Acta agriculturae Slovenica, 119/2, 1–10, Ljubljana 2023 doi:10.14720/aas.2023.119.2.2738 Original research article / izvirni znanstveni članek Establishment of an in vitro method for micropropagation of ironwort, (Sideritis raeseri Boiss. & Heldr.) Valbona SOTA 1, 2 , Donald SHUKA 3 , Shawky BEKHEET 4 , Efigjeni KONGJIKA 5 Received June 22, 2022; accepted April 08, 2023. Delo je prispelo 22. junija 2022, sprejeto 8. aprila 2023 1 Department of Biotechnology, Faculty of Natural Sciences, University of Tirana, Tirana, Albania 2 Corresponding author, e-mail: valbona.sota@fshn.edu.al 3 Department of Biology, University of Vlora “Ismail Qemali” , Vlora, Albania 4 Departament of Plant Biotechnology, National Research Center, Cairo, Egypt 5 Academy of Sciences of Albania, Section of Natural and Technical Sciences, Tirana, Albania Establishment of an in vitro method for micropropagation of ironwort (Sideritis raeseri Boiss. & Heldr.) Abstract: Ironwort / Mountain Tea (Sideritis raeseri Boiss & Heldr.) is an endangered (EN) plant species in Albania. This study aimed to develop a rapid clonal propagation protocol using in vitro methodologies. The ironwort seeds were pre-tre- ated with three concentrations of GA 3 (250, 500, and 1000 mg l -1 ). During the inoculation stage, two types of culture media, Murashige & Skoog (MS) and Woody Plant Medium (WPM), were tested, and the effects of both GA 3 concentration and cul- ture media used were evaluated. For the subculture stage, three cytokinins (6-benzylaminopurine / BAP , kinetin, zeatin) at four concentrations (0.5; 1.0; 1.5; 2.0 mg l -1 ), were compared for the RGR index, while for the rooting stage, two different auxins (1-naphthaleneacetic acid / NAA and indole-3-butyric acid / IBA) at four concentrations (0.5; 1.0; 1.5; 2.0 mg l -1 ) were tested. GA 3 at 500 mg l -1 and MS medium resulted as more effective. The highest value of the RGR index during the subculture stage was obtained in the MS nutrient medium supplemented with BAP at 1.5 mg l -1 . For rhizogenesis response, IBA was more effective for roots and length number. Based on these results, in vitro methodologies can be a promising tool for the mass production of this endangered plant species and with possible applications for enhancing the production of valuable nutra- ceuticals. Key words: mountain tea; micropropagation; seed germi- nation; nutrient medium; GA 3 concentration Vzpostavitev in vitro metode za mikropropagacijo albanskega sklepnjaka (Sideritis raeseri Boiss. & Heldr.) Izvleček: Vrsta Sideritis raeseri Boiss & Heldr. je ogrožena (EN) rastlinska vrsta Albanije, sorodna vrsti Sideritis scardica Gris., poznani kot šarplaninski čaj. Namen raziskave je bil raz- viti protokol hitrega klonskega razmnoževanja te vrste z in vitro metodo. Semena so bila predhodno obdelana s tremi koncen- tracijami giberilinov (GA 3 ; 250, 500, in 1000 mg l -1 ). Na stopnji inokulacije sta bili preiskuševani in ovrednoteni dve vrsti go- jišč, Murashige & Skoog (MS) gojišče in gojišče za lesnate ra- stline (WPM) hkrati z učinki različnih koncentracij giberilinov. V prvi fazi gojenja so bili preiskušeni trije citokinini (6-benzila- minopurin (BAP), kinetin, zeatin) v štirih koncentracijah (0,5; 1,0; 1,5; 2,0 mg l -1 ) in primerjani z indeksom relativne prirasti (RGR). V fazi ukoreninjanja sta bila preiskuševana dva auksi- na (1-naftalen ocetna kislina (NAA) in indol-3-maslena kislina (IBA) v štirih koncentracijah (0,5; 1,0; 1,5; 20 mg l -1 ). Giberilini (GA 3 ) pri koncentraciji 500 mg l -1 in MS gojišče so bili najbolj učinkoviti. Največja vrednost indeksa relativne prirasti (RGR) je bila v prvi fazi gojenja dobljena v gojišču MS z dodatkom BAP 1.5 mg l -1 . Za nastanek korenin je bil IBA bolj učinkovit tako glede števila kot dolžine korenin. Na osnovi teh rezultatov lahko zaključimo, da je in vitro metoda obetajoče orodje za ma- sovno razmnoževanje te ogrožene vrste z možnostjo uporabe pri pospešeni proizvodnji vrednih hranilnih snovi. Ključne besede: albanski sklepnjak; mikropropagacija; kalitev semen; gojišča; GA 3 koncentracija Acta agriculturae Slovenica, 119/2 – 2023 2 V. S O TA et al. 1 INTRODUCTION Mountain tea (Sideritis raeseri Boiss et Heldr.), also known as Ironwort, is an aromatic medicinal na- tive plant of the western Balkans, including south Alba- nia, southeastern parts of North Macedonia, and North Greece (Zekaj et al., 2008). It is considered a Balkan en- demic species due to its restricted distribution range of only three countries. Due to its high quantity of bioac- tive compounds with a high percentage of antioxidants, mountain tea has been widely utilized in alternative medicine since ancient times (Romanucci et al., 2017; Tadić et al., 2021). According to Hodaj et al. (2017), it is also used as an