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Medicinal Biotechnology in the Genus Scutellaria

October 28, 2007

By Cole, Ian B Saxena, Praveen K; Murch, Susan J

Abstract Plant-based medicines have an important role in the lives of millions of people. The ancient knowledge of the use of plants as medicines has led to the discovery of many important western pharmaceuticals, and the popularity of whole plant preparations for a range of therapeutic applications is growing rapidly. However, there are many challenges in the production of plant-based medicines, many of which put both the consumer and the plant populations at risk. Modern biotechnology can be optimized to mass-produce plants of specific chemical composition for use as particular treatments and applications. In this review, we have used one of the most important medicinal plant genera, Scutellaria, as a model to assess the potential of applications of biotechnology for the improvement of medicinal plants.

Keywords Indoleamines * Flavonoids * Baicalin * Melatonin * Bioreactors * Plant secondary metabolism

Introduction

Plants have been and continue to be one of the most important sources of medicines for the treatment of human diseases. The World Health Organization has estimated that approximately 80% of the world’s population relies on plant-based traditional medicines for primary health care (Lambert et al. 1997), of which 85% are plant extracts (Farnsworth 1998). In recent years, an increase in popularity and acceptance of herbal medicines has swept the Western world. Many individuals are now taking greater responsibility for their own health and quality of life, taking confidence in herbal and alternative therapies as “natural, safe and effective.” In 1998, consumers in the USA spent over $12 billion on natural supplements and over $27 billion on alternative medicines (Greenwald 1998). The market for herbal therapies has risen by 4-10% per year throughout the 1990s in both the USA and Europe (Leaman 2001). Many prescription pharmaceuticals and conventional treatments also depend on plants. For example, of the 500 million prescriptions written by physicians in the USA every year, 125 million involve a preparation from a leafy plant (Newman et al. 2003). In Canada, legislation enacted in 2004 has had a major impact on the development, production, and marketing of plant-based medicines. There is an imminent need for development of new technologies and production approaches to improve the safety and efficacy of plant-based medicines and to comply with legal regulations.

The challenges facing the development of plant-based medicines include the need to understand the unique physiology of medicinal plants, identification of active principles and modification of its content in plants, optimized protocols for growth, production, harvest, or handling, the loss of wild germplasm, complex plant chemistry and metabolite pathways, adulteration with misidentified plant species, and contamination with pollutants (Murch et al. 2000). These challenges have potentially serious implications for human health and plant biodiversity. Closely related species of plants are often morphologically similar and mistaken for the species of medicinal interest affecting the purity of plant based medicines. In the late 1990s, 2,700 kg of raw plant material labeled as plantain was imported and distributed over a period of about 2 yr in the USA. The raw material contained Digitalis lantana, and its ingestion resulted in toxic levels of serum digoxin in patients (Slifman et al. 1998). Medicinal plant preparations may also be contaminated with insects, fungi, and bacteria. Many herbal products are prepared from unwashed leaf tissues to preserve surface glands containing bioactive molecules. Such tissues may retain large quantities of soil-bom microbes and other environmental pollutants, which can affect the levels of medicinal metabolites (Lauglin and Munro 1982; Bernath 1986) and place the consumers’ health at risk (Slifman et al. 1998).

Agricultural cultivation of many medicinal plants has proven difficult because of lack of knowledge of their specific requirements for sexual reproduction, seed development, and aspects of seed dormancy, germination, and plant growth (Canter et al. 2005). In addition, the involvement of the industry and the Government in developing agricultural strategies for medicinal plants has been rather limited. Commercial agriculture and horticulture have an extensive history of developing and optimizing production systems to meet the specific environmental requirements of crops, but such systems have not been developed for medicinal plants.

