Short Research Paper Abstract: Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alka- loids content analyses and growth curves showed that lines ob- tained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcul- tures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosyn- thesis of the imidazole alkaloid in P. microphyllus. Key words: P. microphyllus, pilosine, pilocarpine, alkaloids. Introduction Pilocarpine is an imidazole alkaloid used to treat glaucoma, xe- rostomia and to stimulate lachrymal and sweat glands (Abreu et al., 2005). This alkaloid is obtained exclusively from the leaves of species known as Jaborandi (Rutaceae). Pilocarpus microphyllus is the species of the genus which accumulates the largest amount of pilocarpine in leaves and is found mainly in the Amazon region of Brazil (Pinheiro, 1997). In addition to pilocarpine, leaf extracts from this species also contain other alkaloids, such as 3-nor-8(11)-dihydropilocarpine, anhydropi- losine and pilosine, pharmacological activity of which is still unknown (Andrade Neto et al., 1996; Link and Bernauer,1972; Voigtlander, 1978). Similar to other species from this region, it has been extensively exploited and, as a result, it was includ- ed a few years ago in the list of Brazilian endangered plant spe- cies (Pinheiro,1997). Therefore, cell cultures may represent an alternative for the production of these valuable substances. Recent advances in biochemistry, molecular, cellular and ge- nomic methodologies have pushed the study of secondary me- tabolite biosynthesis to a level in which very detailed informa- tion can be obtained, enabling better understanding of the fine control of these compounds and how structural and chemical diversity are created (Facchini, 2001). In this sense, model plants and model plant systems have proved to be valuable tools. Both techniques are keys to the study of alkaloids, con- sidering that, unlike other categories of secondary metabo- lites, many alkaloids are biosynthetically unrelated. They form groups which are unique in structure. So far, perhaps the most studied and more versatile model system is Catharanthus roseus (Madagascar periwinkle), which accumulates monoter- penoid indole alkaloids (Vazquez-Flota et al., 2002). In recent years, plant cell suspension cultures have been stud- ied as an economic alternative to produce alkaloids with po- tential medical applications. The possibility to control all the cultivation conditions, alter medium components and sim- ulate stresses has the potential to turn plant cell cultures into a “pot of gold” to study the biosynthesis of alkaloids from the biochemical, chemical, physiological and molecular biology point of view (Collin, 2001). Many factors can affect the pro- duction of alkaloids in callus and cell suspension cultures, since these can be produced through several biosynthetic routes, according to the class to which they belong (Facchini, 2001). Plant cell cultures also offer the advantage of producing compounds not found in the intact plant, once alternative metabolic routes become active (Dias et al., 1998). Abreu et al. (2005) observed that the production of pilocarpine in calluses from Jaborandi leaves was strongly influenced by media com- position. Simola et al. (1988) reported that the tissue used to originate the calluses and the callus age were important fac- tors for the production of tropine alkaloids in Atropa belladon- na. However, independent of the factors affecting alkaloid pro- duction in cell cultures, the main objective herein is to obtain plant cell lines presenting a high metabolite yield and exhibit- ing a high degree of biochemical and physiological stability (Morris et al., 1989). Although some alkaloids have been isolated and identified in Jaborandi, there is no information about the biosynthesis of pilocarpine in this plant. In a previous study, we analyzed the Cell Suspension as a Tool to Study the Biosynthesis of Pilocarpine in Jaborandi I. N. Abreu 1 , N. L. Andreazza 1 , A. C. H. F. Sawaya 2 , M. N. Eberlin 2 , and P. Mazzafera 1 1 Departamento de Fisiologia Vegetal, Instituto de Biologia, C.P. 6109,13083-970 Campinas, SP, Brasil 2 Instituto de Química, Universidade Estadual de Campinas, C.P. 6109,13083-970 Campinas, SP, Brasil Received: January 18, 2007; Accepted: March 20, 2007 Plant Biol. 9 (2007): 793 – 799 © Georg Thieme Verlag KG Stuttgart · New York DOI 10.1055/s-2007-965250 · Published online August 7, 2007 ISSN 1435-8603 793