Abstract
The histological and histochemical changes in developing seeds of Cypripedium debile Rchb. f., a native slipper orchid species with horticultural potential, were investigated. The effects of timing for seed collection, culture media, and cultural conditions were also examined. The optimum germination percentage occurred when mature seeds were collected and sowed on 1/4 Murashige and Skoog basal medium. Besides, the liquid culture promoted germination of mature seeds. This finding is contrary to most other Cypripedium species, which are relatively easy to germinate with immature seeds. Moreover, two notable cytological changes of C. debile were observed. First, the suspensor cell protruded beyond the micropyle opening of the inner seedcoat, making the inner seedcoat not substantial. Second, Nile red staining indicated that the deposition of cuticular material on the seedcoat was fragmentary. It is proposed that the less hydrophobic nature of the seedcoat makes mature seeds of C. debile easier to obtain water and nutrients for germination.
The genus Cypripedium L. is one of the most fascinating groups of orchids, distributing in the temperate region or the high mountains of the Northern Hemisphere (Cribb, 1997). Species of this genus are of high value in ornamental markets because of their exotic flowers. Cypripedium debile Rchb. f. is a miniature species (≈10 to 12 cm tall), making it ideal as a compact potted plant. Only a few populations could be found in central China, Japan, and Taiwan (Cribb, 1997; Lin, 1987). Like many Cypripedium species, Cypripedium debile is threatened as a result of habitat fragmentation and overcollection. Therefore, it is desirable to establish protocols of seed germination for ex situ conservation and reintroduction programs.
Asymbiotic germination is a useful technique to propagate and conserve several endangered orchid species (Arditti and Ernst, 1993; Fay, 1992; Pritchard, 1989), whereas some terrestrial orchids are relatively difficult to germinate in vitro (Rasmussen, 1995). Seeds of terrestrial orchids usually have rigid seedcoats, enveloping the embryos tightly at seed maturity (Lee et al., 2005; Yamazaki and Miyoshi, 2006). As seeds approach maturity, some chemical compounds, e.g., polyphenols, cuticular materials, or lignin, may deposit on the seedcoat and contribute to the hydrophobic nature of mature seeds (Carlson, 1940; Lee et al., 2005; Yeung et al., 1996). These factors may bring about the impermeability of mature seeds and result in low seed germination percentage. Moreover, the accumulations of high levels of endogenous abscisic acid in the mature seeds also inhibit seed germination (Lee et al., 2007; Van der Kinderen, 1987; Van Waes and Debergh, 1986). Consequently, culturing immature seeds was frequently used to maximize germination the percentage of several terrestrial orchids (De Pauw and Remphrey, 1993; Light and MacConaill, 1990). Our preliminary experiments demonstrated that the near mature seeds of C. debile had better germination percentage than immature seeds. This is contrasting to the germination profiles of most Cypripedium species (De Pauw and Remphrey, 1993; St. Arnaud et al., 1992). Currently, little is known about the seed development and the characteristic of seeds in C. debile. Our previous works have demonstrated that the basic knowledge of seed development in several orchids enables a more efficient means about the asymbiotic germination (Lee et al., 2005, 2008). The aim of this study was to investigate the histological and histochemical changes during seed development of C. debile and their relations to the seed germination in vitro.
Material and Methods
Plant material.
Developing capsules of C. debile were collected from a natural population in the bamboo forest located at Hohuan Mountain, Nantou County, Taiwan. Anthesis usually occurred in early June each year. After capsule setting, developing capsules of different stages were collected at regular intervals for histological and asymbiotic seed germination studies.
Light microscopy.
