Bot. Bull. Acad. Sin. (2004) 45: 1-22

Vanisree et al. Studies on the production of some important secondary metabolites

(Review paper)

Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures

Mulabagal Vanisree1, Chen-Yue Lee2, Shu-Fung Lo2,3, Satish Manohar Nalawade1, Chien Yih Lin3, and Hsin-Sheng Tsay*,1

1Institute of Biotechnology, Chaoyang University of Technology, 168, Gifeng E. Road, Wufeng, Taichung, Taiwan 413

2National Chung Hsing University, Taichung, Taiwan 402

3Taiwan Agricultural Research Institute, Wufeng, Taiwan 413

(Received January 8, 2003; Accepted April 22, 2003)

Abstract. Plants are a tremendous source for the discovery of new products of medicinal value for drug development. Today several distinct chemicals derived from plants are important drugs currently used in one or more countries in the world. Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Plant cell culture technologies were introduced at the end of the 1960's as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using an in vitro system, have been extensively studied to improve the production of plant chemicals. The focus of the present review is the application of tissue culture technology for the production of some important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.

Keywords: Biotransformations; Cell suspension cultures; Hairy root cultures; Pharmaceuticals; Secondary metabolites.

Botanical Bulletin of Academia Sinica, Vol. 45, 2004


Many higher plants are major sources of natural products used as pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and pesticides (Balandrin and Klocke, 1988). The search for new plant-derived chemicals should thus be a priority in current and future efforts toward sustainable conservation and rational utilization of biodiversity (Phillipson, 1990). In the search for alternatives to production of desirable medicinal compounds from plants, biotechnological approaches, specifically, plant tissue cultures, are found to have potential as a supplement to traditional agriculture in the industrial production of bioactive plant metabolites (Ramachandra Rao and Ravishankar, 2002). Cell suspension culture systems could be used for large scale culturing of plant cells from which secondary metabolites could be extracted. The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products.

Discoveries of cell cultures capable of producing specific medicinal compounds (Table 1) at a rate similar or superior to that of intact plants have accelerated in the last few years. New physiologically active substances of medicinal interest have been found by bioassay. It has been demonstrated that the biosynthetic activity of cultured cells can be enhanced by regulating environmental factors, as well as by artificial selection or the induction of variant clones. Some of the medicinal compounds localized in morphologically specialized tissues or organs of native plants have been produced in culture systems not only by inducing specific organized cultures, but also by undifferentiated cell cultures. The possible use of plant cell cultures for the specific biotransformations of natural compounds has been demonstrated (Cheetham, 1995; Scragg, 1997; Krings and Berger, 1998; Ravishankar and Ramachandra Rao, 2000). Due to these advances, research in the area of tissue culture technology for production of plant chemicals has bloomed beyond expectations.

The major advantages of a cell culture system over the conventional cultivation of whole plants are: (1) Useful compounds can be produced under controlled conditions independent of climatic changes or soil conditions; (2) Cultured cells would be free of microbes and insects; (3) The cells of any plants, tropical or alpine, could easily be multiplied to yield their specific metabolites; (4) Automated control of cell growth and rational regulation of metabolite processes would reduce of labor costs and improve productivity; (5) Organic substances are extractable from callus cultures.

