Enhancement of medium components and the effects of cytokinins and auxins on in vitro propagation and successful reintroduction of Thai endangered orchid Dendrobium proteranthum Seidenf

Enhancement of medium components and the effects of cytokinins and auxins on in vitro propagation and successful reintroduction of Thai endangered orchid Dendrobium proteranthum Seidenf | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancement of medium components and the effects of cytokinins and auxins on in vitro propagation and successful reintroduction of Thai endangered orchid Dendrobium proteranthum Seidenf Thanakorn Wongsa, Boworn Kunakhonnuruk, Wittaya Pakum, Charun Maknoi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8260330/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 An efficient in vitro propagation and reintroduction protocol was developed for Dendrobium proteranthum Seidenf., an endemic and rare orchid species of Thailand. Six-month-old seedlings were cultured on full-, half-, and quarter-strength MS media with varying sucrose levels. All treatments showed high survival (86.7–100%). Full-strength MS with 30 g L−1 sucrose was optimal, yielding the highest shoot (3.0), leaf (7.0), and root (7.2) numbers per plantlet. Seedlings were cultured on MS medium with 0–4.0 mg L−1 BA, kinetin, or TDZ for 12 weeks. The highest shoot induction (3.4 shoots and 8.3 leaves/plantlet) occurred with 1.0 mg L−1 BA, while root formation peaked in the control (5.9 roots/plantlet). The combined effects of BA and NAA (0–2.0 mg L−1) were tested. The combination of 0.5 mg L−1 BA + 1.0 mg L−1 NAA achieved 100% shoot formation, the highest shoot number (4.8 shoots/plantlet), and tallest plantlets (8.4 mm). Meanwhile, 0.5 mg L−1 BA + 0.5 mg L−1 NAA yielded 100% leaf and root formation with the most roots (14.4 roots/plantlet). The greatest leaf number (14.7 leaves/plantlet) occurred with 1.0 mg L−1 BA + 1.0 mg L−1 NAA. For ex situ conservation, plantlets acclimatized on sphagnum moss at Ban Romklao Botanical Garden showed 88.5% survival. After reintroduction to Phu Luang Wildlife Sanctuary, 78.7% survived after 40 weeks and completed both vegetative and reproductive stages. This study establishes an effective, reproducible protocol for mass propagation and in situ restoration of D. proteranthum. Dendrobium Propagation Preservation Reintroduction Ex situ conservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Dendrobium proteranthum Seidenf. is an epiphytic orchid species belonging to the family Orchidaceae (Seidenfaden 1985). It is endemic to Thailand and can be found only in specific habitats in Chiang Rai, Chiang Mai, Tak, Phitsanulok, Kamphaeng Phet, and particularly at Phu Luang Wildlife Sanctuary in Loei Province (Kasetluksamee and Ngernsaengsaruay 2010; Prommanut 2017). Unfortunately, this species is currently threatened with extinction due to natural and anthropogenic factors such as global warming, climate change, deforestation, and overexploitation. As a result, it has been listed in CITES Appendix II and included in The Thailand Red Data: Plants as an endangered species (Santisuk et al. 2006). This orchid grows exclusively in montane rainforests at altitudes between 1,400 and 1,500 meters above sea level (Sittisujjatham 2009). The flowers are small and waxy, with pale yellow sepals and petals bearing three dark purplish-red stripes, and a light brownish-yellow labellum marked with deep purplish-red streaks. The inflorescences are dense and long-lasting, remaining open for several days (Prommanut 2017). The species currently faces challenges such as low natural fruit set and ongoing habitat loss from deforestation and climate change, which may further reduce its populations and increase the risk of extinction in the wild (Chamchumroon et al. 2017). Tissue culture techniques have been widely employed not only for the rapid mass propagation of orchids but also for ex situ conservation purposes (Murthy and Pyati 2001). Several successful in vitro propagation and conservation methods have been developed for both large-scale orchid multiplication and in vitro preservation (Teixeira da Silva and Dobránszki 2013). The in vitro micropropagation of orchids, including Dendrobium species, has been extensively studied and optimized (Teixeira da Silva et al. 2015). Successful in vitro propagation depends on several key factors, including explant source, culture medium composition, sucrose concentration, plant growth regulators (PGRs), and environmental conditions (Chugh et al. 2009; Azmi et al. 2016; Zahara et al. 2017). Medium strength and sucrose concentration are among the most critical factors influencing plant development under in vitro conditions. Because the photosynthetic ability of in vitro plantlets is often limited by low CO₂ availability, sucrose serves as a vital energy source that supports growth and morphogenesis (Shin et al. 2013; Martins et al. 2015). The effects of medium strength and sucrose concentration have been investigated in several Dendrobium species such as D . candidum (Yang et al. 2015), D . officinale (Gao et al. 2020), and D . nobile (Arafa et al. 2021). Plant growth regulators such as cytokinins and auxins are also essential components influencing in vitro propagation of Dendrobium species (Chuengpanya et al. 2020; Nguyen et al. 2022). Cytokinins are commonly used to promote bud initiation and shoot regeneration in many plant species, including Dendrobium orchids. Among them, 6-benzylaminopurine (BA), 6-furfurylaminopurine (kinetin), and thidiazuron (TDZ) are the most effective and widely utilized (Teixeira da Silva et al. 2015; Nguyen 2017; Tham et al. 2018; Nguyen et al. 2022). The optimal cytokinin concentration for shoot multiplication varies depending on the Dendrobium species (Shiau et al. 2005; Kong et al. 2007; Sunitibala et al. 2009). Auxins, particularly indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA), are widely used in culture media to promote rooting by regulating cell division, elongation, and differentiation (Finet and Jaillais 2012; Márquez et al. 2016). Auxins can be applied alone or in combination with cytokinins, as their synergistic interaction plays a crucial role in shoot and root development. Such combinations have been effective in various Dendrobium species, including D . palpebrae (Bhowmik and Rahman 2020), D . stratiotes (Purwanto et al. 2023), D . heyneanum (Kaladharan et al. 2024), D . mussauense (Noli and Nur 2024), and D . primulinum (Liu et al. 2025). Because D . proteranthum has a small natural population distributed in limited areas of Thailand, germplasm preservation through ex situ conservation is urgently needed. Ex situ conservation of plants derived from in vitro propagation can be performed under greenhouse conditions (Phillips et al. 2020). However, plantlets obtained from in vitro culture require proper hardening and acclimatization before transfer to natural environments, as high mortality rates are often observed during the introduction and reintroduction stages (Mullin et al. 2022). Therefore, acclimatization is a critical final step in in vitro propagation, enabling plants to transition from aseptic culture conditions to greenhouse or natural habitats (Teixeira da Silva et al. 2017). Successful acclimatization and transfer from in vitro to ex vitro conditions have been reported for several Dendrobium species, including D . densiflorum (Mamu et al. 2022), D . moniliforme (Liu et al. 2023), D . cruentum (Samala and Thipwong 2023), and D . farmeri (Nuammee et al. 2024). Reintroduction of species into their natural habitats is also an important strategy for orchid conservation, contributing to germplasm recovery and biodiversity restoration (Wu et al. 2014). Therefore, a combination of ex situ and in situ conservation, together with reintroduction, represents an effective approach for conserving endangered orchid species threatened with extinction (Oldfield, 2009). To date, no studies have reported the development of an in vitro propagation, ex situ conservation, or reintroduction protocol for D . proteranthum . Therefore, the objectives of this study were to investigate the effects of medium strength and sucrose concentration to determine an optimal culture medium, improve multiplication rates using cytokinins and auxin-cytokinin combinations, and evaluate acclimatization under greenhouse conditions and the reintroduction of this endangered orchid species into its natural habitat. Materials and Methods Capsule preparation and sterilization Mature capsules of D . proteranthum collected from plants growing in Phu Luang Wildlife Sanctuary, Loei Province, were surface-sterilized with 10% (v/v) sodium hypochlorite (NaOCl) containing 1–2 drops of Tween-20 for 15 minutes (Fig. 1A), followed by 2–3 rinses with sterile distilled water. Capsules were cut longitudinally, and the seeds were aseptically removed and cultured on semi-solid Vacin and Went (VW) medium (Vacin and Went 1949) supplemented with 20 g L⁻¹ sucrose, 50 g L⁻¹ potato extract, and 150 mL L⁻¹ coconut water, and solidified with 7.5 g L⁻¹ agar. The pH of the medium was adjusted to 5.2 prior to autoclaving. Cultures were maintained in a growth room at 25 ± 2 °C under warm white LED lamps (40 μmol m⁻² s⁻¹) with a 12-h photoperiod. Seeds germinated asymbiotically, and the resulting seedlings were used as explants for further experiments. Factors affecting in vitro propagation of D. proteranthum Effect of medium strength and sucrose concentration on growth and development of D. proteranthum seedlings Six-month-old seedlings of D . proteranthum derived from germination on VW medium, measuring 1.5–2.0 cm in height with 2-3 leaves and roots, were cultured on semi-solid Murashige and Skoog (MS) medium (Murashige and Skoog 1962) of different strengths: full (MS), half (½MS), and quarter (¼MS). The media were supplemented with sucrose at 0, 10, 20, or 30 g L⁻¹. The pH of all media was adjusted to 5.7 and solidified with 7.5 g L⁻¹ agar before autoclaving at 121°C for 15 minutes. Cultures were maintained in a growth room at 25 ± 2°C under warm white LED illumination (40 μmol m⁻² s⁻¹) with a 12-hour photoperiod for 12 weeks. After the culture period, the survival (%), shoot formation (%), leaf formation (%), root formation, and the number of shoots, leaves, and roots were recorded. Effect of cytokinin on shoot multiplication, growth, and development of D . proteranthum seedlings Six-month-old seedlings of D. proteranthum , 1.5–2.0 cm in height with 2-3 leaves and roots, derived from seed germination, were used in this experiment. In vitro seedlings were cultured on semi-solid MS medium supplemented with 30 g L⁻¹ sucrose and 7.5 g L⁻¹ agar, and supplemented with BA, kinetin, and TDZ at concentrations of 0, 1.0, 2.0, and 4.0 mg L⁻¹. The pH of all media was adjusted to 5.7 prior to autoclaving at 121 °C for 15 minutes. Cultures were maintained in a growth room at 25 ± 2 °C under warm white LED lamps (40 μmol m⁻² s⁻¹) with a 12-h photoperiod. After 12 weeks of culture, the percentages of shoot, leaf, and root formation as well as the number of shoots, leaves, and roots per seedling were recorded. Effect of cytokinin and auxin on growth and development of D. proteranthum seedlings Six-month-old seedlings of D. proteranthum , 1.5–2.0 cm in height with 2–3 leaves and roots, derived from seed germination, were used in this experiment. Two seedlings were transferred to MS medium supplemented with 30 g L⁻¹ sucrose, 0.1 g L⁻¹ myo-inositol, and combinations of benzyladenine (BA) and naphthaleneacetic acid (NAA) at 0, 0.5, 1.0, and 2.0 mg L⁻¹. The medium (25 mL per bottle) was dispensed into 120 mL clear glass bottles, adjusted to pH 5.7, solidified with 7.5 g L⁻¹ agar, and autoclaved at 121 °C for 15 minutes. Cultures were maintained at 25 ± 2 °C under warm white LED lamps (40 μmol m⁻² s⁻¹) with a 12-h photoperiod for 12 weeks. At the end of the culture period, the percentages of survival, shoot, leaf, root, and protocorm-like bodies (PLBs) formation, as well as callus induction and flowering, were recorded. In addition, the number of shoots, leaves, and roots per seedling, plantlet height, leaf length and width, and root length were measured. Ex vitro conservation Transplantation and acclimatization for ex situ conservation in nursery Small clump of D . proteranthum plantlets containing 2–3 shoots with 3–4 cm in height at least 2–3 roots derived from the previous experiment: effect of media strength sucrose concentrations, shoot multiplication and growth development, effect cytokinin and auxins were used to acclimatize for ex situ conservation in greenhouse condition. Selected clumps of plantlets were taken out from culture bottles and rinsed thoroughly with tap water to remove residual nutrients and agar from the plantlets. Two types of supporting materials were used: chopped coconut husk chips and sphagnum moss. The plantlets were acclimatized under two environmental conditions: (1) shaded conditions and (2) light-exposed conditions covered with plastic bags to maintain humidity. The plastic covers were opened daily in the morning (08:00 – 09:00 AM) to release excess heat and moisture, then resealed until the completion of a 4-week acclimatization period. Each treatment consisted of 70 plantlets clumps with three replications. Thereafter, the covers were permanently removed, and survival rate, growth performance, and morphological changes of the seedlings were recorded continuously for up to 16 weeks and ex situ acclimatized in the greenhouse at Ban Romklao Botanical Garden, Phitsanulok with 70–80% relative humidity and about 9–12 h photoperiod, 100–200 µmol m -2 s -1 photosynthetic photon flux density (PPFD; shaded sunlight), and 33 ± 1 to 30 ± 1 °C day/night temperature. Plantlets were sprayed with water twice daily and acclimatized for 16 weeks. The growth, development, and survival of the plantlets were evaluated after 8 and 16 weeks of transplantation. Reintroduction of D . proteranthum plantlets into their natural habitat Regarding genetic diversity, all D. proteranthum clumps used in this experiment were derived from in vitro seed cultures established from mature capsules collected in the natural habitat, without any treatment to induce shoot multiplication. Small clumps, each containing 2–3 shoots (3–4 cm tall) and 2–3 roots, were removed from the culture vessels and rinsed with tap water. The plantlets were then immersed in a 0.1% (v/v) vitamin B1 solution containing rooting hormone for 30 minutes. At the onset of the rainy season in May 2022, the plantlets were directly attached to the branches of phorophytes and reintroduced into the natural habitats of the mother plant populations within Phu Luang Wildlife Sanctuary, Loei Province. A total of 10 plantlets clumps per replicated were used, with three replications. The survival and development of D . proteranthum plantlets were randomly sampled monitored every 10 weeks until 40 weeks after re-establishment (February to March 2023, late winter. After 40 weeks of reintroduction, the survival of plantlets was recorded. Experimental design and statistical analysis All experiments were performed in a completely randomized design (CRD) with three replications. The data were tested for differences between means of each parameter using one-way analysis of variance (one-way ANOVA), followed by Duncan’s Multiple Range Test (DMRT) in IBM SPSS Statistics software version 25 (Armonk, NY). All data normality was assessed with the Kolmogorov–Smirnov test. Results Effect of medium strength and sucrose concentration on the survival, shoot, leaf, and root formation of D . proteranthum plantlets The survival of plantlets was affected by both medium strength and sucrose concentration (Fig. 2A). In full-strength MS medium, survival increased significantly from 86.7% at 0 g L⁻¹ sucrose to 100% at 10 and 20 g L⁻¹, followed by a slight decline to 96.7% at 30 g L⁻¹, with no significant differences among the 10-30 g L⁻¹ treatments. A similar trend was observed in ½MS medium, where survival rose from 86.7% at 0 g L⁻¹ to 100% at 20 g L⁻¹ before decreasing slightly to 96.7% at 30 g L⁻¹. In ¼ MS medium, survival peaked at 100% with 10 g L⁻¹ sucrose and declined to 93.3% at 20 and 30 g L⁻¹. Medium strength and sucrose concentration significantly influenced shoot, leaf, and root formation in D. proteranthum plantlets (Fig. 2B-D). The highest shoot formation (100%) was observed in MS medium supplemented with 10-20 g L⁻¹ sucrose, ½MS medium with 20 g L⁻¹ sucrose, and ¼MS medium with 10 g L⁻¹ sucrose. In contrast, significantly lower shoot formation (83.3%) occurred in both MS and ½MS media without sucrose supplementation (Fig. 2B). Leaf formation ranged from 83.3% to 100% across treatments (Fig. 2C). The highest leaf formation (100%) was achieved in MS medium supplemented with 20 g L⁻¹ sucrose, although differences among treatments were not statistically significant. The lowest leaf formation (83.3%) was observed in MS and ½MS media without sucrose, and ¼MS medium with 20 g L⁻¹ sucrose. Root formation generally increased with sucrose supplementation (Fig. 2D). In both MS and ½MS media, root formation peaked at 93.3% with 20 g L⁻¹ sucrose, significantly higher than in the sucrose-free MS treatment. Similarly, in ½MS medium, root formation at 20 g L⁻¹ (93.3%) was significantly greater than at 0 g L⁻¹ (63.3%) and 30 g L⁻¹ (76.7%). In ¼MS medium, the highest root formation (90.0%) occurred at 10 g L⁻¹ sucrose, which was significantly higher than the control (63.3%). However, higher concentrations (20-30 g L⁻¹) lightly reduced root formation, ranging from 80.0% to 86.7%. Influence of medium strength and sucrose on the number of shoots, leaves, and roots in D . proteranthum plantlets The influence of medium strength and sucrose concentration on plantlets development after 12 weeks of culture are presented in Fig. 3. All treatments supported the formation of new shoots, leaves, and roots; however, their numbers varied significantly depending on medium strength and sucrose level (Table 1). Shoot number increased markedly with sucrose supplementation, reaching a maximum of 3.0 shoots per plantlet in MS and ½MS media with 30 g L⁻¹ sucrose, approximately 2.5 times higher than in ¼MS medium at the same sucrose concentration (1.2 shoots per plantlet). Leaf induction showed a similar trend, with MS medium containing 20 g L⁻¹ sucrose yielding the highest number of leaves (7.2 leaves per plantlet) obtained in MS medium supplemented with 20 g L⁻¹ sucrose, nearly threefold greater than in the sucrose-free control (2.5 leaves per plantlet). In contrast, ¼MS medium produced fewer shoots and leaves over all, with no significant improvement at higher sucrose concentrations. Root number also increased slightly with sucrose supplementation, peaking at 7.2 roots per plantlet in MS medium with 30 g L⁻¹ sucrose, 4.5 times greater than in the sucrose-free MS treatment (1.6 roots per plantlet). Effect of cytokinin on shoot, leaf, and root formation (%) in D . proteranthum plantlets Shoot formation was generally high across all treatments, ranging from 86.7% to 100.0% (Fig. 4A). The highest shoot formation (100%) occurred with 1.0 mg L⁻¹ BA, followed by 4.0 mg L⁻¹ TDZ (96.7%) with no significant difference from the control. In contrast, 1.0 mg L⁻¹ TDZ resulted in the lowest shoot formation (86.7%). Leaf formation of plantlets ranged from 93.3% to 100.0% among treatments (Fig. 4B). All BA concentrations (1–4 mg L⁻¹) promoted complete leaf formation (100%), while slight, non-significant reductions were observed in media supplemented with 2.0 mg L⁻¹ kinetin or 1.0 mg L⁻¹ TDZ (96.7%). The control exhibited the lowest leaf formation (93.3%). Root formation was most pronounced in the control and 1.0 mg L⁻¹ kinetin (90.0%), which was non-significant difference between treatments (Fig. 4C). While the increase in BA (1.0–4.0 mg L⁻¹) and kinetin (2.0-4.0 mg L⁻¹) concentration in the culture medium significantly reduced rooting, with root formation decreasing from 56.7% (1.0 mg L⁻¹) to 33.3% (4.0 mg L⁻¹) and 60.0% (2.0 mg L⁻¹ kinetin) to 53.3% (4.0 mg L⁻¹ kinetin). Whereas TDZ had the strongest reducing root formation for 4.5-fold when compared to control (90.0%) to 20.0% for 1.0 mg L⁻¹ TDZ. Effect of cytokinin on shoot, leaf, and root number of D . proteranthum plantlets Cytokinin added to the culture medium significantly impacted the growth and development of plantlets after 12 weeks, affecting the number of shoots, leaves, and roots (Fig. 5). The highest shoot number (3.4 shoots per plantlet) was achieved on MS medium supplemented with 1.0 or 2.0 mg L⁻¹ BA, which significantly outperformed all TDZ treatments. On the other hand, TDZ at 1.0–4.0 mg L⁻¹ produced the fewest shoots (2.7 shoots per plantlet), significantly lower than both the control and the 1.0–2.0 mg L⁻¹ BA treatments (Fig. 4A). Leaf number was also promoted by BA. Specifically, 1.0 mg L⁻¹ BA yielded the highest value (8.3 leaves per plantlet), which was significantly higher than both the control and TDZ at 2.0–4.0 mg L⁻¹ (Fig. 4B). Higher concentrations of BA (4.0 mg L⁻¹) and kinetin (4.0 mg L⁻¹) reduced leaf numbers to 7.0 and 6.5 leaves per plantlet, respectively. The lowest leaf production (5.9 leaves per plantlet) resulted from TDZ at 4.0 mg L⁻¹, which was 1.4 times lower than the yield observed with 1.0 mg L⁻¹ BA. The root number was greatest in the control (5.9 roots per plantlet), which was significantly higher than in all cytokinin-supplemented treatments (Fig. 4C). Root induction generally declined with increasing concentrations of BA and TDZ. Although kinetin at 1.0 mg L⁻¹ produced a relatively high root number (4.4 roots per plantlet), significantly lower than the control but higher than most BA and TDZ treatments. However, the lowest root induction was observed with 1.0 mg L⁻¹ TDZ, yielding only 0.3 roots per plantlet. Effect of BA and NAA on survival, shoot, leaf, and root formation in D . proteranthum The effect of BA and NAA were investigated after cultured for 12 weeks. Plantlets survival remained high across all treatments, ranging from 93.3 to 100% (Fig. 6A). The control medium exhibited 100% survival, similar to the media supplemented with 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA, 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA, 0.2 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, with no significant difference between treatments. Slight reductions in survival were observed with higher BA or NAA concentrations; for example, 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA or 0.5-2.0 mg L⁻¹ BA combined with 2.0 mg L⁻¹ NAA resulted in 93.3% survival, without statistically significant differences (Fig. 6A). Shoot formation was successfully induced in all treatments, ranging from 90% to 100% (Fig. 6B). The highest response (100.0%) was observed in media supplemented with 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA and 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, with no significant differences among treatments. The lowest shoot formation (90%) occurred in the control and in 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA treatment. Leaf formation was generally high across all treatments, ranging from 90% to 100%, with no statistically significant differences observed (Fig. 6C). The highest leaf formation (100%) was recorded in plantlets grown on 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA and 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA. In contrast, the lowest leaf formation (90%) occurred in media containing 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA and 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA. Root formation varied significantly among treatments (Fig. 6D). The combination of 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA yielded the highest rooting percentage (100%). Increasing NAA concentration to 2.0 mg L⁻¹ caused a marked reduction in rooting. Media supplemented with 0.5–2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA exhibited the lowest rooting percentages (10.0%). Influence of BA and NAA on protocorm-like bodies (PLBs) development, callus formation, and flowering of D . proteranthum PLBs formation was observed as green clumps at the base of the stem and leaf primordia (white arrows; Fig. 7A). Overall, PLBs induction was low across all treatments, ranging from 0.0% to 6.3%. No PLBs were observed in the control medium or in the treatment containing 1.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA (Fig. 7B). The combination of 0.5 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA resulted in the highest PLBs formation (6.3%), although this was not significantly different among treatments. In contrast, lower percentages of PLBs formation (3.3%) were observed in media containing 0.5 mg L⁻¹ BA combined with 0.5 or 1.0 mg L⁻¹ NAA, representing a 1.9-fold reduction compared with the best-performing treatment. Callus formation in plantlets was also affected by the interaction between BA and NAA concentrations. A rough, green, and compact callus typically developed at the cut ends of roots (white arrows; Fig. 7A). No callus was observed in the control medium or in most low-concentration treatments (0.5 mg L⁻¹ BA + 0.5-1.0 mg L⁻¹ NAA, and 1.0 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA) (Fig. 7B). Callus induction first appeared in media supplemented with 0.5 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA (3.3%) and increased with higher BA concentrations. The highest callus formation (13.3%) was obtained with 2.0 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA, which was significantly greater than the control and most other treatments (Fig. 7C). Regarding flowering, small flowers with yellowish-green petals and pale pink lips were observed (Fig. 7A). No flowering occurred in the control treatment or in most combinations of BA and NAA at lower concentrations (Fig. 7D). The highest flowering percentage (20%) was recorded in plantlets cultured on 2.0 mg L⁻¹ BA combined with 2.0 mg L⁻¹ NAA, which was significantly higher than all other treatments. Impact of BA and NAA on shoot, leaf, and root development of D . proteranthum plantlets The effects of different combinations of BA and NAA on plantlet growth and development are shown in Fig. 8 and summarized in Table 2. For shoot number, supplementation with 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA produced the highest value (4.8 shoots per plantlet), which was significantly greater than the control (3.1 shoots per plantlet). This represents approximately a 1.5-fold increase compared with the control medium lacking growth regulators. Leaf production ranged from 8.3 to 14.7 leaves per plantlet. The highest number of leaves (14.7 leaves per plantlet) was obtained in the medium containing 1.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA, which differed significantly from the control treatment. The fewest leaves (8.3 leaves per plantlet) were observed in the control medium. Root production was negatively affected by increasing concentrations of cytokinin and auxin. The maximum root number (14.4 roots per plantlet) was recorded at 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA. However, as BA and NAA concentrations increased, root production decreased markedly, reaching the lowest value (1.7 roots per plantlet) in the treatment with 1.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA. Plantlet height varied between 6.4 and 8.4 mm depending on the treatment. The control and the medium supplemented with 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA resulted in the tallest plantlets (8.4 mm), with no significant difference between these and the treatments containing 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA. In contrast, the shortest plantlets (6.4 mm) were observed in the medium containing 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA, which differed significantly from the control. Leaf size and root length of plantlets were influenced by BA and NAA combinations. The widest leaves (2.3 mm) were observed in plantlets grown on media containing 1.0 mg L⁻¹ BA combined with 0.5 or 1.0 mg L⁻¹ NAA. The longest leaves (13.9 mm) occurred in the control treatment, which did not differ significantly from the 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA treatment (13.8 mm). In contrast, the shortest leaves (11.5 mm) were recorded in plantlets cultured with 1.