全 文 ：Received 23 Oct. 2003 Accepted 12 Mar. 2004
Supported by the National Natural Science Foundation of China (30170061) and Important Project Sponsored by the Department of
Science and Technology of Guangdong Province (2003A2010401).
* Author for correspondence. Tel: +86 (0)20 37201242; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (7): 839-845
In Vitro Megagametophyte Development via Unfertilized Ovule
Culture in Doritis pulcherrima
WU Cheng-Hou, LIANG Cheng-Ye, YE Xiu-Lin*
(South China Institute of Botany, The Chinese Academy of Sciences, Guangzhou 510650, China)
Abstract: The ovule of Doritis pulcherrima Lindl. is anatropous, tenuinucellate and bitegmic. The mature
megagametophyte is composed of seven cells with eight nuclei and its formation belongs to Allium type. Its
embryo sac matures about 45 d after pollination. The megasporogenesis in vitro by ovule culture can be
induced on all media whether with exogenous hormones or not 3 d after inoculation, but the later
megagametogenesis will stop on the medium without hormone or only with auxin or cytokinin. Mature
embryo sac can form from megaspore mother cell on the modified VW medium supplemented with 0.5
mg/L BA and 0.1 mg/L NAA.
Key words: Doritis pulcherrima ; ovule; embryo sac; in vitro ; embryology
Haploids of 21 angiosperm species have been obtained
from in vitro culture of unfertilized ovules and ovaries since
1976, and most of the species are using explants at stages
of uninucleate to mature embryo sac (Wu, 2003). This method
is not only a viable approach of inducing haploids
(Mukhambetzhanov, 1997), but also opening a new way to
genetic research and regulating on the development of
megaspore and megagametophyte, and the embryogenesis
in vitro (Yang and Zhou, 1982). For the great difficulty of in
vitro culture of ovules and ovaries at early stages such as
megasporocyte or archesporial cell, successful reports of
inducing haploids have only been found in lily (Lilium
davidii var. willmottiae (Wilson) Roffill) (Gu and Cheng,
1983) and tobacco (Nicotiana tabacum L.; N. rustica L.)
(Wu and Cheng, 1982; Zhu et al., 1981; 1984) and only
megasporocyte of lily (Gu and Cheng, 1983) developed to
mature embryo sac.
In most orchid species studied to date the ovules are
quite rudimentary prior to pollination. Upon successful
pollination, massive number of ovules is produced in each
ovary. This provides a unique opportunity to study events
of ovule and embryo sac development in angiosperms.
However, our knowledge concerning the ovule and the em-
bryo sac of orchids by now is limited to general develop-
mental patterns and few experimental and cell biological
studies are available (Arditti and Pridgeon, 1997).
Doritis pulcherrima, a member of the subtribe Vandinae
of the family Orchidaceae, is a native orchid in Hainan
Province, China and widely distributed in the Southeast
Asian countries (Ji et al., 1999). It has beautiful flowers and
is a valuable germplasm for the orchid hybridization. The
embryo sac developmental pattern of this species has been
studied by Yasugi (1983) and Tang et al. (1998), but results
of their reports were conflicted. This paper reports the re-
sults on certainty of the embryo sac developmental pattern
and the in vitro culture of unfertilized ovules in D.
pulcherrima.
1 Materials and Methods
Doritis pulcherrima Lindl. plants were collected from
Hainan, China and cultivated in a greenhouse in the South
China Institute of Botany, the Chinese Academy of
Sciences. Two flowers borne on each scape of the plants
were hand-pollinated. Sixty scapes, 120 flowers were used
for this purpose in the years 2001 and 2002.
The medium used throughout the experiments was modi-
fied VW (Vacin and Went, 1949) supplemented with vari-
ous concentrations of a-naphthatenacene acid (NAA),
benzyladenine (BA), coconut milk (CM), 2% (W/V) sucrose,
and 0.8% (W/V) agar powder. The pH of all media was
adjusted to 5.3 before autoclaving. Ovule cultures were
started at 32 d after pollination. The ovaries for in vitro
culture were surface-sterilized, first in 70% (V/V) ethanol
for 1 min followed by a 0.4% (V/V) sodium hypochlorite
solution for 10 min with occasional agitation. Ovary-slice
0.1 cm thickness was used as the explants, three slices per
flask. All cultures were incubated at about (26± 1) ℃ in
darkness.
The x (width) and y (length) axes of median sections of
ovules were measured to the nearest 0.1 mm using a
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004840
calibrated eyepiece micrometer. A minimum of 10 ovules at
each developmental stage from megasporocyte to mature
embryo sac were measured for the purposes of this study.
