Enhanced in vitro propagation of Thymus bovei via nanoparticles and analysis of antibacterial activity

Propagação in vitro aprimorada de Thymus bovei via nanopartículas e análise da atividade antibacteriana

Abstract

Thymus bovei is one of the most well-known medicinal plants in Jordan. Due to the growing demand and limited availability of planting material, it is important to establish an effective protocol for the in vitro propagation of T. bmg/ovei. Therefore, the objective of the current study was to develop a novel in vitro propagation protocol for T. bovei by examining the influence of nanoparticles on its propagation. Microshoots were cultivated in Murashige and Skoog medium (MS) with benzylaminopurine (BA), or in MS medium without any growth regulators, then both treatments were supplemented with different concentrations of nanoparticles: silica celite (SiO₂), copper oxide (CuO), and silica−copper oxide (SiO₂CuO), for a five-week growth period. The maximum multiplication rate of T. bovei (7.3 shoots/explants) was achieved from microshoots proliferated on MS medium augmented with 1.5 to 2.0 mg L⁻¹ BA and 5.0 mg L⁻¹ SiO₂CuO. T. bovei extracts were derived from different plant tissue sources; plants from the field (ex vitro plants), plants cultured in vitro on MS medium (in vitro plants), plants cultured in vitro on MS medium with nanoparticles (nano-plant), and plants cultured in vitro on MS medium with nanoparticles and BA hormone (nano-BA-plant) using acetone, ethanol, and methanol solvents. Then, the antibacterial activity of all plant extracts was tested against Salmonella sp., Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Extracts of T. bovei from both ex vitro and in vitro sources exhibited strong antibacterial activity against all tested bacteria in a varied degree of inhibition zone diameter, ranging from 1.1 to 3.9 cm. The highest inhibition zone was recorded in Salmonella sp. in response to acetone and ethanol nano-plant extract (3.3 cm and 3.9 cm respectively), which was comparable to the positive control meropenem (3.4 cm). S. aureus and B. subitilis exhibited high sensitivity to all plant extracts. While antimicrobial activity against E. coli was reported in response to T. bovei extracts from acetone in vitro plants, methanol and ethanol nano-plant. Consequently, this study showed that SiO₂ and CuO nanoparticles in MS media gave a positive effect on shoot growth of T. bovei in in vitro culture, and highlighted the antibacterial activity of ex vitro plants, in vitro T. bovei extracts.

Keywords:
antibacterial; in vitro propagation; medicinal plant; nanoparticles; silica celite nanoparticles; copper nanoparticle; silica-copper oxide nanoparticles; Thymus bovei

Resumo

Thymus bovei é uma das plantas medicinais mais conhecidas na Jordânia. Devido à crescente demanda e à disponibilidade limitada de material de plantio, é importante estabelecer um protocolo eficaz para a propagação in vitro de T. bovei. Portanto, o objetivo do presente estudo foi desenvolver um novo protocolo de propagação in vitro para T. bovei, examinando a influência de nanopartículas em sua propagação. Microbrotos foram cultivados em meio Murashige e Skoog (MS) com benzilaminopurina (BA) ou em meio MS sem reguladores de crescimento; ambos os tratamentos foram suplementados com diferentes concentrações de nanopartículas – sílica celite (SiO₂), óxido de cobre (CuO) e sílica-óxido de cobre (SiO₂-CuO) – por um período de crescimento de cinco semanas. A taxa máxima de multiplicação de T. bovei (7,3 brotos/explante) foi obtida a partir de microbrotos proliferados em meio MS suplementado com 1,5 a 2,0 mg L⁻¹ de BA e 5,0 mg L⁻¹ de SiO₂-CuO. Extratos de T. bovei foram obtidos a partir de diferentes fontes de tecido vegetal: plantas de campo (plantas ex vitro), plantas cultivadas in vitro em meio MS (plantas in vitro), plantas cultivadas in vitro em meio MS com nanopartículas (nanoplanta) e plantas cultivadas in vitro em meio MS com nanopartículas e hormônio BA (nanoplantas-BA), utilizando os solventes acetona, etanol e metanol. Em seguida, a atividade antibacteriana de todos os extratos vegetais foi testada contra Salmonella sp., Escherichia coli, Bacillus subtilis e Staphylococcus aureus. Os extratos de T. bovei, tanto de fontes ex vitro quanto in vitro, apresentaram forte atividade antibacteriana contra todas as bactérias testadas, com diâmetros de zona de inibição variando de 1,1 a 3,9 cm. A maior zona de inibição foi registrada para Salmonella sp. em resposta aos extratos de nanoplantas com acetona e etanol (3,3 cm e 3,9 cm, respectivamente), valor comparável ao do controle positivo meropenem (3,4 cm). S. aureus e B. subtilis apresentaram alta sensibilidade a todos os extratos vegetais. Embora a atividade antimicrobiana contra E. coli tenha sido relatada em resposta a extratos de T. bovei de plantas in vitro de acetona, metanol e nanoplanta de etanol. Consequentemente, este estudo mostrou que as nanopartículas de SiO₂ e CuO em meio MS tiveram um efeito positivo no crescimento de brotos de T. bovei em cultura in vitro, e destacou a atividade antibacteriana de plantas ex vitro, extratos in vitro de T. bovei.

