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Article

Seed Asymbiotic Germination Morphological Traits and Seedling Development in Cymbidium faberi Rolfe (Orchidaceae)

by
Zhiqing Zhou
1,2,†,
Siyu Han
1,†,
Hao Huang
1,2 and
Zhixiong Liu
1,2,*
1
College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China
2
Hubei Key Laboratory of Spices & Horticultural Plant Germplasm Innovation & Utilization, Yangtze University, Jingzhou 434025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(12), 1491; https://doi.org/10.3390/horticulturae11121491
Submission received: 6 November 2025 / Revised: 2 December 2025 / Accepted: 7 December 2025 / Published: 9 December 2025
(This article belongs to the Special Issue From Structure to Function: Anatomical and Reproductive Studies in Native and Cultivated Horticultural Plants)
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Abstract

Cymbidium faberi Rolfe is a Chinese flower famous due to its beautiful floral pattern and strong floral scent and is also a threatened terrestrial orchid. Moreover, the traditional propagation method through tillers and symbiotic seed germination with the correct fungus under nature conditions could not meet conservation and commercial needs. Here, an efficient procedure for asymbiotic seed germination and in vitro seedlings development of C. faberi was successfully established through evaluation of time of seed collecting, seed pretreatments, light conditions and composition of culture media, respectively. Seed pretreatment with 1% NaClO for 30 min, dark culture on 1/4 MS medium containing 0.5 mg·L−1 6-BA and 0.1 mg·L−1 NAA for 30 days and subsequent long day condition (14 h light/10 h dark photoperiod) culture on this medium for 30 days could obviously enhance the seed germination rate of C. faberi. The highest germination rate (85.0 ± 0.79%) was achieved when seeds were collected at 120 d after cross-fertilization, and then germination percentages progressively decreased. Furthermore, histological analyses from protocorm formation to seedling growth were explored. This study not only offers a reliable and scalable system for mass propagation to meet commercial and conservation demands but also serves as a foundational reference for physiological and molecular studies in Cymbidium and related orchids.

1. Introduction

Cymbidium faberi Rolfe is a potted orchid, very popular due to its beautiful floral pattern and strong floral scent (King of Fragrance), and has been cultivated for centuries in China, Japan, and Korea [1,2,3,4]. As the most famous Chinese flower and a cultural symbol, C. faberi is characterized by its strong floral fragrance, delicate flower shapes, elegant plant structures and is renowned as the ‘Gentleman of flowers’ [5,6]. Due to its extremely high ornamental, cultural, and economic value, commercially important varieties with marvelous floral pattern, rare floral color and abnormal leaf have been developed during several thousand years of domestication. Moreover, C. faberi exhibits various floral morphologies and unique floral scents and vegetation traits, thus attracting the interest of botanists and hobbyists [7]. In the wild, C. faberi is threatened by deforestation, illegal poaching and commercial overexploitation, primarily for their ornamental value [5]. C. faberi was categorized into Level II in the list of National Key Protected Wild Plants (NKPWPs) of China since 2021 [8]. C. faberi is propagated through tillers and seed germination under nature conditions. Orchids produce the smallest seeds by size (0.05–6 mm) or weight (0.31–24 μg) among all flowering plants [9,10]. A single orchid fruit capsule can produce up to 4 million of dust-like, tiny seeds. Most orchid seeds fall close to the mother plant, with only a tiny fraction germinating and eventually becoming an adult plant [9,11]. Orchid seeds are devoid of endosperm and hence lack nutrient resources for seed germination, which are dependent on external nutrition provided by seed sgOMFs (seed germination-promoting orchid mycorrhizal fungi) for germination and subsequent seedling growth under nature conditions [12]. In the wild, orchid seed germination occurs once the correct fungus has entered the seed, with its tiny seedling capable of precisely balancing the amount of nutrients received with the potential pathogenicity of the associated fungus [9]. Due to the obligate association with a mycobiont, the germination niches in orchid species are extremely complex and varied [13]. In addition, many orchid seeds do not germinate or develop without their compatible fungus symbiont because the mycorrhizal specificity associated with the orchid in situ seed symbiotic germination is often limiting [14]. In vitro symbiotic germination has been used for orchid propagation [15]. However, successful propagation needs extensive knowledge of the specific orchid–mycorrhizal interaction, and species-specific fungus isolated and cultured. Furthermore, many orchid mycorrhizal fungi are unculturable axenically, which makes symbiotic germination impossible [16].
Orchid asymbiotic seed germination is a process in which organic nutrients are provided to seeds through an artificial medium and first successfully established by Lewis Knudson in 1922, which is not inhibited by the obstacle of orchid–fungal specificity [16]. Moreover, many orchid seed propagation and production protocols were developed through asymbiotic seed germination, such as Cephalanthera falcata [17], Calopogon tuberosus [18], Coelogyne nervosa [19], Dendrobium [20], Paphiopedilum [21,22,23], Chloraea crispa [24], Pecteilis radiata [25], Gastrochilus matsuran [26], Anacamptis longicornu and Ophrys panormitana [27], Orchis militaris [28], Vanilla planifolia [29,30], Spiranthes ochroleuca [31], Cypripedium subtropicum [32], C. guttatum [33], Eulophia bicallosa [34], etc. Although asymbiotic germination may allow for a more efficient means to propagate orchids from seed, such techniques are not foolproof. The developmental requirements of orchids vary drastically across the family, especially between species in tropical and temperate regions, necessitating the use of diverse methodologies [35].
The protocorm is considered as a unique structure designed to establish symbiotic association with mycorrhizal fungi and with the primary goal to form a shoot apical meristem [36]. From a developmental and functional point of view, orchid protocorm is a unique structure designed to establish symbiotic association with a compatible fungus, and with the primary goal to form a shoot apical meristem (SAM) for plantlet growth. The body plan of a protocorm is established during embryogeny, with cells having distinct cell fates [37]. Meristems make the plants; without a functional SAM, there is no further development into a plantlet [38]. Hence, the present study aimed to develop an efficient in vitro asymbiotic germination protocol for the terrestrial orchid C. faberi through evaluating the effect of seed pretreatments, culture media, and time of seed collecting on seed germination. Our study also documents, through detailed morphological and histological observation, the complete developmental process from protocorm to seedling of this commercially important and threatened orchid. In addition, the present study also a provides practical and useful technique for mass in vitro propagation of C. faberi to meet conservation and commercial needs.

