Effect of Cold Plasma on the Germination and Seedling Growth of Durum Wheat Genotypes

Abstract

Cold atmospheric pressure plasma (CAP) has attracted increased interest in recent years for possible biomedical, environmental and agricultural applications. A wide range of cold plasma treatment effects is observed in agricultural applications, like effects on the seed germination and seedling growth, but more systematic investigations are needed. The aim of this study was to identify the most appropriate combinations of the plasma source and duration of treatment positively affecting seed germination. In addition, the effect of cold plasma on the seedling growth and osmotic stress tolerance was studied. The seeds of three Bulgarian durum wheat cultivars were treated with cold plasma in twelve variants. The results obtained were processed statistically via two-way ANOVA. The treatment of seeds with a plasma torch for 20 s and the treatment with underwater diaphragm discharge for 5 min when the seeds were placed in both cameras in two different positions (relative to the electrodes between which the plasma is supplied, “+” and “−”) have the greatest positive effect on the all traits related to germination. The analysis of variance reveals that the variation in germination energy, shoot length and root length after the cold plasma treatment of seeds is mainly due to the interaction between the genotype and treatment variant and to a small degree due to the genotype. The treatment of seeds with cold plasma improves the osmoregulation ability of cells and therefore increases the drought resistance of genotypes.

1. Introduction

In recent decades, new plasma sources operating at atmospheric pressure and producing plasma with low temperature have been intensively developed. This was the basis for opening new frontiers in plasma applications and technology in fields like biology, medicine, agriculture and ecology. For such applications, it is obligatory to produce plasma with gas temperature T_g—the temperature of the heavy particles (ions, radicals, excited atoms and molecules) low enough to avoid the thermal damage of the treated biological systems (below 40 °C in the best cases), the so-called cold atmospheric pressure plasma (CAP). At the same time, the electrons have much higher energy (corresponding to electron temperature T_e of about 1–5 eV or more) so that the ionization, excitation, reactive particle production and other important processes for sustaining plasma and for the biomedical applications are possible. These non-thermal or non-equilibrium plasmas are far from the thermodynamic equilibrium, and their unique properties are very important for specific applications in biological system treatments. A detailed review of plasma-assisted agriculture can be found in [1].

Depending on the plasma properties and the desired effect, the treatment of the biological system can be performed directly in the active plasma region (direct plasma treatment) or outside the active plasma production region (indirect plasma treatment) in the afterglow region or by using plasma activated water/liquid (PAW) [1,2]. In the direct treatment regime, all active plasma components—the electrons, ions, short- and long-living excited species, radicals, electric fields, electromagnetic radiation (including VUV and UV photons)—act on the treated samples directly and in a synergetic way. The low gas temperature in the active plasma-sustaining region (CAP) can be achieved by applying low electrical power, high frequency or a pulsed regime of operation and an appropriate choice of gas and gas flow rate [3]. Producing plasma at such conditions is possible only for some types of plasma devices. When the gas temperature in the active plasma region is excessive, the afterglow region is used for biological system treatment in order to avoid thermal damage. In the afterglow plasma regions, some of short-living reactive particles are lost, and the VUV-UV radiation is absorbed; the electric fields can be completely zero, and the number and energy of the electrons and ions can be significantly reduced. At the same time, the thermal plasma component is also strongly reduced, which is needed. The long-living reactive oxygen and nitrogen species (RONS) are still available in the afterglow, and they can produce the necessary effects on the treated sample. Thus, although the CAP is used generally for all cases, the choice and optimized plasma source operation for any particular application needs to be investigated.

In agriculture, cold atmospheric pressure plasma treatment is considered to be a fast, cost-effective and pollution-free method for improving seed germination, plant growth and ultimately plant production [4,5,6,7]. An increasing number of reports on the influence of CAP treatment on the germination of seeds of agricultural crops and other valuable plant species can be found in the literature. In [8], CAP treatment was applied to Chenopodium album seeds, and a threefold increase in germination was achieved compared to control seeds. In [9], it is reported that the treatment of wheat and oat seeds does not affect germination but accelerates root growth and alters metabolic processes in young plants.

