You are on page 1of 20

International Journal of Applied Science and Engineering 2004.

2, 1: 29-48

Plant Cell Cultures - An Alternative and Efficient Source for the Production of Biologically Important Secondary Metabolites
Vanisree Mulabagal and Hsin-Sheng Tsay∗ Institute of Biotechnology, Chaoyang University of Technology, Wufeng, Taichung county 413, Taiwan, R.O.C. Abstract: Plant cell culture systems represent a potential renewable source of valuable medicinal compounds, flavors, fragrances, and colorants, which cannot be produced by microbial cells or chemical synthesis. Biotechnological applications of plant cell cultures presents the most updated reviews on current techniques in plant culture in the field. The evolving commercial importance of the secondary metabolites has in recent years resulted in a great interest, in secondary metabolism, and particularly in the possibility to alter the production of bioactive plant metabolites by means of cell culture technology. The principle advantage of this technology is that it may provide continuous, reliable source of plant pharmaceuticals and could be used for the large-scale culture of plant cells from which these metabolites can be extracted. In addition to its importance in the discovery of new medicines, plant cell culture technology plays an even more significant role in solving world hunger by developing agricultural crops that provide both higher yield and more resistance to pathogens and adverse environmental and climatic conditions. This paper describes the callus and suspension culture methods that we have established in our laboratory for the production of bioactive secondary metabolites from medicinal plants. Keywords: bioreactors; callus cultures; secondary metabolites; suspension cultures; pharmaceuticals; precursor feeding. 1. Introduction Medicinal plants are the most exclusive source of life saving drugs for the majority of the world’s population. Bioactive compounds currently extracted from plants are used as food additives, pigments, dyes, insecticides, cosmetics and perfumes and fine chemicals [Balandrin and Klocke, 1988]. These compounds belong to a group collectively known as secondary metabolites. Studies on plant

secondary metabolites have been increasing over the last 50 years. The molecules are known to play a major role in the adaptation of plants to their environment, but also represent an important source of pharmaceuticals [Ramachandra Rao and Ravishankar, 2002]. In recent years, traditional system of medicine has become a topic of global importance. Although modern medicine may be available in developed countries, herbal medicines [phytopharmaceuticals] have often maintained popularity for historical and cultural reasons.
Accepted for Publication: Dec. 23, 2003

Corresponding author: e-mail:

© 2004 Chaoyang University of Technology, ISSN 1727-2394

Int. J. Appl. Sci. Eng., 2004. 2, 1


Vanisree Mulabagal and Hsin-Sheng Tsay

Many of the plant species that provide medicinal herbs have been scientifically evaluated for their possible medical applications. It has been mentioned that natural habitats for medicinal plants are disappearing fast and together with environmental and geopolitical instabilities; it is increasingly difficult to acquire plant-derived compounds. This has prompted industries, as well as scientists to consider the possibilities of investigation into cell cultures as an alternative supply for the production of plant pharmaceuticals. Advances in biotechnology particularly methods for culturing plant cell cultures, should provide new means for the commercial processing of even rare plants and the chemicals they provide. These new technologies will extend and enhance the usefulness of plants as renewable resources of valuable chemicals. There has been considerable interest in plant cell cultures as a potential alternative to traditional agriculture for the industrial production of secondary metabolites [Dicosmo and Misawa, 1995]. Plant cell culture technologies were introduced at the end of 1960s as a possible tool for both studying and producing plant secondary metabolites. Different strategies using cell cultures systems have been extensively studied with the objective of improving the production of bioactive secondary metabolites. Cell culture systems could be used for the large scale culturing of plant cells from which secondary metabolites can be extracted. The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products. The major advantages of cell cultures includes (i) synthesis of bioactive secondary metabolites is running in controlled environment, independently from climatic and soil conditions; (ii) negative biological influences that affect secondary metabolites production in the nature are eliminated (microorganisms and insects); (iii) it is possible to select cultivars with higher production of secondary metabolites; (iv) with automatization of cell growth control and metabolic processes regu30 Int. J. Appl. Sci. Eng., 2004. 2, 1

lation, cost price can decrease and production increase. The scheme of production of some important plant pharmaceuticals produced in cell cultures has been presented in Table 1. The objectives of many industries are to develop plant cell culture techniques to the stage where they yield secondary products more cheaply than extracting either the whole plant grown under natural conditions or synthesizing the product. Although the production of pharmaceuticals using plant cell cultures have been highlighted, other uses have also been suggested as new route for synthesis, for products from plants difficult to grow, or in short supply, as a source of novel chemicals and as biotransformation systems. It is expected that the use, production of market price and structure would bring some of the other compounds to a commercial scale more rapidly and in vitro culture products may see further commercialization. Recent research results indicate that plant cell suspension cells can be used for recombinant protein production under controlled conditions [Fischer et al., 1999]. The aim of the present review is to focus the successful research accomplishments obtained on callus and suspension cultures for production of bioactive secondary metabolites in our research laboratory. 2. Accumulation of secondary metabolites in plant cell cultures For plant cell culture techniques to become economically viable, it is important to develop methods that would allow for consistent generation of high yields of products from cultured cells [Berlin and Sasse, 1985]. Careful selection of productive cells and cultural conditions resulted in accumulation of several products in higher levels in cultured cells. In order to obtain yields in high concentrations for commercial exploitation, efforts have focused on the stimulation of biosynthetic activities of cultured cells using various methods (Ramachandra Rao, 2000; Dixon, 1999; Ravishankar and Venkataraman, 1993; Buite-

Caffeine Cornus kousa Polyphenols Corydalis ophiocarpa Isoquinoline alkaloids Croton sublyratus Kurz Plaunotol Cruciata glabra Anthraquinones Cryptolepis buchanani Roem. 1982. γ-glutamyl derivatives L-Canavanine Capsaicin Anthraquinones Indole alkaloids Catharanthine Furoquinoline alkaloids Culture type Callus Suspension Suspension Callus Suspension Callus Suspension Suspension Callus Suspension Callus Suspension Suspension Suspension Suspension Suspension Callus Suspension Suspension Suspension Suspension Suspension Callus Callus Suspension Callus Callus Suspension Callus Suspension Suspension Suspension Callus Suspension Reference Andrijany et al. Cassia acutifolia Catharanthus roseus Catharanthus roseus Choisya ternata Chrysanthemum folium Chrysanthemum folium Active ingredient Saponins Alkaloids Canthinone alkaloids Alliin Tetrahydroanthracene glucosides Altamisine Rosmarinic acid Canthinone alkaloids Saikosaponins Theamine. 2000. O'Dowd et al. 1987. Morimoto and Murai.. 1986. 1983. 1984. 1985. 1987.An Alternative and Efficient Source for Production of Biologically Secondary Metabolites laar and Tramper. 1987. 1989.. 1982 Heble and Staba. J. Bupleurum falcatum Camellia Sinensis Canavalia ensiformis Capsicum annuum L.. 1999. 1993.. 1999. 1999. 1990. 1986... Rajasekaran et al. Moreno et al. 1993. Yamada and Endo. Table 1. De-Eknamkul and Ellis.Ephedrine D-pseudoephedrine continued … Int. Schripsema et al. 2. cinerariae. Anthraquinones Cinchona succirubra Anthraquinones Citrus sp. Orihara and Furuya.. 1981. 1991. 1990.) Merr.. Koblitz et al. Ramirez et al... 1 31 . Appl. 1983. Johnson et al. Kueh et al. Anderson et al. 1982. Wijnsma et al. Iwasa and Takao.. 1993.Chrysanthemic acid and pyrethrins Cinchona L. Venkateswara et al... 2001. 1983.. Alkaloids Cinchona robusta Robustaquinones Cinchona spe.. 1990. Malpathak and David ..Plant Cell Cultures . Wang and Huang. Cardenolides Dioscorea deltoidea Diosgenin Dioscorea doryophora Hance Diosgenin Duboisia leichhardtii Tropane alkaloids Ephedra spp. Limonin Coffea arabica L. 1980. Sejourne et al. Bioactive secondary metabolites from plant cell cultures Plant name Agave amaniensis Ailanthus altissima Ailanthus altissima Allium sativum L. Huang et al... 1992).. Waller et al... 1985. Naringin. Barthe et al.. Zhao et al. Ishimaru et al. Liu et al. Hagimori et al. Dornenburg and Knorr. Sci. Goleniowski and Trippi . 1986. & Cryptosin Schult Digitalis purpurea L. 1992.. Anderson et al.Pyrethrins cinerariae. Yagi et al. Aloe saponaria Ambrosia tenuifolia Anchusa officinalis Brucea javanica (L. Khouri et al. 1993.. 2004. L. 1996. Eng. 1985.. Nazif et al.

