Comparison of Presowing Wheat Treatments by Low-Temperature Plasma, Electric Field, Cold Hardening, and Action of Tebuconazole-Based Disinfectant

Featured Application

Cereals are the basis of food safety and are common food products worldwide. Improving agricultural practices for growing cereals is a crucial task. In this work, several presowing wheat treatment approaches were compared to determine a possible replacement with alternative, renewable technological solutions instead of chemical-based industrial wheat cultivation.

Abstract

This work compares the presowing treatment of winter wheat seeds with a low-temperature plasma, a constant high-voltage electric field, a plant protection disinfectant, and cold hardening on the resistance of seedlings to freezing and their morphophysiological characteristics at the initial stage of germination. Various treatment combinations were considered, including the effect of the disinfectant jointly with low-temperature plasma treatment. The greatest stimulating effect from the point of view of seedlings’ morphophysiological characteristics was achieved when seeds were cold-hardened. The action of low-temperature plasma is noticeable up to the third day of germination. The treatment with the low-temperature plasma of seeds pretreated and not-pretreated with the disinfectant had a similar effect on the morphophysiological characteristics of seedlings. The plasma treatment and the electric field were combined with each other, i.e., the plasma treatment effects were added to the electric field effects. Resistance to low temperatures was increased with the hardening of seeds treated with the electric field and the disinfectant. Resistance to low temperatures was reduced when treated with the electric field and/or low-temperature plasma after being treated with the disinfectant.

1. Introduction

Cereals are the basis of food safety and are common food products worldwide [1,2]. According to the Food and Agriculture Organization (FAO) of the United Nations, three of them, rice, maize, and wheat, provide 60% of the world’s food energy intake [3]. Moreover, as stated by the FAO [4], “Food and agriculture production systems worldwide are facing unprecedented challenges from an increasing demand for food for a growing population, rising hunger and malnutrition, adverse climate change effects, overexploitation of natural resources, loss of biodiversity, and food loss and waste. …We need to expand and accelerate the transition to sustainable food and agriculture”. Therefore, a steady improvement in agricultural practices for cereal growing is a crucial task [5,6].

Electrophysical technologies are some of the most promising for the intensification of agricultural practices. High-voltage technologies are already widely used in electrostatic precipitators, for the electrosynthesis of ozone, in methods for separating materials in an electric field (ore dressing, redistribution of dumps, etc.), and in many others [7]. At the same time, the high-voltage techniques in the field of the electrophysical preparation of seeds for sowing, despite their long development, have not yet found widespread use in industrial agriculture. Much of this is due to a lack of fundamental understanding to produce certain predictable effects. However, to date, a large amount of patent, scientific, and technical information has been accumulated on the use of electrophysical methods in the presowing preparation of seeds [8]. Moreover, electric machines intended for seed preparation are used in field work.

Electrophysical methods have several advantages. First of all, they are environmentally friendly compared to the most common chemical methods. Secondly, they are technologically advanced, and there are no consumables other than electrical energy. Only a small number of operations with seeds are added. Namely, the seeds are guided through some additional unit or reactor. If a comparable yield increase is achieved, the environmental friendliness and manufacturability of the electrophysical method comes to the fore.

In this work, as the object of our research, we use cereals—in particular, soft wheat. At present, chemical methods of presowing preparation are most often used to protect, sanitize, and improve the resistance of wheat plants. However, some authors indicate [9,10,11,12] that promising plasma techniques will be able to replace chemical methods due to their efficiency. This would make a significant contribution to the transition to environmentally friendly farming. As a successful argument supporting this expectation, the complete suppression of seed contamination by individual pathogens after plasma treatment is ensured in [12,13]. The combined action of a fungicide and low-temperature plasma is discussed in [14].

Potentially, plasma treatment in air or gas with a high oxygen concentration carries risks similar to UV radiation processing. In the working area, ozone is intensively formed. An inevitable necessity, in this case, is the creation of protective devices in such installations for ozone decomposition. As a safer method without generating ozone, it is proposed to use a high-voltage electric field, which is also used for presowing seed preparation [15,16]. It is possible that these two methods can be combined, with an increase in efficiency, using the three-electrode surface discharge system [17], in which a constant electric field is also superimposed on the plasma volume.

However, chemical treatment provides not only a decrease in seed infestation, but it also increases the stress resistance of the plant and its protection in the soil after sowing. It seems that it is impossible to completely abandon such treatment, but it may be possible to reduce the consumption of the chemical agent through a combination of chemical and plasma treatment.

Checking the combination ability of the chemical and plasma treatments is one of the objectives of this work. To test the concept of the compatibility of plasma and chemical treatments, in this work, we chose a tebuconazole-based seed disinfectant, “Bunker” (water suspension concentrate, content of tebuconazole is 60 g/L, JSC “August” Inc., Moscow, Russia ) [18]. There is a great deal of technical information on the use of the disinfectant, including in field studies [19,20,21,22,23]. The effect of disinfectants, including the disinfectant “Bunker”, on the quality of the winter wheat crop Moskovskaya 39 (Triticum aestivum L., variety Moskovskaya 39), cultivated for sowing seed purposes, was determined in [21] in a three-year experiment on plots of 25 m${}^2$. The use of the disinfectant “Bunker” reduced the infection with root rot from 32–36% to 1–14%, accelerated the development of plants by 1–2 days, and increased the field viable germination by 11–14% and winter hardiness by 3%, and the yield average for three years was $3.5$ t/ha (control $3.09$ t/ha). The action of a chemical disinfectant increased the strength of growth, the mass of 100 shoots, the height of the shoots, and the length of the primary roots. Throughout the entire growing season, “Bunker” protected plants from root rot. Long-term protection of the plant from diseases, due to the chemical stability of the agent, is also noted in the work [22]. On the seedlings of spring soft wheat Tulunskaya 12 (Triticum aestivum L., variety Tulunskaya 12) [20] and soft winter wheat Irkutskaya (Triticum aestivum L., variety Irkutskaya) [22], the effect of various concentrations of the “Bunker” disinfectant was studied. On both spring and winter wheat, with an increase in the concentration of the disinfectant, an increase in the growth-inhibiting effect was observed, but when exposed to spring wheat, the growth of both the aboveground and underground parts of the plant slowed down, and when exposed to winter wheat, the growth of the aboveground one slowed down and the growth of the underground one accelerated. The relative frost resistance of etiolated seedlings of Irkutskaya winter wheat became larger with the increase in the concentration of the disinfectant to $1.5$ L/t from $0.5$ L/t [19]. For the “Bunker” disinfectant, a dose of $1.5$ L/t is designated as optimal for Irkutskaya winter wheat in [23].

