You are on page 1of 10

Annals of Botany 98: 1311–1319, 2006 doi:10.1093/aob/mcl222, available online at www.aob.oxfordjournals.


Embryo Development in the Lady’s Slipper Orchid, Paphiopedilum delenatii, with Emphasis on the Ultrastructure of the Suspensor
Y U N G - I L E E 1, E D W A R D C . Y E U N G 2, N E A N L E E 3 and M E I - C H U C H U N G 1,*
1 2

Institute of Plant and Microbial Biology, Academia Sinica, 115, Taipei, Taiwan, Republic of China, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada and 3 Department of Horticulture, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd, 106, Taipei, Taiwan, Republic of China

Received: 22 June 2006 Returned for revision: 22 August 2006 Accepted: 31 August 2006 Published electronically: 20 October 2006 

Background and Aims Owing to large-scale collecting, the lady’s slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species.  Methods Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy.  Key Results The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation.  Conclusions The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady’s slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.
Key words: Cuticular material, embryology, lady’s slipper orchid, Paphiopedilum, suspensor.

Compared with the majority of flowering plants, orchids have an atypical pattern of embryo development (Arditti, 1992; Dressler, 1993). One of the distinctive features of orchid embryos is the diverse morphology of their suspensors. In 1949, Swamy devised a classification scheme for orchid embryo development based on suspensor morphology alone. Depending on the species, the suspensor of orchids may be unicellular or else consist of a few filamentous cells (Swamy, 1949; Clements, 1999). At the light microscope level, the orchid suspensor usually appears as a vacuolated organ such as found in Phaius (Ye et al., 1997) and Cymbidium (Yeung et al., 1996). The suspensor in flowering plants is important to embryo development. Structural and experimental evidence clearly indicates that it can serve as a conduit for nutrient flow and may provide unique metabolites for the growth of the embryo proper (Yeung and Meinke, 1993; Nikitcheva, 2006; Yeung et al., 2006). One of the unique structural features of suspensor cells is that they takes on a ‘transfer cell’ morphology with wall ingrowths and numerous mitochondria nearby. Endoplasmic reticulum (ER) cisternae and specialized plastids can also be abundant within the cytoplasm of suspensor cells. At present, detailed ultrastructural information concerning orchid suspensors is lacking
* For correspondence. E-mail

(see Yam et al., 2002). Owing to the vacuolated nature of the orchid suspensor cells, it is uncertain whether these cells indeed have structural specializations similar to those of other flowering plants. Thus, in order to support the notion that the orchid suspensor serves as the conduit for nutrient flow to the embryo proper, additional structural information is needed. The genus Paphiopedilum comprises a number of commercially important species that have been extensively cultivated and produce a wide range of attractive varieties, cultivars or hybrids as a result of intensive breeding (Cribb, 1998). P. delenatii, an endemic slipper orchid species of southern Vietnam, has pale pink flowers (Averyanov et al., 2003). It is of interest horticulturally, but is also considered to be a threatened species in the wild due to over-collection and habitat destruction. Similar to many terrestrial orchids, Paphiopedilum seeds are very difficult to germinate in vitro (Pierik et al., 1988). For the conservation and commercial production of this endangered species, information concerning its reproductive biology and improved methods of in vitro propagation are thus of great importance. Basic knowledge of embryo and seed development will aid in the design of experiments for asymbiotic seed germination studies, as shown in our previous study of Cypripedium formosanum (Lee et al., 2005). At present, information concerning embryo development in Paphiopedilum species is limited, except

