Micropropagation of Endemic Taxa of the Flora of Turkey: Helichrysum arenarium (L.) Moench subsp. erzincanicum P.H. Davis & Kupicha (Asteraceae)

Micropropagation of Endemic Taxa of the Flora of Turkey: Helichrysum arenarium (L.) Moench subsp. erzincanicum P.H. 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Moench subsp. erzincanicum P.H. Davis & Kupicha (Asteraceae) şeyma önlü, Gülşen AKÇA This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6925544/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study developed a micropropagation protocol using explants grown from seeds of Helichrysum arenarium (L.) Moench subsp. erzincanicum and plant tissue culture methods. Initially, indirect shoot organogenesis was performed with H. arenarium subsp. erzincanicum in vitro plantlets on Murashige and Skoog (MS) and B5 media (abbrev. as MSB) supplemented with 6-benzylaminopurine (BAP), kinetin (Kin) and indole-3-acetic acid (IAA). A maximum number of shoots was induced in 1 mg L -1 BAP (61.2%). Subsequently, the shoots were inoculated in MSB combinations with 0.5–1.5 mg L -1 indole-3-acetic acid and indole-3-butyric acid (IBA), wherein 1.0 mg L -1 IBA (77.8%) recorded the maximum number of roots. Additionally, callus formation was achieved with different concentrations of thidiazuron (TDZ) (0.5, 1.0, 2.0 mg L -1 ) and α-naphthaleneacetic acid (NAA) (1.0 mg L -1 ) and the highest callus formation percentage (80.2%) was observed MSB media combinations with 2.0 mg L -1 TDZ and 1.0 mg L -1 NAA. This study presents the successful development of a micropropagation protocol for H. arenarium subsp. erzincanicum and supports its in vitro propagation via explants grown from seed. Endemic species Helichrysum arenarium subsp. erzincanicum Gibberellic acid Kinetin Micropropagation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Helichrysum arenarium (L.) Moench subsp. erzincanicum P.H. Davis & Kupicha (abbreviated as HAE) is endemic to Turkey, specifically distributed across the provinces of Kastamonu, Ağrı, Erzincan, and Kayseri. It is a perennial, herbaceous species endemic to Turkey, growing in its natural vegetation (Umaz & Umaz, 2020 ; Uz & Savalan, 2024 ). In Turkey, 30 Helichrysum species have been identified, 16 of which are endemic (Eroğlu, 2018 ). Helichrysum is also commonly known as golden grass, an everlasting or immortal flower (Czinner et al., 2000 ). Helichrysum has traditionally been used as a diuretic, cholagogue, hepatoprotective, bile-building, antithrombotic and capillary-sealing agent with effects in the gastrointestinal tract, liver and gallbladder (Liu et al., 2019 ). Many Helichrysum species have been used not only in folk medicine but also for ornamental, cosmetic, and pharmaceutical purposes (Gradinaru et al., 2014 ; Pawelek, 2015 ). With bright yellow color, it is suitable for fresh and dry bouquet arrangements, as an insect repellent, essential oil and aromatic chemical glycosides for cosmetics, pharmaceutical and food industries are used (Dănăilă-Guidea et al., 2022 ). The essential oils and phytochemicals of Helichrysum , such as monoterpenoids, sesquiterpenes, triterpenoids, flavonoids, and pyrones, have important pharmaceutical properties (Jakupovic et al., 1986 ; Leonardi et al., 2018 ). Because of its antiproliferative, anti-inflammatory, antiviral, antifungal, and antimicrobial effects, Helichrysum species need to be protected and produced, especially by in vitro methods. Micropropagation is an efficient plant tissue culture method that has been applied to a very wide range of species. Micropropagation techniques are important in terms of preserving the generation of endemic plants and ensuring their production of bioactive components in vitro . To date, different Helichrysum species have typically been propagated from axillary buds, leaves, or apical meristems. For example, Figas et al. ( 2016 ) used the apical meristems of H. arenarium as explants and determined that the best plant growth regulator (PGR) combination for shoot regeneration was 5.0 mg L − 1 N6-furfuryladenine (KIN) and 0.5 mg L − 1 indole-3-acetic acid (IAA) or 5.0 mg L − 1 KIN alone (Figas et al., 2016 ). Clasquin and Henry ( 2002 ) used shoots and stem nodes as explants for micropropagation of H. arenarium and found that more calli formed in Murashige and Skoog (MS) media supplemented with 1.0 mg L − 1 2,4-D (Clasquin & Henry, 2002 ). Perrini et al. ( 2009 ) used the apical buds and axillary shoots of Helichrysum italicum subsp. microphyllum as explant sources (Perrini et al., 2009 ). Prinsloo and Meyer ( 2006 ) used the young leaves and axillary buds of Helichrysum kraussii , and calli formed in MS nutrient media supplemented with 1 mg L − 1 α-naphthaleneacetic acid (NAA) and 0.05 mg L − 1 KIN (Prinsloo & Meyer, 2006 ). Furthermore, Dimitrova and Nacheva ( 2018 ) reported the use of H. italicum axillary buds as explants for shoot regeneration (Prinsloo & Meyer, 2006 ); Botha et al. ( 2015 ) optimized an in vitro propagation protocol from Helichrysum umbraculigerum leaves (Botha et al., 2015 ); and Uz & Savalan ( 2024 ) used young H. italicum shoots for in vitro production (Uz & Savalan, 2024 ). However, to our knowledge, there are no prior reports on the micropropagation of endemic H. arenarium subsp. erzincanicum . Hence, this study aimed to develop an effective micropropagation method for this subspecies, using explants derived from seeds endemic to and naturally occurring in Turkey. Materials and methods Material H. arenarium (L.) Moench subsp. erzincanicum Davis & Kupicha (Fig. 1 ) seeds were collected from the variety garden of the Muş Alparslan University Faculty of Applied Sciences in October 2021. Then it was identified by Dr. Ümit Bingöl (M.Ü. Bingöl 6829) from the Faculty of Science, Ankara University. Methods Seed sterilization and germination Seeds were disinfected with 7.5% sodium hypochlorite (NaOCl) and placed in an oven at 25°C for 20 minutes. Afterward, 70% ethanol was applied 30 min and then incubated at 42°C for 24 hours. The sterile seeds were rinsed for 5 times with sterile distilled water (Battal et al., 2019 ). After sterilization, the media was additionally supplemented with 1.0 mg L − 1 gibberellic acid (GA 3 ), 1.0 mg L − 1 6-benzylaminopurine (BAP), and 1.0 mg L − 1 amphotericin B. The samples were maintained at room temperature (24 ± 2°C) under white fluorescent light (3000 lux) with a photoperiod of 16 hours of light and 8 hours of darkness. The germination study included three replicates, each containing 20 seeds. Shoot regeneration media After 21 days of germination, the in vitro -obtained plantlets were transferred to MSB media and used as explants. The following nutrient media mixtures were prepared for shoot regeneration: M1 (1.0 mg L − 1 BAP); M2 (2.0 mg L − 1 BAP); M3 (3.0 mg L − 1 BAP); M4 (1.0 mg L − 1 BAP and 0.1 mg L − 1 NAA); M5 (2.0 mg L − 1 BAP and 0.1 mg L − 1 NAA); M6 (3.0 mg L − 1 BAP and 0.1 mg L − 1 NAA); M7 (1.0 mg L − 1 KIN and 0.5 mg L − 1 NAA); M8 (2.0 mg L − 1 KIN and 0.5 mg L − 1 NAA); M9 (3.0 mg L − 1 KIN and 0.5 mg L − 1 NAA); M10 (4.0 mg L − 1 KIN and 0.5 mg L − 1 NAA); and M11 (5.0 mg L − 1 KIN and 0.5 mg L − 1 NAA). Callus growth medium Calli that formed in media supplemented with 1.0 mg L − 1 BAP were used as explants for callus culture in MSB media in the presence of various PGRs. The following media mixtures were prepared for callus culture: 2,4-D (0.5 mg L − 1 , 1.0 mg L − 1 , 1.5 mg L − 1 ); TDZ (1.0 mg L − 1 ) and picloram (0.5 mg L − 1 , 1.0 mg L − 1 , 2.0 mg L − 1 ); NAA (1.0 mg L − 1 ) and TDZ (0.5 mg L − 1 , 1.0 mg L − 1 , 2.0 mg L − 1 ). Rooting of shoots Eight-week-old shoots were transferred to MS medium supplemented with 0.1, 0.5, or 