Castanea is a hardwood forest genus of considerable agro-economic importance for both timber and nut production. Chestnuts are one of the most significant nut crops in the temperate zone. However, this species is threatened by pollution, social factors, economical changes, and two major fungal diseases: ink disease (Phytophthora spp.), and chestnut blight canker (Cryphonectria parasitica). Similar to other wood species, chestnuts are difficult to propagate both generatively by seed and vegetatively by means of grafting or cuttings. Biotechnological methods such as in vitro culture have been developed in the last few years as an alternative to conventional vegetative propagation. Biotechnology plays a very important role not only in the propagation of selected individuals (being used at a commercial level), but also in its short-term preservation, and offers the possibility of preserving the propagated material in the medium-term (cold storage) or long-term using cryopreservation.