Acta agriculturae Slovenica, 121/3, 1–11, Ljubljana 2025 doi:10.14720/aas.2025.121.3.18920 Original research article / izvirni znanstveni članek Successful micropropagation of critically endangered Thymus bovei Benth: A wild medicinal plant from the Jordanian environment Refad Y. ALKHAWALDAH 1, Rida A. SHIBLI 1, 2, 3, Reham W. TAHTAMOUNI 4, Mohamad A. SHATNA- WI 5, Tamadour AL-QUDAH 6, Tamara S. AL-QUDAH 2 Received June 10, 2024, accepted September 02, 2025 Delo je prispelo 10. junij 2024, sprejeto 2. september 2025 1 ,Department of Horticulture and Crop Sciences, Faculty of Agriculture, University of Jordan, Amman, Jordan. 2 Hamdi Mango Center for Scientific Research (HMCSR), University of Jordan, Amman, Jordan. 3 Corresponding author E-mail: r.shibli@ju.edu.jo 4 Department of Social and Applied Sciences, Princess Alia University College, Al-Balqa Applied University. 5 Department of Biotechnology, Faculty of Agricultural Technology, Al-Balqa Applied University, Al-Salt 19117, Jordan 6 Department of Nutrition and Food Technology, Faculty of Agriculture, Mutah University, Karak, Jordan. Successful micropropagation of critically endangered Thymus bovei Benth: A wild medicinal plant from the Jordanian en- vironment Abstract: Thymus bovei Benth. (Lamiaceae), locally re- ferred to as Zateer Barry, is a critically endangered native plant species in Jordan (Taifour and EL-Oqlah, 2014), renowned for its exceptional medicinal properties. This study aims to estab- lish a reliable micropropagation technique to prevent T. bovei from extinction. The research focused on explant establish- ment, shoot multiplication, rooting, callus induction, and ac- climatization of T. bovei. Explants were established in a half- strength Murashige and Skoog (MS) media supplemented with different concentrations of gibberellic acid (GA3). Successful shoot multiplication was achieved by subculturing nodal seg- ments onto half-strength MS medium supplemented with ki- netin (KIN), thidiazuron (TDZ), or benzylaminopurine (BA). Among the treatments, the most effective medium for shoot multiplication was supplemented with 2.5 mg l-1 KIN and 0.5 mg l-1 GA₃, which resulted in an average of 7.50 ± 0.54 micro- shoots per explant and a shoot length of 2.33 ± 0.18 cm. Root- ing was most effective on half-strength MS media with 1.0 mg l-1 indole-3-butyric acid (IBA), yielding an average of 5.29 roots per microshoot. The highest callus development, measured by fresh mass (0.601 g), occurred in half-strength MS media with 2.0 mg l-1 2,4-D, while no callus formed with naphthaleneacetic acid (NAA). Acclimatized plants showed a 90 % survival rate when transferred to greenhouse conditions. Key words: endangered plant; Jordan; medicinal plant; micropropagation; tissue culture; Thymus bovei Uspešna razmnožitev kritično ogrožene vrste materine dušice (Thymus bovei Benth), samonikle zdravilne rastline iz Jorda- nije Izvleček: Vrsta materine dušice, Thymus bovei Benth., (Lamiaceae), lokalno imenovana kot ‘Zateer Barry’ je kritično ogrožena samonikla zdravilna rastlina v Jordaniji, poznana zaradi izrednih zdravilnih lastnosti. V raziskavi je predstavljena zanesljiva tehnika njene mikropropagacije, da bi preprečili nje- no izumrtje. Poudarek raziskave je bil na ohranjanju izsečkov, indukciji kalusa, nastajanju, vkoreninjanju in aklimatizaciji poganjkov. Izsečki so bili gojeni na polovičnem Murashige in Skoogovem (MS) gojišču, ki so mu bile dodane različne kon- centracije giberilinske kisline (GA3). Uspešen razvoj poganjkov je bil dosežen pri gojenju nodijskih segmentov na polovičnem MS gojišču, ki so mu dodali kinetin (KIN), tidiazuron (TDZ) ali benzilaminopurin (BA). Med obravnavanji je bila najbolj učinkovita tvorba poganjkov na gojišču, ki so mu dodali 2,5 mg l-1 KIN in 0,5 mg l-1 GA₃, ko je v poprečju nastalo 7,50 ± 0,54 mi- kropoganjkov na izseček, z dolžino poganjka 2,33 ± 0,18 cm. Vkoreninjanje je bilo najbolj učinkovito na polovičnem MS gojišču z dodatkom 1,0 mg l-1 indol-3-maslene kisline (IBA), kar je