Capsules of different developing stages were sliced and fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.05 M phosphate buffer, pH 6.8, for 24 h at 4 °C. After fixation, the sections were dehydrated in methyl cellosolve for 24 h followed by two changes of 100% ethanol for 24 h each at 4 °C. The samples were infiltrated gradually (3:1, 1:1, and 1:3 100% ethanol: Historesin, 24 h each) with Historesin (Leica Canada, Markham, Ontario, Canada) followed by two changes of pure Historesin. The tissues were then embedded according to Yeung (1999). Median longitudinal serial sections, 3 μm thick, were cut using a Ralph knife on a Reichert-Jung 2040 Autocut rotary microtome. Sections were stained with periodic acid-Schiff’s reaction for total carbohydrate and counterstained with either 0.05% (w/v) toluidine blue O (TBO) in benzoate buffer for general histology or 1% (w/v) amido black 10B in 7% acetic acid for protein. The accumulation of cuticular material was detected by Nile red staining as detailed in Yeung et al. (1996). The Historesin-embedded tissues were stained with 1 μg·mL−1 of Nile red (Sigma Chemical Co., St. Louis, MO) for 10 min, briefly washed in distilled water, and mounted in water containing 0.1% n-propyl gallate (Sigma Chemical Co.), an antifading compound. The fluorescence pattern was examined using an epifluorescence microscope (Axioskop 2; Carl Zeiss AG, Germany) equipped with the Zeiss filter set 15 (546/12 nm excitation filter and 590 emission barrier filter). The sections were viewed and the images were captured digitally using a CCD camera attached to a light microscope (Axioskop 2; Carl Zeiss AG).
Effect of seed collection timing and cultural medium on seed germination.
Capsules of various developing stages were collected for asymbiotic germination experiments. The capsules were taken to the laboratory and were surface-sterilized with a 1% sodium hypochlorite solution for 15 min and rinsed three times with sterile distilled water. After surface sterilization, the capsules were cut open, and then the seeds were scooped out with forceps onto the culture medium. To ensure the seed quality and developmental stages of each capsule, the remaining seeds of each capsule were fixed and examined under a microscope. Approximately 90% of seeds contained fully developed embryos (as confirmed by microscopy).
Two cultural media used in this experiment were the modified Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) and Thomale GD medium (Thomale, 1957). The modified MS medium contained 1/4 strength of macroelements with full strength of microelements, 2 mg·L−1 glycine, 0.5 mg·L−1 niacin, 0.5 mg·L−1 pyridoxine HCl, 0.1 mg·L−1 thiamine, and 100 mg·L−1 myo-inositol. Both the modified MS medium and Thomale GD medium were supplemented with 1 g·L−1 tryptone, 1 g·L−1 yeast extract, 20 g·L−1 sucrose (Sigma Chemical Co.), and 100 mL·L−1 coconut water. Fresh coconut water was taken from a green coconut and was added into the culture medium immediately. The pH value was adjusted to 5.6 before autoclaving. Twenty milliliters of medium were placed into each Erlenmeyer flask (125 mL). In this experiment, the liquid culture was used for seed germination. After sowing, the flasks were placed on an orbital shaker at 70 rpm and incubated in the dark at 25 ± 1 °C growth room.
Effect of liquid and solid cultures on seed germination.
For this experiment, mature seeds were collected and inoculated on liquid or solidified medium. The cultural medium used in this experiment was 1/4 MS medium as described previously. For solid culture, the medium was solidified with 7 g·L−1 agar (Sigma Chemical Co.). After autoclaving, 20 mL of medium was placed into each plastic plate (90 × 15 mm; Scientific Corp., Taiwan) in the laminar flow cabinet. After sowing, the plastic plates were sealed with parafilm and incubated in a growth room at 25 ± 1 °C in constant darkness.
Seedling growth medium for developing protocorms.
After 5 months of seed culture in vitro, the young protocorms were transferred to the seedling growth medium: 1/4 MS medium supplemented with 20 g·L−1 sucrose, 1 g·L−1 activated charcoal, 20 g·L−1 potato homogenate, and 7 g·L−1 agar for future growth as reported by Lee (2010). The pH value was adjusted to 5.6 before autoclaving. One hundred milliliters of medium was placed into a 500-mL culture flask. After transferring the young protocorms, the flasks were incubated in a growth room at 25 ± 1 °C in constant darkness.