In order to obtain high yields suitable for commercial exploitation, efforts have focused on isolating the biosynthetic activities of cultured cells, achieved by optimizing the cultural conditions, selecting high-producing strains, and employing precursor feeding, transformation methods, and immobilization techniques (Dicosmo and Misawa, 1995). Transgenic hairy root cultures have revolutionized the role of plant tissue culture in secondary metabolite production. They are unique in their genetic and biosynthetic stability, faster in growth, and more easily maintained. Using this methodology a wide range of chemical compounds have been synthesized (Shanks and Morgan, 1999; Giri and Narasu, 2000). Advances in tissue culture, combined with improvement in genetic engineering, specifically transformation technology, has opened new avenues for high volume production of pharmaceuticals, nutraceuticals, and other beneficial substances (Hansen and Wright, 1999). Recent advances in the molecular biology, enzymology, and fermentation technology of plant cell cultures suggest that these systems will become a viable source of important secondary metabolites. Genome manipulation is resulting in relatively large amounts of desired compounds produced by plants infected with an engineered virus, whereas transgenic plants can maintain constant levels of production of proteins without additional intervention (Sajc et al., 2000). Large-scale plant tissue culture is found to be an attractive alternative approach to traditional methods of plantation as it offers a controlled supply of biochemicals independent of plant availability (Sajc et al., 2000). Kieran et al. (1997) detailed the impact of specific engineering-related factors on cell suspension cultures. Current developments in tissue culture technology indicate that transcription factors are efficient new molecular tools for plant metabolic engineering to increase the production of valuable compounds (Gantet and Memelink, 2002). In vitro cell culture offers an intrinsic advantage for foreign protein synthesis in certain situations since they can be designed to produce therapeutic proteins, including monoclonal antibodies, antigenic proteins that act as immunogenes, human serum albumin, interferon, immuno-contraceptive protein, ribosome unactivator trichosantin, antihypersensitive drug angiotensin, leu-enkephalin neuropeptide, and human hemoglobin (Hiatt et al., 1989; Manson and Arntzen, 1995; Wahl et al., 1995; Arntzen, 1997; Hahn et al., 1997; La Count et al., 1997; Marden et al., 1997; Wongsamuth and Doran, 1997; Doran, 2000). The appeal of using natural products for medicinal purposes is increasing, and metabolic engineering can alter the production of pharmaceuticals and help to design new therapies. At present, researchers aim

Vanisree et al. Studies on the production of some important secondary metabolites

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Vanisree et al. Studies on the production of some important secondary metabolites

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Taxus cell cultures. Fett-Neto et al. (1995) have studied the effect of nutrients and other factors on paclitaxel production by T. cuspidata cell cultures (0.02% yield on dry weight basis). Srinivasan et al. (1995) have studied the kinetics of biomass accumulation and paclitaxel production by T. baccata cell suspension cultures. Paclitaxel was found to accumulate at high yields (1.5 mg/l) exclusively in the second phase of growth. Kim et al. (1995) established a similar level of paclitaxel from T. brevifolia cell suspension cultures following 10 days in culture with optimized medium containing 6% fructose. Ketchum and Gibson (1996) reported that addition of carbohydrate during the growth cycle increased the production rate of paclitaxel, which accumulated in the culture medium (14.78 mg/l). In addition to paclitaxel, several other taxoids have been identified in both cell and culture medium of Taxus cultures (Ma et al., 1994). Parc et al. (2002) reported production of taxoids by callus cultures from selected Taxus genotypes. In order to increase the taxoid production in these cultures, the addition of different amino acids to the culture medium were studied, and phenylalanine was found to assist in maximum taxol production in T. cuspidata cultures (Fett-Neto et al., 1994). The influence of biotic and abiotic elicitors was also studied to improve the production and accumulation of taxol through tissue cultures (Ciddi et al., 1995; Strobel et al., 1992; Yukimune et al., 1996). The production of taxol from nodule cultures containing cohesive multicultural units displaying a high degree of differentiation has been achieved from cultured needles of seven Taxus cultivars (Ellis et al., 1996). Factors influencing stability and recovery of paclitaxel from suspension cultures and the media have been studied in detail by Nguyen et al. (2001). The effects of rare earth elements and gas concentrations on taxol production have been reported (Wu et al., 2001 and Linden et al., 2001).

Morphine and Codeine

Latex from the opium poppy, Papaver somniferum, is a commercial source of the analgesics, morphine and codeine. Callus and suspension cultures of P. somniferum are being investigated as an alternative means for production of these compounds. Production of morphine and codeine in morphologically undifferentiated cultures has been re

to produce substances with antitumor, antiviral, hypoglycaemic, anti-inflammatory, antiparasite, antimicrobial, tranquilizer and immunomodulating activities through tissue culture technology.

Exploration of the biosynthetic capabilities of various cell cultures has been carried out by a group of plant scientists and microbiologists in several countries during the last decade. In the last few years promising findings have been reported for a variety of medicinally valuable substances, some of which may be produced on an industrial scale in the near future. The aim of the present review is to focus on the importance of tissue culture technology in production of some of the plant pharmaceuticals reported earlier. We will also describe the successful research on tissue cultures for production of bioactive metabolites performed at our own laboratory.