Ó The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email:

giving rise to a four-celled embryo (Fig. delenatii were maintained in the greenhouse of the National Taiwan University. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum three 15-min buffer rinses. plastids. material was dehydrated in an graded ethanol series. At the early globular stage (105 DAP). The basal cell first divided transversely. as were histochemical changes during embryo development. 1A). producing a smaller terminal cell and a larger basal cell (Fig. Jena. Austria). The sections were stained with the PAS reaction for total carbohydrates. MO. at 4  C overnight. and processed for Historesin embedding according to Yeung (1999). Periclinal divisions from the surface layer of the cells gave rise to the protoderm as well as contributing additional cells to the embryo proper (Fig. Although the starch-containing plastids were prominent at this stage. an anti-fading compound. Light microscopy RESULTS Fertilization occurred approximately 60 d after pollination (DAP). At each developmental stage. Darmstadt. small vacuoles and osmiphilic lipid bodies (Fig. A red colour indicates carbohydrates. the material was post-fixed with 1 % OsO4 in the same buffer for 4 h at room temperature and then rinsed in three 15-min changes of buffer. Historesin sections of 3 mm were cut using glass knives on a Reichert 2040 Autocut microtome. distributed at the apical end. and counterstained with amido black 10 B (Merck KGaA. USA). mitochondria. The fluorescence pattern was examined using an epifluorescence microscope (Imager A1. 1A). insigne (Zinger and Poddubnaya-Arnoldi. pH 6Á8. 1D). USA) for 10 min. flowers were hand-pollinated. resulting in the growth of the embryo proper. contained numerous mitochondria. MATERIA L A ND METHODS Plant material Plants of P. 1966). 1B. The sections were examined and photographed using a Philips CM 100 transmission electron microscope at 80 kV. The polar nuclei within the endosperm cavity eventually disintegrated during embryo development (Fig. A distinct protoderm layer was found at approximately 105 DAP (Fig. the zygotes and proembryos could be observed within the capsules. St Louis. (1996). more than 100 embryos were analysed under light microscopy to examine their morphological characteristics. The cytoplasm of the zygote. the material was dehydrated in a graded acetone series. The Historesin-embedded tissues were stained with 1 mg mlÀ1 Nile red (Sigma Chemical Co. The terminal cell had a dense cytoplasm whereas the basal cell was elongated and vacuolated. C). In addition. Mitotic figures were readily observed during the early stages of formation of the embryo proper (Fig. and a prominent vacuole was found toward the micropylar end. 1B). Vienna. the structural pattern of embryo development of P. Histochemical staining of protein and total carbohydrates was performed according to Yeung (1984). additional cell divisions had occurred in the inner tiers (Fig. By the early globular stage (90 DAP). The first division of the zygote was unequal. plastids with starch granules. To ensure a good fruit set and seed quantity. Taiwan. These sections were stained with uranyl acetate and lead citrate. lipid bodies and small vacuoles were readily seen in the cells of the embryo proper (Fig. Anthesis occurs during March–April. PA. Ultrathin sections 60–70 nm thick were cut using a diamond knife on an ultramicrotome (Richert-Jung Ultracut E. delenatii from fertilization to seed maturity was investigated. for work on P. Washington. Germany) for proteins. 1F). After three 15-min buffer rinses. The presence of a cuticle was detected using Nile red as detailed in Yeung et al. at 4  C overnight. the terminal tier of cells towards the chalazal end divided and resulted in the formation of a group of four cells (Fig. 2A). Developing fruits were harvested at regular intervals after pollination. and embedded in Spurr’s resin (Electron Microscope Sciences. In this study. This was soon followed by an anticlinal division occurring in the cell at the terminus. Ultrastructural observations demonstrated an abundance of organelles within cells of the embryo proper at all stages of development. briefly washed in distilled water and mounted in water containing 0Á1 % n-propyl gallate (Sigma). 1I). These organelles tended to congregate around the nucleus. After In the four-celled embryo. they gradually . Taipei. As in other orchid species. 3A). delenatii. These cells eventually gave rise to the embryo proper. pH 6Á8. These sections were stained with the periodic acid–Schiff (PAS) procedure and counterstained with toluidine blue O for general histological examination. 1H.. Approximately 40 developing fruits were gathered for this study. I). and blue indicates proteins. 1G) as well as in the surface layer (Fig. 1E). resulting in a three-celled embryo (Fig. emphasis was placed on the ultrastructural features of the suspensor to determine whether the suspensor indeed has the structural requirements to serve as a nutrient conduit for the developing embryo. These preparations were examined and photographed with a Carl Zeiss Imager A1 light microscope (Carl Zeiss AG. no endosperm was observed in P. Germany). 2B).1312 Lee et al. The zygote appeared as a highly polarized cell (Fig. Transmission electron microscopy Seeds at different developmental stages were fixed with 2 % paraformaldehyde and 2Á5 % glutaraldehyde in 0Á1 M sodium phosphate buffer. At 75 DAP. The nucleus of the zygote was located toward the chalazal end. Carl Zeiss) equipped with the Zeiss filter set 15 (546/12 nm excitation filter and 590 nm emission barrier filter). Following fixation. Development of the embryo proper Seeds at different developmental stages were fixed with 2 % paraformaldehyde and 2Á5 % glutaraldehyde in 0Á1 M sodium phosphate buffer.

