全 文 ：Vol. 29 , No. 4
pp. 545～550 　July , 2003
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 29 卷 第 4 期
2003 年 7 月 　545～550 页
Induction of Abnormal Mitosis and Changes in Protein Compositions after Treat2
ment of Root Meristems with Phosphoric Amide Herbicide APM in Allium
WANG Zhen2Ying1 　CHENGLuo2Gen2 　CHNE Hong1 　PENG Yong2Kang1 , 3
(1 Department of Biology , Tianjin Normal University , Tianjin , 300074 ; 2 Department of Biology , Nanjing Normal University , Nanjing , Jiangsu 210097 , China)
Abstract 　The effect of Amiprophose2methyl (APM) on mitotic cells of the root meristems of Allium cepa was studied at a
range of concentrations (4 —6μmol/ L) , length of treatments (4 —16 h) . The results showed that the mitosis metaphase
index (Met . I ) could be obviously improved from 0. 8 (in control) to 5. 0 or 5. 2 respectively when the root meristems were
treated with APM at a range of concentrations 5 —6μmol/ L for 16 h. The result also indicated that APM treatment (more
than 4μmol/ L) severely affected the cell division. The polypolar divided cells (especially 3 —4 polar) , metaphase chro2
mosome condensed and micronuclei , were revealed in the cells of the root meristems. In 2D SDS2PAGE , 5 new protein
spots with molecular weight of 24 —90 kD , showing p I ranging from 5. 0 —7. 3 , were detected and 2 protein spots with
molecular weight of 24 kD , 40 kD with p I of 4. 8 ,5. 5 were lost in the root meristems treated with APM. Judging from the
relationship between accumulation lost of these protein spots and treatment of APM , these proteins may be related to APM
treatment .
Key words 　Abnormal mitosis ; Multipolar spindle ; APM; 2D PAGE; Allium cepa
磷酰胺除草剂 APM 诱导洋葱根尖分生组织细胞异常有丝分裂和蛋白质
组分变化
王振英1 　程罗根2 　陈　宏1 　彭永康1 , Ξ
(1天津师范大学生物系 ,天津 300074 ;2南京师范大学生物系 ,江苏南京 210097)
摘 　要 　研究了不同浓度 APM(4～6μmol/ L)和不同处理时间 (4～6 h) 对洋葱根尖细胞有丝分裂的影响。结果表明 ,当
用 5. 6μmol/ L APM处理洋葱分生组织 16 h 后 ,中期细胞有丝分裂指数 (Met. Ⅰ)从 0. 8 (对照)分别提高至 5. 0、5. 2。结果
也同时表明 ,当 APM浓度超过 4μmol/ L 时 ,严重影响细胞有丝分裂。在根尖细胞中 ,多极分裂细胞 (尤其是 3～4 级) ,中
期染色体凝集和微核被检测到。用 2D SDS2PAGE分析 ,5 种分子量处于 24～90 kD、p I 在 5. 0～7. 3 的新的蛋白质被检测
到 ,而分子量为 24 kD、40 kD ,p I 为 4. 8、5. 5 的蛋白组分消失。从 APM处理后蛋白质组分消失来分析 ,这些蛋白质变化可
能与 APM处理有关。
关键词 　异常有丝分裂 ;多级纺锤丝 ;APM;2D2PAGE;洋葱
中图分类号 : S633 文献标识码 :A
　　Organophosphorus herbicide is still used in agricul2
ture of China because of its rapidly degraded and little
residual problem to the environment . Amiprophose2Methyl
(APM) is the phosphoric amide herbicide. Many experi2
ments demonstrated that it could block cell division at the
metaphase after root meristems were treated with
APM[1 ,2 ,3 ] . In the past decade , APM had been widely
used in induction mitotic metaphase synchronization in or2
der to isolate mass metaphase chromosomes for further
analysis of biochemical composition[2 ,4 ] , morphologicalΞFoundation :Foundation for university key science and technology by the ministry of education of China (02010) .
Biography : WANG Zhen2Ying , (1966 - ) , female , born in Tianjin. Ph. D of Genetic. Professor of Biology Department . Tianjin Normal University.3 Corresponding author (022) 23540057
Received (收稿日期) : 2002205216 , Accepted(接受日期) :2002212203.

structure[6 ] and in situ hybridization. In the early 1990s ,
although considerable progress had been made concerning
the metaphase chromosome isolation and chromosome mor2
phological structure after treatment with APM , up to now ,
little information has been obtained regarding the changes
in protein compositions and abnormal mitosis after treat2
ment of root meristems with APM. In this paper , the ef2
fect of APM on chromosome structure variation , micronu2
clei formation and protein composition change of root
meristems of Allium cepa , were investigated at a range of
concentrations (4 —6μmol/ L) , length of treatment (4 —
16 h) . The main experiment results were reported and
discussed.
