Download use of tissue culture for the mass propagation of pathogen

USE OF TISSUE CULTURE FOR THE MASS
PROPAGATION OF PATHOGEN-FREE PLANTS
Hsin-Sheng Tsay
Department of Applied Chemistry,
Chaoyang University of Technology
168 Gifeng E. Rd., Wufeng, Taichung 41301, Taiwan
ABSTRACT
Different in vitro culture techniques have been used for rapid plant propagation. Pathogenfree plants have been produced using techniques such as seed culture, meristem culture,
micropropagation using axillary or adventitious shoot buds, and somatic embryogenesis. This
Bulletin discusses the importance of in vitro culture techniques for the production of pathogen-free
medicinal plants such as Anoectochilus formosanus, Limonium wrightii and Corydalis yanhusuo.
INTRODUCTION
Plant tissue culture technology has been
successfully used for the commercial
production of pathogen-free plants (Debergh
and Maene 1981), and to conserve the
germplasm of rare and endangered species
(Fay 1992). Techniques such as meristem
culture (Hu and Wang 1983) and hot-water
treatment of explants before in vitro culture
(Hol and van der Linde 1992, Langens-Gerrits
et al. 1998) have been used to produce plants
free from pathogens. In Taiwan, plant tissue
culture is being used for the mass production
of medical plants that in the wild are fairly
rare, and which are becoming scarcer as time
goes by.
Medicinal plants are important to the
health of many people in developing countries.
Approximately 80% of people in developing
countries still rely on traditional medicine for
their primary health care needs. This usually
involves the use of plant extracts (Vieira and
Skorupa 1993).
Many medicinal plant species are
disappearing at an alarming rate, as a result
of rapid agricultural and urban development,
deforestation and indiscriminate collection.
Plant tissue culture technology may help to
conserve rare and endangered medicinal plants.
Many important Chinese medicinal herbs have
been successfully propagated in vitro, either
by organogenesis (Erdei et al. 1981, Shoyama
et al. 1983, Hatano et al. 1986, Matsumoto et
al. 1986, Hiraoka and Oyanagi 1988, Nishioka
1988, Tsay et al. 1989, Huang et al. 2000,
Chen et al. 2001, Chueh et al. 2001) or by
somatic embryogenesis (Hiraoka et al. 1986,
Kitamura et al. 1989, Tsay and Huang 1998,
Sagare et al. 2000). In vitro propagated
plants of many important medicinal species
were found to be uniform, showing less
variation in their content of secondary
metabolites than their wild/cultivated
counterparts (Yamada et al. 1991). This
paper
describes
the
propagation
of
Anoectochilus formosanus using nodal explants,
Limonium wrightii through adventitious shoot
buds, and Corydalis yanhusuo by means of
somatic embryogenesis.
Keywords: Anoectochilus formosanaus, Corydalis yanhusuo, germplasm conservation, Limonium
wrightii, medicinal plants, micropropagation, pathogen-free plants, plant tissue culture, shoot
morphogenesis, somatic embryogenesis
1
TISSUE CULTURE OF THE ORCHID
ANOECTOCHILUS FORMOSANUS
Micropropagation using nodal explants
Anoectochilus
formosanus
Hayata
(Orchidaceae), is an important medicinal herb.
It belongs to a group of terrestrial orchids
commonly known as “Jewel Orchids”, because
of their attractive foliage (Teuscher 1978). A.
formosanus is an endemic species in Taiwan.
It grows to a height of 20 – 30 cm, and has
velvety-textured leaves, the upper surface of
which is dark green with intricate silver veins.
The lower surface of the leaves is pale purple
in color. A. formosanus is found at an
altitude of 800 – 1500 m in the central
mountain range of Taiwan and the offshore
island of Lanyu. It is also found in the
Japanese Ryukyu Islands, and Fujian province
in Mainland China (Anon 1999).
In Taiwanese folk remedies, the whole
plant of A. formosanus, fresh or dried, is
boiled in water and taken internally to treat
chest and abdominal pains (Hu 1971),
diabetes, nephritis (Chiu and Chang 1995),
fever, hypertension, impotence, liver and
spleen disorders and pleurodynia (Kan 1986).
The fresh herb is applied externally as a
treatment for snake-bite (Kan 1986). The
current market price of the fresh herb
collected from its natural habitat is around
US$320 per kilogram, while the dried herb
sells for US$3200 per kilogram.