Scale bar = 30 mm. the cell division occurs in the inner tier of cells (arrowhead) and the hypophyseal cell (arrow). Scale bar = 60 mm. degenerated synergid. (B) The first cell division of the zygote results in the formation of a smaller terminal cell and a larger basal cell. The basal cell divides first transversely (arrowhead). (A) Light micrograph of the zygote (arrows) after fertilization at 75 DAP. DS. The cell towards the micropylar end is highly vacuolated. suspensor. . the suspensor (S) consists of two cells. suspensor. (E) Additional anticlinal divisions occurring in the second derivative of the terminal cell produce a six-celled embryo. S. IL. Scale bar = 30 mm. four-celled embryo is the product of an anticlinal division occurring in the terminal cell of a three-celled embryo. Scale bar = 30 mm. persistent synergid. Endosperm fails to develop in this species. Scale bar = 60 mm. Soon thereafter. The polar–chalazal complex (arrowhead) includes the chalazal nuclei and the polar nuclei.Lee et al. The additional cell divisions of the basal cell result in the formation of a three-celled suspensor (arrows). Scale bar = 30 mm. Scale bar = 60 mm. the outer layers of the seed coat. The dividing basal cell shows condensed chromosomes at metaphase (arrowhead) just before the periclinal cell division. S. OL. (G) By the early globular stage (90 DAP). Light micrographs of the early stages of Paphiopedilum delenatii embryo development. (I) Light micrograph showing the occurrence of periclinal division (arrowhead) within the embryo proper that results in the formation of the inner tier of cells. Scale bar = 30 mm. PS. Scale bar = 30 mm. 1. (D) A T-shaped. The zygote is highly polarized with a chalazally located nucleus and a prominent vacuole occupying the micropylar end. the suspensor enlarges and elongates towards the micropylar region of the seed. (F) The embryo proper continues to develop by periclinal (arrow) and anticlinal (arrowhead) divisions. resulting in an increase in embryo size. (C) A three-celled embryo resulting from the transverse division of the basal cell of the two-celled embryo. the inner layers of the seed coat. At this stage. (H) Tissue differentiation begins with the formation of the protoderm by anticlinal divisions (arrowhead) in the outmost cell layer. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum 1313 DS PS S D G A OL IL S E H B S C F I F I G . resulting in the formation of the globular-shaped embryo.

N. Storage materials. Wall ingrowth was absent from the walls and an electron-dense line was visible enveloping the embryo proper (Fig.1314 Lee et al. Electron micrographs of the development of Paphiopedilum delenatii embryo proper. (D) Accumulation of storage materials within the embryo proper of the mature seed (210 DAP). mitochondria. There is an electron-dense line (arrow) enveloping the embryo proper. The embryo cells had abundant storage materials deposited in the form of lipid and protein bodies in the cytoplasm (Figs 2D and 3C). inner layers of the seed coat. Scale bar = 5 mm. (E) The part of the embryo proper adjacent to the inner integument at the globular stage. showing the cuticular layers (CL) overlying the surface wall of the embryo proper. and the innermost walls of the inner layers of the seed coat (IL). the osmiphilic lipid bodies (OL) gradually increase in number from the basal region of the embryo proper. lipid bodies (L). cell wall. (A) A portion the zygote tip. i. IL. . M. Scale bar = 2 mm. N. Scale bar = 4 mm. while the plastids contain starch grains.e. disappeared as the embryos reached the mature globular stage of development (Fig. protein body. N. The volume of large vacuoles within the cytoplasm became smaller and the cytoplasm consequentially took on a dense appearance (Fig. 2C). lipid and protein bodies. PB. small vacuoles (V) and lipid bodies (L). The cytoplasm contains several mitochondria (M). Plastids (P) containing starch grains. the embryo was only nine cells long and six cells wide (Fig. nucleus. vacuole. As the embryo reached the mature globular stage (150 DAP). 2. nucleus. mitochondria (M) and numerous small vacuoles (V) are present at this stage. mitotic activity had ceased. which begin to disappear from the embryo proper. 3B). could be detected by histochemical staining at this stage. nucleus. CW. At maturity. Scale bar = 2 mm. (B) The early globular embryo proper (105 DAP) showing subcellular contents. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum V M N L OL V N IL M C V A PB L N P L D M V L IL CL L CW P V B E F I G . 3C). Scale bar = 2 mm. lipid body. It is important to note that storage materials began to accumulate first in the cells near the suspensor (Figs 2C and 3B). V. (C) As the globular embryo approaches maturity (150 DAP). 2B). L.