Palavras-chave:
antibacteriano; propagação in vitro; planta medicinal; nanopartículas; nanopartículas de sílica celite; nanopartículas de cobre; nanopartículas de sílica-óxido de cobre; Thymus bovei

1. Introduction

Jordan wild plants contain diverse bioactive compounds that have been shown to exhibit antimicrobial, antioxidant, anticancer, and antidiabetic properties (Alrayes et al., 2016). Wild thyme (Thymus bovei) is a perennial medicinal plant belonging to the Labiatae (Lamiaceae) family, known for its therapeutic and nutritional qualities. T. bovei contains an essential oil that displays effective anthelmintic, expectorant, antispasmodic, and antiseptic properties (Abdel-Hady et al., 2014). T. bovei suffers from overexploitation, where it exposed to continuous extreme harvesting in its natural habitat for medicinal use (Alkhawaldah et al., 2025). In addition, T. bovei contains numerous compounds with antimicrobial, antifungal, and antioxidant properties. Previous investigations have reported a high inhibitory effect of the T. bovei plant against a diverse spectrum of microbial organisms (Al-Qura’n, 2009; Al-Tabini et al., 2012; Duwayri et al., 2012; Bakhtiar et al., 2016). Chattipakorn et al. (2007) reported that T. bovei plants contain active compounds such as thymol and carvacrol, which possess antimicrobial activity.

Thymus bovei is suffering extinction due to the excessive collection from its natural habitat for its medicinal applications (Alkhawaldah et al., 2025); therefore, this species needs real strategies for protection. Moreover, there has been increasing attention and prompting extensive research initiatives to explore native plant species for their potential to yield innovative bioactive compounds; these pursuits aim to uncover new phytochemicals that could serve as cornerstones for groundbreaking antimicrobial therapies (Shatnawi et al., 2019; Abu-Odeh et al., 2023; Shahrour et al., 2024b). As well as, there is an urgent need for natural products among consumers. Thus, it becomes an important issue to initiate an effective protocol for the propagation of T. bovei. Micropropagation is a technique that offers a good new method for T. bovei multiplication. In Jordan, few attempts were made to produce this plant in vitro. In vitro plants can produce large quantities of bioactive compounds in short periods, enabling rapid and beneficial production of secondary metabolites (Shatnawi et al., 2010). Therefore, there is a need to increase T. bovei production using this technique.

Nanotechnology plays a crucial role in improving human life in the future. It can be used in agriculture, biology, physics, and chemistry (Singh et al., 2011). Nanotechnology has an innovation in the field of in vitro culture due to its impacts on propagation, production of secondary compounds, decontaminators, and development of in vitro culture (Safavi, 2014). Nanoparticles enable plant tissue culture for an increase in number, improving plant quality, and increasing production of secondary metabolites (Wang et al., 2016). On the other hand, supplementation of nanoparticles to the media can improve plant growth and can disinfect the explants (Sarmast and Salehi, 2016). The use of various nanoparticles in in vitro culture media has been studied in many species (Safavi, 2014; Shokri et al., 2014; Khan et al., 2019; Sharma et al., 2023). Bello-Bello and Spinoso-Castillo (2023) reported that incorporating silver nanoparticles in an MS medium could increase the effectiveness of micropropagation. Helaly et al. (2014) observed that MS medium containing 100 mg L⁻¹ Zn NPs showed the highest somatic embryogenesis and the highest rooting, shoot formation, and regeneration in banana. Safavi (2014). Using 50 mg L⁻¹ Ag and TiO₂ on MS medium decreased microbial growth on tobacco and potato plants. Whereas the effect of nanoparticles on T. bovei propagation is not yet recognized. Therefore, the current study was carried out to examine the effects of SiO2, CuO and SiO2CuO nanoparticles on the in vitro growth and multiplication of T. bovei and to assess the antibacterial activity of the extracts obtained from field-grown, in vitro-grown and nanoparticle-treated plants against some pathogenic bacteria.

