Effect of exogenous polyamines on coconut (<em>Cocos nucifera</em> L.) embryogenic callus multiplication

Major bottlenecks of coconut in vitro culture are poor plant regeneration rate, severe browning and premature necrosis of cultured tissue, and heterogeneous response of individual palms and explants to in vitro culture conditions. Among them, tissue browning is a common and often severe problem in coconut in vitro culture systems which results in death of explant/ callus ultimately. This experiment was carried out to enhance the in vitro multiplication of coconut, which is a highly recalcitrant species to in vitro culture through exogenously added polyamines in the media. The polyamines are important for in vitro cell division, cell growth and to delay senescence. In the present study, unfertilized ovary derived calli were cultured on Y3 basal medium supplemented with sucrose (5%), 2,4-D, phytagel (3 gL-1), activated charcoal (2.5 gL-1), and polyamine. Three polyamine types (O.lmM spermine, 1.0 mM putrescine and 0.5mM spermidine) were tested in combination with two 2,4-D concentrations (0.30 and 0.60 mM) in order to enhance coconut in vitro multiplication. All the cultures were incubated in dark at 26±2 °C. The embryogenic structures, embryogenic callusing, non-embryogenic callusing, and browning were recorded separately for each treatment after five weeks from the culture establishment. The polyamine treatments did not have significant effects on frequencies of embryogenic structures, embryogenic callus, non-embryogenic callus and browned callus formation at the initial stages of coconut somatic embryogenesis irrespective of the tested 2,4-D concentrations. Furthermore, the results indicated that decreased 2,4-D levels have significantly reduced browning, resulting 44.79 % browning frequency (0.8-fold lesser browning) in media supplemented with 0.30 mM 2,4-D. However, the potential effects of exogenously added polyamines at the initial stages of coconut somatic embryogenesis could be delivered during the latter stages of the somatic embryo genesis as previously reported in other experiments. Thus, continuous subculture may be necessary.