A Tool for the Production of High Quality Plant-Based Medicines

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Current Pharmaceutical Biotechnology, 2006, 7, 33-49 33 Micropropagation: A Tool for the Production of High Quality Plant-based Medicines

Mousumi Debnath, C.P. Malik and P.S. Bisen*

Tissue Culture Laboratory, Institute of Biotechnology and Allied Sciences, Seedling Academy of Design, Technology And Management, Jaipur-302025, India

Abstract: Medicinal plants are the most important source of life saving drugs for the majority of the world’s population. The biotechnological tools are important to select, multiply and conserve the critical genotypes of medicinal plants. Plant tissue culture techniques offer an integrated approach for the production of standardized quality phytopharmaceutical through mass-production of consistent plant material for physiological characterization and analysis of active ingredients. Micropropagation protocols for cloning of some medicinal plants such as Catharanthus roseus (Apocynaceae), Chloro- phytum borivilianum (Liliaceae), Datura metel (Solanaceae), and Bacopa monnieri (Scrophulariaceae) have been developed. Regeneration occurred via organogenesis and embryogenesis in response to auxins and cytokinins. The integrated approaches of our culture systems will provide the basis for the future development of novel, safe, effective, and high- quality products for consumers.

Key Words: Regeneration, micropropagation, Catharanthus roseus, Chlorophytum borivilianum, Datura metel, Bacopa monieri, medicinal plant.

INTRODUCTION medicinal constituents, or on the development of semi- synthetic drugs, or still again on the active screening of natu- Recent and renewed interest in medicinal plants coupled ral products to yield synthetic pharmacologically-active to developments in information technology has fuelled an compounds. In Germany, for example, over 1500 plant spe- explosion in the range and content of electronic information cies encountered in some 200 families and 800 genera have concerning medicinal plants as a re-emergent health aid. been processed into medicinal products. In South Africa, Medicinal plants, since times immemorial, have been used in likewise, some 500 species are commercialized trade prod- virtually all cultures as a source of medicine. The widespread ucts. Today, Bulgaria, Germany and Poland are recognized use of herbal remedies and healthcare preparations, as those as major exporters of plant-based medicinal products. India described in ancient texts such as the Vedas and the Bible, has about 45,000 plant species; medicinal properties have and obtained from commonly used traditional herbs and me- been assigned to several thousand. The world market for dicinal plants, has been traced to the occurrence of natural herbal derivatives /products is in the increase order of mag- products with medicinal properties. Medicinal plants play a nitude. In the U.S. and Europe the ecological movement has key role in world heath care systems [13]. Herbal medicine brought about a renewed interest in traditional medicines. is one of the most remarkable uses of plant based biodiver- High inflation rates in the third world have caused some citi- sity. As many as 75 to 90% of the worlds’ rural people rely zens to return to or begin using herbal remedies, The pre- on herbal medicine for their primary health care. The success scription drug market is around $40 billion today, suggesting of any health care system depends on the availability of suit- a value of about $10 billion for those drugs containing at able drugs on a sustainable basis. Reserves of herbs and least one compound derived from higher plants. Even today, stocks of medicinal plants in developing countries are di- the World Health Organization estimates that up to 80 per minishing and in danger of extinction as a result of growing cent of people still rely mainly on traditional remedies such trade demands for cheaper healthcare products and new as herbs for their medicines. plant-based therapeutic markets in preference to more expen- sive target-specific drugs and biopharmaceuticals. Such con- Medicine, in several developing countries, using local cerns have stimulated positive legal and economic interest. traditions and beliefs, is still the mainstay of health care. As defined by WHO, health is a state of complete physical, In several industrialized societies, plant-derived pre- mental, and social well being and not merely the absence of scription drugs constitute an element in the maintenance of disease or infirmity. In Europe, some 1500 species of me- health. Medicinal plants are an integral component of re- dicinal and aromatic plants are widely used in Albania, Bul- search developments in the pharmaceutical industry. Such garia, Croatia, France, Germany, Hungary, Poland, Spain, research focuses on the isolation and direct use of active Turkey, and the United Kingdom (Table 1) The practice of traditional medicine is widespread in China, India, Japan, Pakistan, Sri Lanka and Thailand. In China about 40% of the *Address correspondence to this author at the Tissue Culture Laboratory, Institute of Biotechnology and Allied Sciences, Seedling Academy of De- total medicinal consumption is attributed to traditional tribal sign, Technology and Management, Jaipur-302025, India; medicines. In Thailand, herbal medicines make use of leg- E-mail: [email protected] umes encountered in the Caesalpiniaceae, the Fabaceae, and

