ORIGINAL PAPER Polyamines influence morphogenesis and caffeine biosynthesis in in vitro cultures of Coffea canephora P. ex Fr. Vinod Kumar Æ P. Giridhar Æ A. Chandrashekar Æ G. A. Ravishankar Received: 2 June 2006 / Revised: 29 August 2007 / Accepted: 26 September 2007 / Published online: 15 November 2007 Ó Franciszek Go ´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako ´w 2007 Abstract The influence of polyamines, polyamine inhibitors and ethylene inhibitors were tested in Coffea canephora for in vitro morphogenetic response and caf- feine biosynthesis. Coffea canephora produced non- embryogenic and embryogenic calli. Somatic embryos were produced only from the embryogenic callus. Endo- genous polyamine pools were estimated in these tissues. Somatic embryos were subjected to secondary embryo- genesis under the influence of putrescine, silver nitrate and specific inhibitors of polyamine biosynthesis. Estimation of endogenous total polyamines revealed that embryogenic callus contained 11-fold more spermine and 3.3-fold higher spermidine when compared to non-embryogenic callus. Incorporation of polyamines resulted in 58% explant response for embryogenesis when compared to control with 42% response. Incorporation of silver nitrate resulted in 65% response for embryogenesis. Incorporation of poly- amine biosynthetic pathway inhibitors DFMO and DFMA resulted in 83% reduction in embryogenic response with concomitant increase in caffeine levels by two-fold as compared to control. These results have clearly demon- strated that polyamines play a crucial role in embryogenesis and caffeine biosynthesis. Keywords Caffeine Á Coffea canephora Á Polyamines Á S-adenosyl-L-methionine Á Somatic embryogenesis Abbreviations NEC Non-embryogenic callus BA 6-Benzylaminopurine IAA Indole acetic acid AgNO 3 Silver nitrate PA Polyamine Put Putrescence SAM S-Adenosyl methionine DFMA-a DL-Difluromethyl arginine DFMO-a DL-Difluromethyl ornithine Introduction Plant embryogenesis represents the most definitive stages of the plant life cycle, with the overall architectural pattern of the mature organism established during a relatively short interval in many plant species (Thomas 1993). Endogenous and exogenously administered hormones play a crucial role in somatic embryogenesis. Polyamine pools and ethylene pathway are interlinked and known to redirect the cell towards embryogenesis when present in optimum concen- trations (Feirer et al. 1984). Secondary embryogenesis process requires a fine balance in the reprogramming of cells towards differentiation and maturation. Polyamines (PAs) viz. putrescine, spermidine and spermine are known to play important role in various cel- lular processes (reviewed by Bais and Ravishankar 2002). Chemically, they are non-protein, straight chain, aliphatic amines. Polyamines are known to be involved in DNA replication, cell division, protein synthesis, responses to abiotic stress, rhizogenesis, flower development and in vitro flower induction (reviewed by Bais and Ravishankar 2002). Polyamines are reported to inhibit ethylene Communicated by J. Kepczynski. V. Kumar Á P. Giridhar Á A. Chandrashekar Á G. A. Ravishankar (&) Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 575 020, India e-mail: pcbt@cftri.res.in 123 Acta Physiol Plant (2008) 30:217–223 DOI 10.1007/s11738-007-0110-x