1 Introduction

Dendrobium officinale Kimura et Migo, also known as Dendrobium catenatum, is a precious medicinal plant of Orchidaceae that occupies an important position in traditional Chinese medicine, functional foods, and the modern health industry (Li et al., 2024; Wei et al., 2024). It has been used in China for thousands of years, and its dried stems are officially recorded in the Chinese Pharmacopoeia and included in the catalogue of substances with both medicinal and food uses, reflecting both long-standing traditional application and broad consumption potential (Zhang et al., 2023; Wei et al., 2024). Traditional records describe its functions in nourishing yin, benefiting the stomach, promoting fluid production, and supporting general health, while modern studies further show that D. officinale contains diverse bioactive constituents including polysaccharides, bibenzyls, flavonoids, phenanthrenes, alkaloids, and other phenolics (Duan et al., 2022; Zhang et al., 2023). Reviews have reported that these constituents are associated with antioxidant, anti-inflammatory, immunomodulatory, gastrointestinal protective, hepatoprotective, hypoglycemic, neuroprotective, and antitumor activities, which explains why D. officinale has become one of the most intensively studied medicinal species in the genus (Xu et al., 2022). At the broader genus level, China has abundant Dendrobium resources, with dozens of medicinally valuable taxa and at least 78 species reported nationally, but D. officinale remains one of the most prominent research and development targets because of its recognized medicinal value, edible potential, and market appeal. In addition, the plant has expanding value beyond stems alone: recent work indicates that leaves and flowers also contain numerous active compounds and can be developed for food, health-care, and nutraceutical uses, suggesting that the industrial chain of D. officinale is still widening.

The rising medicinal, nutritional, and commercial demand for D. officinale has sharply increased pressure on germplasm resources and seedling supply. Because wild populations were overexploited, the species has been listed as a secondary endangered plant in China, and rational protection together with artificial propagation has become a central issue in its utilization. Although artificial cultivation technology has made progress, current cultivation resources are still mixed, product quality is uneven, and the evaluation system remains imperfect, all of which constrain standardized industrial development. Conventional propagation routes are not sufficient to meet large-scale market demand because they depend on limited source materials and generally cannot provide rapid, uniform, and continuous production of elite seedlings (Xu et al., 2022). This limitation is reflected indirectly by later rapid-propagation studies, which were specifically designed to solve restricted explant availability, low propagation efficiency, and the need for large-batch production. Orchid micropropagation research more broadly identifies low shoot multiplication, clonal instability, poor rooting frequency, and high production cost as major obstacles in medicinal orchid propagation, indicating that propagation efficiency and seedling quality are persistent technical barriers rather than trivial operational details. For D. officinale specifically, the transition from in vitro culture to greenhouse or field conditions is another key bottleneck, because tissue-cultured seedlings often show low survival and weak growth after transplanting. This means that even when aseptic seedlings can be produced in vitro, industrial propagation still fails if rooting, hardening-off, substrate selection, microbial symbiosis, and transplant management are not optimized into an integrated technical chain (Wang, 2021; Li et al., 2024).

For that reason, recent studies on D. officinale tissue culture have shifted from simple seedling induction toward the construction of complete rapid-propagation systems covering explant disinfection, adventitious bud or protocorm induction, proliferation, differentiation, strong-seedling culture, rooting, hardening, and substrate transplanting. Several reports show that appropriate combinations of basal medium, plant growth regulators, activated carbon, and organic additives such as banana juice, potato extract, or coconut juice can markedly improve shoot multiplication, rooting, and seedling vigor. Likewise, transplant survival is highly sensitive to the hardening process and to the choice of substrate (Li et al., 2023). Reported survival outcomes vary by protocol, from 80% in peat-pine bark-macadam mixtures, to 88% in fine wood substrate, to 95% after sphagnum hardening-off and 97% after matrix transplantation, while some planting methods under optimized cultivation management report survival above 98%. Other work suggests that biological assistance can further improve acclimatization, since Mycena dendrobii enhanced seedling survival and growth by promoting stress tolerance and new root formation (Wei et al., 2024). Meanwhile, highly efficient protocorm-like body systems have reported 99% induction, a differentiation coefficient of 50 seedlings per 0.1 g protocorm-like bodies, 100% rooting, and transplant survival above 95%, highlighting the feasibility of industrialized seedling production once the full pathway is optimized.

This study will explore the technical pathways for tissue-culture rapid propagation and improvement of transplant survival rate in Dendrobium officinale, and systematically sort out the key links affecting propagation efficiency and post-bottle establishment, including explant selection, medium screening, hormone regulation, rooting enhancement, hardening-off, substrate configuration, and transplant management. The significance of this study lies not only in increasing the multiplication coefficient and shortening the seedling production cycle, but also in stabilizing seedling quality, improving the survival rate of tissue-cultured plantlets, and providing a technical foundation for standardized and large-scale cultivation. From a broader perspective, efficient rapid propagation technology can reduce dependence on wild resources, support the conservation and sustainable utilization of this endangered medicinal orchid, and better meet the growing demand from the pharmaceutical, functional food, and health-product industries. Since pharmacological development, product diversification, and quality control all depend on a stable and sufficient supply of raw materials, establishing reliable rapid-propagation technology and high-survival transplant technology is a necessary prerequisite for the modern industrialization and high-value utilization of Dendrobium officinale.