herbal tea to treat digestive system dis- orders, coughs and as a dietary supplement for avoiding anemia. The European Medicinal Agency (EMA) has ap- proved the use of mountain tea, due to its medicinal val- ues and its marketing in the market or pharmacy, based on its traditional use for at least 30 years in Europe and particularly in the Balkans, where the population has the experience and accurate information on optimal daily dosing (EMA, 2016). According to Tomasini & Theilade (2019), moun- tain tea is a culturally and commercially significant im- portant species locally consumed as a tea and used for the treatment of flu symptoms and respiratory problems, as well as harvested for trade. However, due to its wide- spread use, it has been subjected to uncontrolled and de- structive harvest practices in its distribution area of oc- currence in Albanian territory, resulting in a decrease in the wild populations of the mountain tea (Bojadzi et al., 2012; Tomasini & Theilade, 2019). Mountain tea populations have been reduced by 50 % in the Prespa area from 1990 until today, and since its population in Albania has been reduced by 30 % and de- structive harvest practices of the natural population con- tinue, it has been assessed as endangered (EN) species by the Albanian government (Shuka & Malo, 2010; MoE, 2013; Shuka et al., 2021). Furthermore, S. raeseri, with its closest relative, S. scardica, is listed as species of high conservation interest for the western Balkan countries (Aneva & Zhelev, 2018). In light of the preceding, it is critical to improving the situation through in situ and ex situ cultivation and conservation techniques. Furthermore, in vitro technolo- gies are effectively applied with the goal of rapid mass production via clonal propagation and the establishment of a genetic collection for the conservation of endangered plant species of economic importance. The medicinal and aromatic plants (MAPs), among others, are the main focus of these techniques’ application because of their importance and widespread use in pharmacy and medi- cine (Neergheen-Bhujun et al., 2017; Moraes et al., 2021). Seasonal variations, growing practices, the expense of production, as well as other factors all impede large- scale phytochemical production from field-grown plants. The application of biotechnological techniques would be of significant interest in this area, not only for biomass production but also for optimizing the production of secondary metabolites (Georgiev et al., 2009; Cardoso & Silva, 2013; Kapoor et al., 2018). Efforts are being made to modify the organogenesis of the secretory structures of MAPs in terms of density or glandular diameter (Van- tu & Gales, 2009; Sota et al., 2019; Sota et al., 2020), as well as the possibility of increased synthesis of essential secondary metabolites, using specific physic-chemical parameters under in vitro conditions (Avato et al., 2005; Tousi et al., 2010; Sharma et al., 2015; Radić et al., 2016; Jamwal et al., 2018).  The success of in vitro stabilization and multipli- cation of plant germplasm is determined by several pa- rameters, including the explant chosen, the physical or chemical treatments applied, and any pretreatment used. Some studies (Shtereva et al., 2015; Papafotiou & Kalantzis, 2009; Danova et al., 2013) employed seeds as primary explants and isolated nodal explants for further multiplication via subcultures for Sideritis sp. In many cases, when seeds are used as initial explants, pretreat- ment with GA 3 is seen as effective in order to enhance their germination and faster proliferation under in vitro conditions (Khuat et al., 2022; Cornea-Cipcigan et al., 2020; Rout et al., 2017; Arabaci et al., 2014; Gashi et al., 2012). This effect is related to the synthesis of α-amylases, essential enzymes that help and promote breaking seed dormancy (Finch-Savage & Leubner-Metzger, 2006). For Sideritis leucantha Cav., a Spanish endemic species, Juan- Vicedo et al. (2021) refined a micropropagation and cry- opreservation strategy using shoot explants. Sarropoulou & Maloupa1 (2015) studied the effects of various dikegu- lac sodium concentrations, a PGR that enhance lateral growth, on in vitro regeneration of S. raeseri using shoot tips as primary explants. In some of these studies, efforts have been made to find a suitable plant growth regula- tors (PGRs) ratio in different stages of micropropagation that enhanced in vitro regeneration with potential uses for other purposes such as conservation or secondary metabolites production.  