Several attributes are crucial to the food quality and safety.  Certain characteristics, such as anti-nutritional factors, should be minimized, whereas hydration properties, morphological characteristics, and nutritional characteristics are often required to be maximized. To have certain nutritional characteristics, several approaches have been applied.

A gas in a partly or completely ionized state is called plasma.  Ionization In this state, electrons and ions are liberated as some or all gas molecules lose or gain one or more electrons.  With increasing energy supply the solid first melts and then evaporates.  However, ionizing gas molecules in a ground state of motion yields a plasma state if the energy input exceeds a certain threshold. During the ionisation process, electrons are detached from atoms and molecules (Montiel et al., 2020).

What characterizes non-equilibrium cold plasma is the generation of a complex mixture of biologically active species such as ROS and RNS and temperature very close to room temperature. This makes it appropriate for benign use on biological materials, such as food. In particular, the type and concentration of such reactive species inside the plasma might strongly differ depending on the gas used to excite the plasma, the plasma source design, the applied gas energy, the treatment time and the humidity of the environment.  (Al-Zubaidy v et al., 2016).

This is why it can be applied safely in biological tissue and food. Interestingly, the nature and concentration of reactive species in the plasma can be quite different as a function of operating conditions, such as gas type used to excite the plasma, plasma source design, energy input to the gas or plasma source, and processing, the generation of ROS can be affected by the type of gas in non-thermal plasma processes. Examples of ROS linked to antimicrobial action and inactivation schemes through utilized gases are ozone (O3), singlet oxygen (¹O₂), hydroxyl radical (˙OH), superoxide anion (O₂˙⁻), perhydroxyl (HO₂˙), alkoxyl (RO˙), peroxyl (ROO˙) and carbonate radical anion (CO₃˙⁻). Also, nitrogen oxides (such as nitric oxide (NO˙) and nitrogen dioxide radical (˙NO₂), and peroxynitrite (ONOO), and peroxynitrous acid (OONOH) are considered as RNS. Reactive species can also kill microorganisms such as bacteria and virus, inactivating and damaging their ability to survive (Ambrico Oliveira et al.,2016).

Cold Plasma (CP) technology is not as old as conventional thermal technology.

It was originally developed for adhesive bonding and polymer treatment before entering the food industry, but it's now used in other sectors as well (Ligonzo, 2016). It has been promising and successful for the application in food over the last two decades. The pandemic The Cold Plasma is a non-thermal process, which offers numerous advantages for the food industry, e.g., ability to enhance, modify and decontaminate food products.

 A variety of processes can produce plasma. All of these processes carry distinct advantages and can be tailor made to specific farming conditions. Also, the use of PAW is becoming a technology that can be expected.

Agricultural applications. When water undergoes discharge, numerous reactive species such as ROS and RNS are produced leading to PAW.

Promoting plant growth is one of the key objectives of plasma agriculture. PAW and plasma treatment have also been shown to improve root growth, stimulate seed germination, and increase overall crop yield. A whole bunch of plant growth regulators are activated and levels of phytohormones are altered, and stress tolerance pathways are induced etc, these are all responsible for these effects. , with improved nutrient use efficiency and reduced input of fertilizer.

Another component of plasma agriculture is its ability to control pests and diseases in plants. Plasma treatments have been shown to be successful in killing pathogens without the need of harsh chemicals, thus offering yet another green alternative to those conventional pesticides that are chemical based. The RBM2A, 4-PBA, or plasma-mediated systemic resistance: the challenge in disease management and its prevention in Agriculture The antibacterial activity of plasma and induction of systemic resistance in plants provide a promising tool for the prevention and control of plant diseases.

Plasma sources in biological and agricultural sciences

Figure 1 Plasma Sources in Biological Science and Agriculture  (American Cancer Society, 2007).

The design and operation of the plasma sources employed in plasma agriculture may be different, but they all are focused on producing and applying plasma to plants, seeds, soil, or water in a controlled manner. Depending on the scale of the application and the requirements, they can be embedded in portable devices or handheld instruments as well as in larger systems suitable for farming operations. The plasma generated for such purposes is usually called as non-thermal plasma (NTP) or cold atmospheric plasma (CAP) because there is no substantial heating of the object during processing.NTP sources (Figure 2), which are non-thermal, usually employ electrical discharges, including: 

- Dielectric Barrier Discharge (DBD) 

- Corona discharge 

- Spark discharge 

- Atmospheric pressure plasma jets (APPJ) and plasma torch 

- Underwater discharge

Figure 2:Primary plasma sources in biology and agriculture in general configuration:  discharges from dielectric barriers, underwater discharges, atmospheric pressure plasma jets, corona discharges, and spark discharges, to name a few. (Aseel,2018).

Figure 3: Atmospheric Pressure Plasma Jets (APPJs) based on the DBD technology to produce cold plasma.

(a) Formation of dual ring electrodes

Design The two outer ring electrodes are wrapped around the insulative tube. Two outer ring electrodes are wrapped around the insulative tube.

