Radio-frequency (RF) room temperature plasma treatment of sweet basil seeds (Ocimum basilicum L.) for germination potential enhancement by immaculation

Graphical abstract

Abstract

Ocimum basilicum L. is an antiviral and immunity boosting medicinal plant and culinary herb. Potential use of sweet basils in COVID 19 prevention and management is making its demand rise. This study is aimed at germination potential enhancement of sweet basil seeds. Reported study is evidenced with scientific data of radio-frequency cold plasma treatment using Ar + O₂ feed gas. O. basilicum seeds, placed inside the rotating glass bottle, were directly exposed to RF (13.56 MHz) plasma produced in Ar + O₂ feed gas. Seed treatment was done using RF source power (60 W, 150 W, 240 W), process pressure (0.2 mbar, 0.4 mbar, 0.6 mbar), and treatment time (5 min, 10 min, 15 min) at different combinations. Results show that, the most efficient treatment provide up to ∼89 % of the germination percentage which is an enhancement by 32.3 % from the control. SEM images revealed slight shrinkage in the seed size with eroded appearance over the seed. Enhancement of lipid peroxidation, show that oxidation of seed coat may propagate internally. Water imbibition analysis, of the treated seeds, was carried out for 2−12 hours. Further analysis of seed weight, on every one hour, after soaking shows enhanced water absorption capability except the treatment at 240 W, 0.6 mbar and 15 min. Plasma treatment enhanced carbohydrate content and protein content which is reported to be due to increased primary metabolism. Whereas, increased activity of secondary metabolism results in the enhancement of enzymatic (catalase) and non-enzymatic antioxidants (proline). Vital growth parameters, such as SVI I and SVI II, got amplified by 37 % and 133 % respectively after treatment. Ameliorative effects of plasma treatment are found highly significant with a positive and significant correlation value (p < 0.01) between germination percentages, SVI I, SVI II, carbohydrate, protein and proline show their interrelationship. Ar + O₂ plasma treatment is found to bring forth significant changes in the O. basilicum seeds which eventually enhanced the germination potential and it could be a very promising technology for the medicinal crop.

1. Introduction

Plant seed is a fundamental component for crop yield enhancement. Technology innovations require the development of a sustainable seed invigoration technique (Charoux et al., 2021) to raise the seed quality and repair the detrimental effects of stress factors (Randeniya and de Groot, 2015). An improved seed is the carrier of technological innovations and serves as an engine for enhanced agricultural productivity and bridging the yield gap. Seed germination characteristics and vigor can be enhanced by various chemicals, fungicides, hormones (Zhang et al., 2011), antibiotics, and biological treatment methods (Reznikov et al., 2016). Bio-stimulant, microbes, plant extracts, and algae extracts (Goussous et al., 2010; Kagale et al., 2004), etc act against a very narrow spectrum of pathogens making biological treatment method limited purpose use. Chemical treatment is expensive due to the involvement of manual workforce cost and heavy use of chemicals even worse, chemicals residue left over the seed surface contribute to soil pollution and disturb the ecology and environment (Martínez-Ballesta et al., 2020).

Treatment using physical agents such as ultrasonic waves (Liu et al., 2016), electromagnetic field (Mildaziene et al., 2018), magnetic fields (Yao et al., 2005), alternating magnetic field (Aguilar et al., 2009), gamma radiation (Al-Bachir, 2007), laser radiation (Iqbal et al., 2019), high-pressure treatment (Blaszczak et al., 2007), etc. can induce an adaptive plant defense response so as to enhance the seed germination and seedling’s vigor. However, these physical treatment methods suffer from certain limitations such as hormetic dose, seed genotype, exposition time, non-uniformity, and environmental conditions for seed irradiation (Araújo et al., 2016; Aguilar et al., 2009). Strong ultrasonic oscillations in ultrasonic treatment may injure the seed cells whereas ion beam scratching might damage the seed (Xu et al., 2012).

Non-thermal plasma or low-temperature plasma (LTP), operating at a temperature below 40 °C, is a modern age technology. LTP sources, produce physical agents such as excited atoms, molecules, UV radiation, RONS (Reactive oxygen and nitrogen species), gaseous ions, radicals, and free electrons (de Groot et al., 2018). LTP method involves dry chemistry and is thus environment friendly as it does not produce contaminated aqueous waste or toxic gas effluent. These produced active species, having a limited capability of only penetrating about 10 nm depth structure, can be used for surface functionalization of even the most inert surface without changing the bulk property of seed (Denes et al., 1999). Germination enhancement and seedling growth has been reported for different seeds such as Zea mays L. (Filatova et al., 2020), Paulownia tomentosa (Puač et al., 2018), Glycine max (Pérez Pizá et al., 2018), cotton seeds (Wang et al., 2017), Cuminum cyminum (Shashikanthalu et al., 2020), O. basilicum (Ambrico et al., 2017) etc. using atmospheric air or other gases. Synergistic effects of physical agents, produced by LTP, sources provide good fungicidal & bactericidal effects (Pérez Pizá et al., 2018; Zahoranová et al., 2016; Adhikari et al., 2020a; Saeedeh and Brodie, 2020; Gaunt et al., 2006), increased water permeability (Bormashenko et al., 2015) through surface poration (Volkov et al., 2019) which results in subsequent stimulation of the seedling growth.

Production of plasma species, RONS and photons are dependent on various factors some of which are type of plasma source, treatment conditions, treatment time, electrical parameters, interaction type and the gas mixture. The plasma source type is broadly categorized as low-pressure plasma and atmospheric pressure plasma. Dielectric barrier discharge plasma (Pérez-Pizá et al., 2020), atmospheric pressure plasma jet (Alves et al., 2016; Silva, 2018; Volkov et al., 2019), are some of the variants of atmospheric pressure plasma source. Whereas, low pressure plasma sources are subcategorized as inductively coupled low-pressure cold plasma (Selcuk et al., 2008; Kitazaki et al., 2012; Holc et al., 2019) and capacitive coupled low-pressure cold plasma (Volin et al., 2000; Dhayal et al., 2006). Low pressure plasma has distinct advantages of rich process chemistry, less gas consumption, plasma uniformity etc. Whereas, atmospheric pressure plasma requires ventilation of working area, limited process chemistry, large gas consumption. There are other plasma generation techniques (Attri et al., 2020) which are reportedly being used for seed germination enhancement and seedling growth such as GlidArc plasma (Pawłat et al., 2018), corona discharge (Lynikiene et al., 2006), microwave plasma (Šerá et al., 2008) and glow discharge air plasma (Dubinov et al., 2000). Summary from the references of this paper for enhancement of germination, disinfection and other vital parameters by using various bio-chemical treatment methods are shown in the Table 1 .

Table 1.

Physical and bio-chemical treatments enhanced germination, disinfection and other vital parameters which are reported in this paper.

