Tagetes Species Research Articles

Chemistry and biology of industrial crop Tagetes Species: a review

In this review, different research works related to Taxonomy, cytogenetics, tissue culture, Biotechnology, Agronomy, phytochemistry, pharmacology and biological activity as well as its commercial uses of Tagetes species including all the biological and chemical activities mainly T. minuta, T. patula and T. erecta have been compiled. The review also highlights the chemical structures and commercial uses of essential oil and its components of its unique biological activity being used as agrochemical, food and colorants and nutritional supplement and cosmetic industry. This review work also focus on commercial cultivation of T. minuta for its oil and T. patula and T. erecta find floriculture use and also raw material for commercial production of several kinds of compounds.

Biocide plants as a sustainable tool for the control of pests and pathogens in vegetable cropping systems

Synthetic pesticides have played a major role in crop protection related to the intensification of agricultural systems. In the recent years, environmental side effects and health concerns raised by an indiscriminate use have led the EU to the ban of many synthetic pesticides. As a result of this drastic revision, currently there is a strong need for new and alternative pest control methods. An interesting source of biorational pesticides may be represented by the biocidal compounds naturally occurring in plants as products of the secondary metabolism. Groups of plant secondary metabolites most promising for the development of pesticidal formulations are glucosinolates, saponins, and more generally terpenoid phytoconstituents, such as essential oil and their constituents. Glucosinolates are thioglucosidic secondary metabolites occurring mainly in the Brassicaceae and, at a less extent, in Capparidaceae families. The incorporation of glucosinolate- containing plant material into the soil results in degradation products highly toxic to soilborne pest, pathogens and weeds. This practice, known as biofumigation, may be considered as an ecological alternative to soil toxic fumigants. Plant-derived saponins are triterpene glycosides present in top and root tissues of plant species of the families Leguminosae, Alliaceae, Asteraceae, Polygalaceae and Agavaceae. Saponins and saponin-rich plant materials have been also reported for a biocidal activity on phytoparasites and soilborne plant pathogens. Essential oils are volatile, natural, heterogeneous mixtures of single substances, mainly terpenes and phenolics, formed as secondary metabolites by aromatic plants belonging to several botanical families. Among terpenes, limonoid triterpenes have been demonstrated to possess interesting insecticidal, nematicidal and antifungal properties. Occurrence of these compounds is mainly limited to Meliaceae and Rutaceae. Alkaloids, phenolics, cyanogenic glucosides, polyacetylenes and polythienyls are further groups of secondary metabolites also known for their biocidal activity and susceptible for the production of natural pesticides. Alkaloids are derived from various botanical families, amongst which the Solacaneae, and include a number of molecules, such as nicotine, veratrine, cevatrine and ryanodine, used as insecticides. Phenolics were found also toxic to insects, fungi, bacteria, nematodes and weeds. Cyanogenic glucosides are amino acidderived secondary metabolites releasing, upon tissue disruption, hydrogen cyanide that suppress insects, fungus, nematodes and weeds. Finally, polyacetylenes and polythienyls, substances mainly present in Tagetes species, are also well known for their insecticidal and nematicidal properties.

Thiophenes of Tagetes Flowers and Partial Purification of Xanthophyll Esters

Lutein is a health promoting xanthophyll that may be extracted from marigold flowers. But the plant is known also for biosynthesis of phototoxic polythiophenes. Extracts of flowers of some Tagetes species grown in Belgorod (RF) T. erecta L. (cv. Gavaii) , T. patula L. (cv. Petit orange), T. tenuifolia Cav. (unknown cv.), and T. lucida Cav. were investigated by thin layer chromatography (TLC) and high-performance chromatography (HPLC) with diode-array (DAD) and mass spectomertic detector (MS). It has been found that C13 bithienyl (instead of C12-derivatives being common for hairy roots) was a major thiophene derivative of all extracts under investigation, and that thiophenes are accumulated mainly in the parts of flowers hidden from sunlight by a flower's sepals. Therefore two methods of petal preparation for lutein diesters extraction were investigated. It has been shown that solid phase extraction on silica is a simple way for lutein diesters purification from thiophenes.

