of rhizosphere formation have been studied incompletely. In this sense, the root exudates are the products of the vital activity of specific root cells, which is called "border cells" (Hawes et al. 2016;Bozhkov et al. 2009), products of destruction of growing cells Print ISSN : 1974ISSN : -1712 Online ISSN : 2230-732X of the root (Bozhkov et al. 2013), and products of vital activity of microorganisms of the rhizosphere (Ohkama-Ohtsu et al. 2010;). The contribution of each of the elements of the excretory system in the process of rhizosphere formation is unclear.
It is well known that a large number of microorganisms (bacteria, fungi and algae) are present in the rhizosphere, which affect the intensity of root growth and alter the composition of root exudates (Zhang et al. 2014;Majeed et al. 2015;Kumar et al. 2014).
In natural cenoses, microorganisms are an integral component of the rhizosphere (Dennis et al. 2010;). The composition of the rhizosphere includes both symbiotic and antagonistic relation to plants microorganisms (Rasmann et al. 2016;Berendsenet al. 2012;Bulgarelli et al. 2013).
It can be assumed that, when plants are growing in a hydroponic or aquatic culture with relatively sterile conditions and pre-sowing seed treatment, the microorganisms will be removed, and their contribution to the rhizosphere formation is insignificant. However, the study of different modes of pre-sowing seed treatment which aimed to remove microorganisms from the surface of the grains was not carried out. At the same time, controlling the number of microorganisms in hydroponic culture is of fundamental importance for both the development of root technologies and solving fundamental questions of plant physiology.
In this context, the present work was designed to determine the intensity of growth of plants from the 1 st to the 3 rd day of cultivation, the rate of rhizosphere formation by content and composition of proteins, free amino acids and total carbohydrates from the 1 st to the 3 rd day of growth, and the amount and intensity of growth of rhizosphere's microorganisms of 2 nd day seedlings, obtained after various regimes of pre-sowing treatment and inactivation of microorganisms. These data can be used to create root biotechnologies aimed at obtaining biologically active compounds.

MATERIALS AND METHODS
In the work wheat seedlings Triticum aestivum were used. Wheat grains were treated in 3 different ways before soaking : The scheme is shown in the Fig. 1.

Fig. 1: Experimental scheme
After washing all variants, the grain was soaked in sterile distilled water for 24 hours at 26°C. 24 hours after soaking, the nested grains from each treatment option were spread by 100 grain in each Petri dish. 7 ml of sterile distilled water was added to each Petri dish. Petri dishes with sprouts were placed in a chamber with round-the-clock lighting and a temperature of 26°C. On the 1 st , 2 nd and 3 rd day of growth, the number of not germinated grains and root length of wheat seedlings were measured.

Collection of root exudates of wheat germs
To characterize root exudates in the 1 st , 2 nd and 3 rd day of growth from each petri dish, the aqueous root exudate was collected and the content of total protein, free amino acids and total carbohydrates was determined.
From the culture medium of the 2 nd day seedlings, 1 ml of culture medium containing root exudates was taken, and from each Petri dish 10 sprouts Print ISSN : 1974ISSN : -1712 Online ISSN : 2230-732X B were selected. The gel cover surrounding the root apexes was removed from the roots of the selected 10 seedlings, as described in (Bozhkov et al. 2013).
The obtained material was used to determine the presence of microorganisms after culturing the samples on meat-peptone agar.

Determination of total protein content
The content of total protein in the root exudates of wheat germs was determined by the method of Lowry which used in experiments (Bozhkov et al. 2013).

Determination of total carbohydrate content in root exudates
The content of carbohydrates in the composition of the root exudates was determined by the method (Kuznetsova 2008). However, 0.5 ml of a 5% aqueous phenolic reagent was added to aliquots of the soluble fraction of the root exudates. The composition of the sample was vigorously stirred, and 2.5 ml of concentrated sulfuric acid was added. After 20 min, the carbohydrate content was determined at a wavelength of 488 nm on a SF-46 spectrophotometer. The content of total carbohydrates was shown by the calibration curve for glucose and it was expressed in mg glucose/100 grains.