A. formosanus is a slow-growing
perennial herb. It reproduces from seed, but
seedlings take 2 – 3 years to reach maturity
and bear seed. It flowers only once a year,
in winter (October to December).
The
indiscriminate collection of plants, often before
they are old enough to bloom, has decimated
the species so that its survival is in question.
A reduction in the population size of A.
formosanus may lead to a reduced gene flow,
inbreeding depression and reduced fitness.
The development of a rapid in vitro
propagation system for A. formosanus would
not only help in conserving the germplasm
and provide new opportunities for commercial
cultivation, but would also help to take some
of the pressure off the natural populations
2
(Shiau et al. 2002).
In Taiwan, A. formosanus is cultivated
on a limited scale by a few farmers. The
species is difficult to cultivate, as it is delicate
and highly susceptible to stem and rhizome
rot caused by Fusarium oxysporum (Hsieh et
al. 1994). This problem could be overcome
by artificial cross-pollination, using elite
individuals of A. formosanus to produce robust
seedlings which are less likely to get infected
by the disease. Another possibility is the in
vitro propagation of elite plants.
Methods for the in vitro mass
propagation of A. formosanus have already
been developed, using shoot tip and/or nodal
explants (Chow et al. 1982, Liu et al. 1987,
Ho et al. 1987, Tai 1987, Tsay 1999) and
seeds from capsules collected from wild (Ho
et al. 1987, Lee et al. 1992) or handpollinated plants (Shiau et al. in press). We
have developed a method of using nodal
explants for the rapid production of diseasefree plants of A. formosanus (Fig. 1).
Method
Healthy, two-year-old plants of A.
formosanus were used as a source of nodal
explants (Fig. 1). Nodal explants 2-4 cm in
length were cultured in liquid Murashige and
Skoog’s (MS) medium (Murashige and Skoog
1962) supplemented with 2.0 mg/LBA (N 6 benzyladenine) and 0.5 mg/1NAA ( α naphthaleneacetic acid) in a 500-mL
Erlenmeyer flask. After two months of
culture, axillary shoot buds began to emerge
from the explants (see arrows in Fig. 2).
Axillary shoots elongated and rooted when
subcultured on agar-gelled half-strength MS
medium with 2.0 mg/1 BA + 0.5 mg/1 NAA
and 0.2% (w/v) activated charcoal and
incubated for 2 months (Fig 3).
Plants with well-developed rhizomes
were washed with tap water, transferred to
plastic trays containing a peat moss:vermiculite
mixture and acclimatized in a growth chamber
(Fig. 4). Over 90% of the plants survived
after being transplanted to the potting mixture.
This method is being used to produce diseasefree elite plants of A. formosanus on a
commercial scale in Taiwan.
Fig. 1
Fig. 3
Fig. 2
Fig. 4
Fig. 1.
Propagation of disease-free plants of Anoectochilus formosanus Hayata,
using stem node explants from elite plants. Healthy, 2-year-old plant of
A. formosanus grown in a growth chamber.
Fig. 2.
Emergence of axillary shoot buds (arrows) from nodal explants of A.
formosanus after 2 months of culture in liquid MS medium with 2.0 mg/1
BA and 0.5 mg/1 NAA in 500-ml Erlenmeyer flask and incubation on an
orbital shaker with a rotary motion of 100 rev/min.
Fig. 3.
Elongation of axillary shoots of A. formosanus after culture on agar-gelled
half-strength MS medium supplemented with 2.0 mg/1 BA + 0.5 mg/1
NAA and 0.2% (w/v) activated charcoal in 500-ml Erlenmeyer flasks.
Fig. 4.
A. formosanus plants with well-developed rhizomes which have been
transferred to 42x31x11 cm plastic trays containing peat moss:vermiculite
(1:1 v/v) and incubated at 25 oC/20oC day/night temperature and light at
44 µE m-2 s-1 in a growth chamber.
