23 Adv. Hort. Sci., 14 (2000): 23 - 27 Received for publication 20 February 2000. Differential thermal analysis, supercooling and cell viability in organs of Olea europaea at subzero temperatures P. Fiorino, S. Mancuso Dipartimento di Ortoflorofrutticoltura, Università di Firenze, Via G. Donizetti 6, 50144 Firenze, Italy. Key words: chilling tolerance, electrolyte leakage, freezing temperature, Olea europaea, supercooling, visual score, vital stain. Abstract: Cold hardiness in different organs and tissues of four different olive (Olea europaea L.) cultivars characterised in a previous work as different in frost tolerance, was assessed by four different methods (diffe- rential thermal analysis, visual score, vital stain and electrolyte leakage) to determine which method is more reliable for estimating freezing injury in olive. Results obtained with the different techniques consistently agreed. Among the four different procedures utilised, DTA was by far the fastest, whereas the visual score method the simplest, although not as quantitative as the other three methods. It would appear from the present study that the order of sensitivity in the different organs of olive is secondary roots > primary roots > apical lea- ves > basal leaves > shoots > vegetative buds. ‘Ascolana’ was the most chilling-tolerant variety, whereas ‘Coratina’ the most chilling-sensitive. The wide range of tolerance showed by olive (i.e. from –11.2 to –15.3°C for the leaves) together with the recurrent danger of frost in many areas of cultivation, made this species an ideal candidate to breeding for low-temperature-tolerant plants. 1. Introduction Temperature extremes are one of the most impor- tant factors limiting plant distribution and producti- vity. For olive trees, low temperatures are more limi- ting than are high temperatures in both ecological and agronomic contexts. In particular, olive cultivars accli- mated to high temperatures maintain 70-80% of their photosynthetic rate at 40°C (Bongi et al., 1987) whe- reas the low temperature tolerance of most cultivars is not particularly noteworthy, as they generally succumb by –12°C (Mancuso, 2000). Olive plants exposed to low temperatures can sur- vive either by avoiding ice formation in their tissues (supercooling ability) or by developing a tolerance for it (frost hardening). Frost hardening is a genetically-controlled trait which is driven by three key environmental factors: temperature, photoperiod and water stress (Tumanov and Krasavtsev, 1959; Christersson, 1978; Levitt 1980). According to the prevalent theory, certain spe- cific genes are activated as a result of changes in these factors. These changes induce a metabolic hardening mechanism, which results in an increase in frost hardi- ness in autumn and a decrease in spring (Weiser, 1970; Repo, 1992). Experiments with different tree species and prove- nances have shown that the changes in frost hardiness at various stages during annual development are gene- tically determined, as well as the minimum hardiness level during the growing season (Sakai and Eiga, 1985; Sakai and Larcher, 1987; Toivonen et al., 1991). Many woody angiosperm species survive winter temperatures by deep supercooling tissue solution to temperatures as low as the homogeneous nucleation temperature of the aqueous solution (near -40°C for plant solution). The freezing of this fraction of water can be observed as an exotherm during cooling at a constant rate by differential thermal analysis (Ishikawa, 1984; Mancuso, 2000). Supercooling as a cold hardiness mechanism has been demonstrated in various temperate plant tissues such as xylem ray parenchyma, leaf buds and flower buds (George et al., 1974; Sakai, 1979; Hong and Sucoff, 1980). In other words, supercooling is usually employed by limited tissues (xylem, buds, seeds). Compared with the majo- rity of plants, olive seems rather particular as will be shown in the present paper that most of its organs, from leaf to roots, use supercooling as a mechanism of cold hardiness.