Journal of Plant Physiology 166 (2009) 1069—1076 Benzyladenine metabolism and temporal competence of Pinus pinea cotyledons to form buds in vitro Milla ´n Cortizo a , Candela Cuesta a , Marı ´a Luz Centeno b , Ana Rodrı ´guez a , Bele´nFerna ´ndez a , Ricardo Orda ´s a,Ã a A ´ rea de Fisiologı ´a Vegetal, Dpto. Biologı ´a de Organismos y Sistemas, Instituto de Biotecnologı ´a de Asturias, Universidad de Oviedo, C/ Catedra´tico Rodrigo Urı ´a s/n, E-33071 Oviedo, Spain b Departamento de Biologı ´a Vegetal, Universidad de Leo ´n, Campus de Vegazana, E-28071 Leo ´n, Spain Received 30 September 2008; received in revised form 28 November 2008; accepted 19 December 2008 KEYWORDS Bud induction; Caulogenesis; Cytokinin; Shoot organogenesis; Stone pine Summary Germination negatively affects adventitious shoot formation induced by cytokinins in pine cotyledons. To investigate the causes of this decrease in the organogenic response, uptake and metabolism of benzyladenine (BA) were studied in stone pine cotyledons (Pinus pinea) isolated from in vitro germinating embryos and cultured in bud induction medium. As embryos grew, cotyledons showed a progressive decrease in the amount of BA taken up from the medium. BA was barely metabolized; however, a BA metabolite previously undescribed in conifers was found. It was identified as a glucoside of the BA riboside, a type of metabolite recently described in other gymnosperms. Data revealed that differences in the organogenic capacity of P. pinea cotyledons associated with embryo germination are related primarily to their ability to absorb BA from the bud induction medium. & 2009 Elsevier GmbH. All rights reserved. Introduction Adventitious shoot formation, also known as caulogenesis, is the means by which many plants can be micropropagated. In conifers, especially in the genus Pinus, shoots can be induced in vitro by culturing excised cotyledons in medium containing cytokinins as the sole inducing agent (Tranvan, 1979; Aitken-Christie et al., 1985; Alonso et al., ARTICLE IN PRESS www.elsevier.de/jplph 0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.12.013 Abbreviations: BA, benzyladenine; [9R]BA, benzyladenosine; [9R-5 0 P]BA, benzyladenosine-5 0 -monophosphate; [9GR]BA, 6-benzylamino-9-[O-glucopyranosyl-(1-3)ribofuranosyl]-purine. Ã Corresponding author. Tel.: +34985104795; fax: +34 985104777. E-mail address: rordas@uniovi.es (R. Orda´s).