2 Technical Basis of Tissue Culture Rapid Propagation of Dendrobium officinale

2.1 Selection of explants of Dendrobium officinale

The choice of explant is the first determinant of rapid propagation efficiency in Dendrobium officinale, because explant type affects contamination risk, induction rate, regeneration pathway, and the uniformity of resulting seedlings. Current Dendrobium aseptic culture protocols are strongly tissue- and genotype-dependent, so explant selection cannot be separated from the later sterilization and culture scheme (Sukmadjaja and Widhiastuti, 2019). In D. officinale, reported explants include seeds, stem segments with nodes, budding stems, stem tips, leaves, and protocorm-like bodies, indicating that the species has multiple available regeneration routes (Nguyen et al., 2022). For large-scale initiation, seeds are especially important because they are abundant and can directly enter asymbiotic germination and protocorm induction systems (Mamun et al., 2018).

Comparative evidence indicates that seeds are often the most suitable explant for protocorm and PLB induction in D. officinale. One study comparing five explant types reported a 100% induction rate and the best growth status from seeds, while leaves, stem fragments, stem tips, and stems with nodes showed lower induction rates of 43%, 25%, 52%, and 31%, respectively. However, for clonal multiplication of selected germplasm, nodal and stem-derived explants have clearer advantages because they preserve elite genotypes and support uniform vegetative propagation (Nguyen et al., 2022). Stem segments with nodes were reported to achieve 93.3% bud induction on 1/2 MS supplemented with 1.5 mg/L 6-BA and 0.5 mg/L NAA, while budding stems have also been used to establish complete rapid-propagation systems (Silva et al., 2017). Therefore, seed explants are more suitable for high-frequency initiation and PLB establishment, whereas nodal or budding-stem explants are more suitable for stable clonal propagation of target lines (Mamun et al., 2018).

2.2 Establishment of an aseptic culture system for Dendrobium officinale

The establishment of an aseptic culture system is the technical prerequisite for all subsequent stages of D. officinale micropropagation, because contamination at culture initiation directly reduces explant survival and later regeneration success. In Dendrobium, aseptic culture success depends on the source, size, age, and physiological state of explants, as well as the preparation and disinfection procedure used before inoculation. Most donor materials are obtained from greenhouse or similar non-sterile environments, so surface sterilization is indispensable before explants can be transferred into in vitro culture (Sukmadjaja and Widhiastuti, 2019). Because different tissues respond differently to disinfectants, no single sterilization procedure is universally optimal for all Dendrobium explants or genotypes.

For D. officinale, ethanol combined with mercuric chloride remains one of the most commonly reported effective sterilization schemes for stem-derived explants. A systematic rapid-propagation study found that 75% ethanol for 30 s followed by 0.1% HgCl2 for 10 min was the best disinfection method for budding stems (Silva et al., 2017). Supporting evidence from another Dendrobium nodal-segment study showed that 0.1% HgCl2 for 10 min produced the lowest contamination rate and the highest healthy culture establishment, while stronger sodium hypochlorite treatment increased explant mortality. In seed culture, aseptic initiation is often simplified by collecting capsules near maturity and releasing seeds directly under sterile conditions, which reduces the exposure of delicate material to harsh disinfectants. Thus, the aseptic culture system of D. officinale should be built around explant-specific sterilization, strict donor material selection, and careful handling during inoculation (Sukmadjaja and Widhiastuti, 2019).

2.3 Optimization of culture media and nutritional conditions for Dendrobium officinale

Optimization of culture media and nutritional conditions is the core of rapid propagation in D. officinale, because different developmental stages require different balances of mineral salts, plant growth regulators, carbon sources, activated carbon, and natural organic additives (Mamun et al., 2018). Across available studies, MS and 1/2 MS are the main basal media, but the optimal formulation varies with explant type and culture objective rather than following a single universal recipe (Silva et al., 2017). For example, 1/2 MS without phytohormones was reported as optimal for aseptic seed germination, whereas protocorm induction and proliferation were improved on MS supplemented with 1.0 mg/L 6-BA, 1.0 mg/L NAA, and 1.0 mg/L KT (Mehbub et al., 2022). In another seed-based system, the most suitable germination medium was 1/2 MS+0.2 mg/L NAA+100 g/L potato+25 g/L sucrose, while the most suitable rooting and seedling medium was 1/2 MS+0.2 mg/L NAA+100 g/L banana+25 g/L sucrose+0.5 g/L activated carbon (Mamun et al., 2018).

For stem- and node-derived explants, cytokinin-auxin combinations are central to shoot induction and multiplication. In D. officinale, effective formulations include MS+1.0 mg/L 6-BA+0.5 mg/L NAA+2 g/L activated carbon for budding stem induction, MS+0.5 mg/L 6-BA+0.8 mg/L 2,4-D+2 g/L activated carbon for proliferation, and 1/2 MS+1.5 mg/L 6-BA+0.5 mg/L NAA for both bud induction and multiple-shoot culture (Mamun et al., 2018). Other studies likewise reported that MS+2.0 mg/L 6-BA+0.1 mg/L NAA gave the highest cluster-shoot proliferation, and that coconut milk, coconut juice, potato extract, banana extract, and activated carbon could further promote PLB propagation, seedling growth, or rooting (Silva et al., 2017; Nguyen et al., 2022). Rooting media generally shift toward reduced salt concentration and stronger auxin support, such as 1/2 MS+2.5 mg/L NAA+15% potato juice+2 g/L activated carbon, MS+0.5 mg/L IBA+0.5 mg/L NAA, or 1/2 MS+0.2 mg/L IBA+0.5% activated carbon+40% potato extract (Mehbub et al., 2022). Rapid propagation of D. officinale depends on a stage-specific medium optimization strategy rather than a single fixed medium, and this staged regulation is the basis for high regeneration efficiency and subsequent strong seedling formation.

