Effects of Low-Temperature Plasma Treatment on Germination, Seedling Development, and Biochemical Parameters of Long-Term-Stored Seeds

Abstract

The promising field of low-temperature plasma treatment, known for its non-invasive and environmentally sustainable nature, is being actively investigated for its ability to enhance germination, emergence, yield, and overall plant development in a broad spectrum of crops. For gene bank requirements, low-temperature plasma technologies can also improve germination parameters and promote the development seeds suitable for long-term storage. Seeds from four selected cultivars of wheat, oats, flax, and rapeseed stored in the gene bank for 1, 10, and 20 years were subjected to plasma treatments for 20, 25, and 30 min. The study evaluated the mean root and shoot length, root–shoot ratio, and seedling vigour index. Additionally, the malondialdehyde level, total polyphenol content, total flavonoid content, and total antioxidant capacity were analysed. Plasma treatment displayed varying effects on the morphological characteristics and antioxidant activity of the tested cultivars, which were influenced by treatment duration and cultivar. A positive effect of plasma treatment on seedling length, seedling vigour index, and root–shoot ratio was observed in flax cultivar ‘N-9/62/K3/B’ in all periods and in variants T2 and T3. Conversely, the wheat cultivar ‘Granny’ showed variable results, and the oat cultivar ‘Risto’ showed variable negative results in regards to mean root length and mean shoot length after plasma treatment. The indicators of oxidative stress and antioxidant capacity were affected in all the cultivars studied. A positive effect of plasma treatment on these indicators was observed in the wheat cultivar ‘Granny’, while flax cultivar ‘N-9/62/K3/B’ exhibited inconsistent results. While in cereals, a decrease in malondialdehyde content after plasma treatment was associated with an increase in polyphenol and flavonoid content as the treatment duration increased, small-seeded species responded somewhat differently. The rapeseed cultivar ‘Skrivenskij’ and flax cultivar ‘N-9/62/K3/B’ showed an increase in polyphenol and flavonoid content following a decrease in malondialdehyde levels. This study highlights the potential of low-temperature plasma treatment for long-term-stored seeds and its applicability to plant genetic resources. The findings emphasize the need for the further optimization of low-temperature plasma treatment conditions for different plant species and cultivars.

1. Introduction

New approaches are increasingly being used to enhance seed germination quality and subsequent plant development while aiming to reduce pesticide use in the environment. According to EU Directive 2009/128/EC on the sustainable use of pesticides, alternative approaches are being explored to reduce the burden of harmful agents [1]. Traditional seed treatment methods, such as seed dressing and the application of fungicides and phytohormones before sowing [2], are gradually being replaced by innovative techniques. One such emerging method is the use of low-temperature plasma (LTP) [3,4].

Numerous studies have demonstrated that LTP influences seed germination and seedling development [2,3]. LTP plasma affects the microstructure of the seed surface, changing the surface tension and enhancing seed wettability, which may accelerate the onset of germination and promote faster seedling growth [5]. Additionally, plasma treatment has an impact on enzyme content and activity [6], and it plays a role in activating enzymes associated with the early stages of germination [7], triggering a range of stress responses.

Selected morphometric parameters, including the measurement of mean root length (MRL), shoot length (MSL), seedling vigour index (SVI), and root–shoot ratio (RSR), are among the established methods of evaluating the influence of LTP on subsequent seedling development [8,9,10]. Šerá et al. [11] used the total seedling length parameter to monitor the effect of gliding arc type LTP on cannabis seedlings, identifying a positive impact of LTP on plant growth and biomass production. Mildaziene et al. [12] observed an increase in MRL in clover incarnate following LTP treatment. Similarly, Magureanu et al. [9] reported a nearly tripling of root length in tomato seedlings after 5 min of dielectric barrier discharge (DBD) plasma treatment compared to the results for the control seedlings. Hui et al. [13] noted accelerated seedling growth in wheat pertaining to both germination and root development following LTP treatment. Perea-Brenes et al. [14] also demonstrated an increase in root diameter after LTP treatment. In contrast, Judickaité et al. [15] found a different outcome, with a reduction in dry weight and plant height of stevia plants after DBD-type plasma treatment.

SVI is used to assess the development of young plants. It is based on seed vigour (vitality) and the quality of young seedlings [16]. Higher SVI values indicate a more vigorous set of seeds within a species subjected to different seed treatments, while a decrease in values reflects a reduction in vigour [10]. SVI was used by Šerý et al. [10] to evaluate early seedling growth in Scots pine and black pine. They observed significant differences between LTP-treated and untreated seeds in black pine.

RSR is derived from seedling growth. Šerá [16] states that the ratio between the underground (root) and above-ground (shoot) parts of the plant must be balanced to ensure stability, water uptake, and efficient use of solar energy. The RSR parameter is influenced by different life forms and species. According to Šerá [16], this ratio can be applied when comparing different growth conditions or seed treatments.