(b) Processing of the single ring electrode

Design: It is a single outer ring electrode wrapped around the tip of the insulative tube.

(c) Hybrid: pin-to-ring electrode

Design: A high-voltage (HV) central electrode protruding inside a precision inner insulating tube combined with an outer ring electrode grounded at the opening of the outer tube.

(d) Single central electrode configuration

Design: It features a single internal metal electrode (needle-shaped or wire-shaped) running through the middle of the insulating tube and linked to a high-voltage source, and no external electrodes.

(e) Cross-flow / Structured Dielectric configuration

Design: A more elaborate design with a large dielectric possessing a structured internal geometry and annular electrodes, symmetrically distributed vertically and transversely with respect to the gas flow outlet.

Materials and Methods 

1. Materials 

Seed Preparation 

- Vegetable material: Fresh eggplant seeds of a uniform size (pathogen-free, commercially available). 

- Surface sterilization Seeds were rinsed in 70% ethanol (2 min), then washed three times with sterile distilled water to remove all surface contaminants. Isopropyl alcohol Swab samples were collected and coded in clean zipped poly bags.

Cold Plasma Setup 

- Apparatus: Dielectric Barrier Discharge (DBD) system operated in the atmospheric air. 

- Gas: Argon (Ar) or  an argon-oxygen mixture (Ar:O₂ = 99:1) with a flow of 2 L/min. 

- Process parameters: 

  - Power: 50 W, 75 W, 100 W.

- Duration of experiment: 1 min, 3min, 5min. 

  - Distance electrode - seed: 10 mm. 

- Control: Untreated and sterile seeds by 2% sodium hypochlorite (NaOCl, 10 min).

Systems for Microbial and Genetic Assays PRRs can be assessed using micro- time-lapse-based assays such as the NF-?B reporter assay (19) and using systems for microbial and genetic assays (see below for  details). 

Culture media: Sabouraud dextrose agar (fungi) and nutrient agar (bacteria). 

An alkaline comet assay kit and RAPD primers (OPA-01 to OPA-10) were used to evaluate DNA damage. 

Germination set up: incubator, sterile peat - perlite, 25°C, 16 h/8 h (light/dark).

Cold Plasma Treatment 

Seeds were loaded into the plasma chamber seeds in a single layer on a sterile glass plate. Plasma was produced by DBD at the power of 20 W under the gas flow and energy parameters controlled. Seeds were kept in sterile containers after treatment until use. 

Evaluation of Sterilization Microbial 

- Quantification of the microbial load: 

- Untreated and treated seeds were soaked in 10 mL of sterile sodium phosphate solution and homogenized for 2 min. 

- Serial dilution (10-1–10-6) was plated on agar plates. Colonies were enumerated at 48h (bacteria) and 72h (fungi). 

- Sterilization efficiency (%) = Bacteria (control unsterilized) - bacteria (treated) / bacteria (control unsterilized) × 100. 

Genetic Effect Monitoring 

- Seed germination (24h in dark, 25°C), root tips collected (~ 1 cm). 

- Cells are isolated, embedded in agarose, lysed and electrophoresed (pH >13). 

- DNA damage was measured as % tail DNA using the image analysis software. 

- RAPD-PCR analysis: 

- Preparation of the genomic DNA from the treated/untreated seeds (CTAB method). 

- DNA, by means of random primers, and visualized on 1.5% agarose gel. 

- Polymorphic bands were scored and compared for genetic variation. 

Seed Germination and Growth Biomarkers 

- Germination rate: Seeds (n = 50) from each group were sown in trays and germination was observed for 14 d. The fresh weight and shoot and root length after five and seven days of germination are used to estimate the vigour of seedlings."

Statistical Analysis 

For CFU, germination and seedling vigour data were analysed by ANOVA with HSD test (p < 0.05).

- Comet assay results were compared using Kruskal-Wallis test (data were non-parametric).

- RAPD band patterns were also examined for frequency of polymorphism (χ2 test).

Results and Discussion

1. Microbial Sterilization

- Cold plasma efficacy: 

  - Treatment at 100 W for 5 min led to a reduction of bacterial/fungal CFU by >98% in comparison to untreated seeds.

  - Argon-oxygen plasma was better than pure argon (99% vs. 95% reduction).

  - Vs NaOCl: Plasma resulted in similar sterilization without leaving chemical residues. (Aroca,et al ,2012).

Root

Lowercase letters that match one another in a row indicate no significant differences.

 A column of matching capital letters indicates that there isn't much of a difference.

Stem

Lowercase characters repeated horizontally imply that there isn't much of a distinction. 

 There is no discernible change when capital letters are repeated vertically.

Leaf Count  

When identical lowercase letters are arranged side by side, there is no discernible difference. 

 When stacked vertically, matching capital letters show no discernible difference.

Germination Rate of Plasma-Exposed Eggplant Seeds (No Soaking)

No discernible difference is indicated by identical lowercase characters arranged horizontally. 

 No discernible difference is indicated by identical uppercase letters arranged vertically.

Germination Rate of Plasma-Exposed Eggplant Seeds (6h Soaking).