Comparative analysis of the germination of barley seeds subjected to drying, hydrogen peroxide, or oxidative air plasma treatments

Received: 17 February 2022 | Revised: 17 May 2022 | Accepted: 24 May 2022 DOI: 10.1002/ppap.202200035 RESEARCH ARTICLE Comparative analysis of the germination of barley seeds subjected to drying, hydrogen peroxide, or oxidative air plasma treatments Alvaro Perea‐Brenes 1,2 |Ana Gómez‐Ramírez 1,3 |Carmen López‐Santos 1,4 | Manuel Oliva‐Ramírez 1,3 |Ricardo Molina 5 |José Cotrino 1,3 | José L. García 2 |Manuel Cantos 2 |Agustín R. González‐Elipe 1 1 Nanotechnology on Surfaces and Plasma Laboratory, Instituto de Ciencia de Materiales de Sevilla (CSIC‐Universidad de Sevilla), Sevilla, Spain 2 Department of Plant Biotechnology, Instituto de Recursos Naturales y Agrobiología de Sevilla (CSIC), Sevilla, Spain 3 Departamento de Física Atómica, Molecular y Nuclear, Universidad de Sevilla, Sevilla, Spain 4 Departamento de Física Aplicada I. Escuela Politécnica Superior, Universidad de Sevilla, Sevilla, Spain 5 Department of Chemical and Surfactants Technology, Plasma Chemistry Group (CSIC), Institute of Advanced Chemistry of Catalonia (IQAC), Barcelona, Spain Correspondence Carmen López‐Santos, Nanotechnology on Surfaces and Plasma Laboratory, Instituto de Ciencia de Materiales de Sevilla (CSIC‐ Universidad de Sevilla), Avda. Américo Vespucio 49, Sevilla 41092, Spain. Email: [email protected] Ana Gómez‐Ramírez, Departamento de Física Atómica, Molecular y Nuclear, Universidad de Sevilla. Avda. Reina Mercedes, Sevilla 41012, Spain. Email: [email protected] Funding information Agencia de Innovación y Desarrollo de Andalucía, Grant/Award Numbers: US‐1380977, US‐1381045; Ministerio de Ciencia e Innovación, Grant/Award Numbers: PID2019‐ 109603RA‐10, PID2019‐110430GB‐C21, PID2020‐114270RA‐I00 Abstract Acceleration in germination time by 12–24 h for barley seeds treated with atmospheric air plasmas may have a significant economic impact on malting processes. In this study, the increase in germination rate and decrease in contamination level upon plasma treatment could not be directly correlated with any significant increase in the water uptake capacity, except for seeds exposed to mild drying treatment. A variety of germination essays have been carried out with seeds impregnated with an abscisic acid solution, a retarding factor of germination, treated with a peroxide solution, and/or subjected to the plasma and drying treatments. Results suggest that plasma and hydrogen peroxide treatments induce the formation of reactive oxygen and nitrogen species that affects the abscisic acid factor and accelerate the germination rate. KEYWORDS ABA factor, barley germination, hydrogen peroxide treatment, plasma surface oxidation Plasma Process Polym. 2022;19:e2200035 www.plasma-polymers.com | 1of17 https://doi.org/10.1002/ppap.202200035 This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2022 The Authors. Plasma Processes and Polymers published by Wiley‐VCH GmbH. 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1|INTRODUCTION In the quest for improving agriculture culture yields, various strategies have been developed to improve germination efficiency and accelerate the first stages of plant growth. One of these strategies consists of treating the seeds with air plasmas. [1] It is already well‐established that a large variety of seeds treated with air plasmas experience an acceleration in germinationrateand/oranincreaseinthepercentageof germination success. [2–12] This rather common behavior presents some exceptions where plasma treatment has been reported to be neutral or even detrimental for germination, [10,13,14] particularly under water scarcity conditions. [14] Common interpretations of these results have related the germination improvement with an increase in seed water permeation and imbibition [15–17] or with the plasma removal of contaminating biological agents. [1,17–21] Other works though relate the acceleration of germination rate with the affectation of dormancy and the effect on growth factors such as the abscisic acid (ABA) and other seed enzymes of reactive oxidative species (ROS) formed at the seed surface upon plasma interaction. [22–30] As a result, the effect of plasmas on germination is still unclear, failing a clear understanding about the incidence of plasma effects on the germination process. This situation is still more puzzling since the actual influence of plasmas on germination may also depend on the characteristics of seeds and their external coat or the treatment time and type of gases utilized to ignite the plasmas, [9,31] with case examples where plasma produced either no effect or a negative effect on water uptake [14] and/ or germination efficiency. [10,13] This complexity has been further jeopardized by the difficulty to compare results and tests carried out in different laboratories and/or with various seed types of the same species. The present work deals with barley seeds, one of the most common cereals around the world that serve as the basis for the animal and, in part, human feeding. [32,33] In addition, barley is a cereal with a high industrial impact on the production of beer and other agrofood products. Particularly for these industrial