2. Materials and Methods

2.1. Plant Material and Sterilization

The variety of Cymbidium faberi ‘Zheng Xiaohe’ (Figure 1a) were cultivated in redware flowerpots (upper Ø 30 cm, base Ø 18.5 cm, height 28.2 cm) filled with orchid substrates on the campus of Yangtze University under natural conditions in Jingzhou City, China. Cross-fertilization among flowers (Figure 1b) from different plants was performed by hand at the second day of flowering (26 March 2024). Capsules (Figure 1c) were harvested at 120 days (120 d), 150 d, 180 d, 210 d after cross-fertilization and then stored at 4 °C until used, respectively. Capsules harvested 120 d after cross-fertilization were washed under running tap water. Subsequently, they were soaked in 70% ethyl alcohol for 30 s and subjected to surface sterilization in a 2% NaClO solution for 10 min. They were then rinsed 7–8 times for 30 s each with sterile water. The seed capsules were then cut longitudinally with a sterile scalpel, and the exposed seeds were transferred into sterile 5 mL centrifuge tubes. The seeds were stored at 4 °C for no more than 30 days until used.

2.2. The Germination of the Seeds and Protocorm Development

In order to improve the germination rate of the seeds, we have mimicked natural germination conditions through dark treatment and have softened the seed coat with NaClO. Different pretreatments (Table 1) were performed to evaluate their effects on the germination of C. faberi seeds harvested above. All treated seeds were rinsed four times with sterile water. The treated seeds were randomly sown on 1/4 MS (Murashige and Skoog, Coolaber, Beijing, China) medium [39] supplemented with 0.1 mg·L−1 naphthaleneacetic acid (NAA, Aladdin, Shanghai, China), 0.5 mg·L−1 6-benzylaminopurine (6-BA, Aladdin, Shanghai, China), 20% coconut water (CW), 3% sucrose, 0.7% agar, and 0.1% activated charcoal (AC) at pH 5.8 for a preliminary screening of treatments for seed germination. Ten culture dishes containing about 100 seeds per dish were used for each treatment and every treatment was repeated three times. For each treatment, the experiment included three independent biological replicates, with each replicate comprising ten culture dishes (each dish containing approximately 100 seeds).
In order to improve the germination rate of the seeds, we have tested four dilutions MS medium (Table 2) and VW medium (Coolaber, Beijing, China) to find the optimal culture medium. Five germination media (Table 2)—MS (Coolaber, Beijing, China), 1/2 MS (Coolaber, Beijing, China), 1/4 MS, 1/6 MS (Coolaber, Beijing, China), and Vacin and Went (VW) [40] were evaluated for their efficacy in supporting seed germination. All media contained 0.5 mg·L−1 6-BA, 0.1 mg·L−1 NAA, 20% CW, 3% sucrose, 0.7% agar, and 0.1% AC, and were adjusted to pH 5.8. Treated seeds were sown onto the respective media. The experimental design consisted of ten culture dishes per treatment, each containing approximately 100 seeds, with three independent replicates. Following sowing, the seeds were subjected to a 30-day dark incubation at 25 °C, then transferred to a 14 h light/10 h dark photoperiod at the same temperature for an additional 30 days.
To evaluate the effect of the seed developmental stage on in vitro germination, capsules were collected at 30-day intervals from 120 to 210 days after cross-fertilization. Seeds were sterilized and pretreated as described in the optimal protocol identified earlier. They were then cultured on 1/4 MS medium supplemented with 0.5 mg·L−1 6-BA, 0.1 mg·L−1 NAA, 20% CW, 3% sucrose, 0.7% agar, and 0.1% activated charcoal (pH 5.8). For each collection time point, approximately 100 seeds were plated per dish, with ten replicate dishes per treatment, and the experiment was repeated three times. All cultures were maintained in darkness for 30 days, followed by 30 days under a 14 h light/10 h dark photoperiod at 25 °C. A protocorm was considered successfully induced when it turned green and reached a minimum diameter of 2 mm. The germination rate was assessed after 60 days of culture using the following formula:
Germination rate (%) = (Number of protocorms induction/Total seeds sown) × 100
To induce proliferation, protocorms were transferred to a 1/4 MS medium. The medium contained 1.0 mg·L−1 NAA and 1.0 mg·L−1 6-BA, 200 g·L−1 potato homogenate, 3% sucrose, 0.7% agar, and 0.1% AC (pH 5.8). Cultures were then incubated for 60 days under a 14 h light/10 h dark photoperiod at 25 °C.

