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虎舌红组织培养中有效无菌材料的获得研究(英文)



全 文 :虎舌红组织培养中有效无菌材
料的获得研究
曾云英 * (江苏开放大学/江苏城市职业学
院,江苏南京 210036)
摘 要 该研究以虎舌红为试材, 探讨外植
体母本的预处理、外植体的采集、灭菌、接种等
处理方式对获得有效无菌材料的途径和方法
的影响。 结果表明: 2 月将母株移入室内,进
行预处理,3 月中旬取当年生枝条和萌蘖条上
的茎尖,用去污剂(肥皂水或洗衣粉水)浸泡并
刷洗后,流水冲洗 1~2 h,用 75%的酒精表面
消毒 30 s, 无菌水冲洗 2~3 次后放入 0.1%升
汞中灭菌,先用升汞消毒 4 min,无菌水冲洗 3
次,再用升汞消毒 4 min,无菌水冲洗 5~8 次
后进行有效接种, 这种方法能有效降低污染
率,提高成活率。
关键词 组织培养;无菌材料;虎舌红
基金项目 江苏开放大学、江苏城市职业学院
“十二五”规划课题经费(12SEW-Y-026)。
作者简介 曾云英 (1976- ),女 ,重庆长寿
人,副教授,硕士,主要从事园林植物与观赏园
艺、生态规划方面的研究,E-mail:yuniny@sina.
com。 * 通讯作者。
收稿日期 2014-04-20
修回日期 2014-05-30
Optimization of the Method to Obtain Effective
Sterile Explants in Tissue Culture of Ardisia
mamillata Hance
Yunying ZENG*
Jiangsu Open University/the City Vocational College of Jiangsu, Nanjing 210036, China
Supported by the Research Funds of Jiangsu Open University/the City Vocational
College of Jiangsu during the 12th Five-Year Plan Period (12SEW-Y-026).
*Corresponding author. E-mail: yuniny@sina.com
Received: April 20, 2014 Accepted: May 30, 2014A
Agricultural Science & Technology, 2014, 15(6): 916-917, 936
Copyright訫 2014, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
A rdisia mamillata Hance, aperennial evergreen shrub ofMyrsinaceae, is a rare orna-
mental plant on the list of endangered
species. It also has medicinal value.
A. mamillata Hance propagates more
by seeds and cuttings than by tissue
culture[1-4]. The small hair on the sur-
face of A. mamillata Hance supplies a
place for a large number of bacteria to
propagate, which causes great diffi-
culties to the surface disinfection of
explants in tissue culture. An incom-
plete disinfection will result in a high-
er pollution rate in laboratory [5]. How-
ever, the vitality of A. mamillata
Hance explants may be lost in a thor-
ough surface disinfection [6-7]. So, ef-
fective sterile explant is the key in tis-
sue culture of A. mamillata Hance. In
the present study, the procedures to
obtain sterile material in tissue culture
of A. mamillata Hance were opti-
mized, with an attempt to provide ref-
erence for further research and pro-
duction of A. mamillata.
Materials and Methods
Pretreatment of parental plant
Parental plant was moved to
greenhouse one month before sprout-
ing to do some fertilizer and water
managements and pest control. Such
pretreatment can reduce the probabili-
ty of infection with microorganism in
the process of tissue culture, and can
keep the consistency among the ex-
perimental materials, so as to improve
the reproducibility of the experiments.
Sampling time
The best time for collecting ex-
plant is a prerequisite for successful
establishment of tissue culture sys-
tem. For A. mamillata Hance, pollution
and browning are two important fac-
tors affecting the survival of the ex-
plants. The explants of A. mamillata
Hance were collected in middle March,
middle May, middle August and middle
October, respectively. Then, the pollu-
Abstract In the present study, we optimized the procedures to pretreat the parental
material, to collect, sterilize and inoculate the explants in the tissue culture of Ar-
disia mamillata Hance, so that more effective sterile explants can be obtained. The
results revealed the optimal procedures. In detail, the parental materials were trans-
ferred and pretreated in laboratory in February. The stem tips of new branches
were collected in middle March, cleaned with eradicator and then rinsed with tap
water for 1-2 h. After that, the explants were surface sterilized with 70% alcohol for
30 s, rinsed with sterile water 2-3 times, sterilized with 0.1% mercuric chloride for
4 min, rinsed with sterile water three times and sterilized with 0.1% mercuric chlo-
ride for 4 min again. Finally, they were inoculated into medium after being rinsed
with sterile water 5-8 times. By this method, the pollution rate of explants can be
greatly reduced while their survival rate can be significantly improved.
