全 文 ：·Rapid Communication·
Effects of Ag_carrying Zirconium Phosphate on the Kinetics of Growth
of the Roots of Culture Artemisia annua
OUYANG Jie , WANG Xiao_Dong , ZHAO Bing , WANG Yu_Chun＊
(State Key Laboratory of Biochemical Engineering , Institute of Process Engineering , The Chinese Academy of Sciences , Beijing 100080, China)
Key words:　Artemisia annua;hairy roots;artemisinin;Ag_carrying zirconium phosphate (ACZP);kinetics
　　Artemisinin is an anti_malarial medicine isolated
from sweet wormwood(Artemisia annua L.), a tradition-
al Chinese herb of the family Compositae.It has a marked
activity against chloroquinine_resistant and chloroquinine_
sensitive strains of Plasmodium falciparum and is useful
in the treatment of cerebral malaria (Klayman , 1985).
Plant tissue and cell culture technique has been used as
an important method for the research of natural medicines
and the production of secondary metabolites (Charlwood
et al , 1990).But there still exist many technical prob-
lems on large_scale culture , especially contamination ow-
ing to microbes.This is the largest obstacle for the indus-
tralization of plant tissue and cell culture.
Preservatives , such as sodium benzoate , potassium
sorbate and ethylparaben are widely used as food addi-
tives.They can prevent microbe growth under certain con-
ditions.ACZP is a kind of Ag_type inorganic antibacterial
agent , which has been used in the field such as tap water
treatment , fabric industry and construction materials ,
etc.These preservatives have remarkable antibacterial ac-
tivity and are quite safe (Xue and Liu , 1985;Feng and
Yan , 1998).It may be a breakthrough to introduce such
preservatives into plant cells , tissues and organ cultures ,
which may inhibit microbe growth without affecting the
growth of plant and the biosynthesis of secondary metabo-
lites.In this study , antibacterial effects of some preserva-
tives on the growth of A.annua roots and culture kinetics
have been investigated so as to provide a practically feasi-
ble method for root culture of A.annua on a large scale.
1　Materials and Methods
1.1　Plant materials
Artemisia annua L.root line induced by the leaf_
disc method was used , and the Agrobacterium rhizogenes
strain ATCC15834 with plasmid pRi15834 was used in
leaf_disc method(Liu et al , 1998).
1.2　Chemicals
ACZP was provided by Prof.CHEN Yun_Fa (Insti-
tute of Process Engineering , The Chinese Academy of Sci-
ences).Sodium benzoate , potassium sorbate , ethyl-
paraben and chitosan were obtained from Beijing Chemical
Co., PRC , and were of analytical grade.
1.3　Culture media
Revised MS medium (Murashige and Skoog , 1962)
was supplemented with 3% sucrose and different kinds of
preservatives at different concentrations.The medium pH
was adjusted to 5.85-5.90 with 1 mol/L NaOH and 1
mol/L HCl.All flask experiments were carried out in 100
mL Erlenmeyer flasks containing 40 mL media.Two gram
fresh weight roots per liter medium was inoculated.The
Erlenmeyer flasks were incubated on a rotary shaker at
110 r/min under 16 h light (with the intensity of 24
μmol·m-2·s-1)per day at 24(±1)℃ for 30 d(Liu et
al , 1997;1998), each treatment was in triplicate.
1.4　Assay methods
To determine fresh weight (FW), all the roots in
flasks were gently pressed on filter papers to remove ex-
cess water and weighed.Roots were then dried in an oven
at 50 ℃for 24 h and the dry weight(DW)was recorded.
Artemisinin determination was performed as described by
Shen et al(1983).The total residual glucose concentra-
tion was determined with the phenol and concentrated sul-
furic acid method , and sucrose solution was used as a
standard(Dubois et al , 1956).The determination of am-
monium was based on Weatherburn(1967)and nitrate to
Wang and Zhang(1988).All determinations were carried
out in triplicate.The change of the biomass of roots , con-
sumption of sucrose , ammonium and nitrate and variation
of pH were followed during the whole culture time.
Contamination of cultures was detected by making
smears and simple staining.For those non_contaminated
cultures , the media were transparent and no microbes de-
tected by simple staining.
2　Results
2.1　Antibacterial effects of different preservatives
Sodium benzoate , potassium sorbate , ethylparaben ,
chitosan and ACZP were added in MS medium without
sterilization.The results of root culture are shown in
Table 1.
As shown in Table 1 , sodium benzoate at 1 500
mg/L , potassium sorbate at 400 mg/L , ethylparaben at
200 mg/L and chitosan at 5 000 mg/L could not inhibit
the growth of microbes , and the roots did not grow.