2.3. In Vitro Seedling Development

Protocorms were subcultured for differentiation on 1/4 MS medium composed of 4.0 mg·L−1 NAA, 1.0 mg·L−1 6-BA, 20% CW, 3% sucrose, 0.7% agar, 50 mg·L−1 myo-inositol (Aladdin, Shanghai, China), 2.0 mg·L−1 glycine (Aladdin, Shanghai, China), and 2.0 mg·L−1 nicotinic acid (Aladdin, Shanghai, China) (pH 5.8). For subsequent root development, shoots were transferred to a rooting medium. This medium shared the same 1/4 MS basal composition and organic additives but was supplemented with 1.0 mg·L−1 6-BA, 0.5 mg·L−1 IBA (Aladdin, Shanghai, China), and 0.1% AC. All cultures were incubated for 60 days at 25 ± 2 °C under a 14 h photoperiod.

2.4. Histological and Morphological Analyses

Development seeds and protocorms at sequential developmental stages were fixed immediately in FAA (38% formaldehyde: acetic acid: 70% ethanol = 1:1:18, by volume) for more than 24 h. The samples were dehydrated using a graded ethanol series, cleared in a xylene series, infiltrated with molten paraffin, and then embedded into a paraffin block according to Li et al. [4]. The embedded samples were serially sectioned at a thickness of 3 µm with a Leica RM2235 rotary microtome. The sections were subsequently stained with Periodic acid–Schiff’s (PAS, Servicebio, Wuhan, China). The sections observed under a CAIKON RCK-40C microscope and photomicrographs were subsequently taken. In our histological methodology, at least three similar individual specimens present a key developmental stage.

2.5. Transplantation and Acclimatization

The in vitro seedlings obtained above were transferred to greenhouse maintained at 25 ± 2 °C under 14 h light/10 h dark photoperiod provided by cool white fluorescence tubes at 150 µmol m−2 s−1 and 80% humidity levels ranged from 70 to 80% for 24 h acclimation with the bottle open, and then transplanted into terracotta pots (upper Ø 20 cm, base Ø 14 cm, height 13 cm, nominal volume 2.5 L) filled with mixed coir, volcanic rock, shattered pine bark, and peat moss (1:1:1:3 v/v/v/v), followed by rinsing in sterilized cooled water to clean the gel attached to roots and leaves. The transplanted seedlings were grown in a greenhouse maintained at 25 ± 2 °C under a 14 h light/10 h dark photoperiod provided by cool white fluorescence tubes at 150 µmol m−2 s−1 and humidity levels ranged from 70 to 80%. The seedlings were irrigated with 0.1% KH2PO4 solution at 3 d intervals. The survival rate of seedling, average height of seedling, and the number of leaves were recorded monthly after 90 days transplanting.
Survival rate = Number of surviving seedlings/Number of transplanted seedlings
Average height of seedling = Total height of seedling/Total number of seedlings transplanted
Average number of leaves = Total number of leaves/Total number of seedlings transplanted

2.6. Data Analysis

All data were expressed as mean ± standard errors. Statistical analysis was performed using the SPSS version 28.0 with the one-way ANOVA. p-value < 0.05 was considered statistically significant.

