全 文 ：Ultrastructure of the Sperm Cell in Lilium davidii
CAI Xue
Institute of Botany , Academia Sinica , Beijing 100093 , China
ABSTRACT　The ultrastructure of the sperm cell in the pollen tube of
Lilium davidii was investigated by electron microscopy.　The two sperm
cells were arranged one behind another in all the pollen tubes examined.
The vegetative nucleus took the lead position near the pollen tube tip and
maintained a certain distance to the heading sperm.　The two newly
formed sperm cells were separated by a common transverse cell wall.
Meanwhile, the cases that two sister sperm cells parted from each other
without any connection were often found.　The cytoplasm of the sperm
cell contained mitochondria , endoplasmic reticulum , dictyosomes ,
numerous ribosomes , lipid bodies and vacuoles.　 No plastid was
observed.　Microtubules were absent.　But in the newly formed sperm
cells , microtubules were arranged as bundles in axial orientation under the
plasma membrane of the sperm cel l.
Keywords:　 sperm cell , pollen tube , ultrastructure , Lilium davidi i
Duchartre
1.INTRODUCTION
Our previous studies indicated that the generative cell divisions in the pollen tubes of Amaryll is
and Li lium were different f rom each other;those dif ferences include the behavior of
chromosomes , the org anization and dist ribution of micro tubules , such as the time of the
kinetochore microtubule formation , the shape of spindle and whether the interphase network of
cortical microtubules being persistence or not during the generative cell division , etc.　However ,
the generative cells in the two kinds of plants possessed some similari ties , such as the absence of
preprophase band of microtubules(PPB)and the presence of cell plate in tolephase[ 1 , 2] .　As the
descendants of the generative cell cy tokinesis , what about the structural dif ference have two kinds
of sperm cells ?We had already reported the ultrastructure features of the sperm cell in Amaryll is
befo re
[ 3] .　In this paper , we described the characters of the sperm cell in Li lium in detail--
focused on its ultrastructural aspect .
2.MATERIALS ANDMETHODS
The flowers of Lilium dav idii Dechartre grow n under natural sunlight w ere obtained from
Peking University.
Semi-vivo culture of pollen tubes w as performed as previously described[ 2] .
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DEVELOPMENTAL &REPRODUCTIVE BIOLOGY
Vol.7 No.2 pp63～ 67 Nov.1998
ISSN1004-6453/CN11-3569
★c 1998 Chin.Dev.&Reprod.Biol.Socs.
Vol.7 No.2 (1998)
After incubation for 11 ～ 18 h , DNA fluorochrome Heochst 33258 (1mg/mL)and acetic acid
o rcein were used to detect the developing stages of the generative and sperm cells through
Olympus BH-RFL-W fluorescence microscope and Olympus BH-2 light microscope , respectively .
The parts of the styles contained the dividing generative cells or sperms were excised and fixed in
2.5% g lutaraldehyde in 0.2 M cocady late buffer , pH 7.0 , for 4h in room temperature and then
rinsed in the same buffer for 1h.　Follow ing post fixation in 1% OsO4 overnight in 4℃, the
samples were dehydrated through an ethanol series and embedded in Epon 812
[ 2] .　Sectioning ,
staining and observations w ere performed as previously described
[ 2] .
3.RESULTS
After 11 ～ 18h of semi-vivo pollination , the generative cells in the pollen tubes already completed
their division and produced two sperm cells.　In all the pollen tubes examined , the sister sperms
arranged one af ter another(Figs.1 ～ 3).　The vegetative nucleus took the lead position and w as
near the pollen tube tip(Figs.2&3).　Sometimes , a DNA association between the paired sperm
nuclei was detected by H33258 staining (Fig.3).　The two new ly formed sperm nuclei were
separated from each o ther (Fig.1)and kept a certain distance (Fig.3).　Occasionally , the
paired sperms were in close proximity to the vegetative nucleus(Fig.2).
Ultrastructural observations show ed that the afore tw o sperms joined by a common cell wall were
surrounded by thei r ow n plasma membrane.　The plasma membranes of the tw o sperms together
w ith the membrane of the vegetative cell fo rmed a periplasm.　The periplasm was somewhat
flat tened.　The common w all was transverse , undulated , reticulate and contained many
cytoplasmic channels(Fig.4).　It w as much thicker than the periplasm.
The two sperm nuclei were elongated ellipsoidal w ith small extensions.　They contained
condensed chromatin and 2 ～ 3 nucleoli (Fig.6).　In the longitudinal sections , the chromatin in
one sperm nucleus w as more condensed than that in another(Fig.5).　I t implies that one sperm
may develop earlier.
In the cytoplasm of sperm cell , the organelles such as mitochondria , endoplasmic reticulum ,
dictyosomes , vesicles , small vascuoles , lipid bodies and abundant ribosomes presented.　No
plastid w as found.　The mitochondria showed w ell-developed cristae of lamellar appearance(Fig.
