Plant Cell, Tissue and Organ Culture 60: 47–53, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 47 Micropropagation of Pothomorphe umbellata via direct organogenesis from leaf explants Ana Maria S. Pereira 1 , Bianca W. Bertoni 1 , Beatriz Appezzato-da-Gl´ oria 2 , Alba R. B. Araujo 1 , Ana Helena Janu´ ario 1 , Miriam V. Lourenço 1 & Suzelei C. França 1 1 Departamento de Biotecnologia Vegetal – UNAERP, 16096-380 Ribeirão Preto, SP Brazil; 2 Departamento de Botânica – ESALQ/USP, Piracicaba, SP Brazil Received 21 June 1999; accepted in revised form 22 March 2000 Key words: adventitious shoots, anti-hepatotoxic, medicinal plant, 4-nerolidylcatechol Abstract The establishment of a micropropagation protocol for Pothomorphe umbellata was carried out using leaf segments cultured on 1/4 strength Murashige and Skoog medium supplemented with 0.5 mg l −1 6-benzyladenine, 0.1 mg l −1 gibberelic acid added with 10 g l −1 sucrose. Rooting was achieved using MS medium devoid of growth regulators. An anatomical study confirmed shoot regeneration via direct organogenesis. Abbreviations: BA – 6-benzyladenine; GA 3 – gibberelic acid; MS – Murashige and Skoog medium; MS/2 – 1/2 strength Murashige and Skoog medium; MS/4 – 1/4 strength Murashige and Skoog medium; NAA – naphtaleneacetic acid Introduction Pothomorphe umbellata (L.) Miq. is described in the Brazilian Pharmacopeia and is commonly known as pariparoba or caapeba. It belongs to the Piperaceae family and extracts of its root are used as colagogue and anti-hepatotoxic agents. Studies conducted by Felzenszwalb et al. (1987) with P. umbellata extracts revealed that it was not mutagenic. Ethanolic extracts of P. umbellata have a high an- tioxidant activity comparable to tocoferol, which is attributed to its content of 4-nerolidylcatechol (Kij- joa et al., 1980; Barros et al., 1996). Also, 4- nerolidylcatechol dimers present in P. peltata extracts, show inhibitory activity against HIV (Gustafson et al., 1992). Several Brazilian laboratories produce phytophar- maceuticals containing P. umbellata. However, there is no commercial production of this species in Brazil and its removal from the wild is leading to a rapid and progressive devastation of the species. Despite being an herbaceous plant, it grows very slowly and its seeds present a low germination index. The object- ive of this work was to establish a micropropagation protocol for P. umbellata, to describe the histology of the stages of culture regeneration and determ- ine whether or not 4-nerolidylcatechol is present in micropropagated plantlets. Material and methods Sterilization Leaves of one year-old P. umbellata plants grown at the University of Ribeirão Preto were cut into 1 cm 2 and washed in tap water for 12 hours, soaked in 1% (w/v) benomyl solution for 4 hours, then in a 0.25% calcium hypochlorite (w/v) solution for 30 minutes and finally immersed in gentamicin (100 mg l −1 ) for 1 hour. Explants were placed directly onto the culture media without washing in water. Inoculation Disinfested explants were inoculated onto MS semi- solid medium (Murashige and Skoog, 1962), contain-