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, showing no significant difference from other high hormone treatments (1.0–2.0 mg L⁻¹ BA combined with 1.0–2.0 mg L⁻¹ NAA). Root length was greatest in the control medium (11.7 mm) and significantly exceeded all cytokinin-auxin treatments. Root length progressively decreased with the addition of BA and NAA, particularly at higher concentrations. For example, 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA reduced root length to 8.1 mm, and further increases in hormone concentrations resulted in even shorter roots. The shortest roots (2.7 mm) were observed in plantlets cultured with 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA. Greenhouse acclimatization of D . proteranthum plantlets for ex situ preservation Plantlets were transplanted for acclimatization in the greenhouse at Ban Romklao Botanical Garden, Phitsanulok Province, for ex situ conservation (Fig. 9A). Two substrates, chopped coconut husks and sphagnum moss, were evaluated for their effects on plantlet growth and survival. After 8 weeks, older leaves had senesced in all plantlets, while new pseudobulbs developed and continued to grow. Survival was highest in plantlets grown on chopped coconut husks (96.4%), whereas those in sphagnum moss showed slightly lower survival (93.2%) (Fig. 9B). After 16 weeks, survival rates declined slightly. The highest survival (88.5%) was observed in plantlets grown in sphagnum moss, compared with 83.8% in those grown on chopped coconut husks (Fig. 9B). Despite these minor differences in survival, overall plantlet growth on both substrates was comparable, with rapid root elongation and the emergence of new shoots, indicating successful acclimatization under ex situ conditions. Reintroduction of plantlets to the natural habitat Reintroduction of D . proteranthum plantlets into their natural habitat for in situ conservation was conducted at Phu Luang Wildlife Sanctuary, Loei Province, in May 2022 (Fig. 10A). In vitro-propagated plantlets were directly attached to phorophyte branches without prior acclimatization, demonstrating strong physiological readiness for field conditions. Within 10 weeks, new shoots, leaves, and roots emerged, and by 20 weeks, mature pseudobulbs had formed, reflecting normal ontogenetic progression. Seasonal growth patterns, including leaf expansion, senescence, and summer flowering, confirmed that reintroduced plantlets completed both vegetative and reproductive cycles in the wild. During the summer of 2023, flowers were produced, followed by capsule and seed formation. After 40 weeks, survival remained high at 78.7% (Fig. 10B), highlighting the effectiveness of direct reintroduction of in vitro-propagated plantlets for insitu conservation. Discussions Medium strength and sucrose concentration effects on plantlet survival and morphological development The present study demonstrated that medium strength and sucrose concentration significantly influenced the survival and growth of D . proteranthum plantlets. Full-strength MS medium supplemented with 30 g L⁻¹ sucrose was the most effective treatment, resulting in the highest survival rate and promoting enhanced shoot, leaf, and root development. These findings highlight the importance of a balanced supply of mineral nutrients and carbohydrates for successful morphogenesis (Rezali et al. 2017; Dönmez et al. 2022). Sucrose supplementation was also critical for plantlet survival and morphogenesis, as in vitro grown orchids generally possess limited photosynthetic capacity and depend on exogenous carbohydrates for energy supply and osmotic regulation (Coruzzi and Bush 2001; Rybczyński et al. 2007; Phillips and Garda 2019). Moreover, the interaction between sucrose concentration and medium strength suggested that an appropriate carbohydrate supply promotes vigorous growth by stimulating cell division and expansion (Demo et al. 2008; Huh et al. 2016; Shah et al. 2019). Comparable results have been reported in other Dendrobium species. In D . bigibbum × D . Thailand Black, MS medium containing 30 g L⁻¹ sucrose yielded the highest average number of leaves (Amarasinghe et al. 2021). Similarly, in D . anosmum , MS medium supplemented with 30 g L⁻¹ sucrose was optimal for inducing maximum shoot, leaf, and root formation in protocorms (Nguyen et al. 2022). Likewise, D . heterocarpum cultured on MS medium with 30 g L⁻¹ sucrose exhibited enhanced shoot number, shoot length, pseudobulb length, and leaf production (Longchar and Deb 2022). Influence of cytokinin on the percentage of shoot, leaf, and root formation in D . proteranthum plantlets Cytokinin type and concentration strongly influenced shoot, leaf, and root development in D. proteranthum plantlets. Overall, high shoot and leaf formation across most treatments indicates that all cytokinins tested effectively promote organogenesis. Exogenous cytokinins stimulate cell division and enlargement, enhancing stem proliferation, shoot bud initiation, and leaf development (George et al. 2008; Teixeira da Silva et al. 2015). At the molecular level, cytokinins regulate gene expression via histidine-phosphotransfer (HPt) proteins, which mediate phosphate signal transfer to promote cell differentiation, proliferation, and regeneration, helping overcome recalcitrance in vitro (Brenner et al. 2012; Hnatuszko-Konka et al. 2021; Yang et al. 2021). Consistent with these mechanisms, BA at low to moderate concentrations (1.0–2.0 mg L⁻¹) was most effective in promoting shoot and leaf proliferation in D . proteranthum . Similar observations have been reported in other orchids, including Cyrtopodium saintlegerianum (Rodrigues et al. 2015), D . moschatum (Ritti et al. 2023), and Eulophia bicallosa (Wongsa et al. 2025), In contrast, TDZ generally reduced shoot and root numbers at higher concentrations, reflecting its dual role in promoting shoot initiation at low levels but inhibiting growth when applied excessively (Çelikel et al. 2021). Root development exhibited an inverse relationship with shoot proliferation. The highest root numbers were observed in the control, whereas elevated cytokinin concentrations, particularly BA and TDZ, suppressed rooting. This pattern reflects the antagonistic interaction between cytokinins and auxins, where high cytokinin levels favor shoot formation but inhibit root induction (Kurepa and Smalle 2022; Li et al. 2021). Similar trends have been reported in other orchids, such as D . bensoniae (Riva et al. 2016), D . ellipsophyllum (Ritti et al. 2017), and D . anosmum (Nguyen et al. 2022). Cytokinin and auxin effects on survival , leaf, and root development of D . proteranthum plantlets The combined application of cytokinin (BA) and auxin (NAA) markedly influenced survival, growth, and development in D. proteranthum plantlets. Survival remained high across treatments, indicating that the tested hormone concentrations were suitable for sustaining plantlet viability over 12 weeks. Shoot and leaf formation were consistently high under most treatments, reflecting the synergistic interaction of cytokinin and auxin in promoting aerial organ development. The combination of 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA was most effective for shoot induction, while low to moderate cytokinin-auxin levels (0.5-1.0 mg L⁻¹ BA and 0.5-1.0 mg L⁻¹ NAA) generally enhanced leaf formation. Higher BA concentrations tended to increase leaf number slightly but could reduce root formation. These findings align with the known roles of cytokinins and auxins in shoot and leaf morphogenesis and the antagonistic interaction between the two hormones, which determines the balance between shoot proliferation and root induction (Wattanawikkit et al. 2011; Novak et al. 2014). Root development was most responsive to low BA and NAA concentrations. The combination of 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA yielded the highest root numbers and length, whereas higher concentrations inhibited root induction. This is consistent with auxin’s role in promoting root initiation and elongation at low levels, while high concentrations can be inhibitory (Vanneste and Friml 2009). Similar patterns have been observed in other orchids, including Orchis catasetum (Baker et al. 2014) and Taprobanea spathulata (Devi et al. 2015). Some species, however, require higher auxin-cytokinin concentrations for optimal rooting, reflecting species-specific responses (Riva et al. 2016). Cytokinin-auxin combinations also influenced protocorm-like bodies (PLBs) formation, callus induction, and flowering. Moderate cytokinin with higher auxin (0.5 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA) favored PLBs formation, likely due to the regulation of auxin-responsive genes controlling cell dedifferentiation and organogenesis (Fang et al. 2016; Guo et al. 2021). Callus induction was highest at 2.0 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA, supporting the role of a balanced auxin-cytokinin ratio in maintaining cells in a meristematic state for proliferation (Wu et al. 2004; Niknejad et al. 2011). Flowering was most pronounced at 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, indicating that higher cytokinin-to-auxin ratios promote reproductive transition, consistent with findings in other orchids (de Melo Ferreira et al. 2006; Cen et al. 2010; Zhao et al. 2012). Morphometric traits, including plantlet height, leaf and root size, were influenced by hormone levels. Moderate cytokinin-auxin concentrations-maintained growth comparable to controls, whereas higher concentrations reduced overall size, likely due to physiological or metabolic stress (Khatun et al. 2010; Rineksane et al. 2021). Greenhouse acclimatization of D . proteranthum plantlets for ex situ preservation The acclimatization of in vitro-derived seedlings to greenhouse conditions is a critical step in large-scale propagation and orchid conservation (Teixeira da Silva et al. 2017). This process enables a gradual transition from the sterile, high-humidity culture environment to the variable and less controlled external conditions (Kumar and Rao 2012; Ehirim et al. 2014). In vitro-grown plantlets typically have underdeveloped cuticles and limited stomatal function, making them highly susceptible to desiccation under ambient conditions (Hazarika 2006; Vahdati and Aliniaeifard 2017; Bertolino et al. 2019). Therefore, maintaining optimal humidity and temperature during the initial acclimatization phase is essential to ensure survival and successful ex vitro establishment (Nasution et al. 2020). For Dendrobium species, substrates such as chopped coconut husks, brick pieces, charcoal, and sphagnum moss are commonly used for hardening. These materials provide suitable physical support, aeration, and water and nutrient availability for root development (Deb and Pongener 2022; Banjare et al. 2023; Nuammee et al. 2024). In this study, ex situ acclimatization of D . proteranthum plantlets was conducted under greenhouse conditions at Ban Romklao Botanical Garden. The results showed that sphagnum moss effectively supported plantlet survival and growth. Plantlets developed new pseudobulbs, indicating successful vegetative growth and a smooth transition from in vitro to ex vitro conditions. Furthermore, plantlets grown on sphagnum moss exhibited healthy morphology, comparable to field-grown parent plants, confirming normal development and physiological stability. Sphagnum moss enhances humidity retention, aeration, and nutrient availability due to its high water-holding capacity, nutrient retention ability, and slightly acidic pH, which collectively support vigorous plant growth (Aubé et al. 2015; Kaveriamma et al. 2019; Nuammee et al. 2024). The Ban Romklao Botanical Garden, where acclimatization was performed, is characterized by mountainous terrain and valleys with elevations ranging from 750 to 1,280 m above sea level conditions similar to the natural montane rainforest habitat of D . proteranthum (1,400-1,500 m a.s.l.) (Sittisujjatham 2009). The area is humid with a cool climate throughout most of the year, providing an environment conducive to plant growth and conservation (Maknoi and Khammai 2015). Thus, the geographical and climatic conditions of Ban Romklao Botanical Garden are well suited for the ex situ preservation of D . proteranthum . These results demonstrate that D . proteranthum can be effectively acclimatized using readily available substrates such as sphagnum moss, supporting their use in ex situ conservation and large-scale propagation programs. Reintroduction of plantlet to the natural distribution area Ex situ and in situ conservation, together with reintroduction, are among the principal strategies for conserving threatened plant species (Oldfield 2009). For species that have lost their natural habitats, ex situ conservation in botanic gardens or controlled environments may provide the only means of survival in the short to medium term (Ren et al. 2014). In contrast, in situ conservation is generally regarded as a more sustainable approach, as it maintains species within their natural ecological contexts. Reintroduction of plants into their native habitats has therefore become an increasingly important component of global conservation efforts, particularly for critically endangered groups such as orchids (Zhao et al. 2021). Several successful orchid reintroductions have been reported, including Vanda coerulea (Seeni and Latha 2000), Dendrobium tosaense (Lo et al. 2004), Renanthera imschootiana (Wu et al. 2014), Cymbidium finlaysonianum (Rittirat et al. 2017), Gastrochilus matsuran (Kang et al. 2020), and Paphiopedilum armeniacum (Wang et al. 2021). However, most of these studies required an acclimatization stage prior to field re-establishment. In the present study, D . proteranthum plantlets were successfully reintroduced directly into their natural habitat without prior acclimatization—the first such report for this species. The successful establishment and survival of plantlets under in situ conditions demonstrate their strong physiological readiness and adaptability to natural environments. The observed ontogenetic progression from vegetative growth and pseudobulb formation to flowering and seed capsule production indicates that the reintroduced plantlets completed a full life cycle in the wild. Moreover, the production of viable capsules and seeds suggests the potential for natural regeneration, which is essential for sustaining wild populations over time. The high survival rate suggests that population decline may not result from unsuitable climate conditions but rather from factors such as pollinator limitation and illegal poaching. Long-term conservation will require further study of the species biology. Conclusion This study presents the first successful protocol for the in vitro propagation and reintroduction of D . proteranthum . Full-strength MS medium supplemented with 30 g L⁻¹ sucrose proved most effective, yielding the highest survival rate and promoting vigorous shoot, leaf, and root development. Among the cytokinin treatments tested, low concentrations of BA (1.0−2.0 mg L⁻¹) were optimal for enhancing shoot regeneration, providing a reliable step for subsequent multiplication. Furthermore, the combined application of BA and NAA (0.5−1.0 mg L⁻¹) exhibited a synergistic effect, significantly improving shoot, leaf, and root induction, as well as key morphometric parameters such as plantlet height and leaf and root size. Successful acclimatization of plantlets was achieved under greenhouse conditions using sphagnum moss, which facilitated the development of new pseudobulbs and roots, indicating physiological readiness for adaptation to ex situ environments. Remarkably, reintroduction of D . proteranthum plantlets into natural habitats was accomplished without a prior acclimatization phase, with plantlets establishing, growing, and reproducing successfully in situ. These findings provide a solid foundation for both ex situ and in situ conservation efforts, contributing to the restoration and long-term survival of this rare and valuable orchid species. Declarations Acknowledgments The authors would like to express their sincere gratitude to the Plant Tissue Research Unit, Department of Biology, Faculty of Science, Naresuan University, Phitsanulok, Thailand, for providing laboratory facilities and technical support throughout the research. Appreciation is also extended to Ban Romklao Phitsanulok Botanical Garden, The Botanical Garden Organization, Ministry of Natural Resources and Environment, Chat Trakan District, Phitsanulok, Thailand, for generously providing the site for greenhouse acclimatization and ex situ preservation. This work was conducted under permission no. 6411101 from the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Thailand. Funding This research was supported by the National Research Council of Thailand (NRCT) under the 2021 fiscal year funding. Author information Authors and Affiliations Plant Tissue Culture Laboratory, Program in Biology, Faculty of Science and Technology, Kamphaeng Phet Rajabhat University, Kamphaeng Phet 62000, Thailand Thanakorn Wongsa Plant Tissue Culture Research Unit, Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand Boworn Kunakhonnuruk, Onrut Sapatee, Julaluk Linjikao & Anupan Kongbangkerd Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand Wittaya Pakum Ban Romklao Phitsanulok Botanical Garden, The Botanical Garden Organization, Ministry of Natural Resources and Environment, Chat Trakan District, Phitsanulok 65170, Thailand Charun Maknoi Contributions Conceptualization, AK, TW, BK, and WP; methodology, AK; validation, TW, BK, AK, TW, and WP; formal analysis, BK and WP; investigation, AK, and TW; resources, AK and CM; data curation, TW, BK, and WP; writing—original draft preparation, AK, TW, BK, WP, OS, and JL; writing—review and editing, JL and AK. 