2004.3-b] quinolinium alkaloids Alkaloids Reserpine Suspension Suspension Callus Suspension Suspension Callus Callus Callus Suspension Suspension Suspension Suspension Suspension Suspension Suspension Suspension Callus Callus Callus Suspension Callus Callus Callus Suspension Callus Callus Suspension Suspension Suspension Suspension Suspension Callus Suspension Callus Callus Suspension Suspension Taniguchi et al. Skrzypczak et al. 1995.. 1988. Tabata and Hiraoka... J. 2003. Eucommia ulmoides Fumaria capreolata Gentiana sp. Ayabe et al.Vanisree Mulabagal and Hsin-Sheng Tsay Eriobotrya japonica Eucalyptus tereticornis SM. 1990. Ikuta and Itokawa... 1986. 1973. Jang et al.... Schroder and Bohm. 1987. 1998.. 1984. 2002. 1999.. 1986.. Carrier et al. R. Kitajima et al.. Wichers et al. 1987. Day et al. Phytolacca americana Picrasma quassioides Bennett Podophyllum hexandrum royle Polygala amarella Polygonum hydropiper Portulaca grandiflora Ptelea trifoliata L. Desbene et al. continued … 32 Int. Ayabe et al. Mantell et al.. 1991. Venkateswara et al. Furuya et al. 1983.. Fukui et al. 1999. Codeine β-Carboline alkaloids Betacyanin Quassin Podophyllotoxin Saponins Flavanoids Betacyanin Dihydrofuro [2. Nothapodytes foetida Ophiorrhiza pumila Panax ginseng Panax notoginseng Papaver bracteatum Papaver somniferum L. 1993. Triterpenes Callus Sterols and Phenolic com. 1985. Eng. 1986. Kitamura et al. Nakao et al.. 1999. 1993. Bassetti et al. Appl. 2. 1998. 1986. 1975. 1998. Fujita et al... 2002. Thengane et al. Yamamoto and Yamada. Furuya et al. Uden et al. Sasse et al. Chattopadhyay et al. Tanahashi and Zenk... 1990... Scragg and Allan. Zenk et al . 1998..... 1 . Wang et al. Rauwolfia sellowii Rauwolfia serpentina Benth.. 1986.. 1982.. 1990.. 1982. glandulifera Hyoscyamus niger Isoplexis isabellina Linum flavum L. Petit-Paly et al. Sakuta et al. Zhong and Zhu. 1976. Yamada and Hashimoto. 1972. Sci. Arrebola et al. Ginkgo biloba Glehnia littoralis Glycyrrhiza echinata Glycyrrhiza glabra var. Brain K. 1991.. Papaver somniferum Peganum harmala L. 1995. Lithospermum erythrorhizon Lithospermum erythrorhizon Lycium chinense Morinda citrifolia Morinda citrifolia Mucuna pruriens Mucuna pruriens Nandina domestica Nicotiana rustica Nicotiana tabacum L. Rech et al. Siah and Doran. Uden et al..Callus pounds Chlorogenic acid Isoquinoline alkaloids Secoiridoid glucosides Ginkgolide A Furanocoumarin Flavanoids Triterpenes Tropane alkaloids Anthraquinones 5-Methoxypodophyllotoxin Shikonin derivatives Shikonin derivatives Cerebroside Anthraquinones Anthraquinones L-DOPA L-DOPA Alkaloids Alkaloids Nicotine Camptothecin Camptothecin related alkaloids Saponins and Sapogenins Ginsenosides Thebaine Alkaloids Morphine . 2003. 1976. 1989.. 1981.

1983. Huang et al. Sci. 1995.. 1972.. baccatin III Taxoids Phenylpropanoid glycosides Berberin Berberin Diterpenoids Saponins Callus & suspension Callus Callus Suspension Suspension Suspension Suspension Suspension Suspension Suspension Suspension Callus Suspension Suspension Suspension Suspension Neera and Ishimaru. Cryptotanshinone Tannin Alkaloids Phenolics Spirostanol saponin Solasodine Solamargine L-DOPA Alkaloids Taxol Taxol.. Orihara et al. The tissue culture cells typically accumulate large amounts of secondary compounds only under specific conditions. Nakagawa et al. 1999). radicans Trigonella foenumgraecum 3-Oxo-rhazinilam Acridone and Furoquinoline alkaloids and coumarins Rosmarinic acid Callus Callus Gerasimenko et al. 1999. 2003 sion Morimoto et al. Lithospermic acid B and Ros.. For example. Callus & suspen... Brain and Williams.. Goleniowski and Trippi. Cusido et al. Optimization of cultural conditions: number of chemical and physical factors like media components.Callus marinic acid Suspension Miyasaka et al.. Wu et al. Until now. ginsenosides by Panax ginseng [Choi et al. 1 33 .. Eng. aeration. 1987. (ii) selecting high yielding cell clones. Culture productivity is critical to the practical application of plant cell culture technology to production of plant-specific bioactive metabolites. Franklin and Int.An Alternative and Efficient Source for Production of Biologically Secondary Metabolites Rauvolfia serpentina x Rhazya stricta Hybrid plant Ruta sp.. 1984. Fett-Neto et al.. Furuya et al.Karam et al. 2003.. 1997. 1989. 2001. 1996. 2000.. Villarreal et al. Taxus baccata Taxus cuspidata Tecoma sambucifolium Thalictrum minus Thalictrum minus Torreya nucifera var. Baumert et al.. Salvia fruticosa Salvia miltiorrhiza Salvia miltiorrhiza Sapium sebiferum Scopolia parviflora Scutellaria columnae Solanum chrysotrichum (Schldl.Plant Cell Cultures ... 1992. 1986. 1999.) Solanum laciniatum Ait Solanum paludosum Stizolobium hassjoo Tabernaemontana divaricata Taxus spp.. Stojakowska and Kisiel. Appl. agitation. Badaoui et al. Several products were found to be accumulating in cultured cells at a higher level than those in native plants through optimization of cultural conditions. (iii) precursor feeding. temperature. Sierra et al.. 1992. 1994.. That means maximization of the production and accumulation of secondary metabolites by plant tissue cultured cells requires (i) manipulating the parameters of the environment and medium.. Ketchum et al. 2004. Chandler and Dodds.. 1994. Pletsch et al. phytohormones. J. 2002. and (iv) elicitation. 1999. 1992. Manipulation of physical aspects and nutritional elements in a culture is perhaps the most fundamental approach for optimization of culture productivity. 1983. Tabata et al. 2. various strategies have been developed to improve the production of secondary metabolites using plant cell cultures. 2001. light affecting production of secondary metabolites has been extensively studied (Lee and Shuler.. 2002. 1993 Kobayashi et al. Wang et al. pH.

2 g/L of the medium and the strain was very stable. ubiquinone-10 by Nicotiana tabacum [Fontanel and Tabata. led to enhancement of volatile monoterpenes in cultures of Perilla frutiscens. 2004. which is an intermediate. 1993. Sci. thereby reducing the process time to attain high product concentrations (Barz et al. by supplying precursor or intermediate compounds. 1991].. Watanabe et al. rosmarinic acid by Coleus bluemei [Ulbrich et al. J. 1982. which grew faster and produced a higher amount of berberin and cultivated the strain in a 14 L bioreactor.. 1987. 1985. Biotic and abiotic elicitors which are classified on their origin are used to stimulate secondary metabolite formation in plant cell cultures. 1984). 1997). 1982].. Cell cloning methods provide a promising way of selecting cell lines yielding increased levels of product.. shikonin by Lithospermum erythrorhizon [Takahashi and Fujita. Eilert. 2002. Widholm. 1970]. 34 Int. amino acids have been added to cell suspension culture media for production of tropane alkaloids. were accumulated in much higher levels in cultured cells than in the intact plants. Increased capsaicin and rosmarinic acid in PEP cell lines of Capsicum annuum were reported (Salgado-Garciglia and Ochoa-Alejo.]. Addition of phenylalanine to Salvia officinalis cell suspension cultures stimulated the production of rosmarinic acid [Ellis and Towers. Moreno et al. glyphosate and biotin have also been studied to select high-yielding cell lines (Amrhein et al. 1993 and 1994]. addition of leucine.. Appl. 1988).. where as addition of geraniol to rose cell cultures led to accumulation of nerol and citronellol (Mulder-Krieger et al. i. A strain of Euphorbia milli accumulated about 7-fold the level of anthocyanins produced by the parent culture after 24 selections [Yamamoto et al. 1985].e. 1987]. Production of many valuable secondary metabolites using various elicitors were reported . 1998). Elicitation: plants produce secondary metabolites in nature as a defense mechanism against attack by pathogens. 1985). berberin by Coptis japonica [Matsubara et al. Elicitors are signals triggering the formation of secondary metabolites. 1974).. stands a good chance of increasing the yield of the final product. producing a high level of berberin even after 27 generations. For example. 1981]. indole alkaloids etc. Cell cloning using cell aggregates of Coptis japonica [Yamada and Sato. Selected cell line increased growth about 6-fold in 3 weeks and the highest amount of alkaloid was produced 1.. Selective agents such as 5-methyltryptophan..Vanisree Mulabagal and Hsin-Sheng Tsay Dixon. 1 This approach is useful when the precursors are inexpensive. 2.. Whitmer et al. Use of elicitors of plant defense mechanisms. Selection can be easily achieved if the product of interest is a pigment (Fujita et al. 1994. has been one of the most effective strategies for improving the productivity of bioactive secondary metabolites (Roberts and Shuler. The concept is based on the idea that any compound. Feeding ferulic acid to cultures of Vanilla planifolia resulted in increase in vanillin accumulation (Romagnoli and Knorr.. in or at the beginning of a secondary metabolite biosynthetic route. Furthermore.. Furuya. 1988). DiCosmo and Tallevi. elicitation. Selection of high-producing strains: plant cell cultures represent a heterogeneous population in which physiological characteristics of individual plant cells are different. 1990).. and obtained strain. have been effective in many cases (Silvestrini et al. Synthesis of several products in high amounts using selection and screening of plant cell cultures have been described by Berlin and Sasse [1985]. Addition of the same precursor resulted stimulation of taxol production in Taxus cultures [Fett-Neto et al. 1989]. 1988. Precursor feeding: exogenous supply of a biosynthetic precursor to culture medium may also increase the yield of the desired product. Eng. Attempts to induce or increase the production of plant secondary metabolites. 1988.

