Quantifying Plasma Dose for Barley Seed Treatment by Volume Dielectric Barrier Discharges in Atmospheric-Pressure Synthetic Air

Abstract

Plasma-assisted treatment is a potentially interesting technology for advanced seed processing. In this work, we address the issue of defining and quantifying the plasma dose during the exposure of seeds to microdischarges formed in a barrier discharge configuration fed with synthetic air at atmospheric pressure. Using advanced imaging and other optoelectrical diagnostics, we identify suitable conditions for the formation of microdischarges developing exclusively between the powered electrode and the seed coat, which allows for the relatively accurate quantification of the plasma dose for an individual barley seed. In addition to determining the microdischarge energy/power consumed to treat a single seed during controlled exposure, we also provide an estimate of the electric field and gas temperature, which are key parameters that can affect seed viability. In this way, each individually exposed seed can be linked to the exact exposure time, total number, energy, and temperature of the microdischarges that came into contact with it. This is fundamentally different from conventional “averaging” approaches based on the simultaneous exposure of many seeds, which makes it virtually impossible to correlate the responses of individual seeds with the corresponding individual plasma dose. Finally, we propose a minimal treatment protocol that could allow for the more direct interpretation of the results of subsequent biological tests to reveal seed responses to specific plasma–chemical stimuli during germination and seedling growth.

1. Introduction

Plasma-assisted seed treatment is currently being investigated as a suitable technology for agricultural purposes, where seeds can be treated either to improve their germinability (i.e., increase the likelihood of early and uniform germination) or to increase their storage shelf life (i.e., reduce the likelihood of crop loss due to various pathogens) [1,2,3,4,5,6,7,8,9,10,11,12,13,14].

As noted by Wascow et al. [15], the main current problem is the lack of standardization of the experimental methodology, which prevents a convincing evaluation of the outcomes of plasma-assisted seed treatments. As a result, it remains challenging to achieve the reproducibility of experimental observations and unambiguously attribute the response (e.g., seed germination parameters and seedling growth) to a specific stimulus (specific parameters of the plasma used to treat the seeds). To achieve this, it is important to provide both the complete characteristics of the plasma device (e.g., characteristics of the high-voltage source, electrode configuration, and corresponding materials) and the parameters of the plasma treatment of seeds (e.g., discharge mode and power, composition and flow rate of the working gas, treatment time and geometry of seed exposure to the plasma, and basic plasma parameters). Since any practical application would have to allow for the efficient and economical treatment of large volumes of seeds, the greatest potential in using a plasma source for seed treatment is with atmospheric-pressure dielectric barrier discharges (DBDs), using air as the working medium [16]. With a few exceptions, DBD configurations produce strongly inhomogeneous streamer-based transient microdischarges (MDs) characterized by very short durations (tens of nanoseconds), making it difficult to accurately characterize the plasma–seed interaction [17,18,19,20,21,22]. The effect of the treatment strongly depends on whether the seed is in direct (MDs directly impinging on treated samples) [23] or indirect (MDs close to but not impinging on treated samples) [24] contact with the MDs or is only remotely exposed to a bath of reactive species produced by the discharge [7]. The characterization of remote treatment is a relatively easy task, as it only requires the measurement of the concentrations of more or less stable species (e.g., O₃ or NO_x) and the temperature/humidity of the working gas mixture. In contrast, the most complex treatment is when the seed is in direct contact with the MDs, as the seed is exposed to an electric field, ionizing electrons, charged and excited species, reactive oxygen and nitrogen species (RONS), and plasma-induced (vacuum ultraviolet, ultraviolet, visible, near-infrared (VUV–UV–Vis–NIR) emissions, which makes this task extremely challenging. Furthermore, depending on the duration and intensity, direct treatment can lead to the etching of the seed coat, which can significantly affect the seed viability [25,26]. A simplified approach and procedure, therefore, appear to be essential in identifying the characteristics and parameters of direct seed treatment.

In this work, we propose a possible approach based on treating a group of seeds one by one to determine the plasma dose with high accuracy (i.e., the number of MDs that reach a given seed). Further treatment characteristics can be obtained by determining the average properties of a typical microdischarge (MD). Using advanced optoelectric diagnostics, we identified suitable conditions for the counting of MDs and the determination of several basic parameters, such as the energy load associated with exposure, the temperature of the gas with which the seed surface comes into contact, exposure to UV-A/B/C light, and the effective electric field. Finally, we propose a minimal treatment protocol that could allow for the more direct interpretation of the results of subsequent biological tests.

