Enhanced shoot organogenesis in Bixa orellana L. in the presence of putrescine and silver nitrate

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 mu M 6-benzyladenine (BA) and 4.9 mu M indole-3-butyric acid (IBA), and supplemented with either 200-1,500 mu M putrescine or 40 mu M silver nitrate (AgNO3). Putrescine at 800 and 1,000 mu M 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 AgNO3 treatments was 70%. This protocol can be used for improving the in vitro regeneration of B. orellana for transformation studies.