1 　Materials and Methods
1. 1 　Plant materials and culture
　　The common onion Allium cepa L. (2 n = 16) was used
as experiment material , equal2sized bulbs were surface steril2
ized with a solution of 0. 1 % HgCl2 for 5 min and washed two
times with tap water. The bulbs were grown in beakers with
distilled water for 24 h , then the bulbs were again placed in
beakers with solution of amiprophos2methyl (APM) ranging
from 4 —6μmol/ L , treatment time from 4 —16 h. All treat2
ments were performed at 23 ℃in darkness ; at the end of the
treatment , root tips of 0. 1 —0. 2 cm in size were excised from
the bulbs. After rinsing three times with distilled water , part
of the roots were kept in ice water for 24 h in order to segre2
gate chromosomes and decrease their stickiness , then fixed in
70 % ethanol for cytological analysis[4 ] , other fresh root tips
(without the fixation ) were used direct for biochemical
analysis.
1. 2 　Cytological observation
The fixative solution was washed away with distilled wa2
ter before collecting root tips. The digestion of meristems was
carried out using an enzyme mixture (2. 5 % cellulase RS and
2. 5 % pectolyase Y—23 diluted in 75 mmol/ L KCl and 7. 5
mmol/ L EDTA ; pH 4. 0) at 23 ℃. After 30 min digestion a
single root tip was placed in a drop of Caebel fuchsin2stained
solution on a slide kept for 2 —3 min at room temperature and
squashed. Mitotic division , chromosome behavior and mi2
cronucleus formation were observed by light microscopy.
Metaphase index (Met . I) was expressed as the percentage of
nuclei undergoing mitosis among the total number of nuclei
scored in a sample. Data are based on a scoring of at least
2000 cells per treatment .
1. 3 　SDS2polyacrylamide gel electrophoresis
One2dimension PAGE was performed according to
Laemmli (1970) [15] , using a 12. 5 % polyacrylamid separat2
ing gel (3 % stacking gel) in the discontinuous Tris2glycine
buffer system (pH 8. 3) . Sample preparations were dissolved
in sample buffer (500mmol/ L Tris —HCl , pH 6. 8 ; 10 %
SDS , 100mmol/ L , β2mercaptoethanol , 10 % glycerol , 0.
1 % bromophenol blue) . Electrophoresis was run for 7 —8 h
at 120 V.
Two2dimensional polyacrylamide gel electrophoresis was
performed according to the method of O’Farrell et al . [16] ,
with the use of a modified procedure : the one dimensional
3 % polyacrymide gel , containing 9 mol/ L urea 0. 98g ; 2 %
nonionic detergent p40 40μL , 30 % polyacrylamide 340μL ,
ampholytes 30μL ( pH 3 —10) and 60 μL ( pH 5 —7 ) ;
ddH2O 1. 09 mL , TEMED 2 μL , 10 % APS 10 μL , were
each cast in glass tubes (120 mm × 3 mm) and were re2
spectively pre2electrophoresed for 15 min at 200 V , 30 min at
300 V and 60 min at 400 V. The protein samples were dis2
solved in the sample buffer which contained ddH2O 4. 0 mL ,
500 mmol/ L Tris2HCl (pH 618) 1. 0 mL , glycerol 0. 8 mL ,
10 % SDS 1. 6 mL ,β2mercaptothanol 0. 4 mL , 10 % (w/ v)
bromophenol blue 0. 2 mL. The first dimension was isoelec2
trofocusing , 60μL protein samples (4 —6 mg/ mL) were lo2
cated in each glass tube ; electrophoresis was run for 14 —16
h at 400 V. After eletrofocusing , the gels were removed from
the tubes by shattering the glass and placing in equilibration
buffer which contained 6 mmol/ L Tris—HCl (pH 6. 8) ,β2
mercaptoethanol , 10 % glycerol , 2 % ( w/ v) SDS for 20
min. The second dimension was a 12. 5 % SDS2PAGE that
was performed according to the method of Laemmli[15] .
The tube gels were placed on top of second dimension
gels , 1 % agarose was overlayered and allowed to polymerize.