3 Protocorm Induction and Proliferation Culture

3.1 Conditions for protocorm induction

Protocorm-like body induction in Dendrobium officinale depends first on a suitable basal medium and a balanced cytokinin-auxin combination. A targeted study using plumules as explants identified 1/2 MS supplemented with 2% sucrose, 10% banana puree, pH 6.0, plus 6-BA 0.3 mg/L and NAA 0.2 mg/L as the optimal induction condition for PLBs (Fan et al., 2016). More broadly in Dendrobium orchids, auxins and cytokinins are the growth regulators most commonly used for PLB induction, and PLBs themselves are considered the most responsive explant type for propagation systems (Arli et al., 2023). Comparative orchid evidence also shows that induction response varies strongly by genotype and regulator type: thidiazuron can be highly effective in some Dendrobium systems, while 2iP, meta-topolins, or BA-NAA combinations outperform alternatives in others, so induction formulas should be treated as species- and material-specific rather than universal (Figure 1) (Cardoso et al., 2020).

Induction efficiency is also shaped by explant condition and culture environment after inoculation. In D. officinale, light-yellow, loose, and plump PLBs proliferated faster than compact or physiologically aged material, indicating that early visual screening of PLB quality improves subsequent culture performance (Fan et al., 2016). Thin Cell Layer approaches in Dendrobium have been reported to outperform larger conventional explants because thinner tissues improve contact with the medium and diffusion into the explant, which is relevant when designing highly responsive induction systems (Arli et al., 2023). At the developmental level, PLBs in D. officinale initially show embryoid-like morphology and later shift toward organogenesis, with most protocorms beginning germination and differentiation after about 35 days, so induction protocols should align subculture timing with this transition (Tang et al., 2024).

3.2 Subculture proliferation technology

After induction, subculture proliferation should prioritize physiological uniformity, inoculum density, and transfer interval. In D. officinale, inoculating ten PLBs with similar physiological status as one group gave better proliferation performance, and the most significant biomass gain occurred after 45 days of culture, when the proliferation rate reached 1008% (Fan et al., 2016). The same study found that laminated culture favored the upper layer for proliferation and weight gain, and also helped rejuvenate PLBs, suggesting that spatial arrangement in the culture vessel can affect oxygen, light, and nutrient access during repeated passage (Fan et al., 2016). These findings support a practical subculture strategy of selecting vigorous, light-colored PLBs, standardizing inoculum size, and transferring at roughly 45-day intervals before visible decline in vigor (Fan et al., 2016).

Hormone and additive optimization remains central during proliferation culture. In a recent D. officinale study, 75 µM melatonin accelerated the appearance of white reticulated structures on PLB surfaces and was more conducive to PLB proliferation than the untreated control, whereas 100 µM inhibited adventitious bud differentiation (Tang et al., 2024). Cross-orchid evidence supports the need for species-specific tuning: 2.5 mg/L NAA or 0.5~2.5 mg/L BAP increased PLB percentage and fresh weight in Dendrobium sp., while BA with IBA or TDZ plus coconut water improved PLB proliferation and plantlet regeneration in other orchids (Hussien et al., 2024). Modified basal media can also raise PLB biomass without relying heavily on growth regulators; in Dendrobium Sabin Blue, a revised half-MS formula increased PLB dry mass by 23% and was proposed to reduce both production cost and the likelihood of somaclonal variation (Chin et al., 2021). Emerging approaches such as liquid shaking culture, temporary immersion systems, and oxygen nanobubbles also appear promising for improving proliferation efficiency, though direct validation in D. officinale remains limited (Cardoso et al., 2020; Mawardi et al., 2024).

3.3 Control of browning and vitrification

Browning is one of the main barriers to successful PLB culture because oxidation of phenolic compounds reduces regeneration capacity, suppresses growth, and can cause tissue necrosis or death (Permadi et al., 2024; Kuluev, 2020). Reviews across plant tissue culture agree on several core controls: presoaking explants in antioxidant solutions, incorporating antioxidants such as PVP or ascorbic acid into the medium, using activated charcoal, shortening subculture intervals, and imposing an initial dark treatment (Amente and Chimdessa, 2021; Permadi et al., 2024). Mechanistic evidence shows that browning is closely linked to PPO, POD, and PAL activity; vitamin C, citric acid, activated charcoal, and PVP can all suppress browning to different degrees, with vitamin C performing best in one mechanistic study by reducing PPO and POD activity and lowering total polyphenols (Xu et al., 2023).

Activated charcoal is especially relevant in orchid culture because it adsorbs inhibitory compounds, toxic metabolites, phenolic exudates, and brown exudate accumulation, although it can also adsorb vitamins and plant growth regulators, so its concentration must be optimized rather than added indiscriminately. Additional evidence from recalcitrant species shows that ascorbic acid can outperform other antioxidants in some systems, while activated charcoal at 150 ~200 mg/L can be better for culture establishment and shoot proliferation in others, reinforcing that anti-browning recipes remain species-dependent (Guntur et al., 2019; Jakhar et al., 2019). Direct vitrification evidence is sparse in the supplied D. officinale corpus, but practical control still follows the same logic as browning prevention: avoid prolonged exposure to excessively high cytokinin levels, maintain moderate inoculum density, and use timely subculture to prevent water-soaked, hyperhydric tissues during rapid proliferation (Fan et al., 2016; Tang et al., 2024). Overall, effective PLB culture in D. officinale requires coupling high-proliferation conditions with antioxidant management and disciplined passage schedules, so that rapid multiplication does not come at the cost of tissue quality.

