全 文 ：Embryological Studies on Facultative Apomixis in Apluda mutica (Gramineae)
MA San_Mei1 ,2 , YE Xiu_Lin1＊ , ZHAO Nan_Xian1 , LIANG Cheng_Ye1
(1.South China Insti tute of Botany , The Chinese Academy of Sciences , Guangzhou 510650 , China;
2.Department of Biology , Yantai Normal University , Yantai 264025 , China)
Abstract:　A cytological study on Apluda mutica L.revealed that this species was a facultative apomict with
60.74%of apomictic embryo sac formation.Its sexual reproduction exhibited the characteristics of polygonum_
type embryo sac formation.After the 3 megaspores at the micropylar end had degenerated , leaving only the
chalazal megaspore in the nucellus , a nucellar cell adjacent to the megaspore differentiated into an aposporous
initial , which later developed into a panicum_type embryo sac.It was an aposporous 4_nucleate embryo sac
without chromosome reduction and composed of an egg , a single synergid and a central cell with 2 polar
nuclei.The chalazal megaspore degenerated before the onset of the division in the aposporous initial cell.
Key words:　Apluda mutica ;facultative apomixis;embryology;apospory
　　The monotypic genus Apluda belongs to the sub_
family Andropogoninae of Gramineae.Apluda mutica is a
perennial grass , widely distributed in the tropical and
south subtropical Asia.It thrives throughout southwest
and South China , and usually occurs along the edges of
fields , in moist places along streams , and meadows on
slopes below 2 000 m.The young plants can be used as
forage grass[ 1 ,2] .A.mutica is a component of Savanna
in Sichuan , Yunnan and Hainan.There is little doubt
that this type of grassland is developed after forest is de-
stroyed[ 3-5] .A.mutica is adapted to the environment in
which it exists.It has some desirable traits and is of con-
siderable importance as forage grass.Thus it seems neces-
sary to study its reproductive characteristics , which may
provide some scientific basis for further exploration and
utilization of A.mutica , especially in relation to the de-
velopment of rangeland industry.
Since Murty[ 6] did the first embryological study in
Apluda mutica var.aristita in 1973 , no further investi-
gations have been carried out.Our knowledge on the em-
bryology of this genus is still very limited.The present
study was undertaken to fill up a part of the large gap in
the embryology of A.mutica.
1　Materials and Methods
All materials were obtained from a population of
Apluda mutica L.on the campus of South China Institute
of Botany , the Chinese Academy of Sciences in the sub-
urb of Guangzhou.Intact inflorescences at different stages
were collected from plants and fixed in formalin_glacial
acetic acid_ethanol(FAA)solution , and then transferred
to 70% alcohol at 4 ℃ for storage.The ovaries were re-
moved from the flowers with a pair of very fine tipped nee-
dles , stained in enriched hematoxylin , dehydrated
through an ethanal gradient series , and embedded in
paraffin.Serial sections of 5 -8 μm in thickness were
cut , observed , and photographed under a Nikon UFX_II
bright field microscope.
2　Results
2.1　Inflorescence and ovule of Apluda mutica
The inflorescence is a terminal panicle , consisting of
many pedunculate racemes.Each raceme has a naviculate
spathe and a joint on the axis.There are three spikelets(1 sessile and 2 pedicellate).The sessile spikelet is
hermaphroditic , which has two florets , but usually only
the second floret can set seed.One of the pedicelled
spikelets is reduced to a small glume;the other one that
has two florets is male or sometimes hermaphroditic.The
sessile fertile spikelet falls with the glume at maturity.
The flower has 3 stamens.The ovary is uni_locular with
one anatropous ovule , which is bitegmic and tenuinucel-
late.The styles are united at the base , with hairy stig-
mas.
2.2　Formation of sexual embryo sac
The female archesporium is always initiated at the
apex of the nucellus in the hypodermic layer in A.muti-
ca.The archesporial cell fails to engender a parietal cell
and it directly functions as the megaspore mother cell.It
becomes conspicuous by its slightly enlarged size and
deeply stained cytoplasm (Fig.1).The inner integument
begins to develop at this time.The ovule is turned at an
angle of approximately 90°to the axis of the floret.The
megaspore mother cell is somewhat elongated parallel to
the nucellar axis.Its nucleus is positioned nearer to the
micropylar pole of the cell(Fig.2).When the megaspore
mother cell is formed , it is covered externally by a single
layer of nucellar cells.The megaspore mother cell divides
by meiosis into four daughter cells as the tetrad(Fig.3).
A higher cytoplasmic concentration in the chalazal pole of
the megaspore tetrad was seen.At this time , the outer in-
tegument is formed.The three micropylar megaspores gen_
erally degenerate early.The chalazal megaspore further
Received:2001-02-05　Accepted:2001-06-26
Supported by the National Natural Science Foundation of China(3970086)and the Ninth_Five_Year Key Project of the Chinese Academy of Sciences(K 2952S1_
112).
