全 文 ：打碗花(旋花科)受精前和受精后的卵器和
中央细胞的超微结构
胡赞民 　胡适宜
(北京大学生命科学学院　北京　100871)
摘要　观察了受精前打碗花(Calystegia hederacea Wall.)成熟胚囊的超微结构及在受精后的变化 , 并描
述了卵细胞 、助细胞与中央细胞在胚囊中部位结构上的关系。证明它们之间的界壁大部分只具质膜 , 表
现与被子植物的大多数在受精的靶区缺少壁相似的特征。在超微结构上 ,雌性生殖单位各组成细胞除
具一般的特征外 ,还表现有一些不寻常的特点:卵细胞核位于合点端的大液泡之上 , 细胞质中含多聚核
糖体;中央细胞不仅在向珠心的一面具壁内突 ,而且在向助细胞一面也具内突。这些特点可能与其受精
周期短和缺少反足细胞相关。作者提出了把打碗花雌性生殖单位作为一个结构与功能相适应的结构 ,
以保证成功的受精的结论。
关键词　雌性生殖单位 ,卵细胞 , 助细胞 ,中央细胞 , 超微结构 ,打碗花
ULTRASTRUCTURES OF THE EGG APPARATUS AND THE
CENTRAL CELL OF CALYSTEGIA HEDERACEA(CONVOLVULACEAE)BEFORE AND
AFTER FERTILIZATION

HU Zan-Min 　HU Shi-Yi
(College of Li fe Sciences , Peking U niversity , Bei jing 100871)
Abstract　Ultrastructures of the mature embryo sac of Calystegia hederacea Wall.and i ts
changes after fertilization are described.The positional org anization of the egg cell , the tw o
synergids and the central cell , as w ell as thei r interrelationships w ere studied.Some regions
of the cell boundaries between the egg cell and the central cell , as well as between the egg
cell and the synergids were devoid of typical cell w all before fertilizat ion , displayed a feature
quite similar to the characteristic absence of the cell w all in the fertilization target zone
occurred in most angiosperms.Besides the general ult rast ructural characteristics of the egg
apparatus and the central cell , there w ere several unusual aspects in C.hederacea , such as
the egg nucleus located above the large vacuole near the chalazal end of the cell , many
poly ribosomes in the cy toplasm of the egg cell and w all ing row ths on bo th sides of the hooks
of the central cell.All these unusual characteristics seemed to be closely associated w ith the
short duration of the fertilizat ion and the absence of antipodal cells in the mature embryo sac.
It is concluded that the female germ unit of C.hederacea is considered to be a topog raphical
and physiological unit to realize thei r functions for successful double fertilization.
Key words 　Female germ unit , Egg cell , Synerg id cell , Central cell , Ultrastructure ,
Calystegia hederacea


Author for correspondence.
Received:1997-08-20 Revised:1997-11-14
Present address:Institute of Genet ics , the Chinese Academy of S ciences.
This research w as supported by the Nat ional Natural Science Foundation of China.
植　物　学　报　1998 , 40(6):487 ～ 492
Acta Botanica Sinica
A body of investigations on the ultrast ructure of mature embryo sac in many
angiosperms have been reported[ 1] .A general feature of the egg , the tw o synergids and the
central cell particularly adapting to reproduction is the absence of a w all in the target zone ,
where the sperms must fuse w ith and enter through.Dumas et al[ 2] proposed the concept of
the female germ unit(FGU).During the last decade the ult rastructural observations on the
mature embryo sac provided some new evidences to support the concept of FG U as a normal
functional unit in double fertilizat ion[ 3 ,4] .When we investigated the transmission of plastids
during the fertilization in several species of Convolvulaceae , we observed some unusual
characteristics of the egg cell , the synergids and the central cell in Calystegia hederacea ,
which have been paid very lit tle attention in o ther species.This paper deals wi th the
ultrastructural changes of the egg apparatus and the central cell before and af ter fertilization
in C.hederacea , for further understanding the role of FGU in fertilization.
