全 文 ：Received 27 Mar. 2003 Accepted 7 Aug. 2003
Supported by the National Natural Science Foundation of China (39980016).
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Embryological Evidence of Apomixis in Eulaliopsis binata
YAO Jia-Ling*, YANG Ping-Fang, HU Chun-Gen, ZHANG You-De, LUO Bin-Shan
(College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China)
Abstract: Embryological investigation was carried out on apomixis in Eulaliopsis binata (Rotz) C. E. Hubb
by using paraffin section method. The results indicated that the development pattern of the embryo sac
was apospory. In the early stage of the ovule development, a few of the nucellar cells developed into
aposporous initial cells, which differentiated later into two forms of mature embryo sac: (1) one form of the
mature embryo sac contained one egg cell, one synergid and two polar nuclei; (2) another form possessed
one egg cell, two synergids and one polar nucleus. The former occupied about 67.6% and the later only
32.4%. The development pattern of the aposporous embryo sac resembled the Panicum type. Multiple
initial cells of apospory might undergo development simultaneously to form two- or multiple-embryo sacs.
The ratio of multiple mature embryo sacs in one ovule was 17.7%. No sexual embryo sac was found in the
observed ovules. The genesis of the embryo could be classified into two types according to their initial
time: (1) the pre-genesis embryo (74%), which originated from unreduced egg cell before the division of the
polar nucleus, was observed at one to two days earlier than anthesis. (2) the late-genesis embryo (26%)
which was observed at one or two days after anthesis and formation of free endosperm nuclei. The
endosperm was derived from the polar nucleus or secondary nucleus without fertilization. The process of
the embryonic development followed the sequence of the sexual embryo. The frequency of polyembryony
observed was 13%.
Key words: apomixis; embryology; apospory; Eulaliopsis binata
Asexual seed production (agamospermy) in flowering
plants via gametophytic apomixis is accomplished by
apomeiosis followed by parthenogenesis. Two major types
of gametophytic apomixis have been described, namely
aposporous and diplosporous apomixis, depending on the
origin of the unreduced megagametophytes. In apospory,
unreduced female gametophytes develop from a cell of the
nucellus, and in diplospory megasporocytes arise directly
from mitotic or mitotic-like divisions of the megasporocyte.
Parthenogenetic development of the unreduced egg cell
gives rise to embryos that are genetically identical to the
maternal parent (Asker and Jerling, 1992), a feature of great
value for plant breeding and seed production. If apomictic
nature could be introduced into sexual crops, it would
greatly simplify breeding schemes and allow the fixation of
any genotype, including that of F1 hybrids (Grossniklaus
et al., 1998; Spillane et al., 2001a). There are more than 400
plant species which reproduce asexual seed by apomixis,
but typically, apomixis is a facultative phenomenon in which
an apomictic plant usually produces a few meiotic embryos
beside a majority of ameiotic embryos (Sherwood et al.,
1980; Spillane et al., 2001b), which variation in the
expressivity of apomixis may create an additional complica-
tion in genetic studies and breeding field. For nearly two
decades, the genetic control of apomixes has been eluci-
dated in very few species. Recently, however, inheritance
studies for several natural apomicts have been published
that shed new light on the genetic control of this important
developmental process (Noyes and Riesseberg, 2000;
Grossniklaus and Nogler, 2001). So, it has become an inten-
sively studied field seeking the genetic resources for obli-
gate apomixis.