2. Material and Methods

2.1. Plant material

Seeds of Wild Thyme (T. bovei) plants were collected from Mersah, Jerash (32° 16' 50.95" N and 35° 53' 57.44" E). In vitro initiation of these plants was carried out in Hamdi Mango Center for Scientific Research, University of Jordan, according to the methods developed by Al Shhab et al. (2022) on hormone-free Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 30 g/L sucrose and 8.0 g/L agar agar.

2.2. Influence of nanoparticles on in vitro growth of Thymus bovei

Microshoots 15 mm were thrived as the previous method used by Alrayes et al. (2016). Explants were subcultured on free hormone MS medium. Then, the media containing nanoparticles were prepared. Nanoparticles were prepared according to Salmeia et al. (2025), which were food-grade diatomaceous earth samples purchased from Celatom, Ep Minerals (Nevada, USA). The Pure silica sample was characterized using scanning electron microscopy (SEM), Wide-angle X-ray diffraction (WAXD), Fourier Transform Infrared Spectra (FT- IR), and ²⁹Si MAS Nuclear Magnetic Resonance (NMR) (Salmeia et al., 2025). Each nanoparticle was sonicated for 20 minutes and stirred for 20 minutes before being added to the media to guarantee homogeneity. The pH of the medium was adjusted to 5.8 and was solidified with 8.0 g/L agar. Microshoots with apical meristem were cultivated in fresh MS medium containing silica celite (SiO₂), or copper oxide (CuO) nanoparticles (0.0, 1.0, 2.0, 5.0, 10, and 50 mg L⁻¹) of each nanoparticle (Chauhan et al., 2021; Natsheh et al., 2023; Kazmi et al., 2025). Every treatment (flask) had five microshoots, and each treatment had five replicated flasks. Cultures were maintained under standard growth room conditions (24 ± 2 C°, 16 h light/8 h dark). After five weeks of growth, data were collected on the number of new shoots/explants, shoot length, fresh shoot weight, and dry shoot weight after drying in the oven at 80-85 °C for 24 h for each one.

2.3. Influence of nanoparticles with growth regulators on in vitro growth of Thymus bovei

The influence of different concentrations of benzyl amino purine (BA) with CuO, and BA with SiO₂CuO nanoparticles on the in vitro growth of T. bovei was evaluated. Microshoots with apical meristem (10-12 mm in length) that were grown on free hormone MS medium were subcultured to fresh MS media containing BA (0.0, 0.5, 1.0, 1.5, and 2.0 mg L⁻¹) with 5 mg L⁻¹CuO, and fresh MS media containing BA (0.0, 0.5, 1.0, 1.5, and 2.0 mg L⁻¹) with 5 mg L⁻¹SiO₂CuO. MS media with nanoparticles were prepared as in the previous section. Every treatment (flask) had five microshoots, and each treatment had five replicated flasks. Cultures were maintained under standard growth room conditions (24 ± 2 C°, 16 h light/8 h dark). After five weeks of growth, data were collected on the number of new shoots/explants, shoot length, fresh weight, and dry shoot weight after drying in the oven at 80- 85 °C for 24 h for each one.

2.4. Antimicrobial activity

2.4.1. Plant material and plant extract preparation

Different T. bovei extracts were prepared from four plant sources for antimicrobial evaluation:

1. Field plant from Mersah, Jerash (32° 16' 50.95" N and 35° 53' 57.44" E) (ex vitro-plant)

2. In vitro cultured plants on MS (in vitro plants)

3. In vitro cultured plants on MS with nanoparticles (nano-plant).

4. In vitro cultured plants on MS with nanoparticles and BA (nano-BA-plant).

Plant samples were oven-dried at 60-65 °C. All dried materials were ground into a fine powder using an electric grinder.

2.4.2. Plant extracts preparation

Five grams of each plant powdered sample were dissolved separately in 100 mL of three solvents (99% ethanol, 99.1% methanol, and 98.9% acetone) and incubated in a shaker for 48–72 h. Extracts were filtered through Whatman filter paper, and solvents were evaporated to obtain pure solvent-free extracts. Plant extracts were weighed and stored at 4 °C in foil-wrapped containers to minimize light-induced degradation. Then, 300 mg of each extract was dissolved in 3 mL of dimethyl sulfoxide (DMSO) in final concentration 100mg mL⁻¹ for antimicrobial evaluation.

2.4.3. Growth of bacteria

Four bacterial strains were cultured on nutrient agar medium; Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Salmonella spp. Bacterial cultures were prepared according to the McFarland standard (10⁸ CFU/mL) and diluted to 10⁶ CFU/mL (except Salmonella, diluted to 10⁷ CFU/mL due to slower growth). Nutrient agar medium was prepared by dissolving 28 g of medium in 1 L of distilled water, sterilized at 121 °C for 20 min, and poured into 20 cm Petri dishes (15 mL/plate; ~4 mm thickness).