Tunklova et al. [8] found that diffuse coplanar surface barrier discharge (DCSBD) plasma treatment affected RSR in the tested durum wheat plants, an important trait for successful germination and biomass allocation to the roots, which enhances drought stress tolerance [3,17]. According to Attri et al. [3], the treatment of wheat seeds with DBD plasma led to an increase in RSR compared to that of the control [18]. This result was also confirmed by Magureanu et al. [9], who evaluated the effect of DBD plasma on ‘Belle F1’ tomato seeds and reported RSR values of 0.51 in untreated seeds, and 0.87 and 0.73 in two plasma-treated variants. In contrast, Velichko et al. [19] argue that the effect of plasma jet treatment on biomass parameters such as MRL, MSL, or RSR is rather ambiguous, and plant response depends on the specific method of plasma application.

Oxygen- or nitrogen-based radicals (ROS or RNS), including superoxide anions (O₂⁻), singlet oxygen (¹O₂), hydroxyl radicals (∙OH), and hydrogen peroxide (H₂O₂), are key triggers for many cellular reactions [20]. These reactive species are primarily produced through respiratory metabolism and can cause various types of cellular damage, but they also function as important signalling molecules [21,22]. However, the production of these radicals is also induced by plasma discharge [18,23].

During dry seed storage, increased water content can trigger respiration, producing H₂O₂ and other ROS [24,25], which may lower abscisic acid levels—normally protective against ROS—and contribute to dormancy loss and senescence [26,27]. In hydrated seeds, elevated respiration leads to O₂⁻ formation via mitochondrial electron leakage, which is then converted to H₂O₂. However, ROS and RNS generated by plasma can reduce phytopathogen load, enhance germination conditions, and improve seed coat permeability, aiding water and nutrient uptake [18,28,29].

Malon-dialdehyde (MDA), a product of membrane lipid peroxidation under oxidative stress, is widely used as an indicator of membrane damage [30,31]. LTP has been shown to reduce MDA content by enhancing antioxidant activity, particularly under drought stress [32,33]. Similarly, Tong et al. [34] observed a decrease in MDA levels following LTP treatment, suggesting reduced oxidative damage. In contrast, Šerý et al. [10] reported increased MDA levels in pine seeds after direct DCSBD plasma treatment, attributed to elevated ROS levels.

Lipid peroxidation, on the other hand, can be inhibited by phenolic compounds [35]. Zielińska et al. [36] reported an increase in total phenolic content (TPC) and total antioxidant capacity (TAC) in okra seeds after LTP treatment. In contrast, Othman et al. [35] observed a decrease in both TPC and total flavonoid content (TFC) following LTP treatment, which they attributed to a possible reduction in seed phenolic compounds. A similar trend was noted by Yodpitak et al. [6] in rice, where both TPC and TAC declined after LTP exposure. However, Judickaité et al. [15] reported no significant effect of LTP on TPC, TFC, or TAC. Interestingly, Ben Othman et al. [35] suggested that a high TAC level might contribute to a reduction in TPC.

The aim of this research was to investigate the effects of LTP treatment on the germination and early development of long-term-stored seeds from two selected large-seeded cultivars, i.e., wheat and oats, and two small-seeded cultivars, i.e., flax, and rapeseed, stored for 1, 10, and 20 years in the Gene Bank, Czech Agrifood Research Center (CARC), Czech Republic. In the research, the influence of different LTP treatment durations (20, 25, and 30 min) on the morphological and biochemical parameters was evaluated. The first research hypothesis is that LTP improves seedling development in seeds stored for 1, 10, and 20 years under gene bank conditions. The second hypothesis is that LTP treatments modulate oxidative stress and antioxidant capacity, reducing MDA levels and enhancing TPC, TFC, and TAC levels, thereby contributing to improved seedling vigour.

2. Materials and Methods

2.1. Plant Material Used in the Study

A set of seeds of selected cultivars of agricultural crops (

Table 1. Characteristics of cultivars used in the experiment.

Cultivar Number	ECN	Plant Species	Cultivar
TA_1	01C0100139	Triticum aestivum	Granny
AS_2	03C0700967	Avena sativa	Risto
LU_3	05X1100390	Linum usitatissimum	N-9/62/K3/B
BN_4	15O0100097	Brassica napus f. napus	Skrivenskij

ECN—national gene bank accession registration code.

) stored in the Gene Bank of CARC was used for the experiments. Selected seed samples were treated by LPT after storage in the gene bank for 1 (P1), 10 (P10), and 20 (P20) years at a temperature of −18 °C. Only intact seeds without visible defects or disease germs were used. The seeds were divided into the following groups: control, non-treated variants, and variants treated with LTP. Each sample included 30 seeds with three replicates.

2.2. Plasma Treatment

The plasma source was a Plasonic AR-550-M device (SurfaceTreat, Turnov, Czech Republic), generating vacuum LTP through a stationary microwave resonator. The process parameters were as follows: microwave discharge pulse duration, 60 microseconds; treatment time, 20 (T1), 25 (T2), and 30 (T3) minutes; air flow as working gas, 50 sccm; and pressure in the vacuum chamber at the moment of discharge ignition, <50 Pa using the Leybold D16A Trivac vacuum rotary pump (Leybold Vacuum Products Inc., Export, PA, USA). The operating power of the magnetron was 500 W, the frequency was 2.45 MHz, and the temperature was 25 °C [37]. The operating pressure was maintained by simultaneously pumping the vacuum chamber and adding air as the working gas using the FV201-Cvh valve system (Bronkhorst High-TechH B.V., Ruurlo, The Netherlands). The working gas flow pushed the active, reactive particles generated in the plasma region toward the treated sample. The LTP treatment was applied to samples stored in the gene bank for periods of 1 year (P1), 10 years (P10), and 20 years (P20). The treated seeds, in glass Petri dishes with a diameter of 9 cm, were inserted into the bottom of the vacuum recipient, approximately 15 cm below the plasma entry portal.