Because many Sideritis species are indigenous to certain places, they are adapted to the native growing conditions in their natural habitats. Hence, adaptation abilities are likely to vary significantly among species in the genus. This study aimed to stabilize an effective micropropagation protocol by using various concentra- tions of GA 3 for enhancing seed germination under in vitro conditions and confronting some PGRs ratios for in Acta agriculturae Slovenica, 119/2 – 2023 3 Establishment of an in vitro method for micropropagation of ironwort ... vitro regeneration and rooting induction on the derived plantlets. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL COLLECTION AND DISIN- FECTION As primary explants were used mature seeds of Si- deritis raeseri Boiss. & Heldr. collected in the National Park of Prespa, Albania. The seeds were left for 30 min in tap water and, after that, were sterilized with 5.20 % sodium hypochlorite solution for 15 minutes. 2.2 GA 3 PRETREATMENT FOR SEED GERMINA- TION ENHANCEMENT  Before inoculation in culture vessels, the seeds were treated for 24 h in GA 3 solution. Three concentrations of gibberellic acid (GA 3 ), specifically I: 250 mg l -1 ; II: 500 mg l -1 ; III: 1000 mg l -1 , were tested and compared. After this treatment, the explants were inoculated in the nutrient medium, and their in vitro cultivation was initiated. 2.3 MEDIA COMPOSITION IN EACH STAGE OF MICROPROPAGATION Inoculation and seeds germination stage: after GA 3 treatment, the explants were inoculated in a nutrient me- dium for their germination. Two different basal media, specifically Murashige-Skoog (MS) medium (Murashige & Skoog, 1962) and Woody Plant Medium (WPM) (Lloyd & McCown, 1980) were compared, each of them supplemented with 1-naphthaleneacetic acid (NAA) at 0.1 mg l -1 and 6-benzylaminopurine (BAP) at 1 mg l -1 . Seeds’ germination started 6 – 7 days of culture, but germination percentage and morphometric parameters (shoot length and leaves number) were evaluated after 30 days of culture. Subculture stage: For shoots regeneration, MS basal medium was used, and the effect of three different cyto- kinins (BAP , kinetin, zeatin) at four concentrations (0.5; 1.0; 1.5; 2.0 mg l -1 ) were tested and compared. The plant material was weighed before inoculation in each treat- ment (initial mass - M1), while after 30 days, the biomass obtained in each treatment was weighed (final mass - M2). After that, the relative growth rate (RGR) following the formula: RGR = (lnM2 – lnM1) / (no. of days) x 100 was evaluated, where ln is the natural logarithm, and FM is the fresh mass (Gatti et al., 2017). Rooting stage: For rhizogenesis induction, MS basal medium was used, and the effect of two different auxins, specifically -naphthaleneacetic acid (NAA) and indole- 3-butyric acid (IBA) at four concentrations (0.5; 1.0; 1.5; 2.0 mg l -1 ), were tested and compared. In this stage, roots number and lengths were evaluated. In all cases, all media were enriched with sucrose at 3 % and agar at 0.57 %. The pH was adjusted to 5.7 prior to autoclaving. 2.4 INCUBATION CONDITIONS The cultures were maintained in the growth cham- ber at a temperature 25 °C ± 2 °C with a 16 h light / 24 h photoperiod with cool, white fluorescent light of inten- sity 43.4 mmol m -2 s -1 . 2.5 STATISTICAL ANALYSES For each treatment, 30 explants were used, and all experiments were repeated at least three times. Experi- mental data was elaborated by the Student’s Test and the analysis of variance (ANOVA) with JMP 7.0 statistical software. Seeds germination, morphometric parameters, and RGR index in each cultivation stage were measured after 30 days of culture. 3 RESULTS 3.1 EFFECT OF GA 3 AND MEDIA TYPE ON IN VITRO SEEDS GERMINATION In this experiment, seed germination and shoots development of S. raeseri affected by GA 3 and the type of culture media were investigated. Seeds started germina- tion after 6 7 days of culture, and shoot / root organo- genesis was observed due to the proliferation of zygotic embryos (Fig. 1 a). Within a week, these organs are dif- ferentiated (Fig. 1 b, c). The obtained results showed that seeds pre-treatment with GA 3 solution gave high germi- nation rates in all concentrations used.  From the variability chart (Fig. 2), it can be ob- served that the differences in this parameter were not in- fluenced by the type of basal media used but only by the GA 3 concentration. The results clearly show no statistical differences between MS and WPM media for the same concentration of GA 3 . Therefore, treatment with GA 3 at 500 mg l -1 was evaluated as the most effective concentra- tion, resulting specifically in an 88.4 % of germination rate for MS medium and 86.4 % for WPM medium. Acta agriculturae Slovenica, 119/2 – 2023 4 V. S O TA et al. After seeds germination, morphological charac- teristics such as shoots length (cm) and the number of leaves were monitored to detect if the pre-treatment of seeds or the basal media used caused any significant