applications, any improvement in germination rate, even if meaning a small decrease in the period of time required to open the seed bark and trigger the plant rooting, may have a considerable economic impact because of an effective reduction in the malting period. [34] The enhancement of germination rate of barley seeds treated with plasmas is a widely studied case example [25,29,35–40] where, similarly to other seeds, the found improvements in germination have been attributed to a variety of chemical and physical effects of the plasma treatments either on the surface of seeds and/or in their internal biochemistry and enzymatic activity. Herein, we investigate the effect of plasmas on the germination and growth of barley seeds and study the influence on other phenomena that may be also affected by plasma and/or other related treatments. Namely, we directly investigate the changes that cold air atmospheric plasmas produce in the water uptake capacity of barley seeds both from the vapor and liquid phases. Indirectly, we also assess its effect on the ABA factor and its influence in the germination rate of seeds treated or nontreated with air plasmas. For comparison, we also study the effect of treating the seeds with an H 2 O 2 solution, a classical treatment known to promote the seed germination capacity. [41–46] Similar analysis has been also carried out with seeds that have been subjected to a severe drying process at room temperature and where an increase in germination rate was also observed. This drying treatment, although not completely equivalent to classical priming treatments, [47,48] has resulted quite efficiently in accelerating the germination rate. The analysis of treated and nontreated barley seeds with techniques such as scanning electron microscopy (SEM) or X‐ray photoelectron spectroscopy (XPS) has been also carried out to determine possible changes in the chemistry and/or surface morphology induced by the plasma treatments. Although these investigations do not permit yet to figure out a holistic model accounting for all phenomena resulting from the atmospheric air plasma treatments of seeds, clear evidence of this investigation is that water uptake capacity results are almost unaffected by the air plasma treatment and that the plasma affectation of the ABA present in the seeds is an important factor contributing to increase the germination rate. 2|EXPERIMENTAL SECTION 2.1 |Seeds type Mature barley seeds (Hordeum vulgare L. var. planet) provided by the company Intermalta S.A. have been used for this study. No special treatment was applied to these commercial seeds. This variety of barley seeds is typical of two‐rowed and spring cycles. It is also rather common for the malting industry due to its high productivity and quality. For agricultural purposes, it is much appreciated because of its adaptability to different zones and agroclimatic conditions. During the time required for the experiments (about 6 months), seeds were kept stored in a closed container under controlled conditions of temperature and humidity. 2of17 | PEREA‐BRENES ET AL. 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2.2 |Plasma reactor and plasma treatment conditions Plasma treatments were carried out in a dielectric barrier discharge reactor with a parallel plate configuration. Two stainless steel electrodes (8 cm diameter) were covered with two quartz plates (0.5 mm thickness and 10 cm diameter to avoid edge discharges). The separation between the two plates was fixed at 4.2 cm and the seeds (usually 35 seeds specimen for each treatment) were placed well separated onto the bottom grounded electrode. The active electrode at the top was activated by a high voltage source from TREK (model PD05034) that, in turn, was connected to a function generator (Stanford Research System, model DS345). V(t) and I(t) signals were recorded with an oscilloscope (TEKTRONIK, model TDS2001C) with a bandwidth of 50 MHz and a sample rate per channel of 500 Ms/s. A 1:1000 high voltage probe and a current probe coil (conversion factor of 0.05 V mA −1 ) were used for this recording. For all treatments, the high voltage source was set at 1 kHz frequency and 8.6 kV voltage. Under these conditions, the current passing through the system was 6.5 mA and the discharge power 5.3 W. A pressure of around 700 mbar of dry air was dosed in the reactor during the discharge for periods of time fixed at 1, 3, and 7 min. Occasionally, longer times up to 15 min or more were also utilized. Further details about the reactor and operating conditions can be found in previous publications. [5,49] 2.3 |Drying procedure Various sets of seeds were subjected to a drying process either in gentle ambient temperature (around 21°C) conditions in a desiccator or to more intensive drying treatments on a stove at 50°C and 70°C for 24 h. After these intensive drying essays, germination results were bad in the sense that only a reduced amount of heated seeds accomplished germination. Therefore, accelerated drying experiments will only be incidentally mentioned in the text. Drying at ambient conditions was carried out by placing sets of 50 seeds in a closed flask containing silica gel (Panreac), avoiding any direct contact between the silica gel granules and the seeds. Usually, after 2/3 weeks in these conditions, seeds experienced a weight decrease of about 4%–6% of its initial weight. We will designate these seeds in the text as “dry”seeds. 