Effects of Plasma and Activated Water on Biological Characteristics of Bromus inermis Seeds Under Different Power Supply Excitation

Abstract

To explore the potential of plasma technology in regulating seed germination, this study compared the effects of direct treatment with needle-plate electrodes using DC and pulse power supplies, and indirect treatment with plasma-activated water on the growth characteristics of Bromus inermis seeds. By comparing different pulse power parameters, including voltage, pulse width, frequency, and duration, it was found that treatments at 15 kV, 2500 ns, 6 kHz, and 10 min significantly increased the surface hydrophilicity and germination performance of the seeds. The best conditions for DC power supply were 15 kV and 10 min. Indirect treatment with plasma-activated water (15 kV, 10 min) effectively broke the seed dormancy by regulating active nitrogen oxygen particle components, increasing the germination percentage by 50%. Analysis of antioxidant enzyme activity showed that in seedlings the activities of superoxide dismutase (SOD) and peroxidase (POD) increased by 75% and 21%, respectively, after treatment, revealing the mechanism of oxidative stress response induced by plasma. This study provides theoretical and technical references for the application of plasma technology in enhancing seed vitality and agricultural practices.

1. Introduction

Plasma technology, as a cutting-edge physical method, has garnered significant attention due to its unique ability to generate high-energy electrons, free radicals, and reactive oxygen and nitrogen species (RONS) [1]. This capability endows it with immense potential for applications across various critical fields, including agriculture, biomedicine, and environmental remediation [2]. In the agricultural sector, plasma technology offers an innovative, green, and highly efficient solution for enhancing seed vitality, promoting seed germination, and increasing yield. The underlying principle is that the high-energy reactive particles generated during plasma discharge can interact with biomolecules on and within the seed, thereby regulating the physiological and metabolic processes of the seed [3]. With the intensification of global climate change, continuous population growth, and the dwindling availability of arable land, agriculture is confronted with unprecedented challenges. Traditional seed treatment methods, such as chemical soaking and high-temperature treatment, often come with complexities in operation, high costs, and significant environmental risks. In contrast, plasma technology stands out with its simplicity, environmental friendliness, and cost-effectiveness, making it a promising candidate for modern agricultural production [4]. In high-altitude regions of Qinghai, overgrazing and harsh climatic conditions have led to the degradation of some meadow vegetation. The need for meadow plants with strong stress resistance and rapid growth is particularly acute. Seeds of Bromus inermis have emerged as an ideal choice for reseeding [5]. Thus, applying plasma technology to study the stress resistance and growth rate of Bromus inermis seeds is highly representative.

The application of plasma technology in agriculture mainly takes two forms: indirect treatment with plasma-activated water (PAW) and direct plasma treatment. By using low-temperature plasma to activate media, reactive radicals from gaseous plasma can be transported into liquid/solid systems. This process generates long-lived reactive oxygen and nitrogen species (e.g., H₂O₂, O₃, NO₃⁻, NO₂⁻) and other active radicals that retain disinfection and sterilization capabilities even after plasma discharge ceases. When water is the activated medium, it is termed plasma-activated water (PAW) [6,7]. These reactive species endow PAW with strong chemical reactivity and redox capabilities, enabling it to penetrate seeds, break dormancy, and promote germination. Direct plasma treatment involves applying plasma discharge directly to the seed surface. High-energy electrons, free radicals, and UV radiation interact physically and chemically with the seed surface, altering its structure and chemical composition to modulate the seed’s physiological properties [8]. Different high-voltage power sources affect the discharge energy injection, stability, and the types of reactive species generated by the corona discharge electrode structures. Corona discharge can be driven by DC, AC, or pulsed power sources [9]. DC power provides stable discharge conditions suitable for continuous treatment [10], while pulsed power sources, characterized by short rise times, narrow pulse widths, and high repetition rates, can generate high-energy-density plasma, enhancing treatment efficiency and effectiveness. Utilizing high-voltage nanosecond pulses to drive atmospheric-pressure low-temperature plasma helps improve energy efficiency and enables multi-channel discharge [11]. Both modes have shown significant effects in promoting seed germination and enhancing seed vitality. However, their mechanisms and applicable scenarios differ, necessitating further in-depth research and systematic comparison.