v poprečju dalo 5,29 korenin na mikropoganjek. Največja tvorba kalusa, merjena kot sveža masa (0,601 g), je nastala na polovičnem MS gojišču z dodatkom 2,0 mg l-1 2,4-D, medtem, ko se kalus pri dodatku naftalenocetne kisline (NAA) sploh ni tvoril. Aklimatizirane rastline so imele pri prenosu v rastlinjak 90 % preživetje. Ključne besede: ogrožene rastline, Jordanija, zdravilne rastline; mikropropagacija, tkivna kultura, Thymus bovei Acta agriculturae Slovenica, 121/3 – 20252 R. Y. ALKHAWALDAH et al. 1 INTRODUCTION In recent years, a surge in global interest in tradi- tional medical systems has been noticed. According to current projections, developing countries account for the vast majority of users worldwide in terms of their dependence on medicinal plants to meet health needs (Al-Qura’n, 2011). Despite the availability of modern medicine in developing countries, the use of medici- nal herbs (phytomedicines) persists due to popular and historical culture (Karim and Al-Qura’n, 1986). Jordan is distinguished by its rich plant diversity, owing to its small size; it contains nearly 2530 wild plant species, (Al- Qura’n, 2011; Royal Botanic Garden (RBG), 2012). The Jordan flora has 485 medicinal plant species divided into 330 genera and 99 families (Al-Eisawi, 1996). Thymus bovei is known as (Zateer Barry) in the Middle East. It is a medicinal and fragrant perennial herb of the genus Thymus with a variety of medicinal and cu- linary uses. Due to its remarkable medicinal properties, T. bovei is known as one of the most famous thyme spe- cies (Hassan et al., 2018). This has been attributed to the anthelmintic, expectorant, antispasmodic, and antiseptic properties of its essential oil, which was prescribed since ancient times to cure respiratory, skin, and blood infec- tions (Tepe et al., 2011; Abdel-Hady et al., 2014). T. bovei essential oil contains phytochemical compounds such as thymol, carvacrol and trans-geraniol and has good anti- oxidant, antibacterial, and anthelmintic properties (Jara- dat et al., 2016). T. bovei is found in different regions of Jordan including Irbid, Petra, Mafraq, Azraq, and Tafila and it has been observed in some localities but not in any protected area in Jordan, where this species is subject to excessive collection by people for food and medicinal uses (Taifour and EL-Oqlah, 2014). One of the most important modern techniques for preserving plants and their genetic resources is tissue culture (Engelmann and Engel, 2002; Shibli, et al., 2006). Now micropropagation is the alternative effective meth- od of plant propagation leaving behind the conventional propagation methods. In vitro propagation will be suit- able for fast plant proliferation, removal of pathogens, germplasm preservation, and production of secondary metabolites (Negash, et al., 2001; Ahmad, 2013); Shatna- wi, et al., 2006). Conservation via tissue culture provides a powerful and diverse set of procedures that can be used when other conservation strategies are not possible. In vitro culture of endangered plants offers several advan- tages, including the capacity to produce species with lim- ited reproductive potential and those found in vulnerable environments (Fay 1992). Previous investigations of Thy- mus sp. using in vitro techniques have been industrial- ized, including Thymus spicata L., T. vulgaris L., T. longi- caulis C.Presl and T. lotocephalus G.López & R.Morales (Ozudogru et al. 2011; Coelho et al., 2012; Tahtamouni et al., 2016). Plant growth regulators (PGRs) play a pivotal role in regulating developmental processes during micropro- pagation (Sabagh et al., 2021). Among these, auxins, cy- tokinins, and gibberellins are the most commonly used. Auxins such as indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA) are es- sential for root initiation and callus formation (Elmongy et al., 2018). Cytokinins, including benzylaminopurine (BA), kinetin (KIN), and thidiazuron (TDZ), stimulate cell division and promote shoot induction and multipli- cation (Ali