At present, the full range of negative consequences has not been determined yet. The need to reduce the amount of chemical plant protection products used in general, and tebuconazole, in particular, is associated with the many undesirable effects that they have on the environment. Research to determine these consequences is being actively carried out [24,25,26].

There are other methods of presowing preparation aimed at increasing the resistance of plants: hydropriming, osmopriming, biological methods, the use of phytohormones, and others [27,28]. Among them, some technologies have great environmental potential and efficiency. It is also necessary to take into account the cross action [28]. This means that the action of certain stress during seed preparation provides resistance not only to this stress but also to other possible stresses acting on plants throughout the entire process of development. This concept fits the data that indicate that plasma treatment and other electrophysical technologies can provide resistance to various stresses (for example, drought, salinity, and phytopathogenic load [29]). However, under natural conditions, with a decrease in daily temperature, winter crops undergo natural hardening at a low temperature and acquire resistance to shock freezing. Pre-hardening by plasma treatment can prevent cold hardening. It is also known that the use of a tebuconazole-containing disinfectant does not interfere with cold hardening, which imitates these natural conditions [19,22].

It is often found that it is very difficult to compare data obtained by different authors, due to the neglect of individual details in the description of experimental procedures. This situation can be resolved by unifying the experimental protocol. To ensure the unity of data interpretation, we followed the methodological suggestions of [30].

Accordingly, in this work, we describe several issues of presowing treatment of soft winter wheat seeds. The following research tasks were set and investigated:

• To determine the effect of a combination of electrostatic field and plasma treatment;
• To determine the effect of the electrostatic field and plasma together with the effect of the disinfectant “Bunker”;
• To determine the effect of seed treatment with the electrostatic field and plasma on the relative frost resistance of hardened seedlings;
• To determine the effect of seed treatment with the electrostatic field and plasma on the relative frost resistance of hardened seedlings in combination with the effect of the disinfectant “Bunker”;
• To compare the effect of the presowing hardening of seeds by the action of low temperatures with the effects of the action of the plasma, electric field, and disinfectant.

2. Materials and Methods

2.1. Statistics and Experimental Design

The experiments to determine the morphophysiological characteristics of seedlings were carried out from 30 January 2021 to 13 August 2021 in 60 sets. In parallel, the relative frost resistance of seedlings was assessed. Due to the need to simultaneously compare a large number of different treatment options, the number of repetitions per option in each set was reduced to one or two. Thus, when calculating the final statistics, the number of repetitions indicated in the tables was obtained by combining the repetitions included in the different sets.

The design of the experiments excluded the possibility of a full comparison of the options with each other, since, in half of the cases, the options intersected no more than two times, and in some cases only once. Each of the variants was reliably compared only with the control (variant without any processing), which was included in each of the datasets. The length of shoots and roots was measured with a measuring ruler with a division value of $0.25$ mm. The weight of the seeds was determined on an analytical balance with an accuracy of up to $0.1$ mg.

In the present study, we considered all treatment techniques as black boxes. The criterion for the quality of processing was the effect on the frost resistance and morphophysiological characteristics of the seedlings. However, the proposed mechanisms of the physiological effects on seeds with a tebuconazole-based seed disinfectant can be found in [22,23]; cold hardening in [19]; electric field in [15,16]; and plasma in [8,9,10,11,12,13,14].

Calculations and statistical analyses of the data were performed in Microsoft Office Excel 2007 ™. The mean values for the samples were calculated with the Excel AVERAGE function, which returns the arithmetic mean of the arguments. The standard deviation from the mean was obtained with the Excel STDEV function. The STDEV function returns the square root of the sum of squares of variation, defined as the difference between each value and the mean, and then divided by the square root of the number of data points minus one. A 95% confidence interval was estimated with the Excel CONFIDENCE function.

2.2. Seed Material and Seedling Handling Methods

High-quality soft winter wheat “Irkutskaya” (Triticum aestivum L., variety Irkutskaya), harvested in 2017, was used as a model object. The seeds were provided from the collections [31] of The Core Facilities Center “Bioresource Center” at The Siberian Institute of Plant Physiology and Biochemistry SB RAS (Irkutsk, Russia). The seeds were cleared of dust and impurities. One portion of the seeds was stored without any treatment. Other seeds were treated with the disinfectant “Bunker” at a dose of $0.5$ and $1.5$ l/(t of seeds). After this, the seeds were stored for approximately two years.

A detailed illustrative diagram of the seedling handling in this research is shown in Figure 1. The main steps for determining the results of the treatments are described below.

After the treatment (in Figure 1) with an electric field or/and plasma, the seeds were placed for germination in a thermostat immediately.

The seeds prepared for the assessment of their morphophysiological characteristics were germinated in a thermostat in the dark at $24\pm 1$ °C and 99% humidity on two layers of filter paper moistened with distilled water. The seeds were laid out at a distance of approximately 5 mm from each other, with five samples per individual container. The morphophysiological characteristics of the seedlings were assessed at the 3rd, 7th, and 9th days of germination. On the 3rd day, half of the sample was taken, and on the 7th and 9th days, one quarter was taken on each. The seed germination layout in the thermostat prepared for the subsequent determination of morphophysiological characteristics is shown in Figure 2. The germination capacity was determined as the ratio of the number of germinated seeds to their total number in the selected sample. For each seedling, the length of the shoots and individual roots was determined. For each part of the sample, the wet and dry (drying at 125 $°$C to constant weight) mass of shoots and roots were determined.

