Asymbiotic Germination and Leaf Explant-Based Regeneration of the Endangered Medicinal Orchid Hemipilia cucullata from Mature Seeds

Summary

Here, we present a protocol to optimize asymbiotic seed germination, seedling development, and shoot elongation in Hemipilia cucullata. The protocol also enables the induction of protocorm-like bodies from sterile leaf explants, facilitating efficient in vitro propagation for the conservation and sustainable use of this endangered medicinal orchid.

Abstract

Hemipilia cucullata (Orchidaceae) is a terrestrial orchid widely valued for its medicinal properties and ornamental appeal, yet its wild populations are endangered due to habitat degradation, low seed viability, and limited pollination success. To support its conservation and sustainable utilization, this study developed an optimized protocol for the asymbiotic germination of mature seeds, protocorm development, shoot elongation, and protocorm-like body (PLB) induction from sterile leaf explants. Seeds cultured in 1/2 MS liquid medium supplemented with 0.5 mg/L NAA showed a germination rate of 72 ± 6%, significantly higher than the control (46%) and solid medium (31%), with germination initiating approximately 21 days earlier in liquid culture. Protocorms were subcultured on B5 medium containing 0.5 mg/L BA, 0.2 mg/L NAA, and 100 mL/L coconut water, leading to efficient proliferation. After 5-6 weeks, plantlets were transferred to shoot elongation medium (B5 supplemented with 0.5 mg/L BA and 1 mg/L NAA), where they exhibited robust shoot elongation and healthy leaf formation. For PLB induction, the highest induction rate (44.3 ± 5.1%) was achieved on MS medium containing BA (3 mg/L) and NAA (0.2 mg/L). This comprehensive and reproducible protocol can be an effective platform for the large-scale propagation and ex situ conservation of H. cucullata, offering valuable support for recovery efforts of this endangered orchid.

Introduction

Hemipilia cucullata (L.) Y.Tang, H.Peng & T.Yukawa, commonly known as cucullate neottianthe orchid, is a wild terrestrial orchid native to temperate Eurasia and is widely distributed from Poland to Japan¹. Initially classified in the genus Neottianthe, this species was recently reassigned to the genus Hemipilia, section Neottianthe, based on molecular evidence². The plant grows from underground tubers and reaches up to 30 cm in height, producing pinkish or purple-red flowers arranged in a one-sided spike. Due to the elegant shape and attractive coloration of its flowers, it possesses significant ornamental value. Additionally, the entire plant is recognized as a medicinal herb in multiple countries, where it is widely utilized for various therapeutic purposes, including the treatment of cardiac conditions, enhancement of blood circulation, relief of bruises, and promotion of fracture healing^3,4. Recent phytochemical studies have identified several anthocyanins in this species, such as cyanidin 3-oxalylglucoside, chrysanthemin, and cyanin, which possess antioxidant and vasoprotective properties and may contribute to its medicinal effects^5,6.

Currently, wild populations of H. cucullata face threats from habitat loss and environmental disturbances, leading to severe population declines and, in some areas, bringing the species close to extinction^3,7,8. However, conservation of this species is particularly challenging due to difficulties in its natural propagation. In the wild, H. cucullata reproduces both clonally and sexually, but both modes are inefficient. Natural clonal propagation is slow and infrequent, while sexual reproduction is limited by poor pollination success and low seed viability^9,10. Moreover, field investigations have revealed a markedly imbalanced age structure, with a scarcity of juvenile individuals and limited natural recruitment, especially under anthropogenic or pyrogenic stress conditions^7,8,11. The seeds of H. cucullata, like many orchids, are minute, lack endosperm, and require specific fungal partners for germination, further complicating natural regeneration. Consequently, despite its broad geographical distribution, the population size and age structure of wild populations are declining, and local extinction risks are increasing⁷. Therefore, the establishment of an efficient method for artificial rapid propagation is essential both for the conservation of germplasm resources and for the sustainable utilization of this orchid species.

Among the available propagation techniques, symbiotic germination -- relying on the association with orchid mycorrhizal fungi -- can closely simulate natural processes but is often difficult to standardize due to its dependency on species-specific fungal partners^12,13,14. In contrast, asymbiotic germination offers a simpler and more reproducible alternative that is widely used in orchid tissue culture systems¹⁵. Although asymbiotic germination and rapid propagation protocols have been developed for several medicinal orchids such as Bletilla, Dendrobium, and Cypripedium^16,17,18,19, species-specific optimization of culture media and tissue culture conditions for H. cucullata is still required.

