Study on asymbiotic germination and acclimatization of Paphiopedilum purpureum

Orchidaceae is one of the largest and most diverse groups of flowering plants in the world, with over 730 genera and more than 29,000 species^[1−3]. The genus Paphiopedilum, one of the most primitive lineages in the Orchidaceae family, is commonly known as the 'lady's slipper orchid' or 'slipper orchid', due to its distinctive pouch- shaped or sac-shaped labellum, exhibiting both significant scientific research value and high ornamental value^[4]. The genus Paphiopedilum comprises 109 species and 35 natural hybrids worldwide, with 33 species currently documented in China^[3,5,6]. Plants of this genus typically grow in clustered populations, making them highly vulnerable to complete extraction through illegal wild harvesting, which can lead to localized extinction^[7]. The genus Paphiopedilum primarily reproduces through two methods: seed propagation, and division, both of which have significant limitations. In natural environments, the seeds must establish a symbiotic relationship with fungi to obtain nutrients for germination. However, even with additional nutrient supply, most mature Paphiopedilum seeds still exhibit poor germination rates^[8]. Vegetative propagation through division is limited by its long cycle and low propagation rate, making it inadequate for meeting large-scale production demands^[9]. The reproductive characteristics of Paphiopedilum have resulted in narrow wild population distributions, and low individual counts. Coupled with habitat degradation and excessive wild harvesting, these orchids have become one of the most endangered groups within the Orchidaceae family^[10]. Currently, all wild Paphiopedilum species are listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), receiving international protection^[11].

Asexual propagation methods for Paphiopedilum species, including axillary bud induction and shoot tip regeneration have been reported in existing literature^[12]. However, the in-vitro propagation of Paphiopedilum is significantly constrained by several technical challenges associated with explant materials, including sterilization difficulties, low induction efficiency, and limited multiplication rates, resulting in inefficient asexual reproduction systems^[12,13]. Most Paphiopedilum species cannot achieve industrialized tissue cloning production like Phalaenopsis orchids^[14]. Seeds represent the optimal source for preserving plant genetic diversity, with their inherent heterogeneity making them particularly suitable for conserving the genetic diversity of wild populations^[15]. Consequently, asymbiotic germination is recognized as the most efficacious method for Paphiopedilum propagation.

Current studies have documented successful in-vitro seedling production via asymbiotic germination in multiple Paphiopedilum species. However, seed germination and protocorm development in these orchids are significantly influenced by multiple factors, including capsule maturity, seed pretreatment protocols, medium composition, and culture conditions^[16]. Studies have shown that different Paphiopedilum species have a relatively optimal embryonic stage after pollination, during which seed germination rate is highest, while fully mature seeds are often difficult to germinate^[17]. In P. bellatulum and P. niveum, the seeds collected 120 to 150 days after pollination (DAP) were the optimum for culture in vitro, while for P. godefroyae, it was 90 to 120 DAP, and for P. henryanum and P. spicerianum, it was 120 to 180 DAP^[18]. The optimal germination period for P. emersonii was approximately 180 DAP^[19], while for P. concolor, the germination rate was relatively higher at around 210 DAP^[20]. Results from different studies on the same species also show variation. For example, the peak germination period for P. bellatulum ranged from 95 to 120 DAP^[13,21]. Furthermore, while immature seeds germinate readily, they were often associated with low seedling establishment rates^[22]. In contrast, mature seeds, despite being more suitable for long-term storage, present the practical challenge of being difficult to germinate. Therefore, precisely determining the optimal germination period during seed development and balancing the trade-off between germination rate and seedling establishment rate remain the core challenges to be addressed in the asymbiotic propagation of Paphiopedilum. Furthermore, due to genotype-specific requirements, optimal asymbiotic germination conditions vary significantly among Paphiopedilum species. Notably, propagation protocols remain underdeveloped for several taxa, including P. purpuratum^[23].

