Botanical Studies (2008) 49: 139-146.
4
Present address: Botany Department, National Museum of
Natural Science, Taichung, Taiwan, ROC.
*
Corresponding author: E-mail: bomchung@gate.sinica.edu.
tw; Tel: 886-2-27892701; Fax: 886-2-27827954.
EMBRYOLOGY
Embryology of Phalaenopsis amabilis var. formosa:
embryo development
Yung-I LEE
1, 4
, Edward C. YEUNG
2
, Nean LEE
3
, and Mei-Chu CHUNG
1,
*
1
Institute of Plant and Microbial Biology, Academia Sinica, 11529, Taipei, Taiwan, ROC
2
Department of Biological Sciences, University of Calgary Calgary, Alberta T2N 1N4, Canada
3
Department of Horticulture, National Taiwan University, No 1, Sec. 4, Roosevelt Rd., 106, Taipei, Taiwan, ROC
(Received November 30, 2006; Accepted October 17, 2007)
ABSTRACT.
Phalaenopsis amabilis var. formosa is an endemic epiphytic orchid variety native to Taitung
and Lanyu of Taiwan. A
-shaped, four-celled embryo is produced by two successive cell divisions of a
zygote. Soon after, two of the four cells toward the micropyle enlarge and divide two more times resulting in
the formation of eight tubular suspensor cells. The suspensor cells are highly vacuolated; the bottom tier of
suspensor cells elongates towards the micropyle, and the upper tier elongates towards the chalazal end of the
seed. During the early stages of embryo development, lipid droplets appear in the elongating suspensor cells
and disappear soon afterwards, indicating the suspensor functions in nutrient uptake and as a temporary food
storage site for the developing embryo. In the mature seed, a differentiated apical zone containing the rela-
tively small cells can be seen in the embryo proper. Protein and lipid bodies are the main storage products in
the embryo proper cells. The results of Nile red staining indicate that a cuticular layer is present only on the
surface walls of the embryo proper, but is absent from the suspensor cell wall Cuticular material is also pres-
ent in the outermost layer of the seed coat and persists through seed maturation.
Keywords: Embryo; Orchid; Phalaenopsis amabilis var. formosa; Suspensor.
INTRODUCTION
The genus Phalaenopsis (Orchidaceae) comprises about
63 species that have produced numerous attractive hybrids
and cultivars (Christenson, 2001). Of these, Phalaenop-
sis amabilis var. formosa is an endemic epiphytic orchid
variety native to Taiwans Taitung and Lanyu Island (lat.
22X N, long. 121X3 E) (Lin, 1988). During the past few
decades, P. amabilis var. formosa has been used extensive-
ly in breeding for Phalaenopsis hybrids, and now it is one
of the most important species for the floriculture industries
in Taiwan.
Orchid seeds are tiny, and most contain a globular-
shaped embryo and lack a well-defined endosperm (Arditti,
1992; Yam et al., 2002). Additionally, the orchid embryos
have a diversified suspensor morphology. This led Swamy
(1949) to propose a classification scheme for orchid em -
bryo development based in part on the form and the pattern
of suspensor development. According to Swamy (1949),
the suspensor of Phalaenopsis contains eight filamentous
cells which elongate toward the chalazal end of the seed,
surrounding the embryo proper. Although the general pat-
tern of embryo developmental is known, detailed structural
information is not available.
The objectives of this study were to document the
anatomical events in the embryo development of P. ama-
bilis var. formosa from fertilization to seed maturity, and
to detail the formation of the suspensor. The information
presented here will provide valuable background informa-
tion for both propagation and future molecular biology
studies on the embryogenesis of Phalaenopsis species.
MATERIALS AND METHODS
Plant Materials
Plants of Phalaenopsis amabilis var. formosa were
grown in greenhouses at National Taiwan University in
Taipei, Taiwan. To ensure a good fruit set and seed quan-
tity, flowers were hand pollinated. Developing fruits were
harvested at regular intervals after pollination. Approxi-
mately 50 developing fruits were gathered for this study.
Light Microscopy
Transverse sections, approximately 2 mm thick of de-
veloping and mature fruits were fixed in 2.5% glutaralde-
hyde and 1.6% paraformaldehyde buffered with 0.05 M
phosphate buffer, pH 6.8, for 24 h at 4XC. After fixation,
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Botanical Studies, Vol. 49, 2008
the sections were dehydrated in methyl cellosolve (BDH
Chemicals) for 24 h, followed by two changes of 100%
ethanol for 24 h each at 4XC. The samples were infiltrated
gradually (3:1, 1:1, and 1:3, 100% ethanol: Historesin,
24 h each) with Historesin (Leica Canada, Markham, On-
tario), followed by two changes of pure Historesin. The
tissues were then embedded according to Yeung (1999).