4 Multiple Shoot Induction and Strong Seedling Cultivation

4.1 Regulation of multiple shoot differentiation

The regulation of multiple shoot differentiation in Dendrobium officinale depends primarily on the coordinated adjustment of basal medium, cytokinin-auxin balance, and the developmental state of the cultured material. Across Dendrobium systems, shoot formation is mainly driven by plant growth regulator composition rather than by any single additive alone, and successful regeneration generally requires species-specific optimization (Pasternak and Steinmacher, 2024; Erkoyuncu, 2026). In related Dendrobium species, BA or BAP combined with low to moderate NAA repeatedly promoted shoot proliferation, including MS+1.0 mg/L BA+1.0 mg/L NAA in D. moniliforme with a 6.1-fold cluster-shoot proliferation coefficient, MS+3.0 mg/L BAP+1.0 mg/L NAA in Dendrobium ‘Red Bull’ with 7.66 shoots per explant, and MS+0.5 mg/L BAP+0.5 mg/L NAA in D. chryseum with 5.8 shoots per explant (Mamun et al., 2018; Pathak et al., 2022; Liu et al., 2023). These findings are consistent with broader orchid evidence that cytokinin-dominant media promote shoot induction, while excess auxin tends to redirect development toward callus or rooting rather than repeated shoot differentiation (Kaladharan et al., 2024).

For protocorm-like body and protocorm-derived cultures, multiple shoot differentiation is also strongly influenced by organic supplements and activated charcoal. In D. crumenatum, MS+15% coconut water produced 96.0% shooting and 9.5 shoots per explant, while activated charcoal further improved leaf and root development, showing that shoot induction and seedling quality can be uncoupled and optimized sequentially (Klaocheed et al., 2021). Similar responses were reported in D. thyrsiflorum, where 0.4 mg/L BA+0.4 mg/L kinetin gave the highest multiplication rate of 4.53 times, and in D. heyneanum, where 1.0 mg/L kinetin produced the highest protocorm-derived micropropagation frequency of 90.20% (Figure 2) (Cuc et al., 2022; Kaladharan et al., 2024). Additional evidence from general orchid tissue culture indicates that light quality modifies shoot proliferation by altering sugar accumulation, chlorophyll synthesis, and antioxidant activity, but the response is species-specific, so light should be treated as a secondary regulatory factor after the hormone regime is fixed (Mehbub et al., 2022; Feng et al., 2025).

4.2 Key measures for strong seedling cultivation

Strong seedling cultivation begins after shoot differentiation and requires synchronized improvement of rooting, leaf expansion, photosynthetic competence, and ex vitro adaptability. In Dendrobium orchids, the most common pattern is to shift from multiplication media to lower-salt or auxin-enriched rooting media, often with activated charcoal or organic additives to improve root number and root elongation. In D. moniliforme, MS with 0.5 mg/L NAA+0.5 mg/L IBA gave the highest rooting rate, while in D. chryseum 2 mg/L IBA gave the highest root number and root length and 2 mg/L NAA performed poorly, indicating that auxin type and concentration affect root quality as much as root induction itself (Liu et al., 2023; Pathak et al., 2022). In Dendrobium sp. derived from PLBs, 2.5 mg/L BAP+5.0 mg/L NAA+0.5 mg/L kinetin produced 6.17 roots and 5.10 cm root length, whereas a lower-NAA combination favored shoot regeneration, reinforcing the need for stage-specific rather than one-step media design.

Hardening and transplant preparation are equally important for producing strong seedlings because in vitro plantlets must shift from high humidity and photomixotrophic growth to autotrophic growth under greenhouse conditions. Reviews of Dendrobium acclimatization show that gradual reduction of humidity and staged increase in light improve stomatal control, root function, and survival after transfer (Silva et al., 2017). Substrate choice also has a large effect: equal-volume pine bark, turfy soil, and peanut shells performed best for D. moniliforme, sterilized coconut husk supported 95% survival in D. crumenatum, tree fern produced 94.8% survival in D. thyrsiflorum, and chopped coconut husk+brick+charcoal+cocopeat gave the best vegetative hardening response in D. nobile plantlets (Klaocheed et al., 2021; Cuc et al., 2022; Liu et al., 2023; Khilari et al., 2023). Even with optimized protocols, survival remains variable across species, ranging from 52.73% in D. heyneanum to 92% in Dendrobium ‘Red Bull’ and 87% in D. heterocarpum, so strong seedling cultivation should be judged by both vigor and post-transplant stability rather than by in vitro growth alone (Mamun et al., 2018; Longchar and Deb, 2022; Kaladharan et al., 2024).

4.3 Quality evaluation of tissue-cultured seedlings

Quality evaluation of tissue-cultured seedlings should integrate morphology, rooting status, physiological performance, acclimatization outcome, and genetic or phenotypic stability. Morphological indicators remain the most practical primary screen, including shoot height, leaf number, leaf size, root number, root length, and overall plant compactness, because these traits respond predictably to medium and environmental optimization (Khilari et al., 2023; Feng et al., 2025). Physiological quality adds another layer: in orchid seedlings, light quality alters chlorophyll, carotenoids, fresh weight, Rubisco activity, and the transition from C3 toward CAM metabolism, showing that visually similar seedlings can differ substantially in photosynthetic readiness for ex vitro growth. General tissue culture evidence also supports the use of soluble sugars, soluble proteins, and antioxidant enzyme activity as indicators linked to proliferation and seedling physiological status (Feng et al., 2025).