It is shown in [10] that the variation in the duration of plasma treatment (from 3 to 10 min) can produce different responses of rapeseed germination and of the initial seedling growth. Cold plasma treatment has significant influence on the plant development and physiological processes, including the promotion of seed germination and seedling growth [8,11], activation of photosynthesis [9,11] and regulation of carbon and nitrogen metabolism [12,13].

So far, there are no in-depth studies to clarify the cause of the stimulating effect of cold plasma on the growth of seeds and young plants. There are studies that attempt to investigate changes in the structure and functions of biopolymers as a possible mechanism to explain these effects [14,15]. According to some authors [8,15,16,17], the increase in seed germination rate is indirectly related to the improvement of water uptake in seeds after cold plasma treatment. In addition, it was found that plasma treatment significantly increased the soluble sugar and protein contents of seedlings in different crops [16,18,19]. The increase in both organic substances can be related to the increasing activity of enzymes, related to their metabolism [20].

The potential of cold plasma seed treatment technology and the still small number of publications in the field of important cereal crops motivated us to undertake research to study the effects of this treatment on durum wheat. The plasma properties and the corresponding effects strongly depend on the type of the plasma source used and the operational conditions. For a given plasma source, the treatment time needs to be optimized. And the treatment time can widely vary for different plasma sources. Two different types of plasma sources were used in this investigation: (i) a microwave (MW) plasma torch and (ii) underwater diaphragm discharge. Both plasma sources can be used in a direct plasma treatment regime when the treated samples are placed in the active plasma-sustaining region.

The aim of this study was to identify the most appropriate combinations of plasma source and duration of treatment positively affecting seed germination as well as to investigate the effect of cold plasma on the seedling growth and osmotic stress tolerance in three durum wheat genotypes.

2. Materials and Methods

This research was conducted with seeds of 3 durum wheat varieties. All used varieties were created at Field Crops Institute, Chirpan, Bulgaria—the older variety Progress; the standard variety, Predel; and one of the newest varieties, Kehlibar.

Variety Progress was created by the combined use of mutation and hybridization breeding and listed in the Bulgarian Official Catalogue of Varieties in 1990. It is characterized by high quality and productivity, and until recently it occupied a large percentage of the cultivated areas of durum wheat in Bulgaria.

Variety Predel is listed in the Bulgarian Official Catalogue of Varieties in 2010. Recently, it became the national standard for yield in the variety testing system in Bulgaria (Executive Agency of Variety Testing Field Inspection and Seed Control). It is characterized by high and stable yields. The variety has a high content of carotenoids, which give a stable color to pasta made from it. Predel carries a marker gene for high gluten quality: gamma gliadin 45.

Variety Kehlibar is one of the newest varieties created at the Field Crops Institute. It is characterized by a high yield potential, high hectoliter weight and grain vitreousness, medium protein content and high wet gluten.

The treatment of the seeds was carried out in the plasma laboratory at Sofia University, and the germination of the seeds and the cultivation of the plants was carried out at the Field Crops Institute, Chirpan.

2.1. Cold Atmospheric Plasma Sources

Two types of cold atmospheric plasma (CAP) sources were used for direct treatment of seeds: a microwave plasma torch and underwater diaphragm discharge.

2.1.1. Microwave Plasma Torch

The microwave plasma torch is produced by an electromagnetic wave at 2.45 GHz frequency in argon gas flowing inside the discharge tube. The electromagnetic wave is excited by a surfatron wave launcher [21,22,23,24] and travels along the discharge tube–plasma interface. When the discharge tube is shortly outside the surfatron, the electromagnetic wave continues traveling along the plasma–air interface, producing the plasma torch in the open space in this way (Figure 1). Usually, the plasma gas temperature T_g of the torch is above 100 °C and depending on the electromagnetic wave power can reach 5000 °C. At low wave power (12 W in our case) and appropriate discharge conditions, the temperature of the plasma is below 40 °C [25], and at such conditions the microwave torch can be used as a CAP source. The treated samples are inside the active plasma-sustaining region in the plasma torch but not in an afterglow region. This is important because, in the active plasma region, all plasma components and short-living particles can react with the sample and produce effects. When they all act on the sample in a synergetic way, the treatment time can be significantly shortened, and stronger effects can be obtained. The treatment time applied in this investigation varied from 5 s up to 1 min (5, 10, 20, 60 s), which is much shorter than usually used with the other types of plasma devices. Adding to this, the low electrical wave power (12 W) makes the MW plasma torch very energetically effective.

