Micropropagation: A Tool for the Production of High Quality Plant-based Medicines

Current Pharmaceutical Biotechnology, 2006, 7, 33-49 33

1389-2010/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

Micropropagation: A Tool for the Production of High Quality Plant-basedMedicines

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

Tissue Culture Laboratory, Institute of Biotechnology and Allied Sciences, Seedling Academy of Design, TechnologyAnd 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. Planttissue culture techniques offer an integrated approach for the production of standardized quality phytopharmaceuticalthrough 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 devel-oped. Regeneration occurred via organogenesis and embryogenesis in response to auxins and cytokinins. The integratedapproaches 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

Recent and renewed interest in medicinal plants coupledto developments in information technology has fuelled anexplosion in the range and content of electronic informationconcerning medicinal plants as a re-emergent health aid.Medicinal plants, since times immemorial, have been used invirtually all cultures as a source of medicine. The widespreaduse of herbal remedies and healthcare preparations, as thosedescribed in ancient texts such as the Vedas and the Bible,and obtained from commonly used traditional herbs and me-dicinal plants, has been traced to the occurrence of naturalproducts with medicinal properties. Medicinal plants play akey role in world heath care systems [13]. Herbal medicineis one of the most remarkable uses of plant based biodiver-sity. As many as 75 to 90% of the worlds’ rural people relyon herbal medicine for their primary health care. The successof any health care system depends on the availability of suit-able drugs on a sustainable basis. Reserves of herbs andstocks of medicinal plants in developing countries are di-minishing and in danger of extinction as a result of growingtrade demands for cheaper healthcare products and newplant-based therapeutic markets in preference to more expen-sive target-specific drugs and biopharmaceuticals. Such con-cerns have stimulated positive legal and economic interest.

In several industrialized societies, plant-derived pre-scription drugs constitute an element in the maintenance ofhealth. Medicinal plants are an integral component of re-search developments in the pharmaceutical industry. Suchresearch focuses on the isolation and direct use of active

*Address correspondence to this author at the Tissue Culture Laboratory,Institute of Biotechnology and Allied Sciences, Seedling Academy of De-sign, Technology and Management, Jaipur-302025, India;E-mail: [email protected]

medicinal constituents, or on the development of semi-synthetic drugs, or still again on the active screening of natu-ral products to yield synthetic pharmacologically-activecompounds. In Germany, for example, over 1500 plant spe-cies encountered in some 200 families and 800 genera havebeen processed into medicinal products. In South Africa,likewise, some 500 species are commercialized trade prod-ucts. Today, Bulgaria, Germany and Poland are recognizedas major exporters of plant-based medicinal products. Indiahas about 45,000 plant species; medicinal properties havebeen assigned to several thousand. The world market forherbal derivatives /products is in the increase order of mag-nitude. In the U.S. and Europe the ecological movement hasbrought about a renewed interest in traditional medicines.High inflation rates in the third world have caused some citi-zens to return to or begin using herbal remedies, The pre-scription drug market is around $40 billion today, suggestinga value of about $10 billion for those drugs containing atleast one compound derived from higher plants. Even today,the World Health Organization estimates that up to 80 percent of people still rely mainly on traditional remedies suchas herbs for their medicines.

Medicine, in several developing countries, using localtraditions and beliefs, is still the mainstay of health care. Asdefined by WHO, health is a state of complete physical,mental, and social well being and not merely the absence ofdisease or infirmity. In Europe, some 1500 species of me-dicinal and aromatic plants are widely used in Albania, Bul-garia, Croatia, France, Germany, Hungary, Poland, Spain,Turkey, and the United Kingdom (Table 1) The practice oftraditional medicine is widespread in China, India, Japan,Pakistan, Sri Lanka and Thailand. In China about 40% of thetotal medicinal consumption is attributed to traditional tribalmedicines. In Thailand, herbal medicines make use of leg-umes encountered in the Caesalpiniaceae, the Fabaceae, and

34 Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 1 Debnath et al.

the Mimosaceae. In the mid-90s, it is estimated that receiptsof more than US$2.5 billion have resulted from the sales ofherbal medicines. And, in Japan, herbal medicinal prepara-tions are more in demand than mainstream pharmaceuticalproducts. In total, 75 species belonging to 42 families havebeen investigated and new ethnomedical information hasbeen obtained for 41 species [67]. Despite the increasing useof medicinal plants, their future, seemingly, is being threat-ened by complacency concerning their conservation. Glob-ally, the early part of the 20th century brought an evolutionof the pharmaceutical industry. With the progress in chemi-cal techniques, crude drugs came to be replaced by purechemical drugs and the developed countries witnessed a de-cline in popularity of medicinal plant therapy. However,during the recent past, the pendulum has swung again andthere is a resurgence of interest in study and use of medicinalplants. Many traditional plant based remedies are back in useand find increasing applications as:

(i) source of direct therapeutic agents,

(ii) as a raw material base for the elaboration of morecomplex semi-synthetic chemical compounds,

(iii) as models for new synthetic compounds, and

(iv) as taxonomic markers for the discovery of newcompounds.

Thus, plants are a tremendous source for the discovery ofnew products of medicinal value for drug development. To-day several distinct chemicals derived from plants are im-portant drugs currently used in one or more countries in theworld. A drug "Memory Plus" has recently been developedusing Brahmi (Bacopa monniera L.) by the scientists of theCentral Drug Research Institute, Lucknow, India. SimilarAyurvedic pills, for rejuvenating humans has come up in thename of "Thirty Plus", which use Ashwagandha (Withaniasomnifera L.) and Ginseng (Panax aquintifolium) as the in-gredients. Pudin Hara, another Ayurvedic drug containingmint (Mentha arvensis L.), which is used for indigestion, isavailable across the counter. The production, consumptionand international trade in medicinal plants, and phytomedici-nes, therefore, are growing and expected to grow in futurequite significantly. Consumption of herbal medicines iswidespread and increasing. Yet harvesting from the wild, themain source of raw material, is causing loss of genetic diver-sity and habitat destruction.

ECONOMIC BENEFIT

World wide, there has been a renewed interest in naturalproducts. The world market for plant-derived chemicals viz.pharmaceuticals, fragrances, flavors, and color ingredients,alone exceeds several billion dollars per year. Classic exam-ples of phytochemicals in biology and medicine includetaxol, vincristine, vinblastine, colchicine ,artemisinin, fork-olin, Saponin etc. Trade in medicinal plants is growing involume and in exports. The $60-billion global herbal marketis growing at a rate of 7 percent per annum and is expectedto generate $5 trillion by 2050. According to a WHO docu-ment, one fourth of the 500 million prescriptions written inthe US each year contain a mention of leafy plant extracts.The Indian climate is conductive to herbal cultivation. De-veloping and emerging countries besides catering to domes-

tic demands have the potential to improve export of plantdrugs. For example, China has attained a leader position inthe export of crude and Chinese drugs to the world marketand it is followed far behind by Singapore and India. Thebotanical market, inclusive of herbs and medicinal plants, inthe USA, is estimated, at retail, at approximately US$1.6billion p.a. China with exports of over 120,000 tonnes p.a.,and India with some 32,000 tonnes p.a. dominate the inter-national markets. It is estimated that Europe, annually, im-ports about 400,000 tonnes of medicinal plants with an aver-age market value of US$ 1 billion from Africa and Asia. Theindustry anticipates growth on the order of 15–20% into thenew millennium. A growing awareness of this new con-tributor to the foreign-exchange reserves of several nationaltreasuries is beginning to emerge. To satisfy growing marketdemands, surveys are being conducted to unearth new plantsources of herbal remedies and medicines at the same timedevelop new strategies for better yield and quality.