In this study, we used mature seeds of H. cucullata obtained through manual cross-pollination of wild individuals (Figure 1A) at Mount Tianmu in eastern China (30°20′ N, 119°22′ E) to establish and optimize an effective protocol for asymbiotic seed germination, seedling development, and shoot elongation. Additionally, we developed a regeneration system from leaf explants via PLBs induction, thereby contributing to the conservation and cultivation of this endangered medicinal orchid.

Protocol

NOTE: Before starting the experiment, wear personal protective equipment, including gloves, a lab coat, and safety goggles. Perform all procedures under a laminar flow hood for sterility. Handle ethanol and sodium hypochlorite with caution and dispose of waste per laboratory safety regulations.

1. Capsule sterilization and seed extraction

1. Place the capsule in a beaker. Add 1-2 drops of detergent and rinse thoroughly with tap water to remove surface contaminants.
2. Wash the capsule under running water for 5 min. Absorb the surface water with sterile absorbent paper.
3. Transfer the capsule to a sterile workbench for further disinfection.
4. Immerse the capsule in 75% (v/v) ethanol for 30 s.
5. Sterilize the capsule by immersing it in 50 mL of 20% (v/v) sodium hypochlorite solution (diluted from a commercial bleach containing ~5% available chlorine), with the addition of 2 drops of undiluted household dishwashing detergent for 10-12 min.
6. Rinse the capsule five times with sterile distilled water to remove residual disinfectant.
7. Using a sterile scalpel, remove approximately 1 mm from both the apex and the base (pedicel end) of the capsule.
8. Make a small incision at the upper end of the capsule to expose the seeds. Place the capsule in a sterile Petri dish for seed extraction.

2. Culture medium preparation

1. Prepare 1/2 MS liquid medium: Use half the macronutrient concentrations of the standard MS medium, retain all other components as in the MS medium, add 20 g/L sucrose and α-naphthaleneacetic acid (NAA, 0.5 mg/L), and adjust the pH to 5.8.
2. After boiling, dispense the medium into 500 mL culture bottles immediately (the number of bottles can be adjusted according to the desired scale of propagation).
3. Place the dispensed medium in an autoclave and sterilize at 121 °C for 15 min. After sterilization, remove the medium from the autoclave and allow it to cool to room temperature before use.

3. Seed germination

1. Use sterile forceps to evenly disperse approximately 500-600 seeds into each bottle of medium (prepared in step 2.3). Allow seeds to absorb water and settle to the bottom of the liquid medium.
2. Maintain cultures at 25 °C with a 12 h light/dark cycle and a light intensity of 36 µmol·m^-2·s^-1.
3. Observe seed germination weekly, record morphological changes, and take photographs for documentation.
4. After approximately 12 weeks, when most seeds have germinated, count the number of germinated seeds and calculate the germination rate. Use a sterile inoculation loop to randomly collect one loopful of liquid medium containing seeds. Transfer the sampled seeds to a petri dish or glass slide and observe under a stereomicroscope.
5. Count the number of seeds that have formed protocorms as germinated. Calculate the germination rate using the following formula:
   Germination rate (%) = (Number of germinated seeds/Total number of seeds in sample) × 100.

4. Protocorm subculture and proliferation

1. Prepare the proliferation medium by supplementing B5 medium (3.21 g/L) with coconut water (100 mL/L), 6-benzylaminopurine (BA, 0.5 mg/L), and NAA (0.2 mg/L). Add 20 g/L sucrose and 7.3 g/L agar, and adjust the pH to 5.8.
2. After boiling, dispense the medium into 150 mL culture bottles immediately (the number of bottles can be adjusted according to the desired scale of propagation).
3. Place the dispensed medium in an autoclave and sterilize at 121 °C for 15 min. Allow the medium to cool to room temperature before use.
4. Aseptically transfer 10-15 germinated protocorms (0.3-1 cm in size) per bottle onto the culture medium prepared in step 4.3.
5. After a 5-week cultivation period, assess the proliferation status and record the number of newly formed protocorms per bottle.
6. Continue culturing the proliferated protocorms for an additional 4-6 weeks to allow development into plantlets with visible shoot apex and rhizoid-like structures prior to transfer for shoot elongation.
   NOTE: Maintain cultures at 25 °C, with a 12 h light/dark cycle and a light intensity of 36 µmol·m^-2·s^-1.