Carotenoids of Therapeutic Significance from Marigold

Use of flowers and plants for worshipping, decoration and personal adornment is an integral part of Indian cultural heritage since pre-historic times. Marigold is a potential ornamental plant grown commercially in different parts of the world and obtained from different species of Tagetes of family Asteraceae. The essential oil from Tagetes erecta flowers has been used in high class perfumery and also acts as antihaemorrhagic, anti-inflammatory, antiseptic, antispasmodic, astringent, diaphoretic and emmenagogue. The oil is very valuable in aromatherapy for its powerful skin healing effects and also possesses fly repellant properties. Marigold varieties have pesticidal value as they destroy ground pests particularly nematodes. The genus is also recognized as a potential source of very interesting biologically active products viz. carotenoids that are currently being used as food colorants, nutritional supplements and poultry feed additives and in ophthalmology for the treatment of age related ocular diseases viz. cataract and dry age related macular degeneration (ARMD). Several species of the genus Tagetes find tremendous application as per the traditional database for the treatment of various ailments and diseases. Carotenoids from Tagetes species The extracts of Tagetes erecta were earlier reported to contain epoxides such as lutein 5, 6-epoxide and other oxidation products of lutein. Some preliminary studies during the same phase demonstrated the predominance of lutein and zeaxanthin (88-92%) out of 17 different separated pigments, while less than 3% were reported to be epoxy pigments [1]. The carotenoid composition of marigold extract product was analyzed later via HPLC whereby epoxides namely violaxanthin and neoxanthin were it should be reported [2]. The saponified Tagetes erecta flower extract was reported for 93% utilizable pigments (detected at 450 nm), consisting of all trans and cis-isomers of zeaxanthin (5%), all trans and cis-isomers of lutein, and lutein esters (88%). Among the lutein isomers, trans-lutein was reported as the major component with several cis-lutein isomers as minor components from the commercial marigold flower extract. The yield of xanthophyll content in well preserved flowers of Tagetes erecta was reported to be 105.19 g/kg in contrast to the unpreserved flower sample (58.87 g/kg), thereby emphasizing the significance of flower preservation during the extraction of xanthophylls (Figures 1 and 2) [3]. Purification of carotenoids

MORPHOLOGY OF FLORAL STRUCTURES RELATED TO ANTITELECHORY DISPERSAL OF FRUITS IN Tagetes moorei H. Rob. var. breviligulata Villarreal

Tagetes moorei var. breviligulata (Asteraceae-Tageteae) es una especie endémica de Querétaro, México. La especie se encuentra en algunos lugares semiáridos y crece en suelos calizos. Hay pocos antecedentes sobre la biología y ecología de esta especie. En este trabajo se describen algunos aspectos de la morfología vegetal de T. moorei, particularmente de la cabezuela floral, relacionados con un mecanismo especial de dispersión de semillas clasificado como autócoro-antitelécoro. En este mecanismo, los aquenios formados en el involucro no caen directamente al suelo durante la deshidratación y secado de la cabezuela, como ocurre en la mayoría de las especies de Tagetes. En este caso, el pedicelo se desintegra y se separa del involucro, y entonces los aquenios se deslizan por dentro del pedúnculo hueco. Este mecanismo de dispersión de aquenios en ambientes semiáridos es poco común en las formas de adaptación de Asteraceae.

Composition and Anti-insect Activity of Essential Oils from Tagetes L. Species (Asteraceae, Helenieae) on Ceratitis capitata Wiedemann and Triatoma infestans Klug

Essential oils from four species of the genus Tagetes L. (Asteraceae, Helenieae) collected in Tucumán province, Argentina, were evaluated for their chemical composition, toxicity, and olfactory activity on Mediterranean fruit fly Ceratitis capitata Wiedemann adults and for repellent properties on Triatoma infestans (Klug) (Chagas disease vector). Yields of essential oils range from 0.2 to 0.8% (v/w). The same main constituents among Tagetes minuta L., Tagestes rupestris Cabrera, and Tagetes terniflora Kunth, (cis-trans)-ocimenes, (cis-trans)-tagetones, and (cis-trans)-ocimenones showed important differences in their relative compositions. Tagetes filifolia Lag. was characterized by the recognized phenylpropanoids methylchavicol and trans-anethole as the main components. LD(50) was ≤20 μg/insect in topical bioassays. T. rupestris was the most toxic to C. capitata females, whereas the other oils presented similar toxicities against males and females. Tagetes rupestris oil attracted both sexes of C. capitata at 5 μg, whereas T. minuta showed opposite activities between males (attractant) and females (repellent). Oils from T. minuta and T. filifolia were the most repellent to T. infestans. The results suggest that compositions of essential oils influence their insecticidal and olfactory properties. The essential oils from Tagetes species show an important potential as infochemical agents on insects' behaviors. This study highlights the chemical variability of essential oils as a source of variation of anti-insect properties.

