American Journal of Plant Sciences, 2011, 2, 325-333
doi:10.4236/ajps.2011.23037 Published Online September 2011 (http://www.SciRP.org/journal/ajps)
325
Distribution of Nuclei of Different Ploidy Levels
during Ovule, Seed and Protocorm Development
in Phalaenopsis aphrodite subsp. formosana
(Orchidaceae)
Goamg-Tyng Jean1, Yu-Lin Kao1, Ching-Yan Tang1, Wen-Huei Chen2
1Department of Life Sciences & Institute of Biotechnology, National University of Kaohsiung, Kaohsiung, Taiwan, Chinese Taipei;
2Orchid Research Center, National Cheng Kung University, Tainan, Taiwan, Chinese Taipei.
Received April 6th, 2011; revised May 8th, 2011; accepted June 2nd, 2011.
ABSTRACT
Distribution of nuclei of different ploidy levels was studied at different developmental stages in the embryonic tissue of
the ovule, seed and protocorm of Phalaenopsis aphrodite subsp. formosana (Miwa) E.A. Christ by a combination of
flow cytometry and fluorescence microscopy with Apo Tome Slider. Three stages of ploidy patterns were identified in
the ovular tissue at different days after pollination (DAP). Firstly, between pollination and fertilization (0 to 50 DAP),
2C nuclei were dominant over 4C nuclei and resulted in low level of cycle value. Secondly, between fertilization and
seed maturation (50 to 110 DAP), amount of 4C nuclei increased rapidly, maintained at a high level and then de-
creased gradually to a low level. Small amount of 8C nuclei was also detected at this stage. Thirdly, at seed maturation
(110 to 130 DAP), 2C nuclei became dominant over 4C nuclei again and the cycle value remained at a low but signifi-
cant level at this stage. After seed sowing, nuclei with ploidy levels of 2C, 4C and 8C were observed in the developing
protocorms as early as at 4 DAS (days after sowing). Nuclei with high ploidy levels (8C and 16C) increased gradually
until 40 DAS in this study. Significant level of cycle value at this stage of protocorm development indicated the presence
of endopolyploidy. 4,6-diamido-2-phenylindol (DAPI) staining showed large and prominent nuclei in the basal portions
of the mature seeds before sowing and in the developing protocorms at 20 DAS. These findings clearly demonstrate the
occurrence of different distribution patterns of nuclei with different ploidy levels during ovule, seed and protocorm de-
velopment in Phalaenopsis aphrodite. These observations will provide fundamental information for further studies in
Phalaenopsis orchids.
Keywords: Moth Orchid, Phalaenopsis, Endopolyploidy, Ovule, Seed, Protocorm, Flow Cytometry
1. Introduction
The normal cell cycle includes different phases such as
gap 1 (G1), DNA synthesis (S), gap 2 (G2) and mitosis
(M). Throughout the processes of DNA duplication and
cell division, the DNA content, or the chromosome num-
ber, remains constant from cycle to cycle. Recent re-
search found that a complicated system was involved in
the regulation of these phases [1-3]. Furthermore, due to
various internal or external factors, the regulation of the
cell cycle may be disturbed and resulted in the duplica-
tion of nuclear DNA without cell division, which is
called endoreduplication [1,4]. Due to endoreduplication,
cells having different ploidy levels occurred in the same
tissue, giving rise to endopolyploidy, which was com-
monly found in various plant species [5]. This kind of
mixture of cells having different ploidy levels in the tis-
sue is also called polysomaty [6,7].
There are different degrees of endopolyploidy in dif-
ferent organs and tissues within a species. In general, the
occurrence of endopolyploidy is lower in actively grow-
ing tissues and higher in more differentiated tissues
[5,8-10]. A high degree of endopolyploidy has also been
observed in certain types of highly specialised cells, such
as root hairs, trichomes, endosperm, suspensors and an-
tipodal cells [5,11,12]. Many reports have shown that the
cell volume and nuclear size were correlated with en-
doreduplication in various plant species [5,13,14].