1E also divided once more. Structural specializations in the suspensor could be detected beginning at the six-cell stage. B). resulting in a smaller upper cell and a larger cell near the micropyle. while the storage materials (arrowhead) begin to appear within the basal regions of the embryo proper. Although lipid is not preserved in this preparation. The embryo proper is approximately nine cells long and six cells across at its widest point. (A) A longitudinal section through a globular embryo (105 DAP). 5A). the spaces between the protein bodies are occupied by storage lipid bodies. 5B). Plasmodesmata were present in the common walls between the suspensor initial and its adjoining cell. As the seeds became fully matured (Fig. 5B). Scale bar = 50 mm. (C) A longitudinal section through a mature seed (210 DAP). The general organization of the cytoplasm in the suspensor remained the same throughout early development of the globular embryo.Lee et al. The suspensor (S) consists of three cells that are well developed and press tightly against the innermost walls of the seed coat. prominent fluorescence enveloped the entire embryo when viewed with a fluorescence microscope (Fig. At the micropylar end of the basal suspensor cell. the outer layers of the seed coat. At this stage. 210 DAP). At maturity. In the cytoplasm surrounding the nucleus. the cuticular layer did not extend to the walls of the suspensor cells (Fig. Such a profile was absent from the cells of the embryo proper (Fig. giving rise to two more suspensor cells. 4A. three suspensor cells were present in this species. In addition to the SER. Another notable feature was the abundance of smooth ER (SER) cisternae in the cytoplasm (Fig. In total. C). IL. the suspensor initial had a prominent vacuole located at the micropylar end of the cell. Nile red staining indicated that a cuticular substance started to appear in the surface wall of cells of the embryo proper at the early globular stage and was clearly absent from the suspensor (Fig. . As the embryo approached maturity (150 DAP). The rough ER tended to congregate around the nucleus. mitochondria. 2E). The nucleus and the associated cytoplasm were located towards the embryo proper. this is regarded as the suspensor initial. the suspensor cells were more vacuolated as compared with cells of the embryo proper. OL. 5C). D). The larger cell near the micropyle as shown in Fig. the fluorescence became brighter over the entire surface of the embryo proper and. 3. (B) As the seed approaches maturity (150 DAP). 4C. again. starch-containing plastids and a few dictyosomes were readily found (Fig. the embryo is enveloped by the shrivelled inner (IL) and outer (OL) layers of the seed coat. This cell divided once unequally. large vacuoles begin to be replaced by small ones. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum 1315 OL OL OL S IL IL IL A B C F I G . The smaller cell subsequently divided once more. Suspensor development The cell nearest to the micropyle of a four-celled embryo gave rise to the suspensor (Fig. Scale bar = 50 mm. Scale bar = 50 mm. By the globular stage (105 DAP). IL. well-developed wall ingrowths were present along the walls of the suspensor abutting onto the seed coat (Fig. 3A). Mitochondria were abundant and located near the wall ingrowths. a notable amount of rough ER also became evident (Fig. The suspensor cells elongated after their formation and often appeared convoluted and compressed as the embryo proper expanded in size. An obviously defined cuticular layer could be observed on the surface wall of the embryo proper (Fig. At the four-celled embryo stage. SER remained abundant at this time. 4E. the inner layers of the seed coat. 4F). 1I). with the resulting upper cell being incorporated into the embryo proper and the lower one becoming the uppermost suspensor cell (Fig. the inner layers of the seed coat. OL. 5C). hence. Protein bodies (arrowhead) of various sizes can be found within the embryo proper. the outer layers of the seed coat. 1D). Throughout embryo development. the vacuolated suspensor cells continued to elongate and often became twisted within the endosperm cavity enclosed by the seed coat (Fig. 2B. wall ingrowths appeared as small papillae along the cytoplasmic face of the cell wall (Fig. Light micrographs of the late stages of Paphiopedilum delenatii embryo development.

