Amino Acids as Biostimulants: Effects on Growth, Chlorophyll Content, and Antioxidant Activity in Ocimum basilicum L.

Abstract

It is necessary to explore possibilities to increase agricultural production in environmentally friendly ways while maintaining the quality standards of plant raw materials. The effect of amino acids on sweet basil (Ocimum basilicum L.) development may stimulate biomass accumulation and enhance the biosynthesis of secondary metabolites. Investigated varieties “Rosie”, “Red Opal”, “Bordeaux”, “Dark Opal”, “Red Rubin”, “Genovese”, “Cinamon”, “Italiano Classico”, “Marseillais”, and “Thai” were cultivated in a controlled-environment growth chamber and the impact of isoleucine, methionine, glutamine, tryptophan, phenylalanine was studied on biomass accumulation, chlorophyll and phenolic content, and antioxidant activity. Five to six true leaves plants were treated once with an aqueous solution containing 100 mg L⁻¹ of the mentioned amino acids or received no treatment. Our results show that methionine or tryptophan improved the most fresh and dry weight of shoot system of sweet basil plants. Methionine increased chlorophyl a content in 6 of 10 sweet basil varieties, while glutamine had the greatest results in chlorophyl b content. Phenylalanine increased total phenolic content in most treated plants, as well as antioxidant activity. Amino acids may be applied as useful biostimulants in modern agriculture, as they play an important role in ensuring sustainable crop productivity, fostering beneficial plant properties.

1. Introduction

For a long time, research in agricultural systems has been oriented towards enhancing yields, with minimal consideration given to product quality and the efficient utilisation of resources. In contrast, the focus is now on product quality and the sustainability of farming systems [1]. Furthermore, agrotechnology planning is increasingly focused on reducing production costs. Agricultural crops frequently demand substantial quantities of fertilisers and pesticides, but the overuse of chemical fertilisers in agriculture has led to environmental degradation and reduced sustainability. In response to these challenges, alternative approaches, such as the application of biostimulants, are gaining attention for their potential to enhance crop yield and quality while minimising environmental harm [2]. The European Union [3] defines biostimulants as natural substances that promote nutrient uptake and efficiency by modulating plant physiological processes. Examples include humic substances, seaweed extracts, protein hydrolysates, amino acids, chitosan and its derivatives, and microbial and bacterial inoculants [4]. The function of biostimulants is to enhance and regulate key physiological processes in plants, the promotion of root and leaf development, improvement of stress tolerance, and support of overall plant growth [5,6]. Amino acids have also been demonstrated to enhance the process of seed germination and to stimulate the biological functions of plants. Environmentally friendly, biostimulants play a crucial role in advancing sustainable agriculture by enabling high productivity with reduced input levels [6,7]. Their use contributes to minimising the reliance on chemical fertilisers and plant protection agents [7,8]. Furthermore, amino acids—which are fundamental units of proteins—serve essential roles in plants, including structural support, metabolic regulation, and nutrient transport [8].

Using growth-promoting substances, as amino acids, for the sweet basil has the potential to increase biomass yield and stimulate the production of secondary metabolites [9]. Ocimum basilicum L. is a widely cultivated aromatic and spice herb that is valued for its role in enhancing the flavour of foods and condiments. This is due to its rich phytochemical profile [10,11]. The plant is renowned for its nutritional and medicinal benefits. It provides essential bioactive compounds, including flavonoids, phenolics, vitamins A and C, and essential minerals such as iron, manganese, and magnesium [12]. The plant has been used traditionally to alleviate ailments such as migraines, sore throats, and asthma. Sweet basil extracts have been shown to have beneficial effects on the immune system and inflammation [13,14].

The application of biostimulants has been shown to enhance the accumulation of photosynthetic pigments in plants, thereby improving photosynthetic efficiency and promoting the biosynthesis of carbohydrates [15]. Exogenously applied amino acids can be readily absorbed through the foliage and utilised in essential physiological processes, as they can be metabolised into plant-available nitrogen forms. Foliar-applied amino acids enter the plant primarily through stomatal openings, subsequently integrating into the nitrogen cycle [16]. These compounds play a crucial role in regulating key physiological functions, including respiration, photosynthesis, and water transport. Moreover, they contribute to elevated levels of ascorbic acid during the early stages of plant development and stimulate protein synthesis [17].