An Alternative and Efficient Source for Production of Biologically Secondary Metabolites (Wang and Zhong. berberine. It depends on extraction from yew trees. This has included production of taxol. L-DOPA. and it also had significant activity in the treatment of patients with malignant melanoma. 2. It has been approved for clinical treatment of ovarian and breast cancer by the FDA. 2004. which is approximately a single dose in the course of a cancer treatment. Hu et al. Lee and Shuler. Int. Taxus mairei calli were induced from needle and stem explants on B5 medium [Gamborg's et al. Case study 1: Production of taxol from Taxus mairei by cell suspension cultures As early as 1969. In 1983.. The plant cell culture of Taxus spp. 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. after precursor feeding and 6 weeks of incubation. taxol was approved to enter phase I clinical trials for ovarian cancer by the Food & Drug Administradon (FDA) in the USA. One of the cell lines. Wani and his colleagues discovered a novel anticancer diterpene amide. Different cell lines were established using stem and needle-derived callus. imperatorin from Angelica dahurica. 2002b. 2002a. 1991]. 3. and the bark is the only commercial source. 1993]. 2000). 2001. lung cancer.4-D or NAA. and the principle components such as docetaxel. Sci. produced 200 mg taxol per liter cell suspension cultures. is considered as one of the approach available to provide a stable supply of taxol and related taxane derivatives [Slichenmyer and Von Horf. Appl. we collected different tissues of Taxus mairei. 1993]. shikonin derivatives. We have successfully established cell cultures for production of taxol from Taxus mairei. Cell culture protocols for production of important secondary metabolites established in our laboratory Most applications of plant cell cultures in biotechnology are aimed at the production of bioactive secondary metabolites. morphine and codeine. Nicolaou et al.. capsaicin. diosgenin. 3. podophyllotoxin...001% of taxol by dry weight basis. gentipicroside and swertiamarin from Gentiana. A century old tree yields an average of 3 kg of bark. and 10-deacetylbaccatin in leaf extract were higher than those in bark extracts [Lee et al. 2001. vincristine and vinblastine [Dicosmo and Misawa. ajmalicine. Eng.. Because of the scarcity of the slow growing trees and the relatively low content of taxol [Cragg et al.. 1971]. "taxol" from the Pacific yew (Taxus brevifolia) extract [Wani et al. baccatin III. 1 35 . and other solid tumors [Wickremesinhe and Arteca.. cryptotanshinone from Salvia miltiorrhiza and the results were described in the present review. Since cell suspension cultures are preferred for large-scale production due to its rapid growth cycles they have been used for generating large amounts of cells for quantitative or qualitative analysis of growth responses and metabolism of novel chemicals. J. we focus on the production of some important pharmaceuticals in plant cell cultures. Taxol is considered as the prototype of a new class of cancer chemotherapeutic agents [Cragg et al. The HPLC analysis revealed that the amounts of taxol and taxol related compounds varies in individual plants. The extracts of bark and leaf tissues were analyzed by using HPLC for the content of taxol and taxol related compounds. Dong and Zhong. 1968] supplemented with 2 mg/l 2.Plant Cell Cultures . corresponding to 300 mg of taxol. The thin bark of yew tree contains 0. alternative sources are needed to meet the increasing demand for the drug. ginsenosides.. However the supply of taxol for clinical use is limited. 1993 and 1994]. 1995]. a species found in Taiwan at an altitude of about 2000m above the sea level.1. For exploiting the source of taxol. diosgenin from Dioscorea doryophora. 1994]. 1995]. 1994. In our laboratory.

(Dioscoreaceae) is frequently used as tonic in Chinese traditional medicine. Influence of auxins on imperatorin production were investigated. 1994]. Sci. especially the nutritional factors optimal for imperatorin production were also investigated. The effects of the ratio of ammonium nitrate to nitrate in the medium had also been studied. the best medium composition was 1/2 strength of MS medium [Murashige and Skoog's. 0. formosana by cell suspension cultures Angelica dahurica var. The possible stimulating effect of elicitors on imperatorin synthesis were studied. and 3% sucrose. Increasing phosphate concentration (1 mM to 2 mM ) promoted imperatorin production.. 1 fructose in terms of their effects on imperatorin production. formosana commonly known as "Bai-Zhi" in Chinese is a valuable medicinal herb used in the treatment of headache and psoriasis in China [Zhou.2. It was found that MS basal media was the best among the media tested on imperatorin production. 2. Case study 2: Formation of imperatorin from Angelica dahurica var. [Tsukamoto et al..0 mg/L 2.. A moderate ammonium nitrate to nitrate ratio (2:1) increased the amount of imperatorin production.4-D. These results showed that the cultured cells possessed the biosynthetic potential of the intact plants from which they were derived. formosana is a perennial and indigenous plant in Taiwan [Chen et al. 4-D and 0. which can be used as a precursor for many important medicinal steroids such as prednisolone. which can be used as an alternative source for the synthesis of steroids [Yeh et al.. Eng. For establishing a rapidly growing and finely dispersed cell suspension culture. Angelica dahurica var. 2004. Callus was successfully induced on MS basal medium supplemented with 2. Al the cultures were routinely sub cultured at an interval of 14 days. The purpose of the present study was to establish the cell suspension cultures of Dioscorea doryophora Hance for the production of diosgenin. dexamethasone.2 mg/L BA. Glucose was found to be bettor carbon source than sucrose and 36 Int. the production of imperatorin was maximum between the 10th and 14th day. The most active ingredient discovered in the tuber is diosgenin. The quantity of imperatorin produced by this treatment (460 μ g/gd. 1994]. Appl. The most significant effect was recorded when vanadyl sulphate at the concentration of 30 mg/L was added to the medium on the 10th day of the culture. w. 3. Cell suspen- . Tsay et al. In a growth cycle of the suspension cells. A rapid and drastic increase in imperatorin synthesis was observed by adding 20 g/L of adsorbent Amberlite XAD-7 to suspension cells on the 10th day of culture. The constituent imperatorin has been suggested as the major active ingredient for curing the skin disease [Zhou et al. J. 1980]. Growth conditions. 1994]. Dioscorea doryophora Hance. The biosynthetic potential could be further facilitated by the addition of proper stimulants to the culture medium. Deletion of auxins from the medium was beneficial to imperatorin production and addition of BA (0.) was 140 folds higher than that produced by the check treatment [Tsay. As the production of Bai-Zhi by conventional cultural method is far short to meet the demand. Case study 3: Diosgenin production from Dioscorea doryophora by cell suspension cultures Dioscorea spp.. 1999. tubers are in high demand as it is used not only as crude drug but also as food. norethisterone and metenolone etc. depending on its concentration and the growth stage of the cells.1 mg/L kinetin.5-1 mg/L) promoted synthesis of imperatorin in suspension cells.3. cell suspension culture methods for production of imperatorin was therefore emphasized.. 1962] supplemented with 1 mg/L 2. 1988]. 1936].Vanisree Mulabagal and Hsin-Sheng Tsay 3. Addition of vanadyl sulphate to cell suspension cultures enhance the accumulation of imperatorin.