Citation	Seeds type	Treatment method	Findings
Adhikari et al., 2020a	Tomato (Solanum lycopersicum)	atm pressure; air plasma jet; water	Seedling growth, defense hormone, modulate plant growth, RONS
Adhikari et al., 2020b	Tomato (Solanum lycopersicum)	atm pressure; air plasma jet; water	Disinfection, plasma vaccination
Aguilar et al., 2009	Maize (Zea mays)	Alternating magnetic field (20, 60, 100 m T); 60 Hz, 7.5, 15, 30 min	21 days after planting; Seedling growth, biostimulation
Al-Bachir, 2007	Aniseed (Pimpinella anisum)	Gamma irradiation ;05,101,520 kGy	Microbial decontamination, color, flavour, test after 12 months storage
Alves et al., 2016	Mulungu (Erythrina velutina)	DBD, atm pressure, plasma jet, He	Hydrophilicity, growth, seed coat modification
Ambrico et al., 2017	Sweet basil (Ocimum basilicum L.)	Surface DBD, air	RONS, decontamination, germination, seedling growth parameters, vigor
Ambrico et al., 2019	Sweet basil (Ocimum basilicum L.)	Surface DBD, air	Micronutrient redistribution, seed coat modification, germination, growth
Araújo et al., 2016	Review	Physical methods, Magneto-priming, Ionizing radiation, UV etc.	Electron paramagnetic resonance, seed invigoration, hermetic dose
Attri et al., 2020	Review	Nonthermal plasma treatment, plasma activated water	Germination, growth, biochemical analysis, post-harvest, lab to industry
Bafoil et al., 2019	Arabidopsis thaliana	DBD, air, atm pressure	Stress defense, seed coat modification, growth, molecular changes
Blaszczak et al., 2007	Cowpea seeds	high-pressure treatment (300−500 MPa) for 15 min	Soluble protein content, not much changes in starch content
Bormashenko et al., 2012	Lentils, bean, wheat	RF plasma, air	Apparent contact angle, germination, yield, oxidation of surface
Bormashenko et al., 2015	Beans	RF, air, vacuum	Water absorption, speed of germination, role of micropyle
Charoux et al., 2021	Review	Different plasma technologies	Food, disinfection, seed performance, germination yield
Cui et al., 2019	Arabidopsis thaliana	Surface DBD, air, 0–10 min, atm pressure	Physio-chemical, germination, seedling growth, surface modification, antioxidant
de Groot et al., 2018	Cotton	Atm pressure, air, DBD, 27 min	Water absorption, germination,hydrophilicity
Dhayal et al., 2006	Carthamus tinctorium L.	Low pressure, RF, Ar	Germination rate, germination time, surface changes
Dubinov et al., 2000	Oat, barley	Air, low pressure, pulsed, continuous, glow discharge	Germination, sprout length,
Filatova et al., 2020	Maize, wheat, lupine	CCP, low pressure, air, 200 Pa, RF	Disinfection, fungi, Fusarium culmorum, growth, disease resistance, metabolism
Gómez-Ramírez et al., 2017	Quinoa	Air plasma (DBD and RF), low and atm pressure	Germination, water uptake reason, micro-nutrient such as potassium, nitrate, surface modification, etching, functionalization
Goussous et al., 2010	Pathogens (Alternaria solani)	Medicinal plant (Syrian marjoram, rosemary, roselle) extracts	Inhibition of mycelia growth
Hayashi et al., 2015	Brassicaceae	Atm pressure, oxygen, air	Germination, growth, antioxidant activity
Holc et al., 2019	Peeled garlic cloves	Low pressure, ICP, RF, oxygen	Root growth, not shoot growth, field performance: plant height, bulb mass
Homa et al., 2021	Fusarium oxysporum f.sp.basilici in sweet basil	Cold plasma jet, cold plasma jet seedlings, DBD plasma	Direct cold plasma on seedlings, DBD plasma, disinfection
Iqbal et al., 2019	Triticum spp.	Laser, low pressure Ar plasma	Germination, fungus resistance; laser : abolishes fungi and enhance water uptake
Kitazaki et al., 2012	Raphanus sativus	RF, low pressure, O2	Oxygen radicals instead ions and photons found effective, growth
Liu et al., 2016	Grass seeds	Ultrasonication	Germination percentage, growth of aged seeds
Lynikiene et al., 2006	Carrot, radish, beet, beetroot, barley	Corona discharge field of continuous current	Increased germination dynamics, viability
Martínez-Ballesta et al., 2020	Review	Osmopriming	Ion homeostasis into seed tissue, seed vigor, radical emergence, yield, abiotic stress effect
Meng et al., 2017	Wheat	DBD plasma using air, Ar, N2, O2	Seed coat modification, germination, early growth, metabolism
Mildaziene et al., 2018	Purple coneflower (Echinacea purpurea)	Low pressure, CCP, RF, air	Secondary metabolites in leaves, height, more leaves, faster germination,
Pawłat et al., 2018	Lavatera thuringiaca L.	DBD plasma jet, atm pressure, N2	Germination, upper cuticle removal, seed coat modification, growth
Pérez Pizá et al., 2018	Soybean seed, Diaporthe/Phomopsis	DBD, atm pressure	Disinfection, reverse oxidative damage by fungi
Pérez-Pizá et al., 2020	Soybean seed	DBD, air, imposed O2/N2	Nitrogenise activity, lehaemoglobin activity in nodules, N2 content in nodules, nodulation
Paužaitė et al., 2017	Norway spruce	Low pressure, CCP, RF, air	Growth, height, branching
Prasad et al., 2018	Artemisia annua L	KCl stress	Decrease in germination percentage, seedling vigor index
Rahman et al., 2018	Wheat	Low pressure DBD, Ar/air, Ar/O2	Ar/O2:Seed H2O2 concentration, biomass, shoot, growth mechanism
Reznikov et al., 2015	Charcoal rot of soybean	Biological product (Trichoderma vitride or bacillus subtilis), chemical treatment (thiophanate methyl + strobin)	Highest yield in chemical treated plot
Roy et al., 2018	Wheat	Medium pressure glow discharge, air, air + O2	Germination, growth, time to growth
Sarinont et al., 2016	Raphanus sativus L.	DBD, Air, O2, NO (10%)+N2, N2, He, Ar	OH and O radicals are key species for growth enhancement
Šerá et al., 2010	Wheat, Oat	After glow plasma, microwave generated, low pressure	Seed coat erosion, metabolism, modified germination
Sadhu et al., 2017	mung beans (Vigna radiata)	Low pressure, air, CCP	Germination rate, radical root length, conductivity, hydrophilic, enzymatic activity
Saeedeh and Brodie, 2020	Botrytis Grey Mould on lentil seeds	Afterglow Ar/Air, atm pressure, microwave	Disinfection, disease management
Selcuk et al., 2008	Grains, legumes	Low pressure, RF, SF6	Complete elimination (Aspergillus spp., Penicillum spp.), germination preserved
Shashikanthalu et al., 2020	Cuminum cyminum L.	DBD, air, atm pressure	Medicinal seed, germination,growth, root length, shoot length, total chlorophyl
Silva, 2018	Hybanthus calceolaria (L.)	He plasma jet, DBD, atm pressure	germination
Singh et al., 2019	Sweet basil (O. basilicum L.)	Low pressure, RF, Ar + O2	Germination, vigor index, metabolism
Stolárik et al., 2015	Pea (Pisum sativum L.)	DCSBD, air, atm pressure	Surface modification, permeability, germination, seedling vigor, water uptake
Sohan et al., 2021	Wheat (Triticum aestivum)	Low frequency glow discharge, medium pressure, Ar + O2	Germination, growth, nutritional traits
Tomeková et al., 2020	Pisum sativum	Diffuse coplanar surface barrier, air, N2 & O2, atm pressure	DNA damage, ameliorative, stress resistance
Volin et al., 2000	Maize (Zea mays), Soybean (Glycine max L.	Low pressure, RF (13.56 MHz), aniline, hydrazine, CF4, ODFD, cyclohexane, rotary drum	Germination modification, speed up/delay germination
Volkov et al., 2019	Pumpkin	Atm pressure, plasma jet, He, Ar	Germination, seedling growth, poration, water uptake, corrugation
Wang et al., 2017	Cotton	DBD, atm pressure, ambient air or nitrogen	Surface etching, oxidising, water permeation, FTIR, exhaust and emission spectra
Yao et al., 2005	cucumber (Cucumis sativus) seedlings	Magnetic field (0, 0.2, 0.45 T)	Sensitivity to UV-B radiation
Zahoranová et al., 2016	Wheat, seeds, seedling	Diffuse coplanar surface barrier discharge, air	Inactivation, F. nivale, F. culmorum, T. roseum, A. flavus, A. clavatus, germination, wettability, vigor, dry weight
Zahoranová et al., 2018	Maize	DCSBD, air, atm pressure	Filamentous fungi disinfection, A. flavus, F. culmorum, wettability, growth

