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In vitro propagation of some important Chinese medicinal plants and their

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Article  in  In Vitro Cellular & Developmental Biology - Plant · March 2004

DOI: 10.1079/IVP2003504

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In Vitro Cell. Dev. Biol.—Plant 40:143–154, March –April 2004 DOI: 10.1079/IVP2003504
q 2004 Society for In Vitro Biology
1054-5476/04 $18.00+0.00

IN VITRO PROPAGATION OF SOME IMPORTANT CHINESE

MEDICINAL PLANTS AND THEIR SUSTAINABLE USAGE

SATISH MANOHAR NALAWADE AND HSIN-SHENG TSAY*

Institute of Biotechnology, Chaoyang University of Technology, Wufeng, Taichung 413, Taiwan

(Received 26 May 2003; accepted 22 August 2003; editor E. C. Pua)

Summary
Medicinal plants are valuable sources of medicinal and many other pharmaceutical products. The conventional
propagation method is the principal means of propagation and takes a long time for multiplication because of a low rate of
fruit set, and/or poor germination and also sometimes clonal uniformity is not maintained through seeds. The plants used in
the phyto-pharmaceutical preparations are obtained mainly from the natural growing areas. With the increase in the
demand for the crude drugs, the plants are being overexploited, threatening the survival of many rare species. Also, many
medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development,
uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and
tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants.
The purpose of the present review is to focus the application of tissue culture technology for in vitro propagation via
somatic embryogenesis and organogenesis and the cell suspension culture with suitable examples reported earlier. An
overview of tissue culture studies on important Chinese medicinal plants and related species is presented.
Key words: Angelica sinensis; cell suspension cultures; Gentiana davidii; organogenesis; Scrophularia yoshimurae; somatic
embryogenesis.

Introduction cultivation, and sustainable usage of important medicinal plant

species for future use.
Plants are an important source of medicines and play a key role In modern medicine, plants are used as sources of direct
in world health (Constabel, 1990). Almost all cultures from ancient therapeutic agents, as models for new synthetic compounds, and as
times to today have used plants as medicine. Today medicinal a taxonomic marker for discovery of new compounds. They serve as
plants are important to the global economy (Srivastava et al., 1995), a raw material base for the elaboration of more complex
as approximately 85% of traditional medicine preparations involve semisynthetic chemical compounds (Akerele, 1992). The synthesis
the use of plants or plant extracts (Vieira and Skorupa, 1993). In the of bioactive compounds chemically is difficult because of their
past few decades there has been a resurgence of interest in the complex structure and high cost (Shimomura et al., 1997). Wide
study and use of medicinal plants in health care and in recognition variations in medicinal quality and content in phytopharmaceutical
of the importance of medicinal plants to the health system preparations have been observed. These are influenced mainly by
(Lewington, 1993; Mendelsohn and Balick, 1994; Hoareau and cultivation period, season of collection, plant-to-plant variability in
DaSilva, 1999). This awakening has led to a sudden rise in demand the medicinal content, adulteration of medicinal preparations with
for herbal medicines, followed by a belated growth in international misidentified plant species, a lack of adequate methods for the
awareness about the dwindling supply of the world’s medicinal production and standardization of the crop, a lack of understanding
plants (Bodeker, 2002). Most of the pharmaceutical industry is of the unique plant physiology or efficacy with human consumption,
highly dependent on wild populations for the supply of raw and consumer fraud. Generally, herbal preparations are produced
materials for extraction of medicinally important compounds. The from field-grown plants and are susceptible to infestation by
genetic diversity of medicinal plants in the world is getting bacteria, fungi, and insects that can alter the medicinal content of
endangered at an alarming rate because of ruinous harvesting the preparations (Murch et al., 2000). It is difficult to ensure the
practices and over-harvesting for production of medicines, with quality control as the medicinal preparations are multi-herb
little or no regard to the future. Also, extensive destruction of the preparations and it is difficult to identify and quantify the active
plant-rich habitat as a result of forest degradation, agricultural constituents (Wen, 2000). Also, there is significant evidence to
encroachment, urbanization, etc. are other factors. Hence there is a show that the supply of plants for traditional medicines is failing to
strong need for proactive understanding in the conservation, satisfy the demand (Cunningham, 1993). An efficient and most
suited alternative solution to the problems faced by the
*Author to whom correspondence should addressed: Email hstsay@mail. phytopharmaceutical industry is development of in vitro systems
cyut.edu.tw for the production of medicinal plants and their extracts.