Determination of the content of amino acids in the composition of root exudates
The content amino acid in the root exudates was determined by the ninhydrin method which used (Bozhkov et al. 2013). in her experiments. Then, an equal volume of 10% trichloroacetic acid was added to the aliquot of the root exudates and the samples were incubated for 12 hours at + 40 °C to precipitate the proteins. The samples were then centrifuged at 3000g for 20 minutes. Aliquots were taken from the supernatant, diluted to 0.6 ml with distilled water, 1 ml of 1% ninhydrin and 1 ml of 10% pyridine were added. Then the samples were placed on a boiling water bath for 30-40 minutes. After cooling, the content of amino acids in the samples, was determined at a wavelength of 570 nm on a SF-46 spectrophotometer. The amino acid content was shown by the calibration curve for glycine and expressed in μg glycine/100 grains.

Cultivation of microorganisms on a universal nutrient medium
For analysis the microbiota of wheat seeds, the collected root exudates were transferred to nutrient agar medium -meat-peptone agar (MPA). The exudates of wheat seeds were applied to the nutrient medium in sterile conditions with a 3 μl micropipette. Then, the samples of microorganisms were cultured in the thermostat at 37 °C for 24 hours.

Determination of the growth dynamics of microorganisms on different media
By cultivating microorganisms on a nutrient agar medium, the growth dynamics of the culture was assessed by the growth of colonies. To do this, every 3 hours the colony was photographed and the area of the colonies was measured using the computer program Axio vision, which was expressed in conventional units.

Statistical processing of results
The experiments were performed in 3 biological replicates with 3 analytical repeats. The mean values of the obtained data were determined and the error of the mean was calculated. The reliability of the differences between the samples was indicated using a nonparametric method using the Mann-Whitney U test (Thomas et al. 2016).

RESULTS AND DISCUSSION
The influence of different methods of presowing seed treatment on germination and root growth intensity The Pre-sowing treatment of seeds ensures the removal of the accompanying microorganisms on the surface of seeds or inactivate them, while it should not affect the germination of seeds and the intensity of root growth.
It was found that, in the control variant, 26% of the seeds did not germinate at the end of the 1 st day, but to the 3 rd day only 6% of seeds are not germinated ( Fig. 2A). Consequently, in the control variant there were seeds which are sprouted in 1 st day, and the others sprouted only in the 3 rd day. Therefor 74% of seeds germinated in the 1 st day, 12% sprouted in the next day, 8% ( In this case, when the wheat seeds were treated with 0.05% solution of potassium permanganate (first method of treatment), the dynamics of seed germination was slightly different from the control variant case. Thus, 72% of the seeds are germinated during the 1 st day, 15% of seeds in the following day, while 3% of them are germinated in the 3 rd day, and 10% of seeds are not germinated, which is 4% more than control (Fig. 2B).
In the other case, when the seeds were treated with 70% alcohol and 5% sodium hypochlorite (second method of treatment), seed germination was decreased 10% if compared with the control variant case. So, in the 1 st day, 64% of seeds are germinated, and up to the 3 rd day the percentage of non-germinated seeds was 9%, which is more than the percentage of non-germinated seeds in the control variant case ( Fig. 2A, B).
Such decline of seed germination can be caused by various reasons: death of the embryo, destruction of the water absorption system in the seeds, inhibition of the rate of the embryo development.
The length of the roots of wheat germs in the control variant case reached to 0.76 cm in the 1 st day of growth, and 1.89 cm to the 2 nd day, and the roots had a length of 2.78 cm in the 3 rd day (Fig. 3A). If we illustrate the growth rate of the roots in the control variant case, the greatest increase can be seen in the 2 nd day, but in the 3 rd day it has somewhat decreased ( If the seeds were treated within the second method (treatment variant with K 2 MnO 4 ), the intensity of root growth would be affected. Roots are increased intensively in the 2 nd day, but later if compared with the control variant case, they were grow slowly and this can be clearly seen in the growth rate (Fig. 3A, B).
There are unexpected results which appeared in the second method of seed treatment, the length of the roots of wheat seeds in this method reached to 0.57 cm in the 1 st day of growth (which means that the length of the roots is 25% less than roots length in the control variant case) (Fig. 3A). In the 2 nd day, the growth of roots was obviously increased (Fig.  3B). While in the 3 rd day the root growth was 25% longer than its length in the control variant case.  2B). If we compare these three experimental variants of pre-sowing seed treatments, it should be noted that the growth rate of the root in the control in 3 rd day increased about 17% in comparison with 1 st day, in the case of the first treatment method, it decreased about 36%, and for the second treatment the growth rate increased about 121% (Fig. 3C). Consequently, in the case of the second method of pre-sowing treatment of wheat seeds, there was an effect of growth inhibition followed by growth stimulation.
Such stimulation of root growth by pre-sowing seed treatment with 70% alcohol and 5% sodium hypochlorite, which comes after the inhibition of seed germination, can be caused by several reasons: 1) the effect of synchronization of cell cycle; 2) effect of hormesis; 3) changes in the ratio between the formed microbiota of the root and its influence on the growth rate.
When talking about the effect of hormesis, it should be noted that small "disturbances" have stimulatory effects (Jia et al. 2015) and in this formula can occur.
Finally, it is known, that the intensity of growth largely depends on the quantitative and qualitative composition of the microorganisms which are formed in the root rhizospher and provided root nutrition for plants. The main components of the microenvironment of the root are carbohydrates, proteins and free amino acids ). So, the root exudates exert direct and indirect (through the regulation of the qualitative and quantitative composition of the microbiota), the influence on the growth rate at the next stage of the work, is that the qualitative and quantitative composition of the root exudates were determined in the case of different methods of pre-sowing treatment of wheat seeds.