1389-2010/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 34 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al. the Mimosaceae. In the mid-90s, it is estimated that receipts tic demands have the potential to improve export of plant of more than US$2.5 billion have resulted from the sales of drugs. For example, China has attained a leader position in herbal medicines. And, in Japan, herbal medicinal prepara- the export of crude and Chinese drugs to the world market tions are more in demand than mainstream pharmaceutical and it is followed far behind by Singapore and India. The products. In total, 75 species belonging to 42 families have botanical market, inclusive of herbs and medicinal plants, in been investigated and new ethnomedical information has the USA, is estimated, at retail, at approximately US$1.6 been obtained for 41 species [67]. Despite the increasing use billion p.a. China with exports of over 120,000 tonnes p.a., of medicinal plants, their future, seemingly, is being threat- and India with some 32,000 tonnes p.a. dominate the inter- ened by complacency concerning their conservation. Glob- national markets. It is estimated that Europe, annually, im- ally, the early part of the 20th century brought an evolution ports about 400,000 tonnes of medicinal plants with an aver- of the pharmaceutical industry. With the progress in chemi- age market value of US$ 1 billion from Africa and Asia. The cal techniques, crude drugs came to be replaced by pure industry anticipates growth on the order of 15–20% into the chemical drugs and the developed countries witnessed a de- new millennium. A growing awareness of this new con- cline in popularity of medicinal plant therapy. However, tributor to the foreign-exchange reserves of several national during the recent past, the pendulum has swung again and treasuries is beginning to emerge. To satisfy growing market there is a resurgence of interest in study and use of medicinal demands, surveys are being conducted to unearth new plant plants. Many traditional plant based remedies are back in use sources of herbal remedies and medicines at the same time and find increasing applications as: develop new strategies for better yield and quality. (i) source of direct therapeutic agents, CONVENTIONAL PLANT-BREEDING AND (ii) as a raw material base for the elaboration of more MICROPROPAGATION complex semi-synthetic chemical compounds, Conventional plant-breeding methods can improve both (iii) as models for new synthetic compounds, and agronomic and medicinal traits. In vitro propagation or tissue (iv) as taxonomic markers for the discovery of new culture of plants holds tremendous potential for the produc- compounds. tion of high-quality plant-based medicines. This can be achieved through different methods including micropropa- Thus, plants are a tremendous source for the discovery of gation [106]. The evolving commercial importance of secon- new products of medicinal value for drug development. To- dary metabolites has in recent years resulted in a great inter- day several distinct chemicals derived from plants are im- est in secondary metabolism, particularly in the possibility of portant drugs currently used in one or more countries in the altering the production of bioactive plant metabolites by world. A drug "Memory Plus" has recently been developed means of tissue culture technology. Plant cell culture tech- using Brahmi (Bacopa monniera L.) by the scientists of the nologies were introduced at the end of the 1960's as a possi- Central Drug Research Institute, Lucknow, India. Similar ble tool for both studying and producing plant secondary Ayurvedic pills, for rejuvenating humans has come up in the metabolites [78]. Different strategies, using an in vitro sys- name of "Thirty Plus", which use Ashwagandha (Withania tem, have been extensively studied to improve the produc- somnifera L.) and Ginseng (Panax aquintifolium) as the in- tion of plant chemicals. gredients. Pudin Hara, another Ayurvedic drug containing mint (Mentha arvensis L.), which is used for indigestion, is Micropropagation has many advantages over conven- available across the counter. The production, consumption tional methods of vegetative propagation, which suffer from and international trade in medicinal plants, and phytomedici- several limitations. There has been significant progress in the nes, therefore, are growing and expected to grow in future use of tissue culture and genetic transformation to alter quite significantly. Consumption of herbal medicines is pathways for the biosynthesis of target metabolites. Obsta- widespread and increasing. Yet harvesting from the wild, the cles to bringing medicinal plants into successful commercial main source of raw material, is causing loss of genetic diver- cultivation include the difficulty of predicting, which ex- sity and habitat destruction. tracts will remain marketable and the likely market preference for what is seen as natural source of extracts. ECONOMIC BENEFIT The most outstanding merits offered by micropropaga- World wide, there has been a renewed interest in natural tion, over the conventional methods are: products. The world market for plant-derived chemicals viz. 1. In a relatively short time and space large number of pharmaceuticals, fragrances, flavors, and color ingredients, plants can be produced, starting from single explant. alone exceeds several billion dollars per year. Classic examples of phytochemicals in biology and medicine include 2. Unlike the conventional methods of plant propagation, micropropagation of even temperature species may be taxol, vincristine, vinblastine, colchicine ,artemisinin, fork- carried out throughout the year. olin, Saponin etc. Trade in medicinal plants is growing in volume and in exports. The $60-billion global herbal market 3. Tissue cultured plants are generally free from fungal and is growing at a rate of 7 percent per annum and is expected bacterial diseases. Virus eradication and maintenance of to generate $5 trillion by 2050. According to a WHO docu- plants in a virus-free-state are also readily achieved in ment, one fourth of the 500 million prescriptions written in tissue culture. the US each year contain a mention of leafy plant extracts. 4. The multiplication rate is greatly increased. The Indian climate is conductive to herbal cultivation. De- veloping and emerging countries besides catering to domes- 5. It also permits the production of pathogen-free material. Micropropagation Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 35

Table 1. Some Important Medicinal Plant Species of Europe and Asia

Species Some active ingredient(s) Action

Acokanthera schimperi Ouabain cardiac

Aconitum napellus Aconitine CNS-active

Adonis vernalis Adonoside cardiotonic

Aesculus hippocastanum Aescin anti-inflammatory

Agrimonia eupatoria Catechin anthelmintic

Ailanthus altissima Quassinoids antimalarial

Ammi visnaga Khellin antiasthmatic

Anabasis aphylla Anabasine antismoking, myorelaxant

Anamirta cocculus Picrotoxin analeptic

Andrographis paniculata Andrographolide, neoandrographolide antidysenteric

Angelica polymorpha Phthalides sedative

Anisodus tanguticus Anisodine anticholinergic

Ardisia japonica Bergenin antitussive

Areca catechu Arecoline anthelmintic

Artemisia annua Artemisinin antimalarial

Artemisia maritima Santonin ascaricide

Aspidosperma spp. Yohimbine aphrodisiac

Belamcamda chinensis Shikonin antibacterial antitussive

Berberis spp. Berberine antidysenteric

Bocconia spp. Sanguinarine antiseptic

Camptotheca acuminata Camptothecin antitumor

Cannabis sativa Delta-9-tetrahydrocannabinol antiglaucomic, antemetic

Carica papaya Papain, chymopapain proteolytic

Catharanthus roseus vincristine,vinblastine anticancerous

Cephaelis ipecacuanha Emetine amebicide, emetic

Cephalotaxus spp. Harringtonine, etc. antitumor

Chondodendron tomentosum Tubocurarine myorelaxant

Chrysanthemum cinerariaefolium Pyrethrins insecticide

Chrysanthemum parthenium Parthenolides antimigraine

Cinchona calisaya Quinine, quinidine antimalarial

Cinnamomum camphora Camphor rubefacient

Cissampelos pareira Cissampeline myorelaxant

Coleus forskohlii Forskolin cardiovascular

Convallaria majalis Convallatoxin cardiotonic

Coptis spp. Berberine, palmatine antidysenteric, antipyretic, detoxicant

Corydalis ambigua Tetrahydropalmatine analgesic, sedative 36 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al.

(Table 1) contd….

Species Some active ingredient(s) Action

Crotalaria sessiliflora Monocrotaline antitumor

Curcuma longa Curcumin choleretic anticoagulant

Cynara scolymus Cynarin choleretic

Daphne genkwa Yuanhuacine yuanhuadine ecbolic, antitumor

Datura stramonium Scopolamine, hyoscyamine, atropine hallucinogenic

Drimys winteri Polygodiol fungicide

Fraxinus rhynchophylla Aesculetin antidysenteric

Gaultheria procumbens Methyl salicylate rubefacient

Gossypium Gossypol male contraceptives

Heliotropium indicum Heliotrine antitumor, hypotensive

Hemsleya amabilis Hemsleyadin antidysenteric, antipyretic

Holarrhena antidysenterica Conessine conkurchine amebicide, anesthetic, hypotensive, vasodilator

Huperzia serrata Huperzine anticholinesterase

Hypericum spp. Hypericin antiretroviral

Juglans spp. Juglone anthelmintic

Justicia adhatoda Vasicine oxytocic, expectorant

Larrea divaricata Nordihydroguariaretic acid antioxidant

Lycoris squamigera Galanthamine cholinesterase-inhibitor

Macleaya cordata Sanguinarine antiplaque

Mentha spicata Menthol rubefacient anesthetic

Momordica charantia Charantin antidiabetic

Montanoa spp. Extracts contraceptive

Morus alba Morin myorelaxant

Mucuna pruriens Bufotenine , L-Dopa anticholinesterase ,antiparkinsonian

Musa paradisiaca Fruit pulp antiulcer

Ocotea glaziovii Glaziovine antidepressant

Olea europaea Olive oil mono-unsaturates

Paeonia albiflora Paeoniflorin antiinflammatory

Peumus boldus Boldine, benzylbenzoate scabicide

Phyllanthus spp. Phyllanthoside antitumor

Picrorrhiza kurroa Kuskin, cinnamic acid, vanillic acid, apocyanin choleretic, laxative

Plantago ovatum Psyllium husks, mucilage hypocholesterolemic

Platycodon grandiflorum Platycodin analgesic, antitussive

Pterocarpus marsupium Epicatechin antidiabetic

Quisqualis indica Quisqualic acid ascaricide

Rheum rhaponticum Anthraquinones emodin Micropropagation Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 37

(Table 1) contd….