143
144 NALAWADE AND TSAY

The in vitro-propagated medicinal plants furnish a ready source of environmental conditions delivers healthy and sterile explants
uniform, sterile, and compatible plant material for biochemical (Sagare et al., 2001, and references cited therein). The physiological
characterization and identification of active constituents (Wakhlu age of the explants, and the explant type and size are the other factors
and Bajwa, 1986; Miura et al., 1987). In addition, compounds from which exercise an influence on formation of organs in vitro (Rout et al.,
tissue cultures may be more easily purified because of simple 2000). A range of explants has been successfully tested to induce
extraction procedures and absence of significant amounts of morphogenetic response in medicinal plants (Table 1). Temperature,
pigments, thus possibly reducing the production and processing photoperiod, light intensity, pH of medium, carbohydrate source,
costs (Chang et al., 1992, 1994). type of gelling agent, plant growth regulator concentrations in the
Plant tissue culture techniques have been increasingly applied to medium, and additional media amendments also play a determining
many medicinal plants in particular for mass propagation, role in the morphogenesis (Narayanaswamy, 1977). The success of
conservation of germplasm, study and production of bioactive any tissue culture protocol depends on the efficient acclimatization of
compounds, and for genetic improvement. Medicinal plants have vast in vitro-obtained plantlets to greenhouse and field conditions.
genetic diversity, which is a valuable source of agronomic gene/s of Plantlets growing under in vitro conditions exhibit no or reduced
interest for the future. Large-scale plant tissue culture is found to be photosynthetic capacity, and during acclimatization there is a need
an attractive alternative approach to the traditional methods of for rapid transition from the heterotrophic to the photoautotrophic
plantations, as it offers a controlled supply of biochemicals state for survival (Preece and Sutter, 1991). Thus, for effective
independent of plant availability and more consistent product quality acclimatization and better adaptation, the in vitro-raised plantlets are
(Sajc et al., 2000). Minimal growth of tissue in culture and gradually exposed to field conditions. Based on the plant species and
cryopreservation have been used to store plant materials from a wide culture conditions, in vitro propagation could be achieved by direct
variety of species (Withers, 1987). Combinations of in vitro and/or indirect shoot organogenesis and/or somatic embryogenesis.
propagation techniques (Fay, 1992) and cryopreservation may help Various reports are available on the production of bioactive
in the conservation of biodiversity of locally used medicinal plants. compounds from the in vitro-grown plants and cell suspension
Cryopreservation is a reliable method for long-term storage of the cultures (Mulabagal et al., 2003, and references cited therein). Cell
germplasm of endangered species (Bramwell, 1990). Several suspension culture systems could be used for large-scale culturing
medicinal plant species have been successfully cryopreserved of plant cells from which secondary metabolites can be extracted.
(Bajaj, 1995; Naik, 1998). Therefore, the principal objective of our The advantage of this method is that it can ultimately provide a
research programs was to standardize the protocols of in vitro continuous, reliable source of natural products.
propagation for the important Chinese medicinal plants. We have The schematic representation of the steps followed in our