The qualitative and quantitative composition of root exudates after different methods of presowing treatment of wheat seeds
Excretory activity of the roots of the control variant repeatedly increased from 1 st to 3 rd days of growth in aquatic culture. So, in the 1 st day 1418 μg of root exudates were detected in the aquatic environment per 100 seeds, at the end of 2 nd day 2739 μg/100 grains, after 3 days there were 5880 μg / 100 grains (Fig. 4). Therefore, during the 2 nd day the amount of exudates increased about 1321 μg /100 grains if compared with 1 st day, while at the 3 rd day in comparison with the 2 nd day about 3141 μg/100 grains, there was almost an exponential increase in the number of root exudates in the case of the control variant/ Results are shown in the Fig. 4.   Fig. 4: Content of the total number of root exudates at the 1 st , 2 nd and 3 rd days of wheat sprouts after different pre-sowing treatments

* -significant differences compared with control
In this case when the seeds were treated with potassium permanganate, the intensity of the root excretion in the 1 st day of growth was 60% higher if compared with the control variant case (Fig.  4). But, at the 2 nd day of growth, the rate of root excretion did not change if compared with control variant (Fig. 4). While in the 3 rd day of growth, the amount of excreted substances increased about 4790 μg/100 roots if compared with 2 nd day and exceeded about 25% more than the excretion of the control variant case (Fig. 4). Consequently, the first method of pre-sowing seed treatment increased the rate of root exudates and influenced the dynamics of this process. A similar nature of excretory activity took place in the case of the 2 nd method of pre-sowing seed treatment (Fig. 4). The difference is only, that the intensity in the 1 st and 2 nd days does not differ practically from the control variant, but in the 3 rd day it is exceeded the control variant about 20% and came close to the first method of treatment at the same time (Fig. 4).
The obtained results allow us to conclude that presowing treatment of wheat seeds can be, firstly, used Print ISSN : 1974-1712 Online ISSN : 2230-732X to induce excretory activity in root biotechnologies because it allows firstly, obtaining up to 7.5-8 mg of root exudates/100 grains. Secondly, influenced the intensity growth and development of plants with regulating the composition and number of components formed microenvironment of the root.
At the same time, the most important characteristics of the influence of seed treatment methods on excretory activity and understanding of the biological role of root exudates is their composition, specially the relationship between the main components of exudates.
Despite the wide range of substances excreted by the root system, more than 90% of them located on 3 main components: carbohydrate, proteins and free amino acids, at least for wheat (Bozhkov et al. 2013).
At the next stage of work, the content of total sugar, proteins and amino acids in the root exudates of wheat seeds was determined after different methods of pre-sowing seed treatment.
The content of carbohydrates in the 1 st day of growth of the roots of the control variant was 950 g of glucose/100 grains, which was 67% of the total amount of root exudates (Table 1). At the next day it increased about 41% when compared with 1 st day, and in 3 rd day the amount of carbohydrates was 3311 g of glucose/100 grains, which was 146% higher if compared with 2 nd day in the control variant case (Table 1).
The dynamics of the excretory activity of carbohydrates in roots in the first and second methods of pre-sowing seed treatment generally was similar to that of common exudates (Fig. 4). Thus, in the 1 st day of growth, especially after the first treatment method, the roots excreted up to 1527.3 μg of glucose/100 grains into the aqueous medium, which was 1.6 times greater than control variant at same time. Later, in the 2 nd day, the sugar content remained the same as the 1 st day, while in the 3 rd day the excretion increased and the carbohydrate content was 4929.3 μg glucose/100 grains, which was 211% more if compared with the 2 nd day of growth, and 30% more than the control variant at that time (Tab 1). The content of sugar in the composition of root exudates after the second method of pre-sowing wheat seed treatment did not differ from the control variant case in the 1 st and 2 nd day of seedlings but it was increased by 37% in 3 rd day of seedlings (Tab 1).