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Subtrop Plant Sci 41:48–50 Tables Table 1. Number of shoots, leaves, and roots of D . proteranthum plantlets after culture on different media strength and sucrose concentrations for 12 weeks. Media strength Sucrose (g L −1 ) Average number per plantlet Shoots Leaves Roots MS 0 1.6 ± 0.2 bc 2.5 ± 1.0 f 1.6 ± 0.2 e 10 2.7 ± 0.5 a 5.8 ± 0.4 bcd 3.2 ± 0.7 cde 20 2.7 ± 0.2 a 7.2 ± 0.3 a 6.3 ± 0.8 ab 30 3.0 ± 0.1 a 7.0 ± 0.3 ab 7.2 ± 0.3 a 1/2MS 0 1.5 ± 0.1 bc 3.5 ± 0.2 ef 2.0 ± 0.4 de 10 2.2 ± 0.4 ab 5.2 ± 0.2 cd 3.9 ± 0.2 cd 20 2.3 ± 0.3 ab 4.5 ± 0.4 de 4.8 ± 1.0 bc 30 3.0 ± 0.4 a 6.1 ± 0.6 abc 5.1 ± 1.4 bc 1/4MS 0 1.3 ± 0.2 c 2.6 ± 0.1 f 1.8 ± 0.1 de 10 1.3 ± 0.1 c 2.8 ± 0.1 f 3.0 ± 0.5 cde 20 1.5 ± 0.1 bc 2.9 ± 0.2 f 3.6 ± 0.4 cde 30 1.2 ± 0.1 c 2.8 ± 0.4 f 3.3 ± 0.5 cde Results are mean ± SE of 3 replicates (30 seedlings per replicate). Means with the same letter(s) in the same column are not significantly different at p ≤ 0.05 according to Duncan’s multiple range test (DMRT). Table 2. Effect of cytokinin and auxin on the number of shoots, leaves, and roots, as well as plantlet height, leaf width, leaf length, and root length of D . proteranthum seedlings after 12 weeks of culture. BA (mg L⁻¹) NAA (mg L⁻¹) Average number per plantlet Plantlet height (mm) Leaf width (mm) Leaf length (mm) Root length (mm) Shoots Leaves Roots 0 0 3.1 ± 0.4 c 8.3 ± 0.9 c 9.2 ± 0.6 b 8.4 ± 0.0 a 1.9 ± 0.0 b 13.9 ± 0.0 a 11.7 ± 0.1 a 0.5 0.5 3.5 ± 0.2 bc 11.2 ± 0.9 b 14.4 ± 0.9 a 8.2 ± 0.1 a 2.1 ± 0.0 ab 13.1 ± 0.0 ab 8.1 ± 0.0 b 1.0 4.8 ± 0.6 a 13.8 ± 0.2 ab 7.0 ± 1.1 bc 8.4 ± 0.0 a 2.2 ± 0.0 ab 13.8 ± 0.0 a 5.8 ± 0.0 c 2.0 4.4 ± 0.5 ab 13.1 ± 1.2 ab 2.4 ± 0.7 e 6.9 ± 0.1 b 2.1 ± 0.0 ab 11.8 ± 0.1 b 4.6 ± 0.1 cd 1.0 0.5 4.0 ± 0.5 abc 13.1 ± 1.1 ab 9.2 ± 2.1 b 7.3 ± 0.0 ab 2.3 ± 0.0 a 12.8 ± 0.1 ab 4.7 ± 0.0 cd 1.0 4.5 ± 0.2 ab 14.7 ± 0.7 a 6.4 ± 1.6 bcd 6.8 ± 0.0 b 2.3 ± 0.0 a 11.6 ± 0.0 b 3.5 ± 0.0 de 2.0 3.9 ± 0.3 abc 11.2 ± 0.7 b 1.7 ± 0.9 e 6.8 ± 0.0 b 2.2 ± 0.0 ab 11.5 ± 0.1 b 3.5 ± 0.1 de 2.0 0.5 3.4 ± 0.1 bc 11.5 ± 0.3 b 5.0 ± 0.8 cde 6.8 ± 0.1 b 2.2 ± 0.0 ab 12.6 ± 0.0 ab 5.1 ± 0.0 cd 1.0 3.7 ± 0.1 abc 11.4 ± 0.8 b 3.1 ± 0.5 de 6.4 ± 0.0 b 2.1 ± 0.0 ab 11.9 ± 0.1 b 2.9 ± 0.0 e 2.0 4.7 ± 0.2 a 12.3 ± 0.7 ab 1.8 ± 0.7 e 6.8 ± 0.0 b 2.1 ± 0.0 ab 12.5 ± 0.1 ab 2.7 ± 0.0 e Results are mean ± SE of 3 replicates (30 seedlings per replicate). Means with the same letter(s) in the same column are not significantly different at p ≤ 0.05 according to Duncan’s multiple range test (DMRT). Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":203345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDendrobium proteranthum\u003c/em\u003e Seidenf.\u003cem\u003e \u003c/em\u003ein its natural habitat at Phu Luang Wildlife Sanctuary, Loei Province. (A) Mature capsule of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e. (B) Seedlings of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e germinated and developed on semi-solid VW medium, used as initial explants for further experiments.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/5aab6aa34115c052e3373636.jpg"},{"id":98629033,"identity":"be526622-7b8a-4594-8b72-635cf78db476","added_by":"auto","created_at":"2025-12-19 17:13:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":246110,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different medium strengths (MS, ½MS, and ¼MS) and sucrose concentrations (10, 20, and 30 g L\u003csup\u003e−1\u003c/sup\u003e) on the percentages of survival, shoot, leaf, and root formation in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets after 12 weeks of culture. Data represent the mean of three replicates (30 seedlings per replicate). Error bars indicate the standard error (SE). Different letters above the bars denote significant differences at \u003cem\u003ep ≤ \u003c/em\u003e0.05 according to Duncan’s Multiple Range Test (DMRT); ns = not significant.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/98411eb2ee1ae72e6e4375d4.jpg"},{"id":98628512,"identity":"93d915e2-c9b5-4835-bbcb-9406421ae4ab","added_by":"auto","created_at":"2025-12-19 17:11:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":498073,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different medium strengths (MS, ½MS, and ¼MS) and sucrose concentrations on the growth and development of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003eplantlets after 12 weeks of culture.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/52cba918ac80e6f0202ce183.jpg"},{"id":98629560,"identity":"c5efc7a2-1a4f-4b94-a883-ea6084758b7f","added_by":"auto","created_at":"2025-12-19 17:14:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":300992,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different cytokinin concentrations (BA, kinetin, and TDZ) on survival rate (%), shoot (A), leaf (B), and root (C) formation, as well as the number of shoots, leaves, and roots in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets after 12 weeks of culture. Data represent the mean of three replicates (30 seedlings per replicate). Error bars indicate the standard error (SE). Different letters above the bars denote significant differences at \u003cem\u003ep ≤\u003c/em\u003e 0.05 according to Duncan’s Multiple Range Test (DMRT); ns = not significant.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/c520bad5ad58fc65f28b1930.jpg"},{"id":98629002,"identity":"2ed2674d-ab19-4bee-ab0a-14dc39784109","added_by":"auto","created_at":"2025-12-19 17:13:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":340075,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different cytokinin concentrations (BA, kinetin, and TDZ) on the growth and development of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003eseedlings after 12 weeks of culture.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/f7eff2423bd78b14cd001ca5.jpg"},{"id":98628442,"identity":"8b738f06-086d-486d-9797-256a778abff3","added_by":"auto","created_at":"2025-12-19 17:11:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":223453,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of cytokinin and auxin on survival (A), leaf formation (B), and root formation (C) in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003eseedlings after 12 weeks of culture. Results are presented as the mean of three replicates (30 seedlings per replicate). Error bars represent the standard error (SE). Different letters on each bar indicate significant differences at \u003cem\u003ep\u003c/em\u003e ≤ 0.05 according to Duncan’s Multiple Range Test (DMRT); ns = non-significant.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/859d6ba0cfcc6838ea3f2167.jpg"},{"id":98628742,"identity":"4e3407c7-48b0-419a-9b41-aba30d5d7aed","added_by":"auto","created_at":"2025-12-19 17:12:20","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":213467,"visible":true,"origin":"","legend":"\u003cp\u003eProtocorm-like bodies (PLBs) formation, callus induction, and flowering of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e (A) on media supplemented with cytokinin and auxin, and the percentage of PLBs formation (%) (B), callus formation (%) (C), and flowering (%) (D) in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003eseedlings after 12 weeks of culture. Data represent the mean of three replicates (30 seedlings per replicate). Error bars indicate the standard error (SE). Different letters above the bars denote significant differences at \u003cem\u003ep ≤ \u003c/em\u003e0.05 according to Duncan’s Multiple Range Test (DMRT).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/8b97fd8503ddbc5c6f2cb28e.jpg"},{"id":98628398,"identity":"f5a340fa-f1f6-4303-8114-23ffabd66f74","added_by":"auto","created_at":"2025-12-19 17:11:27","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":247170,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth and development of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003eplantlets after 12 weeks of culture on MS medium supplemented with combinations of BA (0.5, 1.0, and 2.0 mg L⁻¹) and NAA (0.5, 1.0, and 2.0 mg L⁻¹).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/7cc7157590bec0e9e14abb9b.jpg"},{"id":98628573,"identity":"4689fe8b-d84c-48bd-8a0b-d5826ee7968e","added_by":"auto","created_at":"2025-12-19 17:11:45","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":210338,"visible":true,"origin":"","legend":"\u003cp\u003eEx situ\u003cem\u003e \u003c/em\u003econservation of\u003cem\u003e D. proteranthum \u003c/em\u003eplantlet clumps at Ban Rom Klao Botanical Garden, Phitsanulok Province, Thailand. (A) Plantlet clumps after transplantation to sphagnum moss and coconut husk chips. (B) Survival of plantlets after transplantation for 8 and 16 weeks. Data represent the mean ± SE of three replicates (70 plantlet clumps per replicate).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/b3431b69f443de0e136bf301.jpg"},{"id":98617235,"identity":"12af91cb-b049-49ad-802e-4b280681d127","added_by":"auto","created_at":"2025-12-19 15:30:26","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":351372,"visible":true,"origin":"","legend":"\u003cp\u003eReintroduction of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003eplantlets at Phu Luang Wildlife Sanctuary, Loei Province, Thailand. (A) Plantlets reintroduced into their natural habitat. (B) Survival percentage of plantlets after 40 weeks in the natural habitat. Data represent the mean ± SE of three replicates (10 plantlet clumps per replicate).\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/3c3000981b92dc0f08c1075e.jpg"},{"id":104404480,"identity":"ad99054a-d617-4b06-b021-0fd0ce6a6ee8","added_by":"auto","created_at":"2026-03-11 12:20:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4887000,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8260330/v1/f52e4853-e88f-45ba-bea7-efab7cfd6552.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEnhancement of medium components and the effects of cytokinins and auxins on in vitro propagation and successful reintroduction of Thai endangered orchid Dendrobium proteranthum Seidenf\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eDendrobium proteranthum\u003c/em\u003e Seidenf. is an epiphytic orchid species belonging to the family Orchidaceae (Seidenfaden 1985). It is endemic to Thailand and can be found only in specific habitats in Chiang Rai, Chiang Mai, Tak, Phitsanulok, Kamphaeng Phet, and particularly at Phu Luang Wildlife Sanctuary in Loei Province (Kasetluksamee and Ngernsaengsaruay 2010; Prommanut 2017). Unfortunately, this species is currently threatened with extinction due to natural and anthropogenic factors such as global warming, climate change, deforestation, and overexploitation. As a result, it has been listed in CITES Appendix II and included in The Thailand Red Data: Plants as an endangered species (Santisuk et al. 2006).\u003c/p\u003e\n\u003cp\u003eThis orchid grows exclusively in montane rainforests at altitudes between 1,400 and 1,500 meters above sea level (Sittisujjatham 2009). The flowers are small and waxy, with pale yellow sepals and petals bearing three dark purplish-red stripes, and a light brownish-yellow labellum marked with deep purplish-red streaks. The inflorescences are dense and long-lasting, remaining open for several days (Prommanut 2017). The species currently faces challenges such as low natural fruit set and ongoing habitat loss from deforestation and climate change, which may further reduce its populations and increase the risk of extinction in the wild (Chamchumroon et al. 2017).\u003c/p\u003e\n\u003cp\u003eTissue culture techniques have been widely employed not only for the rapid mass propagation of orchids but also for ex situ conservation purposes (Murthy and Pyati 2001). Several successful in vitro propagation and conservation methods have been developed for both large-scale orchid multiplication and in vitro preservation (Teixeira da Silva and Dobránszki 2013). The in vitro micropropagation of orchids, including \u003cem\u003eDendrobium\u003c/em\u003e species, has been extensively studied and optimized (Teixeira da Silva et al. 2015). Successful in vitro propagation depends on several key factors, including explant source, culture medium composition, sucrose concentration, plant growth regulators (PGRs), and environmental conditions (Chugh et al. 2009; Azmi et al. 2016; Zahara et al. 2017). Medium strength and sucrose concentration are among the most critical factors influencing plant development under in vitro conditions. Because the photosynthetic ability of in vitro plantlets is often limited by low CO₂ availability, sucrose serves as a vital energy source that supports growth and morphogenesis (Shin et al. 2013; Martins et al. 2015). The effects of medium strength and sucrose concentration have been investigated in several \u003cem\u003eDendrobium\u003c/em\u003e species such as \u003cem\u003eD\u003c/em\u003e. \u003cem\u003ecandidum\u003c/em\u003e (Yang et al. 2015), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eofficinale\u003c/em\u003e (Gao et al. 2020), and \u003cem\u003eD\u003c/em\u003e. \u003cem\u003enobile\u003c/em\u003e (Arafa et al. 2021). Plant growth regulators such as cytokinins and auxins are also essential components influencing in vitro propagation of \u003cem\u003eDendrobium\u003c/em\u003e species (Chuengpanya et al. 2020; Nguyen et al. 2022). Cytokinins are commonly used to promote bud initiation and shoot regeneration in many plant species, including \u003cem\u003eDendrobium\u003c/em\u003e orchids. Among them, 6-benzylaminopurine (BA), 6-furfurylaminopurine (kinetin), and thidiazuron (TDZ) are the most effective and widely utilized (Teixeira da Silva et al. 2015; Nguyen 2017; Tham et al. 2018; Nguyen et al. 2022). The optimal cytokinin concentration for shoot multiplication varies depending on the \u003cem\u003eDendrobium\u003c/em\u003e species (Shiau et al. 2005; Kong et al. 2007; Sunitibala et al. 2009).\u003c/p\u003e\n\u003cp\u003eAuxins, particularly indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA), are widely used in culture media to promote rooting by regulating cell division, elongation, and differentiation (Finet and Jaillais 2012; Márquez et al. 2016). Auxins can be applied alone or in combination with cytokinins, as their synergistic interaction plays a crucial role in shoot and root development. Such combinations have been effective in various \u003cem\u003eDendrobium\u003c/em\u003e species, including \u003cem\u003eD\u003c/em\u003e. \u003cem\u003epalpebrae\u003c/em\u003e (Bhowmik and Rahman 2020), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003estratiotes\u003c/em\u003e (Purwanto et al. 2023), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eheyneanum\u003c/em\u003e (Kaladharan et al. 2024), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003emussauense\u003c/em\u003e (Noli and Nur 2024), and \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eprimulinum\u003c/em\u003e (Liu et al. 2025). Because \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e has a small natural population distributed in limited areas of Thailand, germplasm preservation through ex situ conservation is urgently needed. Ex situ conservation of plants derived from in vitro propagation can be performed under greenhouse conditions (Phillips et al. 2020). However, plantlets obtained from in vitro culture require proper hardening and acclimatization before transfer to natural environments, as high mortality rates are often observed during the introduction and reintroduction stages (Mullin et al. 2022). Therefore, acclimatization is a critical final step in in vitro propagation, enabling plants to transition from aseptic culture conditions to greenhouse or natural habitats (Teixeira da Silva et al. 2017).