Final seedling quality should also be verified by performance after transplanting, because survival and normal development are the most direct tests of whether in vitro seedlings are truly strong. In D. crumenatum, regenerated plantlets showed 95% survival and normal greenhouse development, while D. nobile plantlets showed 82.3% survival together with high genetic stability by SCoT and IRAP markers, and D. heterocarpum showed 87% transplant survival with 96.89% monomorphism in fidelity testing (Klaocheed et al., 2021; Longchar and Deb, 2022). New image-based grading approaches in orchids further show that top-view and side-view imaging can identify root and leaf defects with F1-scores up to 90.44%, suggesting that future D. officinale quality evaluation can combine conventional morphological grading with automated digital inspection (Lin et al., 2025). Taken together, high-quality tissue-cultured seedlings of D. officinale should be defined not only by rapid in vitro growth, but by balanced shoot-root development, physiological robustness, high transplant survival, and stable true-to-type performance.

5 Rooting and Pre-Transplant Management

5.1 Optimization of rooting culture medium

The optimization of rooting culture medium in Dendrobium officinale should follow a stage-specific, reduced-salt, auxin-regulated strategy rather than a single universal formula. In direct studies on D. officinale, half-strength MS was repeatedly used as the basal medium for rooting, and the best reported formulations included 1/2 MS+2.5 mg/L NAA+15% potato juice+2 g/L activated carbon, which produced a rooting rate of 90.4%-92.8%, and 1/2 MS supplemented with 10% banana puree+1.0 mg/L NAA, which produced stronger roots and more vigorous plantlets. Another D. officinale optimization study found that two closely related media, both based on 1/2 MS with mashed potatoes, low BA, low NAA, activated carbon, Huabao No.1, and 3% sucrose, were the best overall for rooting and seedling growth promotion. Evidence from related D. officinale propagation systems also shows that banana-containing media are useful in the seedling-root growth stage, reinforcing the value of organic additives during rooting culture (Turnip, 2023).

Auxin type and concentration strongly affect the architecture and later usefulness of the root system, but the optimal regulator is not identical across orchid taxa. In D. officinale, invigorating culture showed no major vigor difference across 0~2.5 mg/L exogenous auxin overall, yet IBA at 0.75 mg/L or NAA at 0.5 mg/L improved uniformity and vigor, while NAA at 1.0 mg/L with banana puree improved root performance. In other Dendrobium species, NAA often increased root number whereas IBA more often promoted root elongation, although some studies reported the reverse depending on concentration and genotype (Mirani et al., 2017; Tini et al., 2025). For example, D. aqueum produced 8.75 roots per shoot on 1/2 MS+IBA 5 mg/L, but the longest roots were obtained with NAA 7 mg/L. By contrast, Dendrobium cv. Sonia Earsakul and Phalaenopsis amabilis both responded best to IBA at 1 mg/L for root length and rooting percentage, while D. chrysotoxum showed stronger rooting under relatively high IBA or NAA combined with low cytokinin levels (Sabastian et al., 2022; Alghanimy and Alamery, 2025). These comparisons indicate that for D. officinale, medium screening should prioritize half-strength salts, moderate auxin supply, activated carbon, and organic additives, while avoiding the assumption that more auxin always gives better roots (Seliem et al., 2020; Poniewozik et al., 2021).

5.2 Evaluation of root development quality

The evaluation of root development quality in D. officinale should go beyond rooting percentage and include root number, root length, uniformity, thickness, vigor, and the subsequent capacity to support acclimatization. Orchid studies consistently identify in vitro rooting as the stage most directly linked to successful transplantation, because acclimatization performance depends primarily on the roots produced before bottle removal (Mirani et al., 2017; Poniewozik et al., 2021). Accordingly, root quality should be judged as a compound trait. In D. officinale, the best media were described not only by rooting rate but also by stronger growth, better vigor, and more uniform seedlings. In D. anosmum, activated charcoal-containing medium improved shoot length, leaf number, root number, and root length simultaneously, illustrating that a usable transplantable seedling requires balanced whole-plant quality rather than roots assessed in isolation (Nguyen et al., 2022).

Cross-species evidence helps clarify which root traits are most meaningful for pre-transplant assessment. In D. nobile, NAA alone outperformed IBA alone for root number, length, and thickness, but combined NAA+IBA reduced rooting and induced callus at the shoot base, showing that visually abundant rooting is not necessarily desirable if root bases are abnormal (Mirani et al., 2017). In Paphiopedilum insigne, 1 mg/dm³ IAA or IBA produced many rooted, good-quality plantlets, and the best later acclimatization followed in vitro rooting treatment plus a suitable substrate, indicating that root quality must be validated by ex vitro performance (Poniewozik et al., 2021). In Phalaenopsis, nano-selenium plus 0.5 mg/L NAA increased rooting percentage, root length, and root number and then improved acclimatization, again linking root metrics to transplant readiness (Seliem et al., 2020). Practical evaluation in D. officinale should therefore classify high-quality roots as numerous enough for anchorage, sufficiently elongated, evenly distributed, free of basal callus or browning, and associated with vigorous leaves and stems, because these combined features are the best predictors of survival after transfer (Mirani et al., 2017; De Stefano et al., 2022).