2.3. Morphometric Parameters of Germinated Seeds

The mean root length (MRL) and mean shoot length (MSL) were measured according to the methods of Roy et al. [38], and simultaneously, the number of germinated seeds was determined on each evaluation date (day 1, 5, and 7 after sowing in Petri dishes). All germinated seedlings were measured continuously using a non-destructive method with a standardized millimetre gauge (type 28101, Koh-i-noor Hardmuth, a.s., Prague, Czech Republic).

The seedling vigour index (SVI) was assessed according to the methods of Šerá [16] to assess both seed germination (SG) and the quality of the young seedlings. The calculation of SVI, combining the SG parameter with the total seedling length, including root and shoot length, was used. The SG parameter used for this calculation was evaluated in a previously published article [39].

The root–shoot ratio (RSR), as described by Šerá [16], is used to compare different cultivation conditions or seed treatment types. An equilibrium value of 1 indicates a balance between root and shoot length. Values <1 indicate shorter roots, while values >1 indicate shorter shoots.

2.4. Biochemical Parameters of Germinated Seeds

2.4.1. Determination of Malondialdehyde

The thiobarbituric acid assay was used to determine MDA as the final product of lipid peroxidation in the seeds. The seed samples (0.5 g) were homogenized in liquid nitrogen and extracted with 10.5 mL of 80% ethanol. The estimation of MDA (expressed in nmol/g) was modified according to the methods of Du and Bramlage [40]. The extract was mixed with 2-thiobarbituric acid in trichloroacetic acid with toluene butylhydroxide. The mixture was then heated in a water bath (WNB22; Memmert; Büchenbach, Germany) at 95 °C for 15 min.

To stop the reaction, the tubes were placed in an ice bath, followed by centrifugation at 10,000 rpm for 1 min in a Frontier 5718R device (Ohaus Europe GmbH; Nänikon, Switzerland). The absorbance of the supernatant was measured using a UV/Vis spectrophotometer (Evolution 201; Thermo Scientific; Waltham, MA, USA) at 440, 532, and 600 nm, with water as a blank. The measured values at particular wavelengths were used for the calculation of the MDA content in nmol·g⁻¹ of fresh weight (FW), according to the methods of Du and Bramlage [40].

2.4.2. Extraction and Determination of Total Phenols

TPC was analysed from dry matter using a method slightly modified from that of Singleton and Rossi [41]. The extract used for MDA determination was mixed with 10-fold diluted Folin–Ciocalteu reagent, and 7% sodium carbonate was added after 5 min of incubation. After 90 min, the absorbance of the samples was measured at 765 nm using the same UV/Vis spectrophotometer (Evolution 201; Thermo Scientific; Waltham, MA, USA). TPC was expressed as the gallic acid equivalent (mg·g⁻¹ FW).

2.4.3. Extraction and Determination of Total Flavonoids

TFC from dry matter was measured using the method described by Tsanova-Savova et al. [42]. An aliquot of the ethanolic extract from the MDA assay was mixed sequentially with 5% NaNO₂, 10% AlCl₃, and finally 1 mol/L NaOH. The absorbance of the mixture was measured using a UV/Vis spectrophotometer at 415 nm against a blank, and TFC was expressed as the quercetin equivalent in mg·g⁻¹ FW.

2.4.4. Extraction and Determination of Total Antioxidant Capacity

TAC from dry weight was determined using a method adapted from Prieto et al. [43]. The same ethanolic extract as that used in previous assays was mixed with a reagent solution containing 0.6 mol/L H₂SO₄, 28 mmol/L Na₃PO₄, and 4 mmol/L (NH₄)₆Mo₇O₂. The mixture was heated in a water bath at 95 °C for 90 min, and the absorbance was measured at 695 nm against a blank using the same UV/Vis spectrophotometer. TAC was expressed as the ascorbic acid equivalent in mg·g⁻¹ FW.

2.4.5. Statistical Analyses

The results were adjusted for statistical evaluation using SW R (version 3.6.3), and statistical analysis was performed in RStudio (version 1.2.5019). A one-way analysis of variance (ANOVA) was conducted to determine whether the treatments and periods exerted a significant effect. Statistically significant differences were identified using the post hoc Tukey HSD test at a significance level of p < 0.05. The results are presented as mean values with standard deviations for each characteristic. Statistically significant differences are indicated by letter indexes in the figures and tables. To evaluate the relationships between variables, a correlation matrix based on Pearson’s correlation coefficient at a significance level of p < 0.05 was used. For visualization of the correlation matrix, the function “rquery.cormat()” in R software was employed [44].

