Embryology of two mycoheterotrophic orchid species, Gastrodia elata and Gastrodia nantoensis: ovule and embryo development
© The Author(s) 2016
Received: 14 June 2016
Accepted: 3 August 2016
Published: 8 August 2016
Gastrodia elata, a famous herbal medicine, has been received great attention on its treatments of headache, vertigo and epilepsy. Gastrodia nantoensis is a newly described species from central Taiwan with potential medicinal value. Gastrodia species are fully mycoheterotrophic orchids, and the courses of their seed development are more rapid as compared to the chlorophyllous orchids. A better understanding of their reproductive biology would provide insights into the propagation and conservation of the mycoheterotrophic orchid species.
Based on the histological and histochemical investigations, we observed some notable features in ovule and embryo development. First, only the archesporial cell and/or megasporocyte are present within their ovaries at the time of anthesis. Second, their suspensors consist of a single cell and their mature embryos consist of a gradient of small to large cells. Nile red staining of a globular embryo reveals the presence of cuticular material in the surface wall of embryo proper and the lateral walls of suspensor cell, indicating that the basal wall of suspensor cell is the major route for nutrient supply from maternal tissues to embryo proper. Third, their seed coats are derived from a single integument, and lignin but not cuticular material is present in the outer most layer of seed coat and persists through seed maturation.
The faster seed development of Gastrodia species is due to the speedy courses of ovule and embryo development. In the mature seeds, the presence of a differentiated apical zone in embryo proper suggests the easy-to-germinate character. This study provides basic knowledge for further molecular studies on embryo development and symbiotic germination of Gastrodia species.
The tiny orchid seed has a rudimentary embryo and lacks endosperm; therefore, the germination of orchid seed requires the mycorrhizal association, which supplies nutrients for the germinating seed until the seedling differentiates green leaves and becomes autotrophic (Rasmussen 1995). Although a large amount of orchids are chlorophyllous, some orchids remain achlorophyllous and reply on their mycorrhizal partners for nutrient supplies throughout the entire life cycle, and they are known as mycoheterotrophic plants (Leake 1994; Merckx 2013; Lee et al. 2015). For many fully mycoheterotrophic orchids, their growth patterns are unique. They stay underground year-round, but after sprouting, the phases of blooming and then fruit setting complete within a few weeks. The course of seed development is more rapid than chlorophyllous orchids (Afzelius 1954; Arekal and Karanth 1981).
The genus Gastrodia comprises more than 50 species, distributing mainly in Asia and Oceania regions (Hsu 2008). All Gastrodia species are fully mycoheterotrophic, and they are considered as the largest mycoheterotrophic genus in Orchidaceae. Gastrodia elata, a representative Gastrodia species, is an important traditional Chinese medicine (known as Tian Ma) for treatment of headache, vertigo and epilepsy (Xu and Guo 2000). Besides G. elata, there are more than 20 Gastrodia species native in Taiwan (Hsu and Kuo 2010). Some of them may have medicinal values and are under the threats of over-collection for the medicinal market. Gastrodia. nantoensis is a newly described endemic species with only a few known populations in Taiwan. As compared to the growth of stalk of G. elata, the growth of stalk of G. nantoensis is quite different. G. nantoensis has a very short stalk at the time of anthesis, and the further elongation of stalk is triggered by pollination. Although the structure of G. elata embryology has been documented in previous studies by the line drawing or paraffin sections (Kusano 1915; Abe 1976; Liang 1981, 1984), information concerning the development of ovule and embryo of G. nantoensis is lacking. For the commercial production and the conservation works of Gastrodia species, information concerning its reproductive biology is essential. Basic knowledge of seed development will assist in the improvement of artificial propagation, as demonstrated in our previous studies of Cypripedium formosanum (Lee et al. 2005) and Cypripedium macranthos (Zhang et al. 2013).
The objectives of this study are to document the key anatomical events in the embryogenesis of two Gastrodia species during the courses of megasporogenesis, megagametogenesis, fertilization and seed maturity. In this study, we used the resin-embedded sections, providing high resolution, light microscopic interpretations of various developmental stages. The observations would shed light on the embryogenesis of Gastrodia species and add to the current literature.
Plants of G. elata were cultivated in the greenhouse at Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China, while plants in a natural population of G. nantoensis in the bamboo forest located at Nantou County, Taiwan were selected for this study. Anthesis of G. elata occurred from April to May, and anthesis of G. nantoensis occurred from September to October. To ensure a good fruit set and seed quantity, the flowers were hand-pollinated. Developing ovaries and fruits were harvested at regular intervals after pollination. Approximately 30 developing ovaries and fruits of each species were gathered for this study.
Light microscopy and histochemical observations
Development ovaries and capsules were fixed with 2 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 6.8, at 4 °C overnight. After fixation, the samples were dehydrated using an ethanol series, and embedded in Technovit 7100 (Kulzer & Co., Germany) as described by Yeung and Chan (2015). Serial, 3 µm-thick sections were cut with glass knives using a Reichert-Jung 2040 Autocut rotary microtome. These sections were stained with Periodic acid–Schiff’s reaction for total insoluble carbohydrates, 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 (Yeung 1984). The presence of cuticular material was detected using Nile red as detailed in Lee et al. (2006). The sections were stained with 1 μg ml-1 of Nile red (Sigma Chemical Co., St. Louis, Mo.) for 5 min, briefly washed in distilled water for 1 min, and mounted in a solution containing 0.1 % n-propyl gallate (Sigma Chemical Co.), an antifading compound. The fluorescence signal was examined using an epifluorescence microscope (Axioskop 2, Carl Zeiss AG) equipped with the Zeiss filter set 15 (546/12 nm excitation filter and 590 emission barrier filter). These sections were viewed and the images were captured digitally using a CCD camera attached to the light microscope.