The majority of medicinal plants are harvested from the wild rather than field or greenhouse production, as in traditional agriculture and horticulture, causing loss of genetic diversity and habitat destruction. Increased popularity of medicinal plants is frequently accompanied by a surge in the number of gatherers indiscriminately removing the plants from the wild and placing the species in danger of extinction. It is estimated that 10,000 medicinal plant species are endangered worldwide (Leaman 2001), and many of these species are exported from developing countries. In Germany, approximately 1,560 medicinal species are imported; however, only 50-100 species are produced commercially (Schippmann 2001). In the UK, 25% of residents use herbal medicines regularly at a market value of over Pounds 88 million, with only 10% in commercial production (Edwards 2004; Vines 2004). In 1990 alone, traditional Chinese medicine (TCM) practitioners used 700,000 tons of plant material, 80% of which was collected from the wild (Srivastava et al. 1996), placing immense strain on plant populations and sensitive ecosystems and risking the loss of valuable germplasm. Threatened medicinal plants such as Echinacea tennesseensis are federally listed in the USA (Catling and Porebski 1998), and similar examples exist in many different countries. Goldenseal (Hydrastis canadiensis), one of the most commonly sold North American medicinal plant, is an example of a species listed as endangered as a result of overharvest of wild germplasm. The preparations of this species may also endanger human health, as they have been found to contain high levels of aluminum, lead, cadmium, nickel, and other metals (Liu et al. 2004).

The value of medicinal plants is in their unique phytochemicals commonly known as secondary metabolites, the compounds that are not normally required for plant growth and development. Secondary metabolites mediate many vital functions in plant survival against herbivory, predators, environmental stress, and competition. Plant secondary metabolites also serve as attractants and pigments aiding reproduction and may effect growth and development of the plants themselves as well as of the other organisms (Seigler 1996; Dewick 2002). To date, very little is known about the genetics of plant secondary metabolism, as the genes of most pathways have not been identified and little is understood of their regulation and function. By contrast, the agriculture and horticulture sectors have benefited tremendously from the vast information and technologies currently available for model species such as rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana), with fully mapped genomes. No such model species exists for medicinal plants or plant species with complex secondary metabolite production. Therefore, the emerging biotechnologies have an enormous potential for genetic improvement of medicinal plants through the development of methods for mass propagation in controlled environment, chemical profiling, metabolic engineering, and understanding the regulation and role of secondary metabolism in plant development. As well, the economic potential in a field desperately lacking in optimized commercial production protocols and facilities is huge.

The Genus Scutellaria

The genus Scutellaria is a diverse and widespread genus within the family Lamiaceae, the mint family. With over 350 species worldwide, these herbaceous shrubs, commonly called skullcaps, are found from Siberia to the tropics of South America, the Rocky Mountains of North America and Canada, on the islands of Japan, and throughout much of Europe and Asia. Scutellaria has been used by many cultures to treat a variety of different ailments, including cancers, hepatitis, cirrhosis, jaundice, anxiety, and nervous disorders. Scutellaria would be an optimal choice for the development of a medicinal model species because of an extensively documented chemical profile and a relatively small genome size. The nucleus of Scutellaria racemosa is about 0.75 pg (Cole et al., in preparation). Therefore, Scutellaria species are excellent model systems to study the biochemistry and genetic regulation of secondary metabolism in medicinal plants.

One of the most commonly prescribed herbs in TCM and in Japanese Kampo medicine is huang-qin (Scutellaria baicalensis Georgi; Watanabe et al. 2002; Murch et al. 2004). More than 275 scientific reports have appeared since 2000, outlining the effectiveness of extracts of huang-qin in the inhibition of metabolic processes of various medical conditions, including reduced prostate cancer (Hsieh et al. 2002), inhibition of enzymatic activity of HIV (HIV-1/RT) in lymphoid cell lines and peripheral blood mononuclear cells (Li et al. 1993), suppressed proliferation of human hepatoma cell lines (Chang et al. 2002), protection against aflatoxin -B1-induced liver mutagenesis (de Boer et al. 2005), inhibition of liver fibrosis (Nan et al. 2002), inhibition of hemin-nitrite-H^sub 2^O^sub 2^ induced liver damage (Zhao et al. 2006), attenuation of inflammation- mediated degeneration of dopaminergic neurons (Li et al. 2004), and reduction of symptoms of type 1 allergic reactions (Lim 2003). Figure 1. Common medicinal compounds of Scutellaria sp.