Tissue Cultures Producing Pharmaceutical Products of Interest

Research in the area of plant tissue culture technology has resulted in the production of many pharmaceutical substances for new therapeutics. Advances in the area of cell cultures for the production of medicinal compounds has made possible the production of a wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavanoids, and amino acids. Successful attempts to produce some of these valuable pharmaceuticals in relatively large quantities by cell cultures are illustrated.


Taxol (plaxitaxol), a complex diterpene alkaloid found in the bark of the Taxus tree, is one of the most promising anticancer agents known due to its unique mode of action on the micro tubular cell system (Jordan and Wilson, 1995). At present, production of taxol by various Taxus species cells in cultures has been one of the most extensively explored areas of plant cell cultures in recent years owing to the enormous commercial value of taxol, the scarcity of the Taxus tree, and the costly synthetic process (Cragg et al., 1993; Suffness, 1995). In 1989, Christen et al. reported for the first time the production of taxol (placlitaxel) by

Vanisree et al. Studies on the production of some important secondary metabolites

ported (Tam et al., 1980; Yoshikawa and Furuya, 1985). Removal of exogenous hormones from large-scale culture systems could be implemented using a two-stage process strategy by Siah and Doran (1991). Without exogenous hormones, maximum codeine and morphine concentrations were 3.0 mg/g dry weight and 2.5 mg/g dry weight, respectively, up to three times higher than in cultures supplied with hormones. Biotransformation of codeinone to codeine with immobilized cells of P. somniferum has been reported by Furuya et al. (1972). The conversion yield was 70.4%, and about 88% of the codeine converted was excreted into the medium.


The root of Panax ginseng C.A. Mayer, so-called ginseng, has been widely used as a tonic and highly prized medicine since ancient times (Tang and Eisenbrand, 1992a). Ginseng has been recognized as a miraculous promoter of health and longevity. The primary bioactive constituents of ginseng were identified as ginsenosides, a group of triterpenoid saponins (Huang, 1993a; Proctor, 1996; Sticher, 1998). Among them, ginsenoside Rg1 is one of the major active molecules from Panax ginseng (Lee et al., 1997). Chang and Hsing (1980a) obtained repeatable precocious flowering in the embryos derived from mature gin

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

seng root callus cultured on a chemically defined medium. Also, plant regeneration through somatic embryogenesis in root-derived callus of ginseng has been reported (Chang and Hsing, 1980b). In recent years ginseng cell culture has been explored as a potentially more efficient method of producing ginsenosides. The effect of medium components like carbon (Furuya et al., 1984; Choi et al., 1994), nitrogen (Franklin and Dixon, 1994), and phosphate (Zhang and Zhong, 1997) concentrations and plant growth hormones (Furuya, 1988) were thoroughly studied to increase the production of ginsenosides. Influence of potassium ion was also studied (Liu and Zhong, 1996). Large-scale suspension culture of ginseng cells was first reported by Yasuda et al. (1972). Later on an industrial-scale culture process was initiated by Nitto Denko Corporation (Ibaraki, Osaka, Japan) in the 1980s using 2000 and 20000-1 stirred tank fermentors to achieve productivities of 500-700 mg/l per day (Furuya, 1988; Ushiyama, 1991). This process is considered an important landmark in the commercialization of plant tissue and cell culture on a large scale. In addition to this, Agrobacterium tumefaciens infected root cultures were introduced, productivity of which was found to exceed the callus of normal roots threefold (Choi et al., 1989). Other types of tissue cultures, such as embryogenic tissues (Asaka et al., 1993) and hairy roots transformed by Agrobacteria (Yoshikawa and Furuya, 1987; Hwang et al., 1991; Ko et al., 1996) have been examined. Yu et al. (2000) reported ginsenoside production using elicitor treatment. These developments indicate that ginseng cell culture process is still an attractive area for commercial development around the world and it possesses great potential for mass industrialization. Concentration of plant growth regulators in the medium influences the cell growth and ginsenoside production in the suspension cultures (Zhong et al., 1996). Recent studies have shown that addition of methyl jasmonate or dihydro-methyl jasmonate to suspension cultures increases the production of ginsenosides (Wang and Zhong, 2002). Also, jasmonic acid improves the accumulation of gensinosides in the root cultures of ginseng (Yu et al., 2002).


