Abstract This study aimed to identify biological contaminants of banana in vitro cultures. We also tested the effect of Zn or ZnO nanoparticles on elimination of some bacterial and fungal contaminants and their influence regeneration. Nine strains of bacterial contaminants (Cellulomonas uda, Cellulomonas flarigena, Corynebacterium panrometabolum, Bacillus megaterium, Staphylococcus spp., Klebsiella spp., Erwinia cypripedii, Pseudomonas spp. and Proteus spp.) and four fungal (Fusarium spp., Aspergillus spp., Penicillium spp. and Candida spp.) contaminants were identified in nanoparticles-free media of banana in vitro cultures. They eventually led the explants death. The contamination-free cultures of banana in vitro cultures were obtained as a result of application of nano Zn and ZnO particles to the culture MS media, with no negative effect on regeneration. The callus growth decreased while total proline as well as SOD, CAT and POX activities were increased significantly, when the nanoparticles doses increased. The highest percent of somatic embryogenesis was observed in MS media supplemented with 100 mg/L nano Zn followed by nano ZnO. Excellent shooting, rooting and regenerated plantlets were observed also in MS+100 mg/L nano Zn and ZnO. Regenerated plantlets were successfully acclimatized with about 98% efficiency for the experimental period (one month). Nanoparticles treated somaclones accumulated more proline, chlorophyll, antioxidant enzymes activity and developed more dry weight accumulation than the control. In conclusion, the microbial contaminants in banana in vitro culture can effectively be eliminated by incorporation of nano Zn and nano ZnO particles on growth media at different concentrations. However, 100 mg/L dose was preferable because it showed the best effects on increasing the regeneration of plantlets with well-formed root systems. Further studies are needed to investigate the mechanisms and the side effects of nanoparticles on genetic stability of banana in vitro cultures
