Enhancing viability and vigor of deteriorated true shallot seeds (Allium cepa var. ascalonicum) through ultra-fine bubble and plasma-activated water priming

ABSTRACT:

Ultra-fine bubbles (UFB) and plasma-activated water (PAW) are well-known water treatment technologies that have recently been explored for seed priming. Most results demonstrated improvements in seed vigor and viability, hypothesized to be due to reactive oxygen species (ROS). This study aimed to determine the optimum priming technique using UFB and PAW for deteriorated true shallot seeds (TSS). The TSS with various expiration dates and low viability (germination rate <80%) were primed for 24 hours with UFB water (dissolved oxygen levels of 18 and 24 mg.L^-1) and PAW (five ozone concentrations, 0; 0.1; 0.5; 1.0; 4.0 mg.L^-1 and three exposure times (10, 20, and 30 min) at separated experiment. The results showed that UFB and PAW effectively improved seed physiology. It increased the germination rate and radicle emergence to >80%, similar to fresh TSS. The UFB water treatment dissolved oxygen levels resulting in similar improvement, whereas PAW with 0.1 mg.L^-1 ozone was the optimum treatment level. The exposure duration demonstrated variability in the PAW priming effectivity. Internal seed ROS alterations due to UFB- and PAW-produced ROS improved TSS pre-germinative metabolism, i.e., hydrolytic enzyme activity, respiration rate, and membrane integrity. Thus, it potentially underlined the viability and vigor enhancement of deteriorated TSS.

Index terms:
amylase activity; cold plasma; nanobubble; ozone; pre-germinative metabolism

RESUMO:

Bolhas ultrafinas (UFB) e água ativada por plasma (PAW) são tecnologias de tratamento de água bem conhecidas que foram recentemente exploradas para osmocondicionamento de sementes. A maioria dos resultados demonstrou melhorias no vigor e viabilidade das sementes, hipotetizadas como sendo devido a espécies reativas de oxigênio (EROs). Este estudo determinou a técnica ideal de preparação usando UFB e PAW para sementes deterioradas de Allium cepa var. ascalonicum. Sementes com baixa viabilidade (taxa de germinação <80%) foram preparadas por 24 horas com água UFB (níveis de oxigênio dissolvido de 18 e 24 mg.L^-1) e PAW (cinco concentrações de ozônio: 0; 0,1; 0,5; 1,0; 4,0 mg.L^-1) e três tempos de exposição (10, 20 e 30 min) em experimentos separados. UFB e PAW aumentaram a taxa de germinação e a emergência da radícula para >80%, semelhante ao controle. O nível ideal de PAW foi 0,1 ppm de ozônio. A duração da exposição ao plasma demonstrou variabilidade na eficácia da preparação. As alterações internas de EROs da semente devido aos EROs produzidos pelo UFB e PAW melhoraram o metabolismo pré-germinativo das sementes, ou seja, o equilíbrio de EROs, a respiração e a integridade da membrana. Isso pode preparar o cenário para a viabilidade e o aumento do vigor das sementes deterioradas.

Termos para indexação:
atividade da amilase; plasma frio; nanobolhas; ozônio; metabolismo pré-germinativo

INTRODUCTION

The shallots (Alium cepa) become a staple herbs and spices in many culinary and medicinal culture. Its provide the macro- and micro-nutrients and also provide high amount of anti-oxidant (Adeyemo et al., 2023). In Indonesia, shallots are a crucial horticultural crop for the Indonesian economy because its influence on national inflation rate. Shallot productivity in Indonesia over the last five years has been stagnant and showed declining trend (Indonesia, 2020). True shallot seed (TSS) is proposed as a strategy to increase productivity (Rosliani et al., 2016). TSS has advantages over vegetative tubers, such as cheaper storage and distribution costs, more uniform planting, more stable productivity, and lower risk of seed-borne diseases (Van Den Brink and Basuki, 2012; Rosliani et al., 2016).

However, the implementation of TSS as a planting material in Indonesia presents several challenges. TSS production in Indonesia is encountered by many obstacles, including the environment and cultivation technique (e.g. high rainfall and daily temperature average, short photoperiod, low pollinator, severe disease incidence) that limit the flowering and seed formation (Sayaka et al., 2021). Indonesia’s shallot seed supply chain shows that TSS is generally used to produce mini bulbs (Sayaka et al., 2020). Therefore, TSS may not be routinely applied during each planting season. Seed storage conditions at the farm level are generally uncontrolled and can potentially reduce seed viability. TSS quickly deteriorate when stored under uncontrolled conditions (Hornke et al., 2020).

Deterioration causes viability loss and affects other germination properties, such as imbibition and metabolic reactivation. It could disrupt germination and reduce the uniformity of germination (Zhang et al., 2021). Priming is a technique applied to seed using various agent (including water-hydropriming) to increase their germination capability through alleviating seed damage and recovering seed-repairing ability (Corbineau et al., 2023). Priming is known to increase and enhance the seed genome repair activity, hydrolytic enzyme synthesis, and seed reserve mobilization which occur when water imbibition is in phase II, before radicle protrusion (Pagano et al., 2023).

Ultra-fine bubbles and plasma-activated water are well-known technologies used for water treatment. UFBs are bubbles lower than 1 nm that are retained for an extended period in water and can impart unique characteristics to UFB-containing water, such as enhanced gas solubility, increased bubble surface area, and production of reactive oxygen species (ROS) (Liu et al., 2016; Tanaka et al., 2020). Plasma-activated water (PAW) is water subjected to plasma exposure. Plasma is produced from electricity that ionizes gas (Boulos et al., 2016). In recent years, UFB and PAW have been explored for use as seed-priming agents. Treatment of soybeans (Glycine max L.) (Guragain et al., 2021), tomato (Solanum lycopersicum L.) (Vichiansan et al., 2023), and lettuce (Lactuca sativa L.) (Stoleru et al., 2020) with PAW led to an increase in seed viability and vigor. The increase in seed viability and vigor from UFB and PAW priming is known to be caused by the ROS present in UFB and PAW.

ROS is a by-product from reduction of oxygen (O₂) on aerobic metabolism inside the cell, and consisted of the free radical (e.g. superoxide anion) and nonradical species (e.g. hydrogen peroxide). In the seed, ROS is produced through non-enzymatic pathway by lipid oxidation and after imbibition started, the enzymatic pathway is engaged inside mitochondria (Bailly, 2023). At specific concentrations, ROS in seeds plays a vital role in cell signaling pathways and positively affects seed metabolism (Bailly, 2019). This study aimed to investigate the UFBW and PAW priming effectiveness for TSS and evaluate the mechanism underlying the TSS viability and vigor enhancement after priming.