Chemistry of Scutellaria Species

The most important compounds isolated from Scutellaria species to date are baicalin, baicalein, and wogonin (Fig. 1). Crude extract of root tissue of S. baicalensis, also known as Wogon or Scutellaria radix, has many known pharmacological actions, which include its use as a diuretic laxative (Kumazaki 1958), anti-inflammatory (Koda et al. 1982; Hirai et al. 1983; Koda 1987), anti-allergy (Koda 1973, 1987, Koda et al. 1970a, b, c, d; Kubo et al. 1981, 1984, 1985), and modulator of lipid (Aonuma et al. 1957; Kimura et al. 1981a, b, 1982, 1985; Yamahara et al. 1981) and arachidonate metabolism (Kimura et al. 1985). Malikov and Yuledashev (2002) have reported 208 phenolic compounds isolated from Scutellaria species. S. baicalensis with the highest number of known phenolic compounds (>62) represents the most studied species in the genus (Malikov and Yuledashev 2002). Baicalin and baicalein are the most commonly investigated compounds in Scutellaria preparations (Horvath et al. 2005). Baicalin is a flavonoid nonnucleoside reverse transcriptase inhibitor (NNRTI), and clinical studies have revealed many pharmacological activities. Baicalein inhibits platelet 12- lipoxygenase-1 (Sekiya and Okuda 1982), lipid peroxidation (Gao et al. 1996), and cell growth of human hepatocellular carcinoma cell lines (Matsuzaki et al. 1996). Additional reports have shown the pharmacological actions and efficacy of other compounds found in Scutellaria sp., such as scutellarin, wogonin, and the human neurohormone melatonin. Hattori was reported to be the first to discover and isolate wogonin in roots of S. baicalensis in 1930. Recent studies have outlined the therapeutic potential of wogonin and recommended for an increased use in western medicine (Tai et al. 2005). This is mainly because of the large body of literature about pharmacological effects of wogonin and an extraordinary safety record in eastern medicine (Tai et al. 2005). Wogonin has been found to be a potent natural neuroprotective compound, which inhibits inflammatory action of microglia (Lee et al. 2003). The same study also found that wogonin attenuated the death of neurons and inhibited inflammation in animal models (Lee et al. 2003). In the past, scutellarin has been used to treat various cardiovascular diseases, sleep disorders, depression, migraines, and memory impairment (Pouzet 2002; Gamer et al. 2003; Goh et al. 2005); however, current treatments in China utilize scutellarin for the dilation of blood vessels, improvement of microcirculation, decreasing the viscosity of blood, and reducing blood platelet count (Hong and Liu 2004; Liu et al. 2005). Additionally, S. baicalensis has been shown to contain high concentrations of melatonin (Murch et al. 1997, 2004, Cole et al., in preparation). Melatonin is a potent antioxidant, an important regulator of circadian rhythms in many organisms and acts as a hormone in several neurological conditions such as migraines and depression. Many Japanese Kampo medicine treatments are applied to relieve complaints that may be related to dysfunction of circadian rhythms, such as seasonal affective disorders and the nocturnal polyuria of diabetes (Watanabe et al. 2002) and use S. baicalensis as a component. The presence of melaonin has been reported in 108 plant species used in TCM (Chen et al. 2003). The medical efficacy of melatonin, wogonin, baicalin, baicalein, and scutellarin emphasizes the importance of medicinal plants as an invaluable source for the development of novel anti- cancer, anti-inflammatory, and neuroprotective drugs.

Metabolomics/Metabonomics: New Tools to Unravel Plant Chemistry

Metabolomics is the untargeted identification and measure of all of the small compounds in a given sample (Hall et al. 2002). In plant tissues, such measures provide information on the whole spectrum of primary and secondary metabolites at a specific time and in a specific environment. Further studies of metabonomics sequentially follow the changing metabolite profile of a tissue through time and treatment. For medicinal plants, these studies are new opportunities to examine many of the old paradigms in secondary metabolism and the opportunity for the discovery of new medicinal metabolites in plants used for the treatment of disease. Recently, metabolomic analysis of 5. baicalensis found over 2,400 different compounds including 781 with possible medicinal importance and novel amino derivatives of baicalin and wogonin (Murch et al. 2004). Most interesting in this study was the putative identification of hyperforin in these plant tissues. The identification of hyperforin was later confirmed by other, traditional HPLC techniques (Murch and Saxena 2006). These data provide a dramatic representation of how much there is to learn about medicinal plant chemistry. Hyperforin has recently been identified as an entirely new class of antidepressant that acts in human brain and muscle tissues by nonselective activation of calcium channels. The problem of synergy has plagued medicinal plant research for decades, and attempts to isolate a single “silver bullet” pharmaceutical compound from traditional medicinal plants have not been successful in many instances. Metabolomic analysis allows for these complex questions to be addressed in a more complete manner.