Thiophene occurrence in differentTagetesspecies: agricultural biomasses as sources of biocidal substances

Tagetes species produce thiophenes, polyacetylenic compounds that possess strong biocidal activity, thus making Tagetes plants very useful for suppressing nematode populations in the soil and as sources of natural pesticides. In the present study, Tagetes species (T. erecta, T. filifolia, T. lucida, T. minuta, T. patula and T. tenuifolia) grown in northern Italy were evaluated for their morphophenological parameters and thiophene pattern in different plant parts (roots, shoots and flowers). Roots had the highest diversity and contents of thiophenes (from 64 to 100% of the total thiophene amount), with 5-(3-buten-1-ynyl)-2,2'-bithienyl (BBT) as the main component followed by 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), 2,2':5',2''-terthienyl (alpha-T) and 5-(4-hydroxy-1-butynyl)-2,2'-bithienyl (BBTOH). Tagetes lucida and T. tenuifolia possessed the highest amounts of total thiophenes (6717.3 and 6452.5 mg kg(-1) dry weight respectively), while T. minuta had the highest total thiophene yield (518.8 mg m(-2)), with BBT accounting for 98% of the total. Considering both thiophene concentrations and biomass yields, T. minuta and T. lucida appeared to be the most promising Tagetes species, with high potential for use as biocidal crops for the implementation of pest control practices that are less harmful to human health and natural resources.

Flowers of marigold (Tagetes) species as a source of xanthophylls

The accumulation of xanthophylls in flowers of marigold (Tagetes) species cultivated under conditions of the Belgorod region has been studied. Five cultivars representing three marigold species were investigated, including T. erecta (Rhodes and Orange Snow cultivars), T. patula (Bolero and Harmony) and T. tenuifolia (Red Gem). The overall xanthophyll content in the petals of flowers has been determined spectrophotometrically, while the composition of lutein diesters in each Tagetes species as well as the composition of anthocyanins in the flowers with claret spots have been studied by reverse phase HPLC. It is established that the total content of xanthophylls and their composition are close to the published data for analogous species growing in other regions of the world. It is shown that more than 90% of xanthophylls in flowers are retained upon drying and the content of lutein diesters in the dry material can exceed 15 mg/g.

Potential toxicity of new genotypes of Tagetes (Asteraceae) species against stored grain insect pests

Essential oils of three genotypes of Tagetes minuta, six of Tagetes patula and 15 of Tagetes erecta were screened for potential fumigant and contact toxicity against three stored product beetle species, Callosobruchus maculatus (Fabricius), Sitophilus oryzae (Linnaeus) and Tribolium castaneum (Herbst). Genotype 1 of T. minuta (TM-1), genotype 2 of T patula (TP-2) and genotype 13 of T erecta (TE-13) were the most toxic. These three genotypes were then evaluated for adult toxicity, oviposition deterrence, and ovicidal and population reduction activities. Essential oils of genotypes TM-1 and TP-2 induced 100% adult mortality for all three insect species at dosages of 50,000 ppm and 500 μgg/insect in fumigant and contact toxicity bioassays, respectively. Adults of C. maculatus and S. oryzae were highly susceptible to the essential oils of the three genotypes in both fumigant and contact toxicity bioassays. The essential oil of genotype TM-1 deterred oviposition in T. castaneum by 81% and suppressed its egg hatchability by 91% when applied at a dosage of 70,000 ppm on filter paper.