Distribution of Nuclei of Different Ploidy Levels during Ovule, Seed and Protocorm Development in
326
Phalaenopsis aphrodite subsp. formosana (Orchidaceae)
Endoreduplication has been reported in some groups
of orchids [15-18]. Lee et al. [19] observed that endore-
duplication occurred at different stages of floral devel-
opment in Phalaenopsis aphrodite, Phalaenopsis eques-
tris and Oncidium varicosum. Their study showed that
the degree of endoreduplication was positively correlated
with the stage of floral development. When the flower
had fully expanded, endoreduplication and flower fresh
weight had ceased to increase. In addition, cell size was
highly correlated to the number of cycles of endoredu-
plication. Quantitative changes in nuclear DNA content
were studied in developing embryos of Vanda sanderi-
ana, using a cytophotometric technique [20]. It was
found that DNA content was positively correlated with
cell size and with the distance of the nucleus from the
meristem of the embryo.
For Phalaenopsis orchids, the pathways of mega-
sporogenesis and microsporogenesis and the structure of
the embryo sac are similar to most of the angiosperms
[21]. However, pollination is required to stimulate ovary
growth, ovule development and megasporogenesis in this
orchid. This process may take 40 to 80 days depending
on the species [21-23]. After fertilization, seeds with
incomplete embryos lacking endosperm are developed.
However, information on the occurrence of endopoly-
ploidy during ovule, seed and protocorm development is
not available in this orchid.
Flow cytometry and fluorescence microscopy were
used in the present study to investigate the distribution of
nuclei of different ploidy levels at different developmen-
tal stages of the ovule, seed and protocorm in Phalaenop-
sis aphrodite subsp. formosana (Miwa) E.A. Christ. (syn.
Phalaenopsis amabilis var. formosa Shimadzu) to pro-
vide fundamental information for further studies in this
group of orchids.
2. Materials and Methods
2.1. Plants and Culture Conditions
Flowering plants of diploid Phalaenopsis aphrodite su-
bsp. formosana purchased from orchid nurseries in Tai-
wan were grown in a culture room at a temperature of
23˚C - 25˚C, under white fluorescent light (FL40D, TOA
Lighting, Taiwan) with a 16/8 h L/D photoperiod. Self-
pollination was carried out to initiate the growth of the
ovaries and capsules that were used as materials for this
study. Seeds were harvested from the mature capsules
and stored at 4˚C.
Before sowing, seeds were surface-sterilised with 5%
CloroxTM (containing 6% sodium hypochlorite) for 5
minutes and rinsed three times with sterilised distilled
water. Soon after, they were sown on modified Mura-
shige and Skoog (MS) [24] basal medium containing 1/4-
strength MS salts (1.1 g·dm–3), supplemented with 1
g·dm–3 tryptone, 20 g·dm–3 sucrose, 65 g dm–3 homoge-
nised potato and 8.5 g·dm–3 agar [25]. Petri dishes con-
taining the seeds were placed in darkness in a culture
room at 25˚C ± 2˚C for one week and then cultured at the
same temperature with 20 μmol ·m–2·s–1 of light provided
by white fluorescent lights.