Feed gas is an important input for the generation of plasma species and reactive particles which eventually modifies the seed property. Gases affect the seed biochemistry, molecular events and physical characteristics in different ways due to RONS and the reactive species interaction of a specific gas with a particular seed for a chosen plasma source type, in Ar + O₂ gas mixture, has been found useful for germination enhancement (Singh et al., 2019)

O. basilicum is an essential oil-bearing medicinal plant and culinary herb from the family Lamiaceae. It contains Linalool, Methyleugenol, Phenolic acid– Rosemarinic acid, Methyl cinnamate; luteolin apigenin, quercetin, kaempferol, isoeugenol, flavanoids, Calcium, Phosphorous, Vitamin A, C, Beta – carotene as bioactive chemical constituents. It improves taste perception and is used to pacify vata, kapha. Therapeutically it is getting used to work for lung complaints, cold & influenza related fever), nausea, poor digestion, insomnia, intestinal parasites, migraine etc. Potential application of sweet basil seeds in COVID 19 prevention and management is making its demand rise. Ambrico et al. (2019) have demonstrated the capability of air plasma treatment and also its efficacy in decontamination (Homa et al., 2021) is proved. This work is aimed at further improvement in the previously reported results with a more detailed study.

This work is aimed at germination potential enhancement of sweet basil seeds using radio-frequency cold plasma treatment in Ar + O₂ feed gas. Vital changes in physical, chemical and biological parameters were analyzed and their correlation coefficient was computed statistically. Various combinations of RF source power (60 W, 150 W, 240 W), process pressure (0.2 mbar, 0.4 mbar, 0.6 mbar), and treatment time (5 min, 10 min, 15 min) were applied for the seed treatment.

2. Material and methods

2.1. Experimental apparatus

Room temperature plasma exposure of seeds was performed in, M/s Diener electronic GmbH Germany (Model: PICO) make, commercial equipment, with a radio frequency (13.56 MHz, 300 W) powered electrode centrally positioned along the axis. Seeds were placed inside the rotating glass bottle containing the powered electrode (in the centre) with a gas inlet at the end of electrode (Singh et al., 2019). During all treatment flow of the gas was controlled by mass flow controllers (MFC) connected to the gas inlet of the chamber while the exhaust line was connected to the rotary vane pump (model D16BCS) of Oerlikon. Seeds (approximately 500 in numbers) were directly exposed and got uniform treatment due to tumbling inside the glass bottle. The process gases Ar & O₂ are fed through the powered electrode via an inlet cap.

2.2. Treatment conditions

Sweet basil seeds were directly exposed with the Ar + O₂ plasma with a large set of process conditions obtained from the various combinations of process parameters: such as RF power at W1 (60 W), W2 (150 W) and W3 (240 W); Process pressure at P1 (0.2 mbar), P1 (0.4 mbar) and P3 (0.6 mbar); and Treatment time at T1 (5 min), T2 (10 min) and T3 (15 min). A combination of high purity Oxygen & Argon gases, in the ratio of 9:1, was fed through MFC. RF power was applied after the stabilization of process pressure at a set value for the fixed process time. The temperature inside the chamber during exposure time was predicted by the farnell make thermocouple and it was at approximately 27−28 °C.

2.3. Experimental design

The seed collection, experimental trial, characterization and analysis were done in the year 2019−2020. Sampling was done in random way with four replications so as to eliminate any biasing factor. Seeds were arranged on top of filter paper (TP) having a diameter of 15 cm. The seeds were soaked with sterile distilled water placed inside the Petri dishes (16 cm diameter x3 cm deep) at temperatures of 25 °C at 16 h light (180 lx)/8 h dark daily regimes with 70–80 % relative humidity. A total of 100 seeds were positioned on each filter paper and initially it was hydrated with 10 mL distilled water. For maintaining sufficient moisture for germination during the experimental process, distilled water of 5 mL quantity on every other day was added to the Petri dishes. On the 6th day, observations on the shoot and root length, germination percentage, SVI I and SVI II were recorded (Kumar, 2012). The 6th day was previously established as a final count day for germination studies in O. basilicum (Kumar, 2012). Numbers of normal seedlings were used for data analysis.

2.4. Determination of germination percentage, seedling vigor index I and vigor index II

Germination percentage, SVI I, and SVI II were calculated according to Kumar (2012).

2.5. Determination of biochemical assays

One gram of seedlings from each treatment on 7th day of germination were crushed in an ice-cold mortar and pestle with liquid nitrogen, further add 3 mL of 50 mM Phosphate buffer saline at neutral pH to prepare extract. Now this extract was centrifuged for 10 min at 10,000×g and supernatant were kept at −20 °C until other biochemical parameters (carbohydrate, protein and catalase) were tested. The extraction for total phenolic content, proline and lipid peroxidation were done separately (Prasad et al., 2018).