Characterization and Yield Evaluation of Essential Oils from Different Tagetes Species

Six species of Tagetes (T. erecta, T. filifolia, T. lucida, T. minuta, T. patula and T. tenuifolia), grown in northern Italy, were evaluated for their morpho-phenological characteristics, biomass yield and essential oil composition. The species showed marked differences in plant height, shape, flower size, habit and vegetative cycle length as found for T. filifolia which finished the vegetative cycle without reaching the flowering stage. The leaves always showed fresh material yields many fold higher than flowers (on average 26.8 and 4.4 t ha,−1 respectively). Tagetes erecta and T. patula produced significantly higher amounts of flowers than the other species (7.3 and 6.4 t ha,−1 respectively), whereas Tagetes lucida and T. filifolia were those with the highest yields of leaves (41.9 and 33.3 t ha,−1 respectively). The oils isolated by steam distillation from the flowers and leaves, were analyzed by GC and GC/MS. The flowers and the leaves of each species showed very similar qualitative oil compositions and the leaves, on average, were richer in oil contents. The oil yields between the flowers and leaves showed differences highly significant (on average 1.8 and 150.3 kg ha,−1 respectively). Tagetes erecta, T. minuta, T. patula and T. tenuifolia comprised the same pool of components (dihydrotagetone, tagetones, ocimenones and piperitone) which showed different and typical ratios in each species and, piperitone, (E)-tagetone, terpinolene and (E)-ocimenone were those more abundant respectively. Tagetes filifolia and T. lucida had methyl chavicol as the main compound.

GROWTH ANALYSIS AND EVOLUTION IN MARIGOLD AND FRENCH MARIGOLD (Tagetes erecta y Tagetes patula)

Mexico is considered as a diversity center for major Tagetes (Asteraceae) species. Since prehispanic age Tagetes erecta and Tagetes patula traditionally have been used as ceremonial flowers, in addition today they have promissory uses in sustentable agriculture, food, medicine, industry and ornamental, however domestication knowledge of these species is limited. Domestication of wild plant is able to change the morphology and development of plant species. In this study, growth analysis technique was used contrasting Tagetes erecta and Tagetes patula species, and regarding to their domestication level. Based on the apical and lateral meristems, it was determined that the flower initation (FI) ocurred 56 days after planting; in addition, flower development (FI to blooming) required 84 days in Tagetes erecta and 104 days in Tagetes patula. Tagetes erecta showed a more uniform seed germination, less branching in stems, lower biomass yield per plant, a shorter life cycle, higher values of the root/stem ratio and higher rates of relative growth and net assimilation than Tagetes patula. For this reasons, Tagetes erecta was considered to be in a higher level of domestication than Tagetes patula.

Xanthobacter tagetidis sp. nov., an organism associated with Tagetes species and able to grow on substituted thiophenes.

Members of the marigold genus of flowering plants (the genus Tagetes), which synthesize and accumulate thiophene compounds in their roots, were investigated as potential sources of bacteria able to degrade substituted thiophenes. Batch and continuous enrichment cultures inoculated with compost from root balls of Tagetes patula and Tagetes erecta reproducibly produced the same predominant type of bacterium when they were supplied with thiophene-2-carboxylate (T2C) or thiophene-2-acetate (T2A) as a carbon and energy substrate. This organism was a yellow-pigmented, neutrophilic, mesophilic, gram-negative, pleomorphic, rodshaped bacterium, which we classify as a new species of the genus Xanthobacter, Xanthobacter tagetidis; strain TagT2C (= DSM 11105) is the type strain. Strain TagT2CT (T = type strain) grew on simple thiophenes, such as T2C, thiophene-3-carboxylate, and T2A, on analogs of these compounds (pyrrole-2-carboxylate and furan-2-carboxylate), and on the condensed thiophene dibenzothiophene. X. tagetidis was facultatively autotrophic, fixing carbon dioxide by means of ribulose bisphosphate carboxylase, and was able to grow on hydrogen, thiosulfate, or sulfide as an energy substrate. It also grew on a wide range of other heterotrophic, chemolithotrophic, and methylotrophic substrates. Its growth on T2C was optimal at 28 to 31 degrees C and pH 7.6 to 7.8, and the maximum growth rate in batch culture was 0.22 h-1. The DNA base composition of X.tagetidis is 68 mol% G + C. A 16S ribosomal DNA sequence analysis of strain TagT2CT showed that this organism represents a distinct lineage within the Aquabacter-Azorhizobium-Xanthobacter cluster of the alpha-2 subclass of the Pro-teobacteria. Discrimination of X. tagetidis from the other genera in this group and from other Xanthobacter species is discussed.

