全 文 ：Scanning Electron Microscope Observation of Three
Species on Pollen Grain Morphology and Leaf Epi-
derm Shape in Gramineae Triticeae
ZHANGShan-shan1 , ZHOUYin-fu1＊ , LIN Xiao-hu1 , QINSu-ping1 , ZHANG Yan1 , ZHANG Zhi-wen1 , WANG Qiu-tao2
1.Hebei Normal University of Science and Technology , Qinhuangdao 066004;2.Baoding Vocational and Technical College, Baoding 071000
Abstract　The pollen grain morphology and leaf epiderm shape andmicrostructural of Rye(Secale cereal), commonwheat and Octoploid Trititrigia were ob-
served with the scanning electronmicroscope(SEM).The results revealed that there were great differences between pollen and leaf epidermmicrocosmic mor-
phology in three species.Such characteristics of Octoploid tritical were between those of commonwheat and Rye.Themorphology of pollengrain and leaf epi-
derm shape counld be used asan index for taxonomy for genera and species, and had somewhat scientific references to identifynew mult iploid species created by
chromosome engineering.
Keywords　Gramineae;Pollen grain;Leaf epiderm shape;SEM
Received:January 4 , 2009　　Accepted:April 15 , 2009
Supported by Key Projects in the National Science &Technology Pillar
Program in the Eleventh Five-year Plan Period(2006BAD02A08-9);the
Science and Technology Research and Development Program of Hebei
Province(06230105);Program of Science and Technology from Educa-
t ion Department of Hebei Province(2006425);the Science and Technol-
ogy Research and Development Program of Tangshan City(08120201A-
8).
＊Corresponding author.E-mail:zhouy infu@126.com
　　The Triticum aestivum(2n=6x=42)is taken as female par-
ent to hybridize with Secale cereale (2n =2x =14)and chromo-
some is doubled to obtainOctoploid tritical (2n=8x=56)[ 1] .The
botanic conformation of Octoploid tritical is between parents.In re-
cent 10 years , there are many reports about ultrastructure of
plants
[ 2-5] , however , the report about wheat plants is not toomuch.
The SEM was used to observe pollen-morphological characteristics
and structure of leaf epidermis ofSecalecereale , Triticumaestivum
andOctoploid tritical , through the comparisonof their micro-morpho-
logical characteristics , their genome ploidy and genetic relationships
were explored to provide reference for identifying polyploidy new
species and studying relationship of different species.
Materials and Methods
The experimental Triticum aestivum , Secale cereale andOc-
toploid tritical were introduced from WANG Hong-gangs subject
group in National Wheat Improvement Center and these materials
were planted in experimental plot of Analytical and Testing Center of
Hebei Normal University of Science and Technology.The leaves of
all materials were cut into small pieces and fixed by FAA then
washed clean.They were dehydrated step by step with alcohol
(30%, 50%, 70%, 85%, 95%and 100%)and every dehydra-
tionwas lasted10 to15minthen weredried naturally and adhered to
double-sided adhesive.The dried pollens were directly adhered to
double-sided adhesive and treated by ion sputtering equipment of
KYKY-2800 SEM , then observed under SEMand analyzed by image
analysis system.5 pollens of every wheat were determined and the
valuedwas average , besides , the typical pollenswere taken photos.
Results and Analysis
Pollen-morphological characteristics
Size and shapeof pollen　It was concluded from the result of SEM
that the pollensize of Octoploid tritical was betweenthat ofTriticum
aestivum and Secale cereale.The pollen size of Secale cereale
was biggest and that of Triticum aestivum was smallest.The pollens
of three wheat were subcircular and that of Secale cereale was long
oval , while that of Octoploid tritical and Triticum aestivum were el-
lipse or round.Some pollens had shrinkage , showing wizened triangle
or irregular shape , the phenomenon might be related to dialysismeth-
ods[ 6-7] .
Character of aperture of pollengrain　The character of aperture
of pollen grain was one of main basis for identifying pollen grain[ 2] .