In this work, the plasma was produced in a quartz tube with an outer diameter of 7 mm and inner diameter of 3 mm at an Ar (99.99999%) gas flow of 5 L/min.

2.1.2. Underwater Diaphragm Discharge

The underwater diaphragm discharge used in this study is shown in Figure 2 [3,26]. The polycarbonate camera is separated by a dielectric membrane (diaphragm, denoted by “D”) in two containers. The containers were filled with water (water solutions) with 50 mL volume each. The membrane has a thickness of 1 mm, and a pin-hole with a 0.6 mm diameter was produced in its center. This configuration is also called “pin-hole discharge” [26]. Two planar, high-voltage (HV) electrodes were installed at fixed positions on both sides in the container so that they were immersed in the water. The high-frequency (15 kHz) voltage of 5 kV was applied to the red electrode (denoted by “+”). The black electrode (denoted by “−“) was grounded. These notations are used in

Table 1. Variants of cold plasma durum wheat seed treatment.

No	Type of Plasma Treatment	Plasma Treatment Time
1	Plasma torch	5 s
2	Plasma torch	10 s
3	Plasma torch	20 s
4	Plasma torch	60 s
5	Underwater “+”	5 min
6	Underwater B “+”	5 min
7	Underwater “−“	5 min
8	Underwater B “−“	5 min
9	Underwater “+”	10 min
10	Underwater B “+”	10 min
11	Underwater “–“	10 min
12	Underwater B “−“	10 min
13	Control W 50 °C	0
14	Control W 65 °C	0
15	Control	0

.

The treatment of seeds is organized in discharge regimes variants presented in Table 1. Variants 1–4 correspond to different treatment times by the plasma torch. The variants 5–12 correspond to treatment of seeds immersed in 50 mL distilled water in the underwater discharge cameras. The variants of seeds treated in the camera connected to the powered (red in Figure 2) electrode are denoted by “+” (No. 5, 6, 9 and 10); the variants of seeds in the camera connected to the grounded (black in Figure 2) electrode are denoted by “−“ (No. 7, 8, 11 and 12). In variants No. 5, 7, 9 and 11, the seeds are in one camera, and the other is filled with water only. In variants No. 6, 8, 10 and 12, the seeds are treated simultaneously in both cameras and are denoted additionally by “B” with a corresponding sign “+” or “−“.

The non-treated control is listed as No. 15 in Table 1. Because, during the treatment in the underwater discharge cameras, the water temperature increased up to 50 °C for 5 min treatment and up to 65 °C for 10 min treatment, two additional controls of seeds immersed in water with the corresponding temperature for 5 min and 10 min were included in this investigation (variants 13 and 14 in Table 1 denoted by “Control W 50 °C” and “Control W 65 °C”.

2.2. Preliminary Experiment

Twelve variants of different discharge methods and treatment times, presented in Table 1, were carried out for the seeds of the three varieties. Different combinations of the two plasma sources—the plasma torch (Figure 1) and underwater diaphragm discharge (UDD) (Figure 2)—and treatment time were used. The durations of exposure to the plasma torch were 5, 10, 20 and 60 s and 5 and 10 min for the underwater discharge, and different locations of the seeds were used relative to the electrodes in the containers. Three control variants, one dry control and two wet controls, with different water temperatures, were also used.

The experiment was conducted in two replicates for each cultivar and treatment variant, each replicate consisting of 50 seeds. The controls 13, 14 and 15 consist of 100 seeds each. The number of seeds for the whole experiment is quite high. In view of the accuracy of the experiment, all treatments and later the germination tests and the corresponding measurements had to be performed within the same day. This would not be possible with a larger number of replicates or a limited number of personnel performing the study.