L-3,4-dihydroxyphenylalanine, is an important intermediate of secondary metabolism in higher plants and is known as a precursor of alkaloids, betalain, and melanine, isolated from Vinca faba (Guggenheim, 1913), Mucuna, Baptisia and Lupinus (Daxenbichler et al., 1971). It is also a precursor of catecholamines in animals and is being used as a potent drug for Parkinson's disease, a progressive disabling disorder associated with a deficiency of dopamine in the brain. The widespread application of this therapy created a demand for large quantities of L-DOPA at an economical price level, and this led to the introduction of cell cultures as an alternative means for enriched production. Brain (1976) found that the callus tissue of Mucuna pruriense accumulated 25 mg/l DOPA in the medium containing relatively high concentrations of 2,4-D. Teramoto and Komamine (1988) induced callus tissues of Mucuna hassjoo, M. Pruriense, and M. deeringiana and optimized

the culture conditions. The highest concentration of DOPA was obtained when M. hassjoo cells were cultivated in MS medium with 0.025 mg/l 2.4 -D and 10 mg/l kinetin. The level of DOPA in the cells was about 80 mmol/g-f.w.


Berberine is an isoquinoline alkaloid found in the roots of Coptis japonica and cortex of Phellondendron amurense. This antibacterial alkaloid has been identified from a number of cell cultures, notably those of Coptis japonica (Sato and Yamada, 1984), Thalictrum spp. (Nakagawa et al., 1984; Suzuki et al., 1988), and Berberis spp. (Breuling et al., 1985). The productivity of berberine was increased in cell cultures by optimizing the nutrients in the growth medium and the levels of phytohormones (Sato and Yamada, 1984; Nakagawa et al., 1984, 1986; Morimoto et al., 1988). By selecting high yielding cell lines, Mitsui group produced berberine on a large scale with a productivity of 1.4 g/l over 2 weeks. Other methods for increasing yields include elicitation of cultures with a yeast polysaccharide elicitor, which has been successful with a relatively low producing T. rugosum culture (Funk et al., 1987). The influence of spermidine on berberine production in Thalictrum minus cell cultures has been reported by Hara et al. (1991).

Vanisree et al. Studies on the production of some important secondary metabolites


Diosgenin is a precursor for the chemical synthesis of steroidal drugs and is tremendously important to the pharmaceutical industry (Zenk, 1978). In 1983, Tal et al. reported on the use of cell cultures of Dioscorea deltoidea for production of diosgenin. They found that carbon and nitrogen levels greatly influenced diosgenin accumulation in one cell line. Ishida (1988) established Dioscorea immobilized cell cultures, in which reticulated polyurethane foam was shown to stimulate diosgenin production, increasing the cellular concentration by 40% and total yield by 25%. Tal et al. (1983) have been able to obtain diosgenin levels as high as 8% in batch-grown D. deltoidea cell suspensions. However, the daily productivity was only 7.3 mg/l. Several other groups have also attempted cell cultures for diosgenin production (Heble et al., 1967; Brain and Lockwood, 1976; Jain and Sahoo, 1981; Jain et al., 1984; Emke and Eilert, 1986; Huang et al., 1993). Kaul et al. (1969) studied the influence of various factors on diosgenin production by Dioscorea deltoidea callus and suspension cultures. The search for high-producing cell lines coupled to recent developments in immobilized cultures and the use of extraction procedures, which convert furostanol saponins to spirostanes such as diosgenin, should prove useful in increasing productivity in the years to come.

veloped for the production of capsaicin from C. frutescens cells (Lindsey et al., 1983). Holden et al. (1988) have reported elicitation of capsaicin in cell cultures of C. frutescens by spores of Gliccladium deliquescens. The effects of nutritional stress on capsaicin production in immobilized cell cultures of Capsicum annum were studied thoroughly by Ravishankar et al. (1988). Biotransformation of externally fed protocatechuic aldehyde and caffeic acid to capsaicin in freely suspended cells and immobilized cells cultures of Capsicum frutescens has also been reported (Ramachandra Rao and Ravishankar, 2000).