2.4.4. Antimicrobial assay

Antimicrobial activity was tested using the well diffusion method. A 100-µL aliquot of each microbial culture was spread uniformly over agar plates. Three plates were used for each treatment. Four wells (6–8 mm) were aseptically drilled in each plate, and 100 µL of each plant extract (100mg mL⁻¹) was introduced into each well. Meropenem (10 mg, antibiotic) served as the positive control, while DMSO was used as the negative control. Plates were incubated at 37 °C for 24 h, and inhibition zones were measured in cm.

2.5. Statistical analysis and experimental design

Statistical analyses were performed using the SPSS software package (version 2019) (SPSS, 2017). The experimental design was arranged in a completely randomized design (CRD). Each experiment (treatment) included five replicates, with five explants per replicate. The reported means were calculated based on 25 observations per treatment (n = 25). Data were statistically analyzed using one-way analysis of variance (ANOVA), and mean comparisons were performed at p ≤ 0.05 using Tukey’s HSD test.

3. Results

3.1. Influence of nanoparticles on in vitro growth of Thymus bovei

3.1.1. Silica celite nanoparticles (SiO₂)

MS media with different SiO2 concentrations differed in shoot length (Table 1; Figure 1). The maximum number of shoots (4.3) with the use of MS augmented with 5.0 mg L⁻¹SiO₂ nanoparticles. Shoot length was also maximized (45.4 mm) at 5.0 mg L⁻¹of SiO₂ compared to the control treatment (44.9 mm), while the shortest shoots (11.1 mm) were obtained at 10.0 mg L⁻¹of SiO₂. In addition, for fresh and dry weight, a statistical difference was observed between MS medium treatments with different SiO2 concentrations (Table 1). Maximum fresh and dry weight was obtained on MS medium added with 5.0 mg L⁻¹SiO₂ nanoparticle.

Table 1
Influence of various concentrations of silica celite (SiO₂) nanoparticles on the in vitro growth of Thymus bovei after a five-week growth period.

Silica Celite (SiO2) (mg L−1)	Number of axillary shoots/explants	Shoot length (mm)	Fresh weight/five explants (mg)	Dry weight/five explants(mg)
0.0	1.9 ± 0.3 b	30.6 ± 6.8 b	678.7 ± 6.2 b	69.6 ± 5.2 c
1.0	1.9 ± 0.3 b	13.1 ± 0.6 c	140.9 ± 6.6 c	23.1 ± 8.4 d
2.0	1.8 ± 0.3 b	13.6 ± 1.1 c	150.4 ± 6.3 c	27.2 ± 7.7 d
5.0	4.3 ± 0.9 a	45.4 ± 9.2 a	1242.5 ± 9.5 a	184.3 ± 5.7 a
10.0	1.8 ± 0.2 b	11.1 ± 0.4 c	175.1 ± 8.9 c	24.7 ± 9.9 d
50.0	1.9 ± 0.4 b	22.9 ± 3.92bc	767 ± 6.4 b	131.3 ± 5.8 b

• Means within a column with the same characters are not significantly different based on Tukey’s HSD test at P ≤ 0.05.

Figure 1
In vitro growth of Thymus bovei under different concentrations of silica celite nanoparticles after five weeks of culture. (A) T. bovei plantlets growing inside the culture flask containing 50 mg L⁻¹ silica celite nanoparticles, showing vigorous shoot elongation and leaf development. (B) Representative in vitro plantlets after removal from the medium, displaying morphological variations among treatments in terms of shoot height, leaf number, and root formation. The bars represent 1.0 cm.

3.1.2. Copper oxide (CuO)

The different treatment of CuO nanoparticles in MS media gave a variety in shoot growth (Table 2). The highest numbers of axillary shoots were recorded at 10 mg L⁻¹and 50 mg L⁻¹of CuO nanoparticles (5.6 and 5.5 shoots per explant, respectively). Shoot length was significantly affected by CuO concentration on MS medium. The maximum shoot length (44.0 mm) was observed at 10 mg L⁻¹of CuO, whereas at 50 mg L⁻¹produced the shortest shoots (15.9 mm). The highest fresh and dry weights were significantly different in MS medium supplemented with 5.0 mg L⁻¹CuO (801.6 and 111.6 mg, respectively) (P≤0.05) (Table 2).

Table 2
Influence of various concentrations of copper oxide nanoparticles on the in vitro grown Thymus bovei after a five-week growth period.