5C). The fluorescence is never observed in the suspensor wall (arrowhead). Nile red staining indicated that a cuticular substance was absent over the walls of the suspensor cells throughout their development and maturation (Fig. Scale bar = 50 mm. 4B. (C) Light microscopy showing a P. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum IL IL S S IL A IL C E IL IL B D F F I G . (D) Nile red staining fluorescence micrograph of an orchid seed at a stage similar to that in C. Scale bar = 30 mm. suspensor.1316 Lee et al. (B) Nile red staining fluorescence micrograph of an orchid embryo at the same stage as that seen in A. delenatii embryo (150 DAP) with an elongated suspensor (S). 5B–D). S. (E) Light micrograph showing a longitudinal section through a mature seed (210 DAP). IL. The surface wall of the early globular embryo reacts weakly to the stain (arrow). Scale bar = 50 mm. Scale bar = 30 mm. D). which twists and tightly presses against the innermost walls of the seed coat. delenatii embryo is progressing with formation of the globular-shaped embryo. the inner layers of the seed coat. The cytoplasmic components had broken down and appeared disorganized (Fig. the organelles were not clearly defined at this stage. IL. Note that the fluorescence of the suspensor wall (arrowhead) is absent. Plastids of different configurations were readily seen throughout the cytoplasm (Fig. 5D). the inner layers of the seed coat. 5D). The suspensor has degenerated and the mature embryo is enveloped by the shrivelled inner layers (IL) of the seed coat. the suspensor degenerated (Fig. Both integuments were composed of two layers of highly vacuolated parenchyma . The cytoplasm became less electron-dense and. while the innermost walls of the inner layers of the seed coat (IL) also react positively to the stain. The innermost walls of the inner layers of the seed coat (IL) and the surface wall (arrow) of the embryo proper fluoresce brightly. except for the wall ingrowths. 4C). Scale bar = 50 mm. Ultrastructural observations indicated that the electron-dense cuticular layer that had accumulated on the surface walls of the embryo proper was absent from the suspensor cell wall (Fig. (F) Nile red staining fluorescence micrograph of an orchid seed at a stage similar to that in E. (A) The developing P. Scale bar = 50 mm. the cell wall stained pink with toluidine blue O. At the final stages of seed maturation (210 DAP). 4. indicating that phenolic compounds were not present in the wall (Fig. Light micrographs showing the fluorescence outline of the developing Paphiopedilum delenatii embryo after Nile red staining. The innermost walls of the inner layers of the seed coat (IL) and the surface wall (arrow) of the embryo proper fluoresce brightly. Changes in the integumentary tissues The seed coat originated from the inner and outer integuments of the ovule. Moreover.