Consequently, pre-sowing seed treatment with potassium permanganate increased the rate of carbohydrate excretion in the medium on the 1 st and 3 rd day of seedlings.
The protein content of root exudates in wheat seeds in the control variant case increased from the 1 st to the 3 rd day of cultivation (Table 1). So, its number in the control variant increased from 1 st to 3 rd day about 3, 5 times (Table 1).
Pre-sowing treatment of seeds with potassium permanganate increased the protein content in the composition of root exudates on the 1 st day of seedlings about 75%, but on the 2 nd day the protein content was 40% less than its content in the control variant. The seed treatment with ethyl alcohol and sodium hypochlorite reduced the protein content on the 2 nd day about 45% if compared with control case (Table 1).
Pre-sowing seed treatment did not affect the content of free amino acids in the composition of root exudates (Table 1). At the same time, the content of amino acids in the composition of root exudate increased in 7.9-8 times from 1 st to 3 rd day of growth regardless of the methods of pre-sowing seed treatment.
It should be noted that the intensity of amino acid excretion increased linearly from the 1 st to the 3 rd day of cultivation, but the content of proteins and sugars increased in nonlinear way during cultivation. Results are shown in the Fig. 5. Such differences in the dynamics of excretion of different components led to a change in the ratio between its components in the 1 st , 2 nd , and 3 rd dayold seedlings, which must be taken into account when obtaining root exudates. So, different methods of pre-sowing seed treatment lead to a change in the composition of carbohydrates and proteins, especially expressed on 1 day of growth. Results are shown in the Fig. 6.
T h e m o s t i m p o r t a n t c o m p o n e n t s o f t h e microenvironment of the root, along with proteins, amino acids and carbohydrates is a microbiota. As is known, microorganisms of the root rhizosphere are not only utilize the root exudates, but also excrete their exudates and as a consequence, they change the microenvironment of the root and can affect the intensity of root growth and the composition of the obtained root exudates. Fig. 6: Change in the content of carbohydrate, total protein and free amino acids (in relation to control, which is taken as 100%) in 1 st , 2 nd and 3 rd days of root exudates with different presowing treatments: first method -0.5% solution of potassium permanganate; the second method is 70% alcohol and 5% sodium hypochlorite At the next stage of the work, the content of the total number of microorganisms was determined in the 2 nd day of wheat sprouts after different methods of pre-sowing seed treatment and in the cultivation of seedlings in the same culture with the observance of aseptic conditions. The effect of pre-sowing seed treatment on the number of microorganisms in the 2 nd day of wheat sprouts in the aquatic culture Cultivation of inoculants, obtained from the rhizosphere of wheat seedlings on nutrient agar medium, showed that: 1. In the composition of root exudates there are microorganisms which can actively proliferate on nutrient agar medium.
2. In the case of the control variant, the stationary phase of the culture growth arrived at 24 hours with the attainment of the relative area of the colonies of about 4 thousand usl unit.
3. In the case of the first method of treatment, the yield to the stationary phase of microorganism also occurred at 24 hours, but the relative area of the colonies was less about 20-25% compared with the control variant.
4. In the case, when an inoculant was prepared in the 2 nd day of seedling after second method of pre-sowing seed treatment, the output to the stationary phase was carried out at 6 hours, and the relative area of the colonies was 2 times less than the relative area in the control variant case.
Results are shown in the Fig. 7. Consequently, pre-sowing treatment of wheat seeds with 0.05% of potassium permanganate insignificantly inactivated located microorganisms on the seed's surface. The seed treatment with 70% ethyl alcohol and 5% sodium hypochlorite inactivated most of the microorganisms which were on the seed's surface and delayed the germination of seeds.
