This study describes an optimization of a plant regeneration protocol from mesophyll protoplasts in N. glauca that should enable further research with this species, which has shown interesting features for plant resistance to nematodes. Protoplasts were isolated from leaf mesophyll of in vitro-grown N. glauca plants and cultured on Mprot medium supplemented with 0.45 uM 2, 4-dichlorophenoxyacetic acid 2, 4-D, 4.44 uM 6- enzylaminopurine (BAP), and 5.37 uM alpha-naphthaleneacetic acid (NAA). After five weeks, microcalli were subcultured individually in MS medium supplemented with 0.27 uM NAA and 17.76 uM BAP for shoot regeneration. The 0.5 cm high shoots that appeared after eight weeks were transferred to MS medium without plant growth regulators for root induction. Plant acclimatization began 18 weeks after protoplast isolation and lasted for three weeks, after which they were transferred to soil. Shoot regeneration efficiency was 80-90%, and rooting efficiency was 86%.