In this study we used chestnut clones selected for their resistance to ink disease and propagated them under photoautotrophic conditions during both proliferation and adventitious rooting stages. For successful proliferation, shoots were first exposed to decreasing sugar levels for some months, to enable their survival in sucrose-free medium. In vitro rooting in photoautotrophic conditions was applied to 15 chestnut genotypes. More than 6000 shoots were tested, and average values for rooting and acclimation were better than in conventional micropropagation.