3
CULTIVATION OF A PERENNIAL HERB
Propagation of Limonium wrightii
(Hance) Ktze. (Pumbaginaceae), an
important traditional medical plant,
using adventitious shoot buds
Limonium wrightii is a herbaceous
perennial plant. It is found on the Bonin
Islands and the Ryukyu Islands of Japan, in
the southern part of Taiwan, and on the
Taiwanese islands of Lanyu and Lutao (Li
1978). It grows among rocks along the
seashore. In Taiwan, L. wrightii is cultivated
from seeds on a limited scale by a few
farmers on Lutao Island. However, it is not
possible to produce a large number of elite
plants within a short period using seeds,
because it flowers only in the autumn, and
seed set and germination are very poor.
The dried plants with the leaves
removed are used to make traditional Chinese
medicinal preparations. These are mainly used
for the treatment of asthma, tuberculosis,
colds, hypertension and backache and also as
a fortifying agent (Kan 1978). Because of
the increasing demand and high price, L.
wrightii plants have been collected
indiscriminately from their natural habitat, and
now are very rare in the wild.
To conserve the germplasm and
encourage commercial cultivation of L.
wrightii, we have recently developed a method
of rapid in vitro plant propagation using the
shoot tip, leaf base and inflorescence node
explants (Huang et al. 2000). Fig. 5 shows
the propagation of L. wrightii using leaf base
explants. Healthy plants of L. wrightii were
collected from farmers on Lutao Island and
raised in a growth chamber. Leaves were
taken from the plants in the growth chamber.
Their surface was disinfected, and the leaves
were cultured on MS medium supplemented
with 2.0 mg/1 BA and 0.2 mg/L NAA for
two months. Well-developed adventitious
shoots formed on the leaf base explants (Fig.
6). Adventitious shoots were multiplied by
subculturing on 2x-strength MS medium
supplemented with 2.0 mg/LBA and 0.2 mg/L
NAA (Fig. 7). When shoot multiplication
medium was used, an average of eight shoot
buds could be produced per shoot. Shoots
were easily rooted on MS basal medium with
1.0 mg/Lindole-3-butyric acid (IBA) (Fig. 8).
4
Using this protocol, it is possible to produce a
large number of healthy, uniform plants of
this endangered species.
PRODUCTION OF MEDICINAL TUBERS
Propagation of disease-free tubers from
somatic embryo-derived plants of
Corydalis yanhusuo W.T. Wang
(Fumariaceae)
The dried and pulverized tubers of
Corydalis yanhusuo are used in traditional
herbal remedies in China, Japan, and Korea.
C. yanhusuo is also known as Rhizoma
corydalis (Tang and Eisenbrand 1992). The
tubers are used in traditional Chinese medicine
to treat gastric and duodenal ulcers, cardiac
arrhythmia (Kamigauchi and Iwasa 1994),
rheumatism and dysmenorrhea (Tang and
Eisenbrand 1992). C. yanhusuo is cultivated
as an annual crop, using the tubers.
However, the plants are susceptible to fungal
diseases, especially downy mildew caused by
Peronospora corydalis de Bary. This infects
the tubers, and may cause losses of 30 –
50% when conditions are suitable for fungal
growth (Gao et al. 1991).
To improve the yield and quality of the
tubers, it is necessary to have pathogen-free
planting material. Pathogen-free plants of C.
yanhusuo can be obtained from seed, but the
seeds have a low germination rate (Hu and
Liang 1996).
They also take a long time to germinate,
because of the extremely immature state of
the zygotic embryo at the time of dispersal.
Both warm and cold stratification treatments
are required to induce the germination of
seeds.
Furthermore, the growth of the
seedling is very slow. During the first year,
the plant forms only a small, immature tuber.
Mature tubers can be obtained only in the
following year, after a period of dormancy
(Hu and Liang 1996).
Recently, we have reported an efficient
method of plant regeneration by means of
somatic embryogenesis from tuber-derived
callus (Sagare et al. 2000). Using this
protocol, it is possible to produce a large
number of disease-free plants and tubers of C.
yanhusuo in a short time (Fig. 9).
Mature, healthy tubers were used to
establish embryogenic callus cultures of C.
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 5.
Rapid in vitro propagation of Limonium wrightii (Hance) Ktze.
mature plant of L. wrightii in 14-cm diameter pot
Healthy,
Fig. 6.
Direct regeneration of shoots from the basal part of a leaf explant
cultured on MS medium, supplemented with 2.0 mg/LBA and 0.2 mg/1
NAA after two months of incubation.
Fig. 7.