The cold hardening of seeds and seedlings was carried out according to a two-stage technique [32]. We used this technique in our previous work [33]. Seeds and seedlings were hardened in a climatic chamber in the dark in two stages – (in Figure 1). The first stage was 7 days at $+2$ $°$C. The second stage was 3 days at $-4$ $°$C.

Determination of the relative frost resistance of seedlings was carried out as follows. The seedlings after germination in a thermostat were divided into samples of 50 pieces in gauze bags . After the hardening – , the seedlings were subjected to freezing for one day at temperatures of $-14$, $-16$, $-18$, and $-20$ $°$C consistently. One quarter of the seedlings were taken out on one day at $-14$ $°$C ; after the second day at $-16$ $°$C, the second quarter of the seedlings was taken out , the third one a day later at $-18$ $°$C , and, finally, the last quarter after another day at $-20$ $°$C . After this, they were exposed to defrosting , thawing , and completion of growing . The relative frost resistance of seedlings was determined as the proportion of seedlings that survived freezing. To determine the proportion of surviving seedlings after the thawing stage , the seedlings were laid out in groups of samples on one layer of filter paper and germinated in the dark in distilled water for up to 7 days. We placed up to 20 samples in one container (Figure 3). Seedlings that started growing within 7 days were classified as survivors. A sample count example is shown in Figure 4.

The seed contamination was determined in the Laboratory of Mycology and Phytopathology at the All-Russian Institute of Plant Protection (FSBSI VIZR, Saint-Petersburg, Russia) [34]. Identification of phytopathogenic fungi was performed on potato sucrose agar media. Before filling Petri dishes with the potato sucrose agar media, a solution of a mixture of HyClone ™ antibiotics at a concentration of 1 mL/L (to reduce the growth of bacteria) and a Triton × 100 solution at a concentration of $0.004$ $\mathsf{\mu }$L/L (to reduce linear growth of filamentous fungi) were introduced into the media previously cooled to 55 $°$C.

For surface sterilization, the seeds were previously soaked in “BIOREBA” ©extraction bags in water with the addition of surfactants, and then washed in running water and sterilized with a 3% dichlorine disinfectant for 1–3 min. After using the sterilizing agent, the seeds were carefully washed first by tap water, and then sterile water. In the laminar box, the dried, surface-sterilized seeds were subjected to a burner flame and laid out in groups of 10 seeds on the potato sucrose agar surface in Petri dishes.

After 10 days of incubation in the dark at 24 $°$C, the identification of phytopathogenic fungi and species populations was performed. Seed contamination (%) was calculated as the number of seeds that had been allocated to a certain taxonomic fungi group, to the total number of seeds. The total amount of micromycetes could exceed the value of the number of analyzed seeds, since several micromycetes may be released from one seed.

2.3. Experimental Setup and Seed Treatment

The experimental setup and all its main components, such as the high-voltage direct current (HVDC) source, high-voltage alternating current (HVAC) generator, and others, were designed and produced by members of the authors’ team.

Seed treatment with low-temperature plasma was carried out in a surface dielectric-barrier discharge reactor. The electrode configuration is presented in Figure 5. The electrode system consisted of nine parallel copper foil strips (20 $\mathsf{\mu }$m copper foil) on one side, with a corundum ceramic barrier (${\mathrm{Al}}_2{\mathrm{O}}_3$ plate with thickness 1 mm). Each strip’s length was 130 mm. A return electrode covered the entirety of the opposite side of the dielectric barrier plate.

Two variants of applied voltage were used with the grounded return electrode (Figure 5, Var a) or with a constant high-voltage bias of $+5$ kV at the return electrode (Figure 5, Var b). The connecting scheme of the power sources to the electrodes is shown in Figure 6. The constant high-voltage bias potential was applied to the return electrode through the output winding of the output transformer (see Figure 6). The geometric capacitance of the electrode system was 298 pF. A measuring capacitor of $22.6$ nF was introduced into the grounded return electrode circuit for the determination of the discharge energy by the Volt–Coulomb characteristics procedure, as in [35]. Voltage was measured with a 1:1000 Tektronix P6015A high-voltage probe. The additional extraction plane electrode was separated by a 10 mm air gap from the dielectric surface. Seeds were placed on it in one layer. Thus, in the scheme with a combination of the action of surface discharge and a constant high-voltage field, the seeds were placed always on a grounded plate. In one experiment in each configuration, no more than 20 g of seeds could be processed. Plasma treatment was carried out for 1 min.

The power completely determined the parameters of the plasma in the system. The plasma covered the area between the strip electrodes with a thin layer. The effect on seeds was determined by the field, ion flow, and other factors. The parameters of plasma, ion flow to the substrate, and optical characteristics in the system designed by the authors’ team can be found in [35,36,37].

The treatment with a high-voltage electrostatic field was carried out in a plane-parallel configuration with a distance between the electrodes of 10 mm. The seeds were arranged in one layer on a grounded aluminum electrode. A positive voltage of 5 kV was applied to the second electrode. Treatment with the electric field was carried out for 1 and 10 min.

Regarding the choice of exposure time, short times of 1 and 10 min were chosen for more preferable implementation in practical applications. Treatment with plasma and discharge products with long exposure times of up to 60 min were previously studied by our group [38,39]. However, processing reasonable amounts of seeds for use in real applications is difficult at long exposure times.

In all cases, the seeds did not move in the electrode system. In the case of the action of the electric field, if individual seeds were unfolded or thrown up due to volume polarization, the experiment was rejected.