＊ Author for correspondence.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2002 , 44(3):259-263
260　 植物学报　Acta Botanica Sinica　Vol.44　No.3　2002
undergoes an over_all enlargement , maintaining a relative-
ly large size and developing into a uni_nucleate embryo
sac(Fig.4).The nucleus of the chalazal megaspore di-
vides and the daughter nuclei occupy positions at the poles
of the cell , leaving a vacuole between them.This is the
two_nucleate stage of the embryo sac and the nuclei them-
selves are designated as the primary micropylar and the
primary chalazal nucleus respectively , depending on their
respective position (Fig.5).Both nuclei divide twice.
The first division results in a group of two nuclei at either
end of the embryo sac (Figs.6A , 6B).The second divi-
sion results in a group of four nuclei at either end of the
embryo sac (Fig.7).The organized embryo sac shows a
typical 8_nucleate , 7_celled structure.
2.3　Formation of the apospory embryo sac
The development of some polygonum embryo sacs is
prevented in A.mutica.When the only one chalazal
megaspore exists in the nucellus , a nucellar cell situated
often close to the megaspore grows considerably in size as-
sociated with gradual vacuolation of the cytoplasm.Such a
cell functions as the initial of an aposporous embryo sac(Figs.8A , 8B).Moreover the location of the cells is
variable.Nucellar cells which are located in the immedi-
ate neighbourhood of the megaspore and the cells that are
remotely located in the nucellus , all could function as the
initial cells for aposporous development.In all these cas-
es , the megaspore degenerates before the onset of division
in the initial cell that is destined to develop into an
aposporous embryo sac.The nucleus of the aposporous
initial cell directly embarks on mitosis resulting in the for-
mation of unreduced (that is , diploid)nuclei.The
daughter nuclei never separate from one another but stay
in one polar for a significant period (Figs.9-14).The
first division results in a group of two nuclei at micropylar
end of the embryo sac (Figs.9 ,10).The second division
results in a group of four nuclei also at micropylar end of
the embryo sac(Figs.11 ,12).The organized embryo sac
shows a typical 4_nucleate , 3_celled structure.The four
nucleate embryo sacs had an egg apparatus consisting of
an egg , a single synergid and two polar nuclei(Figs.13 ,
14).The wall of the aposporous embryo sac is less clear
than that of the polygonum embryo sac.
2.4　Degree of apospory
A total of 242 ovules with mature embryo sac was
observed in the experiment , among which 95 ovules have
polygonum embryo sac and 147 ovules have apospory em-
bryo sac.The frequency of ovules with unreduced embryo
sac is 60.74%.
3　Discussion
3.1　Differentiation of the aposporous initial cell
In Pennisetum squamulatum , during the period of
formation or degeneration of the megaspore or the triad a
number of nucellar cells around the degenerated sexual
cell became much enlarged.Frequently , one of the en-
larging nucellar cells near the micropylar end became vac-
uolated and then developed into an aposporous uninucleate
embryo sac
[ 7] .Cytological studies on guinea grass(Pan-
icum maximum)indicate all megaspores usually degener-
ate;one or more adjacent somatic cells of the nucellus
will grow and divide into aposporous embryo sacs after a
normal reduction division.Initial cell is placed in the im-
mediate neighbourhood of megaspore , or remotely located
in the nucellus in Poa pratensis , Brachiaria decumbens ,
Panicum maximum and Pennisetum ciliare[ 8-10] .The lo-
cation and the time of differentiation of initial cell in A.
mutica have not been investigated in previous studies.
The data presented in this paper show that initial cell of
aposporous embryo sac is differentiated at the stage of only
one chalazal megaspore existing in the nucellus.It is lo-
cated about 8μm from the megaspore.Therefore the loca-
tion and the time of differentiation are variable in different
species.
3.2　Two kinds of embryo sacs
Murty[ 6] found the reproduction mode in A.muti-
ca , but he did not report the formation of embryo sac.A
higher cytoplasmic density in the chalazal pole of the
megaspore tetrad was observed in the present research.
This polarity may be related to the selection of the func-
tional megaspore.There is a large central vacuole in poly-
gonum embryo sac from the 2_nucleate stage.The wall of
embryo sac is very clear from the 2_nucleate to the 8_nu-
cleate stage.Apospory is the type of apomixis that occurs
when the embryo sac is formed from the initial cell.The
first division is at the right angles to the longitudinal axis
of the cell and occurs very close to the micropylar end.
The second division of the two daughter nuclei forms four
nuclei , all of which remain at the micropylar end of the
embryo sac.Large central vacuole separating the nuclei is
not found in the aposporous 2_nucleate to aposporous 4_
nucleate stages.The absence of the large central vacuole
separating the nuclei may be used as indication for
screening apospory.Previous observation did not mention
the wall of the embryo sac.However , in the species ex-
amined in this study , the wall of aposporous embryo sac is
less clear than that of the polygonum embryo sac.