Scale bar = 2 mm. (C) The micropylar end of the fully developed suspensor at the globular stage. vacuole. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum V L P WI V M M N 1317 A D RER D P B M WI P IL WI M P C D F I G . showing the formation of the wall ingrowth (WI. L. only a few mitochondria (M) still remain visible in the cytoplasm. and plastids (P). arrows) along the basal wall. Cells of the inner layers (derived from the inner integuments) of the seed coat took on a slender shape. The cuticular material is absent from the suspensor wall and the adjoining innermost walls of the inner layers of the seed coat (IL) toward the micropylar end. The outer layers reacted negatively to the Nile red stain. 4D). indicating the absence of cuticular material in the wall. lipid body. starch-containing plastids (P) and a few dictyosomes (D). M.Lee et al. The wall ingrowths (WI) are well developed. At maturity. 3A). The cells are interconnected by the plasmodesmata (arrowhead). By the globular stage. Scale bar = 2 mm. whereas those of the outer layers (derived from the outer integuments) were broader (Fig. 3C). cells. V. As the embryo matured. The basal cell contains a large vacuole. the rough endoplasmic reticulum (RER) begins to appear. At the early globular stage. V. Nile red staining demonstrated that cuticular material was present over the innermost walls enclosing the endosperm cavity (Fig. The wall ingrowths (WI) persist but have become shadowy. the entire seed coat became dehydrated and constricted into a thin layer that enveloped the entire embryo (Fig. (A) The basal cell of a four-celled embryo showing cellular contents. which is in the form of long strands surrounding the nucleus. 4B) although the fluorescence was less intensive toward the micropylar end. dictyosomes (D) and mitochondria (M) are abundant. Scale bar = 2 mm. One of the notable features in the development of the seed coat was the deposition of cuticular material in the innermost walls. 1B). (B) The micropylar end of the suspensor at the six-celled embryo stage. (D) The degenerating suspensor at 210 DAP showing cellular contents. 2C). At this stage. mitochondrion. . Scale bar = 2 mm. In the limited cytoplasm. N. 5. Electron micrographs of the suspensor development of the Paphiopedilum delenatii embryo. the outermost layer of the seed coat started to compress and shrivel gradually (Fig. Most organelles have collapsed and become disorganized. The cytoplasm is filled with the tubular smooth endoplasmic reticulum system. vacuole. nucleus. there are several mitochondria (M). Ultrastructural observations further confirmed the presence of an electron-dense layer in the inner surface of the seed coat (Fig. the fluorescence became brighter and more distinctive (Fig.