Germination percentage calculation and data analysis.
Each plate or flask was examined at 1-month intervals for 5 months in culture by using a Carl Zeiss stereomicroscope (10× magnification). Germination was defined as emergence of the embryo from the testa. The germination percentage was calculated as the percentage of the number of seeds germinated among the total countered number of seeds with embryos. Experiments were performed in a randomized design. Twelve replicates were taken for each treatment in the experiments of seed germination in vitro. The data were statistically analyzed using analysis of variance followed by Fisher’s protected least significant difference test.
Results
Structural and histochemical study.
After fertilization (≈2 months after anthesis), the zygotes and proembryos could be observed within the capsules. The zygote was highly polarized with a large vacuole located toward the micropylar end (Fig. 1A). The cytoplasm at the zygote tip contained several tiny starch granules congregating around the nucleus. In the six-celled proembryo, the cells of embryo proper had dense cytoplasms, whereas the basal cell was highly vacuolated (Fig. 1B). By the early globular stage (≈3 months after anthesis), protoderm had formed (Fig. 1C). Numerous starch granules were found to accumulate in the cytoplasm of the developing embryo proper and the suspensor. In this species, the suspensor consisted of a single cell that was highly vacuolated. At this stage, the suspensor cell had protruded beyond the micropyle opening of the inner seedcoat and grew into the lumen enclosed by the outer seedcoat. As the embryo reached the globular stage (≈4 months after anthesis), mitotic activity had ceased and the suspensor cell began to degenerate (Fig. 1D). At this stage, the cytoplasm of embryo proper still contained numerous starch granules. At maturity (≈5 months after anthesis), the embryo was only eight cells long and five cells wide (Fig. 1E). The embryo cells had abundant storage products in the form of lipid and protein bodies in the cytoplasm, whereas the starch granules became absent as the embryo approached maturity.
The histological study of the embryo development of Cypripedium debile. (A) A median longitudinal section showing a zygote. The nucleus was located toward the chalazal end and a large vacuole occupied the micropylar end. The cytoplasm of zygote contained several tiny starch grains (arrow) surrounding the nucleus. At this stage, the outer seedcoat (OS) has not completely covered the embryo sac, and starch grains of various sizes could be found within cells of seedcoat. DS = degenerated synergid; IS = inner seedcoat. Scale bar = 20 μm. (B) A median longitudinal section showing a six-celled embryo. The cytoplasm of embryo proper contained several tiny starch grains (arrow), and the lower cell toward the micropylar end will differentiate into the suspensor (S). The degenerating endosperm (arrowhead) could be observed within the cavity of embryo sac. Scale bar = 20 μm. (C) Light micrograph showing a longitudinal section through an early globular embryo. The cytoplasm of embryo proper contained a number of starch grains (arrow), whereas the suspensor cell (S) was highly vacuolated. At this stage, the inner seedcoat (arrowheads) has gradually compressed into a thin layer, and the suspensor cell had protruded beyond the micropyle opening of the inner seedcoat and grew into the lumen enclosed by the outer seedcoat (OS). Scale bar = 40 μm. (D) Light micrograph showing a longitudinal section through a globular embryo. The size of starch grains (arrow) within the embryo proper became larger, whereas the size of suspensor (S) reduced and began to degenerate. Scale bar = 40 μm. (E) As the seed approached maturity, the suspensor (arrow) has just degenerated and left an empty space at the micropylar end. The inner seedcoat (arrowhead) has compressed into a thin layer covering the embryo proper except for the micropylar end. Scale bar = 40 μm. (F) Nile red staining fluorescence micrograph of an orchid seed at the stage similar to E. The surface wall (arrow) of the embryo proper, inner seedcoat (arrowhead), and outer seedcoat (double arrowhead) reacted positively. Note that the fluorescence of the degenerated suspensor at the micropylar end is absent. Scale bar = 40 μm. (G) Light micrograph showing a longitudinal section through a mature seed. Protein bodies (arrowhead) of various sizes can be found within the embryo proper. Although lipid is not preserved in this preparation, the translucent vesicles (arrow) in the cytoplasm of the embryo cell indicate the location of the lipid deposits. At maturity, the embryo is enveloped by the shriveled inner seedcoat (IS) and the outer seedcoat (OS). Scale bar = 40 μm. (H) Nile red staining fluorescence micrograph of an orchid seed at the stage similar to G. The surface wall (arrow) of the embryo proper and the shriveled inner seedcoat (arrowhead) and outer seedcoat (double arrowhead) reacted positively. Scale bar = 40 μm.