Copper oxide Concentration (mg L−1)	Number of axillary shoots/explant	Shoot length (mm)	Fresh weight/five explants (mg)	Dry weight/five explants(mg)
0.0	1.9 ± 0.6 cd	31.7 ± 6.2 c	583.2 ± 7.4 b	96.5 ± 9.1 b
1.0	2.5 ± 0.3 c	28.1 ± 3.8 cd	433.4 ± 9.1 b	88.2 ± 9.8 b
2.0	3.1 ± 0.7 b	32.2 ± 4.4 c	529 ± 3.8 b	95.4 ± 2.4 b
5.0	3.9 ± 1.1 b	57.8 ± 5.3 a	801.6 ± 2.2 a	111.6 ± 6.1 a
10.0	5.6 ± 1.1 a	44.0 ± 6.6 b	796.1 ± 9.7 a	106 ± 2.5 a
50.0	5.5 ± 0.2 a	15.9 ± 1.8 d	280 ± 3.4 c	33.6 ± 6.4 c

• Means within a column with the same characters are not significantly different based on Tukey’s HSD test at P ≤ 0.05.

3.2. Influence of nanoparticles with growth regulators on in vitro growth of T. bovei

3.2.1. BA with CuO nanoparticles

The effect of combination BA with 10 mg L⁻¹CuO nanoparticles on the in vitro growth of T. bovei is presented in Table 3. The highest number of axillary shoots (3.7 shoots per explant) and the maximum shoot length (62.4 mm) were recorded in the treatment containing 5 mg L⁻¹CuO without BA. Increasing BA concentration in the presence of 5 mg L⁻¹CuO did not improve shoot proliferation or shoot elongation (Table 3). Similarly, fresh and dry weight recorded the maximum measures in the control treatment (1339.2 and 268.7 mg, respectively). Whereas the lowest values were observed at 0.5 mg L⁻¹BA with 5 mg L⁻¹CuO (Table 3).

Table 3
Influence of various concentrations of BA with 5 mg L⁻¹CuO nanoparticles on the in vitro growth of Thymus bovei after a five-week growth period.

BA (mg L−1)	CuO (mg L−1)	Number of axillary shoot/explant	Shoot length (mm)	Fresh weight/five explants (mg)	Dry weight/five explants(mg)
0.0	5	3.7 ± 1.5 a	62.4 ± 4.1 a	1339.2 ± 7.2 a	268.7 ± 7.2 a
0.5	5	1.1 ± 0.18 b	15.5 ± 0.6 b	185.6 ± 4.6 d	59.5 ± 9.5 d
1.0	5	3.2 ± 1.1 a	17.6 ± 1.1 b	528.1 ± 6.7 b	232.1 ± 4.7 b
1.5	5	1.2 ± 0.6 b	16.5 ± 0.4 b	301.1 ± 3.4 c	91.5 ± 6.4 c
2.0	5	1.3 ± 0.4 b	17.5 ± 0.5 b	350.4 ± 3.9 c	108.5 ± 4.1 c

• Means within a column with the same characters are not significantly different based on Tukey’s HSD test at P ≤ 0.05.

3.2.2. BA with SiO2 and CuO nanoparticles

The effect of BA with 5.0 mg L⁻¹SiO2CuO nanoparticles on the in vitro growth of T. bovei is presented in Table 4. Among different concentrations of BA, the MS medium containing 1.5 to 2.0 mg L⁻¹BA with 5.0 mg L⁻¹SiO2CuO nanoparticles, formed higher multiplication rate (7.2-7.3 shoots/explants) than any other concentration tried for micropropagation. Shoot length was negatively affected by the presence of BA at various concentrations, they ranged from 16.1 to 25.5 mm (Table 4). Treatments with 1.5 mg L⁻¹BA and with 5.0 mg L⁻¹SiO2CuO nanoparticles resulted in a maximum fresh weight of 1041.5 mg, and a dry weight of 152 mg of T. bovei biomass (Table 4).

Table 4
Influence of various concentrations of BA with 5.0 mg L⁻¹SiO₂CuO nanoparticles on the in vitro grown T. bovei after a five-week growth period.

BA (mg L−1)	SiO2CuO (mg L−1)	Number of axillary shoot/explant	Shoot length (mm)	Fresh weight/five explants (mg)	Dry weight/five explants(mg)
0.0	5.0	2.95 ± 0.4 b	29.2 ± 4.6 a	574.4 ± 60.5 b	103.6 ± 21.7 b
0.5	5.0	3.25 ± 0.5 b	24.7 ± 1.8 b	407.1 ± 76.8 c	93 ± 16.5 b
1.0	5.0	3.30 ± 0.3 b	16.1 ± 0.9 c	205.4 ± 67.2 d	56.1 ± 17.5 c
1.5	5.0	7.20 ± 0.8 a	23.25 ± 1.6 b	1041.5 ± 93.1 a	152 ± 8.4 a
2.0	5.0	7.30 ± 1.2 a	25.5 ± 2.5 b	590.1 ± 79.3 b	131.9 ± 6.3 ab

• Means within a column with the same characters are not significantly different based on Tukey’s HSD test at P ≤ 0.05.