The seeds were transported and placed in Petri dishes for germination according to generally accepted methods after the treatment. The seeds of each variant and genotype were placed in 9.2 cm diameter Petri dishes between two layers of filter paper and moistened with 10 mL of distilled water. The Petri dishes were kept at a temperature of 24 °C for 7 days. The following traits were observed: germination energy (%), germination rate (%), fresh seedling weight (g), shoot length (mm) and root length (mm).

The germinating energy GE is determined by the following formula:

GE (%) = Number of germinated seeds per 3 days/total number of seeds planted

Germination rate G is determined by the following formula:

G (%) = Number of germinated seeds per 7 days/total number of seeds planted

2.3. Experiment for Evaluation of Seedling Growth and Osmotic Stress Tolerance after the Cold Plasma Treatment of Seeds

On the basis of the results from the preliminary experiment, we have chosen the best discharge conditions and treatment time for investigation of the effect of plasma treatment on the seedling growth and osmotic stress tolerance. The seeds of the three above-mentioned varieties were treated with cold plasma, using two plasma sources with three variants—a plasma torch for 20 s (denoted as Var. 2 in this experiment), underwater diaphragm discharge “+” for 5 min (Var. 4) and diaphragm discharge “−” for 5 min (Var. 5)—which were identified as the most appropriate in the preliminary experiment. For the underwater discharge, a different arrangement of the seeds was applied to the electrodes in the water bath. Two control variants were also used: a dry control, Var. 1, and a control with seeds soaked in water, Var. 3.

The experiment was performed in two replications for each treatment variant and genotype. For each variant, 2 × 50 seeds were treated.

After the treatment, the seeds were dried, transported and placed for germination on moistened filter paper, which was wrapped in rolls. The length of roots and shoots was measured on the 5th day after they were set for germination. Four replications were prepared from each variety and from each variant, and each replication contained 10 seeds. The rolls were placed in boxes with distilled water in a thermostat at temperature 24 °C. To determine the tolerance to osmotic stress of cold-plasma-treated varieties, the method of depression in seedling growth was used with an osmotic solution.

The methodology is described in detail in a previous publication [27]. Depression in seedling growth was calculated according to the formula [28]:

% of depression = [(A − B)/A × 100]

where

A—average length of shoots/roots in water;

B—average length of shoots/roots in an osmotic solution.

The obtained results of both experiments were statistically processed by two-way analysis of variance (ANOVA). Statistical calculations were carried out by means of the program package Statistica (TIBCO Statistica 13.3).

3. Results and Discussion

3.1. Preliminary Experiments to Achieve a Positive Effect on Seed Germination by the Most Appropriate Combinations of Plasma Source and Duration of Treatment

Firstly, we compared the results for germination energy and germination rate for the three controls, the one without any treatment (Var. 15, Table 1) and the two wet controls (Var. 13, seeds for 5 min in water with a temperature of 50 °C, and Var. 14, for 10 min in water at 65 °C, Table 1). To facilitate the comparison and analysis, results are presented in two ways: grouped by varieties (Figure 3a,b) and grouped by controls (Figure 3c,d). The results presented in Figure 3a,c show that the two wheat varieties Progress and Kehlibar have almost the same germination energy, while it is lower for the variety Predel. In comparison to the dry control, the wet controls of the variety Progress have significantly lower germination energy. The other two varieties are less sensitive, and their germination energies are very close for the dry and wet controls. The situation changes for the germination rate (Figure 3b,d). The dry controls of all three varieties have the same germination rate, which remains almost the same for the wet controls of varieties Predel and Kehlibar. The variety Progress seems to be more sensitive; its wet controls have a lower germination rate, and it decreases with the water temperature and the soaking time.