Camptothecin, a potent antitumor alkaloid was isolated from Camptotheca acuminata. Sakato and Misawa (1974) induced C. acuminata callus on MS medium containing 0.2 mg/l 2,4-D and 1 mg/l kinetin and developed liquid cultures in the presence of gibberellin, L-tryptophan, and conditioned medium, which yielded camptothecin at about 0.0025% on a dry weight basis. When the cultures were grown on MS medium containing 4 mg/l NAA, accumulation of camptothecin reached 0.998 mg/l (Van Hengal et al., 1992). 10-Hydroxycamptothecin, a promising derivative of camptothecin is in clinical trials in the US.

Vinblastine and Vincristine

The dimeric indole alkaloids vincristine and vinblastine have become valuable drugs in cancer chemotherapy due to their potent antitumor activity against various leukemias and solid tumors. These compounds are extracted commercially from large quantities of Catharanthus roseus. Since the intact plant contains low concentrations (0.0005%), plant cell cultures have been employed as an alternative to produce large amounts of these alkaloids. Vinblastine is composed of catharanthine and vindoline. Since


Capsaicin, an alkaloid, is used mainly as a pungent food additive in formulated foods. It is obtained from fruits of green pepper (Capsicum spp.). Capsaicin is also used in pharmaceutical preparations as a digestive stimulant and for rheumatic disorders (Sooch et al., 1977). Suspension cultures of Capsicum frutescens produce low levels of capsaicin, but immobilizing the cells in reticulated polyurethane foam can increase production approximately 100-fold (Lindsey and Yeoman, 1984). Further improvements in productivity can be brought about by supplying precursors such as isocapric acid (Lindsey and Yeoman, 1984). Lindsey (1985) reported that treatments which suppress cell growth and primary metabolism seem to improve capsaicin synthesis. A biotechnological process has been de

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

the cultured cells of C. roseus was used as an enzyme source. The reaction mixture contained catharanthine, vindoline, Tris buffer, Ph 7.0, and the crude enzyme; the mixture was incubated at 300C and for 3 h. The products of the reaction were various dimeric alkaloids including vinamidine, 3(R)-hydroxyvinamidine, and 3, 4-anhydrovinblastine. Dimerization using ferric ion catalyst in the absence of enzyme resulted in anhydrovinblastine and vinblastine in 52.8% and 12.3% yields, respectively. The yield of vinblastine via chemical coupling was improved in the presence of ferric chloride, oxalate, maleate, and sodium borohydride. Influence of various parameters like stress, addition of bioregulators, elicitors and synthetic precursors on indole alkaloids production were studied in detail by Zhao et al. (2001a and b). Also, metabolic rate-limitations through precursor feeding (Morgan and Shanks, 2000) and effect of elicitor dosage on biosynthesis of indole alkaloids (Rijhwani and Shanks, 1998) in Catharanthus roseus hairy root cultures have been reported.


Tanshinones are a group of quinoid diterpenoids believed to be active principles of Danshen (Salvia miltiorrhiza), a well known traditional Chinese medicine. Tanshinone I and cryptotanshinone prevent complications of myocardial ischemia; tanshinone II A has undergone

vindoline is more abundant than catharanthin in intact plants, it is less expensive. Misawa et al. (1988) established an economically feasible process consisting of production of catharanthine by plant cell fermentation and a simple chemical or an enzymatic coupling. The significant influence of various compounds, like vanadyl sulphate, abscisic acid, and sodium chloride on catharanthin production have been described by Smith et al. (1987). Endo et al. (1988) attempted synthesis of anhydrovinblastine (AVLB from catharanthine and vindoline through enzymic coupling followed by sodium borohydride reduction). A crude preparation of 70% ammonium sulphate precipitated protein from

Vanisree et al. Studies on the production of some important secondary metabolites

successful clinical trials for the treatment of angina pectoris in China (Bruneton, 1995). Plant cell and organ culture technology provide an alternative means of producing these active ingredients. Nakanishi et al. (1983) established a cell line containing abundant amounts of cryptotanshinone from S. miltiorrhiza. Adventitious root cultures of S. miltiorrhiza and the culture conditions for high yield production of tanshinones in the adventitious roots were reported by Shimomura et al. (1991). Diterpenoid production in Ti-transformed root or hairy root cultures of S. miltiorrhiza has also been established by Hu and Alfermann (1993). In these cultures, although relatively high tanshinone production was achieved, the morphological characteristics of the hairy roots require special bioreactors for the cultivation, which has hindered the scale-up of such processes.