CONVENTIONAL PLANT-BREEDING ANDMICROPROPAGATION

Conventional plant-breeding methods can improve bothagronomic and medicinal traits. In vitro propagation or tissueculture of plants holds tremendous potential for the produc-tion of high-quality plant-based medicines. This can beachieved through different methods including micropropa-gation [106]. The evolving commercial importance of secon-dary metabolites has in recent years resulted in a great inter-est in secondary metabolism, particularly in the possibility ofaltering the production of bioactive plant metabolites bymeans of tissue culture technology. Plant cell culture tech-nologies were introduced at the end of the 1960's as a possi-ble tool for both studying and producing plant secondarymetabolites [78]. Different strategies, using an in vitro sys-tem, have been extensively studied to improve the produc-tion of plant chemicals.

Micropropagation has many advantages over conven-tional methods of vegetative propagation, which suffer fromseveral limitations. There has been significant progress in theuse of tissue culture and genetic transformation to alterpathways for the biosynthesis of target metabolites. Obsta-cles to bringing medicinal plants into successful commercialcultivation include the difficulty of predicting, which ex-tracts will remain marketable and the likely market prefer-ence for what is seen as natural source of extracts.

The most outstanding merits offered by micropropaga-tion, over the conventional methods are:

1. In a relatively short time and space large number ofplants can be produced, starting from single explant.

2. Unlike the conventional methods of plant propagation,micropropagation of even temperature species may becarried out throughout the year.

3. Tissue cultured plants are generally free from fungal andbacterial diseases. Virus eradication and maintenance ofplants in a virus-free-state are also readily achieved intissue culture.

4. The multiplication rate is greatly increased.

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


(Table 1) contd….


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


(Table 1) contd….


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, includingmany medicinal plants, has been reported. Propagation fromexisting meristems yields plants that are genetically identicalwith the donor plants. Tissue culture technique has beenwidely accepted as a tool for biotechnology for vegetativepropagation of plants of agricultural, horticultural and for-estry importance [14, 20]. Biotechnological tools are impor-tant for multiplication and genetic enhancement of the me-dicinal plants by adopting techniques such as in vitro regen-eration and genetic transformations. It can also be harnessedfor production of secondary metabolites using plants asbioreactors. The Centre for Science and Technology of theNon-Aligned and other Developing Countries in India orga-nized an international workshop on Tissue Culture of Eco-nomic Plants in April, 1994, as a better and quicker means ofusing modern biotechnological techniques to nurture andconserve medicinal plants.

This paper reviews the achievements and advances in theapplication of tissue culture for the in vitro regeneration ofmedicinal plants from various explants and enhanced pro-duction of secondary metabolites.

PRODUCTION OF SECONDARY METABOLITES

Pharmaceuticals from Natural Plants Progress in the fieldof pharmaceuticals have been spectacular. Most valuablephytochemicals are products of plant secondary metabolism.

In the search for alternatives to production of desirable me-dicinal compounds from plants, biotechnological ap-proaches, specifically, plant tissue cultures, are found to havepotential as a supplement to traditional agriculture in theindustrial production of bioactive plant metabolites. Secon-dary metabolites are compounds produced in plants that arenot necessary for the plants basic functions although someact as chemical defenses against microbes and animals. Sec-ondary metabolites are used in the pharmaceutical industryas flavorants and dyes, and in perfumery. The developmentand commercialization of medicinal plant-based bioindus-tries in the developing countries is dependent upon the avail-ability of facilities and information concerning upstream anddownstream bioprocessing, extraction, purification, andmarketing of the industrial potential of medicinal plants. Asconventional growing of medicinal plants is relatively ex-pensive, production of medicinal and/or other compoundscan be elicited in vitro.

The production of secondary metabolite in vitro can bepossible through plant cell culture. Successful establishmentof cell lines capable of producing high yields of secondarycompounds in cell suspension cultures have been reported[107].

Cell suspension culture systems could be used for largescale culturing of plant cells from which secondary metabo-lites could be extracted. The advantage of this method is that


it can ultimately provide a continuous, reliable source ofnatural products. Discoveries of cell cultures capable of pro-ducing specific medicinal compounds at a rate similar orsuperior to that of intact plants have accelerated in the lastfew years. New physiologically active substances of medici-nal interest have been found by bioassay. It has been demon-strated that the biosynthetic activity of cultured cells can beenhanced by regulating environmental factors, as well as byartificial selection or the induction of variant clones. Some ofthe medicinal compounds localized in morphologically spe-cialized tissues or organs of native plants have been pro-duced in culture systems not only by inducing specific orga-nized cultures, but also by undifferentiated cell cultures. Thepossible use of plant cell cultures for the specific biotrans-formations of natural compounds has been demonstrated[32,33]. Due to these advances, research in the area of tissueculture technology for production of plant chemicals hasbloomed beyond expectations. The major advantages of acell culture system over the conventional cultivation ofwhole plants are:

(1) Useful compounds can be produced under controlledconditions independent of climatic changes or soilconditions;

(2) Cultured cells would be free of microbes and insects;

(3) The cells of any plants, tropical or alpine, could easilybe multiplied to yield their specific metabolites;

(4) Automated control of cell growth and rational regula-tion of metabolite processes would reduce of laborcosts and improve productivity;

(5) Organic substances are extractable from callus cul-tures.

Plant cell culture technologies were introduced at the endof the 1960’s as a possible tool for both studying and pro-ducing plant secondary metabolites. Different strategies,using an in vitro system, have been extensively studied toimprove the production of plant chemicals. In order to obtainhigh yields suitable for commercial exploitation, efforts havefocused on isolating the biosynthetic activities of culturedcells, achieved by optimizing the cultural conditions, select-ing high-producing strains, and employing precursor feeding,transformation methods, and immobilization techniques.

Advances in the area of cell cultures for the production ofmedicinal compounds has made possible the production of awide variety of pharmaceuticals like alkaloids, terpenoids,steroids, saponins, phenolics, flavanoids, and amino acids.Successful attempts to produce some of these valuablepharmaceuticals in relatively large quantities by cell culturesare discussed below. The accumulation of secondary prod-ucts in plant cell cultures depends on the composition of theculture medium, and on environmental conditions. Strategiesfor improving secondary products in suspension cultures,using different media for different species, have been re-ported [88,69]. The production of secondary metabolites inplant cell suspension cultures has been reported from variousmedicinal plants. The production of solasodine from calli ofSolanum eleagnifolium, and pyrrolizidine alkaloids from rootcultures of Senecio sp. are examples. Cephaelin and emetinewere isolated from callus cultures of Cephaelis ipecacuanha.Scragg et al. [88] isolated quinoline alkaloids in significant

quantities from globular cell suspension cultures of Cin-chona ledgeriana. Enhanced indole alkaloid biosynthesis inthe suspension culture of Catharanthus roseus has also beenreported [26, 57,71]. Ravishankar and Grewal [82] reportedthat the influence of media constituents and nutrient stressinfluenced the production of diosgenin from callus culturesof Dioscorea deltoidea. Parisi et al. [69] obtained high yieldsof proteolytic enzymes from the callus tissue culture of gar-lic (Allium sativum L.) on MS medium supplemented withNAA and BAP. Pradel et al . [73] observed that the biosyn-thesis of cardenolides was maximal in the hairy root culturesof Digitalis lanata compared to leaf. The production ofazadirachtin and nimbin has been shown to be higher in cul-tured shoots and roots of Azadirachta indica compared tofield grown plant. Pande et al. [68] reported that the yield oflepidine from Lepidium sativum Linn. depends upon thesource and type of explants.

Research in the area of plant tissue culture technologyhas resulted in the production of many pharmaceutical sub-stances for new therapeutics. Many of the drugs sold todayare simple synthetic modifications or copies of the naturallyobtained substances. The evolving commercial importanceof secondary metabolites has in recent years resulted in agreat interest in secondary metabolism, particularly in thepossibility of altering the production of bioactive plant me-tabolites by means of tissue culture technology. Transgenichairy root cultures have revolutionized the role of plant tis-sue culture in secondary metabolite production. They areunique in their genetic and biosynthetic stability, faster ingrowth, and more easily maintained. Using this methodologya wide range of chemical compounds have been synthesized.