Characterization of Tagete spatula cDNA Clones Obtained by Differential Screening in Relation to Thiophene Biosynthesis

In an attempt to isolate genes involved in thiophene metabolism, a cDNA library was constructed from poly(A) + RNA of a thiophene-producing root culture of Tagetes patula and differentially screened against a cDNA probe prepared from poly(A) + RNA of leaves, which are low in thiophenes. Five cDNA clones were selected for expression and sequence analysis. Two clones, containing previously unknown sequences, revealed the accumulation of gene transcripts in roots and hypocotyls but not in leaves of both Tagetes species. Since thiophenes predominantly accumulated in roots and hypocotyls, it is concluded that with respect to transcript distribution, the genes corresponding to these two clones are expressed as expected for genes of thiophene biosynthesis. However, in roots, the steady-state levels of the transcripts detected by both clones did not reflect the differences in thiophene content and thiophene synthesizing capacity, indicating that the corresponding genes are not involved in rate-limiting steps of the secondary pathway. Three cDNA clones showing homology to pathogenesis-related proteins of the Betvl -type revealed the accumulation of transcripts exclusively in roots of T. patula and T. erecta .

Toxicity of Extracts from Three Tagetes Against Adults and Larvae of Yellowfever Mosquito and Anopheles stephensi (Diptera: Culicidae)

Whole-plant Soxhlet extractions for the three Tagetes species showed that T. minuta had the greatest biocidal effect on the larvae and adults of Aedes aegypti (L.) and Anopheles stephensi Liston. Bioassays of simultaneous steam distillation extractions of the various parts of T. minuta found extracts from the flowers provided LD90s of 4 and 8 ppm against the larvae and 0.4 and 0.45% against the adults of A. aegypti and A. stephensi, respectively. Further research on T. minuta floral extracts as new biorational insecticides are discussed.

Content of Thiophenes in Transformed Root Cultures of Argentinian Species ofTagetes

The presence of thiophenes in four Argentinian species of TAGETES was studied. T. TERNIFLORA HBK and T. MINUTA L. seedlings contain 5-(4-hydroxy-1-butynyl)-2-2'-bithienyl (BBTOH); 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), while T. CAMPANULATA Griseb and T. LAXA Cabrera seedlings also accumulated BBT and alpha-T. From the four TAGETES species tested only T. LAXA was able to produce transformed roots when infected with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones of transformed roots were obtained in which the total thiophene content present showed considerable variations (277 to 1773 microg/g FW). The thiophene spectrum, however, was similar between different clones. In addition, the thiophene patterns in these transformed clones differed from that formed in the parent plants.

Control of thiophene accumulation in calli of two Tagetes species

The relation between root differentiation and accumulation of biocidal thiophenes was studied in stem calli of two Tagetes species. Disorganized tissues of T. erecta were low in thiophene production. A sharp rise in thiophene content coincided with the emergence of roots on the calli. Root regeneration and the amount of thiophenes produced were found to be quantitatively related. Callus tissues of T. minuta did not differentiate into organs. Nevertheless, they accumulated thiophenes throughout the incubation period. Light at 12 W m‐ stimulated thiophene production in T. erecta without having an apparent effect on root regeneration. In T. minuta even low irradiance levels (2 W m∼2) strongly inhibited thiophene accumulation. Under favorable conditions thiophene concentrations in calli of both species were comparable to or somewhat lower than the levels in roots on the plants and in excised, cultured roots of T. erecta. We conclude that in calli of T. erecta thiophene accumulation is coupled to root regeneration whereas a different control mechanism allowing for accumulation in disorganized tissues is operative in T. minuta.

Distribution and Accumulation of Thiophenes in Plants and Calli of DifferentTagetesSpecies

Ketel, D. H. 1987. Distribution and accumulation of thiophenes in plants and calli of different Tagetes species.—J. exp. Bot. 38: 322-330. The diversity of thiophenes (natural biocides) and the differences between the concentrations of these compounds in the leaves and roots of Tagetes erecta L., T.patula L. cv. Nana furia, and T. minuta L. (marigolds) indicated the presence of at least two different sites of accumulation: leaves and roots. Leaf expiants of Tagetes, however, are used by preference to obtain callus cultures. Once subcultured, secondary (C2) calli of T. patula obtained from leaves of 4 to 7-week-old plants, contained higher amounts of accumulated thiophenes (up to 80% of the amounts in the leaves) than original (C,) or twice subcultured calli (C3). The concentrations of thiophenes in C2 calli of T. minuta were about half those of C! calli, while the concentrations of thiophenes of C, calli amounted to 1-2% of the leaf values. Most of the C3 calli of T. minuta did not contain thiophenes at all. Although C3 calli of T. erecta also contained considerable amounts of thiophenes, the C2 calli died, most likely owing to high levels of accumulated polyphenolic compounds. The combination of species effects and the physiological state of plants and calli provides adequate information to decide whether Tagetes calli are able to produce thiophenes or not. It is concluded that the ability to produce thiophenes does not depend on the organ used, but on the genetic information present in the species, and on the physiological state of plants and calli, particularly their age.