2.2. Method for Flow Cytometric Analyses of
Nuclear DNA Content
Fresh tissue samples from ovules or protocorms, weigh-
ing 10 mg - 40 mg, were individually chopped with a
sharp razor blade to pieces < 1 mm in size in a 6-cm
glass Petri dish containing 100 μdm3 extracting buffer
(solution A of the CyStain UV Precise P kit, Partec, Mü-
nster, Germany). After chopping, 400 μdm3 of 4,6-Dia-
midino-2-phenylindol (DAPI) staining buffer (solution B
of the kit) were added. The suspension was filtered through
a 30-μm nylon mesh (CellTricsTM, Partec). For each
sample, 2500 - 5000 nuclei were analysed, using a Partec
PA-I (Münster, Germany) flow cytometer equipped with
an HBO-100 mercury lamp. To determine the DNA con-
tent of the samples, young leaves of in vitro plantlets of
diploid Phalaenopsis aphrodite were used as reference
for the 2C DNA content, which is 2.80 pg 2C–1 [15]. In
this study, 1C represents the nuclear DNA content of
gametes with 19 chromosomes in Phalaenopsis aphro-
dite. Ploidy patterns were represented by the distribution
of nuclei with different C values (e.g., 2C, 4C, 8C). Cy-
cle values, which indicate the mean numbers of endore-
duplication cycles representing the degree of endopoly-
ploidy in the tissue, were calculated after Barow and
Meister [10] as follows:
Cycle value
= (0 · n2C + 1 · n4C + 2 · n8C + 3 · n16C ···)
÷(n2C + n4C + n8C + n16C ···)
where n2C, n4C, n8C, n16C ··· are the numbers of nuclei with
the corresponding C-values (2C, 4C, 8C, 16C ···).
2.3. Determination of Nuclear DNA Contents of
Ovules and Seeds during Ovary and
Capsule Development
After pollination, developing capsules (Figure 1(a)) were
collected at 7-day intervals from 35 days after pollination
(DAP) until 99 DAP. Three capsules were studied at
each interval. Between 35 and 64 DAP, parts of the har-
vested capsules were sectioned for microscopic examina-
tion, as shown in the last part of this section. The re-
maining parts of the capsules were dissected longitudi-
nally. The white ovular tissues (Figure 1(b)) on the sur-
faces of the placentae were carefully removed with a scal-

Distribution of Nuclei of Different Ploidy Levels during Ovule, Seed and Protocorm Development in
Phalaenopsis aphrodite subsp. formosana (Orchidaceae)
327
secting microscope equipped with an image capturing
device. Tissues were fixed in a fixative containing 95%
alcohol and glacial acetic acid (3:1, v:v) overnight and
then stored in 70% alcohol for further treatment. Fixed
tissues were put in 3-mdm3 microcentrifuge tubes con-
taining 1 mdm3 of DAPI staining solution (double-dis-
tilled water and Partec UV staining buffer, 1:4, v:v).
They were then placed in a sealed plastic container
evacuated by an air pump, until the air pressure reached
600 mmHg for 40 minutes. The DAPI-stained capsule
sections were then examined with an inverted fluores-
cence microscope (Zeiss Axio Observer). DAPI-stained
seeds and protocorms were examined with the same mi-
croscope, equipped with an Apo Tome Slider system to
obtain serial images of the optical sectioning from the top
surface to the bottom of the tissue.
pel, for flow cytometric analysis. After 78 DAP, seeds
which detached easily from the placentae were collected
for the above analysis. From 110 DAP, mature seeds
were collected at 10-day interval until 130 DAP.
2.4. Determination of Nuclear DNA Contents of
the Germinating Seeds
Samples of developing protocorms were collected at 4,
10, 20, 30 and 40 days after sowing (DAS) for micro-
scopic investigation and for the analysis of ploidy pattern
by flow cytometry.
2.5. Microscopic Examination and Nuclear
Staining
Seeds, protocorms and transverse sections (less than 1 mm
thick) of the young capsules were examined under a dis-
Figure 1. Ovary and developing ovules and seeds inside the ovary of Phalaenopsis aphrodite at different days after pollination
(DAP): (a) outer appearance of the ovary at 35 DAP (bar = 1 cm); (b) light micrograph of transverse sections of the ovary at
35 DAP (white tissues are developing ovules) ; (c) DAPI-stained fluorescence micrographs of developing ovules at 50 DAP; (d)
developing seeds at 57 DAP; (e) at 78 DAP; (f) at 99 DAP (all bars in micrographs = 100 μm).