Species Some active ingredient(s) Action

Rhododendron molle Rhomitoxin tranquilizer, hypotensive

Rorippa indica Rorifone antitussive

Salix alba Salicin, salicylic acid analgesic

Saussurea lappa Saussurine bronchiorelaxant

Silybum marianum Silymarin antihepatotoxic

Simarouba glauca Glaucarubin antiamebic

Stephanie tetrandra Tetrandrine hypotensive

Tabebuia spp. Lapachol antitumor

Taxus brevifolia Taxol antitumor

Theobroma cacao Theobromine, caffeine CNS, stimulant

Thymus vulgaris Thymol spasmolytic

Trichosanthes kirilowii Trichosanthin abortifacient

Tripterygium wilfordii Wilfordine (antitumor

Urginea maritima Scillaren A cardiotonic

Valeriana officinalis Valepotriates tranquilizer

Vinca minor Vincamine cerebrotonic, hypotensive

Warburgia ugandensis Polygodiol antifeedant antiyeast

Zea mays Cornsilk diuretic

Zingiber officinale zingerone analgesic, anti-inflammatory, antipyretic

Micropropagation of various plant species, including In the search for alternatives to production of desirable me- many medicinal plants, has been reported. Propagation from dicinal compounds from plants, biotechnological ap- existing meristems yields plants that are genetically identical proaches, specifically, plant tissue cultures, are found to have with the donor plants. Tissue culture technique has been potential as a supplement to traditional agriculture in the widely accepted as a tool for biotechnology for vegetative industrial production of bioactive plant metabolites. Secon- propagation of plants of agricultural, horticultural and for- dary metabolites are compounds produced in plants that are estry importance [14, 20]. Biotechnological tools are impor- not necessary for the plants basic functions although some tant for multiplication and genetic enhancement of the me- act as chemical defenses against microbes and animals. Sec- dicinal plants by adopting techniques such as in vitro regen- ondary metabolites are used in the pharmaceutical industry eration and genetic transformations. It can also be harnessed as flavorants and dyes, and in perfumery. The development for production of secondary metabolites using plants as and commercialization of medicinal plant-based bioindus- bioreactors. The Centre for Science and Technology of the tries in the developing countries is dependent upon the avail- Non-Aligned and other Developing Countries in India orga- ability of facilities and information concerning upstream and nized an international workshop on Tissue Culture of Eco- downstream bioprocessing, extraction, purification, and nomic Plants in April, 1994, as a better and quicker means of marketing of the industrial potential of medicinal plants. As using modern biotechnological techniques to nurture and conventional growing of medicinal plants is relatively ex- conserve medicinal plants. pensive, production of medicinal and/or other compounds This paper reviews the achievements and advances in the can be elicited in vitro. application of tissue culture for the in vitro regeneration of The production of secondary metabolite in vitro can be medicinal plants from various explants and enhanced pro- possible through plant cell culture. Successful establishment duction of secondary metabolites. of cell lines capable of producing high yields of secondary compounds in cell suspension cultures have been reported PRODUCTION OF SECONDARY METABOLITES [107]. Pharmaceuticals from Natural Plants Progress in the field Cell suspension culture systems could be used for large of pharmaceuticals have been spectacular. Most valuable scale culturing of plant cells from which secondary metabo- phytochemicals are products of plant secondary metabolism. lites could be extracted. The advantage of this method is that 38 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al. it can ultimately provide a continuous, reliable source of quantities from globular cell suspension cultures of Cin- natural products. Discoveries of cell cultures capable of pro- chona ledgeriana. Enhanced indole alkaloid biosynthesis in ducing specific medicinal compounds at a rate similar or the suspension culture of Catharanthus roseus has also been superior to that of intact plants have accelerated in the last reported [26, 57,71]. Ravishankar and Grewal [82] reported few years. New physiologically active substances of medici- that the influence of media constituents and nutrient stress nal interest have been found by bioassay. It has been demon- influenced the production of diosgenin from callus cultures strated that the biosynthetic activity of cultured cells can be of Dioscorea deltoidea. Parisi et al. [69] obtained high yields enhanced by regulating environmental factors, as well as by of proteolytic enzymes from the callus tissue culture of gar- artificial selection or the induction of variant clones. Some of lic (Allium sativum L.) on MS medium supplemented with the medicinal compounds localized in morphologically spe- NAA and BAP. Pradel et al. [73] observed that the biosyn- cialized tissues or organs of native plants have been pro- thesis of cardenolides was maximal in the hairy root cultures duced in culture systems not only by inducing specific orga- of Digitalis lanata compared to leaf. The production of nized cultures, but also by undifferentiated cell cultures. The azadirachtin and nimbin has been shown to be higher in cul- possible use of plant cell cultures for the specific biotrans- tured shoots and roots of Azadirachta indica compared to formations of natural compounds has been demonstrated field grown plant. Pande et al. [68] reported that the yield of [32,33]. Due to these advances, research in the area of tissue lepidine from Lepidium sativum Linn. depends upon the culture technology for production of plant chemicals has source and type of explants. bloomed beyond expectations. The major advantages of a Research in the area of plant tissue culture technology cell culture system over the conventional cultivation of has resulted in the production of many pharmaceutical sub- whole plants are: stances for new therapeutics. Many of the drugs sold today (1) Useful compounds can be produced under controlled are simple synthetic modifications or copies of the naturally conditions independent of climatic changes or soil obtained substances. The evolving commercial importance conditions; of secondary metabolites has in recent years resulted in a (2) Cultured cells would be free of microbes and insects; great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant me- (3) The cells of any plants, tropical or alpine, could easily tabolites by means of tissue culture technology. Transgenic be multiplied to yield their specific metabolites; hairy root cultures have revolutionized the role of plant tis- (4) Automated control of cell growth and rational regula- sue culture in secondary metabolite production. They are tion of metabolite processes would reduce of labor unique in their genetic and biosynthetic stability, faster in costs and improve productivity; growth, and more easily maintained. Using this methodology a wide range of chemical compounds have been synthesized. (5) Organic substances are extractable from callus cultures. Bioreactors [100] are the key step towards commercial production of secondary metabolites by plant biotechnology. Plant cell culture technologies were introduced at the end Bioreactors have several advantages for mass cultivation of of the 1960’s as a possible tool for both studying and pro- plant cells: ducing plant secondary metabolites. Different strategies, using an in vitro system, have been extensively studied to i. It gives better control for scale up of cell suspension improve the production of plant chemicals. In order to obtain cultures under defined parameters for the production of high yields suitable for commercial exploitation, efforts have bioactive compounds; focused on isolating the biosynthetic activities of cultured ii. Constant regulation of conditions at various stages of cells, achieved by optimizing the cultural conditions, select- bioreactor operation is possible; ing high-producing strains, and employing precursor feeding, transformation methods, and immobilization techniques. iii. Handling of culture such as inoculation or harvest is easy and saves time; Advances in the area of cell cultures for the production of iv. Nutrient uptake is enhanced by submerged culture con- medicinal compounds has made possible the production of a ditions, which stimulate multiplication rate and higher wide variety of pharmaceuticals like alkaloids, terpenoids, yield of bioactive compounds; and steroids, saponins, phenolics, flavanoids, and amino acids. Successful attempts to produce some of these valuable v. Large number of plantlets are easily produced and can pharmaceuticals in relatively large quantities by cell cultures be scaled up. are discussed below. The accumulation of secondary prod- Since the biosynthetic efficiency of populations varies, a ucts in plant cell cultures depends on the composition of the high yielding variety should be selected as a starting mate- culture medium, and on environmental conditions. Strategies rial. The fundamental requirement in all this is a good yield for improving secondary products in suspension cultures, of the compound, and reduced cost compared to the natural using different media for different species, have been re- synthesis by the plants. ported [88,69]. The production of secondary metabolites in plant cell suspension cultures has been reported from various The bioreactor system has been applied for embryogenic medicinal plants. The production of solasodine from calli of and organogenic cultures of several plant species. Significant Solanum eleagnifolium, and pyrrolizidine alkaloids from root amounts of sanguinarine were produced in cell suspension cultures of Senecio sp. are examples. Cephaelin and emetine cultures of Papaver somniferum using bioreactors. Ginseng were isolated from callus cultures of Cephaelis ipecacuanha. root tissue cultures in a 20 tonne bioreactor produced 500 Scragg et al. [88] isolated quinoline alkaloids in significant mg/L/day; of the saponin that is considered as a very good Micropropagation Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 39 yield. Bioreactors offer optimal conditions for large-scale 25kg/year. These figures represent the local as well as export plant production for commercial manufacture. Much pro- market demands. Vinblastine (Fig. 1) is the first drug of gress has been achieved in the recent past on optimization of choice in many forms of leukemia and since the 1950s’ it has these systems for the production and extraction of valuable increased the survival rate of childhood leukemias by 80%. medicinal plant ingredients such as ginsenosides and Another antileukemic drug, Vincristine (Fig. 1) has been shikonin. Roots cultivated in bioreactors have been found to discovered in the Madagascar periwinkle. Companies like release medicinally active compounds, including the antican- Cipla and Tamilnadu Dada and Tamilnadu Herbals Ltd.of cer drug isolated from various Taxus species, into the liquid India produce currently about 15-18 kg of vinblastine. In media of the bioreactor, which may then be continuously addition compounds like vindolin can be isolated but the extracted for pharmaceutical preparations. Conventional exact market potential of vindolin is still untapped. practices require the harvest of the bark of trees, all ap- Plant regeneration from shoot and stem meristems has proximately 100 years old, to obtain 1 kg of the active com- yielded encouraging results in Catharanthus roseus [60]. pound taxol. Research over the last two decades has estab- Rapid in vitro multiplication of plants from mature nodal lished efficient protocols for isolated cell cultures and a explants of Catharanthus roseus have been reported [56]. large-scale bioreactor system. The acceptance of this process The effects of auxins, cytokinins, different nutrient media for the industrial production of this invaluable compound has and explant sources on callus induction on shoot multiplica- recently been established and will significantly impact the tion of Catharanthus roseus L.G. Don plant have been stud- production of the tumor-inhibiting pharmaceutical. ied [3]. In vitro and in vivo cultivation has been a source to study alterations in the growth of a Catharanthus roseus L. MICROPROPAGATION OF SELECTED MEDICINAL callus tissues [106] and cytological investigation [45] have PLANTS also been studied. Tissue culture is basically defined as in vitro growth of plantlets from any part of the plants in suitable nutritive cul- N ture medium. It is also known as ‘micropropagation’ in sci- CH2CH3 entific technology. Callus-mediated organogenesis and regeneration through somatic embryogenesis are the usual OH mode of regeneration by tissue culture. The induction of N R2 callus growth and subsequent differentiation, organogenesis H and somatic embryogenesis are accomplished by the differ- H3CO ential application of growth regulators and the control of N conditions in the culture medium. With the stimulus of en- dogenous growth substances or by addition of exogenous N H CH CH growth regulators to the nutrient medium, cell division, and R1 2 3 cell growth and tissue differentiation are induced Micro- H OH O CO CH3 propagation systems have been successfully developed for R2 many medicinal species such as Periwinkle (Catharanthus 1 2 roseus), Safed Musli (Chlorophytum borivilianum) Brahmi Vinblastine: R =CH3, R =COOCH3 1 2 (Bacopa monieri) and Thorn apple (Datura metel). Vincristine: R =CHO, R =COOCH3