GC‐MS analysis of the essential oils of four tagetes species and the anti‐microbial activity of Tagetes minuta

AbstractThe chemical composition of essential oils from the flowering plants of four Tagetes species was examined by GC‐MS. Of the species examined only T. minuta L. was rich in β‐ocimene. The other major constituents of this species and of T. tenuifolia Cav. were dihydrotagetone, tagetone, and (E)‐ocimenone. These two species only contained small amounts of (Z)‐ocimenone but, in contrast, the essential oil of T. patula L. contained equal amounts of (Z)‐and (E)‐ocimenone. The oil from T. patula also contained limonene and β‐caryophyllene. The essential oil from T. erecta L. differed largely from the other three species containing mainly limonene, β‐caryophyllene, and piperitone. In agar‐diffusion experiments the essential oil of T. minuta inhibited the multiplication of fungi, Gram‐positive and Gram‐negative micro organisms.

Characterization of Cutaneous Photoxicity Induced by Topical Alpha-Terthienyl and Ultraviolet A Radiation

Alpha-terthienyl (alpha-T), a phototoxic thiophene compound isolated from marigolds (Tagetes species), affects cell membranes and does not appear to induce cytogenetic damage. This study was undertaken to investigate topical delivery of alpha-T and characterize its cutaneous phototoxicity in combination with long-wave UV radiation (UVA) in comparison with locally (intradermal) administered alpha-T. Percutaneous penetration (PC) of 0.1% and 1% alpha-T in a 3% Azone gel vehicle was studied in guinea pig skin in vitro and quantitated by UV fluorescence microscopy. Dose-dependent PC of epidermis, adnexae, and superficial dermis was demonstrated in vitro. Alpha-terthienyl (0.1% and 1%) in this vehicle was applied topically in vivo and irradiated with 30 J/cm2 UVA at intervals of 10 min-24 h. Maximum sensitization was achieved with irradiation 1 h following drug application. The clinical response was dose-dependent consisting of erythema, edema, crusting, erosion, and inhibition of hair growth and was observed 72 h to 7 days postirradiation. A comparable dose-dependent phototoxic response was observed when 5-500 micrograms alpha-T were injected intradermally and irradiated with UVA. These results indicated that low-dose topical alpha-T in a nonirritating vehicle can rapidly produce cutaneous photosensitization. Topical alpha-T/UVA may provide a selective and safer alternative approach for the photochemotherapy of psoriasis and other cutaneous diseases.

Inorganic Nutrition of Callus Tissues of Tagetes Species: The Effects on Morphogenesis and Accumulation of Thiophenes and other Non-polar Secondary Metabolites

Summary The formation and morphology of callus were studied on leaf explants of Tagetes patula and Tagetes minuta , inserted into three different solid media with the inorganic composition of media used for soilless culture of intact plants, or into MS-medium Callus formation responded differently to the type of medium and plant species. On all «plant media» callus formation occurred in only one of the two species. Which species died and which species proliferated depended on the type of medium. Growth of calli was curtailed on plant culture media as compared with MS-medium. However, transfer of secondary calli from plant culture media to fresh MS-medium restored a high rate of growth. Calli of T. patula had a higher level of non-polar metabolites and thiophenes (natural biocides from Tagetes plants) than calli of T. minuta . In T. patula calli the content of non-polar metabolites increased from primary to secondary callus and was highest when MS-medium was used. In contrast, T. minuta calli showed a decrease in non-polar metabolite content upon subculturing and this content was lowest on MS-medium. These trends of the content of non-polar secondary metabolites paralleled the trends in morphological differentiation that were observed when calli were cultured continuously on MS-medium (Ketel, Physiol. Plant. 66 [1986], 392) and confirm the positive correlation between morphological and biochemical differentiation. It is concluded that the inorganic composition of the medium affected secondary metabolism only to a quantitative extent and that a correct inorganic composition of the medium together with the choice of the most suitable species are essential in the production of desired secondary metabolites in vitro.