2. Experimental Setup, Materials, and Methods

Figure 1a displays the compact and modular 3D-printed reactor with a volume dielectric barrier discharge (VDBD) geometry utilized in this study. The reactor is composed of two circular (Ø = 2.54 cm) alumina dielectrics (AD-96; CoorsTek, Inc., Golden, CO, USA) attached to polylactide (PLA) holders sealed in a main PLA reactor body [26,27]. The opposite sides of the disks are covered by silver-based electrodes (Ø = 2.1 cm), with the discharge generated in the volume formed by the alumina disks. The movable holders (Figure 1b) allow the easy insertion of treated samples, the simple replacement and/or cleaning of the dielectric or electrodes, and, most importantly, the adjustment of the discharge volume gap. The gap size is set using an appropriate spacer (Figure 1b), which is inserted first to ensure the correct position of the outer collar. This slip-on collar restricts the insertion of the grounded electrode into the chamber, allowing for accurate and reproducible electrode placement during repeated insertions and seed replacements after treatment.

The reactor was powered by a combination of a TG1010A Function Generator (Aim-TTi, Ltd., Huntingdon, Cambridgeshire, UK), Powertron Model 1000A RF Amplifier (Industrial Test Equipment Co., Inc., Port Washington, NY, USA) and a high-voltage step-up transformer. The sinusoidal HV waveform (f = 5 kHz) is modulated by a square waveform (f_M = 500 Hz) with a fixed duty cycle of D = T_ON/(T_ON + T_OFF) = 40%, resulting in four consecutive HV-sine waves (T_ON = 0.8 ms), followed by a cooling period with T_OFF = 1.2 ms. The burst mode of the applied AC voltage appears to be suitable for heating heat-sensitive samples, as it guarantees the maintenance of constant treatment conditions (especially a low gas temperature) at low airflow rates for longer exposure times (several minutes) [26,27]. In all reported plasma treatments, synthetic air (Scientific, Messer, Bad Soden, Germany; H₂O < 2 ppm, CO + CO₂ < 0.2 ppm) was used as the working gas, with a fixed airflow of 1 slm. A fast-digitizing oscilloscope Tektronix DPO5204 (Tektronix, Inc., Beaverton, OR, USA) (bandwidth 2 GHz, sampling rate 5 GS/s) was used to sample the discharge characteristics, with an average of 2048 individual samples to reduce noise. The applied voltage, current, and transferred charge were measured using a Tektronix P6015A high-voltage probe (1000:1@1 MΩ, bandwidth 25 MHz), a Pearson Electronic m. 2877 current probe (Pearson Electronics, Inc., Palo Alto, CA, USA) (bandwidth 200 MHz, risetime 2 ns), and a 490 nF capacitor connected in series. Two quartz side windows allow for the sampling of optical emissions from the discharge area. An Andor iStar ICCD (intensified charge-coupled device) camera DH740i-18U-03 (Oxford Instruments, Abingdon, UK) was coupled with an iHR-320 spectrometer (Jobin-Yvon, Horiba Instruments Inc., Edison, NJ, USA) equipped with 300, 1200, and 3600 G mm⁻¹ dispersion gratings. The visualization of streamer formation in the reactor gap was realized using a four-channel XXRapidFrame ICCD camera (Stanford Computer Optics, Inc., Berkeley, CA, USA) focused on the entire discharge area. The power source, oscilloscope, and ICCD detectors were synchronized using an external trigger provided by a digital delay/pulse generator BNC Model 575 (Berkeley Nucleonics Corporation, San Rafael, CA, USA). The ozone and NO_x concentrations, as the principal plasma products, were measured using a non-dispersive UV absorption ozone monitor (Advanced Pollution Instrumentation Model 450, Teledyne API, San Diego, CA, USA) and chemiluminescence NO_x analyzer (Model T200H, Teledyne API, San Diego, CA, USA), respectively.