Periwinkle (Catharanthus roseus) Fig. (1). Vinblastine and vincristine. Catharanthus roseus (Apocynaceae) originated from Madagascar, but has now been spread throughout the tropics In our lab also callus induction (Fig. 2A) from nodal explants was observed on Murashige and Skoog [62] MS me- and subtropics by human activity.This plant is cultivated as -1 -1 an ornamental plant throughout tropical and subtropical ar- dium supplemented with NAA (0.2 mgl ) and KN(2mgl ). Multiple shoot proliferation (Fig. 2B) and shoot elongation eas and many parts of the world. Catharanthus is a plant (Fig. ) was observed on MS medium supplemented belonging to the family, well known for being rich in alka- 2C,D with NAA (0.5mgl-1) and KN(2mgl-1). These shoots when loids. All parts of plant, especially, the leaves contain anti- transferred to MS medium supplemented with IBA (2mgl-1) neoplastic bisindole alkaloids, among those are vinblastine resulted in rooting. For alkaloid extraction, plant and vincristine, which are widely employed as chemothera- in vitro multiplication is an ideal approach to produce leaf material peutic agents against cancer. The absolute levels of vin- in large quantity. heavy metal stress on callus culture blastine and vincristine are considered far too low to explain In vitro [105] and regeneration of pathogen free [40.59] healthy the activity of crude extracts of Catharanthus. Various stud- plants through callus culture have been reported. ies show the presence of other antineoplastic alkaloids [72,86] in the plant. Though its primary traditional use was A number of nutritional factors as well as the growth for people with diabetes, Catharanthus also has anticancer factors 2,4-D and IAA were studied to determine their influ- effects. In the intact plant, vindoline is localized in the leaves ence on growth and alkaloid formation [36,57,58,71,88] in and catharanthine is found in both the leaves and roots. Ex- Catharanthus roseus suspension cultures. Zhao J, et al. [109] tracts obtained from the plants like Catharanthus roseus studied the effects of stress factors, bioregulators, and syn- have found a tremendous export potential. The demand for thetic precursors on indole alkaloid production in compact vinblastine [$75/gm] and vincristine [135 $/gm] isolated callus clusters cultures of Catharanthus roseus. Large scale from Periwinkle or Catharanthus roseus is around 20- fermentation and alkaloid production of cell suspension cul- 40 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al.

A B

C D

Fig. (2). Catharanthus roseus. (A) Callus induction from nodal segments, cultured on MS +(in mgl-1) NAA (0.2) + KN (2). (B) Multiple shoot proliferation on subculturing on MS +(in mgl-1) NAA (0.5)+ KN (2). Multiple shoots after 4 weeks; (C)Shoot elongation and subculturing on same media, after 5 weeks; (D) Plantlets,6 weeks old. tures has also been reported [26, 86,109]. Reports on various tion by transformed root cultures [70] and use of elicitors factors on the release of secondary metabolites from roots of [26] has also been performed. Quantitative analysis of vari- Datura stramonium, Catharanthus roseus, and Tagetes ous alkaloids produced in vitro and in vivo has been exten- patula cultured in vitro like effect of the medium pH [85], sively done [10, 16,19,25] in Catharanthus roseus. sucrose [53] have been reported. Enhanced alkaloid produc- Micropropagation Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 41