1.0 mg L − 1 IAA or IBA for root formation. Statistical analysis The data were statistically analysed using SPSS for Windows (SPSS Inc., Chicago, IL, USA). Differences in means ± standard errors (SEs) were evaluated for significance using Duncan’s one-way analysis of variance (ANOVA) at the P < 0.05 level. Results Seed germination rate The effects of the PGRs; BAP and GA 3 on the germination of H. arenarium subsp. erzincanicum seeds on MSB medium are shown in Table 1 . The highest germination rate (27.22%) was obtained in MSB media supplemented with 1.0 mg L − 1 GA 3 and 1.0 mg L − 1 BAP. Table 1 Effects of the plant growth regulators BAP and GA 3 on the seed germination rate of H . arenarium subsp. erzincanicum in MSB media Concentration of PGRs Germination rate (%) Control 6.6 ± 1.6 d GA 3 (mg L − 1 ) BAP (mg L − 1 ) 1.0 0.0 21.31 ± 2.1 b 1.0 1.0 27.22 ± 2.6 a 1.0 2.0 14.76 ± 1.2 c Different letters within the same column indicate a significant difference between means at the 0.05 level, and (±) denotes the standard error . Shoot proliferation The sterile plantlets obtained from the seeds were transferred to nutrient media containing three different concentrations of growth regulator (0.5, 1.0, and 2.0 mg L − 1 BAP) at the end of the 21st day (Table 2 ). The best shoot regeneration was observed MSB media supplemented with 1.0 mg L − 1 BAP (61.2%, P < 0.05). Table 2 Effects of different concentrations of BAP in MSB media on H. arenarium subsp. erzincanicum shoot formation 8 weeks after culture initiation Plant growth regulator (mg L − 1 ) Rate of shoot formation from the explants (%) BAP 0.5 19.8 ± 1.6 c 1.0 61.2 ± 4.2 a 2.0 38.0 3 ± 2.8 b Different letters in the same column indicate a significant difference between means at the 0.05 level, and (±) indicates the standard error . The shoots obtained from media supplemented with 1.0 mg L − 1 BAP were transferred to MSB media supplemented with different combinations of KIN and IAA (Figas et al., 2016 ). By the end of the 30th day, the plantlets had grown in height (Fig. 2 ). Among the six different combinations of KIN and IAA tested for shoot regeneration, 5.0 mg L − 1 KIN and 0.5 mg L − 1 IAA resulted in the highest percentage of shoot formation, reaching 60% (Table 3 ). Table 3 Response of shoot formation of HAE on MSB media supplemented with different concentrations of KIN and IAA PGR combination (mg L − 1 ) Shoot formation (%) KIN IAA 0.0 0.0 26.6 ± 1.3 c 1.0 0.5 - 2.0 0.5 - 3.0 0.5 53.3 ± 2.7 a 4.0 0.5 46.7 ± 2.3 b 5.0 0.5 60 ± 3.1 a Callus formation and structure The structures and colours of the calli obtained are given in Table 4 , and the treatments yielded significantly different calli formation rates (p < 0.05). The highest rate of callus formation (80.2%) was obtained with MSB media supplemented with 2.0 mg L − 1 TDZ and 1.0 mg L − 1 NAA (Fig. 3 ). Table 4 Effects of callus growth of HAE on MSB media with different concentrations of 2,4-D, TDZ, NAA, and picloram PGR(s) Callus structure and colour Callus formation rate (%) 2,4-D (mg L − 1 ) 0.5 Brown, compact 27.1 ± 2.4 b 1.0 Brown, friable 38.7 ± 3.1 a 2.0 Brown, compact 17.8 ± 1.6 c TDZ (mg L − 1 ) Picloram (mg L − 1 ) 1.0 0.5 Brown, compact 26.7 ± 2.8 a 1.0 1.0 Brown, friable 33.3 ± 3.1 a 1.0 2.0 Brown, compact 20.0 ± 2.4 b TDZ (mg L − 1 ) NAA (mg L − 1 ) 0.5 1.0 Brown, compact 26.7 ± 4.8 c 1.0 1.0 Yellowish, friable 60.1 ± 10.2 b 2.0 1.0 Green, friable 80.2 ± 16.8 a Different letters in the same column indicate a significant difference between means at the 0.05 level, and (±) indicates the standard error . Rooting The elongated shoots were subsequently transferred to MSB media supplemented with 0.5, 1.0, or 1.5 mg L − 1 IBA or IAA for rooting, and roots developed within four weeks of culture. The highest percentage (77.8%) of roots were found in the 1.0 mg L − 1 IBA treatment group, in which new adventitious shoots formed (Figs. 4 and 5 ). Discussion The sterilization of the H. arenarium subsp. erzincanicum seeds was challenging due to their small size and hairy structure. Battal et al. ( 2019 ) reported the sterilization method that was successfully applied in this study (Battal et al., 2019 ). While low amounts of sodium hypochlorite (7.5%) did not damage the seeds' structure, increasing the temperature to 42°C removed the bacteria, fungi, and spores that may have been present on the seeds' hairy surface. In addition to physical factors such as light, temperature, and humidity, the addition of an endogenous growth factor (GA 3 ) is also required for seed germination. It has been reported that in Arabidopsis , GA 3 release causes the seed coat to weaken by stimulating the expression of genes involved in cell expansion and modification (Gupta & Chakrabarty, 2013 ). Moreover, in their organogenesis studies on argan plants, Amghar et al. ( 2021 ) reported that GA 3 increased the germination rate and that supplementing MS media with 1.0 mg L − 1 BAP with 2.0 mg L − 1 GA 3 increased the number of adventitious shoots formed (Amghar et al., 2021 ). First, the seed germination rate in our study was 6.6% in MSB media in the absence of growth regulators. Thus, different concentrations of the PGRs BAP and GA 3 were subsequently used to promote germination and increase the germination rate. The best result (27.22%) was obtained in MS/B5 media supplemented with 1.0 mg L − 1 BAP and 1.0 mg L − 1 GA 3 . This study revealed that GA 3 ensures germination by breaking dormancy in the seeds while the hormone BAP increases shoot length. Since it is difficult to germinate the seed of Helichrysum species, micropropagation studies have generally been performed on aerial parts (Clasquin & Henry, 2002 ; Figas et al., 2016 ). These results demonstrate that seed sterilization is applicable under certain physical conditions and that germination is possible using different plant growth regulators (PGRs), such as BAP and GA 3 . Second, H. arenarium subsp. erzincanicum shoot development was optimal in MSB media supplemented with 5.0 mg L − 1 KIN and 0.5 mg L − 1 IAA (60%). The experiment was stopped at the optimum concentration and that further testing might be needed to fully explore the dose-response relationship, and to determine if higher concentrations might be even more effective. According to Figas et al. ( 2016 ), supplementing MS media with the PGRs KIN and IAA positively affects height as well as shoot formation (Figas et al., 2016 ). On the other hand, Dimitrova and Nacheva ( 2018 ), in their micropropagation study on H. italicum , reported that the highest percentage of shoot regeneration occurred in DKW media supplemented with 5 µM KIN (Dimitrova & Nacheva, 2018 ). Botha et al. ( 2015 ) optimized an in vitro propagation protocol from H. umbraculigerum leaves and found that shoots were produced most efficiently on MS media containing a specific concentration of TDZ (Botha et al., 2015 ). Uz & Savalan ( 2024 ) reported that the highest shoot formation percentage from young H. italicum shoots occurred in MS media supplemented with 0.5 mg L − 1 BAP, 1.0 mg L − 1 GA 3 , and 0.2 mg L − 1 NAA, while the lowest percentage occurred in MS media without hormone supplementation (Uz & Savalan, 2024 ). In another study, Giovannini et al. (2003) micropropagated H. stoechas and H. italicum plants and reported that the number of shoots increased twofold in response to supplementation with BAP (Giovannini et al., 2001 ). In our protocol, calli were formed via indirect organogenesis in media supplemented with 1.0 mg L − 1 BAP. The calli were subsequently transferred to MSB media supplemented with different concentrations of the PGRs 2,4-D, TDZ, NAA, and picloram. Green, friable calli grew best on MSB media supplemented with 