5. Shoot elongation

1. Prepare the medium for shoot elongation by supplementing B5 medium (3.21 g/L) with BA (0.5 mg/L) and NAA (1 mg/L). Add 20 g/L sucrose and 7.3 g/L agar, and adjust the pH to 5.8.
2. After boiling, dispense the medium into 150 mL culture bottles (the number of bottles can be adjusted according to the desired scale of propagation).
3. Select healthy plantlets (1-2 cm in size, without obvious blackening or complete yellowing) for shoot elongation. Using sterilized and cooled forceps, aseptically transfer 8-12 plantlets into each 150 mL culture bottle containing fresh medium prepared in Step 5.1. Ensure that the plantlets are evenly distributed within each bottle.
4. After a 5-week shoot elongation period, select plantlets with fully developed shoots and healthy leaves for leaf explant sampling.
   NOTE: Maintain cultures at 25 °C, with a 12 h light/dark cycle and a light intensity of 36 µmol·m^-2·s^-1.

6. Induction of PLBs from leaf explants

1. Prepare the PLBs induction medium by supplementing MS basal medium (4.43 g/L) with BA (3 mg/L) and NAA (0.2 mg/L). Add 20 g/L sucrose and 7.3 g/L agar, and adjust the pH to 5.8.
2. After boiling, dispense the medium into 150 mL culture bottles (the number of bottles can be adjusted according to the desired scale of propagation).
3. Select healthy plantlets after 5 weeks of shoot elongation (step 5.4). Excise fully expanded sterile leaves, remove the apical region and margins, and cut each leaf into approximately 0.5 cm × 0.5 cm segments using a sterile scalpel under aseptic conditions.
4. Place the 10 leaf segments onto the PLB induction medium with the adaxial surface facing upward, ensuring firm contact with the medium surface.
5. After 5 weeks of culture, assess the number of successfully induced PLBs and calculate the induction rate (%). Additionally, record the average bud height (cm) and the number of buds formed per explant.
   NOTE: Maintain cultures at 25 °C, with a 12 h light/dark cycle and a light intensity of 36 µmol·m^-2·s^-1.

Results

The protocol steps presented in this study represent the optimized methodology developed through a series of comparative experiments aimed at improving asymbiotic seed germination, protocorm proliferation, shoot elongation, and PLB induction in H. cucullata. For example, in the selection of PGRs to promote seed germination, NAA enhances embryo development and water uptake, while BA promotes bud differentiation and cell division. Their synergistic effect has been shown to improve germination rates and early seedl...

Discussion

Orchid capsules typically contain numerous seeds, often exceeding ten thousand, but their natural germination is extremely limited due to the absence of endosperm and their high dependency on symbiotic fungal associations and specific environmental cues²⁰. Asymbiotic germination protocols have thus been widely adopted to bypass these limitations and enable efficient seedling propagation under controlled conditions. For instance, Chen et al¹⁵ reported a germination rate of 9...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1/2 MS Base Salts with vitamins	Coolaber	PM1061
6-Benzylaminopurine	Shanghai Macklin Biochemical Technology Co., Ltd.	1214-39-7
Agar Power	Beijing Chembase Technology Co., Ltd.	1400
Clean Bench	Suzhou Purification Equipemnt Co., Ltd.	SW-CJ-2G
Coconut Water	GOOD FARMER		https://e.tb.cn/h.6F8GTrHG7pif7Tj?tk=jFTyVPU3OBs
Detergent	Liby Science and Technology	6920174756784
Gamborg B5 Medium	PhytoTechnology Laboratories	G398
Glass Bottle	Morebetter Biotechnology Co., Ltd.	MBT-BL-240
Murashige & Skoog Basal Salts	PhytoTechnology Laboratories	M519
Pipette	Eppendorf	3123000268
Precision Balance	METTLER TOLEDO	30834109
SPSS	International Business Machines Corporation		Software used for statistical significance testing
Sucrose	Sinopharm chemical Reagent Co., Ltd.	20210802
Thidiazuron	Shanghai Macklin Biochemical Technology Co., Ltd.	51707-55-2
α-Naphthaleneacetic Acid	Shanghai Macklin Biochemical Technology Co., Ltd.	86-87-3