7).　Sometimes , they dist ributed in linear under the plasma membrane (Fig.7).　In general ,
the micro tubules w ere absent in the sperm , while in the new ly formed cell , the microtubules
arranged as longitudinal bundles w ere observed in the thin cy toplasm parallel to the long axis
(Fig.8).　It w as f requent ly found that the tw o sperm cells w ere separated wi thout any
connect ion.　We preferred to believe that the disappeared common wall accompanied the prolong
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Developmental &Reproductive Biology
development of sperms.
Figs.1 ～ 3.Light(Figs.1&2)and hoechst 33258 fluorescence(Fig.3)micro scopic graphs ,
showing the tw o sperm nuclei (SN)in a pollen tube.　Note the vegetative nucleus(VN) as the
lead position.　Fig.1×450 , The others×600.
Fig.4.A par t of a pollen tube in a longisection , the cell pla te(CP)between the two sperm cells
was show n.×16040.
Figs.5&6.The leading sperm cell(SC1)and a part of another sperm cell(SC2)in a pollen tube
(Fig.5)and the other part of SC2 (Fig.6)in the same pollen tube.　Note the two sperm cells
were tho roughly separated without any connection.×2500.
Figs.7&8.A par t of sperm cell in longitudinal section , showing the organelles in the cytoplasm(Fig.7)and the micro tubule distribution(Fig.8).×23520.
LB Lipid body;Va Vacuole;M Mitochondrium;CH Chro-matin;MT Microtubule;ER
Endoplasmic reticulum;Ve Vesicle.
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Vol.7 No.2 (1998)
4.DISCUSSION
Our observations revealed that the tw o sperm cells in the pollen tube of Lilium dav idii were
connected by a common transverse cell w all.　This phenomenon w as similar to that of most
plants [ 1 , 4-6] .　Our previous studies[ 2] demonst rated that the comportment of the chromosomes
in the generat ive cell of Li lium davidii was not like that of Amaryl lis vi ttata in w hich they
acted in typical mitosis[ 1] , but like that of Tradescant ia v irginiana in w hich chromosomes
distributed along the long axis of the cell in mito tic metaphase , and the cy tokinesis w as completed
w ithout cell plate
[ 7] .　Combining the present data with previous repo rts , we were conceivable
that the generative cell division in li lium dav idii is like that of Nicotiana tabacum[ 8] , which
maybe the intermediate types between Amary llis and Tradescant ia.
The male germ unit (MGU)is a universal feature in tricellular and bicellular pollen grains[ 3～ 6] .
However , no apparent MGU occurred in pollen g rains and pollen tubes of Helleborus foetidus[ 9]
and Zea mays[ 10] .　In our present study , MGU was no t found in ultra-thin sections although a
few cases that the vegetative nucleus w as really in close proximity to the sperm cell and DNA
association between the two sperms were observed by light microscopy.　This fact reflected that
MGU is a dynamic and temporal structure.　On the other hand , MGU was based on the
embayment of the vegetat ive nucleus w ith the extensions of the reproduct ive cell (generative or
sperm cell )and the cytoskeleton in the reproduct ive cell , i .e.microtubules may play an
important role in maintaining the cohesion of the reproductive cell w ith the vegetative
nucleus[ 3 ～-6] .　In Lil ium , in contrast to the generative cell , the microtubules in the sperm cell
w ere absent , and similar results w ere also obtained in Plumbago[ 11] and Populus[ 12] .　This
situation maybe one of the reasons that MGU was not found betw een the paired sperms and the
vegetative nucleus in Lilium dav idii .
REFERENCES
1.　 Cai X., Hu S.Y.and Zhu C.:　Ultrastructural changes of the microtubule in the generative cell of
Amaryllis vittata Ait.during mitosis.Acta Bot S in , 36:745 ～ 750 , 1994.
2.　Cai X.and Hu S.Y .:　Changes of microtubular skeleton in the generative cell in pollen tube during mitosis
of Lilium davidii .Acta Bot S in , 38:761 ～ 766 , 1996.
3.　Cai X.and Wei L.B.:　Ultrastructural features of sperm cell in Amaryllis vittata.　Developmental and
Reproductive B iology , 5:49 ～ 54 , 1996.
4.　 McConchie C.A., et al.:Quantita tive cytology of the sperm cells of Brassica campestris and B.
Oleracea.Planta , 170:446 ～ 452 , 1987.
5.　Wagner V.T .and M ogensen H.L .:The male germ unit in the pollen and pollen tubes of Petunia hybrida:
ult rastructural , quantitative and three-dimensional features.Protoplasma , 143:101 ～ 110 , 1988.
6.　 Taylor P., et al.:　The male germ unit of Rhododendron:quantitative cytology , three-dimensional
reconstruction , isolation and detection using fluorescent probes.Sex Plant Reprod , 2:254 ～ 264 , 1989.
