全 文 ：Rapid Propagation of Pteris vittata L . via
Tissue Culture
Liqiong LUO1, Jun ZHANG1, Xu LUO1, Bing YANG2*
1. Bioscience and Biopharmaceutical College, Guangdong Pharmaceutical University, Guangzhou 510006, China;
2. South China Institute of Environmental Science, Ministry of Environment Protection, Guangzhou 510655, China
Supported by National Natural Science Foundation of China (30900158); Research
Foundation for Guangdong Pharmaceutical University (43553006).
*Corresponding author. E-mail: andyang@tom.com
Received: June 27, 2011 Accepted: November 21, 2011A
Abstract [Objective] The study had developed a means of rapid propagation Pteris
vittata L.by tissue culture. The species was a perennial fern belonging to the genus
Pteris. [Method] The leaf bud of P. vittata collected in field conditions as explantsand
the 1/2 MS + 3% sucrose + 0.7% agar as the basic medium were used to screen
the medium formula of the phytohormone ratio for callus induction and subculture of
P. vittata. [Result] The best medium formula for each step was list below: 1/2 MS +
3% sucrose + 0.7% agar + 0.5 g/L PVP + 0.1 mg/L KT + 0.5 mg/L 2, 4-D for in-
ducing the callus from explants; 1/2MS + 3% sucrose + 0.7% agar + 0.5 g/L PVP +
1.0 mg/L KT + 0.01 mg/L 2,4-D for inducing the GGB from callus and the seedlings
from GGB. In addition, 1/2 MS + 3% sucrose + 0.7% agar + 0.5 g/L PVP + 0.5
mg/L 2,4-D for the subculture could make the continued proliferation of callus. [Con-
clusion] This study makes an applicable procedure by the direct use of field materi-
als, for propagating P. vittata in a simplified and rapid mode.
Key words Differentiation; Phytohormone; Plant tissue culture; Pteris vittata L.
P teris vittata L. is ancient peren-nial ferns, with asexual sporesfor its reproduction[1]. It can ab-
sorb arsenic, thus having a broad appli-
cation prospects in the purification of ar-
senic[2-3]. Arsenic is a toxic metalloid ele-
ment. Arsenic and its compounds are
widely distributed and utilized for the
development of pesticides, herbicides,
pesticides and alloys. There is arsenic
found in human body, however, ex-
cessive arsenic intake can lead to ar-
senic poisoning. With the widespread
application of arsenic in industry, ar-
senic contamination has become a
serious problem that can not be ig-
nored. Rapid seed propagation, char-
acterized by rapid, single source and
single genetic background, is the most
widely used and most effective tech-
nology in plant tissue culture. Rapid
propagation in vitro is tens of thou-
sands of times faster than the con-
ventional methods[4]. Existing researc-
hes on tissue culture of P. vittata
mainly use the gametophyte generat-
ed by spores to induce callus, which is
complicated and difficult. In this study,
the leaf buds of pinnately compound
leaf of P. vittata collected in the field
were used as materials to simplify the
procedures.
Materials and Methods
Reagents
The reagents used were as fol-
lows: MS medium, 6-KT (6-Furfury-
laminopurine), 2,4-D (2,4 - dichloroph-
enoxyacetic acid), PVP (polyvinylpyrr-
olidone), 0.1% HgCl2, 75% alcohol and
8% -10% calcium hypochlorite. 6-KT
and 2,4-D were prepared to mother
liquor with the concentration of 1 g/L,
while PVP was prepared to 50 g/L.
Materials
The leaf buds of P. vittata with a
length of about 3-6 cm were collected
in the field. After the pubescence was
carefully removed from the surface of
leaves, the leaves were washed with
tap water for more than 3 h. The leaf
buds were first soaked in 75% alcohol
for 10 s, and then soaked in 10% cal-
cium hypochlorite solution and placed
in shaker oscillation for 10 min or
above. After disinfection, the leaf buds
were washed with sterile water for
more than six times. Cleaning opera-
tion and the following steps were car-
ried out in a sterile room. The dissec-
tion tray, forceps, and scalpel blades
were sterilized. Then, the sterilized
explants were cut from the middle of
itself, and the curved part at the top of
explants was selected and cut into
small pieces of about 2 -3 mm. The
explants were placed in the medium
for dark culture after re-sealing with the
wound surface stick to the medium.
Culture conditions
MSmedium [5] was used as the ba-
sic medium for callus induction, the
medium used in this study was: 1/2MS+
3% sucrose +0.7% agar + 0.5 g/L PVP +
0.1 mg/L KT + 0.5 mg/L 2,4-D. Then, the
pH was adjusted to 5.8-6.0 with NaOH
and HCl. Autoclave sterilization. Incu-
bation temperature was of 18 -25 ℃ .
Dark culture is utilized.