An Alternative and Efficient Source for Production of Biologically Secondary Metabolites sion cultures was successfully established by subculturing callus into a liquid medium containing MS basal medium supplemented with 0. formosana (Hayata) T. the dried roots of Salvia miltiorrhiza Bunge. prevent obesity and aging. commonly known as Long-dan in Chinese is a perennial herb indigenous to Taiwan. The maximum content of the two principles. Differences in the quality and diosgenin content of the induced callus were observed among the six source organs. 1996]..5. nourishing blood and tranquilizing the mind and cooling blood to relieve carbuncles [Verpoorte. 1 37 . collected from the wild habitat. 2004.. davidii var. Regular consumption of Long-dan has been believed to enhance memory. Dan-shen.2 mg/ L 2. where as. Using this standard protocol.Ho cell suspension cultures Gentiana davidii var.2 mg/L kinetin and 3% sucrose. Case study 5: Production of cryptotanshinone from callus cultures of Salvia miltiorrhiza Bunge. As the amount of diosgenin obtained from tuber derived cell suspension is high and comparable with that found in the intact tuber [Chen. The major active principles of the organic extract of Dan-shen are tanshinones. 1996 and Zheng et al.33 μ E m-2 s-1 at 25 ± 1 ℃ . under 80-100 rev/min shaker speed. We have optimized the conditions for establishment of cell suspension cultures of G.5%). Analysis by HPLC revealed that both stem-node and microtuber derived suspension cells contained diosgenin.Plant Cell Cultures . The entire dried herb. pH between 4. which justify the use of most species of this family in traditional medicine or for the preparation of bitter tonic [Rodriguez et al. Although 6% sucrose was an optimum concentration for the growth and increasing dry weight of the cell suspension culture. 2.. 3. Bitter principles of Gentianaceae constitute many pharmacologically important compounds. davidii for the production of gentipicroside and swertiamarin.2-5. Fast growing cell suspension cultures were established by subculturing the callus in MS basal medium supplemented with 0. 1998]. 4-D. is one of the most popular Chinese medicines and widely used for promoting blood circulation to remove blood stasis. cell suspension culture could be used for production of diosgenin. Callus was initiated by culturing stem explants of G.2% diosgenin per gram dry weight. it may be possible to study the effect of precursor feeding on the content of active principles. is used as a crude drug in traditional Chinese medicine in Taiwan. Case study 4: Establishment of Gentiana davidii var. J. formosana (Hayata) T. formosa on MS basal medium supplemented with 0. N. Sci. and protect the liver by removing toxic metabolites [Lou and Chin.4.2 mg/L kinetin and 3% sucrose [Chueh et al. 900 species in the family Lamiaceae and some species of Salvia have been cultivated worldwide for use in folk medicine.3-3. Eng. 1985]. The content of microtuber derived cell suspension culture contains 3. 1997]. 3% sucrose gave better results of the diosgenin production. 2000]. the quinoid Int. Optimal cell growth was obtained when callus cultured in 25 mL liquid MS basal medium supplemented with 0. clearing away heat. Secoiridoid glycosides are the main compounds with medicinal properties in roots of Gentiana species [Skrzypczak et al.3%.2 mg/L kinetin and 1. 3. swertiamarin and gentipicroside.2. Ho (Gentianaceae).. N.0 mg/L α -naphthaleneacetic acid (NAA). Salvia is an important genus consisting of ca. the stem-node derived cultures contains only 0. Callus from microtuber showed yellowish color and granular in appearance and contained the highest diosgenin content (3. relieving vexation. Sucrose concentration optimal for diosgenin production was investigated. 1993]. the two pharmacologically important compounds. Appl. in cell suspension were obtained after 12 and 24 days of culture respectively.. incubated in light 2.

2003]. [ 3] Anderson. The increased use of genetic tools and an emerging picture of the structure and regulation of pathways for secondary metabolism will provide the basis for the production of commercially acceptable levels of product. A glyphosate-tolerant plant tissue cultures. J. Primary callus was induced by culturing leaf explants on Murashige and Skoog's (MS) basal medium supplemented with 1. Roberts. Studies on Ailan- . miltiorrhiza). References [ 1] Amrhein. Miyasaka et al.4-D and 0. The increased appeal of natural products for medicinal purposes coupled with the low product yields and supply concerns of plant harvesation has renewed interest in large-scale plant cell culture technology. 1989 and Shimomura et al. In: Neumann.05 mg/g dry wt. These results shows that plant cell culture systems have potential for commercial exploitation of secondary metabolites.. is also likely to be a significant step towards making cell cultures more generally applicable to the commercial production of secondary metabolites. Johanning. Berlin: Springer-Verlag: 355-361.4-D) in darkness. 0. Sci. F. Barz.0 mg/L of BA was significantly higher than the marketed crude drug (processed underground parts of S. Hay.4-dichlorophenoxyacetic acid (2.4-D from the medium resulted in a marked increase in the content of cryptotanshinone in callus.0 mg/L of 2. C. D. Because of the complex and incompletely understood nature of plant cells in in vitro cultures.1.2 mg/L BA for sixty days [Wu et al. C. Studies on Ailanthus altissima cell suspension cultures. E. strategies are needed to develop an information based on a cellular and molecular level.5. The callus proliferated further on MS basal medium containing 1. 2004. 1. M. Maximum yield of cryptotanshinone (4.5 mg/L BA and was analyzed for cryptotanshinone by high performance liquid chromatography (HPLC). “Primary and Secondary Metabolism of Plant Cell Cultures”. and 2. and consequently. C. Plant Cell Reports. so as to produce transgenic cultures and to effect the expression and regulation of biosynthetic pathways. 2.0 mg/L 2... [ 2] Anderson.. Since Dan-shen preparations constitute a basis for considerable commercial activity.Vanisree Mulabagal and Hsin-Sheng Tsay diterpenes [Bruneton. Roberts. editors. The HPLC results indicated that it contained small amounts of cryptotanshinone (0. In this experiment effect of N 6.. K. 1995]. H. Omission of 2. A..2. and Phill ipson.. W..26 ± 0. In our continuing study of the application of tissue culture of medicinal plants to the production of bioactive secondary metabolites. 1985. D... L. 1991]. A. 0. A. Knowledge of biosynthetic pathways of desired compounds in plants as well as in cultures is often still in its infancy. The HPLC analysis revealed that the content of cryptotanshinone in the callus cultured on the MS basal medium supplemented with 0. J.09 mg/g dry wt. 1993. Reinhard.0. J. and Phillipson. Appl. Conclusions The use of plant cell culture for the production of chemicals and pharmaceuticals has made great strides building on advances in 38 Int.. miltiorrhiza through callus cultures.) was observed in the callus cultured on MS basal medium supplemented with 0. there is a continued interest in development of biotechnology-based approaches to the production of tanshinones [Hu and Alfermann.benzyladenine (BA) on cryptotanshinone formation in callus cultures of Salvia miltiorrhiza was examined.. L.59 ± 0.. and Smart. Eng. 5: 387-390. we have adopted an approach to the production of cryptotanshinone from S. N. 4.). F. The introduction of the techniques of molecular biology.. M. case-by-case studies have been used to explain the problems occurring in the production of secondary metabolites from cultured plant cells. D. 1987. 1 plant science. 1986.

Baumert. A. M. C. In: Bajaj. “Plant Cell Biotechnol- [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] ogy”. Balandrin. and Soehono. In: Pais. G. 1999..... F. Journal of Biotechnology. Barz. Elicitation and metabolism of phytoalexins in plant cell cultures. J. Appl. D. Sci. Novais. Takano.. M.. K. Brain.. K.. and Henry. J. J. 1976. S. T. S. Medicinal. L. Ringbom. T. 1990. Phytochemistry... and Furuya. J. M. Surfactant-induced non-lethal release of anthraquinones from suspension cultures of Morinda citrifolia. Mancini.. H. R. 1986. H. Bhojwani. 9: 181-184. 522. L. Medicinal and Aromatic plant”. B. A. NATO ASI Series. Ayabe. Arrebola.. S. M.. 1983. Journal of Bioscience and Int. H.. S. and Williams. V. W.. S. Groger. “Biotechnology in Agriculture and Forestry.. P. Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells. D. H. 10: 256-259. 1 39 . magnesium. Buitelaar. Bruneton. Indrayanto. 2: 69-72.. Chattopadhyay.. Y. 28: 159-162. Coulombe. 1992.. Plant Cell Tissue and Organ Culture. and Bisaria. Plant Cell Reports. 1988.. Hinderer. Medicinal plants. Fujita. Ayabe. S. M. Kessmann... K.An Alternative and Efficient Source for Production of Biologically Secondary Metabolites [ 4] [ 5] [ 6] [ 7] [ 8] [ 9] [10] [11] thus altissima cell suspension cultures. 1983. G. M.. Secondary metabolites produced by callus cultures of various Ruta species. V.. Accumulation of L-DOPA in cultures from Mucuna pruriens. S. and Johannes. and Reisch. U. 1992. T. 1996. and Mansell. and Dodds. A. 2002. Jaques.. J. Daniel. D. 31: 99-132. Heidelberg: 1-36. Production of solamargine by in vitro cultures of Solanum paludosum. 4. Jourdan. T. glandulifera. Weber. Triterpenoid biosynthesis in tissue cultures of Glycyrrhiza glabra var. 1988.Plant Cell Cultures . and Takahashi. 39: 149-155. and Verpoorte. 55: 103-108. Muguet. 2: 7-10. Naringin and limonin production in callus cultures and regenerated shoots from Citrus sp. and Archambault. 6: 239-241. Berlin: Springer-Verlag: 211-230. 3: 186-189. 1995. Evidence for an alternative route from sterol to sapogenin in suspension cultures from Trigonella foenumgraecum. 127: 55-65. R.. J. Detection of ginkgolide A in Ginkgo biloba cell cultures. Springer-Verlag. N.. McIntosh.. and Tiemann.. Badaoui. Plant Cell Reports. Intercept UK. and Klocke. 2. Neufeld. Plant Cell Reports. J.. Hagendoorn. Chandler. S. A. Bassetti. P. Kuzovkina. Selection and screening techniques for plant cell cultures. 52: 1283-1286. 1995. P. 23: 111-143. Eng. 45: 123-127. 1999. Anthraquinones from Isoplexis isabelliana cell suspension cultures. Berlin. R. E1. K.. 1985.. K....L. T. S. J. and Tramper.. Mavituna. S. L. M. R. Berlin. Iida. R. Plant Cell Tissue and Organ Culture.. Carrier. Chauret. Solasodine production in rapidly proliferating tissue cultures of Solanum laciniatum Ait. Koster. Pharmacognosy. Plant Cell Reports. Srivastava. Hirota. Plant Cell Reports.. S. N. F. Andrijany. M. The effect of basal media on growth and alkaloid production. J. editors. 2004. A. Strategies to improve the production of secondary metabolites with plant cell cultures: a literature review. M. Plant Cell Reports. J. aromatic and industrial materials from plants. Phytochemistry. Journal of Plant Physiology. H. 1991. Journal of Biotechnology.. copper and cobalt on sapogenin steroids content in callus cultures of Agave amaniensis.. and Sasse. 7: 157-161. 1987. Plant Science Letters. Barthe. Simultaneous effect of calcium. Brain. Plant Cell Tissue and Organ Culture. Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor. Advanced Biochemistry and Engeneering. J. I. W. editor. R.

