Great advances have been made but some cultured plantlets still having a poor performance during the ex vitro stages and more specifically during the acclimatization and the nursery establishment stages. The problem is highly complex and requires the fast adaptation of plants growing in artificial conditions when transferred to the natural conditions. Until now, there is no consensus on the better strategy to obtain a higher efficiency of the propagation protocols while optimising acclimatization success. In this study we present the results of growth analysis of in vitro-regenerated chestnut hybrid plantlets (Castanea sativa x C. crenata), during the acclimatization stage, using two CO2 concentrations (350 µLL-1 and 700 µLL-1) at 250 µmol m- 2 s-1 as irradiance level (PPFD). Elevated CO2 did not affect the survival rate and it was susceptible to increase progressive autotrophy, expressed by a significant increase in relative growth, shoot/root ratio and leaf area ratio (LAR). For both CO2 concentrations, the plants are successfully acclimatized and they are in good conditions to be transferred to a greenhouse to continue their development for the rest of the season, and in the next winter/spring they can go to the field. The plants under elevated CO2 showed a higher stomatal frequency but the new leaves developed at the end of acclimatization revealed a gradual normal stomatal morphology and they reduced the stomatal frequency. Their morphology showed an effective water loss control, which is one of the most important problems during this critical phase of the autotrophic competence acquiring process. The net photosynthesis rate (A) was similar in both treatments but the plants acclimatized at elevated CO2 showed an increase in maximum photosynthetic rate (Amax), and this can lead to a better physiological development. The different analysed leaf types showed a marked increment of the maximum photosynthetic rate as the new leaves developed during the acclimatization stage. Net photosynthesis rate and the maximum photosynthetic rate are light dependent, and are positively affected by the highest irradiance level. We think that the gains that we have achieved with the use of elevated CO2 can be more significant if a higher light intensity can be used instead because they have a better response capacity to an increment of the level of irradiance
