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    Production of useful biochemicalsby higher-plantcell cultures: biotechnological and economic aspects

    A. SASSONUnited Nations Educational, Scientific and Cultural Organization(UNESCO), 7 P lace de Fontenoy,75700PARIS, FRANCE

    SUMMARY - Plant secondary metabolites re economically important, insuch different fields as drugs, ragrances, pigments, foodadditives and pesticides. An alternative to their extraction from the plant, s to synthesize them from in vitro cell cultures. After abrief revision of the historical background of the in vitro production, ts technical aspects according to its diverse forms. along witha number of applications including industrial-scale products. illustrate this process. Economic data, with the indication of limitingfactors and an analysis of potential valuable developments, complement he assessment of this new process. The market share, theinvestment-profit ratio, the competition ith other applications will also be valuable data fordecision-making.Key words: Secondary metabolites Tissue culture - Elicitor cell suspension culture Bioconversion - Biomass - Immobilized cells -Pigments - Fragrances - Pharmaceutical compounds.

    R E S U M E - Production de substances bio chin ziq~ ~es intrt par culture de cellules delantesuprieures : aspectsbioteclmologiq ues et conom iques. Les mtabolites secondaires ch ez les vgtaux prsen tent un grand intrt cononziqrle. dans desdomaines aussi divers que la plzarnzacie, les armes, les pigments, les additqs dimentaires et les pesticides. Ulie alternative c leurextraction d partir de la plante, consiste c les faire produ irepar des cultures cellulairesin vitro. Aprs un bref rappel Izistorique de cetteproduction in vitro, les aspects techniques de ses diverses for mes ainsi que de nom breu x exemples allant jusquc des productionsind~utrielles dans les divers donlaines illustrent le procd. Des donnes con on~ ique s ccompagnes dune mise en vidence desfacteurs linlitants et dune analyse des dveloppenzents positifs possibles compltent lvahmtion deette voie nouvelle de production.Le volwne du ~nrclz , les appoyls i~lvestissenzents-b~z~c e,a conzptitivit a vec dautres voies devro nt gale men t tre des lnzerztsimportants de dcision.Mots-cls :Mtabo lites secondaires - Culture de tissus - Susp emio ns cellulaires lic ite~ ~rs Biocolzversion - Bionlasse -Cellulesinmobilises - Pigments - Armes - Substances pha rn~a ceut iq ~~es .

    Plant secondary metabolitesThe distribution of secondary metabolites in plantsis far more restricted than that of primary metabolites;

    a compound is often only found in a few species, oreven within a few varieties within a species. Thoughtheir function in plant metabolism is unclear,nevertheless they may have an ecological role, e.g. assexual attractants for pollinating insects or in defencemechanisms against predators (Grisebach, 1988).Secondary metabolites often accumulate in the plant insmall quantities, sometimes in specialized cells. Hencetheir extraction is often difficult. Among them aremanycompounds which are commercially important asmedicinal substances, fragrances, food additives(pigments, flavouring andaromatic compounds) andpesticides (Heble and Chadha, 198513: Kurz, 1989).

    In spite of the progress made in organic synthesis orsemi-synthesis of a wide range of compounds similar tothose produced by the plants, extraction of secondarymetabolites from plants is still of considerablecommercial importance. A large number of thesemetabolites are difficult or virtually impossible tosynthesize at economic values. In several cases, thenatural product is more easily accepted by consumersthann artificially produced one. Both reasons,objective and subjective, explain that natural extractionstill applies to a large number of aromas or fragranceswhich are he result of a mixture of hundreds ofdifferent compounds as is the case of jasmine andstrawberry, oro biochemicals that have compIexmolecular structures (e.g. some alkaloids andglycosides).

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    There is great interest in developing alternatives tothe intact plant for the production of plant secondarymetabolites. This originally had centred on the use oftissue and cell cultures thoughhe most recentapproaches involve applying molecular biologytechniques to enhance the metabolic pathways leadingto specific compounds. During the past three decades,research has concentrated on the use of plant cell andtissue cultures, particularly in Japan and Germany, butalso to a lesser extent in the USA, for the commercialproduction of a wide range of secondary metabolites, injust the same way as bacteria and fungi have been usedfor antibiotic or amino-acid production (Sasson, 1988;Kurz, 1989).

    Plant cell cultures:advantagesand drawbacksPlant tissue cultures were first established in 1939-1940. However, it was only in 1956 that the first patentfor the production of metabolites by mass cell cultureswas filed by theAmericanpharmaceutical company,Pfizer Inc. (Ptiard and Bariaud-Fontanel, 1987). Thepotential of plant cell cultures to roduce usefulcompounds, especially forrugevelopment, wasperceived in the late 1960s. Thus Kaul and taba (1967)and Heble et al. (1968) isolated visnagin and diosgeninrespectively from cell cultures in larger quantities than

    from he whole plant.However,a argenumber ofcultures failed to synthesize products characteristic ofthe parent plant. For instance, morphinan, tropane andquinoline alkaloids are synthesized only at extremelylow levels in cell cultures (Berlin, 1986). That is why,after a surge of interest, the trendof research declined.In 1976, at an internationalcongress held in Munich,Zenk and his co-workers demonstrated the outstandingmetabolic capacities of plant cells and highlighted thespontaneous variability of plant cell biosyntheticcapacity, which could explain the contradictory resultsobtained earlier. This natural variability is exploited toidentify high-yielding cultures for use on an industrial

    scale (Tabata et al., 1976: Zenk et al., 1977; Zieg et al.,1983; Yamada, 1984; Benjamin et aZ., 1986). Since thelate 1970s, research and development in this area hasseen high increase inheumber of patentapplications filed, especially by the scientific andcorporate sectors in the Federal Republic of Germanyand Japan. In 1983, for the first time, a dye, shikonin,with anti-inflammatory and anti-bacterial properties,was produced by plant cell cultures on an industrialscale by Mitsui Petrochemical Industries Ltd (Fujita etOZ 1982). However, although this was thought to be amajorbreakthrough, shikonin production is still *(in1990) the only plantproduct to be produced on acommercial scale by cell cultures.

    Confronted with having to increase the amount ofsecondary metabolites in plant cell cultures, the need

    for greater biochemical and molecular research on thesecondary metabolism of plants has been requentlyemphasized (Fowler, 1981, 1984a,b; Misawa, 1985;Berlin, 1986; Stafford et al., 1986). The results ofresearchn this area could lead tohe successfulmanipulation of secondary metabolism and significantlyincrease the amounts of the compound(s) sought.

    It is now thought that any substance of plant origincan be produced by cell cultures. Thus t should bepossible to achieve the synthesis of a wide range ofcompounds such as alkaloids, flavonoids, terpenes,steroids, glycosides, etc., i.e. a total of several hundredswith complex chemical structures, using plant cellculture technology. It also seems possible to identifycell lines that canproduce amounts of compounds equalor even higher than those in the plant from which theyderive. Furthermore, new molecules which have notbeen ound previously in plants or have even beensynthesized chemically, have beenproduced by cellcultures. Thus this technology constitutes a genuinelynew means of achieving production of novelmetabolites.

    Finally, plant cells can transform natural orartificialcornpounds, introduced nto he cultures, throughavariety of reactions such as hydrogenation,dehydrogenation, isomerization, glycosylation,hydroxylation, opening of a ring and addition of carbonatoms (Rajnchapel-Messai, 1988). On the other hand,growing plant cells ona large scale would permitastricter control of the quality of the products as well astheir regular production without dependence on hevariations of natural production resulting from climateand socio-political changes in theircountries of origin.