Longitudinal serial sections, 3 gm thick, were obtained us-
ing Ralph knives on a Reichert-Jung 2040 Autocut rotary
microtome. Sections were stained with the periodic acid-
Schiff s (PAS) reaction for total insoluble carbohydrates
and counter-stained 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 sections were viewed, and the images
were captured digitally using a CCD camera (Cool Snap
fx, Photometrics, Tucson, AZ) attached to a microscope
(Axioplan, Carl Zeiss AG, Germany).
For detecting the storage lipid, some tissues were
post-fixed in 1% OsO
4
in the same buffer for 4 h at room
temperature and then rinsed in three 15-min changes of
buffer. Following fixation, the materials were dehydrated
in an acetone series and embedded in Spurrs resin (Elec-
tron Microscope Sciences, Washington, PA). Sections, 1
gm thick, were obtained by using a diamond knife on an
Ultracut E ultramicrotome, and were stained with 0.1%
(w/v) alkaline TBO in benzoate buffer for 1 min at 60XC
on a hot plate. The grayish color indicated the presence of
unsaturated lipid (Yeung, 1990).
The presence of a cuticle was detected by using Nile
red as detailed in Yeung et al. (1996). The sections were
stained with 1 gg ml
-1
of Nile red (Sigma Chemical Co.,
St. Louis, Mo.) for 10 min, briefly washed in distilled wa-
ter, and mounted in water containing 0.1% n-propyl gallate
(Sigma Chemical Co., St. Louis, Mo.), an antifading com-
pound. The fluorescence pattern was examined using an
epifluorescence microscope (Imager A1, Carl Zeiss AG)
equipped with the Zeiss filter set 15 (546/12 nm excitation
filter and 590 emission barrier filter).
RESULTS
The main structural changes occurring within the ovary
of Phalaenopsis amabilis var. formosa from 60 days after
pollination (DAP) until seed maturity at 150 DAP are
summarized in Table 1. At 60 DAP, most of the ovules
had been fertilized and embryo development had just com-
menced. During the early stages of embryo development
(60-90 DAP), the seeds are white and moist. As the seeds
approach maturity (120 DAP), they are beginning to turn
yellowish white and desiccate. At maturity (150 DAP),
they are yellowish brown and become dry and detached
from placentas.
After fertilization, the zygote had an ovoid shape (Fig-
ure 1). The nucleus which had a prominent nucleolus was
located toward the chalazal end. Judging from the location
of the mitotic apparatus, the first division of the zygote
was unequal (Figure 2). This division produced a smaller
terminal cell and a larger basal cell (Figure 3). The termi-
nal cell eventually gave rise to the embryo proper while
the basal cell formed the suspensor and also contributed
cells to the embryo proper. The endosperm nuclei and the
chalazal nuclei formed a complex and appeared as a dis-
Table 1. Major developmental events occurring in developing
fruits of Phalaenopsis amabilis var. formosa after fertilization.
DAP
*
Developmental Stage
Seed color
60 Zygote, and 2 to 4 celled embryo White
70 Proembryo
White
80 Early globular to globular embryo White
90 Globular embryo
White
120 Late globular embryo, and the
suspensor starts to degenerate
A mixture of
yellowish white
and brown seeds
150 Dry, mature seed
Yellowish brown
*
DAP = days after pollination.
Figures 1-9. (1) Light micrograph of a zygote (arrow) after fertilization at 60 DAP. The zygote has a dense cytoplasm and a prominent
nucleus located at the chalazal end of the cell. Endosperm fails to develop in this species. The polar-chalazal complex (arrowhead)
includes the degenerating chalazal nuclei and the polar nuclei. Scale bar = 20 gm; (2) Light micrograph showing the first cell division
of the zygote. The mitotic apparatus (arrowhead) is located toward the chalazal end of the cell. Scale bar = 20 gm; (3) The first cell
division of the zygote results in the formation of a smaller terminal cell and a larger basal cell. Scale bar = 20 gm; (4) Light micro-
graph showing a further cell division within the two-celled embryo. The basal cell divides first (arrowhead) and results in two cells of
different sizes. Scale bar = 20 gm; (5) Light micrograph showing a three-celled embryo resulting from the transverse division of the
basal cell. The cell towards the micropylar end is larger with a prominent nucleus. This section was stained with PAS-TBO. Scale bar
= 20 gm; (6) An anticlinal division occurring in the basal cell of a three-celled embryo. Scale bar = 20 gm; (7) Light micrograph show-
ing a four-celled embryo stage. The two cells toward the chalazal end remain small and have a dense cytoplasm while the other two
cells toward the micropylar end continue to enlarge. Scale bar = 20 gm; (8) Additional cell divisions (arrowhead) occurring in the cells
toward the micropylar end at 70 DAP. The daughter cells subsequently differentiate into suspensor cells. Vacuoles also become more
prominent in the cytoplasm of these dividing cells. Scale bar = 20 gm; (9) The cells of the embryo proper divides obliquely (arrow-
head), which signals the formation of the globular shaped embryo. At this stage, the suspensor cells (S) enlarge and elongate rapidly
within the endosperm cavity towards both the micropylar and chalazal ends of the seed cavity. SC = seed coat. Scale bar = 20 gm.