Scutellaria: A Model for Application of Biotechnology to Medicinal Plants

Biotechnology has played an important role in the development of modern agriculture and horticulture but has not been applied to many medicinal plants or medicinal plant production systems. Biotechnology for improvement of an agricultural or horticultural crop is commonly defined as the development of a new cultivar with improved yield, disease resistance, salt tolerance, flowering, colors, or morphological traits. These characters are selected through the isolation of desirable lines with altered plant secondary metabolism. In the case of medicinal plants, improvement must be defined by the increase in the yield of a specific phytochemical or combinations of phytochemicals. These traits are not easily discernable and require rigorous chemical analyses. Therefore, medicinal plant varieties need to be developed with optimized specific chemistry and high levels of desirable compounds. However, to ensure the stability of such lines, methods for clonal propagation and large-scale in vitro production are required.

Mass-propagation and controlled environment production systems. The importance of Scutellaria in the Chinese and Japanese medicine has spurred research projects developing in vitro propagation protocol with special emphasis on metabolite production. The first report of tissue culture of Scutellaria was by Trofimova in 1982 but with rather limited details of specific methods and data. Uhring (1982) developed a protocol for the micropropagation of Scutellaria costaricana using liquid medium, which produced 30% more shoots compared to the solid medium. Later, in vitro protocols for propagation of undifferentiated S. baicalensis tissues to be used in the quantification of flavonoids were reported in a series of studies by Yamamoto et al. (1986a, b, c, d). Callus growth was achieved by culturing stem tissue in the presence of indole acetic acid (IAA) and kinetin in the dark with quantification of 11 flavonoids (Yamamoto et al. 1986a, b, c, d). Comparative studies of wogonin in the traditional root preparation and callus tissue showed similar levels of the flavonoid (Yamamoto et al. 1986a). Studies then sought to modify the flavonoid content of S. baicalensis callus tissue by investigating the effects of different carbon sources substituted for sucrose in the culture media. For example, the production of flavonoids, specifically baicalin, wogonin, and wogonin-7-O-glucuronide, was most affected in cells grown in the presence of maltose (Yamamoto et al. 1986b). These studies revealed a logarithmic callus growth habit and gradual increases of flavonoid content with the highest values of baicalin and wogonin-7-0- glucuronide after 60 d of cultivation (Yamamoto et al. 1986e).

The effect of plant growth regulators on the callus growth and flavonoid content led to the discovery and utilization of a two- stage protocol to maximize callus growth and production of flavonoids (Yamamoto et al. 1989a). Further work with the callus tissue explored the effects of various growth inhibitors including 3- (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a urea herbicide), abscissic acid, L-phenylalanine, p-fluorophenylalanine (PFP), streptomycin, chloramphenicol, and the protein and nucleic acid synthesis inhibitor cycloheximide (Yamamoto et al. 1987). The addition of these compounds to the culture medium and cell growth in light and dark conditions profoundly influenced the production of flavonoids, especially baicalin (Yamamoto et al. 1987). This work was followed by studies investigating the effects of earlier carbon sources and growth regulators on cultures grown in liquid medium and several different basal media (Yamamoto et al. 1989a). The best callus growth was observed in Linsmaier and Skoog (LS) basal medium containing 10^sup -7^ M IAA, and the highest flavonoid content was observed in cells grown on White’s medium containing 10^sup -6^ M IAA and 10^sup -5^ M kinetin. Addition of 5% maltose was found to increase cell growth by 52% and flavonoid content (baicalin, wogonin, 7-0-glucuronide) by 92% (Yamamoto et al. 1989a). Together, these studies (Yamamoto et al. 1989a, b) resulted in the development of a two-stage liquid medium protocol for efficient production of chemically rich S. baicalensis cells with increased baicalin and wogonin 7-0-glucuronide from 4.0 and 1.5%, respectively, in ordinary cultures to 9.07 and 6.33%. This is one of the earliest, and most detailed biotechnology studies of S. baicalensis with special emphasis on medicinal compound production. The quantification of flavonoids by Yamamoto et al. followed methods described previously by Tomimori et al. (1982, 1983, 1986a, b) and included methanol extractions and silica gel chromatography to isolate and identify 11 flavonoids (Yamamoto et al. 1991).