Amino acids play diverse and essential roles in plant physiology, not only serving as building blocks for proteins but also acting as precursors for key biomolecules that regulate growth, development, stress responses, and overall plant health. Isoleucine is a branched-chain amino acid that plays integral roles in plant physiology, particularly in protein synthesis and stress signalling. Its involvement in synthesising jasmonic acid (JA), specifically its conjugate with isoleucine (JA-Ile), is crucial for plant defence responses. [18]. Methionine plays a multifaceted role in plant biology: it is precursor for S-adenosylmethionine (SAM) (critical in the biosynthesis of ethylene. Ethylene is regulating various physiological processes, such as flowering and leaf senescence in plants), enhancing stress tolerance, contributing to antioxidant defences, and improving nutritional quality [19,20]. Glutamine, an amino acid prevalent in plants, plays a critical role in nitrogen metabolism and serves as a vital nitrogen carrier essential for amino acid and protein biosynthesis. It promotes plats growth and improving overall health. Applications of glutamine can enhance leaf expansion and chlorophyll content [21]. Tryptophan serves as a critical precursor for the biosynthesis of indole-3-acetic acid (IAA), one of the principal plant hormones known as auxin, which regulates plant growth, cell elongation, and differentiation, root architecture, shoot development [22,23,24]. Phenylalanine is a pivotal amino acid in plants, acting as the foundation for the synthesis of lignin, flavonoids, and salicylic acid [25]. These substances are vital for reinforcing the plant’s physical structure and strengthening its immune responses. Moreover, its function in the synthesis of flavonoids and salicylic acid has been shown to facilitate protection against environmental stress and the incursion of pathogens [8,26].

The hypothesis holds that spraying amino acids on in sweet basil can enhance biomass yield and promote the accumulation of secondary metabolites during plants cultivation. To evaluate it, the impact of amino acids on biomass accumulation, chlorophyll, phenolic content, and antioxidant activity were investigated.

2. Materials and Methods

2.1. Research Conditions

In 2023 an investigation took place at the Agriculture Academy of Vytautas Magnus University, using various sweet basil (Ocimum basilicum L.) cultivars: “Rosie”, “Red Opal”, “Bordeaux”, “Dark Opal”, “Red Rubin”, “Genovese”, “Cinamon”, “Italiano Classico”, “Marseillais” and “Thai”. The sowing medium comprised a mixture of raised bog peat and sand in a 3:1 proportion. The substrate had a pH of 5.5–6.5 and was supplemented with 1.0 kg m⁻³ of a compound mineral fertiliser (N-P₂O₅-K₂O, 14:16:18), providing all essential macro- and micronutrients required for optimal plant growth. Plastic pots measuring 12 × 10 cm (diameter × hight) were used for sowing seeds. The basil plants were cultivated in climate-controlled growth chambers under a 16 h light and 8 h dark photoperiod, with day/night temperatures maintained at 25/22 °C and a light intensity of 150 µmol m⁻² s⁻¹. All variants and replicates were irrigated with equal amounts of water (100 mL pet pot) to ensure uniform and optimal growing conditions, thereby preventing drought stress or overwatering. Once the seedlings reached the cotyledon stage, they were thinned to five plants per pot. When plants developed 5–6 true leaves, they were split into six treatment groups. Each five groups, except the sixth, was treated once with an aqueous solution (25 mL per 5 plants) containing 100 mg L⁻¹ of one of the following amino acids: isoleucine, methionine, glutamine, tryptophan, or phenylalanine. The sixth group received no treatment. Samples were taken seven weeks after germination for analysis. The experiment was replicated three times.

2.2. Research Methods

Chlorophyll was extracted from 0.2 g of fresh mature leaves using 80% acetone, and absorbance at 663 and 647 nm was measured in the supernatant after centrifugation at 3000 rpm applying a Spectro UV–VIS Dual Beam spectrophotometer (Labomed Inc., Los Angeles, CA, USA). Chlorophyll a and b levels were evaluated in accordance with the methodology of Sims and Gamon [27].

Upon completion of the experiment, all above-ground parts of the basil plants were harvested at the soil level. The fresh biomass of the shoots was recorded using a digital scale, followed by drying in a thermostat at 55 °C for 48 h. The dry weight was then measured with the same device.

The radical scavenging capacity of phenolic compounds was determined in 96% ethanolic extracts, prepared by homogenising 0.3 g of plant material in 10 mL of 96% ethanol. The homogenates were centrifuged at 4500× g for 30 min, and the resulting supernatant was collected for further analysis. Total phenolic content (TPC) was quantified using the Folin–Ciocalteu method. Briefly, 100 µL of plant extract was mixed with 300 µL of 0.2 M Folin–Ciocalteu reagent in a test tube and incubated for 10 min at room temperature in the dark. Subsequently, 5 mL of 7.5% Na₂CO₃ solution was added, and the mixture was incubated for an additional 30 min under the same conditions. The absorbance was measured at 765 nm using a spectrophotometer (Spectro UV–VIS Dual Beam, Labomed Inc., Los Angeles, CA, USA). A calibration curve was constructed using gallic acid standards, and results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight. All analyses were performed in triplicate.