Eulaliopsis binata is a perennial grass, which belongs
to the subtribe Apocopidinae in Gramineae, widely distrib-
uted in the south part of Qinling moutain in China (Liu, 1988),
and has been used in the conservation of water and soil for
its thrive roots. It is also an excellent material in the paper
making industry for its long vegetable fiber (Zhou,1990;
Zhang et al., 1996). Recently, apomixis phenomenon was
observed in E. binata (Zhang et al., 1996). However, the
information about its reproduction is still limited. Whether
or not the sexual reproduction and apomixis coexists in E.
binata, and whether the stimulation of pollination is neces-
sary to the development of the embryo and endosperm is
really not known. In order to reveal the pattern and degree
of apomixis (facultative or obligate) in E. binata, careful
Acta Botanica Sinica
植 物 学 报 2004, 46 (1): 86-92
87YAO Jia-Ling et al.: Embryological Evidence of Apomixis in Eulaliopsis binata
embryological investigation is needed. This article presents
the results of our embryological investigation.
1 Materials and Methods
All materials were obtained from a population of slen-
der-leaf and red haulm ecotype Eulaliopsis binata (Rotz)
C. E. Hubb which were grown in the experimental field of
Huazhong Agricultural University. At the flowering stage,
samples were collected every 1-3 h. Each time five panicles
(about 350 to 400 flowers) were harvested and fixed in
Carnoy’s Fluid (95% alcohol : acetic acid, 3:1) then stored
in 70% alcohol solution. Before anthesis and after blossom,
samples were collected and fixed once every day. Ovaries
were peeled and stained with Ehrlich’s haematoxylin. Se-
quential paraffin sections 8-10 mm thick were made. Obser-
vation and photography were conducted under an Olympus
Vanox AH3 microscope.
2 Results
2.1 The origin and development process of embryo sac
The inflorescence is a digitiform panicle, consisting of
many pedunculate spikelets. There are two kinds of florets
in one ear of E. binata, one is hermaphroditic and the other
one is single male. The flower has three stamens and pos-
sesses a unilocular ovary with an anatropous ovule, which
is bitegmic and tenuinucellatae.
In the early stage of ovule development, one or more
nucellar cells could be distinguished by its slightly enlarged
size and obvious nuclei and nucleoli (Figs.1, 2). These spe-
cialized nucellar cells were found usually in the 4th to 6th
cell layer of the nucellar tissue. They were round in shape
at first and then, gradually changed into a long rectangular
shape with an increase in cell volume. With the cytoplasm
tenuating gradually, the nuclear and nucleoli seemed to be
more obvious and distinctive (Figs.1, 2). Later, the special-
ized cells developed into aposporous embryo sac. Though
the sexual archesporial cell and megasporocyte was ob-
served under the epidermis of nucellus in very few (about
1%) ovules, they degenerated soon without any further
development (Fig.3).
The primary aposporous cells continued to enlarge with
the nucleus in the center until many small vacuoles emerged
in cytoplasm and later combined into a large vacuole. Thus
the aposporous uninucleate embryo sac was formed (Figs.
4, 5). Later on, the nucleus moved toward the micropylar
end and underwent the first division to form a two-nucleate
embryo sac. The mitosis usually occurred in a perpendicu-
lar direction to the vertical section of the embryo sac and
the two nuclei were located side by side near the micropyle
(Fig.6). After another mitosis division, 4-nucleate embryo
sac was formed (Fig.7). Then, two types of mature embryo
sacs were produced as follows: one type of mature embryo
sac contained one egg cell, one synergid and one central
cell of two polar nuclei (Figs.10,11); while another type
possessed one egg cell, two synergids and one central cell
of single nucleus (Fig.12). The former occupied about
67.6% and the latter only 32.4%. Antipodal cells were com-
pletely lacking in the both types. The mature embryo sac
had a polarity with micropyle or integument end located
egg apparatus and the centralized polar nucleus (Figs. 13-
15). Along with the development of aposporous embryo
sac, the adjacent nucellar cells continued to disintegrate
and resulted in an enlargement of the cavity in the embryo
sac. Development of the sexual embryo sac was not ob-
served in the observed 2 680 ovules.
