Establishment of an in vitro method for micropropagation of ironwort, ( Sideritis

: 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-treated 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 culture media used were evaluated. For the subculture stage, three cytokinins (6-b enzylaminopurine / 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-n aphthaleneacetic acid / NAA and i ndole-3-butyric acid / I BA) 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.


INTRODUCTION
Mountain tea (Sideritis raeseri Boiss et Heldr.), also known as Ironwort, is an aromatic medicinal native plant of the western Balkans, including south Albania, southeastern parts of North Macedonia, and North Greece (Zekaj et al., 2008). It is considered a Balkan endemic species due to its restricted distribution range of only three countries. Due to its high quantity of bioactive 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 disorders, coughs and as a dietary supplement for avoiding anemia. The European Medicinal Agency (EMA) has approved the use of mountain tea, due to its medicinal values 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), mountain tea is a culturally and commercially significant important 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 widespread use, it has been subjected to uncontrolled and destructive harvest practices in its distribution area of occurrence 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 destructive harvest practices of the natural population continue, 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 technologies 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 medicine (Neergheen-Bhujun et al., 2017;Moraes et al., 2021).
Seasonal variations, growing practices, the expense of production, as well as other factors all impede largescale 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 ( Vantu & 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 multiplication of plant germplasm is determined by several parameters, 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, pretreatment 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 cryopreservation strategy using shoot explants. Sarropoulou & Maloupa1 (2015) studied the effects of various dikegulac 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 regulators (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 concentrations of GA 3 for enhancing seed germination under in vitro conditions and confronting some PGRs ratios for in vitro regeneration and rooting induction on the derived plantlets.

PLANT MATERIAL COLLECTION AND DISIN-FECTION
As primary explants were used mature seeds of Sideritis 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.

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.

MEDIA COMPOSITION IN EACH STAGE OF MICROPROPAGATION
Inoculation and seeds germination stage: after GA 3 treatment, the explants were inoculated in a nutrient medium 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 cytokinins (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 treatment (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.

INCUBATION CONDITIONS
The cultures were maintained in the growth chamber at a temperature 25 °C ± 2 °C with a 16 h light / 24 h photoperiod with cool, white fluorescent light of intensity 43.4 mmol m -2 s -1 .

STATISTICAL ANALYSES
For each treatment, 30 explants were used, and all experiments were repeated at least three times. Experimental 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.

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 germination after 6 7 days of culture, and shoot / root organogenesis was observed due to the proliferation of zygotic embryos (Fig. 1 a). Within a week, these organs are differentiated ( Fig. 1 b, c). The obtained results showed that seeds pre-treatment with GA 3 solution gave high germination rates in all concentrations used.
From the variability chart (Fig. 2), it can be observed that the differences in this parameter were not influenced 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 concentration, resulting specifically in an 88.4 % of germination rate for MS medium and 86.4 % for WPM medium. V. SOTA et al. After seeds germination, morphological characteristics 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 difference 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 solution at 500 mg l -1 . However, significant differences were observed between MS and WPM medium for this concentration (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 breaking seed dormancy. From our data, it is clear that GA 3 positively affects in vitro organogenesis by stimulating 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 parameters. So, it is evidenced that the GA 3 beneficial role in promoting seed development of S. raeseri.

BIOMASS PRODUCTION UNDER DIFFERENT CONCENTRATIONS AND TYPES OF CYTO-KININS
The proliferated plantlets were subcultured for further multiplication, whereas before inoculation in the nutrient medium, the roots were removed, and small shoots were used for this purpose. A few days after cultivation in the subculture stage, new shoots and leaves  were formed in all concentrations or types of cytokinins supplemented in the nutrient media ( Fig. 1 d). The initial 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 effectiveness of the three cytokinins used, in all other concentrations 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 concentrations within the same type of cytokinin (Fig. 4b) for kinetin and BAP, the best results for RGR value were obtained 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.

IN VITRO ROOT FORMATION USING DIFFER-ENT CONCENTRATIONS OF IBA AND NAA
Root formation is a crucial stage for the micropropagation 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 investigated. 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 parameter, and the best result was obtained at 2.0 mg l -1 of IBA, with a respective value of 14.21 roots/plantlet.

DISCUSSION
Seed germination is a complex process, and GA 3 plays a crucial role in controlling and encouraging germination in many plant species. In this respect, exogenous 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. Furthermore, the results revealed that the seed treatments significantly 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 report, Cornea-Cipcigan et al. (2020) concluded that exogenous applications of GA 3 stimulated not only the germination 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 germination of Ramonda serbica Pančić seeds grown in Petri dishes 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. Similarly, 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, Nikam & 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 differences 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 Sideritis 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. 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 affecting the in vitro multiplication of plant propagules. In the present work, variations in the response of the multiplication 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 multiplication in the present study was obtained by enhancing shootlets' fresh mass, which is crucial in employing tissue culture techniques for Sideritis raeseri micropropagation. Similar to our results, other authors, when confronting 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 micropropagation 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 require a medium supplemented with essentially auxinspecific 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, 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.

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 stage, 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 concentration 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 induction, IBA was more effective than NAA for roots length and number. Effective clonal growth enables the creation 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 important medicinal species.

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 Innovation) in Albania.