The effects of the plasma treatment on the germination energy using different plasma devices and varying the discharge conditions, including treatment time, are presented in Figure 4. The results of each variant of treatment are compared with the corresponding controls: the MW torch treatment with the dry control (Figure 4a); the underwater discharge treatment for 5 min with the 5 min wet control (Figure 4b) and the underwater discharge treatment for 10 min with the 10 min wet control (Figure 4c). It can be seen from Figure 4a that the MW plasma treatment effect on germination energy is different for the three varieties and can lead to an increase (Predel) or decrease (Progress and Kehlibar) in comparison to the corresponding control. A drastic reduction in the GE is observed at the longest treatment time (60 s) only for the more sensitive genotype Progress. For all three genotypes, the best results for GE (equal or higher compared to the controls) are obtained with 20 min treatment time.

The obtained results of underwater discharge treatment are seen in Figure 4b (5 min treatment) and Figure 4c (10 min treatment). The comparison between them shows better effects for all three varieties in the 5 min treatment. It is important to note that the positive effects are produced by the synergetic action of all active plasma components but not by the warm water. As we saw in Figure 3, the soaking of the seeds in warm water decreases their germination energy more significantly for the genotype Progress, while the treatment by the underwater discharge not only compensates this but can significantly increase the GE.

As a whole, the most significant effect in this indicator was observed after treatment Var. 6 (underwater discharge simultaneous treatment in both containers for 5 s)—100% for variety Progress. For the control variants, this germination energy is in the range between 83% and 95%. A significant decrease in this indicator is observed in treatment variants 4, 5, 7 (Progress), 5, 9 and 12 (Predel) compared to the corresponding controls. Variety Progress responded with the greatest increase in germination energy after treatment with the most appropriate treatment variant and variety Predel with the smallest increase.

Such effects can be seen to an even stronger degree in Figure 5, where the germination rate after the plasma treatment is presented under the same conditions as for the GE in Figure 4.

The germination rate is expressed as the percentage of germinated seeds. As a result of MW plasma treatment, the germination rate increases with the treatment time except for the 60 s treatment of variety Progress, where a significant reduction (G = 86%) is observed (as was also the case of GE). A positive effect is obtained for all varieties at 20 s treatment time (Figure 5a).

It is difficult to see any regularity of the effects after underwater discharge treatment. The response of different varieties to the 5 min and 10 min underwater discharge treatments is in a wide interval from 87% up to 100% germination rate (Figure 5b,c).

Generally, the genotypes studied respond differently to cold plasma treatment. All seeds germinated (100%) after the treatment at variants 4 and 6 for the Kehlibar variety; after variants 2, 6 and 10 for the Progress variety; and after variants 2, 3, 4, 7 and 9 for the Predel variety (see Table 1). The lowest percentage of germinated seeds was obtained for the same Var. 4 but for the Progress variety (86%). The next lowest germination rates were those of Var. 7 (87%) and the wet control at 10 min (Var. 14), again for the Progress variety.

In order to show the effects more clearly and make comparison easier, the difference between the values of germination energy (GE) or germination rate (G) for plasma treatment variants (average of all varieties) and the corresponding controls are presented in Figure 6a (GE) and Figure 6b (G), respectively.

The increase in the given indicator relative to the corresponding control is shown by a positive value in Figure 6 and the decrease by a negative value. Both the germination energy and rate the 20 s MW plasma torch treatment have a positive effect (Var. 3). The same is true for the underwater discharge treatment at 5 min for Variants 6 and 8 and for 10 min for Var. 11 (see Table 1).

The effect of cold plasma treatment on the initial seedling growth, expressed by the roots and shoot length (Figure 7), is observed. As in the previous cases, the results depend significantly on the variety. Generally, a stimulating effect on the length of shoots compared to the control untreated variants was observed for treatment options 2 and 3 (MW plasma torch treatment for 10 s and 20 s), while options 9 and 12 (underwater for 10 min) suppressed their growth.

Treatment options 1 to 3 (MW plasma torch, 5 to 20 s treatment time) and options 6 and 8 (underwater discharge, 5 min) stimulated root growth, and the highest root length was observed in comparison with corresponding controls. The treatment variants 10 and 12 (underwater plasma treatment, 10 min) inhibited the root growth in comparison with both wet controls.