3. Results

3.1. Effect of Different Pretreatments, Media, and Time of Seed Collecting on Seed Germination

The effects of 1/4 MS medium containing 0.5 mg·L−1 6-BA and 0.1 mg·L−1 NAA for protocorms induced of C. faberi seeds were investigated in different pretreatments (Table 1). The highest germination rate (85.0 ± 0.79%) was observed in T3 treatment, while the lowest germination rate (10.0 ± 0.39%) was in CK (Figure 2a), which suggested that dark incubation and seeds pretreated with 1% NaClO could significantly enhance the germination rate of C. faberi seeds. However, seeds pretreated with 1% NaClO or dark incubation only resulted in the germination rate to 20.0 ± 0.49% and 25.0 ± 0.57%, respectively (Figure 2a). Moreover, the effects of different media (MS, 1/2 MS, 1/4 MS, 1/6 MS and VW) for germination were also evaluated (Figure 2b). The 85.0 ± 0.79% of germination rate on the 1/4 MS medium was observed and exhibited superior performance compared to the MS, 1/2 MS, 1/6 MS and VW media, respectively. The germination rate of 69.73% was observed on the VW medium. The germination rate of 45.0 ± 2.85% and 65.0 ± 1.43% was observed on 1/2 MS and 1/6 MS, respectively. However, only 25.0 ± 1.22% of germination rate was observed on the MS medium. In addition, the germination of seed was obtained under different time of seed collecting after 60 d culture (Figure 3c). The highest germination rate (85.0 ± 0.79%) was observed at 120 d after cross-fertilization. Moreover, germination percentage progressively decreased for seeds collected at 120 d or later. The 65.13 ± 0.63% and 25.06 ± 0.74% germination rate were obtained at 150 d and 180 d after cross-fertilization, respectively. Only 14.46 ± 0.67% germination rate was observed at 210 d after cross-fertilization.

3.2. Embryo Development

The development of the embryo in seeds of C. faberi was revealed by the paraffin sectioning method (Figure 3). At 120 d after pollination, the globular embryo is very close to the seed coat (SC) with the 4.36 ± 1.63 μm gap between the inner integument and the embryo, and the cell nuclei stained obviously (Figure 3a). The globular embryo had no further morphological changes at 150 d after pollination, but the gap between the inner integument and the embryo was visible at this stage (approximately 19.6 ± 2.07 μm, Figure 3b). The embryo had fully developed, and starch continued to accumulate at 180 d after pollination, the gap between the inner integument and the embryo increased gradually (approximately 33.35 ± 3.56 μm, Figure 3c). At 210 d after pollination, and the embryo became more intensely stained and the gap between the inner integument and the embryo was 38.52 ± 5.65 μm (Figure 3d).

3.3. Morpho-Anatomical Changes from Seed to Protocorm

We monitored the morpho-anatomical changes in C. faberi with seed germination and protocorm proliferation by the paraffin sectioning method and histological analyses. Germination was detected 60 days after sowing (including the initial 30 d dark incubation and subsequent 30 d light culture) on 1/4 MS medium supplemented with 0.5 mg·L−1 6-BA, 0.1 mg·L−1 NAA, 20% coconut water (CW), 3% sucrose, 0.7% agar, and 0.1% activated charcoal (pH 5.8). At 0 d culture, a seed with intact testa (Figure 4a)—a single globular embryo that lacks endosperm and is tightly enclosed by a thin-walled seed coat—was observed (Figure 4b). And then, the pregermination was the imbibed seed and embryo enlargement at 21 d culture (Figure 4c). The cell number increased significantly, and starch grain continued to accumulate (Figure 4d). Mature protocorm formatted with size elongation, rhizoids emergency, and seed coat disappeared at 60 d culture (Figure 4e), and protocorm cells accumulated abundant starch grains (Figure 4f). Subsequently, protocorms obtained were transferred to 1/4 MS medium which contained 1.0 mg·L−1 NAA and 1.0 mg·L−1 6-BA, 200 g·L−1 potato homogenate, 3% sucrose, 0.7% agar, and 0.1% AC (pH 5.8). During 10 days of proliferation, mature protocorm elongate gradually increased in size (Figure 4g), and the development of vascular system (VS) emerged (Figure 4h). Lots of green short buds differentiated from protocorm after 40 d culture (Figure 4i), the shoot apical meristem (SAM) emerged, and vascular system development were recorded with typical characteristics: dense cytoplasm, small size, and sparse starch grains (Figure 4j).

3.4. Morpho-Anatomical Changes from Protocorm to Seedling

Protocorm differentiation was observed across 60 days culture. Morpho-anatomical changes from protocorm to seedling of C. faberi were documented in detail. Protocorms were subcultured for differentiation culture on 1/4 MS medium composed of 4.0 mg·L−1 NAA, 1.0 mg·L−1 6-BA, 20% CW, 3% sucrose, 0.7% agar, 50 mg·L−1 myo-inositol, 2.0 mg·L−1 glycine, and 2.0 mg·L−1 nicotinic acid (pH 5.8). At 10 d culture, the white shoots emerged from protocorm (Figure 5a), and shoot apical meristem (SAM) and early leaf primordia emerged, gradually maturing into a typical dome-shaped structure, where the thin-walled cells containing an obvious nucleus push up against one another and the cell staining was intense (Figure 5b). The shoot turned from white to green at 20 d culture (Figure 5c), and cells in the SAM region exhibited a spindle-shaped morphology (Figure 5d). The first true leaf emerges from the shoot at 30 d culture (Figure 5e), and starch granules are directionally translocated from protocorm storage tissues to emerging leaves (Figure 5f). Finally, full leaf expansion and entry into the maturation stage occurs at 60 d culture (Figure 5g), and lots of starch accumulation in the new leaves was observed. The endogenous basal meristem initiates outward protrusion (Figure 5h). Then, seedlings were transferred to culture on 1/4 MS supplemented with 0.1 mg·L−1 NAA, 1.0 mg·L−1 6-BA, 0.5 mg·L−1 IBA, 20% CW, 3% sucrose, 0.7% agar, 50 mg·L−1 myo-inositol, 2.0 mg·L−1 glycine, and 2.0 mg·L−1 nicotinic acid (pH 5.8). After 60 d of robust culture, this study successfully obtained healthy autotrophic seedlings with fully expanded leaves and intact root systems (Figure 5i).