et al., 2022). Gibberellins, particularly gibber- ellic acid (GA₃), are mainly involved in shoot elongation and enhancing overall plantlet vigor (Shah et al., 2023). The interaction and concentration of these hormones greatly influence the success of each micropropagation stage, including explant establishment, shoot multiplica- tion, rooting, and acclimatization (Grzelak et al., 2024). Due to the significance of T. bovei as a medicinal herb, and because it is one of the many rare plants found in only a few locations within the Jordanian environ- ment, this study was conducted to develop a reliable pro- tocol for its preservation. T. bovei is native to the eastern Mediterranean region and, unfortunately, is subject to se- vere overharvesting and the impacts of climate change in Jordan. The aim of this study was to establish a depend- able method for the mass propagation of T. bovei through tissue culture, one of the most widely adopted in vitro conservation techniques for maintaining plant genetic resources. 2 MATERIALS AND METHODS Research was carried out at the Hamdi Mango Cent- er for Scientific Research (HMCSR) in the plant tissue culture labs of the University of Jordan. The plant sam- ples (aerial parts) were gathered from a naturally grow- ing solitary plant found in Almafraq, Jordan, (Latitude: 315282E, Longitude: 355425N) (Figure 1). 2.1 ESTABLISHMENT OF THYMUS BOVEI EX- PLANTS Nodal segments of Thymus bovei were subjected to surface sterilization to ensure aseptic conditions for in vitro culture. Initially, the explants were washed with a few drops of mild detergent and rinsed thoroughly under running tap water for 15 minutes to remove surface de- bris. The segments were then immersed in a 1.5 % (v/v) Acta agriculturae Slovenica, 121/3 – 2025 3 Successful micropropagation of critically endangered Thymus bovei Benth: A wild medicinal plant from the Jordanian environment sodium hypochlorite (NaOCl) solution for 10 minutes under a laminar airflow cabinet. Afterward, they were rinsed three times with sterile distilled water, with each rinse lasting 5 minutes. This was followed by a 30 second immersion in 70 % ethanol (v/v), and finally, the explants were again rinsed three times with sterile distilled water (5 minutes each) under aseptic conditions in the laminar airflow cabinet. The sterilized explants were transferred into Erlenmeyer flasks containing 100 ml of half-strength solid Murashige and Skoog (MS) medium (Murashige & Skoog, 1962), supplemented with 30 g  l-1 sucrose and 8 g  l-1 agar. Different concentrations of gibberellic acid (GA₃) (0, 0.5, and 1.0 mg l-1) were added to the medium to promote bud growth and to determine the optimal GA₃ concentration for shoot multiplication when com- bined with other micropropagation growth regulators. The rate of shoot emergence was measured after 4 weeks. Cultures were grown under specific conditions (16/ 8 h of light/dark period at 24 ±2 °C). 2.2 SHOOT MULTIPLICATION OF THYMUS BOVEI Two-centimeter nodal segments (each with two nodes) from previously established tissue culture plants were placed in 100 ml of MS medium (Murashige and Skoog, 1962), supplemented with 30 g l-1 sucrose, 8 g l-1 agar, and varying concentrations (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg l-1) of 6-benzyladenine (BA), kinetin (KIN), or thidiazuron (TDZ), along with 0.5 mg l-1 GA₃. The ex- plants were cultured for four weeks at 24 ± 2 °C under a 16/8 h (light/dark) photoperiod. Growth parameters, including the number of microshoots and shoot height, were recorded after four weeks of culture. 2.3 ROOTING OF THYMUS BOVEI Shoot tips were subcultured in solid MS media con- taining varying concentrations (list here from scheme) of auxin indole acetic acid (IAA), or indole-3-butyric acid (IBA), or 1-naphthalene-acetic acid (NAA). The cultures were kept at 24 ± 2 °C for four weeks, under 16 hours of light and 8 hours of darkness. Growth parameters, in- cluding root number, root length, and shoot height, were recorded after four weeks of incubation with the corre- sponding growth regulators. 