\u003c/p\u003e\n\u003cp\u003eSuccessful acclimatization and transfer from in vitro to \u003cem\u003eex vitro\u003c/em\u003e conditions have been reported for several \u003cem\u003eDendrobium\u003c/em\u003e species, including \u003cem\u003eD\u003c/em\u003e. \u003cem\u003edensiflorum\u003c/em\u003e (Mamu et al. 2022), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003emoniliforme\u003c/em\u003e (Liu et al. 2023), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003ecruentum\u003c/em\u003e (Samala and Thipwong 2023), and \u003cem\u003eD\u003c/em\u003e. \u003cem\u003efarmeri\u003c/em\u003e (Nuammee et al. 2024). Reintroduction of species into their natural habitats is also an important strategy for orchid conservation, contributing to germplasm recovery and biodiversity restoration (Wu et al. 2014). Therefore, a combination of ex situ and in situ conservation, together with reintroduction, represents an effective approach for conserving endangered orchid species threatened with extinction (Oldfield, 2009).\u003c/p\u003e\n\u003cp\u003eTo date, no studies have reported the development of an in vitro propagation, ex situ conservation, or reintroduction protocol for \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e. Therefore, the objectives of this study were to investigate the effects of medium strength and sucrose concentration to determine an optimal culture medium, improve multiplication rates using cytokinins and auxin-cytokinin combinations, and evaluate acclimatization under greenhouse conditions and the reintroduction of this endangered orchid species into its natural habitat.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCapsule preparation and sterilization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature capsules of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e collected from plants growing in Phu Luang Wildlife Sanctuary, Loei Province, were surface-sterilized with 10% (v/v) sodium hypochlorite (NaOCl) containing 1–2 drops of Tween-20 for 15 minutes (Fig. 1A), followed by 2–3 rinses with sterile distilled water. Capsules were cut longitudinally, and the seeds were aseptically removed and cultured on semi-solid Vacin and Went (VW) medium (Vacin and Went 1949) supplemented with 20 g L⁻¹ sucrose, 50 g L⁻¹ potato extract, and 150 mL L⁻¹ coconut water, and solidified with 7.5 g L⁻¹ agar. The pH of the medium was adjusted to 5.2 prior to autoclaving. Cultures were maintained in a growth room at 25 ± 2 °C under warm white LED lamps (40 μmol m⁻² s⁻¹) with a 12-h photoperiod. Seeds germinated asymbiotically, and the resulting seedlings were used as explants for further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFactors affecting in vitro propagation of \u003cem\u003eD. proteranthum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of medium strength and sucrose concentration on growth and development of \u003cem\u003eD. proteranthum\u003c/em\u003e seedlings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-month-old seedlings of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e derived from germination on VW medium, measuring 1.5–2.0 cm in height with 2-3 leaves and roots, were cultured on semi-solid Murashige and Skoog (MS) medium (Murashige and Skoog 1962) of different strengths: full (MS), half (½MS), and quarter (¼MS). The media were supplemented with sucrose at 0, 10, 20, or 30 g L⁻¹. The pH of all media was adjusted to 5.7 and solidified with 7.5 g L⁻¹ agar before autoclaving at 121°C for 15 minutes. Cultures were maintained in a growth room at 25 ± 2°C under warm white LED illumination (40 μmol m⁻² s⁻¹) with a 12-hour photoperiod for 12 weeks. After the culture period, the survival (%), shoot formation (%), leaf formation (%), root formation, and the number of shoots, leaves, and roots were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of cytokinin on shoot multiplication, growth, and development of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e seedlings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-month-old seedlings of \u003cem\u003eD. proteranthum\u003c/em\u003e, 1.5–2.0 cm in height with 2-3 leaves and roots, derived from seed germination, were used in this experiment. In vitro seedlings were cultured on semi-solid MS medium supplemented with 30 g L⁻¹ sucrose and 7.5 g L⁻¹ agar, and supplemented with BA, kinetin, and TDZ at concentrations of 0, 1.0, 2.0, and 4.0 mg L⁻¹. The pH of all media was adjusted to 5.7 prior to autoclaving at 121 °C for 15 minutes. Cultures were maintained in a growth room at 25 ± 2 °C under warm white LED lamps (40 μmol m⁻² s⁻¹) with a 12-h photoperiod. After 12 weeks of culture, the percentages of shoot, leaf, and root formation as well as the number of shoots, leaves, and roots per seedling were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of cytokinin and auxin on growth and development of \u003cem\u003eD. proteranthum\u003c/em\u003e seedlings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-month-old seedlings of \u003cem\u003eD. proteranthum\u003c/em\u003e, 1.5–2.0 cm in height with 2–3 leaves and roots, derived from seed germination, were used in this experiment. Two seedlings were transferred to MS medium supplemented with 30 g L⁻¹ sucrose, 0.1 g L⁻¹ myo-inositol, and combinations of benzyladenine (BA) and naphthaleneacetic acid (NAA) at 0, 0.5, 1.0, and 2.0 mg L⁻¹. The medium (25 mL per bottle) was dispensed into 120 mL clear glass bottles, adjusted to pH 5.7, solidified with 7.5 g L⁻¹ agar, and autoclaved at 121 °C for 15 minutes. Cultures were maintained at 25 ± 2 °C under warm white LED lamps (40 μmol m⁻² s⁻¹) with a 12-h photoperiod for 12 weeks. At the end of the culture period, the percentages of survival, shoot, leaf, root, and protocorm-like bodies (PLBs) formation, as well as callus induction and flowering, were recorded. In addition, the number of shoots, leaves, and roots per seedling, plantlet height, leaf length and width, and root length were measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx vitro conservation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransplantation and acclimatization for ex situ conservation in nursery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmall clump of \u003cem\u003eD\u003c/em\u003e.\u003cem\u003e\u0026nbsp;proteranthum\u003c/em\u003e plantlets containing 2–3 shoots with 3–4 cm in height\u0026nbsp;\u003cstrong\u003eat least\u0026nbsp;\u003c/strong\u003e2–3 roots derived from the previous experiment: effect of media strength sucrose concentrations, shoot multiplication and growth development, effect cytokinin and auxins were used to acclimatize for ex situ conservation in greenhouse condition. Selected clumps of plantlets were taken out from culture bottles and rinsed thoroughly with tap water to remove residual nutrients and agar from the plantlets.\u0026nbsp;\u003cstrong\u003eTwo types of supporting materials were used: chopped coconut husk chips and sphagnum moss. The plantlets were acclimatized under two environmental conditions: (1) shaded conditions and (2) light-exposed conditions covered with plastic bags to maintain humidity. The plastic covers were opened daily in the morning (08:00\u003c/strong\u003e–\u003cstrong\u003e09:00 AM) to release excess heat and moisture, then resealed until the completion of a 4-week acclimatization period. Each treatment consisted of 70 plantlets clumps with three replications. Thereafter, the covers were permanently removed, and survival rate, growth performance, and morphological changes of the seedlings were recorded continuously for up to 16 weeks and\u0026nbsp;\u003c/strong\u003eex situ acclimatized in the greenhouse at\u0026nbsp;Ban Romklao Botanical Garden, Phitsanulok\u0026nbsp;with 70–80% relative humidity and about 9–12 h photoperiod, 100–200 µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e photosynthetic photon flux density (PPFD; shaded sunlight), and 33 ± 1 to 30 ± 1 °C day/night temperature. Plantlets were sprayed with water twice daily and acclimatized for 16 weeks. The growth, development, and survival of the plantlets were evaluated after 8 and 16 weeks of transplantation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReintroduction of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets into their natural habitat\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegarding genetic diversity, all \u003cem\u003eD. proteranthum\u003c/em\u003e clumps used in this experiment were derived from in vitro seed cultures established from mature capsules collected in the natural habitat, without any treatment to induce shoot multiplication. Small clumps, each containing 2–3 shoots (3–4 cm tall) and 2–3 roots, were removed from the culture vessels and rinsed with tap water. The plantlets were then immersed in a 0.1% (v/v) vitamin B1 solution containing rooting hormone for 30 minutes. At the onset of the rainy season in May 2022, the plantlets were directly attached to the branches of phorophytes and reintroduced into the natural habitats of the mother plant populations within Phu Luang Wildlife Sanctuary, Loei Province. A total of 10 plantlets clumps per replicated were used, with three replications. The survival and development of \u003cem\u003eD\u003c/em\u003e.\u003cem\u003e\u0026nbsp;proteranthum\u003c/em\u003e plantlets were randomly sampled monitored every 10 weeks until 40 weeks after re-establishment (February to March 2023, late winter. After 40 weeks of reintroduction, the survival of plantlets was recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design and statistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in a completely randomized design (CRD) with three replications. The data were tested for differences between means of each parameter using one-way analysis of variance (one-way ANOVA), followed by Duncan’s Multiple Range Test (DMRT) in IBM SPSS Statistics software version 25 (Armonk, NY). All data normality was assessed with the Kolmogorov–Smirnov test.\u003c/p\u003e\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n"},{"header":"Results ","content":"\u003cp\u003e\u003cstrong\u003eEffect of medium strength and sucrose concentration on the survival, shoot, leaf, and root formation of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival of plantlets was affected by both medium strength and sucrose concentration (Fig. 2A). In full-strength MS medium, survival increased significantly from 86.7% at 0 g L⁻¹ sucrose to 100% at 10 and 20 g L⁻¹, followed by a slight decline to 96.7% at 30 g L⁻¹, with no significant differences among the 10-30 g L⁻¹ treatments. A similar trend was observed in ½MS medium, where survival rose from 86.7% at 0 g L⁻¹ to 100% at 20 g L⁻¹ before decreasing slightly to 96.7% at 30 g L⁻¹. In ¼ MS medium, survival peaked at 100% with 10 g L⁻¹ sucrose and declined to 93.3% at 20 and 30 g L⁻¹.\u003c/p\u003e\n\u003cp\u003eMedium strength and sucrose concentration significantly influenced shoot, leaf, and root formation in \u003cem\u003eD. proteranthum\u003c/em\u003e plantlets (Fig. 2B-D). The highest shoot formation (100%) was observed in MS medium supplemented with 10-20 g L⁻¹ sucrose, ½MS medium with 20 g L⁻¹ sucrose, and ¼MS medium with 10 g L⁻¹ sucrose. In contrast, significantly lower shoot formation (83.3%) occurred in both MS and ½MS media without sucrose supplementation (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eLeaf formation ranged from 83.3% to 100% across treatments (Fig. 2C). The highest leaf formation (100%) was achieved in MS medium supplemented with 20 g L⁻¹ sucrose, although differences among treatments were not statistically significant. The lowest leaf formation (83.3%) was observed in MS and ½MS media without sucrose, and ¼MS medium with 20 g L⁻¹ sucrose.\u003c/p\u003e\n\u003cp\u003eRoot formation generally increased with sucrose supplementation (Fig. 2D). In both MS and ½MS media, root formation peaked at 93.3% with 20 g L⁻¹ sucrose, significantly higher than in the sucrose-free MS treatment. Similarly, in ½MS medium, root formation at 20 g L⁻¹ (93.3%) was significantly greater than at 0 g L⁻¹ (63.3%) and 30 g L⁻¹ (76.7%). In ¼MS medium, the highest root formation (90.0%) occurred at 10 g L⁻¹ sucrose, which was significantly higher than the control (63.3%). However, higher concentrations (20-30 g L⁻¹) lightly reduced root formation, ranging from 80.0% to 86.7%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of medium strength and sucrose on the number of shoots, leaves, and roots in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe influence of medium strength and sucrose concentration on plantlets development after 12 weeks of culture are presented in Fig. 3. All treatments supported the formation of new shoots, leaves, and roots; however, their numbers varied significantly depending on medium strength and sucrose level (Table 1).\u003c/p\u003e\n\u003cp\u003eShoot number increased markedly with sucrose supplementation, reaching a maximum of 3.0 shoots per plantlet in MS and ½MS media with 30 g L⁻¹ sucrose, approximately 2.5 times higher than in ¼MS medium at the same sucrose concentration (1.2 shoots per plantlet).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLeaf induction showed a similar trend, with MS medium containing 20 g L⁻¹ sucrose yielding the highest number of leaves (7.2 leaves per plantlet) obtained in MS medium supplemented with 20 g L⁻¹ sucrose, nearly threefold greater than in the sucrose-free control (2.5 leaves per plantlet). In contrast, ¼MS medium produced fewer shoots and leaves over all, with no significant improvement at higher sucrose concentrations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRoot number also increased slightly with sucrose supplementation, peaking at 7.2 roots per plantlet in MS medium with 30 g L⁻¹ sucrose, 4.5 times greater than in the sucrose-free MS treatment (1.6 roots per plantlet).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of cytokinin on shoot, leaf, and root formation (%) in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u0026nbsp;\u003c/em\u003eplantlets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShoot formation was generally high across all treatments, ranging from 86.7% to 100.0% (Fig. 4A). The highest shoot formation (100%) occurred with 1.0 mg L⁻¹ BA, followed by 4.0 mg L⁻¹ TDZ (96.7%)\u0026nbsp;with no significant difference from the control.\u0026nbsp;In contrast, 1.0 mg L⁻¹ TDZ resulted in the lowest shoot formation (86.7%).