MATERIAL AND METHODS

Seed material

TSS var. Lokananta with four different expired date labels and initial germination rates (Table 1) were used in this experiment. The L1 is acquired in a fresh pack prior to use while L2, L3, and L4 previously have been stored in controlled room (20 °C temperature and 50-60% relative humidity (RH)) for various storage times inside sealed aluminum bags.

Table 1
Seed lot characteristic used in this experiment.

Seed lot	Expired date on packaging label	Initial Germination Rate (%)
L1	December 2023	89.4
L2	December 2021	88.1
L3	January 2022	63.5
L4	August 2020	68.2

Experiment 1: TSS Priming technique using UFBW and PAW

Experiment design and treatment: The first experiment was performed in triplicate using a factorial, completely randomized design (CRD). The first factor is the seed lot with four different levels of viability (L1-L4). The second was a priming treatment consisting of untreated seeds (control), treatment with ultrafine-bubble water (UFBW) containing 18 mg.L^-1 (UFB18) and 24 mg.L^-1 (UFB24) dissolved oxygen, and treatment with plasma-activated water from three exposure durations, e.g., 10, 20, and 30 min (PAW10-PAW30) which contains 0.1 mg.L^-1 of ozone. The 30 g.L^-1 KNO₃ was used as a positive control according to Muruli et al. (2016).

Priming solution production: UFB and PAW were produced in distilled water. The UFB generator (UltrafineGaLF FZ1N-10, IDEC Japan) was operated with oxygen gas injection for 55 min and 70 min to make UFB containing 18 mg.L^-1 and 24 mg.L^-1 dissolved oxygen (DO), respectively. A plasma-fine bubble generator with a dielectric barrier discharge plasma source generated the PAW. Water was exposed to plasma for 10, 20, and 30 min. This device also produces fine bubbles. UFBW and PAW characteristics (temperature, pH, and conductance) was measured in triplicate. KNO₃ solution were made with 30 g.L^-1 concentration.

Seed Priming treatment: Approx. 3 g of TSS were primed with 400 mL of each priming solution in covered glass jar. Priming was carried out for 24 hours in 20 °C room. The seeds then were dried back to initial moisture level (8-10 hours in 20 °C room). Seeds were stored in controlled room (20 °C; 50% relative humidity (RH)) inside impermeable aluminum bags prior to germination assay.

Germination assay: Seeds were germinated in triplicate (100 seeds per replication) with top paper method using two-layer moistened filter paper as a substrate. The assays were done using plastic box (16.5 cm × 11.0 cm × 4.7 cm) in 20 °C and 50% RH room (ISTA, 2018). Germination parameters were observed are: radicle emergence RE (≥ 2 mm length) percentage on 72 hours after germination (Kamanga et al., 2021); Germination rate (GR): percentage of total normal seedling at first and final count (6 and 12 days after germination (DAG) (ISTA, 2018); Speed of germination (SG) based on Copeland and McDonald (2001); and seedling dry weight (SDW), obtained from oven-drying (24 hours; 80 °C) the normal seedlings from 12 DAG.

Experiment 2: PAW ozone concentration effect on seed viability

Experiment design and treatment: This experiment was done using one-factor CRD with ozone concentration in PAW assigned as factor with five ozone concentration level (in mg.L^-1), namely 0 (untreated control); 0.1; 0.5; 1.0; and 4.0. Lot L3 was used as a subject because it had relatively lower initial quality compared to the other lot and it demonstrated good response after treated with UFB and PAW in Experiment 1. The PAW containing those ozone level were created using distilled water activated with 20 min plasma exposure. Priming treatment were done with same procedure on Experiment 1.

Germination and physiology assay: A triplicate germination test (100 seeds per replication) was performed using the same procedure as in Experiment 1. The following germination parameters were examined: RE, FCG, GR, SG, SDW, seedling respiration rate (SRR) and electrical conductivity (EC). EC tests were performed in for using method described by Dias et al. (2006). Fifty seeds were immersed with 50 ml of deionized water and incubated for 24 hours in 20 °C room. The test was done in four replicate and two blanks (deionized water only). EC is calculated according to ISTA (2018):

$\text{EC}\left(\text{μS}\text{.}{\text{cm}}^{\text{-1}}\text{.}{\text{g}}^{\text{-1}}\right)\text{= [}\text{seed conductivity}\text{(μS}\text{.}{\text{cm}}^{\text{-1}}\text{) -}\text{blank solution conductivity}\text{]/}\text{seed weight}\text{(g)}$

Experiment 3: Priming effect on seed pre-germinative metabolism

Experiment design and treatment: This experiment was carried out using one-factor CRD. Invigoration treatment assayed were UFBW24 and PAW20 with 0.1 mg.L^-1 ozone with addition of untreated control. Seed lot L3 was used as a seed subject. Priming solutions (UFBW and PAW) were made using same method as in Experiment 1 with corresponding treatment level. Approx. 3 g of TSS were primed using each priming solution, following the procedure in Experiment 1.

Seed physiology assay: The following germination parameters were examined: seedling respiration rate (SRR), and electrical conductivity (EC). The SRR was observed using the titration method. Fifty seeds were germinated using the top-paper method with moistened two-layer filter paper in an airtight container (volume 1.2 L). A small bottle containing 30 mL of 0.2 M KOH was placed in the center of the container. The containers were then incubated in a germination room at 20 °C and 50% RH for seven days (±184 hours). Containers containing the same bottle of KOH solution, but without seeds, were also incubated under the same conditions. After incubation, KOH inside the bottle was titrated. First, two drops of phenolphthalein were added so that a pink color appeared on the solution, and titration was carried out using 0.5 M HCl until the solution became colorless. Subsequently, two drops of methyl orange were added. Titration was repeated until a red-orange color appeared in the solution. SRR was calculated using the following formula:

${\text{CO}}_{\text{2}}\left({\text{mg.h}}^{\text{-1}}\right)\text{= [(a - b) × N × 44]/T}$

Which a is total volume of HCl used for seed-container titration; b total volume of HCL used for seedless-container titration; N molarity of HCl; and T incubation time (hour). Seed’s EC after priming in this experiment is measured using method similar to Experiment 2.