5.3 Grading of seedlings before bottle removal

Seedlings should be graded before bottle removal because pre-transplant selection improves the consistency of acclimatization management and reduces losses from weak or non-uniform plantlets. The acclimatization literature treats transfer from bottle to external substrate as a critical stage and an important indicator of tissue culture success, so seedlings entering this stage should first be separated by vigor and developmental completeness (Faradilla et al., 2022; Sari et al., 2023; Turnip, 2023). In D. officinale, reports describing improved vigor and uniformity under selected rooting treatments imply that uniform seedlings are a legitimate management target before transplanting. Studies on other orchids show that post-culture evaluation commonly records plantlet height, leaf number, root length, plant height, SPAD, NDVI, and related phenotypes during acclimatization, providing a practical basis for pre-bottle grading criteria (Mullin et al., 2022).

A practical grading system for D. officinale before bottle removal should therefore distinguish at least three classes: seedlings suitable for immediate transplanting, seedlings needing continued strengthening, and inferior seedlings to be discarded. Seedlings selected for removal should have a well-developed root system and balanced shoot growth, because such plantlets consistently show higher survival in later hardening studies (Poniewozik et al., 2021; Sarmah et al., 2024). Rooted orchid plantlets grown on 1/2 MS and then shifted briefly to sucrose-free medium acclimatized successfully with 96% survival in one study, suggesting that seedlings should ideally be physiologically prepared, not merely rooted, before transfer. Substrate studies further show that survival varies with media and seedling quality, with favorable outcomes reported in brick+charcoal, coconut coir+charcoal, wood sawdust, and husk-charcoal-containing systems (De Stefano et al., 2022; Sari et al., 2023; Turnip, 2023). Emerging orchid work on automated grading using image-based detection of leaf and root defects achieved F1-scores of 84.44% to 90.44%, indicating that future D. officinale production could combine conventional morphological grading with digital inspection for more objective seedling selection (Lin et al., 2025).

6 Acclimatization and Transplant Preparation

6.1 Effects of acclimatization on survival rate

Acclimatization directly determines the ex vitro survival of tissue-cultured Dendrobium plantlets because seedlings transferred abruptly from culture vessels to greenhouse conditions rapidly desiccate, wilt, and often die. Gradual adaptation to lower relative humidity and higher light improves survival by allowing plantlets to correct in vitro-induced anatomical and physiological abnormalities, including weak stomatal regulation, poor cuticle function, and incomplete autotrophic competence (Silva et al., 2017). Structural evidence from orchids further shows that hardening strengthens the cuticle, epidermis, mesophyll, and vascular tissues, and these changes were associated with 100% survival in one ex vitro hardening study (Manokari et al., 2023). For D. officinale specifically, domestication management during the seedling-training period raised survival during hardening to about 95%, compared with a traditional level of 70%, and post-transplant survival after training exceeded 98%.

Survival after acclimatization also depends strongly on seedling vigor, rooting quality, and biological support. D. officinale seedlings generally show low survival and growth after transfer from in vitro conditions, but inoculation with Mycena dendrobii significantly enhanced both survival and growth by increasing stress tolerance and promoting new root formation. Other Dendrobium studies similarly indicate that pre-hardening on sucrose-free medium or prolonged in vitro strengthening improves later survival, with reported values of 96% in D. aqueum and 94% in tea after enhanced in vitro hardening (Bag et al., 2019). Across orchid acclimatization trials, suitable substrates can further stabilize survival, with values of 88.33% in cocopeat, 90% in peat-perlite-bark or river-soil mixtures, and 100% in some wood sawdust or coconut coir systems, showing that acclimatization success reflects both environmental transition and substrate matching (Turnip, 2023; Alghanimy and Alamery, 2025; Luthfi et al., 2026).

6.2 In-bottle and ex-bottle acclimatization measures

In-bottle acclimatization should begin before plantlets are removed from culture vessels, because partial hardening under controlled in vitro conditions reduces transplant shock after transfer. General ex vitro establishment research shows that lowering relative humidity and increasing light intensity before bottle removal can partially harden plantlets in vitro (Mahendra et al., 2020). Prolonged culture also improves transplant readiness: extending the strengthening period increased plantlet growth and raised acclimatization survival from 46% to 94% in one micropropagation system (Bag et al., 2019). In Dendrobium, transfer to sucrose-free medium before potting has been used as a transitional step to promote autotrophic adjustment, and D. aqueum plantlets cultured for several weeks on sugar-free 1/2 MS subsequently achieved 96% survival after greenhouse transfer. For D. officinale, strong in vitro seedlings with better vigor and uniformity were obtained under moderate auxin regulation, which provides a better material basis for later acclimatization.

Ex-bottle acclimatization should then proceed by staged humidity reduction, light regulation, and use of porous, moisture-retentive substrates. Reviews of Dendrobium acclimatization recommend greenhouse hardening under fog or mist conditions with progressive reduction of humidity and gradual increase of light using shading management (Silva et al., 2017). Related hardening systems commonly maintain initial relative humidity near 95% with plastic covers or inverted containers, then gradually reduce moisture and nutrient supply as seedlings move to shade-house conditions (Mahendra et al., 2020). Potting mixtures successful in orchids include brick plus charcoal covered with moss and enclosed with polyethylene bags, peat-perlite-bark combinations, cocopeat, husk charcoal, and bark-based media, all of which improve aeration, drainage, and root spread during the transition period (Alghanimy and Alamery, 2025; Luthfi et al., 2026). In D. officinale, bark and moss-based transplant substrates were specifically identified as suitable, and one rapid propagation system further combined bark-moss substrate with gibberellin and Epulorhiza sp. during transplanting.