The fresh seedling weight for each genotype after the treatment with the MW plasma torch and underwater discharge for 5 min and 10 min together with the corresponding controls is presented in Figure 8. One can see that the results are almost the same for the three varieties in the treatment variants 3 (MW 20 s) and 7 (underwater “−“, 5 min) despite large differences between varieties in the controls. The highest weight (average of all genotypes) is observed in treatment variants 1, 2 and 9 and the lowest in variants 6 and 10. The seedling weight from all plasma torch treatment variants is higher compared to the weight of seedling from the dry control.

The analysis of variance for three genotypes and 12 variants of cold plasma treatment and three control variants showed that all the studied factors—genotype, cold plasma treatment and the interaction between both—had a statistically significant influence on the variation in all studied traits (

Table 2. Analyses of variance of the studied traits.

Traits	Source of Variation and % of Total Variation
	Genotype (G)		Treatment (T)		Interaction (GxT)
	MS	η2,%	MS	η2,%	MS	η2,%
Germination energy	267.0 **	8.24	114.4 *	24.7	77.2	33.3
Shoot length	144.2 ***	16.9	23.8 **	19.6	28.7 *	47.08
Root length	134.1 ***	8.98	74 ***	34.7	52.4 ***	49.4
Seedling weight	1.659 ***	41.95	0.0646 *	11.47	0.0925 **	32.7

*** p < 0.001, ** p < 0.01, * p < 0.05.

). The variation in germination energy, shoot length and root length is mostly due to the interaction between the genotype and treatment and insignificantly due to the genotype. This fact illustrates the different responses of the used varieties to the cold plasma treatment options.

The results of the analysis of variance and Duncan’s Multiple Range test reveal that the differences in germination energy are statistically significant (see Table 2).

3.2. Experiment for Evaluation of Seedling Growth and Osmotic Stress Tolerance after the Cold Plasma Treatment of Seeds

In this experiment, the effect of cold plasma seed treatment on the growth of seedlings-shoots and roots and their tolerance to osmotic stress was studied. Only three treatment variants were used, for which the best results were obtained in the preliminary experiment and two control variants (see Section 2). The experiment was conducted using two replicates for each cultivar and treatment variant, with each replicate consisting of 50 seeds.

The effect of cold plasma seed treatment on root growth in durum wheat cultivars is shown in Figure 9. The treatment of seeds with a plasma torch—Var. 2—has a slight inhibitory effect on the root growth on day 5 after germination in the varieties Predel and Progress, while in variety Kehlibar there is a slight stimulating effect compared to the dry control—Var. 1.

The seed treatment with underwater diaphragm discharge “+” and “−” also has a negative effect on root growth in the cultivars Kehlibar and Progress. In cultivar Predel, in both variants of underwater discharge treatment (Var. 4 and 5), the roots grew by about 25% more compared to the control variant Var. 3.

Plasma torch treatment (Var. 2) produces a slightly positive effect on the growth of shoots in Predel and Kehlibar, and they increased by 11.4% in Predel and by 4.8% in Kehlibar in comparison with the control (Figure 10). Both variants of treatment with underwater diaphragm discharge “+” (Var. 4) and “−” (Var. 5) had a positive effect on the growth of seedlings only in the variety Predel. On the fifth day after germination, the shoots at Var. 4 were 24.4 mm (65%) longer compared to these of the wet control (Var. 3) and in Var. 5 were 16.2 mm (43.3%) longer. In cultivar Kehlibar, a positive effect was found only in Var. 5, as the seedlings were 13.1 mm longer (33.2%) compared to those in the wet control (Var. 3). In cultivar Progress, Var. 5 did not affect the growth of the seedlings, while Var. 4 had a suppressive effect, and the shoots remained 20.6 mm (66.9%) shorter than those in the wet control.