Reactive Oxygen Species (ROS) are a series of highly reactive oxygen derivatives produced during normal cellular metabolism. These include O₂⁻, H₂O₂, ·OH, and others. ROS play important roles in cellular signaling, defense mechanisms, and apoptosis. However, when ROS production exceeds the cell’s antioxidant capacity, oxidative stress may occur, leading to damage to lipids, proteins, and DNA in cells and potentially causing various diseases. Therefore, maintaining a balance of ROS levels is crucial for normal cellular function [12]. Reactive oxygen and nitrogen species (RONS) are key to how plasma affects seedling growth characteristics. The concentration, types, and discharge mode and power are closely related to these reactive species [13]. Laurita et al. used a high-voltage pulse source with an amplitude of 20 kV, single energy of 50 mJ, and a repetition frequency of 1 kHz to generate atmospheric-pressure low-temperature plasma through dielectric barrier discharge to treat 60 mL of deionized water. After 2.5 min, the pH value dropped to around 3.0, with little further change upon continued treatment [14]. Man et al. optimized pulse parameters, using a sub-nanosecond pulse power source with a fast rise time and narrow pulse width to generate plasma, achieving a maximum NO₂⁻ concentration of 0.12 mM in the activated water [15]. Jiang et al. studied the effects of different discharge power helium plasmas on wheat growth, finding that a discharge power of 80 W significantly improved the hydrophilicity of wheat seeds and the chlorophyll content of seedlings [16]. Vichiansan et al. investigated the impact of plasma-activated water on tomato seed growth, discovering that H₂O₂ and NO₃⁻ concentrations were related to discharge power, with the fastest growth rate of tomato seedlings at a discharge power of 20 W [17]. Clearly, the reactive species generated at different discharge powers significantly influence the growth of the seedling from the germinated seed characteristics. Further optimization of discharge power is needed to meet the growth requirements of various plants. Currently, research on plasma technology in seed treatment has yielded several important results. For example, Li et al. treated seed potato with dielectric barrier discharge plasma-activated water, finding that plant fresh weight increased by 25.9%, chlorophyll content by 31.7%, and yield by 17.5%, demonstrating the significant advantages of PAW in promoting crop growth [18]. Pang et al. discovered that pH regulation could optimize the storage stability of reactive species in PAW, providing new ideas for enhancing its application effects [19]. Yang et al. used carbon cloth to enhance the falling film DBD plasma to prepare activated water, significantly increasing the concentrations of H₂O₂ and O₃ in water, and enhancing the sterilization performance and biological activity of PAW [20]. Seed dormancy is an adaptive physiological mechanism formed by plants during long-term evolution to cope with adverse environmental conditions. In a dormant state, the metabolic activities within the seed are relatively stagnant, resulting in low germination percentages and slow growth [21]. Reactive oxygen metabolism plays a key role in regulating seed dormancy and germination. Moderate oxidative stress can activate antioxidant enzymes (SOD, POD, CAT, etc.) within the seed [22]. These enzymes can remove reactive oxygen free radicals within the seed, reduce oxidative damage, and regulate the physiological and metabolic processes within the seed to accelerate germination. Viakas et al. found that PAW treatment could increase seed germination percentage, vigor index, and mean germination time. Compared with the control, the root and shoot of cultivated plants treated with PAW were longer, and their fresh and dry weights, as well as chlorophyll, sugar, and protein concentrations, were higher [3]. The activity of antioxidant enzymes (SOD, POD, CAT) in the PAW treatment group significantly increased, especially in the roots. This indicates that PAW pre-treatment of seeds has potential for promoting germination and plant growth. Li et al. treated wheat seeds with DBD plasma and also found that the activity of antioxidant enzymes was enhanced, an etching coating was formed on the seed coat surface, and both the seed germination index and seedling growth height were improved [23]. Combining PAW with 0.1% chitosan nanocoating raises the germination percentage to 85%, compared with 70% for PAW alone, by providing a sustained release matrix for RONS. Similarly, pre-soaking seeds in 50 µM gibberellin followed by pulsed plasma increases drought-survival by 40%, indicating additive signalling pathways [24]. At present, there are few systematic comparative studies on the synergistic effects of different power parameters and direct and indirect treatments. Moreover, research on the induction of the growth of the seedling from the germinated seed characteristics and antioxidant enzyme systems by plasma to enhance seed vitality is also relatively insufficient.

This study employed needle-plate electrodes powered by DC and pulsed power sources to conduct direct plasma treatment on Bromus inermis seeds. Simultaneously, plasma-activated water (PAW) was prepared using a pulsed needle-plate electrode for indirect treatment. By adjusting power parameters and treatment durations, we compared and analyzed the effects of different power sources and treatment methods on seedling growth characteristics and antioxidant enzymes. The study compared the surface hydrophilicity (via a contact angle measurement), germination percentage, chlorophyll content, and antioxidant enzyme differences in Bromus inermis seeds under three treatment methods. Additionally, we measured the pH value and concentration changes of H₂O₂, O₃, NO₃⁻, and NO₂⁻ in PAW to explore the mechanisms by which plasma treatment affects plant seeds. By optimizing power parameters and exploring synergistic effects, this study provides experimental data and theoretical support for the wide application of plasma technology in enhancing seed vitality and green agriculture.

2. Materials and Methods

2.1. Experimental Setup

Corona discharge is a type of gas discharge phenomenon that occurs under specific conditions. It is characterized by the accumulation of charge at the tips of sharp electrodes, such as needles, edges of plates, or fine wires, under the influence of a high-voltage electric field. This accumulation leads to local ionization and the formation of corona discharge.

The experimental setup, as depicted in Figure 1, includes a direct current (DC) power supply module and a pulsed power supply module for plasma-activated water (PAW) generation. The DC power supply module consists of a power control console transformer T1, which inputs 220 V alternating current (AC) voltage. This voltage is then stepped up by a no-local-discharge test transformer T2 (30 kVA/200 kV). Subsequently, a rectifier circuit composed of a high-voltage silicon stack D1 (300 kV/0.2 A), a filter capacitor C1 (10,000 pF), and a current-limiting resistor R1 (5 kΩ) provides a negative DC voltage. The current limiting modules R1 (5 kΩ), R2 (30 kΩ), R3 (15 kΩ), and R4 (5 kΩ) can reduce current.