3.3. Antibacterial activity of T. bovei

3.3.1. Acetone crude plant extract

The antibacterial activity of acetonic crude extracts of T. bovei derived from ex vitro plants, in vitro plants, nano-plants, and nano-BA-plant against Salmonella, E. coli, B. subtilis, and S. aureus is presented in Table 5. The acetone extracts exhibited clear antibacterial potential with inhibition zone diameters ranging from 1.7 to 3.7 cm. For Salmonella, the nano-plant acetone extract produced the largest inhibition zone (3.3 cm), comparable to the positive control meropenem (3.4 cm). In the case of E. coli, antibacterial activity was detected only with the in vitro plant acetone extract (1.7 cm), while no inhibition was observed for ex vitro, nano-plant, or nano-BA-plant extracts. For B. subtilis, all acetone extracts demonstrated observable antibacterial activity. The ex vitro acetone extract showed an inhibitory effect of 3.3 cm, exceeding that of the positive control, meropenem (2.7 cm). Similarly, the acetone extract derived from nano-plants exhibited a pronounced inhibitory effect against S. aureus, producing the largest inhibition zone (3.7 cm), which was slightly higher than the positive control (3.6 cm).

Table 5
Antibacterial activity of the acetonic crude extract of different Thymus bovei plant extracts using the agar well diffusion method (inhibition zone in cm).

Bacteria Strain	Inhibition zone (cm)
	Acetone extracts				Positive control	Negative control
	ex vitro-plant	in vitro plants	nano-BA-plant	nano-plant	Meropenem	DMSO
Salmonella sp	1.9 ± 0.03 c	2.8 ± 0.6 b	2.4 ± 0.03 b	3.3 ± 0.12 a	3.4 ± 0.1 a	0.0 ± 0.0 d
Escherichia coli	0.0 ± 0.0 c	1.7 ± 0.03 b	0.0 ± 0.0 c	0.0 ± 0.0 c	3.9 ± 0.03 a	0.0 ± 0.0 c
Bacillus subitilis	3.3 ± 0.03 a	2.1 ± 0.06 c	2.1 ± 0.03 c	2.1 ± 0.09 c	2.7 ± 0.1 b	0.0 ± 0.0 d
Staphylococcus aureus	3.2 ± 0.03 b	2.8 ± 0.03 b	2.2 ± 0.09 c	3.7 ± 0.06 a	3.6 ± 0.0 a	0.0 ± 0.0 d

• Means within a row with the same characters are not significantly different based on Tukey’s HSD test at P ≤ 0.05.

3.3.2. Methanolic crude plant extract

The methanoicl extracts from T. bovei possessed different antimicrobial activity against Salmonella, E. coli, B. subtilis, and S. aureus (Table 6). The highest antibacterial activity againstS. aureus (2.5 cm) was found using the ex vitro plant. For Salmonella, inhibition zones ranged from 2.0 to 2.2 cm, with the highest activity observed in the ex vitro and nano-plant extracts. For B. subtilis, all methanolic extracts showed inhibitory activity, with inhibition zones ranging from 1.5 to 2.33 cm. The nano-plant extract produced the largest inhibition zone (2.3 cm), exceeding that of the positive control and highlighting the superior antimicrobial efficacy of extracts derived from nano-treated plants. Likewise, S. aureus exhibited high sensitivity to the methanolic extracts, with the ex vitro plant extract producing the largest inhibition zone (2.5 cm), followed by the nano-plant extract (2.2 cm). In contrast, E. coli demonstrated notable resistance to the methanolic extracts. No antibacterial activity was detected for the ex vitro, in vitro, or nano-BA-plant extracts (0.0 cm), whereas only the nano-plant extract showed a limited inhibitory effect (1.3 cm). These results indicate that T. bovei methanolic extracts are particularly effective against Gram-positive pathogens.

Table 6
Antibacterial activity of the methanolic crude extract of different types ofnextract plants using the agar well diffusion method (inhibition zone in cm).