3. Results and Discussion

3.1. Experimental Data

The results of measuring the morphophysiological characteristics of seedlings on the third, seventh, and ninth days of seedling development are shown in

Table 1. Morphophysiological characteristics of seedlings on the 3rd, 7th, and 9th days of development after seed treatment with cold hardening, disinfectant, plasma, and electric field. Control denotes variants without any treatment; cold hardening refers to cold hardening of the seeds with two stages; Bunker 0.5 indicates samples treated with the “Bunker” disinfectant at a dose of $0.5$ L/t and Bunker 1.5 with $1.5$ L/t; Bunker 0.5, 16 kHz/1.5 kV indicates variants with seeds pretreated with “Bunker” disinfectant at a dose of $0.5$ L/t and subsequently treated by discharge at $1.5$ kV and 16 kHz for 1 min; Bunker 1.5, 5 kV/cm 1 min denotes variants with seeds pretreated with “Bunker” disinfectant at a dose of $1.5$ L/t and subsequently treated with electric field of 5 kV/cm for 1 min; and Bunker 1.5, 16 kHz/1.5 kV +5 kV denotes variants with seeds pretreated with “Bunker” disinfectant at a dose of $1.5$ L/t and subsequently treated by discharge at $1.5$ kV and 16 kHz with bias voltage of 5 kV for 1 min.

	Control	Cold Hardening	Bunker 0.5	Bunker 1.5	Bunker 0.5 16 kHz/1.5 kV	Bunker 1.5 5 kV/cm 1 min	Bunker 1.5 16 kHz/1.5 + 5 kV
3rd day of Germination
Shoot length $\pm d95$% (mm)	$10.1\pm 0.4$	$14.4\pm 0.8$	$10.6\pm 0.4$	$9.5\pm 0.4$	$12\pm 0.8$	$9.8\pm 0.8$	$11.9\pm 0.8$
Root length $\pm d95$% (mm)	$18.6\pm 0.3$	$24.3\pm 0.7$	$16.5\pm 0.4$	$18.0\pm 0.4$	$18.6\pm 0.7$	$15.6\pm 0.8$	$18.3\pm 0.7$
Total root length $\pm d95$%  (mm)	$53.3\pm 1.5$	$70.7\pm 3.2$	$49.4\pm 1.8$	$53.7\pm 1.4$	$54\pm 3.1$	$43.4\pm 3.6$	$53.4\pm 3.4$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$8.1\pm 1.4$	$12.5\pm 2.1$	$8.4\pm 2.2$	$6.8\pm 1.1$	$8.4\pm 2.6$	$6.9\pm 2.3$	$8.6\pm 1.7$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$1.5\pm 0.2$	$2.0\pm 0.4$	$1.4\pm 0.3$	$1.5\pm 0.2$	$1.4\pm 0.3$	$1.3\pm 0.3$	$1.6\pm 0.3$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$9.6\pm 2.3$	$15.5\pm 2.4$	$5.8\pm 1.1$	$4.2\pm 0.8$	$6.1\pm 0.8$	$5.2\pm 0.4$	$6.5\pm 1.4$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$1.7\pm 0.3$	$2.3\pm 0.3$	$1.5\pm 0.2$	$1.6\pm 0.1$	$1.3\pm 0.2$	$1.2\pm 0.07$	$1.3\pm 0.2$
Germination $\pm d95$%	$0.73\pm 0.07$	$0.75\pm 0.18$	$0.80\pm 0.10$	$0.91\pm 0.04$	$0.75\pm 0.10$	$0.70\pm 0.19$	$0.81\pm 0.10$
Number of sets, pcs	16	6	5	4	4	2	4
Total number of plants, pcs	767	233	347	366	135	80	130
7th day of Germination
Shoot length $\pm d95$% (mm)	$94.5\pm 3.1$	$103\pm 7.6$	$83\pm 3.1$	$80\pm 2.6$	$91.5\pm 4.8$	$73.4\pm 5.3$	$89\pm 4.6$
Root length $\pm d95$% (mm)	$64.1\pm 1.6$	$55.5\pm 3.0$	$49\pm 1.7$	$48\pm 1.7$	$42.2\pm 2.4$	$26.6\pm 1.2$	$47.6\pm 2.6$
Total root length $\pm d95$%  (mm)	$261.2\pm 9.2$	$222\pm 16.4$	$217\pm 10.5$	$220\pm 10.2$	$197\pm 17.0$	$131\pm 7.9$	$203\pm 15.8$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$67.0\pm 4.3$	$70.5\pm 5.3$	$60\pm 7.0$	$54\pm 9.7$	$62.6\pm 5.3$	$52.1\pm 10.4$	$60.8\pm 4.2$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$7.2\pm 0.8$	$7.2\pm 0.7$	$6.9\pm 0.8$	$6.6\pm 0.6$	$7.1\pm 0.9$	$5.6\pm 0.2$	$6.9\pm 0.9$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$26.7\pm 7.4$	$44.9\pm 9.8$	$35.8\pm 13$	$24\pm 12.1$	$30.5\pm 16.0$	$14.9\pm 11.2$	$31.1\pm 9.9$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$4.0\pm 0.5$	$4.2\pm 0.6$	$4.3\pm 0.8$	$4.2\pm 0.6$	$3.9\pm 1.3$	$2.8\pm 0.5$	$4.1\pm 1.2$
Germination $\pm d95$%	$0.80\pm 0.02$	$0.65\pm 0.33$	$0.86\pm 0.05$	$0.90\pm 0.04$	$0.80\pm 0.10$	$0.69\pm 0.32$	$0.84\pm 0.11$
Number of sets, pcs	11	3	5	4	4	2	2
Total number of plants, pcs	446	98	287	246	126	73	138
9th day of Germination
Shoot length $\pm d95$% (mm)	$117\pm 3.3$	$144\pm 10.5$	$116\pm 3.5$	$110\pm 3.4$	$115\pm 7.5$	$112\pm 10.8$	$121\pm 6.4$
Root length $\pm d95$% (mm)	$85\pm 1.7$	$106\pm 5.9$	$62\pm 2.3$	$60\pm 2.1$	$64\pm 3.8$	$51\pm 5.2$	$80.4\pm 4.1$
Total root length $\pm d95$%  (mm)	$343\pm 9.1$	$431\pm 29.7$	$278\pm 14.9$	$270\pm 12.5$	$272\pm 19$	$236\pm 30.2$	$336\pm 25$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$77.0\pm 6$	$96.9$	$73\pm 6.0$	$67\pm 4.0$	$71.5\pm 2.3$	$68.4$	$76.8\pm 5.5$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$7.7\pm 0.6$	$9.9$	$7.9\pm 0.4$	$7.7\pm 0.4$	$6.9\pm 0.4$	$8.5$	$8.6\pm 1.9$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$39.5\pm 9.8$	$76.7$	$42\pm 23.6$	$35\pm 18.2$	$30.7\pm 5$	$28.3$	$38.2\pm 22$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$4.7\pm 0.3$	$6.6$	$4.7\pm 1.2$	$4.8\pm 0.8$	$4.2\pm 0.6$	$4.6$	$5.3\pm 1.3$
Germination $\pm d95$%	$0.90\pm 0.03$	$0.88$	$0.90\pm 0.05$	$0.90\pm 0.01$	$0.88\pm 0.10$	$0.70$	$0.88\pm 0.09$
Number of sets, pcs	11	1	5	4	3	1	3
Total number of plants, pcs	543	46	266	253	91	31	104