1　MATERIALS AND METHODS
The experimental material Calystegia hederacea Wall.was g row n in the plant garden of
Agricultural Institute of Haidian Dist rict , Beijing.Flow ers at different developmental stages
w ere sampled for elect ron microscopy.Collections included unpollinated flow ers w hich w ere
emasculated one day before the time when their petals w ere just opened in the morning , and
pollinated flow ers at 4 , 8 ,10 and 12 h af ter hand pollination.Ovules were immediately placed
in 0.2 mol/L sodium cacody late buf fer with 3% glutaraldehyde , pH 7.2 , for 4 to 6 h at
room temperature.After w ashed w ith the same buffer fo r 3 times (each for 30 min), the
ovules w ere placed in 1% osmium tetroxide for 0.5 to 1 h at room temperature and then
overnight at 4 ℃.The ovules were then rinsed 4 times in sodium cacodylate buf fer each fo r
30 min , dehydrated in a graded alcohol series , and immediately polymerized at 60 ℃ for 24
h.Ultrathin sections w ere cut wi th a diamond knife on a LKB ult ratome V and stained w ith
urany l acetate and lead citrate.Sections were observed under a JEM-100CX transmission
electron microscope at 100 kV.
2　RESULTS
2.1　Positional organization of the egg apparatus and the central cell
The posi tional organization of the egg apparatus and the central cell in the mature
embryo sac(ES)of Calystegia hederacea is illust rated in Fig.1.The ant ipodal cells have
degenerated at the mature ES stage.The chalazal end of the ES is not show n in this figure.
Two synergids are highly polarized by the presence of a large vaculole at each chalazal end.
They are at tached to the ex treme micropy lar end of the ES.A thick t rident-like filiform
apparatus(FA)is jointly formed by both synergids f rom each own micropylar end of the w all
(Pl.Ⅰ:1).The egg cell and tw o synergids are in a t rianglar arrangement.The egg cell is
also highly polarized by a large vacuole , which is unusually located at the chalazal end of the
cell , thus it s nucleus in the dense cy toplasm is located above the large vacuole(Pl.Ⅰ:2).I t
is considered that the egg cell has a point at tached to the embryo sac w all at a certain distance
aw ay from the micropylar end of the tw o synergids.At the chalazal end of the egg
apparatus , there is no typical cell wall betw een the egg cell and the synergids , as well as the
egg cell and the central cell.The spaces between these cells are only lined with two plasma
membranes(Pl.Ⅰ :2;Pl.Ⅱ:5).Although these spaces are alw ay s narrow , they may be
chequered w ith some expanded reg ions , in w hich a few fibrous materials or small vesicles can
be observed(Pl.Ⅱ:3).The large and elongate shaped central cell is also highly polarized , in
w hich a sing le large vacuole occupies the position f rom the chalazal end to the two thirds of
the cell , and its cytoplasm is rest ricted to the periphery and to the neighbourhood of the egg
488　 植　　物　　学　　报 40 卷
apparatus.Polar nuclei are located near the egg apparatus and surrounded by the dense
cytoplasm(Pl.Ⅱ:6).The micropy lar part of the central cell surrounds the egg apparatus to
make the appearance of two hook-like structures (hooks in brief)at both sides of the
synergids in the longitudinal section of the embryo sac(Fig .1).
By viewing the topology of the egg cell , the synergids and the central cell as a w hole , i t
seems rational to consider that they are closely related to the female germ unit during
fertilization.
2.2　Ultrastructural characteristics of synergids before and after fertilization
Generally , one of the tw o synergids usually degenerates prior to o r after the arrival of
the pollen tube in angiosperm .C.hederacea belongs to the lat ter case.At anthesis , tw o
synergids are similar in structure.Each has a prominent nucleus with a single nucleolus(Pl.
Ⅰ:1).The cy toplasm of each synergid is rich in organelles:Plastids are abundant , each
containing one to several starch grains , and mitochondria w ith well developed cisternae are
numerous;many rough endoplasmic reticulum (RER)are present , their single or several
cisternae array in parallel;coated vesicles are numerous , which seem to be formed from the
sw ollen profiles of RER(Pl.Ⅰ :1;Pl.Ⅱ:7 , 8).All organelles in both synergids have no
obviously regional dist ribution.The ult rastructures of these organelles indicate that both
synergids may function actively prio r to fertilization.Moreover elect ron dense materials
probably secreted by the synergids are deposited in the space betw een the membranes of the
tw o synergids(Pl.Ⅱ:4).