2.4 |Sowing tests and statistics Germination tests were carried out under two different conditions: in plastic Petri dishes and in soil substrate. For the germination tests in the plastic Petri dishes, sets of 50 seeds (control seeds or seeds previously subjected to different treatments) were carefully placed on a double Whatman filter paper located in Petri dishes of 9 cm diameter. A watering of 4 ml per dish with miliQ water was applied to each dish. Petri dishes were then placed in the dark in a bacteriologic incubator (Selecta model PREBATEM 80L) at a fixed temperature of 20°C. After successive 12 h the Petri dishes were carefully inspected to identify seeds that might have germinated. A seed was considered “germinated”when the radicle had traversed the seed cover or upon the emergence of the coleoptile. Germinated seeds were counted and placed in another Petri dish. This process was repeated every 12 h up to completing a period of 72 h. The procedure utilized for the germination tests in Petri dish is deemed to be close to the industrial malting process of barley seeds. For comparison, germination tests were also carried out in the soil. These tests were carried out by sowing individual seeds into the alveols of horticultural cell trashes (20 seeds per test, one seed per cell at an approximate depth of 1 cm from the soil surface) filled with a commercial substrate provided by Klasmann‐ Deilmann GmbH 49744 Geeste, Germany (peat FLORABELLA consisting of a mixture of clay and Sphagnum blonde and black soil, this latter frozen). Physical and chemical characteristics of this soil were an electric conductivity of 40 mS m −1 (±25%), pH (H 2 O): 5.5–8.5 and added fertilizer NPK: 14–16–18 in a proportion of 1.5 kg m −3 . The ensemble was placed in an illuminated climatic chamber (111 μEm −2 s −1 of photosynthetic photon flux density [PPFD] and 16 h of photoperiod) at a temperature of 24°C and constant humidity (80%). A watering of 5 ml per cell was applied three times per week (Monday, Wednesday, and Friday). By this test, seeds were considered germinated when the aerial part of the plant had just emerged from the substrate. It is noteworthy that this evaluation of germination percentage is subjected to larger uncertainties than the equivalent evaluation in Petri dish as the sowing depth in soils cannot be exactly replicated from one alveol to another. In parallel to the assessment of germination rate in the substrate, taken as the emergence time of the plant, the heights of the plants were also measured after 7 days from the planting day. Germination in Petri dishes and germination in the substrate of seeds subjected to the different treatments carried out in this study (i.e., plasma, drying, H 2 O 2 , ABA, etc., see Sections 2.2 and 2.7), water uptake and contamination tests were carried out with sampling sets of 50, 20, 30, and 10 seeds, respectively. An error bar corresponding to the standard deviation over two exact replicates of each experiment has been assumed for the obtained results. When data refer to the height of plants, PEREA‐BRENES ET AL. | 3of17 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License we utilize the standard error of the mean value to assess the accuracy of the obtained results. 2.5 |Contamination tests To determine the contamination degree of the seeds and its evolution with time, a set of 10 seeds was located in a laminar flux cabinet in Petri dishes of 9 cm in diameter. The utilized sterilized Petri dishes were filled with 40 ml of a mixture of agar and water (0.6%) previously sterilized in an autoclave. The seeds were examined every 24 h to verify the evolution of contamination at the seeds as identified by the presence of a fungus mycelium or a bacteria stream/colony on it. 2.6 |Water uptake tests The capacity of seeds to incorporate water in their interior was determined following two procedures consisting of their exposure to water vapor at 100% relative humidity and to liquid water, respectively. In both cases, the uptake capacity was determined by weighing sets of 20 or 50 seeds exposed to water, vapor, or liquid respectively, for increasing periods of time. For the water vapor uptake test, seeds were placed on a Petri dish floating onto liquid water contained in a closed flask. The temperature was kept constant at 21°C. These conditions provide a water vapor pressure of 18.7 Torr (i.e., vapor pressure of water at this temperature) and relative humidity of 100%. For the liquid water uptake test, seeds were immersed in liquid water (milliQ quality) for given periods of time and then weighted to determine the percentage of weight increase in each case. Before measuring the weight, the seeds were left to dry on a filter paper in air for 1 min. An additional test aiming at carefully determining the evolution with time of the uptake of water was carried out at a constant temperature of 21°C, relative humidity of about 80%, and one seed in each trial. The analysis was carried out at constant temperature (21°C) in a DSC‐TGA SDT 0600 (TA Instruments) microbalance for