Bioreactors [100] are the key step towards commercialproduction of secondary metabolites by plant biotechnology.Bioreactors have several advantages for mass cultivation ofplant cells:

i. It gives better control for scale up of cell suspensioncultures under defined parameters for the production ofbioactive compounds;

ii. Constant regulation of conditions at various stages ofbioreactor operation is possible;

iii. Handling of culture such as inoculation or harvest iseasy and saves time;

iv. Nutrient uptake is enhanced by submerged culture con-ditions, which stimulate multiplication rate and higheryield of bioactive compounds; and

v. Large number of plantlets are easily produced and canbe scaled up.

Since the biosynthetic efficiency of populations varies, ahigh yielding variety should be selected as a starting mate-rial. The fundamental requirement in all this is a good yieldof the compound, and reduced cost compared to the naturalsynthesis by the plants.

The bioreactor system has been applied for embryogenicand organogenic cultures of several plant species. Significantamounts of sanguinarine were produced in cell suspensioncultures of Papaver somniferum using bioreactors. Ginsengroot tissue cultures in a 20 tonne bioreactor produced 500mg/L/day; of the saponin that is considered as a very good


yield. Bioreactors offer optimal conditions for large-scaleplant production for commercial manufacture. Much pro-gress has been achieved in the recent past on optimization ofthese systems for the production and extraction of valuablemedicinal plant ingredients such as ginsenosides andshikonin. Roots cultivated in bioreactors have been found torelease medicinally active compounds, including the antican-cer drug isolated from various Taxus species, into the liquidmedia of the bioreactor, which may then be continuouslyextracted for pharmaceutical preparations. Conventionalpractices require the harvest of the bark of trees, all ap-proximately 100 years old, to obtain 1 kg of the active com-pound taxol. Research over the last two decades has estab-lished efficient protocols for isolated cell cultures and alarge-scale bioreactor system. The acceptance of this processfor the industrial production of this invaluable compound hasrecently been established and will significantly impact theproduction of the tumor-inhibiting pharmaceutical.

MICROPROPAGATION OF SELECTED MEDICINALPLANTS

Tissue culture is basically defined as in vitro growth ofplantlets from any part of the plants in suitable nutritive cul-ture medium. It is also known as ‘micropropagation’ in sci-entific technology. Callus-mediated organogenesis and re-generation through somatic embryogenesis are the usualmode of regeneration by tissue culture. The induction ofcallus growth and subsequent differentiation, organogenesisand somatic embryogenesis are accomplished by the differ-ential application of growth regulators and the control ofconditions in the culture medium. With the stimulus of en-dogenous growth substances or by addition of exogenousgrowth regulators to the nutrient medium, cell division, andcell growth and tissue differentiation are induced Micro-propagation systems have been successfully developed formany medicinal species such as Periwinkle (Catharanthusroseus), Safed Musli (Chlorophytum borivilianum) Brahmi(Bacopa monieri) and Thorn apple (Datura metel).

Periwinkle (Catharanthus roseus)

Catharanthus roseus (Apocynaceae) originated fromMadagascar, but has now been spread throughout the tropicsand subtropics by human activity.This plant is cultivated asan ornamental plant throughout tropical and subtropical ar-eas and many parts of the world. Catharanthus is a plantbelonging to the family, well known for being rich in alka-loids. All parts of plant, especially, the leaves contain anti-neoplastic bisindole alkaloids, among those are vinblastineand vincristine, which are widely employed as chemothera-peutic agents against cancer. The absolute levels of vin-blastine and vincristine are considered far too low to explainthe activity of crude extracts of Catharanthus. Various stud-ies show the presence of other antineoplastic alkaloids[72,86] in the plant. Though its primary traditional use wasfor people with diabetes, Catharanthus also has anticancereffects. In the intact plant, vindoline is localized in the leavesand catharanthine is found in both the leaves and roots. Ex-tracts obtained from the plants like Catharanthus roseushave found a tremendous export potential. The demand forvinblastine [$75/gm] and vincristine [135 $/gm] isolatedfrom Periwinkle or Catharanthus roseus is around 20-

25kg/year. These figures represent the local as well as exportmarket demands. Vinblastine (Fig. 1) is the first drug ofchoice in many forms of leukemia and since the 1950s’ it hasincreased the survival rate of childhood leukemias by 80%.Another antileukemic drug, Vincristine (Fig. 1) has beendiscovered in the Madagascar periwinkle. Companies likeCipla and Tamilnadu Dada and Tamilnadu Herbals Ltd.ofIndia produce currently about 15-18 kg of vinblastine. Inaddition compounds like vindolin can be isolated but theexact market potential of vindolin is still untapped.

Plant regeneration from shoot and stem meristems hasyielded encouraging results in Catharanthus roseus [60].Rapid in vitro multiplication of plants from mature nodalexplants of Catharanthus roseus have been reported [56].The effects of auxins, cytokinins, different nutrient mediaand explant sources on callus induction on shoot multiplica-tion of Catharanthus roseus L.G. Don plant have been stud-ied [3]. In vitro and in vivo cultivation has been a source tostudy alterations in the growth of a Catharanthus roseus L.callus tissues [106] and cytological investigation [45] havealso been studied.

Fig. (1). Vinblastine and vincristine.

In our lab also callus induction (Fig. 2A) from nodal ex-plants was observed on Murashige and Skoog [62] MS me-dium supplemented with NAA (0.2 mgl-1) and KN(2mgl-1).Multiple shoot proliferation (Fig. 2B) and shoot elongation(Fig. 2C,D) was observed on MS medium supplementedwith NAA (0.5mgl-1) and KN(2mgl-1). These shoots whentransferred to MS medium supplemented with IBA (2mgl-1)resulted in rooting. For alkaloid extraction, in vitro plantmultiplication is an ideal approach to produce leaf materialin large quantity. In vitro heavy metal stress on callus culture[105] and regeneration of pathogen free [40.59] healthyplants through callus culture have been reported.

A number of nutritional factors as well as the growthfactors 2,4-D and IAA were studied to determine their influ-ence on growth and alkaloid formation [36,57,58,71,88] inCatharanthus roseus suspension cultures. Zhao J, et al. [109]studied the effects of stress factors, bioregulators, and syn-thetic precursors on indole alkaloid production in compactcallus clusters cultures of Catharanthus roseus . Large scalefermentation and alkaloid production of cell suspension cul-

N

N

OH

CH2CH3

R2

H

H3CO

N

N

HOOHR2

CH2CH3R1

CO CH3

H

Vinblastine: R1=CH3, R2=COOCH3

Vincristine: R1=CHO, R2=COOCH3


tures has also been reported [26, 86,109]. Reports on variousfactors on the release of secondary metabolites from roots ofDatura stramonium, Catharanthus roseus, and Tagetespatula cultured in vitro like effect of the medium pH [85],sucrose [53] have been reported. Enhanced alkaloid produc-

tion by transformed root cultures [70] and use of elicitors[26] has also been performed. Quantitative analysis of vari-ous alkaloids produced in vitro and in vivo has been exten-sively done [10, 16,19,25] in Catharanthus roseus.

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) Multipleshoot proliferation on subculturing on MS +(in mgl-1) NAA (0.5)+ KN (2). Multiple shoots after 4 weeks; (C)Shoot elongation and subcul-turing on same media, after 5 weeks; (D) Plantlets,6 weeks old.


Efficient development and germination of somatic em-bryos are prerequisites for commercial plantlet production.Lowering of growth regulator concentrations in culture me-dia has improved embryo development and germination ofmany medicinal plants. Germination of the somatic embryosis achievable on MS medium without the growth regulator.High frequency plant regeneration from anther-derived cellsuspension cultures via somatic embryogenesis in Ca-tharanthus roseus [41] as well as somatic embryogenesis andindole alkaloid production [72] have been observed in vitro.The suspension culture of Catharanthus roseus from stemand leaf explants on medium containing NAA and kinetinhas been established by Zhao et al. [110]. Somatic hybridi-zation and long term stability of electromanipulated proto-plasts of Glycine max var. Acme and a Catharanthus roseusmutant [85] has been reported. The effects of various heavymetals cell suspension cultures like cobalt (II) and nickel (II)[111], aluminum [55, 110] and the regulation of DNA syn-thesis and cell division by polyamines in Catharanthusroseus suspension cultures [54] has been studied.