7.　Palevitz B.A.and Cresti M .:Cytoskeletal changes during generative cell division and sperm formation in
Tradescantia virginiana.Protoplasma .150:54 ～ 71 , 1989.
8.　Palevitz B.A.:　Organization of the mitotic apparatus during generative cell division in Nicotiana tabacum .
Protoplasma , 174:25 ～ 35 , 1993.
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Developmental &Reproductive Biology
9.　Heslop-Harrison J., Heslop-Harrison J.J.and Heslop-Harrison Y.:　The comportment of vegetative
nucleus and generative cell in the pollen and pollen tubes of Helleborus f uetidus L .Ann Bot , 58:1 ～ 12 ,
1986.
10.　McConchie C.A ., Hough T.and Knox R.B.:　Ultrastructural analysis of the sperm cells of mature pollen
of maize , Zea mays.Protoplasma , 139:9 ～ 19 , 1987.
11.　Russell S.D.and Cass D.D.:　Ultrastructure of the sperms of Plumbago zeylanica I.Cytology and
association with the vegetative nucleus.Protoplasma , 107:85 ～ 107 , 1981.
12.　Rougier M ., et al.:　Male gametophyte development and formation of the male germ unit in Populus
deltoides follow ing compatible pollination.Protoplasma , 162:140 ～ 150 , 1991.
提要
川百合精细胞的超微结构
蔡　雪
中国　北京 100093　中国科学院植物研究所
川百合与朱顶红花粉管中的生殖细胞分裂行为非常不同。诸如:染色体行为 、微管的组
织形式和分布 、包括着丝点 、微管形成的时间 , 纺锤体的形状及间期周质微管网络在生殖细
胞分裂过程中消失与否等 。但这两种细胞具有某些共性 , 包括在有丝分裂前期缺乏早前期带
微管(PPB), 末期形成细胞板等。这两种植物精细胞的结构应有较大差异。我们曾报道了朱
顶红精细胞的超微结构 , 本文详细从超微结构方面描述了川百合精细胞的特征。
川百合花粉管的萌发采用半离体-活体培养方式 。11 ～ 18小时后 , DNA 荧光染料Hoechst
33258和醋酸地衣红染色检查花粉管中生殖细胞和精细胞发育时期。切取含有分裂的生殖细
胞和精细胞的花柱部分 , 按曾报道的方法固定 、包埋 、切片 、染色及观察 。
在所有检查的花粉管中 , 两精子均前后排列(Fig.1 ～ 3), 营养核前导并靠近花粉管顶端
(Fig.2 , 3)。H33258 染色可见两精核间以 DNA 联系(Fig.3)。两个新形成的精核彼此分离
(Fig.1), 后来又相互靠近 , 并维持一定距离(Fig.3)。偶尔一对精子与营养核靠近(Fig.2)。
两精细胞被一共同的细胞壁连接 , 他们不仅被自己的质膜也被营养细胞的质膜包围构成
周质 。周质平坦光滑 。共同壁横向 、弯曲 、网状具胞质通道(Fig.4), 厚度明显大于周质 。两精
核为长的椭圆形 , 具有小的突起和浓缩的染色质 , 含 2 ～ 3个核仁(Fig.6)。有的精核中染色
质凝集的程度更大些(Fig.5), 可能意味着一个精子发育的早些。精细胞质中具有线粒体 、内
质网 、高尔基体 、脂体和大量核糖体。无质体。线粒体具有发育完好的条带状的脊 , 有时线粒
体沿质膜排成一排(Fig.7)。通常精细胞缺乏微管 , 而在新形成的精细胞中 , 微管呈纵向束排
列于稀薄的胞质中并平行于细胞长轴(Fig.8)。常常发现两精细胞彼此分离无联系 , 可以认为
是伴随精细胞的继续发育 , 共同壁消失了。
与朱顶红等植物的染色体行为遵循典型有丝分裂方式不同 , 川百合生殖细胞与紫露草相
同 , 它的染色体在有丝分裂中期沿细胞长轴分布 , 胞质分裂时没有细胞板出现。可以认为:川
百合象烟草一样是介于朱顶红和紫露草之间的中间类型。雄性生殖单位(MGU)在三细胞和
二细胞花粉中普遍存在 。尽管本工作观察到营养核与精细胞紧密联系 , 以及两精子与 DNA
联系的例子 , 但 MGU在超薄切片中并未见到 , 有可能 MGU 是一个动态的和时间上的暂时
结构。另一方面 , MGU的建立是以性细胞(生殖细胞或精细胞)的突起和营养核的裂瓣相互
环绕为基础的 , 而性细胞中的细胞骨架(即:微管)可能对维持其与营养核的附着起重要作
用。缺乏微管 , 可能是川百合精细胞不存在 MGU的原因之一 。
(Received 4 November , 1997)
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