a plasma, dry, and control seed each time. The microbalance was operated in the following way: the corresponding seed was placed in one of the arms of the device. The enclosure was then closed and nitrogen let flow through the chamber for 5 min at a flow rate of 10 ml min −1 .At this point (t= 0 for the experiments), an airflow of 150 ml min −1 , after bubbling through water at 21°C, was mixed with the dry nitrogen flow, and weight measurements were taken automatically and in a continuous way for the next 6 h and 15 min. Taking into account the used nitrogen and water vapor saturated air flows, this mixture renders a relative humidity of 93% for the tests. After the indicated period in humid conditions, the flow of air was closed and dry nitrogen continued flowing through the weighting chamber for more than 12 h at the same temperature. It is noteworthy that the exposure time of the seeds to the atmosphere before their placement in the thermogravimetric balance was minimized as much as possible, but was different depending on the seed. The dry seed was taken directly from the desiccator and placed in the setup that was subsequently closed (exposure time to atmosphere estimated in 3–4 min). The plasma‐treated seed was taken from the plasma chamber, placed in a closed tube, and brought to the microbalance, all these processes entailing a longer total exposure time to the atmosphere of 5–6 min. During this handling period, some water adsorption from the environment cannot be discarded. The control seed, stored in a plastic bag without any special precaution, was placed directly into the microbalance arm. 2.7 |ABA and hydrogen peroxide tests A series of experiments have been carried out to assess the possible role of ABA in the retardation of germination and whether plasma and drying treatments may influence this retarding enzymatic factor. Experiments consisted of following the germination of seeds previously subjected to various ABA treatments to reinforce its effect in dormancy and germination retarding, or to lessen this function by subjecting the seeds to treatments that supposedly contribute to the removal of ABA. Plasma treatments in this context consisted of the standard treatment for a period of 3 min. A brief description of these treatments and the labeling utilized for the treated seeds in each case is included next. Seeds were immediately sown after the treatments: (a) ABA: Seeds were soaked in a 100 µM ABA solution for 30 min. The seeds taken from the solution were placed on a double sheet of Whatman filter paper for 30 min and then left dry in air for 20 h. (b) ABA‐plasma: Seeds were subjected to the treatment in (a) and then subjected to the selected plasma treatment. (c) H 2 O 2 : Seeds were soaked in a 6% water solution of H 2 O 2 for 30 min, taken off from the solution, and left dried as in (a) before sowing. (d) ABA‐H 2 O 2 : Seeds were first soaked in an ABA solution as in (a) and then introduced in an H 2 O 2 solution as in (c). Seeds were sown after applying the same drying process than in (c). 4of17 | PEREA‐BRENES ET AL. 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (e) H 2 O: Seeds were soaked in pure milliQ water for 30 min and then dried as in cases (a–c). (f) H 2 O 2 ‐plasma: Seeds were subjected to the same treatment as in (c), then dried for 20 h and then subjected to a plasma treatment like in (b). (g) Dry‐ABA: Seeds were dried in the desiccator for 72 h, a period after which the seeds had lost 4.5% of their weight. Then there were subjected to the same treatment than in (a) followed by their sowing. (h) ABA‐dry: Seeds were subjected to the same treatment as in (a). Then they were stored in a desiccator for a period of 192 h before their sowing. The drying period in the desiccator entailed a weight decrease of 12.3% with respect to the seeds treated with the ABA solution (note that seeds will likely accumulate water during the ABA treatment). 2.8 |Assessment of the content of peroxo‐like species in the seeds A central hypothesis of this study is that peroxo‐like species may affect the ABA and thus induce changes in dormancy and, consequently, germination rate. [23–30,35–45,48] It is, therefore, important to assess the content of peroxo‐like species incorporated in the seeds after their treatment with a plasma of air or with a solution of H 2 O 2 .Theperoxo‐like content in the seeds was estimated by means of the classical colorimetric procedure proposed by Soares et al. [50] According to this procedure peroxo‐like species formed or incorporated into the seeds can be extracted by applying the following procedure: (i) grinding 1 g of seeds in a 10 ml phosphate‐buffered solution (50 mmol l −1 , pH=6,5) with 1 mmol l −1 hidroxilamine; (ii) the resulting slurry is then centrifuged at 6.000 g n for 25 min; (iii) 3 ml of the separated liquid is then mixed with a 1 ml of titanium sulfate solution at 0.1% in a volume ratio of 20%; (iv) this mixture is centrifuged at 6.000 g n for 25 min and the separated transparent liquid is then optically analyzed with a spectrometer. Using the Lambert‐Beer law, the content of peroxo‐like species was taken proportional to the absorbance at 410 nm, considering an extinction coefficient of 0.28 µmol −1 cm −1 and a cuvette path length