. and Grever. Sagare. 3: 425-453. Dicosmo. S. Biochemical engineering Journal. T. Appl. A. Day. Science Monthly. 13 . S. Mallol. 1993. Chang. I.. G.T. Chueh. editors. 146: 101-107. Ellis. 43: 79-85. J. and Tallevi. Plant natural products: the molecular genetic basis of biosynthetic diversity. H. Significant improvement of taxane production in suspension cultures of Taxus chinensis by combining elicitation with sucrose feed. J. Palazon.. 3: 110-111. M. K. E... 1985. 1985. 62: 923-926. H. S. M. and Pinol. A... 1999.. J. Ahn. H. 1995. C.. J. N. New NCI policies for handling the large-scale production of novel natural product anticancer and anti-HIV agents.. Russian Journal of Plant Physiology. Plant regeneration and thebaine content of plants derived from callus culture of Papaver bracteatum. Sakata. In: Constabel. F. Analysis of diosgenin constituent of plants from Taiwan. J. Chang.. H. formosana II.. Morales. Y. M. and Towers. Trends in Biotechnology. 2. Plant Science. 8: 145-150. G. Dong. Melanson.... Journal of Chinese Medicine. Eilert. C. Effects of macronutrients on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis. F. 5: 123-134. Mayer). Dixon. J.. Journal of Natural Products. Dornenburg. 1994. C. Journal of Biotechnology. 5: 471-474. Dicosmo. 10: 192-197. B. Biotechnology.Vanisree Mulabagal and Hsin-Sheng Tsay [22] [23] [24] [25] [26] [27] [28] [29] Bioengineering. M. J. Study on application of diosgenin-I. formosana by high performance liquid chromatography. Biogenesis of rosamrinic acid in Mentha. O. Desbene. R. M. and Tsay. De-Eknamkul. Shoyama. 2004. Suffuess. Journal of Biochemistry. F. and Knorr. H. S. W. Production of ginseng saponin in tissue culture of ginseng (Panax ginseng C.. Navia-Osorio. “Cell Culture and Somatic Cell Genetics of Plants”. U. Schepartz. G. G. Biotechnology Advances. C.. Draper. Bonfill. C. J.. Chen. 1985. and Tsay. F. 56: 1657-1668. Establishment of cell suspension culture and evaluation of cultural conditions. Y. 1999. The taxol supply crisis.. B. 50: 55-62. and Lacaille-Dubois.. and Park.. 1987. A. K. and DiCosmo. 1970. Fett-Neto. and Zhong. S. D. Eng. Production of taxol and baccatin III by a selected Taxus baccata callus line and its derived cell suspension culture. A. Int. 1999. D.2: 215-220. 1995. 41: 784-788. Studies on the tissue culture of Angelica dahurica var. Biologically active triterpene saponins from callus tissue of Polygala amarella. Journal of Natural Products. Plant Cell Reports. 1996. S. A. 67: 70-73. G. M. 1 [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] Plant Cell Reports. Chen. A. F. Elicitation: methodology and aspects of application. I. Plant cell cultures and microbial insult: interactions with biotechnological potential.. 118: 291-297.. Vasil. 4: 46-49. Quantitative determination of secoiridoid glucoside in in vitro propagated plants of Gentiana davidii var.. San Diego: Academic Press: 153-196. Plant cell and tissue culture: Alternatives for metabolite production.. H. and Smith. B. B. and DiCosmo. M. Sci. Fett-Neto A. 11: 731-734.. S. H. D. H. Wagner. E.. Hanquet.. J. Cragg.. 2000. 1993. S. 4. 93 . Cusido R. and Ellis. Current Opinion in Biotechnology. Effect of white light 40 . 1986. A. F. T. M. Planta Medica. Pennington.A. and Misawa. Chen. R. Semicontinuous processes for anthraquinone production with immobilized Cruciata glabra cell cultures in a three-phase system. P. C. K. Choi. Improved growth and taxol yield in developing calli of Taxus cuspidata by medium composition modification.. 1994.

K. and Obi.. B.. 1990. 1981. K. R. C. Sci. R.. Initiation and maintenance of callus and cell suspension cultures. Fujita. and Oda. A.. H. K... Matsumoto. Kim. 1988. [52] Gerasimenko. and Staba... In: Vasil. grown in liquid media. and Suga. Kojima. and Tabata. 2nd edition. Hara. S. Liao. III. I. J.. R. Murthi.. 1: 161-166. A. Oxford: 1-25. A. GenInt. cuspidata. Nicholson. Uotani. A. R J. J.. Takahashi. 1 41 . 56: 215-218 [54] Hagimori. Nadizadeh. Sheludko. C. Ikuta. Shindo. K. Chemical & Pharmaceutical Bulletin. Y. 47: 70-75. T.. Y. J.. F. M. S.. S. 1994. and Trippi. P. Y. K.. Selection of cell lines with high productivity of shikonin derivatives through protoplasts of Lithospermum erithrorhizon. A. 1: 59-60. Syono.. 44: 967-971. Zhang. Plant Cell Reports.. H. A... Stewart. Biediger. Somoza.. Eng. Y. J. 2001. 3-Oxo-rhazinilam: A new indole alkaloid from Rauwolfia serpentina x Rhazya stricta hybrid plant cell cultures. Effects of nutrients on digitoxin formation by shoot-forming cultures of Digitalis purpurea L. 5. Fontanel. T. [50] Furuya.. P. M. Alkaloids from callus cultures of Papaver somniferum. In: Dixon. Tani. 146: 584-590. 64: 114-116. Biotechnology Bioengineering. 7: 1205-1211... 1973. Studies on the production of Digitalis cardenolides by plant tissue culture. T. Tao. R. R. Pennington. Induction of shikonin biosynthesis by endogenous polysaccharides in Lithospermum erythrorhizon cell suspension cultures. and Stockigt. Fujita. E.. 2004. V. P. 1982.. 1968. 380: 825-839. O. and DiCosmo. H. M. Accumulation in cell cultures of Taxus cuspidata Sieb and Zucc. “Cell Cultures and Somatic Cell Genetics of Plants”. Hoffmann. Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. Saponins (ginseng saponins). and Yamada.. and Emans. 1994. IRL Press.. [48] Furuya.. y. 1980. and Tabata. C... Kim. Y. T.. Tang... 2. Fett-Neto. A. D. Isolation of saponin and sapogenins from callus tissue of Panax ginseng. [55] Heble. L. (Ed.. Journal of Plant Physiology. Fukui. and Dixon. 1987.. M. Fischer. Plant Cell Tissue and Organ Culture. 23... and Syono. S. Biological Chemistry. T. (eds. and. M. Ishi. R. and Ojima. Journal of Natural. “Plant Cell Culture-A Practical Approach”. Smith. Nishio. M..). Products. A. Experimental Cell Ressearch.. M. 21: 98-101. Academic Press. Vu. N. Ogino.An Alternative and Efficient Source for Production of Biologically Secondary Metabolites [40] [41] [42] [43] [44] [45] [46] [47] on taxol and baccatin III... C. Steroid metabolism in stationary phase cell suspensions of Dioscorea deltoidea.. Suzuki. T. J. Orihara. Liang. Proceedings of European congress Biotechnology. Schillberg. Y. 1984. [53] Goleniowski. Appl. Studies of the culture conditions for Panax ginseng cells in jar fermentors. Boatman. K. Molecular farming of recombinant antibodies in plants. CA: 213-234.. S. Gonzales.. Plant Cell Physiology. 11: 3041-3044.K. [56] Holton. 1984. Nestle Research News: 92-103.. [51] Gamborg. H. Phytochemistry. Miller. G.. F. Journal of Natural Products. Nutrient requirements of suspension cultures of soyabean root cells.. H. I.. A. Planta Medica Supplement: 124-128. 1972. M. T. 9: 73-76.. [49] Furuya. C. M. I. T. Improved taxol yield by aromatic carboxylic acid and and amino acid feeding to cell cultures of T.Plant Cell Cultures . C. Yoshikawa. Furuya. K. S. R. 50: 151158. Production of secondary metabolites from plant tissue and cell cultures. Plant Cell Reports.). C. Y. 1999. Effect of growth medium composition on psilostachyinolides and altamisine production. Y. 1999. San Diego. Franklin.