1.0 mg L − 1 NAA and 2.0 mg L − 1 TDZ. Similarly, 81.3% green and friable calli of H. pallasii were obtained by using 1.0 mg L − 1 TDZ (Yurteri et al., 2021 ). The addition of different concentrations of different PGRs to calli growth media encourages root or shoot development to yield plantlets (Evans et al., 1981 ). The auxin PGRs IAA, IBA and NAA are the most frequently PGRs reported in the literature for rooting. Notably, both root induction and root elongation are extremely sensitive to auxin concentration (Koca, 2015 ). Moreover, the addition of auxins positively affected root and adventitious shoot formation in our study. The ideal mixture for rooting in our study was MS/B5 media supplemented with 1.0 mg L − 1 IBA (77.8%). Although the rooting rate varies depending on the plant species in terms of a hormonal or nonhormonal environments, it has generally been reported that the growth regulator IBA promotes rooting and increases shoot length (Yurteri et al., 2021 ). Conclusion This is the first report of the successful micropropagation of H. arenarium subsp. erzincanicum using plant tissue culture techniques. The protocol described here uses BAP, KIN, and IAA to induce the proliferation of multiple shoots on tissue explants, and BAP supplementation produced the most robust organogenic response via indirect organogenesis. Following shoot production, plantlets treated with IBA developed root systems. This micropropagation protocol provides an alternative to seed propagation and contributes to the conservation of H. arenarium subsp. erzincanicum , a rare and endemic plant species. Additionally, this study contributes to the development of in vitro methods for propagating other Helichrysum and rare plant species. Abbreviations Gibberellic acid (GA 3 ) Indole-3-acetic acid (IAA) Murashige and Skoog (MS) 6-benzylaminopurine (BAP) N6-furfuryladenine (kinetin (KIN)) Thidiazuron (TDZ) α-naphthaleneacetic acid (NAA) Plant growth regulator (PGR) Sodium hypochlorite (SH) Declarations Author contribution statement G.A : Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – review & editing. Ş.Ö: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft. Funding This research work was financed by the Mus Alparslan University Scientific Research Coordination Unit, 21-FEF-4901-04, Mus, Turkey. Data availability All data produced or analyzed during this study are included in this article. Data will be available upon request from the corresponding author. Conflict of interest The authors have declared that they have no conflict of interest. Acknowledgements The authors would like to thank Dr. Ahmet Yenikalaycı for plant material. References Amghar, I., Diria, G., Boumlik, I., Gaboun, F., Iraqi, D., Labhilili, M., Mentag, R., Meziani, R., Mazri, M. A., & Ibriz, M. (2021). An efficient regeneration pathway through adventitious organogenesis for the endangered Argania spinosa (L.) Skeels. Vegetos , 34 , 355-367. Battal, A., Görmez, G., & Türker, M. (2019). An effective germination method for medically important Althaea officinalis : notching the seed coat. Bitlis Eren University Journal of Science 8 (3), 843-851. Botha, K., Crampton, B., & Heyman, H. (2015). The in vitro propagation of Helichrysum umbraculigerum . Planta Medica , 81 (16), PM_236. Clasquin, S., & Henry, M. (2002). Micropropagation d' Helichrysum arenarium (L.) Moench. Acta botanica gallica , 149 (2), 189-195. Czinner, E., Lemberkovics, É., Bihátsi-Karsai, E., Vitányi, G., & Lelik, L. (2000). Composition of the essential oil from the inflorescence of Helichrysum arenarium (L.) Moench. Journal of Essential Oil Research , 12 (6), 728-730. https://doi.org/https://doi.org/10.1080/10412905.2000.9712202 Dănăilă-Guidea, S. M., Eremia, M. C., Dinu, L. D., & Miu, D.-M. (2022). Helichrysum arenarium: From cultivation to application. Applied Sciences , 12 (20), 10241. Dimitrova, N. G., & Nacheva, L. (2018). Micropropagation of Helichrysum italicum (ROTH) G. Don–a medicinal plant with ornamental value. Journal of BioScience and Biotechnology , 7 (2-3), 97-101. Eroğlu, H. E. (2018). Türkiye Helichrysum taksonlarının Türkçe ve diğer dillerdeki isimleri. Avrasya Terim Dergisi , 6 (1), 26-34. Evans, D. A., Sharp, W. R., & Flick, C. E. (1981). Growth and behavior of cell cultures: embryogenesis and organogenesis. Plant tissue culture: methods and applications in agriculture , 45-113. Figas, A., Tomaszewska-Sowa, M., Sawilska, A., & Keutgen, A. J. (2016). Improvement of in vitro propagation and acclimation of Helichrysum arenarium L. Moench. Acta scientiarum Polonorum. Hortorum cultus , 15 (4). Giovannini, A., Amoretti, M., Savona, M., Di Guardo, A., & Ruffoni, B. (2001). Tissue culture in Helichrysum spp. I International Symposium on Acclimatization and Establishment of Micropropagated Plants 616, Gradinaru, A. C., Silion, M., Trifan, A., Miron, A., & Aprotosoaie, A. C. (2014). Helichrysum arenarium subsp. arenarium : phenolic composition and antibacterial activity against lower respiratory tract pathogens. Natural product research , 28 (22), 2076-2080. Gupta, R., & Chakrabarty, S. (2013). Gibberellic acid in plant: still a mystery unresolved. Plant signaling & behavior , 8 (9), e25504. Jakupovic, J., Kuhnke, J., Schuster, A., Metwally, M., & Bohlmann, F. (1986). Phloroglucinol derivatives and other constituents from South African Helichrysum species. Phytochemistry , 25 (5), 1133-1142. Koca, A. S. (2015). Çeltikte (Oryza sativa L.) endosperm destekli olgun embriyo kültüründe kallus oluşumu ve bitki rejenerasyonuna tohum ıriliğinin etkisi Ankara Universitesi (Turkey)]. Leonardi, M., Giovanelli, S., Ambryszewska, K. E., Ruffoni, B., Cervelli, C., Pistelli, L., Flamini, G., & Pistelli, L. (2018). Essential oil composition of six Helichrysum species grown in Italy. Biochemical Systematics and Ecology , 79 , 15-20. Liu, X., Jing, X., & Li, G. (2019). A process to acquire essential oil by distillation concatenated liquid-liquid extraction and flavonoids by solid-liquid extraction simultaneously from Helichrysum arenarium (L.) Moench inflorescences under ionic liquid-microwave mediated. Separation and Purification Technology , 209 , 164-174. https://doi.org/https://doi.org/10.1016/j.seppur.2018.07.028 Pawelek, J. (2015). Water management in Poland in view of water supply and sewage disposal infrastructure development. Infrastruktura i Ekologia Terenów Wiejskich (II/2). Perrini, R., Morone-Fortunato, I., Lorusso, E., & Avato, P. (2009). Glands, essential oils and in vitro establishment of Helichrysum italicum (Roth) G. Don ssp. microphyllum (Willd.) Nyman. Industrial Crops and Products , 29 (2-3), 395-403. Prinsloo, G., & Meyer, J. (2006). In vitro production of phytoalexins by Helichrysum kraussii . South African Journal of Botany , 72 (3), 482-483. Umaz, A., & Umaz, K. (2020). Determination and comparison of volatile compounds of gold grass (Helichrysum arenarium) from two different locations. Gümüşhane University Journal of Science , 10 (3), 592-600. Uz, İ., & Savalan, Ş. (2024). In vitro Micropropagation of Immortelle Grass ( Helichrysum italicum (ROTH) G. DON). Tekirdağ Ziraat Fakültesi Dergisi , 21 (1), 246-255. Yurteri, E., Aksu, M., Küplemez, H., Özcan, A., & Seyis, F. (2021). Effects of Thidiazuran and Zeatin on Plant Regeneration in Helichrysum pallasii . