The global population's continuous growth necessitates enhanced horticultural systems for vegetable production. Hydroponic systems offer advantages such as reduced pest problems and high productivity. However, maintaining food safety requires nutritious produce free of contaminants. High-yield agriculture relies on high-vigor seeds, often treated with chemical biostimulators. Non-thermal plasma (NTP) technology offers a potential eco-friendly alternative to chemical treatments. Cold plasma applications in agriculture include microbial control, pesticide reduction, and stress mitigation. Studies on plasma-activated water (PAW) have demonstrated its effectiveness in removing organic contaminants and generating reactive species such as H₂O₂, NOx, and NO₂⁻. These reactive species, particularly nitrogen oxides, influence water conductivity and pH, impacting plant growth. While NTP's positive effects on seed germination and early plant growth are known, its influence on yield remains less explored. Lettuce (*Lactuca sativa* L.), a widely consumed vegetable, is often grown hydroponically on nutrient-poor substrates. This study aims to evaluate the dynamic influence of PAW treatment on Shangore F1 lettuce, assessing seed germination, seedling growth, chlorophyll content, and nitrite/nitrate levels.

Existing literature highlights the benefits of hydroponic systems for vegetable production, emphasizing the importance of nutrient supply and food safety. The use of chemical fertilizers and biostimulators in agriculture is widespread, but their environmental impact is a concern. Non-thermal plasma (NTP) technologies have emerged as potential alternatives to conventional treatments, showing promise in controlling microorganisms and reducing pesticide residues in various agricultural settings. Several studies demonstrate that PAW can promote seed germination and early plant growth. Previous research on the effects of PAW on lettuce has shown mixed results, possibly due to variations in PAW concentration, application methods, and plant species. The role of reactive species generated by PAW, such as hydrogen peroxide and nitrogen oxides, in influencing plant growth is also an area of ongoing research. The accumulation of nitrates in lettuce, a key nutritional consideration, is affected by various factors including light intensity and cultivar. The European Commission regulates maximum allowable nitrate levels in lettuce.

This study utilized a spray water plasma reactor to generate PAW. Two PAW samples (PAW I and PAW II) were produced with different nitrite (1.5 mg L⁻¹ and 3.0 mg L⁻¹ respectively) and hydrogen peroxide concentrations. Shangore F1 lettuce seeds were soaked, disinfected, and germinated in Petri dishes. After 48 hours, PAW I or PAW II (or distilled water for the control) was applied to the seeds. Germination rates were monitored. At 10 days after sowing (DAS), seedlings were transferred to trays with Kekkilla peat substrate, and later transplanted into pots of 400 cm³ (V1) and 3200 cm³ (V2). Plants were grown in a greenhouse with controlled conditions. Biometric parameters (radicle/hypocotyl length, leaf area, leaf length/width, foliar/radicular weight) were measured at 50, 57, and 64 days after transplanting (DAT). Chlorophyll content was determined using a CCM-200 plus chlorophyll meter. Nitrite and nitrate levels were measured in dried samples using spectrophotometry. Statistical analysis was performed using one-way and two-way ANOVA with Tukey's post-hoc test.

PAW treatment did not significantly influence lettuce seed germination. However, PAW I (1.5 mg L⁻¹ NO₃⁻) significantly promoted radicle and hypocotyl growth at 8 DAS. PAW II (3.0 mg L⁻¹ NO₃⁻) significantly increased chlorophyll content in lettuce plants grown in larger pots (V2) at 64 DAT. No significant differences were found in root weight among treatments. However, foliar weight and area were significantly higher in plants treated with PAW II in larger pots (V2) at all harvest times. Dry weight was significantly higher for PAW-treated lettuce in larger pots at 57 DAT and smaller pots at 64 DAT. Lettuce grown in larger pots exhibited significantly lower nitrite levels but significantly higher nitrate levels compared to plants in smaller pots, regardless of PAW treatment. A positive correlation was observed between foliar area and leaf weight (99.85%), and between leaf length and width with foliar area (approximately 99%).

The findings suggest that PAW can positively impact lettuce growth and yield, particularly when applied at a concentration of 3.0 mg L⁻¹ NO₃⁻ in larger pot volumes, which likely promotes better root development and nutrient uptake. The increased chlorophyll content in PAW II treated plants in larger pots indicates enhanced photosynthetic activity, contributing to increased biomass. The higher nitrate levels in lettuce grown in larger pots might be attributed to the larger substrate volume facilitating better nitrate absorption from the PAW solution. The lack of significant differences in root weight may suggest that the positive effects are primarily on above-ground growth. The lower nitrite content in larger pots is interesting, but further investigation is needed to explore this observation. The study's results support the potential of PAW as a sustainable alternative to chemical fertilizers, particularly for specific plant species and growth conditions. The impact of different PAW concentrations and substrate volumes on plant growth warrants further exploration.

This study demonstrates the potential of PAW as a plant growth promoter for lettuce. PAW treatment, particularly at higher concentrations (3.0 mg L⁻¹ NO₃⁻) and with larger pot volumes, positively influences chlorophyll content, foliar weight, and area, leading to improved yield. However, the higher nitrate content in larger pots necessitates careful consideration of optimal harvest timing. Further research should investigate the long-term effects of PAW on plant health and the underlying mechanisms of its plant growth promotion effects. Optimizing PAW application parameters and exploring its effectiveness across different plant species and growing systems is also recommended.

The study was conducted under controlled greenhouse conditions, and the results might not be directly transferable to field conditions. The study focused on a single lettuce cultivar, and the responses of other cultivars to PAW treatment may differ. Further research with a wider range of PAW concentrations and longer experimental periods is needed to fully understand the long-term effects of PAW on plant growth and nutrient accumulation. The study considered only two pot volumes, and additional investigation is required to optimize substrate volume for enhanced plant growth. Finally, more detailed investigation into the interactions between reactive species generated by PAW and plant metabolic processes could improve understanding of the observed results.