Seed biochemical assay: For crude extract preparation, approx. 0.2 g of primed TSS was imbibed between two layers of filter paper moistened with deionized water in a 20 °C room for 18 and 24 hours. The imbibed seeds were homogenized in addition of 15 mL of cold phosphate buffer (0.1 M pH 7). The homogenate was then double-centrifuged, first at 4,427 x g for 6 min, then at 13,250 x g for 5 min. Supernatants were extracted and used as crude enzyme in amylase activity and total protein content assays. Amylase activity was measured using the method described by Bernfeld (1955). The total protein content was quantified according to Lowry et al. (1951).

ROS histochemical localization: Seed ROS (superoxide type) after 24 hours of imbibition was localized using nitro blue tetrazolium (NBT) staining. Imbibed seeds were longitudinally cut but not until separated, then immersed with 0.1 mM NBT solution inside 50 mM phosphate buffer (pH 7) for (60 min, dark, 30 °C). Superoxide inside the seed is depicted as a dark blue insoluble stain (Beyer and Fridovich, 1987). The stained seeds were observed using a stereomicroscope.

Statistical analysis: ANOVA was performed for all parameters observed in each experiment. If the result was significant (p <0.05), the means were subjected to Duncan’s multiple range test (DMRT) ɑ = 0.05, to determine the difference between means. Student’s t-test was also performed to compare amylase activity and total protein data from the two imbibition duration.

RESULT AND DISCUSSION

PAW generation influenced water pH and conductivity. The water pH displayed a decrease trend as the duration increased (Figure 1A). Water pH decreased after plasma exposure for 10, 20, and 30 min to 6.9, 6.3, and 6.0, respectively, compared with untreated water (pH 7.3). Those range remained safe for general physiological and metabolic process. Water conductivity increased after plasma exposure to water (Figure 1B). At 10 and 20 min exposure time, water conductivity is significantly higher (10.5 μS.cm^-1 and 10.3 μS.cm^-1, respectively) than distilled water (5.6 μS.cm^-1). The conductivity slightly decreased after 30 min but remained higher than the distilled water. UFB did not show any influence to water pH and conductivity.

Figure 1
The pH (A) and conductivity (B) of water after plasma exposure (ozone conc. 0.1 mg.L^-1). Different lowercase within same parameter denote difference by DMRT 5%.

The UFB and plasma-fine bubble generator used in this experiment produced heat that increases the UFBW and PAW temperatures from 26 °C to 27-33 °C. Both UFB and PAW is increased water DO from 8 mg.L^-1 (distilled water) to 18 and 24 mg.L^-1 (UFBW from 55 and 70 min running duration, respectively) and 14-16 mg.L^-1 (PAW). UFB generator in this experiment produced constant fine bubble concentration (10⁸-10⁹ mL^-1) regardless the operational duration.

Changes in pH and conductivity on PAW are possible because of the production of ions such as H⁺, NO₂ ^-, and NO₃ ^- around the plasma, which diffused into the water. These ions are generally acidic (Mizoi et al., 2022). Ions and ROS accumulation in PAW water also impacts water conductivity. Wong et al. (2023) revealed a positive correlation between the PAW conductivity and the presence of ions and ROS. In this experiment, UFB generation duration influenced the UFBW DO concentration. It occurred because the DO concentration in the UFBW was positively correlated with running duration. Other factors affect UFBW DO concentration are initial water DO, gas type, and injection rate (Tekile et al., 2016; Hanam et al., 2022).

The priming method and seed lot interaction significantly influenced the TSS viability and vigor (Table 1). PAW and UFBW priming significantly enhanced the viability of aged and deteriorated seed lots (Table 2). The poorly viable L3 and L4 lots demonstrated improvements based on GR and the value was similar to the fresh seeds. The GR increased to >80%, equivalent to fresh seed GR (89%). The SDW of the L4 lot was significantly higher (0.120-0.135 g) than the control (0.102 g) after UFBW and PAW priming.

Table 2
Interaction between seed lot and priming treatment on TSS viability parameters.

Lot	Priming treatment
	Control	PAW10^	PAW20	PAW30	UFB18	UFB24	KNO3
	Seedling dry weight (g)
L1‡	0.129 Aa*	0.127 Aab	0.113 Bd	0.128 ABab	0.120 Aabc	0.118 ABcd	0.123 Babc
L2	0.127 Aab	0.128 Ab	0.125 ABab	0.134 Aa	0.124 Aab	0.114 Bab	0.113 Cb
L3	0.115 Aa	0.128 Aa	0.129 Aa	0.122 Ba	0.132 Aa	0.128 Aa	0.135 Aa
L4	0.102 Ac	0.125 Aab	0.120 ABb	0.135 Aa	0.125 Aab	0.122 ABb	0.130 ABab
	Germination rate (%)
L1	89.39 Aab	90.88 Aa	81.59 Ab	88.96 Aab	88.20 Aab	88.55 Aab	88.79 Aab
L2	88.13 Aa	84.02 Aa	82.44 Aa	87.81 Aa	82.01 Aa	84.37 Aa	79.33 Ba
L3	63.47 Bb	91.36 Aa	91.19 Aa	87.90 Aa	85.56 Aa	89.65 Aa	86.39 Aa
L4	68.19 Bb	88.73 Aa	85.04 Aa	90.08 Aa	88.00 Aa	89.06 Aa	88.99 Aa

• *Values with different uppercase (column) and lowercase (row) are significantly different by DMRT 5%. ^‡L1-L4 = Seed lot with different expired date; L1 (Dec 2023), L2 (Dec 2021), L3 (Jan 2022), L4 (Aug 2020). ^PAW10-30 = PAW from 10, 20, and 30 min exposure; UFB18 = UFBW with 18 mg.L^-1 DO; UFB24 = UFBW with 24 mg.L^-1 DO; KNO₃ = 30 g.L^-1 KNO₃ solution.

Compared to the untreated seed, the deteriorated seed (L2-L4) vigor was enhanced after primed with UFBW and PAW. Some treatment even made the deteriorated seed vigor became comparable to that fresh seeds (Table 3). Lot L2 had FCG with initial value of 16% increased to 44-49% and 24-30% after primed with all PAW and UFBW treatment levels, respectively. Overall, priming with UFBW and PAW at all treatment levels did not significantly influence the physiological quality of fresh seeds (L1), and it tended to negatively impact their viability and vigor. The PAW20 and UFB24 treatments decreased the fresh seed SDW from 0.129 g in untreated seed to 0.113 g and 0.118 g, respectively. The SG and RE parameters on the same seed lot were not significantly different from the untreated control after priming with UFBW and PAW.

Table 3
Interaction between seed lot and priming treatment on TSS vigor parameters.

