1. Introduction
Cremastra appendiculata var.
variabilis (Blume) I.D. Lund, commonly known as single-leaf or warty lip cremastra, is a terrestrial orchid belonging to the genus
Cremastra that possesses pseudobulbs. Single-leaf cremastra (SLC) is distributed in central and southern China, Korea, southern Sakhalin and Kuril Islands, Japan, Thailand, and northern Vietnam. It occurs in mountain streams, hillsides, and forests [
1]. SLC has variegated leaves and beautiful fragrant flowers [
2] and can be used as an ornamental pot and garden plant. It is one of the rare, threatened species in Korea, and the natural population of SLC has decreased recently due to deforestation, environmental condition (climate change), urbanization, and illegal collection [
3]. The conventional methods (sexual and asexual) used in the propagation of SLC are inefficient due to the low seed germination rate and the shortage of pseudobulbs (tubers). Thus, alternative propagation methods are required for its conservation and commercial production.
In vitro propagation methods have been used to multiply endangered, rare, threatened, and commercial plants [
4,
5]. There are several reports detailing the in vitro propagation of
Cremastra appendiculata [
6,
7,
8]. Zhang et al. [
8] studied the impact of endophyte extract and plant growth regulators (PGR) on protocorm initiation using apical buds. The protocorms of
C. appendiculata obtained on Murashige and Skoog [
9] (MS) medium containing 10.0 mg/L endophyte extract, 2.0 mg/L 6-Benzyle adenine (BA) and 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) were sprouted and rooted best on half-strength MS medium containing 10.0 mg/L endophyte extract, and 0.5 mg/L of each α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA). Gao et al. [
7] tested the influence of symbiotic fungus on in vitro seed germination of
C. appendiculata. Mycorrhizal fungus (
Coprinellus disseminatus) promoted higher germination (71.61%) of
C. appendiculata seeds as compared with asymbiotic germination (9.68%). On the other hand, Yang et al. [
8] obtained a high frequency (76.42%) of asymbiotic germination when the
C. appendiculata seeds were cultured in MS medium with 1.0 mg/L NAA and 75 g/L potato extract. However, there are no reported protocols for the propagation of SLC (
Cremastra appendiculata var.
variabilis) using in vitro culture techniques.
Asymbiotic in vitro-seed germination method has been extensively used for the mass production of terrestrial orchids such as
Bletilla striata [
10],
Cyrtopodium paludicolum [
11], and
Spathoglottis plicata [
12]. Numerous orchid species, including
C. appendiculata (D. Don). Makino [
7,
8] has also been multiplied by symbiotic or asymbiotic in vitro-seed germination [
13,
14,
15,
16]. Thus, asymbiotic germination of SLC seeds in vitro permits higher multiplication rates than conventional propagation methods. Indeed, the rate of asymbiotic seed germination in orchids is associated with the age of the seeds [
17], culture media [
18], activated charcoal (AC) [
19], organic extracts [
20], and PGR [
21,
22]. In this study, we described an in vitro propagation protocol of SLC. We investigated the effect of seed age, culture media, AC, and PGR on asymbiotic seed germination, protocorm multiplication (secondary protocorm formation), and SLC development. The optimized method described here will be helpful in the large-scale propagation of SLC.
2. Materials and Methods
2.1. Plant Materials and Surface Sterilization
Seed capsules collected from hand-pollinated greenhouse-grown SLC plants at three distinct ages (132, 159, and 210 days) were thoroughly washed in running tap water and then rinsed with distilled water (DH2O). After air drying, capsules of SLC were sterilized in ethanol (EtOH; 70%) for 2.5 min, sodium hypochlorite (NaClO; 3.0%) for 16 min, and mercuric chloride (HgCl2; 0.2%) for 12 min. After being treated with EtOH, NaClO, and HgCl2, the capsules of SLC were washed with autoclaved DH2O 2, 4, and 3 times respectively.