The cultured ovary-slice was fixed each day through a
period of 14 d. Ovaries were taken at two day intervals from
25 d to 60 d after pollination for the observation of embryo
sac development in vivo. All materials were fixed in modi-
fied FAA solution (formalin:acetic acid:50% alcohol = 5:6:
89, by volume) for 2-3 d, stored in the same solution at 4
℃, stained in Ehrlich’s haematoxylin, washed thoroughly
in several changes of distilled water and then tap water,
each for 1-2 h. The stained materials were passed through
a routine procedure of paraffin method, sectioned to 8 mm
and, after being dewaxed with xylol, mounted directly in
Canada balsam.
2 Results
2.1 Development of embryo sac in vivo
The ovary of Doritis pulcherrima consists of three pla-
cental ridges. Twenty-five days after pollination, hundreds
of ovular primordia that composed of a single axial row of
cells covered by a layer of epidermal cells have grown on
each of the placenta ridges. A single archesporial cell that
differs from its neighbor by its larger size and a denser
cytoplasm is differentiated from the outermost cell of the
axial row in the ovular primordium 28 d after pollination.
The archesporial cell enlarges further and differentiates into
the megasporocyte. The megasporocyte is distinguished
by an elongated cell axis and a prominent nucleus (Fig.1).
The start of inner integument is prominent in epidermal
cells of ovule. The outer integument initiates its develop-
ment later but it grows faster. The ovule is anatropous,
tenuinucellate and bitegmic.
The megasporocyte divides and gives rise to a rela-
tively equal dyad (Fig.2). The chalazal dyad cell enlarges
and differentiates into a functional megaspore while the
micropylar one degenerates (Fig.3).
The functional megaspore begins to divide. No cell wall
is laid down between the nuclei after the second meiotic
division so that a 2-nucleate embryo sac is formed directly
from it with a large vacuole separating the two nuclei at
each end (Fig.4). The large central vacuole aids to the ex-
pansion of the embryo sac. Both nuclei divide and a 4-
nucleate embryo sac forms (Fig.5). The nuclei are of a simi-
lar size at the 4-nucleate stage. All nuclei divide again to
result in the formation of a 8-nucleate embryo sac (Fig.6).
The three nuclei at the micropylar end are organized into an
egg apparatus, consisting two synergids and an egg cell.
The three chalazal nuclei are organized into antipodal cells.
The two polar nuclei migrating from the opposite end of the
embryo sac fuse and a central diploid nucleus forms. Ma-
ture embryo sac of a 7-celled, 8-nucleate is formed about 45
d after pollination. The embryo sac development belongs
to a bisporic pattern— Allium type.
2.2 Unfertilized ovule in vitro cultures
2.2.1 Effects of hormones on the development of embryo
sac in vitro Most of the ovules in D. pulcherrima appear
at megaspore mother cell stage and a few at archesporial
cell stage 32 d after pollination. Exogenous hormones are
dispensable in the initial cultures. Three days after cultured,
megasporogenesis was capable of taking place on all me-
dia whether with exogenous hormones or not. When cul-
tured 7 d, ovules on the medium without hormone could
not continue the following megametogenesis procedure
and most of them degenerated. When the media only con-
tained auxin or cytokinin the megametogenesis procedure
stopped halfway. Ovules cultured on media with NAA 0.5
mg/L, BA 0.1 mg/L, BA 0.1 mg/L+NAA 0.1 mg/L, and BA
0.5 mg/L respectively, a few of 2-nucleate embryo sacs were
capable of undergoing the first mitotic division and some
4-nucleates were formed but the second mitotic division
did not take place. Mature embryo sac would form from
megasporocyte only on the medium with BA 0.5 mg/L and
NAA 0.1 mg/L (Table 1).
2.2.2 Embryo sac development in vitro The
megasporocyte continued to enlarge along the cell axis and
contained a prominent nucleus and dense cytoplasm when
cultured in vitro (Fig.7). The polarity of the megasporocyte
in vitro was as that in vivo. After the first meiotic division,
a relatively equal dyad formed (Fig.8). The chalazal dyad
cell enlarged and differentiated into the functional me-
gaspore at the expense of the micropylar one (Fig.9). The
functional megaspore after the second meiotic division so
that a 2-nucleate embryo sac came to expand. A large vacu-
ole separated the two nuclei (Fig.10). Both nuclei divided
and a 4-nucleate embryo sac formed, two nuclei displayed
front and behind at the micropylar end and two nucleus
stained more deeply due to the presence of more hetero-
chromatin within the nucleoplasm (Fig.11). One further di-
vision took place and the embryo sac became 8-nucleate.
