Initiation of tissue culture of many plant species is a very difficult stage due to appearance of many contaminations. The other problem might be a choice of media for regeneration. Initiation of grass species tissue cultures are thought to be very difficult. Therefore, a research was undertaken to evaluate the use of nano-silver particles for plant material disinfection and to estimate a medium Pennisetum alopecuroides. The plant material were buds and nodal explants that were disinfected in 2% NaOCl for 30 min or 0.1% HgCl2 for 1 min. Half of the explants disinfected with NaOCl were soaked in 50, 100 or 250 mg·dm Ag–3 NPs for 1 hour. Explants not soaked in nano-silver were placed on media with Ag NPs at concentrations of 4, 8 or 16 mg·dm–3. An influence of growth regulators on Pennisetum alopecuroides was evaluated in vitro. Regenerated shoots were placed on MS media with: 3 mg·dm–3 BA + 0.3 mg·dm–3 IBA, 3 mg·dm–3 KIN + 0.3 mg·dm–3 IAA, 1 mg·dm–3 BA + 0.1 mg·dm–3 IBA. It was observed that the use of nano-silver particles lowered the level of contamination. The best results were obtained when Ag NPs was used at concentration of 100–250 mg·dm–3 alone or as a supplementation of the media, at concentration of 4 mg·dm–3 for nodes and 16 mg·dm–3 for adventitious buds. The use of nodal explants allowed to obtain less contamination. Regeneration depended on a media content. The most regenerated shoots were obtained on the MS media supplemented with 1 mg·dm–3 BA and 0.1 mg·dm–3 IBA.