The common characters of aperture of pollengrain of Octoploid triti-
cal and Triticum aestivum and Secale cereale were that around ap-
erture had uplifts and l ids , however , the sizes of aperture and lids as
well as the shape of lids were different.The aperture of Triticum
aestivum was bigger than that ofOctoploid tritical and Secale cere-
ale and its l id hadcrack , besides , aperture was convex out.Theap-
erture of Secale cereale was smaller than that of Triticum aestivum
andOctoploid tri tical , however its lid had no crack.The aperture of
Octoploid tritical was between that of Secale cereale and Triticum
aestivum , however , processes of aperture , lid size and crack were
different among different varietieswithout any regularity(Fig.1).
Surface characteristics of pollen grain　The exine sculpture of
pollen grain was granular sculpture.The sculpture particle with single
processwas called singleparticle sculpture, while sculptureparticle with 2
or more than2processes andarranged likezigzag or petal was called com-
plex particle sculpture[3] .Among 3 kinds of observed materials , although
pollensurfaceswereall particles sculpture , however , there weremany
obviously differences(size , type and son on)among sculpture.Oc-
toploid tritical and Secale cereale were single particle sculpture and
the arrangement was relatively loose , while the particle was small.
TheparticlesofSecalecereale were uniformand all were single par-
ticle , while there were some complex particles were among single
Agronomy
Agricultural Science &Technology , 2009 , 10(2):86-89
Copyright 2009, Informat ion Institute of HAAS.All rights reserved.
DOI :10.16175/j.cnki.1009-4229.2009.02.007
particles of Octoploid tritical (Fig.1).Among Octoploid tritical ,
surfaces of some varieties had complex particles , however , Triticum
aestivumhad complex particle sculpture while the particles were big
and the arrangement was close(Table 1).
A:Triticum aestivum;B:Secale cereale;C:Octoploid tritical (jinsong5);D:Octoploid tritical(jinsong49).
Fig.1　Electronmicroscope scanning observat ion on pollengrain of Triticum aestivum , Secale cereale and Octoploid tritical
Table 1　Pollen grain morphological features of Triticum aestivum , Octoploid tritical and Secale cereale
Material Opercular shape feature of aperture Surface feature Size∥μm×μm
Triticum aestivum Aperture was convex out and lids had cracks Rough, many were complex big particles and the ar-
rangement was close
72.85× 44.29
Octoploid tritical Aperture was convex out while some lids had cracks
and some had not
Many particleswere single part iclesor complex parti-
cles, andthe particles were not uniformwith loose ar-
rangement
72.78× 52.85
Secale cereale Aperture was convex out and lids hadmanycracks All were single small part icles with loose arrange-
ment.
61.42×51.43
Structural features of leaf epidermis
The lower epidermis of all materials were observed and the
common features of three materials were stomatal apparatus , long
cells and the parallel between cell arrangement and vein.The ar-
rangement of epidermal cells of Triticum aestivum were tidy and
smooth, while the stomatal arrangement was l inear , so the dumbbell
shapedguard cells were observed , however , theywere not as obvi-
ous as theseof other 2 materials , besides , setaswere not same long(Fig.2).Compared withTriticumaestivumandSecale cereale , the
arrangement of epidermal cells of Octoploid tritical was not tide ,
some folds around stomata wereobviously shownup , so stomata was
looked l ike rectangle.The stomatal arrangement was linear and
guard cells were obvious , besides , setas were few and short.Its
single stomata was one time bigger than that of other two materials ,
whichmight be related to its chromosome number[ 6] .Compared with
other 2materials , thecell arrangement of Secale cereale was the ti-
diest and smooth , besides , the stomatal interval was relatively big
and the guard cells were obvious(Table 2).
A:Leaf epidermis of Triticum aestivum;B:Stoma of Triticum aestivum;C:Leaf epidermisof Octoploid tritical;D:Stoma of Octoploid tritical;E:
Leaf epidermis of Secale cereale;F:Stoma of Secale cereale.