2.5.1. Determination of carbohydrate content

An aliquot (0.1 mL) of the supernatant was diluted with 1 mL of 50 mM phosphate buffer saline and 1 mL of 5% phenol (aqueous w/v) then 5 mL of H₂SO₄ were added rapidly and mixed after that Incubated at 37 °C for 10 min. The colour development was read at 490 nm using a UV–vis spectrophotometer. The reagent without the sample served as a blank. The amount of carbohydrate was estimated using standard graph prepared with different concentration of d-glucose ranging from 0.1 to 1 mg/mL (Dubois et al., 1956).

2.5.2. Estimation of protein content

To an aliquot (50 μl) of the supernatant,1 mL of extraction buffer and 5 mL of Coomassie brilliant blue (CBB) G-250 was added and mixed thoroughly. The absorbance was read at 595 nm in a spectrophotometer against a reagent blank. The amount of protein was calculated using standard prepared with different concentrations of bovine serum albumin (BSA) ranging from 0.1 to 1 mg/mL according to Bradford (1976).

2.5.3. Determination of catalase activity

Catalase activity was determined by Chandlee and Scandalies method (1984) with slight modifications, the reaction mixture containing 0.2 mL of enzyme extract, 2.5 mL of NaPO₄ and 0.1 mL of 10 mM H₂O₂ was added to observe the catalase activity. The absorbance was recorded at 230 nm upto 75 s at every interval of 15 s. The enzyme activity was expressed in U/mg protein (U = 1 mM of H₂O₂ reduction per min per mg of protein).

2.6. Statistical analysis

All data are presented as the mean value of four replicates. At the end of the experiment, data were subjected to analysis of variance (ANOVA) and mean separation. The statistical analysis was done using GenStat® Release 15.1. For comparing the means of different test parameters, the least significant difference (LSD) at the 5% level was used.

3. Results

The present study revealed that variation in pressure (P), wattage (W), time (T), and their interactions [(P × W), (P × T), (W× T) and (P × W ×T)] were found to be highly significant for the germination percentage, SVI I, carbohydrate and protein content (Table 2 ). Content of proline was recorded highly significant for pressure (P), time (T) and interaction of (P×W). Due to the highly significant (P × W), (P × T), (W× T) and (P × W ×T) interactions, it is imperative to identify the effect of different doses of plasma treatment (Table 2). A significant change over the control was observed for all of the studied physical and biochemical parameters for all sets of applied treatment throughout the experiment except the content of carbohydrate at T1 & T2 of applied pressure P1 & P3 and proline at P1W1T1 along with activity of catalase at P1W3T3, P2W2T2, P3W1T3, P3W3T2 & P3W3T3 (Table 3 ).

Table 2.

Analysis of variance for radio frequency treatment over seed germination percentage (G), seedling vigor index I (SVI I) and seedling vigor index II (SVI II), carbohydrate, protein, catalase, lipid peroxidation (LPO) and proline.

Source of variation	DF	Germination (%)	Seedling vigor index I	Seedling vigor index II	Carbohydrate	Protein	Catalase	LPO	Proline
Replication	2	44.61	29.50	0.000	31.875 **	0.008	0.986	0.000	0.001
P	2	3565.39 **	115955.90 **	0.998	792.190 **	7.483 **	0.507	0.005	12.760 **
W	2	4076.39 **	112334.80 **	1.579	354.270 **	9.087 **	0.802	0.008	0.170
P X W	4	1573.49 **	68102.88 **	0.480	193.220 **	8.975 **	0.855	0.007	2.970 **
T	3	451.76 **	10704.78 **	0.703	121.577 **	15.320 **	0.899	0.004	5.540 **
P X T	6	572.41 **	17260.38 **	0.192	92.912 **	1.064 **	0.897	0.001	1.540
W X T	6	778.81 **	20733.19 **	0.267	53.586 **	2.233 **	0.883	0.001	0.216
P X W X T	12	371.73 **	12896.35 **	0.090	29.202 **	1.916 **	0.971	0.002	0.431
Error	72	7.83	34.35	0	3.266	0.005	0.995	0	0.001
Total	107

(*p < 0.05, **p < 0.01).

Table 3.

Effect of different doses of pressure (P), RF power (Watt) and time (T) on mean seed germination percentage (G%), seedling vigor index I (SVI I) and seedling vigor index II (SVI II), carbohydrate, protein, catalase, lipid peroxidation (LPO) and proline of O. basilicum variety ‘CIM-Saumya’.

Treatment	Germination (%)	Seedling vigour index I	Seedling vigour index II	Carbohydrate	Protein	Catalase	LPO	Proline
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P1W1T1	77 fg	378.75 f	1.07 f	31.68 fg	0.448 pqr	0.004 hi	0.320 ghijk	0.518 n
P1W1T2	87 ab	447.80 c	1.186 c	31.65 fg	0.952 n	0.005 ef	0.325 fgh	0.753 m
P1W1T3	88.66 a	494.04 b	1.276 b	35.596 d	1.686 j	0.005 de	0.344 c	0.822 L
Control	67 i	360.17 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P1W2T1	75 fgh	317.04 L	0.376 o	37.343 d	1.886 i	0.006 cd	0.356 b	0.977 k
P1W2T2	45 j	206.056 m	0.485 m	38.023 cd	2.170 h	0.006 bc	0.339 cd	0.824 L
P1W2T3	42.666 j	170.946 n	0.486 m	32.423 ef	2.314 g	0.006 b	0.331 def	0.826 L
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P1W3T1	41.666 jk	169.38 n	0.267 q	24.32 ij	0.709 o	0.007 a	0.322 ghij	0.8313 L
P1W3T2	37.333 kl	123.956 p	0.397 n	20.47 k	0.466 pq	0.004 hi	0.326 efg	0.829 L
P1W3T3	26.666 m	117.346 p	0.287 p	19.256 k	0.190 s	0.003 klm	0.307 mno	0.717 m
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P2W1T1	77.666 ef	358.063 i	0.941 j	35.36 de	1.037 n	0.002 lmn	0.276 p	1.463 g
P2W1T2	81.666 cde	368.836 gh	1.358 a	36.073 d	1.238 m	0.003 ij	0.318 hijkl	1.502 g
P2W1T3	83.66 bc	378.686 f	1.029 g	36.94 d	1.577 k	0.003 i	0.334 de	1.637 f
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P2W2T1	84 bc	427.723 d	0.970 i	46.133 a	1.736 j	0.003 jk	0.3143 klm	1.935 d
P2W2T2	85 abc	431.016 d	1.107 e	43.26 ab	2.571 f	0.003 kl	0.32 ghijk	2.422 c
P2W2T3	88 ab	514.166 a	1.263 b	41.186 b	3.271 e	0.002 lmn	0.3246 fghi	2.742 b
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P2W3T1	87 ab	442.153 c	1.187 c	41.076 b	3.322 de	0.002 mn	0.315 jklm	2.987 a
P2W3T2	84.333 abc	427.427 d	1.006 h	41 b	3.428 cd	0.002 no	0.317 ijkl	2.979 a
P2W3T3	85 abc	406.853 e	0.963 i	40.71 bc	3.533 bc	0.001 p	0.275 p	2.977 a
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P3W1T1	82.333 cd	398.223 e	1.133 d	35.136 de	1.4013 L	0.001o	0.43 a	1.463 g
P3W1T2	81.666 cde	377.94 fg	1.067 f	37.066 d	1.66 jk	0.002 lmn	0.4253 a	1.680 f
P3W1T3	83.665 bc	397.883 e	1.267 b	31.5 fg	3.61 b	0.0032 kl	0.362 b	1.828 e
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P3W2T1	79 def	372.373 fg	1.094 e	31.223 fg	3.92 a	0.004 fg	0.319 ghijkl	1.251 h
P3W2T2	75.333 fgh	357.3 i	0.976 i	29.333 gh	3.557 b	0.005 f	0.316 jkl	1.187 i
P3W2T3	73 gh	338.64 j	0.942 j	26.9 hi	2.104 h	0.004 fg	0.313 klmn	1.166 i
Control	67 i	360.173 hi	0.542 L	29.34 gh	0.368 qr	0.003 kl	0.306 no	0.490 n
P3W3T1	71.66 h	328.59 k	0.813 k	25.933 i	0.543 p	0.004 gh	0.311 lmn	1.060 j
P3W3T2	33 L	143.034 o	0.233 r	21.4 jk	0.345 r	0.003 jk	0.307 mno	0.231 o
P3W3T3	23.666 m	120.023 p	0.162 s	20.323 k	0.088 s	0.003 kl	0.303 o	0.063 p
Gen. Mean	68.990	340.436	0.784	32.093	1.474	0.003	0.322	1.169
C.D. 5%	4.555	9.539	0.014	2.941	0.111	0.000	0.008	0.044