\u003c/p\u003e\n\u003cp\u003eLeaf formation of plantlets ranged from 93.3% to 100.0% among treatments (Fig. 4B). All BA concentrations (1–4 mg L⁻¹) promoted complete leaf formation (100%), while slight, non-significant reductions were observed in media supplemented with 2.0 mg L⁻¹ kinetin or 1.0 mg L⁻¹ TDZ (96.7%). The control exhibited the lowest leaf formation (93.3%).\u003c/p\u003e\n\u003cp\u003eRoot formation was most pronounced in the control and 1.0 mg L⁻¹ kinetin (90.0%), which was non-significant difference between treatments (Fig. 4C). While the increase in BA (1.0–4.0 mg L⁻¹) and kinetin (2.0-4.0 mg L⁻¹) concentration in the culture medium significantly reduced rooting, with root formation decreasing from 56.7% (1.0 mg L⁻¹) to 33.3% (4.0 mg L⁻¹) and 60.0% (2.0 mg L⁻¹ kinetin) to 53.3% (4.0 mg L⁻¹ kinetin). Whereas TDZ had the strongest reducing root formation for 4.5-fold when compared to control (90.0%) to 20.0% for 1.0 mg L⁻¹ TDZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of cytokinin on shoot, leaf, and root number of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eD\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e. \u003cem\u003eproteranthum\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eplantlets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytokinin added to the culture medium significantly impacted the growth and development of plantlets after 12 weeks, affecting the number of shoots, leaves, and roots (Fig. 5). The highest shoot number (3.4 shoots per plantlet) was achieved on MS medium supplemented with 1.0 or 2.0 mg L⁻¹ BA, which significantly outperformed all TDZ treatments. On the other hand, TDZ at 1.0–4.0 mg L⁻¹ produced the fewest shoots (2.7 shoots per plantlet), significantly lower than both the control and the 1.0–2.0 mg L⁻¹ BA treatments (Fig. 4A).\u003c/p\u003e\n\u003cp\u003eLeaf number was also promoted by BA. Specifically, 1.0 mg L⁻¹\u0026nbsp;BA yielded the highest value (8.3 leaves per plantlet), which was significantly higher than both the control and TDZ at 2.0–4.0 mg L⁻¹\u0026nbsp;(Fig. 4B). Higher concentrations of BA (4.0 mg L⁻¹) and kinetin (4.0 mg L⁻¹) reduced leaf numbers to 7.0 and 6.5 leaves per plantlet, respectively. The lowest leaf production (5.9 leaves per plantlet) resulted from TDZ at 4.0 mg L⁻¹, which was 1.4 times lower than the yield observed with 1.0 mg L⁻¹ BA.\u003c/p\u003e\n\u003cp\u003eThe root number was greatest in the control (5.9 roots per plantlet), which was significantly higher than in all cytokinin-supplemented treatments (Fig. 4C). Root induction generally declined with increasing concentrations of BA and TDZ. Although kinetin at 1.0 mg L⁻¹ produced a relatively high root number (4.4 roots per plantlet), significantly lower than the control but higher than most BA and TDZ treatments. However, the lowest root induction was observed with 1.0 mg L⁻¹ TDZ, yielding only 0.3 roots per plantlet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of BA and NAA on survival, shoot, leaf, and root formation in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of BA and NAA were investigated after cultured for 12 weeks. Plantlets survival remained high across all treatments, ranging from 93.3 to 100% (Fig. 6A). The control medium exhibited 100% survival, similar to the media supplemented with 0.5\u0026nbsp;mg L⁻¹ BA + 0.5\u0026nbsp;mg L⁻¹ NAA, 0.5\u0026nbsp;mg L⁻¹ BA + 1.0\u0026nbsp;mg L⁻¹ NAA, 0.2 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, with no significant difference between treatments. Slight reductions in survival were observed with higher BA or NAA concentrations; for example, 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA or 0.5-2.0 mg L⁻¹ BA combined with 2.0 mg L⁻¹ NAA resulted in 93.3% survival, without statistically significant differences (Fig. 6A).\u003c/p\u003e\n\u003cp\u003eShoot formation was successfully induced in all treatments, ranging from 90% to 100% (Fig. 6B). The highest response (100.0%) was observed in media supplemented with 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA and 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, with no significant differences among treatments. The lowest shoot formation (90%) occurred in the control and in 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA treatment.\u003c/p\u003e\n\u003cp\u003eLeaf formation was generally high across all treatments, ranging from 90% to 100%, with no statistically significant differences observed (Fig. 6C). The highest leaf formation (100%) was recorded in plantlets grown on 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA and 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA. In contrast, the lowest leaf formation (90%) occurred in media containing 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA and 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA.\u003c/p\u003e\n\u003cp\u003eRoot formation varied significantly among treatments (Fig. 6D). The combination of 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA yielded the highest rooting percentage (100%). Increasing NAA concentration to 2.0 mg L⁻¹ caused a marked reduction in rooting. Media supplemented with 0.5–2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA exhibited the lowest rooting percentages (10.0%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of BA and NAA on protocorm-like bodies (PLBs) development, callus formation, and flowering of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePLBs formation was observed as green clumps at the base of the stem and leaf primordia (white arrows; Fig. 7A). Overall, PLBs induction was low across all treatments, ranging from 0.0% to 6.3%.\u0026nbsp;No PLBs were observed in the control medium or in the treatment containing 1.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA (Fig. 7B). The combination of 0.5 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA resulted in the highest PLBs formation (6.3%), although this was not significantly different among treatments. In contrast, lower percentages of PLBs formation (3.3%)\u0026nbsp;were observed in media containing 0.5 mg L⁻¹ BA combined with 0.5 or 1.0 mg L⁻¹ NAA, representing a 1.9-fold reduction compared with the best-performing treatment.\u003c/p\u003e\n\u003cp\u003eCallus formation in plantlets was also affected by the interaction between BA and NAA concentrations. A rough, green, and compact callus typically developed at the cut ends of roots (white arrows; Fig. 7A). No callus was observed in the control medium or in most low-concentration treatments (0.5 mg L⁻¹ BA + 0.5-1.0 mg L⁻¹ NAA, and 1.0 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA) (Fig. 7B). Callus induction first appeared in media supplemented with 0.5 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA (3.3%)\u0026nbsp;and increased with higher BA concentrations. The highest callus formation (13.3%)\u0026nbsp;was obtained with 2.0 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA, which was significantly greater than the control and most other treatments (Fig. 7C).\u003c/p\u003e\n\u003cp\u003eRegarding flowering, small flowers with yellowish-green petals and pale pink lips were observed (Fig. 7A). No flowering occurred in the control treatment or in most combinations of BA and NAA at lower concentrations (Fig. 7D). The highest flowering percentage (20%)\u0026nbsp;was recorded in plantlets cultured on 2.0\u0026nbsp;mg L⁻¹\u0026nbsp;BA combined with 2.0\u0026nbsp;mg L⁻¹\u0026nbsp;NAA, which was significantly higher than all other treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of BA and NAA on shoot, leaf, and root development of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of different combinations of BA and NAA on plantlet growth and development are shown in Fig. 8 and summarized in Table 2.\u0026nbsp;For shoot number, supplementation with 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA produced the highest value (4.8 shoots per plantlet), which was significantly greater than the control (3.1 shoots per plantlet). This represents approximately a 1.5-fold increase compared with the control medium lacking growth regulators.\u003c/p\u003e\n\u003cp\u003eLeaf production ranged from 8.3 to 14.7 leaves per plantlet. The highest number of leaves (14.7 leaves per plantlet) was obtained in the medium containing 1.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA, which differed significantly from the control treatment. The fewest leaves (8.3 leaves per plantlet) were observed in the control medium.\u003c/p\u003e\n\u003cp\u003eRoot production was negatively affected by increasing concentrations of cytokinin and auxin. The maximum root number (14.4 roots per plantlet) was recorded at 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA. However, as BA and NAA concentrations increased, root production decreased markedly, reaching the lowest value (1.7 roots per plantlet) in the treatment with 1.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA.\u003c/p\u003e\n\u003cp\u003ePlantlet height varied between 6.4 and 8.4 mm depending on the treatment. The control and the medium supplemented with 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA resulted in the tallest plantlets (8.4 mm), with no significant difference between these and the treatments containing 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA. In contrast, the shortest plantlets (6.4 mm) were observed in the medium containing 2.0 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA, which differed significantly from the control.\u003c/p\u003e\n\u003cp\u003eLeaf size and root length of plantlets were influenced by BA and NAA combinations. The widest leaves (2.3 mm) were observed in plantlets grown on media containing 1.0 mg L⁻¹ BA combined with 0.5 or 1.0 mg L⁻¹ NAA. The longest leaves (13.9 mm) occurred in the control treatment, which did not differ significantly from the 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA treatment (13.8 mm). In contrast, the shortest leaves (11.5 mm) were recorded in plantlets cultured with 1.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, showing no significant difference from other high hormone treatments (1.0–2.0 mg L⁻¹ BA combined with 1.0–2.0 mg L⁻¹ NAA).\u003c/p\u003e\n\u003cp\u003eRoot length was greatest in the control medium (11.7 mm) and significantly exceeded all \u0026nbsp;cytokinin-auxin treatments. Root length progressively decreased with the addition of BA and NAA, particularly at higher concentrations. For example, 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA reduced root length to 8.1 mm, and further increases in hormone concentrations resulted in even \u0026nbsp;shorter roots. The shortest roots (2.7 mm) were observed in plantlets cultured with 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGreenhouse acclimatization of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eD\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.\u003cem\u003e\u0026nbsp;proteranthum\u003c/em\u003e plantlets\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;for ex situ preservation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlantlets were transplanted for acclimatization in the greenhouse at Ban Romklao Botanical Garden, Phitsanulok Province, for ex situ conservation (Fig. 9A). Two substrates, chopped coconut husks and sphagnum moss, were evaluated for their effects on plantlet growth and survival. After 8 weeks, older leaves had senesced in all plantlets, while new pseudobulbs developed and continued to grow. Survival was highest in plantlets grown on chopped coconut husks (96.4%), whereas those in sphagnum moss showed slightly lower survival (93.2%) (Fig. 9B). After 16 weeks, survival rates declined slightly. The highest survival (88.5%) was observed in plantlets grown in sphagnum moss, compared with 83.8% in those grown on chopped coconut husks (Fig. 9B). Despite these minor differences in survival, overall plantlet growth on both substrates was comparable, with rapid root elongation and the emergence of new shoots, indicating successful acclimatization under ex situ conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReintroduction of plantlets to the natural habitat\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReintroduction of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets into their natural habitat for in situ conservation was conducted at Phu Luang Wildlife Sanctuary, Loei Province, in May 2022 (Fig. 10A). In vitro-propagated plantlets were directly attached to phorophyte branches without prior acclimatization, demonstrating strong physiological readiness for field conditions. Within 10 weeks, new shoots, leaves, and roots emerged, and by 20 weeks, mature pseudobulbs had formed, reflecting normal ontogenetic progression. Seasonal growth patterns, including leaf expansion, senescence, and summer flowering, confirmed that reintroduced plantlets completed both vegetative and reproductive cycles in the wild. During the summer of 2023, flowers were produced, followed by capsule and seed formation. After 40 weeks, survival remained high at 78.7% (Fig. 10B), highlighting the effectiveness of direct reintroduction of in vitro-propagated plantlets for insitu conservation.\u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003e\u003cstrong\u003eMedium strength and sucrose concentration effects on plantlet survival and morphological development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study demonstrated that medium strength and sucrose concentration significantly influenced the survival and growth of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets. Full-strength MS medium supplemented with 30 g L⁻¹ sucrose was the most effective treatment, resulting in the highest survival rate and promoting enhanced shoot, leaf, and root development. These findings highlight the importance of a balanced supply of mineral nutrients and carbohydrates for successful morphogenesis (Rezali et al. 2017; Dönmez et al. 2022).\u003c/p\u003e\n\u003cp\u003eSucrose supplementation was also critical for plantlet survival and morphogenesis, as in vitro grown orchids generally possess limited photosynthetic capacity and depend on exogenous carbohydrates for energy supply and osmotic regulation (Coruzzi and Bush 2001; Rybczyński et al. 2007; Phillips and Garda 2019). Moreover, the interaction between sucrose concentration and medium strength suggested that an appropriate carbohydrate supply promotes vigorous growth by stimulating cell division and expansion (Demo et al. 2008; Huh et al. 2016; Shah et al. 2019).