and

Table 2. Morphophysiological characteristics of seedlings on the 3rd, 7th, and 9th days of development after seed treatment with plasma and/or electric field. Control denotes variants without any treatment; 4.4 kHz/1.5 kV denotes variants treated by discharge at $1.5$ kV and 4 kHz for 1 min; 4.4 kHz/2.4 kV by discharge at $2.4$ kV and 4 kHz for 1 min; 16 kHz/1.5 kV denotes variants treated by discharge at $1.5$ kV and 16 kHz for 1 min; 16 kHz/2.4 kV by discharge at $2.4$ kV and 16 kHz for 1 min; 5 kV/cm 1 min treated by electric field of 5 kV/cm for 1 min; 5 kV/cm 10 min treated by electric field of 5 kV/cm for 10 min; and 16 kHz/1.5 kV +5 kV denotes variants treated by discharge at $1.5$ kV and 16 kHz with bias voltage of 5 kV for 1 min.

	Control	4.4 kHz/1.5 kV	4.4 kHz/2.4 kV	16 kHz/1.5 kV	16 kHz/2.4 kV	5 kV/cm 1 min	5 kV/cm 10 min	16 kHz /1.5 + 5 kV
3rd day of Germination
Shoot length $\pm d95$% (mm)	$10.6\pm 0.4$	$9.6\pm 0.7$	$8.8\pm 0.6$	$12\pm 0.4$	$9.8\pm 0.7$	$9.9\pm 0.7$	$10.8\pm 0.5$	$10.6\pm 0.5$
Root length $\pm d95$% (mm)	$19.3\pm 0.4$	$18.3\pm 0.7$	$17\pm 0.5$	$21\pm 0.4$	$18.7\pm 0.7$	$19.6\pm 0.73$	$18.4\pm 0.5$	$20.0\pm 0.5$
Total root length $\pm d95$%  (mm)	$55.5\pm 1.6$	$51.7\pm 3.3$	$48\pm 2.4$	$61\pm 1.8$	$52.7\pm 3.2$	$57.1\pm 3.3$	$52.7\pm 2.2$	$57.1\pm 2.3$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$8.1\pm 1.3$	$6.0\pm 3.2$	$5.7\pm 2.2$	$9.1\pm 1.75$	$6.1\pm 1.6$	$6.6\pm 2.4$	$7.7\pm 1.4$	$7.6\pm 1.4$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$1.4\pm 0.2$	$1.4\pm 0.5$	$1.2\pm 0.1$	$1.4\pm 0.2$	$1.5\pm 0.3$	$1.3\pm 0.4$	$1.3\pm 0.3$	$1.5\pm 0.4$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$8.0\pm 2.1$	$5.2\pm 3.1$	$6\pm 3.0$	$8.3\pm 2.6$	$5.1\pm 3.4$	$5.0\pm 3.6$	$7.4\pm 2.5$	$7.0\pm 1.9$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$1.5\pm 0.2$	$1.1\pm 0.4$	$1.1\pm 0.3$	$1.4\pm 0.3$	$1.3\pm 0.2$	$1.2\pm 0.3$	$1.3\pm 0.4$	$1.4\pm 0.3$
Germination $\pm d95$%	$0.75\pm 0.06$	$0.72\pm 0.18$	$0.70\pm 0.11$	$0.80\pm 0.07$	$0.77\pm 0.11$	$0.79\pm 0.13$	$0.70\pm 0.12$	$0.69\pm 0.10$
Number of sets, pcs	16	4	4	12	3	3	8	8
Total number of plants, pcs	743	207	207	650	166	164	363	364
7th day of Germination
Shoot length $\pm d95$% (mm)	$92.8\pm 2.8$	$84.6\pm 3.8$	$87\pm 4.0$	$90\pm 3.2$	$86.3\pm 4.2$	$83.7\pm 3.8$	$88.5\pm 3.2$	$91.8\pm 3.0$
Root length $\pm d95$% (mm)	$59.7\pm 1.4$	$58.5\pm 2.2$	$64\pm 2$	$63\pm 1.7$	$54.7\pm 2.2$	$65.2\pm 2.2$	$65.8\pm 2.0$	$60.4\pm 1.7$
Total root length $\pm d95$%  (mm)	$247.2\pm 7.8$	$239\pm 12.3$	$255\pm 11.3$	$265\pm 10$	$230\pm 12.7$	$258\pm 10.7$	$263\pm 11.2$	$248\pm 10.1$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$67.4\pm 3.9$	$62.2\pm 2.1$	$61\pm 7.0$	$66\pm 2.7$	$60.4\pm 1.8$	$60\pm 3.1$	$64\pm 4.8$	$65.0\pm 5.1$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$7.1\pm 0.7$	$5.9\pm 0.7$	$6.4\pm 0.9$	$6.9\pm 0.5$	$6.2\pm 0.4$	$6.8\pm 0.8$	$6.8\pm 0.8$	$6.8\pm 0.9$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$26.5\pm 6.1$	$25\pm 3.0$	$17\pm 3.9$	$27\pm 6.5$	$21.7\pm 6.6$	$30\pm 10.4$	$29\pm 5.2$	$31.1\pm 6.1$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$3.9\pm 0.5$	$3.2\pm 0.4$	$3.7\pm 0.5$	$4.1\pm 0.7$	$3.3\pm 0.4$	$3.8\pm 0.4$	$3.8\pm 0.4$	$4.1\pm 0.9$
Germination $\pm d95$%	$0.82\pm 0.07$	$0.89\pm 0.06$	$0.90\pm 0.05$	$0.90\pm 0.06$	$0.90\pm 0.03$	$0.90\pm 0.04$	$0.86\pm 0.10$	$0.79\pm 0.09$
Number of sets, pcs	13	4	5	8	4	5	6	9
Total number of plants, pcs	535	198	233	347	182	237	244	395
9th day of Germination
Shoot length $\pm d95$% (mm)	$115.9\pm 3.3$	$109\pm 6.1$	$109\pm 4.9$	$119\pm 3.6$	$106\pm 6.1$	$109\pm 5.3$	$113\pm 5.0$	$122\pm 5.5$
Root length $\pm d95$% (mm)	$77.9\pm 1.7$	$71.9\pm 2.6$	$76\pm 2.5$	$82\pm 1.8$	$62.5\pm 2.6$	$68.3\pm 2.4$	$78\pm 2.5$	$81\pm 2.7$
Total root length $\pm d95$%  (mm)	$319.5\pm 8.9$	$286\pm 14$	$303\pm 14.8$	$336\pm 10.1$	$261\pm 12.9$	$282\pm 12.6$	$322\pm 14.7$	$347\pm 17.1$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$76.2\pm 4.2$	$70.8\pm 5.8$	$69\pm 3.7$	$77\pm 6.0$	$67.8\pm 4.3$	$70.5\pm 6.5$	$74\pm 6.1$	$75.5\pm 6.0$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$7.5\pm 0.5$	$7.1\pm 1.0$	$7.3\pm 0.4$	$7.7\pm 0.7$	$7.1\pm 0.4$	$7.2\pm 0.8$	$7.2\pm 0.8$	$8.0\pm 1.1$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$35.8\pm 9.9$	$26.9\pm 8.9$	$29\pm 10.8$	$42\pm 10.0$	$19.9\pm 6.7$	$26.7\pm 9.2$	$32.3\pm 7.7$	$32.9\pm 7.3$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$4.4\pm 0.4$	$4.0\pm 0.3$	$4.5\pm 1.0$	$4.8\pm 0.7$	$3.7\pm 0.5$	$3.6\pm 0.6$	$4.2\pm 0.7$	$4.5\pm 1.3$
Germination $\pm d95$%	$0.89\pm 0.02$	$0.91\pm 0.05$	$0.90\pm 0.04$	$0.90\pm 0.02$	$0.88\pm 0.02$	$0.92\pm 0.01$	$0.88\pm 0.12$	$0.84\pm 0.15$
Number of sets, pcs	12	4	5	11	4	5	6	5
Total number of plants, pcs	555	177	246	501	170	211	231	230