At maturity, therefore, the embryo sac was seven-celled.
The embryo sac developmental pattern was the same type
as in vivo, but the polar nucleus migrated to the chalazal
end and the antipodal cells degenerated quicker than those
formed in vivo (Fig.12).
The difference of ovule width (X-axis) was little between
in vivo and in vitro at megasporocyte, dyad, 4-nucleate
and mature embryo sac stages when cultured on the
WU Cheng-Hou et al.: In Vitro Megagametophyte Development via Unfertilized Ovule Culture in Doritis pulcherrima 841
Figs.1-6. Embryo sac development of Doritis pulcherrima in vivo. The micropylar end is oriented downwards in all figures. 1.
Megaspore mother cell (*), ×550. 2. Dyad, ×550. 3. The micropylar dyad cell has degenerated and the enlarged chalazal one differenti-
ates into the functional megaspore, ×550. 4. Bi-nucleate embryo sac with a large vacuole (V) in the middle of the cell, ×550. 5.
4-nucleate embryo sac with a large vacuole (V) in the middle of the cell, ×550. 6. Mature embryo sac with 8-nuclei in seven cells, this
section showing two synergids (S), an egg cell (E), one polar nucleus (P) and two antipodal cells (*), ×550.
Table 1 Effects of hormones on the in vitro development of embryo sac in Doritis pulcherrima
Hormones
Cultured
(mg/L)
days

Ovules at different stages
BA NAA Σ m.m.c meio.Ⅰ dyad meio.Ⅱ 2-n mit.Ⅰ 4-n mit.Ⅱ m.s
0 0 3 124 94 8 11 1 10
7 45 32 8 2 0 3
0 0.1 3 130 106 5 7 1 11
7 12 10 2 0 0 0
0 0.5 3 150 75 15 42 1 17
7 69 48 8 5 0 7 0 1
0.1 0 3 166 98 7 49 1 11
7 71 36 6 14 1 13 1
0.1 0.1 3 138 125 6 6 0 1
7 41 26 2 7 0 4 0 2
0.1 0.5 3 146 130 5 8 0 3
7 31 25 0 4 0 2
0.5 0 3 122 93 10 8 1 10
7 54 33 5 9 0 5 0 2
0.5 0.1 3 187 146 11 11 1 18
7 114 13 3 8 0 75 4 7 1 3
*The basic medium is VW medium. m.m.c., megaspore mother cell; meio., meiosis; mit., mitosis; 2-n, 2-nucleate embryo sac; 4-n, 4-nucleate
embryo sac; m.s, mature embryo sac.
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004842
medium with 0.5 mg/L BA+0.1 mg/L NAA (Table 2). At the
2-nucleate embryo sac stage, however, the width of ovule
cultured in vitro was longer than that in vivo.
The ovule length (Y-axis) was longer in vitro on the
medium with 0.5 mg/L BA+0.1 mg/L NAA than that in vivo
and the differences were more obvious at the developing
Figs.7-14. In vitro embryo sac development in Doritis pulcherrima via ovule cultures. 7. Megaspore mother cell, ×550. 8. Dyad just
forms, ×550. 9. Dyad, showing the micropylar cell is degenerating and the chalazal one (*) is developing into the functional megaspore,
×550. 10. Bi-nucleate embryo sac, ×550. 11. 4-nucleate embryo sac, showing two nucleus display front and behind at the micropylar
end, ×550. 12. Mature embryo sac, this section showing two synergids, an egg cell (E), one polar nucleus (*) migrates to the chalazal
end for three antipodal cells have degenerated, ×550. 13. Abnormality in the first meiosis, showing the chromosomes display along the
longitude of the embryo sac, ×550. 14. 4-nucleate embryo sac, showing two nuclei at the chalazal end have degenerated, ×550.
WU Cheng-Hou et al.: In Vitro Megagametophyte Development via Unfertilized Ovule Culture in Doritis pulcherrima 843
stages than at mature embryo sac stage (Table 2).
Other abnormities occurred in vitro. The nucellar cell at
the micropylar end that should degenerate at 4-nucleate
embryo sac in vivo (Fig.5) degenerated at 2-nucleate em-
bryo sac stage in vitro (Fig.10). The orientation of the
spindle in meiosis became oblique results in the chromo-
somes displayed along the longitude direction of the em-
bryo sac (Fig.13). Two chalazal nuclei degenerated at 4-
nucleate embryo sac stage in vitro (Fig.14).