Cylindrical gels were run at a constant voltage of 80 V for 515
h in a Bio2Rad unit . Gels were stained with 0. 4 % AgNO3
solution. [17]
2 　Result
2. 1 　Metaphase cell arrest
　　The data on Met . I was given in Table 1 after treatment
with APM (4 —6μmol/ L) for various periods. Met . I reached
a maximum (510 and 512 respectively) after treatment with
5 —6μmol/ L APM for 6 h , but it was only 0. 8 in the con2
645 　　　作 　　物 　　学 　　报 29 卷 　

trol . However ,the Met . I was decreased to 018 —019 when
the root meristems were treated with 6μmol/ L APM for 16 h
(Table 1 ) , even though many metaphase cells were not
scored because of the losing of typical phase due to spindle
function disturbance. These abnormal metaphase cells would
discussed below.
Table 1 Metaphase index ( Met. I) after
treatment of the root meristems with APM
Content of APM
(μmol/ L)
Treatment times(h)
4 5 6 10 16
Control 0. 7 0. 9 0. 8 1. 0 0. 8
4 2. 1 2. 2 2. 3 2. 2 0. 9
5 2. 9 3. 1 5. 2 1. 7 0. 9
6 2. 7 3. 0 5. 0 0. 9 0. 8
2. 2 　Abnormal metaphase cells
Many abnormal metaphase cells were induced in the root
meristems after treated with APM. These abnormal metaphase
cells could be divided into two kinds as follows.
2. 2. 1 　Multipolar spindle cells
APM induced spindle disturbance in metaphase cell and
manifested aberrant anaphase with multipolar spindle in root
meristems and this situation. It was observed very easily ,
more detailed data on the concentration course of the induc2
tion of multipolar spindle cells listed in Table 2. It was ap2
parent from Table 2 that multipolar spindle cells index showed
a slightly increasing trend with the increase of treatment con2
centration. 32polar , 42polar spindle cells appeared when root
meristems were treatment with 4 μmol/ L APM. When the
concentration increased to 5 —6μmol/ L , the frequency of the
cells with spindle disturbance increased very fast and 52polar ,
62polar spindle cells could also be seen easily (Table 2) .
Table 2 The formation of multipolar spindle cells after treatment
of root meristems with APM at 4 —6μmol/ L for 16 h
Concent of APM(μmol/ L) 32polar 42polar 52polar 62polar
Control - - - -
4 5 4 - -
5 15 14 11 3
6 17 17 12 7
　　Typical multipolar spindle cells were observed in treated
root meristems(Fig. 1) , and it was clearly that the chromo2
somes lost regular arrangement on the spindle. The chromo2
some groups of 4 , 5 or more chromosomes including separated
single chromosome or fragments were formated in some cells
( Fig. 1a) , and arranged in a wide ring or in a star like shape
( Fig. 1b , c , d ) . The existence of the multipolar spindle
cells in the control was not observed. Metaphase chromosome
condensation was observed in the other abnormal mitosis cells
(Fig. 1e) .
Metaphase chromosome condensation was showed in
some cells of treated root meristems with 4μmol/ L APM for 6
h( Fig. 1e) . The less efficient in chromosome condensation
was observed at the low concentration (below 4μmol/ L) , but
the abnormal mitosis frequency of chromosome condensation
increased with the increases of APM from 4 —6 μmol/ L.
Metaphase chromosome condensation reached a maximum of
1715 % at the 6μmol/ L APM for 6 h. It was thought to be
related to the meristems treatment with APM.
2. 2. 2 　Micronuclei formation
The cells with micronuclei were found in the treated root
meristems. The result was also thought to be directly related
to the meristems treatment with APM. The existence of the
cells with micronuclei has not been shown in the control , and
the frequency was about 3. 2 % at 16 h after APM treatment
(4 μmol/ L) , and gradually increased with the up of APM
concentration.
The maximum frequency was about 11 % when the root
meristems were treated with 6μmol/ L APMfor 16 h. The mi2
cronuclei per cell generally varied from 2 —9 , 2 —3 micronu2
clei could be observed per cell in most cells ( Fig. 1f) , more
than 9 micronuclei were only observed in a few cells.
2. 3 　Changes in protein species
To identify the changes of protein species existing in the
treated root meristems , the protein compositions were ana2
lyzed by SDS2Polyacrylamide gel electrophoresis. One2dimen2
sional SDS2PAGE results showed that there were 16 protein
species existing in the root tips cultured on the distilled wa2
ter , with a molecular weight of 122110 kD. Out of 16 pro2
teins , 12 protein species with the molecular weight of 14 kD ,
17 kD , 21 kD , 31 kD , 37 kD , 40 kD , 55 kD , 67 kD , 74
kD , 88 kD , 95 kD , 110 kD were abound. 35 kD protein
species was lost in the treated root meristems. It can be seen
clearly in the scanning (Fig. 2c) indicated by arrows.
