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Cold acclimation and freezing in Douglas-fir seedlings Timmis, Roger

Abstract

Five major investigations were conducted on the cold hardiness of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings: (1) to define a hardiness measurement technique; (2) to determine environmental control and inducibility of hardiness at different parts of the first seasonal growth cycle; and (3) to examine the independence of acclimation, loss of hardiness and flushing in climatically "split" plants. Genetically identical material from hardy and nonhardy branches of the split plants was then used in conductometric and calorimetric studies of (4) the freezing process, and (5) the energy of tissue water, to see if adaptive changes in these could account for the induced hardiness differences. The measurement technique involved freezing samples of excised needles under controlled conditions to various temperatures, injury was estimated as the degree of browning after 7 days, and hardiness defined as the temperature causing 50% injury. Injury to excised needles was correlated with injury to attached needles, which was in turn broadly related to ultimate survival of whole plants frozen at the same temperature. Random error and possible bias in hardiness estimates both increased in hardier populations. Ten percent hardiness differences (e.g., two degrees in twenty) between populations could be detected with fifteen-plant samples; changes of 1°C could be followed during the course of treatment of an individual seedling. Controlled environment studies in the first growth cycle showed that germinants (1 week) were unable to attain any freezing tolerance under 8-hr days at 2°C even after 9 weeks, but were killed whenever ice formed. However, seedlings older than 3 weeks (1 to 2 cm of epicotyl) could develop true hardiness under the influence of either short days (less effective) or low temperatures (above 0°C), independently of lignification, bud setting or entry into rest. Ability to acclimate increased gradually with age, and was inversely related to growth and maturation, apparently because the latter processes had higher temperature optima. Photoperiod affected growth and bud formation only above about 15°C, but influenced hardiness at 1°C with a longer inductive daylength at low than at high light intensities (12 and 8 hours respectively). Interruption of the long inductive dark period with 15 min of red light (650 nm) caused a decrease in hardiness and bud set, and an increase in growth. Far-red interruptions (750 nm) alone had no effect, but enhanced the red light effect when applied immediately afterwards. Night frosts (-7°C) caused significant dehydration, and rapidly increased hardiness, only if both the warm short-day, and chilling treatments had been given in sequence first, and the daily supply of light continued. These results are in general agreement with the hypothesis that cold acclimation takes place in three physiologically distinct stages under natural conditions. Studies on 3-year-old seedlings were carried out by exposing each branch of a forked plant to a different temperature (2° or 20°C) but similar light conditions for periods of one to five months. The chilling stimulus for breaking rest and inducing hardiness was confined to the chilled branch, but the warm branch apparently transmitted a factor which prevented full hardening in the chilled one. A factor moving in the same direction also promoted flushing in branches chilled only at night from December to June (and receiving greenhouse temperatures and natural photoperiods by day). This was not replaceable by a single injection of gibberellic acid. Factors from the expanding shoot caused loss of short-day-induced hardiness in previous year's foliage and stimulated cambial division. Chilling at night prevented the dehardening but did not prevent cambial activity. The dehardening factor was translocated to an opposite branch whereas movement of cambium stimulator was strictly basipetal. These results suggest that promoter-inhibitor levels controlling dormancy are independently regulated, and that a two-stage dehardening process might protect against premature loss of hardiness in nature. The progress of freezing in needles of the hardy/nonhardy branch pairs was recorded simultaneously by differential thermal analysis and the conductance of low voltage direct electric current. The results of both methods exhibited the same major patterns. Freezing in immature leaves was nonequilibrium and intracellular. Freezing in needles cold-acclimated under short days was an equilibrium process preceded by a short non-equilibrium freezing of the free intercellular water fraction. This pattern did not change in leaves more deeply cold-acclimated by low temperatures. Thawing in mature needles was characterized by a greater proportion of ice (than during freezing) at all temperatures, with indications that not all the original cell water was reabsorbed. Freezing records are interpreted as showing that the cell membrane became more permeable to ions after injurious slow freezing but retained its essential integrity, whereas rapid freezing caused immediate membrane damage. No features of the freezing or thawing curves of first or subsequent freeze-thaw cycles were useful as predictors of injury to needles by slow freezing. Energy of water in hardy/nonhardy needle pairs was compared by two methods. Heats of vapourization (Δ H[sub v]) of weighed increments of water, removed from excised needles under vacuum, were estimated from the calibrated vapourization endotherms recorded on a differential thermal analyser. In the second method, needle water contents were measured gravimetrically after equilibration with lithium chloride solutions of known desiccating energy. It was found that Δ H[sub v], a proposed measure of water binding near surfaces, increased as the proportion of water remaining in the leaf decreased. For each increment removed, Δ H[sub v] was significantly higher in hardy needles, notwithstanding various possible sources of error. Hardy needles also retained more water non-osmotically (than nonhardy needles) after equilibration with LiCl isopeistic with their frost-killing temperature. The date suggest that avoidance of dehydration, principally by non-osmotic lowering of cell water potential, can account for almost half of the 25 centigrade degree difference in hardiness between branches.

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