The pulsed power supply employs a voltage, frequency, and pulse-width adjustable device (HVP-20P, Xi’an Ling feng yuan Electronics Technology Co., Ltd., Xi’an, China). The corona plasma generation device with a multi-needle-column-plate configuration was developed in-house. The discharge electrodes are made of brass, with the upper and lower plates measuring 120 mm in length and width. The thickness of the upper and lower plates is 15 mm and 10 mm, respectively. The corona discharge device consists of eight needle electrodes surrounding a column electrode, forming a discharge unit in an array configuration. Each discharge unit has an area of 30 mm × 30 mm, and there are a total of 16 discharge units. The distance from the needle tip to the center of the column electrode is 10 mm. The cylindrical part of the needle electrode has a radius of 2 mm, and the curvature radius of the needle tip is 0.12 mm. The column electrode has a radius of 5 mm, with a 0.3 mm straight chamfer at the tip due to actual machining conditions. The electrode gap of the discharge device is adjusted to 30 mm, and the discharge container is a 200 mL cylindrical glass beaker filled with deionized water.

For electrical characteristics, a voltage probe (Tektronix P6015A, Tektronix, Beaverton, OR, USA) is used to measure the discharge voltage, while a current probe (Tektronix P6015A, Tektronix, Beaverton, OR, USA) collects the discharge current. An oscilloscope (Tektronix MSO4404-BW-200, Tektronix, Beaverton, OR, USA) is employed to record and save the voltage and current waveforms. For optical characteristics, a spectrometer (Fu xiang Optics PG4000, Fu xiang Optics, Shanghai, China) is used to measure the discharge spectrum, and a digital camera is utilized to detect the plasma morphology.

2.2. Experimental Process

The experiment was set up with four groups: DC power supply, pulse power supply, activated water, and a control group for comparison. For the DC power supply, different voltages (15 kV, 16 kV, 17 kV) (Excessively high voltage can damage seeds) and treatment durations (10 min and 20 min) were applied by changing the applied voltage and treatment time. For the pulse power supply, more power supply parameters were altered, including different voltages (14 kV, 15 kV, 16 kV, 17 kV), pulse widths (1500 ns, 2000 ns, 2500 ns, 3000 ns), frequencies (4 kHz, 5 kHz, 6 kHz, 7 kHz), and treatment times (10 min, 20 min, 30 min). The activated water experimental group was set up with different voltages (10 kV, 15 kV, 17 kV, 20 kV) and treatment durations (3 min, 5 min, 10 min, 15 min, 20 min).

The seeds used in the experiment were Bromus inermis seeds. Seeds of Bromus inermis were harvested in 2023 from the College of Ecological and Environmental Engineering of Qinghai University, and stored at 4 °C in darkness prior to use. Fifty seeds of similar mass and volume were placed in each group. After direct and indirect treatments, they were cultivated in Petri dishes lined with filter paper and placed in a constant temperature incubator (12 h light at 25 °C, 45% RH, 60% light intensity; 12 h dark at 10 °C, 45% RH). The number of germinated seeds (embryo roots ≥ 2 mm were counted as germinated) was recorded daily. When the seedlings reached a length of ≥5 mm (on the 7th day), they were planted. All seeds were exposed to the same light conditions. Each dish received a daily supplementation of 2 mL of the respective aqueous solution to maintain consistent moisture levels. For the control and direct plasma treatment groups (DC and Pulsed), tap water was used throughout the germination period. In contrast, seeds in the PAW treatment group were both treated with PAW and continuously irrigated with the same PAW during the entire germination process to ensure sustained exposure to plasma-generated reactive species. The number of germinated seeds was determined. To avoid the randomness of experimental data, the experiment was repeated three times.

In this study, a two-phase experimental strategy was employed to systematically evaluate the effects of different plasma treatments on Bromus inermis seeds. In the first phase, seeds were treated under a range of electrical parameters (e.g., voltage, frequency, pulse width, duration) for each treatment modality (DC, Pulsed, and PAW). The initial screening was based on key physiological responses including surface hydrophilicity (contact angle) and germination percentage. For PAW treatments, the concentration of reactive particles was also considered. From these assessments, the optimal treatment conditions within each modality, those that most effectively improved seed performance, were selected for further analysis. In the second phase, seeds treated under these optimized conditions along with a non-treated control group were subjected to in-depth biological characterization. This included quantitative measurements of antioxidant enzyme activities (SOD, POD, and CAT), chlorophyll content, and α-amylase activity. These analyses aimed to elucidate the physiological and biochemical mechanisms underlying the enhanced germination and growth observed in plasma-treated seeds.

2.3. Determination of Plant Seed Biological Characteristics

2.3.1. Contact Angle Measurement

The surface hydrophilicity of the seeds, a critical factor influencing water uptake during germination, was assessed by measuring the contact angle using a JC2000DM (Beijing Zhongyi Kexin Technology Co., Ltd., Beijing, China) Contact Angle Meter. The contact angle is defined as the angle formed at the three-phase (air) contact line between the tangent to the liquid air interface and the interface. A smaller contact angle indicates greater surface hydrophilicity.

For the measurement, individual seeds were fixed on a horizontal stage with the test surface facing upward and leveled. A 1–2 μL droplet of medical-grade deionized water was dispensed onto the seed surface using a manual micro-syringe. The droplet image was captured in real-time via the instrument’s built-in USB CCD camera. The left and right contact angles were automatically calculated using the accompanying JC2000DM (POWEREACH^®2.0) software, and the reported value is the average of the two. The instrument’s measurement range and resolution are 0–180° and 0.01°, respectively.

2.3.2. Germination Percentage

The germination percentage (GP) was calculated to evaluate seed viability and emergence. It was defined as the percentage of seeds that successfully germinated after a specified period and was calculated according to Equation (1):

$$\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} (\mathrm{G}\mathrm{P})={\displaystyle \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}}}\times 100\%$$

(1)

2.3.3. Determination of RONS Concentrations in PAW

The concentrations of key reactive oxygen and nitrogen species (RONS)—hydrogen peroxide (H₂O₂), nitrate (NO₃⁻), nitrite (NO₂⁻), and ozone (O₃)—in the plasma-activated water (PAW) were determined using standardized colorimetric methods [25].