Rapid multiplication of adventitious shoots in a 500 ml Erlenmeyer flask
with 2x-strength MS medium supplemented with 2.0 mg/LBA and 0.2 mg/
LNAA.
Fig. 8.
Rooted axillary shoots after two months of culture in MS basal medium,
supplemented with 1.0 mg/1 IBA.
5
Fig. 11
Fig. 9
+
Fig. 10
Fig. 9.
Fig. 12
Production of disease-free plants and tubers of Corydalis yanhusuo through
somatic embryogenesis. Embryogenic callus derived from mature, healthy
tubers after three months of culture on MS medium supplemented with
2.0 mg/LBA and 0.5 mg/LNAA and incubation in darkness.
Fig. 10. Tuber-derived embryogenic callus showing the formation of a cotyledonarystage somatic embryo (C) and converted somatic embryo (arrows) after
five weeks of culture on medium supplemented with 1.0 mg/L zeatin
riboside.
Fig. 11. Converted somatic embryos with well-developed shoot and root after
culture in half-strength liquid MS medium which contains 1.0 mg/L zeatin
riboside and has been on a rotary shaker for two weeks.
Fig. 12. Somatic embryo-derived tubers (arrows) formed after 9 months of culture
on half-strength MS medium supplemented with 3% sucrose.
6
yanhusuo. Embryogenic callus was induced
by culturing mature tuber pieces on MS
medium, supplemented with 2.0 mg/LBA and
0.5 mg/1 NAA, and incubating them in
darkness for three months. Somatic embryos
were induced by subculturing the primary
callus on MS medium supplemented with 1.0
mg/L zeatin, with two weeks of culture in
light. Fig. 10 shows a cotyledonary-stage
somatic embryo (c) and a converted somatic
embryo (arrow) formed on primary callus.
Somatic embryos converted into plantlets when
they were transferred into liquid MS medium
supplemented with 1.0 mg/L zeatin riboside
(Fig. 11). Converted somatic embryos were
cultured on half-strength MS medium,
supplemented with 3% sucrose to promote the
further development of plantlets and tubers.
Well-developed, healthy tubers were obtained
after six months of culturing somatic embryoderived plants (Fig. 12).
These tubers can be used as disease-free
planting materials. Tubers derived from
somatic embryos of C. yanhusuo have recently
been shown to contain pharmaceutically
important compounds, such as D,Ltetrahydropalmatine and D-corydaline (Lee et
al. 2001).
CONCLUSIONS
Tissue culture technology makes it
possible to produce a large number of diseasefree and uniform plants of medicinally
important species. Such plants can be used
for the extraction of medicinally important
compounds, or for pharmacological studies.
They can also help in germplasm
conservation, and can be reintroduced into
protected habitats such as National Parks.
Pathogen-free plants maintained under in vitro
conditions can also be used for the safe
exchange of germplasm across national
borders.
REFERENCES
Anon. 1999. Anoectochilus formosanus
Hayata. In: Chung-Hua-Ben-Tsao, Vol. 8.
Shanghai Science Technology Press,
Shanghai, pp. 672-673. (In Chinese).
Chen, C.C., S.J. Chen, A.P. Sagare and H.S.
Tsay.
2001.
Adventitious shoot
regeneration from stem internode explants
of Adenophora triphylla (Thunb.) A.DC.
(Campanulaceae) – an important medicinal
herb. Botanical Bulletin of Academia
Sinica 42:1-7.
Chiu, N.Y. and K.H. Chang.
1995.
Anoectochilus formosanus Hayata. In: The
Illustrated Medicinal Plants of Taiwan,
Vol. 4 SMC Publishing Inc., Taipei,
Taiwan, pp. 282-283. (In Chinese).
Chow, H.T., W.C. Hsieh and C.S. Chang.
1982.
In vitro propagation of
Anoectochilus formosanus. Journal of
Science and Engineering 19: 155-165.
(In Chinese).
Chueh, F.S., C.C. Chen, A.P. Sagare and H.S.
Tsay. 2001. Quantitatiave determination
of secoiridoid glucosides in in vitro
propagated plants of G. davidii var.
formosana by high performance liquid
chromatography. Planta Medica 67: 7073.
Debergh, P.C. and L.J. Maene. 1981. A
scheme for commercial propagation of
ornamental plants by tissue culture.