As sample seeds, we used spring barley, a medium–late variety belonging to the malting variety group (Hordeum vulgare L., cv. Malz, country of origin: Czech Republic). Germination tests were performed using an agar water medium placed in a Petri dish. In terms of seed treatment, three basic categories (direct, indirect, and remote treatment) have been established in a recent review [7]. These specific approaches differ in the complexity of the interaction between the treated biosample and the plasma source. In the case of direct treatment, treated samples come into direct contact with electric fields, energetic electrons/ions, excited species, radicals, and the full spectrum of radiation (including VUV and UV photons) produced within active plasma regions. On the other hand, in the case of indirect treatment, the samples are not in direct contact with the active plasma region; however, the distance between the active plasma and samples is sufficiently short that the samples are still exposed to UV photons, diffusing ions, and short-lived radicals (energetic electrons and VUV can be neglected). Finally, in remote treatment, only long-lived plasma products, such as reactive oxygen and nitrogen species, can reach the treated samples. In this work, we utilized the direct treatment approach, meaning that the MDs produced by the VDBD geometry came into direct contact with the seed. In this study, seeds were loosely placed on the dielectric of a grounded electrode (direct treatment), leaving sufficient space between the seeds to safely distinguish MDs associated with a given seed in the acquired images.

3. Results

3.1. Basic Discharge Characteristics

Figure 2a shows one DBD driving an HV burst composed of four consecutive AC waves and the corresponding current and transferred charge waveforms. While the evolution of the voltage is symmetric with regard to the voltage polarity (the amplitudes of the positive and negative half-cycles are comparable), the transferred charge exhibits a noticeably larger amplitude in positive polarity, which is more than twice as high as the negative one. The observed charge asymmetry (Section 3.1) can be attributed to the sinusoidal HV waveform settings specified in Section 2, where the function generator and RF amplifier’s modulation may influence the MD formation dynamics (e.g., different mechanisms of MD formation at positive and negative polarities and/or residual charge deposited on the surface of the seeds and alumina substrate).

The energy dissipated in the reactor gap during the HV burst was calculated using the Q-V plot (or Lissajous figure) method [28] as follows:

$$E_d=\oint UdQ,$$

(1)

where the surface enclosed by the Q-V plot of one AC wave (U and Q are the applied voltage and transferred charge, respectively) is equal to the dissipated energy during the same period. The characteristic Q-V plot of all four AC waves for a discharge gap of 3 mm is shown in Figure 2b. The sine waves 2–4 are almost perfectly symmetrical (with regard to the polarity switch) and almond-shaped, which is typical of surface DBDs [29,30,31] rather than volume DBDs [26,32,33,34], with an average dissipated energy of 1.27 mJ per wave. However, the Q-V plot corresponding to the first sine exhibits noticeable asymmetry in positive polarity and a resulting increase in the total dissipated energy of roughly 10% (to 1.39 mJ). This indicates that the first discharge event per HV burst is the most intense, and this property is true for all measured conditions.

The aforementioned symmetry and regular shape of the Q-V plots allow us to easily utilize the equivalent circuit approach [35,36,37] to infer further discharge properties from the Q-V plots. In the simplest approach, the equivalent circuit considers the reactor to consist of a dielectric (with effective capacitance C_d) and a gas gap (C_g) connected in parallel, together forming the total reactor capacitance (C_cell). The two distinct slopes on the Q-V plot ($C={\displaystyle \frac{dQ}{dV}}$) then indicate the two values of effective discharge capacitance: C_cell for the phase without discharge, when the reactor behaves as a pure capacitor, and C_d for the discharge phase, when the air gap becomes conductive and only the dielectric barrier contributes to the capacitance. However, in most cases, this is not a valid assumption, and the slope of the active phase (usually marked ζ_d) serves as the bottom estimate of C_d [35,38]. Additionally, in all phases, parasitic capacitances, which are basically unavoidable due to the high-voltage connections and cables, together with stray capacitances, influence the capacitances measured by the equivalent circuit method.

