Causes of variation in cold hardiness in the autumn were assessed among closely related, fast-growing clones of willow of northern/continental and southern/maritime origins, under controlled regimes and natural conditions. Cold hardiness was assessed by controlled freezing followed by injury analysis, based on measurements of chlorophyll fluorescence (stems) and electrolyte leakage (leaves). During growth at a given temperature, the cold hardiness of the clones' stems was negatively correlated with their rate of growth. This apparently phenotypic variation was independent of temperature and, hence, the absolute growth rate. At later stages, cold hardiness of stems varied mainly with respect to genetic differences in the timing and rate of cold hardening. Cold hardening began up to 7 weeks earlier in northern/continental clones, and their rates of hardening in cool temperature regimes were up to three times higher than in southern/maritime clones. Ranking of clones with respect to rates was essentially the same whether natural or abrupt reductions of day length were used to trigger cold hardening. Results closely agreed with those of a previous field trial. Comparisons of rates at cool and warm temperatures suggest that cold hardening became increasingly dependent on cool temperatures with time. Increasing sucrose-to-glucose ratios, and especially dry-to-fresh weight ratios, paralleled early cold hardening. Before leaves were shed in the autumn they underwent cold hardening in parallel with stems, eventually allowing them to tolerate temperatures down to ----10 °°°° C.

Closely related, fast-growing clones of willows from northern/continental and southern/maritime origins were assessed for their levels of cold hardiness. Assessments were made during active growth and, subsequently, during cold hardening at mean temperatures of 3C (the COLD regime) and 8C (the MILD regime). The onset of hardening was triggered simultaneously in all clones by administering a drastic day length reduction on the first day. The northern/continental clones showed consistently higher rates of hardening than the southern/maritime ones. This was particularly true under the COLD regime, suggesting that their hardening was less sensitive to low temperatures. The stems' visible and near-infrared absorption spectra, and concentrations of ten major soluble sugars, were also determined. Multivariate analysis revealed that spectral data could predict up to 96% of the variation in cold hardiness, when the analysis was restricted to the MILD regime and the data corrected for irrelevant systematic information. Possible direct links between spectral changes and chemical changes are discussed. Multivariate analysis also revealed that sugar concentrations could be used to predict up to 73% of the variation in cold hardiness. Different sugars displayed different patterns of variation during hardening. Concentrations of mannose and myo-inositol both decreased, whereas concentrations of galactose, sucrose, maltose, raffinose and stachyose all increased, but at different times. Dry matter increased markedly during hardening, so expressing the concentrations of sugars relative to dry matter does not provide an accurate measure of the amounts present.

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