H₂O₂ was quantified via the titanium sulfate method. H₂O₂ reacts with titanium sulfate in an acidic medium to form a yellow-orange peroxotitanium complex, the absorbance of which was measured at 405 nm [18].

NO₂⁻ was determined using the Griess reaction. In an acidic medium, NO₂⁻ diazotizes with sulfanilamide and subsequently couples with N-(1-naphthyl)ethylenediamine dihydrochloride to form a reddish-purple azo dye, the absorbance of which was measured at 540 nm [26].

NO₃⁻ was measured indirectly by first reducing it to NO₂⁻ using a copper cadmium reduction column. The resulting NO₂⁻ concentration was then determined by the Griess method as described above, with the NO₃⁻ concentration calculated by subtracting the initial NO₂⁻ concentration [26].

O₃ was quantified using the indigo trisulfonate decolorization method. Indigo carmine, which reacts stoichiometrically with O₃, undergoes decolorization. The concentration of O₃ was determined spectrophotometrically by measuring the decrease in absorbance of the indigo carmine solution at 600 nm [27].

2.3.4. Determination of Antioxidant Enzyme Activities

The activity of superoxide dismutase (SOD) was measured using the WST-8 method. All kits use Shanghai Yizhui liquid chromatography detection technology. Detailed operation can be found in the kit manual (Shanghai Uplc-MS Testing Technology Co., Ltd., Shanghai, China, UPLC-MS-4533, 4539, 5048, 4170, c112). The following is the calculation formula:

$$\mathrm{S}\mathrm{O}\mathrm{D} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} ({\mathrm{U} \mathrm{g}}^{-1} \mathrm{F}\mathrm{W})={\displaystyle \frac{20\times \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}/(1-\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n})}{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{g}\right)}}$$

(2)

The activity of peroxidase (POD) was measured through the oxidation of guaiacol. POD catalyzes the dependent oxidation of guaiacol to a brownish product, which has a maximum light absorption at 470 nm. The absorbance is recorded at 470 nm at 30 s (${\mathrm{A}}_1$) and at 1 min 30 s (${\mathrm{A}}_2$). $∆\mathrm{A}={\mathrm{A}}_2-{\mathrm{A}}_1$ is calculated. The crude enzyme preparation was performed in the same manner as for SOD.

$$\mathrm{P}\mathrm{O}\mathrm{D} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} ({\mathrm{U} \mathrm{g}}^{-1} \mathrm{F}\mathrm{W})={\displaystyle \frac{9800\times \Delta \mathrm{A}}{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{g}\right)}}$$

(3)

The activity of catalase (CAT) was measured using the UV method. CAT decomposes H₂O₂, and the rate of absorbance decrease at 240 nm reflects the enzyme activity. The crude enzyme preparation was carried out as described for SOD.

$$\mathrm{C}\mathrm{A}\mathrm{T} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} ({\mathrm{U} \mathrm{g}}^{-1} \mathrm{F}\mathrm{W})={\displaystyle \frac{459\times \Delta \mathrm{A}}{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{g}\right)}}$$

(4)

The activity of α-amylase (α-AL) was measured using the DNS method. α-AL hydrolyzes starch to produce reducing sugars, which reduce 3,5-dinitrosalicylic acid to a reddish-brown product. The absorbance of this product is measured at 540 nm.

$$\mathsf{\alpha }-\mathrm{A}\mathrm{L} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} (\mathrm{m}\mathrm{g} {\mathrm{m}\mathrm{i}\mathrm{n}}^{-1}{\mathrm{g}}^{-1} \mathrm{F}\mathrm{W})={\displaystyle \frac{2\times [\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{s}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{r}](\mathrm{m}\mathrm{g} \mathrm{m}{\mathrm{L}}^{-1})}{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{g}\right)}}$$

(5)

The chlorophyll content was determined using spectrophotometric extraction. Chlorophyll a and b exhibit characteristic peaks at 645 and 663 nm, respectively.

$$\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l} (\mathrm{m}\mathrm{g} {\mathrm{g}}^{-1} \mathrm{F}\mathrm{W})={\displaystyle \frac{2\times (20.21\times {\mathrm{A}}_{645}+8.02\times {\mathrm{A}}_{663})\times 10}{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{g}\right)}}$$

(6)

All measurements were performed in triplicate and expressed as mean ± SD. One-way ANOVA followed by Tukey’s test (SPSS 26.0) was applied; different lowercase letters (a–d) indicate that there is a significant difference between groups. To minimize variability, all enzyme activities and pigment contents were calculated on a fresh weight basis without protein normalization.

3. Experimental Results

3.1. Discharge Characteristics

Figure 2A,B show the voltage and current waveform characteristics of the Trichel pulse discharge under the action of DC power supply and pulse power supply. The DC power supply presents a high-frequency narrow pulse current characteristic in the negative half-cycle, with a single pulse width of about the microsecond level, corresponding to the instantaneous conduction of the micro-discharge channel, presenting a filamentary discharge morphology. It can be observed that the current amplitude in the negative half-cycle is approximately −1.5 mA, significantly higher than −0.5 mA in the positive half-cycle, which is closely related to the secondary emission mechanism of electron avalanche in negative corona discharge. While the discharge current of the pulse power supply is mainly concentrated at the rising and falling edges of the voltage pulse, its sub-microsecond rapid electric field change significantly increases the electron density, thereby suppressing the random expansion of the discharge channel [28,29].