Efficient development and germination of somatic em- safed musli goes into over 200 ayurvedic preparations, it is bryos are prerequisites for commercial plantlet production. known in countries like Saudi Arabia as an effective Indian Lowering of growth regulator concentrations in culture me- Herbal Viagra. dia has improved embryo development and germination of The origin of Safed Musli can be traced back in the old- many medicinal plants. Germination of the somatic embryos est mountain ranges on the continent, the Aravalis from is achievable on MS medium without the growth regulator. where it spread to the near-by areas of the sub-continent, High frequency plant regeneration from anther-derived cell presently grown in the states of Gujrat, Rajasthan, Madhya suspension cultures via somatic embryogenesis in Ca- Pradesh and the Central Deccan Plateau [38] and is thus en- tharanthus roseus [41] as well as somatic embryogenesis and demic to India. The genus includes about 300 species, which indole alkaloid production [72] have been observed in vitro are distributed throughout the tropical and subtropical parts .The suspension culture of Catharanthus roseus from stem of the world. Tropical and subtropical Africa is the probable and leaf explants on medium containing NAA and kinetin Centre of Origin of the genus, where about 85% of the spe- has been established by Zhao et al. [110]. Somatic hybridi- cies are found. It is now widely cultivated in different parts zation and long term stability of electromanipulated proto- of India on commercial basis. In India, about 13 species are plasts of var. Acme and a Glycine max Catharanthus roseus reported [42], of which seven species (C. tuberosum, C. mutant [85] has been reported. The effects of various heavy arundinaceum, C. breviscapum, C. attenuatum, C. laxum, C. metals cell suspension cultures like cobalt (II) and nickel (II) borivilianum and C. malabaricum) are commonly observed. [111], aluminum [55, 110] and the regulation of DNA syn- Nowadays, there is a vast demand of C. borivilianum all over thesis and cell division by polyamines in Catharanthus the world (specially gulf countries and cold countries). There suspension cultures [54] has been studied. roseus are around 256 varieties of Chlorophytum in the world, which are known yet. The dried roots of C. borivilianum are Safed musli, Chlorophytum borivilianum the products of commerce. Dried powdered roots or its ex- Chlorophytum borivilianum (Liliaceae) commonly tracts are widely used in several herbal tonics [78]. The plant known as Safed musli is among the few rare medicinal plants yields a flavonone glycoside, which is a powerful uterine [65] witnessing steadily growing use in pharmaceutical, stimulant. Dried roots of Chlorophytum contain 42% carbo- phyto-pharmaceutical and nutraceutical products. The Me- hydrate, 80–89% protein, 3–4% fiber and 2–17% saponin dicinal Plants Board has recognized Safed Musli as sixth [104]. Research studies on Chlorophytum conducted in India among the 28 selected priority medicinal plants, important and elsewhere indicate that saponins [38] are responsible for herb to be protected and promoted. The Board encourages medicinal properties. A saponin (Fig. 3) is a category of mainstream cultivation of Safed Musli by farmers by ex- phytonutrients (plant nutrients) found abundantly in many tending a subsidy of 20% through National Horticultural beans, and other plants such as ginseng, alfalfa, yucca, aloe, Board on project cost. The steadily growing demand for quinoa seed and also in Safed musli. Saponins in safed musli Safed Musli has caused its price to soar to Rs.1,500 per Kg are known to ensure the defense of the plant against micro- in the market [21], resulting in over exploitation through bial or fungal attack. Several drugs owe their anti- wild harvesting pushing the plant to the verge of extinction. inflammatory and antiedemic properties to saponins. While the demand for Safed Musli is over 35,000 tons per annum, the supply is only to the tune of 5,000 tons a year. Chlorophytum borivilianum Santapau & Fernandes is an important herbaceous, medicinal plant found in the forests of Madhya Pradesh and Gujarat states of India. It is a member COO Gic of the Liliaceae family and perennates by fleshy roots at- 28 tached to a condensed stem disc, which remains dormant in 3 the soil for about 7 months. These are fascicled, sessile, cy- Gic O lindrical, variable in number and length, brown to black skinned and white after peeling. The fleshy roots are the economically important part, which contain saponins and are Fig. (3). Saponin. used for the preparation of many Ayurvedic tonics prescribed against general debility. The roots fetch an attractive Similarly, many saponin containing drugs are tradition- market price at present (Rs. 900–1200/kg of dry roots). In ally used for their antitussive and expectorant properties. nature, the plant regenerates soon after or immediately be- Low seed set, poor seed viability, high dormancy and low fore the rainy season (May–June) from the previous year’s percentage of seed germination are some of the problems in dormant root present in the soil. propagation of some medicinal plants. Seeds set are ex- tremely poor in safed musli. Recently, Fusarium solani was The many therapeutic applications of Safed Musli in- identified for the first time as causing a tuber-rot in safed clude as a restorative and a health-giving tonic; a curative for musli [77]. To encourage its adoption as a crop plant, propa- pre-natal and post-natal illness; and a remedy for diabetes, gation and cultural methods have been investigated [44]. To arthritis etc. Safed Musli supports physiological balance or overcome this problem, recently one of the rapidly expand- homeostasis. It improves access to energy stores, helps to ing areas of pharmacognosy has involved the application of improve stamina and resistance to stress and tiredness, helps tissue culture of plant cells, tissues and organs in the study of in mobilization of the bodys’ nutritional resources, helps to medicinal plants [103]. Therefore attempts were made to normalize various indices such as blood pressure. While propagate the material by in vitro methods. This method 42 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al. holds tremendous potential for the production of high-quality grows naturally in wet soil, shallow water, and marshes. The plant-based medicine. herb can be found at elevations from sea level to altitudes of Extensive work has been done on several ornamental 4,400 feet, and is easily