developed protocols for mass propagation of valuable medicinal laboratory in the in vitro study of medicinal plants is given in Fig. 1.
herbs, Limonium wrightii, Adenophora triphylla, Gentiana davidii The aim of the present paper is to focus on the importance of in vitro
var. formosana, Anoectochilus formosanus, Scrophularia yoshimurae, propagation of medicinal plants and cell suspension cultures in
Pinellia ternata, Bupleurum falcatum, Zingiber zerumbet, Dendro- production of some of the plant pharmaceuticals with suitable
bium linawianum, and Fritillaria hupehensis through shoot examples as reported earlier.
morphogenesis, and Angelica sinensis and Corydalis yanhusuo Case Study 1: somatic embryogenesis in Angelica sinensis. So-
through somatic embryogenesis, as well as established cell matic embryogenesis refers to the formation of an embryo from a cell
suspension cultures of Taxus mairei, Angelica dahurica, Angelica other than a gamete or a direct product of gamete fusion.
sinensis, Dioscorea doryophora, Gentiana davidii, and Bupleurum Development of normal embryos and whole plants from undiffer-
falcatum. In this review we have cited an example each for entiated somatic cells in culture is a unique quality of many plant
organogenesis, somatic embryogenesis, and cell suspension studies species. Somatic embryos develop directly into differentiated plants
in Chinese medicinal plants. through characteristic physiological changes resembling zygotic
embryo development (Dudits et al., 1995). Direct and indirect
In Vitro Studies of Traditional Chinese Medicinal Plants somatic embryogenesis has been reported in many Chinese
medicinal plants from a wide range of explants (Table 1). An
In vitro propagation refers to the true-to-type propagation of optimized system of plant regeneration via repetitive somatic
selected genotypes using in vitro culture techniques. It is an embryogenesis in Angelica sinensis using the immature embryo-
alternative method of propagation (George and Sherrington, 1984) derived callus has been described herein. Angelica sinensis
and is being used widely for the commercial propagation of a large (Umbelliferae), also known as Dang Guei, is a very valuable herb
number of plant species, including many medicinal plants. used in traditional Chinese medicine prescriptions since ancient
In vitro propagation has been achieved in several medicinal plants times (Chen et al., 1994). It has a wide range of pharmaceutical
using tissue culture techniques (Rout et al., 2000; Nalawade et al., applications, and is commonly used in the treatment of hypertension,
2003). rheumatism, ulcers, anemia, constipation, to regulate menstruation,
Various factors are responsible for in vitro morphogenesis. The and to relieve pain (Zhang and Cheng, 1989). Dang Guei also
source of the explant cultured is important in determining the contains compounds that act to stimulate the central nervous system,
morphogenetic and regenerative potential, which are significantly and is also used as a mild energizer for people (Chen, 1973). This
influenced by the phytosanitary and physiological conditions of the medicinal herb was not originally grown in Taiwan, but has now been
donor plant (Debergh and Maene, 1981; Read, 1988). Prior to the successfully cultivated here. Traditionally the plants are propagated
establishment of aseptic culture, meticulous selection, identification, from seeds; the disadvantage of this method is that it is difficult
and maintenance of stock plants used as the source of explants to control seed quality, which may greatly affect the medicinal
is necessary. Maintaining the donor plants in clean and controlled property of the adult plant. If tissue culture replaces traditional
 IN VITRO PROPAGATION OF CHINESE MEDICINAL PLANTS 145