2.4 CALLUS INDUCTION AND MULTIPLICATION OF THYMUS BOVEI Shoot tips were transferred to solid MS media supplemented with 3 % sucrose, and 0.8 % agar, with different concentrations of auxins 1-naphthalene-acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.0, 1.0, 2.0, and 3.0 mg l-1). Half of the cultures were grown under 16-hour light/8-hour dark cycle, while the other half were grown under dark conditions. The cul- tures were maintained at 24 ± 2 ⁰C for 6 weeks. After six weeks, the callus was harvested for determination of fresh weight. 2.5 EX VITRO ACCLIMATIZATION OF THYMUS BOVEI Once the plantlets had developed roots, they were exposed for 3 days in test tubes in laboratory before transferring to the greenhouse environment where tem- peratures were maintained at 24 ± 2 during the day and 20 ± 2 ºC at night, with intermittent misting. The plant- lets were grown in trays filled with 1 part peat moss and 1 part perlite. 2.6 DATA ANALYSIS In the current study, a completely randomized de- sign (CRD) was applied. For the shooting percentage, one explant was placed per replicate, with a total of 20 replicates for each GA₃ concentration. For shoot mul- tiplication experiments, each treatment was performed 5 times (4 explants per replicate). Rooting experiments Figure 1: Wild Thymus bovei collected in Almafraq, Jorden, (Latitude: 315282E, Longitude: 355425N). Blue bar represents 10 cm. Acta agriculturae Slovenica, 121/3 – 20254 R. Y. ALKHAWALDAH et al. included 10 replicates (one explant per replicate in test tubes), while callus experiments had 10 replicates (2 explants per replicate in petri dishes). For acclimatiza- tion experiments, 10 plants were used for each hormone treatment. SPSS software was used to analyze the results. The standard error of the mean was calculated, and anal- ysis of variance (ANOVA) was performed for each ex- periment. The Tukey HSD test was used to differentiate means at p ≤ 0.05. 3 RESULT AND DISCUSSION A comprehensive in vitro propagation protocol was successfully developed for Thymus bovei, a critically endangered medicinal plant. The study demonstrated that the addition of gibberellic acid (GA₃), particularly at 0.5 mg  l-1, significantly improved explant establish- ment. Among the cytokinins tested, kinetin (KIN) at 2.5 mg  l-1 yielded the highest shoot multiplication rate and shoot number, outperforming both benzylaminopurine (BA) and thidiazuron (TDZ). For root induction, indole- 3-butyric acid (IBA) at 1.0 mg l-1 was the most effective, producing the greatest number of roots and root length, while naphthaleneacetic acid (NAA) was ineffective un- der light conditions. Callus induction was successfully achieved using 2, 4-D at 2.0 mg l-1, whereas NAA failed to induce callus under the same conditions. The regener- ated plantlets were successfully acclimatized with a high survival rate, confirming the feasibility of this protocol for conservation and large-scale propagation of T. bovei. 3.1 GA₃ ENHANCES SUCCESSFUL IN VITRO ES- TABLISHMENT OF THYMUS BOVEI Thymus bovei is a critically endangered plant spe- cies that requires efficient propagation methods for its conservation. To improve explant establishment in vitro, we tested the effect of different concentrations of gib- berellic acid (GA₃) on shoot tip culture. The most im- portant result showed that culturing shoot tips on MS medium supplemented with 0.5 mg  l-1 GA₃ led to the highest production of new microshoots (90 %) (Table 1, Figure 2). GA₃ is a well-known plant hormone that pro- motes growth by stimulating seed germination and the transition from meristem to shoot development (Gupta et al., 2013). Similar positive effects of GA₃ on shoot proliferation have been reported in Thymus satureioides Coss. (Aicha et al., 2013) and Mentha x piperita L. (Is- lam and Alam, 2018). These findings indicate that GA₃ at 0.5 mg/L effectively enhances the in vitro establishment of T. bovei and supports its micropropagation efforts. 