Safed musli, Chlorophytum borivilianum

Chlorophytum borivilianum (Liliaceae) commonlyknown as Safed musli is among the few rare medicinal plants[65] witnessing steadily growing use in pharmaceutical,phyto-pharmaceutical and nutraceutical products. The Me-dicinal Plants Board has recognized Safed Musli as sixthamong the 28 selected priority medicinal plants, importantherb to be protected and promoted. The Board encouragesmainstream cultivation of Safed Musli by farmers by ex-tending a subsidy of 20% through National HorticulturalBoard on project cost. The steadily growing demand forSafed Musli has caused its price to soar to Rs.1,500 per Kgin the market [21], resulting in over exploitation throughwild harvesting pushing the plant to the verge of extinction.While the demand for Safed Musli is over 35,000 tons perannum, the supply is only to the tune of 5,000 tons a year.

Chlorophytum borivilianum Santapau & Fernandes is animportant herbaceous, medicinal plant found in the forests ofMadhya Pradesh and Gujarat states of India. It is a memberof the Liliaceae family and perennates by fleshy roots at-tached to a condensed stem disc, which remains dormant inthe soil for about 7 months. These are fascicled, sessile, cy-lindrical, variable in number and length, brown to blackskinned and white after peeling. The fleshy roots are theeconomically important part, which contain saponins and areused for the preparation of many Ayurvedic tonics pre-scribed against general debility. The roots fetch an attractivemarket price at present (Rs. 900–1200/kg of dry roots). Innature, the plant regenerates soon after or immediately be-fore the rainy season (May–June) from the previous year’sdormant root present in the soil.

The many therapeutic applications of Safed Musli in-clude as a restorative and a health-giving tonic; a curative forpre-natal and post-natal illness; and a remedy for diabetes,arthritis etc. Safed Musli supports physiological balance orhomeostasis. It improves access to energy stores, helps toimprove stamina and resistance to stress and tiredness, helpsin mobilization of the bodys’ nutritional resources, helps tonormalize various indices such as blood pressure. While

safed musli goes into over 200 ayurvedic preparations, it isknown in countries like Saudi Arabia as an effective IndianHerbal Viagra.

The origin of Safed Musli can be traced back in the old-est mountain ranges on the continent, the Aravalis fromwhere it spread to the near-by areas of the sub-continent,presently grown in the states of Gujrat, Rajasthan, MadhyaPradesh and the Central Deccan Plateau [38] and is thus en-demic to India. The genus includes about 300 species, whichare distributed throughout the tropical and subtropical partsof the world. Tropical and subtropical Africa is the probableCentre of Origin of the genus, where about 85% of the spe-cies are found. It is now widely cultivated in different partsof India on commercial basis. In India, about 13 species arereported [42], of which seven species (C. tuberosum, C.arundinaceum, C. breviscapum, C. attenuatum, C. laxum, C.borivilianum and C. malabaricum) are commonly observed.Nowadays, there is a vast demand of C. borivilianum all overthe world (specially gulf countries and cold countries). Thereare around 256 varieties of Chlorophytum in the world,which are known yet. The dried roots of C. borivilianum arethe products of commerce. Dried powdered roots or its ex-tracts are widely used in several herbal tonics [78]. The plantyields a flavonone glycoside, which is a powerful uterinestimulant. Dried roots of Chlorophytum contain 42% carbo-hydrate, 80–89% protein, 3–4% fiber and 2–17% saponin[104]. Research studies on Chlorophytum conducted in Indiaand elsewhere indicate that saponins [38] are responsible formedicinal properties. A saponin (Fig. 3) is a category ofphytonutrients (plant nutrients) found abundantly in manybeans, and other plants such as ginseng, alfalfa, yucca, aloe,quinoa seed and also in Safed musli. Saponins in safed musliare known to ensure the defense of the plant against micro-bial or fungal attack. Several drugs owe their anti-inflammatory and antiedemic properties to saponins.

Fig. (3). Saponin.

Similarly, many saponin containing drugs are tradition-ally used for their antitussive and expectorant properties.Low seed set, poor seed viability, high dormancy and lowpercentage of seed germination are some of the problems inpropagation of some medicinal plants. Seeds set are ex-tremely poor in safed musli. Recently, Fusarium solani wasidentified for the first time as causing a tuber-rot in safedmusli [77]. To encourage its adoption as a crop plant, propa-gation and cultural methods have been investigated [44]. Toovercome this problem, recently one of the rapidly expand-ing areas of pharmacognosy has involved the application oftissue culture of plant cells, tissues and organs in the study ofmedicinal plants [103]. Therefore attempts were made topropagate the material by in vitro methods. This method

O

COO

Gic

Gic

3

28


holds tremendous potential for the production of high-qualityplant-based medicine.

Extensive work has been done on several ornamentalspecies of family Liliaceae [46,89,102] but very few medici-nal plants of this family have been cultured in vitro [39].Biotechnological research on Chlorophytum borivilianumhas been supported previously by central funding agencies.In nature it is a slow growing plant. Perusal of literatureshows only a few reports on in vitro studies in C. borivil-ianum [20,74,75,99]. Initially different explants like leafbase segments, stem disc with shoot meristem, sliced roottuber and young shoot buds were used as explants [37].

In our laboratory, regeneration of C. borivilianum fromboth young shoot buds as well as inflorescence axis bearingan axillary bud gave good result [22]. Regeneration was suc-cessful both from the inflorescence axis (Fig. 4A) and youngshoot buds (Fig. 4B) on MS basal medium supplementedwith (1-5 mg l-1) BAP .In some cases, callusing and multipleshoot proliferation was also observed. These cultures latergave rise to 24 -35 shoots per culture from segments of inflo-rescence axis (Fig. 4C) and 10-13 shoots per culture fromyoung shoots buds (Fig. 4D) on MS medium containing4mgl-1 BAP. The healthy shoots were transferred to 1/2 MSmedium containing auxins for rooting (Fig. 4E) at variousconcentrations (0.5-2.5mgl-1) of IBA [23]. Rooted plantletson agar solidified media were removed and subjected to dif-ferent modes of hardening and acclimatization (Fig. 4F).

Somatic embryogenesis in callus culture of Chlorophy-tum borivilianum was first observed in immature zygoticexplants and intact ovules by Purohit et al. [76]. Callus in-duction was observed in embryos on the 10th day after theirinoculation on the MS medium containing 4.52µm-22.6µmof 2,4-D.However the best response was observed in thepresence of 4.52µm of 2,4-D. Subculture to MS mediumcontaining low concentration 2,4-D (1.13µm) and 567.0µmof ascorbic acid initiated faster growth of embryogenic cul-tures. Mature somatic embryos when transferred to mediumwithout growth regulators, they germinate only when kept inlight. The inoculated embryos gave rise to shoots and ulti-mately roots along with a mixed population of precociouslygerminating embryos showing profuse rhizogenesis. Encap-sulation of young shoots buds and plantlets regenerationhave been reported by Dave et al. [21]. in Chlorophytumborivilianum. Four millimeter long shoot buds encapsulatedin 3.0%sodium alginate matrix polymerized by 100mM so-lution of CaCl2.2H2O yielded best results. Recently, Fluores-cence In Situ Hybridization (FISH) technique has been ap-plied on somatic chromosomes and extended DNA fibers inthe medicinally important species of Chlorophytum to eluci-date physical localization and measurement of the rDNAsites using two rRNA multigene families homologous to 45Sand 5S rDNA [47].