Scientia Horticulturae 14: 335-345.
Erdei, I., Z. Kiss and P. Maliga. 1981.
Rapid clonal multiplication of Digitalis
lanata in tissue culture.
Plant Cell
reports 1: 34-35.
Fay, M.F. 1992. Conservation of rare and
endangered plants using in vitro methods.
In: Vitro Cellular and Developmental
Biology. pp. 1-4. 28pp.
Hatano, K., Y. Shoyama and I. Nishioka.
1986. Multiplication of Pinellia ternate
by callus culture of leaf segment.
Shoyakugaku Zasshi 40: 188-192.
Hiraoka, N., T. Kodama, M. Oyanagi, S.
Nakano, Y. Tomita, N. Yamada, N.
Yamada, O. Iida and M. Satake. 1986.
Characteristics of Bupleurum falcatum
plants propagated through somatic
embryogenesis of callus cultures. Plant
Cell Reports 5: 319-321.
Hiraoka, N. and M. Oyanagi. 1988. In vitro
propagation of Glehnia littoralis from
shoot-tips. Plant Cell Reports 7: 39-42.
Ho, C.K., S.H. Chang and Z.Z. Chen. 1987.
Tissue culture and acclimatization in
Anoectochilus formosanus Hay. Bulletin
of Taiwan Forestry Research Institute 2:
83-105. (In Chinese).
Hol, G.M.G.M. and P.C.G. van der Linde.
1992. Reduction of contamination in
7
bulb-explant cultures of Narcissus by a
hot-water treatment of parent bulbs. Plant
Cell, Tissue and Organ Culture 31: 75-79.
Hsieh, S.P.Y., W.R. Chuang, S.F. Huang.
1994. Stem rot of Taiwan Anoectochilus
and its causal organism. Plant Pathology
Bulletin 3: 140-146. (In Chinese).
Hu, S.Y. 1971. The Orchidaceae of China.
Quarterly Journal of the Taiwan Museum
24: 67-103.
Hu, C.Y. and P.J. Wang. 1983. Meristem,
shoot tip, and bud cultures. In: Evans,
D.A., Sharp, W.R., Ammirato, P.V. and
Y. Yamada (Eds.). Handbook of Plant
Cell Culture: Techniques for Propagation
and Breeding.
Vol. 1. Macmillan
Publishing Co., New York, pp. 177-227.
Huang, C.L., M.T. Hsieh, W.C. Hsieh, A.P.
Sagare and H.S. Tsay. 2000. In vitro
propagation of Limonium wrightii (Hance)
Ktze.
(Plumbaginaceae), an ethnomedicinal plant, from shoot-tip, leaf- and
inflorescence-node explants. In vitro
Cellular and Developmental Biology –
Plant 36: 220-224.
Kamigauchi, M. and K. Iwasa. 1994. VI
Corydalis spp.: In vitro culture and the
biotransformation of protoberberines. In:
Biotechnology in Agriculture and Forestry,
Y.P.S. Bajaj (Ed.). Vol. 26. SpringerVerlag, Berlin Heidelberg, pp. 93-105.
Kan, W.S. 1978. Manual of Vegetable
Drugs in Taiwan. Part 2. Medicine
Publishing Inc., Taipei, Taiwan, pp. 43-44.
Kan, W. S. 1986. Anoectochilus formosanus
Hay.
In: Pharmaceutical Botany.
National Research Institute of Chinese
Medicine, Taipei, Taiwan, pp. 647. (In
Chinese).
Kitamura, Y., H. Miura and M. Sugii. 1989.
Plant regeneration from callus cultures of
Swertia pseudochinensis. Shoyakugaku
Zasshi 43: 256-258.
Langens-Gerrits, M., M. Albers and G.J. De
Klerk. 1998. Hot-water treatment before
tissue culture reduces initial contamination
in Lilium and Acer. Plant Cell, Tissue
and Organ Culture 52: 75-77.
Lee, S.G., C.L. Huang and C.C. Lin. 1992.
Development of natural crude drug
resources from Taiwan (XI) – study on
economic
cultural
practices
in
Anoectochilus culture. Journal of Chinese
Medicines 3: 53-60. (In Chinese).
8
Lee, Y.L., A.P. Sagare, C.Y. Lee, H.T. Feng,
Y.C.Ko, J.F. Shaw and H.S. Tsay. 2001.