Lot	Priming treatment
	Control	PAW10^	PAW20	PAW30	UFB18	UFB24	KNO3
	First count germination (%)
L1‡	27.0 Ac*	43.0 Ab	31.0 Bc	52.3 Aa	22.0 Ac	25.0 Ac	51.0 Aab
L2	16.3 BCd	48.0 Aa	43.7 Aa	49.0 Aa	24.3 Ac	29.7 Abc	34.0 Bb
L3	18.0 Bbc	24.3 Bb	18.7 Cbc	22.3 Bb	11.0 Bc	23.7 Ab	52.3 Aa
L4	12.0 Cf	30.7 Bcd	35.0 ABbc	47.7 Aa	13.3 Bef	23.3 Ade	44.0 ABab
	Speed of germination (%.24h-1)
L1	13.54 Aa	12.08 Acd	11.88 Ad	12.02 Acd	12.89 Ab	12.59 Abc	13.99 ABa
L2	12.58 Ba	11.37 ABb	11.65 Aab	11.97 Aab	11.96 Bab	11.76 Bab	11.99 Cab
L3	11.54 Dbc	10.78 Bcd	10.88 Bc	10.54 Bd	12.56 ABb	11.89 Bb	14.35 Aa
L4	12.08 Cb	10.79 Bcd	11.36 ABc	12.09 Ab	12.09 Bb	12.13 Bb	13.34 Ba
	Radicle emergence (%)
L1	91.33 Aa	82.00 Ab	83.67 ABb	90.33 Aa	93.33 Aa	93.67 Aa	93.67 Aa
L2	83.67 Ba	72.67 Ab	81.67 Ba	84.67 ABa	87.67 Ba	86.33 Ba	89.33 Ba
L3	71.67 Cd	77.00 Acd	81.33 Bbc	77.67 Bcd	89.33 ABab	88.00 Bab	94.00 Aa
L4	75.00 Cc	79.00 Ac	89.33 Aab	85.67 ABb	91.00 ABa	90.00 ABab	90.33 Bab

• *Values with different uppercase (column) and lowercase (row) are significantly different by DMRT 5%. ^‡L1-L4 = Seed lot with different expired date; L1 (Dec 2023), L2 (Dec 2021), L3 (Jan 2022), L4 (Aug 2020). ^PAW10-30 = PAW from 10, 20, and 30 min exposure; UFB18 = UFBW with 18 mg.L^-1 DO; UFB24 = UFBW with 24 mg.L^-1 DO; KNO₃ = 30 g.L^-1 KNO₃ solution.

Plasma exposure duration during PAW generation demonstrated variability in seed viability enhancement. The positive effect of prolonged plasma exposure demonstrated that the FCG and SG value of the L4 lot with PAW30 was 36% and 6% (for FCG); 55% and 12% (for SG) higher than PAW10 and PAW20, respectively. In contrast, prolonged plasma exposure negatively influenced SG on the L3 lot, where PAW30 had a lower SG than PAW10 or PAW20. The PAW20 treatment considered given better enhancement than PAW10. Although the 30 min exposure (PAW30) demonstrate similar positive effect with PAW20, it took much longer time to produce and had relatively higher temperature among the treatment level. DO concentration in UFBW generally did not significantly influence the UFBW priming effect. Both DO levels shared similar enhancement ability in viability parameters on L3 and L4, but not for all vigor parameters at those lots. Only the RE parameter that demonstrated enhancement on L3 and L4 after UFB18 and UFB24 treatment.

Different responses from plasma exposure have been observed in other species. Application of DBD plasma to lettuce seeds revealed that 10-20 min plasma exposure resulted higher germination than the untreated seed, and the germination is insignificantly different than control on prolonged exposure (25-30 min) (Than et al., 2022). Eggplant seeds primed with PAW showed germination improvement from the control until PAW generated from 6 min exposure and stagnated until 8 min (Rashid et al., 2023). Plasma exposure altered ROS concentrations in PAW and influence seed internal ROS dynamics. Vigna mungo seeds demonstrated increase of ROS (H₂O₂ and NO) on extended plasma exposure, with 9 min exposure level negatively influence the germination (Sajib et al., 2020).

KNO₃ priming effectively enhanced the germination of a wide range of seeds, including shallots/onions (Muruli et al., 2016). In this experiment, UFBW and PAW priming improved the viability and vigor of deteriorated seed lots with comparable value to KNO₃ treatment (Tables 2 and 3). The L3 and L4 lots had similar GR values as the KNO₃ primed seed after primed with all UFBW and PAW treatment levels, with GR values reaching >80%. The same phenomenon was also observed in SDW, where the L4 SDW value increased by 24% (0.120 - 0.135 g range) and 21% (0.123 g) after primed with UFBW and PAW, respectively, and did not significantly differ with KNO₃ (0.130 g).

Ozone (O₃) is one of the ROS in PAW generated from the plasma-fine bubble generator used in this experiment. It has a relatively strong oxidation capacity and longevity in water. The effect of ozone on seed germination is influenced by its concentration and the species (Pandiselvam et al., 2020). PAW20 priming containing several level of ozone concentrations (0.1, 0.5, 1.0, and 4.0 mg.L^-1) on L3 lot revealed that 0.1 mg.L^-1 ozone concentration is the optimal level for priming based on FCG and SG parameter (Figure 2). The value on both parameters is significantly higher on 0.1 mg.L^-1 ozone concentration compared with untreated control, then decreased on the 0.5-4.0 mg.L^-1 ozone concentration. The 0.1 mg.L^-1 ozone concentration significantly increase the RE compared to the control (Figure 2A). However, the value is statistically similar with 0.5 mg.L^-1 and 1.0 mg.L^-1 ozone concentration (89-90%). The 0.1-1.0 mg.L^-1 range of ozone concentration provide better seed membrane repair capacity illustrated with lower EC value (33.36-36.59 μS.cm^-1.g^-1) than untreated control (77.58 μS.cm^-1.g^-1) and 4 mg.L^-1 ozone concentration treatment (42.34 μS.cm^-1.g^-1) (Figure 2D). All ozone concentration applied did not affect seed viability (GR and SDW) where the value is not significantly differed with control (Figure 2E and 2F).

Figure 2
Effect of different ozone concentration inside PAW (PAW20) on TSS vigor (A, B, C, D) and viability (E, F) parameter. Values are mean ± SE (n = 3). Different lowercase within same parameter denote difference by DMRT 5%. Lot L3 was used as the seed subject.