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
The histological study of the embryo development of Cypripedium debile. (A) A median longitudinal section showing a zygote. The nucleus was located toward the chalazal end and a large vacuole occupied the micropylar end. The cytoplasm of zygote contained several tiny starch grains (arrow) surrounding the nucleus. At this stage, the outer seedcoat (OS) has not completely covered the embryo sac, and starch grains of various sizes could be found within cells of seedcoat. DS = degenerated synergid; IS = inner seedcoat. Scale bar = 20 μm. (B) A median longitudinal section showing a six-celled embryo. The cytoplasm of embryo proper contained several tiny starch grains (arrow), and the lower cell toward the micropylar end will differentiate into the suspensor (S). The degenerating endosperm (arrowhead) could be observed within the cavity of embryo sac. Scale bar = 20 μm. (C) Light micrograph showing a longitudinal section through an early globular embryo. The cytoplasm of embryo proper contained a number of starch grains (arrow), whereas the suspensor cell (S) was highly vacuolated. At this stage, the inner seedcoat (arrowheads) has gradually compressed into a thin layer, and the suspensor cell had protruded beyond the micropyle opening of the inner seedcoat and grew into the lumen enclosed by the outer seedcoat (OS). Scale bar = 40 μm. (D) Light micrograph showing a longitudinal section through a globular embryo. The size of starch grains (arrow) within the embryo proper became larger, whereas the size of suspensor (S) reduced and began to degenerate. Scale bar = 40 μm. (E) As the seed approached maturity, the suspensor (arrow) has just degenerated and left an empty space at the micropylar end. The inner seedcoat (arrowhead) has compressed into a thin layer covering the embryo proper except for the micropylar end. Scale bar = 40 μm. (F) Nile red staining fluorescence micrograph of an orchid seed at the stage similar to E. The surface wall (arrow) of the embryo proper, inner seedcoat (arrowhead), and outer seedcoat (double arrowhead) reacted positively. Note that the fluorescence of the degenerated suspensor at the micropylar end is absent. Scale bar = 40 μm. (G) Light micrograph showing a longitudinal section through a mature seed. Protein bodies (arrowhead) of various sizes can be found within the embryo proper. Although lipid is not preserved in this preparation, the translucent vesicles (arrow) in the cytoplasm of the embryo cell indicate the location of the lipid deposits. At maturity, the embryo is enveloped by the shriveled inner seedcoat (IS) and the outer seedcoat (OS). Scale bar = 40 μm. (H) Nile red staining fluorescence micrograph of an orchid seed at the stage similar to G. The surface wall (arrow) of the embryo proper and the shriveled inner seedcoat (arrowhead) and outer seedcoat (double arrowhead) reacted positively. Scale bar = 40 μm.