3.5. Transplantation and Acclimatization of Seedling

A total of 100 seedlings selected from the optimal germination culture medium were used in the transplantation experiment. The seedlings of C. faberi obtained were transplanted into terracotta pots filled with mixed coir, volcanic rock, shattered pine bark and peat moss (1:1:1:3 v/v/v/v) under a greenhouse. After 90 days of acclimatization (Figure 6a), the survival rate of seedling, the average height of seedling, and the number of leaves were recorded (Table 3). The average height of seedling increased to 7.84 ± 0.32 cm and the number of leaves increased to 4.91 ± 0.15, and the survival rate of seedlings was 80 ± 0.32%. The velamen radicum emerged from the outer surface of the root and base of the shoots after 30 days since transplantation (Figure 6b), which suggested that the new root began to develop after transplantation. In addition, the root diameter and seedling height were significantly increased after 90 days acclimatization (Figure 6c). In our study, about 80 ± 0.32% of seedlings were successfully acclimatized to greenhouse conditions and can be used for ornamental and conservation purposes.

4. Discussion

In terrestrial orchids, successful seed germination largely depends on compatible mycorrhizal fungi in nature conditions. Asymbiotic germination shows high germination seed percentage and is more practical for mass in vitro propagation of orchids than symbiotic germination, which needs to balance germination and pathogenesis [20]. However, an efficient in vitro propagation system through asymbiotic seed germination of terrestrial orchids is subjected to several limitations, such as the timing of seed collection [17,23], seed pretreatments [23,29,31], light conditions [24,34], composition of culture medium [24,33], etc.
Previous studies suggested that seed germination and subsequent protocorm development of terrestrial orchids are significantly influenced by capsule maturity or timing of seed collection. The seed germination frequency declined with the progress of seed maturity on the mother plant of C. falcate, and the highest frequency (39.8%) of seed germination was obtained with seeds collected 70 d after pollination [17], which suggested that immature seeds are more suitable for asymbiotic germination than mature seeds of C. falcate. A similar result was also observed in P. armeniacum, where the highest germination percentage (96.2%) was obtained with immature seeds harvested 95 d after pollination and was significantly higher than all other collection periods [23]. In C. subtropicum, the germination percentage was the highest (31.21%) for immature seeds collected at 105 d after pollination, but the germination percentage progressively decreased for seeds collected at 90 d or earlier, and 120 d or later, respectively [32]. Similar results were also observed in our study: the highest frequency (85.0 ± 0.79%) of protocorm formation was obtained with mature seeds harvested 120 d after cross-fertilization. Moreover, the germination rate significantly decreased for seeds collected at 150 or later.
Pretreatment of sodium hypochlorite (NaClO) solutions may erode the testa and disrupt cell wall integrity, thus increasing the permeability of the seed to oxygen and nutrients [23]. Soaking mature seeds in 4% sodium hypochlorite solution from 75 to 90 min significantly increased germination of V. planifolia [29]. Germination and protocorm development of P. armeniacum could also be stimulated by suitable pretreatment with NaClO, and the highest seed germination percentage were obtained with mature seeds (180 d after pollination) in 1.0% NaClO for 90 min [23]. Interesting, Spiranthes seeds germination occurred when seeds were scarified for 3 min in 10% NaClO, while embryos of seeds from the 10 min treatment were all damaged and/or dead upon visual inspection [31]. Moreover, a 10 min treatment with 1% NaClO proved to be the appropriate treatment to promote the germination of C. guttatum [33]. In present study, the seed germination rate of C. faberi increased obviously with 1.0% NaClO treatment for 30 min. Generally, continuous darkness could promote seed germination of terrestrial orchid C. guttatum [33] and C. crispa [24]. Under light treatment conditions, germination did not occur in C. guttatum [33]. In the present study, dark treatment for 30 days and subsequent 14 h light/10 h dark photoperiod treatment for 30 days could enhance the seed germination of C. faberi.