Distribution of Nuclei of Different Ploidy Levels during Ovule, Seed and Protocorm Development in
328
Phalaenopsis aphrodite subsp. formosana (Orchidaceae)
3. Results
3.1. Ovule and Seed Development after
Pollination
After pollination, the ovary of Phalaenopsis aphrodite
subsp. formosana grew rapidly for about 40 days, reach-
ing approximately 6 cm in length and 1 cm in width (Fig-
ure 1(a)). The expansion then slowed down and levelled
off. Different stages of ovule development were investi-
gated in free-hand cross-sections with or without DAPI
staining and observed under the microscope. At 35 DAP,
placentae became fork-like structures with white and
loosely arranged tissue on the surface, indicating the de-
velopment of ovule primordia (Figure 1(b)). At 43 and
50 DAP, the globular structures of mature ovules were
observed (Figure 1(c)). At 57 and 78 DAP, these ovules
elongated and became seeds with ellipsoidal structures
(Figures 1(d) and (e)). At this stage, nuclei were more or
less evenly distributed throughout the structures. At 85
DAP, striped coats appeared on the surfaces of seeds,
which easily departed from the ends of the placentae. By
92 - 99 DAP, seeds with seed coats and embryos were
developed (Figure 1(f)). Nuclei inside the seeds were
concentrated in the embryos proper, while the nuclei in
the seed coats disappeared at this stage, indicating the
occurrence of embryo development. It was also observed
that the nuclei of the embryos proper appeared to be un-
evenly distributed, with more nuclei at one end of the
elongated seeds. The seed coats became brownish-yellow
by 110 - 120 DAP, indicating seed maturation at this stage.
3.2. Flow Cytometric Analysis of Nuclear DNA
Contents during Ovule and Seed
Development
Frequency distributions of nuclei having different ploidy
levels were investigated by means of flow cytometry in
ovules and seed tissues at different stages of develop-
ment are shown in Table 1 and Figure 2. Different pat-
terns of distribution at three stages were identified. First,
between 35 and 50 DAP, 2C nuclei with high frequency
were dominant over 4C nuclei (Figure 2(a)). The 1C
nuclei appeared in small numbers at 43 and 50 DAP and
then disappeared in the later stages. Second, from 57 to
92 DAP, 4C nuclei increased rapidly and remained at a
high level (Figure 2(b)). 8C nuclei were observed at this
stage, ranging from 1.48% to 3.5%. Then the proportion
of 4C nuclei decreased gradually and was replaced by an
increase in 2C nuclei until 110 DAP (Figure 2(c)). Third,
between 120 and 130 DAP, 2C nuclei remained at high
level and became dominant over 4C nuclei again (Figure
2(d)). 8C nuclei were observed at very low frequency at
this stage of seed maturation. The cycle values, repre-
senting the mean numbers of endoreduplication cycles,
are shown in Table 1. Cycle values of the ovular tissue
were low from 0 to 50 DAP, then increased at 57 DAP
and remained high until 110 DAP, indicating the occur-
rence of increased amounts of endoreduplicated nuclei at
this stage. Later, these values decreased to a low level at
120 - 130 DAP.
Table 1. Percentages of nuclei with various nuclear DNA contents and cycle value of the ovules and seeds of Phalaenopsis
aphrodite at different days after pollination (DAP). All values are means ± SD of three capsules, each with three observations.
DAP 1C 2C 4C 8C Cycle valuea
35 0 85.7 ± 0.9 14.3 ± 0.9 0 0.14 ± 0.01 e
43 8.2 ± 3.2 78.2 ± 3.9 13.7 ± 1.4 0 0.15 ± 0.02 e
50 9.1 ± 4.1 63.6 ± 6.4 27.3 ± 6.0 0 0.30 ± 0.06 d
57 0 27.6 ± 5.2 69.6 ± 3.6 2.8 ± 2.7 0.75 ± 0.07 ab
64 0 24.3 ± 1.7 74.7 ± 2.0 1.8 ± 2.2 0.78 ± 0.03 a
71 0 25.8 ± 2.9 72.7 ± 3.3 1.5 ± 2.3 0.76 ± 0.04 ab
78 0 28.9 ± 7.4 68.7 ± 9.5 2.4 ± 2.7 0.73 ± 0.06 ab
85 0 29.7 ± 8.3 67.7 ± 9.0 2.6 ± 4.0 0.73 ± 0.09 ab
92 0 29.4 ± 3.6 67.1 ± 4.9 3.5 ± 3.0 0.74 ± 0.04 ab
99 0 38.8 ± 3.2 59.2 ± 3.6 2.0 ± 2.9 0.63 ± 0.05 bc
110 0 49.5 ± 12.6 48.7 ± 12.2 1.8 ± 2.9 0.52 ± 0.14 c
120 0 73.9 ± 9.8 25.7 ± 10.3 0.4 ± 1.3 0.26 ± 0.10 de
130 0 78.5 ± 6.4 20.7 ± 5.3 0.7 ± 2.1 0.22 ± 0.07 de
aCycle values followed by the same letter within a column are not significantly different at α = 0.05 (Duncan’s multiple-range test).