Podophyllotoxin is an antitumor aryltetralin lignan found in Podophyllum peltatum and Podophyllum hexandrum. It also serves as a starting material for the preparation of its semisynthetic derivatives, etoposide and teniposide, widely used in anti-tumor therapy (Issell et al., 1984). These plants, which grow very slowly, are collected from the wild and are thus increasingly rare. This limits the supply of podophyllotoxin and necessitates the search for alternative production methods. Cell cultures of P. peltatum for production of podophyllotoxin was first attempted by Kadkade et al. (1981, 1982). To increase the yield of podophyllotoxin, Woerdenberg et al. (1990) used a complex of a precursor, coniferyl alcohol, and b-cyclodextrin to P. hexandrum cell suspension cultures. The addition of 3 mM coniferyl alcohol complex yielded 0.013% podophyllotoxin on a dry weight basis, but the cultures without the precursor produced only 0.0035%. Smollny et al. (1992) reported that callus tissues and suspension culture cells of Lilium album produced 0.3% podophyllotoxin. Several other tissue culture approaches have been studied to in

crease the yields (Berlin et al., 1988; Van Uden et al., 1989; Hyenga et al., 1990). Since 5-methoxypodophyllotoxin, an analogue of podophyllotoxin, has strong cytostatic activity (Berlin et al., 1988), many researchers have tried to improve its yield through tissue cultures (Van Uden et al., 1990; Wichers et al., 1990).

Studies on In Vitro Cultures and Production of Important Secondary Metabolites in the Author's Laboratory

Even though several types of cell culture methods are being used to produce important bioactive secondary metabolites, use of cell suspension cultures is preferred for large-scale production due to its rapid growth cycles. Thus cell suspensions are used for generating large amounts of cells for quantitative or qualitative analysis of growth responses and metabolism of novel chemicals. Based on the exciting results in production of medicinal compounds reported above using cell suspension cultures, we have successfully established cell suspension cultures for the production of taxol from Taxus mairei, imperatorin from Angelica dahurica, and diosgenin from Dioscorea doryophora at our research center. We have also succeeded in propagating some of the valuable Chinese medicinal herbs and estimating their active ingredients quantitatively using high performance liquid chromatography (HPLC). The work carried out at our research centers is summarized in the following sections.

Production of Taxol from Taxus mairei by Cell Suspension Cultures

Taxol, a complex diterpene alkaloid, is an anticancer drug found in 1971, by Wani et al. from the Pacific yew tree, Taxus brevifolia (Wani et al., 1971). At present the drug is approved for clinical treatment of ovarian and breast cancer by the Food and Drug Administration (FDA, USA). It also has significant activity against malignant melanoma, lung cancer, and other solid tumors (Wickremesinhe and Arteca, 1993, 1994). However, the supply of taxol for clinical use is limited. It depends on extraction from yew trees, and the bark is the only commercial source. The thin bark of the yew tree contains 0.001% taxol on a dry weight basis. A century-old tree yields an average of 3 kg of bark, corresponding to 300 mg of taxol, approximately a single dose in the course of a cancer treatment. Because of the scarcity of the slow growing trees and the relatively low taxol content (Cragg et al., 1993), alternative sources are needed to meet the increasing demand for the drug. The total synthesis of taxol on an industrial-scale seems economically unrealistic due to the complexity of the chemical structure of this molecule (Holton et al., 1994; Nicolaou et al., 1994). The plant cell culture of Taxus spp. is considered one possible approach to providing a stable supply of taxol and related taxane compounds (Slichenmyer and Von Horf, 1991).

To exploit the source of taxol, we collected different tissues of Taxus mairei, a species found in Taiwan at an alti

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tude of about 2,000 m above sea level. The extracts of bark and leaf tissues were analyzed using HPLC for the content of taxol and taxol related compounds. The HPLC analysis revealed that amounts of taxol and taxol related compounds varies in individual plants, and the principle components such as docetaxel, baccatin III, and 10-deacetylbaccatin in leaf extract were higher than those in bark extracts (Lee et al., 1995). Taxus mairei calli were induced from needle and stem explants on Gamborg's B5 medium supplemented with 2 mg/l 2,4-D or NAA. Different cell lines were established using stem and needle derived callus. One of the cell lines, after precursor feeding and 6 weeks of incubation, produced 200 mg taxol per liter of cell suspension culture.

