Pyrethrins, the economically most important of natural insecticides, are currently derived from the pyrethrum plant. Some biotechnology companies study the possibilities of industrial production. Whether they are able to overcome the biological and technical constraints is still unclear. But if so, the production of hundreds of thousands of pyrethrum farmers in the South will be threatened.

Due to an increased resistance of pests to synthetic pesticides, stricter environmental legislation, and mounting R&D costs of chemical insecticides, interest in natural insecticides has been expanding continuously in recent years. The plant world is not only a very important source of natural insecticidal compounds, but also provides core structures from which new and more effective insecticidal agents can be synthesized. Today, the most economically important natural plant compounds used as insecticides are the pyrethrins, a group of six, chemically closely related, complex esters.

Pyrethrum
The principal source of pyrethrins is pyrethrum (Chrysanthemum cinerariaefolium). Pyrethrum is a tufted perennial herb of temperate origin, with white­yellow flower heads. It is cultivated mainly at higher altitudes in tropical countries such as Kenya, Tanzania, Rwanda, and Ecuador (see also Monitor no. 13). Pyrethrins are found in all above­ground parts of the pyrethrum plant, but predominantly in the flower heads. The harvesting of flowers is very labour intensive, which has resulted in a decreasing cultivation in some parts of the world. Japan, for example, which used to be a large producer, has abandoned cultivation, while in India only 100 to 200 hectares are presently under cultivation.
Powder and extracts prepared from the dried flower heads have been used as insecticides for many years. The natural pyrethrins have some of the qualities of an ideal pest­control agent. They are very effective against a broad range of insects, while little resistance has developed. Especially valued is their rapid paralysation of insects resulting in low damage levels, and their low toxicity to mammals and other warm­blooded animals. Another application is as a repellent to protect foods. Pyrethrins are non­inflammable and leave no oily residue.
In spite of their superior environmental qualities, the general instability of the natural pyrethrins has restricted their application as a multi­purpose insecticide. Since pyrethrins are useful only under conditions without extensive exposure to light and air, they are too unstable outdoors to control pests of agricultural crops and forests efficiently. However, by mixing with antioxidants or stabilizers (including the natural plant compounds tannic acid and hydroquinone) and synergists (including the natural plant compounds sesamin and mysristicin), the stability has been improved, and pyrethrins have become economically viable insecticides. Today, the natural pyrethrins are used predominantly in domestic insecticide sprays.
The instability of an otherwise very powerful insecticide led to the development of pyrethrin­like synthetic compounds, called pyrethroids. In the late 1960s and early 1970s the first synthetic compounds were produced, and several of these compounds were registered. The greater stability means that the time the insecticide is present in the field after spraying is much longer than with the natural pyrethrins. This could cause the build­up of insect resistance and increases the potential of toxic and/or carcinogenic effects on mammals, which are seen today as the major drawbacks of synthetic pyrethroids. Pyrethroids are commonly used in crop spraying.

Biotechnological alternatives
The disadvantages of the synthetic pyrethroids, together with the labour intensity of conventional pyrethrum production, the commercial importance, the high demand, and the often unstable supply of pyrethrum has stimulated research in alternative production of the natural pyrethrins. Efforts have been made by several research groups and biotechnological companies to generate pyrethrins by in vitro cultures of Chrysanthemum cinerariaefolium (callus cultures, cell suspension cultures, shoot and root cultures) and, more recently, bioconversions of pyrethrins precursors.

In vitro cultures
The aim of the culture systems was the establishment of one or more highly pyrethrin­producing lines, which would eventually be cultivated in bioreactors. It appeared that callus cultures, cell suspension and root cultures do not produce pyrethrins, while callus does produce some of the pyrethrins precursors. Studies of pyrethrum plant­derived in vitro cultures revealed that unorganized tissue cultures do not have the secondary metabolism characteristics of the corresponding intact plant, and that accumulation of the pyrethrins occurred only after shoot morphogenesis. Consequently, only organized shoot cultures could be considered for pyrethrin production. Biological as well as technological obstacles, however, have prevented the development of a large­scale industrial process based on shoot cultures so far.
The only serious attempt to develop such a process was initiated by McLaughlin Gormley King Co., Minneapolis, Minnesota, USA, a leading importer and processor of pyrethrum, at the beginning of the 1980s. It funded research at the International Plant Research Institute, San Carlos, California, USA, to develop an in vitro culture process for the production of pyrethrins. Small­scale prototype bioreactors were developed, but the project was discontinued after several years by the lack of economic and technological feasibility. The levels of pyrethrin production in shoot cultures appeared to be too low. Pyrethrin yields were raised to approximately 0.5 per cent of dry weight in differentiated tissues, but these were too low, in comparison with 2 per cent in the flower heads of the field grown plant, to be economically feasible. A major technological obstacle was encountered when novel bioreactor design and configurations appeared to be necessary for the large­scale cultivation of shoots.