6.3 Cleaning and disinfection after bottle removal

After bottle removal, the first operation should be careful cleaning of the root system to remove residual medium, because attached medium can retain excess moisture and increase contamination risk during transplant establishment. General hardening reviews state that regenerated plantlets are usually separated completely from the MS medium and the roots are thoroughly washed before planting (Mahendra et al., 2020). This step is especially important because newly exposed roots and basal tissues are highly vulnerable to soil microorganisms during early acclimatization, and sudden exposure to microbial communities is a major cause of mortality in tissue-cultured plants (Bag et al., 2019). Accordingly, many systems use initially sterilized or disinfected substrates to reduce pathogen pressure during establishment (Bag et al., 2019). In D. officinale seedling domestication practice, management after bottle and subsequent field planting are treated as distinct technical links, underscoring the importance of post-bottle cleaning and handling for maintaining high survival.

Disinfection after bottle removal should balance microbial control with avoidance of phytotoxic injury. In orchid systems, chemical disinfestation with commercial bleach has been used for re-cultivation, and one study found no significant growth difference between plantlets transferred in autoclaved vessels and those managed with bleach-based disinfection (Fontes et al., 2017). However, disinfectant concentration is critical, because increasing sodium hypochlorite levels can reduce germination or increase toxicity, while lower concentrations can still control contamination. For orchid explants more broadly, effective sterilization has been reported with 70% ethanol followed by sodium hypochlorite or HgCl2, but stronger or prolonged treatments can increase mortality (Kajol et al., 2024). Evidence from hydrogen peroxide treatments suggests that H₂O₂ is more effective against fungal than bacterial contamination, so it should not be relied on alone when bacterial pressure is high (Mtd et al., 2026). Therefore, post-bottle preparation of D. officinale should combine root washing, removal of damaged tissues, transplanting into clean porous substrate, and cautious use of low-to-moderate disinfection intensity to reduce contamination without compromising seedling viability.

7 Transplant Survival Improvement Techniques

7.1 Selection and formulation of transplant substrates

The transplant substrate is a primary determinant of post-bottle establishment in Dendrobium officinale because it controls aeration, water retention, root anchorage, and microbial exposure during the most vulnerable recovery period. Direct D. officinale studies identified several effective formulations, including vermiculite:perlite:humus soil at 1:1:5, which was selected as the corresponding transplant medium in an industrialized regeneration system, and bark-based matrices, with pure bark giving the highest survival in one rapid propagation study. Another D. officinale protocol recommended a bark-moss base combined with Epulorhiza sp., while substrate metabolomics work showed that pine bark, coconut coir, and their 1:1 mixture all support cultivation, but they produce different metabolic outcomes, underscoring that substrate choice affects both survival and seedling quality (Zhang et al., 2024).

Broader orchid evidence supports using porous, moisture-buffering substrates with stable structure during acclimatization. Cocopeat gave the highest survival at 88.33% in one orchid study, coconut coir outperformed a sphagnum-charcoal-bark combination for seedling height and leaf number in another, and wood sawdust or coconut coir produced 100% early survival in a hardening experiment (Silva et al., 2017; Longchar and Deb, 2022; Han et al., 2025). For Dendrobium specifically, sterilized coconut husk supported 95% survival in D. crumenatum, tree fern supported 94.8% survival in D. thyrsiflorum, and D. nobile performed best in a mixed substrate of chopped coconut husk, brick, charcoal, and cocopeat. Together, these results suggest that D. officinale transplant substrates should prioritize bark- or coir-based materials with good drainage and moderate moisture retention, while local optimization remains necessary because performance differs by genotype and cultivation objective (Rachmawatı et al., 2024).

7.2 Temperature, humidity, light, and ventilation management

Environmental management after transplanting should follow a gradual transition rather than an abrupt exposure to ambient conditions. In Dendrobium, acclimatization improves survival by progressively lowering relative humidity and increasing light, allowing plantlets to shift from heterotrophic or photomixotrophic growth to autotrophic growth while improving stomatal control and root function (Silva et al., 2017). More general orchid hardening guidance similarly recommends maintaining relative humidity near 95% at first, then gradually reducing moisture and nutrient supply while moving seedlings into shaded conditions, which promotes wax formation, thicker leaves, and more stable water relations (Zhang et al., 2022). In D. officinale domestication practice, greenhouse seedling training for about three months produced thicker leaves, darker color, stronger stems, more robust roots, and post-transplant survival above 98%, indicating that controlled environmental transition materially improves transplant success (Rachmawatı et al., 2024).

Light and ventilation must be coordinated with seasonal temperature control because excessive heat and stagnant humidity increase stress and disease risk. A D. officinale cultivation study reported that plantlets were properly shaded, cooled, and ventilated during summer and autumn, linking these measures to improved growth quality under cultivation (Zhang et al., 2024). Orchid precision-greenhouse research further shows that intelligent environmental management can track growth status and support decisions on greenhouse factors, with recognition accuracy reaching 98.6%, which supports future refinement of temperature, humidity, light, and ventilation control in orchid nurseries. Physiological evidence also indicates that appropriate light and potassium treatments can increase anthocyanin content and that different cultivation modes affect active compound accumulation, so environmental management during recovery should aim not only at survival but also at maintaining medicinal quality (Zhang et al., 2024).