More recent examples of S. baicalensis tissue culture systems investigated the effects of different growth hormones and culture conditions such as light, dark, and temperature. For example, Stojakowska et al. (1999) found that most efficient production of auxiliary buds was accomplished with Murashige and Skoog (MS) basal medium (Murashige and Skoog 1962) supplemented with 0.5 [mu]M naphtaleneacetic acid (NAA) and 2.5 [mu]M kinetin. Later, a two- stage procedure was established to increase the multiplication rate. First, thidiazuron (TDZ: [N-phenyl-N'-(l,2,3-thidiazol-5-ylurea)]) (0.1-0.5 [mu]M; Fig. 2a) was used to induce organogenic callus, and shoots were then regenerated on medium containing 2.2 [mu]M BA with an efficiency of over 20 shoots per expiant (Fig. 2b-d). Li et al. (2000) observed the effectiveness of TDZ for induction of de novo shoot organogenesis from intact seedlings and the leaf and petiole expiants of S. baicalensis. More regenerants were formed along hypocotyls of intact seedlings (20 shoots/expiant) than were observed on excised hypocotyls (9.7 shoots/expiant), indicating that the isolation of expiants was not essential for optimal regeneration and the surrounding tissues provided the necessary endogenous metabolites for shoot initiation. A successful protocol was also developed for the establishment of plantlets in the greenhouse from liquid media, semisolid media, and sterilized peat pellets (Fig. 2e; Li et al. 2000). More than 95% of de novo regenerants in this study formed roots and had the capacity to be established in the greenhouse (Li et al. 2000). Inoculation of micropropagated plants of S. integrifolia with arbuscular mycorrhizal fungi has been shown to improve root growth and overall plant development (Joshee et al. 2007).

Figure 2. An example of the practical application of biotechnology in the production of the medicinal herb Scutellaria with special emphasis on chemical profile. Many problems exist in current production methods of medicinal plants, such as threat of extinction of wild harvested species, misidentification of collected plant material, contamination by processing methods and environmental parameters, and lack of understanding of the unique and complicated chemistry of medicinal plants. The following represents several steps in the production of Scutellaria. (a) Field collection of Scutellaria involves removal of a finite amount of material from the wild giving relief to wild populations. (b) Establishment of sterile, axenic, in vitro cultures allows for micropropagation and provides material for chemical analysis. (c) Shoot organogenesis of Scutellaria provides material for propagation and chemical analysis. (d) Mass propagation of Scutellaria in temporary immersion bioreactor systems. (e) Selection of unique germplasm lines based on chemical profile and growth in a controlled environment.

Flavonoids and other secondary metabolites have also been separated and quantified in callus tissues of S. columnae (Stojakowska and Kisiel 1998) and S. baicalensis (Yamamoto et al. 1986a, b, c, d; Morimoto et al. 1995). Seo et al. (1993) reported flavonoid (baicalin and wogonin-7-0-glucuronic acid) yield of 2.5 g/ l in 5. baicalensis cell Suspension cultures, which was twice as high as obtained with root tissue. Further studies showed that changing the temperature from 30 to 25[degrees]C increased baicalin production from 2.5 to 4.2 g/l (Seo et al. 1993) and the use of SH medium increased S. baicalensis cell growth and baicalin production by 1.5 and 1.67 times, respectively (Seo et al. 1996). Micropropagation of several other species of Scutellaria, e.g., 5. lateriflora, S. racemosa, S. scordifolia, S. montana, S. integrifolia, and numerous Chinese species utilized in TCM has been mentioned in both scientific and popular literature often without details. The tissue culture procedures described for Scutellaria species show the complexity of medicinal plant and secondary metabolite production.