dif- ference in these monitored parameters. For the above, the obtained results were interesting (Fig. 3). Regarding shoots length (cm), no differences were observed caused by the basal medium type. Even for this parameter, the highest results were obtained in GA 3 solution at 500 mg l -1 , precisely 2.31 cm for MS medium and 2.16 cm for WPM medium. The lowest results were obtained in GA 3 solution at 1000 mg l -1 for both basal media used (Fig. 2 a). The same trend was observed even for the number of leaves, where the best results were obtained in GA 3 solu- tion at 500 mg l -1 . However, significant differences were observed between MS and WPM medium for this con- centration (precisely, 17.01 in MS medium and 14.22 in WPM medium). For the other GA 3 concentrations, no differences were observed between MS and WPM media for the leaves number parameter (Fig. 2 b). In an overall analysis, we conclude that pretreatment with GA 3 at 500 mg l -1 is the most effective concentration, and cultivation in MS basal medium is more advantageous than WPM. GA 3 is the plant hormone that is crucial in break- ing seed dormancy. From our data, it is clear that GA 3 positively affects in vitro organogenesis by stimulat- ing the proliferation of embryos within the seed to give shoots and roots. In all treatments with GA 3 , is observed not only the germination rate at high values but also the increase of biomass of the monitored biometric param- eters. So, it is evidenced that the GA 3 beneficial role in promoting seed development of S. raeseri. 3.2 BIOMASS PRODUCTION UNDER DIFFERENT CONCENTRATIONS AND TYPES OF CYTO- KININS The proliferated plantlets were subcultured for fur- ther multiplication, whereas before inoculation in the nutrient medium, the roots were removed, and small shoots were used for this purpose. A few days after cul- tivation in the subculture stage, new shoots and leaves Figure 1: Sideritis raeseri Boiss. & Heldr. micropropagation a) Seed germination under in vitro conditions b, c) Shoot and root differentiation d) Shoots regeneration during subculture e) Rhizogenesis induction Figure 2: Variability chart for germination rate depending on the GA 3 concentration and basal media used Acta agriculturae Slovenica, 119/2 – 2023 5 Establishment of an in vitro method for micropropagation of ironwort ... were formed in all concentrations or types of cytokinins supplemented in the nutrient media (Fig. 1 d). The ini- tial and final mass results indicated that both type and cytokinin concentration affected in vitro regeneration of plantlets during this stage (Tab. 1). Regarding the variability for the RGR index between different cytokinins for the same concentration (Fig. 4a), it can be said that except for the lowest concentration used of 0.5 mg l -1 where kinetin showed the highest ef- fectiveness of the three cytokinins used, in all other con- centrations the highest value of RGR was obtained from the use in the nutrient medium of BAP. In most cases, kinetin and zeatin are very close to each other in their ef- fectiveness concerning the value of RGR according to the measurements performed, except for the RGR value at 1.5 mg l -1 of cytokinins concentration, where zeatin gave the lowest value. Regarding comparing different concen- trations within the same type of cytokinin (Fig. 4b) for kinetin and BAP , the best results for RGR value were ob- tained when using 1.5 mg l -1 of each cytokinin, precisely 5.72 for kinetin and 6.11 for BAP. While for zeatin, the most optimal concentration resulted the one at 2.0 mg l -1 . An overall analysis of the obtained data showed that the most effective treatment, depending on the cytokinin type and concentration, was the use of BAP at 1.5 mg l -1 , where the RGR value obtained is equal to 6.11. Figure 3: Oneway Analysis of a) Shoots length (cm); b) Leaves number; depending on the GA 3 concentration and basal media used Table 1: Plantlets’ weight before and after subculture stage Kinetin Zeatin BAP Concentration (mg l -1 ) 0.5 1 1.5 2 0.5 1 1.5 2 0.5 1 1.5 2 Initial mass (g) (M1) 1.04 1.12 1.08 1.20 1.10 1.08 1.28 1.15 1.12 1.04 1.10 1.13 Final mass (g) (M2) 3.38 4.21 6.01 5.77 2.81 3.92 4.49 5.57 3.07 4.31 6.88 5.98 RGR index 3.93 4.41 5.72 5.23 3.13 4.30 4.18 5.26 3.36 4.74 6.11 5.55 Acta agriculturae Slovenica, 119/2 – 2023 6 V. S O TA et al. 3.3 IN VITRO ROOT FORMATION USING DIFFER- ENT CONCENTRATIONS OF IBA AND NAA Root formation is a crucial stage for the micropro- pagation of plants reproduced in vitro. In this part of the study, the effect of IBA and NAA added separately in four concentrations on in vitro rooting of S. raeseri was in- vestigated. The rooting response was observed at a high rate in all treatments (Fig. 1 e), but even in this stage was observed that the