    The techniques of plant cell cultures include thefollowing sequential stages or developments: selectionamong wild plants of a high-producing one, in-vitroculture or callogenesis, which involves the selection andstabilization of producing calli with a view to identifyinga high-producing line or strain; maximizing callus or cellsuspension, culture conditions and isolation of the best-producing line; industrial scaling-up, mass cultivation inbioreactors; downstream processing, i.e. extraction andpurification of the compounds sought.

    Means fo r increasingplantsecondarymetabolitesTherere several means of increasing theproduction of secondary metabolites by plant cell

    cultures or suspensions. These are:use of biotic r abiotic elicitors that couldstimulate the metabolic pathways as in the intactplant;addition of a precursor of the desired compoundin the culture medium with a view to increasing

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    its production or inducing changes in the flux ofcarbon o favour the expression of pathwaysleading to theompound(sj sought, i.e.alteration of controls of secondary metabolismpathways;

    - production of new genotypes through protoplastfusion or genetic engineering but thispresupposes the identification of the genesencoding key enzymes of secondary metabolicpathways and their expression once introducedin the plant cells;

    - use of mutagens to increase the variabilityalready existing in living cells;- use of root cultures.The use of mutagens could increase the naturally-occurring diversity among the cell clones and induce thecreation of new higher-producing lines or strains. Alsocell fusion and gene transfer could improve thesynthesis capacity of cells. However, the results of theseefforts depend on he knowledge of the metabolicpathways and their genetic control.

    1. Alteration of controls of secondarymetabolism pathwaysFor the majority of secondary metaboIism pathways,

    the roposed biosynthesis, deduced from chemicalconsiderations and feeding experiments, must awaitverification by the identification of the correspondingenzyme reactions. Thus, direct manipulations of thepathways areot possible dueohe lack ofenzymological background (Berlin, 1988).Enzymological knowledge relating to secondarymetabolism pathways has been drawn from the studiesof cell suspension systems (Hahlbrock and Grisebach,1979; Zenk, 1980; Zenk et al., 1985j but organ culturesand intact plants are excellent sources for suchenzymes.

    Another issue related to the possible alteration ofsecondary metabolism pathways concerns theidentification of those enzymes which are regulatory orrate-limiting. This is most probably the case forenzymes at the beginning of a sequence or atbranchingpoints of metabolic pathways, especially when theirabsence prevents de novo formation of the compoundsor when manifold increases in he activity of theseenzymes are observed under conditions promotingamajor increase in product formation. t is also necessaryto know whether the otherbiosynthetic enzymes are co-induced with the proposed regulatory enzyme or arepermanently presentven in non-producing cellcultures. Such knowledge helps to evaluatehepossibility of specifically manipulating these secondarymetabolism pathway(sj to increase the concentration ofthe desired product.

    It is generally assumed that he regulatory genesoccur as gene families and that each gene within onefamily is separately controlled and is thus expressed bya different signal. Thus, direct alterations of suchcontrols would be difficult to achieve (Berlin, 1988j. Itis, however, possible to introduce a desired gene into anintact plant through suitable vectors and have the geneexpressed in a specific organ such as the chloroplasts(Broeck et al., 1985; Fraley et al., 1985; Klee et al., 1985;Potrykus et al., 1985; Abel et al., 1986; Eckes et al., 1986;Schocher et al., 1986; Shah et al., 1986). The gene codingfor chalcone synthase (CHSj could be the first plantgene to be used in such a ransformation process tostudy its interference with a secondary metabolismpathway. Veltkamp and Mol (1986) have carried outexperiments aimed at repairing anthocyaninbiosynthesis in mutants deficient in CHS throughgenetic transformation.

    Although there is no example of altering theregulatory controls of secondary metabolism pathwaysthrough genetic engineering (with a view to improvingthe roductionates in cell cultures of a desiredcompound), this seems to behe only promisingapproach. To achieve this end, it is crucial that moregene coding for biosynthetic enzymes with a regulatoryfunction in secondary metabolism pathways should beidentified. Anotherapproach which might be morerelevant biotechnologically would be to transfer plantgenes into bacteria ando express themorstereospecifically difficult biotransformations (insteadof organic synthesis). Once the genes for the relevantenzymes are isolated and cloned, it could then bedecided whether transformed plant cells, transformedmicro-organisms or immobilized enzymes are the bestfor increasing the production of a specific compound(Berlin, 1988).

    2. Effectsof elicitorsThe use of abiotic and biotic elicitors is a promisingtool to improve the yields of products in cell-culturesystems (DiCosmo and Misawa, 1985j. Biosynthesis of

    flavonoids is induced by light via phytochrome orlandUV-photoreceptorsnd by infection withphytopathogenic organisms or compounds which inducethe synthesis of antimicrobial compounds in plants. Theaddition of an extract of Verticillium, a parasitic fungusof plants, has induced the synthesis of gossypol by cellsuspensions of Gossypiunz arboreum (Heinstein, 1984,intiardndBariaud-Fontanel, 1987). See alsoCramer et al. (1985).It can be predicted that very specific elicitors shouldbe found which will significantly improve the amounts

    of morphinan, tropane, or quinoline alkaloids in plantcell cultures (Berlin, 198Sj. The increase in theproduction of morphinic alkaloids by poppy cells couldbe likewise increased but this is still an empirical

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    procedurend a single generaluleannot beformulated. The elicitor needs obeadded regularlyand this therefore complicates its use.

    3. BioconversionPlant cells can transform a wide range of substratesand thus perform several reactions such as oxidation,

    hydroxylation, reduction, methylation, glucosylation,acylation and amino-acylation (Furuya, 1978; Heble etal., 1983: Heble and Chadha, 1985b).

    One could cite the transformation of steviol by cellsof Stevin rebaudiana into a glycoside, steviobioside,which is 300 times sweeter than sucrose and which isused as a sweetener in Japan. Another example is thatof salicylic acid which is glycosylated by cell cultures ofMallotus japonica to give aproduct having a higheranalgesic power and better olerated n he stomachthan aspirin (acetylsalicylate).

    In the case of Digitalis Ianata, two cardiotoniccompounds are isolated from the leaves: digitoxin inlarge quantities and digoxin in small quantities. Onlydigoxin has interesting pharmaceutical properties, but itcannot be produced either chemically or by microbialbioconversion. A bioconversion of highlypharmaceutical interest is the 12-hydroxylation of thecardiotonic drug beta-methyl-digitoxin into hemoredesirable, less toxic drug, beta-methyl-digoxin, by cellcultures of Digitalis lanata (Reinhard and Alfermann,1980; Alfermann et al., 1985). Using selectiontechniques, higher-yielding cell lines have been isolatedand he most effective bioconversion achieved so farhas been g/litre during a 28-day cultivation period(Heble et al., Bio-Organic Division, BhabhaAtomicResearch Centre, Trombay,Bombay, India).

    4. Root culturesAfter he pioneering work of White (1939) who

    established tomato root cultures capable of unlimitedgrowth in media containing macro- and micronutrients,sucrose and yeast extract, Dawson, in 1942, succeededin growing the roots of the tobacco plant. In 1957, Soltobserved that the increase of nicotine concentrations intobacco root cultures closely paralleled the increase inroot tissue as measured through root length, numberofbranches and dry weight accumulation (in Flores et al.,1987). It was also shown that root cultures of henbane(Hyoscyamus niger) synthesize hyoscyamine (in Floreset al., 1987).