.. Plant Cell Reports. G.. Ueno.. Appl. Improvement of Panax notoginseng cell cultures for production of ginseng saponin and polysaccharide by high-density cultivation of pneumatically agitated bioreactors.. Williams. 2002. Stockigt. J.. Induction of furanocoumarin biosynthesis in Glehnia littoralis cell suspension cultures by elicitor treatment. 1 [66] [67] [68] [69] [70] [71] [72] [73] saicin production by immobilized cells and placental tissues of Capsicum annuum L. Ooe. 7: 21432145. 32. H. H. N.. Callus induction from different source organs and the measurement of diosgenin content. In vitro capInt. H. 5: 1193-1197. 2003. N. M. 1993. B. Polyphenol production in cell cultures of Cornus kousa.. C. 1987.. 1988. 2: 151-160. U. A.. Alkaloids of tissue cultures of Nandina domestica. and Groger. H. 1.. 1982. Ikenaga. M. Koblitz. H. Hu. Diterpenoid production in hairy root cultures of Salvia miltiorrhiza. R. S. A. 1993. H. Phytochemistry. Kitajima. S. Ohsawa. 5: 423-426. and Aimi. 3: 611-614. M. and Takao. Kueh. V. 6: 779-791.. U. Production of chry- 42 .. 1: 107-111. W. 18: 252-254. 62 (6): 901-909. J. Fukui.. M. Biotechnology Progress.W. Taxus metabolites: methyl jasmonates preferentially induces production of taxoids oxygenated at C-13 in Taxus media cell cultures. H.. 1990. 1993. 1998. 1983. Phytochemistry. Production of a hepatoprotective cerebroside from suspension cultures of Lycium chinense. Schmauder. J. H.. J. T. and Chan. D. T. D. S. W.. E. M. Plant Cell Tissue and Organ Culture. W. 1998. Ikuta. Phytochemistry. Eng.. 116: 1597-1600. M. Takayama. Jang. 2. 17: 838-846. Kim... 21. H.. C. Jawad.. Plant Cell Reports. B.. Effects of nutritional factors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra.. Arakawa. K. M. Koblitz. and Rideau. Hu. W. 32: 699-703. and Alfermann. and Itokawa. First total synthesis of taxol... Yeh. Y. P. A. and Liu. S. J. grown in liquid medium. Functionalization of the B ring. Chinese Medicinal College Journal. 2. F. K. N.. 1994. Studies on tissue cultures of the genus Cinchona L.Vanisree Mulabagal and Hsin-Sheng Tsay [57] [58] [59] [60] [61] [62] [63] [64] [65] tile. 70: 223-229. B. A.. 1: 113-117. Phytochemistry. and Huh. and Mizukami. L. H... H. Y. Plant Cell Reports. R. Kitamura.. J. and Zhong. J. Ketchum. 1985. Formation of alkaloids in Corydalis ophiocarpa callus cultures. M. and Pattenden. Anthraquinone from Ophiorrhiza pumila tissue and cell cultures. and root cultures of wild Salvia fruticosa.. Y. MacKenzie. Phytochemistry. growth and rosmarinic acid accumulation in callus. H. K.. P. Sci. C. Hiraoka. Yao. Kim. S. Rithner. Ishimaru.. 30. R. S. 1998. Qiu. F. Shen.. Arikat. 27. Y. M. 1986. N. T. 2004.. Ravishankar. W. Johnson. Nakamura. Huang. alkaloid production in cell suspension cultures. An immobilized cell culture system for berberine production by Thalictrum minus cells. Huang. Fischer. Plant Science.. 2001. 48. and Shibli. Enzyme and Mmicrobial Technology. H. Journal of American Chemical Society. D. 2: 122-125.. R. and Tabata. S. Unger. Development of bioreactor operation strategy for L-DOPA production using Stizolobium hassjoo suspension culture. 73: 117-121. Cheng. N. Karam. Tissue culture of Dioscorea doryophora HANCE 1. S. 2003.. Iwasa. cell suspension. G.. Phytochemistry. Z... Y. R. D. Lee. and Venkataraman. Y. 6: 185-186. 48. and Neera. H. Plant Cell Reports. Phytochemistry. N. and Tsay. Y. Kobayashi. and Croteau. C.. H. Y. Ibrahim. E. L. A. I. Khouri..

... and Tabata. K.S. M. Beijing Medical University and Peking Union Medical College Press. Miyasaka. 1999. J. Plant Cell Reports. C. R. T. N. Yoshioka.. 1986. Verpoorte. Liu. R.430. J. Beijing.. and Tsay.. Journal of Natural Products. [82] [83] [84] [85] [86] [87] [88] [89] [90] 1989. Neera. S. 1986. S. Roberts. F. April 21.C. Nakagawa. Yang. Morimoto. P. T. 8: 210-213. Journal of Chemical Technology and Biotechnology. 31. L. H. C. Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus. 1993. In “Species Systematization and Quality Evaluation of Commonly Used Chinese Traditional Drugs”.. Yang. Shigekazu. Plant Cell Reports. Z. and Ishimaru. T. Wang. Flavor formation in tissue cultures of garlic (Allium sativum L. 9: 261-263. 1992. Medicinal and Aromatic Plants II ed. 4: 118-119. B. L. Nasu. Lin. SpringerVerlag Berlin. S. H. L. A review. 3: 833-836. Z. 18: 759-763.. K. 57. Moreno. L. Biotechnology Bioengineering. M. 1996. D. H... P. Plant Cell Reports... Production of canthin-6-one alkaloids by cell suspension cultures of Brucea javanica (L. and Shuler. 1962. Svendse. and Yamada. Sci. H.Plant Cell Cultures . Lou. 5: 446-447. The effect of gelling agents on plaunotol accumulation in callus cultures of Croton sublyratus Kurz. C. R. Murashige. S. and Verpoorte.) Merr. Yang. Appl. The effect of inoculum density and conditioned medium on the production of ajmalcine and catharanthine from immobilized Catharanthus roseus cells. Plant Cell Reports. Liu. K. 1988. Phytochemistry. 15: 473-497. Lee. and Smith. 2: 73-33. S. D. J. 5: 69-71. K. and David. van der Heijden. The effect of initial phosphate and sucrose levels on nicotine accumulation in batch suspension cultures of Nicotiana tabacum L.P.. Y. Heidelberg: 417.. Ono. Fujita. K. Goto. Nakao. Morimoto. Plant Cell Reports. Mulder-Krieger. 12: 702-705.. 1995. on development and utilization of resources of medicinal plants in Taiwan. Y. and Chin. Tannin production in cell cultures of Sapium sebiferum.. L. Production of lithospermic acid B and rosmarinic acid in callus tissue and regenerated plantlets of Salvia miltiorrhiza. Taiwan. 2. W. K.. By Bajaj.. S.. Lee. and Murai. P. K. Mantell. M. 6: 817-823. The effect of calcium on flavanol production in cell suspension cultures of Polygonum hydropiper. Fukui. Salvia miltiorrhiza: In vitro production of cryptotanshinone and feruginol. In “Biotechnology in agriculture and forestry”. F. Y.. Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus. 7. China. 2004.. Taiwan Agricultural Research Institute. R. H. Ueno. Pearson.. M. Eng. and Skoog. A revised medium for rapid growth and bioassays with tobacco tissue cultures. J. H.. Malpathak. North-China edition. Production of essential oils and flavours in plant cell and tissue cultures. 3: 437-504. and Takio. Physiologia Plantarum. F. S. J. P. H. 1983. Nicolaou. 46: 61-69. C. Matsubara. TARI Special Publication 48: 137-148. Y. H. F. Y..An Alternative and Efficient Source for Production of Biologically Secondary Metabolites [74] [75] [76] [77] [78] [79] [80] [81] santhemic acid and pyrethrins by tissue cultures of Chrysanthemum cinerariaefolium. Plant Cell Reports. Wei. Plant Cell Reports. 1 43 . 1995. A. S. 1994. Hiegh density culture of Coptis japonica cells increases berberine production. H. and Phillipson.. K. and Shoyama. 1989. H. 1989. Longdan. Taxol production by cell cultures of Taxus mairei. T.. Plant Cell Reports. T.). Y.. 67: 61-71. Plant Cell Tissue and Organ Culture. 13: 85-114. K.. 2000. 1990. W. Int. and Scheffer.. Hazell. Proceeding of Symposium... and Yoneda. C..