The sections from figures 1-9, excluding figure 8, were stained with PAS-TBO, and that from figure 8 was stained with PAS- amido
black 10B.
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var.
formosa
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Botanical Studies, Vol. 49, 2008
Figures 10-15. (10) Light micrograph showing a longitudinal section through an early globular embryo at 80 DAP. The suspensor
cells (S) are well developed and surround the embryonic mass. In this Spurrs resin section, it is worth noting that the suspensor cells
contained several lipid droplets (arrowhead) after TBO staining. SC = seed coat. Scale bar = 40 gm; (11) At 90 DAP, the cells at the
chalazal region of the embryo proper divide (arrowhead) more frequently while those at the micropylar region begin to enlarge. A
distinct protoderm layer can also be found at this stage. The suspensor cells (S) are highly vacuolated and continue to expand toward
the chalazal and micropylar ends. SC = seed coat. Scale bar = 40 gm; (12) At 120 DAP, cell division has ceased within the embryo
proper. The embryo proper consists of two cell types: smaller cells in the chalazal region and larger cells in the micropylar end
near the suspensor. Starch grains (arrowhead) are abundant and tend to congregate around the nucleus. The suspensor cells (S) are
undergoing the dehydration and the compression in the late stage of embryo development. Scale bar = 40 gm; (13) Light micrograph
showing a longitudinal section through a mature seed at 150 DAP. Numerous tiny protein bodies (arrowhead) can be found within the
embryo proper. Although lipid could not be preserved in this preparation, the spaces between the protein bodies are supposed to be the
storage lipid bodies. At this stage, the suspensor cells have degenerated, and the embryo is enveloped by the shriveled seed coat. Scale
bar = 40 gm. The sections of figures 10 and 11 are Suprrs-embedded specimens that have been postfixed with osmium tetroxide, and
the sections are stained with alkaline TBO. The sections of figures 12 and 13 are stained with PAS-amido black 10B; (14) Nile red
staining fluorescence micrograph of an orchid seed at the stage similar to Figure 9. A weak fluorescence indicates a positive staining
in the seed coat (arrowhead). Only a trace of fluorescence is noted at the surface of the embryo proper. Scale bar = 40 gm; (15) Nile
red staining fluorescence micrograph of an orchid seed at the stage similar to Figure 11. As the embryo matures, the surface wall of the
embryo proper fluoresces brightly (arrowhead). Note that the fluorescence of the suspensor wall (arrow) is absent during development.
Scale bar = 40 gm.
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LEE et al. X Embryology of
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formosa
143
tinct structure within the endosperm cavity (Figures 1 and
3). In this species, the endosperm failed to develop. Soon
after, the developing embryo occupied the endosperm cav-
ity, and the endosperm nuclei and the chalazal nuclei were
eventually absorbed by the embryo.
Suspensor development
A transverse division occurred first in the basal cell
giving rise to a three-celled embryo (Figure 4). The
micropylar end cell of the 3-celled embryo enlarged more
than the two chalazal end cells (Figure 5). It then divided
once anticlinally (Figure 6) resulting in a -shaped,
4-celled embryo. Subsequently, two of the four cells to-
ward the micropyle enlarged further through the process
of vacuolation while the remaining two cells located at the
terminus remained small with a dense cytoplasm (Figure
7). The two larger cells at the base continued to divide
(Figure 8) and resulted in the formation of eight filamen -
tous suspensor cells. The suspensor cells elongated and
expanded rapidly by the process of vacuolation (Figure 9
and 16). The four cells at the very base grew towards the
micropyle. The other four cells grew towards the chalazal
end of the seed, surrounding the embryo proper (Figure
10). It is worth noting that the suspensor cells contained
several dark blue bodies after osmium post-fixation and
TBO staining from the proembryo to the early globular
stage (Figures 9 and 10). From the TEM observation
(unpublished data), these dark blue bodies are highly
osmiophilic, indicating they are most likely lipid droplets.