Ethylparaben has limited antibacterial effect , the media
containing 500 mg/L and 800 mg/L ethylparaben were
contaminated by bacteria after 10_day and 17_day culture ,
respectively .It is obvious that the above preservatives
Received:2002-07-31　Accepted:2002-09-29
＊Author for correspondence.Tel:+86(0)10 62561815;Fax:+86(0)10 62561813;E_mail:.
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Acta Botanica Sinica　2003 , 45(2):136-139 http://www.chineseplantscience.com
Table 1　Growth and contamination of roots in MSmedia containing
preservatives under nonsterile conditions
Preservative
Concentration of
preservative
(mg/ L)
The day
beginning
contamination
The kinds
of microbes
Control 0 2 Bacteria , Fungi
Sodium
benzoate
500
1 000
1 500
2
2
2
Bacteria , Fungi
Potassium
sorbate
200
300
400
2
2
2
Bacteria
Ethylparaben 200
300
400
500
800
4
5
7
10
17
Bacteria
Chitosan 500
1 000
5 000
2
2
2
Bacteria
ACZP 40
50
5
over 30
Bacteria
-
could not meet the requirement of the culture of A.an-
nua roots.Though microbes were not detected in 30_day
culture of the roots in the media with 50 mg/L ACZP , the
roots grew to a limited extent.Therefore , particular atten-
tion was paid to examining antibacterial effects of different
low ACZP concentrations on root growth and artemisinin
accumulation.
2.2　Effects of ACZP at low concentrations on the
growth of roots and artemisinin biosynthesis
ACZP at different concentrations were added into MS
medium which was autoclaved for 20 min at 121 ℃.The
root cultures were inoculated and incubated for 30 d.Re-
sults are presented in Table 2.The roots grew more rapid-
ly in the medium containing 30 mg/L ACZP than those
with higher concentrations of ACZP , its DW (11.74
g/L)and artemisinin yield (162.30 mg/L)were about
93% and 83% of control (12.63 g/L and 196.07
mg/L), respectively.It was observed that during many
batch cultures(10 batches×10 Erlenmeyer flasks), the
contamination incidence of media was zero , but in media
without ACZP , the contamination incidence was 10%-
20%.Therefore , addition of ACZP in the media for the
culture of A.annua roots , especially for large_scale cul-
ture in a bioreactor , can be used for general practice.
2.3　Culture kinetics
Based on the above observation , the culture
Table 2　Effects of ACZP on root growth and artemisinin biosynthe-
sis in sterile media
Concentration
of ACZP(mg/L)
FW(g/L) DW(g/L) Artemisinincontent(%) Artemisininyield(mg/L)
Control(0) 150.5±58.32 12.63±1.12 1.54±0.14 196.07±34.93
30 144.51±10.29 11.74±1.21 1.37±0.12 162.30±30.67
40 78.92±3.83 6.75±0.42 1.31±0.11 88.89±12.93
50 21.71±2.02 2.29±0.23 0.97±0.08 22.40±4.07
Values are means of triplicate standard deviation.
kinetics , including the changes of the root biomass ,
medium pH , residual sucrose , consumption of ammonia
and nitrate with time were studied.
2.3.1　Changes of the biomass of A.annua root　
The growth rate and final biomass of the hairy roots are
important factors when the addition of preservatives is
considered.The comparative results of changes in biomass
vs time between control and the culture with 30 mg/L
ACZP are shown in Fig.1.Control as well as ACZP con-
taining cultures grew in a similar trend.In lag phase and
exponential phase , the root growth rates are identical ,
only in stationary phase , there is slight difference.This
indicated that the addition of 30 mg/L ACZP did not af-
fect the root growth remarkably.Final biomass of the roots
reached 11.74 g/L after 30_day culture , which was about
93%of control(12.63 g/L).
Fig.1.　Biomass of roots vs time in control and in the culture with
30 mg/L ACZP.
2.3.2 　pH variation and consumption of nitrogen
sources　Figure 2 shows that the pH value in the control
declined slowly from 5.85 to 4.63 during the first 15
days , then increased to 7.50 at the 30th day.The pH
variation was closely related to the utilization of different
nitrogen sources.The changes of ammonia and total ni-
trate content(5 mmol/L NH4NO3 with 20 mmol/L KNO3
together)with culture time were presented in Figs.3 and
4.The pH value declined at the beginning of culture ow-
ing to NH+4 consumption was higher than that of NO-3 in
NH4NO3 consumption.After 15 d , total NO-3 consump-
tion was higher than NH+4 and K+ consumption , leading
to the sharp increase of pH.Before 15 d , KNO3 con-
sumption had no remarkable effect on pH change because
both K+ and NO-3 in KNO3 consumption were basically
the same.This phenomenon was the same as what ob-
served in other plant tissue culture studied(Fetto_Neto et
al , 1994).The pH of the medium containing 30 mg/L
ACZP declined from 5.85 to 4.87 , slower than that of
control at the beginning , then increased to 7.34 at the
30th day.It might be that the rates of utilization of am-
monium at the beginning were slightly lower and after 15 d
OUYANG Jie et al:Effects of Ag_carrying Zirconium Phosphate on the Kineties of Growth of the Roots of Culture Artemisia annua L. 137　
were slightly higher than those of control.