of 1 cm. Three types of samples were used to determine the content of peroxo‐like species: control seeds without any treatment, seeds treated with plasma for 3 min, and seeds soaked for 30 s in a 4.5% hydrogen peroxide solution. The estimated amount of peroxo‐like species determined for the H 2 O 2 treated seeds was corrected by dividing the result obtained in the measurement by 1.026. This correction stems from the fact that after 30 s of immersion of the seeds in the 4.5% H 2 O 2 solution, they increase their weight by 2.65% and this weight percentage should be taken into account to estimate the actual weight of the sample before soaking. For example, absorption spectra recorded for liquids extracted from seeds subjected to various treatments are reported in the Supporting Information: Figure S1. 2.9 |SEM‐X‐ray detector (EDX) and XPS analysis A Hitachi S4800 SEM‐FEG field emission microscope working at 2 kV has been used for the morphological analysis of seed surface and cross‐section cuts, without applying any metallization protocol. Elementary and compositional maps have been studied with an EDX in an EDX‐Bruker‐X Flash‐4010 analyzer working at 20 kV. Chemical state characterization of the surface state of seeds, including an estimation of the atomic concentration of surface elements, has been performed by XPS analysis in a SPECS spectrometer provided with a hemispherical analyzer (DLSEGD‐Phoibos‐Hsa3500). Nonmonochromatic Al Kα radiation was employed to record the spectra at the normal configuration, which were obtained in the constant pass energy mode at a value of 50 eV for the general survey and 30 eV for high‐resolution spectra. Calibration in binding energy (BE) has been done at the carbon functional C–Hand C–C bonding groups appearing at 284.5 eV in the C1s zone. 3|RESULTS AND DISCUSSION 3.1 |Germination tests in Whatman filters and in vitro contamination test In Figure 1a, we show results of a germination essay in a Whatman filter that illustrate found differences in the emergency of the coleoptile of the radicle. The bar plots show the evolution of the germination rate as a function of time after the treatment with an air plasma for different periods of time and, for comparative purposes, the evolution of control seeds and the set of dry seeds, these latter are subjected to drying at ambient temperature, as described in the experimental section. Several issues deserve comment in this figure. First, plasma treatments for 1 or 3 min are quite effective in promoting the germination of barley seeds with around a 20% and a 70% germination rate after 12 and 18 h, respectively, against 8% and 44% for the control seeds after the same periods of time. At 24 h, germination rates reached 76% and 68%, respectively. A small difference remains after 36 h, while for longer times only a little difference of 98% versus 94% remained between seeds plasma treated for 3 min and the control. Taking into PEREA‐BRENES ET AL. | 5of17 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License account the error limits considered in our analysis, it can be concluded that up to 24 h after sowing, positive differences in germination rate are clearly observed for the 1 and 3 min plasma treated seeds with respect to the control. It is also apparent from this plot that plasma treatments for 7 min have a certain deleterious effect in stimulating the germination rate. The images in Figure 1b illustrate the germination process at different times after sowing for seeds treated with plasma for 3 min. The progression in the emergence of the radicles is clearly seen in these images. Regarding the plasma treatment time, data in Figure 1a reveal differences within the statistical error, although a maximum 98% germination was achieved for 3 min plasma treatment. Pictures from Figure 1c corroborate the good development of the radicle as an effect of the plasma treatment compared to the control one. In the rest of this study, most experiments correspond to plasma treatment times for 3 min. Overall, the observed differences in germination rate can be of great significance when thinking in industrial manipulation where a small reduction of the malting time may have a significant impact on the cost of the process. [34] Surprisingly, for the 24 first hours after seeding, the drying treatment produces a clear acceleration in germination rates with percentages of successful germination that were higher than those of plasma seeds. The observed differences become small after 48 h or longer times since the sowing, although plasma treated and dry seeds presented germination percentages between 92% and 96% against 92% for the control seeds. After 72 h these percentages amounted to 94%–100% for the plasmatreated and dry seeds against 92% for the control. Regarding this drying treatment, it is relevant that weighting the seeds before their storage in the desiccator and just before sowing revealed a weight loss of 4.5%, most likely due to some loss of intrinsically absorbed water. At this point, it is relevant to comment that seeds subjected to a similar desiccation process in an oven at 50°C and 70°C showed weight losses of 2.0% and 5.9% (a) (b) (c) FIGURE 1 (a) Bar diagrams of the germination rate (in percentages) in a Whatman filter of barley seeds subjected to plasma treatments at various times. Results for control and dry seeds are included for comparison. (b) Images of Petri dishes for the seeds subjected to 3 min of plasma treatment for increased time periods after the treatment. (c) Images of Petri dishes for the seeds subjected to different plasma treatments at 24 and 48 h after treatments. 