Germination Percentage – G%; Seedling vigor index I – SVI I; Seedling vigor index II – SVI II; P-Pressure, W-Watt, T-Time, P1, P2, P3 = 0.2,0.4,0.6 mbar; W1, W2, W3 = 60,150,240 W; T1, T2, T3 = 5, 10, 15 min. Means followed by the same letter are not significantly different at P < 0.05 by the Tukey test.

At applied pressure P1 (0.2 mbar) and power W1 (60 W), germination percentage, SVI I, SVI II, content of carbohydrate, protein, activity of catalase and lipid peroxidation were found to be increasing significantly over the control in a time dependent manner. Carbohydrate, protein, catalase, LPO and proline followed the same pattern of significance when compared to control, for applied pressure P1 and power W2 (150 W). However, germination percentage, SVI I and SVI II was found to be decreasing significantly over the control in time dependent manner for applied treatment P1W2 except for germination percentage at P1W2T1 (Table 3) (Fig. 1 a, b, c). Out of three studied physical parameters content, the carbohydrate content was also found to be decreasing in time dependent manner at P1W3. Proline and protein were also increasing significantly over the control except at P1W1T1 while at P1W3T3 the content of protein was recorded to be decreasing significantly. A significant increase in all the studied physical and biochemical parameters were recorded for all three applied wattages (W1, W2, W3) and time treatments (T1, T2, T3) at P2 (0.4 mbar) excepting the activity of catalase enzyme. Whereas, SVI I and LPO was found to be decreasing significantly at P2W1T1 and P2W3T3 respectively, over the control. At pressure P3 (0.6 mbar) with power W1 and W2, germination percentage, SVI II, protein, LPO and proline were found to be increasing significantly for all the three applied time treatments, over the control. At P3W1, SVI I and carbohydrate content were increasing significantly from control for T1, T2 and T3 (Table 3). However, at P3W2 SVI I got increased significantly up to P3W2T1 and got decreased further while carbohydrate content got increased non-significantly up to P3W2T1 and further got decreased, over to the control. At applied pressure P3 with W3, germination percentage, SVI II, protein, LPO and proline were found to be increasing significantly up to T1 and got decreased further for T2 and T3. Whereas, at all three applied time treatments of LPO and P3W3T2, P3W3T3 of catalase activity the change was non-significant, over the control. At P3W3, SVI I and carbohydrate content were found to be decreasing significantly from control in time dependent manner. The studied enzymatic parameter i.e., the activity of catalase throughout the treatment was increasing significantly over the control, except at P1W3T3, P2W2T2, P3W1T3, P3W3T2 and P3W3T3 where the activity was at par to the control and was non-significant. The enzymatic activity of catalase was found to be decreasing significantly at P2W1T1, P2W2T3 and P3W1T2, over the control (Table 3). Plasma exposure enhances germination percentage (GP) by approximately 32.8 % and other parameters such as SVI I by 43.8 %, SVI II by 57.44 %, carbohydrate by 40.26 %, and protein content by 879.67 %. Lipid peroxidation (LPO) has been found to increase by 40.0 % with significant growth in enzymatic anti-oxidant (Catalase) and non-enzymatic anti-oxidant (Proline) properties. SEM images (Fig. 2 ) shows modification of seed coat property which regulates the water imbibitions as shown in Table 4 .

Fig. 1.

a Germination percentage of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment. b Seedling vigor index I (SVI I) of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment. c Seedling vigor index II (SVI II) of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment. d Carbohydrate content of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment. e Protein content of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment. f Catalase activity of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment. g Lipid peroxidation activity of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment. h Proline content of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar signifies the standard deviation for each treatment.

Fig. 2.

Scanning electron microscopy (SEM) images of O. basilicum seeds. A and D are control sample while B,C,E,and F are the cold plasma treated seeds– P2W1T1 (B, E), P3W3T3 (C, F) P- Pressure, W- Watt, T- Time, P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min, Scale bars: 1 mm (A, B, C), 100 μm (D, E, F).

Table 4.

Water absorption capacity of radiofrequency cold plasma treatment in sweet basil seeds as a function of soaking time.