From the discharge phenomenon observed in Figure 2C–E, the DC discharge shows obvious instability: the discharge area presents a flickering bright white light spot, and the spectral analysis in Figure 3 shows strong N2(C³Πu→B³Πg, 337 nm) and continuous radiation, indicating the transformation of local high-temperature transient filamentary discharge to streamer discharge. While the pulse discharge is characterized by a uniform and diffuse pale purple glow, with the main emission lines being N+(B²Σu⁺→X²Σg⁺, 391 nm) and O (777 nm), corresponding to stable glow discharge at low gas temperature. For plasma-activated water discharge, due to the dielectric constant of water being significantly higher than that of air, the effective electric field in the discharge gap is enhanced, causing the discharge to be concentrated within a 5 mm range at the water air interface, accompanied by enhanced emissions of OH (309 nm) and Hα (656 nm) [30,31].

3.2. Contact Angle and Germination Percentage Under Different Treatments

Germination potential and germination percentage reflect seed vigor. Under controlled conditions (12 h light at 25 °C, 45% RH, 60% light intensity; 12 h dark at 10 °C, 45% RH), the germination dynamics of treated seeds were monitored for 15 days. (Soil ingredients: Latvian peat with coarse fiber, pine bark, perlite, etc.; pH value: 5.5–7)

• DC Plasma Treatment

The influence of voltage and treatment duration on the surface wettability and germination performance of Bromus inermis seeds was systematically investigated under DC plasma treatment. As shown in Figure 4A, when the treatment time was fixed at 10 min, the contact angle initially decreased and then increased with increasing voltage (14–17 kV), reaching a minimum value of 36.7° ± 0.5 at 15 kV. Correspondingly, the germination percentage also exhibited a trend of first increasing and then decreasing, peaking at 15 kV. Because below 15 kV, electric field strength is insufficient to activate surface reactions; Above 15 kV, excessive energy cleaves organic molecular chains, exposing hydrophobic alkyl groups (-CH₃, -CH₂-). This inverse correlation between contact angle and germination percentage suggests that enhanced hydrophilicity promotes water uptake and germination initiation [32]. Furthermore, at a fixed voltage of 15 kV, the contact angle after 10 min of treatment was significantly lower than that after 20 min, and the germination percentage was higher. This indicates that prolonged treatment may lead to surface damage or thermal effects, thereby reducing treatment efficacy. Therefore, the optimal parameters for DC plasma treatment were determined to be 15 kV and 10 min. The germination percentage rose from 20% to 36%.

2.

Pulsed Plasma Treatment

For pulsed plasma treatment, four parameters—voltage, frequency, pulse width, and duration—were comprehensively evaluated. As illustrated in Figure 4B, the contact angle showed a consistent trend of first decreasing and then increasing with rising voltage (14–17 kV), frequency (4–7 kHz), and pulse width (1500–3000 ns). The germination percentage correspondingly increased initially and then decreased, demonstrating that moderate plasma treatment significantly improves surface hydrophilicity and germination capacity, while excessive energy input inhibits these effects. Regarding treatment duration, 10 min remained the most effective, consistent with the results from DC treatment. Based on these findings, the optimal pulsed plasma parameters were identified as 15 kV, 6 kHz, 2500 ns, and 10 min. The germination percentage rose from 20% to 38%

As shown in Figure 5, contact angle comparisons demonstrate significant hydrophilicity enhancement by plasma modification:

• Control group: Initial contact angle 89° ± 1.5 indicates intrinsic hydrophobicity;
• DC-optimal group (15 kV/10 min): Angle further decreased to 36.7° ± 0.5, significantly surpassing pulsed group;
• Pulsed-optimal group (14 kV/6 kHz/2500 ns/10 min): Angle reduced to 38° ± 0.8 attributed to micro-nano structures and polar groups induced by periodic plasma impulses.

3.3. Trends in PAW

PAW contains various active particles such as H₂O₂, O₃, NO₂⁻, and NO₃⁻. The changes in particle concentrations in PAW were observed (Figure 6) to reveal the correlation mechanism between plasma energy input and the generation of liquid-phase active substances.

• Effect of Treatment Time as shown in Figure 6A:

H₂O₂: The concentration of H₂O₂ initially increased and then decreased with increasing treatment time, indicating that its generation percentage was dominated by the continuous electron collision dissociation of H₂O.

O₃: The concentration reached a peak at 3 min and then gradually decreased due to the self-decomposition of O₃ and its reaction with OH⁻ in the liquid phase.

NO₂⁻: The concentration reached its maximum at 5 min and then decreased due to oxidation to NO₃⁻.

NO₃⁻: The concentration remained stable, suggesting that the generation and consumption rates reached a dynamic equilibrium within the experimental duration.

• Effect of Treatment Voltage as shown in Figure 6B:

When the voltage was below the breakdown voltage of 14 kV, the concentration of RONS in water did not change significantly, indicating that the plasma did not effectively break through the interface. When the voltage reached 14 kV:

H₂O₂: The concentration continued to increase with voltage, attributed to the enhanced H₂O dissociation and ·OH recombination promoted by the increased high-energy electron flux.

O₃: The concentration reached a peak at 15 kV and then decreased due to the accelerated thermal decomposition of O₃ caused by overheating in the discharge zone.

NO₂⁻: The concentration increased monotonically with voltage, due to the enhanced dissociation of N₂/O₂ in the gas phase and the oxidation path of NO in the liquid phase.