The consumption of nitrogen sources in the medium
containing 30mg/L ACZP was lower than that of the con-
trol , this was related to the growth of A.annua roots in
both media.
Fig.2.　Variation of pH vs culture time.
Fig.3.　Time course of residual ammonium in the medium.
2.3.3　Changes of residual sucrose　Sucrose in the
medium was hydrolyzed to glucose and fructose during au-
toclaving and the growth of roots , so the residual hexose
was detected as residual sucrose.In the control medium ,
sucrose was utilized gradually along with the growth of
roots.Sucrose was depleted at the end of culture , which
was related to the largest biomass.As shown in Fig.5 ,
the consumption of sucrose in the medium containing 30
mg/L ACZP was similar to that of control , but the con-
sumption rate was lower than that of the control.
3　Discussion
The antibacterial principle of ACZP is an photoin-
ductive effect , ACZP has strong inhibitory activity against
gram positive and negative bacteria under light(Feng and
Yan , 1998).Sodium benzoate was changed into benzoic
acid at low pH , thus inhibits the activity of enzymes , es-
pecially the condensation of acetyl coenzyme A.Potassium
sorbate links with -SH of enzyme and thus to destroy the
function of many important enzymes.Ethylparaben in-
hibits the activity of respiratory enzyme and electronic
transport enzyme and destroys membrane structure (Xue
and Liu , 1985).Sorbate inhibits the uptake of pheny-
lalanine , decreases the rate of protein synthesis , and al-
ters the patterns of phosphorylated nucleotide accumula-
tion , resulting in increased intracellular concentrations of
GTP , ppGpp , and an unidentified compound (possibly
pppGpp)(Ronning and Frank , 1987).
Fig.4.　Time course of residual nitrate in medium.
Fig.5.　Time course of residual sucrose in medium.
The effects of preservatives on plant tissues and cells
are not very clear till now.There are big differences from
cell wall and membrane structure between plant cells and
microbial cells , so the effects of preservatives differ re-
markably.It is possible to find a kind of suitable preser-
vative , which can inhibit the growth of microbes to some
extent without affecting the growth of plant tissue and
biosynthesis of secondary metabolites evidently.In this
138　 植物学报　Acta Botanica Sinica　Vol.45　No.1　2003
study , the optimum preservative used in A.annua root
culture was found to be ACZP , which at the concentration
of 30 mg/L can lower the contamination incidence of
medium without major effects on the growth of root and
artemisinin biosynthesis.
The result may be instructive for large_scale culture
of A.annua root in a bioreactor.It is a practical method
using ACZP in a medium to lower contamination and get
better economic benefit.
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(Managing editor:ZHAO Li_Hui)
银型磷酸锆盐抗菌粉对青蒿根生长的影响及生长动力学研究
欧阳杰　王晓东　赵　兵　王玉春＊
(中国科学院过程工程研究所生化工程国家重点实验室 , 北京 100080)
摘要:　在植物组织和细胞培养过程中 , 尤其是在生物反应器培养中的染菌问题 , 一直是制约植物细胞培养工业化
的难题。通过比较各种防腐剂的抑菌效果 ,确定银型磷酸锆盐抗菌粉为青蒿根培养的最佳防腐剂。 银型磷酸锆盐
抗菌粉在浓度为 30 mg/L时 , 既能降低培养液的染菌几率 , 又不明显抑制青蒿根的生长及青蒿素的生物合成。在添
加 30 mg/L抗菌粉的培养液中进行的青蒿根生长 、pH值变化以及残糖 、铵离子和硝酸根离子消耗的动力学研究表
明 ,在 40 d 内青蒿根在培养液中生长良好 , 营养成分的消耗和对照呈相似的趋势。
关键词:　青蒿;毛状根;青蒿素;银型磷酸锆盐抗菌粉;生长动力学
中图分类号:S432.1　　　文献标识码:A　　　文章编号:0577-7496(2003)02-0136-04
收稿日期:2002-07-31　接收日期:2002-09-29
＊通讯作者。Tel:010_62561815;Fax:010_62561813;E_mail:。
(责任编辑:赵丽惠)
OUYANG Jie et al:Effects of Ag_carrying Zirconium Phosphate on the Kineties of Growth of the Roots of Culture Artemisia annua L. 139