The results of the work showed that; in the soft shortterm pre-sowing treatment (treat with potassium permanganate) of wheat seeds there was a slight delay in the growth rate of seedlings, an increase in the intensity of excretion of root exudates, a change in the composition of root exudates, and a decrease in the number of microorganisms in the rhizosphere of the wheat's root in aquatic culture. The change in these out comes depended on the method of pre-sowing seed treatment. The results indicate the existence of a relationship between growth characteristics and excretory system function. It is possible to manage the process of obtaining root exudates by using different methods of pre-sowing seed treatment.
So, in particular, the pre-sowing seed treatment slightly delayed the growth rate of seedlings in the 1 st day of germination, and this delay was accompanied by a pronounced increase in the specific growth rate of seedlings in the 2 nd day. Then it should be noted that in the 3 rd day the specific growth rate again decreased (Fig. 3B).
Consequently, the specific growth rate had rhythmic feature. The pre-sowing treatment of seeds increased the amplitude of fluctuations in the rhythmic nature of the specific growth rate from 1st to 3rd day of germination.
We believe that, the effect of delayed growth rate with the stimulation of seedling growth can be considered as a symptoms of the hormesis effect on the seedling model. As it is known, the hormesis effect is known as the stimulation of biological processes by small doses of toxic substances or physical factors and the effect is described for various types of radiation, that was shown in the model of action of heavy metal ions and other chemical compounds (Jia et al. 2015;Tang et al. 2015;Bozhkov et al. 2010).
It should be noted that in this paper there is a direct relationship between the degree of "delay" of root length and the subsequent increase in specific growth rate of seedlings when using these methods of pre-sowing seed treatment. So, the soft way method (with potassium permanganate) showed less effect than the hard way (70% ethyl alcohol and 5% sodium hypochlorite) of seed treatment (Fig. 3B).
So, the hormone's effect can be realized by various mechanisms. It can be realized by the synchronizing passage of the cell cycle of cells of an intensively growing root.
As it is known that, the first mitoses of cells of sprouts occur relatively synchronously, but in the future there is an asynchronous occurrence of the cell cycle (Moubayidin et al. 2010;Marshall et al. 2012;Hayashi et al. 2013). The delay in root growth after pre-sowing seed treatment may be associated with a short-term delay in forming rate of the cell cycle. The subsequent "removal" of the inhibitory effect of the components of pre-sowing seed treatment is accompanied by the synchronous entry of cells into the cycle, which is manifested with increase in the growth rate in the 2 nd day of growth. Further, in the 3 rd day of growth there is again an asynchrony occurrence of the cell cycle, and as a consequence a decrease in the specific growth rate of the seedlings.
Of course, the synchronization of the cell cycle is not only the factor in the nonlinear growth rate of seedlings, but also an appreciable contribution in this process which can also be made by the process Print ISSN : 1974ISSN : -1712 Online ISSN : 2230-732X of "stretching" in root cells, and an increase in their size. However, the basis of this process, of course, is the rate and the direction of the metabolism of the cells of the growing root.
One of the indicators of metabolic activity is the characteristic of the excretory system of the root.
Earlier, it was shown that an increase in the secretion density of the root exudates on a root inhibition model by phytotoxicant -sodium fluoride (Bozhkov et al. 2009, Bozhkov et al. 2013. The slow growth rate of wheat sprouts, which comes from the natural storage of the seeds, was also accompanied by an increase in the excretory activity of the root. Consequently, when the root grows more slowly, at least from 1st to 3rd day, it excites more active exudates into the culture (Bozhkov et al. 2009;Bozhkov et al. 2013).