cultivated if adequate water is avail- species of family Liliaceae [46,89,102] but very few medici- able. Flowers and fruit appear in summer and the entire plant is used medicinally. nal plants of this family have been cultured in vitro [39]. Biotechnological research on Chlorophytum borivilianum Bacopa monniera is traditionally prescribed in Ayurveda, has been supported previously by central funding agencies. a holistic system of medicine originating from India. Practi- In nature it is a slow growing plant. Perusal of literature tioners of Ayurveda classify Bacopa as a natural sedative and shows only a few reports on in vitro studies in C. borivil- mood stabilizer, which is most often used to mildly relax the ianum [20,74,75,99]. Initially different explants like leaf Central Nervous System (CNS) as well as, like focus and base segments, stem disc with shoot meristem, sliced root memory. Traditionally, it was used to support healthy cogni- tuber and young shoot buds were used as explants [37]. tive functions, brain tonic to enhance memory development, learning, and concentration, and to provide relief to patients In our laboratory, regeneration of C. borivilianum from both young shoot buds as well as inflorescence axis bearing with anxiety or epileptic disorders. The plant has also been an axillary bud gave good result [22]. Regeneration was suc- used in India and Pakistan as a cardiac tonic, digestive aid, and to improve respiratory function in cases of bronchocon- cessful both from the inflorescence axis (Fig. 4A) and young striction [15]. Brahmi may be useful for people who want to shoot buds (Fig. 4B) on MS basal medium supplemented -1 improve mental function and concentration particularly un- with (1-5 mg l ) BAP .In some cases, callusing and multiple der pressure or in stressful conditions Research on anxiety, shoot proliferation was also observed. These cultures later epilepsy, bronchitis and asthma, irritable bowel syndrome, gave rise to 24 -35 shoots per culture from segments of inflo- and gastric ulcers also supports the Ayurvedic uses of Ba- rescence axis (Fig. 4C) and 10-13 shoots per culture from copa. Bacopa’s antioxidant properties may offer protection young shoots buds (Fig. 4D) on MS medium containing 4mgl-1 BAP. The healthy shoots were transferred to 1/2 MS from free radical damage in cardiovascular disease and certain types of cancer. All available medical literature [93, 94] medium containing auxins for rooting (Fig. 4E) at various concentrations (0.5-2.5mgl-1) of IBA [23]. Rooted plantlets confirms that there has been no toxicity report associated with the use of Bacopa as a dietary supplement in children or on agar solidified media were removed and subjected to dif- adults [18]. ferent modes of hardening and acclimatization (Fig. 4F). Brahmi is especially suitable for students as it enhances Somatic embryogenesis in callus culture of Chlorophy- the minds ability to learn and to focus [95]. In India the plant tum borivilianum was first observed in immature zygotic is used for all sorts of skin problems-eczema, psoriasis, ab- explants and intact ovules by Purohit et al. [76]. Callus in- th scess, ulceration, it is said to stimulate the growth of skin, duction was observed in embryos on the 10 day after their hair and nails [51]. studies on anticancer studies of inoculation on the MS medium containing 4.52mm-22.6mm In vitro of 2,4-D.However the best response was observed in the Bacopa monniera [28] have also been performed. Other studies have confirmed that Brahmi has a calming effect for presence of 4.52mm of 2,4-D. Subculture to MS medium stress and has become the herb of choice for attention deficit containing low concentration 2,4-D (1.13mm) and 567.0mm of ascorbic acid initiated faster growth of embryogenic cul- disorder A.D.D. in hyperactive children. The results of re- tures. Mature somatic embryos when transferred to medium cent laboratory studies suggest that, in addition to its mood- stabilizing effects, Bacopa also exhibit activity, without growth regulators, they germinate only when kept in adaptogenic light. The inoculated embryos gave rise to shoots and ulti- which is thought to naturally increase the bodys’ resistance mately roots along with a mixed population of precociously to emotional stress and mental exhaustion [101]. Researchers germinating embryos showing profuse rhizogenesis. Encap- have categorized multiple phytochemical compounds derived from extract, including (Fig. ), sulation of young shoots buds and plantlets regeneration Bacopa monniera Bacosides 5 . The active constituents in Brahmi have been reported by Dave et al. [21]. in Chlorophytum Alkaloids and Glycosides are the all important steroidal saponins [79,80,81,83,91]. borivilianum. Four millimeter long shoot buds encapsulated in 3.0%sodium alginate matrix polymerized by 100mM so- The ontogenic and seasonal variation in accumulation of lution of CaCl2.2H2O yielded best results. Recently, Fluores- bacoside A in Bacopa monniera has been categorized [29]. cence In Situ Hybridization (FISH) technique has been ap- A new 13,14-seco-steroid Bacosterol and bacosine, a new plied on somatic chromosomes and extended DNA fibers in triterpene from Bacopa monniera [2] have also been iso- the medicinally important species of Chlorophytum to eluci- lated. A simple sensitive HPTLC method have been devel- date physical localization and measurement of the rDNA oped for the analysis of bacoside A in the plant Bacopa sites using two rRNA multigene families homologous to 45S monnieri and in its commercial monoherbal capsule formu- and 5S rDNA [47]. lation. The proposed HPTLC method provides a faster and cost effective qualitative control for routine analysis of baco- Water Hyssop, Bacopa monniera side in formulations containing Bacopa monnieri saponins. Bacopa monniera, also referred to as Bacopa monnieri, An effective protocol for mass propagation of Bacopa Herpestis monniera, water hyssop, and “Brahmi”, has been monnieri (L.) Pennell, an important medicinal plant [90] was used in the Ayurvedic system of medicine for centuries. Ba- developed using shoot tips and nodal segments as explants. copa monniera, a member of the Scrophulariaceae family, is Shoot regeneration and somatic embryogenesis from differ- a small, creeping herb with numerous branches, small oblong ent explants of Brahmi.The explants were cultured on Mura- leaves, and light purple flowers. In India and the tropics it shige and Skoog's medium supplemented with various aux- Micropropagation Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 43