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6925544","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476345597,"identity":"cd21d2be-6d33-401b-ad1a-fc6254f20335","order_by":0,"name":"şeyma önlü","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-2005-1019","institution":"Muş Alparslan Üniversitesi: Mus Alparslan Universitesi","correspondingAuthor":true,"prefix":"","firstName":"şeyma","middleName":"","lastName":"önlü","suffix":""},{"id":476345598,"identity":"92345751-14d7-4d04-968c-85e4c6484389","order_by":1,"name":"Gülşen AKÇA","email":"","orcid":"","institution":"Mus Alparslan University: Mus Alparslan Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Gülşen","middleName":"","lastName":"AKÇA","suffix":""}],"badges":[],"createdAt":"2025-06-18 19:09:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6925544/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6925544/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85657845,"identity":"e9e2a48c-e3b4-4ed7-aa7b-05e18ad3de82","added_by":"auto","created_at":"2025-06-30 11:03:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":596351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHelichrysum arenarium \u003c/em\u003e(L.) Moench subsp. \u003cem\u003eerzincanicum\u003c/em\u003eDavis \u0026amp; Kupicha aerial parts from the variety garden of the Muş Alparslan University Faculty of Applied Sciences.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6925544/v1/a0eeb9e49f251c5890dcf8db.png"},{"id":85657474,"identity":"fd5b93f9-9969-4e25-be80-abeb8b520ce2","added_by":"auto","created_at":"2025-06-30 10:55:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393256,"visible":true,"origin":"","legend":"\u003cp\u003eShoot regeneration (A) on the first day after placing in MSB media supplemented with 1 mg L\u003csup\u003e-1\u003c/sup\u003e BAP and (B) transfer of the plantlets after 8 weeks. (C) Images of plantlets transferred from media supplemented with 1.0 mg L\u003csup\u003e-1\u003c/sup\u003e BAP to media supplemented with 5.0 mg L\u003csup\u003e-1\u003c/sup\u003e KIN with 0.5 mg L\u003csup\u003e-1\u003c/sup\u003e NAA (C) on the first day and (D) after 30 days.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6925544/v1/4641779d236942676e136be6.png"},{"id":85657473,"identity":"4e5123af-6b8a-4463-ba13-342953b1ca9c","added_by":"auto","created_at":"2025-06-30 10:55:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529737,"visible":true,"origin":"","legend":"\u003cp\u003eCallus formation after 60 days in MSB media supplemented with (A) 1 mg L\u003csup\u003e-1\u003c/sup\u003e TDZ and 0.5 mg L\u003csup\u003e-1\u003c/sup\u003e Pic; (B) 1 mg L\u003csup\u003e-1\u003c/sup\u003e TDZ and 1.0 mg L\u003csup\u003e-1\u003c/sup\u003e NAA; and (C) 2 mg L\u003csup\u003e-1\u003c/sup\u003e TDZ and 1.0 mg L\u003csup\u003e-1\u003c/sup\u003e NAA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6925544/v1/39fa614e4d4c3e86d1a4d9d5.png"},{"id":85657479,"identity":"c4a86f91-648a-4b80-b4cd-f131769ec22e","added_by":"auto","created_at":"2025-06-30 10:55:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":763617,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of plantlets transferred to media supplemented with 1.0 mg L\u003csup\u003e-1\u003c/sup\u003e IBA for rooting (A) on the first day and (B) after 30 days.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6925544/v1/fd66a765e3bc81e0a74348c0.png"},{"id":85657478,"identity":"529a3266-d7cd-43c1-bdb4-fb5538c47f5a","added_by":"auto","created_at":"2025-06-30 10:55:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":51444,"visible":true,"origin":"","legend":"\u003cp\u003eRoot formation rates (%) after 30 days.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6925544/v1/3ae4e397f79536a1ffdc6233.png"},{"id":87489197,"identity":"7d4244f7-1b2f-4414-904d-6ae0c3864349","added_by":"auto","created_at":"2025-07-24 11:33:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3725887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6925544/v1/517e4fcb-22dc-4c49-8cde-a6f687b0c01e.pdf"}],"financialInterests":"","formattedTitle":"Micropropagation of Endemic Taxa of the Flora of Turkey: Helichrysum arenarium (L.) Moench subsp. erzincanicum P.H. Davis \u0026amp; Kupicha (Asteraceae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eHelichrysum arenarium\u003c/em\u003e (L.) Moench subsp. \u003cem\u003eerzincanicum\u003c/em\u003e P.H. Davis \u0026amp; Kupicha (abbreviated as HAE) is endemic to Turkey, specifically distributed across the provinces of Kastamonu, Ağrı, Erzincan, and Kayseri. It is a perennial, herbaceous species endemic to Turkey, growing in its natural vegetation (Umaz \u0026amp; Umaz, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Uz \u0026amp; Savalan, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In Turkey, 30 \u003cem\u003eHelichrysum\u003c/em\u003e species have been identified, 16 of which are endemic (Eroğlu, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eHelichrysum\u003c/em\u003e is also commonly known as golden grass, an everlasting or immortal flower (Czinner et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). \u003cem\u003eHelichrysum\u003c/em\u003e has traditionally been used as a diuretic, cholagogue, hepatoprotective, bile-building, antithrombotic and capillary-sealing agent with effects in the gastrointestinal tract, liver and gallbladder (Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany \u003cem\u003eHelichrysum\u003c/em\u003e species have been used not only in folk medicine but also for ornamental, cosmetic, and pharmaceutical purposes (Gradinaru et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pawelek, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). With bright yellow color, it is suitable for fresh and dry bouquet arrangements, as an insect repellent, essential oil and aromatic chemical glycosides for cosmetics, pharmaceutical and food industries are used (Dănăilă-Guidea et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The essential oils and phytochemicals of \u003cem\u003eHelichrysum\u003c/em\u003e, such as monoterpenoids, sesquiterpenes, triterpenoids, flavonoids, and pyrones, have important pharmaceutical properties (Jakupovic et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Leonardi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Because of its antiproliferative, anti-inflammatory, antiviral, antifungal, and antimicrobial effects, \u003cem\u003eHelichrysum\u003c/em\u003e species need to be protected and produced, especially by \u003cem\u003ein vitro\u003c/em\u003e methods. Micropropagation is an efficient plant tissue culture method that has been applied to a very wide range of species. Micropropagation techniques are important in terms of preserving the generation of endemic plants and ensuring their production of bioactive components \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo date, different \u003cem\u003eHelichrysum\u003c/em\u003e species have typically been propagated from axillary buds, leaves, or apical meristems. For example, Figas et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) used the apical meristems of \u003cem\u003eH. arenarium\u003c/em\u003e as explants and determined that the best plant growth regulator (PGR) combination for shoot regeneration was 5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e N6-furfuryladenine (KIN) and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indole-3-acetic acid (IAA) or 5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN alone (Figas et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Clasquin and Henry (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) used shoots and stem nodes as explants for micropropagation of \u003cem\u003eH. arenarium\u003c/em\u003e and found that more calli formed in Murashige and Skoog (MS) media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 2,4-D (Clasquin \u0026amp; Henry, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Perrini et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) used the apical buds and axillary shoots of \u003cem\u003eHelichrysum italicum\u003c/em\u003e