To evaluate total antioxidant activity, extracts prepared from ground basil leaves using 96% ethanol were utilised. Antioxidant activity was quantified and expressed as milligrams of Trolox equivalents (TE) per gram of dry weight. The ABTS^•+ radical scavenging capacity of the basil extracts was assessed using the ABTS^•+ (2,20-azino-bis(3-ethylbenzothiazoline−6-sulfonic acid)) cation decolourisation assay. The ABTS^•+ radical was generated by mixing a 2 mM ABTS stock solution with 0.0095 g of potassium persulfate and allowing the mixture to incubate in the dark at room temperature for 16 h. Prior to testing, this ABTS^•+ solution was diluted with ethanol to obtain an absorbance of 0.8 ± 0.03 at 734 nm. For each analysis, 20 µL of the ethanolic basil extract was added to 3 mL of the ABTS^•+ working solution, and the reaction was carried out in the dark for 60 min. The absorbance of the reaction mixture was then measured at 734 nm. The calibration curve for the ABTS^•+ assay was obtained using Trolox standard solutions, and is described by the linear equation

y = −0.2896x + 0.7859; R² = 0.9895

(1)

where y means the absorbance and x corresponds to the Trolox concentration (mg mL⁻¹).

Total antioxidant capacity of the basil extracts was assessed through the ABTS^•+ radical scavenging method, as outlined by Yim and Nam [28].

2.3. Statistical Analysis of Research Data

The experiment was designed using a completely randomised layout, and all analyses were conducted in triplicate. TIBCO Statistica version 10 (TIBCO Software Inc., Palo Alto, CA, USA) was used to perform the data analysis. The statistical difference (p < 0.05) among the means was analysed by Tukey’s post hoc test. Data are expressed as means ± standard deviations for at least three independent measurements. Statistical analysis was performed by one-way ANOVA. Tukey’s honestly significant difference (HSD) test was applied to assess significant differences (p < 0.05) among the samples. The principal analysis was performed to in order to evaluate the relationships between the application of amino acids on the evaluated parameters, the principal analysis was performed using XLSTAT software version 2021.3.1 (Addinsoft, Paris, France).

3. Results and Discussion

3.1. Effect of Amino Acid Treatment on Shoot System Fresh and Dry Weight

The exogenously effect of amino acids resulted in an increasing in fresh and dry weight in seven out of ten Ocimum basilicum L. varieties (

Table 1. Fresh weight (g) of shoot system of basil plants under different amino acids treatment.

Amino Acid Treatment	Varieties
	“Rosie”	“Red Opal”	“Bordeaux”	“Dark Opal”	“Red Rubin”	“Genovese”	“Cinamon”	“Italiano Classico”	“Marseillais”	“Thai”
No treatment	4.85 ± 0.13 c	5.82 ± 0.24 b	6.37 ± 0.11 bc	7.85 ± 0.81 bc	5.99 ± 0.65 b	18.15 ± 0.93 a	10.41 ± 0.92 a	8.03 ± 0.10 b	7.71 ± 0.51 b	9.65 ± 0.48 a
Isoleucine	5.67 ± 0.56 bc	5.14 ± 0.51 b	4.83 ± 0.18 cd	4.99 ± 0.43 d	4.30 ± 0.08 c	8.70 ± 0.12 d	5.70 ± 0.13 c	6.67 ± 0.13 c	11.14 ± 0.81 a	7.97 ± 0.85 bc
Methionine	8.91 ± 0.01 a	7.17 ± 0.67 a	9.04 ± 0.35 a	7.21 ± 0.02 c	4.58 ± 0.33 c	12.14 ± 1.68 bc	8.50 ± 0.65 b	6.64 ± 0.35 c	8.02 ± 0.63 b	6.32 ± 0.41 d
Glutamine	6.05 ± 0.01 b	5.86 ± 0.52 b	8.90 ± 0.63 a	9.87 ± 0.33 ab	6.52 ± 0.23 b	10.59 ± 0.52 cd	8.47 ± 0.91 b	7.14 ± 0.30 c	11.40 ± 0.58 a	6.71 ± 0.33 cd
Tryptophan	5.87 ± 0.21 c	5.42 ± 0.53 b	7.89 ± 1.14 ab	10.20 ± 0.30 a	7.67 ± 0.35 a	13.18 ± 0.62 b	8.52 ± 0.56 b	9.54 ± 0.10 a	8.66 ± 0.59 b	8.18 ± 0.46 abc
Phenylalanine	3.13 ± 0.12 d	3.48 ± 0.48 c	3.20 ± 0.53 d	6.98 ± 0.56 c	3.83 ± 0.31 c	8.23 ± 0.31 d	5.14 ± 0.26 c	5.31 ± 0.08 d	8.33 ± 0.93 b	8.99 ± 0.21 ab

Note: Data are expressed as mean ± standard error. Different lowercase letters (a, b, c and d) in the columns represent significant differences between amino acid treatment (p < 0.05).

and

Table 2. Dry weight (g) of shoot system of basil plants under different amino acids treatment.