2.2. In Vitro Seed Germination
Seeds extracted from the surface-sterilized SLC capsules were placed on Murashige and Skoog (MS) [
9], half-MS, Knudson C [
23] (KC), and Hyponex [(N-P-K, 6.5-6-19) 3 g/L (Hyponex Japan Corp. Ltd., Osaka, Japan) with peptone (2 g/L), banana (30 g/L), and apple (20 g/L)] media contained sucrose (30 g/L) and plant agar (8 g/L) to examine the effect of media on seed germination. To study the usefulness of activated charcoal (AC) in improving asymbiotic germination, the seeds of SLC were placed on a culture medium (MS contained 30 g/L sucrose and 8 g/L plant agar) with 0–2000 mg/L AC. To examine the effectiveness of PGRs in improving in vitro germination, the seeds of SLC were placed on a culture medium with 500 mg/L AC and 0–10 µM indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or α-naphthaleneacetic acid (NAA) and then supplemented with 0–8 µM kinetin (KIN) and 3.0 µM NAA. Before autoclaving at 123 °C for 23 min, the pH of all seed germination medium was adjusted to 5.7–5.8 using potassium hydroxide (0.1 N) or hydrochloric acid (0.1 N). The SLC seed containers (500 mL bottles) were maintained for 90 days at 23 ± 2 °C under 5 µmol m
–2 s
–1 for 12 h with white light emitting diodes (WLED) light. Seeds of SLC randomly removed from each culture bottle were fixed and observed under a light microscope. For each treatment, ten 500 mL culture bottles (more than 1500 seeds placed in each bottle) were used, and the experiment was repeated twice. The seed germination percentage was calculated after 90 days as the number of germinated seeds/total number of seeds × 100 [
24].
2.3. Secondary Protocorm Formation and Conversion
The protocorms developed from SLC seeds were placed on a culture medium containing 2, 4, or 8 µM 6-benzyladenine (BA) combined with 1, 2, or 4 µM KIN and maintained for 60 days at 23 ± 2 °C under 30 µmol m–2 s–1 for 16 h with WLED light. For each treatment, fifteen 500 mL culture bottles (5 protocorms placed in each bottle) were used, and the experiment was repeated twice. The number of secondary SLC protocorms and their conversion rate were recorded after 60 days.
2.4. Protocorm Conversion and SLC Development
For conversion and SLC development, the protocorms were placed on a culture medium containing 0–3 µM gibberellic acid (GA3) and maintained under 10 µmol m–2 s–1 for 12 h with WLED light for 45 days and then placed under 45 µmol m–2 s–1 for 16 h with WLED light for 45 days at 23 ± 2°C. Five protocorms were placed in each of the fifteen 500 mL culture bottles for each treatment, and the experiment was repeated twice. The protocorm conversion rate was recorded after 90 days.
2.5. Statistical Analysis
Analysis of variance (ANOVA) of the collected data was done using the SAS (SAS Institute, NC, USA) statistical software release 9.4. The averages were separated by Duncan’s multiple range test (DMRT) at the 5% level of significance.
4. Discussion
Surface sterilization of plant materials is a crucial step in plant tissue culture [
25]. Asymbiotic germination of orchid seed in vitro was commonly impeded by contamination of the culture medium. In this study, a method for surface sterilization of SLC seeds in order to accomplish asymbiotic germination in vitro. Frequently, the composition of asymbiotic culture medium affects the germination of terrestrial orchid seeds. In this study, the MS medium produced the highest germination rate overall. On the other hand, the germination rate of SLC seeds that were grown on half-strength MS and KC media was similar (
Table 1). The MS medium can promote SLC seed germination in vitro due to its high nutrition content. Reducing the nutrients in the MS medium had a negative effect on SLC seed germination. This result indicates that SLC seeds require a high concentration of mineral nutrients in order to germinate in vitro. The total mineral nutrient concentration of MS, half-strength MS and KC media were 96.02 (mM), 48.01 (mM), and 46.72 (mM), respectively [
26]. The seed germination of various terrestrial orchids such as
C. appendiculata [
8,
27],
Paphiopedilum bellatulum,
Paphiopedilum godefroyae,
Paphiopedilum helenae,
Paphiopedilum henryanum,
Paphiopedilum niveum,
Paphiopedilum spicerianum [
28],
Pecteilis radiata [
22], and
Phaius tancarvilleae [
29] was attained best using MS medium. The age of the seed also affects the asymbiotic germination rate of orchids [
17,
30]. However, the effect of seed age on non-symbiotic
Cremastra germination has not been reported. In this study, 159-day-old SLC seeds showed higher germination frequency than 132-day-old and 210-day-old seeds. The result indicates that seed age is critical in enhancing the germination rate in SLC. The germination rate in 210-day-old SLC seeds significantly decreased (
Table 1). Several factors may contribute to the low germinability of mature seeds of various orchids, such as the presence of chemical inhibitors [
31], impermeable testa [
30], and lack of germination-promoting phytohormones [
32].