The results suggest that TSS may exhibit sensitivity to ozone exposure, similar to other plant species. As depicted in Figure 2, most of the TSS vigor parameter values began to decline at ozone concentrations exceeding 0.1 mg.L^-1, despite remaining above those of the untreated seeds. The same phenomenon depicted on tomato seed where priming with ozone-containing water with 0.001-0.1 g ozone.g^-1 seed concentration for 20 min according to Sudhakar et al. (2011) had positive influence on seed germination and higher ozone concentration (1 g ozone.g^-1 seed) reduced the germination percentage and increased seed’s abscisic acid content. Other species, e.g. maize, chilli, and sunflower had higher tolerance to seed ozone exposure because their germination still remained high after exposed to high ozone concentration (>20 mg.L^-1) (Violleau et al., 2008; Rodrigues et al., 2015; Sharaf-Eldin et al., 2022).

Seed priming treatment is known for its ability to provide a series of pre-germination metabolisms to seeds, what could underlie the seed viability and vigor improvement of the primed seed. In this experiment, UFB24 and PAW20 containing 0.1 mg.L^-1 ozone were applied to initially low vigor seed (L3) to study priming effect on pre-germination metabolism of deteriorated seeds.

Both UFBW and PAW effectively improved seed amylase activity and increased the overall protein content (Figure 3). UFBW- and PAW-primed seeds showed higher amylase activity than the control during both the imbibition periods. UFBW increased seed amylase activity by 78% and 81% compared to the control after 18 and 24 hours imbibition periods, respectively. PAW provided a slightly smaller increase (47% and 60% after 18 and 24 hours imbibition), however, it did not significantly differ from UFBW. Total protein observed at 18 and 24 hours imbibition periods demonstrated that UFBW and PAW priming enhanced protease activity. Both treatments resulted in higher total protein levels compared to the control. At 18 hours period, PAW showed higher total protein content than UFBW, and the value was not significantly different from the 24 hours imbibitional period value (0.110 mg protein.mg^-1 dw and 0.119 mg protein.mg^-1 dw, respectively).

Figure 3
Priming effect on pre-germinative metabolism. Amylase activity (A), total protein content (B), respiration rate (C), and seed membrane integrity (D) in control and primed seed. Values are mean ± SE (n = 3). Different lowercases denote difference between treatment by DMRT 5% (for A and B, comparison is done for each imbibition duration). Ns: non-significant (p>0.05), **: significant (p<0.01) by t-student test for value between imbibition duration in each treatment (for A and B). Lot L3 was used as the seed subject.

UFBW priming showed a better seed respiration rate (0.126 mg CO₂.h^-1) compared to the control (0.110 mg CO₂.h^-1), where the seed respiration rate on PAW-primed seeds (0.116 mg CO₂.h^-1) did not significantly differ between the two treatments (Figure 2A). UFBW and PAW reinforced seed membrane integrity, as revealed by the EC observations (Figure 2B). The seed EC values was reduced from 60.96 µS.cm^-1. g^-1 (untreated control) to 41.85 µS.cm^-1. g^-1 and 36.34 µS.cm^-1.g^-1, after treated with UFBW and PAW, respectively. ROS (superoxide type) localization with NBT staining revealed that internal seed ROS production was higher in UFBW- and PAW-primed seeds than in untreated seeds (Figure 4). UFBW- and PAW-primed seeds showed more intense and evenly distributed dark blue-purple stain across the embryo and endosperm. In control seeds, a dark blue-purple stain only appeared on a small, scattered part of the endosperm and embryos with a lighter intensity than primed seeds.

Figure 4
Longitudinal cut of TSS. NBT-unstained control seed (A); NBT-stained, control and primed seed (B). Scale bar = 1 mm. White dash: embryo. Red arrow: radicle end. Lot L3 was used as the seed subject.

Priming is proven to improve seed viability and vigor in many species and alleviate deleterious effects on stored seed (Paparella et al., 2015; Pereira et al., 2022)seed morphology and physiology, different priming treatments can be applied, all of them triggering the so-called ‘pre-germinative metabolism’. This physiological process takes place during early seed imbibition and includes the seed repair response (activation of DNA repair pathways and antioxidant mechanisms. Priming allows early water imbibition and metabolism resumption without inducing radicle protrusion. Several metabolic activities were reactivated when water rapidly permeates the seed on phase I of the imbibition, including repairing response, DNA and RNA transcription, and mitochondria reactivation that promotes seed respiration. Those processes are continued in phase II of the imbibition and then followed by seed reserve mobilization. The dry-back applied on the primed seed restricted the seed moisture content from reaching favorable to germination (phase III). Those processes induced better seed physiological and metabolic readiness, promoting more rapid and better pre-germinative metabolism when sowed (Fabrissin et al., 2021).

The priming potential of UFBW and PAW is due to their ability to produce ROS. Continuous UFB collapse induced oxygen gas dissociation inside the UFB, becoming an oxygen molecules and triggering ROS production, such as O₂ ^-, OH^-, and H₂O₂ (Yasui et al., 2019). Liu et al. (2016) showed that oxygen injected UFBW had an oxidation capability to 0.5 mM H₂O₂, 0.25 μM OH^-, and 50 μM O₂ ^-. The ROS contained in PAW comes from the production of ROS gas-phase through cold plasma that diffuses into water. PAW also contained reactive nitrogen species (RNS) that combined with ROS generating the reactive oxygen and nitrogen species (Priatama et al., 2022). The external ROS produced by PAW or UFBW can manipulate the equilibrium of seed internal ROS and improve the germination process. ROS localization by NBT staining (Figure 4) showed that seed internal ROS was altered by ROS-containing UFBW and PAW. This phenomenon has also been documented in barley seeds, where UFBW-primed seeds had darker blue staining, and higher germination compared to untreated seed (Liu et al., 2016). Other studies by Ellouzi et al. (2021) on cauliflower seed and Li et al. (2022) on Jatropha curcas seeds revealed that priming with external ROS (using H₂O₂) increased the production of internal ROS and triggered better germination.

ROS are crucial messengers in cell signalling pathways during seed pre-germinative metabolism. ROS during germination induced the inhibition of germination regulators, activating the GA synthesis along with the suppression of ABA production. Furthermore, GA induced the production of hydrolytic enzymes, such as amylase and protease, that mobilize the seed reserve to support embryo cell elongation and division (Amri et al., 2016). Figure 3 shows that priming using UFBW and PAW promoted higher amylase activity, total protein content, and seed respiration rate in deteriorated TSS than in untreated controls. These advancements enhance the germination capabilities of deteriorated seeds and are comparable to fresh, viable seeds.

CONCLUSIONS

The UFBW and PAW application as priming agents for TSS can enhance their viability and vigor. Stored seeds (init. GR 64-68%) demonstrated high responsiveness to priming, with the GR increased to 85-91%, comparable to fresh seeds (89%). The RE also increased from <75% to >81%. Priming of fresh seeds priming did not considerably influence the viability and vigor.