Fig.2　Electronmicroscope scanning observat ion on leaf epidermis of Triticum aestivum , Secale cereale andOctoploid tritical
Discussion
(1)The pollen grains observed in this experiment are almost
round , however , there are still some wrinkled and wizened triangle
pollenswhich may be related to preparationmethod.CAIXiu-zhen[6] ,
CHENJing-ying[ 7] , and XUBai-sen[ 8] et al have reported the pollen
morphology under SEM through different sample preparation method ,
however , there aredifferent conclusions of pollenswith different treat-
ments under SEM.Therefore , before observing pollen grains through
SEM , it is better to compare the results of different treatments and
87
ZHANG Shan-shan et al.Scanning ElectronMicroscopeObservation of Three Species on Pollen GrainMorphology and Leaf EpidermShape in Gra-
mineae Triticeae
pick out the optimummethod for observing.
Table 2　Leaf epidermis morphological features of Trit icum aest ivum, Octoploid tritical andSecale cereale
Material Leaf epidermis feature Stoma size∥μm×μm Stomata feature Seta
Triticum aestivum stomatal apparatus, long cells,
smooth and tidy arrangement of hair
cells
44.29×10.00 Slender , ellipse , linear arrangement ,
relatively big cell interval, obvious
guard cells
Relat ively few and not
same long
Octoploid tritical stomatal apparatus, long cells, not
smooth and tidy arrangement of hair
cells
97.29×30.00 Rectangle , generally linear arrange-
ment , big cell interval and obvious
guard cells
Few and short
Secale cereale stomatal apparatus, t idy and smooth
arrangement of long cells
45.71×17.14 Ellipse , rather big cell interval and
obviousguard cells
More and long
　　(2)It is concluded from the comparison that there are obvious
differences on surface sculptures of Triticum aestivum , Octoploid
tritical and Secalecereale.The aperture of Triticum aestivu is big-
gest and that of Secale cereale is smallest , while that of Octoploid
tritical is betweenthem.However , the surfacesculptures among dif-
ferent genera and varieties are different.
(3)During observing leaf epidermal characters , the lower epi-
dermis , stomatal size and stomatal characters of Octoploid tritical
are obviously different from these of Triticum aestivum and Secale