A B

C D

E F

Fig. (4). Chlorophytum borivilianum (A) Inflorescence axis bearing axillary node on MS + (in mgl-1) BAP(5), after 2 weeks. (B) Young shoot buds on MS + (in mgl-1) BAP(4), after 2 weeks. (C) Multiple shoot proliferation (24 -35 shoots per culture) and elongation on MS + (in mgl-) BAP(4) from segments of inflorescence axis. (D) Shoot proliferation (10-13 shoots per culture) from young shoots buds and elongation on the same medium. (E) The healthy shoots were transferred for rooting to 1/2 MS medium + (in mgl-1) IBA(2). (F) Plants at pre-flowering stage. ins, cytokinins either alone or with coconut milk and auxins generated shoots developed roots on the same medium. In plus cytokinins. Multiple shoots were obtained on MS me- our lab, similar observation was noticed. The initiation of dium supplemented with auxins or/and cytokinins with or shoot proliferation (Fig. 6A,B,C) from nodal explants was without coconut milk. Maximum number of plants were ob- observed on MS medium supplemented with BAP(1mgl-1) tained on medium containing KN/2-ip (0.1 mg/l) and KN (1 and KN (4mgl-1). When these were subcultured on MS me- -1 mg/1) in shoot tip and nodal cultures, respectively. The re- dium supplemented with BAP (5mgl ) multiple shoot. Bud 44 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al. proliferation was further enhanced .They were found to effects of the drug. Another type of Datura, called Atlinan by originate ,associated with or without roots. Shoot elongation the Aztecs, enjoyed a particularly sacred status. It was re- (Fig. 6D) was observed on the same medium. These shoots garded as the sister of Ololuiqui hallucinogenic plant. These when transferred to 1\2 MS medium supplemented with IBA plants were so sacred that only the priests were allowed to (2mgl-1) resulted in rooting. Regenerated plantlets were use them. With their help they held counsel with the Gods - transferred to soil after a very brief period of hardening. The divining the outcome of future events, discovering the regenerated plants are dark green in colour and more robust whereabouts of lost or stolen objects and prognosticating the in their growth than the normal plants. These characters can causes of diseases, especially if black magic was suspected. be exploited further to determine somaclonal variations. As a medicinal remedy they prepared an ointment for cracked soles and injured feet, made plasters for ulcers, pus- tules and infected wounds and skin sores, and used it for poultices to treat rheumatic aches and pains. Here too, the OH ancient use of Datura as a powerful aphrodisiac is clearly implied. O All species of Datura contain powerful alkaloids, which in sufficient quantities have the power to kill. The main alkaloids represented are Scopolamine, Hyoscyamine and At- HO ropine (Fig. 7). Self-experimentation is not recommended O HO O HO O O and must be strictly avoided by anyone who suffers any kind O HO of heart condition. O OH O HO The effects are stimulating on the central nervous system and simultaneously depressing on the peripheral nerves. Symptoms include an increased heart rate, drying up of the HO OH mucus membranes, a dry throat and sometimes cramps. At first the effects are arousing, sometimes manifesting as un- Fig. (5). Bacoside. controlled talking or laughing, forgetfulness and indulging in senseless repetitive activities. Vivid hallucinations and de- Morphogenetic response [8] and isozymes of Bacopa lirious illusions may also occur. Occasionally the effects can monnieri cultivars grown under different conditions like salt stress [6], bioaccumulation and biochemical effects of mer- produce extreme violence and destructive urges. The period of agitation is usually followed by a deep prolonged sleep cury [98], copper stress [7], influence of cadmium [4] and accompanied with vivid dreams and hallucinations, often of zinc [33] on growth and photosynthesis of Bacopa monniera a sexual nature. Upon awakening one might experience a cultivated in vitro has been identified. The application of high-performance liquid chromatography coupled to nuclear distinct hangover and a total lack of memory as to what ac- tually happened during the state of altered consciousness. magnetic resonance spectrometry, mass spectrometry and bioassay has been performed for the determination of active All parts of the plant are anodyne, antispasmodic, hallu- saponins in Bacopa monniera [37]. A tracer technique have cinogenic, hypnotic and narcotic. It has been used in the past been used to evaluate bacoside A, in various Bacopa mon- as a painkiller and also in the treatment of insanity, fevers niera accessions [30]. Dammarane- type triterpenoid sapon- diarrhoea and skin diseases. The plant contains several alka- ins [31] and Bacopasaponin D, a pseudojujubogenin glyco- loids, the most active of which is scopolamine. This is a po- side [32,49,52]. tent cholinergic-blocking hallucinogen, which has been used to calm schizoid patients. The leaves contain 0.52% sco- Thornapple, Datura metel polamine, the calices 1.08%, the stems 0.3%, the roots 0.39%, the fruits 0.77%, the capsules 0.33%, the seeds Since time immemorial various Datura (Solanaceae) spe- 0.44% and the whole plant 0.52 - 0.62%. Any use of this cies have been referred sacred visionary plants by practically plant should be with extreme caution and under the supervi- all cultures that have come into contact with it. Their distri- sion of a qualified practitioner since the toxic dose is very bution spans all warm and tropical regions of the world. close to the medicinal dose. Daturas usually grow as herbaceous annuals/perennials. The most striking feature, shared by all species are the beautiful Tissue cell culture study of Datura metel [24] has been trumpet-like flowers, ranging in color from white to pinkish performed extensively. In vitro plant regeneration was purple, and in some varieties even to bright golden yellow achieved in Datura metel from nodal explants collected from and red. The flowers exude a beautiful, narcotic scent, espe- both in vitro germinated seedlings and field grown plants (in cially at night. The seed capsules of the Datura species are vivo). The explants were cultured on MS medium with BAP typically the size of a walnut and are covered with thorns (0.5-3.0 mgl-1) and NAA (0.5 mgl-1). The nodal explants that may become quite sharp and spiky as the plant matures. isolated from in vivo source exhibited a greater number of The appearance of these seed-capsules has given rise to the healthy multiple shoots than in vitro. BAP at 3 mgl-1 with English common name, ‘Thorn apple’. It was used as a pain- NAA at 0.5 mgl-1 was found to be optimal for regeneration of killer in certain initiation rituals and given as a narcotic to shootlets [63]. Arockiasamy, D. I., B. Muthukumar, et al. [9] the ritual sacrifices. For this purpose the preferred method of observed in vitro plant regeneration from internodal seg- administration was either by enema or as a rolled-up leaf ments of Datura metel L. Effect of carbon source on Datura suppository, which reduces some of the less pleasant side metel microspore embryogenesis and the growth of callus Micropropagation Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 45