1996). and the abundance of mitochondria. These structural features suggest that the suspensor serves as a conduit to supply the necessary nutrients for the growth of the embryo proper (Yeung and Clutter. Thompson et al. may ensure moisture retention by the embryo cells. 2006). delenatii as reported herein. The vacuolated appearance suggests that orchid suspensors may not have further functional specializations. it is clear that structural specializations are indeed present in the suspensor of P.. Nile red staining of developing P. The formation of a prominent cuticle on the surface of the embryo proper. In this study. However. The suspensor cells are still vacuolated and functional (Fig. The rapid development of a cuticle could represent an adaptation to the unique environment surrounding the developing orchid embryo. oils and volatile essential oils (Gunning and Steer. delenatii. Yeung et al. ER and plastids clearly indicate that the suspensor of P. Some recent findings regarding embryo development in flowering plants (e. SER appears early during embryo development. In P. In the present study. the seed coat is thin and may not offer protection to the embryo. The presence of cuticular material in the walls of the embryo proper and the inner walls of the seed coat creates a protective covering of the embryo proper. delenatii suspensor cells therefore support the notion that the suspensor is the nutrient uptake site for the developing orchid embryo. Furthermore. 1998. a cuticle is well developed in the embryo proper as early as the globular stage of development. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum DISCUSSION ER and plastids are additional structural features found in suspensor cells of flowering plants (Zhukova. The wall ingrowths are strategically located on the side walls abutting the maternal seed coat. As a result of these combined developmental events. Physically. In orchids. optimal asymbiotic seed germination of P. clearly signifying the importance of transfer cells in nutrient uptake. Lee. In Cymbidium sinense (Yeung et al. The presence of wall ingrowths and the abundance of mitochondria near the ingrowths in P. nutrient uptake is confined to the micropylar end of the seed via the suspensor. Furthermore. 2002) and the suspensor generally appears as a vacuolated organ. similar to that reported for suspensor cells of Stellaria (Newcomb and Fowke. i.e. between the embryo and the maternal seed coat. delenatii embryos also indicates the absence of cuticular substances in the suspensor wall. the suspensor attaches the embryo proper to the seed coat and there are no direct symplastic connections. this appears to be the most opportune time for in vitro germination to occur for this species. histological observation from this study indicates that the embryo is fully developed in size and yet the cuticular layer has not fully formed. As a result. The increase in the surface area of the suspensor cell supports the idea of enhanced nutrient uptake at this location. 1997). it is often found in a variety of gland cells secreting fats. especially in terms of moisture retention. 3B). delenatii occurred at 150 DAP (68 %). Ultrastructural studies are rare for orchid embryos and the few studies available have tended to focus on changes in the embryo proper. delenatii. 1996. Asymbiotic seed germination is often difficult for fully mature orchid seeds. 1979). 1978. Moreover. and the germination percentage decreased drastically at 210 DAP when the seeds were fully mature (31 %. In our previous study.. The cuticular material may also offer physical protection to the embryo during germination. 1996) have indicated the presence of cuticular substances on the surface of the embryo proper and their absence in the suspensor cell walls. 1993). as well as in the innermost layer of the seed coat. The ‘transfer cell’ morphology is a common structural feature found in the suspensor of flowering plants (Yeung and Meinke. At 150 DAP. delenatii indeed has structural specialization similar to those of other flowering plants. albeit that the wall ingrowths are not as elaborate as those reported in species such as Phaseolus coccineus (Yeung and Clutter. pointing out that the main function of transfer cells in seeds is the uptake of the solutes as sugars and amino acids for the production of storage product. Fig. 3C). Yeung and Meinke. one of the potential drawbacks of having a well-developed cuticle and a tightly fitted seed coat with a thick cuticular layer is that the seeds are difficult to germinate in vitro. recent studies indicate that symporter proteins are localized in transfer cells of Vicia faba (Harrington et al. 1979). the presence of a large amount of SER and the varied morphologies of the plastids and their absence in the embryo proper further indicate the unique nature of the embryo suspensor. The presence of SER suggests that besides serving as a nutrient conduit. Rodkiewicz et al. the findings of the present study corroborate the hypothesis that the suspensor is the major site of nutrient uptake for the developing embryo. the absence of cuticular substance in the suspensor would allow for apoplastic continuity between the maternal tissues and the embryo. 1993). an endosperm fails to develop during seed development. plasmodesmata. Furthermore. A majority of structural studies of orchid embryo development have focused on changes at the light microscope level (see Yam et al. in particular on storage product deposition (see Yam et al. the suspensor may provide unique nutrients that are necessary for the growth of the embryo proper.. the cuticular material may also serve as a barrier for nutrient uptake. which is similar to the suspensor of P. The suspensor takes on a ‘transfer cell’ morphology. 1996) as well as in P. Knowledge of embryo development will help facilitate success in asymbiotic seed germination and the conservation of endangered orchid species. These observations clearly indicate structural and functional differences between the two parts of the embryo. 2002). (2001) have summarized the development and functions of seed transfer cells in a recent review. Lackie and Yeung.1318 Lee et al.g. 1974) and Phaseolus (Yeung and Clutter. 1994. In conclusion. This would also facilitate nutrient movement from the maternal tissues to the embryo proper via the suspensor.. delenatii. . The presence of wall ingrowths. especially those of the terrestrial orchids... The suspensor is a short-lived embryonic organ of flowering plants. Although the role of SER is not well understood.