6of17 | PEREA‐BRENES ET AL. 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (note that seeds were exposed to air before weighting and that they may have recovered some of the water lost during heating), but their germination was severely affected with germination rates of only 78% and 22% after 24 h (see the Supporting Information: Figure S2). As confirmed by Soares et al. [50] this behavior agrees with the known decrease in germination capacity of barley grains subjected to overheating at temperatures above 40°C. Therefore, to ensure any overheating damage, drying treatments in our work have been carried out at ambient temperature. An important factor affecting seed germination and plant growth refer to their contamination degree, [1,18–21,51] a feature that can be positively decreased by the plasma treatments as a prevention method. [5,18,19,49] Figure 2a shows the contamination degree, defined and determined as described in the experimental section, for the plasmatreated seeds, the control set, and the dry seeds. An example of the appearance of contamination in seeds is reported in Figure 2b. Clearly, the plasma treatment produces a positive effect and contributes to drastically reducing the contamination degree with respect to the controlseeds,with contamination percentages around 80% and 40% after 110 h for control and plasma treated seeds, respectively. Particularly interesting was the fact that the contamination degree of plasma‐treated seeds remained under a manageable value lower than 50% even 110 h after the beginning of thetest,muchlowerthanthevaluefoundforthecontrol seeds. Interestingly, the set of dry seeds presented an even lower contamination degree of 10% after this period of time. A hypothesis for this outstanding behavior is that the avidity of these seeds for water (see water adsorption experiments in Section 3.3) would deprive the surface of seeds of the conditions (i.e., basically hydroxylation degree and amount of adsorbed water) required for efficient deployment of fungi or bacteria. Clearly, plasma and drying treatments induce a net reduction in contamination degree with the expected benefits for seeds storage and conservation and/or their germination success. [18,19,51–53] However, whether equivalent factors are involved in reducing the contamination degree in both situations is still a subject of debate, although we presume that the oxidative character of the plasma should play a specific role in the plasma case. 3.2 |Germination rate in the substrate The positive effect of the plasma treatment on the germination rate was less apparent for the germination essays in the substrate. It has been noted in the experimental section that small differences in the sowing depth for each seed and the fact that plants have to emerge from the soil to determine a germination event makes this analysis intrinsically less accurate than that in a Petri dish. Nonetheless, a certain favorable effect in increasing germination rate can still be noted for plasma‐treatedseedat72 and 96 h after sowing (note that the criterion defining germination, in this case, is different than in the Whatman filter test and that no information is available for periods of time shorter than 72 h). According to Figure 3a, seeds treated with an air plasma for 1 and 3 min depicted a germination rate of 80% and 75% after 72 h after sowing, against 65% for the control and 7 min plasma treated seeds and only 30% for the dry seeds. Although the reasons for this slow growth rate of dry seeds are still unclear, we speculate that plant emergency is slower for these seeds (note that plant emergency is the criterion adopted to count germinated seeds in soil), while radicle emergency might occur more readily. Differences between plasma treated and control seeds became smaller after 96 and 120 h, although the germination rate was still slightly higher for the former. An evaluation of the height of the plants after 7 days from sowing revealed a slight favorable tendency for the (a) (b) FIGURE 2 (a) Evolution with time of the contamination degree (in percentage) in vitro of barley seeds subjected to plasma treatments for various times. Results for control and dry seeds are included for comparison. Results of plasma 1 min and plasma 3 min experiments present similar values. (b) Image of a contaminated seed depicting the typical black stains that denote contamination. PEREA‐BRENES ET AL. | 7of17 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License plasma‐treated seeds with respect to control (see Figure 3b and Supporting Information: Figure S3)andasignificant decrease in height for the dry seeds, in line with the observation of slower germination in soil. This latter result shed doubts about the interest of the drying treatment for the plant growing in soil, although it may still represent a clear advantage for industrial malting processes where the germination protocol presents clear similarities with the Whatman filter germination procedure. [34] For the treatments and experiments carried out in the rest of this study, as well as for the discussion of results, the reference procedure will be that of the Petri dish experiments. 