subsp. \u003cem\u003emicrophyllum\u003c/em\u003e as explant sources (Perrini et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Prinsloo and Meyer (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) used the young leaves and axillary buds of \u003cem\u003eHelichrysum kraussii\u003c/em\u003e, and calli formed in MS nutrient media supplemented with 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e α-naphthaleneacetic acid (NAA) and 0.05 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN (Prinsloo \u0026amp; Meyer, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, Dimitrova and Nacheva (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported the use of \u003cem\u003eH. italicum\u003c/em\u003e axillary buds as explants for shoot regeneration (Prinsloo \u0026amp; Meyer, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e); Botha et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) optimized an \u003cem\u003ein vitro\u003c/em\u003e propagation protocol from \u003cem\u003eHelichrysum umbraculigerum\u003c/em\u003e leaves (Botha et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); and Uz \u0026amp; Savalan (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) used young \u003cem\u003eH. italicum\u003c/em\u003e shoots for \u003cem\u003ein vitro\u003c/em\u003e production (Uz \u0026amp; Savalan, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, to our knowledge, there are no prior reports on the micropropagation of endemic \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e. Hence, this study aimed to develop an effective micropropagation method for this subspecies, using explants derived from seeds endemic to and naturally occurring in Turkey.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial\u003c/h2\u003e \u003cp\u003e \u003cem\u003eH. arenarium\u003c/em\u003e (L.) Moench subsp. \u003cem\u003eerzincanicum\u003c/em\u003e Davis \u0026amp; Kupicha (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) seeds were collected from the variety garden of the Muş Alparslan University Faculty of Applied Sciences in October 2021. Then it was identified by Dr. \u0026Uuml;mit Bing\u0026ouml;l (M.\u0026Uuml;. Bing\u0026ouml;l 6829) from the Faculty of Science, Ankara University.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSeed sterilization and germination\u003c/h2\u003e \u003cp\u003eSeeds were disinfected with 7.5% sodium hypochlorite (NaOCl) and placed in an oven at 25\u0026deg;C for 20 minutes. Afterward, 70% ethanol was applied 30 min and then incubated at 42\u0026deg;C for 24 hours. The sterile seeds were rinsed for 5 times with sterile distilled water (Battal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After sterilization, the media was additionally supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e gibberellic acid (GA\u003csub\u003e3\u003c/sub\u003e), 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 6-benzylaminopurine (BAP), and 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e amphotericin B. The samples were maintained at room temperature (24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) under white fluorescent light (3000 lux) with a photoperiod of 16 hours of light and 8 hours of darkness. The germination study included three replicates, each containing 20 seeds.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eShoot regeneration media\u003c/h3\u003e\n\u003cp\u003eAfter 21 days of germination, the \u003cem\u003ein vitro\u003c/em\u003e-obtained plantlets were transferred to MSB media and used as explants. The following nutrient media mixtures were prepared for shoot regeneration: M1 (1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP); M2 (2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP); M3 (3.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP); M4 (1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP and 0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA); M5 (2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP and 0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA); M6 (3.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP and 0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA); M7 (1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA); M8 (2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA); M9 (3.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA); M10 (4.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA); and M11 (5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA).\u003c/p\u003e\n\u003ch3\u003eCallus growth medium\u003c/h3\u003e\n\u003cp\u003eCalli that formed in media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP were used as explants for callus culture in MSB media in the presence of various PGRs. The following media mixtures were prepared for callus culture: 2,4-D (0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); TDZ (1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and picloram (0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); NAA (1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and TDZ (0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRooting of shoots\u003c/h2\u003e \u003cp\u003eEight-week-old shoots were transferred to MS medium supplemented with 0.1, 0.5, or 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA or IBA for root formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were statistically analysed using SPSS for Windows (SPSS Inc., Chicago, IL, USA). Differences in means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors (SEs) were evaluated for significance using Duncan\u0026rsquo;s one-way analysis of variance (ANOVA) at the \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSeed germination rate\u003c/h2\u003e \u003cp\u003eThe effects of the PGRs; BAP and GA\u003csub\u003e3\u003c/sub\u003e on the germination of \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e seeds on MSB medium are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The highest germination rate (27.22%) was obtained in MSB media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GA\u003csub\u003e3\u003c/sub\u003e and 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of the plant growth regulators BAP and GA\u003csub\u003e3\u003c/sub\u003e on the seed germination rate of \u003cem\u003eH\u003c/em\u003e. \u003cem\u003earenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e in MSB media\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eConcentration of PGRs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGermination rate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGA\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eBAP (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e27.