After pollination for 4 h , the pollen tube has entered one of the synerg ids , which
becomes deeply stained and degenerated (not show n in the figure).Eight hours after the
pollination the persistent synergid remains in ultrast ructurally unchanged as compared w ith
that before pollination.However , dictyosomes seem to be more active and result to form
numerous vesicles.The amount of RER and coated vesicles remains relatively abundant , but
starch grains disappeared in the plastids (Pl.Ⅱ:9).The persistent synergid begins to
degenerate 12 h after pollination , when a proembryo with a few cells has formed.Wall
ingrow ths of FA in both synergids are obviously impeded and lose thei r o riginal feature (Pl.
Ⅳ:19).
Fig.1　Diagrammatic reconstruction o f the positional
organization of the female germ unit in
Calystegia hederacea
CC.Cent ral cell;E.Egg cell;FA.Filiform apparatus;
PN.Polar nucleus;SY.Synergid;V.Vacuole
2.3　The egg cel l before fertilization and the
zygote at early developmental stage
At anthesis , the egg nucleus and the
majori ty of the cy toplasm are located above the
large vacuole in the cell;only a thin layer of
the cytoplasm surrounds the large vacuole(Pl.
Ⅰ:2).The cy toplasm exhibits a complement
of o rganelles. Mitochondria are relatively
abundant.Plastids are somewhat less than
mitochondria , but only a few contain several
starch g rains (Pl.Ⅲ:10).Ribosomes are
abundant and polyribosomes are obvious (Pl.Ⅲ:11).A few parallel RER are present.The
nucleus has a sing le nucleolus and dispersed
chromatin(Pl.I:2).Some nuclear pores may
be seen on the nuclear envelope (Pl.Ⅲ:11).
By 8 h af ter pollinat ion , fusion of the
sperm and the egg is completed.The most
notable ultrastructural changes from the egg to
a zy gote are :(a)Elect ron-opaque deposits appear in the expanded space reg ions betw een the
6 期 胡赞民等:打碗花(旋花科)受精前和受精后的卵器和中央细胞的超微结构(英) 489　
egg cell and the persistant synergid , as well as betw een the egg cell and the central cell(Pl.
Ⅲ:12 to 14);(b)The polarity of the egg cell becomes unclear , because of an increase of the
cytoplasm and the segregation of the sing le large vacuole into a number of small ones
distributed evenly in the cytoplasm.As a result , the nucleus is pushed tow ards the center of
the cell(Pl.Ⅲ:12);(c)The amount of cy toplasm including org anelles is increased.The
o rganelles are distributed all over the cy toplasm , but more concentrated arround the nucleus.
The starch-g rain-bearing plastids and poly ribosomes are obviously increased(Pl.Ⅲ:12 ,13);
(d)Highly dense nuclear pores appear on the nuclear envelope of the zy gote (Pl.Ⅲ:15)
af ter pollination for 12 h.
2.4　The central cell before and after fertil ization
In each of the hook-like st ructures of the central cell seen in the longitudinal section w all
ingrow ths not only form from the lateral w all bordered to the nucellar cells , but also project
from its wall bordered to the synergids(Pl.Ⅳ:16 to 18), where the wall ing row ths of the
lat ter ex tend from the FA to the tw o thirds of the leng th of the synergid.However those on
the lateral w all of the central cell ex tend from the level of the center of the egg apparatus
further to a plane over the polar nuclei.All the w all ing row ths project f rom bo th sides
tow ards the cy toplasm of the central cell.In the central cell numerous mitochondria , ER and
dictyosomes are present , but no t as rich as those in the synergids.Amyloplasts are
abundant , especially dist ributed in the cytoplasm around the polar nuclei(Pl.Ⅱ:6).
After pollination for 8 h , primary endosperm nucleus divides to form several f ree nuclei.
At this stage , more org anelles are fo rmed w ith the increase of endosperm cy toplasm.The
number of amyloplasts and lipid bodies also increased(Pl.Ⅳ:20).The w all ingrow ths in the
central cell have become indistinct(Pl.Ⅳ:19)at the proembryonic stage.