3.2 SHOOT MULTIPLICATION IS SIGNIFICANTLY INFLUENCED BY TYPE AND CONCENTRA- TION OF CYTOKININS, BA PROMOTES MOD- ERATE SHOOT PROLIFERATION BUT LESS EFFECTIVELY THAN KIN Cytokinins are widely used to promote shoot multiplica- tion in plant tissue culture. To evaluate the effect of ben- zylaminopurine (BA) on Thymus bovei, nodal segments were cultured on MS medium supplemented with 2.5 mg  l-1 BA. This treatment resulted in a moderate shoot proliferation rate, producing an average of 4.3 shoots per explant with the shortest shoot length of 2.0 cm (Table 2). Notably, no callus formation was observed under these conditions. BA is known to stimulate protein synthesis, which enhances cell division and growth (Royani et al., 2021). Our findings align with previous reports of BA’s role in promoting shoot multiplication in Lamiaceae species, such as patchouli (Pogostemon cablin (Blanco) Benth.) (Swamy et al., 2016). Therefore, while BA posi- tively influences shoot proliferation in T. bovei, its effect GA3 Conc. (mg l -1) Shoots (%)* 0.0 10 ± 1.0 bx 0.5 90 ± 10.0 a 1.0 20 ± 1.5 b Table 1: Effect of GA3 concentration on the percentage of emerging shoots of Thymus bovei. *Shooting (%): Represents the mean percentage of explants that pro- duced shoots. One explant was placed per replicate, with a total of 20 replicates for each GA₃ concentration. xThe values presented are means ± standard error, N = 20.. Figure 2: Microshoot formation of Thymus bovei cultured on MS medium supplemented with 30 g  l-1 sucrose and 8 g  l-1 agar. (A) Control MS medium without growth regulators. (B) MS medium supplemented with 0.5 mg l-1 GA₃. Cultures were maintained for 2 weeks at 24 °C under a 16/8 h light/dark pho- toperiod. The blue bar represents 1.0 cm. Acta agriculturae Slovenica, 121/3 – 2025 5 Successful micropropagation of critically endangered Thymus bovei Benth: A wild medicinal plant from the Jordanian environment is less pronounced compared to kinetin. 3.3 KIN AT 2.5 MG L-1 ACHIEVES THE HIGHEST SHOOT MULTIPLICATION RATE Cytokinins play a crucial role in stimulating shoot proliferation in plant tissue culture. To determine the ef- fectiveness of kinetin (KIN) on Thymus bovei, explants were cultured on MS medium supplemented with 2.5 mg  l-1 KIN. This treatment resulted in the highest shoot multiplication rate, with an average of 7.5 shoots per ex- plant, surpassing the control (Table 3). Additionally, shoot length was significantly increased compared to lower KIN concentrations, and no callus formation was observed. These results are consistent with Teshome et al. (2016), who reported the longest shoot length (6.33 cm) in lemon- scented thyme (Satureja punctata (Benth.) R.Br. ex Briq.)) at the same KIN concentration. Similar positive effects of KIN were also documented in kidney tea plants (Ortho- siphon aristatus (Blume) Miq.))) (Jayakumar et al., 2013). Overall, 2.5 mg  l-1 KIN is highly effective in promoting shoot multiplication in T. bovei. 3.4 TDZ INDUCES SHOOT FORMATION BUT RE- SULTS IN SHORTER, STUNTED SHOOTS Thidiazuron (TDZ) is known for its strong cytoki- nin-like activity in promoting shoot multiplication. To evaluate its effect on Thymus bovei, explants were cultured on MS medium supplemented with varying TDZ concen- trations. The highest number of microshoots per explant (4.85) was observed at 0.5 mg l-1 TDZ (Table 4). However, increasing TDZ concentrations led to a decline in shoot multiplication and significantly reduced shoot length, with the shortest shoots (2.08 cm) recorded at 3.0 mg l-1. While TDZ has shown excellent multiplication performance in some Lamiaceae species, such as wild mint (Mentha ar- vensis L.) where it promoted both high shoot numbers and length (Faisal et al., 2014), it can also have adverse effects. For instance, negative impacts of TDZ were reported in Sideritis athoa Papan. & Kokkini.and Thymus moroderi Pau ex Martinez (Papafotiou and Kalantzis, 2009; Marco-Medina and Casas, 2015). These results suggest that although TDZ can induce shoot formation in T. bovei, its higher concentra- tions may lead to stunted growth, limiting its practical ap- plication. 