    In Datura cells, alkaloid production was inverselycorrelated to the growth rate of callus cultures (Lindseyandeoman, 1983, in Flores etal., 1987).Undifferentiated calli of Atropa bellodonna do not

    produceheropane alkaloid hyoscyamine butproduction does occur when roots form on the callus(Bhandary et al., 1969, in Flores et al., 1987). Thecardiotonic glycosides of Digitalis are produced whencalli are induced to undergo embryogenesis followingtreatment with plant growth regulators (Kuberski et al.,1984, in Flores et al., 1987). All these observations haveled researchers to use continuously growing, organizedplant tissue cultures or even cultures of organs such asroots for the productionf secondary metabolites.

    Chilton et al. (1982) discovered thathe soilmicro-organism, Agrobacteriunz rhizogenes, caused ahairy root disease affecting a wide range ofdicotyledonous species and resulting in theproliferationof fast-growing adventitious roots at he host woundsite (due to the stable integration of a portion of the Kiplasmid of A. rhizogenes into the plant genome). Hairyroots were then established as aseptic cultures aftertreatment with antibiotics. Flores and Filner (1985,cited in Flores et al., 1987) were the first to demonstratethat hairy roots showed stable growth and producedsecondary metabolites, namely the tropane alkaloids ofHyoscyamus. Researchers of the Plant BiotechnologyGroup,griculturendood Research CouncilInstitute of Food Research, Norwich Laboratory,United Kingdom, used transformed root cultures as agenetically and biochemically stable system for hestudy andproduction of secondary metabolites. Theresulting stable high-producing lines may potentially beexploited directly as cultures in bioreactors or as plantsin the field following plant regeneration (Rhodes et al.,1988).

    Axenic transformedoot cultures of Nicotianarustica and Datura stramonium (which respectivelyproduce nicotine andheropane alkaloidshyoscyamine and scopolamine) were developedfollowing inoculation of plantaterial withAgrobacterium rhizogeness train LBA9402. The exactnature of the factors leading to root formation are stillpoorly understood.Transformed roots grow fast byplantstandards; they show a logarithmic pattern ofgrowth with doubling times which can be less than 48hours nd re characterized by a high degree ofbranching. This growth is associated with theproduction of the characteristic secondary metabolitesthat resemble the parent plant species in terms of bothabsolute amounts and spectrum of products (Rhodes etal., 1988).

    More than twenty hairy root clones of Hyoscyamusnzuticzrs (Flores and Filner, 1985, cited in Flores et al.,1987) producedropane alkaloids athe sameconcentrations as in the whole plant or in normal rootcultures. Alkaloid production has been stable n twoselected clones forover 40 monthly passages, butdecreased markedly when roots were induced to formcalli, and then reappeared when calli underwent rootdifferentiation (Flores et al., 1987). Furthermore,biomass production was very high: from an initial- 2-

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    inoculum of 2 to 4 mg (1 to 2 root tips), a typical hairyroot clone of Hyoscyanms nuticz:~ grown in batchculture over three weeks showed a 2,500 to 5,000-foldincrease, i.e. a higher increase than that obtained withthe fastest cell suspensions. The results withHyoscyanz~~smuticus oots were extended o Daturaand Scopolia species (Flores et al., 1987).

    It has been shown that hairy root cultures of Betavulgaris and Nicotiana ustica synthesized betalainsand nicotine plus anabasine, respectively, and that asignificant portion of tobacco alkaloids was found inthe growth medium of a 20-day batch culture. Hairyroots of Atropa elladonna were shown to formatropinendcopolaminetoncentrationscomparable with those found in field-grown plants andhigherhanhoseoundnntransformedootcultures (Kadama et al., 1986, in Flores et al., 1987).Marro et al. (1986, in Flores et al., 1987) examined 29hairy root clones of Scopolia japonica and isolated twohighly productive clones: clone S1 which accumulatedscopolamine to 0.5% (wlw) dry weight and clone S22which synthesized hyoscyamine at 1.3% (wlw) dryweight. Normal root cultures of several Hyoscyanzusspecies accumulated hyoscyamine and scopolamine inthe range of 0.04% to 1.1% (wlw) dry weight and0.06% to 0.3% (w/w) dry weight, respectively(Hashimoto et al., 1986, in Flores et al., 1987).

    The production of alkaloids erivedrom thetropane ring by root cultures, as well as that of beta-xanthine, is not exceptional. Hairy root cultures havebeen used to produce thesecondary metabolites of thefamily Asteraceae (1,000 generand ver 15,000species), i.e. the sesquiterpeneactonesnd thepolyacetylenes. Theatter re very active againstbacteria, fungi and nematodes. They are found in theroots and their synthesis may be elicited by infectionwith fungal pathogens. During 1986, over 30 normaland hairy root clones from hegenera Anzbrosia,Bidens,Rudbeckia and Tagetes wereestablished byFlores et al . (1987). These clones grew faster than theirnormalounterparts,roducedhiophene-likecompounds similar to those of normal roots and werestable for several monthly passages. It was concludedtherefore hat heseorgancultures could synthesizepolyacetylenes and heir cyclic derivatives (Flores etal., 1987).

    According toesultsbtained at CornellUniversity, Ithaca, New York, cultures of onion rootsin bioreactors are more favourable for the productionof onionromahan cell suspensions. Thesedifferentiated tissues are able to produce directly thecomplex chemical aromatic compounds that are thenreleasedntoheulturemediumndan besubsequentlysolated by high-pressure liquidchromatography. Thirteen different species of onionsfrom Europe, Japan and North Americahavebeentestedndeveraleem to e valuableorheindustrialroduction of onionroma.ulture

    conditions have been studied and it was reported thatthe final aroma could be modified by changing theprecursors n the culture medium. The onion aromaproduced by onion roots in culture would be muchmore related to the natural one than urrent availablepreparations (in Biofutur, no. 71, September 1988,p. 13).

    Root culturesouldhusecome commercialsources of many secondary metabolites following theexample fromRhodes et al . (1986, inFlores et al.,1987) who reported he production of nicotine byhairy root cultures of Nicotiana rustica in a two-stagebatchlcontinuous-flow system. These cultures showeda rapid growth rate and released a major portion ofnicotine into the culture medium. A prototype for alarge-scale bioreactor has now beendeveloped forgrowing hairy roots of H y o s c y a ~ m s u tic us (in Floreset al., 1987).

    Transformed roots are also being used to elucidatetheontrol mechanisms of secondary metabolismpathways with a view to enhancing the production ofmetabolites.For nstance,Rhodes et al. (1988) havebeen developing anpproacho manipulate themetabolic pathway at the level of the individual geneand thus the individual enzyme. It involves identifyinggenes coding for enzymes which are under-expressedin culture, then to isolate the gene and to re-introduceit nto heplantunder he control of de-regulated,high-expression promoters to increase arbon fluxthrough the pathway. In addition to putrescinemethyl-transferasePMT),rnithine decarboxylase(ODC) limits thepart of themetabolic pathwaycommon to nicotineand hyoscyamine biosynthesis.Thus, Rhodes et al. (1988) have transferred the genecoding for ODC from Saccharomyces cerevisiae intotransformed oots of Nicotinna rustica where t isunder the regulatory control of the cauliflower mosaicvirus 35s promoter. The gene was integrated into theplant genome and was successfully transcribed into afully active enzyme. The pattern of expression of otherenzymes involved in biosynthesis of the alkaloids wasunaffected in the transformed cells.