Distribution of Nuclei of Different Ploidy Levels during Ovule, Seed and Protocorm Development in 329
Phalaenopsis aphrodite subsp. formosana (Orchidaceae)
Figure 2. Flow cytometric histograms of nuclei distribution in ovular tissues of Phalaenopsis aphrodite at different days after
pollination (DAP): (a) at 35 DAP; (b) at 57 DAP; (c) at 99 DAP; (d) at 120 DAP.
3.3. Protocorm Development and Nuclear DNA
Contents during Seed Germination
The mature seed of Phalaenopsis aphrodite subsp. for-
mosana, observed under the dissecting microscope, was
composed of a yellow embryo proper and a transparent
seed coat (Figure 3(a)). At 4 days after sowing (DAS),
the embryo proper expanded, and the seed coat broke
after 10 DAS. White absorbing hairs appeared at the base
of the protocorm by 20 DAS (Figure 3(b)). Frequency
distributions of nuclei having different ploidy levels in
protocorms at different stages of development after seed
germination are shown in Table 2. Nuclei having 4C and
8C DNA contents were observed at 4 DAS and afterward.
As the DAS increased, there was a tendency for 2C nu-
clei to decrease and for 8C nuclei to increase. Nuclei
with 16C nuclear DNA content appeared after 30 DAS.
The percentages of 4C nuclei remained almost the same
at moderate levels throughout development. The occur-
rence of high percentages of 4C and 8C nuclei indicated
that endoreduplication took place early in germination.
The cycle values increased gradually from 0.48 to 0.69 as
the DAS increased (Table 2).
The optical sectioning of the seeds and protocorms
was examined by an inverted fluorescence microscope
equipped with the Apo Tome Slider system. The largest
nuclei were found at the basal region of the embryo in
the seeds before sowing (Figure 3(c)). These large nuclei
were found mostly in the region 4.55 μm beneath the
seed coat. They occupied about three-quarters of the total
length of the embryo, while the rest of the embryo was
occupied by smaller nuclei. A similar situation was ob-
served in the protocorm at 20 DAS (Figure 3(d)). Cells
with large nuclei were located in the basal portion of the
protocorm, in the region about 7.15 μm beneath the sur-
face.

Distribution of Nuclei of Different Ploidy Levels during Ovule, Seed and Protocorm Development in
330
Phalaenopsis aphrodite subsp. formosana (Orchidaceae)
Figure 3. Micrographs of seed and protocorm development of Phalaenopsis aphrodite at different days after seed sowing
(DAS): (a) light micrograph of mature seeds before sowing; (b) protocorm at 20 DAS; (c) DAPI-stained fluorescence micro-
graph of the optical section 4.55 μm depth within the seed at 140 DAP before sowing, arrows showing large nuclei; (d)
DAPI-stained image of the optical section at 7.15 μm depth within the protocorm at 20 DAS, arrows showing large nuclei
(bar = 100 μm).
Table 2. Percentages of nuclei with various nuclear DNA contents and cycle values of the protocorms of Phalaenopsis aphro-
dite at different days after sowing (DAS). All values are means ± SD of 10 replications.