Several initial cells of apospory embryo sacs could be
observed coexisting in one ovule and co-developed syn-
chronously or asynchronously (Figs.1, 2, 4-9). According
to the statistics, two or more mature multiple embryo sacs
were observed simultaneously in 17.7% of the ovules. The
membranes of the aposporous embryo sacs are not so eas-
ily identified. In this case, there should be one mature em-
bryo sac near the micropylar end, while others distributed
randomly, they might be observed either at the chalazal
end or in the center or in elsewhere in the ovules (Figs.13-
15).
2.2 The genesis and development of embryo and en-
dosperm
The egg cell became intensively vacuolized, and en-
larged with strict polarity. The nucleus was near the cha-
lazal end while the large vacuole was located in the micro-
pylar end (Figs. 12,16). According to the initial time, the
embryogenesis could be divided into two types. One was
the pre-generated embryo (74%), which happened 1-2 d
earlier than anthesis. Another type was the late-generated
embryo (26%), which could be found 1-2 d later than
anthesis. The pre-generated embryo was derived from the
unreduced egg cell which produced pro-embryo (Fig.16)
before division of the polar nucleus. When the globular
embryo formed the polar nucleus had not divided yet (Fig.
17). As to the latter, when the egg was about to divide, the
polar nucleus had already divided into several free en-
dosperm nuclei, and meanwhile, the synergids had disap-
peared as a result of degeneration (Fig.18). No matter when
they were produced, both types of the embryos derived
from egg cells would undergo development following the
process similar to sexual embryo differentiation (Figs. 19,
20). At the last, the matured embryo (Fig.21) possessed
Acta Botanica Sinica植物学报 Vol.46 No.1 200488
Figs.1-12. 1. Aposporous initial cells (arrows), × 630. 2. Enlarged aposporous initial cells (arrows), × 630. 3. A degenerated
archesporial cell, ×630. 4. An aposporous initial cell (arrow) and 1-nucleate aposporous embryo sac with a vacuole (V), ×630. 5. Two
1-nucleate aposporous embryo sac (arrow), ×630. 6. A 2-nucleate aposporous embryo sac (T) and two 1-nucleate embryo sacs
(arrows), ×600. 7. A 4-nucleate embryo sac (F) with a vacuole and two degenerated cavities (D), ×400. 8. Showing three nuclei (arrows)
in one 4-nucleate embryo sac, while three cells in another embryo sac, ×400. 9. Adjacent optical sections 9A and 9B. 9A shows a polar
nucleus (P), synergids (S), one degenerated 2-nucleate embryo sac (T), and a degenerated cavity (D); 9B shows an egg in the mature
embryo sac, ×400. 10. A mature embryo sac with two polar nuclei (P) and one synergid in degeneration (DS), one egg cell (E) is division,
×400. 11. A mature embryo sac with one polar nucleus (P), one egg cell (E) and one synergid(S), ×400. 12. Sections 12A and 12B are
adjacent optical sections. 12A shows one polar (P) and the nuclei of two synergids (S and arrows), 12B shows the egg cell, ×540.
89YAO Jia-Ling et al.: Embryological Evidence of Apomixis in Eulaliopsis binata
shoot apex, coleoptile, radicle, coleorhiza, scutellum, etc.
The endosperm derived from polar nucleus or
secondary polar nucleus. While the embryo was in the pear-
shaped stage, the free endosperm nuclei started to form
Figs.13-21. 13. Multiple mature embryo sacs (ES1), (ES2) and a disintegrated embryo sac (ES3), ×200. 14. 14A showing multiple
mature embryo sacs ES1, ES2, a 2-nucleate embryo sac （T） and a degenerated embryo sac (D); 14B showing the egg cell (E), synergid
(S) and polar nucleus (P) in ES2; 14C showing the other mature embryo sac (ES3) located in the micropylar end, ×200. 15. Showing
three embryo sacs, two of them at mature stage (ES1 and ES2) located in micropylar end and chalaza end respectively, and in another
embryo sac (ES3), two polar nuclei (P) were seen, ×200. 16. Showing synergid (S), the 2-celled pro-embryo（EM）, ×480. 17. A
globular embryo（EM） and polar nucleus (P), ×400. 18. Specialized egg cell (E) and several free endosperm nuclei (arrows), ×400.