Figure 3a,b display Lissajous figures for two electrode distances (3 and 5 mm) and various fillings of the reactor with barley seeds (0, 1, 3, and 5). For an electrode gap of 5 mm, at least one seed must be present for discharge to occur under the applied voltages. For a 3 mm gap, in which the barley seed is in contact (or nearly in contact) with both electrodes, the differences in shape between the Lissajous figures are marginal, with the slope of the discharge phase being independent of seed presence (ζ_d = 15.2 ± 0.6 pF) and a slope without discharge exhibiting a slight increase with seed presence. As the reactor gap is packed with an increasing number of seeds with a higher dielectric constant than that of air, the reactor capacitance increases accordingly. The dielectric permittivity of seeds generally lies in the range of 2–6, depending on the frequency of the applied voltage and the moisture content in the seeds [39,40,41]. The total capacitance increases by 30% (from 2.28 to 2.99 pF) when the electrode gap is fully filled with seeds (5 seeds) in comparison with that of the empty gap. The seeds also affect the onset of the discharge, as evidenced by the fact that the change in slope to the discharge phase occurs at lower voltages when at least one seed is present. The seeds or any other packing material serve as an artificial inhomogeneity (shortening) in the electrode gap and cause the local intensification of the electric field [42]. Therefore, MDs tend to form predominantly in the seed–electrode gaps and subsequently propagate on the seed surface in the form of a surface ionization wave [43,44]. However, the relatively constant value of the dielectric capacitance ζ_d for all displayed conditions shows that the discharge eventually covers the same area irrespective of the seed presence because, for sufficiently high voltages or short gas gaps (as in the case of a 3 mm gap), MDs also form beside the packing particles [45]. It should be noted that the equivalent circuit provides only a general description of the reactor and discharge properties, which are averaged over the entire volume, with all local effects (discussed in Section 3.2) presumed here.

When the electrode gap is increased to 5 mm, the seeds are no longer in close contact with the upper electrode. This leads to a change in the behavior of both phases. In the phase without a discharge, the capacitance once again increases with the seed number; however, the change is even less notable than for a shorter electrode gap, as the increase in reactor volume (by 66%) means that the seeds’ dielectric permittivity replaces a smaller ratio of air in relative terms. A more significant change occurs in the discharge phase, which requires a higher voltage than that in the 3 mm gap and does not exhibit a single, distinctive slope but has a rather gradual transition to the “full discharge” phase (when the slope reaches its maximum). The maximal value of ζ_d also directly depends on the number of seeds in the reactor, unlike the 3 mm gap case, where it was independent. This indicates that the presence of MDs is directly tied to the seeds, with MDs being initiated only in the seed–electrode gap and then spreading around the seed surface; thus, only this area, which directly scales with the number of seeds, contributes to the measured slope of the discharge phase, while the remaining space retains its air capacitance (hence the lower value of the measured ζ_d). The gradual onset of the discharge and, thus, the continuous change in slope in the Lissajous figures is probably the result of the uneven height of the seeds, leading to the unequal distribution of the first MDs.

Figure 4a,b display the dependence of the discharge power and ozone concentration in the outlet gas on the amplitude of the applied voltage and the number of seeds in the reactor for electrode gaps of 3 and 5 mm, respectively. For a 3 mm gap, the power is independent of the number of seeds and increases almost linearly with the applied voltage. This power independence also shows that, despite the changes in discharge morphology (discussed earlier in Figure 3a,b), the average surface of the Lissajous figure remains approximately constant, only slightly changing in shape. The ozone concentration, however, decreases with the seed content. This is likely a result of the surface interactions of O radicals with the seeds, which inhibit ozone production. The dependence on the applied voltage of the discharge power and the ozone concentration changes in the case of a longer 5 mm gap (Figure 4b), where MDs form only in the seed–electrode gap. Thus, the number of MDs and, therefore, the discharge power are tied to the available seed surface and increase with the number of seeds in the reactor. The ozone concentration also appears to be dependent on the seed content. However, when it is normalized to the discharge power (i.e., recalculated to the yield), it remains roughly constant, meaning that the synthesis of ozone in a 5 mm gap is directly controlled only by the amount of dissipated energy, as the larger reactor volume allows the gas particles to more easily avoid coming into contact with the seed surface. Similar to ozone, the NO_x generation is also noticeably weaker in the case of a 5 mm gap, where it does not exceed 3 ppm, while, for a shorter gap (3 mm), the concentration reaches levels as high as 30 ppm [26].