TABLE 1

IN VITRO MORPHOGENESIS IN SOME OF THE IMPORTANT TRADITIONAL CHINESE MEDICINAL PLANTS AND RELATED SPECIES

Plant Explant used Reference

In vitro direct shoot morphogenesis

Aconitum carmichaeli Debx Shoot tip, axillary bud Hatano et al., 1988
Adenophora triphylla Thunb Stem internode Chen et al., 2001
Alpinia galanga Willd. Rhizome bud Borthakur et al., 1999a
Angelica acutiloba Kitagawa Stem Watanabe et al., 1998
Anoectochilus formosanus Hayata Stem node Huang et al., 1991
Shoot tip Liu et al., 1987; Du et al., 1998
Capsule Shiau et al., 2002
Astragalus membranaceus Bunge Shoot tip Fujioka et al., 1983
Atractylodes japonica Koidz Shoot tip Hatano et al., 1990
Atractylodes lancea DC. Flower bud Hiraoka et al., 1984
Atractylodes ovata DC. Shoot tip Hatano et al., 1990
Camptotheca acuminata Decaisne Axillary bud, shoot tip Liu and Li, 2001
Cnidium officinale Makino Shoot tip Shimomura et al., 1980
Corydalis pallida Stem Ikuta et al., 1974
Curcuma longa Linn. Rhizome bud Nadgauda et al., 1978; Shirgurkar et al., 2001;
Salvi et al., 2002
Immature floral bud, inflorescence segment Salvi et al., 2000
Dendrobium linawianum Reichb. F Leaf blade Chen et al., 1995
Digitalis lanata Shoot tip Erdei et al., 1981
Dioscorea bulbifera L. Stem node Forsyth and van Staden, 1982
Gentiana davidii var. formosana Stem node Chueh et al., 2001
Gentiana lutea L. Shoot meristem, shoot tips, axillary bud Viola and Franz, 1989
Shoot tip, inflorescence bud Feijoo and Iglesias, 1998
Gentiana scabra Bunge Axillary bud Yamada et al., 1991
Gentiana triflora Leaf, shoot, root Hosokawa et al., 1996
G. triflora £ G. scabra Nodal segment Hosokawa et al., 1998
Glehnia littoralis F. Schimidt ex Miq Shoot tip Hiraoka and Oyanagi, 1988
Glycyrrhiza glabra L. Shoot tip, axillary buds Thengane et al., 1998
Holarrhena antidysenterica Wall. Axillary bud Raha and Roy, 2001
Houttuynia cordata Thumb Axillary bud Borthakur et al., 1999b
Kaempferia galanga Rhizome buds Vincent et al., 1992
Limonium wrightii (Hance) Shoot tip, leaf inflorescence segments Huang et al., 2000
Mentha spp. Axillary buds Rech and Pires, 1986
Shoot tip, stem internodes Reed, 1999
Lonicera tatarica Nodal segments Palacios et al., 2002
Murraya koenigii Intact seedling Bhuyan et al., 1997
Papaver bracteatum Intact seedling Ikuta et al., 1974
Papaver somniferum L. Primary somatic embryo Ovecka et al., 2000
Pinellia ternata (Thunb.) Breitenbach Tuber segments Shoyama et al., 1983a, b; Hatano et al., 1986;
Nishioka, 1988
Leaf blade, petiole, tubers Seong et al., 1988
Leaf blade, bulbils, petiole Tsay et al., 1989
Piper longum Shoot tip, leaf, stem node, stem internode Sarasan and Nair, 1991
Platycodon grandiflorum A. DC. Shoot tip axillary bud Yonemitsu et al., 1998
Plumbago zeylanica L. Nodal segments Selvakumar et al., 2001
Podophyllum peltatum L. Rhizome Moraes-Cerdeira et al., 1998
Rehmannia glutinosa Liboschitz Shoot tip Shoyama et al., 1983c; Nishioka, 1988
Leaf segment Matsumoto et al., 1986
Shoot apical meristem, nodal buds Paek et al., 1995
Rheum rhaponticum L. Shoot meristem Walkey and Matthews, 1979
Rheum emodi Wall. Shoot tip leaf Lal and Ahuja, 1989
Saussurea lappa Cotyledons leaf Arora and Bhojwani, 1989
Scopolia japonica Max. Shoot tip Shimomura et al., 1981b
Scrophularia yoshimurae Yamazaki Shoot tip, node buds, stem segments, leaf Lin et al., 1998; Sagare et al., 2001
Scutellaria baicalensis Georgi. Intact seedling, etiolated hypocotyl, stem segments Li et al., 2000
Stevia rebaudiana Nodal segments Yang et al., 1981
Vitex negundo L. Axillary buds Sahoo and Chand, 1998
Zingiber officinale Roscoe Rhizome bud Hosoki and Sagawa, 1977
Zingiber zerumbet Smith. Shoot tip Hsu et al., 1991
In vitro indirect shoot morphogenesis
Artemisia annua L. Leaf Veegauwe et al., 1998
Bupleurum falcatum L. Leaf segment Wang and Huang, 1982
Cnidium officinale Makino Shoot tip Shimomura et al., 1981c
Curculigo orchioides (Gaertn.) Leaf, rhizome Augustine and D’Souza, 1997
Foeniculum vulgare Miller Hypocotyls Anzidei et al., 2000
146 NALAWADE AND TSAY