Formation of protoberberine-type alkaloids
by the tubers of somatic embryo-derived
plants of Corydalis yanhusuo. Planta
Medica 67:839-842..
Li, H.L. 1978. Plumbaginaceae. In: Flora
of Taiwan, H.L. Li, T.S. Liu, T.C. Huang,
T. Koyama and C.E. Devol (Eds.). Vol.
4. Epoch Publishing Co., Ltd. Taipei,
Taiwan, Republic of China, pp. 90-93.
Liu, S.Y., H.S. Tsay, H.C. Huang, M.F. Hu
and C.C. Yeh. 1987. Comparison of
growth characteristics and nutrient
composition
between
plants
of
Anoectochilus
species
from
mass
vegetative propagation by tissue culture
techniques.
Journal of Agricultural
Research of China 36: 357-366. (In
Chinese).
Matsumoto, M., M. Nagano, Y. Shoyama and
I. Nishioka. 1986. New vegetative
propagation method of Rehmannia
glutinosa. Shoyakugaku Zasshi 40: 193197. (In Japanese).
Murashige, T. and F. Skoog. 1962. A
revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiologia Plantarum 15: 473-497.
Nishioka, I. 1988. Clonal multiplication of
medicinal plants by tissue culture.
Shoyakugaku Zasshi 42: 1-11.
Sagare, A.P., Y.L. Lee, T.C. Lin, C.C. Chen
and H.S. Tsay. 2000. Cytokinin-induced
somatic
embryogenesis
and
plant
regeneration in Corydalis yanhusuo
(Fumariaceae) – a medicinal plant. Plant
Science 160: 139-147.
Shiau, Y.J., U.C. Chen, S.R. Yang, A.P.
Sagare
and
H.S.
Tsay.
2002.
Conservation of Anoectochilus formosanus,
a medicinally important terrestrial orchid,
by synchronizing flowering, handpollination, and in vitro culture of seeds.
Bot. Bull. Acad. Sin. 43: 123-130.
Shoyama, Y., K. Hatano and I. Nishioka.
1983. Clonal multiplication of Pinellia
ternate by tissue culture. Planta Medica
49: 14-16.
Tai, K.S. 1987. In vitro propagation of
Anoectochilus formosanus (Hayata).
Journal of the Agricultural Association of
China 137: 42-54. (In Chinese).
Tang, W. and G.Eisenbrand. 1992. Corydalis
turtschaninovii Bess. F. yanhusuo Y.H.
Chou et C.C. Hsü. In: Chinese Drugs of
Plant Origin, Chemistry, Pharmacology,
and Use in Traditional and Modern
Medicine, W. Tang and G. Eisenbrand
(Eds.).
Springer- Verlag, Berlin
Heidelberg, pp. 377-393.
Teuscher, H. 1978. Erythrodes, Goodyera,
haemaria
and
Macodes,
with
Anoectochilus. American Orchid Society
Bulletin 47: 121-129.
Tsay, H.S.
1999.
Tissue culture of
Anoectochilus formosanus.
Paper
presented at the 16th World Orchid
Conference, Vancouvor, Canada.
Tsay, H.S., T.G. Gau and C.C. Chen. 1989.
Rapid clonal propagation of Pinellia
ternate by tissue culture. Plant Cell
Reports 8: 450-454.
Tsay, H.S. and H.L. Huang. 1998. Somatic
embryo formation and germination from
immature embryo-derived suspensioncultured cells of Angelica sinensis (Oliv.)
Diels. Plant Cell Reports 17: 670-674.
Vieira, R.F. and L.A. Skorupa.
1993.
Brazilian medicinal plants gene bank.
Acta Hortculturae 330: 51-58.
Yamada, Y., Y. Shoyama, I. Nishioka, H.
Kohda, A. Namera and T. Okamoto.
1991.
Clonal micropropagation of
Gentiana scabra Bunge var. buergeri
Maxim.and
examination
of
the
homogeneity
concerning
the
gentiopicroside content. Chemical and
Pharmaceutical Bulletin 39: 204-206.
Yonemitsu, H., M. Kusube and M. Noguchi.
1998. Studies on propagation by tissue
culture of Platycodon grandiflorum A.DC.
Natural Medicines 52: 368-371.
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