3.2. Four-Channel High-Speed Imaging

The discharge morphology variations across different DBD gaps are a direct consequence of our uniquely designed reactor (Section 2), allowing the precise adjustment of the electrode distance, thereby affecting MD development. Time-resolved images of the discharge luminosity evolution during an AC HV cycle provide crucial information about the discharge morphology. The 4-Picos optics splits the photon flux collected from the DBD gap and provides four equivalent images formed at the splitter output, with each output image sampled by a single ICCD detector [46]. The ICCD detectors capture MD light by integrating the broadband optical emission (determined by the transmittance of the collection and imaging optics and the quantum efficiency of the MCP S20 photocathode) at time intervals defined by adjusting the MCP gate for each of the four ICCDs separately. Here, we used the same MCP gate (5 microseconds) for all ICCDs synchronized in such a way as to cover a 20-microsecond time interval continuously, without any dead time. We then zoomed in on each AC cycle within the burst where MDs occurred and recorded characteristic images for each half-cycle sequentially one after the other.

To find suitable discharge conditions that allowed the better quantification of the plasma dose, we acquired phase-resolved images to reveal the MD morphology in different DBD gaps, ranging from 3 to 6 mm. In the case of the smallest DBD gap, the seeds almost touched the surface of the top-powered DBD electrode, while, in the case of the largest gap, the seed surface was about 3 mm away from the top electrode. The discharge morphology was inspected with a microsecond time resolution for each AC half-cycle within an AC burst. The most important results obtained at a fixed AC HV amplitude are shown in Figure 5, Figure 6, Figure 7 and Figure 8 (t₀ = 0 represents the unifying trigger time of the entire setup and corresponds to the start of the HV burst).

Figure 5 shows the MDs in the shortest DBD gap (3 mm) propagating directly between the powered and grounded electrodes. These MDs pass around the seeds at different distances, and the intensity of their interaction with the seed surface is practically impossible to quantify since this MD “shower” is highly unstable in spatiotemporal terms and because individual seeds vary slightly in both shape and size. Seeds treated under such conditions are likely to be relatively homogeneously exposed to UV radiation and discharge products such as ozone or NO_x, but exposure to charged particles and the electric field is apparently random. However, with larger DBD gaps, MDs (for a given AC amplitude and frequency) cease to develop directly between the electrode surfaces and start to propagate toward and across the seed surface. This “uncertainty” is due to small individual differences between the three seeds, so that each seed is exposed to its own individual plasma dose. However, all of the energy of the discharge is already directed toward the seeds, as all MDs develop between the powered electrode and the top of the seed and then slide along the seed coat toward the grounded electrode below.

The images shown in Figure 7 (DBD gap 5 mm) reveal that, with a larger gap, MDs develop similarly to the previous case (DBD gap 4 mm), but with a notably higher temporal spread in the onset of MD formation. This spread is well captured in the fourth frame in Figure 7 (t₀ + 545 μs), where the seed in the center is hit by two MDs, while the MDs that developed toward the left and right seeds are already extinguished (compare with the first three frames at t₀ + 530, t₀ + 535, and t₀ + 540 μs). With the largest DBD gap (6 mm) captured in Figure 8, each seed is hit by one MD during one AC half-cycle, and, sometimes, even the seed on the right remains without being hit by an MD. The MDs also do not propagate further along the seed surface, as is the case for smaller gaps (Figure 5, Figure 6 and Figure 7); thus, only the top of each seed is directly treated by plasma.

It follows from the above that, for given external discharge parameters (AC amplitude/frequency, duty cycle, and gas flow), we can “tune” the DBD gap so that seeds of a given size and shape (in this case, malting barley) are hit by a defined number of MD filaments. The total number of MDs can then be easily determined from the optical signal obtained from the gap above a given seed. Furthermore, the given MD count number can be linked to the results obtained from the electrical and spectrometric measurements presented in the previous (Section 3.1) and following (Section 3.3) sections, respectively.

3.3. Optical Emission Spectroscopy

Optical emission spectra were monitored around a seed located on the axis of the DBD gap from a region precisely defined by lenses and iris diaphragms pre-set using a red diode laser (see inset of Figure 9). The spectral range between 200 and 500 nm is of primary interest because the emission bands of molecular nitrogen (N₂) and nitrogen ions (N₂⁺) allow for the estimation of fundamental plasma parameters [47]. In addition, UV light likely plays an important role in the process of triggering germination and the decontamination of seed surfaces contaminated with various pathogens.