3 Discussion
Yasugi (1983) reported that the embryo sac develop-
mental pattern of Doritis pulcherrima is monosporic mode.
Tang et al. (1998) reported that the pattern belongs to Al-
lium type, one of the bisporic modes. However, Yasugi
(1983) only provided figures of dyad and mature embryo
sac stages but no tetrad and other stages at all. The func-
tional megaspore is always differentiated from the chalazal
dyad cell in our results whether in vitro or in vivo, sug-
gesting that the embryo sac developmental pattern of D.
pulcherrima should be bisporic, Allium type.
Megasporogenesis of D. pulcherrima is capable of tak-
ing place on the medium without hormone cultured in the
first three days. This suggests endogenous hormones make
the effects. Zhou and Yang (1981) have also discovered the
same phenomenon in the culture of unpollinated ovaries of
rice (Oryza sativa). The exogenous hormones are indis-
pensable for the female gametophyte development in the
later cultures. Recent physiological and molecular biology
studies indicate that ethylene in the presence of auxin is
required for ovule development and differentiation (Zhang
and O’Neil, 1993). In the culture of unfertilized ovules of D.
pulcherrima, the ovules do not develop well when the media
only contain auxin. Mature embryo sac can form from
megasporocyte when the medium contains 0.5 mg/L BA
and 0.1 mg/L NAA. Thus, it may be reasonable to suggest
that cytokinin also plays an important role in the ovule
development. According to these information, we can un-
derstand more phenomena that the ovule initiates quite
prior to pollination in most orchid species. It may be
reasonable to suggest that the pistil induces the endog-
enous hormones to promote the ovule genesis and later
megagametophyte development.
Mature embryo sac could not form from the
megasporocyte of a Phalaenopsis hybrid for the integu-
ment cells degenerated quickly in vitro (Wu, 2003). In D.
pulcherrima ovule cultures, the embryo sac also do not
develop well when the media contain no hormone or the
hormones not enough because the integument cells de-
generate quickly. The embryo sacs develop well in vitro
when the medium contains 0.5 mg/L BA and 0.1 mg/L NAA
and the integument cells containing obvious nucleolus and
dense cytoplasm keep well. From this result we may sug-
gest that the integument cells which are kept well by exog-
enous hormones should be important for the embryo sac
development, and the mechanism still need research.
Haploid plants have induced from megaspore or cells of
the female gametophyte in some angiosperm species via in
vitro cultures of unfertilized ovules and ovaries (Yang and
Zhou, 1982; Mukhambetzhanov, 1997). In the cultured
young ovaries of rice, the processes of megagametophyte
development could be switched to the formation of various
abnormally organized embryo sacs and then to the initia-
tion of synergid apogamy (Li and Yang, 1986). There are
two developmental pathways of megasporocyte in lily (Gu
et al., 1983) and tobacco (Zhu et al., 1984) via unfertilized
ovary cultures; one is abnormal that forms haploid plants
and the other is normal development of the embryo sac.
Mature embryo sac of lily (Gu et al., 1983) formed in vitro
and then degenerated for nonfertilization while in tobacco
(Zhu et al., 1981) free nuclear embryo sac which could not
form mature embryo sac developed to cell masses. Au-
tonomous endosperm has also induced from in vitro formed
embryo sacs in unfertilized ovule or ovary culture of rice
(Zhou and Yang, 1981), Voila odorata (Wijowska et al.,
1999), Lupinus luteus, Helleborous niger and Melandrium
albium (Mól et al., 1995). Abnormities could be induced in
unfertilized ovule culture in D. pulcherrima (Figs.5, 6), but
most of the embryo sac in D. pulcherrima only developed
as the routine in vivo. This indicates that different species
Table 2 Differences between in vivo and in vitro of ovule dimensions in Doritis pulcherrima
Developmental stages
X-axis* Y-axis*
In vivo In vitro In vivo In vitro
Megasporocyte 51.5± 5.3 52.0± 4.2 71.5± 6.3 97.0± 14.6
Dyad 53.0± 6.8 53.6± 6.9 78.5± 8.5 104.3± 12.1
2-Nucleate embryo sac 57.5± 3.6 65.5± 10.7 105.0± 13.4 130.0± 28.2
4-Nucleate embryo sac 66.3± 6.4 66.0± 11.2 107.5± 12.6 131.5± 28.6
Mature embryo sac 66.5± 5.5 66.3± 12.6 127.0± 10.9 131.5± 30.0
*X,Y± SD in mm.
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004844
(Managing editor: WANG Wei)
make different results in the embryo sac development in
vitro.
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