3　DISCUSSION
The concept of FGU is further developed af ter its o riginal proposition.The adaptations
promo ted successfully double fertilizat ion in FGU has been summerized by Huang and
Russell[ 3] .One of the adaptations they illustrated is the positioning of the egg nucleus near
the chalazal end before fertilization.However , the egg nucleus of Calystegia hederacea we
observed is not posi tioned tow ard the chalazal end for keeping a distance from the polar nuclei
befo re fertilization.According to this configuration , when tw o sperm cells are discharged to
the degenerated synergid , they will move separately in different di rections to contact the egg
nucleus and polar nuclei respectively.It is possible that there are tw o sites for gamete fusion
in the FG U.Because in C.hederacea both sites for g amete fusion are the same in the
absence of cell w all.Huang and Russell[ 5] recent ly observed two distinct bands of actin , in
the tabacco ES during synergid degeneration.One band is in the interfaces betw een the egg
cell and the central cell , the o ther is at the chalazal end of the degenerated synergid.They
proposed a hypo thesis of short-distance transport of sperm cells prior to gamete fusion.In our
observat ion , it may also be explained that there are two w ays to facilitate the access of male
gametes to the egg and central cell separately.Of course , this hypo thesis of two sites for
g amete fusion in the FGU should be proved wi th more facts.
The rapid development of embryo sac before and af ter fertilization must be supplemented
w ith nutrients by different w ays in dif ferent tax a.A most prominent feature in the FGU of
C.hederacea is the presence of w all ingrow ths from bo th sides of the central cell hooks ,
mainly fo r the enhancement of absorpt ion f rom the nucellus.Wall ingrow ths have been
reported to occur at the micropy lar end of the matured central cell in several ang iosperms ,
such as Helianthus annus[ 6] , Glycine max[ 7] , Brassica campestris[ 8] , Oryza sativa [ 9] and
Pelargonium hortorum[ 10] .To our best know ledge , all the previous repo rts have claimed
that the w all ing row ths are developed only on the lateral w all of the central cell , bordering to
490　 植　　物　　学　　报 40 卷
the nucellar cells.Wall ingrow ths in C.hederacea , fo r instance , occurred on both sides of
the central cell is very rare.These w all ing row ths are typically the same as that in a t ransfer
cell, served as an eff icient site of increasing movement of metabolites.The vigorous
development of w all ing row ths on the central cell of C.hederacea prio r to fertilization seems
to be closely associated w ith the short duration of the fertilization and early degenerat ion of
the antipodal cells before the ES maturation.The duration f rom pollination to the f irst
division of the zygo te and primary endosperm nucleus is only 8 h and a rapid supply of
metabolites f rom the nucellus to the central cell , and then to the egg apparatus for their
development is neccessary before fertilization.Vigorous development of w all ingrow ths in the
central cell is an appropriate w ay to increase transport between the apoplast and the
symplast
[ 11] .In this observation , there are three aspects of evidence that may support this
viewpoint:(1)The distribution of w all ing row ths at bo th sides of the central cell reflects an
increasing abso rption f rom the nucellus.(2) Accumulation of starch grains w ith a
considerable number in the cytoplasm of the central cell may be observed prio r to
fertilization.(3)Ultrast ructural characteristics of the act ive organelles in the immediate
vicini ty of the wall ing row ths , such as the numerous mitochondria and ER cisternae , seem to
be in relevance with the function of the w all ingrow ths.
The short duration of fertilization in C.hederacea ref lects also in the state of the egg
cell prior to fertilizat ion.Unfertilized egg cell is physiologically or synthetically inactive , and
i ts metabolism is w eak in many plants
[ 3 , 9] .However , according to the ult rast ructural
observat ion of some plants , such as Beta[ 12] , Glycine[ 8] , and Pelargonium [ 10] , organelles
in the unfertilized egg cell unusually appear to be physiologically active suggest ing that the
mature egg cell appears to bear the physiological potential of synthesis[ 13] .In the unfertilized
egg cell of C.hederacea , the characteristic features such as the presence of polyribosomes in
i ts cy toplasm , the dispersive chromatin and irregular nucleolus in it s nucleus , and the obvious
nuclear pores on i ts nuclear envelope suggest that the egg cell has some activi ty in protein
synthesis as an adaptat ion to its rapid development.
In conclusion , the unusual pat tern of the egg cell , the development of strong w all
ingrow ths of the central cell and some synthetic activities of the egg cell prio r to fertilization
show that the members of the FGU of C.hederacea not only have their topog raphical
relationships , but also present their physiological interactions to realize their function in
successful double fertilizat ion.