The composition, type, concentration, and ratio of macronutrients and micronutrients in the culture medium are critical factors that influence seed germination and seedling development of terrestrial orchids. In general, terrestrial orchids only require a low concentration of nutrients or more diluted media [20]. For E. bicallosa, protocorm survival was highest (100.0%) on 1/2 MS medium and shoot formation was the most (93.3%) pronounced on 1/2 MS, which was significantly higher than on all other media [34]. Most Paphiopedilum species prefer a low-salt medium for seed germination and the most appropriate medium for seed germination was 1/8 MS (94.3%) or 1/4 MS (96.2%), but 1/8 MS was more suitable for subsequent protocorm development [23]. Similarly, the most appropriate medium for seed germination of C. faberi was 1/4 MS (85%). Interestingly, seed germination of C. faberi was also observed on culture media both with high- and low-salt media. Growth regulators are essential to induce orchid germination, and the response of orchid seeds to exogenous hormones varies from genus to genus and even from one species to another [41]. For E. bicallosa, 1.0 mg·L−1 BA was the most effective for promoting shoot formation and increasing the number of shoots and leaves, and a moderate concentration of 0.5 mg·L−1 IBA was particularly effective in maintaining high survival rates while supporting overall plantlet growth [34].
Previous study suggested that treatment with 0.5 mg·L−1 NAA resulted in the longest average root length (2.06 cm) and the highest average number of roots (3.22 roots) of Paphiopedilum [42]. However, higher NAA concentrations (1.0–2.0 mg·L−1) did not enhance rooting rates or root numbers. The application of 1 mg·L−1 NAA also proved to promote germination, protocorm formation, and the development of C. guttatum [33]. In C. goeringii, 9.0 mg·L−1 NAA in combination with 9.0 mg·L−1 6-BA and 3.0 mg·L−1 IBA achieved a rhizome proliferation rate of 35.17%, but high 6-BA concentrations (10 mg·L−1) in combination with lower NAA concentrations (0.1 mg·L−1) achieved a 100% differentiation (3.93 buds per explant) [43]. In C. faberi, the present study shows that 1 mg·L−1 NAA concentrations in combination with 1 mg·L−1 6BA were suitable for protocorm proliferation, but high NAA concentrations (4 mg·L−1) in combination with 1 mg·L−1 6BA were the most effective for protocorm differentiation. In addition, 0.1 mg·L−1 NAA in combination with 1 mg·L−1 6BA and 0.5 mg·L−1 IBA achieved the highest root induction rate. These findings suggested that the culture media are tailored to the species-specific requirements in order to achieve successful in vitro propagation of terrestrial orchids.
The superior germination efficacy of seeds harvested at 120 DAP aligns with the well-documented phenomenon that immature orchid seeds possess a higher germination potential [44]. This can be largely attributed to their physiological state at this developmental window: the endogenous abscisic acid (ABA) level, a key germination inhibitor, remains low, and the physical barriers such as a heavily lignified seed coat and carapace are not fully developed [45]. In our study, the decrease in the germination rate of seeds collected after 120 DAP may be associated with the increase in abscisic acid (ABA) in seeds. Our histological observations revealed a distinct apical–basal polarity within the protocorms, a phenomenon that is not random but rather the manifestation of the pre-patterned protocorm body plan established during embryogeny [46,47]. This structural gradient is indicative of distinct cell fates: the small, densely cytoplasmic apical cells are destined for meristem formation, while the enlarged basal cells are speculated to facilitate nutrient uptake [48]. The de novo formation of the shoot apical meristem (SAM), the pivotal event transitioning a protocorm into a true plantlet, was successfully captured in our sections. The initiation was marked by cytological landmarks—including cells with a high nucleus-to-cytoplasm ratio and a distinctive angular shape—that are consistent with classical descriptions of meristem formation in orchids [49]. The subsequent emergence of the first leaf primordium, creating a characteristic ‘dimple-shaped’ apex, further confirms the establishment of a functional SAM, without which further development is impossible [36]. However, the molecular genetic mechanisms underpinning SAM initiation in C. faberi remain elusive, presenting an intriguing avenue for future research. In conclusion, by systematically optimizing culture conditions, we have established a robust protocol for the mass propagation of the threatened C. faberi. More importantly, leveraging this reliable system, we have provided the first comprehensive histological account of its post-germinative development, delineating the critical stages from a polarized protocorm to a seedling with a functional SAM. This work not only addresses conservation needs but also furnishes a fundamental developmental framework for future physiological and molecular studies on Cymbidium and related orchids.