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
The histological study of the embryo development of Cypripedium debile. (A) A median longitudinal section showing a zygote. The nucleus was located toward the chalazal end and a large vacuole occupied the micropylar end. The cytoplasm of zygote contained several tiny starch grains (arrow) surrounding the nucleus. At this stage, the outer seedcoat (OS) has not completely covered the embryo sac, and starch grains of various sizes could be found within cells of seedcoat. DS = degenerated synergid; IS = inner seedcoat. Scale bar = 20 μm. (B) A median longitudinal section showing a six-celled embryo. The cytoplasm of embryo proper contained several tiny starch grains (arrow), and the lower cell toward the micropylar end will differentiate into the suspensor (S). The degenerating endosperm (arrowhead) could be observed within the cavity of embryo sac. Scale bar = 20 μm. (C) Light micrograph showing a longitudinal section through an early globular embryo. The cytoplasm of embryo proper contained a number of starch grains (arrow), whereas the suspensor cell (S) was highly vacuolated. At this stage, the inner seedcoat (arrowheads) has gradually compressed into a thin layer, and the suspensor cell had protruded beyond the micropyle opening of the inner seedcoat and grew into the lumen enclosed by the outer seedcoat (OS). Scale bar = 40 μm. (D) Light micrograph showing a longitudinal section through a globular embryo. The size of starch grains (arrow) within the embryo proper became larger, whereas the size of suspensor (S) reduced and began to degenerate. Scale bar = 40 μm. (E) As the seed approached maturity, the suspensor (arrow) has just degenerated and left an empty space at the micropylar end. The inner seedcoat (arrowhead) has compressed into a thin layer covering the embryo proper except for the micropylar end. Scale bar = 40 μm. (F) Nile red staining fluorescence micrograph of an orchid seed at the stage similar to E. The surface wall (arrow) of the embryo proper, inner seedcoat (arrowhead), and outer seedcoat (double arrowhead) reacted positively. Note that the fluorescence of the degenerated suspensor at the micropylar end is absent. Scale bar = 40 μm. (G) Light micrograph showing a longitudinal section through a mature seed. Protein bodies (arrowhead) of various sizes can be found within the embryo proper. Although lipid is not preserved in this preparation, the translucent vesicles (arrow) in the cytoplasm of the embryo cell indicate the location of the lipid deposits. At maturity, the embryo is enveloped by the shriveled inner seedcoat (IS) and the outer seedcoat (OS). Scale bar = 40 μm. (H) Nile red staining fluorescence micrograph of an orchid seed at the stage similar to G. The surface wall (arrow) of the embryo proper and the shriveled inner seedcoat (arrowhead) and outer seedcoat (double arrowhead) reacted positively. Scale bar = 40 μm.
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
The seedcoat of C. debile has two layers: the inner and outer seedcoats that were derived from the inner and outer integuments of the ovule separately. Both of them were composed of two layers of highly vacuolated parenchyma cells (Fig. 1A). As the seeds approached maturity, the cells of the seedcoat dehydrated and constricted into a thin layer, which enclosed the embryo (Fig. 1E). At maturity, staining by using Nile red demonstrated that cuticular materials were present over the surface wall of embryo proper and the radial wall of outer seedcoat, whereas the fluorescence was less intensive in the inner seedcoat (Figs. 1F and 1H). Moreover, only the radial wall of the outer seedcoat stained greenish blue with the TBO stain, indicating the presence of phenolic compounds in the radial wall.
Seed germination in vitro and seedlings development.
In C. debile, high germination (72.6% to 75.0%) was obtained with the seeds collected in the globular and mature stages (≈4 to 5 months after anthesis). On the contrary, no germination was obtained with the immature seeds collected from the zygote to early globular stages. In addition, the germination of mature seeds was higher in 1/4 MS medium than in Thomale GD medium (75.0% and 72.6%, respectively; Table 1). In the liquid culture, the germination rate was higher and more uniform than those in the solid culture (Figs. 2 and 3A–B). For both liquid and solid cultures, the germination increased sharply after 2 months in culture and reached the maximum after 5 months in culture. The roots began to develop from young protocorms after 1 month of culture in the seedling growth medium (Fig. 3C). After 2 months of culture, the formation of shoots from seedling could be observed (Fig. 3D).