V. 1993. 60. Rech. T. Current opinion in Biotechnology... Schripsema. M.. C. and Yoshikawa. Yang. Nazif.. Montagu. P. 12: 705707. 2. C.. V. Petit-Paly. and Shuler. N. 8: 617-620. J. Formation of L-canavanine in in vitro cultures of Canavalia ensiformis (L) DC. Y.3-b] quinolinium alkaloids in cultured cells of Ptelea trifolia L.. D.. Callus production. Plant Cell Tissue and Organ Culture... Large scale plant cell culture... G. Pletsch. J. G. M... Dihydrofuro [2. E. 34 (1): 161-165. G. J.. T. Wolfender. Physiologia Plantarum. N. J. B. Rajendran. a new biosidic ester secoiridoid from Halemia corniculata. Phytochemistry.. 1987. radicans. U.. Romagnoli. N. M. Viel.. Abietane diterpenoid from suspension cultured cells of Torreya nucifera var. 2002.. Verpoorte.. K.. F. Ravishankar. 2004. Y. Paulvannan. Eng. A.. and Int. M. 9: 65-68. 1998. 1993.. 4: 73-102. Appl. Rodriguez. Plant Cell Tissue and Organ Culture. 2002. S. G. L. C. M. 1990. 8: 154-159. 71: 34-40.. M. Food Biotechnology. Influence of nutrient stress on pyrethrin production by cultured cells of pyrethrum (Chrysanthemum cinerariaefolium). Biotechnology Advances. S. Odontuya. A. Salgado. 54: 61-63. 2: 93-104. Orihara. Plant Cell Reports. 1994. 1 [100] [101] [102] [103] [104] [105] [106] [107] [108] Oropeza. Garciglia. Quiroz. Ramachandra Rao. 1990. 2000. G. 79: 363-370.. Increased capsaicin content in PFP resistant cells of chilli pepper (Capsicum annuum). A. 1996. 34: 149-155. L. S. 367: 630-634. 20: 101-153. Plant Cell Reports.. R. O. Pizza. Orihara.. O' Dowd. L. and Ochoa-Alejo.. Plant Cell Tissue and Organ Culture. Cell cultures of Rauwolfia sellowii: growth and alkaloid production. Effects of nitrogen source on betacyanin accumulation and growth in suspension cultures of Phytolaca americana. A. G. suspension culture and in vitro alkaloid yields of Ephedra. Effects of ferulic acid treatment on growth and flavour development of cultured Vanilla planifolia cells.. 1987.. Takagi. and Sorensen. Plant cell cultures: Chemical factories of secondary metabolites.. Current Science. Plant Cell Reports. Sakuta. New Delhi: Oxford IBH Press: 255-274. Fitoterapia. C. The accumulation of phenylpropanoid glycosides in tissue cultures of Tecoma sambucifolium. Corniculoside. Couladouros. Helvtica Chimica Acta. G. and Charlwood. T. 1993. Phytochemistry. J. E. 59: 385-389. F. B. L. and Chenieux. Total synthesis of taxol. S.. A. and Knorr. McCauley. Ramirez..F. and Seif E1-Nasr.. Hostettmann. Rideau. S. K. M.. V.. 30: 231-235. K. Heckenberg. and Ravishankar. L. 6: 309-312. Role of plant cell cultures in food biotechnology: commercial prospectus and problems. Roberts. 1988. L. and Henriques. J. T. R. N. and Purev. Koge. T. 1997. M.A. W.. 2000. Stimulation of anthraquinone production in suspension cultures of Cassia acutifolia by salt stress. J. Rajasekaran. Production of theanine and other γ-glutamyl derivatives by Camellia sinensis cultured cells. Ravishankar.. S. P. D.. 71: 459-463. S. Journal of Biochemistry Molecular Biology Biophysics. V. C..Vanisree Mulabagal and Hsin-Sheng Tsay [91] [92] [93] [94] [95] [96] [97] [98] [99] 44 H. Alpizar. G. and Berlin. Piacente. C. Sci. Guy. and Wilson. Ramachandra Rao. 1991.. G. Batista. Biotechnological production of phyto-pharmaceuticals. J. Rady. K. R. Claiborne. and Venkataraman. Renaud.. and Venkataraman. L. M. Accumulation of β-carboline alkaloids and serotonin by cell cultures of . Nantermet. Komiya. Richardson. and Furuya. H. M.. 1992.. Sasse. Nature. and Komamine. R. J. 1982.

1 45 ... Kitazawa. M.. Kulkarni.. 1981. Taniguchi. I. 11: 949-955. Shimura. and Hiraoka. Takamatsu.. 10: 349-353. T. C.. T. 69: 400-404. Tabata. and Bohm. Y. Plant Cell Reports.. C. H. Silvestrini. R. 1999. 2004. R. and Verpoorte. 70: 324-325..An Alternative and Efficient Source for Production of Biologically Secondary Metabolites [109] [110] [111] [112] [113] [114] [115] [116] [117] Peganum harmala L. M.. V.. 1991. N. Ramos Valdivia. 2. regeneration. Tokuda. Eng.. 1991. M. 3: 14-17. M. 1991.. Gentiana species XII: In vitro culture. B. Sejourne. 59: 315-323. Tanahashi. Thengane. Schripsema. M. Phytochemistry. Viel. K. B. 40: 749-753.. 51: 55-60. Effect of alkaloid precursor feeding on a Camptotheca acuminata cell line. Sugita. Phytochemistry. H. 1984. 1976. 2003.. and Doran... Sierra. . M. Plant Physiology and Biochemistry. editors. S. 1992. 4: 96-99. Monacelli. 2: 353-355. and Dicosmo.. Bruneton. Production of bioactive triterpenes by Eriobotrya japonica calli. 5: 356-359.. N. Misawa. Betacyanin concentrations in young cell cultures from Portulaca grandiflora an analysis of variation. Medicinal and aromatic plants IV. Isoquinoline alkaloids from cell suspension cultures of Fumaria capreolata. Ito. In: Bajaj. L. M. C.. J. 2002. P. .P. Komamine. and Fjita. 21. A. Tabata. Cosmetic materials: 72-78. In: “Plant Cell Culture in Japan”. J. Imayoshi. A. Tanshinone production in adventitious roots and regenerates of Salvia miltiorrhiza. A. K. Plant Cell Tissue and Organ Culture. Botta. Plant Physiology.. A. and Kisiel.. R. P. and Von Horf. Physiology Plantarum 38: 19-23. L. editor. 2: 519-530. 1999.. Stability of alkaloid production in cell suspension cultures of Tabernaemontana divaricata during longterm subculture. D. R. S. Influence of medium composition on callus induction and camptothecin(s) accumulation in Nothapodytes foetida. Nishino. 54: 1583. and Chenieux. A.. Tsay. Phytochemistry. and Skrzypczak. van der Heijden. T. J.. V. Fitoterapia.. Plant Cell Reports. W. Berlin. N.. Pasqua. H. and Verpoorte. 1985. 72: 247-251. M. D.H. Y. Growth and furoquinoline alkaloid production in cultured cells of Choisya ternata. M. 28: 59-68. Anti-Cancer Drugs. Journal of Natural Products. Plant Cell Reports.. J. and Verpoorte. and Konoshima. Plant Cell Tissue and Organ Culture. 20. Robustaquinones. and Krishnamurthy. Phytochemistry. Organization and alkaloid production in tissue cultures of Scopolia parviflora. K. 1993. J.. Y.. Tissue culture technology of medicinal herbs and its appliInt. S. T. A. Variation of alkaloid production in Nicotiana rustica callus cultures. Schroder.. Siah.. J. T. Heijden. Rideau. E. Sci. G. Skrzypczak. Leer.. Sakagami. Appl.. and Yoshida. A. B. Heidelberg: Springer-Verlag: 172-186. Enhanced codeine and morphine production in suspended Papaver somniferum cultures after removal of exogenous hormones. and Zenk. “Biotechnology in Agriculture and Forestry”.. Wesolowska. Yamamoto. R. and [118] [119] [120] [121] [122] [123] [124] [125] [126] production of secoirridoid glucosides. W. H. and Allan.. Scragg. Shrikhande. H. D.Plant Cell Cultures . Joshi. D. W. Hiraoka. Kobayashi. 1972. D. Y... Shimomura.S. Plant Cell Reports. Okamura. Takahashi. Stojakowska. H. Sonawane. F. Hatano. S.. Slichenmyer. M. R. H. E. 2002. K. 1999. 1991. Production of triterpenoid quassin and cell suspension cultures of Picrasana quassioides Bennett. E. Secondary metabolites from a callus culture of Scutellaria columnae. 1986. and Yagi.. H. novel anthraquinones from an elicited Cinchona robusta suspension culture. S. Taxol: a new and effective anticancer drug.