DAS 2C 4C 8C 16C Cycle valuex
4 55.5 ± 5.8 41.3 ± 4.5 3.3 ± 4.5 0 0.48 ± 0.09 c
10 56.2 ± 3.3 34.4 ± 2.8 9.4 ± 2.2 0 0.53 ± 0.05 c
20 53.0 ± 3.7 35.0 ± 2.7 12.1 ± 2.8 0 0.59 ± 0.06 bc
30 49.5 ± 3.1 34.7 ± 3.2 12.6 ± 1.9 3.2 ± 3.5 0.69 ± 0.11 ab
40 44.1 ± 2.5 37.3 ± 2.3 14.1 ± 2.4 4.6 ± 2.5 0.79 ± 0.06 a
xCycle values followed by the same letter within a column are not significantly different at α = 0.05 (Duncan’s multiple-range test).
4. Discussion
Ovary development of Phalaenopsis orchids is pollina-
tion dependent, and the growth pattern is biphasic [22].
This kind of developmental pattern is also true in the
flowers of Phalaenopsis aphrodite, as shown in our study.
Our observations showed that two factors initiated the
growth and development of the ovary: pollination, and
meiosis of the megaspores and fertilization. Rapid growth
of the ovary and extensive differentiation of the ovular
tissue after pollination, as well as the dominance of 2C
nuclei over 4C nuclei before 50 DAP, suggests that the
growth of the ovular tissue might come from rapid cell
division as in the normal cell cycle. In addition, 1C nu-
clei appeared between 43 and 50 DAP, indicating the

Distribution of Nuclei of Different Ploidy Levels during Ovule, Seed and Protocorm Development in 331
Phalaenopsis aphrodite subsp. formosana (Orchidaceae)
occurrence of meiosis in the megaspores and the entrance
of pollen tubes into the ovules. Fertilization might take
place immediately after this stage because 1C nuclei
were not found in the later stages of development. As in
other angiosperms, the ovule of the Phalaenopsis orchid
mainly consists of integuments, the nucellus and the
megaspore, which becomes the embryo sac after fertili-
zation. Through a series of cytological studies, Zhang
and O’Neill [22] showed that the growth of the ovule at
this stage is mainly due to an increase in the number of
cells in the peripheral tissues surrounding the mega-
gametophyte, i.e., the integuments and nucellus. Similar
to other actively growing tissues such as the shoot tip in
seedlings of Brassica species [26], endopolyploidy did
not occur at the early stage of ovule development in
Phalaenopsis aphrodite before pollination. Evidence of
low level of endoreduplication was also indicated by the
low cycle values (0.14 - 0.15) at this stage. An organ
with a cycle value below 0.1 was not considered to have
endoreduplication [10].
After fertilization and the formation of the embryo sac,
the growth pattern of the ovule changed. Besides the
morphological changes in the ovules, the distribution of
the nuclei changed from being evenly distributed early in
development to being localized in the embryo when the
seed became mature. Flow cytometric analysis indicated
that 4C nuclei increased rapidly after fertilization, main-
tained at a high level and then decreased by the time of
seed maturation. These 4C nuclei might come from two
possible sources: G2 nuclei of cells having 2C nuclear
DNA content in the normal mitotic cycle and G1 nuclei
of cells derived from first endoreduplication of 2C nuclei
[6]. Right after fertilization, rapid nuclear and cell divi-
sion might occur in the cells of the pro-embryo and the
peripheral tissues, including the integuments and nucel-
lus. Because of the extensive synthesis and duplication of
DNA during S- and G2-phases of the cell cycle, 4C nu-
clei increased rapidly at this stage of development. Com-
parison of the nuclei distribution of the ovular structure
at 50 and 57 DAP (Figures 1(c) and (d)) indicated that
the early stage of seed development was mainly through
the increase in the number of cells. The growth of seeds
of Phalaenopsis aphrodite included the development of
seed coat from the integuments, suspensor cells and the
embryo proper. While the embryos continued to grow by
cell division, the cells of the integuments and suspensors
elongated and became differentiated tissues to perform
their specific function [23]. The nuclei of these differen-
tiated cells might shift to the endoreduplication cycle and
maintained as 4C nuclei. The occurrence of small amount
of 8C nuclei during seed development (Table 1) and
relative high level of cycle values supported that part of
the 4C nuclei were the G1 nuclei resulted from endore-
duplication. Occurrence of endopolyploidy in integu-
ments and suspensors during seed development have also
been reported in other plant species [5,27,28]. When the
seeds of Phalaenopsis aphrodite became mature, the
inner integument and suspensor degenerated, while the
outer integument turned into the shrivelled seed coat [23].