3.3 |Water uptake capacity Unlike the hydrophobic character of pristine seeds, seeds subjected to plasma treatment depicted a hydrophilic behavior (see the Supporting Information: Figure S4) that lasted several weeks after the plasma exposure. This behavior is commonly encountered for different kinds of seeds and experimental conditions and has contributed to the rather widespread idea that the water uptake capacity of plasma‐treated seeds becomes exalted. [14,16,17] However, we believe that this behavior is by no means common and very likely depends on seed treatment and manipulation conditions. Actually, in previous works of our group and others working with soybean, wheat, quinoa, nasturtium, or cotton seeds it has not been found a clear increase in water uptake capacity after plasma treatments similar to those applied in the present work, while differences were quite dependent on manipulation conditions or water availability in the environment. [7,14,50,54] For the barley seeds investigated in the present study, we have carried out a systematic investigation of their water uptake capacity either from a vapor or liquid phases, as represented in Scheme 1. The number of used seeds depended on the water uptake test: from one in the microbalance to 20 and 50 seeds in water vapor exposure and liquid water immersion experiments, respectively. The initial steps of water incorporation from the vapor phase have been also investigated in a precision thermogravimetric balance following the methodology explained in the experimental section. Recent and past publications have systematically dealt with the problem of water uptake either from liquid and vapor phases due to its importance for germination, seed storage, or longterm preservation. [55–57] Our purpose herein is more limited and aims at establishing possible differences in the water uptake capacity depending on seed treatment. Figure 4shows the evolution of water uptake from the vapor phase for barley seeds subjected to plasma treatment, the control group, and the set of dry seeds subjected to the drying process. The curves, obtained by weighting according to the protocol explained in the experimental section show progressive incorporation of water from the vapor phase in a continuous process extending for more than 144 h (8640 min). This progressive incorporation of water is rather similar for the control and plasma‐treated seeds, though, depending on treatment time, plasma‐treated seeds seem to uptake 2%–3% more water than the control seeds after similar periods of time. In turn, the dry seeds were much more effective to incorporate water in a process that is quite noticeable already after 24 h of exposure (weight increase by a 4.25% for a 2.7% for the control), presenting a clear difference after 144 h of exposure to water vapor when a weight increase of 35% was found for the dry seeds against 27.5% for the control. It is noteworthy that the germination process may also occur by exposure to humidity under the same conditions as in this experiment (see Supporting Information: Figure S5), although the germination time was longer than 168 h and (a) (b) FIGURE 3 (a) Bar diagram of the germination rate in the substrate (in percentages) of barley seeds subjected to a plasma treatment at various times. Results for control and dry seeds are included for comparison. (b) Photographs of plant emergency in the trash with most plants emerged corresponding to barley seeds treated with plasma for 1, 3, and 7 min. 8of17 | PEREA‐BRENES ET AL. 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License presented a well‐differentiated behavior depending on seed treatment. The incorporation of water from the vapor phase was also investigated in a model experiment using a highly sensitive thermogravimetric balance operated as explained in the experimental section. The curves in Figure 5show a comparison of the evolution of the relative weight of a control seed, a seed subjected to plasma treatment, and a dry seed (i.e., stored in the desiccator for 30 days). These curves show the relative weight evolution (i.e., [W+ΔW]/W, where Wrepresents the weight of the seed before the test) of each seed exposed first to humid air and then to the dried nitrogen flow as explained in the experimental section. We attribute the weight increase/decrease curves in this figure to water uptake/release processes. The three curves depict quite different profiles. In agreement with the evolution in Figure 5, the enhancement in the relative weight of the seed is maximum for the dry seed (i.e., 3.5%) followed, at a much lower level, for the control (0.9%) and finally for the plasma‐treated seed (0.3%). Conversely, the weight loss of the seeds when closing the humid air flow, once reached the maximum weight, shows that water is more effectively retained in the dry seed (weight loss by 1.2% after 1000 min) and more easily released in the control (weight loss by 1.4%) and, particularly, in the