We obtained characteristic UV–vis–NIR spectra by averaging the emissions in the positive and negative half-cycles for all four AC cycles within the burst. The spectral analysis revealing the dominant UV-A exposure leverages the sensitivity of the iHR-320 spectrometer in the UV range, as outlined in our methodology (Section 2), ensuring the accurate measurement of plasma-induced UV emissions. The spectra in the UV range were produced exclusively by two dipole-allowed transitions, the second positive system (SPS) N₂ (transition) and the first negative system (FNS) N₂⁺ (transition), while the vis–NIR range (Figure 9) was used to detect one sequence (∆v = +2) of the first positive system (FPS) and the potential atomic emission of neutral oxygen. The oxygen triplet O^I(3s ⁵S⁰→3p ⁵P) at 777 nm overlaps with the weak satellite branches of the FPS(2,0) band and is usually the most intense transition of neutral oxygen in the vis–NIR range [47,48].

Its apparent absence in all detected spectra (Figure 9) in a 5 mm gap indicates the limited dissociative excitation of molecular oxygen, which is in contrast to a previous study that reported a shorter 3 mm gap, in which the 777 nm triplet was clearly detectable [26]. This can also further explain the limited synthesis of NO_x and O₃ noted for larger gaps (Figure 4), for which the oxygen radical is an important precursor [49,50,51].

Both the SPS and FNS are often used to estimate the gas temperature in MD filaments (a crucial parameter in the plasma-assisted processing of heat-sensitive samples such as seeds) by analyzing the at least partially resolved structure of the strongest vibronic bands. Furthermore, the ratio between the intensities of the FNS and SPS bands allows, under certain conditions, for the estimation of the reduced electric field (E/N) characterizing the MD filaments [52,53,54]. The smallest residual between the measured and synthetic spectra was achieved for the rotational temperature of the N₂(C) state T_rot = 330 ± 20 K (Figure 10) for both voltage polarities (which limits the maximum gas temperature to approximately 360 ± 20 K), indicating the limited heating of the seed surface (and inside the seed) if plasma treatment is applied for a short period of time (see discussion of overtreatment in Section 3.4). The vibrational distribution functions (VDFs) of the N₂(C³Π_u) and N₂⁺(B²Σ⁺_u) states in the positive/negative half-cycles in Figure 11 reveal marginal differences between both polarities, with only N₂⁺ exhibiting a close-to-FCF-like distribution [55]. However, the ratios between the FNS and SPS band intensities and the reduced electric field E/N (Figure 12) differ significantly, with the E/N in the positive half-cycle being twice that in the negative half-cycle.

After determining the rotational temperatures and vibrational distributions of the N₂(C³Π_u) and N₂⁺(B²Σ⁺_u) states from the emission registered in the spectral interval of 300–450 nm and verifying that no NO-γ and NO-β bands are detectable in the UV region, we can better analyze the exposure of seeds to whole UV radiation, which is important for seed germination (dormancy breaking) [56,57] but also for the microbiota residing on seeds (e.g., bacteria and fungi) [58,59,60] and possibly for the evolution of germinating seeds and seedlings (not addressed in this work) [61,62,63]. The experimental emission spectra in the range of 300–450 nm were extended using the synthetic SPS and FNS models toward lower and higher wavelengths and subsequently integrated from 200 nm to 500 nm. In Figure 13, we show the dependence of the integral values on the upper limit of the integral for the UV–vis spectra characteristics of both polarities. Note that the curves are normalized for a wavelength of 400 nm in order to easily visualize the UV-A (320–400 nm), B (290–320 nm), and C (200–290 nm) fractions from the total UV load. Under the conditions studied, UV-A exposure evidently dominates, while the UV-C fraction remains below 0.1% (which by no means implies a negligible effect on biological samples). At higher energy densities (not the case in this study), the weight of the UV-C contribution can be significantly increased by the band emission of nitric oxide molecules from the three lowest electronically excited doublet states (A²Σ⁺, B²Π_r, and C²Π_r), which are usually produced by the recombination of atomic species. Furthermore, in the case of humid air, the UV-B contribution can, in principle, be enhanced by the OH band emission around 308 nm.