Results and discussion
Major microscopic structural events in the developing fruits of G. elata and G. nantoensis
Days after pollination
Archesporial cell and megasporocyte
Megasporogenesis and megagametogenesis
Mature embryo sac
Fertilization and zygote
Late globular embryo
Changes in cell dimensions during embryo sac development of G. elata and G. nantoensis
21.7 ± 0.7
29.4 ± 1.3
20.4 ± 0.6
29.1 ± 1.5
20.8 ± 1.0
26.6 ± 1.7
20.5 ± 0.8
28.7 ± 1.1
2-nucleate embryo sac
25.1 ± 1.7
35.7 ± 2.5
26.4 ± 1.1
36.1 ± 2.2
4-nucleate embryo sac
28.5 ± 1.2
42.8 ± 1.8
27.3 ± 1.7
44.6 ± 2.6
Mature embryo sac
31.0 ± 1.1
53.1 ± 2.4
31.7 ± 1.5
54.6 ± 1.9
In G. elata, an anticlinal divisions in the terminal cell of the three-celled embryo resulted in the formation of a four-celled embryo (Fig. 6d), and in G. nantoensis, the occurrences of an anticlinal division in the terminal cell and transverse division in the subterminal cell of the three-celled embryo gave rise to a five-celled embryo (Fig. 7d). Additional divisions in the inner cell tiers of embryo proper resulted in the formation of an early globular embryo (Figs. 6e, 7e). Further anticlinal and periclinal cell divisions in the inner cell tiers of embryo proper gave rise to a globular embryo (Figs. 6f, 7f). Meanwhile, a large amount of starch grains accumulated in the developing embryo proper. As the embryo approached maturity (Figs. 6g, 7g), cell division had ceased within the embryo proper, and the accumulation of large starch grains was prominent. The composition and structure of these large starch grains remained to be investigated. At maturity, the embryo of G. elata took on an ovoid shape and was only seven cells long and four cells wide (Fig. 6h), while in G. nantoensis, the embryo had an ellipsoidal shape and was eight cells long and four cells wide (Fig. 7h). Like the mature seeds of many orchids, in G. elata and G. nantoensis, most starch grains had disappeared; instead, protein bodies and lipid bodies became the major storage products in their embryos (Yam et al. 2002). It is noteworthy that the mature embryos of G. elata and G. nantoensis consisted of a gradient of small to large cells (Figs. 6h, 7h). The marked gradient of cell size in the embryo proper can be observed in the easy-to-germinate species, such as Phalaenopsis amabilis var. formosa (Lee et al. 2008). On the contrary, in the difficult-to-germinate species, such as Calypso bulbosa (Yeung and Law 1992) and C. formosanum (Lee et al. 2005), no marked gradient of cell size within the embryo proper is observed. The presence of marked gradient of cell size within the globular embryo in orchids may speed the germination process. In the asymbiotic cultures, mature seeds of G. elata and G. nantoensis could readily germinate and reached the average germination percentage over 80 %.
Seed coat development
In most orchids, their ovules are covered by the outer and inner integuments (known as bitegmic) (Law and Yeung 1993; Mayer et al. 2011), while in G. elata and G. nantoensis, their seed coats developed from a single integument (known as unitegmic), which were composed of two-celled layers (Figs. 6c, 7c). Species with simple ovule structures, e.g. the unitegmic ovule and the lack of a distinct micropyle are common features in some mycoheterotrophic orchids (Tohda 1967; Abe 1976). It has been proposed that the unitegmic ovules might be more advanced than the bitegmic ovules (Abe 1972). After fertilization, the cells of seed coat elongated rapidly and enclosed the embryo sac entirely. The cells of seedcoat were highly vacuolated and contained several starch grains (Figs. 6a, 7a). At maturity, the seed coat became dehydrated and compressed into a thin layer that enveloped the embryo (Figs. 6h, 7h). In the developing seed of G. nantoensis, the outermost wall of seed coat gave a weak fluorescence from Nile red staining, suggesting the presence of little cuticular material (Fig. 8a, b). However, the weak fluorescence in the seed coat can be easily quenched by pre-staining of sections with TBO, indicating that a distinct cuticle may be absent (see Holloway 1982; Yeung et al. 1996). TBO is a cationic dye that binds to negatively charged groups in cells and gives two main spectra of reaction, i.e. pinkish purple for carboxylated polysaccharides such as pectin and greenish blue for aromatic substances such as lignin (Pradhan Mitra and Loqué 2014). Under fluorescence microscopy, if TBO staining causes the fluorescence quenching of the cell wall by UV-excitation, we would suggest the accumulation of lignin rather than cuticular material in the cell wall. In this study, the compressed seed coat stained greenish blue with TBO, indicating the lignified cell wall of the seed coat (Figs. 6h, 7h). In orchids, the lignified seed coat could protect the minute globular embryo at the time of seed dispersal.
The present study illustrates the key anatomical events in the seed development of G. elata and G. nantoensis and identifies a number of features that are not yet described clearly in the previous studies, such as the initial differentiation of ovule at the time of anthesis, the histochemical characters of the cell wall of suspensor and seed coat, and the changes of storage products within the embryo proper. This study provides basic knowledge for further molecular studies on embryo development and symbiotic germination of Gastrodia species.
LYY carried out the experimental work and drafted the manuscript; CXM, GSX and LYI provided funding, planned the study and revised the manuscript. All authors read and approved the final manuscript.
This work was supported by grants from the National Natural Science Foundation of China to Shun-Xing Guo; from National Museum of Natural Science, Taiwan, to Yung-I Lee.
The authors declare that they have no competing interests.
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