.

Table 3. Relative frost resistance of two-stage hardened seedlings after various seed treatments. mean $\pm d95$% indicates the mean value with 95% confidence interval, NR indicates the number of repetitions. Designations of variants coincide with designations in Table 1 and Table 2.

	$-14$ $°$C		$-16$ $°$C		$-18$ $°$C		$-20$ $°$C
	mean$\pm \mathit{d}\mathbf{95}$%	NR	mean$\pm \mathit{d}\mathbf{95}$%	NR	mean$\pm \mathit{d}\mathbf{95}$%	NR	mean$\pm \mathit{d}\mathbf{95}$%	NR
Control	$0.69\pm 0.06$	64	$0.55\pm 0.09$	52	$0.34\pm 0.08$	55	$0.25\pm 0.08$	62
Bunker 0.5	$0.70\pm 0.25$	13	$0.69\pm 0.15$	20	$0.53\pm 0.16$	16	$0.41\pm 0.15$	14
Bunker 1.5	$0.91\pm 0.07$	10	$0.78\pm 0.20$	5	$0.73\pm 0.18$	3	$0.58\pm 0.27$	4
Bunker 0.5; 16 kHz/1.5 kV	$0.90\pm 0.05$	5	$0.53\pm 0.18$	5	$0.20\pm 0.18$	9	$0.12\pm 0.13$	10
Bunker 0.5; 5 kV/cm 1 min	$0.52\pm 0.45$	3	$0.04\pm 0.03$	4	$0.01\pm 0.02$	4	$0.01\pm 0.01$	6
Bunker 0.5; 16 kHz/1.5 + 5 kV	$0.87\pm 0.13$	2	$0.62\pm 0.54$	3	$0.11\pm 0.18$	3	$0.12\pm 0.14$	4
4.4 kHz/1.5 kV	$0.83\pm 0.11$	7	$0.47\pm 0.26$	7	$0.39\pm 0.19$	7	$0.29\pm 0.24$	6
4.4 kHz/2.4 kV	$0.57\pm 0.13$	23	$0.50\pm 0.09$	28	$0.41\pm 0.12$	32	$0.36\pm 0.11$	36
16 kHz/1.5 kV	$0.75\pm 0.11$	27	$0.56\pm 0.15$	15	$0.35\pm 0.12$	25	$0.31\pm 0.13$	27
16 kHz/2.4 kV	$0.74\pm 0.18$	7	$0.80\pm 0.17$	9	$0.53\pm 0.35$	7	$0.48\pm 0.35$	8
5 kV/cm 1 min	$0.88\pm 0.09$	9	$0.80\pm 0.19$	13	$0.50\pm 0.19$	13	$0.51\pm 0.21$	15
5 kV/cm 10 min	$0.69\pm 0.15$	11	$0.47\pm 0.22$	11	$0.38\pm 0.19$	13	$0.40\pm 0.19$	14
16 kHz/1.5 kV + 5 kV	$0.59\pm 0.16$	11	$0.64\pm 0.15$	16	$0.63\pm 0.19$	17	$0.51\pm 0.15$	17

shows the results of the measurement of the relative frost resistance of seedlings.

Even after the long-term storage of wheat seeds treated with the “Bunker” disinfectant at a dose of $0.5$ and $1.5$ L/t, they retained high germinating capacity at all stages of germination considered.