19. Two embryos, one of which was pro-embryo (EM1), another was in differentiation (EM2), ×200. 20. Two embryos（EM1 and
EM2） in differentiation, ×200. 21. A mature embryo (EM), ×100.
Acta Botanica Sinica植物学报 Vol.46 No.1 200490
endosperm cell from the micropylar end firstly then to other
positions. In about 7.6% ovules, no endosperm generated,
which caused the arrest of the embryo development, lead-
ing to shrunken seeds.
Because in observed more than two thousand ovules,
no symptoms of fertilization, such as entrance of pollen
tube into embryo sac, were observed, and moreover, the
synergids remained integrating while the egg cell initiated
mitotic division. These results indicated that the embryo
derived by parthenogenesis and the endosperm formed by
division of polar nucleus autonomously in E. binata. The
seed formation was fertilization independent.
Poly-embryo phenomena were easily found in apospory
plants. In E. binata, poly-embryo was observed at a fre-
quency of 13%. They originated from the egg cells in vari-
ous embryo sacs. They might be in different development
stage (Figs.19, 20).
3 Discussion
3.1 Characteristics of the genesis and development of
apospory embryo sac in Eulaliopsis binata
As documented in other aposporous plants, the initial
cell of apospory embryo sac usually originated from nucel-
lar cell (Nogler, 1984; Dujardin and Hanna, 1984; Li et al.,
1996; Yao et al., 1997; Wen et al., 1998). Cytological studies
on guinea grass (Panicum maximum) indicate all me-
gaspores degenerate; one or more adjacent somatic cells of
the nucellus will grow and divide into aposporous embryo
sacs after a normal division (Warmks, 1954). However, while
initial cells of the aposporous embryo sac formed, archespo-
rial cell and megasporocyte of sexual embryo sac were sel-
dom observed in Eulaliopsis binata. The possible reason
might be that they degenerated at very early stage.
Therefore, the genesis modes of apospory embryo sac might
be variable in different species.
According to Brown and Emery (1958), apospory em-
bryo sac in Gramineae could be divided into two types:
Panicum type and Hieracium type. Based on our
investigation, the generation of aposporous embryo sac in
E. binata belonged to the former. One or more specialized
nucellar cells vacuolized at first, then the nuclei divided
twice to produce 4-nucleus embryo sac. With further
differentiation, two types of mature embryo sac were con-
structed as mentioned above, which is in accordance with
the report in Panicum maximum (Warmks, 1954). However,
in Pennisetum squamulatum and P. ciliare (Hanna, 1992;
Wen et al., 1998), another species of the Panicum type,
only the second type of mature embryo sac was formed.
Why are there two kinds of apospory embryo sacs in E.
binata simultaneously and what kinds of mechanism are
there in E. binata? Koltunow (1995) described the enor-
mous variability existing in apomoctic processes. In
Hieracium, apomictically derived embryo sacs usually re-
produce through apospory. However, several loci modify
the timing of apomictic initiation, the frequency at which
apomictic embryo sacs are formed, and the mode of pro-
gression of apomictic development (Koltunow, 2000). These
findings indicate that the major locus associated with apo-
mixis might create a competence for a variety of reproduc-
tive developmental processes in the ovule. Our results add
support to this opinion. The developmental mechanisms
employed by different apomoctic species, however, are re-
markably varied, possibly reflecting the apparent polyphyl-
etic origin of this trait among flowering plants.