Bioconversion
Recently, bioconversion of readily available precursors by isolated plant enzymes or genetically engineered micro­organisms has emerged as another alternative to the conventional production of pyrethrins. In 1984 a patent was granted to McLaughlin Gormley King Co. for the enzymatic synthesis of pyrethrins. This patent describes a process for the production of radioactively­labelled pyrethrins. The process comprises: (a) preparation of a cell­free homogenate (homogenized plant tissues) containing enzymes and cofactors (non­protein substances essential for one or more related enzyme reactions) of the pyrethrin pathway of Chrysanthemum spp.; and (b) an incubation of the homogenate with radioactively­labelled mevalonic acid or isopentyl pyrophosphate, which are both pyrethrin precursors. However, the composition of the cell­free homogenate regarding enzymes and cofactors is not defined in the patent. Large­scale industrial production with such a process is questionable, particulary because of possible variations in composition and enzymatic activity of a cell­free homogenate.

The synthesis of precursors and/or its bioconversion to pyrethrins, could also be carried out by the use of genetically engineered micro­organisms. Based on knowledge of the pyrethrins biosynthetic pathway, certain steps can be selected as candidates for bioconversion of readily available precursors. Enzyme(s) catalysing desired step(s) have to be isolated and described, after which the gene(s) responsible for the synthesis of these enzyme(s) have to be identified and characterized. Besides the complexity of these steps, the process is further complicated by the fact that the target, the pyrethrins, is not a single compound but a mixture of six closely related, but different, complex esters.
The biotechnology company AgriDyne Technologies Inc., Utah, USA, is active in this field. The company uses a genetically engineered micro­organism containing the plant gene coding for chrysanthemyl diphosphate synthase to develop a key active intermediate product normally produced in the pyrethrum plant. This intermediate product could be further converted to pyrethrins. According to the research manager of the pyrethrum developmental programme at AgriDyne, scientists are currently testing the activity of the transferred gene in the micro­organism.
Chrysanthemyl diphosphate synthase is, however, just one of the enzymes acting in the biosynthesis of pyrethrins. Even if it would catalyse the most difficult step in the pathway, there are other steps in the biosynthetic route that will require enzymatic or chemical catalysis. Ultimately, the success of this approach will not only be determined by the level of enzyme expression in micro­organisms, but also by other aspects, such as price and availability of necessary precursor(s), and the potential need for partial chemical synthesis or modification(s) of intermediates. It is unclear if and when industrial production of pyrethrins based on bioconversions of readily available precursors will become reality. At present, however, this approach seems to be the only attractive biotechnological alternative for industrial development.

Commercialization and possible impact
For the successful commercialization of a biotechnological process for the production of pyrethrins, the ultimate criterion is that it must be less expensive to make pyrethrins by alternative biotechnological means than to extract it from field­grown plants. At present, it is impossible to assess the economic feasibility of biotechnological production of pyrethrins. Pertinent data on product yields are scarce while data on productivity are virtually non­existent. Important factors such as market demand, price fluctuations and dumping, alternative supply sources, and socio­political aspects will have to be considered as well.
Nevertheless, there are economic potentials making research in the biotechnological production of pyrethrins feasible. The annual world market for the natural pyrethrum insecticide has been estimated to be as high as US$ 400 million (1992), while the conventional production of natural pyrethrins is still below global market demand. Therefore, even with a moderate capture of 10 per cent of the world market, biotechnological pyrethrins could reach annual sales of US$ 40 million, far more than the suggested threshold for commercialization of US$ 10 million per year.
The pyrethrins are not attractive for large­scale production based on shoot cultures, not only because of biological and technological bottlenecks, but also because the market price of plant­extracted pyrethrins (approximately US$ 400 per kg) is below the estimated bottom price level of US$ 500 per kg. for products produced by plant cell/tissue cultures. The bioconversion process under development at AgriDyne seems to be more promising economically. The company estimate that, if successful, they will produce bio­pyrethrum within a price range of US$ 110 to US$ 150 per kg and sales would potentially reach US$ 100 million by the late 1990s. However, the technological feasibility of this approach is still uncertain. If AgriDyne Technologies Inc. could overcome the technological constraints, bio­pyrethrum would cost less than East African pyrethrum and capture a substantial share of the natural pyrethrum market. This development could prove to be economically very damaging to an estimated 200,000 East African small­scale farmers who cultivate pyrethrum flowers. However, as described above, this perspective seems to be still remote.
Srdjan Jovetic

Sources
G.J. Kudakasseril and E.J. Staba (1988), "Insecticidal Phytochemicals." In: F. Constabel and I.K. Vasil (eds.), Cell Culture and Somatic Cell Genetics of Plants, Vol.5. New York: Academic Press.

H. Shand (1992), "Genetic Engineering of Pyrethrins: Early warning for East African pyrethrum farmers". RAFI Communique, June 1992.

E.J. Staba and S.W. Zito (1985), "The production of pyrethrins by Chrysanthemum cinerariaefolium (Trev) Boccone." In: Neuman, Barz and Reinhard (eds.), Primary and Secondary Metabolism of Plant Cell Cultures. Berlin­Heidelberg: Springer­Verlag.

Personal communication with O. Sahai (ESCA Genetics Corporation, USA), L.V. Venkataraman (Central Food Technological Research Institute, India) and G. King (AgriDyne Technologies Inc., USA).