Paphiopedilum purpuratum is a perennial herbaceous species belonging to the genus Paphiopedilum, primarily distributed in southern China, including Guangdong, Guangxi, Hainan, and Yunnan provinces, and is also found in Vietnam^[24]. Through investigation and research, it has been found that due to habitat destruction and human excavation, the wild population of P. purpuratum is extremely scarce, placing the species in an endangered state. There is an urgent need for artificial assistance in its propagation and the reconstruction of its wild populations. Currently, P. purpuratum is classified as a Class I protected plant in China^[25]. Although studies on its conservation biology, chloroplast genome, taxonomy and community ecology have been reported in recent years^[26−29], systematic research on its artificial propagation and cultivation techniques remains inadequate. To address the inadequacies in aseptic germination techniques for P. purpuratum, this study focuses on investigating its seed asymbiotic germination. The research aims to elucidate critical stages and influencing factors in its reproductive process, thereby providing theoretical foundations for optimizing sterile propagation protocols. Furthermore, this work contributes to the conservation and utilization of P. purpuratum resources, holding significant implications for the preservation of its germplasm.

The specimens of P. purpuratum used in this study were derived from individuals originally introduced from southwestern China approximately two decades ago and are currently maintained at the Orchid Conservation Research Center of Shenzhen, Guangdong Province. Capsules obtained from artificial cross-pollination in November 2023 were collected as experimental materials.

Methods

Capsule collection and sterilization

Capsules were randomly collected at 140, 170, 200, 210, 220, 230, 240, 250, 260, and 270 DAP (three capsules per time point; 10 sampling stages total). Under sterile conditions, capsules were surface-disinfected by wiping with 75% ethanol-soaked gauze, followed by immersion in a 20% sodium hypochlorite (NaClO) solution for 30 min. After five rinses with sterile water, surface moisture was removed using autoclaved filter paper prior to capsule dissection.

Aseptic sowing

The sterilized seeds were evenly distributed onto a germination medium composed of 1/4 MS basal salts (macronutrients at 1/4 strength) supplemented with 100 mL coconut water. Each capsule was sown in five bottles. Cultures were maintained under dark conditions at 25 ± 2 °C. Germination status was monitored periodically. Upon germination, the cultures were transferred to light conditions, and formal assessments of germination rate and mortality conducted after 3 months of cultivation. At each stage, nine 1 × 1 cm squares were randomly selected, and germination within each square was observed and recorded. The germination rate was determined based on protocorm formation (embryo emergence through the seed coat)^[30], while the mortality rate was calculated according to the incidence of protocorm browning or necrosis post-germination initiation. Germination initiation time was recorded at the first observed embryo protrusion through the seed coat.

Protocorm differentiation induction culture

To induce the differentiation of protocorms into leaves and roots, germinated protocorms were transferred to a medium consisting of 1/2 MS + 0.5 mg/L 6-BA + 0.2 mg/L NAA + 0.5 g/L activated charcoal (AC) and cultured for three months. The culture conditions included a temperature of 25 ± 2 °C, photosynthetic photon flux density (PPFD) 66.13 µM·M⁻²·S⁻¹, and a photoperiod of 14 h light/10 h dark.

Rooting and strengthening of seedlings

When the seedlings of P. purpuratum had differentiated 2–3 leaves and reached a height of 1 cm, they were transferred to a culture medium consisting of 1/2 MS + 0.2 mg/L 6-BA + 0.5 mg/L NAA + 1 g/L AC supplemented with 50 mg/L (mL/L) of potato, banana, or coconut water, respectively. The control group (CK) was cultured in 1/2 MS + 0.2 mg/L 6-BA + 0.5 mg/L NAA + 1 g/L AC without any organic additives. This setup aimed to investigate the effects of different organic additives on the rooting and strengthening of P. purpuratum seedlings. Seven culture bottles were inoculated for each formulation, with three seedlings per bottle, and the experiment was repeated three times. The culture conditions included a temperature of 25 ± 2 °C, PPFD of 66.13 µM·M⁻²·S⁻¹, and a photoperiod of 14 h light/10 h dark.

To investigate the impact of different light intensities on the rooting and strengthening of P. purpuratum seedlings, the seedlings were cultured in a medium consisting of 1/2 MS + 0.2 mg/L 6-BA + 0.5 mg/L NAA + 1 g/L AC + 50 mg/L potato used as the rooting and strengthening medium under varying PPFD (4.67 µM·M⁻²·S⁻¹ and 66.13 µM·M⁻²·S⁻¹). Potato extract was added as an organic supplement to promote rooting and seedling strengthening^[31]. Six culture bottles were inoculated for each light intensity treatment, with three seedlings per bottle, and the experiment was repeated three times. The culture temperature was maintained at 25 ± 2 °C, with a photoperiod of 14 h light/10 h dark.