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csub\u003eDifferent letters within the same column indicate a significant difference between means at the 0.05 level, and (\u0026plusmn;) denotes the standard error\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eShoot proliferation\u003c/h2\u003e \u003cp\u003eThe sterile plantlets obtained from the seeds were transferred to nutrient media containing three different concentrations of growth regulator (0.5, 1.0, and 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP) at the end of the 21st day (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The best shoot regeneration was observed MSB media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP (61.2%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of different concentrations of BAP in MSB media on \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e shoot formation 8 weeks after culture initiation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant growth regulator (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRate of shoot formation from the explants (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBAP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/b\u003e \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.0 3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csub\u003eDifferent letters in the same column indicate a significant difference between means at the 0.05 level, and (\u0026plusmn;) indicates the standard error\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe shoots obtained from media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP were transferred to MSB media supplemented with different combinations of KIN and IAA (Figas et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). By the end of the 30th day, the plantlets had grown in height (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the six different combinations of KIN and IAA tested for shoot regeneration, 5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA resulted in the highest percentage of shoot formation, reaching 60% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResponse of shoot formation of HAE on MSB media supplemented with different concentrations of KIN and IAA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePGR combination (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eShoot formation (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKIN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eIAA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/b\u003e \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCallus formation and structure\u003c/h2\u003e \u003cp\u003eThe structures and colours of the calli obtained are given in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, and the treatments yielded significantly different calli formation rates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest rate of callus formation (80.2%) was obtained with MSB media supplemented with 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TDZ and 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of callus growth of HAE on MSB media with different concentrations of 2,4-D, TDZ, NAA, and picloram\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePGR(s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCallus structure and colour\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCallus formation rate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2,4-D (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown, compact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown, friable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown, compact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTDZ (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePicloram (mg L\u003c/b\u003e\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown, compact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown, friable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown, compact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTDZ (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNAA (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown, compact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYellowish, friable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreen, friable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e80.2\u003c/b\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;16.8\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csub\u003eDifferent letters in the same column indicate a significant difference between means at the 0.05 level, and (\u0026plusmn;) indicates the standard error\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRooting\u003c/h2\u003e \u003cp\u003eThe elongated shoots were subsequently transferred to MSB media supplemented with 0.5, 1.0, or 1.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IBA or IAA for rooting, and roots developed within four weeks of culture. The highest percentage (77.8%) of roots were found in the 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IBA treatment group, in which new adventitious shoots formed (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe sterilization of the \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e seeds was challenging due to their small size and hairy structure. Battal et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported the sterilization method that was successfully applied in this study (Battal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While low amounts of sodium hypochlorite (7.5%) did not damage the seeds' structure, increasing the temperature to 42\u0026deg;C removed the bacteria, fungi, and spores that may have been present on the seeds' hairy surface. In addition to physical factors such as light, temperature, and humidity, the addition of an endogenous growth factor (GA\u003csub\u003e3\u003c/sub\u003e) is also required for seed germination. It has been reported that in \u003cem\u003eArabidopsis\u003c/em\u003e, GA\u003csub\u003e3\u003c/sub\u003e release causes the seed coat to weaken by stimulating the expression of genes involved in cell expansion and modification (Gupta \u0026amp; Chakrabarty, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Moreover, in their organogenesis studies on argan plants, Amghar et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that GA\u003csub\u003e3\u003c/sub\u003e increased the germination rate and that supplementing MS media with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP with 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GA\u003csub\u003e3\u003c/sub\u003e increased the number of adventitious shoots formed (Amghar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFirst, the seed germination rate in our study was 6.6% in MSB media in the absence of growth regulators. Thus, different concentrations of the PGRs BAP and GA\u003csub\u003e3\u003c/sub\u003e were subsequently used to promote germination and increase the germination rate. The best result (27.22%) was obtained in MS/B5 media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP and 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GA\u003csub\u003e3\u003c/sub\u003e. This study revealed that GA\u003csub\u003e3\u003c/sub\u003e ensures germination by breaking dormancy in the seeds while the hormone BAP increases shoot length. Since it is difficult to germinate the seed of \u003cem\u003eHelichrysum\u003c/em\u003e species, micropropagation studies have generally been performed on aerial parts (Clasquin \u0026amp; Henry, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Figas et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These results demonstrate that seed sterilization is applicable under certain physical conditions and that germination is possible using different plant growth regulators (PGRs), such as BAP and GA\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eSecond, \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e shoot development was optimal in MSB media supplemented with 5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA (60%). The experiment was stopped at the optimum concentration and that further testing might be needed to fully explore the dose-response relationship, and to determine if higher concentrations might be even more effective. According to Figas et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), supplementing MS media with the PGRs KIN and IAA positively affects height as well as shoot formation (Figas et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). On the other hand, Dimitrova and Nacheva (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), in their micropropagation study on \u003cem\u003eH. italicum\u003c/em\u003e, reported