3. Results

In total, three variants of low-temperature vacuum plasma treatment were tested on seed samples of selected crops. A detailed overview of the morphometric and biochemical parameters and their statistical evaluation is provided in Tables S1 and S2 (Supplementary Data File).

3.1. Evaluation of Morphometric Parameters in Germinated Seeds

The morphological traits of young seedlings, specifically MRL and MSL, were among the evaluation criteria for assessing the effect of LTP on seed and young plant development. These traits also formed the baseline for evaluating the previously mentioned SVI and RSR traits. Young plants showed a more positive response regarding MSL, with greater variability observed among individual cultivars.

The evaluation of the spring wheat cultivar ‘Granny’ is presented in Figure 1. The assessment of MRL and MSL characteristics indicates a similar response to LTP treatment across all periods. The longest roots and shoots were consistently observed in the control seedlings for all periods. LTP treatment had a negative effect on all treated variants (T1, T2, and T3). A statistically significant increase in MRL was only observed among the treated variants with increasing exposure time to LTP (T1: 11.5 mm; T3: 39.6 mm in P1). Similar trends were observed for MSL. More uniform results were recorded in periods P10 and P20, where no statistically significant differences were found between the control (P10: 21.3 mm) and the treated variants T2 (P10: 21.7 mm) and T3 (P10: 16.8 mm). For this parameter as well, the most pronounced influence of LTP was recorded in variant T1 across all periods, similar to the results for MRL. Based on the evaluation of SVI, the least variable results were observed in period P20, where a significant decrease in SVI values was recorded for all treated variants compared to the results for the control. A different outcome was noted in P10, where a significant reduction in SVI was found only in variant T2 (4003.7) compared to the results for the other variants. The RSR parameter was least affected in P10, where no statistically significant differences were observed between the control and the treated variants. In contrast, the most significant reductions in RSR values were recorded in P1 and P20, particularly in variant T2 (0.9 in P1 and 1.27 in P20).

The evaluation of morphometric characteristics of the oat cultivar ‘Risto’ is presented in Figure 2. The cultivar ‘Risto’ demonstrated a negative effect of LTP on root development, making it impossible to assess treatments T2 and T3 in period P10, and partially in P20, because the seeds did not germinate (Figure 2a). This limitation also extends to MSL, as treatments T2 and T3 could not be evaluated in P10 and to a lesser extent, in P20 (Figure 2b). Consequently, SVI reflected these deficiencies, with both treatments T2 and T3 exhibiting a statistically significant decrease in all periods (Figure 2c). These observations are further supported by the MRL and MSL and are also reflected in the RSR. It is evident that this parameter was similarly affected by the reduced root and shoot lengths observed in treatments T2 and T3 across all periods.

The overall evaluation of the flax cultivar ‘N-9/62/K3/B’ (Figure 3) revealed that LTP treatment influenced both MRL and MSL development, with changes in these characteristics reflected in SVI and RSR. It can be concluded that LTP treatment exerted a positive effect on MRL (Figure 3), as an increase in root length was observed in treatment T3 across all assessed periods. LTP treatment affected treatment T1 in all periods, while T2 and T3 showed values comparable to those of the control in periods P1 and P10. Moreover, treatments T2 and T3 demonstrated higher MRL values compared to those of T1 (21.4 mm) in period P20 (24.0 mm). MSL showed a similar trend in both P1 and P10, with a statistically significant decrease in value observed in variant T1 (P1: 7.4 mm; P10: 15.2 mm). Evaluation of the SVI characteristic (Figure 3) indicated that the greatest influence of LTP was observed in treatment T1, consistent with the responses of other traits (MRL and MSL) in this cultivar. Treatment T1 exhibited significantly lower values compared to those of the control across all periods. In contrast, treatment T3 showed values comparable to the control throughout all periods. RSR indicated a statistically significant increase in shoot length in period P1 under treatment T1 (RSR = 2.53), suggesting that the shoot length exceeded the root length. A similar trend was observed for treatment T1 in periods P10 and P20, although the differences were less pronounced. The largest RSR difference was recorded in P1, where the gap between the lowest value (control) and the highest (T1) was 1.67. It can be concluded that the RSR values of treatment T2 across all periods most closely reflected a balanced ratio between the root and shoot lengths, with values fluctuating around 1 (Figure 3).

The results of the MRL, MSL, SVI, and RSR assessments for the rapeseed cultivar ‘Skrivenskij’ are presented in Figure 4. The MRL evaluation showed a statistically significant increase in values for variant T1 during periods P1 (33.4 mm) and P10 (36.9 mm) compared to those for the other variants. The MSL parameter showed more consistent results, with a decrease observed only in variant T1 during periods P1 (15.7 mm) and P10 (14.1 mm). The SVI evaluation revealed a statistically significant effect of LTP only in period P1 for variant T1 (4912) compared to the results for the control (2565). In the other periods, no statistically significant differences were observed between the treated variants. The RSR parameter showed more variable results. In periods P1 and P10, a statistically significant increase in RSR values was recorded compared to the results for the control. In contrast, in P20, a decrease was observed. In period P1, all treated variants showed a significant increase in RSR values compared with the levels of the control. In P20, however, the effect of LTP was reversed. Despite this, a statistically significant decrease compared to the results for the control (2.4) was observed only in variant T2 (1.6).