After 90 d of culture, observations and records were made on the rooting and strengthening of the seedlings, and measurements were taken for chlorophyll content, soluble sugar content, and root activity. Chlorophyll content was quantified through ethanol extraction^[32], while soluble sugar content was determined by anthrone-sulfuric acid colorimetry^[33]. Root system vitality was assessed via the 2,3,5- triphenyltetrazolium chloride (TTC) reduction assay^[34].

Acclimatization and transplantation

The in vitro seedlings of P. purpuratum, approximately 2 cm in height with three fully developed leaves and 3–5 robust roots, were transferred to a greenhouse for 7 d. Subsequently, the bottle caps were opened for an additional 7-d acclimatization period to complete the hardening process. During transplantation, the culture medium adhering to the roots was rinsed off with clean water, and the seedlings were air-dried before being transplanted into three distinct substrate mixtures with varying volume ratios: bark: limestone (1:1), in-situ substrate: limestone (1:1), and bark: volcanic rock (1:1). Eight plants were transplanted into each substrate, with the experiment repeated three times. The culture environment parameters were as follows: PPFD 120.6–136.7µM·M⁻²·S⁻¹, humidity 70%, daytime temperature 24−28 °C, and nighttime temperature 20−24 °C. The characteristics of these substrates are detailed in Table 1. After 90 d, parameters including survival rate, crown width increment, chlorophyll content, and soluble sugar content were measured.

Data statistics and analysis

Experimental data were organized using Microsoft Excel 2019 and subjected to analysis of variance (ANOVA) and t-tests with SPSS Statistics 27.0.

As shown in Fig. 1, seeds at different developmental stages exhibited distinct color variations in their longitudinal sections. At 140 d after pollination (DAP), the seeds displayed white coloration and were tightly attached to the placenta, making it difficult to separate individual seeds from the placental tissue (Fig. 1a). The 170 DAP seeds retained their white pigmentation while exhibiting reduced placental adhesion compared to 140 DAP specimens, facilitating easier detachment of individual seeds (Fig. 1b). At 200 DAP, the capsule apex developed light brown pigmentation while maintaining white coloration in remaining regions, with seeds exhibiting significantly enhanced dispersibility (Fig. 1c). The 210 DAP capsules exhibited pronounced darkening of brown pigmentation at the apex, with progressive extension of browning towards the median capsule region, while seeds adjacent to the pedicel maintained a characteristic yellowish-white coloration (Fig. 1d). Longitudinal sections revealed a gradual browning of seeds from the ovarian apex to the base. At 220 DAP, the capsule seeds exhibited more extensive browning compared to the 210 DAP specimens, with only a few remaining yellowish-white seeds (Fig. 1e). At 230 DAP, the seeds had turned tan with occasional dark brown specimens (Fig. 1f). From 240 to 270 DAP, the seeds developed uniform brown coloration and became loosely attached, easily dislodged from the capsules (Fig. 1g-j).

Figure 1. 

Longitudinal sections of P. purpuratum capsules at different developmental stages (scale bars = 1 cm). (a) 140 DAP, (b) 170 DAP, (c) 200 DAP, (d) 210 DAP, (e) 220 DAP, (f) 230 DAP, (g) 240 DAP, (h) 250 DAP, (i) 260 DAP, and (j) 270 DAP.

Effects of capsule maturity on seed germination in P. purpuratum

Statistical analysis of initial germination time and germination rate was conducted for P. purpuratum seeds at different developmental stages (Table 2). The results showed that 140-DAP seeds turned brown 20 d after sowing without germination. Seeds at 170 DAP achieved a relatively high germination rate of 65.77%, but 68.39% of the resulting protocorms browned and failed to develop into seedlings. For seeds at 200−210 DAP, the germination rates ranged from 42.64% to 48.27%, with corresponding protocorm mortality rates between 33.93% and 47.71%. The highest germination rate of 80.58% was observed in 230-DAP seeds, and all germinated protocorms developed normally into seedlings. Germination rates gradually declined with increasing capsule age, decreasing to 45.28% at 240 DAP and 42.25% at 250 DAP. Complete loss of germinability occurred by 260 DAP. The germination initiation time remained consistent at 51−53 d across all developmental stages, showing no discernible pattern.