Also, we noted in E. binata that there were several ma-
tured embryo sacs presented in the same one ovule with
the frequency of 17.7%. Among the apospory species, one
or more initial cells in one nucellus undertake development
at the same time. Usually, only mature embryo sac is lo-
cated in one nucellus, but the descriptions about poly-
embryo sacs were reported in Pennisetum and Poa (Nogler,
1984). Producing more female gametophytes may be a re-
productive strategy to apomictic plants.
Dujardin and Hanna (1984) and Wen et al. (1998)con-
sidered Pennisetum ciliare and P. squamulatum as an ob-
ligate apospory plant that only the aposporous embryo
sac would develop further, whereas the sexual embryo sac
would degenerate in certain stage. In more than two thou-
sand ovules, the development of sexual embryo sac was
not observed at all, only the aposporous embryo sac was
generated in E. binata. Therefore, based on the develop-
ment of embryo sac, it might be suggested that E. binata
should be an obligate apospory species.
3.2 Development of embryo and endosperm in Eulaliopsis
binata
In aposporous plants, the embryo could develop with-
out stimulation of fertilization. The unreduced egg cell ini-
tiated embryogenesis in the absence of fertilization
(parthenogenesis) in E. binata as well as in other
aposporous species. Interestingly, the initial time of par-
thenogenesis of egg cell was different, which was consis-
tent with the reports in Pennisetum aquamulatum (Wen et
al., 1998).
For agricultural applications, it is essential that en-
dosperm development in engineered apomicts is normal.
Most apomictic seed formation requires fertilization of the
central cell (pseudogamy) (Nogler, 1984; Asker and Jerling,
1992; Spillane et al., 2001b). We had paid much attention to
91YAO Jia-Ling et al.: Embryological Evidence of Apomixis in Eulaliopsis binata
making sure if fertilization be involved in endosperm forma-
tion in E. binata. However in two years’ investigation, we
had not find evidence to support this usual opinion. Dur-
ing flowering time, we fixed samples every 1-3 h but we
thoroughly failed to observe any symptom of fertilization.
We did not find trace of pollen tube or sperm nucleus in
embryo sac. We also applied fluorescence method to check
if pollen tube entered the embryo sac. We found that only
few of pollen could germinate on stigma but none of them
could elongate further and stretch into ovule (data not
shown). This result also suggested that no stimulation of
male gamete and no pseudo-fertilization were involved in
egg cell division and endosperm formation. Similar results
were reported in Hieracium, Alnus rugosa, Allium nutans,
Malus hupehensis and Chondrilla spp. (Nogler, 1984;
Grossniklaus et al., 2001), but no records from Gramineae.
Our work provided an exception in apomictic monocots.
Koltunow reported that a dominant locus is required for
apospory and autonomous embryo/endosperm formation
in apomictic Hieracium, and the dissection of this locus is
a major objective in their laboratory (Koltunow et al., 2000).
Why do the autonomous apomictics not need a sexual
endosperm, and how do they manage without one? Is the
genetic regulation in E. binata identical with dicots or not?
Further work is necessary in order to understand its repro-
ductive characteristic.
In our study, polyembryony was observed with the fre-
quency of 13%, which came from different embryo sacs in
the same ovule. Moreover, the embryo and endosperm may
come respectively from different embryo sacs. The results
interrelate with the poly-mature embryo sac. After seed
germination, twin- or multiple-seedlings were observed in a
frequency of 8.4%, which suggested that most of the poly-
embryos could develop into mature stage.
Although the conclusion that Eulaliopsis binata should
be an obligate apomixis plant needs to be corroborated
through genetic and cytological investigation, it will be
another new valuable genetic resource in Gramineae in ad-
dition to species, such as Pennisetum ciliare, Tripsacum
dactyloides, which has shown great potential in crop
breeding.
Acknowledgements: We would like to thank Prof. Anna
KOLTUNOW of CSIRO Plant Industry Horticulture Unit
(Waite Campus, Australia) for her critical comments on this
investigation, and Dr. June HAMMOND of Sydney Uni-
versity for her kind amending of this manuscript in English.
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