Treatment	Seed weight	Seed weight-2 h	Seed weight-3 h	Seed weight-4 h	Seed weight-5 h	Seed weight-6 h	Seed weight-7 h	Seed weight-8 h	Seed weight-9 h	Seed weight-10 h	Seed weight-11 h	Seed weight-12 h
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.655 mn	1.613 pq	1.438 r	1.387 q
P1W1T1	0.064 cdef	1.021 f	1.142 op	1.246 op	1.315 u	1.477 L	1.543 lm	1.724 j	1.868 gh	1.905 h	1.832 f	1.811 f
P1W1T2	0.068 bcd	1.203 ab	1.284 f	1.376 jk	1.469 no	1.521 jk	1.672 h	1.791 h	1.581 pq	1.572 r	1.527 p	1.521 o
P1W1T3	0.067 bcde	0.903 L	1.165 mno	1.213 q	1.357 t	1.464 lm	1.601 k	1.597 o	1.668 lm	1.627 p	1.761 hi	1.757 h
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.657 mn	1.613 pq	1.438 r	1.387 q
P1W2T1	0.062 ef	0.975 ghij	1.192 jkl	1.394 ij	1.472 no	1.581 i	1.625 j	1.635 m	1.761 I j	1.803 k	1.8 g	1.785 g
P1W2T2	0.063 def	0.895 L	1.476 c	1.636 e	1.889 d	1.964 cd	1.996 c	2.072 c	2.100 d	2.225 b	2.175 a	2.132 a
P1W2T3	0.065 cde	0.966 hijk	1.126 p	1.257 op	1.434 q	1.475 lm	1.587 k	1.620 mn	1.524 s	1.493 s	1.502 q	1.493 p
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.657 mn	1.613 pq	1.438 r	1.387 q
P1W3T1	0.059 f	0.964 ijk	1.185 klm	1.235 pq	1.314 u	1.446 m	1.522 n	1.571 p	1.557 r	1.692 n	1.714 lm	1.701 jk
P1W3T2	0.068 bcd	1.013 fg	1.472 c	1.775 bc	1.991 b	1.981 c	2.141 a	1.964 d	2.151 c	2.264 a	2.174 a	2.126 a
P1W3T3	0.074 a	1.016 fg	1.273 fg	1.478 f	1.556 ij	1.655 g	1.733 f	1.686 k	1.708 k	1.822 j	1.774 h	1.728 i
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.657 mn	1.613 pq	1.438 r	1.387 q
P2W1T1	0.074 abc	1.094 de	1.242 h	1.469 fg	1.589 g	1.762 f	1.803 e	1.843 f	1.922 f	2.075 e	1.984 c	1.99 c
P2W1T2	0.069 abcd	1.025 f	1.65 a	1.871 a	2.091 a	2.243 a	2.152 a	2.255 a	2.269 a	2.148 c	2.102 b	2.097 b
P2W1T3	0.068 bcd	0.975 ghij	1.202 jk	1.373 jk	1.502 L	1.606 hi	1.649 i	1.568 pq	1.644 n	1.745 L	1.742 jk	1.702 jk
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.655 mn	1.613 pq	1.438 r	1.387 q
P2W2T1	0.067 bcde	1.176 bc	1.213 ij	1.251 op	1.326 u	1.456 lm	1.539 mn	1.665 L	1.796 i	1.79 k	1.798 g	1.78 g
P2W2T2	0.07 abc	1.087 e	1.552 b	1.797 b	1.935 c	2.054 b	2.148 a	2.114 b	2.154 c	2.036 f	2.111 b	2.003 c
P2W2T3	0.067 bcde	1.074 e	1.19 jklm	1.383 ijk	1.576 gh	1.55 j	1.624 j	1.557 pq	1.574 q	1.715 m	1.705 m	1.685 kl
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.655 mn	1.613 pq	1.438 r	1.387 q
P2W3T1	0.066 bcde	1.229 a	1.334 e	1.439 h	1.542 j	1.624 h	1.741 f	1.864 e	1.858 h	1.865 i	1.826 f	1.832 ef
P2W3T2	0.062 ef	1.199 ab	1.473 c	1.663 d	1.766 f	1.838 e	1.944 d	1.824 g	2.197 b	2.125 d	1.756 hij	1.719 ij
P2W3T3	0.043 g	0.937 jkl	1.093 q	1.324 m	1.523 k	1.585 i	1.622 j	1.512 r	1.653 n	1.73 lm	1.745 ijk	1.703 jk
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.655 mn	1.613 pq	1.438 r	1.387 q
P3W1T1	0.068 bcd	1.235 a	1.365 d	1.444 gh	1.565 hi	1.656 g	1.694 g	1.763 i	2.016 e	1.955 g	1.923 d	1.926 d
P3W1T2	0.065 cde	0.909 L	1.033 r	1.263 no	1.409 r	1.479 L	1.593 k	1.553 q	1.591 p	1.655 o	1.625 o	1.606 n
P3W1T3	0.067 bcde	1.004 fghi	1.171 lmn	1.334 m	1.477 mn	1.509 k	1.646 i	1.563 pq	1.617 o	1.745 L	1.679 n	1.634 m
Control	0.065 cde	0.928 kl	1.158 L no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.655 mn	1.613 pq	1.438 r	1.387 q
P3W2T1	0.065 cde	1.175 bc	1.292 F f	1.347 lm	1.457 op	1.582 i	1.668 h	1.732 j	1.705 k	1.719 m	1.729 kl	1.714 ij
P3W2T2	0.068 bcd	0.922 kl	1.249 gh	1.402 i	1.491 lm	1.512 k	1.644 i	1.611 n	1.677 L	1.607 q	1.763 h	1.663 L
P3W2T3	0.068 bcd	0.899 L	1.235 hi	1.367 kl	1.416 r	1.459 lm	1.560 L	1.404 s	1.459 t	1.472 t	1.451 r	1.408 q
Control	0.065 cde	0.928 kl	1.158 no	1.364 kl	1.448 pq	1.595 i	1.673 h	1.725 j	1.655 mn	1.613 pq	1.438 r	1.387 q
P3W3T1	0.065 cde	1.181 b	1.233 hi	1.286 n	1.352 t	1.471 lm	1.598 k	1.716 j	1.875 g	1.804 jk	1.885 e	1.844 e
P3W3T2	0.067 bcde	1.011 fgh	1.152 no	1.325 m	1.391 s	1.399 n	1.493 o	1.513 r	1.552 r	1.57 r	1.618 o	1.597 n
P3W3T3	0.071 ab	1.132 cd	1.491 c	1.751 c	1.813 e	1.951 d	2.023 b	1.804 h	1.861 gh	1.881 i	1.827 f	1.789 g
Gen. Mean	0.065	1.015	1.247	1.416	1.529	1.629	1.711	1.723	1.756	1.765	1.707	1.673
C.D. 5%	0.005	0.044	0.025	0.025	0.015	0.028	0.017	0.016	0.014	0.019	0.018	0.022

Germination Percentage – G%; Seedling vigor index I – SVI I; Seedling vigor index II – SVI II; P-Pressure, W-Watt, T-Time, P1, P2, P3 = 0.2,0.4,0.6 mbar; W1, W2, W3 = 60,150,240 W; T1, T2, T3 = 5, 10, 15 min. Means followed by the same letter are not significantly different at P < 0.05 by the Tukey test.

Absorption of water in treated and non-treated seeds (control) has been recorded up to 12 h of soaking (Table 4). Treatment at P1W1T3 condition shows water absorption for the maximum period of time i.e., 11 h with a highest germination percentage throughout the experiment. Germination percentage at P2W2T3 was recorded at par to the maximum germination percentage i.e., at P1W1T3 by absorbing water up to 10 h. However, least germination percentage was recorded for P3W3T3 with increasing water absorption up to 7 h. Increase in water imbibitions was recorded for a period up to 11 h on treatment conditions P1W1T3, P1W3T1, P2W2T1, P3W2T2, P3W3T1 and P3W3T2 (Table 4). Absorption of water was observed to be increasing up to 10 h in treatments P1W3T2, P1W3T3, P1W1T1, P2W1T3, P2W2T3, P2W3T1, P3W1T2 and P3W1T3. Seeds imbibed water till 9 h of soaking when treated with P1W1T2, P2W1T2, P2W2T2, P2W3T2 and P3W1T1. Non-treated seeds of sweet basil along with seeds treated with P1W2T3 and P3W2T1 shows increase in water absorption up till 8 h of soaking (Table 4) (Fig. 3 ).