Effect of collection timing and cultural medium on seed germination in vitro of C. debile.z
Mean germination percentage of C. debile mature seeds on liquid (●) and solid (○) 1/4 Murashige and Skoog (MS) medium. Error bars represent se (n = 3).
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
Mean germination percentage of C. debile mature seeds on liquid (●) and solid (○) 1/4 Murashige and Skoog (MS) medium. Error bars represent se (n = 3).
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
Mean germination percentage of C. debile mature seeds on liquid (●) and solid (○) 1/4 Murashige and Skoog (MS) medium. Error bars represent se (n = 3).
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
Asymbiotic seed germination and protocorm development of Cypripedium debile. (A) The uniform germination in the liquid medium after 4 months of culture. Scale bar = 1 cm. (B) After 4 months of culture in the solid medium, different stages of developing protocorms could be observed. Scale bar = 1 mm. (C) The young protocorms with elongating roots after 1 month of culture in the seedling growth medium. Scale bar = 5 mm. (D) The seedling with developing shoots s after 2 months of culture in the seedling growth medium. Scale bar = 5 mm.
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
Asymbiotic seed germination and protocorm development of Cypripedium debile. (A) The uniform germination in the liquid medium after 4 months of culture. Scale bar = 1 cm. (B) After 4 months of culture in the solid medium, different stages of developing protocorms could be observed. Scale bar = 1 mm. (C) The young protocorms with elongating roots after 1 month of culture in the seedling growth medium. Scale bar = 5 mm. (D) The seedling with developing shoots s after 2 months of culture in the seedling growth medium. Scale bar = 5 mm.
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
Asymbiotic seed germination and protocorm development of Cypripedium debile. (A) The uniform germination in the liquid medium after 4 months of culture. Scale bar = 1 cm. (B) After 4 months of culture in the solid medium, different stages of developing protocorms could be observed. Scale bar = 1 mm. (C) The young protocorms with elongating roots after 1 month of culture in the seedling growth medium. Scale bar = 5 mm. (D) The seedling with developing shoots s after 2 months of culture in the seedling growth medium. Scale bar = 5 mm.
Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1495
Discussion
In the present study, the optimum germination of C. debile was found in mature seeds (Table 1). This feature is contrary to previous reports of most Cypripedium species, in which mature seeds are more difficult to germinate in vitro than immature seeds (De Pauw and Remphrey, 1993; Lee et al., 2005). From the results of histological and histochemical studies, two notable cytological changes were observed during the course of seed formation. First was the protruding suspensor cell beyond the micropyle opening of the inner seedcoat at the globular stage. Second was the fragmentary deposition of cuticular material on the seedcoat.
Most terrestrial orchids have firm seedcoats protecting their embryos at maturity (Arditti, 1967; Rasmussen, 1995). In the difficult-to-germinate species, besides the outer seedcoat, there is one more layer of seedcoat: the inner seedcoat (also known as “carapace”) that formed a rigid envelope enclosing the embryo at seed maturity (Carlson, 1940; Lee et al., 2005; Veryet, 1969; Yamazaki and Miyoshi, 2006). In C. debile, during the early stages of embryo development (Fig. 1A–B), the proembryo was growing in the lumen enveloped by the inner seedcoat. At the early globular stage, the suspensor cell protruded outside the micropylar opening of the inner seedcoat (Fig. 1C). On the contrary, in the more difficult-to-germinate species such as C. formosanum Hayata (Lee et al., 2005) and Cephalanthera falcate (Thunb.) Blume (Yamazaki and Miyoshi, 2006), their suspensor cells never grew beyond the inner seedcoat, and consequently their embryos were enveloped more tightly by the inner seedcoat as the seed approached maturity. In C. debile, because the suspensor cell wall is primary in nature (Fig. 1E–F), the micropylar site of the inner seedcoat may remain the character of the primary cell wall as the protruded suspensor cell and the inner seedcoat compressed into a thin layer at seed maturity. This flexible micropylar site of the inner seedcoat may imply that there are fewer obstacles for the embryos to absorb water and nutrients when sowing the mature seeds on culture media.