Bioreactor production of Scutellaria. Bioreactors provide highly controlled environments for the rapid multiplication of cells, tissues, and plantlets. Growing tissues in this manner allows production of secondary metabolites, until the media is depleted of nutrients or some other culture variable is changed. Six different systems were evaluated for biomass and phytochemical production of S. baicalensis (Zobayed et al. 2004). The greatest accumulation of biomass occurred in a temporary immersion bioreactor with liquid media, and the most effective system for the production of specific phytochemicals was a large vessel under forced aeration on gelled medium. In addition, plantlets grown in this system with supplemental CO2 produced the largest plantlets with some evidence of enhanced photosynthesis (Zobayed et al. 2004). This work shows the potential of the application of biotechnology and analytical chemistry for the production of chemically optimized medicinal plant tissues.

Selection of elite germplasm of Scutellaria. Murch et al. (2004) demonstrated the usefulness of in vitro manipulation to select natural and induced variations for harvesting chemical diversity. The first step in this study utilized the protocol developed by Li et al. (2000) for de novo shoot organogenesis, followed by treatment of the seedling with the mutagen 2.5 [mu]M l^sup -1^ ethylnitrosurea (ENU). Treated and untreated seedlings were subcultured in the light for 28 d on basal media. More than 300 germplasm lines were selected based on morphological growth characteristics. These lines were then screened for antioxidant potential, and the compounds of medicinal interest (baicalin, baicalein, wogonin, and melatonin) were extracted and quantified from tissues by high performance liquid chromatography (HPLC)-mass spectrometry MS/MS (Murch et al. 2004; Cao et al. 2006). The result was 26 chemically distinct lines resulting from naturally occurring and chemically induced mutations. These lines showed a greater than tenfold (1201,270 [mu]g) variability in antioxidant potential. The line with the highest potential was a naturally occurring variation rather than a mutated line. Melatonin content in lines showed the greatest variability with a 5,000-fold (9 nmol-44,362 nmol g^sup -1^) variation between lines. Baicalin, baicalein, and wogonin were all significantly altered with 10-, 39-, and 30-fold variations observed. The lines with the highest baicalin and baicalein content had lower melatonin, as did other lines rich in flavonoids, although no correlation was found between antioxidant potential and flavonoid production. The fastest growing line showed moderate antioxidant potential, baicalin, and baicalein, with low levels of wogonin and the lowest reported concentration of melatonin. Conversely, the slowest growing line showed the highest levels (5,000-fold) melatonin (Murch et al. 2004). Natural or induced polyploidy is a common and extremely beneficial practice in commercial agriculture. Gao et al. (2002) showed successful production of tetraploids from colchicine-treated callus and selected seven new varieties for commercial breeding programs (Gao et al. 2002). Tetraploid tissues showed slightly smaller baicalin content than that in the diploid controls but showed higher productivity and thicker and larger leaves, stems, and roots increasing overall yield by about 40% (Gao et al. 2002). Similar breeding programs should be established for developing plants with a specific chemical profile and growth habit for commercial production. Recent studies have also shown genetic stability in nuclear DNA contents and ploidy levels of elite S. baicalensis germplasm lines maintained in vitro (Alan et al. 2007). The successful development and maintenance of elite germplasm lines with increased flavonoid production and vigorous growth habits could be very useful in further commercial production programs.