Amino Acid Treatment	Varieties
	“Rosie”	“Red Opal”	“Bordeaux”	“Dark Opal”	“Red Rubin”	“Genovese”	“Cinamon”	“Italiano Classico”	“Marseillais”	“Thai”
No treatment	0.42 ± 0.01 cd	0.42 ± 0.04 b	0.58 ± 0.02 bc	0.63 ± 0.03 bc	0.52 ± 0.04 ab	2.01 ± 0.23 a	1.56 ± 0.18 a	0.86 ± 0.15 ab	0.98 ± 0.15 ab	1.20 ± 0.24 a
Isoleucine	0.52 ± 0.06 bc	0.48 ± 0.02 b	0.46 ± 0.09 cd	0.55 ± 0.05 c	0.33 ± 0.01 c	1.36 ± 0.10 b	0.56 ± 0.01 d	0.69 ± 0.03 bc	1.28 ± 0.11 a	0.82 ± 0.03 bc
Methionine	0.94 ± 0.01 a	0.68 ± 0.04 a	0.96 ± 0.05 a	0.66 ± 0.01 bc	0.47 ± 0.02 bc	1.46 ± 0.22 b	1.12 ± 0.24 abc	0.74 ± 0.04 abc	0.88 ± 0.09 b	0.69 ± 0.04 c
Glutamine	0.60 ± 0.09 b	0.45 ± 0.04 b	0.88 ± 0.05 a	0.71 ± 0.02 b	0.61 ± 0.11 a	1.44 ± 0.03 b	1.31 ± 0.07 ab	0.81 ± 0.06 ab	1.20 ± 0.07 ab	0.63 ± 0.08 c
Tryptophan	0.53 ± 0.09 bc	0.45 ± 0.05 b	0.65 ± 0.04 b	0.91 ± 0.04 a	0.64 ± 0.03 a	1.41 ± 0.15 b	0.96 ± 0.22 bcd	0.97 ± 0.07 a	1.02 ± 0.09 ab	1.03 ± 0.09 ab
Phenylalanine	0.31 ± 0.02 d	0.34 ± 0.05 c	0.35 ± 0.02 d	0.63 ± 0.11 bc	0.34 ± 0.01 c	1.48 ± 0.16 b	0.74 ± 0.12 cd	0.54 ± 0.05 c	1.09 ± 0.23 ab	1.14 ± 0.07 ab

Note: Data are expressed as mean ± standard error. Different lowercase letters (a, b, c and d) in the columns represent significant differences between amino acid treatment (p < 0.05).

). In the absence of amino acid treatment, the fresh weight of the studied plants ranged from 4.85 g to 18.15 g (Table 1), and the dry weight ranged from 0.42 g to 2.01 g (Table 2) among the different varieties. The influence of methionine showed the highest fresh and dry weights of varieties “Rosie”, “Red Opal”, and “Bordeaux”, whereas the greatest fresh and dry weights of ‘Dark Opal’, ‘Red Rubin’, and ‘Italiano Classico’ were recorded following tryptophan treatment. The highest fresh weight of cultivar “Marseillais” was determined under the glutamine impact, while the highest dry weight was determined under the isoleucine impact; however, these differences were not found to be significant. Amino acids, due to their involvement in numerous cellular metabolic pathways, have been widely applied to enhance plant growth and productivity [29,30]. In the present study, the positive impact of methionine on sweet basil growth parameters may be attributed to its multifaceted physiological roles. Methionine serves as a vital source of nitrogen, carbon, energy, and as a building block for enzymes and coenzymes [31,32]. Furthermore, as a biosynthetic precursor of the plant hormone ethylene, methionine may contribute to increased cell division and expansion, ultimately leading to greater biomass accumulation, as reflected in elevated fresh and dry weights. Studies have proven the benefits of using methionine to increase the onions, lettuce, broccoli mass [33,34,35]. In addition, scientific research has also determined that tryptophan has a beneficial effect on the fresh and dry mass accumulation of guar, caraway, Echinacea purpurea plants [36,37,38]. Research shows that tryptophan application positively influences plant productivity and biomass accumulation through its role as a precursor for auxins, particularly for indole-3-acetic acid (IAA) [39,40].