Osmoregulation is one of the most important cellular mechanisms for adaptation to drought. This basic cellular response, which occurs during drought, avoids cell dehydration and reduced yields. In drought conditions, plants with better osmoregulation capacity show better growth and higher yields. The ability of plants for osmoregulation can be measured by indirect methods, one of which is the growth depression of seedlings placed in an osmotic environment. The lower the depression rate is, the better the osmoregulation capacity and higher the drought resistance. The osmotic stress applied in this study, simulated by 1 M sucrose solution and applied on the third day after seed germination, caused seedling growth inhibition in the control and CAP treatment variants in all genotypes (Figure 11). Different degrees in depression rates were observed between treated and untreated seeds in all studied varieties. For the Predel variety, the coefficients of depression for the plasma-treated group Var. 2 and underwater diaphragm discharge Var. 5 “−” are lower in comparison with the corresponding control variants. For cultivar Kehlibar, a decrease in the coefficient of depression was observed in all variants of CAP in comparison with the control variants. The most significant reduction in this coefficient is by 21% and was observed in Var. 4. Cultivar Progress again has the weakest response to the CAP treatment, and a reduction in the coefficient of depression of seedling growth is observed only in Var. 4 by 14.8%.

The results obtained in the preliminary experiment are in good agreement with the results in [8]. They reported that the treatment with CAP of wheat and oat seeds insignificantly affects germination but on the other hand has an accelerating effect on root growth and altering metabolic processes in young plants.

It was found in our preliminary experiments that the plasma torch treatment of seeds for 20 s and the two configurations of the underwater discharge treatment for 5 min when the seeds are placed in the two containers (“+” and “−”) have the greatest positive effect on germination, germination energy and indicators related to the initial growth. The increase in the treatment time (to 60 s for the MW torch and 10 min for the underwater discharge) produces negative effects, which are connected to the temperature increase and thermal damage. Such negative effects can be seen even in the wet controls, which were not treated by plasma but immersed in water with a temperature of 50 °C for 5 min and 65 °C for 10 min. The treatment time should sufficient for the stimulating processes to take place and not excessive in order to avoid thermal damage.

The variation in germination energy, shoot length and root length is mainly due to the interaction between the genotype and treatment variant and to a small degree due to the genotype. This fact illustrates the different reactions of the studied durum wheat cultivars to the treatment variant [29]. The different reactions of different plant species to cold plasma treatment depend on various factors such as seed size, the hardness of the seed coat and the presence of some plant proteins and polymers such as keratin, suberin or lignin [29]. It can be assumed that some of these factors determine the different reactions of durum wheat cultivars during germination after the treatment of seeds with cold plasma. This large and statistically significant interaction between the genotype and cold plasma treatment should be take into account by developing commercial technologies for cold plasma seed treatment.

Based on the results obtained, it can be concluded that the treatment of seeds with cold plasma can improve the ability of cells for osmoregulation and therefore increase the drought resistance of genotypes.

Osmoregulation is one of the most important cell adaptation mechanisms and occurs only when dehydration begins. To minimize the loss of water from the cells and to sustain the cell functions in case of water deficit, soluble materials are stored in them. By this main cell response, occurring during drought, dehydration of cells and a decrease in the yields are avoided [28].

There are a limited number of studies that also show improved tolerance to abiotic stress after plasma treatment. The treatment of seeds of various plant species with cold plasma improves seed germination and soluble sugars and protein content in sprouts [9,10,30]. It is well known that the improved resistance to abiotic stress correlates with increased content of soluble sugar and protein in plants.

4. Conclusions

As a result of this study, the most appropriate combinations of plasma source and duration of treatment, positively affecting seed germination and seedling growth in three durum wheat cultivars, are identified. It was found that the treatment of seeds with a plasma torch for 20 s and the treatment with underwater discharge for 5 min when the seeds are placed in both containers in two different positions (relative to the electrodes between which the plasma is supplied, “+” and “−”) have the most positive effect on germination, germination energy and indicators related to the initial growth of seedlings.

The variation in germination energy, shoot length and root length after the cold plasma treatment of seeds is mainly due to the interaction between the genotype and treatment variant and to a small degree due to the genotype.

The treatment of seeds with cold plasma can improve the osmoregulation ability of cells and therefore increase the drought resistance of genotypes.

In the future investigation, due to the different effects of the plasma treatment on the seed varieties studied, new types of plasma sources, discharge conditions and greater number of replications must be included.