    Another approach is to study the co-ordinatedregulation of the expression of the entire pathway inorder to understand how the expression of the groupof genes is temporally and developmentally regulated.This would lead to the manipulation of this regulationto optimize expression of the whole pathway (Rhodeset al., 19SS).

    To sum up, the use of root cultures as a commercialsource of secondary metabolites will depend on thescaling-up of production and recovery techniques, onthe further knowledge of the regulatory signals whichinduce the production of compounds at over 10% dryweight, and onhe identification of high-valuebiochemicals in the roots (Sasson, 1988).

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    Industrial productionof usefulbiochemicals by higher-plant cellcultures1. Market-value estimations

    Economic considerations govern themportanceattached to the production of natural substances andbiochemicals (see Table). The estimated annual marketvalue of pharmaceutical products of plant origin inindustrialized countries was over US $20 billion in themid-1980s. The annual market value of codeine and ofthe anti-tumour alkaloids, vinblastine and vincristine,has been stimated at bout US $100 million perproductPtiard ndBariaud-Fontanel, 1987). Theworld-wide market value of aromas and fragrances has

    expected to rise to US $6 billion in 1990 (Rajnchapel-Messai, 1988).

    In 1988, the stimated nnualmarket value ofshikonin (for details see below) was about US $600,000which is far romhe US $20 to US $50 millioninvestment of the original research and developmentwork. However, the final cost of the product fell to US$4,000 per kg which compares with US $4,500 per kg forthe substance extractedrom the roots ofLithospermurnerythrorhizon. It should be noted hatKanebo, the apanese cosmetics corporation, whichdeveloped lipsticks containing shikonin, realized aturnover of about US $65 million over two years inJapan hrough the sale of 5 million lipsticks, eachlipstick selling for US $13. In the Republic f Korea andChina, Mitsui Petrochemicals Ltd todav intends tobeenestimated at over US $4 billion in r980 and is market the product itself (Rajnchapel-Mesiai, 1988).

    Economic data forome substances of plant origin

    SUBSTANCE AND USE

    Pharmacyajmalicinecodeinedigoxindiosgeninvinblastinevincristine

    Food-additives and fragrancesjasmine oilmint oil

    natural vanillin

    Cosmeticsshikonin

    ANNUALNEEDS

    3- 5 tonnes80-150 tonnes6 tonnes200 tonnes. 5-10kg

    100 kg3,000 tonnes30 tonnes

    150 kg

    INDUSTRIALCOST(US$PER KG )

    1,500650-9003,00020-405 million

    5,00030

    2,500

    4,000

    ESTIMATED ANNUALMARKET VALUE(IN US$ MILLION)

    4.5-7.552-135184-825-50

    0.59075

    0.6Sources: Fontanel, A.; Tabata, M. 1987. Production of secondary metabolites by plant tissue and cell cultures. Present aspects andprospects. Nestl Research News 1986-1987,pp.93-103.

    Rhodes, M.J.C.; Robins, R.J.: Hamill, J.; Parr, A.J. 1986. Potential for the production of biochemicals by plant cellcultures.New Zealmzd Journal of Technology, 2, pp.59-70.Scragg, A. 1986. The potentialof plant cell cultures in biotechnology.B L , pp.44-7.Rajnchapel-Messai (1988).

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    that of bacteria. In general, cell multiplication andmetabolite synthesis are uncoupled, the latter occurringat the end f the growth phase.3. Achievements

    Significant progress has been made to overcome themajor constraints of industrial production since the late1960s. Heble et al. of the Bio-Organic Division of theBhabha Atomic Research Centre, Trombay, Bombay,India, are carrying out research on mass cultivation in 20-litre capacity bioreactors of selected cellines ofRauwolfiaerpentina (ajmaline, reserpine), Papaversomniferunz (thebaine, codeine, morphine), Artemisiaannua (artemisinin), and other plant species (Chadha etd., 1988).

    In Japan, the Nitto Denki corporation uses a non-continuous process for biomass production of Panaxginseng in 20,000-litre bioreactors, the yield being 500mg dry matter per itre per day (Rajnchapel-Messai,1988). Hashimoto et al. (1982, in Ptiard and Bariaud-Fontanel, 1987) succeeded in cultivating tobacco cells in20,000-litre bioreactors for over two months with yieldsbeing 5.82 g dry matter per litre per day. Fujita et al.(1982) selected a highly productive cell line ofLithospermun erythrorlzizon (Ko-shikon), anddeveloped a two-stage growth and production methodfor shikonin which is an anthraquinone extensively usedin Japan for its anti-inflammatory andantibacterialproperties and also as a dye. This substance is found inthe roots of the plant which accumulates up to 2 to 3%of its dry weight as shikonin, the plant taking 5 to 7years to reach a size useful for commercial production.No method is yet available to synthezise shikonin. In1983, Mitsui Petrochemical Industries Ltd reported atechnique forhe industrial-scale production ofshikonin by cell cultures. The process involved twostages: (a)plant cells were first grown in a 200-litrecapacity bioreactorand(b)the resulting biomass wasthen transferred into a second bioreactor in which thecomposition of theulture medium favouredhesynthesis of shikonin. Even though the capacity of thesecond bioreactor was only 750 litres, theJapanesetechnique markedan mportant urning point in thebio-industrial application of plant tissue cultures(Fowler, 1984b). In a 23-day culture period, cells grownin the 750-litre bioreactor accumulated 23% of their dryweight as shikonin (Curtin, 1983). The productivity ofL. erythrorhizon cell cultures was 60 mg per g of cellsper week, that is 1,000 times higher than that of theplant roots which required a longer time period of 5 to 7years.

    The success of shikonin production was due to theselection of a cell line which accumulated a ten-foldhigher level of shikonin than that found in roots of themature lant. This achievement resultedromnempirical, labour-intensive search for optimal growth

    conditions andproduction media whichwas furtherfacilitated by a visual selection for overproducing cells.Cell cultures have now thus become the majorcommercial source of shikonin (Flores et al., 1987).

    In addition to he work on shikonin, Japanesescientists were ableoobtain higher quantities ofberberine from growing cells of Coptis berberica. Thisplant species accumulates significant amounts ofberberine in its oots n our to six years; similarconcentrations could be obtained in four weeks usingtissue culture. Hara et al. of Mitsui PetrochemicalIndustries Ltd have isolated a cell line of Coptisjaponica that contains 10% of berberine (dry weight)and which could produce bout 1,500mgf thisantibacterialand antipyretic alkaloid per itre n14days. Analysis of cell lines by flow cytometry indicatesthat the increase in berberine production resulting fromcell selection is related to the ncrease of the number ofcells with a high content of berberine, rather than to anoverall increase of this content in all the cells. Thus, thehigh-yielding line is most probably heterogeneous (inBiofutur, 110.83, October 1989, p.17). Industrialproduction of geranio1 is also being developed by thecosmetic company Kanebo (Rajnchapel-Messai, 1988).