plasma‐treated seed (1.9%). These curves sustain that the dry seed presents a high avidity for water, much higher than that of the control and plasma‐treated seed. In contrast, water release was maximum from the plasma‐treated seed where it seems to occur readily in dry ambient conditions. We speculate that the new oxygen functional groups generated onto the surface and their hydrophilic behavior induce by the plasma treatment seem to favor water permeability, particularly under dry environmental conditions. However, at this stage, it is not justified to make a quantitative analysis of these curves because there is no data regarding the water uptake/release of seeds during their handling in the atmosphere before their placement in the set‐up, time inside the plasma chamber or simply the relative humidity of the air during manipulation outside its storage container. Despite this, a qualitative evaluation of the found results is illustrated in the cartoon in Figure 5b showing this different behavior: the dry seed is more prone than the other to uptake water, while in the plasma‐treated seed water released in dry conditions is maximum. Concerning the plasma treatment experiments, these results prove the high sensitivity of plasmatreated seeds to vapor pressure and the need to keep strict control of the ambient humidity when handling seeds in air to reach reliable results. The water uptake capacity of seeds was higher and occurred more rapidly when they were immersed in liquid water. Figure 6shows the evolution of weight for the seeds immersed in water for increasing periods of time. The comparison between the plasma‐treated, control, and dry seeds reveal a rather similar water uptake capacity for the control and plasma treated seeds and a higher capacity for the dried seeds, manifested SCHEME 1 Water uptake experimental configurations, from vapor and liquid phases PEREA‐BRENES ET AL. | 9of17 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License results from liquid or vapor sources indicate that water incorporation is quite similar in the plasma‐treated seeds and in the control, although it significantly increases in the seeds dried at ambient temperature. These results suggest a possible affectation of the germination rate because of a plasma‐induced modification of the water incorporation capacity, but also that this factor might contribute to the observed increase in germination in the plasma‐treated seeds. Surface analysis of seeds subjected to plasma treatments indicates small changes in chemistry and topology that comply with partial surface oxidation of seeds and suggest the incorporation of RONS due to seed‐plasma interactions. Based on the hypothesis that these oxidative species may affect the ABA germination retarding factor present in all the seeds, a series of treatments of seeds with ABA solutions, hydrogen peroxide solutions, and plasma and drying processes, have clearly confirmed the retarding role of ABA regarding germination and that this effect is released if the seeds are subjected to an oxidative treatment, either with H 2 O 2 or air plasma. The obtained results have effectively confirmed that oxidative plasmas behave in a rather similar way to hydrogen peroxide regarding both the triggering of germination and the incorporation of oxidative species into the seeds. In this way, a general conclusion of this study is that atmospheric pressure plasmas accelerate the germination rate of barley seeds because they affect the biochemistry of seeds, very likely contributing to reducing the concentration of ABA germination retarding factor. ACKNOWLEDGMENTS We acknowledge the financial support of the Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía through first the project US‐ 1381045, and second the US‐1380977, as well as the EU through cohesion fund and FEDER 2014–2020 programs for financial support. We also thank the AEI‐MICINN (PID2019‐110430GB‐C21, PID2019‐109603RA‐10, and PID2020‐114270RA‐I00 funded by MCIN/AEI/10.13039/ 501100011033 and by “ERDF (FEDER) A way of making Europe,”by the “European Union.”The contribution of Arquimea Research for the realization of parts of this study is also acknowledged. We thank Intermalta S.A. for the seeds supply. This article frames within COST Action PIAgri‐CA19110, supported by COST (European Cooperation in Science and Technology), www.cost.eu. Carmen López‐Santos thanks the support of the University of Seville through the VI PPIT‐US and the Ramon y Cajal Spanish National programs funded by MCIN/AEI/ 10.13039/501100011033. Manuel Oliva‐Ramírez thanks a contract grant within the Paidi 2020 of the EU social fund‐University of Seville. We also thank effusively the support of Dr. Pedro E. Sánchez‐Jiménez during the thermogravimetric microbalance experiments. DATA AVAILABILITY STATEMENT Data are available on request from the authors. 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García, M. Cantos, A. R. González‐Elipe, Plasma Processes Polym.2022;19:e2200035. https://doi.org/10.1002/ppap.202200035 PEREA‐BRENES ET AL. | 17 of 17 16128869, 2022, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ppap.202200035 by Universidad De Sevilla, Wiley Online Library on [10/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License