This process would result in the decrease in the propor-
tion of 4C and 8C nuclei in the seed at this stage as
shown in our data. From these observations, endopoly-
ploidy in cells of the integument and suspensor might
play a role not only in the expansion of the size of ovules,
but also as transitional tissue for nutrient storage and
transfer for the growth of the embryo as the seeds mature
[5,23,27,29]. Direct evidence of the occurrence of en-
doreduplicated nuclei in these cells is worthy for further
investigation.
In this study, observations of 8C nuclei at 4 DAS and
16C nuclei at 10 DAS provide evidence for endopoly-
ploidy at the early stage of seed germination in Pha-
laenopsis aphrodite. The level of endopolyploidy in-
creased as the protocorms developed. Similar observation
was also reported in Vanda orchid [16]. However, the
occurrence of endopolyploidy in the protocorm at 4 DAS
in this study is the earliest comparing to other studies on
the development of protocorms of other orchids [16-18].
Although the embryos in the seeds of Phalaenopsis
orchid lacked the defined tissue pattern observed in other
flowering plants, smaller cells were seen in the apical
zone of the embryo proper in the mature seeds of Pha-
laenopsis aphrodite [23]. Large nuclei located at the
basal portion of mature seeds and developing protocorms
were observed in this study. Together with the data of the
flow cytometry, these cells containing large nuclei would
be the result of endopolyploidy since cell size was shown
to be positively correlated with the degree of endopoly-
ploidy [5,13,14,19]. In addition, we also found a higher
frequency of endopolyploid nuclei in the basal portion of
the protocorms than that in the apical portion which con-
tained the meristem [30]. The occurrence of endopoly-
ploidy in the seeds of Phalaenopsis aphrodite before
germination might be an early indication of tissue dif-
ferentiation of the embryo making this group of orchids
easy to germinate, as in Epidendron ibaguense [23]. In a
study of the developing embryo of Vanda sanderiana
after germination [20] a gradient of small to large nuclei
was observed from the meristematic region to the poste-
rior suspensor region. The amount of DNA in the nuclei
was shown to increase in direct proportion to the distance
of the nuclei from the meristem. The author proposed
that the posterior region of the protocorm was part of the
embryo differentiated to serve as the absorbing organ for

Distribution of Nuclei of Different Ploidy Levels during Ovule, Seed and Protocorm Development in
332
Phalaenopsis aphrodite subsp. formosana (Orchidaceae)
the transfer of nutrients, as in the endosperm of the al-
buminous seeds. Our observation of the early occurrence
of endopolyploidization in protocorm development would
suggest a role of endopolyploidy similar to that in Vanda
sanderiana [20].
In conclusion, this study showed that different distri-
bution patterns of nuclei of different ploidy levels oc-
curred in ovules after fertilization; in mature seeds before
germination and in early protocorm development in Ph-
alaenopsis aphrodite. The occurrence of endopolyploidy
during embryo development of the seed and at early
stage of protocorm development might perform specific
functions related to organ differentiation. Further studies
of the role of endopolyploidy during the development of
seeds and protocorms of Phalaenopsis orchids are wor-
thy to be pursued.
5. Acknowledgements
The authors would like to thank Miss W. W. Liu for her
preliminary investigation of this study. This research was
supported by the National Science Council, Taiwan (NSC-
96-2317-B-390-004; NSC-99-2324-B-390- 002-CC1).
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