DO concentration did not significantly influence UFBW priming effect, where both concentrations were effective in enhanced stored seed viability and vigor. Ozone concentration in PAW plays a more vital role than plasma exposure duration in seed physiological enhancement. The 0.1 mg.L^-1 ozone concentration is considered the optimal level for priming. Plasma exposure duration on the PAW generation variously influence the seed viability and vigor enhancement. Several levels of UFBW and PAW treatments showed comparable efficacy to KNO₃ in improving TSS viability. Related to vigor, KNO₃ was still superior to both treatments, particularly in terms of the seedling growth rate.

The increased production of internal ROS in primed seeds positively impact the pre-germinative metabolism (i.e., amylase and protease activity. seed respiration, and membrane integrity). These may provide a potential mechanism for the physiological improvement on deteriorated TSS.

ACKNOWLEDGMENTS

The authors express their gratitude to the Seed Laboratory Team at Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, for supporting the experiments preparation.

REFERENCES

• ADEYEMO, A.E.; OMOBA, O.S.; OLAGUNJU, A.I.; JOSIAH, S.S. Assessment of nutritional values, phytochemical content, and antioxidant properties of Shallot (Allium ascalonicum L.) leaf and bulb. Measurement: Food, v.10, p.100091, 2023. https://doi.org/10.1016/j.meafoo.2023.100091.
  » https://doi.org/https://doi.org/10.1016/j.meafoo.2023.100091
• AMRI, B.; KHAMASSI, K.; ALI, M.B.; TEIXEIRA DA SILVA, J.A.; BETTAIEB BEN KAAB, L. Effects of gibberellic acid on the process of organic reserve mobilization in barley grains germinated in the presence of cadmium and molybdenum. South African Journal of Botany, v.106, p.35-40, 2016. https://doi.org/10.1016/j.sajb.2016.05.007.
  » https://doi.org/https://doi.org/10.1016/j.sajb.2016.05.007
• BAILLY, C. The signalling role of ROS in the regulation of seed germination and dormancy. Biochemical Journal, v.476, p.3019-3032, 2019. https://doi.org/10.1042/BCJ20190159.
  » https://doi.org/https://doi.org/10.1042/BCJ20190159
• BAILLY, C. ROS in seed germination. In: Advances in Botanical Research [s.l.] Elsevier, v.105, p.177-204, 2023. https://doi.org/10.1016/bs.abr.2022.10.003.
  » https://doi.org/https://doi.org/10.1016/bs.abr.2022.10.003
• BERNFELD, P. [17] Amylases, α and β. In: Methods in enzymology I. [s.l: s.n.]. v.1, p.149-158, 1955. https://doi.org/10.1016/0076-6879(55)01021-5.
  » https://doi.org/https://doi.org/10.1016/0076-6879(55)01021-5
• BEYER, W.F.; FRIDOVICH, I. Assaying for superoxide dismutase activity: Some large consequences of minor changes in conditions. Analytical Biochemistry, v.161, p.559-566, 1987. https://doi.org/10.1016/0003-2697(87)90489-1.
  » https://doi.org/https://doi.org/10.1016/0003-2697(87)90489-1
• BOULOS, M.I.; FAUCHAIS, P.L.; PFENDER, E. The plasma state In: BOULOS, M.I.; FAUCHAIS, P.L.; PFENDER, E. (Ed.). Handbook of Thermal Plasmas Cham: Springer International Publishing, p.1-53, 2016. https://doi.org/10.1007/978-3-319-12183-3_1-2.
  » https://doi.org/https://doi.org/10.1007/978-3-319-12183-3_1-2
• COPELAND, L.O.; MCDONALD, M.B. Principles of seed science and technology Boston: Springer US, 2001. https://doi.org/10.1007/978-1-4615-1619-4.
  » https://doi.org/https://doi.org/10.1007/978-1-4615-1619-4
• CORBINEAU, F.; TASKIRAN-ÖZBINGÖL, N.; EL-MAAROUF-BOUTEAU, H. Improvement of seed quality by priming: concept and biological basis. Seeds, v.2, p.101-115, 2023. https://doi.org/10.3390/seeds2010008.
  » https://doi.org/https://doi.org/10.3390/seeds2010008
• DIAS, D.C.F.D.S.; BHERING, M.C.; TOKUHISA, D.; HILST, P.C. Teste de condutividade elétrica para avaliação do vigor de sementes de cebola. Revista Brasileira de Sementes, v.28, p.154-162, 2006. https://doi.org/10.1590/S0101-31222006000100022.
  » https://doi.org/https://doi.org/10.1590/S0101-31222006000100022
• ELLOUZI, H.; OUESLATI, S.; HESSINI, K.; RABHI, M.; ABDELLY, C. Seed-priming with H2O2 alleviates subsequent salt stress by preventing ROS production and amplifying antioxidant defense in cauliflower seeds and seedlings. Scientia Horticulturae, v.288, p.110360, 2021. https://doi.org/10.1016/j.scienta.2021.110360.
  » https://doi.org/https://doi.org/10.1016/j.scienta.2021.110360
• FABRISSIN, I.; SANO, N.; SEO, M.; NORTH, H.M. Ageing beautifully: can the benefits of seed priming be separated from a reduced lifespan trade-off? Journal of Experimental Botany, v.72, p.2312-2333, 2021. https://doi.org/10.1093/jxb/erab004.
  » https://doi.org/https://doi.org/10.1093/jxb/erab004
• GURAGAIN, R.P.; PRADHAN, S.P.; BANIYA, H.B.; PANDEY, B.P.; BASNET, N.; SEDHAI, B.; DHUNGANA, S.; CHHETRI, G.K.; JOSHI, U.M.; SUBEDI, D.P. Impact of plasma-activated water (PAW) on seed germination of soybean. Journal of Chemistry, v.2021, p.1-12, 2021. https://doi.org/10.1155/2021/7517052.
  » https://doi.org/https://doi.org/10.1155/2021/7517052
• HANAM, E.S.; SOFIA, D.R.; AZHARY, S.Y.; PANATARANI, C.; JONI, I.M. Effect of gas sources on the oxygen transfer efficiency produced by fine bubbles generator. Journal of Physics: Conference Series, v.2376, p.012004, 2022. https://doi.org/10.1088/1742-6596/2376/1/012004.