Effects of different organic supplements on seedling strengthening and rooting in P. purpuratum

Seedlings with 2−3 leaves and approximately 1 cm in height were transferred to culture media containing 1/2 MS + 0.2 mg/L 6-BA + 0.5 mg/L NAA supplemented with either 50 g/L potato, coconut water, or banana. The results demonstrated significant variations in the effects of different organic supplements on seedling strengthening and root development in P. purpuratum. As presented in Table 3, marked differences were observed across multiple growth parameters among the treatment groups. Regarding crown width expansion, all organic supplement treatments demonstrated statistically superior performance compared to the control group (CK), with the banana-supplemented group exhibiting optimal results. The banana treatment achieved a crown width increment of 4.53 cm, showing a significant advantage over both the potato and coconut water treatments. In terms of root proliferation, the banana treatment group demonstrated optimal performance, reaching 4.78 roots per seedling, which was significantly higher than other treatment groups. The coconut water and potato treatments showed intermediate results with 3.65 and 3.61 roots, respectively, though no statistically significant difference was observed between these two groups. Regarding total root length increment, both banana and coconut water treatment groups showed statistically significant differences compared to the CK, with increases of 31.85 and 27.52 cm, respectively. Although the coconut water treatment group performed slightly inferior to the banana group in terms of both root proliferation and elongation, it induced the development of more robust root systems (Fig. 2). Physiological index analysis (Table 4) revealed that the potato treatment group achieved optimal performance in total soluble sugar content, reaching a maximum value of 89.44 mg/g, which was significantly higher than all other groups, while no significant differences were observed among other physiological indices. In contrast, the coconut water treatment group showed lower values in total soluble sugar content compared to other treatments. In summary, in the cultivation of robust P. purpuratum seedlings, banana effectively promotes the enlargement of crown width and root growth, coconut water significantly enhances root diameter, while potato facilitates the accumulation of soluble sugars in the leaves.

Figure 2. 

Effects of different organic additives on robust seedling growth of P. purpuratum (scale bars = 2 cm). (a) CK, (b) potato, (c) banana, (d) coconut water.

Effects of light intensity on seedling vigor of P. purpuratum

Plantlets with 2−3 leaves and approximately 1 cm in height were transferred to a seedling-strengthening medium composed of 1/2 MS basal salts supplemented with 0.2 mg/L 6-BA, 0.5 mg/L NAA, and 50 g/L potato extract. The cultures were then subjected to distinct light conditions: PPFD 4.67 µM·M⁻²·S⁻¹ (representing indoor diffused light) and 66.13 µM·M⁻²·S⁻¹ (achieved using dual 18W LED lamps) for cultivation. The results presented in Table 5 demonstrate significant differences in canopy expansion and root elongation of P. purpuratum under different light conditions, while no effect was observed on root proliferation. Under high PPFD (66.13 µM·M⁻²·S⁻¹), the crown width growth rate was significantly greater than that under low PPFD (4.67 µM·M⁻²·S⁻¹). Conversely, root elongation was significantly enhanced under low light conditions compared to high light intensity. Neither light treatment showed significant effects on chlorophyll content, root activity, or soluble sugar content (Table 6). Therefore, high light conditions promote canopy expansion, whereas low light favors root elongation growth.

Effects of different cultivation substrates on transplant survival of P. purpuratum

In-vitro seedlings after robust growth cultivation were acclimatized and then transplanted into various growth media, followed by cultivation in greenhouse conditions. The results demonstrated that the in-situ substrate mixture of limestone (1:1) achieved the highest survival rate of 75.93%, followed by bark: volcanicrock (1:1) at 63.89%, while bark: limestone (1:1) showed the lowest survival rate of merely 36.11% (Table 7). In addition, the results showed no statistically significant differences in crown width (Fig. 3), soluble sugar content, and chlorophyll content among the different cultivation substrate treatments.

Figure 3. 

Effects of different cultivation substrates on transplantation of P. purpuratum (scale bars = 2 cm), (a) bark: limestone (1:1), (b) in-situ substrate: limestone (1:1), (c) bark: volcanic rock (1:1).

Seeds, as commonly used explants for in vitro culture in Paphiopedilum species exhibit a germination rate that was directly influenced by capsule maturity and generally follows a dynamic unimodal pattern, initially increasing before declining^[18]. Among terrestrial orchids in the family Orchidaceae, near-mature seeds typically exhibited higher germination rates compared to fully mature seeds^[22]. The study found that P. purpuratum took about 280 DAP from pollination to capsule dehiscence, but achieved optimal germination and seedling establishment at 230 DAP. This phenomenon was not an isolated case. Similar germination patterns have been reported in multiple other Paphiopedilum species, including P. armeniacum^[8], P. spicerianum^[35], and P. delenatii^[36]. The consistent germination trend observed in P. purpuratum, as a member of this genus, suggests it may reflect a sophisticated and evolutionarily conserved reproductive strategy within Paphiopedilum.