Two dimensional SDS2PAGE patterns of proteins extract2
ed from root meristems were given on Fig. 3. The result indi2
cated that more than 90 protein spots were detected (Fig. 3a)
in the meristems cultured on the distilled water , but the com2
745　4 期 WANG Zhen2Ying et al . : Induction of Abnormal Mitosis and Changes in Protein Compositions after Treatment . . . 　　　

Fig. 1 The effect of APM on chromosome structure
a. The chromosome groups of 4 , 5 or more chromosomes including separated single chromosome ; b2d. chromosome groups in
ring or a star like shape ; e. chromosome condensation ; f . micronuclei formation(Bar equals 10μmol/ L)
Fig. 2 SDS2PAGE analysis of protein compositions of root meristeims
a. molecular weight marker ; b. control ; c. treatment with APM
845 　　　作 　　物 　　学 　　报 29 卷 　

Fig. 3 2D SDS2PAGE analysis of protein composition of root meristems
a. control ; b. treatment with 6μmol/ L APM for 16 h
　
positions obviously changed in the meristems treated with 6
μmol/ L APM for 16 h. 5 new protein spot , at least , which
had molecular weight and p I of 26 kD/ p I 5. 0 , 30 kD/ p I
514 , 78 kD/ p I 6. 9 , 76 kD/ p I 7. 3 , 90 kD/ p I 6. 8 , re2
spectively , were found in the treated meristems by using 2D
SDS2PAGE analysis (Fig 3b indicated by arrows) . 2 protein
spots , which located in low and middle molecular weight re2
gion (24 kD/ p I 4. 8 , 40 kD/ p I 5. 5) disappeared in the
treated meristems.
3 　Discussion
APM was a phosphoric amide herbicide , also a specific
drug that directly interrupts microtubule dynamics in plant
cells[18] . Therefore , it could disturb the spindle function in
division cells and was used widely to induce mitotic
metaphase synchromization[1 ] . Although phosphoric amide
herbicide was widely used in agriculture , little information on
cytological and biochemical toxicology had been obtained
when plants were treated with the herbicide. In this paper ,
the cytotoxicity and biochemtoxicity of APM on division cells
were investigated in order to safely and suitable using herbi2
cide in agriculture. In our experiment , root tips were treated
with 4 μmol/ L APM (induction mitotic metaphase synchro2
nization concentration) and 5 —6 μmol/ L APM , the results
showed that metaphase index (Met . I) could be obviously in2
creased when root tips were treated with 4 —6μmol/ L APM
for 4 —10 h (Table1) . Met . I reached 5. 0 ,5. 2 respectively
at 5 —6μmol/ L APM for 6h , but only 0. 8 —0. 9 Met . I was
observed when root tips were treated with 6μmol/ L APM for
16h. It was thought treatment time was more important than
concentration. Although the same results were reported in
culture cell lines[3 , 6 , 8～14] , root tips was used directly as ex2
periment materials to examine the cytotoxicity and biochem2
toxicity of division cells of the root tips treated with 4—6
μmol/ L APM for 4 —16 h in this study. The result indicated
that APM could induce spindle disturbance in metaphase cells
and ma2nifested aberrant metaphases with tri2 or multipolar
spindles , chromosome groups in ring or a star like shapes as
well as cells with micronuclei when root tips were treated with
4 —6 μmol/ L APM for 16 h. The occurrence frequency of
aberrant chromosome cells got up with increase of APM con2
centration. The changes of protein compositions was analyzed
by using SDS2PAGE method in order to further study the poi2
son mechanism of APM on plant cell division at biochemical
level by using 2D PAGE analysis. More than 90 protein
species at least were identified and more than 30 protein
species were abundant in the control root meristems , but 5
new protein species (24 kD/ p I 510 , 30kD/ p I 514 , 78 kD/
p I 619 , 76 kD/ p I 713 , 90 kD/ p I 618) were found , and 2
protein species (24 kD/ p I 418 , 40 kD/ p I 515) were lost in
the meristems treated with 6μmol/ L APM for 16 h.
945　4 期 WANG Zhen2Ying et al . : Induction of Abnormal Mitosis and Changes in Protein Compositions after Treatment . . . 　　　

Up to now , only the tubulin studies have been made
concerning the effect of APM on cell division in higher
plants[18] , little other biochemical information exists concern2
ing the protein composition change in plants which have been
treated with APM[7 ] . In this paper , 5 new protein species
were induced and 2 protein species were lost when root meris2
tems were treated by APM at 6μmol/ L for 16 h , although we
have not understood the exact function of these changed pro2
tein species , it may provide valuable information in relation to
possible the poisen mechanism of crops.
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055 　　　作 　　物 　　学 　　报 29 卷