monitored morphometric parameters (number of roots and their length) are highly affected by the type and auxin concentration (Fig. 5 a; b). From an overall evaluation, IBA resulted more effective than NAA for both parameters under evaluation. Regarding roots length, the most effective treatment resulted in the use of IBA at 1.5 mg l -1 , a value (3.51 cm) statistically different from all the other values obtained, followed by the use of IBA at 1 mg l -1 and NAA at 1.5 mg l -1 , with mean values respectively 3.38 and 3.35. As for roots number, all the treatments with IBA showed higher values of this param- eter, and the best result was obtained at 2.0 mg l -1 of IBA, with a respective value of 14.21 roots/plantlet. 4 DISCUSSION Seed germination is a complex process, and GA 3 plays a crucial role in controlling and encouraging ger- mination in many plant species. In this respect, exog- enous applications of GA 3 are primarily used during in vivo or in vitro plant cultivation for enhancing seeds germination. Our study showed that adding GA 3 to the culture medium, regardless of concentration, increased the percentage of in vitro Sideritis raeseri germination, indicating the role of GA in breaking dormancy. Further- more, the results revealed that the seed treatments sig- nificantly affected the germination and seedling growth parameters. Maximum germination and other seedling growth parameters were observed with 500 mg l -1 of GA 3 . Otherwise, seedlings derived from GA 3 -treated seeds showed normal morphology. In this regard, in their re- port, Cornea-Cipcigan et al. (2020) concluded that exog- enous applications of GA 3 stimulated not only the germi- nation of Cyclamen sp. but also higher rates of biometric parameters in the obtained plantlets. Similarly, Gashi et al. (2012) found that using 1000 mg l -1 GA 3 + 0.3 % KNO 3 highly stimulated the germina- tion of Ramonda serbica Pančić seeds grown in Petri dish- es under controlled physical conditions. Also, Arabaci et al. (2014) mentioned that the pre-treatment of Sideritis perfoliata L. seeds with GA 3 at 100 mg l -1 for two hours resulted in a 100 % of germination rate. Furthermore, in their study on seed germination of Vasconcellea stipulate V .M. Badillo, Vélez-Mora et al. (2015) found effective the use of GA 3 at 1.44 µM in Nitch & Nitch basal medium, which significantly stimulated seeds germination. Simi- larly, Ake et al. (2007) obtained positive results on in vitro germination of coconut embryos by supplementing the semi-solid medium with GA 3 at 4,6 µM. Meanwhile, Ni- kam & Barmukh (2009) found effective the soaking of Santalum album L. seeds at 4 mM of GA 3 solution and obtained an 80.67 % of germination rate after seeds in vitro inoculation in MS medium.  In our study, there are no observed significant dif- ferences between MS and WPM basal media used for most of the results. Regarding the efficiency of MS media for in vitro regeneration of S. raeseri plantlets under in vitro conditions, our results are similar to those reported by other authors for micropropagation of different Sideri- tis species (Juan-Vicedo et al., 2021; Sevindik et al., 2019; Shtereva et al., 2015; Papafotiou & Kalantzis, 2009). On the other hand, Yavuz (2016) found compelling the use of B5 medium for in vitro regeneration of Sideritis stricta Benth. plantlets. Figure 4: Variability for RGR index between a) different cyto- kinins for the same concentration b) different concentrations for the same cytokinin Acta agriculturae Slovenica, 119/2 – 2023 7 Establishment of an in vitro method for micropropagation of ironwort ... Cytokinins have been used to stimulate plant growth and development as they favor cell division and cytokinesis, thus stimulating lateral shoots growth. In tissue culture, the types and concentrations of cytokinin added to culture media are the most important factors af- fecting the in vitro multiplication of plant propagules. In the present work, variations in the response of the mul- tiplication parameters of Sideritis raeseri were observed depending on the type and concentration of cytokinin. Among three types of cytokinins, i.e., BAP, kinetin, and zeatin, used for in vitro shoot multiplication, BAP at 1.5 mg l -1 was the most effective treatment. Shoot multipli- cation in the present study was obtained by enhancing shootlets’ fresh mass, which is crucial in employing tis- sue culture techniques for Sideritis raeseri micropropa- gation. Similar to our results, other authors, when con- fronting different types or concentrations of cytokinins, also have reported the effective use of BAP for in vitro multiplication of Sideritis sp. (Yavuz, 2016; Papafotiou & Kalantzis, 2009). Meanwhile, Juan-Vicedo et al. (2021) evidenced that for S. leucantha, the best