→
Figs.1-7.　 Formation of polygonum embryo sac.1.Megaspore mother cell stage , showing the megaspore mother cell(arrow).Note the
development of inner integument(hollow arrow), ×750.2.Megaspore mother cell at a later stage of development.Note the much elongated
cell—its nucleus is positioned nearer to the micropylar pole(arrow), ×700.3.Linear tetrad of megaspores(arrowheads), showing the de-
velopment of outer integument(hollow arrow), ×750.4-7.Stages in the formation of polygonum embryo sac.4.Uninucleate embryo sac ,
showing the functional megaspore in the middle of nucellus(arrow)and the remnants of the three micropy lar megaspores (hollow arrow),
×600.5.Polygonum binucleate embryo sac with a large vacuole in the middle of the embryo sac , ×600.6A , 6B.Polygonum four_nucleate
embryo sac in two adjacent sections.There is a large vacuole in the middle of the embryo sac, ×600.7.Polygonum 8_nucleate embryo sac
before wall formation.There is a large vacuole in the middle of the embryo sac , ×600.
MA San_Mei et al:Embryological Studies on Facultative Apomixis in Apluda mutica (Gramineae) 261　
262　 植物学报　Acta Botanica Sinica　Vol.44　No.3　2002
Embryological investigations of apospory in A.mutica re-
vealed some new characteristic feature of apospory.These
morphological criteria of the panicum type of embryo sac
may also be used for screening apospory.
3.3　The frequency of aposporous embryo sac
The frequency of apomictic embryo sac estimated in
the experiment is 60.74%.When sexual and asexual re-
production occur simultaneously , can environmental factor
influence the frequency of aposporous embryo sac ?No in-
vestigation has been undertaken.Further work is neces-
sary in order to understand its reproductive character.
Acknowledgements　We express our sincere gratitude to
HU Qi_Ming for correcting the manuscript.
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水蔗草兼性无融合生殖胚胎学研究
马三梅1 ,2　叶秀 1＊　赵南先1　梁承邺1
(1.中国科学院华南植物研究所, 广州 510650;2.烟台师范学院生物学 , 烟台 264025)
摘要:　对水蔗草(Apluda mutica L.)的生殖方式进行研究 ,结果表明水蔗草进行兼性无融合生殖。胚囊发育分为两
种类型 ,即有性生殖的蓼型和无孢子生殖的大黍型。无融合生殖胚囊频率为 60.74%。 在大孢子母细胞发育至四
分体后 ,珠孔端的 3 个大孢子解体。合点端的大孢子未解体时 ,邻近大孢子的 1 个珠心细胞开始特化 , 形成无融合
生殖的原始细胞 ,由该原始细胞发育形成有 1 个卵细胞 、1 个助细胞和 2 个极核的四核胚囊。
关键词:　水蔗草;兼性无融合生殖;胚胎学;无孢子生殖
中图分类号:Q944.47　　　文献标识码:A　　　文章编号:0577-7496(2002)03-0259-05
收稿日期:2001-02-05　接收日期:2001-06-26
基金项目:国家自然科学基金(3970086);中国科学院“九五”重点项目(K2952S1_112)。
＊通讯作者。
(责任编辑:梁　燕)
→
Figs.8-14.　 Formation of the apospory embryo sac.8A.The functional megaspore is still visible in the photograph , which degenerates
sooner or later as in the first section(arrow).Note the remnants of three micropylar megaspores(hollow arrow), ×700.8B.An aposporous
initial cell in the third section(arrow), which is a somatic cell usually from the center of the nucellus , ×700.9.An aposporous binucleate
embryo sac of early stage(arrow).There is not a large vacuole in the middle of embryo sac , ×700.10.An aposporous binucleate embryo sac
of later stage(arrow).There is not a large vacuole in the middle of the embryo sac , ×600.11A , 11B.An aposporous 4_nucleate embryo sac
in early stage;there is not a large vacuole in the middle of the panicum embryo sac.×400.12.An aposporous 4_nucleate embryo sac in later
stage.Four nuclei remain at the micropylar end of the embryo sac.×400.13A , 13B.Mature unreduced aposporous embryo sac , showing the
mature aposporous embryo sac with an egg cell(E), a synergid and two polar nuclei(P).Synergid starts degeneration(arrowhead)and there
is no antipodals in the panicum embryo sac.×300.14.An egg cell and two polar nuclei in the aposporous embryo sac, showing one degener-
ated synergid in the embryo sac.There is no antipodals in the embryo sac, ×300.
Abbreviations:DS , degenerating synergid cell;E , egg cell;P , polar nucleus.
MA San_Mei et al:Embryological Studies on Facultative Apomixis in Apluda mutica (Gramineae) 263