3.5 AUXIN TYPE DETERMINES ROOTING EFFI- CIENCY IN T. BOVEI MICROSHOOTS; NAA SHOWS LIMITED ROOT INDUCTION POTEN- TIAL IN T. BOVEI Auxins like naphthalene acetic acid (NAA) are often BA Conc. (mg -1) Microshoot number/ explant Shoot length (cm) 0.0 2.85 ± 0.28 cx 3.41 ± 0.10 a 0.5 2.85 ± 0.31 c 2.60 ± 0.07 b 1.0 3.90 ± 0.19 ab 2.40 ± 0.06 bc 1.5 3.85 ± 0.18 ab 2.35 ± 0.08 bcd 2.0 3.80 ± 0.20 ab 2.33 ± 0.18 bc 2.5 4.30 ± 0.21 a 2.00 ± 0.00 d 3.0 3.20 ± 0.26 bc 2.11 ± 0.04 cd Table 2: Effect of BA concentration on shoot multiplication in vitro of Thymus bovei after four weeks’ growth period xThe values are means ± standard error, N = 5. KIN Conc. (mg l-) Microshoot number/ explant Shoot length (cm) 0.0 2.85 ± 0.28 cx 3.41 ± 0.10 d 0.5 2.85 ± 0.29 c 4.55 ± 0.11 a 1.0 4.30 ± 0.26 b 4.00 ± 0.16 b 1.5 4.60 ± 0.23 b 3.90 ± 0.06 c 2.0 4.70 ± 0.23 b 3.86 ± 0.11 c 2.5 7.50 ± 0.54 a 2.33 ± 0.18 e 3.0 6.50 ± 0.32 b 3.50 ± 0.12 d Table 3: Effect of kinetin level on shoot multiplication in vitro of Thymus bovei after four weeks’ growth periods x The values presented represent the means ± standard error, N = 5 TDZ Conc. (mg l-1) Microshoot number/explant Shoot length (cm) 0.0 2.85 ± 0.28 cx 3.41 ± 0.10 a 0.5 4.85 ± 0.58 a 2.08 ± 0.02 c 1.0 4.05 ± 0.32 ab 2.41 ± 0.09 bc 1.5 4.10 ± 0.33 ab 2.48 ± 0.07 b 2.0 3.75 ± 0.41 ab 2.28 ± 0.06 bc 2.5 2.95 ± 0.26 c 2.19 ± 0.04 bc 3.0 2.60 ± 0.31 c 2.32 ± 0.09 bc x The values presented represent the means ± standard error, N = 5 Table 4: Effect of thidiazuron (TDZ) level on in vitro shoot multiplication of Thymus bovei after four weeks’ growth period Acta agriculturae Slovenica, 121/3 – 20256 R. Y. ALKHAWALDAH et al. used to induce rooting in plant tissue culture. To assess its effect on Thymus bovei, explants were treated with various concentrations of NAA; however, no root formation was observed at any tested concentration (Table 5, Figure 3). These findings are consistent with Teshome et al. (2016), who also reported the absence of rooting in lemon-scented thyme (Satureja punctata) under similar NAA treatments. In contrast, Sarropoulou and Maloupa (2019) observed rooting induction at 0.5 mg l-1 NAA in Satureja thymbra L., indicating that rooting response to NAA may be species- specific within the Lamiaceae family. Interestingly, shoot height increased with higher NAA concentrations, pos- sibly due to enhanced shoot apical dominance, as noted by Teshome and Soromessa (2015) in Satureja abyssinica (Hochst. ex Benth.) Briq.. Therefore, while NAA does not promote rooting in T. bovei, it may influence shoot elonga- tion. 3.6 IBA AT 1.0 MG  L-1 INDUCES OPTIMAL ROOT- ING AND ROOT DEVELOPMENT Indole-3-butyric acid (IBA) is widely recognized as an effective auxin for promoting root initiation in plant tissue culture. To determine its effect on Thymus bovei micro- shoots, various concentrations of IBA were tested for root- ing efficiency. Root formation increased with IBA concen- tration up to 1.0 mg l-1, where the highest average number NAA conc. (mg l-1) Roots number Roots length (cm) Shoot height (cm) 0.0 0 0 2.53 ± 0.14 bx 0.5 0 0 3.40 ± 0.37 ab 1.0 0 0 3.25 ± 0.24 ab 1.5 0 0 3.69 ± 0.31 a 2.0 0 0 3.41 ± 0.33 ab Table 5: Effect of 1-naphthalene acetic acid (NAA) level on the number of roots, root length, and shoot length, of in vitro grown T bovei after four weeks. xThe values presented are the means ± standard error, N = 10 IBA conc. (mg l-1) Roots number Roots length (cm) Shoot height (cm) 0.0 0.00 d 0.00 cx 2.53 ± 0.14 c 0.5 2.33 ± 0.16 c 0.10 ± 0.0 c 2.98 ± 0.11 bc 1.0 5.29 ± 0.42 a 0.43 ± 0.06 a 6.16 ± 0.64 a 1.5 4.20 ± 0.17 b 0.26 ± 0.02 b 4.26 ± 0.41 b 2.0 2.67 ± 0.06 c 0.27 ± 0.01 b 3.58 ± 0.51 bc Figure 3: Effect of 2.0 mg l-1 NAA on roots of in vitro grown T bovei. The blue bar represents 2.0 cm. Table 6: Effect of indole-3-butyric acid (IBA) level on rooting, root length, and shoot