NO₃⁻: The concentration remained stable, possibly due to the saturation of the redox potential in the liquid phase.

The water treated by plasma typically becomes acidic, with a decrease in pH value. This is due to the redox reactions of active particles (such as H₂O₂ and O₃) and NO₃⁻ and NO₂⁻ generated during plasma treatment. Under the treatment conditions of 15 kV and 10 min, the pH value decreased to approximately 3.0.

During the pulsed discharge plasma treatment of water, particle transitions such as OH, N₂, and N₂⁺ occur, generating new reactive species in the liquid phase. These include long-lived particles like H₂O₂, NO₂⁻, and NO₃⁻, as well as short-lived radicals (e.g., ·OH, H·) and ONOOH [33]. Measurements of H₂O₂, O₃, NO₂⁻, and NO₃⁻ concentrations in PAW reveal that the concentrations of hydrogen peroxide (H₂O₂) and ozone (O₃) increase with longer treatment times. This is due to high-energy electrons colliding with water molecules (H₂O), producing radicals (e.g., ·OH, H·):

$${\mathrm{H}}_2\mathrm{O}+e^{-}\to \cdot \mathrm{OH}+\mathrm{H}\cdot +e^{-}$$

(7)

·OH radicals recombine to form H₂O₂:

$$\cdot \mathrm{OH}+\cdot \mathrm{OH}\to {\mathrm{H}}_2{\mathrm{O}}_2$$

(8)

Oxygen (O₂) in the air is dissociated into oxygen atoms (O·) by high-energy electrons, which then combine with O₂ to form O₃:

$$O_2+e^{-}\to 2\mathrm{O}\cdot +e^{-}\to \mathrm{O}\cdot +O_2\to O_3$$

(9)

As the treatment time increases, the plasma continuously inputs energy, leading to more complete generation and recombination reactions of reactive radicals (·OH, O·), resulting in the accumulation of H₂O₂ and O₃.

In contrast, the concentrations of nitrate (NO₃⁻) and nitrite (NO₂⁻) initially increase and then decrease due to competing oxidation and reduction reactions in the plasma: H₂O₂ and O₃, being strong oxidants, can further oxidize NO₂⁻ to NO₃⁻:

$${\mathrm{NO}}_2^{-}+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{NO}}_3^{-}+{\mathrm{H}}_2\mathrm{O}$$

(10)

The water treated by plasma is usually acidic (pH decreases). Under low pH conditions, H⁺ can react with NO₃⁻/NO₂⁻, leading to reduction reactions and the formation of gaseous products (e.g., NO, N₂O):

$${\mathrm{NO}}_3^{-}+2{\mathrm{H}}^{+}+e^{-}\to {\mathrm{NO}}_2+{\mathrm{H}}_2\mathrm{O}$$

(11)

High-energy electrons can directly break the chemical bonds of NO₃⁻/NO₂⁻, generating N₂ gas and escaping (denitrification effect):

$${2\mathrm{NO}}_3^{-}+10{\mathrm{H}}^{+}+8e^{-}\to {\mathrm{N}}_2+5{\mathrm{H}}_2\mathrm{O}$$

(12)

As the treatment time increases, these reactions continue, leading to a decrease in the total amount of NO₃⁻ and NO₂⁻.

For plant seeds, it is not the case that the higher or lower the concentration, the better. Low concentrations of hydrogen peroxide (H₂O₂) have a positive effect on seeds, as they can act as signal molecules to activate the antioxidant system of seeds (such as SOD and CAT enzymes), break dormancy and promote germination. High concentrations can cause oxidative damage, destroying cell membrane lipids, proteins and DNA, leading to a decline in seed vitality or even seed death.

Low concentrations of ozone (O₃) have antibacterial effects, reducing pathogenic microorganisms on the surface of seeds and lowering the risk of diseases. High concentrations can damage the integrity of seed cell membranes, inhibit mitochondrial respiration, and lead to delayed or failed germination. NO₃⁻ and NO₂⁻ are important nitrogen sources for plants, participating in the synthesis of amino acids and chlorophyll. Their deficiency can lead to restricted growth. The decrease in NO₃⁻/NO₂⁻ content may reduce the nitrogen supply required for seed germination, but the active nitrogen generated by plasma treatment (such as NH₄⁺ or organic nitrogen) may compensate for this effect and further detection is needed.

Based on the concentration detection and analysis, the impact on seeds shows a dual nature: low concentration promotes and high concentration inhibits. 10 min was selected as the optimal processing duration to measure the influence of different voltages. The results showed that below 15 kv (the halo voltage), there was no discharge, only the electrostatic field acted, and the electric field intensity was insufficient to ionize gas molecules. It was found that the water did not change at all. When the voltage began to break down, local breakdown occurred near the needle tip, generating high-energy electrons to collide with H₂O₂ and O₃, triggering a chain reaction. The continuous increase in voltage leads to the instability of the discharge channel, forming local arcs and uneven energy distribution. After seed germination, it was found that the results were obvious. Therefore, PAW (15 kv, 10 min) was selected for the subsequent experiment.

The seeds treated with PAW were cultured for 15 days under the same culture conditions as mentioned above, and germination was as shown in Figure 7.

Owing to the significant changes in the concentration of reactive species in PAW, its effect on seed germination was more pronounced compared to direct plasma treatments. As illustrated in Figure 7, when the voltage was fixed at 15 kV and the treatment duration varied, the germination percentage reached its peak after 10 min of treatment—consistent with the optimal time observed in both DC and pulsed plasma groups.

