Tagetes species were used by ancient civilizations like the Aztecs for various purposes (). The pigments of the flowers were used as a dye and in chicken feed, oil was extracted from the leaves and used as an ingredient of perfumes, and the roots were also assumed to have interesting properties. Field tests in the USA in the 1930s showed that larvae of a root-knot nematode entered the roots of marigolds, but usually failed to develop and neither reached the adult stage nor produced eggs (). In 1953, a Dutch bulb breeder () reported the biological activity of common garden marigolds (Tagetes patula) against root rot in Narcissus caused by free-living nematodes. The latter finding was an incentive for a scientific analysis of the effect of Tagetes plants by the crop protection industry and the academic world. A few years after the initial report by Van de Berg-Smit (), Uhlenbroek and Bijloo () isolated and described some active principles from Tagetes plants. These chemicals belonged to a group of heterocyclic sulphur-containing compounds, the thiophenes. The thiophene oe-terthienyl, which occurs in Tagetes and related species, was first synthesized in 1941 () and isolated from plants in 1947 ().
In the past three decades, much more has been discovered about these bioactive molecules. Recently (), a conference was held on the chemistry and biology of naturally occurring acetylenes and related compounds, with an emphasis on thiophenes. The proceedings of this conference report on the various aspects of thiophenes, e.g., their ecological significance, their biological activity, and their potential applications. Although various patents have been granted, no large-scale commercial production and application of thiophenes has been achieved. The 1988 conference proceedings make clear that many fundamental hurdles are still to be crossed. Therefore, the area is still a subject of active interdisciplinary research in Europe, North America, and Asia.
The biocidal activity of thiophenes has also prompted the study of thiophene metabolism in cell and tissue culture: on the one hand, to have access to a relatively simple and reproducible biological model system and on the other, to eventually incorporate these compounds in a process based on modern plant biotechnology, either to enhance the resistance against plant diseases or to produce specific compounds by cultured plant cells. Although species related to Tagetes and other compounds than thiophenes were part of these studies, emphasis seems to be on thiophene accumulation in a few Tagetes species. In this chapter we will summarize the work on cell and tissue culture of Tagetes spp. and where appropriate we will refer to studies with intact plants.
Characteristics of Tagetes Plants
Tagetes species belong to the Asteraceae family. This family comprises ca. 10% of all flowering plant species and contains many tribes with significant levels of polyacetylenes and related compounds, such as thiophenes (). Especially the subtribe Tageteae (tribe Helenieae) is rich in thiophenes. Probably because of its widespread use as an ornamental plant, the research on natural thiophenes has focused on a few of the ca. 20 Tagetes species, mainly Tagetes patula () and Tagetes erecta.
Tagetes spp. are strongly scented herbaceous or woody annual and perannual plants, with South-central Mexico as the area of the greatest diversity. Within the genus, there is a great variation in plant size, from a few decimeters in cultivated marigolds to many meters in the bushes of Tagetes minuta in Central America. In a greenhouse in Holland, the latter species could attain a height of more than 5 m.
In small-scale horticulture, marigolds are used for crop protection. Their use has been reported as a source of insecticides in Mexico () and for medicinal purposes in the Philippines (), China (), India and Pakistan (), Malaysia (), and South America (). In a review on the ethnobotany of Tagetes, Neher () reports that these species are used in folk medicine as an antiseptic, analgesic, carminative, diuretic, expellant, stimulant, vermifuge, vermin repellant, and aphrodisiac. Apart from agriculture and medicine, the plants are used for a variety of mystical and magical ceremonies in Latin America, India, and East Africa. It may well be that the alleged properties, already exploited in history, relate to the same specific phytochemicals which are studied nowadays in cell and tissue culture.
Conclusions and Prospects
It may be assumed that the use of Tagetes plants in ancient civilizations was partly based on their content of acetylenic compounds like thiophenes. Many centuries later in the 1940s, oc-terthienyl was synthesized and isolated from plant tissue. In the 1930s and 1950s, the biological activity of Tagetes against nematodes was (re?)dis-covered, followed by chemical analysis and unsuccessful nematicidal trials by the crop protection industry in the late 1950s.
The ascent of plant biotechnology and the need to use less hazardous biocides in agriculture has resulted in a new wave of interest for Tagetes and thiophenes in the 1980s. At present, various laboratories use Tagetes cell and tissue cultures to study thiophene accumulation for various reasons, ranging from potential application to academic attractiveness. Time will show whether fermentor-produced thiophenes will be competitive with chemically synthesized thiophenes and with other natural biocides. In Table “Summary of the protocol to obtain cell cultures and root cultures of Tagetes spp. and the production of thiophenes by these cultures”, the best protocols and results from our work in terms of growth and thiophene content have been summarized.
The in vitro culture of Tagetes species has proven to be an interesting and manageable alternative for intact plants to further the understanding of the regulation of secondary metabolism in general and that of naturally occurring acetylenes and thiophenes in particular. Hitherto most of the research has been rather descriptive and empirical. It is encouraging to note that during the last years, the first enzymes, related to thiophene biosynthesis have been characterized, and that the study of the biosynthetic pathway of thiophenes and their acetylenic precursors is reinforced. Very recently the effects of genetic transformation of Tagetes by Agrobactericum species have prompted a molecular analysis of the transformation process and its consequences for growth, morphology and thiophene metabolism. It is to be expected that the methods currently available to study and influence gene expression will be directed toward the understanding and exploitation of thiophene accumulation by in vitro cultures.
The success of the molecular approach, however, may be curtailed by the problems of instability of growth and metabolite production detailed in the present review, and the lack of knowledge of biochemical key processes, key enzymes, and their regulatory controls. Also, more basic knowledge of the relationship between thiophene metabolism and the metabolism of related classes of compounds, e.g., polyunsaturated fatty acids, other acetylenes and other polar and nonpolar S-compounds, has to be generated before the altered expression of genes involved in one or a few steps of thiophene metabolism will result in higher rates of metabolite production. Finally, it may well be that the knowledge of thiophene metabolism in vitro may in the next century be applicable to improve the defense system of crops against their predators, rather than to use chemicals from in vitro cultures for practical purposes.
Selections from the book: “Medicinal and Aromatic Plants IV”, 1993.