The results of this study confirm what we have explained above, and they also show the relation between the period of growth and excretion is not strong enough, but it is a passageway or the range of wide interconnections, which must be taken into consideration in the technology of obtaining exudates.
Thus, a small delay in root growth after presowing seed treatment in a "soft" way (potassium permanganate) was accompanied by an increase in excretory activity in the 1 st day of growth. So, in 1st day of seedlings, the rate of excretion increased to 60%, and at the same time the ratio between root exudates is changed. Acceleration of the growth rate in the 2nd day was accompanied by a decrease in the rate of excretion of the root exudates. But the decrease in the specific growth rate in the 3rd day was accompanied by a 25% increase in the rate of excretion of the root exudates.
At the same time, in the "hard" treatment of seeds with alcohol and sodium hypochlorite, the process did not exert a pronounced effect of stimulation of excretory activity.
Consequently, the process of excretion is regulated by a various factors and probably there are specific mechanisms of excretion, as well as general mechanisms which reflect the overall metabolic level in the root cells.
As we know, the growing root, from the first hours of growth, forms its own specific microenvironment. The study of the highly specific "root-microenvironment" system can be carried out on the model of the water culture of seedlings. The knowledge of the specifics formation of the "root-microenvironment" system is important for the fundamental principles of plant physiology, for obtaining biologically active compounds and for the development of root technologies.
It should be noted that, the components of the microenvironment of the root are formed from several sources: 1 -specific excretory system of the root represented by the border cells of the root (Hawes et al. 2016;Bozhkov et al. 2013); 2 -products of degradation of cells of a growing root; 3 -by inhabiting microorganisms for the rhizosphere of the root, as well as by the products of their vital activity Berendsen et al. 2012).
There are enzymes and microorganisms in the microenvironment, its composition dynamically changes. Furthermore, the "contribution" of these constituent components in the microenvironment during the growth of the root will vary greatly.
If (in the 1 st -3 rd day) the main contribution to the microenvironment is made by exudates, then in the future, during the growth process, the contribution of the components of cell degradation and metabolites of microorganisms will increase.
Consequently, from 1 st to 3 rd day of growth of seeds in aquatic culture under maintaining sterile growth conditions, the main components of the microenvironment of the root are sugars, proteins and amino acids that are actively excreted into the aquatic environment.
The intensity and composition of these root exudates, during this period, depend on the conditions of cultivation, and species composition of the culture, and they can be obtained in industrial quantities. The pre-sowing treatment of wheat seeds allows to increase the yield of dry biomass of root exudates from 5.8 mg / 100 roots in the control to 7.5-8.0 mg/100 roots. As shown earlier, such root exudates have biological effect and can be used in biotechnology (Bozhkov et al. 2013).
It should be noted that, a small amount of microorganisms was exist in the composition of wheat exudates in the water culture, which grew only on a universal nutrient medium, but they did not grow in the composition of root exudates. The pre-sowing seed treatment reduced the growth rate of these microorganisms, which suggests that they are exist on the surface of grain, and they did not have any significant contribution in the excretory root system in aquatic culture.

CONCLUSION
However, wheat sprouts, in the 1 st -3 rd day in aquatic culture, have excretory activity, but the amount of excreted substances in the medium and the composition of the components are affected by presowing seed treatment. By using different methods of pre-sowing seed treatment and collecting exudates in the 1 st , 2 nd , and 3 rd day of wheat sprouts, it is possible to obtain different amounts of exudates with different compositions.
Concomitant microorganisms do not make any significant contribution to the quantitative and qualitative composition of the exudates in the aquatic culture from 1 st to 3 rd day of germination.