3.2. Assessment of Biochemical Parameters in Germinated Seeds

In the spring wheat cultivar ‘Granny’, the effect of all treatment variants on the content of all biologically active compounds (MDA, TPC, TFC, and TAC) was evident (Figure 5). In the case of MDA, a decrease in content was observed in all storage periods after LTP treatment. The most significant difference was observed in P1 between the control and T1 (26.8 and 9.7 nmol·g⁻¹ FW, respectively). In contrast, the LTP treatment led to an increase in TPC and TFC contents across all periods. TAC showed some variability across storage periods; in P1 and P10, TAC decreased following LTP treatment, whereas in P20, an increase in TAC activity was observed after LTP application.

The oat cultivar ‘Risto’ (Figure 6) showed a similar response pattern to LTP treatment as that of the wheat cultivar ‘Granny’. A significant decrease in MDA content was observed between the control and T1 treatment samples across all storage periods, with the highest difference recorded in P1 (20.6 for control, 4.7 for T1; Figure 6). The amount of TPC increased significantly with the extension of LTP exposure time in all periods. While the TFC content in oats increased with longer LTP exposure, as for wheat, the opposite trend was observed in flax and rapeseed. TAC showed slightly variable results: in P1, TAC decreased after LTP treatment, whereas in P10 and P20, a significant decrease in activity was observed with prolonged LTP exposure.

In flax cultivar ‘N-9/62/K3/B’ (Figure 7), similar to the results for the previous species, a decrease in MDA content was observed after LTP treatment in P10 and P20. An increase in content was recorded in P1 between the control and T1 variants (34.6 and 39.7 mmol/g⁻¹ FW, respectively). Regarding TPC levels, a gradual increase with longer LTP exposure was noted in P1 and P20, while in P10, the highest increase between the control and T1 samples was observed (by 5.22 mmol/g⁻¹ FW). While TFC content increased with prolonged LTP exposure in the wheat and oat cultivars, flax (as well as rapeseed) showed a significant decrease. The largest difference was observed in P20 between the control and T1 samples (8.6 and 2.0 mmol/g⁻¹ FW, respectively; Figure 7).

In the rapeseed cultivar ‘Skrivenskij’, the highest MDA levels were observed in the control variant across all storage periods, i.e., P1, P10, and P20 (82.7, 76.6, and 61.7 mmol/g⁻¹ FW, respectively; Figure 8), representing the highest MDA content among all evaluated cultivars. The TPC content showed variable results: in P1, a decrease was observed after LTP treatment in T1 and T2, while T3 statistically matched the results for the control. In P10, the trend was different—a significant increase in TPC was detected after LTP treatment, although no significant differences were found among the individual treatment variants (T1–T3). In P20, TPC also significantly increased compared to the results for the control after LTP exposure, with the highest statistically significant value recorded in T3. As in flax, TFC content significantly decreased after LTP treatment compared to the results for wheat and oats. The most pronounced reduction in TFC between the untreated and treated variants (control and T1) was recorded in P1 (9.37 mmol/g⁻¹ FW; Figure 8). TAC followed a similar trend to that noted in the previous cultivars, showing reduced activity after LTP application. The highest TAC value was observed in T3 (35.34 mmol/g⁻¹ FW) and the lowest in T1 (32.84 mmol/g⁻¹ FW) in P20.

3.3. Comparative Correlation Analysis of Morphometric and Biochemical Traits

The correlation analysis revealed species-specific responses to LTP seed treatment across morphometric and biochemical parameters. While wheat, oats, and flax generally exhibited negative correlations for traits such as SVI and RSR, rapeseed showed distinct patterns, including negative associations between MRL and MSL. Biochemical parameters such as MDA, TAC, TPC, and TFC also displayed predominantly negative correlations, with notable variation among cultivars and treatment conditions.

The results indicate that the relationships between morphometric parameters across the evaluated periods (P1–P20) in the wheat cultivar ‘Granny’ are generally positive for traits such as MRL and MSL (Figure 9). However, for the RSR and especially SVI traits, period P10 shows a negative correlation with the other traits. A similar trend can be observed in the correlation analysis between treatment variants (control, T1–T3), where RSR and SVI in variant T1 exhibit negative correlations with the remaining evaluated traits. Moreover, both MDA and TAC exhibit negative correlations with the remaining evaluated parameters, as illustrated in Figure 9.

The oat cultivar ‘Risto’ exhibits distinct results (Figure 10). The correlation analysis of morphometric traits across the evaluated periods (P1–P20) revealed positive relationships among all traits; however, the correlations for SVI and RSR were relatively weak. The assessment of morphometric traits across treatment variants (control–T3) showed variable outcomes, but overall, SVI and RSR tended to exhibit negative correlations with other traits, specifically MRL and MSL. The evaluation of the content and activity of biochemically active compounds revealed a strong association between MDA and TAC. Both TPC and TFC showed strong negative correlations with MDA and TAC. A similar trend was observed in the correlation analysis across treatment variants (control–T3), where TPC and TFC were frequently negatively correlated with MDA and TAC (Figure 10). Furthermore, a negative correlation was also found between TFC and TPC, with a high correlation coefficient.