3.2. Effect of Amino Acid Treatment on the Content of Primary Photosynthetic Pigments

Amino acid treatment has been demonstrated to enhance chlorophyll a content in Ocimum basilicum L. plants, except for the “Italiano Classico” variety (

Table 3. Chlorophyll a content (mg g⁻¹, FW) in sweet basil plants under different amino acids treatment.

Amino Acid Treatment	Varieties
	“Rosie”	“Red Opal”	“Bordeaux”	“Dark Opal”	“Red Rubin”	“Genovese”	“Cinamon”	“Italiano Classico”	“Marseillais”	“Thai”
No treatment	2.37 ± 0.002 b	2.30 ± 0.005 d	2.38 ± 0.012 ab	2.31 ± 0.005 e	2.38 ± 0.003 e	2.22 ± 0.001 e	2.28 ± 0.004 c	2.33 ± 0.001 a	1.86 ± 0.018 d	2.45 ± 0.003 b
Isoleucine	2.29 ± 0.001 d	2.40 ± 0.013 b	2.40 ± 0.005 a	2.23 ± 0.019 f	2.44 ± 0.001 c	2.07 ± 0.001 f	2.14 ± 0.001 e	1.99 ± 0.001 e	2.35 ± 0.015 b	2.49 ± 0.001 a
Methionine	2.32 ± 0.012 c	2.46 ± 0.003 a	2.41 ± 0.004 a	2.53 ± 0.001 a	2.58 ± 0.001 a	2.47 ± 0.001 a	2.33 ± 0.004 b	2.29 ± 0.001 b	2.15 ± 0.007 c	2.49 ± 0.011 a
Glutamine	2.37 ± 0.006 b	2.31 ± 0.008 cd	2.32 ± 0.066 bc	2.43 ± 0.001 c	2.42 ± 0.001 d	2.41 ± 0.001 b	2.35 ± 0.003 a	2.14 ± 0.002 c	2.40 ± 0.002 a	2.44 ± 0.004 c
Tryptophan	2.37 ± 0.001 b	2.32 ± 0.004 c	2.30 ± 0.003 c	2.39 ± 0.002 d	2.38 ± 0.002 e	2.32 ± 0.002 d	2.05 ± 0.004 f	2.02 ± 0.012 d	1.93 ± 0.021 c	2.38 ± 0.001 d
Phenylalanine	2.52 ± 0.011 a	2.27 ± 0.002 e	2.39 ± 0.001 ab	2.49 ± 0.001 b	2.48 ± 0.010 b	2.36 ± 0.006 c	2.23 ± 0.003 d	1.95 ± 0.001 f	2.13 ± 0.011 c	2.42 ± 0.010 c

Note: Data are expressed as mean ± standard error. Different lowercase letters (a, b, c, d, e and f) in the columns represent significant differences between amino acid treatment (p < 0.05).

). The chlorophyll a content in plants cultivated with no treatment ranged from 1.86 mg g⁻¹ FW to 2.45 mg g⁻¹ FW depending on the cultivars. The impact of methionine significantly improved chlorophyll a content in cultivars “Red Opal”, Bordeaux”, “Dark Opal”, “Red Rubin” and “Thai”, while in cultivars “Cinamon” and “Marseillais” chlorophyll a content was statistically significant increased by the influence of glutamine. Phenylalanine had the most positive effect on “Rosie” cultivars’ chlorophyll a content. The results indicated that methionine treatment enhanced the chlorophyll a content in the majority of tested sweet basil varieties. Although all amino acids serve as sources of carbon and nitrogen, methionine also contains sulphur, which is essential for protein biosynthesis and the synthesis of glutathione (GSH), an important antioxidant. Additionally, methionine may contribute to the biosynthesis of chlorophyll a by supporting the synthesis and activity of enzymes such as glutamyl-tRNA reductase, magnesium chelatase, and protochlorophyllide oxidoreductase [41,42,43,44]. The present study corroborates extant scientific research demonstrating the capacity of methionine to enhance photosynthetic pigments in green onions, sweet basil, and corms [45,46,47].

Except for the “Italiano Classico” variety, amino acid treatment increased chlorophyll b content in most Ocimum basilicum L. plants (

Table 4. Chlorophyll b content (mg g⁻¹, FW) in sweet basil plants under different amino acids treatment.