length, of in vitro grown T bovei after four weeks. xThe values presented are the means ± standard error, N = 10 Figure 4: Effect of 1.0 mg l-1 IBA on roots of T bovei. The blue bar represents 1.0 cm. Acta agriculturae Slovenica, 121/3 – 2025 7 Successful micropropagation of critically endangered Thymus bovei Benth: A wild medicinal plant from the Jordanian environment of roots per microshoot (5.29) and root length (0.43 cm) were recorded (Table 6, Figure 4). However, concentrations above 1.0 mg l-1 caused a significant decline in both root number and length, and the control group without IBA showed no rooting. The positive effect of IBA is attributed to its ability to elevate endogenous auxin levels, enhancing stability and reducing catabolism through conjugation with growth inhibitors (Frick & Strader, 2018). Similar results were reported by Mustafa and Weal (2022) in Thymus ser- pyllum L., where 1.0 mg l-1 IBA yielded the highest rooting rates. These findings confirm that 1.0 mg l-1 IBA is optimal for inducing rooting and root development in T. bovei mi- croshoots. 3.7 IAA PROMOTES ROOTING BUT LESS EFFEC- TIVELY COMPARED TO IBA Indole-3-acetic acid (IAA) is another commonly used auxin known to stimulate root formation in plant tissue cultures. To evaluate its effectiveness in Thymus bovei, dif- ferent concentrations of IAA were added to the MS medi- um. The highest rooting response was observed at 1.0 mg l-1 IAA, resulting in an average of 3.5 roots per explant and a root length of 0.32 cm (Table 7, Figure 5). Although IAA positively influenced rooting, its effect was less pronounced compared to IBA under similar conditions. Comparable re- sults were reported in Origanum elongatum (Bonnet) Emb. & Maire by Benkaddour et al. (2022), where IAA promoted adventitious root formation. Furthermore, Sagharyan et al. (2021) demonstrated that increasing IAA concentrations enhanced rooting in Nepeta binaloudensis Jamzad, with the highest rooting observed at 1.5 mg l-1. These findings sug- gest that while IAA facilitates root development in T. bovei, it is less effective than IBA, and optimal concentrations vary among species. 3.8 CALLUS SUCCESSFULLY INDUCED USING 2,4- D BUT NOT NAA: 2,4-D AT 2.0 MG L-1 YIELDS THE HIGHEST CALLUS BIOMASS Callus induction is a crucial step in plant tissue cul- ture for regeneration and secondary metabolite produc- tion. To determine the optimal auxin concentration for callus formation in Thymus bovei, different levels of 2,4-di- chlorophenoxyacetic acid (2,4-D) were tested under light conditions. The highest callus biomass was obtained with 2.0 mg l-1 2,4-D, producing a fresh mass of 0.601 g, which was significantly greater than the control (Table 8, Figure 6). This result confirms the positive role of 2,4-D in pro- moting callus growth, consistent with previous findings in Thymus persicus (Ronniger ex Rech. f.) Jalas reported by Bakhtia et al. (2016). Similar callus induction effects of 2,4- D have been documented in several medicinal plants such as Achyranthes aspera L: (Sen et al., 2014), Vitex negundo L. (Choudhury et al., 2011), Aquilaria agallocha Roxb. (Debnath, 2013), and Centella asiatica (L.) Urban (Bira- dar, 2017). Additionally, Suhartanto et al. (2022) observed that callus fresh mass was higher in cultures grown in the dark (115.1 mg) compared to those grown under light (96.3 mg), highlighting the influence of light conditions on cal- IAA conc. (mg l-1) Roots number Roots length (cm) Shoot height (cm) 0.0 0.0 d 0.00 dx 2.53 ± 0.14 b 0.5 1.0 ± 0.0 c 0.20 ± 0.0 bc 2.76 ± 0.11 ab 1.0 3.5 ± 0.26 a 0.32 ± 0.04 a 3.18 ± 0.13 a 1.5 3.0 ± 0.0 a 0.30 ± 0.06 ab 2.79 ± 0.26 ab 2.0 2.4 ± 0.17 b 0.15 ± 0.01 c 2.38 ± 0.84 b Table 7. Effect of indole acetic acid (IAA) level on the rooting, root length, and shoot length, of in vitro grown T bovei after four weeks. xThe values presented are the means ± standard error, N = 10 Figure 5: Effect of 1.0 mg l-1 IAA on roots of T bovei. The blue bar represents 1.0 cm. Acta agriculturae Slovenica, 121/3 – 20258 R. Y. ALKHAWALDAH et al. lus development. Overall, 2,4-D at 2.0 mg l-1 under light is effective for inducing substantial callus biomass in T. bovei. 