7.3 Disease prevention and management during the seedling recovery period

Disease prevention during the recovery period should begin with sanitation and microbial risk reduction because newly transplanted tissue-cultured seedlings are highly susceptible to pathogen attack. General acclimatization studies note that a major cause of mortality is the rapid exposure of aseptically raised roots to soil microbial communities, especially fungi, and sterilized soil is therefore often used at the beginning of establishment (Rachmawatı et al., 2024). Post-bottle handling should include thorough washing to remove residual medium from the roots before planting, since retained medium can favor excess moisture and contamination (Silva et al., 2017). These sanitation steps fit D. officinale domestication systems, which explicitly treat post-bottle handling and post-field management as separate technical links within survival-oriented nursery management (Rachmawatı et al., 2024).

Biological management also appears important for disease resistance and recovery vigor in D. officinale. Mycena dendrobii significantly enhanced survival and growth after transplanting, and proteomic analysis indicated that the induced proteins included defense-and stress-response proteins, supporting a disease-preventive role through improved stress tolerance rather than simple growth promotion alone. Other orchid mycorrhizal studies found that fungal symbiosis can raise antioxidant enzyme activity, improve drought and disease resistance, and increase root and leaf growth, biomass, and medicinal polysaccharides, although effects differ by fungal strain and development stage (Zhang et al., 2020; Zhang et al., 2022). During the recovery period, disease management should therefore combine clean substrate, careful watering, adequate ventilation, and targeted use of beneficial fungi, while avoiding overly complex fungal mixtures because synthetic combinations did not show synergistic effects and sometimes produced offset effects (Wu et al., 2025).

8 Conclusions and Prospects

Tissue culture rapid propagation is the most practical basis for producing high-quality Dendrobium officinale seedlings because conventional propagation provides limited offspring and does not support industrial-scale seedling supply, whereas in vitro systems can establish continuous regeneration from germination, protocorm induction, proliferation, rooting, hardening, and transplanting. Direct studies on D. officinale have shown that stage-specific optimization of medium composition can support efficient germination, shoot proliferation, rooting, and transplant preparation, although the best formula differs by explant and culture phase. The same body of work indicates that high-quality seedlings are not defined only by multiplication rate, but by uniformity, strong rooting, and later transplant competence, which is why complete propagation systems are more valuable than isolated induction protocols. Broader Dendrobium evidence further supports this conclusion by showing that tissue culture is already an established propagation route for the genus and also provides the technical basis for germplasm conservation, clonal fidelity control, synthetic seed, bioreactor, and transformation technologies. In related medicinal Dendrobium species, optimized protocols produced about 87% transplant survival with 96.89% monomorphism after transfer, while D. nobile showed high genetic stability and 82.3% acclimatization survival, indicating that rapid propagation can support both scale and seedling quality when protocol stability is maintained.

Improving the transplant survival rate of D. officinale requires multi-stage coordination because losses arise not from a single step, but from cumulative mismatches among rooting quality, acclimatization intensity, substrate properties, and post-transplant stress response. Reviews of Dendrobium acclimatization show that plantlets must be gradually adapted to lower humidity and higher light so they can restore stomatal regulation, root function, and autotrophic growth before field or greenhouse establishment. Direct D. officinale studies also show that survival changes markedly with substrate and transplant management, with high-performing systems reported for pure bark, bark-moss bases, and vermiculite:perlite:humus soil mixtures, while broader orchid studies similarly favor porous, moisture-buffering media rather than overly compact substrates. Biological coordination is another important layer, because Mycena dendrobii significantly enhanced survival and growth of D. officinale seedlings by inducing defense- and stress-response proteins and promoting new root or mycorrhizal formation. Mycorrhizal evidence further shows that fungal effects are development-dependent and strain-specific: some fungi promote germination, others favor biomass, rooting, tillering, or polysaccharide accumulation, while synthetic fungal combinations do not necessarily produce synergistic effects. Therefore, future survival-improvement strategies for D. officinale should integrate strong-seedling culture, graded acclimatization, substrate matching, and targeted microbial assistance into one continuous technical chain rather than optimizing each step in isolation.

Standardization and intelligent management appear to be the clearest future directions for D. officinale seedling propagation because current orchid tissue-culture research is increasingly focused on medium optimization, endophytic-fungal regulation, and systematic solutions to contamination, browning, and vitrification, yet protocol fragmentation still limits reproducibility across genotypes and production sites. Recent bioreactor work in Dendrobium shows that liquid culture, temporary immersion systems, anti-browning additives such as ascorbic acid, and banana-extract-based regeneration media can markedly improve multiplication efficiency and may provide a foundation for standardized commercial systems, although genotype-specific optimization remains necessary. Orchid studies outside D. officinale also show that medium formulation depends on cultivation system and that combining optimized media with temporary immersion technology can reduce cost while balancing regeneration efficiency and genetic stability, which is directly relevant to future seedling industrialization. At the management level, intelligent greenhouse technology can already identify orchid growth status with 98.6% recognition accuracy and support environmental control and decision-making, suggesting practical value for digital monitoring of seedling vigor, environmental fluctuations, and transplant recovery. Taken together, the future of D. officinale seedling propagation lies in building standardized, data-driven technical systems that combine rapid propagation, clonal quality control, microbial regulation, bioreactor scaling, and intelligent greenhouse management, so that seedling production becomes more stable, economical, and suitable for large-scale medicinal cultivation.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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