TABLE 1 – Continued

Plant Explant used Reference

Gentiana scabra Bunge Leaf Jomori et al., 1995

Linum usitatissimum Root, cotyledons, hypocotyls McHughen and Swartz, 1984
Macleaya cordata Shoot tip Ikuta et al., 1974
Papaver bracteatum Seedling Day et al., 1986
Papaver somniferum L. Hypocotyl Yoshikawa and Furuya, 1985, 1983
Stem, root, capsule Ikuta et al., 1974
Pinellia ternata (Thunb.) Breitenbach Tuber segments Hatano et al., 1986; Su, 1989
Leaf, stem, rhizome Wan et al., 1995
Rehmannia glutinosa Liboschitz Petiole stem Xu and Davey, 1983
Ruta graveolens Stem, leaf, root Abou-Mandour, 1982
Scopolia japonica Max. Stem segments Shimomura et al., 1981a
Direct somatic embryogenesis
Corydalis ambigua Chem. and Schlecht Tuber Hiraoka et al., 2002
Shoot tip Economou and Spanoudaki, 1985
Gardenia jasminoides Ellis Axillary buds George et al., 1993
Hyoscyamus niger Zygotic embryo Tu et al., 1996
Flower bud, stem, leaf Kishira et al., 1992
Panax ginseng C. A. Meyer Cotyledons Choi and Soh, 1997; Choi et al., 1998b, 1999c,d
Papaver somniferum L. Capsule Ovecka et al., 2000
Indirect somatic embryogenesis
Aconitum carmichaeli Debx. Anther Hatano et al., 1987
Angelica acutiloba Kitagawa Pedicle Nakagawa et al., 1982
Flower bud Miura et al., 1988
Angelica sinensis (Oliv.) Diels Immature embryo Huang et al., 1997; Tsay and Huang, 1998
Aralia cordata Thunb. Inflorescence bud Lee et al., 2002
Bupleurum falcatum L. Leaf segment Wang and Huang, 1982; Hiraoka et al., 1983, 1986
Bupleurum scorzonerifolium Willd Stem node Xia et al., 1992
Coptis japonica Pedicle Nakagawa et al., 1982
Corydalis yanhusuo Mature tuber piece Sagare et al., 2000
Primary somatic embryo Kuo et al., 2002
Cuminum cyminum L. Hypocotyls, leaf Tawfik and Noga, 2002
Dysosma pleiantha Hance. Immature seed, zygotic embryo Chuang and Chang, 1987a
Rhizome, leaf Chuang and Chang, 1987b
Eleutherococcus senticocu Maxim Hypocotyl Choi et al., 1999a, 2002
Somatic embryos Choi et al., 1999b
Foeniculum vulgare Miller Petiole Miura et al., 1987
Hypocotyls Anzidei et al., 2000
Panax ginseng C. A. Meyer Roots Chang and Hsing, 1980
Flower bud Shoyama et al., 1988
Zygotic embryos Choi et al., 1998a; Arya et al., 1993
Panax japonicus C. A. Meyer Flower buds, rhizomes Fujioka et al., 1986
Flower buds, leaf, stem, rhizomes, roots Shoyama et al., 1987
Panax notoginseng (Burk.) F. H. Chen Flower buds Shoyama et al., 1997
Papaver somniferum L. Hypocotyl Nessler, 1982; Schuchmann and Wellmann, 1983;
Wakhlu and Bajwa, 1986, 1987;
Laurain-Matter et al., 1999
Saposhnikovia divaricata Schischkin Leaf Hiraoka et al., 1987
Swertia pseudochinensis Hara Root, leaf Kitamura et al., 1988
Intact seedling, hypocotyl root Kitamura et al., 1989

methods of cultivation, it will be possible to raise the quality of germinating somatic embryos during the culture. Embryogenic
A. sinensis by means of propagating large quantities of superior competence was maintained by culturing the callus in liquid media,
somatic seedlings throughout the year without seasonal constraints. supplemented with 0.5 or 1.0 mg l21 (2.26– 4.52 mM) 2,4-
Immature embryo axis explants from the surface-sterilized flower dichlorophenoxyacetic acid (2,4-D). In this medium the embryo-
buds, collected 1 – 2 wk after pollination, were aseptically removed genic callus remained as cells and cell clumps. The cells and cell
and cultured on MS basal medium (Murashige and Skoog, 1962). clumps grew and developed into somatic embryos upon subculture
The explants developed embryogenic callus (Fig. 2A), as well as on MS basal medium devoid of 2,4-D (Fig. 2B). A shaking speed of
normal and abnormal plantlets, after 4 wk of culture in the light. 80 rpm was found to be optimal for the initiation of suspension
The growth of embryogenic callus was more rapid on MS basal cultures, while 100 rpm produced more somatic embryos and
medium than on B5 or White medium, and full-strength MS medium germinating embryos (Fig. 2C) with an initial cell density of 0.2 ml
and half-strength MS basal medium were optimal for the packed cell volume per 25 ml of medium. Addition of 0.3% Difco
proliferation of embryogenic callus. Embryogenic callus was used agar to the liquid medium also stimulated the formation of somatic
to establish a suspension culture and somatic embryos and embryos and germinating embryos. Forty percent of the somatic
 IN VITRO PROPAGATION OF CHINESE MEDICINAL PLANTS 147