The power was the control parameter for the treatments with plasma. The discharge power values determined by the Volt–Coulomb characteristics method [35] at two frequencies of sinusoidal applied voltages of $4.4$ and 16 kHz are shown in

Table 4. Discharge power for various plasma treatment regimes.

$U_{RMS}$, kV	$0.5$	$0.75$	1	$1.5$	$1.75$	2	$2.1$	$2.4$
Frequency	Power, W
4.4 kHz	$0.3$	$0.7$	$1.3$	$3.2$	$4.3$	$6.3$	$7.7$	$9.4$
16 kHz	$0.7$	$1.9$	3	$7.4$	$12.6$	$15.9$	$22.4$	$32.2$

.

In experiments comparing the effect of plasma and an electric field on the resistance to negative temperatures of unhardened wheat seedlings, no distinct protective effect was found. In the control unhardened variants, no surviving seedlings were found when exposed to temperatures in the entire range from $-14$ $°$C to $-20$ $°$C. In variants with treatments at a temperature of $-14$ $°$C, single surviving plants were found.

An important factor in plasma and electric field treatments is whether the seed layer is placed on a high-voltage or on a grounded electrode and whether plasma is present above this seed layer. Previously, we published data on the effect of a constant high-voltage field on spring wheat seeds [40]. The best morphophysiological characteristics were obtained for seeds in contact with a metal electrode under a high positive voltage. Moreover, in Section 2.3, we mentioned the turning of seeds in a high-voltage field. The turning of seeds was observed in [40] under the action of polarization forces (see Figure 1 in [40]). In this work, with such contact and with the presence of plasma, the qualitative characteristics of germination were always inhibited. The best results were obtained for seeds on a grounded electrode. Furthermore, at high DC bias voltages, no such turning was observed in the presence of plasma above the seed layer.

In some cases, the treatment did not work uniformly. Not every replication achieved the same effect: some of them fluctuated around the control level, while some were wider. Thus, the confidence interval could be wider than the control variant.

3.2. Chemical Treatment

The effect of the disinfectant “Bunker” in doses of $0.5$ and $1.5$ L/t on the shoot and root growth of the winter wheat green seedlings (Triticum aestivum L., variety Irkutskaya, grown by imitation day/night on Knop’s solution) on the third, seventh, and ninth days of development was compared in [22]. In particular, the shoot length, total root length, raw mass of shoots, and wet and dry weight of shoots and roots were determined. A comparison of the results for the seventh and ninth days with our results is given in

Table 5. Morphophysiological characteristics of seedlings on the 7th and 9th days of development. Comparison of current work’s data with reference work [22]. Designations of variants coincide with designations in Table 1.

	Reference [22]			Current Values
7th day of Germination
	Control	Bunker 0.5	Bunker 1.5	Control	Bunker 0.5	Bunker 1.5
Shoot length $\pm d95$% (mm)	$121\pm 11$	$87\pm 6$	$69\pm 2$	$94\pm 3$	$83\pm 3$	$80\pm 3$
Total root length $\pm d95$%  (mm)	$228\pm 16$	$211\pm 7$	$211\pm 9$	$261\pm 9$	$217\pm 11$	$220\pm 10$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$69.2\pm 13.8$	$56.6\pm 4.0$	$63.5\pm 11.9$	$67\pm 4.3$	$60\pm 7.0$	$54\pm 9.7$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$7.0\pm 1.1$	$6.3\pm 0.7$	$6.6\pm 1.0$	$7.2\pm 0.8$	$6.9\pm 0.8$	$6.6\pm 0.6$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$30.9\pm 6.0$	$31.3\pm 7.7$	$35.6\pm 7.6$	$26.7\pm 7.4$	$35.8\pm 13.0$	$24\pm 12$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$2.2\pm 0.1$	$2.5\pm 0.2$	$2.5\pm 0.3$	$4.0\pm 0.5$	$4.3\pm 0.8$	$4.2\pm 0.6$
9th day of Germination
Shoot length $\pm d95$% (mm)	$168\pm 12$	$125\pm 8$	$103\pm 8$	$117\pm 3$	$116\pm 4$	$110\pm 3$
Total root length $\pm d95$%  (mm)	$269\pm 14$	$295\pm 13$	$309\pm 21$	$341\pm 9$	$278\pm 15$	$270\pm 13$
Raw mass of shoots
per plant $\pm d95$%  (mg/pcs)	$118.8\pm 11$	$105.2\pm 4.5$	$107.0\pm 6.5$	$77\pm 6$	$73\pm 6$	$67\pm 4$
Dry mass of shoots
per plant $\pm d95$% (mg/pcs)	$11.2\pm 1.2$	$10.4\pm 0.2$	$10.3\pm 0.6$	$7.7\pm 0.6$	$7.9\pm 0.4$	$7.7\pm 0.4$
Raw mass of roots
per plant $\pm d95$% (mg/pcs)	$34.4\pm 3.8$	$41.2\pm 4.0$	$45.7\pm 8.6$	$39.5\pm 9.8$	$42\pm 23.6$	$35\pm 18$
Dry mass of roots
per plant $\pm d95$% (mg/pcs)	$2.5\pm 0.2$	$2.9\pm 0.1$	$3.5\pm 0.5$	$4.7\pm 0.3$	$4.7\pm 1.2$	$4.8\pm 1.8$

. Treatment with a disinfectant increased germination on the third and seventh days of development. On the seventh day of development, the main tendencies found in [22] were reproduced during germination in the dark.

The disinfectant inhibited the shoot length; with an increase in the dose of the disinfectant, the inhibition became stronger. The length of the root system was also inhibited in the control variant. There was no difference in the fresh mass of shoots and roots. The dry mass of the shoot and roots did not respond to the treatment with the disinfectant. By the ninth day, on seedlings grown in the dark, tendencies towards the dose-dependent inhibition of the shoot length and the length of the root system and the invariability of the dry mass of shoots and roots persisted. It could be seen that this was not the case for green seedlings, where the root systems of plants treated with the disinfectant developed more intensively.