cereale.There are many ridgy fringes around stomata , which make
every stomata and guardcells obvious , besides its singlestomata are
nearly one time bigger than that ofTriticumaestivum and Secalece-
reale .LINAG Han-xing[ 9] , LIU Ping[ 10] et al have identified wheat
with different chromosome numbers by observing stomatal size and
concluded that stomatal size is related to ploidy of wheat , which is
similar to the result of this experiment.
(4)The result of this experiment has shown that SEM can be
used as an auxiliary method to identify the species differences and
provide certain reference for identifying new polyploid species and
studying species relationship , however , the identificationof cytologi-
cal techniques ismore accurate to confirm ploidy of genome.
References
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[ 2] SHAOLX, ZHANGJP, LIUY.Observationof leavesof eight Cupressaceae
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Responsible editor:ZHENG Dan-dan 　　Responsible translator:LI Zhu-le 　　Responsible proofreader:WU Xiao-yan
禾本科 3个物种花粉粒及叶片扫描电镜观察
张珊珊1 ,周印富1＊ ,林小虎1 ,秦素平1 ,张 燕1 ,张志雯1 ,王秋涛2 　
(1.河北科技师范学院 ,河北秦皇岛 066004;2.河北省保定职业技术学院 ,河北保定 071000)
　　以普通小麦(2n=6x=42)作母本 ,与二倍体黑麦(2n=2x=
14)进行杂交 ,并进行染色体加倍 ,所获得的新物种就是八倍体
小黑麦(2n=8x=56)。八倍体小黑麦的植物学形态介于双亲之
间 ,兼有两者的性状 。
1　材料与方法
供试二倍体黑麦 、普通小麦及八倍体小黑麦引自国家小麦
改良中心泰安分中心王洪刚课题组 ,种植于河北科技师范学院
分析测试中心实验地 。取各类型材料的叶片 ,剪成小块 ,用
FAA 固定 ,清洗 ,乙醇逐级梯度脱水 (30%、50%、70%、85%、
95%、100%),每次 10 ～ 15 min ,自然干燥后 ,粘于双面胶上;干
燥的花粉粒直接粘于双面胶上 ,用 KYKY-2800型扫描电子显
微镜配套的离子溅射仪喷金处理后 ,置扫描电镜(工作电压 15
88 Agricultural Science &Technology Vol.10 , No.2 , 2009
kV)下观察 ,观察图像直接进入扫描电镜连机图像分析系统 。3
种小麦各测定 5粒花粉 ,取平均值 ,并取有代表性的花粉拍照。
2　结果与分析
2.1　花粉粒形态特征
2.1.1　花粉粒的大小及形状。二倍体黑麦的花粉粒最大 ,普通
小麦的花粉粒最小 ,八倍体小黑麦花粉粒的大小介于二者之间 。
3种小麦的花粉粒大都为近圆形 ,其中二倍体黑麦的花粉粒为
长椭圆形 ,而八倍体小黑麦与普通小麦的花粉粒相似 ,为椭圆或
近圆形 。部分花粉粒有皱缩现象 ,呈瘪三角形或不规则形 ,这可
能与所采用的制片方法有关。
2.1.2　花粉粒萌发孔的特征。3种试材的花粉粒萌发孔共有的
特征是:萌发孔为近圆形 ,孔口周围隆起 ,孔口有萌发孔盖 ,但孔
口和孔盖的大小以及孔盖的形状特征(如裂缝、凸凹)有一定的
差别。普通小麦的萌发孔最大 ,且孔盖出现裂缝 ,萌发孔外凸明
显;二倍体黑麦的萌发孔最小 ,孔盖没有裂缝;而八倍体小黑麦
的萌发孔大小介于其他 2种之间 ,孔口的突起程度、孔盖大小及
裂缝在不同品种中也不尽相同 ,没有一定的规律性(图见第 87
页 Fig.1:普通小麦 、二倍体黑麦和八倍体小黑麦的花粉粒扫描
电镜观察 ,图中 A.普通小麦;B.二倍体黑麦;C.八倍体小黑麦劲
松 5号;D.八倍体小黑麦劲松 49号)。
2.1.3　花粉粒表面特征 。八倍体小黑麦和二倍体黑麦多为单