results for shoot morphogenesis were obtained on a nutrient medium supplemented with 0.44 μM 2-isopentenyladenine. On the other hand, Shtereva et al. (2015) stated that for mi- cropropagation of S. scardica, the use of zeatin at 2 mg l -1 combined with 0.2 mg l -1 indole-3-acetic acid (IAA) was the best combination for shoot proliferation. For in vitro root formation, most plant species re- quire a medium supplemented with essentially auxin- specific PGRs. Usually, IBA, IAA, or NAA are used for the rhizogenesis of plant microshoots. In the present study, IBA was found to be superior over the NAA for in vitro root formation of S. raeseri since the highest values of root numbers and lengths were observed with IBA. Furthermore, the obtained roots’ appearance was healthy and suitable for successful acclimatization. Our findings on the effect of IBA in rhizogenesis induction of S. raeseri are in line with the ones reported by Yavuz (2016), who achieved the best rooting rate of S. stricta on B5 medium supplemented with 4.5 mg l -1 IBA. On the other hand, Figure 5: Oneway Analysis of a) Roots length (cm); b) Roots number, depending on the auxin type and/or concentration Acta agriculturae Slovenica, 119/2 – 2023 8 V. S O TA et al. for Sideritis leucantha the best auxin for rooting response resulted 1-naphthaleneacetic acid (Juan-Vicedo et al., 2021). In this respect, Ragavendran et al. (2012) rooted in vitro raised shoots of Passiflora foetida L. by use of IBA. 5 CONCLUSIONS An effective micropropagation method of Sideritis raeseri Boiss. & Heldr. was recognized using seeds as the initial explant. The in vitro germination rate and shoots length were strongly affected by the GA 3 concentration, where the treatment with GA 3 at 500 mg l -1 gave the best results. Many plants were obtained in the subculture sta- ge, where the most effective cytokinin was found to be the cytokinin BAP . For the monitored parameters (RGR), it can be concluded that the concentration of 1.5 mg l -1 of BAP and kinetin was the most optimal concentrati- on that strongly influences the in vitro regeneration of plantlets. At the same time, zeatin was found effective at the concentration of 2 mg l -1 . For rhizogenezis inducti- on, IBA was more effective than NAA for roots length and number. Effective clonal growth enables the creati- on of a plant collection with the potential for utilization in conservation programs, which are very important to apply to this endangered plant species. Also, this may further extend studies toward optimizing protocols for in vitro production of secondary metabolites from this im- portant medicinal species. 6 ACKNOWLEDGEMENTS This research is realized in the framework of two projects “Assessment of rare and endangered species of plants and invertebrates as well as their habitats in the mountainous areas of Korça, Berat, and Vlora Regions” , and “Enrichment of Plant Tissue Cultures Laboratory, FNS, UT in service of mass and quality production of fruit species required by farmers” , both financed by NAS- RI (National Agency for Scientific Research and Innova- tion) in Albania. 7 REFERENCES Aké, A. P., Maust, B., Orozco-Segovia, A., Oropeza, C. (2007). The effect of gibberellic acid on the in vitro germination of coconut zygotic embryos and their conversion into plant- lets. In vitro Cellular & Developmental Biology – Plant, 43, 247-253. https://doi.org/10.1007/s11627-006-9018-1 Aneva, I., & Zhelev, P . (2018). The ecological and floristic char - acteristics of populations of Sideritis scardica Griseb. in Olympus Mts., Greece. Ecologia Balkanica, 10(2), 93-99. Arabaci, O., Öğretmen, N. G., Tan, U., Yaşa, F. (2014). Effect of some seed treatments on germination of Sideritis perfoliata L. Trakya University Journal of Natural Sciences, 15(2), 83- 87. Avato, I. M., Fortunato, I. M., Ruta, C., D’ elia, R. (2005). Glan- dular hairs and essential oils in micropropagated plants of Salvia officinalis L. Plant Science, 169, 29–36. https://doi. org/10.1016/j.plantsci.2005.02.004 Bojadzi, A., Brajanoska, R, Stefkov, Gj., Fotiadis, G., Shumka, S., Avukatov, V . (2012). Conservation Action Plan for Moun- tain tea in the Prespa Lakes Watershed (Final Report). UNDP/GEF project “Integrated ecosystem management in the Prespa lakes basin” 2012, pp 66. Cardoso, J. C., Silva, J. A. T . (2013). Micropropagation of Zeyhe- ria montana Mart. (Bignoniaceae), an endangered endemic medicinal species from the Brazilian cerrado biome. In vitro Cellular & Developmental Biology-Plant, 49, 710-716. https://doi.org/10.1007/s11627-013-9558-0 Cornea-Cipcigan, M., Pamfil, D., Sisea, C. R., Mărgăoan, R. (2020). Gibberellic acid can improve seed germination and ornamental quality of selected Cyclamen species grown under short and long days. Agronomy, 10, 