that the highest percentage of shoot regeneration occurred in DKW media supplemented with 5 \u0026micro;M KIN (Dimitrova \u0026amp; Nacheva, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Botha et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) optimized an \u003cem\u003ein vitro\u003c/em\u003e propagation protocol from \u003cem\u003eH. umbraculigerum\u003c/em\u003e leaves and found that shoots were produced most efficiently on MS media containing a specific concentration of TDZ (Botha et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Uz \u0026amp; Savalan (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that the highest shoot formation percentage from young \u003cem\u003eH. italicum\u003c/em\u003e shoots occurred in MS media supplemented with 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP, 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GA\u003csub\u003e3\u003c/sub\u003e, and 0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA, while the lowest percentage occurred in MS media without hormone supplementation (Uz \u0026amp; Savalan, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In another study, Giovannini et al. (2003) micropropagated \u003cem\u003eH. stoechas\u003c/em\u003e and \u003cem\u003eH. italicum\u003c/em\u003e plants and reported that the number of shoots increased twofold in response to supplementation with BAP (Giovannini et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our protocol, calli were formed via indirect organogenesis in media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP. The calli were subsequently transferred to MSB media supplemented with different concentrations of the PGRs 2,4-D, TDZ, NAA, and picloram. Green, friable calli grew best on MSB media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA and 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TDZ. Similarly, 81.3% green and friable calli of \u003cem\u003eH. pallasii\u003c/em\u003e were obtained by using 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TDZ (Yurteri et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The addition of different concentrations of different PGRs to calli growth media encourages root or shoot development to yield plantlets (Evans et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1981\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe auxin PGRs IAA, IBA and NAA are the most frequently PGRs reported in the literature for rooting. Notably, both root induction and root elongation are extremely sensitive to auxin concentration (Koca, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, the addition of auxins positively affected root and adventitious shoot formation in our study. The ideal mixture for rooting in our study was MS/B5 media supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IBA (77.8%). Although the rooting rate varies depending on the plant species in terms of a hormonal or nonhormonal environments, it has generally been reported that the growth regulator IBA promotes rooting and increases shoot length (Yurteri et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is the first report of the successful micropropagation of \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e using plant tissue culture techniques. The protocol described here uses BAP, KIN, and IAA to induce the proliferation of multiple shoots on tissue explants, and BAP supplementation produced the most robust organogenic response via indirect organogenesis. Following shoot production, plantlets treated with IBA developed root systems. This micropropagation protocol provides an alternative to seed propagation and contributes to the conservation of \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e, a rare and endemic plant species. Additionally, this study contributes to the development of \u003cem\u003ein vitro\u003c/em\u003e methods for propagating other \u003cem\u003eHelichrysum\u003c/em\u003e and rare plant species.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGibberellic acid (GA\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003eIndole-3-acetic acid (IAA)\u003c/p\u003e\n\u003cp\u003eMurashige and Skoog (MS)\u003c/p\u003e\n\u003cp\u003e6-benzylaminopurine (BAP)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eN6-furfuryladenine (kinetin (KIN))\u003c/p\u003e\n\u003cp\u003eThidiazuron (TDZ)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-naphthaleneacetic acid (NAA)\u003c/p\u003e\n\u003cp\u003ePlant growth regulator (PGR)\u003c/p\u003e\n\u003cp\u003eSodium hypochlorite (SH)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.A\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Data curation, Formal analysis, Methodology, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eŞ.\u0026Ouml;:\u003c/strong\u003e Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing \u0026ndash; original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was financed by the Mus Alparslan University Scientific Research Coordination Unit, 21-FEF-4901-04, Mus, Turkey.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data produced or analyzed during this study are included in this article. Data will be available upon request from the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors have declared that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Ahmet Yenikalaycı for plant material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmghar, I., Diria, G., Boumlik, I., Gaboun, F., Iraqi, D., Labhilili, M., Mentag, R., Meziani, R., Mazri, M. A., \u0026amp; Ibriz, M. (2021). An efficient regeneration pathway through adventitious organogenesis for the endangered\u003cem\u003e Argania spinosa\u003c/em\u003e (L.) Skeels. \u003cem\u003eVegetos\u003c/em\u003e,\u003cem\u003e 34\u003c/em\u003e, 355-367. \u003c/li\u003e\n\u003cli\u003eBattal, A., G\u0026ouml;rmez, G., \u0026amp; T\u0026uuml;rker, M. (2019). An effective germination method for medically important \u003cem\u003eAlthaea officinalis\u003c/em\u003e: notching the seed coat. \u003cem\u003eBitlis Eren University Journal of Science 8\u003c/em\u003e(3), 843-851. \u003c/li\u003e\n\u003cli\u003eBotha, K., Crampton, B., \u0026amp; Heyman, H. (2015). The in vitro propagation of \u003cem\u003eHelichrysum umbraculigerum\u003c/em\u003e. \u003cem\u003ePlanta Medica\u003c/em\u003e,\u003cem\u003e 81\u003c/em\u003e(16), PM_236. \u003c/li\u003e\n\u003cli\u003eClasquin, S., \u0026amp; Henry, M. (2002). Micropropagation d\u0026apos;\u003cem\u003eHelichrysum arenarium\u003c/em\u003e (L.) Moench. \u003cem\u003eActa botanica gallica\u003c/em\u003e,\u003cem\u003e 149\u003c/em\u003e(2), 189-195. \u003c/li\u003e\n\u003cli\u003eCzinner, E., Lemberkovics, \u0026Eacute;., Bih\u0026aacute;tsi-Karsai, E., Vit\u0026aacute;nyi, G., \u0026amp; Lelik, L. (2000). Composition of the essential oil from the inflorescence of \u003cem\u003eHelichrysum arenarium\u003c/em\u003e (L.) Moench. \u003cem\u003eJournal of Essential Oil Research\u003c/em\u003e,\u003cem\u003e 12\u003c/em\u003e(6), 728-730. https://doi.org/https://doi.org/10.1080/10412905.2000.9712202 \u003c/li\u003e\n\u003cli\u003eDănăilă-Guidea, S. M., Eremia, M. C., Dinu, L. D., \u0026amp; Miu, D.