As the globular embryo formed, these droplets vanished
from the suspensor cells (Figure 11). Eventually, the sus-
pensor cells extended beyond the micropyle opening of
the inner integument and grew into the lumen enclosed by
the outer integument (Figure 11). However, the suspensor
never extended beyond the micropyle of the outer integu-
ment (Figure 12). During embryo development, the sus-
pensor cells were always tightly appressed against the seed
coat. As the embryo matured, the suspensor cells began to
collapse as they became dehydrated (Figure 13).
Embryo proper development
In the four-celled embryo (Figure 7), the terminal cy-
toplasmic cells eventually gave rise to the embryo proper.
Oblique cell divisions were readily observed during the
early stages of embryo proper formation, resulting in the
formation of a spheroidic embryo proper (Figure 9). At
90 DAP, a distinct protoderm layer of the embryo proper
had formed (Figure 11). At the same time, additional
cell divisions occurred in the inner cell tiers. It should be
noted that the cells toward the chalazal end divided more
frequently than the cells toward the micropylar end. At
maturity, an ellipsoidal embryo consisted of different cell
sizes: the cells toward the chalazal end are smaller than
those toward the micropylar end (Figure 12).
Storage products
During the early stages of embryo development, the
Figure 16. The Nomarski image showing a developing embryo
at the stage similar to Figure 11. It is clear that the suspensor
(arrow) contains eight filamentous cells. Scale bar = 40 gm.
cytoplasm reacted strongly with amido black 10B, a
protein stain. However, no distinct protein body-like
structure is observed within the cytoplasm of the embryo
proper. Starch grains are not detected in the proembryo
stage. After the cells had ceased to divide (120 DAP),
starch grains began to accumulate first within the embryo
proper (Figure 12) and they tended to congregate around
the nucleus of the cells. At the same time, small protein
bodies began to appear and accumulate within the
cytoplasm of the embryo proper. As the seed approached
maturity (150 DAP), the large vacuoles had broken
down and the starch grains had vanished (Figure 13).
Concomitant with the disappearance of the large vacuoles,
lipid bodies began to accumulate within the cytoplasm. In
the cytoplasm of suspensor cells, no storage products were
found. At the time of fruit dehiscence, protein and lipid
bodies were the major storage products within the embryo
proper (Figure 13).
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Botanical Studies, Vol. 49, 2008
Testa
The mature seed coat was only composed of the outer
integument and was about two-three cells thick. The cells
of the inner integument gradually degenerated during the
early stages of embryo proper formation (Figures 9 and
10), and their cell content was presumably absorbed by
the embryo. At the proembryo stage (Figure 9), the radial
walls of the outermost layer of the seed coat were stained
greenish blue with the TBO stain, indicating the presence
of a secondary wall. The autofluorescence of the seed
coat indicated that lignin was also present in the secondary
wall. In addition, the secondary walls reacted positively
to the Nile red stain (Figures 14 and 15). At maturity, the
cells of the seed coat became dehydrated and compressed
into a thin layer (Figure 13).
The pattern of Nile red staining indicates that a cuticu-
lar substance was absent over the walls of the suspensor
cells through their development and maturation (Figures
14 and 15). The cell wall also gave a purple color when
stained with toluidine blue O, indicating the absence of
phenolic compounds in the wall.
DISCUSSION
One of the unique characteristics of orchid embryos
is the diverse morphology of their suspensors. Based on
the cell division pattern of the developing embryo and
the suspensor morphology, orchid embryos have been
classified into five types (Swamy, 1949). Phalaenopsis
and some genera of the Vandoids group in the orchid
family belonged to the type IV group (Swamy, 1949;
Poddubnaya-Arnoldi, 1967). In this group, a set of eight
suspensor cells is formed following three divisions by
the suspensor initial cell. At the 3-celled embryo stage,
the larger basal cell of the 3-celled embryo divides verti-
cally, resulting in the formation of a
-shaped four-celled
embryo (Figure 6). After two further divisions of the two
basal cells, eight suspensor cells are formed, and they
elongated rapidly towards the micropylar and chalazal end
of the developing embryo proper. In the Nun orchid, cy-
toskeletal elements, i.e. microtubules and actin filaments
play an important role in the morphogenesis of the suspen-
sor cell (Ye et al., 1997). One would predict that the cyto-
skeletal elements may also be involved in the growth and
elongation of the suspensor cells in Phalaenopsis amabilis.