3.9 NAA FAILS TO INDUCE CALLUS FORMATION IN LIGHT CONDITIONS Callus induction is influenced by both plant growth regulators and environmental factors such as light. In this study, no callus formation was observed at any con- centration of naphthalene acetic acid (NAA) under light conditions. However, under dark conditions, callus de- veloped successfully, with the highest fresh massof 1.124 g recorded at 3.0 mg  l-1 NAA (Table 9, Figure 7). This indicates that the effect of NAA on callus induction in Thymus bovei is dependent on light exposure. Supporting these findings, Niloofar et al. (2020) reported increased callus production in Salvia tebesana Bunge when NAA was added to MS media. Similarly, Bahrames (2005) observed enhanced callusing in Trigonella corniculata (L.) L. leaf explants with NAA treatment. These results highlight the importance of optimizing both hormonal and environmental conditions to achieve efficient callus induction. 3.10 ACCLIMATIZED PLANTS SHOW HIGH SUR- VIVAL RATES UNDER GREENHOUSE CONDI- TIONS Successful acclimatization is a crucial step for the transition of in vitro cultured plants to soil conditions. In this study, Thymus bovei plantlets were efficiently accli- matized, achieving a high survival rate of 90% for plants treated with 1.0 mg l-1 IBA and IAA (Figure 8). Moreover, 2,4-D (mg l-1) Dark Light % of callusing* Fresh mass (g) % of callusing Fresh mass (g) 0.0 0 c 0 dx 0 d 0 d 1.0 100 ± 0 a 0.240 ± 0.01 b 75 ± 15 b 0.343 ± 0.1 b 2.0 100 ± 0 a 0.343 ± 0.01 a 100 ± 0 a 0.601 ± 0.14 a 3.0 50 ± 5 b 0.113 ± 0.02 c 62.5 ± 10 c 0.184 ± 0.0 c Table 8: Effect of 2,4-D level on callus induction of Thymus bovei after six weeks’ growth period. % Callusing = (Number of explants that produced callus/Total number of explants) × 100 xThe values presented represent the means ± standard error. N = 10 Figure 6: Callus induction from leaf discs on 2,4-D (2.0 mg l-1) under light. The bar represents 1.0 cm. NAA. (mg l-1) Dark Light % of callusing* Fresh mass (g) % of callusing Fresh mass (g) 0.0 0 0 0 0 1.0 100 ± 0 0.675 ± 0.24 bx 0 0 2.0 100 ± 0 0.655 ± 0.11 b 0 0 3.0 100 ± 0 1.124 ± 0.24 a 0 0 Table 9: The effect of 1-naphthalene-acetic (NAA) level on callus induction of Thymus bovei after six weeks growth period. % Callusing = (Number of explants that produced callus/Total number of explants) × 100 xThe values presented represent the means ± standard error. N = 10 Acta agriculturae Slovenica, 121/3 – 2025 9 Successful micropropagation of critically endangered Thymus bovei Benth: A wild medicinal plant from the Jordanian environment these plantlets exhibited robust health and vigor under greenhouse conditions. These results are consistent with previous findings by Sarropoulou and Maloupa (2019), who reported successful acclimatization of other Lamia- ceae tissue-cultured plants such as Satureja thymbra L. Similarly, Teshome and Soromessa (2015) observed ex- cellent ex vitro adaptation and survival rates between 88 % and 96 % in plantlets of various Satureja species, including S. abyssinica. These findings emphasize the ef- fectiveness of the acclimatization protocols used and sug- gest promising potential for large-scale propagation of T. bovei. 4 CONCLUSIONS T. bovei was successful multiplicated in vitro for the first time in Jordan. MS medium with 2.5 mg l-1 KIN and 0.5 mg l-1 GA3 was reported to be the most effective for generating new microshoots. Meanwhile, rooting was the most successful in shoot tips treated with 1.0 mg l-1 IBA. Callus formation occurred under both light and dark conditions in media supplemented with 2.0 mg  l-1 2,4-D. However, when NAA was used, callus induction was observed only under dark conditions. Meanwhile, most plants were successfully acclimatized after being transferred to greenhouse conditions. 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