Formation of Imperatorin from Angelica dahurica var. formosana by cell Suspension Cultures

Angelica dahurica var. formosana commonly known as "Bai-Zhi" in Chinese is a valuable medicinal herb used in the treatment of headache and psoriasis in China (Zhou, 1980). The constituent imperatorin is believed to be the major active ingredient for curing skin disease (Zhou et al., 1988). Angelica dahurica var. formosana is a perennial and indigenous plant in Taiwan (Chen et al., 1994). We have studied cell suspension cultures of Angelica dahurica var. formosana for the production of imperatorin. Angelica dahurica var. formosana plants were obtained from their natural habitat in the Yang-Ming National Park of Taiwan. The callus was induced from petiole explants on a medium supplemented with 1 mg/l 2, 4-D and 0.5 mg/l kinetin. The resultant callus was used in establishing the cell suspension culture. By increasing the phosphate concentration in the basal medium to 2 mM and using an ammonium to nitrate ratio of 2:1, it was possible to increase the production of imperatorin in cell suspension cultures. Glucose was found to be a better carbon source than sucrose and fructose. The addition of 0.5-1 mg/l of BA to the culture medium increased imperatorin yield, while addition of auxins to the culture medium decreased it. Supplementing the medium with 20 g/l of the adsorbent Amberlite XAD-7 increased imperatorin yield 140-fold (Tsay et al., 1994; Tsay, 1999).

Production of Diosgenin from Dioscorea doryophora by Cell Suspension Culture

Dioscorea spp. (Dioscoreaceae) are frequently used as a tonic in Chinese traditional medicine. Dioscorea doryophora Hance tubers are in high demand as they are used not only as crude drug but also as food. The most active ingredient discovered in the tuber is diosgenin, which can be used as a precursor for many important medicinal steroids, such as prednisolone, dexamethasone, norethisterone, and metenolone (Tsukamoto et al., 1936).

In order to increase diosgenin yield and facilitate the purification process, we have established a cell suspension culture of Dioscorea doryophora Hance (Yeh et al., 1994). Cell suspension cultures were obtained from microtuber and stem node-derived callus in liquid culture medium supplemented with 0.1 mg/l 2,4-D, 3% sucrose and incubated on a rotary shaker at 120 rpm. Although 6% sucrose was found to be optimum for the growth of cell suspension culture, cells cultured in a 3% sucrose medium produced more diosgenin. Analysis by HPLC revealed that both stem-node and microtuber derived suspension cells contained diosgenin. The microtuber derived cell suspension culture contains 3.2% diosgenin per gram dry weight while the stem-node derived cultures contain only 0.3%. As the amount of diosgenin obtained from a tuber-derived cell suspension is high and comparable with that found in the intact tuber (Chen, 1985), a cell suspension culture can be used to produce diosgenin.

Formation and Analysis of Corydaline and Tetrahydropalmatine from Tubers of Somatic Embryo-Derived Plants of Corydalis yanhusuo

The genus Corydalis (Fumariaceae or Papaveraceae) comprises about 320 species, widely distributed in the northern hemisphere, of which around seventy species have been used in traditional herbal remedies in China, Japan, and Korea (Kamigauchi and Iwasa, 1995). The dried and pulverized tubers of C. yanhusuo, also called Rhizoma Corydalis or yan-hu-suo are a rich source of several pharmacologically important alkaloids (Huang, 1993b). These are used in traditional Chinese medicine to treat gastric and duodenal ulcer, cardiac arrhythmia disease (Kamigauchi and Iwasa, 1995), rheumatism and dysmenorrhea (Tang and Eisenbrand 1992b). Corydalis yanhusuo is a slow-growing herb susceptible to fungal diseases which cause serious crop loss and also affect tuber quality. To achieve high productivity, homogeneity, and good quality tubers, pathogen-free planting material must be obtained (Sagare et al., 2000). Plant regeneration via in vitro culture of C. yanhusuo would be useful for quick, mass propagation of this important medicinal plant.