Process Biochemistry. Yeno. R. and Malingre. Plant antitumor agents VI. P. Q. F.. a novel antileukemic and antitumor agent from Taxus 46 . Detection and identification of Podophyllotoxin produced by cell cultures derived from Podophyllum hexandrum royle. K. 1936.. E. C. W. 12: 93-97. Tokyo: 71-72. 168: 32-48. Sankara Rao. 1987. J. Journal of Biosciences Bioengineering.. H. Yu. Uden. T. Biochemical Engineering Journal.. Plant Cell Reports. (ed. E.. T. Tsay. 20: 81-87. Y.. W. Drug Discovery Today. J. Ramirez. S. Academia Sinica. 3: 232-238.. Exploration of nature’s chemodiversity: the role of seconInt. Sankara Rao. J. 1990. Villarreal. H. Venkateswara. D. N. Arias. Reinhardt (eds). Wani. 1986. The isolation and structure of taxol. dioscoreasapooxin and diosgenin.Vanisree Mulabagal and Hsin-Sheng Tsay [127] [128] [129] [130] [131] [132] [133] [134] cation of medicinal herbs and its application in Taiwan... M. N. Wall.. W. 6: 291-293. Cell suspension culture of Solanum chrysotrichum (Schldl. SpringerVerlag (Berlin): 293-303. Appl. In primary and secondary metabolism of plant cell cultures. Y. Y... 1985. Biodiversity and allelopathy: from organisms to ecosystems in The Pacific. G. B. and Vaidyanathan. 50: 39-44.. Feria-Velasco. R.. C. Ulbrich. L. Production of 5 methoxypodophyllotoxin in cell suspension cultures of Linum flavum L. Vossebeld. and Quintero. 1999. J.) Plant Tissue Culture. Zhang. J. 1983. editors). Glycosides of Dioscorea tokoro I.. 8: 165-168.. R. 35: 479-483. J.R. Z. C. M. Wang. 3: 231-233. Z. and Suzuki. Taipei. 93: 154-159. and McPhail.. and Chen. 1994.H. Mol.. C. 2002a. Wang. Wang. M.. Venkateswara. S. P. 1982. The production of imperatorin from Angelica dahurica var. P. S. Plant Cell Tissue and Organ Culture. J. Production of saikosaponins by callus and redifferentiated organs of Bupleurum falatum L. and Reinhard. High production of caffeine and related enzyme activities in callus cultures of Coffea arabica L. M. and Malingre. J.A plant producing an antifungal spirostanol saponin. T. Combination of conditioned medium and elicitation enhances taxoid production in bioreactor cultures of Taxus chinenesis cells. W. In: Chou... M. J. Du. L.. and Huang. 1998. Chang. 56: 135. Accumulation of chlorogenic acid in cell suspension cultures of Eucommia ulmoides. C. C. MacVean. T. H. Plant Cell Tissue and Organ Culture.. T. J. Uden.. G. Pras. formosana by cell suspesion culture.. Plant Cell Tissue and Organ Culture. R. Plant Cell Reports.. S. 2004. Journal of Agricultural Association China. Journal of Pharmaceutical Society Japan. J. 1 [135] [136] [137] [138] [139] [140] [141] [142] dary metabolites as leads in drug development. Diocin. O. Manipulation of ginsenoside heterogeneity in cell cultures of Panax notoginseng by addition of jasmonates. and Arens. 2: 109-112 Wang. I.. N. C. Chen. H.. M. H. T. Plant Cell Reports.. T. 2002b. (Neumann. C. Weisner. In: Fujiwara A.. Cryptosin-a new cardenolide in tissue culture and intact plants of Cryptolepis buchanani Roem. and Ohta. Phytochemical constituents of cultured cells of Eucalyptus tereticornis SM.. A. and Zhong. Pras. 1989. S. 1997. R. Taylor. Maruzen.Waller. X.. N. 1971. Significant improvement of taxane production in suspension cultures of Taxus chinensis by sucrose feeding strategy. & Schult. D. and Zhong. Verpoorte. W. and Vaidyanathan. Taiwan: 137-144. J. A. Liao. M. C. 74: 193-195. Waller. Plant Cell Reports. and Chang. E. 2.) . H. Wang. Eng. C. J. Sci. Tsukamoto. Coggon. K... and Zhong. Visser.. 2003.

E. M. R. Y. 1994. and Hashimoto. 5: 50-53. R... N. Plant Cell Reports.. C. Biological and Pharmaceutical Bulletin. and Hu. 35: 181-193. B. and Tsay. Wickremesinhe. A. J. D. an active quinoid diterpene formed in the callus of Salvia miltiorrhiza Bunge. Q. 2004. cells. Physiology Plantarum. Y.. C. Weerden. 20: 545 -547.. U. Phytochemicals. I. Selection of cultured plant cell lines producing high levels of biotin. H. 1993. 93: 2325-2327. T. 21: 513-516. Production of berberine in cultured cells of Coptis japonica. Establishment of suspension culture and the measurement of diosgenin content. Y. Occurrence of L-DOPA and dopamine in plants and cell cultures of Mucuna pruriens and effects of 2... 4: 241-244... Y. J. Stimulation of taxol production and excretion in Taxus spp cell cultures by rare earth chemical lanthanum. and Endo. and Arteca. P. Wickremesinhe. 1998. Y. F. Shoyama. Goncalves. Zhao. Whitmer. 1982. H. 1986. Free tryptophan levels and inhibition of anthranilate synthetase.. and Yamada.. 1982. Theoretical and Applied Genetics.. R. C. Production of tropane alkaloids in cultured cells of Hyoscyamus niger. Chinese Agronomy Journal. R. N. Yamamoto. Go.. Anthraquinones as phytolexins in cell and tissue cultures of Cinchona sp. 61: 113-116. L.. C. S. and Svendsen. C. K.. Verpoorte. R. and Verpoorte. J. Huang. 2. 1974.Plant Cell Cultures . Wijnsma. Wichers. and Sato. A. growth optimization. 2001. Yagi. Wataneba.. J. 26. Selection of a high and stable pigment-producing strain in cultured Euforbia millii cells. T. Visser. Yamamoto. Canel. T. Plant Cell Reports. T. C. Formation of tetrahydroanthracene glucosides by callus tissue of Aloe saponaria. Phytochemistry.. 1984. R. Y. Huizing. F.. Cheng. R. Satish M. 33: 259-264. El-Shagi. A. 6: 845-848. and Yamada.. C. R. 1993. [152] [153] [154] [155] [156] [157] [158] [159] [160] Chen. Y. Plant Physiology. H. 85: 67-73. N. W.S. N. T. Tropane alkaloids in cultured cells of Duboisia leichhardtii. Journal of Plant Physiology. Yamada. W. Y..An Alternative and Efficient Source for Production of Biologically Secondary Metabolites [143] [144] [145] [146] [147] [148] [149] [150] [151] brevifolia. Production of reserpine and its optimization in cultured Rauwolfia serpentina Benth. C. 1: 101-103.. 1 47 . E. Appl. A. 1975. A. Yeh. Wang.. X. Yang. Zhu. Plant Cell Reports. Taxus cell suspension cultures: optimizing growth and production of taxol. 6: 14831484. Widholm. 2001.. Anthraquinone production by cell suspension cultures of Morinda citrifolia. Vanisree M. 1994. J... K. 144: 183-188. Yano.. S. M. Wu. Plant Cell Tissue and Organ Culture. Sci. Hallard. 4-D and NaCl on these compounds. Yamada. Y. H. O. and Yamada. 4: 257-268. 22. Mizuguchi. 1982. Wu. II. and Arteca. Eng. and Schulte. 3: 186-188. Yamada. S. 1981. 30: 323326. H J. J. Journal of Biotechnology. Planta Medica Supplement: 79101. Influence of precursor availability on alkaloid accumulation by transgenic cell lines of Catharanthus roseus. 116: 853-857. 1983. and Nishioka. Enhanced catharanthine production in Catharanthus roseus cell cultures by Int. Na. F. N. Isolation and quantitative analysis of cryptotanshinone. characterization and taxol production. M. Plant Cell Tissue and Organ Culture. and Mei. 1985.. W.. H. Taxus callus cultures: Initiation.. T. van I. and Pras.. Journal of American Chemical Society. F.. Plant Cell Reports. J. Zenk. Harkes. and Tsay. M.. 2003. Evidence for compartmentation of tryptophan in cultured plant tissues. Tissue culture of Dioscorea doryophora Hance. Phytochemistry.

Q. J.Vanisree Mulabagal and Hsin-Sheng Tsay combined elicitor treatment in shake notoginseng. H. G. In Modern Study of TraHsieh Ping. [163] Zhou. Sci.. ditional Chinese Medicine. M. (in Chinese) Beijing Xue Yuan Press. Longdan. J. 1988. C. 1 .. and Zhu. H. Applied Biochemistry Bioflasks and bioreactors. Eng. 28: 673-681. Studies on useful [161] Zheng. 2. 2: 1398-1408. (in Chinese) 48 Int. Studies on Bai-Zhi. of initial phosphate concentration on cell The toxicity test of useful compounds. crobial Technology. Effect Mun. G. Y.. T.. C. Z. J. 2004. Z. G. 8: tion by suspended cultures of Panax 220-221. M. 1995. Beijing. 1980. Enzyme and Mitechnology. and [162] Zhong. 4: 33. Yu.. [164] Zhou. growth and ginsenoside saponin producMedical Journal of China Hospital. Han. Dong. Chung-Chen-Yau Research. C. J. compounds of Bai-Zhi for healing Yin1997. X. China. IV. Appl. 55: 241-246.. and She.