Further analysis under a fixed duration of 10 min revealed that discharge did not occur below 15 kV, resulting in negligible improvement in germination. Once the breakdown threshold of 15 kV was attained, the germination percentage sharply increased to a maximum of 70%, which represents a 50% enhancement compared to the control group. This remarkable improvement underscores the efficacy of PAW in breaking seed dormancy. However, beyond this optimal voltage, further increase led to a decline in germination percentage, likely due to oxidative damage caused by excessive reactive species.

Therefore, the optimal PAW treatment parameters were determined to be 15 kV and 10 min. Notably, under these conditions, PAW not only achieved the highest germination percentage among all treatment modalities but also demonstrated superior performance compared to both direct DC and pulsed plasma treatments, highlighting its potential as an efficient and scalable approach for seed enhancement.

3.4. Seedling Growth

As shown in Figure 8, plasma treatment markedly improved the seedling growth vigor of Bromus inermis at 15 days after sowing. To quantitatively evaluate these treatment effects, further growth parameters of the resulting seedlings, including root length, shoot length, and biomass, were measured and are summarized in

Table 1. Growth performance of 15 days seedlings under different plasma treatments. The table summarizes key morphological and biomass parameters (root length, shoot length, root number, fresh and dry weight) for the control and treatment groups (DC, Pulsed, PAW). Values are mean ± SD (n = 3).

Parameter	PAW (15 kv, 10 min)	Pulsed (15 kv, 6 kHZ, 2500 ns 10 min)	DC (15 kv, 10 min)	Control
Root length (mm)	9.5 ± 0.7	11.5 ± 0.7	10.5 ± 0.7	9.5 ± 2.1
Seedling length (mm)	37.5 ± 0.7	38.0 ± 3.0	40.0 ± 1.4	36.5 ± 0.7
Root number (per plant)	1.5 ± 0.7	1.5 ± 0.7	1.5 ± 0.7	1.5 ± 0.7
Fresh weight (mg/plant)	44.5 ± 3.5	36.5 ± 1.8	34.0 ± 4.2	27.5 ± 3.5
Dry weight (mg/plant)	7.0 ± 1.4	6.5 ± 0.7	5.5 ± 0.7	5.5 ± 0.7

.

• PAW biomass superiority: Fresh weight 44.5 mg (+60%), dry weight 7.0 mg (+27%), indicating enhanced photosynthesis.
  Unchanged root length but higher biomass suggests lateral root proliferation.
• Pulsed root specificity: Root length 11.5 mm (+21%) due to ROS-activated auxin polar transport.
  No biomass gain implies energy allocation to elongation over accumulation.
• DC contradiction: Temporary height increase (40.0 mm) with low dry matter conversion (unchanged dry weight).

3.5. Measurement of Enzyme Activity in Seedlings

After 30 days of growth of Bromus inermis seeds, to further investigate the internal biological characteristics of the seedlings grown from seed directly and indirectly plasma treated, the activities of antioxidant enzymes including SOD, POD, and CAT, as well as chlorophyll content and α-amylase activity were measured. The experimental results are shown in Figure 9 and Figure 10.

3.5.1. Changes in Antioxidant Enzyme Activity

SOD activity: Compared with the control group of 242.78 U/g, PAW treatment significantly increased to 526.81 U/g, an increase of 117%. Aligns with observations by Rathore, V et al. [3] in pea seedlings. Pulse treatment and DC treatment increased by 42%.

POD activity: The PAW treatment group reached 144,363.40 U/g, increasing by 46.78%. The DC treatment group was 120,138.00 U/g, increasing by 22%, while the pulse treatment group was 93,808.15 U/g, slightly lower than the control group.

CAT activity: PAW treatment significantly increased to 217.78 U/g, with a fresh weight increase of 65.65%, while pulse treatment led to a sharp decrease in CAT activity to 30.40 U/g fresh weight, and the DC treatment group was close to the control group.

3.5.2. Photosynthetic Pigments and Metabolic Activities

Total chlorophyll content: The chlorophyll content in the direct current treatment group was the highest at 7.98 mg/g, increasing by 9.2% as compared to control. The chlorophyll content in the PAW treatment group slightly decreased by 3.3%, and that in the pulse treatment group significantly decreased by 11.8%.

α-amylase activity: The PAW treatment group increased to 0.21 mg/min/g, growing by 31.3%, while the activities in both the pulse and direct current treatment groups were lower than those in the control group.

PAW treatment significantly promoted the antioxidant system (SOD, POD, CAT) and metabolic activities (α-amylase) of Bromus inermis seeds, while pulse and direct current treatments showed inhibitory effects or no obvious changes on the activities of some enzymes and photosynthetic pigments.

4. Discussion

This study compared the effects of direct plasma treatment using direct current (DC) and pulsed discharge, as well as indirect treatment using pulsed plasma-activated water (PAW), on the germination characteristics of Bromus inermis seeds. Numerous studies have shown that discharge plasma is an effective technique for enhancing seed germination and promoting plant growth [34]. Corona discharge generates a significant amount of plasma and reactive nitrogen oxides (RONS). Ion wind, which is the movement of neutral particles driven by high-energy electrons produced during gas discharge [35], increases with voltage and can alter the contact angle of seed surfaces, thereby changing their hydrophilicity [32].