A protocol for complete plant regeneration via somatic embryogenesis from tuber derived callus, and production of bioactive compounds such as D, L-tetrahydropalmatine and D-corydaline from the tubers of somatic embryo-derived plants has been standardized in our laboratory (Lee et al., 2001). Primary callus was induced by culturing ma

Vanisree et al. Studies on the production of some important secondary metabolites

ture tuber pieces on a medium supplemented with 2.0 mg/l BA and 0.5 mg/l NAA in darkness. Somatic embryos were induced by subculturing the primary callus on medium supplemented with various concentrations of cytokinins, within 2 weeks of culture in light. The converted somatic embryos of C. yanhusuo were cultured for one month on different treatments (growth regulators) in order to promote tuberization and access their effect on accumulation of protoberberine alkaloids. After one and six months of culture in different treatments, the alkaloid contents in the tuber were analyzed by HPLC. The analysis revealed that, somatic embryos cultured on 0.1 mg/l GA3 for six months showed high amounts of both D, L-tetrahydropalmatine and D-corydalin in the tubers among these treatments. The highest corydalin content was about 3.8 mg/g dry weight after six months of culture on 0.5 mg/l paclobutrazol. The supplementation of an amino acid precursor such as tyrosine (Staba et al., 1982; Kamigauchi and Iwasa, 1995) to the culture medium may further improve the production of these compounds.

In Vitro Synthesis of Harpagoside, an Anti -Inflammatory Irridoid Glycoside from Scrophularia yoshimurae Yamazaki

Scrophularia yoshimurae Yamazaki, belonging to the family Scrophulariaceae, is an herbaceous perennial plant 40-60 cm tall that is indigenous to Taiwan. Scrophularia yoshimurae is used as "Xuanshen," a substitute for S. ningpoensis, in traditional Chinese medicine in Taiwan (Chiu and Chang, 1998). In view of Scrophularia's medici

nal value, an efficient protocol for micropropagation of Scrophularia yoshimurae (Scrophulariaceae) has been developed at our laboratory (Sagare et al., 2001). Multiple shoot development was achieved by culturing the shoot tip, leaf base, stem-node and stem-internode explants on Murashige and Skoog (MS) medium supplemented with 4.44 M N6-benzyladenine (BA) and 1.07 M a-naphthaleneacetic acid (NAA). The shoots were multiplied by subculturing on the same medium used for shoot induction. Shoots were rooted on growth regulator-free MS basal medium, transferred to a soil:peat moss:vermiculite (1:1:1 v/v/v) mixture, and acclimatized in the growth chamber. The content of harpagoside, an anti-inflammatory iridoid glucoside, in different plant materials was determined by HPLC. Harpagoside content in the aerial and underground parts of S. yoshimurae was significantly higher than in the marketed crude drug (underground parts of S. ningpoensis) and varied with the developmental stage of the plant.

Gentipicroside and Swertiamarin from In Vitro Propagated Plants of Gentiana davidii var. formosana (Gentianaceae)

The genus Gentiana (Gentianaceae) comprises about 400 species distributed throughout the world (Skrzypczak et al., 1993). The bitter principles of Gentianaceae constitute many pharmacologically important compounds, explaining the use of most species of this family in traditional medicine and in the preparation of bitter tonics (Rodriguez et al., 1996). Secoiridoid glucosides are the main compounds with medicinal properties in roots of Gentiana species (Skrzypczak et al., 1993). Gentiopicroside and swertiamarin are two important secoiridoid glucosides found in Gentianaceae, the former being quantitatively predominant (Tang and Eisenbrand, 1992c). We have developed a highly reproducible and simple protocol for in vitro propagation of Gentiana davidii var. formosana (Chueh et al., 2000). Induction of multiple shoots (6.3 shoots per explant) was achieved in the axillary buds of the stem node explants (5 mm long) cultured on Murashige and Skoog (MS) medium supplemented with 4.44 M N6-benzyladenine (BA) for a period of two months. A more than twofold increase in the number of shoots per explants

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used to explain the problems occurring in the production of secondary metabolites from cultured plant cells. A key to the evaluation of strategies to improve productivity is the realization that all the problems must be seen in a holistic context. At any rate, substantial progress in improving secondary metabolite production from plant cell cultures has been made within last few years. These new technologies will serve to extend and enhance the continued usefulness of higher plants as renewable sources of chemicals, especially medicinal compounds. We hope that a continuation and intensification efforts in this field will lead to controllable and successful biotechnological production of specific, valuable, and as yet unknown plant chemicals.

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