FIG . 1. Schematic representation of the procedure for conservation of germplasm and sustainable utilization of important Chinese
medicinal herbs.

embryos converted into plantlets after culturing on filter paper (Lin et al., 1998; Sagare et al., 2001). S. yoshimurae (Scrophular-
moistened with liquid half-strength MS basal medium containing iaceae) is an indigenous herbaceous perennial plant of Taiwan and is
3% sucrose (Fig. 2D). These developed into healthy plantlets on a substitute for S. ningpoensis in traditional Chinese medicine. The
transfer to flasks containing the same medium (Fig. 2E). The plants roots of S. ningpoensis have been used for the treatment of
flowered and showed normal growth after transfer to the soil inflammation, laryngitis, tonsillitis, abscesses of carbuncles, and
(Fig. 2F). The embryogenic cell suspension has been maintained constipation (Qian et al., 1992). It can lower blood pressure and
successfully, without any loss in embryogenic competence, for the blood sugar levels and also has antibacterial and antioxidant effects
past 10 yr in our laboratory. This regeneration system could be and is usually used in combination with other herbs as nutrient and
useful for mass production of A. sinensis plants. health-strengthening agents (Huang, 1993; Anonymous, 1999). The
Case Study 2: shoot organogenesis in Scrophularia yoshimur- root contains iridoid glycosides, which are active principles, and
ae. Organogenesis refers to the initial and subsequent de novo small amounts of essential oils, alkaloids, flavanoids, and
growth of organs such as shoots, roots, leaves, or flowers directly from p-methoxycinnamic acid (Huang, 1993). For the crude drug, the
the explants (direct) or via an intervening callus phase (indirect). It roots of S. yoshimurae are collected from plants growing naturally in
is a developmental process that is in some ways unique to plants. the mountains of Taiwan. Proper cultivation of the plant is needed,
There are many reports of direct as well as indirect organogenesis in as the supply is insufficient to meet the local drug demand. In order
Chinese medicinal plants (Table 1). As an example, herein we to aid commercial cultivation and conservation of the germplasm of
describe a direct de novo shoot organogenesis protocol of this medicinally important species, a rapid in vitro propagation
Scrophularia yoshimurae Yamazaki standardized in our laboratory system for S. yoshimurae has been developed.
148 NALAWADE AND TSAY

FIG . 2. Somatic embryogenesis and plant regeneration in Angelica sinensis. A, Embryogenic callus obtained from the immature
embryos cultured on MS basal medium for 1 mo. B, Induced somatic embryos from the embryogenic callus in the suspension cultures
containing MS basal medium devoid of growth regulators. C, Germinating somatic embryos on the liquid MS basal medium. D, Germinated
somatic embryos converted into plantlets after culturing on filter paper moistened with liquid half-strength MS basal medium containing
3% sucrose. E, A somatic embryo-derived plantlet with well-developed roots and shoot after 1 mo. of culture on half-strength MS medium.
F, Flower formation in somatic embryo-derived plant in pot.

To obtain healthy explants for in vitro organogenesis, plants were directly from the cut end of the stem internode and leaf base
collected from the natural habitat and grown in controlled hygienic explants without an intervening callus phase in 1 mo. of culture on
conditions. Various explants, such as shoot tip, leaf base, stem MS basal medium supplemented with 4.44 mM N6-benzyladenine
node, and stem internode taken from the actively growing shoots of (BA) and 1.07 mM a-naphthaleneacetic acid (NAA). All stem node
these plants, were tested for the induction of the morphogenetic and shoot tip explants induced multiple shoots, whereas leaf base
response. Multiple shoots were induced not only from the pre- explants were least responsive. Figure 3A shows the induction of
existing meristems of shoot tip and stem node explants, but also multiple shoots from the internode explants of S. yoshimurae.
 IN VITRO PROPAGATION OF CHINESE MEDICINAL PLANTS 149