Bacteria Strain	Inhibition zone (cm)
	Methanol				Positive control	Negative control
	ex vitro-plant	in vitro plants	nano-BA-plant	nano-plant	Meropenem	DMSO
Salmonella sp	2.2 ± 0.12 b	2.0 ± 0.0 b	2.0 ± 0.1 b	2.2 ± 0.12 b	3.4 ± 0.1 a	0.0 ± 0.0 c
Escherichia coli	0.0 ± 0.0 c	0.0 ± 0.0 c	0.0 ± 0.0 c	1.3 ± 0.03 b	3.9 ± 0.03 a	0.0 ± 0.0 c
Bacillus subitili	2.1 ± 0.03 b	1.6 ± 0.03 c	1.5 ± 0.06 c	2.3 ± 0.12 b	2.7 ± 0.1 a	0.0 ± 0.0 d
Staphylococcus aureus	2.5 ± 0.03 b	2.0 ± 0.0 c	2.0 ± 0.06 c	2.2 ± 0.12 bc	3.6 ± 0.0 a	0.0 ± 0.0 d

• Means within a row with the same characters are not significantly different based on Tukey’s HSD test at P ≤ 0.05.

3.3.3. Ethanolic crude plant extract

The ethanolic extracts exhibited noticeable antibacterial activity against Salmonella, B. subtilis, and S. aureus. For Salmonella, inhibition zones ranged from 1.9 to 3.9 cm. The maximum inhibition zone was recorded for the nano-plant extract (3.9 cm), which exceeded the positive control meropenem (3.4 cm). B. subtilis, inhibition zones varied between 1.3 and 2.9 cm, and the nano-plant extract again showed the strongest antibacterial effect (2.9 cm). Similarly, S. aureus was highly sensitive to ethanolic extracts, with the nano-plant extract producing the largest inhibition zone (3.1 cm). In contrast, E. coli exhibited a high degree of resistance to ethanolic extracts. No inhibitory activity was observed for E. coli with ex vitro, in vitro, or nano-AB-plant extracts, whereas only the nano-plant extract demonstrated weak activity (1.1 cm).

4. Discussion

4.1. In vitro propagation

In vitro propagation has been widely used for forming a large number of plants in a short period (Al-Alouni et al., 2016; Mherat et al., 2022; Shahrour et al., 2024a; Al-Shayeb et al., 2025). Recently, the influence of different nanoparticles and hormones was also evaluated on in vitro growth for Dianthus chinensis and Chrysanthemum morifolium (Sreelekshmi et al., 2022; Kazmi et al., 2025). In the current investigation, nanoparticle use affects the in vitro growth of T. bovei. High concentrations of nanoparticles affected the number of new shoots, shoot length, and biomass (Fresh weight and dry weight) (Tables 1 to 4). Among different concentrations of BA at 1.5 to 2.0 mg L⁻¹ with 5.0 mg L⁻¹SiO₂CuO nanoparticles, a high multiplication rate (7.2-7.3 shoots/explants) was obtained. Nanoparticles can enter the cell wall and cell membranes of plants and can be passed to soft tissues to enhance shoot formation (DeRosa et al. 2010). This is similar to previous results obtained by Parzymies et al. (2019) and Pour et al. (2019), who reported the use of nanotechnology for improving seed germination, plant growth improvement, genetic modification, and crop protection. In addition, the supplementation of nanoparticles to the medium affected plant growth by altering effectiveness, gene expression, antioxidant enzyme activity, ROS production, and inhibition of ethylene production (Manickavasagam et al., 2019; Sreelekshmi et al., 2022). The use of nanoparticles in the current study demonstrated a positive influence on the in vitro growth of the T. bovei plant. Nanoparticles may increase water, nutrient, hormone, and nanoparticle uptake, which may stimulate metabolic activity in the plants. Moreover, these nanoparticles may act as a non-fertilizer, cause increased in cell division in the plants. Furthermore, nanoparticles may increase growth because they act as hormone-like effects on the plants (Tripathi et al., 2017; Rizwan et al., 2019; Bello-Bello and Spinoso-Castillo, 2023). These NPs may enhanced nutrient uptake and physiological efficiency, perhaps by improved membrane transport and metabolic activity (Tripathi et al., 2017). Lee et al. (2025) indicated that NPS can rise oxidative balance by enhancing antioxidant enzymes, which can assist maintain reactive oxygen species homeostasis at optimal levels. Thus, MS medium augmented with SiO2CuO nanoparticles is significant for in vitro propagation methods, and can significantly increase the efficiency of tissue culture techniques. The existences of nanoparticles improves the absorption of nutrients which leads to increase in vitro growth of T. bovei. Adding of cytokinins and nanoparticles is vital for developing in vitro propagation methods, as these additives can outstandingly increase the efficiency of in vitro culture. The application of NPs in T. bovei tissue culture exhibited a optimistic influence on development, emphasizing the optimistic effect of NPS in micropropagation.