一颗粒型雕纹 ,排列相对较疏松 ,颗粒较小。比较而言 ,二倍体
黑麦的颗粒大小整齐、均一 ,全为单一颗粒;而在八倍体小黑麦
的单一颗粒中 ,却能看到间杂在其中的复合颗粒(图见 Fig.1)。
在八倍体小黑麦中 ,也有个别品种的表面颗粒为复合型。普通
小麦为复合颗粒型雕纹 ,颗粒较大 ,排列较紧密(表1)。
表 1　 普通小麦、二倍体黑麦和八倍体小黑麦的花粉粒形态特征
材料 萌发孔孔盖形状特征 表面特征 大小∥μm×μm
普通小麦 萌发孔外凸 ,孔盖有裂缝 粗糙 ,多为复合颗粒 ,颗粒较大,排列紧密 72.85×44.29
八倍体小黑麦 萌发孔外凸 ,孔盖有或无裂缝 为单颗粒或复合颗粒 ,颗粒大小不一 ,排列较松散 72.78×52.85
二倍体黑麦 萌发孔外凸 ,孔盖有许多裂缝 单颗粒 ,颗粒较小 ,排列松散 61.42×51.43
2.2　叶表皮结构特征　该试验均对所有材料的下表皮进行观
察 ,总体来看 3种材料的共同特征是有气孔器 ,长细胞和毛细
胞 ,细胞排列与叶脉平行 。普通小麦的表皮细胞排列较为整齐
且光滑;气孔排列也呈直线 ,可以看到哑铃状的保卫细胞 ,但没
有其他 2个材料明显;有刺毛但长短不一(图见第 87页 Fig.2:
普通小麦 、二倍体黑麦和八倍体小黑麦叶表皮的扫描电镜观察 ,
图中 A .普通小麦叶表皮;B.普通小麦气孔;C.八倍体小黑麦叶
表皮;D.八倍体小黑麦气孔;E.二倍体黑麦叶表皮;F.二倍体黑
麦气孔)。八倍体小黑麦表皮细胞的排列相对于普通小麦和二
倍体黑麦来说很不整齐 ,可以明显地看到气孔周围有许多隆起
的褶皱 ,使气孔看起来呈长方形;气孔排列总体也呈直线形 ,保
卫细胞非常明显 ,有刺毛但极少且短;单个气孔很大 ,比其他 2
个材料要大近 1倍 ,这可能与其是八倍体有很大的缘故。二倍
体黑麦与其他 2个材料相比 ,排列最为整齐且光滑 ,气孔间间隔
较大 ,保卫细胞也比较明显(表 2)。
表 2 　普通小麦、二倍体黑麦和八倍体小黑麦的叶表皮形态特征
材料 叶表皮特征 气孔大小∥μm×μm 气孔特征 刺毛
普通小麦 有气孔器,长细胞、毛细胞排列整齐 ,较
光滑 44.29×10.00
细长 ,椭圆形 ,直线排列 ,间隔较大,保卫
细胞明显
较少且长短不一
八倍体小黑麦 有气孔器 , 长细胞, 毛细胞排列不很整
齐 ,不光滑 97.29×30.00
长方形 ,大体呈直线排列 , 间隔大 ,保卫
细胞很明显
很少且短
二倍体黑麦 有气孔器,长细胞排列整齐 ,光滑 45.71×17.14 椭圆形 ,间隔很大 ,呈直线排列 ,保卫细胞较明显
多而长
基金项目　“十一·五”国家重大科技支撑计划项目(2006BAD02A08-9);河北省科学技术研究与发展计划项目(06230105);河北省教育厅科学研究计
划项目(2006425);唐山市科学技术研究与发展计划项目(08120201A-8)。
作者简介　张珊珊(1984-),女 ,河北保定人 ,硕士研究生 ,研究方向:作物遗传育种。 ＊通讯作者。
收稿日期　2009-01-04　　修回日期　2009-04-15
(上接第 85页)
后显著高于烘前和加热 2 d的现象外 ,其他 3种蔬菜供试种子
在不同加热时间之间均无显著性差异 。种子中脱氢酶活性和
SOD活性是衡量种子生活力的两个重要生理生化指标 ,反映
细胞代谢过程中还原能力的强弱以及种胚损伤劣变程度 ,也是
检测种子受损程度的重要参数 。酶活性越高 ,表明种子受损程
度越小 。试验中种子脱氢酶及 SOD活性在 50 ℃加热超干处
理后 ,除萝卜种子外 ,均未发现有显著下降的趋势 ,其原因有待
于进一步研究 。
2.3　超干适宜含水量　4种不同农作物的种子 ,其适宜含水
量可以分为高和低 2种类型 ,豇豆和笋玉米属于含水量较高的
一类 ,萝卜和油菜种子则属于含水量较低的一类(表见第 84页
Table 5:大量种子加热超干燥基础含水量及适宜含水量)。
3　讨论
研究中 4种大量作物种子经试验证实 ,运用 50 ℃加热干
燥的方法均能在不同天数干燥后使种子含水量降低到适宜超
干贮存的范围 。经萌发和两种酶活性的测定分析 ,不同超干燥
程度的样品对 50 ℃加热干燥处理大多有良好的反应:笋玉米 、
油菜 、萝卜等种子耐热耐干性极强 ,加热干燥后有较好的贮存
效应;而豇豆种子加热干燥时间宜控制在 2 ～ 4 d ,否则种子活
力下降 。
基金项目　北京市科技新星计划(2008B37);北京市人事局留学人员
科技活动市级重点资助项目(20080006);北京市优秀人才
培养资助项目(20071D020500052)。
作者简介　孟淑春(1973-),女 ,北京市人 ,助理研究员 ,硕士 ,从事蔬
菜种质资源和蔬菜遗传育种研究。 ＊通讯作者。
收稿日期　2009-02-08　　修回日期　2009-03-16
89
ZHANG Shan-shan et al.Scanning ElectronMicroscopeObservation of Three Species on Pollen GrainMorphology and Leaf EpidermShape in Gra-
mineae Triticeae