Results and Analysis
Inoculation and induction of callus
The conventional method used to
inhibit browning is to use the basic
medium supplemented with PVP. 1/2
MS basic medium supplemented with
0.5 g/L PVP can effectively inhibit
browning. On this basis, the hormones
were added with the KT concentration
of 0.1-0.5 mg/L and 2,4-D concentra-
tion of 0.2-4.0mg/L. The results showed
that when the concentration of KT was
less than that of 2,4-D, the callus
would be generated. The best hor-
mone combination was: 0.1 mg/L 6-KT+
Agricultural Science & Technology, 2012, 13(1): 68-70
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agricultural Biotechnology
DOI:10.16175/j.cnki.1009-4229.2012.01.029
Agricultural Science & Technology2012
Vol.13, No.1, 2012 Agricultural Science & Technology
Fig.5 Regenerated plant in the soil
0.5 mg/L 2,4-D, which could induce
callus within 20 d with the average in-
duction rate of 90%. The induced cal-
lus was irregular and loose. The thick-
ness of callus was about 2 mm, and
the callus was green with out brown-
ing, as well as easy to be peeled and
transferred (Fig.1).
Callus proliferation
The primary callus was cut into
small pieces with the side length of
about 2 mm, followed by transfer to 1/2
MS + 0.5 g/L PVP medium, with the
2,4-D concentration of 0.5 mg/L. The
results showed that 90% of the callus
could proliferate successfully with
rapid growth rate (Fig.2).
Callus differentiated as GGB (Green
Globular Body)
The callus was cut into small
pieces and plated into medium 1/2
MS+0.5 g/L PVP + 1.0 mg/L KT + 0.01
mg/L 2,4-D. One week later, the regu-
lar and round cell clusters were grown
on its surface, with the color of yellow
green or dark green. The GGB was
constantly produced during the prolif-
eration process of callus (Fig.3).
GGB proliferation
If the dark culture was used in the
differentiation process from GGB to
seedlings, the differentiated adventi-
tious buds would be dedifferentiated to
GGB again, thus resulting in the un-
limited proliferation of GGB.
Differentiation of GGB into seedl-
ings
GGB was cut into pieces with the
diameter of about 2 mm and plated to
1/2 MS + 0.5 g/L PVP + 1.0 mg/L KT +
0.01 mg/L 2,4-D medium. After dark
cultured for 7 d, the buds of about 5
mm were differentiated. Then, the light
culture at 3 000 lx was carried out for
2-3 d, and the buds could be grown in-
to leaf-shaped. After 30 d of light cul-
ture, the seedlings with the plant
height of about 5 cm would be grown
(Fig. 4 a, b and c).
Hardening-seedling
The adventitious buds were sepa-
rated into dozens of seedlings. Seedl-
ings were planted in the soil and cov-
ered with plastic wrap to prevent mois-
ture loss, followed by placed under
light for hardening-seedling. The sur-
vival rate was up to 95%. 7 d later, the
growth conditions of seedlings were
good. Then, they could be planted in
field or directly applied to the destina-
tion field (Fig.5).
Conclusion and Discussion
P. vittata has great prospects in
controlling arsenic contaminated soil
or water. It belongs to gymnosperm
and has lower germination rate, so the
traditional spore-reproduction was
time-consuming. The use of plant tis-
sue culture for rapid propagation of
seedlings is a good solution to this
problem. The selection of explants and
plant hormones is the key factor af-
fecting plant tissue culture. Different
plant hormone levels will regulate the
process of differentiation and dediffer-
entiation in plants[6].
In this study, the young buds were
used as explants, and the medium
was supplemented with 0.5 g/L PVP to
avoid of browning of explants. We tried
to use the young leaves of P. vittata as
explants, and the results showed that
under the same conditions, the
browning was more serious and only
one of three young leaves produced a
small amount of callus under different
hormone combinations.
The medium formula for callus in-
duction and subculture of P. vittata
were successfully screened through
the hormone combinations test. The
optimal medium formula for induction
of callus from explants was: 1/2 MS +
3% sucrose + 0.7% agar + 0.5 g/L
PVP + 0.1 mg/L KT + 0.5 mg/L 2,4 -D;
the optimal medium formula for induc-
tion of GGB from callus and the induc-
tion of seedlings from GGB was: 1/2
MS+3% sucrose +0.7% agar +0.5 g/L
PVP + 1.0 mg/L KT+0.01 mg/L 2,4-D.