The flax cultivar ‘N-9/62/K3/B’ exhibited a similar trend to that observed in the previously assessed cultivars (Figure 11). A negative relationship was observed between SVI, MRL, MSL, and RSR across the evaluated periods (P1–P20). RSR in all treatment variants (control–T3) negatively correlated with the other assessed morphometric parameters. Additionally, SVI in variant T2 showed a negative correlation with both RSR and MRL in variant T1, but a strong positive correlation with MSL and SVI in variant T3. The correlation of biochemically active parameters in flax across periods (P1–P20) was variable (Figure 11). In the evaluation of correlations among treatment variants (control–T3), MDA exhibited a strong negative correlation with TPC, while a weak negative association was observed between MDA and both TFC and TAC.

Compared to the other cultivars, the rapeseed cultivar ‘Skrivenskij’ exhibited distinctly different correlation patterns (Figure 12). Within the evaluated periods (P1–P20), in addition to the negative correlations typically observed for RSR and SVI in other cultivars, negative relationships were also detected between MRL and MSL, particularly between periods P1 and P10. A negative trend was also observed among MRL values across different periods, with MSL showing a similar pattern. Correlation analysis across treatment variants (control–T3) revealed a strong negative association between MSL in variants T1 and the control and the remaining morphometric parameters. This trend was consistent with the observed correlations between MSL in the control and T1 variants. The assessment of biochemical parameters across periods (P1–P20) indicated a strong negative correlation between TPC in periods P10 and P20 and the other evaluated parameters. A similar trend was observed for TAC in period P1. The correlation analysis among treatment variants (control–T3) further demonstrated predominantly negative relationships between TPC across all variants, as well as for TFC in variants T1 and T2, and for MDA in the control and variant T3 (Figure 12).

4. Discussion

LTP seed treatment has previously been tested primarily on agriculturally important species such as wheat [13], barley [3], and basil [23], as well as on model plants such as Arabidopsis thaliana [45]. Currently, due to varying regional climatic conditions, there is a growing potential for the use of existing local varieties in breeding programs aimed at developing modern cultivars with resistance to drought, diseases, and pests [46]. For these varieties, it is essential to ensure sufficient germination and further development to preserve valuable plant materials in gene banks [47]. For the purposes of this and the previous study [39], LTP was used, which was determined to be more suitable for application to small-seeded and oilseed crops.

Selected morphometric approaches, including measurements of MRL, MSL, SVI, and RSR, are among the established methods for assessing the effects of LTP on seed response. A positive effect of LTP was observed only in flax cultivar ‘N-9/62/K3/B’ during all periods for the MRL trait in variant T3 and for the MSL trait in period P1 and P10 in variants T2 and T3. The remaining results for these two traits indicated a more negative influence, which contrasts with the findings of Šerá et al. [11]. In the case of the wheat cultivar ‘Granny’, LTP treatment led to a reduction in both shoot and root length. Furthermore, a pronounced effect of LTP was observed for the oat cultivar, with significant reductions in MRL and MSL values recorded in the treated variants T2 and T3. Similar conclusions were reached by Judickaité et al. [15], who found that DBD-type LTP treatment tended to reduce both dry biomass and plant height in candyleaf.

In contrast, an increase in root length was reported in crimson clover following LTP Treatment [12]. Similarly, a positive effect of LTP on MRL was observed in the flax cultivar ‘N-9/62/K3/B’ across all evaluated storage periods (P1, P10, and P20), with the most pronounced effect recorded in variant T3. In contrast to the findings of Mildaziene et al. [12] and Tamošiūnė et al. [48], LTP had the opposite effect on the oat and wheat cultivars in storage periods P1 and P20, where longer exposure durations negatively affected MRL and MSL values.

Dobrin et al. [18] demonstrated a positive effect of LTP treatment on the root length of seedlings grown from treated seeds. Similarly, Magureanu et al. [9] reported that a 5 min treatment with DBD plasma discharge resulted in an almost threefold increase in tomato root length compared to the results for the control seedlings. Comparable results were observed by Mildaziene et al. [12] and Šerá et al. [11], as well as in the present study for the flax variety ‘N-9/62/K3/B’, where the highest MRL values were recorded in variant T3 across all gene bank storage periods (P1–P20). A comparison of MRL and MSL values for the cultivar indicates that LTP treatment resulted in root elongation (MRL), while MSL decreased in all P20 periods for variants T1 and T3 compared to the results for the control. This suggests a differential sensitivity of root and shoot tissues to plasma exposure at later developmental stages. However, these findings contrast with those of Matra et al. [49], who observed a statistically significant increase in shoot length following LTP treatment. This discrepancy may be attributed to differences in plant species, plasma parameters, or the physiological state of the seeds during treatment.

Tong et al. [34] reported that seedlings of green chiretta were up to 16% smaller than untreated control seedlings. In the present study, rapeseed responded variably to LTP treatment in terms of MRL and MSL, and the findings do not fully support the conclusions of Magureanu et al. [9], as the values of MRL, MSL, and SVI was largely not statistically significant. Notable changes were observed in long-stored seeds during period P20 in the wheat, oat, and flax cultivars, with a decline in SVI recorded across all cultivars, most prominently in the oat cultivar ‘Risto’.