  » https://doi.org/https://doi.org/10.1088/1742-6596/2376/1/012004
• HORNKE, N.F.; GADOTTI, G.I.; CAPILHEIRA, A.F.; CAVALCANTE, J.A.; NADAL, A.P.; SILVA, J.G. DA. Physiological potential of onion seeds stored in different packings and environments. Horticultura Brasileira, v.38, p.312-318, 2020. https://doi.org/10.1590/s0102-053620200312.
  » https://doi.org/https://doi.org/10.1590/s0102-053620200312
• ISTA. International Seed Testing Association. International Rules for Seed Testing Edition 2018. ISTA, Bassersdorf, Switzerland, 2018, 298p.
• KAMANGA, B.M.; PALUPI, E.R.; WIDAJATI, E.; ILYAS, S. Development of seed vigour testing method using single count of radicle emergence for true seed of shallot (Allium ascalonicum B.). International Journal of Food Science and Agriculture, v.5, p.152-162, 2021. https://doi.org/10.26855/ijfsa.2021.03.019.
  » https://doi.org/https://doi.org/10.26855/ijfsa.2021.03.019
• LI, W.; NIU, Y.; ZHENG, Y.; WANG, Z. Advances in the understanding of reactive oxygen species-dependent regulation on seed dormancy, germination, and deterioration in crops. Frontiers in Plant Science, v.13, p.1-9, 2022. https://doi.org/10.3389/fpls.2022.826809.
  » https://doi.org/https://doi.org/10.3389/fpls.2022.826809
• LIU, S.; OSHITA, S.; MAKINO, Y.; WANG, Q.; KAWAGOE, Y.; UCHIDA, T. Oxidative capacity of nanobubbles and its effect on seed germination. ACS Sustainable Chemistry and Engineering, v.4, p.1347-1353, 2016. https://doi.org/10.1021/acssuschemeng.5b01368.
  » https://doi.org/https://doi.org/10.1021/acssuschemeng.5b01368
• LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L.; RANDALL, R.J. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, v.193, p.265-275, 1951. https://doi.org/10.1016/S0021-9258(19)52451-6.
  » https://doi.org/https://doi.org/10.1016/S0021-9258(19)52451-6
• MIZOI, K.; RODRÍGUEZ-GONZÁLEZ, V.; SASAKI, M.; SUZUKI, S.; HONDA, K.; ISHIDA, N.; SUZUKI, N.; KUCHITSU, K.; KONDO, T.; YUASA, M.; FUJISHIMA, A.; TESHIMA, K.; TERASHIMA, C. Interactions between pH, reactive species, and cells in plasma-activated water can remove algae. RSC Advances, v.12, p.7626-7634, 2022. https://doi.org/10.1039/D1RA07774K.
  » https://doi.org/https://doi.org/10.1039/D1RA07774K
• MURULI, C.N.; BHANUPRAKASH, K.; CHANNAKESHAVA, B.C. Impact of seed priming on vigour in onion (Allium cepa L.) seeds. Journal of Applied Horticulture, v.18, p.68-70, 2016. https://doi.org/10.37855/jah.2016.v18i01.15.
  » https://doi.org/https://doi.org/10.37855/jah.2016.v18i01.15
• PAGANO, A.; MACOVEI, A.; BALESTRAZZI, A. Molecular dynamics of seed priming at the crossroads between basic and applied research. Plant Cell Reports, v.42, p.657-688, 2023. https://doi.org/10.1007/s00299-023-02988-w.
  » https://doi.org/https://doi.org/10.1007/s00299-023-02988-w
• PANDISELVAM, R.; MAYOOKHA, V.P.; KOTHAKOTA, A.; SHARMILA, L.; RAMESH, S.V.; BHARATHI, C.P.; GOMATHY, K.; SRIKANTH, V. Impact of ozone treatment on seed germination - a systematic review. Ozone: Science & Engineering, v.42, p.331-346, 2020. https://doi.org/10.1080/01919512.2019.1673697.
  » https://doi.org/https://doi.org/10.1080/01919512.2019.1673697
• PAPARELLA, S.; ARAÚJO, S.S.; ROSSI, G.; WIJAYASINGHE, M.; CARBONERA, D.; BALESTRAZZI, A. Seed priming: state of the art and new perspectives. Plant Cell Reports , v.34, p.1281-1293, 2015. https://doi.org/10.1007/s00299-015-1784-y.
  » https://doi.org/https://doi.org/10.1007/s00299-015-1784-y
• PEREIRA, A.A.S.; NERY, F.C.; FERREIRA, R.A.; SILVA, V.N.D.; BERNARDES, M.M.; SANTOS, H.O.D.; BICALHO, E.M. Can priming with ascorbic acid or nitric oxide improve the germinability of stored sunflower seeds? Journal of Seed Science, v.44, p.e202244012, 2022. https://doi.org/10.1590/2317-1545v44256600.
  » https://doi.org/https://doi.org/10.1590/2317-1545v44256600
• PRIATAMA, R.A.; PERVITASARI, A.N.; PARK, S.; PARK, S.J.; LEE, Y.K. Current advancements in the molecular mechanism of plasma treatment for seed germination and plant growth. International Journal of Molecular Sciences, v.23, p.4609, 2022. https://doi.org/10.3390/ijms23094609.
  » https://doi.org/https://doi.org/10.3390/ijms23094609
• Indonesia. Kementerian Pertanian, Pusat Data dan Sistem Informasi Pertanian. Outlook bawang merah: komoditas pertanian subsektor hortikultura Jakarta: Pusat Data dan Sistem Informasi Pertanian Kementeria Pertanian, 2020.
• RASHID, M.; RASHID, M.M.; ALAM, M.S.; TALUKDER, M.R. Enhancement of growth, enzymes, nutrition and yield of eggplant: combined effects of plasma treatments. Plasma Chemistry and Plasma Processing, v.43, p.163-181, 2023. https://doi.org/10.1007/s11090-022-10301-y.
  » https://doi.org/https://doi.org/10.1007/s11090-022-10301-y
• RODRIGUES, V.O.; COSTA, F.R.; NERY, M.C.; CRUZ, S.M.; MELO, S.G.F. DE; CARVALHO, M.L.M. DE. Treating sunflower seeds subjected to ozonization. Journal of Seed Science , v.37, p.202-210, 2015. https://doi.org/10.1590/2317-1545v37n3148582.
  » https://doi.org/https://doi.org/10.1590/2317-1545v37n3148582