II. In Vvitro Cellular and Developmental Biology—Plant 35: 137–143. Taiwan. RLM. Frommer WB. 1996. In: Pridgeon AM. Suspensor.Lee et al. 202–208. Yeung EC. Yeung EC. Clutter ME. Genera Orchidacearum. New Hampshire: Science Publishers. Clements MA. Yeung EC. 1994. 2. Zhukova GY. Yeung EC. Plant Cell 5: 1371–1381. Seed. 2003. Embryo development of Cypripedium formosanum in relation to seed germination in vitro. Poddubnaya-Arnoldi VA. and from the Natural Sciences and Engineering Research Council of Canada to E. Wang XD. ed.. Plant cell biology. vol. OR: Dioscorides Press. Zee SY. 2006. Fowke LC. Stellaria media embryogenesis: the development and ultrastructure of the suspensor. eds. Arabidopsis. National Taiwan University. Sexual Plant Reproduction 7: 287–289. Yeung EC. In: Kull T. Lee N. 1996. Seed. 2005. Spatial and temporal expression of sucrose transport-related genes in developing cotyledons of Vicia faba L.Y. Embryological studies in the Orchidaceae. 2nd edn. Orlando: Academic Press. Orchid embryos.L. 1993. Lackie S. 1978. Annals of Botany 78: 105–110. Yeung EC. 2006. 1997. eds. The Royal Botanic Gardens. Harrington GN. Dressler RL. 1984. Zee SY. 2001. 1998. Phaius tankervilliae. Seed. 1999. 2. Suspensor functions. Ye XL. Embryology of Phaseolus coccineus: the ultrastructure and development of the suspensor. Chlorophyll and cutin in early embryogenesis in Capsella. 1998. The Royal Botanic Gardens. Zygotic embryo development in Daucus carota. New York: John Wiley and Sons. 1979. The use of histology in the study of plant tissue culture systems—some practical comments. Application of histochemical techniques to the study of embryonic processes in certain orchids. Yeung EC. Dordrecht: Kluwer. Hueros G. Sprenkels PA. Gunning BES.. 38–66. 1999. Fundamentals of orchid biology. Phytomorphology 16: 111–124. Yeung EC. Nikitcheva ZI. 208–212. Ye XL. 1993. Seed germination and further development of plantlets of Paphiopedilum ciliolare Pfitz. 2. 2002. 1988. New Hampshire: Science Publishers.C. Embryology. In: Batygina TB. Cell culture and somatic cell genetics of plants. Histological and histochemical staining procedures. The American Midland Naturalist 41: 202–232. Szczuka E. Yeung EC. Journal of the American Society for Horticultural Science 130: 747–753. Maitz M. Protoplasma 200: 35–50. Zinger NV. Embryology of flowering plants. Kew: Compass Press Limited. Cribb P. The genus Paphiopedilum. 1949. . Kew: Natural History Publications. 1996. Portland. 1974. 1966. vol. Canadian Journal of Botany 52: 607–614. Plant Science 160: 775–783. Becker HA. to M-C. vol. vol. Nussbaumer Y. 1997. Scientia Horticulturae 34: 139–153. Pierik.C. The study of embryo development and seed germination in vitro in slipper orchids. MS Thesis. Ye XL. Loc PK. Lee YI. Ultrastructural characteristics of suspensor. Franceschi VR. Oxford: Oxford University Press. 689–697. Cribb PJ. Lee YI. Zee XY. 2006. Yeung EC. Suspensor development in the nun orchid. 8th edn. Thompson RD. ed. Phylogeny and classification of the orchid family. in vitro. van der Harst B. from Academia Sinica. Chung MC. Embryogenesis in angiosperms: development of the suspensor. In: Batygina TB. Rodkiewicz B. International Journal of Plant Sciences 158: 704–712. Meinke DW. Orchid biology: reviews and persectives. Embryogeny of Phaseolus coccineus: growth and microanatomy. Swamy BGL. Protoplasma 94: 19–40. Development and functions of seed transfer cells. Chase MW. Embryology of flowering plants. In: Batygina TB. Steer MW. Embryology. Canadian Journal of Botany 74: 990–998. Canadian Journal of Botany 57: 120–136. Tegeder M. Boston: Jones and Bartlett Publishers. 287–385. Embryology of flowering plants. Inc. ed. Yam TW. and Stellaria investigated by fluorescence microscopy. In: Vasil IK. ROC. Averyanov L. Cribb P. 1992. Yeung EC. — Embryo Development and Suspensor Ultrastructure in Paphiopedilum ACKNOWLEDGEMENTS This work was supported by grants from the Council of Agriculture of Taiwan to N. Rasmussen FN. 198–202. 1319 LITERATURE CITED Arditti J. Fyk B. Hiep NT. ed. Zee SY. 1. van der Meys QG. Arditti J. Embryology of Cymbidium sinense: embryo development. Clutter ME. Taiwan. Newcomb W. Ye XL. Arditti J. Slipper orchids of Vietnam. et al. New Hampshire: Science Publishers.