-M. (2022). Helichrysum arenarium: From cultivation to application. \u003cem\u003eApplied Sciences\u003c/em\u003e,\u003cem\u003e 12\u003c/em\u003e(20), 10241. \u003c/li\u003e\n\u003cli\u003eDimitrova, N. G., \u0026amp; Nacheva, L. (2018). Micropropagation of \u003cem\u003eHelichrysum italicum\u003c/em\u003e (ROTH) G. Don\u0026ndash;a medicinal plant with ornamental value. \u003cem\u003eJournal of BioScience and Biotechnology\u003c/em\u003e,\u003cem\u003e 7\u003c/em\u003e(2-3), 97-101. \u003c/li\u003e\n\u003cli\u003eEroğlu, H. E. (2018). T\u0026uuml;rkiye \u003cem\u003eHelichrysum \u003c/em\u003etaksonlarının T\u0026uuml;rk\u0026ccedil;e ve diğer dillerdeki isimleri. \u003cem\u003eAvrasya Terim Dergisi\u003c/em\u003e,\u003cem\u003e 6\u003c/em\u003e(1), 26-34. \u003c/li\u003e\n\u003cli\u003eEvans, D. A., Sharp, W. R., \u0026amp; Flick, C. E. (1981). Growth and behavior of cell cultures: embryogenesis and organogenesis. \u003cem\u003ePlant tissue culture: methods and applications in agriculture\u003c/em\u003e, 45-113. \u003c/li\u003e\n\u003cli\u003eFigas, A., Tomaszewska-Sowa, M., Sawilska, A., \u0026amp; Keutgen, A. J. (2016). Improvement of in vitro propagation and acclimation of \u003cem\u003eHelichrysum arenarium\u003c/em\u003e L. Moench. \u003cem\u003eActa scientiarum Polonorum. Hortorum cultus\u003c/em\u003e,\u003cem\u003e 15\u003c/em\u003e(4). \u003c/li\u003e\n\u003cli\u003eGiovannini, A., Amoretti, M., Savona, M., Di Guardo, A., \u0026amp; Ruffoni, B. (2001). Tissue culture in \u003cem\u003eHelichrysum\u003c/em\u003e spp. I International Symposium on Acclimatization and Establishment of Micropropagated Plants 616, \u003c/li\u003e\n\u003cli\u003eGradinaru, A. C., Silion, M., Trifan, A., Miron, A., \u0026amp; Aprotosoaie, A. C. (2014). \u003cem\u003eHelichrysum arenarium \u003c/em\u003esubsp. \u003cem\u003earenarium\u003c/em\u003e: phenolic composition and antibacterial activity against lower respiratory tract pathogens. \u003cem\u003eNatural product research\u003c/em\u003e,\u003cem\u003e 28\u003c/em\u003e(22), 2076-2080. \u003c/li\u003e\n\u003cli\u003eGupta, R., \u0026amp; Chakrabarty, S. (2013). Gibberellic acid in plant: still a mystery unresolved. \u003cem\u003ePlant signaling \u0026amp; behavior\u003c/em\u003e,\u003cem\u003e 8\u003c/em\u003e(9), e25504. \u003c/li\u003e\n\u003cli\u003eJakupovic, J., Kuhnke, J., Schuster, A., Metwally, M., \u0026amp; Bohlmann, F. (1986). Phloroglucinol derivatives and other constituents from South African \u003cem\u003eHelichrysum\u003c/em\u003e species. \u003cem\u003ePhytochemistry\u003c/em\u003e,\u003cem\u003e 25\u003c/em\u003e(5), 1133-1142. \u003c/li\u003e\n\u003cli\u003eKoca, A. S. (2015). \u003cem\u003e\u0026Ccedil;eltikte (Oryza sativa L.) endosperm destekli olgun embriyo k\u0026uuml;lt\u0026uuml;r\u0026uuml;nde kallus oluşumu ve bitki rejenerasyonuna tohum ıriliğinin etkisi\u003c/em\u003e Ankara Universitesi (Turkey)]. \u003c/li\u003e\n\u003cli\u003eLeonardi, M., Giovanelli, S., Ambryszewska, K. E., Ruffoni, B., Cervelli, C., Pistelli, L., Flamini, G., \u0026amp; Pistelli, L. (2018). Essential oil composition of six Helichrysum species grown in Italy. \u003cem\u003eBiochemical Systematics and Ecology\u003c/em\u003e,\u003cem\u003e 79\u003c/em\u003e, 15-20. \u003c/li\u003e\n\u003cli\u003eLiu, X., Jing, X., \u0026amp; Li, G. (2019). A process to acquire essential oil by distillation concatenated liquid-liquid extraction and flavonoids by solid-liquid extraction simultaneously from \u003cem\u003eHelichrysum arenarium\u003c/em\u003e (L.) Moench inflorescences under ionic liquid-microwave mediated. \u003cem\u003eSeparation and Purification Technology\u003c/em\u003e,\u003cem\u003e 209\u003c/em\u003e, 164-174. https://doi.org/https://doi.org/10.1016/j.seppur.2018.07.028 \u003c/li\u003e\n\u003cli\u003ePawelek, J. (2015). Water management in Poland in view of water supply and sewage disposal infrastructure development. \u003cem\u003eInfrastruktura i Ekologia Teren\u0026oacute;w Wiejskich\u003c/em\u003e(II/2). \u003c/li\u003e\n\u003cli\u003ePerrini, R., Morone-Fortunato, I., Lorusso, E., \u0026amp; Avato, P. (2009). Glands, essential oils and in vitro establishment of \u003cem\u003eHelichrysum italicum\u003c/em\u003e (Roth) G. Don ssp. \u003cem\u003emicrophyllum\u003c/em\u003e (Willd.) Nyman. \u003cem\u003eIndustrial Crops and Products\u003c/em\u003e,\u003cem\u003e 29\u003c/em\u003e(2-3), 395-403. \u003c/li\u003e\n\u003cli\u003ePrinsloo, G., \u0026amp; Meyer, J. (2006). In vitro production of phytoalexins by \u003cem\u003eHelichrysum kraussii\u003c/em\u003e. \u003cem\u003eSouth African Journal of Botany\u003c/em\u003e,\u003cem\u003e 72\u003c/em\u003e(3), 482-483. \u003c/li\u003e\n\u003cli\u003eUmaz, A., \u0026amp; Umaz, K. (2020). Determination and comparison of volatile compounds of gold grass (Helichrysum arenarium) from two different locations. \u003cem\u003eG\u0026uuml;m\u0026uuml;şhane University Journal of Science\u003c/em\u003e,\u003cem\u003e 10\u003c/em\u003e(3), 592-600. \u003c/li\u003e\n\u003cli\u003eUz, İ., \u0026amp; Savalan, Ş. (2024). In vitro Micropropagation of Immortelle Grass (\u003cem\u003eHelichrysum italicum\u003c/em\u003e (ROTH) G. DON). \u003cem\u003eTekirdağ Ziraat Fak\u0026uuml;ltesi Dergisi\u003c/em\u003e,\u003cem\u003e 21\u003c/em\u003e(1), 246-255. \u003c/li\u003e\n\u003cli\u003eYurteri, E., Aksu, M., K\u0026uuml;plemez, H., \u0026Ouml;zcan, A., \u0026amp; Seyis, F. (2021). Effects of Thidiazuran and Zeatin on Plant Regeneration in \u003cem\u003eHelichrysum pallasii\u003c/em\u003e. \u003cem\u003eUluslararası Tarım ve Yaban Hayatı Bilimleri Dergisi\u003c/em\u003e,\u003cem\u003e 7\u003c/em\u003e(3), 533-539. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endemic species, Helichrysum arenarium subsp. erzincanicum, Gibberellic acid, Kinetin, Micropropagation","lastPublishedDoi":"10.21203/rs.3.rs-6925544/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6925544/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study developed a micropropagation protocol using explants grown from seeds of \u003cem\u003eHelichrysum arenarium\u003c/em\u003e (L.) Moench subsp. \u003cem\u003eerzincanicum\u003c/em\u003e and plant tissue culture methods. Initially, indirect shoot organogenesis was performed with \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum in vitro\u003c/em\u003e plantlets on Murashige and Skoog (MS) and B5 media (abbrev. as MSB) supplemented with 6-benzylaminopurine (BAP), kinetin (Kin) and indole-3-acetic acid (IAA). A maximum number of shoots was induced in 1 mg L\u003csup\u003e-1\u003c/sup\u003e BAP (61.2%). Subsequently, the shoots were inoculated in MSB combinations with 0.5\u0026ndash;1.5 mg L\u003csup\u003e-1\u003c/sup\u003e indole-3-acetic acid and indole-3-butyric acid (IBA), wherein 1.0 mg L\u003csup\u003e-1\u003c/sup\u003e IBA (77.8%) recorded the maximum number of roots. Additionally, callus formation was achieved with different concentrations of thidiazuron (TDZ) (0.5, 1.0, 2.0 mg L\u003csup\u003e-1\u003c/sup\u003e) and α-naphthaleneacetic acid (NAA) (1.0 mg L\u003csup\u003e-1\u003c/sup\u003e) and the highest callus formation percentage (80.2%) was observed MSB media combinations with 2.0 mg L\u003csup\u003e-1\u003c/sup\u003e TDZ and 1.0 mg L\u003csup\u003e-1\u003c/sup\u003e NAA. This study presents the successful development of a micropropagation protocol for \u003cem\u003eH. arenarium\u003c/em\u003e subsp. \u003cem\u003eerzincanicum\u003c/em\u003e and supports its \u003cem\u003ein vitro\u003c/em\u003e propagation via explants grown from seed.\u003c/p\u003e","manuscriptTitle":"Micropropagation of Endemic Taxa of the Flora of Turkey: Helichrysum arenarium (L.) Moench subsp. erzincanicum P.H. Davis \u0026amp; Kupicha (Asteraceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 10:55:11","doi":"10.21203/rs.3.rs-6925544/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f4f0d83-a446-4fc6-91ff-d127695ccf90","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-24T11:25:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-30 10:55:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6925544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6925544","identity":"rs-6925544","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}