4.2. Antimicrobial activity

The current study showed that ex vitro, in vitro plant, nano-plan, and nano-AB-plant of T. bovei displayed observable antimicrobial activity (Tables 5, 6 and 7). Ex vitro and in vitro plants extract of T. bovei using different solvent systems showed noticeable activity against the tested organisms. In addition, the current investigation provides evidence that T. bovei could be a source of new antibacterial agents even against Salmonella, B. subtilis, and S. aureus. Acetone, ethanol, and methanol extracts showed different antibacterial activity (Tables 5, 6 and 7). This finding agrees with those previously reported by Kakasy et al. (2001), as the extracts exhibited an acidic nature (pH values ranging between 5.0 and 5.5). The acidity, combined with the presence of bioactive components in the extracts, might enhance their antimicrobial activity against the bacteria. Vassiliou et al. (2023) reported that the thymus contains many ingredients that show different remedial properties. Demirci et al. (2018) reported that thymol (carvacrol) has potent antimicrobial and antifungal properties. Thyme oil carvacrol exists in many wild plants (Dahham et al., 2021). In addition, Tural and Turhan (2017) mention that the antibacterial activity of the thyme extract was associated with thymol, carvacrol, and cinnamic acid compounds. In addition, Walsh et al. (2019) reported that thymol extracts containing phenolic compounds such as thymol, carvacrol, and eugenol have a strong antimicrobial activity at high levels. Similarly, Burt (2004) reported that T. bovei was used as an antimicrobial agent against bacteria. These valuable properties are associated with the presence of thymol, carvacrol, p-cymene, and γ-terpinene (Rasooli and Mirmostafa, 2002).

Table 7
Antibacterial activity of the ethanolic crude extract of different types of Thymus bovei plant extracts using agar well diffusion method (inhibition zone in cm).

Bacteria Strain	Inhibition zone (cm)
	Ethanol				Positive control	Negative control
	ex vitro-plant	in vitro plants	nano-BA-plant	nano-plant	Meropenem	DMSO
Salmonella sp	1.9 ± 0.3 c	1.9 ± 0.07 c	2.5 ± 0.1 b	3.9 ± 0.1 a	3.4 ± 0.1 a	0.0 ± 0.0 d
Escherichia coli	0.0 ± 0.0 c	0.0 ± 0.0 c	0.0 ± 0.0 c	1.1 ± 0.1 b	3.9 ± 0.1 a	0.0 ± 0.0 c
Bacillus subitili	1.3 ± 0.3 b	1.5 ± 0.1 b	1.6 ± 0.1 b	2.9 ± 0.2 a	2.7 ± 0.1 a	0.0 ± 0.0 c
Staphylococcus aureus	2.1 ± 0.2 b	2.2 ± 0.1 b	2.2 ± 0.1 b	3.1 ± 0.1 a	3.6 ± 0.1 a	0.0 ± 0.0 c

• Means within a row with the same characters are not significantly different based on Tukey’s HSD test at P ≤ 0.05.

Thymols can destroy the integrity and function of biological membranes of microbial cells that make microorganisms sensitive to thymol oils (Lambert et al. 2001). Moreover, there are high quantities of secondary metabolites in plants, such as polyphenols resulted in different degrees of bacteria suppression due to many factors, such as the solvent used, the quantity of phytochemicals that have been extracted, the amount of extraction, and the multiplicity of inhibitory mixtures extracted (Obeidat et al., 2011). The current study showed high antimicrobial activity of the acetone, ethanol, and methanol extracts in the presence of nanoparticles in medium growth. This refers to the presence of a high quantity of phytochemicals in acetone, ethanol, and methanol extracts, which disrupted bacteria growth. In addition to that, nanoparticles can catalyze the formation of ROS, which may damage proteins and lipids of bacterial cells, leading to cell death (Safavi, 2014; Chauhan et al., 2021).

5. Conclusions

The present study showed favorable influence of SiO2 and CuO nanoparticles in MS medium on shoot growth of T. bovei in in vitro culture. Maximum rate of shoot formation (5.6) was recorded on MS medium with CuO at 10.0 mg L⁻¹ and maximum shoot length of 62.40 mm was obtained in 0.0 mg L⁻¹ BA with 5.0 mg L⁻¹ CuO. Bacterial activity was seen in ex vitro plants, in vitro plants, nano-plants and nano-BA-plant extracts. Water extracts did not exhibit any reasonable action against Salmonella, E. coli, B. subtilis and S. aureus (data not shown). The maximum inhibitory zone was obtained by utilizing nano-plant extracts, it was 3.7 cm for S. aureus by acetonic extracts and 3.9 cm for Salmonella sp. by ethanolic extracts. The present inquiry described a simple and stable protocol for successful in vitro plant propagation of T. bovei. Moreover, it showed the antibacterial activity of T. bovei plant extracts.

Data Availability Statement

All the data is available in the article, and any additional data will be available at request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  31 Aug 2026
• Date of issue
  2026

History

• Received
  11 May 2026
• Accepted
  29 June 2026