Key words Tissue culture; Sterile material; Ardisia mamillata Hance
DOI:10.16175/j.cnki.1009-4229.2014.06.002
Agricultural Science & Technology2014
Table 1 Pollution rate, browning rate and survival rate of the explants collected in different
months
Month Pollution rate∥% Browning rate∥% Survival rate∥%
Mid March 29.73 20 38.76
Mid May 44.87 42.58 20.35
Mid August 65.69 50.92 10.92
Mid November 23.98 9.07 14.37
Table 2 Sterilization of explants with 75% alcohol for different time
Test No. Treatment time∥s Mortality rate∥% Pollution rate∥% Survival rate∥%
1 30 4 9 87
2 45 24 5 71
3 60 95 0 5
Table 3 Treatments for explants disinfection with 0.1% HgCl2
Treatment Time∥min Details
One-step sterilization 5 The explants were sterilized with 0.1% HgCl2 for 5 min once.
8 The explants were sterilized with 0.1% HgCl2 for 8 min once.
10 The explants were sterilized with 0.1% HgCl2 for 10 min once.
Two-step sterilization 3+2 The explants were sterilized with 0.1% HgCl2 for 3 min at first, rinsed with sterile water three times
and then sterilized with 0.1% HgCl2 again for 2 min.
4+4 The explants were sterilized with 0.1% HgCl2 for 4 min at first, rinsed with sterile water three times
and then sterilized with 0.1% HgCl2 again for 4 min.
5+5 The explants were sterilized with 0.1% HgCl2 for 5 min at first, rinsed with sterile water three times
and then sterilized with 0.1% HgCl2 again for 5 min.
tion rate, browning rate and survival
rate of the explants were measured
(Table 1).
Sterilization of explants
Cleaning explant The stem tips of
new branches were cut off and soaked
in soapy water or detergent water.
Then we cleaned the explants gently
with a soft brush to remove the dust
and bacteria that attached to the ex-
plant surface. At last the explants were
washed with tap water for 1-2 h.
Sterilizing the explants with alcohol
As 75% alcohol can easily penetrate
the plant cells and kill them [2 ] , so a
suitable disinfection time is the key to
keep explants activity. In this experi-
ment, after the explants were rinsed
with sterile water 2-3 times, they were
disinfected in 75% alcohol for 30, 45
and 60 s in a super-clean bench
(Table 2).
Sterilizing explants with mercuric
chloride Mercuric chloride (HgCl2)
has been considered as the best fungi-
cide in tissue culture. At room temper-
ature, 0.1% HgCl2 can effectively kill
bacteria and spores attached on the
surface of explants within 10 min. But
the residual Hg + must be removed
completely because it is highly toxic to
plants. The explants were sterilized
with 0.1% HgCl2 for different times by
one step or two steps, as shown in
Table 3.
Inoculation of explants
The stem tips were cut off from
the sterilized explants those main-
tained biological activity and immedi-
ately inoculated in the culture medium
prepared in advance. The explants
were not all buried in the culture medi-
um, only the morphological bottom
was inserted into the culture medium,
while the growth point was out of the
culture medium.
Results and Analysis
The optimal sampling time
As shown in Table 1, the pollution
rate of explants collected in middle
August was the highest, and that of the
explants collected in middle November
was the lowest. The phenolic content
and polyphenol oxidase activity in the
explants of A. mamillata Hance were
the lowest in autumn, and then gradu-
ally increased over time in winter. The
phenolic content and polyphenol oxi-
dase activity reduced again in springs
and then increased in summer. So, the
browning rate of the explants collected
in middle August was the largest. The
highest survival rate was found in the
explants collected in middle March,
when the plants have sufficient nutri-
ents and bud growth points are in the
active period; at the same time, the
phenolic content and oxidation activity
of polyphenol oxidase in the explants
have not yet reached their peaks, so
the stem apex and segments have
high survival rate[1].
Optimal conditions for explant
sterilization with alcohol
The explants were sterilized with
75% alcohol for 30, 45 and 60 s. As
shown in Table 2, none of the explants
sterilized for 60 was polluted, but their
mortality reached 95% , only 5% of
them were survived, while the survival
rate of the explants sterilized for 30 s
was the highest. So, 30 s is the best
time for sterilization of A. mamillata
Hance explants with alcohol.
Optimal conditions for explant
sterilization with
The explants were sterilized with
0.1% HgCl2 for different times by one
step or two steps, as shown in Table 3.
As can be seen from Fig.1, in the
same treatment time, the survival rate
of explants sterilized by two steps was
significantly higher than that of the ex-
plants sterilized by one step. The the
survival rate reached peak when the
explants were sterilized for 8 min by
one step or 3+5 min by two steps. As
shown in Fig.2, the pollution rate of the
explants decreased along with the in-
creasing of sterilization time. For the
explants sterilized for 10 min by one
step or 5+5 min by two steps, the pol-
lution rate was decreased greatly but
the survival rate was also decreased.
The possible reason is that the dam-
age to explants caused by 0.1% HgCl2
increased in a long time of sterilization.