5. Conclusions

Cymbidium faberi is a very popular potted orchid and has been cultivated for centuries in East Asia. In the wild, C. faberi is threatened by deforestation, illegal poaching, and commercial overexploitation, primarily due to their ornamental and economic value. Moreover, C. faberi is propagated through tillers and symbiotic seed germination with the correct fungus under nature conditions, which cannot meet conservation and commercial needs. Here, an efficient procedure for asymbiotic seed germination and in vitro seedling development of C. faberi was successfully established through evaluation of time of seed collecting, seed pretreatments, light conditions, composition of culture media. The highest germination rate (85.0 ± 0.79%) was achieved when seed collected at 120 d after cross-fertilization, and then germination percentages progressively decreased. Seed pretreatment with 1% NaClO for 30 min, dark culture could obviously enhance the seed germination of C. faberi. In addition, histological analyses from protocorm formation to seedling growth were also explored. Acclimatization in pots for 90 days resulted in 80 ± 0.32% survival, the average height of seedling increased to 7.84 ± 0.32 cm, and the number of leaves increased to 4.91 ± 0.15. Here, we provide key morphological traits from protocorm formation to seedling growth. In addition, our work also establishes an effective in vitro propagation system for the large-scale propagation of C. faberi to meet the commercial needs and conservation of this threatened orchid species.

Author Contributions

Writing—original draft preparation, Z.Z.; methodology, Z.Z., S.H., and H.H.; writing—review and editing, supervision, Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Horticulturae 11 01491 g001
Figure 1. (a) Cymbidium faberi Rolfe ‘Zheng Xiaohe’; (b) flower of ‘Zheng Xiaohe’; (c) capsule. Scale bars: (a–c) 2 cm.
Figure 1. (a) Cymbidium faberi Rolfe ‘Zheng Xiaohe’; (b) flower of ‘Zheng Xiaohe’; (c) capsule. Scale bars: (a–c) 2 cm.
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Figure 2. Effects of different pretreatments, media, and time of seed collecting on seed germination rate of C. faberi. (a) The seed germination rate treated by different pretreatments, CK (control); (b) the seed germination rate on different media (MS, 1/2 MS, 1/4MS, 1/6 MS and VW) with T3 pretreatment; (c) the seed germination rate at different time of seed collecting. In panel (b,c), lowercase letters (a, b, c, d and e) on the bars denote statistically distinct groups. The asterisks on the bars denote the significant difference between CK and treatment group. One asterisk (*) represents a significance level of 0.05. Three asterisks (***) represents a significance level of 0.001.
Figure 2. Effects of different pretreatments, media, and time of seed collecting on seed germination rate of C. faberi. (a) The seed germination rate treated by different pretreatments, CK (control); (b) the seed germination rate on different media (MS, 1/2 MS, 1/4MS, 1/6 MS and VW) with T3 pretreatment; (c) the seed germination rate at different time of seed collecting. In panel (b,c), lowercase letters (a, b, c, d and e) on the bars denote statistically distinct groups. The asterisks on the bars denote the significant difference between CK and treatment group. One asterisk (*) represents a significance level of 0.05. Three asterisks (***) represents a significance level of 0.001.
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Figure 3. The embryo development of C. faberi seeds. (a) 120 d after pollination; (b) 150 d after pollination; (c) 180 d after pollination (tr); (d) 210 d after pollination. Seed coat (SC). Scale bars: (a–d) 50 μm. Gap width was measured using CsaeViewer software version 2.3 (n = 5 specimens per developmental stage).
Figure 3. The embryo development of C. faberi seeds. (a) 120 d after pollination; (b) 150 d after pollination; (c) 180 d after pollination (tr); (d) 210 d after pollination. Seed coat (SC). Scale bars: (a–d) 50 μm. Gap width was measured using CsaeViewer software version 2.3 (n = 5 specimens per developmental stage).
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Figure 4. Morpho-anatomical changes from seed to protocorm in C. faberi. (a,b) No germination seed; (c,d) enlarged seeds with testa ruptured; (e,f) mature protocorm with seed coat disappeared; (g,h) protocorm elongation with vascular strands (VS) emergence; (i,j) lots of protocorm produced with vascular strands and shoot apical meristem (SAM). Scale bar: (a) 50 um; (b) 100 um; (c,d,h,j) 250 um; (e,f,g) 1 mm; (i) 2 mm.
Figure 4. Morpho-anatomical changes from seed to protocorm in C. faberi. (a,b) No germination seed; (c,d) enlarged seeds with testa ruptured; (e,f) mature protocorm with seed coat disappeared; (g,h) protocorm elongation with vascular strands (VS) emergence; (i,j) lots of protocorm produced with vascular strands and shoot apical meristem (SAM). Scale bar: (a) 50 um; (b) 100 um; (c,d,h,j) 250 um; (e,f,g) 1 mm; (i) 2 mm.