Fig. 3.

Water absorption capacity of control and treated, O. basilicum, seeds at different plasma conditions - P1, P2, P3 = 0.2, 0.4, 0.6 mbar; W1, W2, W3 = 60, 150, 240 W; T1,T2,T3 = 5, 10, 15 min. Error bar above the column signifies the standard deviation for each treatment at every hour of experiment.

An array of correlations among physical, biochemical, and antioxidant parameters of O. basilicum by radio-frequency cold plasma treatment is depicted in Table 5 . A positive and highly significant correlation was found for germination percentage, SVI I, SVI II and carbohydrate with all the studied physical, biochemical and antioxidant parameters excepting catalase and lipid peroxidation. Moreover, an affirmative and highly significant correlation was found between protein and proline for the studied variety (Table 4). However, the correlation was analyzed to be non-significant for all physical, biochemical, and antioxidant parameters with catalase and lipid peroxidation (Table 5).

Table 5.

Correlation among germination and biochemical parameters in variety ‘CIM-Saumya’ of O. basilicum during radio frequency room temperature plasma exposure.

	Seeedling vigor I	Seeedling vigor II	Carbohydrate	Protein	Catalase	Lipid peroxidation	Proline
Germination percentage	0.961**	0.855**	0.743**	0.493**	0.239	0.167	0.594**
Seeedling vigor I		0.793**	0.705**	0.410**	0.313	0.072	0.522**
Seeedling vigor II			0.65**	0.610**	0.159	0.256	0.663**
Carbohydrate				0.605**	0.164	0.186	0.749**
Protein					0.045	0.174	0.782**
Catalase						0.055	0.334
Lipid peroxidation							0.129

(*p < 0.05, **p < 0.01).

4. Discussion

Results show that radio frequency plasma treatment had a notable ameliorative effect on germination potential of sweet basil seeds. Plasma species such as, RONS and reactive species (Zahoranová et al., 2018), are the main agents for the amelioration. The atoms, molecules, radicals, metastables and various emissions from UV, visible, and near IR spectral range (Adhikari et al., 2020b) are much dependent on the interaction of feeding gas with electrons produced by plasma sources. Sarinont et al. (2016) has reported the plasma spectra obtained in dry air, O₂, NO + N₂, N₂, He and Ar for DI water treatment of seed.

Ar + O₂ plasma is reported (Rahman et al., 2018) to produce nitrogen second positive in spectral range of 294−380 nm, OH in the 306−312 nm spectral range, Nitrogen first negative system in 391−405 nm, atomic oxygen and spectra for argon in 737.21–810.37 nm. Falahat et al. (2018) has reported that at higher concentration of O₂, in Ar + O₂ mixture, the intensity of emitted spectra from oxygen increases while intensity of emitted spectra from argon would decrease. Plasma emission spectrum of oxygen shows atomic oxygen lines at 777.1 nm and 845 nm which plays crucial role in growth enhancement (Kitazaki et al., 2012).

Air plasma is reported to emit strong band of second positive system (Adhikari et al., 2020b; Ambrico et al., 2019) of N₂ exists in various regions of UV—B and UVA— band, whose intensity increases when increasing the ratio of N₂ and decreasing the ratio of O₂ in N₂+O₂ mixture. Nitrogen first negative system, in UV—A and Visible spectra in 390−440 nm region, is visible in air and in N₂+O₂ plasma but not in pure O₂ and pure O₂. Emissions in the range of visible- NIR i.e., 620−750 nm spectral range is produced from the first positive system of nitrogen in spectra of N₂, O₂ (20 %) + N₂ (80 %) and ambient air plasma (Tomeková et al., 2020). OH, radicals get originated by collision of water molecules with N₂ or any other energetic charged particles producing UV—B emission in the 306–309 nm spectral range. Atomic nitrogen (742–868 nm) and atomic oxygen (777 nm and 845 nm) is also produced. However, UV light irradiation from air plasma has insignificant effect in growth enhancement (Sarinont et al., 2016). Nitrogen second positive, OH radicals, nitrogen first negative, UV and visible-NIR emissions are most commonly found in Ar + O₂, Air and N₂+O₂ plasmas as mentioned the above study. The comparison of the emission spectra shows that air/O₂ and Ar + O₂ generate many common plasma species though their intensities could be different.

The choice of gas for effective seed treatment is much dependent on the type of seed and its variety. Oxygen plasma treatment, using atmospheric pressure plasma jet, enhanced more significant plant growth of Brassicaceae when using O₂ gas in comparison to air (Hayashi et al., 2015). Significant improvement in the analysis of the plants grown from treated wheat seeds for shoot length, iron content, total soluble sugar and protein content was found more efficient in comparison with the controls due to Ar/O₂ plasma than that for Ar/Air (Rahman et al., 2018). Whereas, Meng et al. (2017) saw no visible enhancement in the wheat seeds, after DBD plasma treatment created using oxygen gas. Root and shoot length got enhanced in the wheat seeds when treated with air, nitrogen and argon gases using the specified plasma system. Ar + O₂ plasma irradiation induced growth enhancement is achieved by following basic mechanisms in cells: 1) Induced oxidation reactions in the metabolic cycle. 2) UV/or ions induced chemical reactions. 3) Attachment of O atoms on seed surface. 4) Oxidative and physical etching of the seed coat. Step 3 and step 4 helps in the water imbibition process. The increased water uptake of seeds, after plasma treatment (Bormashenko et al., 2012), can be possible through the mechanical process such as etching (Stolárik et al., 2015; Pawłat et al., 2018), combination of both chemical and mechanical process (Gómez-Ramírez et al., 2017) or mainly dominated by the chemical process. Ar ion and O atoms, produced in the Ar + O₂ plasma, play crucial role in the seed treatment. Low process pressure during exposure is useful in the physical etching by ion bombardment as well as faster evaporation of the formed by products. Ions are more directional, due to less collision effect, as compared to atmospheric pressure plasma. Vacuum based technique has rich process chemistry and it can be useful for modified seed germination by etching or chemical deposition process for different coating thickness using various gas mixtures (Volin et al., 2000). Whereas, atmospheric pressure air plasma (Zahoranová et al., 2016) has limited chemistry and also it requires ventilation. Low pressure plasma is uniform as compared to the atmospheric pressure plasma and process parameters are controlled. SEM images revealed slight shrinkage in sweet basil seed size with eroded appearance over the surface confirming the etch process. Topography of the seed surface shows gradual thinning of the protruding tip which is looking less prominent in the P3W3T3 (0.6 mbar, 240 W, 15 min) treated seeds.

