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?19 ��C, indicating that the plants had developed significant freezing tolerance before the start of the warming treatment (Fig.?3). Between October 1 and mid-January freezing, tolerance increased in successively later sampling dates reaching a maximum of on average ?27 and ?24 ��C in ��Titania�� and ��Narve Viking��, respectively. From mid-January to mid-March, freezing tolerance decreased or tended to decrease in both cultivars and treatments, while in April it remained stable in ��Narve Viking�� and in ��Titania�� at ambient temperature, but decreased further in ��Titania�� at elevated temperature. In late May, at the end of the experiment, stem freezing tolerance had decreased to ?5 to ?6 ��C in both cultivars. In both cultivars and treatments, LT50 values determined UNC2881 after initiation of the warming treatment correlated well with the average temperatures at 20 cm since the last sampling date (r = 0.76 for ��Titania�� at ambient and elevated temperatures, r = 0.77 for ��Narve Viking�� at ambient temperature and r = 0.90* for ��Narve Viking�� at elevated temperature, *P learn more at ambient temperature. However, differences were not statistically significant. Figure?3. Seasonal changes in cold hardiness estimated as temperatures representing 50 % REL (LT50) of stems of R. nigrum ��Titania�� (left) and ��Narve Viking�� (right) exposed to ambient or slightly elevated winter temperatures. LT ... None of the viability tests were suitable for assessing cold injury of buds. Regardless of collection date, none of the buds examined exhibited visible browning of the floral primordia after exposure to lethal temperatures. Floral primordia generally appeared turgid, Fluorouracil purchase but some buds, especially those subjected to the most severe freezing temperatures, exhibited signs of water soaking of tissues. Following incubation in TTC the majority of primordia developed coloured formazan, irrespective of the freezing temperature, making it impossible to distinguish between vital, subvital and dead floral primordia. Previous attempts to quantify floral injury in R. nigrum by visible browning (Takeda et al. 1993) or TTC (Warmund et al. 1991) were also unsuccessful. Carbohydrates The concentrations of starch and five soluble carbohydrates, i.e. glucose, fructose, sucrose, mannose and raffinose, were analysed in buds and stems (Figs?4 and ?and5,5, Table?2). Concentrations of carbohydrates varied to different extents depending on the nature of the carbohydrate considered, the organ, the treatment and the cultivar.