The disinfectant increased the relative frost resistance (Table 3) at a dose of $0.5$ L/t at temperatures of $-16$ and $-18$ $°$C and at a dose of $1.5$ L/t over the entire range of considered negative temperatures. This is consistent with literature data [19,23]. Thus, the plant, after treating the seeds with the disinfectant at a dose of $0.5$ L/t, still retained the potential for a further increase in frost resistance. Based on these results, we carried out a combination of treatments with plasma and an electric field with the disinfectant treatment at the dose of $0.5$ L/t.

The action of the disinfectant had the expected decontaminating effect. The results of measuring the contamination of seeds with fungi in the control variant and the variant treated with the disinfectant are shown in

Table 6. Contamination of seeds with fungi.

Seedborne Fungi	Seed Contamination, %
	Control	Bunker 0.5
Alternaria	92	85
Bipolaris	2	0
Cladosporium	1	0
Epicoccum	8	5
Fusarium	6	0
Mucor	1	0
Penicillium	5	4
Trichothecium	2	0

. If the main route through which the gas discharge affects germination is a decrease in contamination, then, in the variant with the combined action of the disinfectant and the discharge, the efficiency of the gas-discharge treatment should decrease.

3.3. Cold Hardening

Seedlings obtained from hardened seeds developed more intensively than others. The increase was observed in most of the monitored indicators on the third, seventh, and ninth days of development, but was not observed afterwards in any of the treatments considered. However, germination did not increase at any stage of development.

3.4. Constant Electric Field

There was a clear effect of the constant electric field on the morphophysiological characteristics with the exposure time. At 10 min of exposure, in most cases, the morphophysiological characteristics were higher than at 1 min. On the third day of development, the seedlings showed root and shoot lengths close to those of the control plants, but they showed a lower mass both in the roots and in the shoots. On the seventh day, the length of the root system was greater than that of the control, and that of the shoot was lower than that of the control. On the ninth day, there was no difference from the control variant.

From the point of view of relative frost resistance, the opposite exposure effect was observed. The action of the electric field at an exposure time of 1 min increased the relative frost resistance in the entire range of freezing temperatures. At 10 min of exposure, there was no effect.

The action of the constant electric field was not combined with the use of the disinfectant. Electric field treatment of seeds pretreated with the disinfectant inhibited almost all morphophysiological characteristics of seedlings and, most importantly, lowered the germination capacity at all stages below not only the level of germination for the treatment with the disinfectant but below the control variant values on the seventh and ninth days. Moreover, this combination reduced the relative frost resistance of seedlings. At $-14$ $°$C, resistance decreased, and with a further decrease in temperature, plant survival dropped to almost zero.

It is likely that, with an increase in the time of exposure of seeds to an electric field, it will be possible to stimulate the development of seedlings. However, even a 1 min exposure time appears to be too long when mass-processing seeds for industrial agriculture field work. The desired optimum time for industrial farming is a few seconds. A positive result is that it was possible to influence the stability precisely at shorter times and, probably, this effect will persist with such a short processing time.

3.5. Plasma Treatment

The effect of plasma exposure was observed only on the third day of plant development in the mode 16 kHz/$1.5$ kV (discharge at RMS voltage of $1.5$ kV and 16 kHz for 1 min). In other modes, either no effect was observed, or it was inhibitory in nature. The action of the discharge in all considered modes did not affect the relative frost resistance.

When the action of the plasma and the constant electric field (mode 16 kHz/$1.5+5$ kV) was combined, the effects were combined. The obvious stimulating effect on the morphophysiological characteristics of seedlings was lost on the third day, but at the same time, the relative frost resistance increased at freezing temperatures of $-18$ and $-20$ $°$C, similar to the variant in which only a constant electric field was applied.

The mode 16 kHz/$1.5$ kV was researched in combination with the disinfectant as this mode had the greatest influence on the development of seedlings. The effect was found to be similar to that on untreated seeds. On the third day, stimulation of seedling development was observed. On the third and seventh days, the indicators did not differ significantly from the control ones. Frost resistance of seedlings obtained from seeds treated with both the disinfectant and plasma at a freezing temperature of $-14$ $°$C was higher than in the control plants and higher than that of seedlings treated only with the disinfectant. At all other temperatures, the stability was comparable to the control plants but lower than that of the variant treated only with the disinfectant.

The constant electric field in combination with the action of a gas discharge in the 16 kHz/$1.5$ kV mode, applied to seeds pretreated with the disinfectant, did not change the values regarding treatment without the application of a constant field.

Electrophysical treatment with plasma and a constant electric field were combined, at least for short exposure times, and applied to wheat seeds. This allows the use of these techniques in real industrial solutions, where it is necessary to minimize the time of seed treatment. However, the combination with preliminary chemical seed preparation with a disinfectant has obvious limitations. It could be assumed that the aggressive action of the discharge products destroys the active substance of the disinfectant, but, under the action of a constant electric field, the negative effect is more pronounced. Thus, the reasons for this negative interaction reside in physiological processes. Moreover, the electric field plays an important role in this effect. The solution to this problem may be to change the sequence of seed treatment, i.e., the seeds should be treated with a disinfectant after treatment with electrophysical methods.

4. Conclusions

Treatments of wheat seeds with a gas discharge and an electric field can be combined, with the preservation of the effects of the techniques at exposure times of no longer than 1 min.

Electrophysical techniques increase the morphophysiological characteristics of seedlings with treatment with a plant protection disinfectant such as “Bunker”. This effect is similar to the effect on seeds not treated with a disinfectant. Under the action of plasma on seeds treated with the disinfectant, the relative frost resistance drops critically. Plasma effects are reduced for the lowest temperatures with subsequent freezing. It may be possible to obtain positive effects if the treatment sequence is changed, i.e., first applying the electrophysical treatment, and then using the disinfectant.

Two-stage hardening of seedlings and seeds with low positive and negative temperatures has been shown to be the best treatment to increase the resistance of seedlings to low temperatures and intensify the development of wheat plants at the initial stages of germination. If the seedlings are not hardened, none of the considered methods give a positive effect on the relative frost resistance.