Forestry plantations with evergreen broadleaf species in Mediterranean climate sites usually perform poorly in the field. Holm oak
(Quercus ilex ssp. ballota (Desf.) Samp.), cork oak (Quercus suber L.), wild olive tree (Olea europaea L. ssp. europaea var. sylvestris)
and lentisk tree (Pistacia lentiscus L.) are extensively used for such plantations. In order to determine the environmental factor that
induces cold hardening most effectively and the mechanisms that are involved in this process, seven month-old nursery seedlings were
taken to three growth chambers during the hardening phase and submitted to: 22/17ºC (day/night) and decreasing photoperiod from 12 to
8 h (Ph chamber), 12 h photoperiod and decreasing temperature from 22/17ºC to 8/3ºC (T chamber); and progressive reductions in
temperature and photoperiod (PhT chamber). The variation of morpho-physiological traits was assessed. Reducing the photoperiod by up
to 8 h did not stop the growth in height and diameter. Air temperatures below 8ºC reduced substantially height growth, mainly in Quercus
species, but not diameter growth. Reducing the temperature proved much more effective for cold hardening than reducing the photoperiod.
Low temperatures induced seedlings to accumulate non-structural carbohydrates (soluble sugars in the leaves and roots, and starch in the
roots), and to improve cold hardiness. Differences among species were observed in cold hardiness, with Q. ilex > Q. suber ≥ P. lentiscus ≥
O. europaea. The maximum level of cold hardiness achieved in this experiment by Q. suber and Q. ilex was reached when seedlings
accumulated 775-800 h8 (hours ≤ 8°C), 750 h8 in O. europaea and 725 h8 in P. lentiscus. Finally, measuring chlorophyll fluorescence
(Fv/Fm) after a freezing test was useful in estimating the cold hardiness of these species, providing results in less than two days for plant quality purposes.