In some orchid species, the addition of AC to the seed-cultivation medium enhanced the germination rate [
19,
20,
22,
24]. The germination rate of SLC seedlings was increased by 500 mg/L of AC added to the medium (
Table 2). It has been reported that AC absorbs chemical inhibitors and nutrients from the growth media [
33], thereby improving SLC seeds’ germinability in vitro. Besides culture media, seed age, and AC, another vital factor, PGR, has also shown a significant effect on SLC seed germination (
Table 3 and
Table 4). In asymbiotic in vitro propagation, various PGRs have often been used to increase the seed germination rate of several orchids [
14,
21,
22,
32], including
C. appendiculata [
8,
27]. Yang et al. [
8] examined the effects of different levels of auxins (IAA, IBA, and NAA) in MS medium on the germination of
C. appendiculata seeds and found that the greatest rate (76.42%) of asymbiotic germination with 5.4 µM of NAA. Similarly, NAA at 3.0 µM yielded the best (73.4%) germination rate in SLC. However, a combined treatment of KIN (2 µM) and NAA (3 µM) was most effective at enhancing (91.9%) germination of SLC seed. In contrast, Gao et al. [
7] noticed a low germination rate (9.68%) when the
C. appendiculata seeds were cultured in MS medium with 1.0 mol/L NAA and 1.0 mol/L BA.
Protocorms obtained from the orchid seeds are often used as explants for the mass production of seedlings. The multiplication and conversion of protocorms are often influenced by PGR [
32]. This study observed protocorm multiplication only on cytokinin-containing MS medium. Similarly, cytokinin has been used to achieve protocorm multiplication in
Cymbidium aloifolium [
34],
Gastrochilus matsuran [
24],
Gastrochilus japonicus [
35], and
Gastrodia pubilabiata [
36]. Yang et al. [
8] examined the effects of BA, IBA, and NAA on PLB multiplication in
C. appendiculata. They found the best protocorm proliferation rate (170.07%) with a half-MS medium containing 1 mg/L each of BA and NAA and 0.2 g/L AC. In this study, the multiplication of SLC protocorm was achieved best on MS medium with 4 µM BA and 2 µM KIN, but the conversion of protocorm is low. Protocorms, on the other hand, were best converted to shoots on half-MS with 2.0 mg/L thidiazuron and 0.2 mg/L NAA. The requirement of PGR and their concentrations for the multiplication and conversion of protocorm can vary with orchid species [
8,
14,
17,
22].
Auxins, cytokinins, GA
3, and chemical additives have been found to convert protocorms into seedlings [
10,
14,
22,
24,
32,
35]. In this study, SLC-protocorm converted best (78.7%) when the medium contained 1.0 µM GA
3. GA
3 has been shown to promote in other orchids, such as
Cattleya tigrine [
37] and
Thrixspermum japonicum [
38]. However, when significant levels of GA
3 are added to the medium, the conversion rate decreases. High GA
3 levels have also been shown to have adverse effects on protocorm conversion in
C. tigrine [
37] and
T. japonicum [
38].
5. Conclusions
In this study, we investigated the effects of seed age, culture medium, AC, and PGR on the asymbiotic seed germination of SLC, a rare, threatened Korean ornamental plant. We found that seed age and culture medium had a significant effect on SLC seed germination. The optimal seed age and culture media for asymbiotic germination were 159-days and MS medium, respectively. The addition of AC and PGR in the culture medium increased the rate of SLC seed germination. The in vitro-derived protocorms multiplied best in cytokinin-containing media. Protocorm conversion, seedlings development, and pseudobulb were observed when the secondary protocorms were grown in the presence of 1.0 µM GA3. Acclimatization and reintroduction of SLC seedlings to their natural environment will be the subject of future research.