Correlation between the optimal germination period and embryo developmental stage

The optimal germination period for seeds of the Paphiopedilum genus often corresponds to specific stages of embryonic development. For P. spicerianum, the highest germination rates occurred 180−210 DAP, when the embryo reached the late globular stage with initial suspensor degeneration^[35]. Research on P. barbigerum similarly confirmed that seeds collected 255 DAP achieved the highest germination rate of 90.71%, at which stage the embryos were in the globular phase, and the suspensors had not yet fully degenerated^[37]. In this study, while P. purpuratum seeds at 170 DAP were capable of germination, they failed to develop into seedlings, suggesting that the embryos might not have completed their morphological and physiological construction at this stage. In contrast, seeds at the near-maturity stage of 230 DAP exhibited the highest germination vigor and seedling potential, which may be related to the complete development of their embryos.

Seed coat maturation leads to a decline in germination rate

Germination rates declined rapidly after reaching a peak, which may be attributed to the maturation and strengthening of the seed coat structure. In P. armeniacum, the seed coats of fully mature seeds became increasingly dense, which impedes the absorption of water and nutrients, thereby suppressing germination^[13]. In P. appletonianum and P. hirsutissimum, the decline in germination rates following the development of the embryo into the ellipsoidal stage was similarly related to the progressive accumulation of lignin-like substances in the seed coat, which reduced its water permeability^[38]. In this study, the decline in germination rates observed in P. purpuratum after 240 DAP was likely attributable to the similar progressive deposition of lignin-like substances within the seed coat. This physical dormancy induced by seed coat thickening serves as a protective mechanism against adverse environmental conditions in natural settings. However, in in-vitro cultivation, it becomes a primary barrier that must be overcome. To address this obstacle in artificial conservation and propagation, the germination rate of seeds could be significantly enhanced through sodium hypochlorite treatment^[39].

Interspecific variations and the implications for conservation practices

Although a common germination pattern exists, the optimal germination period varies among species within the Paphiopedilum genus, reflecting potential adaptive responses to their respective native habitats. For instance, P. delenatii reached its peak germination rate of 79.3% at 210 DAP^[36]. Germination in P. villosum commenced at 150 DAP, with the highest germination rate of 31.33% observed at 200 DAP^[40]. For P. wardii, studies have reported that the optimal germination period occurs at 180 DAP^[22]. In this study, the optimal germination period for P. purpuratum was determined to be 230 DAP. Such variations highlight the necessity of identifying and selectively targeting the specific seed age when developing in-vitro propagation protocols for the endangered Paphiopedilum species.

Effects of light on the physiological responses of P. purpuratum

Adaptation to low-light conditions and chlorophyll stability

Paphiopedilum purpuratum is naturally distributed in southeast-facing forest understories with heavy canopy shade, where its growth requirements are met by moderate diffuse light conditions^[28]. Although studies have reported low light intensity in the natural habitat of P. purpuratum, the effects of varying light intensities on its growth remain poorly understood. Studies have demonstrated that P. emersonii can autonomously regulate leaf chlorophyll content to maintain consistent levels within a certain light intensity range, thereby optimizing the capture and utilization of diffuse light^[41]. The present study revealed that chlorophyll content in P. purpuratum exhibited no statistically significant variation during vigorous seedling cultivation under two distinct PPFD conditions of 4.67 and 66.13 µM·M⁻²·S⁻¹. This finding aligns with the previously mentioned research results and suggests that the Paphiopedilum genus may possess the ability to autonomously regulate chlorophyll metabolism to stabilize pigment content, thereby effectively capturing and utilizing light energy in varying light environments.