The accumulation of cuticular material is commonly observed in the epidermal tissue, forming a vital hydrophobic barrier over the aerial surfaces, preventing water loss and gaseous exchanges (Esau, 1977). In orchid seeds, because their embryos are rudimentary and are enveloped by thin seedcoats, the presence of cuticular material in the protoderm of globular embryos and seedcoat may help to protect the embryos from early desiccation (Lee et al., 2006; Yeung et al., 1996). The thick cuticular layer covering the surface of the inner and outer seedcoats has been detected in several difficult-to-germinate orchid species. In C. falcata, the accumulation of lignin and cuticular material in the inner seedcoat has been reported (Yamazaki and Miyoshi, 2006). Additionally, in C. formosanum (Lee et al., 2005), the intense staining of Nile red in both the inner and outer seedcoats indicates the heavy accumulation of cuticular material. The results of these studies suggest that the cutinization and lignification of inner seedcoat may play a regulatory role in controlling the seedcoat-imposed dormancy. On the contrary, in the easy-to-germinate epiphytic orchids, i.e., Phalaenopsis Blume (Lee et al., 2008) and Bulbophyllum Thouars (Lee and Yeung, 2010), the seedcoat cells with discontinuous thickened walls in the radial direction may not block the uptake of water and nutrients for germination. From the results of Nile red staining in C. debile seeds (Figs. 1F and 1H), it is worthy to note that the cuticular material is primarily present in the embryo surface, and it is weak and discontinuous in the radial wall of the outer seedcoat. This feature suggests the seedcoat of C. debile has a less hydrophobic nature than the seedcoat of C. formosanum, which makes the mature seeds of C. debile easier to germinate. In the previous report by Tomita and Tomita (2002), a decrease of germination percentage was found in mature seeds of C. debile. It is possible that the populations of C. debile in the north temperate area have deeper seedcoat-imposed dormancy. In the future study, it would be interesting to investigate if the seeds of C. debile in Japan have a more substantial seedcoat and hydrophobic nature.
In this study, the increased germination percentage and germination rate were found in liquid culture (Figs. 2 and 3A). Liquid culture has been shown to improve the seed germination of slipper orchids such as C. calceolus (L.) var. pubescens (Willd.) Correll (Chu and Mudge, 1994) and Paphiopedilum primulinum M. Wood & P. Taylor (Lee and Lee, 2001). Liquid culture may facilitate the entrance of water and nutrients into seeds and help diffuse exudates from seeds.
Unlike most Cypripedium species, C. debile is not perennial. Under the natural condition, the plant of C. debile can survive no longer than three years (our unpublished data). As compared with most Cypripedium species, the seedcoat of C. debile does not look substantial and is less hydrophobic, which makes the mature seeds easier to obtain water and nutrients for germination. The speedy generation of numerous young seedlings annually may provide another strategy for maintaining the populations in natural habitats.