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Figure 5. Morpho-anatomical changes from protocorm to seedling in C. faberi. (a–d) morphological changes from protocorm to seedling; (e–h) histological changes from protocorm to seedling; (i) complete seedlings after robust culture. leaf (L), shoot apical meristem (SAM). Scale bars: (a,b) 1 mm; (e–h) 200 um; (c,d,i) 1 cm.
Figure 5. Morpho-anatomical changes from protocorm to seedling in C. faberi. (a–d) morphological changes from protocorm to seedling; (e–h) histological changes from protocorm to seedling; (i) complete seedlings after robust culture. leaf (L), shoot apical meristem (SAM). Scale bars: (a,b) 1 mm; (e–h) 200 um; (c,d,i) 1 cm.
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Figure 6. Acclimatization of C. faberi seedlings. (a) Seedlings in terracotta pot after 90 days of acclimatization; (b) the velamen radicum (vr) emerged from root and base of shoots after 30 days of acclimatization; (c) the diameter of roots (r) and height of seedling were increased after 90 days of acclimatization. Scale bars: (a–c) 3 cm.
Figure 6. Acclimatization of C. faberi seedlings. (a) Seedlings in terracotta pot after 90 days of acclimatization; (b) the velamen radicum (vr) emerged from root and base of shoots after 30 days of acclimatization; (c) the diameter of roots (r) and height of seedling were increased after 90 days of acclimatization. Scale bars: (a–c) 3 cm.
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Table 1. Different treatments on C. faberi seed germination.
Table 1. Different treatments on C. faberi seed germination.
TreatmentDescription
CKincubation under 14 h light/10 h dark photoperiod at 25 °C for 60 days
T1surface sterilization with 1% NaClO for 30 min + incubation under 14 h light/10 h dark photoperiod at 25 °C for 60 days
T2dark incubation for 30 d + incubation under 14 h light/10 h dark photoperiod at 25 °C for 30 days
T3surface sterilization 1% NaClO for 30 min + dark incubation for 30 d + incubation under 14 h light/10 h dark photoperiod at 25 °C for 30 days
Table 2. Nutrient component of the germination media used for the asymbiotic seed germination of C. faberi.
Table 2. Nutrient component of the germination media used for the asymbiotic seed germination of C. faberi.
Nutrient ComponentMS1/2 MS1/4 MS1/6 MSVW
Macronutrient (mg·L−1)
Ammonium Nitrate1650825412.5275
Ammonium Sulfate 500
Calcium Chloride440166.111055.37
Tricalcium Phosphate 200
KH2PO41708542.528.3250
Magnesium Sulfate180.790.3540.1830.12
Potassium Nitrate1900950475316.7525
Micronutrients (mg·L−1)
Boric Acid6.26.26.26.2
Copper Sulfate0.0250.0250.0250.025
Cobalt Chloride0.0250.0250.0250.025
Manganese Sulfate16.916.916.916.95.68
Potassium Iodide0.830.830.830.83
Sodium Molybdate0.250.250.250.25
Zinc Sulfate8.68.68.69.6
Iron source (mg·L−1)
Ferrous Sulfate000027.8
Na2EDTA36.736.736.736.737.26
Organics (mg·L−1)
Glycine2222
Myo-Inositol100100100100
Nicotinic Acid0.50.50.50.5
Pyridoxine HCl0.50.50.50.5
Thiamine HCl0.10.10.10.10.4
Table 3. The survival rate, average height of shoot, and average number of leaves of C. faberi seedlings after 90 days of acclimatization.
Table 3. The survival rate, average height of shoot, and average number of leaves of C. faberi seedlings after 90 days of acclimatization.
Acclimatization TimeSurvival Rate (%)Average Height of Shoot (cm)Average Number of Leaves
0 d100 a5.15 ± 0.17 c3.33 ± 0.14 c
30 d89.58 ± 1.44 b5.41 ± 0.17 c3.5 ± 0.15 c
60 d86.25 ± 1.52 b6.18 ± 0.19 b4.17 ± 0.17 b
90 d80 ± 1.23 c7.84 ± 0.32 a4.91 ± 0.15 a
Note: Values represent means ± S.E. Different lowercase letters show significant difference (p < 0.05 by Duncan’s multiple range test).
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Zhou, Z.; Han, S.; Huang, H.; Liu, Z. Seed Asymbiotic Germination Morphological Traits and Seedling Development in Cymbidium faberi Rolfe (Orchidaceae). Horticulturae 2025, 11, 1491. https://doi.org/10.3390/horticulturae11121491

AMA Style

Zhou Z, Han S, Huang H, Liu Z. Seed Asymbiotic Germination Morphological Traits and Seedling Development in Cymbidium faberi Rolfe (Orchidaceae). Horticulturae. 2025; 11(12):1491. https://doi.org/10.3390/horticulturae11121491

Chicago/Turabian Style

Zhou, Zhiqing, Siyu Han, Hao Huang, and Zhixiong Liu. 2025. "Seed Asymbiotic Germination Morphological Traits and Seedling Development in Cymbidium faberi Rolfe (Orchidaceae)" Horticulturae 11, no. 12: 1491. https://doi.org/10.3390/horticulturae11121491

APA Style

Zhou, Z., Han, S., Huang, H., & Liu, Z. (2025). Seed Asymbiotic Germination Morphological Traits and Seedling Development in Cymbidium faberi Rolfe (Orchidaceae). Horticulturae, 11(12), 1491. https://doi.org/10.3390/horticulturae11121491

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