Water Hyssop, Bacopa monniera

Bacopa monniera, also referred to as Bacopa monnieri,Herpestis monniera, water hyssop, and “Brahmi”, has beenused in the Ayurvedic system of medicine for centuries. Ba-copa monniera, a member of the Scrophulariaceae family, isa small, creeping herb with numerous branches, small oblongleaves, and light purple flowers. In India and the tropics it

grows naturally in wet soil, shallow water, and marshes. Theherb can be found at elevations from sea level to altitudes of4,400 feet, and is easily cultivated if adequate water is avail-able. Flowers and fruit appear in summer and the entire plantis used medicinally.

Bacopa monniera is traditionally prescribed in Ayurveda,a holistic system of medicine originating from India. Practi-tioners of Ayurveda classify Bacopa as a natural sedative andmood stabilizer, which is most often used to mildly relax theCentral Nervous System (CNS) as well as, like focus andmemory. Traditionally, it was used to support healthy cogni-tive functions, brain tonic to enhance memory development,learning, and concentration, and to provide relief to patientswith anxiety or epileptic disorders. The plant has also beenused in India and Pakistan as a cardiac tonic, digestive aid,and to improve respiratory function in cases of bronchocon-striction [15]. Brahmi may be useful for people who want toimprove mental function and concentration particularly un-der pressure or in stressful conditions Research on anxiety,epilepsy, bronchitis and asthma, irritable bowel syndrome,and gastric ulcers also supports the Ayurvedic uses of Ba-copa. Bacopa’s antioxidant properties may offer protectionfrom free radical damage in cardiovascular disease and cer-tain types of cancer. All available medical literature [93, 94]confirms that there has been no toxicity report associatedwith the use of Bacopa as a dietary supplement in children oradults [18].

Brahmi is especially suitable for students as it enhancesthe minds ability to learn and to focus [95]. In India the plantis used for all sorts of skin problems-eczema, psoriasis, ab-scess, ulceration, it is said to stimulate the growth of skin,hair and nails [51]. In vitro studies on anticancer studies ofBacopa monniera [28] have also been performed. Otherstudies have confirmed that Brahmi has a calming effect forstress and has become the herb of choice for attention deficitdisorder A.D.D. in hyperactive children. The results of re-cent laboratory studies suggest that, in addition to its mood-stabilizing effects, Bacopa also exhibit adaptogenic activity,which is thought to naturally increase the bodys’ resistanceto emotional stress and mental exhaustion [101]. Researchershave categorized multiple phytochemical compounds derivedfrom Bacopa monniera extract, including Bacosides (Fig. 5),Alkaloids and Glycosides. The active constituents in Brahmiare the all important steroidal saponins [79,80,81,83,91].

The ontogenic and seasonal variation in accumulation ofbacoside A in Bacopa monniera has been categorized [29].A new 13,14-seco-steroid Bacosterol and bacosine, a newtriterpene from Bacopa monniera [2] have also been iso-lated. A simple sensitive HPTLC method have been devel-oped for the analysis of bacoside A in the plant Bacopamonnieri and in its commercial monoherbal capsule formu-lation. The proposed HPTLC method provides a faster andcost effective qualitative control for routine analysis of baco-side in formulations containing Bacopa monnieri saponins.

An effective protocol for mass propagation of Bacopamonnieri (L.) Pennell, an important medicinal plant [90] wasdeveloped using shoot tips and nodal segments as explants.Shoot regeneration and somatic embryogenesis from differ-ent explants of Brahmi.The explants were cultured on Mura-shige and Skoog's medium supplemented with various aux-


ins, cytokinins either alone or with coconut milk and auxinsplus cytokinins. Multiple shoots were obtained on MS me-dium supplemented with auxins or/and cytokinins with orwithout coconut milk. Maximum number of plants were ob-tained on medium containing KN/2-ip (0.1 mg/l) and KN (1mg/1) in shoot tip and nodal cultures, respectively. The re-

generated shoots developed roots on the same medium. Inour lab, similar observation was noticed. The initiation ofshoot proliferation (Fig. 6A,B,C) from nodal explants wasobserved on MS medium supplemented with BAP(1mgl-1)and KN (4mgl-1). When these were subcultured on MS me-dium supplemented with BAP (5mgl-1) multiple shoot. Bud

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) Youngshoot buds on MS + (in mgl-1) BAP(4), after 2 weeks. (C) Multiple shoot proliferation (24 -35 shoots per culture) and elongation on MS + (inmgl-) BAP(4) from segments of inflorescence axis. (D) Shoot proliferation (10-13 shoots per culture) from young shoots buds and elongationon 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-floweringstage.


proliferation was further enhanced .They were found tooriginate ,associated with or without roots. Shoot elongation(Fig. 6D) was observed on the same medium. These shootswhen transferred to 1\2 MS medium supplemented with IBA(2mgl-1) resulted in rooting. Regenerated plantlets weretransferred to soil after a very brief period of hardening. Theregenerated plants are dark green in colour and more robustin their growth than the normal plants. These characters canbe exploited further to determine somaclonal variations.

Fig. (5). Bacoside.

Morphogenetic response [8] and isozymes of Bacopamonnieri cultivars grown under different conditions like saltstress [6], bioaccumulation and biochemical effects of mer-cury [98], copper stress [7], influence of cadmium [4] andzinc [33] on growth and photosynthesis of Bacopa monnieracultivated in vitro has been identified. The application ofhigh-performance liquid chromatography coupled to nuclearmagnetic resonance spectrometry, mass spectrometry andbioassay has been performed for the determination of activesaponins in Bacopa monniera [37]. A tracer technique havebeen used to evaluate bacoside A, in various Bacopa mon-niera accessions [30]. Dammarane- type triterpenoid sapon-ins [31] and Bacopasaponin D, a pseudojujubogenin glyco-side [32,49,52].

Thornapple, Datura metel

Since time immemorial various Datura (Solanaceae) spe-cies have been referred sacred visionary plants by practicallyall cultures that have come into contact with it. Their distri-bution spans all warm and tropical regions of the world.Daturas usually grow as herbaceous annuals/perennials. Themost striking feature, shared by all species are the beautifultrumpet-like flowers, ranging in color from white to pinkishpurple, and in some varieties even to bright golden yellowand red. The flowers exude a beautiful, narcotic scent, espe-cially at night. The seed capsules of the Datura species aretypically the size of a walnut and are covered with thornsthat may become quite sharp and spiky as the plant matures.The appearance of these seed-capsules has given rise to theEnglish common name, ‘Thorn apple’. It was used as a pain-killer in certain initiation rituals and given as a narcotic tothe ritual sacrifices. For this purpose the preferred method ofadministration was either by enema or as a rolled-up leafsuppository, which reduces some of the less pleasant side

effects of the drug. Another type of Datura, called Atlinan bythe Aztecs, enjoyed a particularly sacred status. It was re-garded as the sister of Ololuiqui hallucinogenic plant. Theseplants were so sacred that only the priests were allowed touse them. With their help they held counsel with the Gods -divining the outcome of future events, discovering thewhereabouts of lost or stolen objects and prognosticating thecauses of diseases, especially if black magic was suspected.As a medicinal remedy they prepared an ointment forcracked soles and injured feet, made plasters for ulcers, pus-tules and infected wounds and skin sores, and used it forpoultices to treat rheumatic aches and pains. Here too, theancient use of Datura as a powerful aphrodisiac is clearlyimplied.

All species of Datura contain powerful alkaloids, whichin sufficient quantities have the power to kill. The main al-kaloids represented are Scopolamine, Hyoscyamine and At-ropine (Fig. 7). Self-experimentation is not recommendedand must be strictly avoided by anyone who suffers any kindof heart condition.

The effects are stimulating on the central nervous systemand simultaneously depressing on the peripheral nerves.Symptoms include an increased heart rate, drying up of themucus membranes, a dry throat and sometimes cramps. Atfirst the effects are arousing, sometimes manifesting as un-controlled talking or laughing, forgetfulness and indulging insenseless repetitive activities. Vivid hallucinations and de-lirious illusions may also occur. Occasionally the effects canproduce extreme violence and destructive urges. The periodof agitation is usually followed by a deep prolonged sleepaccompanied with vivid dreams and hallucinations, often ofa sexual nature. Upon awakening one might experience adistinct hangover and a total lack of memory as to what ac-tually happened during the state of altered consciousness.