516. https://doi. org/10.3390/agronomy10040516 Danova, K., Evstatieva, L., Todorova, M., Trendafilova, A., Wol- fram, E. (2013). Optimization of in vitro culture system for biomass and polyphenolics production in Inula britannica and Sideritis scardica Sofia 2 cultivar. Planta Medica 79 - PN21. https://doi.org/10.1055/s-0033-1352365 European Medicine Agency (EMA) (2016). Assessment report on Sideritis scardica Griseb.; Sideritis clandestina (Bory & Chaub.) Hayek; Sideritis raeseri Boiss. & Heldr.; Sideri- tis syriaca L., herba Final Report, 2 February 2016 EMA/ HMPC/39455/2015 Committee on Herbal Medicinal Prod- ucts (HMPC). Finch-Savage, W . E. and Leubner-Metzger, G. (2006). Seed dor- mancy and the control of germination. New Phytologist, 171, 501-523. https://doi.org/10.1111/j.1469-8137.2006.01787.x Gashi B., Abdullai K., Mata V ., Kongjika E. (2012). Effect of gi- bberellic acid and potassium nitrate on seed germination of the resurrection plants Ramonda serbica and Ramonda nathaliae. African Journal of Biotechnology, 11(20), 4537- 4542. Gatti E., Sgarbi E., Ozudogru E. A., Lambardi M. (2017). The effect of Plantform TM bioreactor on micropropagation of Quercus robur in comparison to a conventional in vitro culture system on gelled medium, and assessment of the microenvironment influence on leaf structure. Plant Biosy- stems, 151, 1129-1136. https://doi.org/10.1080/11263504.2 017.1340356 Georgiev, M. I., Weber, J., Maciuk, A. (2009). Bioprocessing of plant cell cultures for mass production of targeted compo- unds. Applied Microbiology and Biotechnology, 83, 809-823. https://doi.org/10.1007/s00253-009-2049-x Hodaj, E., Tsiftsoglou, O., Shuka, L., Abazi, S., Hadjipavlou- Litina, D., Lazari, D. (2017). Antioxidant activity and chemical composition of essential oils of some aroma- tic and medicinal plants from Albania. Natural Product Communications (NPC), 12(5), 785-790. https://doi. org/10.1177/1934578X1701200525 Acta agriculturae Slovenica, 119/2 – 2023 9 Establishment of an in vitro method for micropropagation of ironwort ... Jamwal, K., Bhattacharya, B., Puri, S. (2018). Plant growth re- gulator mediated consequences of secondary metabolites in medicinal plants. Journal of Applied Research on Medicinal and Aromatic Plants, 9, 26-38, https://doi.org/10.1016/j.jar- map.2017.12.003 Juan-Vicedo, J., Ramírez-Luna, J. E., Piqueras, A. et al. (2021). Micropropagation and cryopreservation by vitrification of the Spanish endemic medicinal plant Sideritis leucantha Cav. subsp. leucantha (Lamiaceae). In Vitro Cellular & Developmental Biology – Plant, 57, 1057-1065. https://doi. org/10.1007/s11627-021-10173-5 Kapoor, S., Raghuvanshi, R., Bhardwaj, P., Sood, H., Saxena, S., Chaurasia, O. P. (2018). Influence of light quality on growth, secondary metabolites production and antioxidant activity in callus culture of Rhodiola imbricate. Journal of Photochemical and Photobiology B: Biology, 183, 258-265. https://doi.org/10.1016/j.jphotobiol.2018.04.018 Khuat, Q. V., Kalashnikova, E. A., Kirakosyan, R. N., Nguyen, H. T., Baranova, E. N., Khaliluev, M. R. (2022). Improve- ment of in vitro seed germination and micropropagation of Amomum tsao-ko (Zingiberaceae Lindl.). Horticulturae, 8, 640. https://doi.org/ 10.3390/horticulturae8070640 Lloyd, G. & McCown, B. (1980). Commercially-feasibible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Combined Proceedings - Internatio- nal Plant Propagators' Society (USA), 30, 421-427. MoE. (2013). For approving of the Red List of wild Flora and Fauna of Albania. Approved by Minister of Environment (MoE), order nr. 1280, dt. 20.11.2013 and published in the official gazette, Nr. 197, dt. 18.12.2013. Moraes, R. M., Cerdeira, A. L., Lourenço, M. V. (2021). Us- ing micropropagation to develop medicinal plants into crops. Molecules, 26(6), 1752. https://doi.org/10.3390/mol- ecules26061752 Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cul- tures. Physiologia Plantarum, 15, 473–497. https://doi. org/10.1111/j.1399-3054.1962.tb08052.x Neergheen-Bhujun, V., Awan, A. T., Baran, Y., Bunnefeld, N., Chan, K., Dela Cruz, T. E., et al. (2017). Biodiversity, drug discovery, and the future of global health: Introduc- ing the biodiversity to biomedicine consortium, a call to action. Journal of Global Health, 7(2), 020304. https://doi. org/10.7189/jogh.07.020304 Nikam, T. D. & Barmukh, R. B. (2009). GA 3 enhances in vi- tro seed germination in Santalum album. Seed Science and Technolog y, 37(2), 276-280(5). https://doi.org/10.15258/ sst.2009.37.2.02 Papafotiou, M. & Kalantzis, A. (2009). Seed germination and in vitro propagation of Sideritis athoa. Acta Horticulturae, 813, 471-476. https://doi.org/10.17660/ActaHortic.2009.813.62 Radić, S., Vujčić, V ., Glogoški, M., Radić-Stojković, M. (2016). Influence