To sum up, the best approach is to
sterilize the explants with 0.1% HgCl2
for 4 min, wash them with distilled wa-
(Continued on page 936)
917
Agricultural Science & Technology 2014
Responsible editor: Qingqing YIN Responsible proofreader: Xiaoyan WU
ter 3 times and sterilize them again
with 0.1% HgCl2 for 4 min.
Conclusion
Both disinfectant type and steril-
ization time obviously affect the sur-
vival rate of explants, as sterilization
for a too lone time may lead to brown-
ing of explants and reduce the survival
rate, but sterilization for a too short
time may not be able to damage all the
bacteria. The small hair on the surface
of A. mamillata Hance supplies a place
for a large number of bacteria to prop-
agate, which causes great difficulties
to the surface disinfection of explants
in tissue culture. It is the reason why
many A. mamillata Hance explants are
polluted and die during tissue culture.
Our results revealed the optimal
procedures to obtain the sterile ex-
plants for during tissue culture: stem
tips of new branches were collected in
middle March, washed with detergent
water, rinsed with tap water for 1-2 h,
surface sterilized with 70% alcohol for
30 s, and then with 0.1% mercuric
chloride for 4 min, rinsed with sterile
water three times and sterilized with
0.1% mercuric chloride for 4 min again,
rinsed with sterile water 5 -8 times,
and inoculated into medium. By this
method, the pollution rate of explants
can be greatly reduced while their
survival rate can be significantly im-
proved.
References
[1] ZHONG J, YE M, ZHUANG P, et al. A
review of Ardisia mamillata Hance [J].
Northern Horticulture, 2008(5): 65-69.
[2] DU MH, LI YY, TIAN L, et al. Establish-
ment of high-frequency regeneration
system for stem apexes of Ardisia
mamillata Hance [J]. Journal of North-
east Forestry University, 2007,35 (3):
21-23.
[3] ZENG YY, SUN Y. Study on the tech-
nique system of tissue culture and rapid
propagation of Ardisia mamillata [J].
Journal of Anhui Agricultural Sciences,
2009, 37(13): 5875-5876.
[4] LUO JF, CHENG ZY, LONG CL. Tissue
culture of Ardisia mamillata [J]. Plant
Physiology Communication, 2004, 40
(4): 465.
[5] ZENG YY. Advances in Tissue Culture
of Ardisia mamillata Hance [J]. Northern
Horticulture, 2009(8): 155-157.
[6] KANG ML. Study on the technique sys-
tem of tissue culture of Ardisia mamilla-
ta[D]. Master’s degree thesis of Si-
chuan Agricultural University, 2003.
[7] ZHANG YJ. The Research on effective
non-bacteria material obtained among
tissue culture[J]. Practical Forestry Te-
chnology, 2004(6): 20-21.
Fig.1 Survival rate of explants sterilized
with 0.1% HgCl2 for different time by
one step or two steps
Fig.2 Pollution rate of explants sterilized
with 0.1% HgCl2 for different time by
one step or two steps
(Continued from page 917)
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Responsible editor: Na LI Responsible proofreader: Xiaoyan WU
After falling on the ground after spin-
ning, they can be wiped out together.
Actively carrying out physical con-
trol to make the larvae die inside
the adults Since the S. cinereari
have the characteristics of phototaxis,
the light trapping method can be used
to control the pests, making the larvae
die inside the adults.
Taking remedial measures of
chemical control First is to kill the
young larvae of no more than 3 instars
by spraying the 50% diflubenzuron III
which is diluted to 1 000-2 500 times
by adding water. Second is to apply
the 5% phoxim granules under the tree
with the application amount of 3 -5 g
per square meters, followed by one
time of shallow hoeing, which can
make the drug particles enter into the
soil, thereby killing the larvae pupate.
Conclusion
The most significant characteris-
tics of the development and infestation
of S. cinereari are causing bare
branch after damaging the leaves,
seriously affecting the urban land-
scaping and beauty; covering the
sidewalks with frass and devoured
leaves, which is also seriously affect
the urban landscape. Therefore, well
controlling S. cinereari is one of the
most important measures to realize
the two protections (protect ecologi-
cal environment and protect the ap-
pearance of the city ) , and strength -
ening the control and prevention of
S. cinereari is of great significance to
beautify urban environment.
References
[1] LIU YQ (刘永齐 ). The development of
Semiothisa cineraria and its control (太
原市国槐尺蠖的发生特点与防治试验 )
[J]. Shanxi Forestry Science and Tech-
nology, 2008(1): 20-21.
[2] DENG H (邓辉). Analysis on the devel-
opment and control of Semiothisa
cineraria(国槐尺蠖的发生与防治技术分
析 ) [J]. Chinese Horticulture Abstract,
2011(3): 105-106.
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