• ROSLIANI, R.; HIDAYAT, I.M.; SULASTRINI, I.; HILMAN, Y. Dissemination of technology for shallot (Allium ascalonicum L.) seed production using true shallot seed (TSS) in Indonesia. Acta Horticulturae, v.1143, p.345-352, 2016. https://doi.org/10.17660/ActaHortic.2016.1143.49.
  » https://doi.org/https://doi.org/10.17660/ActaHortic.2016.1143.49
• SAJIB, S.A.; BILLAH, M.; MAHMUD, S.; MIAH, M.; HOSSAIN, F.; OMAR, F.B.; ROY, N.C.; HOQUE, K.M.F.; TALUKDER, M.R.; KABIR, A.H.; REZA, M.A. Plasma activated water: the next generation eco-friendly stimulant for enhancing plant seed germination, vigor and increased enzyme activity, a study on black gram (Vigna mungo L.). Plasma Chemistry and Plasma Processing , v.40, p.119-143, 2020. https://doi.org/10.1007/s11090-019-10028-3.
  » https://doi.org/https://doi.org/10.1007/s11090-019-10028-3
• SAYAKA, B.; PASARIBU, S.M.; DERMOREDJO, S.K. Prospect for farmers’ adoption of true shallot seed. Forum penelitian Agro Ekonomi, v.38, p.53, 2020.
• SAYAKA, B.; SWASTIKA, D.K.S.; SUNARSIH. Assessing the national seed production system of potato, shallot, and garlic. IOP Conference Series: Earth and Environmental Science, v.892, p.012097, 2021. https://doi.org/10.1088/1755-1315/892/1/012097.
  » https://doi.org/https://doi.org/10.1088/1755-1315/892/1/012097
• SHARAF-ELDIN, M.A.; ALSHALLASH, K.S.; ALHARBI, K.R.; ALQAHTANI, M.M.; ETMAN, A.A.; YASSIN, A.M.; AZAB, E.S.; EL-OKKIAH, S.A.F. Influence of seed soaking and foliar application using ozonated water on two sweet pepper hybrids under cold stress. Sustainability, v.14, p.13453, 2022. https://doi.org/10.3390/su142013453.
  » https://doi.org/https://doi.org/10.3390/su142013453
• STOLERU, V.; BURLICA, R.; MIHALACHE, G.; DIRLAU, D.; PADUREANU, S.; TELIBAN, G.-C.; ASTANEI, D.; COJOCARU, A.; BENIUGA, O.; PATRAS, A. Plant growth promotion effect of plasma activated water on Lactuca sativa L. cultivated in two different volumes of substrate. Scientific Reports, v.10, p.20920, 2020. https://doi.org/10.1038/s41598-020-77355-w.
  » https://doi.org/https://doi.org/10.1038/s41598-020-77355-w
• SUDHAKAR, N.; NAGENDRA-PRASAD, D.; MOHAN, N.; HILL, B.; GUNASEKARAN, M.; MURUGESAN, K. Assessing influence of ozone in tomato seed dormancy alleviation. American Journal of Plant Sciences, v.02, p.443-448, 2011. https://doi.org/10.4236/ajps.2011.23051.
  » https://doi.org/https://doi.org/10.4236/ajps.2011.23051
• TANAKA, S.; NARUSE, Y.; TERASAKA, K.; FUJIOKA, S. Concentration and dilution of ultrafine bubbles in water. Colloids and Interfaces, v.4, p.50-65, 2020. https://doi.org/10.3390/colloids4040050.
  » https://doi.org/https://doi.org/10.3390/colloids4040050
• TEKILE, A.; KIM, I.; LEE, J.-Y. Extent and persistence of dissolved oxygen enhancement using nanobubbles. Environmental Engineering Research, v.21, p.427-435, 2016. https://doi.org/10.4491/eer.2016.028.
  » https://doi.org/https://doi.org/10.4491/eer.2016.028
• THAN, H.A.Q.; PHAM, T.H.; NGUYEN, D.K.V.; PHAM, T.H.; KHACEF, A. Non-thermal plasma activated water for increasing germination and plant growth of Lactuca sativa L. Plasma Chemistry and Plasma Processing , v.42, p.73-89, 2022. https://doi.org/10.1007/s11090-021-10210-6.
  » https://doi.org/https://doi.org/10.1007/s11090-021-10210-6
• VAN DEN BRINK, L.; BASUKI, R.S. Production of true seed shallots in Indonesia. Acta Horticulturae , v.958, p.115-120, 2012. https://doi.org/10.17660/ActaHortic.2012.958.12.
  » https://doi.org/https://doi.org/10.17660/ActaHortic.2012.958.12
• VICHIANSAN, N.; CHATMANIWAT, K.; SUNGKORN, M.; LEKSAKUL, K.; CHAOPAISARN, P.; BOONYAWAN, D. Effect of plasma-activated water generated using plasma jet on tomato (Solanum lycopersicum L. var. cerasiforme) seedling growth. Journal of Plant Growth Regulation, v.42, p.935-945, 2023. https://doi.org/10.1007/s00344-022-10603-7.
  » https://doi.org/https://doi.org/10.1007/s00344-022-10603-7
• VIOLLEAU, F.; HADJEBA, K.; ALBET, J.; CAZALIS, R.; SUREL, O. Effect of oxidative treatment on corn seed germination kinetics. Ozone: Science & Engineering , v.30, p.418-422, 2008. https://doi.org/10.1080/01919510802474631.
  » https://doi.org/https://doi.org/10.1080/01919510802474631
• WONG, K.S.; CHEW, N.S.L.; LOW, M.; TAN, M.K. Plasma-activated water: physicochemical properties, generation techniques, and applications. Processes, v.11, p.2213, 2023. https://doi.org/10.3390/pr11072213.
  » https://doi.org/https://doi.org/10.3390/pr11072213
• YASUI, K.; TUZIUTI, T.; KANEMATSU, W. High temperature and pressure inside a dissolving oxygen nanobubble. Ultrasonics Sonochemistry, v.55, p.308-312, 2019. https://doi.org/10.1016/j.ultsonch.2019.01.013.
  » https://doi.org/https://doi.org/10.1016/j.ultsonch.2019.01.013
• ZHANG, K.; ZHANG, Y.; SUN, J.; MENG, J.; TAO, J. Deterioration of orthodox seeds during ageing: Influencing factors, physiological alterations and the role of reactive oxygen species. Plant Physiology and Biochemistry, v.158, p.475-485, 2021. https://doi.org/10.1016/j.plaphy.2020.11.031.
  » https://doi.org/https://doi.org/10.1016/j.plaphy.2020.11.031

Publication Dates

• Publication in this collection
  15 Nov 2024
• Date of issue
  2024

History

• Received
  11 May 2024
• Accepted
  29 Sept 2024