All parts of the plant are anodyne, antispasmodic, hallu-cinogenic, hypnotic and narcotic. It has been used in the pastas a painkiller and also in the treatment of insanity, feversdiarrhoea and skin diseases. The plant contains several alka-loids, the most active of which is scopolamine. This is a po-tent cholinergic-blocking hallucinogen, which has been usedto calm schizoid patients. The leaves contain 0.52% sco-polamine, the calices 1.08%, the stems 0.3%, the roots0.39%, the fruits 0.77%, the capsules 0.33%, the seeds0.44% and the whole plant 0.52 - 0.62%. Any use of thisplant should be with extreme caution and under the supervi-sion of a qualified practitioner since the toxic dose is veryclose to the medicinal dose.

Tissue cell culture study of Datura metel [24] has beenperformed extensively. In vitro plant regeneration wasachieved in Datura metel from nodal explants collected fromboth in vitro germinated seedlings and field grown plants (invivo). The explants were cultured on MS medium with BAP(0.5-3.0 mgl-1) and NAA (0.5 mgl-1). The nodal explantsisolated from in vivo source exhibited a greater number ofhealthy multiple shoots than in vitro. BAP at 3 mgl-1 withNAA at 0.5 mgl-1 was found to be optimal for regeneration ofshootlets [63]. Arockiasamy, D. I., B. Muthukumar, et al. [9]observed in vitro plant regeneration from internodal seg-ments of Datura metel L. Effect of carbon source on Daturametel microspore embryogenesis and the growth of callus

OHOHO

OH

HO

OOHO

O

O

O

OHHO

HO

O

OH

O


raised from microspore-derived embryos [11] have beeninitiated. They have also studied the effects of using acti-vated charcoal on microspore embryogenesis in Datura metel[12].

High frequency plant regeneration from anther derivedcell suspension culture via somatic embryogenesis was ob-served in Datura metel. Somatic embryos were found to beinitiated from anther at the uninucleate stage in liquid MSmedium supplemented with 2,4-D (0.2-0.4mgl-1). When sub-cultured after 3 days on semisolid liquid MS medium sup-plemented with 2,4-D (0.2-0.4mgl-1). These embryos becamegreen and shoot buds (Fig. 8A) stated initiating. Multipleshoot proliferation (Fig. 8B) was observed on subculturingon MS medium supplemented with BAP (2-5mgl-1) aloneand in combination of NAA (0.2-0.5mgl-1) and Kinetin (2-

4mgl-1). Shoot elongation was observed on MS medium sup-plemented with NAA (0.5mgl-1) and KN(2mgl-1). Theseshoots when transferred to MS medium supplemented withIBA (1-2mgl-1) resulted in rooting. For alkaloid extraction, invitro plant multiplication is an ideal approach to produce leafmaterial in large quantity.

The characterization of the chemical constituents of Da-tura metel Linn.genotypes and alkaloid contents of Daturametel varieties [5] revealed the variation of scopolamine andatropine in different parts of the plant during development[1] and new withanolides, daturametelins C, D, E, F and G-Ac from Datura metel L. [92]. The structures of atropine,scopolamine [64], withametelin and isowithametelin, witha-nolides [96] from Datura has been characterized. Pharma-cognostical studies, variation of alkaloid contents in the

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 ofculture. (B) Multiple shoots, after 3 weeks. (C) Further growth of B, after 4 weeks. (D) On subculturing on MS+ (in mgl-1) BAP(5) multipleshoot were further found to originate ,associated with or without roots. Shoot elongation was observed on the same medium.


Fig. (7). Hyosycamine, Atropine, Scopolamine.

A.

B.

Fig. (8). Datura metel: (A) Young embryos originated from anther (uninucleate stage) in liquid MS + (in mgl-1) 2-4-D (0.2) was subculturedafter on semisolid liquid MS+ (in mgl-1) 2,4-D (0.4). These embryos became proliferative and led to shoot development. (B) Multiple shootproliferation along with callusing was observed on MS + (in mgl-1) NAA (0.5) and Kinetin (4).

N H

CH3

HO

O

OH

N H

CH3

HO

O

OHO

CH

CH2OH

OC

O

CH

N H

CH3

H

C17H23NO3


seeds [66] and toxic effects of alkaloid extracts of Strychnosnux-vomica, Datura metel and Argemone mexicana on thebehaviour of chromatophores and mucous glands of Indianminor carp [96] have been done. In vitro production of tro-pane alkaloids was also observed in Datura metel [17]. Hairyroot lines of Datura metel were established following infec-tion of aseptic stem segments with Agrobacterium rhizogenesstrain A4 and cultured in hormone-free B5 solid medium.The growth and production of hyoscyamine and scopolamine(mg/g dry wt.) of these root cultures was encouraged by us-ing B5 liquid medium with half-strength salts. Application ofshort GC column [50] in the analysis of some tropane alka-loids in plants have also been tried.

CONCLUSION

Micropropagation holds tremendous potential as a toolfor the production of high quality plant-based medicines.Medicinal compounds from plants represent one of the larg-est and most diverse groups of plant secondary metabolites.The advent of advanced bioinformatics tools and moderngenetic technology allowed manipulation of biosyntheticpathways with the potential of generating novel chemicalentities. First, public databases of secondary metabolite re-lated enzymes were interrogated to identify relevant plantgenes from the plant and other species for the production ofhigh-quality plant-based medicine. Protocols have been de-veloped for clonal multiplication of hundreds of plant spe-cies, which include trees, medicinal and aromatic plants andendangered species from several laboratories across thecountry. Tissue culture protocols have been developed forseveral plants but there are many other species, which areover exploited in pharmaceutical industries and need conser-vation. It is now quite apparent that India has made bigstrides in the realm of biotechnology. The galloping rate, atwhich this advancement is continuing, holds out greatpromise for a better future.

In vitro propagation of medicinal plants with enrichedbioactive principles and cell culture methodologies for se-lective metabolite production is found to be highly useful forcommercial production of medicinally important com-pounds. The increased use of plant cell culture systems inrecent years is perhaps due to an improved understanding ofthe secondary metabolite pathway in economically importantplants. Advances in plant cell cultures could provide newmeans for the cost-effective, commercial production of evenrare or exotic plants, their cells, and the chemicals that theywill produce. Knowledge of the biosynthetic pathways ofdesired compounds in plants as well as of cultures is oftenstill rudimentary, and strategies are consequently needed todevelop information based on a cellular and molecular level.Because of the complex and incompletely understood natureof plant cells in in vitro cultures, case-by-case studies havebeen used to explain the problems occurring in the produc-tion of secondary metabolites from cultured plant cells. Akey to the evaluation of strategies to improve productivity isthe realization that all the problems must be seen in a holisticcontext. At any rate, substantial progress in improving sec-ondary metabolite production from plant cell cultures hasbeen made within last few years. These new technologieswill serve to extend and enhance the continued usefulness of

higher plants as renewable sources of chemicals, especiallymedicinal compounds.

Advances in tissue culture, combined with improvementin genetic engineering, specifically transformation technol-ogy, has opened new avenues for high volume production ofpharmaceuticals, neutraceuticals, and other beneficial sub-stances. Recent advances in the molecular biology, enzy-mology, and fermentation technology of plant cell culturessuggest that these systems will become a viable source ofimportant secondary metabolites. Genome manipulation isresulting in relatively large amounts of desired compoundsproduced by plants infected with an engineered virus,whereas transgenic plants can maintain constant levels ofproduction of proteins without additional intervention.Large-scale plant tissue culture is found to be an attractivealternative approach to traditional methods of plantation as itoffers a controlled supply of biochemicals independent ofplant availability. We hope that continuation and intensifica-tion efforts in this field will lead to controllable and success-ful biotechnological production of specific, valuable, and asyet unknown plant chemicals.