of pH and plant growth regulators on seconda- ry metabolite production and antioxidant activity of Stevia rebaudiana (Bert). Periodicum Biologorum, 118(1), 9–19. https://doi.org/10.18054/pb.2016.118.1.3420 Ragavendran, C., Kamalanathan, G., Reena, G., Natarajan, D. (2012). In vitro propagation of nodal and shoot tip explants of Passiflora foetida L. An exotic medicinal plant. Pelagia Research Library, 2(6), 707–711. Romanucci, V., Di Fabio, G., D’ Alonzo, D., Guaragna, A., Sca- pagnini, G., Zarelli, A. (2017). Traditional uses, chemical composition and biological activities of Sideritis raeseri Bo- iss. & Heldr. Jornal of the Science of Food and Agriculture, 97, 373–383. https://doi.org/10.1002/jsfa.7867 Rout, S., Beura, S., Khare, N., Patra, S. S., Nayak, S. (2017). Ef- fect of seed pre-treatment with different concentrations of gibberellic acid (GA3) on seed germination and seedling growth of Cassia fistula L. Journal of Medicinal Plants Stu- dies, 5(6), 135-138. Sarropoulou, V . & Maloupa, M. (2015). Effect of exogenous di- kegulac on in vitro shoot proliferation of Sideritis raeseri L. – Greek mountain tea species. Agriculture & Forestry, 61(4), 153-159. https://doi.org/10.17707/.AgricultForest.61.4.16 Sevindik, B., Tütüncü, M., İzgü, T., Tagipur, E. M., Çürük, P., Kaynak, G., Yilmaz, Ö., Mendi, Y . Y . (2019). Micropropoga- tion of Sideritis pisidica Boiss. et Heldr. Apud Bentham. Acta Horticulturae, 1242, 581-586. https://doi.org/10.17660/Ac- taHortic.2019.1242.85 Sharma, M., Ahuja, A., Gupta, R., Mallubhotla, S. (2015). En- hanced bacoside production in shoot cultures of Bacopa monnieri under the influence of abiotic elicitors. Natural Product Research, 29, 745-749. https://doi.org/10.1080/147 86419.2014.986657 Shtereva, L. A., Vassilevska-Ivanova, R. D., Kraptchev, B. V. (2015). In vitro cultures for micropropagation, mass mul- tiplication and preservation of an endangered medicinal plant Sideritis scardica GriseB. Botanica Serbica, 39(2), 111 – 120. Shuka, L. & Malo, S. (2010). The transboundary important plant areas as conservation units of European green belt (Eastern Albanian zone). Journal of Environmental Protecti- on and Ecology, 11(3), 866–874. Shuka, L., Shuka, D., Diku, A. (2021). Bimët endemike dhe ato me përhapje të kufizuar, Parku Kombëtar “Prespa”, Gent Grafik, Tiranë, Albania, 150 pp. (in Albanian). Sota V., Çuko B., Kongjika E. (2020). Micropropagation of Myrtus communis L. and comparison of epidermal glan- dular trichomes characteristics between in vivo and ex vitro plantlets. Journal of Environmental Protection and Ecology, 21(2), 535–543. Sota V., Themeli S., Kongjika E., Zekaj Zh. (2019). Exogenous cytokinins application induces changes in stomatal and glandular trichomes parameters in rosemary plants regen- erated in vitro. Journal of Microbiology, Biotechnology and Food Sciences, 9(1), 25 – 28. https://doi.org/10.15414/jmb- fs.2019.9.1.25-28 Tadić V., Bojović D., Arsić I., Dorđević S., Aksentijevic K., Stamenić M., Janković S. (2012). Chemical and antimicro- bial evaluation of supercritical and conventional Sideritis scardica Griseb., Lamiaceae extracts. Molecules (Basel, Swit- zerland), 17(3), 2683 – 2703. https://doi.org/10.3390/mol- ecules17032683 Tomasini, S. & Theilade, I. (2019). Local ecological knowledge Acta agriculturae Slovenica, 119/2 – 2023 10 V. S O TA et al. indicators for wild plant management: Autonomous local monitoring in Prespa, Albania. Ecological Indicators, 101, 1064–1076. https://doi.org/10.1016/j.ecolind.2019.01.076 Tousi, S. E., Radjabian, T., Ebrahimzadeh, H., Niknam, V. (2010). Enhanced production of valerenic acids and vale- potriates by in vitro cultures of Valeriana officinalis L. Inter- national Journal of Plant Production, 4(3), 1735-6814. V antu, S. & Gales, R. C. (2009). Structural characteristics of Chry- santhemum morifolium Ramat (Romica cultivar) regenerat- ed in vitro. Analele Științifice ale Universității ‘ Al I Cuza’ din Iași, 10(2), 43-50. https://doi.org/10.1055/s-0029-1234451 Vélez-Mora, D. V ., González, R. A., Zimmermann, M. J. (2015). Enhancement of germination, hyperhydricity control and in vitro shoot formation of Vasconcellea stipulata Badillo. Revista Colombiana de Biotecnologia, 12(2), 16-21. https:// doi.org/10.15446/rev.colomb.biote.v17n2.43611 Yavuz, D. Ö. (2016). Optimization of regeneration conditions and in vitro propagation of Sideritis stricta Boiss & Heldr. International Journal of Biological Macromolecules, 90, 59- 62. https://doi.org/10.1016/j.ijbiomac.2015.10.064 Zekaj, Zh., Shuka, L., Malo, S. (2008). Kariological and histolo- gical variation to some populations of Sideritis raeseri Bo- iss. & Heldr., specie in Abania. Proceedings of ICBES. 26-28 September. Tirana, Albania, p. 112-119. (in Albanian).