Amino Acid Treatment	Varieties
	“Rosie”	“Red Opal”	“Bordeaux”	“Dark Opal”	“Red Rubin”	“Genovese”	“Cinamon”	“Italiano Classico”	“Marseillais”	“Thai”
No treatment	1.14 ± 0.001 c	1.03 ± 0.004 e	1.18 ± 0.016 d	1.08 ± 0.005 d	1.24 ± 0.013 e	0.88 ± 0.001 d	0.91 ± 0.002 d	1.13 ± 0.002 a	1.10 ± 0.004 b	1.44 ± 0.016 b
Isoleucine	0.99 ± 0.006 e	1.29 ± 0.005 c	1.38 ± 0.001 b	0.98 ± 0.010 f	1.52 ± 0.004 b	0.76 ± 0.001 c	0.96 ± 0.004 c	0.78 ± 0.002 c	0.71 ± 0.017 d	1.52 ± 0.007 a
Methionine	1.06 ± 0.001 d	1.39 ± 0.001 b	1.43 ± 0.003 a	1.04 ± 0.001 e	1.30 ± 0.012 d	1.38 ± 0.009 c	0.99 ± 0.003 c	0.92 ± 0.004 b	0.74 ± 0.003 d	1.34 ± 0.033 c
Glutamine	1.23 ± 0.018 b	1.04 ± 0.003 e	1.18 ± 0.028 d	1.44 ± 0.001 a	1.34 ± 0.005 c	1.30 ± 0.014 a	1.06 ± 0.018 a	0.77 ± 0.006 c	1.23 ± 0.005 a	1.49 ± 0.011 a
Tryptophan	1.22 ± 0.011 b	1.07 ± 0.003 d	1.04 ± 0.004 e	1.21 ± 0.001 c	1.18 ± 0.004 f	1.01 ± 0.002 e	0.60 ± 0.002 e	0.72 ± 0.016 d	0.68 ± 0.007 e	1.19 ± 0.003 d
Phenylalanine	1.39 ± 0.008 a	1.56 ± 0.006 a	1.23 ± 0.003 c	1.24 ± 0.008 b	1.56 ± 0.004 a	0.99 ± 0.012 d	0.91 ± 0.015 d	0.60 ± 0.009 e	0.86 ± 0.009 c	1.10 ± 0.002 e

Note: Data are expressed as mean ± standard error. Different lowercase letters (a, b, c, d, e and f) in the columns represent significant differences between amino acid treatment (p < 0.05).

). In plants with no amino acid treatment, chlorophyll b content exhibited a range of 0.88 mg g⁻¹ FW to 1.44 mg g⁻¹ FW, varying among cultivars. Foliar application of glutamine expanded chlorophyll b content in the cultivars “Dark Opal”, “Genovese”, “Cinamon” and “Marseillais”, whereas the highest accumulation of chlorophyll b of “Rosie”, “Red Opal” and “Red Rubin” was determined under the phenylalanine impact. The foliar spraying with isoleucine positively influenced the cultivar “Thai”, while methionine significantly increased chlorophyll b content of variety “Bordeaux”. The role of glutamine in enhancing chlorophyll b content in plants has been supported by various studies demonstrating its positive impact on photosynthetic pigments and overall plant health.

It was determined that glutamine application to strawberry plants improved their physiological parameters, hinting at higher chlorophyll levels leading to improved photosynthesis efficiency [48], foliar applications of glutamine were found to significantly increase the total chlorophyll content in lettuce leaves, rice, sorghum [49,50,51]. Glutamine application positively affects chlorophyll b content in plants, mainly through its role in nitrogen metabolism and enhancing physiological growth conditions.

3.3. Effect of Amino Acid Treatment on the Total Phenolic Content

The exogenous application of amino acids led to a significant increase in the TPCs of most of the Ocimum basilicum L. varieties tested but had no effect on the “Italiano Classico” variety tested. The “Red Opal” and “Dark Opal” varieties had the highest TPCs when amino acid isoleucine was applied to the plant’s leaves (

Table 5. Total phenolic content (mg g⁻¹, DW) in sweet basil plants under different amino acids treatment.