A B

C D

Fig. (6). Bacopa monniera: (A) Nodal segments cultured on MS + (in mgl-1) BAP (1)+ KN (4). Appearance of shoot buds after 2 weeks of culture. (B) Multiple shoots, after 3 weeks. (C) Further growth of B, after 4 weeks. (D) On subculturing on MS+ (in mgl-1) BAP(5) multiple shoot were further found to originate ,associated with or without roots. Shoot elongation was observed on the same medium. raised from microspore-derived embryos [11] have been 4mgl-1). Shoot elongation was observed on MS medium sup- initiated. They have also studied the effects of using acti- plemented with NAA (0.5mgl-1) and KN(2mgl-1). These vated charcoal on microspore embryogenesis in Datura metel shoots when transferred to MS medium supplemented with [12]. IBA (1-2mgl-1) resulted in rooting. For alkaloid extraction, in High frequency plant regeneration from anther derived vitro plant multiplication is an ideal approach to produce leaf material in large quantity. cell suspension culture via somatic embryogenesis was observed in Datura metel. Somatic embryos were found to be The characterization of the chemical constituents of Da- initiated from anther at the uninucleate stage in liquid MS tura metel Linn.genotypes and alkaloid contents of Datura medium supplemented with 2,4-D (0.2-0.4mgl-1). When sub- metel varieties [5] revealed the variation of scopolamine and cultured after 3 days on semisolid liquid MS medium sup- atropine in different parts of the plant during development plemented with 2,4-D (0.2-0.4mgl-1). These embryos became [1] and new withanolides, daturametelins C, D, E, F and G- green and shoot buds (Fig. 8A) stated initiating. Multiple Ac from Datura metel L. [92]. The structures of atropine, shoot proliferation (Fig. 8B) was observed on subculturing scopolamine [64], withametelin and isowithametelin, witha- on MS medium supplemented with BAP (2-5mgl-1) alone nolides [96] from Datura has been characterized. Pharma- and in combination of NAA (0.2-0.5mgl-1) and Kinetin (2- cognostical studies, variation of alkaloid contents in the 46 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al.

CH3

N H CH OH 3 N H

H O H CH3 C O H OC CH N OH H O O CH2OH

C H NO 17 23 3 H O O Fig. (7). Hyosycamine, Atropine, Scopolamine.

Fig. (8). Datura metel: (A) Young embryos originated from anther (uninucleate stage) in liquid MS + (in mgl-1) 2-4-D (0.2) was subcultured after on semisolid liquid MS+ (in mgl-1) 2,4-D (0.4). These embryos became proliferative and led to shoot development. (B) Multiple shoot proliferation along with callusing was observed on MS + (in mgl-1) NAA (0.5) and Kinetin (4). Micropropagation Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 47 seeds [66] and toxic effects of alkaloid extracts of Strychnos higher plants as renewable sources of chemicals, especially nux-vomica, Datura metel and Argemone mexicana on the medicinal compounds. behaviour of chromatophores and mucous glands of Indian Advances in tissue culture, combined with improvement minor carp [96] have been done. In vitro production of tro- in genetic engineering, specifically transformation technol- pane alkaloids was also observed in Datura metel [17]. Hairy ogy, has opened new avenues for high volume production of root lines of Datura metel were established following infec- pharmaceuticals, neutraceuticals, and other beneficial sub- tion of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. stances. Recent advances in the molecular biology, enzy- The growth and production of hyoscyamine and scopolamine mology, and fermentation technology of plant cell cultures suggest that these systems will become a viable source of (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts. Application of important secondary metabolites. Genome manipulation is short GC column [50] in the analysis of some tropane alka- resulting in relatively large amounts of desired compounds loids in plants have also been tried. produced by plants infected with an engineered virus, whereas transgenic plants can maintain constant levels of production of proteins without additional intervention. CONCLUSION Large-scale plant tissue culture is found to be an attractive Micropropagation holds tremendous potential as a tool alternative approach to traditional methods of plantation as it for the production of high quality plant-based medicines. offers a controlled supply of biochemicals independent of Medicinal compounds from plants represent one of the larg- plant availability. We hope that continuation and intensifica- est and most diverse groups of plant secondary metabolites. tion efforts in this field will lead to controllable and success- The advent of advanced bioinformatics tools and modern ful biotechnological production of specific, valuable, and as genetic technology allowed manipulation of biosynthetic yet unknown plant chemicals. pathways with the potential of generating novel chemical entities. First, public databases of secondary metabolite re- ACKNOWLEDGEMENT lated enzymes were interrogated to identify relevant plant genes from the plant and other species for the production of We are grateful to the CEO and Director, Mr. Sandeep high-quality plant-based medicine. Protocols have been de- Bakshi for providing the facilities for pursuing the research veloped for clonal multiplication of hundreds of plant spe- work. cies, which include trees, medicinal and aromatic plants and endangered species from several laboratories across the ABBREVIATIONS country. Tissue culture protocols have been developed for BA = 6-Benzylaminopurine several plants but there are many other species, which are over exploited in pharmaceutical industries and need conser- NAA = a -Naphthaleneacetic acid vation. It is now quite apparent that India has made big IAA = Indole-3 acetic acid strides in the realm of biotechnology. The galloping rate, at which this advancement is continuing, holds out great 2iP = 6-(g -Dimethylallylamino) purine promise for a better future. 2,4-D = 2,4-Dichlorophenoxyacetic acid In vitro propagation of medicinal plants with enriched KN = Kinetin bioactive principles and cell culture methodologies for se- lective metabolite production is found to be highly useful for REFERENCES commercial production of medicinally important com- [1] Afsharypuor, S. and Mostajeran, A. (1995) Planta Med., 61(4), pounds. The increased use of plant cell culture systems in 383-384. recent years is perhaps due to an improved understanding of [2] Ahmed, B. and Rahman, A. (2000) Indian J. Chem., 39B(8), 620- the secondary metabolite pathway in economically important 25. plants. Advances in plant cell cultures could provide new [3] Akcam, E. and Yurekli, A.K. (1995) Turkish J. Botany, 19(6), 569-572. means for the cost-effective, commercial production of even [4] Ali, G., Srivastava, P. S. and Iqbal, M. 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