An efficient protocol for direct shoot organogenesis in Bixa orellana, known as achiote or annatto or Latkhan (India), has been developed. Using nodal shoot-tip explants, significant organogenetic responses, mean shoot number and shoot elongation were observed when these were incubated on Murashige and Skoog (MS) medium containing 6.66 lM 6-benzyladenine (BA) and 4.9 lM indole-3-butyric acid (IBA), and supplemented with either 200-1,500 lM putrescine or 40 lM silver nitrate (AgNO 3 ). Putrescine at 800 and 1,000 lM promoted the highest mean shoot length and mean shoot number per explant, respectively. Moreover, various concentrations of putrescine induced callus development. Incorporation of a polyamine biosynthesis inhibitor Difluro-Methyl Ornithine (DFMO) inhibited in vitro shoot multiplication and also altered the endogenous polyamine pool of B. orellana shoots. The field survival of in vitro-derived plants of putrescine and AgNO 3 treatments was 70%. This protocol can be used for improving the in vitro regeneration of B. orellana for transformation studies.