Plant Cell, Tissue and Organ Culture 54: 61–63, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands. 61 Research note Cell cultures of Rauwolfia sellowii: growth and alkaloid production S.B. Rech ∗ , C.V.F. Batista, J. Schripsema 1 , R. Verpoorte 1 & A.T. Henriques Graduated Course in Pharmaceutical Sciences, UFRGS, Av. Ipiranga 2752, 90.610.000, Porto Alegre, RS, Brazil; 1 Center for Biopharmaceutical Sciences, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands ( ∗ requests for offprints) Received 3 April 1997; accepted in revised form 23 July 1998 Key words: apocynaceae, indole alkaloids, tissue culture, Rauwolfia Abstract Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium supplemented with 1 mg l −1 2,4-dichlorophenoxyacetic acid, 0.2 mg l −1 kinetin and 30 g l −1 sucrose. The growth cycle of suspension cultures was completed in ca. 22 days and the maximum specific growth rate was 0.0098 h −1 with a doubling time of 71 h. The cultures accumulated the same major alkaloids as in the leaves of the parent plant, such as sellowiine, 19α,20α-epoxyakuammicine, vomilenine, picrinine and 12-demethoxytabernulosine. The alkaloid contents of leaves, callus and cell suspension cultures were quantitatively compared by HPLC. Abbreviations: BA – benzylaminopurine; 2,4-D – dichlorophenoxyacetic acid; t d – doubling time; NAA – napthaleneacetic acid Species of Rauwolfia are a source for bioactive indole alkaloids and in vitro cultures have been shown to produce alkaloids of pharmaceutical interest (Stöckigt et al., 1981). Many compounds have been produced from R. serpentina cell cultures of which ajmaline, serpentine and reserpine are the most important (Roja et al., 1987). Rauwolfia sellowi Müll. Arg., commonly known as ‘Jasmim-Grado’, is a rare tree reaching up to 15 m high, which is native to forests from South East to South Brazil (Markgraff, 1968). Several indole alkaloids have been previously isolated from the root bark (von Poser et al., 1988) and from the leaves of this species (Batista et al., 1996). The aim of this study was to investigate the potential of obtaining pharma- ceutical important metabolites from in vitro cultures of Rauwolfia sellowii. Axillary buds derived from a tree growing in the Botanical Garden (Porto Alegre, Brazil) were washed with water, followed 1% NaOCl for 10 min, then with 70% EtOH for 5 min and finally washed three times with sterile H 2 O. Callus tissue was induced on either Gamborg B5 (Gamborg, 1968) medium supplemented with 30 g l −1 or 40 g l −1 of sucrose and various 2,4- D and kinetin concentrations (0.2 mg l −1 , 0.5 mg l −1 , 1 mg l −1 and 2 mg l −1 each) or on Murashige and Skoog medium, supplemented with 30 g l −1 sucrose, 2 mg l −1 NAA and 0.2 mg l −1 BA or with 5 mg l −1 2,4-D and 1 mg l −1 kinetin. After 4 weeks, cal- lus material which has developed at the edge of the explant was excised and transferred to fresh medium. The callus was incubated in 12-h photoperiod, 2000 lux, from cool white fluorescent tubes at 25 ◦ C and subcultured every 4 weeks. The callus developed on all MS medium compositions were hard and unsuit- able for the initiation of suspension cultures. The best medium for callus growth was Gamborg B5 medium supplemented with 1 mg l −1 2,4-D, 0.2 mg l −1 kinetin and with 30 g l −1 sucrose. This medium was used for routine propagation. After a period of five subcultures, a light-green to yellowish coloured friable callus was obtained and used for initiation of suspension cultures. Callus culture (5 g l −1 dwt) was transferred to a 250 ml flask containing 50 ml of liquid B5 medium sup- plemented with 1 mg l −1 2,4-D, 0.2 mg l −1 kinetin ticu2621.tex; 21/10/1998; 10:24; p.1 M:Disk CP PIPS NO.:186243 (ticukap:bio2fam) v.1.15