UV exposure is important not only in maintaining seed quality during storage but also for seed germination and vigor [62,64,65]. Therefore, we consider information on seed exposure to individual UV fractions to be of the utmost importance and propose that these data form an integral part of standard protocols describing plasma-assisted seed treatment experiments.

3.4. Advantages and Consequences of Controlled Seed Treatment

To demonstrate the benefits of the controlled treatment proposed and implemented in this work (i.e., quantifying the exposure of isolated barley seed as outlined in the previous sections), we performed a simplified experiment to verify the effect of MDs on seed germination. Barley seeds were treated in batches of three (see Figure 5, Figure 6, Figure 7 and Figure 8) for several exposure times (20, 60, and 180 s) using a DBD gap of 5 mm. The treatment for each exposure time was performed in quadruplicate (12 seeds altogether for each exposure). After the treatment, the four groups (three exposure times + control) were placed separately on four Petri dishes containing water agar medium and observed under a USB microscope for 6 days. The results of the germination test are shown in the microscopic images in Figure 14. The first two columns (untreated controls and seeds after 20 s of exposure) show that the seed viability remained unaffected, i.e., 100% after the shortest treatment. In contrast, seeds treated for 60 and 180 s (third and fourth columns) did not germinate at all. The images clearly demonstrate the existence of a threshold beyond which the seed viability was severely reduced by an overdose of plasma exposure, which was formed by a combination of an electric field, heating, ozone (and other chemically active products), and UV emission. In particular, the seed heating caused by plasma needs to be considered in studying the reduced viability of seeds as different varieties of the same seed species can have vastly different responses to heat stress [66]. While certain barley varieties can withstand elevated temperatures similar to those measured in this study (≈70–80 °C) for a short period with a minimal drop in viability [67], other varieties can exhibit total germination inhibition as a result of the same heat stress [66]. This is very important when considering the plasma-assisted treatment of seeds as a potential pre-sowing priming technique.

Figure 15 illustrates how the treatment affects the surface properties of the seed coat and the part of the seed (micropyle) through which water enters during germination. The two frames in the figure were extracted from a time-lapse video capturing the imbibition process triggered by placing a small drop (volume of approximately 2 μL) of distilled water on two seeds. The first drop was placed on the reference (untreated) sample, and, immediately afterward, another drop was placed on the treated seed (treatment time of 15 s). The top frame (T₀) represents the first frame after placing the drop on the treated sample, and the bottom frame shows the situation after ten seconds (T₀ + 10 s). While the drop practically did not change in shape or volume in the reference sample, in the treated sample, the drop immediately spread upon impact and, within ten seconds, was completely soaked into the seed coat. This demonstrates a significant change in the wettability of the seed surface. Before the treatment, the surface is hydrophobic (contact angle > 105 degrees), while, after a short treatment, it becomes superhydrophilic (contact angle < 35 degrees). A collateral effect caused by the absorption of a single drop of water is the mechanical stress in the coat layer, which induces expansion in the micropyle/pedicel regions of the seed. This is highlighted by the two zooms on the left, which clearly show the opening of the seed tip (by a factor of 1.5). It is important to note that the micropyle is a narrow pore or passage through which water enters the seed during germination. The expansion of this pore might allow for faster imbibition and, consequently, the faster germination of the seed.

4. Discussion and Conclusions

In this study, we demonstrate that, under well-defined conditions, there is a clear transition (between treatment times of 20 and 40 s) in seed viability after treatment, where germination is not affected at exposures of 20 s or less, whereas, at exposures equal to or greater than 40 s, the treatment has a clearly lethal effect on germinability. We show that, by appropriately combining the DBD gap size with the AC amplitude/frequency with respect to the seed size/shape, it is possible to achieve a discharge regime in which all of the discharge energy is directed to the treated seeds. When working with barley seeds and an AC frequency of 5 kHz (peak-to-peak amplitude slightly above 20 kV), a DBD gap of 4–5 mm appears to be suitable for generating MDs developing exclusively toward the seeds, as shown in particular by diagnostics based on fast phase-resolved imaging and electrical properties inferred from equivalent circuit models. For larger electrode gaps (6 mm), MD formation is not guaranteed (see the rightmost seed in Figure 8) and MDs do not propagate beyond the top of each seed, resulting in a spatially inconsistent plasma treatment. On the other hand, short electrode distances (3 mm) lead to MD formation and the position being almost independent of the seed presence. This then leads to the wasteful and unfocused dissipation of discharge energy out of direct reach of the seeds.