The suspensor, a short-lived embryonic organ is essen-
tial to embryo development in flowering plants (Yeung and
Meinke, 1993). Since symplastic connections are absent
between the developing embryo and the maternal tissues,
at the very least, the suspensor can serve as the nutrient
conduit between these two compartments. In Cymbidium
sinense (Yeung et al., 1996) as well as in this present
study, the tubular suspensor cells are highly vacuolated,
indicating that they can store water and other dissolved
substances for embryo development. In addition, the
cuticular substance is absent in the suspensor wall. This
would facilitate nutrient transport and uptake from the
maternal tissues through its walls as no apoplastic barrier
is present. In Paphiopedilum delenatii, we clearly dem-
onstrated that its suspensor takes on transfer cell morphol-
ogy as wall ingrowths are present (Lee et al., 2006). In
developing seeds, the main function of transfer cells is
the uptake of solutes such as sugars and amino acids for
storage product synthesis (Thompson et al., 2001). In this
study, although wall ingrowths are not detected at the light
microscope level, lipid droplets appear in the elongating
suspensor cells at the globular stage and disappear soon af-
terwards, indicating the suspensor can function in nutrient
uptake and as a temporary site of food storage for the
developing embryo. The transient accumulation of stor-
age products, e.g. starch grains in the suspensor cells, has
been observed during embryo development in a number of
orchid species (Yeung and Law, 1992; Lee et al., 2007). In
the Nun orchid Phaius tankervillae the positive staining of
protein can be seen within the vacuolar sap of the enlarged
suspensor cell (Yeung and Lee, unpublished results). All
this evidence indicates that the suspensor can play a nu-
tritive role supporting the development of the embryo
proper. However, it is important to note that in orchids
such as Spiranthes australis (Clements, 1999), which lack
a structurally defined suspensor, further studies concerning
nutrient acquisition by the developing embryo proper are
needed.
In mature orchid embryos, a defined tissue pattern
similar to that in other flowering plants cannot be found.
Usually, only a protoderm is present and, depending on the
species, a gradient of small to large cells can be seen with
the smaller cells located at the future shoot pole (Andron-
ova, 2006). This may be an indication of structural differ-
entiation within the embryo. It is interesting to note that
in difficult-to-germinate species such as Calypso bulbosa,
there is no marked differentiation within the embryo
proper (Yeung and Law, 1992). The embryo proper has
cells of similar sizes. In contrast, the easy-to-germinate
species Epidendrum ibaguense has a well-differentiated
apical zone in the embryo proper. In the present study,
a marked gradient of cell size also exists in the embryo
proper of the mature seeds of P. amabilis var. formosa.
The seeds of this species readily germinate upon in vitro
culture and have an average germination percentage above
90%. The absence of distinct differentiation within the
embryo proper may further prolong the germination pro-
cess as additional events might be needed leading to the
differentiation of a shoot pole.
During embryo development, cuticular materials can
accumulate in different layers of the seed coat as reported
in Cymbidium (Yeung et al., 1996), Cypripedium (Lee et
al., 2005), Calanthe (Lee et al., 2007), and Paphiopedilum
(Lee et al., 2006). The differences in accumulations of
cuticular materials may influence asymbiotic seed ger-
mination. In those difficult-to-germinate species, the
accumulation of cuticular materials usually formed a uni-
form layer enveloping the embryo proper. In our previous
reports (Lee et al., 2005; Lee et al., 2007), both the outer
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LEE et al. X Embryology of
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formosa
145
and inner layers of the seed coat fluoresce strongly in
Cypripedium formosanum after Nile red staining, while
one layer of the outer walls of the seed coat fluoresces
brightly in Calanthe tricarinata. In these two terrestrial
species, the accumulation of cuticular substances in the
testa appears to be the main reason for the low germination
percentage observed. The tightly fitted cuticular coating
could form a physical barrier restricting embryo growth.
On the other hand, for the easy-to-germinate species, such
as P. amabilis var. formosa, the cuticular materials form a
discontinuous layer covering the embryo proper. This may
enable the embryo to access water and nutrients from the
environment and involve fewer physical constraints on
embryo growth and development.
In conclusion, the key anatomical events in the embryo
development of P. amabilis var. formosa from fertilization
to seed maturity are detailed in this report. The major
developmental events will provide valuable data for
further molecular studies on embryo development of
Phalaenopsis.
Ackonwledgements. This work was supported by grants
from the Council of Agriculture of Taiwan to Nean Lee of
the Academia Sinica, Taiwan and to Mei-Chu Chung, and
by grants from the Natural Sciences and Engineering Re-
search Council of Canada to Edward C. Yeung.
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