Bozhanova et al. [50] mention that variability in MRL and MSL after seed treatment with LTP is primarily caused by the interaction between the genotype and the treatment variant. This observation is supported by the results of this experiment, as the majority of the tested crop species responded differently to varying durations of LTP treatment. The claim by Bozhanova et al. [50] can be further expanded to include the effect of seed storage duration in a gene bank, as this also influenced all examined species. In the oat variety ‘Risto’, longer storage duration in the gene bank led to a disruption in the uniform development of root and shoot lengths during periods P10 and P20 after LTP treatment. In the P10 samples of variants T2 and T3, LTP treatment even resulted in the inhibition of root and shoot development. A comparison of the declining MRL, MSL, and SVI values with increasing LTP treatment duration in this variety provides evidence of a negative effect of LTP.

Since SVI is regarded as an indicator of seedling vitality, a decrease in this index suggests reduced vitality. It is evident that the effect of LTP on seeds, seedlings, and further plant development depends not only on the plant species but also on the specific cultivar. According to Šerá [16], higher SVI values indicate a more vigorous seed sample within a species subjected to different seed treatments.

In the study by Singh et al. [51], the application of radio-frequency plasma discharge on basil resulted in a significant increase in SVI, nearly doubling it at a pressure of 0.2 mbar. In the present study, the effects of LTP treatment were also evident, particularly in relation to treatment duration and seed storage period in the gene bank. With increasing treatment duration, a significant decline in SVI values was observed, notably in wheat in P20 and oats in all periods under the T1 variant. These results are consistent with the findings of Singh et al. [51], suggesting that while plasma treatment can enhance SVI under specific conditions, prolonged exposure or interactions with storage duration may lead to adverse effects on seedling development.

The condition of young plants following LTP treatment is well illustrated by RSR, which tends to increase when one of the evaluated traits differs markedly from the others. A significant increase in this ratio was observed in the rapeseed variety ‘Skrivenskij’ in variant T3 across all three storage periods. Similar behaviour was reported by Magureanu et al. [9] in tomato seedlings and by Šerá et al. [37] in wheat seeds treated with LTP, where the effect was associated with enhanced stress tolerance in wheat. In contrast, for the oat varieties tested in this study, it can be concluded that longer treatment durations (T2 and T3) may also exert an inhibitory effect.

Perea-Brenes et al. [14] and Tong et al. [34] clearly describe a positive effect of LTP on the growth rate of germinated plants. Perea-Brenes et al. [14] specifically mention a faster biomass increase in treated germinated seeds, which is consistent with the findings of Strejčkova et al. [52] in experimental rapeseed plots. The results of this study support the conclusions of the authors in relation to RSR, a trait derived exclusively from the morphological characteristics of seedlings. It is generally accepted that the belowground part of the plant should be balanced with the aboveground part in terms of stability, water uptake, and sunlight exposure [16], and thus, this parameter can be evaluated independently of other traits. In comparison with the findings of Perea-Brenes et al. [14], it can be concluded that the RSR trait was affected in the wheat variety, although each variety exhibited a differential response.

However, Dobrin et al. [18] reached the opposite conclusion, reporting that RSR values were higher in treated seeds compared to in the controls. Similarly, in the present study, a significant increase in RSR was observed for the rapeseed variety in variant T3 across all three storage periods. This outcome, however, reflects the fact that one of the two parameters contributing to the RSR ratio (MRL or MSL) was lower. It can therefore be inferred that if root growth increased more intensively, the shoot growth was conversely affected, resulting in lower values. Tunklová et al. [8] emphasize that strengthening the root system at the expense of above-ground seedling parts may provide an advantage under drought stress conditions, a finding also supported by Attri et al. [3] and Mildaziene et al. [17]. According to these authors, such a shift may positively influence germination under suboptimal sowing conditions.

In addition to standard parameters used for evaluating seed quality and value, this study examined the effect of plasma treatment on the activity of biochemicals associated with germination.

Ongrak et al. [53] found that the highest MDA content was observed in water spinach seeds treated with a 20 min dielectric barrier discharge plasma exposure, while shorter treatment durations led to increased but statistically insignificant MDA levels compared to those of the control. A similar trend was reported for seeds treated with 10 min of LTP [53]. The findings of the present study partially align with those of Ongrak et al. [53], but contrary to their results, a general trend observed in this study was a decrease in MDA content following LTP treatment. Similar results were reported by Seleiman et al. [54], who used plasma-activated water (PAW) as irrigation for already developed seedlings and found a significant reduction in MDA content in both the leaves and roots.