Amino Acid Treatment.	Varieties
	“Rosie”	“Red Opal”	“Bordeaux”	“Dark Opal”	“Red Rubin”	“Genovese”	“Cinamon”	“Italiano Classico”	“Marseillais”	“Thai”
No treatment	10.99 ± 0.23 b	11.86 ± 0.17 a	9.42 ± 0.23 c	8.65 ± 0.04 c	7.84 ± 0.17 c	9.77 ± 0.23 c	10.21 ± 0.05 d	12.90 ± 0.34 a	11.89 ± 0.50 cd	13.31 ± 0.06 a
Isoleucine	11.63 ± 0.11 ab	12.56 ± 0.43 a	12.30 ± 0.19 a	10.12 ± 0.15 a	8.93 ± 0.19 b	10.57 ± 0.05 c	12.53 ± 0.09 b	11.83 ± 0.18 abc	11.49 ± 0.10 d	12.24 ± 0.02 bc
Methionine	11.28 ± 0.35 b	11.73 ± 0.24 ab	10.69 ± 0.30 b	9.59 ± 0.59 ab	9.19 ± 0.10 b	13.31 ± 0.18 a	12.77 ± 0.12 b	11.00 ± 0.12 c	12.69 ± 0.20 b	11.59 ± 0.46 c
Glutamine	9.76 ± 0.28 c	9.85 ± 0.58 c	10.67 ± 0.02 b	10.04 ± 0.44 a	9.79 ± 0.15 a	12.59 ± 0.13 ab	12.63 ± 0.15 b	12.74 ± 0.23 ab	12.34 ± 0.04 bc	13.02 ± 0.06 ab
Tryptophan	11.06 ± 0.17 b	10.84 ± 0.14 bc	12.11 ± 0.16 a	8.04 ± 0.29 c	10.05 ± 0.12 a	11.61 ± 0.78 b	11.82 ± 0.09 c	11.76 ± 0.87 bc	13.58 ± 0.22 a	12.15 ± 0.30 bc
Phenylalanine	12.25 ± 0.18 a	10.81 ± 0.17 bc	12.93 ± 0.67 a	8.82 ± 0.13 bc	9.13 ± 0.16 b	12.90 ± 0.21 a	13.61 ± 0.49 a	10.97 ± 0.08 c	12.23 ± 0.16 bc	13.49 ± 0.49 a

Note: Data are expressed as mean ± standard error. Different lowercase letters (a, b, c and d) in the columns represent significant differences between amino acid treatment (p < 0.05).

). The total phenolic content (TPC) in cultivars grown without amino acid supplementation varied from 7.84 mg g⁻¹ DW to 13.31 mg g⁻¹ DW depending on the cultivar The application of tryptophan resulted in a significant enhancement of total phenolic content in the cultivars “Red Rubin” and “Marseillais”, whereas phenylalanine increased TPCs in “Rosie”, “Bordeaux”, “Cinamon”. Exogenously applied methionine significantly increased the total phenolic content in variety “Genovese”. Phenolic compounds derived from phenylalanine significantly enhance the flavour and aroma of plants, particularly in culinary herbs such as basil. They are known to contribute to the sensory attributes of these plants, influencing their market value and culinary application [52]. Studies have documented that elicitors such as phenylalanine can stimulate the production of flavonoids and other phenolic compounds, including rosmarinic acid, caffeic acid, and chicoric acid. These compounds may contribute to the enhancement of aroma and flavour profiles in sweet basil, although certain phenolics can also impart an astringent or bitter taste depending on their concentration and composition [26,53,54,55,56,57,58]. Phenylalanine ability to improve flavour and aroma further emphasizes the importance of this amino acid in agricultural practices, especially in the cultivation of aromatic herbs like basil.

3.4. Effect of Amino Acid Treatment on the Antioxidant Activity

Amino acid treatment increased antioxidant activity in all Ocimum basilicum L. plants (

Table 6. Antioxidant activity of basil leaf extracts determined by ABTS^•+ assay (mg TE g⁻¹ DW) in sweet basil plants under different amino acids treatment.

Amino Acid Treatment	Varieties
	“Rosie”	“Red Opal”	“Bordeaux”	“Dark Opal”	“Red Rubin”	“Genovese”	“Cinamon”	“Italiano Classico”	“Marseillais”	“Thai”
No treatment	11.64 ± 0.32 cd	10.76 ± 0.24 d	10.48 ± 0.39 c	9.26 ± 0.03 c	8.94 ± 0.74 e	10.52 ± 1.27 b	11.24 ± 0.30 d	13.45 ± 0.25 a	12.82 ± 0.30 bc	14.08 ± 0.67 a
Isoleucine	12.70 ± 0.54 ab	13.51 ± 0.32 a	13.00 ± 0.18 a	11.43 ± 0.05 a	9.41 ± 0.19 de	11.50 ± 0.70 b	11.71 ± 0.14 cd	12.21 ± 0.66 b	12.26 ± 0.23 c	13.39 ± 0.48 ab
Methionine	12.67 ± 0.13 ab	12.75 ± 0.03 b	11.42 ± 0.08 b	9.86 ± 0.04 b	9.84 ± 0.12 cd	14.38 ± 0.02 a	12.16 ± 0.32 c	11.37 ± 0.15 b	13.51 ± 0.10 b	12.94 ± 0.31 b
Glutamine	10.84 ± 0.08 d	12.45 ± 0.16 b	11.38 ± 0.12 b	11.32 ± 0.07 a	10.71 ± 0.11 bc	13.34 ± 0.27 a	13.70 ± 0.38 b	13.58 ± 0.20 a	13.63 ± 0.15 b	14.02 ± 0.15 ab
Tryptophan	12.14 ± 0.29 bc	12.57 ± 0.08 b	13.36 ± 0.16 a	9.46 ± 0.23 c	11.61 ± 0.12 a	13.62 ± 0.03 a	13.23 ± 0.25 b	12.25 ± 0.16 b	14.60 ± 0.50 a	13.73 ± 0.07 ab
Phenylalanine	13.57 ± 0.27 a	11.83 ± 0.21 c	13.31 ± 0.06 a	9.91 ± 0.07 b	10.79 ± 0.08 ab	13.99 ± 0.12 a	14.57 ± 0.17 a	11.71 ± 0.26 b	13.45 ± 0.16 b	14.18 ± 0.59 a