The induced shoots were further proliferated by subculturing them in vitro propagation of selected, elite plants. In vitro-propagated
in fresh medium of similar composition (Fig. 3B). The plants of many species showed less variation in the content of
micropropagated shoots were elongated and rooted by subculturing secondary metabolites than their wild/cultivated counterparts
on growth regulator-free MS basal medium. The plantlets were (Yamada et al., 1991, and references cited therein). The protocol
transplanted to soil and acclimatized in the growth chamber under obtained could be also used for rapid micropropagation,
high humidity conditions. The cultures could be maintained for commercial cultivation, and germplasm conservation of this
over 2 yr without losing their morphogenetic potential. The content medicinal plant.
of harpagoside, a quantitatively predominant iridoid glycoside, in Case Study 3: cell suspension cultures of Gentiana davidii var.
different plant materials was determined by high performance formosana. The production of secondary metabolites from the cell
liquid chromatography (HPLC). The analysis revealed that the suspension culture system is one of the most promising methods in
content of harpagoside in the aerial and underground parts of the medicinal industry. Not only do the cells from the callus or
S. yoshimurae was significantly higher than the marketed crude suspension grow faster, but the products produced can be extracted
drug. For extraction of secondary metabolites and pharmacological more easily than from the cells of the intact plants, and the quantities
studies it is necessary to have plants with high quality and uniform of the product produced in some plants species are much higher than
content of active principles. Genetically homogeneous plants with those of the field-grown plants (Whitaker et al., 1986). Tissue and
uniform content of secondary metabolites could be obtained by cell suspension culture techniques have made industrial production

FIG . 3. A, Induction of multiple shoots from the internode explants of Scrophularia yoshimurae. B, Shoot proliferation from the node
explants of Scrophularia yoshimurae. C, Callus induced from stem segments cultured on MS basal medium with 3% sucrose, 1 mg l21
(0.18 mM) NAA, and 0.2 mg l21 (0.04 mM) kinetin for 8 wk. D, Established suspension cultures from the stem-derived callus.
150 NALAWADE AND TSAY

of some natural products possible. Using cell cultures as metabolic genotypes of medicinal plants, it is possible to produce healthy and
factories, instead of whole plants, one can circumvent environmental disease-free plants which could be released to their natural habitat
vagaries associated with field-grown plants and these viable or cultivated on a large scale for the pharmaceutical product of
protocols may prove rewarding (Pande et al., 2000). With the aim interest. These are novel methods of conserving the natural
of obtaining yields in concentrations high enough for commercial populations of medicinal plants, reducing the risk of their
exploitation, efforts have focused on the isolation of biosynthetic extinction. In vitro propagation techniques impart vigor for the
activities of cultured cells achieved by optimizing the cultural conservation process of the medicinal plants and also maintain the
conditions, selection of high-producing strains, precursor feeding, clonal uniformity not achieved by using seeds. The methods could
transformation methods, and immobilization techniques (Dicosmo also be used for gene manipulation for crop improvement or more
and Misawa, 1995). We have successfully established suspension specifically to alter the expression of gene/s important for the
cultures of Gentiana davidii. The genus Gentiana (Gentianaceae) biosynthesis of bioactive compounds. Various strategies for using in
comprises about 400 species distributed throughout the world vitro systems are being studied extensively with the objective of
(Skrzypczak et al., 1993). Bitter principles of Gentianaceae improving the production and qualitative consistency of plant
constitute many pharmacologically important compounds, which chemicals. Due to these advances, research in the area of tissue
justify the use of most species of this family in traditional medicine culture technology for production of plant chemicals has bloomed
or for the preparation of bitter tonics (Rodriguez et al., 1996). In beyond expectations.
Taiwan, 11 species and two varieties of the genus Gentiana have
been identified (Chen and Wang, 1999). Among the species in Acknowledgments
Taiwan, Gentiana davidii var. formosana is the most widespread,
ranging from low to high elevation throughout the central mountain This research was supported by a grant (NSC-91-2313-B324-002, NSC-
range of the island (Chen and Wang, 1999). The whole dried herb, 92-2313-B324-003, and NSC-91-2313-B324-001) from the National
collected from its wild habitat, is used as a crude drug in traditional Science Council of Taiwan.
medicines in Taiwan. However, the plant is fully protected by law in
Taiwan and collection of plants from the wild habitat is illegal. In References
spite of its importance, a general method for commercial cultivation
Abou-Mandour, A. A. Studies on Ruta graveolens spp. divaricata. (1)
has not been reported for G. davidii. A cell suspension culture could Communication: initiation and cultivation of callus cultures and
be a more suitable alternative for the pharmaceutical industry for induction of shoot. Planta Med. 46:105–109; 1982.
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