    In the Federal Republic of Germany, Alfermann etal. (1985) of Boehringer Mannheim A G were able togrow cells of Digitalis lanatain 200-litre bioreactors andobtain 500 g of beta-methyl-digoxin in three months;the bioconversion rate of beta-methyl-digitoxin wasvery high, up to 93.5%, if the non-used substrate wasrecycled. Ulbrich et al. (1985) cultured Coleus blumeicells in a 42-litre bioreactor itted with the modulespiral stirrer; using this system with aeration, theyreported high yields of rosmarinic acid (5.5 g per litre),representing 21% dry weight of cells. Heble ndChadha (1985a, b) reported the successful cultivation ofCatharanthus Y O S ~ L ~ Sells in 7 to 20-litre capacitybioreactors, modified to provide air lift and agitation, insingle and multiple stages. The cells produced highlevels of total alkaloids comprising ajmalicine andserpentine as the major components. It was shown thatplant cells could withstand shear to some extent, andthat judicious use of air lift and low agitation wasadvantageous. Researchers at Ciba-Geigy AG, Basel,Switzerland, have produced he alkaloid scopolaminefrom cell cultures of Hyoscyamus aegypticus grown inair-lift bioreactors (in McGraw-HillsBiotechnologyNewswatch, 21 January 1984).

    Sanguinarine is a benzophenantridin alkaloid ex-tracted from the oots of Papaver sonzniferunz.Three tofour years are needed for plant maturation before thesubstance can be extracted. Cell cultures have beenused to produce large quantities of this alkaloid, whichis used in toothpastesandmouth lotions to combatdental plaque and tooth decay. Commercial productionwill be the result of a joint venture between the PlantBiotechnology Institute of theNational Research

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    Council of Canada,Saskatoon,andVipont ResearchLaboratories Inc., Fort Collins, Colorado, USA. Themanufacturing process will use the eliciting power ofextracts of a fungus, an unspecified Botrytis, that inducethe synthesis of sanguinarine and dihydrosanguinarineby plant cells. Pilot experiments have shown that insemi-continuous cultures, which could be elicited twice,theproduction rate of alkaloids reached 3% of drybiomass (in Biofutzlr., no.79, May 1989, p.13).

    These examples and others show that industrialproduction of plant cell biomass and secondarymetabolites is possible with equipment and processesanalogous to those used with micro-organisms. From theeconomic viewpoint, Zenkmadean estimate of US$500/kg for the production of a drug by cell cultures at arate of 1 g per litre. Goldstein etal. (1980) of MilesLaboratories Inc. (Bayer AG) also analyzed theeconomics of plant-cell culture methods and suggestedthat products costing more han US $1,00O/kg weresuitable. With higher-yielding cultures, the productioncost could be much lower. For a 10% dry weightyielding product, he cost couId come down to US$228/kg. This could be further reduced if immobilizedcells were used (Sahai and Knuth, 1985; Heble andChadha. 1986). According to a study carried out inJapan, any substance of plant origin with a valueexceeding orequal o US $80/g could be profitablyproduced bycell or tissue cultures. This would evenapply to substances of which the retail price variesbetween US $250 and US $5OO/kg, i.e. many rawpharmaceutical products, aromatic compounds,condiments and fragrances (Fowler, 1984b; Vasil, 1987,1988).

    Immobilizing cells in a gel, which is permeable to themolecules of the nutrient medium or on polymers (witha view to preserving their metabolic capacity and tousing them several times), has the advantage ofextending the production time of cells (over six months)and of making the cells catalyse the same reactionalmost indefinitely. Active research has been carried outin this area since the early 1980s. Thus, he eam ofFurusaki of theDepartment of Engineering, TokyoUniversity, has developed a bioreactor for theproduction of codeine, which is mainly used as an anti-cough compound in pharmacy. Poppy cells have beenimmobilized in calcium alginate beads and they catalyzethe conversion of codeinone into codeine. Thistechnique enabled the Japanese researchers toovercome the drawbacks of plant-cell bioreactors arisingfrom the instability of cells and the low yields of thedesired compounds. They decreased the size of cellclusters (2.5 mm in diameter), thereby increasing theirlife-span and obtaining yields of codeine that were equalto those in non-immobilized cells ( i n Biqfutzw. no . 79,May 1989,p.14).

    The use of immobilized cells should bypass thedirect extraction of the compounds from the biomass asthe products now arise in the medium itself. Examples

    of this approach include theproduction of caffeine,capsaicin and berberine. Many metabolites howeverstill appear to accumulate in the cell vacuoles and it istherefore important to further gain information on howthese metabolites may be made to diffuse out into theculture medium. Although immobilized cell technologyis a promising technique, especially aimed at decreasingproduction costs, clearcut examples still donot existwhich would demonstrate a gain of productivity on anindustrial scale (Ptiard and Bariaud-Fontanel, 1987).4. Prospects

    With the onset of the 1990s, only Japan and, to alesser extent, heFederal Republic of Germanyarereally engaged in the industrial production of secondarymetabolites by plant cell cultures. The only marketedproduct (as of 1990) remains shikonin. In Japan, sevenprivate corporations have created a common subsidiaryin research and development on plant cell cultures. ThePlant Cell Culture Technology (PCC Technology) hasbeenet up with theupport of theapan KeyTechnology Centre (JKTC) byKyowa Hakko KogyoCo., Mitsui Petrochemical Industries Ltd, Mitsui ToatsuChemical Inc., Hitachi Ltd, Suntory Ltd. Toa NenryoKogyo Co. and Kirin Breweries Co. Ltd. By contrast,most North American and Europeancompanies are notenthusiastic about he prospects regarding profitableindustrial production (Rajnchapel-Messai, 1988).

    Several factors or constraints could explain thissituation. Firstly, the time needed or selection andstabilization of cell lines is about two to three years dueto the difficulty of controlling and directing somaclonalvariation. Consequently, there is a need for tedious andtime-consuming screening of a large number of lines.Secondly, the lack of knowledge concerningbiosynthetic pathways of secondary metabolitesexplains certain failures. For instance, it has not beenpossible to isolate a cell line with a good level ofproduction of dimeric alkaloids of Cntlmrmltlulus roseu s,although these alkaloids areresent in minutequantities in the plant (one tonne of dry leaves yields0.5 to 2 g of these compounds). Thirdly, the difficultiesof extraction of the desired compounds are serious,especially when the compounds accumulate ascombined substances; this probably explains why theFrench company Sanofi-Elf-Bio-industries hasabandoned the production of codeine and morphine bycells of Papaver somniferzm. However, according toSteck of Sanofi-Elf-Bio-industries, technical andscientific difficulties can be overcome in fairly shortperiods resulting in lower industrial production costs. Itis, though, essential that a worthwhile target beidentified in order to secure the necessary investments(Rajnchapel-Messai, 1988).

    Thus the real difficulties are identification of targetcompounds as well as economic and legalconsiderations. Production of secondary metabolites by

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    plant cell or tissue cultures must, of course, becompetitive with other conventional means ofproduction such as extraction from the field-grownplants, alternative enzymatic processes, chemicalsynthesis or semi-synthesis, microbial fermentation orimprovement of the plant itself through somaclonalvariation, genetic engineering, etc., followed by theregeneration of the plant from in-vitro cultures.

    Production byell cultures could be justified,however, for rare products that are costly and difficultto obtain through other means. According to Ptiard ofthe French company Francereco, this approach wouldbe feasible only for roducts whose world annualpotential market would be US $20 to US$50million,with a minimum selling price of about US $400to US-$500 per kg. Thus, ajmalicine and jasmine oil, in spite oftheir high selling price - US $1,500 and US $5,000 perkg,espectively - are notttractiveor industrialproduction by cell cultures because of the size of theirmarket, i.e. US $8 million for ajmalicine and only US$500,000 for jasmine oil,whichwould not permit theamortization of the investments to be made in researchanddevelopment. On heother hand, mint aroma,which represents an annual market of US $90 million,has a selling price which is too low - US $30 per kg - tobe worth lowering further through cell-cultureproduction. The challenge lies therefore with theidentification of economically profitable targets(Rajnchapel-Messai, 1988). But as many companiesthroughout the world have considered these aspects, itmay be concluded that such target compounds that mayhave been identified will remain closely guarded secretsfor some time o come.