Note: Data are expressed as mean ± standard error. Different lowercase letters (a, b, c, d and e) in the columns represent significant differences between amino acid treatment (p < 0.05).

). As demonstrated in Table 6, the antioxidant activity determined using the ABTS^•+ method in plants cultivated without amino acid treatment varied from 8.94 to 14.08 mg TE g⁻¹ DW. A significant increase in antioxidant activity was recorded in the “Red Opal” and “Dark Opal” cultivars following foliar application of isoleucine, while the highest activity in “Bordeaux”, “Red Rubin”, and “Marseillais” was induced by tryptophan treatment. A statistically significant increase in antioxidant activity was observed for the “Rosie”, “Cinamon” and “Thai” cultivar when phenylalanine was applied. The foliar spraying of methionine had effect in increasing antioxidant activity in the cultivar “Genovese”, while in the “Italiano Classico” cultivar, the highest accumulation of antioxidant activity was obtained by spraying with the amino acid glutamine. The connection between phenolic compound concentration and antioxidant activity has been consistently demonstrated across various studies. Previous studies have announced a strong linear relationship between total phenolic content and antioxidant activity in ginger extracts, medicinal herbs and spices, indicating that as phenolic content increases, so does the antioxidant capability [59,60,61]. In sweet basil, bioactive compounds such as phenolic acids (e.g., rosmarinic and caffeic acid), flavonoids (e.g., quercetin and luteolin), and essential oils (e.g., eugenol and linalool) have been identified as significant contributors to its antioxidant activity [62,63,64].

3.5. Principal Component Analysis

Principal component analysis (PCA) was used to analyse variations in the morphological and bioactive properties of Ocimum basilicum L. varieties when treated with a foliar spray of five amino acids: isoleucine, methionine, glutamine, tryptophan and phenylalanine (Figure 1). The following parameters were assessed: fresh and dry biomass, chlorophyll content, concentration of phenolic compounds, and antioxidant activity, which was measured using the ABTS^•+ method. The PCA results showed that the first two principal components (F1 and F2) together explained 70.27% of the total variance in the data. The first component (F1, 44.22%) represented the productivity and bioactivity axis, which was primarily influenced by fresh and dry mass, chlorophyll content. The second component (F2, 26.05%) was associated with antioxidant abundance. The PCA indicates that the highest fresh mass, dry mass was associated with cultivars “Genovese” and “Marseillais” sprayed with isoleucine. Chlorophyll a and chlorophyll b content was associated with cultivars “Rosie” and “Red Opal” sprayed with phenylalanine. The total phenolic content and antioxidant activity were identified with cultivar “Cinnamon” sprayed with phenylalanine.

4. Conclusions

The foliar treatment of amino acids has emerged as a promising approach to enhancing plant productivity and quality. In the present research, the influence of amino acid application on the Ocimum basilicum L. morphological parameters, chlorophyll and phenolic compound content, antioxidant activity were evaluated. It was determined that methionine and tryptophan increased the fresh and dry weight of shoot system of “Rosie”, Red Opal”, “Bordeaux” and “Dark Opal”, “Red Rubin”, and “Italiano Classico”, respectively. Methionine increased chlorophyll a content in “Red Opal”, “Bordeaux”, “Dark Opal”, “Red Rubin”, “Genovese”, and “Thai” sweet basil varieties, while glutamine had the greatest results in chlorophyl b content for “Dark Opal”, “Genovese”, “Cinamon”, “Marseillais”, and “Thai” varieties. Phenylalanine increased total phenolic content in “Rosie”, “Bordeaux”, and “Cinamon” plants, as well as antioxidant activity determined by ABTS^•+ assay. Aromatic amino acid phenylalanine is the precursor of phenylopropanoid biochemical pathway which leads to flavonoid biosynthesis. The findings clearly demonstrated that amino acids could be useful biostimulants in modern agriculture.