Literature Cited
Arditti, J. 1967 Factors affecting the germination of orchid seeds Bot. Rev. 33 1 97
Arditti, J. & Ernst, R. 1993 Micropropagation of orchids. Wiley, New York, NY
Carlson, M.C. 1940 Formation of the seed of Cypripedium parviflorum Bot. Gaz. 102 295 301
Chu, C.C. & Mudge, K.W. 1994 Effects of prechilling and liquid suspension culture on seed germination of the yellow lady’s slipper orchid (Cypripedium calceolus var. pubescens) Lindleyana 9 153 159
Cribb, P.J. 1997 The genus Cypripedium. Timber Press, Portland, OR
De Pauw, M.A. & Remphrey, W.R. 1993 In vitro germination of three Cypripedium species in relation to time of seed collection, media and cold treatment Can. J. Bot. 71 879 885
Esau, K. 1977 The epidermis. In: Anatomy of seed plants. 2nd Ed. Wiley, New York, NY
Fay, M.F. 1992 Conservation of rare and endangered plants using in vitro methods In Vitro Cell. Dev. Biol. Plant 28 1 4
Lee, Y.I. 2010 Micropropagation of Cypripedium formosanum Hayata through axillary buds from mature plants HortScience 5 1369 1372
Lee, Y.I. & Lee, N. 2001 Effect of capsule maturity, medium composition and suspension culture on in vitro germination of Paphiopedilum primulinum seed J. Chinese Soc. Hort. Sci. 47 147 156
Lee, Y.I., Lee, N., Yeung, E.C. & Chung, M.C. 2005 Embryo development of Cypripedium formosanum in relation to seed germination in vitro J. Amer. Soc. Hort. Sci. 130 747 753
Lee, Y.I., Lu, C.F., Chung, M.C., Yeung, E.C. & Lee, N. 2007 Developmental changes in endogenous abscisic acid concentrations and asymbiotic seed germination of a terrestrial orchid, Calanthe tricarinata Lindl J. Amer. Soc. Hort. Sci. 132 246 252
Lee, Y.I. & Yeung, E.C. 2010 Embryo development and in vitro seed germination of Bulbophyllum fascinator Acta Hort. 878 243 250
Lee, Y.I., Yeung, E.C., Lee, N. & Chung, M.C. 2006 Embryo development in the lady’s slipper orchid, Paphiopedilum delenatii with emphases on the ultrastructure of the suspensor Ann. Bot. (Lond.) 98 1311 1319
Lee, Y.I., Yeung, E.C., Lee, N. & Chung, M.C. 2008 Embryology of Phalaenopsis amabilis var. formosa: Embryo development Bot. Stud. (Taipei, Taiwan) 49 139 146
Light, M.H.S. & MacConaill, M. 1990 Characterization of the optimal capsule development stage for embryo culture of Cypripedium calceolus var. pubescens. North American Native Terrestrial Orchid Propagation and Production Conference Proc., Mar. 1989. Chadds Ford, PA. p. 92
Lin, T.P. 1987 Native orchids of Taiwan. Vol. 3. Southern Materials Ctr., Taipei, Republic of China
Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 15 473 497
Pritchard, H.W. 1989 Modern methods in orchid conservation: The role of physiology, ecology and management. Cambridge University Press, Cambridge, MA
Rasmussen, H.N. 1995 Terrestrial orchids: From seed to mycotrophic plant. Cambridge University Press, Cambridge, MA
St. Arnaud, M., Lauzer, D. & Barabe, D. 1992 In vitro germination and early growth of seedling of Cypripedium acaule (Orchidaceae) Lindleyana 7 22 27
Thomale, H. 1957 Die Orchideen. Eugen Ulmer, Stuttgart, Germany
Tomita, M. & Tomita, M. 2002 In vitro germination of Cypripedium debile Rchb. f. in relation to culture media and seed maturity Propag. Ornam. Plants 2 22 24
Van der Kinderen, G. 1987 Abscisic acid in terrestrial orchid seeds: A possible impact on their germination Lindleyana 2 84 87
Van Waes, J.M. & Debergh, P.C. 1986 In vitro germination of some Western European orchids Physiol. Plant. 67 253 261
Veryet, Y. 1969 La structure des semences des orchidaceae et leur aptitude a la germination in vitro en cultures pures. Musee d’Histoire Naturelle de Paris Travaux du Laboratoire La Jaysinia 3 89 98
Yamazaki, J. & Miyoshi, K. 2006 In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae) Ann. Bot. (Lond.) 98 1197 1206
Yeung, E.C. 1999 The use of histology in the study of plant tissue culture systems—Some practical comments In Vitro Cell. Dev. Biol. Plant 35 137 143
Yeung, E.C., Zee, S.Y. & Ye, X.L. 1996 Embryology of Cymbidium sinense: Embryo development Ann. Bot. (Lond.) 78 105 110