    In the pharmaceutical area,heumber ofeconomically-profitable targets also appears toerather limited.Many plant substances - or theirderivatives from semi-synthesis - are used in drugs, andtheir production cost is often very high; their individualeconomic weight is nevertheless rather low. They areused in small quantities in a medicine whose final priceis due more to the considerable investments in researchanddevelopment han to he cost of raw materials.Furthermore, the plant production approach is opento the strong competition of chemical synthesis that isvery efficient in developing new molecules with highlyspecificctivity. Thus, the development of newsynthetic cardiotonic compounds in theUSA hasresulted in the decreasedemandor Digitaliscardiotonic glycosides. It is also one of the causes forthe non-commercialization of beta-methyl-digoxinproduced by Digitalis cell cultures (Rajnchapel-Messai,1988). However, commercial production was envisagedfor vincristine and vinblastine from Ccrtlzarc~nthusoseuscell cultures by Eli Lilly & Co., ubiquinone fromNicoticrm (Matsumoto et al., 1982), L-dopa fromM ucu~ z na prur iem (Wichers and Pras, 1984) anddigoxin from Digitdis h a f e / (Alfermann et al., 1985).

    Prospects are more promising for the production ofaromatic substances, flavouring compoundsandfoodadditives, and basic materials for fragrances by plantcell or tissue cultures. These products, although lessvaluable than pharmaceuticals, have larger markets.This explains the recent re-orientation of research, as iswitnessed by the four-fold multiplication of the numberof publications devoted to food additives and cosmeticsthat were ubmitted tohe 1986 Congress of theInternational Association of Plant Tissue Culture, heldin Minneapolis, USA.

    In this area, however, the difficulties relate to thevery low investments made in research anddevelopment by the relevant companies involved withagriculture, food and perfumeries. In addition, technicaldifficulties should not be underestimated because mostaromas and fragrances, with a few exceptions such asvanillin and irone (which is a violet fragrance extractedfrom iris rhizomes that needs to be stored for one tothree years) are mixtures of a large number ofcompounds, ome ofwhich are present in minuteamounts but which are nevertheless vital for the finalproduct to be accepted. It is therefore very difficult toreproduce such mixtures exactly by cell cultures. Theevaluation of productive cell lines usually must rely onthe smell and taste of the perfumer or aromatician thusnecessitating that such work be carried out in locationshaving easy access to such people. Consequently it isalmost impossible to conduct such work outsideindustrial companies. These companies, however, areusually very conservative in their outlook and with onlyone or wo exceptions have usually set their face againstthe feasibility of producing aromas, fragrances andflavours by anything other than conventional means. Atfirst sight, this appears to be a very narrow approachbutt should be remembered that many of thesecompanies rely on suppliers and growers for a numberof their products. To seek to produce one product byalternativemeans (i.e. plant cell culture) couldjeopardize the future supply of a number of otherproducts simply by the supplier or grower now refusingtodo urther business. Thushe balance betweenproducer and seller isa subtle one and is one whichneither party wishes to see upset.

    Other issues are: what will be the legal status ofthese new products? Will they be labelled natural?Would itbe possible tomarkethem without acomplementary toxicological study, as is currently thecase for all substances xtractedrom new plantvarietiesred and cultivated with conventionalmethods? The modification of the genetic heritage of.thesevarieties is, however, much greater han hechanges which may occur in cell suspensions (Ptiardand Bariaud-Fontanel, 1987).

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    Industrialproduction of food additivesand fragrancesFood additives contribute to making foodstuffs

    palatableandattractive by enhancing or improvingtheir flavour, colour and texture. Food technologies tryto respond to these criteria especially with regard to thetexture, taste and aroma of the foodstuff. The need tohave the same taste and aroma in apecific foodstuff inorder to suit the consumers tastes makes it compulsory

    to use natural or artificial aromas, especially in certainproductswhere they may be indispensable. A fewdecades ago, aromas were extracted romplant rawmaterials transformed by fermentation, grinding andheating. Later, chemical compounds, purer asmixtures, completed theange of plantromas.Nowadays, new compounds are added to the existingonesnd they usually originate from enzymaticreactions, orre produced by biotechnologicalmethods, i.e. by microbial andplant cells (Spinnler,1989). Fermentation techniques arereferredochemical synthesis if they are morecost-effective, e.g. intheroduction of citric acid and monosodiumglutamate. Microbial synthesis is also the only means toproduce the thickening additive xanthan.

    Since the late 1950s, many food additives have beenquestioned mainly by nationalndnternationalregulatory authorities, about their safety for long-termuse and consumption. Athe same time, theassociations of consumers, aware of the inclusion ofadditives in foodstuffs, have been exerting pressure ongovernmental bodies to have chemical or artificialadditives replaced by natural additives from plant o ranimal tissues, or be synthesized by micro-organisms orplant cell cultures. New production processes wouldallow food industries to respond to he favourableopinion about natural aromas and also to overcome thepresent constraints related to the climatic and politicalvagaries of supply in he producing countries and otake advantage of the difference in price betweennaturally- and chemically-produced compounds. Forinstance, in the case of vanillin. its price is about 20,000FF per kg if it is extracted from the anilla pod, whereasit costs only 50 FF per kg if it is produced by chemicalsynthesis from lignin. Although this difference isconsiderable, it is not possible to differentiate the twoBinds of vanillin molecules through chemical or physicalanalyses. Hopefully for aromaswith a restricted market,eventual fraud will be easily detectable and presumablywill be rigourously monitored by those industrialistswho will benefit most from conserving their existingsales (Spinnler, 1989).

    Pigments are also food additives but their use hasbeen strongly criticized by the associations ofconsumers in he 1970s, because most of themareproduced by chemical synthesis and are unrelated toany naturally occurring material. InheEuropean

    Economic Community, 24 pigments are currentlyauthorized of which 10 are not of natural origin. It istrue that the olour of foodstuffs is associated with theiracceptance and the pleasure they procure. Thus, the listof pigments used for this purpose is bound to becomelonger but the trend s to replace artificial colourings bynaturalones.Market forces however will determinewhether the public will pay increased costs fornatural products. The biotechnological methods usedfor hepurpose of producing natural ood colorantsconsist of growing micro-organisms, micro-algae andhigher-plant cells (Langley-Danysz, 1987).

    Extraction of pigments from plants is an oldtechnique, e.g. anthocyanins from red berries, betaninefrom beet and curcumine from saffron. Some pigmentscannot be extracted before the plant reaches the adultstage. This is the case of rocou, a pigment extractedfrom heseeds of ashrub,or of shikonin, extractedfrom the roots of Lithospernmm erythrorhizon. In thecase of shikonin, plant cell cultures have been verysuccessful in the industrial production of this dye (seeabove).1. Aromas and fragrances

    Naturalromas are a mixture of numerouscompounds: more han 500 have been identified inroasted coffee beans and 200 in apple. Natural aromasare susceptible tohe conservation processes offoodstuffs such as sterilization, pasteurization, freezing,etc. Some aromas are altered by enzymatic or chemicalreactions and usually disappear if stored ora longperiod. This iswhy substitutes have been sought forthem since the nd of the 19th century. Artificialaromas were manufactured from coal or oil derivativesand were added n very low concentrations (10-6 orppm, and 10-9 or ppb). The present trends are either toproduce synthetic molecules, which are identical tonatural molecules, or to use biotechnological methods.The first category of aromas have the advantage of aconstant composition, of not depending on the seasonand of being manufactured n such a way that heproduction is adapted to the market. Their drawback isof not being mixtures of substances that resemble thenatural ones. This iswhy the biotechnological routesare now being increasingly preferred (Langley-Danysz,1987). For instance, Dziezak (1986, in Langley-Danysz,1987) reported that the characteristic aromas of cocoaand coffee have beenproduced by cell cultures ofTlaeobroma cacao and Coffea arabica, respectively.