In addition, the use of 1/2 MS+3% su-
crose+0.7% agar + 0.5 g/L PVP+0.5
mg/L 2,4-D for subcultured could result
Fig.1 Callus induced from frond bud on
1/2 MS + 0.5 g/L PVP + 0.1 mg/L
KT + 0.5 mg/L 2,4-D for three
weeks
Fig.2 Callus for subculture
Fig.3 GGB induced from callus on 1/2
MS + 0.5 g/L PVP + 1 mg/L KT +
0.01 mg/L 2,4-D for 7 d
a: Multiple shoots induced from GGB after 5 weeks; b, c: Seedlings grown after 9 weeks
Fig.4 Differentiation process of GGB to seedlings
69
Agricultural Science & Technology 2012
Agricultural Science & Technology Vol.13, No.1, 2012
(Continued from page 59)
细枝木麻黄组织培养和转基因研究
林永生 1,2，蒋 晶 2，乔桂荣 2，李海营 2，邱文敏 2，卓仁英 2* （1.福建省莆田市涵江区江口镇农业服务中心，福建莆田 351115；2.中国林科院亚热带
林业研究所，浙江富阳 31140）
摘 要 [目的]对细枝木麻黄进行组织培养和转基因研究。[方法]以耐寒性细枝木麻黄为试验材料，探讨了愈伤组织诱导、不定芽分化、农杆菌介
导转化 3种条件对转化效率的影响。[结果]对细枝木麻黄不定芽诱导分化适宜的激素组合为 DCR+6-BA 5.0 mg/L+NAA 0.5 mg/L；转基因抗生素
选择压力为潮霉素，共培养时间 3 d；以初步建立的转基因体系利用农杆菌介导法获得 94株转基因木麻黄，通过 PCR检测，其中 61株为 PCR阳
性植株。[结论]该研究为细枝木麻黄的组织培养和转基因研究奠定了基础。
关键词 细枝木麻黄；耐寒性；组织培养；转基因
基金项目 浙江省自然科学基金项目(Y306072)资助。
作者简介 林永生（1973-），男，福建莆田人，工程师，从事植物生物学研究。*通讯作者，E-mail: zhuory@gmail.com。
收稿日期 2011-10-18 修回日期 2011-11-15
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[6] KANG JF(康杰芳), WANG ZZ(王喆之).
Primary studies on function of Cepha-
losporins antibiotics in transgenic tobac-
co(头孢霉素类抗生素在转基因烟草中
作用的初步研究)[J]. Acta Botanica Bo-
reali-Occidentalia Sinica (西北植物学
报), 2003, 23(1): 60-63.
[7] PHELEP M, PETIT A, MARTIN L, et al.
Transformation and regeneration of a
nitrogen-fixing tree, Allocasuarina verfi-
cillata Lam[J]. Biotechnology, 1991, 9:
461-466.
[8] LE QV, BOGUSZ D, GHERBI H, et al.
Agrobacterium tumefaciens gene trans-
fer to Casuarina glauca, a tropical nitro-
gen-fixing tree[J]. Plant Science, 1996,
118: 57-69.
Responsible editor: Xiaohui FAN Responsible proofreader: Xiaoyan WU

凤尾蕨科植物蜈蚣草的组织培养
罗利琼 1，张 军 1，罗 旭 1，杨 兵 2*
（1.广东药学院生命科学与生物制药学院, 广东广州 510006; 2.环境保护部华南环境科学研究所, 广东广州 510655）
摘 要 [目的]对凤尾蕨科植物蜈蚣草的组织培养方法进行研究。[方法]以野外采集的叶芽为外植体，以 1/2 MS + 3％蔗糖+ 0.7％琼脂为基本培
养基，通过激素配比试验，筛选蜈蚣草愈伤组织诱导和继代培养的培养基配方。[结果]从外植体诱导出愈伤组织最佳培养基配方为 1/2 MS + 3％
蔗糖+ 0.7％琼脂 + 0.5 g/L PVP + 0.1 mg/L KT + 0.5 mg/L 2,4-D；从愈伤组织诱导出 GGB和从 GGB诱导出幼苗最佳培养基配方为 1/2MS +
3％蔗糖 + 0.7％琼脂+ 0.5g/L PVP + 1.0 mg/L KT + 0.01 mg/L 2,4-D；另外，使用 1/2 MS + 3％蔗糖+ 0.7％琼脂 + 0.5 g/L PVP + 0.5 mg/L
2,4-D做继代培养可以使愈伤组织持续增殖。[结论]研究直接使用野外材料组织培养，简化了试验步骤，是一种方便快速的蜈蚣草组培方法。
关键词 分化；植物激素；组织培养技术；蜈蚣草
基金项目 国家自然科学基金青年项目(NO. 30900158)资助，广东药学院内科研基金项目（43553006）资助。
作者简介 罗利琼（1974-），女，湖北荆州人，实验师，从事生物技术相关试验研究，E-mail：llq-200010@163.com。*通讯作者, 助理研究员，博士，
从事污染生态学研究，E-mail：andyang@tom.com。
收稿日期 2011-06-27 修回日期 2011-11-21
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in continued proliferation of callus.
It was found that light played an
important role in tissue culture of P. vit-
tata. In the process of callus induction,
the explants should be dark cultured
after inoculation, otherwise, the brown-
ing would be speed up. Because the
light would accelerate the metabolism
of explants, thus resulting in the in-
creasing of polyphenol oxidase activi-
ty, leading to enzymatic browning.
More importantly, in the process of the
induction of adventitious buds, after 7
d of culture, the buds should be se-
lected and placed for light culture
when the length was about 10 mm. If
prematurely taken out, the buds would
be short and less in amount, thus af-
fecting the yield. If it was too late (20
d), the buds would be dedifferentiated
into GGB.
In this study, the direct use of field
material for tissue culture had simpli-
fied the test procedure and could pro-
duce a large number of seedlings with-
in two months.
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