Carbon metabolism balance under different light intensities and research limitations

Paphiopedilum purpuratum exhibited marginally higher soluble sugar content under high light intensity compared to low light conditions, though the difference was not statistically significant. This suggests the species may possess an efficient carbon metabolism system capable of maintaining metabolic homeostasis across varying light regimes. Under low light conditions, P. purpuratum likely maintains soluble sugar content by reducing respiratory rate or enhancing photoassimilate accumulation, whereas under high light intensity, it may stabilize sugar levels through increased respiratory rate or promoted translocation and utilization of photoassimilates to dissipate excess photosynthetic products. This carbon metabolic regulation pattern may be closely related to the photosynthetic characteristics of variegated leaves in P. purpuratum. The dark-colored regions possess higher chlorophyll content and serve as the primary sites for light energy absorption, whereas the light-colored regions, although lower in chlorophyll content, exhibit higher energy capture efficiency of the photosystem II (PSII) reaction center, enabling better utilization of low light for photosynthesis^[42]. This study established two distinct light intensity gradients, which preliminarily revealed the stability of physiological indicators in P. purpuratum. However, this experimental design still exhibits evident limitations.

Effect of cultivation substrate on the growth of P. purpuratum

Influence of substrate physical structure on transplant survival

Substrate type is one of the key factors determining the transplant survival of slipper orchids. Commonly used substrates for slipper orchids include volcanic rock, pine bark, peat soil, and their mixtures^[43]. Volcanic rock exhibits high porosity, combining superior aeration and water retention properties, thus serving as an ideal substrate for orchid cultivation. Limestone, characterized by its porous structure, excellent drainage properties, and moderate hardness, is a commonly used component in mixed cultivation substrates for terrestrial orchids. P. hirsutissimum exhibited the highest transplant survival rate of 97% in shredded bark cultivation substrate, demonstrating uniform root development and vigorous growth^[44]. In contrast, P. emersonii achieved a 75.93% survival rate in a mixed substrate composed of bark: volcanic rock: humus soil (1:1:1)^[45]. For P. villosum, a substrate mixture of charcoal, brick fragments, and soil at a 1:1:1 ratio, topped with a layer of moss, proved most suitable for seedlings, demonstrating satisfactory survival rates up to 63.1%^[46].

In this transplantation experiment of P. purpuratum, a 1:1 (v/v) mixture of in-situ substrate and limestone fragments was identified as the optimal growing medium, achieving a high survival rate of 75%. The in-situ substrate was collected from the P. purpuratum population in Zhongshan, primarily composed of humus and leaf litter. This substrate is rich in rhizosphere fungi and their metabolites specific to P. purpuratum, with high soil fertility that maintains excellent nutrient availability. When combined with limestone fragments (1:1 ratio), the mixed medium provides optimal water retention without waterlogging, effectively preventing root rot and consequently achieving the highest survival rate.

Influence of substrate pH and mineral nutrients on survival rate

Both volcanic rock and limestone exhibit large interparticle voids with loose, aerated structures, making them common orchid cultivation substrates when combined with bark or humus. However, in this experiment, P. purpuratum showed markedly different survival rates: merely 36.11% in bark: limestone (1:1) substrate vs 63.89% in bark: volcanic rock (1:1). This significant difference may be related to the contrasting pH-regulating capacities of the two substrates. Measured data show that the irrigation water used in the experiment had a pH of 7.5, while the leachate from the bark: volcanic rock substrate had a pH of 6.8, which was lower than the pH of 7.1 from the bark: limestone substrate. This indicates that volcanic rock likely possesses a greater buffering or acidifying capacity, enabling it to modulate the substrate's pH to some extent and better align with orchids' requirements for their growth environment. In addition to the pH effect, volcanic rock is rich in iron, calcium, potassium, magnesium, manganese, phosphorus, and other trace elements. These nutrients can be gradually absorbed by roots through irrigation, thereby enhancing the growth and development of P. purpuratum. This study did not investigate the synergistic effects of combining native habitat substrate with volcanic rock on P. purpuratum growth. Future research could explore the potential optimization benefits of such mixed substrates.

This study successfully developed a methodology for asymbiotic seed germination and acclimatized transplantation of P. purpuratum. Key findings include: (1) 230 DAP capsules showed the highest germination rate, with all protocorms developing into normal seedlings; (2) organic additives demonstrated specific effects—banana promoted shoot expansion and root growth, coconut water increased root diameter, while potato enhanced soluble sugar accumulation in leaves; (3) light intensity differentially influenced growth—high light favored shoot expansion, whereas low light promoted root elongation; (4) a 1:1 mixture of in-situ substrate and limestone proved optimal for transplanting tissue-cultured seedlings. These results provide a comprehensive technical framework for large-scale propagation, supporting both ex-situ conservation and wild population restoration of this endangered orchid species.