    The value of the world market oraromasandfragrances was estimated a t about US $3 billion in 1988(Vaisman, 1988). In 1987, the en first world leadingcompanies representedmore han two-thirds of thetotalnnualurnover, whereas in 1981 the sameproportion was attributed o he first 20 companies(Cors, 1989):

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    International Flavors and Fragrances (IFF, USA) US$ ..............................................................Quest (Unilever NV, UK and Netherlands)..................................................................................Takasago Perfumery (Japan) ...........................................................................................................Firmenich (Federal Republic of Germany) ....................................................................................Dragoco ...............................................................................................................................................FDO (BASF AG, Federal Republic of Germany) ........................................................................PFW (Hercules, USA) ......................................................................................................................Mane (France)....................................................................................................................................Robertet (France) .............................................................................................................................Sanofi-Elf-Bio-industries France) ..................................................................................................Florasynth (France) ...........................................................................................................................T. Hasegawa Flavor Co. (Japan) .....................................................................................................Ogawa (Japan) ...................................................................................................................................

    Givaudan (Hoffmann-La Roche AG, Switzerland) ......................................................................Haarmann and Reimer(Bayer AG, FRG) ...................................................................................Roure (Hoffmann-La Roche AG, Switzerland)............................................................................

    According to the conclusions of a study carried outin France by a consulting firm, Prcepta, the market ofaromas and fragrances is soaring, compared with that offoodstuffs in general which is progressing a t a slowerpace. In the aroma andfragrance sector, 5% to 6%, andsometimes up to 12% of the total turnover, is devotedtoesearchnd development, i.e. onerder ofmagnitude more than in the food sectorCors, 1989).

    In Japan, he growth of the aroma and fragrancemarket was rather slow during the 1970s butheincreasing consumption of perfumed home products(air purifiers) induced a more rapid growth rate ofthe marketwhich was evaluated at aboutUS$0.8 billionin 1988. Several industrial companies have emphasizedtheir diversification efforts in this sector: TakasagoPerfumery, T. Hasegawa Flavor Co., SodaAromaticCo., Shiono Koryo Kaisha Ltd, and Ogawa. Thus, SodaAromatic Co. controls themarket of deodorantsubstances and 80% of the Japanese market related tothe deodorization of natural gas (Vaisman, 1988).

    Plant cell cultures are promising means ofproduction of aromasnd fragrances inapan(Vaisman, 1988). Thus, Kirin Breweries Co.Ltd,Kyowa Hakko Kogyo Co., Mitsui Toatsu Chemical Inc.,Mitsui Petrochemical Industries Ltd, Hitachi Ltd andToaNenryo Kogyo Co. have concluded co-operativeagreements to producearomatic substances by plantcell cultures. The expertise of Kirin Breweries in themanufacture of fragrances from humulane, a by-product of the processing of hops, and that of MitsuiPetrochemical Industries Ltdnhe large-scaleproduction of shikonin are major contributions to thesuccess of this association. Ajinomoto Co. has also filedapatenton heproduction of safranal (whichgivessaffron its spicy taste) by cultures of cells from the pistilof Crocus sativus (similar research work was carried out

    1987

    746 M635462441333327250125120100837266

    in the laboratory of Yeoman in Edinburgh). KurarayCo. is currently producing lavando1 by cultures oflavender cells, while researchers at Kitasato Universityare using Eucalyptzls cell cultures to catalyze thebioconversion of alpha-menthol (Vaisman, 1988). Allthese products arepromoted by emphasizing theirnatural origin to counteract the consumers reluctanceto use artificial products.

    In France,he consumption of medicinal andaromatic plants has trebled n he past 20 years: aquarter of theroduction being utilised as rawmaterials (flavourings and infusions or steeping ofherbs), and hreequarters being processed for hepharmaceutical, food, fragrance and cosmeticsindustries. In 1986, consumption of these plants inFrance reached 30,000 tonnes. Domestic productionwas only 13,000 tonnes, the balance being achieved byimportation. The trade deficit was valued at 272 millionFF (Comar, 1988).Initiatives have therefore been taken o promote theFrench production of medicinal and aromatic plants,

    while ensuring better protection of the consumers. Thishas involved direct help to the farmers to enable themto respond o the ncreasing demand and also to achievebetter post-harvest operations. Another initiative is thatof a new biotechnology company, Biophytec,established in Lyon, which ai ms to applybiotechnologies to heproduction of medicinal andaromatic plants, with a view to marketing products withmedicinal properties and food additives. Finally, effortshave been made to obtain accelerated authorization tomarket certain plants or plant extracts having medicinalproperties (Comar, 1988).

    The world leading pharmaceutical groups, such asBayerAG,BASFAG and Hoffmann-La Roche AG,have been increasingly involved in this sector and have

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    bought many smaller companies. In France, Sanofi-Elf-Bio-industries has made large investments, whereasagrobusiness companies such as Pernod-Ricard orchemical corporations such as Unilever NV and Procter&r Gamble Inc., have been trying to integrate this newactivity into heir conventional ones. Otherpotentialparticipants are he petrochemical companies such asEsso and Royal Dutch-Shell (Cors, 1989).

    This new situation will limit the negotiating capacityof small firms which are mainlynvolvedn theprocessing of natural products. As they have been slowto diversify their activities, they now must make a choicebetween necessary growth and confrontation with morepowerful companies. Old corporations that supply theindustry with the natural raw materials have to face thecompetition of biotechnological products: the only hopefor them is to concentrate their activities increasingly onthe supply of high-value added products (Cors, 1989).

    However, the future role of the small- and medium-size companies in the sector of aromas and fragrancescould be analogous to hat of small biotechnologycompanies which concluded agreements with the largeand powerful groups, e.g. the British company, ImperialBiotechnology, whichhas research agreements withNestl, Tate & Lyle, and Beecham plc (merged in 1989with the American pharmaceutical corporationSmithkline Beckman into the new group Smithbee). If

    the biotechnological production of aromasndfragrances proves cost effective, the small companieswhich have mastered the relevant biotechnologies willbecome the natural partners of the large food, chemicaland pharmaceutical groups. Such new ventures rpartnerships will be promoted by the rend whichmakes therontiersetweenromas, fragrances,pharmaceuticals, medicines and biochemicalsprogressively disappear. This is, for instance,substantiated by the esearch programmes of Nestlwhich link together activities in nutrition, health anddietetics. An areawhich seems promising and which canbe a driving factor or he industrial production offragrances, including by plant cell cultures, is that ofaromachology.2. Aromachology: new prospectsor fragrance

    production and commercializationSince the early 1980s, Shiseido, the leading Japanesecosmetics company, andthermanufacturers offlavours and fragrances have been carrying out researchand experiments in a new area named aromachology,noto be confused with aromatherapy which usesessential oils for therapeutic purposes.This new scienceaims at studying the effects of scents and fragrances onthe physical and mental conditions of human beings,

    andt using these substances to modify theseconditions. In 1989, in co-operation with KajimaConstruction Company, Shiseido was testing the effect

    on working conditions of the release of a specificfragrance through the air-conditioning system into theoffices at certain hours of the day. The objective, asstated by Shiseido, was to try to eliminate the stress dueto work and to improve the effectiveness andproductivity of employees. Other similar experimentsare being conducted by Shiseido with another civilengineering company, Ujima, and with Taltasuna andShimizu Construction Company. Furthermore,at he1989 automobile show in Tokyo, a prototype car wasdisplayed with an air-conditioning systemhichreleases a jasmine fragrance to keep the driver awake(Leventer, 1990).