ACKNOWLEDGEMENT

We are grateful to the CEO and Director, Mr. SandeepBakshi for providing the facilities for pursuing the researchwork.

ABBREVIATIONS

BA = 6-Benzylaminopurine

NAA = α-Naphthaleneacetic acid

IAA = Indole-3 acetic acid

2iP = 6-(γ -Dimethylallylamino) purine

2,4-D = 2,4-Dichlorophenoxyacetic acid

KN = Kinetin

REFERENCES

[1] Afsharypuor, S. and Mostajeran, A. (1995) Planta Med., 61(4),383-384.

[2] Ahmed, B. and Rahman, A. (2000) Indian J. Chem., 39B(8), 620-25.

[3] Akcam, E. and Yurekli, A.K. (1995) Turkish J. Botany, 19(6),569-572.

[4] Ali, G., Srivastava, P. S. and Iqbal, M. (2000) Biol. Plant, 43(4),599-601.

[5] Ali, M. and Shuaib M. (1996) Indian J. Pharm. Sci., 58(6), 243-245.

[6] Ali, G., Purohit, M., Saba, Iqbal M. and Srivastava, P. S. (1997).Phytomorphology, 47, 97-108.

[7] Ali, G., Srivastava, P. S. and Iqbal, M. (1998) Plant Sci., 138, 191-96.

[8] Ali, G., Srivastava, P. S. and Iqbal, M (1999) Biol. Plantarum., 42,89-97.

[9] Arockiasamy, D. I. and Muthukumar, B. et al. (1999) Adv. PlantSci., 12(1), 227-231.

[10] Atta, U. and Ali, I. (1988). J. Chem. Soc. Perkin Transactions,1(8), 2175-2178.

[11] Babbar, S. B. and Gupta S. C. (1986a) Biochem. Physiol. Pflanzen,181(5), 331-338.

[12] Babbar, S. B. and Gupta S. C. (1986b) Physiol. Plantarum, 66(4),602-604.


[13] Bajaj, M. and Williams J. T. (1995) in Healing Forests - HealingPeople (report of workshop on medicinal plants, Calicut), IDRC,New Delhi, pp. 62.

[14] Cha. I. Y. E. and Kurtz, S. L. (1990) in Handbook of plant cellculture (Ammirato, P. V., Evans. D. A., Sharp, W. R., Bajaj. Y. P.S., Eds) Vol. 5. New York: McGraw hill, pp. 126-164.

[15] Chopra, R., Chopra, I., Handa, K. and Kapur, L. (1994) in Indige-nous Drugs of India, Academic Publishers, Calcutta, India.

[16] Chu, I. H. and Bodnar J. A. (1997) J. Liquid Chromatogr. RelatedTechnol., 20(8), 1159-1174.

[17] Cusido, R. M. and Palazon, J. (1999) Planta Med., 65(2), 144-148.[18] Dar, A. and Channa, S. (1997) Phytotherapy Res., 11, 323-25.[19] Datta, A. and Srivastava P. S. (1997) Phytochemistry, 46(1), 135-

137.[20] Dave, A. and Purohit S. D. (2002) Oikoassay, 15, 19-27.[21] Dave, A., Joshi, N. and Purohit, S. D. (2004) Eur. J. Hort. Sci.,

69(1), 37-42.[22] Debnath, M. (2005) In Vitro Plantlet Production System for Chlo-

rophytum Borivilianum, A Rare Indian Medicinal Herb, 92nd IndianScience Congress Association, Ahmedabad, pp. 106-107.

[23] Debnath, M., Malik, C. P. and Bisen, P. S. (2005) (Communica-ted)

[24] Del, S. J. and Laguna, A. (1987) Interferon Biotecnologia, 4(2),179-185.

[25] Drapeau, D. and Blanch, H. W. (1987) Planta Med., 53(4), 373-376.

[26] Eilert, U. and De L. V. (1987) Arch. Biochem. Biophys., 254(2):491-497.

[27] Eilert, U. and Kurz, W. G. W. (1987) Protoplasma, 140(2-3), 157-163.

[28] Elangovan, V.; Ramamoorthy, N.; Balasubramanian, K. and Go-vindswamy, S. (1995) Fitoterapia, 66(3), 211-15.

[29] Ganjewala, D. and Srivastava, A. K. and Luthara, R. (2001a) J.Med. Aromatic Plant Sci., 23(1A), 233-37.

[30] Ganjewala, D., Srivastava, A. K. and Luthara, R. (2001b) J. Med.and Aromatic Plant Sci., 22(4A), 241-43.

[31] Garai, S.; Mahato, S B.; Ohtani, K.; and Yamasaki, K. (1996)Phytochemistry, 43(2), 447-49.

[32] Garai, S., Mahato, S. B., Ohtani, K. and Yamasaki, K. (1996) Phy-tochemistry, 42(3), 815 - 20.

[33] Gayor, A., Srivastava, P. S. and Iqbal, M. (1999) Plant Sci., 143,187-93.

[34] Gupta, S. S. (1962) Indian J. Physiol. Pharmacol., 6, 25- 27[35] Hackman, R. (1998) Nutr. Sci. News, 10, 530-38.[36] Hirata, K. and Horiuchi, M. (1990) J. Ferment. Bioengineering,

70(3), 193-195.[37] Jain, P. and Kulshreshtha, D K. (1993) Phytochemistry, 33(2), 449-

51.[38] Jat R. D. and Bordia P. C. (1990) in Current Status & Emerging

Challenges, (Chaudhury, B. L., Aery, N. C. and Katewa, S. S.,Eds.) Proceedings of National Symposium on Advances in PlantSciences, pp. 46.

[39] Jha, S., Mitra G. C. and Sen S. (1984) Plant Cell Tissue & OrganCulture, 3, 91-100.

[40] Kaur, K. and P. Lodha (1996) J. Phytol. Res., 9(1), 25-28[41] Kim, S. W. and Song, N. H. (1994) Plant Cell Reports, 13(6), 319-

322.[42] Kirikar K. R. and Basu, B.D. (1975) in Indian Medicinal Plants

(Kirtikar K. R. and Basu B. D. Eds) Periodical Experts BookAgency, New Delhi, pp. 2508-2509.

[43] Kohl, W. and Witte, B. (1984) Planta Med., 50(3), 242-244.[44] Kothari S. K. and Singh K. (2003) J. Hort. Sc. Biotech., 78(2), 261-

264.[45] Kretovics, J. E. and L. Szilagyi (1990) Herba Hungarica, 29(1-2),

63-68.[46] Krikorian, A. D. and Kann R. P. (1986) in Cell Cultures and So-

matic Cell Genetics of plants, (Vasil, I. K. Ed.), Academic Press;New York pp. 187-205.

[47] Lavania U. C., Basu S., Srivastava S. S., Mukai Y. and. Lavania S.(2005) J. Heredity, 96(2), 155-160.

[48] Lodha, R, and Bagga, A. (2000) Ann. Acad. Med., 29(1), 37-41.[49] Mahato, S. B., Garai, S. and Chakravarty, A. K. (2000) Phyto-

chemistry, 53(6), 711-14[50] Mandal, S. and Naqvi, A. A. (1999) J. Med. Aromatic Plant Sci.,

21(3), 672-674.

[51] Martis, G., Rao, A. and Karanth, K. S. (1992) Fitoterapia, 63(5),399-404.

[52] Mathur, S., Gupta, M. M. and Sushil, K. (2000-2001) J. Med. Aro-matic Plant Sci., 22(4A), 320-26.

[53] Merillon, J. M. and Rideau, M. (1984) Planta Med. 50(6), 497-501.[54] Minocha, R., Minocha, S. C., Komamine A. and Shortle W. C.

(1991) Plant Cell Rep., 10, 126-130.[55] Minocha, R., Minocha, S. C., Komamine A. and Shortle W. C.