In the plasma treatment process, parameters such as power supply voltage and treatment duration can significantly influence plant seeds. Figure 4 and Figure 5 show that in DC treatment, with voltages of 14 kV, 15 kV, 16 kV, and 17 kV, and treatment durations of 10 and 20 min, the contact angle first decreased and then increased with rising voltage. Plasma treatment alters the surface hydrophilicity of seeds, as evidenced by the change in contact angle, which in turn affects the germination percentage. Experiments confirmed a negative correlation between germination percentage and contact angle. The germination percentage first increased and then decreased with rising voltage, with 15 kV identified as the optimal treatment voltage. Additionally, prolonged treatment can erode seeds, reducing germination percentages. Considering both germination and growth promotion, the best treatment was 15 kV for 10 min, which increased the germination percentage from 20% to 36%.

In pulsed treatment, with more parameters, we set voltages from 14 to 17 kV, frequencies from 4 to 7 kHz, pulse widths from 1500 to 3000 ns, and treatment durations from 10 to 30 min. Based on measurements of contact angle and germination percentage, the optimal conditions were 15 kV, 6 kHz, 2500 ns, and 10 min, which increased the germination percentage from 20% to 38%.

For indirect treatment using PAW, where water was treated first and then used to treat seeds, we set different voltage levels and treatment durations. Since water participates in the discharge process, we measured the concentrations of H₂O₂, O₃, NO₃⁻, and NO₂⁻ in the water [36,37]. As shown in Figure 6, different voltages and durations have varying effects. Since particle concentration is not always better when higher, studies have shown a low-concentration promotion and high-concentration inhibition effect [18]. Comparing germination percentages, the optimal treatment was 15 kV for 10 min, which increased the germination percentage from 20% to 70%. Compared with direct treatment, the increase in germination percentage was very significant, as can be seen in Figure 8. This also proves that ion wind and RONS generated during discharge are important for seed growth promotion.

In recent years, more and more studies have shown that the germination of many crop seeds is closely related to RONS [38]. The redox level in plants is determined by RONS and antioxidants, such as per-oxidase (POD), catalase (CAT), and super-oxide diastase (SOD) [39]. This study further measured the enzyme activities after 30 days of seedling growth, as shown in Figure 9. Compared to the control, PAW treatment significantly increased the activities of SOD, POD, and CAT by 117%, 46%, and 65%, respectively. Pulsed plasma treatment increased SOD activity by 42%, but decreased POD and CAT activities by 4% and 77%, respectively. DC treatment led to a 42% increase in SOD, a 22% increase in POD, and a 3.8% decrease in CAT activity. The PAW treatment, which demonstrated the strongest germination-promoting ability, concurrently induced the most pronounced up-regulation of antioxidant enzymes in the seedlings, suggesting that the enhanced capacity to scavenge reactive oxygen species may contribute to improved germination.

It should be noted that the observed biological effects of PAW (e.g., enhanced germination) result from the synergistic action of RONS and acidic environment, rather than low pH alone. Although acidic conditions can break dormancy [40], treatment with strong acids (e.g., HCl, HNO₃) may cause embryonic damage and growth suppression, while failing to deliver the oxidative signaling and nitrogen nutrients provided by RONS. In contrast, PAW treatment not only avoids the toxicity risks of strong acids but also enables safer and more efficient promotion of seed germination and seedling stress resistance through multi-component regulation. While our data demonstrate the efficacy of PAW treatment, the individual contributions of RONS and low pH to the overall effect cannot be definitively disentangled in the present experimental design. Future studies should include controls with acidified water (e.g., adjusted to pH 3.0 using HCl or HNO₃) to isolate and quantify the specific role of pH versus plasma-generated RONS.

To summarize, different discharge modes and treatment methods have different effects on plant seeds. In direct treatment, ion wind erodes the seed surface, changing the contact angle and thereby enhancing seed hydrophilicity and germination percentage. In indirect treatment, the concentration of RONS in the liquid phase is changed to alter the seed growth environment and promote seed germination [41]. For all three treatments, the germination percentage first increased and then decreased with rising voltage, with the optimal voltage for all treatments being 15 kV. At lower voltages, plasma channels are not fully formed and cannot effectively influence plant seeds, while higher voltages can damage the seed surface. The optimal treatment duration was 10 min. RONS generated by plasma treatment affect the inside of the seeds. After plasma treatment, SOD, POD, and CAT all changed, indicating that the seed’s antioxidant defense system is activated in response to plasma-induced oxidative stress. This shows that moderate oxidative stress can act as a positive stress signal, breaking dormancy and promoting germination by triggering downstream signaling pathways.

5. Conclusions

This study compared the effects of direct and pulsed power sources on the characteristics of Bromus inermis germination and seedling growth treated directly and indirectly via PAW. Based on the experimental results, the following conclusions were drawn:

(1)

Different power sources and voltage parameters have distinct effects on seeds. The optimal conditions for direct current (DC) treatment were 15 kV for 10 min; for pulsed treatment, 15 kV, 6 kHz, 2500 ns, and 10 min; and for PAW treatment, 15 kV for 10 min.

(2)

Discharge plasma treatment can break seed dormancy and promote germination. Among the three treatments, PAW showed the most significant increase in germination percentage, with a 50% improvement. The pulsed treatment group saw an 18% increase, while the DC treatment group had a 16% increase.

(3)

Discharge plasma treatment can enhance seed germination and promote growth. Regarding the activities of SOD, POD, and CAT, PAW demonstrated the most pronounced effects, with increases of 117%, 46%, and 65%, respectively. In contrast, the effects of DC and pulsed treatments were relatively similar, with SOD increasing by 42% in both cases and POD, DC increased by 22%. Pulse slightly decreased, while CAT decreased in both treatments.