In the present study, an increased MDA production was observed only in a few cases, specifically in flax, but limited to period P1 for ‘N-9/62/K3/B’ in T1. The remaining variants exhibited a further decrease compared to the results for the control. According to Ongrak et al. [53], MDA can serve as an indicator of membrane lipid peroxidation; thus, elevated levels of MDA may signal increased membrane degradation. However, this was not confirmed in the current experiment, where most of the cultivars exhibited a reduction in MDA content following LTP treatment. Ongrak et al. [53] also acknowledged that lower levels of MDA following short-term DBD plasma exposure (5–10 min) may be linked to an increase in the activity of catalase (CAT) or superoxide dismutase (SOD), which are key enzymes involved in the oxidative and reductive metabolic pathways [53]. A similar result was also reported by Guo et al. [55], and these findings were further confirmed in the study by Matějovič et al. [39], for which the present work is a continuation. A similar effect was reported in tomato seedlings, where MDA levels decreased following LTP treatment [33].

Following LTP treatment of carob seeds, a reduction in TPC and TFC was observed compared to the results for the control [35]. A similar decrease in TPC was noted in this study for the rapeseed cultivar ‘Skrivenskij’ (P1). In contrast, other large-seeded cultivars (wheat and oats) and the flax cultivar ‘N/62/K3/B’ exhibited an increase in TPC. The TFC content showed increased values of this parameter with longer LTP treatment duration. This aligns with the findings of Mildaziene et al. [12], who reported the most significant changes in TFC content in crimson clover roots following LTP treatment, with treated variants showing up to a 1.6 times higher TFC content than that of the control. However, prolonged LTP treatment resulted in a decrease in TFC content by up to 1.3 times compared to that of the control [12]. This phenomenon has been documented in several other studies [6]. The results of this study also corroborate the findings of Rithichai et al. [56], who observed higher levels of TFC and TPC in sunflower seeds following LTP treatment. Conversely, in the present study, higher TPC content was detected in the wheat, oat, and flax cultivars, which aligns with the findings of Mildaziene et al. [57], who reported an increase in TPC content with prolonged LTP treatment in eastern purple coneflower. Based on the findings of Guragain et al. [58], who reported an increase in TFC content following LTP treatment, it is important to note that this study confirms such an increase only in cereal cultivars classified as large-seeded species, where a significant rise in TFC content was observed after LTP application. In contrast, for small-seeded flax and rapeseed cultivar, TFC content decreased compared to that of the control. Regarding seed size, small-seeded cultivars exhibited a reduction in flavonoid compound content when compared to the levels in cultivars with larger seeds, consistent with the findings of Guragain et al. [58].

Both enzymatic (e.g., SOD, CAT) and non-enzymatic mechanisms contribute to the total antioxidant capacity (TAC) in plants. These mechanisms serve as biochemical markers of a plant’s response to ongoing environmental changes, as they assess the redox status of the plant [59]. Bussmann et al. [59] applied plasma-treated water (PTW) to the leaves and roots of barley under drought stress conditions. The PTW treatment influenced plant responses by increasing TAC both under control conditions and stressed conditions, where a significant increase in total antioxidant activity (TAC) was observed. However, following LTP treatment, a decrease in TAC was observed in the cultivars of wheat, oats, flax, and rapeseed (P10 and P20 periods), which contrasts with the findings of Bussmann et al. [59]. In contrast to the study by Bussmann et al. [59], where stress factors potentially led to increased TAC, the plants in this study did not experience the same stress. Instead, this study focused on the effect of LTP treatment on seeds and their responses after long-term storage in gene banks, a condition that itself may act as a stress factor.

5. Conclusions

In recent years, plasma seed treatment technologies have been receiving increasing attention in agricultural practice; however, their application to seeds subjected to long-term storage in gene banks remains insufficiently explored. Gene banks play a crucial role in preserving plant genetic resources for future use, and enhancing the viability and vitality of stored seeds is becoming increasingly important. This study presents preliminary research on the effects of LTP treatment on long-term-stored seeds obtained from gene banks and outlines key questions and directions for further investigation. Four plant species with differing seed sizes and distinct morphological and anatomical characteristics were selected for this study. The results indicate that LTP treatment can lead to both beneficial and adverse responses, depending on the species and treatment parameters, including duration and storage period prior to treatment. Balanced responses in early seedling development after LTP treatment were observed in flax and rapeseed, particularly in the morphological traits of MRL and MSL, as well as for SVI in flax. These outcomes could be interpreted as positive or neutral responses. In contrast, wheat exhibited variable reactions to LTP treatment, while oats consistently showed negative responses across all morphological parameters, indicating species-specific variability regarding sensitivity to plasma treatment. Biochemical analyses further revealed that in wheat and oats, higher MDA content generally correlated negatively with parameters such as TPC and TFC, suggesting responses to oxidative stress. A negative correlation between phenolics and MDA was also observed in all time points for oats. In flax and rapeseed, the correlations extended to TFC in all variants, indicating species-specific metabolic adjustments following LTP treatment. In wheat, most morphometric parameters were positively correlated, except for RSR ratio and SVI, which showed strong negative correlations, whereas in the other species, the correlations extended to MRL and MSL, highlighting the broad impact of LTP on seedling architecture. Overall, this study confirms that LTP displays the potential to modulate seedling development and metabolic processes, contributing to improved germination and SVI of plant genetic resources stored in gene banks. At the same time, the findings underscore the need for the cultivar-specific optimization of treatment conditions to mitigate adverse effects and fully leverage the benefits of plasma technologies for ex situ conservation strategies.