(1992) Physiol. Plant., 85, 417-424[56] Mitra, A. and. Khan, B. (1998) Planta Med., 64(4), 390.[57] Miura, Y. and Hirata, K. (1987) Agricul. Biol. Chem., 51(2), 611-

614.[58] Miura, Y. and Hirata, K. (1988) Planta Med., 54(1), 18-20.[59] Moellers, C. and Sarkar S. (1989) Plant Sci., 60(1), 83-90.[60] Moreno, P. R. H. and Van, D. H. R. (1995) Plant Cell Tissue and

Organ Culture 42(1), 1-25.[61] Mumtaz, N. and Choudhary, Q. F. (1990) Sarhad J. Agricul., 6(5),

467-470.[62] Murashige, T. and Skoog, F. (1962) Physiol. Plant., 15, 473-497.[63] Muthukumar, B., Arockiasamy, D. I. and Natarajan, E (2004) Ind.

J. Biotech., 3, 449-451.[64] Naqvi, A. A. and Mandal, S. (1998) Phytochem. Anal., 9(4), 168-

170.[65] Nayar, M. P. and Sastry A. R. K. (1988) in Red Data Book of In-

dian Plants, (Nayar MP & Sastry ARK, Eds), Botanical Survey ofIndia, Calcutta, India Vol 2, pp. 142.

[66] Noro, Y. and Hisata, Y. (1999) Nat. Med., 53(3), 130-133.[67] Nyman, U., Joshi, P., Madsen, L. B., Pedersen, T. B., Pinstrup, M.,

Rajasekharan, S., George, V. and Pushpangadan, P. (1998) J. Eth-nopharmacol., 60(3), 247-63.

[68] Pande, D., Malik, S., Bora, M. and Srivastava, P. S. (2002) In-vitroCell Dev. Biol. Plant, 38, 451-5

[69] Parisi M., Moreno S. and Fernandez G. (2002) In-vitro Cell Dev.Biol. Plant, 38, 608-12.

[70] Parr, A. J. and Peerless, A. C. J. (1988) Plant Cell Reports, 7(5),309-312.

[71] Perez, S. N. and Echevarria, A. (1987) Interferon Biotecnologia,4(2), 170-178.

[72] Piovan, A. and Filippini, R. (2000) Plant Biosystems, 134(2), 179-184.

[73] Pradel, H., Dumkelehmann, U., Diettrich, B. and Luckner, M.(1997) J. Plant Physiol., 151, 209-15.

[74] Purohit, S. D., Dave, A. and Kukda, G. (1994a) Ind. J. Pl. GeneRes., 7(1), 65-71.

[75] Purohit, S. D., Dave A. and Kukda, G. (1994b) Plant Cell Tissueand Organ Culture, 39, 93-96.

[76] Purohit, S. D., Dave A. and Tiagi, Y. D. (1994c) Rheedea, 4, 113-115.

[77] Raghavendra, V. B., Lokesh, S. and Vasanth, T. K. (2005) Austra-lasian Plant Pathol., 34(2), 275–276.

[78] Ramawat, K. G., Sharma, R. and Soni, S. S. (2000) in Biotechnol-ogy:Secondary metabolites (Ramawat, K. G. and Merillon, J. M.Ed.), Oxford and IBH Publishing Co, Pvt. Ltd. pp. 356-356.

[79] Rastogi, S. and Kulshreshtha, D. K. (1999) Indian J. Chem. Sect.,38, 353 -56.

[80] Rastogi, R. P. and Dhar, M. L. (1960) J. Sci. Ind. Res., 19, 455 -58.[81] Rastogi, S., Pal, R. and Kulshreshtha, D. K. (1994) Phytochemistry,

36(1), 133- 37.[82] Ravishankar, G. A. and Grewal, S. (1991) Biotechnol. Lett., 13,

125-30.[83] Renukappa, T.,Roos, G., Klaiber, I., Vogler, B. and KrausW.

(1999) J. Chromatogr. A, 847(1), 109-116[84] Saenz, C. L. A. and Maldonado, M. I. E. (1993) Appl. Biochem.

Biotechnol., 38(3), 257-267.[85] Sayavedra, S. L. and Krikorian, A. (2000) J. Plant Physiol., 156(1),

137-140.[86] Schiel, O. and Berlin, J. (1987) Plant Cell Tissue And Organ Cul-

ture, 8(2), 153-162.[87] Scragg, A.H. (1992) in Plant Biotechnol (Fowler M. W. and War-

ren G. S. Eds) Oxford, Peragmon Press.[88] Scragg, A. H. and Cresswell, R. (1988) Enzyme and Microbial

Technology, 10(9), 532-536.[89] Shah, R. R., Patel, D. B., Dalal, K. C. and Gupta, R. (1988) in

Proceedings of the national seminar on plant tissue culture, (Tata,S. N., ed) New Delhi Publication and Information Directorate, In-dian Council of Agricultureal Research, pp. 98-103.


[90] Shalini, Mathur and Sushilkumar. (1998) J. Med. and AromaticPlant Sci., 20, 1056-59.

[91] Shashi, B.M., Saraswati, G. and Chakravarty, A. K. (2000) Phyto-chemistry, 53(6), 711-14.

[92] Shingu, K. and Furusawa, Y. (1989) Chem. Pharm. Bull., 37(8),2132-2135.

[93] Shukla, S. P. (1983) Nagarjun, 267, 154-56.[94] Singh, H. K. and Dhawan, B. N. (1982) J. Ethnopharmacol., 5(2),

205-14.[95] Singh, H. K. and Dhawan, B. N. (1997) Indian J. Pharmacol.,

29(5), 5359-65.[96] Sinha, M. K. and Sinha, A. K. (1999) J. Environ. Biol., 20(4), 293-

298.[97] Sinha, S. C. and S. Kundu, S. (1989) Tetrahedron, 45(7), 2165-

2176.[98] Sinha, S., Gupta, M. and Prakash, C. (1996) Environ. Toxicol.

Water Qual., 1996, 11(2), 105-112.[99] Suri, S. S., Jain, S. K., Arora, D. K. and Ramawat, K. G. (1999),

Gartenbauwissenschafi, 64(3), 106-110.[100] Tripathi, L. and Tripathi, J. N. (2003) Trop. J. Pharm. Res., 2(2),

243-253.[101] Vaidya, A D B. (1997) Indian J. Pharmacol., 29(5), 340-S343.

[102] Varshney, A., Dhawan, V. and Srivastava, P. S. (2000) In vitro CellDev. Biol. Plant, 36, 383-391.

[103] Vasil, I. K. (1991) in Scale-up and Automation of Plant Propaga-tion (Vasil I. K, Ed), Academic Press Inc., London, pp 1-5.

[104] Wagle, A., Kelkar, G. D. and Heble, M. R. (2000) in Biotechnol-ogy: Secondary metabolites (Ramawat, K. G. and Merillon, J. M.Eds.) Oxford and IBH Publishing Co, Pvt. Ltd. pp 219-220.

[105] Wajda, L. and Kuternozinska, W. (1989) Environ. Exp. Botany,29(3), 301-306.

[106] Yushkova, E. V. and Skuratova, E. V. (1998) Biotekhnologiya, 6,42-47.

[107] Zenk, M. H (1978) in Frontiers of Plant Tissue Culture (Thorpe T.A. Ed.), University of Calgary, Int Ass for Plant Tissue Culture, pp1- 13.

[108] Zhao, J., Zhu, W. H., Hu, Q. and Guo, Y. Q. (2001) In-vitro CellDev. Biol. Plant., 37, 68-72.

[109] Zhao, J. and Zhu, W. H. (2000) Biotechnol. Lett., 22(6), 509-514.[110] Zhou, X.-H.; Minocha R. and Minocha S. C. (1994) J. Plant

Physio., 145, 277-284.[111] Zhu, L. and Cullen W. R. (1993) Chinese J. Environ. Sci., 14(5),

69-71.

Documents

Micropropagation: A Tool for the Production of High Quality Plant-based Medicines