\u003c/p\u003e\n\u003cp\u003eComparable results have been reported in other \u003cem\u003eDendrobium\u003c/em\u003e species. In \u003cem\u003eD\u003c/em\u003e. \u003cem\u003ebigibbum\u003c/em\u003e × \u003cem\u003eD\u003c/em\u003e. Thailand Black, MS medium containing 30 g L⁻¹ sucrose yielded the highest average number of leaves (Amarasinghe et al. 2021). Similarly, in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eanosmum\u003c/em\u003e, MS medium supplemented with 30 g L⁻¹ sucrose was optimal for inducing maximum shoot, leaf, and root formation in protocorms (Nguyen et al. 2022). Likewise, \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eheterocarpum\u003c/em\u003e cultured on MS medium with 30 g L⁻¹ sucrose exhibited enhanced shoot number, shoot length, pseudobulb length, and leaf production (Longchar and Deb 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of cytokinin on the percentage of shoot, leaf, and root formation in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytokinin type and concentration strongly influenced shoot, leaf, and root development in \u003cem\u003eD. proteranthum\u003c/em\u003e plantlets. Overall, high shoot and leaf formation across most treatments indicates that all cytokinins tested effectively promote organogenesis. Exogenous cytokinins stimulate cell division and enlargement, enhancing stem proliferation, shoot bud initiation, and leaf development (George et al. 2008; Teixeira da Silva et al. 2015). At the molecular level, cytokinins regulate gene expression via histidine-phosphotransfer (HPt) proteins, which mediate phosphate signal transfer to promote cell differentiation, proliferation, and regeneration, helping overcome recalcitrance in vitro (Brenner et al. 2012; Hnatuszko-Konka et al. 2021; Yang et al. 2021). Consistent with these mechanisms, BA at low to moderate concentrations (1.0–2.0 mg L⁻¹) was most effective in promoting shoot and leaf proliferation in \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e. Similar observations have been reported in other orchids, including \u003cem\u003eCyrtopodium saintlegerianum\u003c/em\u003e (Rodrigues et al. 2015), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003emoschatum\u003c/em\u003e (Ritti et al. 2023), and \u003cem\u003eEulophia bicallosa\u003c/em\u003e (Wongsa et al. 2025), In contrast, TDZ generally reduced shoot and root numbers at higher concentrations, reflecting its dual role in promoting shoot initiation at low levels but inhibiting growth when applied excessively (Çelikel et al. 2021).\u003c/p\u003e\n\u003cp\u003eRoot development exhibited an inverse relationship with shoot proliferation. The highest root numbers were observed in the control, whereas elevated cytokinin concentrations, particularly BA and TDZ, suppressed rooting. This pattern reflects the antagonistic interaction between cytokinins and auxins, where high cytokinin levels favor shoot formation but inhibit root induction (Kurepa and Smalle 2022; Li et al. 2021). Similar trends have been reported in other orchids, such as \u003cem\u003eD\u003c/em\u003e. \u003cem\u003ebensoniae\u003c/em\u003e (Riva et al. 2016), \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eellipsophyllum\u003c/em\u003e (Ritti et al. 2017), and \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eanosmum\u003c/em\u003e (Nguyen et al. 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokinin and auxin effects on survival\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eleaf, and root development of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe combined application of cytokinin (BA) and auxin (NAA) markedly influenced survival, growth, and development in \u003cem\u003eD. proteranthum\u003c/em\u003e plantlets. Survival remained high across treatments, indicating that the tested hormone concentrations were suitable for sustaining plantlet viability over 12 weeks. Shoot and leaf formation were consistently high under most treatments, reflecting the synergistic interaction of cytokinin and auxin in promoting aerial organ development. The combination of 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ NAA was most effective for shoot induction, while low to moderate cytokinin-auxin levels (0.5-1.0 mg L⁻¹ BA and 0.5-1.0 mg L⁻¹ NAA) generally enhanced leaf formation. Higher BA concentrations tended to increase leaf number slightly but could reduce root formation. These findings align with the known roles of cytokinins and auxins in shoot and leaf morphogenesis and the antagonistic interaction between the two hormones, which determines the balance between shoot proliferation and root induction (Wattanawikkit et al. 2011; Novak et al. 2014).\u003c/p\u003e\n\u003cp\u003eRoot development was most responsive to low BA and NAA concentrations. The combination of 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA yielded the highest root numbers and length, whereas higher concentrations inhibited root induction. This is consistent with auxin’s role in promoting root initiation and elongation at low levels, while high concentrations can be inhibitory (Vanneste and Friml 2009). Similar patterns have been observed in other orchids, including \u003cem\u003eOrchis catasetum\u003c/em\u003e (Baker et al. 2014) and \u003cem\u003eTaprobanea spathulata\u003c/em\u003e (Devi et al. 2015). Some species, however, require higher auxin-cytokinin concentrations for optimal rooting, reflecting species-specific responses (Riva et al. 2016).\u003c/p\u003e\n\u003cp\u003eCytokinin-auxin combinations also influenced protocorm-like bodies (PLBs) formation, callus induction, and flowering. Moderate cytokinin with higher auxin (0.5 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA) favored PLBs formation, likely due to the regulation of auxin-responsive genes controlling cell dedifferentiation and organogenesis (Fang et al. 2016; Guo et al. 2021). Callus induction was highest at 2.0 mg L⁻¹ BA + 0.5 mg L⁻¹ NAA, supporting the role of a balanced auxin-cytokinin ratio in maintaining cells in a meristematic state for proliferation (Wu et al. 2004; Niknejad et al. 2011). Flowering was most pronounced at 2.0 mg L⁻¹ BA + 2.0 mg L⁻¹ NAA, indicating that higher cytokinin-to-auxin ratios promote reproductive transition, consistent with findings in other orchids (de Melo Ferreira et al. 2006; Cen et al. 2010; Zhao et al. 2012).\u003c/p\u003e\n\u003cp\u003eMorphometric traits, including plantlet height, leaf and root size, were influenced by hormone levels. Moderate cytokinin-auxin concentrations-maintained growth comparable to controls, whereas higher concentrations reduced overall size, likely due to physiological or metabolic stress (Khatun et al. 2010; Rineksane et al. 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGreenhouse acclimatization of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eD\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.\u003cem\u003e\u0026nbsp;proteranthum\u003c/em\u003e plantlets\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;for ex situ preservation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe acclimatization of in vitro-derived seedlings to greenhouse conditions is a critical step in large-scale propagation and orchid conservation (Teixeira da Silva et al. 2017). This process enables a gradual transition from the sterile, high-humidity culture environment to the variable and less controlled external conditions (Kumar and Rao 2012; Ehirim et al. 2014). In vitro-grown plantlets typically have underdeveloped cuticles and limited stomatal function, making them highly susceptible to desiccation under ambient conditions (Hazarika 2006; Vahdati and Aliniaeifard 2017; Bertolino et al. 2019). Therefore, maintaining optimal humidity and temperature during the initial acclimatization phase is essential to ensure survival and successful \u003cem\u003eex vitro\u003c/em\u003e establishment (Nasution et al. 2020).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eDendrobium\u003c/em\u003e species, substrates such as chopped coconut husks, brick pieces, charcoal, and sphagnum moss are commonly used for hardening. These materials provide suitable physical support, aeration, and water and nutrient availability for root development (Deb and Pongener 2022; Banjare et al. 2023; Nuammee et al. 2024). In this study, ex situ acclimatization of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets was conducted under greenhouse conditions at Ban Romklao Botanical Garden. The results showed that sphagnum moss effectively supported plantlet survival and growth. Plantlets developed new pseudobulbs, indicating successful vegetative growth and a smooth transition from in vitro to ex vitro conditions. Furthermore, plantlets grown on sphagnum moss exhibited healthy morphology, comparable to field-grown parent plants, confirming normal development and physiological stability.\u003c/p\u003e\n\u003cp\u003eSphagnum moss enhances humidity retention, aeration, and nutrient availability due to its high water-holding capacity, nutrient retention ability, and slightly acidic pH, which collectively support vigorous plant growth (Aubé et al. 2015; Kaveriamma et al. 2019; Nuammee et al. 2024). The Ban Romklao Botanical Garden, where acclimatization was performed, is characterized by mountainous terrain and valleys with elevations ranging from 750 to 1,280 m above sea level conditions similar to the natural montane rainforest habitat of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e (1,400-1,500 m a.s.l.) (Sittisujjatham 2009). The area is humid with a cool climate throughout most of the year, providing an environment conducive to plant growth and conservation (Maknoi and Khammai 2015). Thus, the geographical and climatic conditions of Ban Romklao Botanical Garden are well suited for the ex situ preservation of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e. These results demonstrate that \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u0026nbsp;\u003c/em\u003ecan be effectively acclimatized using readily available substrates such as sphagnum moss, supporting their use in ex situ conservation and large-scale propagation programs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReintroduction of plantlet to the natural distribution area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEx situ and in situ conservation, together with reintroduction, are among the principal strategies for conserving threatened plant species (Oldfield 2009). For species that have lost their natural habitats, ex situ conservation in botanic gardens or controlled environments may provide the only means of survival in the short to medium term (Ren et al. 2014). In contrast, in situ conservation is generally regarded as a more sustainable approach, as it maintains species within their natural ecological contexts. Reintroduction of plants into their native habitats has therefore become an increasingly important component of global conservation efforts, particularly for critically endangered groups such as orchids (Zhao et al. 2021).\u003c/p\u003e\n\u003cp\u003eSeveral successful orchid reintroductions have been reported, including \u003cem\u003eVanda coerulea\u003c/em\u003e (Seeni and Latha 2000), \u003cem\u003eDendrobium tosaense\u003c/em\u003e (Lo et al. 2004), \u003cem\u003eRenanthera imschootiana\u003c/em\u003e (Wu et al. 2014), \u003cem\u003eCymbidium finlaysonianum\u003c/em\u003e (Rittirat et al. 2017), \u003cem\u003eGastrochilus matsuran\u003c/em\u003e (Kang et al. 2020), and \u003cem\u003ePaphiopedilum armeniacum\u003c/em\u003e (Wang et al. 2021). However, most of these studies required an acclimatization stage prior to field re-establishment.\u003c/p\u003e\n\u003cp\u003eIn the present study, \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets were successfully reintroduced directly into their natural habitat without prior acclimatization—the first such report for this species. The successful establishment and survival of plantlets under in situ conditions demonstrate their strong physiological readiness and adaptability to natural environments. The observed ontogenetic progression from vegetative growth and pseudobulb formation to flowering and seed capsule production indicates that the reintroduced plantlets completed a full life cycle in the wild. Moreover, the production of viable capsules and seeds suggests the potential for natural regeneration, which is essential for sustaining wild populations over time. The high survival rate suggests that population decline may not result from unsuitable climate conditions but rather from factors such as pollinator limitation and illegal poaching. Long-term conservation will require further study of the species biology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents the first successful protocol for the in vitro propagation and reintroduction of \u003cem\u003eD\u003c/em\u003e.\u003cem\u003e\u0026nbsp;proteranthum\u003c/em\u003e. Full-strength MS medium supplemented with 30 g L⁻\u0026sup1; sucrose proved most effective, yielding the highest survival rate and promoting vigorous shoot, leaf, and root development. Among the cytokinin treatments tested, low concentrations of BA (1.0\u0026minus;2.0 mg L⁻\u0026sup1;) were optimal for enhancing shoot regeneration, providing a reliable step for subsequent multiplication. Furthermore, the combined application of BA and NAA (0.5\u0026minus;1.0 mg L⁻\u0026sup1;) exhibited a synergistic effect, significantly improving shoot, leaf, and root induction, as well as key morphometric parameters such as plantlet height and leaf and root size.\u003c/p\u003e\n\u003cp\u003eSuccessful acclimatization of plantlets was achieved under greenhouse conditions using sphagnum moss, which facilitated the development of new pseudobulbs and roots, indicating physiological readiness for adaptation to ex situ environments. Remarkably, reintroduction of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets into natural habitats was accomplished without a prior acclimatization phase, with plantlets establishing, growing, and reproducing successfully in situ. These findings provide a solid foundation for both ex situ and in situ conservation efforts, contributing to the restoration and long-term survival of this rare and valuable orchid species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to the Plant Tissue Research Unit, Department of Biology, Faculty of Science, Naresuan University, Phitsanulok, Thailand, for providing laboratory facilities and technical support throughout the research. Appreciation is also extended to Ban Romklao Phitsanulok Botanical Garden, The Botanical Garden Organization, Ministry of Natural Resources and Environment, Chat Trakan District, Phitsanulok, Thailand, for generously providing the site for greenhouse acclimatization and ex situ preservation. This work was conducted under permission no. 6411101 from the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Thailand.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Council of Thailand (NRCT) under the 2021 fiscal year funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant Tissue Culture Laboratory, Program in Biology, Faculty of Science and Technology, Kamphaeng Phet Rajabhat University, Kamphaeng Phet 62000, Thailand\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanakorn Wongsa\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant Tissue Culture Research Unit, Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoworn Kunakhonnuruk, Onrut Sapatee, Julaluk Linjikao \u0026amp; Anupan Kongbangkerd\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWittaya Pakum\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBan Romklao Phitsanulok Botanical Garden, The Botanical Garden Organization, Ministry of Natural Resources and Environment, Chat Trakan District, Phitsanulok 65170, Thailand\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharun Maknoi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, AK, TW, BK, and WP; methodology, AK; validation, TW, BK, AK, TW, and WP; formal analysis, BK and WP; investigation, AK, and TW; resources, AK and CM; data curation, TW, BK, and WP; writing—original draft preparation, AK, TW, BK, WP, OS, and JL; writing—review and editing, JL and AK. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Anupan Kongbangkerd\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data related to the findings of this research are available upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmarasinghe P, Panapitiya D, Leelarathne N, Priyadarshan A, Senanayake SP, Center FR (2021) Effect of edible sugar on \u003cem\u003ein vitro\u003c/em\u003e growth and organogenesis of \u003cem\u003eDendrobium bigibbum\u003c/em\u003e x \u003cem\u003eDendrobium\u003c/em\u003e Thailand Black. 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Plant Cell Tissue Organ Cult 77:107\u0026ndash;109 https://doi.org/10.1023/B:TICU.0000016492.45075.a3\u003c/li\u003e\n\u003cli\u003eWu K, Zeng S, Lin D, Teixeira da Silva JA, Bu Z, Zhang J, Duan J (2014) \u003cem\u003eIn vitro\u003c/em\u003e propagation and reintroduction of the endangered \u003cem\u003eRenanthera imschootiana\u003c/em\u003e Rolfe. PLoS ONE 9(10):1\u0026ndash;12. https://doi.org/10.1371/journal.pone.0110033\u003c/li\u003e\n\u003cli\u003eYang F, Wei NN, Gao R, Piao XC, Lian ML (2015). Effect of several medium factors on polysaccharide and alkaloid accumulation in protocorm-like bodies of \u003cem\u003eDendrobium candidum\u003c/em\u003e during bioreactor culture. Acta Physiol Plant 37(5):94. https://doi.org/10.1007/s11738-015-1843-6\u003c/li\u003e\n\u003cli\u003eZahara M, Datta A, Boonkorkaew P, Mishra A (2017) The effects of different media, sucrose concentrations and natural additives on plantlet growth of \u003cem\u003ePhalaenopsis\u003c/em\u003e hybrid \u0026lsquo;Pink\u0026rsquo;. Braz Arch Biol Technol 60:1\u0026minus;15. https://doi.org/10.1590/1678-4324-2017160149\u003c/li\u003e\n\u003cli\u003eZhao DK, Li CF, Chen ZY, Yang JB (2012) \u003cem\u003eIn vitro\u003c/em\u003e flowering and conservation of \u003cem\u003eDendrobidium strongylanthum\u003c/em\u003e Rchb.f. Subtrop Plant Sci 41:48\u0026ndash;50\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Number of shoots, leaves, and roots of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e plantlets after culture on different media strength and sucrose concentrations for 12 weeks.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"97%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 17px;\"\u003e\n \u003cp\u003eMedia\u003c/p\u003e\n \u003cp\u003estrength\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 19px;\"\u003e\n \u003cp\u003eSucrose\u003c/p\u003e\n \u003cp\u003e(g L\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63px;\"\u003e\n \u003cp\u003eAverage number per plantlet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003eShoots\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eLeaves\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eRoots\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003eMS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1.6 \u0026plusmn; 0.2\u0026nbsp;bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2.5 \u0026plusmn; 1.0\u0026nbsp;f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e1.6 \u0026plusmn; 0.2\u0026nbsp;e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2.7 \u0026plusmn; 0.5\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e5.8 \u0026plusmn; 0.4\u0026nbsp;bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e3.2 \u0026plusmn; 0.7\u0026nbsp;cde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2.7 \u0026plusmn; 0.2\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e7.2 \u0026plusmn; 0.3\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e6.3 \u0026plusmn; 0.8\u0026nbsp;ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e3.0 \u0026plusmn; 0.1\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e7.0 \u0026plusmn; 0.3\u0026nbsp;ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e7.2 \u0026plusmn; 0.3\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1/2MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1.5 \u0026plusmn; 0.1\u0026nbsp;bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e3.5 \u0026plusmn; 0.2\u0026nbsp;ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e2.0 \u0026plusmn; 0.4\u0026nbsp;de\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.4\u0026nbsp;ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e5.2 \u0026plusmn; 0.2\u0026nbsp;cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e3.9 \u0026plusmn; 0.2\u0026nbsp;cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e2.3 \u0026plusmn; 0.3\u0026nbsp;ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e4.5 \u0026plusmn; 0.4\u0026nbsp;de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e4.8 \u0026plusmn; 1.0\u0026nbsp;bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e3.0 \u0026plusmn; 0.4\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e6.1 \u0026plusmn; 0.6\u0026nbsp;abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e5.1 \u0026plusmn; 1.4\u0026nbsp;bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1/4MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1.3 \u0026plusmn; 0.2\u0026nbsp;c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2.6 \u0026plusmn; 0.1\u0026nbsp;f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e1.8 \u0026plusmn; 0.1\u0026nbsp;de\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1.3 \u0026plusmn; 0.1\u0026nbsp;c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2.8 \u0026plusmn; 0.1\u0026nbsp;f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e3.0 \u0026plusmn; 0.5\u0026nbsp;cde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1.5 \u0026plusmn; 0.1\u0026nbsp;bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2.9 \u0026plusmn; 0.2\u0026nbsp;f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e3.6 \u0026plusmn; 0.4\u0026nbsp;cde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e1.2 \u0026plusmn; 0.1\u0026nbsp;c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2.8 \u0026plusmn; 0.4\u0026nbsp;f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e3.3 \u0026plusmn; 0.5\u0026nbsp;cde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults are mean \u0026plusmn; SE of 3 replicates (30 seedlings per replicate). Means with the same letter(s) in the same column are not significantly different at \u003cem\u003ep \u0026le;\u0026nbsp;\u003c/em\u003e0.05 according to Duncan\u0026rsquo;s multiple range test (DMRT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Effect of cytokinin and auxin on the number of shoots, leaves, and roots, as well as plantlet height, leaf width, leaf length, and root length of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003eproteranthum\u003c/em\u003e seedlings after 12 weeks of culture.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7px;\"\u003e\n \u003cp\u003eBA\u003c/p\u003e\n \u003cp\u003e(mg L⁻\u0026sup1;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7px;\"\u003e\n \u003cp\u003eNAA\u003c/p\u003e\n \u003cp\u003e(mg L⁻\u0026sup1;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 35px;\"\u003e\n \u003cp\u003eAverage number per plantlet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003ePlantlet height (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003eLeaf width (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003eLeaf length (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003eRoot length (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eShoots\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eLeaves\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eRoots\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3.1 \u0026plusmn; 0.4 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e8.3 \u0026plusmn; 0.9 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e9.2 \u0026plusmn; 0.6 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e8.4 \u0026plusmn; 0.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.9 \u0026plusmn; 0.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e13.9 \u0026plusmn; 0.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.7 \u0026plusmn; 0.1 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3.5 \u0026plusmn; 0.2 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.2 \u0026plusmn; 0.9 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e14.4 \u0026plusmn; 0.9 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e8.2 \u0026plusmn; 0.1 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.1 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e13.1 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e8.1 \u0026plusmn; 0.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4.8 \u0026plusmn; 0.6 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e13.8 \u0026plusmn; 0.2 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e7.0 \u0026plusmn; 1.1 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e8.4 \u0026plusmn; 0.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e13.8 \u0026plusmn; 0.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e5.8 \u0026plusmn; 0.0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4.4 \u0026plusmn; 0.5 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e13.1 \u0026plusmn; 1.2 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.4 \u0026plusmn; 0.7 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.9 \u0026plusmn; 0.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.1 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.8 \u0026plusmn; 0.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e4.6 \u0026plusmn; 0.1 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4.0 \u0026plusmn; 0.5 abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e13.1 \u0026plusmn; 1.1 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e9.2 \u0026plusmn; 2.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e7.3 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.3 \u0026plusmn; 0.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e12.8 \u0026plusmn; 0.1 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e4.7 \u0026plusmn; 0.0 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4.5 \u0026plusmn; 0.2 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e14.7 \u0026plusmn; 0.7 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.4 \u0026plusmn; 1.6 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.8 \u0026plusmn; 0.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.3 \u0026plusmn; 0.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.6 \u0026plusmn; 0.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3.5 \u0026plusmn; 0.0 de\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3.9 \u0026plusmn; 0.3 abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.2 \u0026plusmn; 0.7 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.7 \u0026plusmn; 0.9 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.8 \u0026plusmn; 0.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.5 \u0026plusmn; 0.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3.5 \u0026plusmn; 0.1 de\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3.4 \u0026plusmn; 0.1 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.5 \u0026plusmn; 0.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e5.0 \u0026plusmn; 0.8 cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.8 \u0026plusmn; 0.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e12.6 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e5.1 \u0026plusmn; 0.0 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3.7 \u0026plusmn; 0.1 abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.4 \u0026plusmn; 0.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3.1 \u0026plusmn; 0.5 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.4 \u0026plusmn; 0.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.1 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.9 \u0026plusmn; 0.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.9 \u0026plusmn; 0.0 e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4.7 \u0026plusmn; 0.2 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e12.3 \u0026plusmn; 0.7 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.8 \u0026plusmn; 0.7 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.8 \u0026plusmn; 0.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.1 \u0026plusmn; 0.0 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e12.5 \u0026plusmn; 0.1 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.7 \u0026plusmn; 0.0 e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults are mean \u0026plusmn; SE of 3\u0026nbsp;replicates (30\u0026nbsp;seedlings per replicate). Means with the same letter(s) in the same column are not significantly different at \u003cem\u003ep\u003c/em\u003e \u0026le; 0.05 according to Duncan\u0026rsquo;s multiple range test (DMRT).\u003c/p\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":"Dendrobium, Propagation, Preservation, Reintroduction, Ex situ conservation","lastPublishedDoi":"10.21203/rs.3.rs-8260330/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8260330/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"An efficient in vitro propagation and reintroduction protocol was developed for Dendrobium proteranthum Seidenf., an endemic and rare orchid species of Thailand. Six-month-old seedlings were cultured on full-, half-, and quarter-strength MS media with varying sucrose levels. All treatments showed high survival (86.7–100%). Full-strength MS with 30 g L−1 sucrose was optimal, yielding the highest shoot (3.0), leaf (7.0), and root (7.2) numbers per plantlet. Seedlings were cultured on MS medium with 0–4.0 mg L−1 BA, kinetin, or TDZ for 12 weeks. The highest shoot induction (3.4 shoots and 8.3 leaves/plantlet) occurred with 1.0 mg L−1 BA, while root formation peaked in the control (5.9 roots/plantlet). The combined effects of BA and NAA (0–2.0 mg L−1) were tested. The combination of 0.5 mg L−1 BA + 1.0 mg L−1 NAA achieved 100% shoot formation, the highest shoot number (4.8 shoots/plantlet), and tallest plantlets (8.4 mm). Meanwhile, 0.5 mg L−1 BA + 0.5 mg L−1 NAA yielded 100% leaf and root formation with the most roots (14.4 roots/plantlet). The greatest leaf number (14.7 leaves/plantlet) occurred with 1.0 mg L−1 BA + 1.0 mg L−1 NAA. For ex situ conservation, plantlets acclimatized on sphagnum moss at Ban Romklao Botanical Garden showed 88.5% survival. After reintroduction to Phu Luang Wildlife Sanctuary, 78.7% survived after 40 weeks and completed both vegetative and reproductive stages. This study establishes an effective, reproducible protocol for mass propagation and in situ restoration of D. proteranthum.","manuscriptTitle":"Enhancement of medium components and the effects of cytokinins and auxins on in vitro propagation and successful reintroduction of Thai endangered orchid Dendrobium proteranthum Seidenf","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-19 15:30:19","doi":"10.21203/rs.3.rs-8260330/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":"8b62e790-6c21-43a2-85ec-cdd49ebaf1ad","owner":[],"postedDate":"December 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-08T07:41:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-19 15:30:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8260330","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8260330","identity":"rs-8260330","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}