    In 1984, the world leading company InternationalFlavors and Fragrances (IFF) filed the first patent on amethod which induces the decrease of physiologicalandlor subjective reactions to stress in human beings.But it was only in 1988-1989 thathe Americancompany could master a methodovaluate inquantitative terms and in a reliable way the changes ofhumournd mental state. This method combinesphysiological measures such as heart rhythm, bloodpressure and brain waveswith the fillingn ofquestionnaires such as those used in psychiatry toevaluate he effects of medicines. According to thestatements made by the executive in charge of technicaldevelopment at IFF, the statistical results obtained areconsidered tobe satisfactory and can supportheeffectiveness of the products tested. However, there is aplacebo effect of about 60% which IFF has, of course,tried toeduceLeventer, 1990). The Japaneseresearchers are also using the variations of physicalindicators as well as encephalographic tests (measuresof alpha- nd beta-waves). According to TakasagoPerfumery, a manufacturer of aromas, the methodologyis considered to be valid and it has been developed inco-operation with Japanese universities and scientificinstitutions (Leventer, 1990).

    New products with specific effects are to bemarketed. n 1984, Shiseido marketed two Cologne-water perfumes for men, Because Psyche Refresh andBecause Psyche Cool. The Japanese company assertsthat heseare he first products in he world whosestimulating or sedative effects have been establishedscientifically. In 1988-1989, the same companydeveloped with Hattori Seiko andmarketed anautomatic alarm clock which releases before ringing aperfume made of pine and eucalyptus fragrances whichshould stimulatehe body. Kanebo,he secondJapanese cosmetics company, developed a fragrancecontaining lavender, camomila, anise, among others,whichhas sedative and sleeping effects. MatsushitaElectric sells cards, which release sedative orstimulating fragrances when they are ntroduced ntosmall bread toasters. Kanebo isow marketinghandkerchieves with microcapsules interspersedbetween the textile fibres which release the fragrancewhen used (Leventer, 1990).

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    Cosmetic products with the same objectives as thoseproduced by IFF have also been marketed by Avon inco-operation with Takasago Perfumery, and by theAlthough the companies thatre investing inaromachology claim that they do not wish to enter intothe medical research area and that for the time beingthey are focussing their efforts on relaxing orstimulating compounds, it can be assumed that this is apromising areanot only for those companies whichmanufacture fragrances but also for those which wouldlike to seize this opportunity to widen their share of thefragrance market. It might not be unusual that in thefuture people buy a perfume not only because of itsscent but also because of its relaxing, stimulating orphysiologically-specific effect.

    French world leading cosmetics company, LOral.

    In this respect, it is worth mentioning thereportmade jointly by the Fragrance Foundation, IFF and theUniversity of Cincinnati about their discovery offragrances that can increase vigilance. Elsewhere, atDuke University, research is being carried out on theeffect of fragrances on violence. At the University ofTsukuba, in collaboration with Shiseido, rates ofrecovery of athletes after strenuous activity are beingmonitored using certain fragrances to be breathed inbefore or after the exercise (Vaisman, 1988; Leventer,1990).Very little information has yet been reported on thereaction of the general public to these developments.Will they accept the well-being afforded by the newproducts, or will consumers associations, trade unionsand public bodies in charge of health regulations reactto the potential side-effects of the products on physicaland mental health?

    ConclusionsOne should not conclude hastily that cell cultureshave no future in thearea of production of usefulsubstances. Many laboratories carry out their researchand development programmes because the possibilities

    exist to increase the yields of their production processes.Furthermore, even if certain compounds are not worthdeveloping up to a commercial stage, some biosyntheticcompounds could be used as valuable precursors fororganic synthesis, or could themselves constitute entirelynew products. The market size for the end product, thecodbenefit ratio of the production technology, thecompetition with substitutes and the existence of othersources of supply are major factors which influence thechoice of the ppropriate manufacturing technique,especially when deciding in favour of plant cell or tissuecultures. Thus, it is probable that metabolites,synthesized through simple enzymatic reactions underthe control of a single gene, could be more efficientlyproduced by genetically-engineered microbial cellsrather than by plant cell or tissue cultures (in United

    States Office of Technology Assessment, OTA, 1983).Finally, it is not easy to identify economically profitabletargets because of industrial secrecy or confidentialitywhich prevails in this area and because of the infrequentcontacts between researchers and companies working inthe field of aromatic and medicinal plants (Rajnchapel-Messai, 1988).

    Japanese scientists and companies however arerather optimistic for the future of plant cell culture andthey are probably right. In 1989, the Japanese Ministryof Agriculture, Fisheries and Forestry launched,throughheir Forestry Agency, a new programmenamed Green Spirit Project. With a budget of about110million yen, this project aims at producing essentialoils, resins and glycosides out of plant residues (wood,branches, bark, leaves). Potential applications of thecompounds tobe produced areood additives [inparticular sweeteners), medicines and fragrances (inBiofutur, no. 79, May 1989,p.8).

    In Europe,Canada and theUSA, a number ofcompanies do carry out research in this area and closelymonitor current progress elsewhere. For instance, inFrance, Sanofi-Elf-Bio-industries maintains a reducedresearch activity in this area and has supported heresearch work of Chnieux and Rideau onheproduction of bioconversion of ellipticine - an anti-tumour alkaloid - by cell cultures of Ochrosia ellipticn(Apocynaceae). The same group is involved throughtheMero Company in the production of fragrancecompounds (flower and fruit) by Ambid and his co-workers atheNational School of Agriculture ofToulouse. Nestls subsidiary, Francereco, carries on itsresearch programme on the production of metabolitesby plant cells and has established fruitful co-operationwith many university teams such as that of Cosson atthe Faculty of Pharmacy, Chatenay-Malabry, in thesouth of Paris. An industrial project awaits a decisionthat will be taken on economic grounds rather than onscientific or technical ones. The pharmaceuticalcompany, Roussel-Uclaf, is interestednhe workcarried out at the University of Montpellier on theproduction of diosgenin. When the French chemicalcompany Rhne-Poulenc boughtheermanphytopharmaceutical firm Nattermann, it dissolved theplant cell-culture project group and its laboratory - oneof the largest plant cell-culture laboratories in he world- in late 1988. Rhne-Poulenc is nevertheless lookingfor an appropriate policy in the field of plant cellcultures (Rajnchapel-Messai, 1988).At theEuropean level, all these issues werediscussed athe Symposium on Bioproduction ofmetabolites by plant cell cultures, held in Paris inSeptember 1988 and organized by the InternationalAssociation of Plant Tissue Culture and theFrenchAssociation pour la Promotion Industrie-Agriculture(APRIA. Association for he Promotion of Industry-Agriculture). The organizers expected that at least two

    or three targetscould be identified in order to launch aresearchprogrammeat heEuropean level and that-71-

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    government support would be forthcoming in an areawhere financial isk is important, but where profitscould be large.References

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