Ar + O₂ plasma enhanced water uptake mainly due to enhanced wettability capabilities. Whereas, Ambrico et al. (2019) have reported that air plasma treatment may improve germination through water retention which is because of the redistribution and diffusion of micronutrients, such as potassium, into seed interior. Seed coat works as water regulator and it allows the controlled entry of water inside the seed so as to avoid imbibition damage. Plasma treatment modify the waxy seed coat so as to enhance, the seed coat, permeability for increased water uptake (Pawłat et al., 2018). While, no visible changes happened in germination of the plasma treated seeds that are devoid of seed coat (Bafoil et al., 2019). It shows the applicability of plasma treatment for the seeds that are having seed coat. Results show that water uptake is very high in the beginning two hours which is followed by reaching a saturation level and then it starts decreasing. It is observed that the water imbibition characteristics are changed after Ar + O₂ plasma treatment. P3W3T3(0.6 mbar, 240 W, 15 min) shows maximum water imbibition up to initial 7 h and then it starts decreasing whereas control seeds imbibe water till initial 8 h. Most of the other treated samples imbibe water up till longer duration. Early increased water imbibition and poor germination potential for P3W3T3 treated seeds show that the treated seeds might have got damaged for this condition. It might be due to penetration of higher amount of water which adversely affects the embryo as evidenced by Molina et al. (2018). An increased level of water absorption elevates ROS species produced during the treatment and it creates oxidative damage that leads to the deterioration of cell/tissue (Sohan et al., 2021).

Oxygen on the surface is not only useful for increase in wettability but may also help in seed respiration. Oxygen delivery over seed by fumigation had been found assisting in plant growth. O2 and NO + O₂ plasma treatment are found more efficient as compared to fumigation because of the directionality of the species delivery (Sarinont et al., 2016). Comparatively O₂ admixture is found more beneficial instead of air (Rahman et al., 2018). Thus, Ar + O₂ plasma can efficiently modify the seed not only for wettability increase but may also benefit the growth parameters. Results obtained show that oxidation of the seed coat may propagate internally so as to make changes in the lipid peroxidation which can further aid in the signal transduction. Chemical modification of the seed coat, through lipid peroxidation may lead to aleurone layer breakdown by carbonylation of protein and thus making it susceptible for cleavages (Weber et al., 2015). Thus, ROS produced in the Ar + O₂ plasma can change the seed from dormant to nondormant. Scientific evidences show (Rahman et al., 2018), that plasma induced H₂O₂, found in hydrated seeds, is involved in water imbibition and early seed germination. Higher concentration of H₂O₂ was reported in Ar + O₂ then Air + O₂ gas mixtures. Increased H₂O₂ concentration had better growth parameters. Cui et al. (2019) has reported that H₂O₂ concentration is also affected by the presence of other species such as NO etc. due to the conversion mechanism. Mildaziene et al. (2018), has reported the correlation between positive and negative effects of H₂O₂ concentration on seed germination.

Ar + O₂ treatment results suggested that content of carbohydrate and protein got increased significantly over control. Enhancement of enzymatic activities, such as α-amylase, and proteases (Berry and Downton, 1982) could be due to increased aggregation of carbohydrate and protein contents by plasma exposition (Bradford, 1976). Plasma exposure expedites the mobilization and depletion rate of seed reserves which is essential for embryo growth and successful establishment of seedlings. Interaction of seeds with plasma dosage might activate seed germination-related enzymes and enhances seed nutrient disintegration which increased the seed reserve utilization for an optimal percentage of seedling vigor index and seed germination (Sadhu et al., 2017).

Some of ROS species produced during the plasma treatment process are diffused inside the seed through the micropores (Souza and Marcos-Filho, 2001). H₂O₂ can diffuse directly through the pores whereas superoxide can breakdown into hydroxyl molecule and singlet oxygen for getting deeper inside the seed (Grene, 2002). ROS can move down deeper into the plant tissue and get accumulated in the chloroplast (Seol et al., 2017). Superoxide, hydroxyl molecules and NO get converted into H₂O₂ eventually (Cui et al., 2019). Concentrations of hormones get modulated by the existing ROS in the plasma (Adhikari et al., 2020a). Hormones activate the primary and secondary metabolism. Increased sugar and protein content are produced from primary metabolism which is used in the growth. Whereas, secondary metabolism alleviates stress condition by increased enzymatic antioxidants like catalase and nonenzymatic antioxidants like proline. It is evidenced in the results obtained for plasma treated seeds.

Proline gets accumulated in the cytoplasm, to cope with the stress conditions such as water deficit, UV radiation, heavy metal exposure, salinity, and low temperature. Inside the cytoplasm it functions as molecular chaperons for stabilizing the protein structure and its assemblage buffers systolic pH and maintains cell redox status (Hayat et al., 2012). Exposure to stress, EMF or UV has been observed to cause increased free radical production in the cellular environment. Cells have anti-oxidative mechanisms, such as lipid peroxidase (LPO) and catalase (CAT) which get alleviated in order to maintain the damage caused by ROS and their products (Calcabrini et al., 2017).

A highly significant correlation with germination to SVI I, SVI II, carbohydrate, protein and proline show that as the germination changes the vigor/performance of seed got affected positively and significantly in the same manner. Proper water uptake, responsible for better germination, led to increase the rate of breakdown of metabolic contents of plants like carbohydrates and proteins. Positive and significant correlation, as obtained in the results, between germination percentage, SVI I and SVI II shows the crucial role of proline to provide the cell with enough energy to sustain rapid growth (Mattioli et al., 2009).

5. Conclusions

Sweet basil seeds, of variety ‘CIM-Saumya’, were directly exposed to Ar + O₂ radio-frequency plasma produced inside the rotating glass bottle. The treated seeds were analysed for the percentage of germination, seedling vigor index (SVI-I, SVI-II) and morphological changes in the seed coat. SEM images revealed slight shrinkage in the seed size. Plasma treatment modifies the seed coat by physical and oxidative etching which thinned out the protrusion over the surface. The modified seed coat enhanced the water imbibition characteristics. The most efficient treatment increased the percentage germination for the process P1W1T3 (0.2 mbar, 60 W, 15 min) and P2W2T3 (0.4 mbar, 150 W, 15 min). Whereas, treatment condition P3W3T3 (0.6 mbar, 240 W, 15 min) exhibit the inhibitory effect on the germination potential and other vital parameters. Plasma exposition made significant enhancement in carbohydrate content, protein content, lipid peroxidation and the antioxidant activity such as catalase and proline. Enhanced lipid peroxidation and proline can be attributed to the entry of ROS deeper down inside the plant tissue. Positive effects of the plasma treatment can be ascribed to the highly significant amelioration in the germination potential and vital growth parameters. High correlation exists in the germination percentage and biochemical properties of the treated seeds. This work extends the existing knowledge of physio-biochemical changes in the Ar + O₂ plasma treated seed germination and growth which would be much beneficial for the seed treatment.

Declaration of Competing Interest

Authors have no conflict of interest.