Genetic modification of Scutellaria. Agrobacterium-induced genetic transformation in S. baicalensis has revealed several interesting characteristics that could be beneficial to a large- scale production program involving medicinal species. For example, Nishikawa and Ishimaru (1997) utilized Agrobacterium rhizogenes Al 3 to insert the beta-glucuronidase (GUS) gene into clones of S. baicalensis and found that glucuronide-type flavonoids were fairly low when compared to nontransformed roots (Nishikawa and Ishimaru 1997). Zhou et al. (1997) investigated the effects of five different culture media for proliferation of hairy root cultures, the best of which was B5 liquid medium. The same group isolated 15 known flavonoids, 5 known phenylethanoids, and 1 novel flavone glucoside, 5,7,2′,6′-tetrahydroxyflavone 2′-0-beta-D-glucopyranoside (Zhou et al. 1997). Nishikawa et al. (1999) followed their previous study by comparing the glucuronide-type flavonoid content of two clones of S. baicalensis, one of which was transformed with the beta- glucuronidase (GUS) gene, the other a control. They reported three times the flavonoid content in the untransformed clone (clone C) and accumulation of acetoside in the roots and leaves of in vitro cultured plants (Nishikawa et al. 1999). Hirotani et al. (2000) showed successful cloning and expression of the uridine diphosphate (UDP)-glucose flavonoid 7-0-glucotransferase (UBGT) from hairy root cultures of S. baicalensis, and the enzyme activities of recombinant UBGT expressed in Escherichia coli were detected toward flavonoids such as baicalin, wogonin, apigenin, scutellarin, 7,4′- dihydroxyflavone, kampherol, and phenolic compounds (Hirotani et al. 2000). In 2001, Kuzovkina et al. showed 20-30 times increase in weight of hairy roots after 4 wk of culture and elicitation of hairy roots with 100 [mu]M methyl jasmonate for 72 h increased the flavonoid content per flask and per root dry weight by 1.8 and 2.3 times, respectively (Kuzovkina et al. 2001). Kovacs et al. (2004) developed liquid Chromatographie methods for determination of the main flavonoid contents of hairy roots cultures and found that the baicalin and wogonin content of transformed roots was consistently higher than intact plants from Siberia (Kovacs et al. 2004). Further work by Kuzovkina et al. (2005) found that the flavonoid content of cultures of transformed roots were threefold lower than in the roots of intact (untransformed) 5-yr-old plants. They also showed that flavonoid ratios in roots cultured on BS or MS media changed but the overall concentration remain unchanged and treatment of 3-wk-old cultured roots with methyl ether of jasmonic acid (MeJa), a stress response, doubled the total concentration of major flavones (Kuzovkina et al. 2005). Recent work by Hwang (2006) evaluated and maximized the root growth and baicalin content of hairy roots on Schenk and Hildebrandt medium supplemented with 4 or 6% sucrose. Baicalin accumulation was enhanced through the addition of the elicitors such as methyl jasmonate, salicylic acid, and various concentrations of fungal cell wall elicitors to the growth medium. Baicalin content ranged from 10.5 to 18.3 mg/g dry weight of roots, which was 1.5- to threefold greater man the controls (Hwang 2006). Conclusions and Future Directions

S. baicalensis is an excellent model system for the investigation of medicinal plant species. The plant itself grows vigorously in a wide range of habitats, across the globe, and possesses a complex chemical complement that includes baicalin, baicalein, wogonin, scutellarin, and the neurohormones serotonin and melatonin. In vitro culture research has shown successful alteration of flavonoid content in tissues and many cases of successful genetic transformation. In addition, the genome size of this medicinal plant species is similar in size to Arabidopsis and small enough for genomic sequencing to be successful and affordable (Cole et al., in review). As a model system for medicinal plants, studies with Scutellaria will lead to (a) effective technologies for medicinal plant production, (b) a greater understandings of medicinal plant secondary metabolism and the role of plants in human health, (c) increased consumer confidence as it becomes more possible to apply biotechnology approaches to the production of regulated, consistent, high quality medicinal plant tissues, and (d) the introduction of new varieties through both biotechnological and traditional breeding programs and the development of elite germplasm lines with greater medicinal efficacy.

Received: 4 February 2007/Accepted: 25 May 2007 / Published online: 6 July 2007 / Editor: P. Lakshmanan

(c) The Society for In Vitro Biology 2007

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Chemistry, LK. Barber School of Arts and Sciences,

University of British Columbia Okanagan,

3333 University Way,

Kelowna, British Columbia V1V 1V7, Canada

e-mail: susan.murch@ubc.ca

P. K. Saxena

Department of Plant Agriculture, University of Guelph,

Guelph, Ontario N1G 2W1, Canada

Copyright Society for In Vitro Biology Jul/Aug 2007

(c) 2007 In Vitro Cellular & Developmental Biology; Plant. Provided by ProQuest Information and Learning. All rights Reserved.




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