Acknowledgement　We appreciate the help of Prof.Zhu C.for crit ically reading and
revising the manuscript.
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Explanation of Plates
a.Amyloplast　CC.Cen tral cell　cv.Coated vesicle　d.Dictyosome　E.Egg cell 　en.Egg nucleus　fa.Filiform
apparatus　m.Mitochondrion　n.Nucleus　p.Plastid　PN.Polar nucleus　 r.Ribosome 　rer.Rough endoplasmic
reticulum　Sy.Synergid　v.Vacuole　w i.Wall ingrow th　Z.Zygote
Plate Ⅰ 　Fig.1.A micropylar port ion of the embryo sac show ing tw o synergids before fert ilizat ion.A prominent f iliform
apparatus is presen t in each cell.×3 600　Fig.2.A mature egg cell show ing it s boundery betw een the egg cell and the
adjacent cells(arrow heads).Note the egg nucleus located at the lateral side of the large vacuole in the cell.×3 800　The
inserted figure show s an extended space of the boundary between the egg cell and the central cell.×5 200
Plate Ⅱ　Fig.3.T he extended spaces between the unfertilized egg cell and the cent ral cell, show ing small vesicles in the
w ide space(arrowhead).×18 000　Fig.4.A portion of the synergids and a small port ion of the egg cell before fertilization.
Note the elect ron dense materials deposited in the intercellular space betw een two synergids.×5 100　Fig.5.A portion of
the boundary region betw een the unfertilized egg cell and the adjacent cent ral cell.Note that there i s no typical w all in this
region.×6000　 Fig.6.A portion of an unfertilized cent ral cell adjacent to the egg apparatus.Note the absence of typical
cell wall betw een the egg cell and the cent ral cell.×5 100　Fig.7.A port ion of the synergid before fert ilizat ion show ing the
organelles in it s cytoplasm.×11 000　 Fig.8.A portion of tw o synergids below the FA before fertilization.Arrow heads
indicate the relat ive thin wall separating the tw o cells.Note the parallel RER and many coated vesicles.×2 000　Fig.9.
A portion of the persistent synergid af ter pollination for 8 h , show ing the organelles.×7 300
Plate Ⅲ　 Fig.10.A portion of the cytoplasm in an egg cell before fertilization , show ing mitochondria , starch-grain-bearing
plastid , rough endoplasmic ret iculum and numerous ribosomes.×12 000　 Fig.11.A portion of another egg cell before
fert ilization , show ing polyribosomes and conspicuous nuclear pores on the nuclear envelope (arrow head).×18 000　 Fig.
12.A portion of a fertilized egg 8 h af ter pollination.T he elect ron dense materials(arrow heads)are deposited betw een the
membranes of the zygote and synergid.The micropylar end is at the base of the microg raph.×4 300　Fig.13.A portion of
the zygote and the synergid , showing the elect ron dense materials (arrow head) deposited in the space betw een the
membranes of tw o cells.×18000　Fig.14.The chalazal part of the egg cell af ter pollination for 4 h , showing the electron
dense materials(arrow head)deposi ted in the space betw een the egg cell and the cent ral cell.×5200　Fig.15.A port ion of
the zygote show ing the presence of numerous nuclear pores(arrow head)on it s nuclear envelope.×8 000
Plate Ⅳ　 Fig.16.A portion of the micropylar end of the mature embryo sac , showing the wall ingrow ths on both sides of
a cent ral cell hook.×4 000　Fig.17.A port ion of the cent ral cell bordering to the embryo sac w all , show ing the details of
w all ingrowths.×10 200　 Fig.18.A portion of the magnification of Fig.16 , neighbouring to the filif orm apparatus ,
show ing the details of w all ingrow ths.×10 200　 Fig.19.A portion of the cent ral cell neighbouring to a degenerated
f iliform apparatus of a synergid af ter pollinat ion for 16 h , showing the degenerated w all ingrow ths.×6 000　Fig.20.The
cytoplasm of the early stage endosperm after pollination for 8 h , showing the increased organelles and numerous amyloplast s.
×6 000
492　 植　　物　　学　　报 40 卷