The appropriately chosen conditions can subsequently be used to evaluate other treatment parameters, the most important of which is the total energy deposited by the MDs into an individual seed during the entire exposure time. Furthermore, using phase-resolved optical emission spectroscopy, we can obtain detailed MD characteristics, such as the average gas temperature in MD microfilaments, characteristic UV-A/B/C fractions, and averaged reduced electric field E/N, which are probably the most important factors affecting not only the surface but also the entire volume of seeds, thus determining the viability of treated seeds.

A detailed analysis of the emission spectra obtained from the interaction area of MDs with a single seed can provide other semi-quantitative treatment characteristics, such as the total exposure of the seed to UV–vis–NIR light, the presence of electronically excited neutral atomic N/O species, and eventually ions. The determination of stable species (O₃ and NO_x) can then complete the treatment picture, which can be correlated with parameters obtained from the subsequent analysis of the seeds during germination and the development of new plantlets.

Lastly, to facilitate easy comparison and reproducibility across plasma treatment experiments reported by different groups, we summarize the basic characteristics of the plasma reactor and seed samples, along with a list of the electrical and plasma parameters characterizing the treatment and post-treatment testing, in

Table 1. Specifications of used plasma device and seed type.

Plasma Reactor Characteristics and Treated Samples
Discharge configuration	Volume DBD in parallel plane geometry, symmetric electrodes
Electrode materials	Alumina dielectric plates Ø = 25.4 mm, d = 0.5 mm Embedded silver powered/grounded electrodes Ø = 21 mm, d = 0.05 mm
Discharge gap	Variable: 3/4/5/6 mm
Discharge power	Periodic AC (5 kHz) bursts at 500 Hz repetition frequency, HV amplitude 21 kV (peak-to-peak)
Working gas	Synthetic air (scientific, Messer), 1 slm at 20 °C and 760 Torr
Treated samples	Spring barley (Hordeum vulgare L., cv. Malz, country of origin: Czech Republic), 0/1/3/5 seeds freely placed on the grounded side
Sample preparation	No soaking or sterilization (base moisture content 11.5%)
Treatment approach	Direct (filamentary microdischarges directly impacting seeds)
Treatment time	ttr = 0, 20, 40, and 180 s at a discharge duty cycle of 0.4
Discharge and Treatment Characteristics (gap 5 mm)
Average discharge power	0.28 ± 0.03 W
Exposure energy per seed	1.9 (ttr = 20 s), 5.6 (ttr = 40 s), and 16.8 (ttr = 180 s) [J/seed]
Number of MDs per seed	16 ± 3 (ttr = 20 s), 32 ± 5 (ttr = 40 s), and 48 ± 8 (ttr = 180 s) [104 NMD/seed]
Average energy per MD	35 ± 7 [μJ]
Exposure to temperature	≤360 ± 20 K (estimation based on the time-averaged rotational temperature of the N2(C3Πu) state)
Exposure to electric field	Positive half-cycle: 430 ± 60 Td, negative half-cycle: 870 ± 70 Td (E/N estimated spectroscopically using FNS/SPS ratio method)
Exposure to gas products	O3: 200–300 ppm, NOx: <3 ppm
Exposure to UV	≤0.1% * (UV-C): 7.5% (UV-B):92.5% (UV-A)
Post-Treatment Testing
Contact angle	≈105° (no treatment), <35°(ttr ≥ 20 s) (droplet of distilled water right after treatment, registered time lapse)
Germinability	100% (ttr = 0 and 20 s), 0% (ttr = 40 and 180 s) (in Petri dish with water agar for 6 days, registered time lapse)

* UV-C detection is limited to approximately 225 nm by the photocathode of the ICCD and the efficiency of the dispersion grating used to detect the spectra.

. The structure and content that we introduce in Table 1 follow the proposals for a unified treatment protocol/checklist put forth by Waskow et al. [15].

Work is underway to expand on the post-treatment characteristics through a detailed analysis of the morphological and chemical changes induced by the direct impact of MD filaments on the barley surface.