Plant Cell, Tissue and Organ Culture 78: 63–68, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 63 In vitro culture and temporary immersion bioreactor production of Crescentia cujete Susan J. Murch 1 , Chunzhao Liu 2 , Rosaura M. Romero 3 & Praveen K. Saxena 4,∗ 1 Institute for Ethnobotany, National Tropical Botanical Garden, 3530 Papalina Rd., Kalaheo, Hawaii 96741, USA; 2 Institute of Process Engineering, Chinese Academy of Science, Beijing 100080, China; 3 Escuela de Quimica, Universidad de Costa Rica, San Pedro. Apdo 2060, Costa Rica and Bioprospecci´ on, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica; 4 Department of Plant Agriculture, University of Guelph, Bovey Bldg., Guelph, Ontario N1G 2W1, Canada ( ∗ requests for offprints: Fax: +1-519-767-0755; E-mail: psaxena@uoguelph.ca) Received 3 April 2003; accepted in revised form 3 November 2003 Key words: axenic cultures, Calabash tree, Crescentia cujete, medicinal plant, temporary immersion bioreactor, thidiazuron Abstract Crescentia cujete L. is a widely distributed medicinal tree with a diverse range of phytochemicals used as medicinal compounds. Seedlings of wild-harvested C. cujete were established in vitro and used as the starting material for the establishment of axenic cultures. Shoots were proliferated from nodal segments and were maintained over a period of more than 2 years by sequential subculture on a medium containing 1.0 μmol l -1 kinetin. De novo regeneration was induced on petiole sections cultured onto a medium containing thidiazuron in combination with 2,4-dichlorophenoxyacetic acid. Axenic cultures were also used to test the efficiency of three different cultivation systems for production of biomass of C. cujete. Growth of plantlets in a tempo- rary immersion bioreactor resulted in significant increases in biomass, leaf number, shoot height and trans- plant efficiency. Plantlets grown in the bioreactors were acclimatized under greenhouse conditions. Together, these experiments have established optimized parameters for propagation and growth of C. cujete plantlets in a sterile controlled environment for biochemical characterization and production of high-quality medicinal products. Introduction Crescentia cujete, the Calabash tree, is an evergreen tree that is used throughout the world as a source of medicines as an aperient, diuretic and febrifuge, to clean wounds, and for the treatment of headaches. Extracts of the plant have been found to be active against Gram positive bacteria but not significantly against Gram negative bacteria and allelopathic ef- fects have been reported. The hemorrhagic effects of Bothrops atrox venom may be neutralized by extracts of C. cujete (Otero et al., 2000). In Vietnam, dried fruit of the Calabash tree are sold by the name ‘Dao Tien’ as a laxative and an expectorant (Kaneko et al., 1998). Medicinally active constituents from C. cujete include: furanonaphthoquinones (Heltzel et al., 1993), iridoids, iridoid glucosides (Kaneko et al., 1997), n- alkyl glycosides and p-hydroxybenzoyloxy glucose (Kaneko et al., 1998). Crescentia cujete is commonly grown throughout Costa Rica but it is not native to North America and is grown only in a relatively small number of nurseries in the United States and Canada. Growth of medic- inal plants for production of high-quality extracts requires that the plants be grown in an envi- ronment with consistent light, nutrition and tem- perature as well as in isolation from abiotic and biotic contaminants (Murch et al., 2001; Saxena, 2001). To date, there have been no reports of growth of C. cujete in a sterile, controlled envi- ronment for production of high-quality tissues for biochemical analyses and production of medicinal