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A time-course study was conducted to characterize seasonal patterns of cold hardiness (CH) and protein profiles in the leaf tissue of five Rhododendron cultivars (`Grumpy Yellow', `Vulcan's Flame', `Autumn Gold', `Chionoides', and `Roseum Elegans'). Leaf samples were collected monthly (starting in mid September) and leaf discs were subjected to controlled freezing and thawing regimes. CH (LT50 defined as temperature causing 50% injury) was assessed by electrolyte leakage and visual observations. Data indicate that cultivars varied in their CH in nonacclimated state and in their ability to cold acclimate. Results obtained in September showed `Grumpy Yellow' to be least hardy (about –3°C) and `Roseum Elegans' to be most hardy (about –7°C). All cultivars exhibited successive increases in CH during fall and winter. Maximum CH in all cultivars occurred by December/January with `Chionoides' being most hardy (about –31°C) while `Grumpy Yellow' was least hardy (about –20°C). LT50 based on electrolyte leakage was highly correlated with visual rating. Seasonal changes of protein profiles and relationship of specific stress proteins to cultivars' CH and cold acclimation ability are discussed.
`Danka' black currant floral buds produce multiple low temperature exotherms (LTEs). However, the absence of visual injury symbtoms in the buds after exposure to subfreezing temperatures make it difficult to assess injury in these buds. A 2,3,5-triphenyltetrazolium chloride (TTC) reduction assay was used to determine whether LTEs corresponded to freezing injury of individual floral primordia or to the entire floral axis. Intact buds were cooled at 3C/n, removed at 3C intervals from -12 to -33C, and thawed on ice for 24 h. Duplicate samples were subjected to differential thermal analysis. Freeze injury Could not be measured with TTC in thawed, intact buds. However, incubation of excised floral primordia in TTC resulted in an all or nothing response. The number of LTES did not correspond to the number of floral primordia killed within a floral bud, but the median LTE did correspond with the temperature at which lethal injury of the whole inflorescence occurred. Therefore, preliminary results indicate that TTC reduction assay of individual floral buds is a fast, reliable technique to assess bud injury.
Dehardening resistance and rehardening capacity in late winter and spring are important factors contributing to the winter survival of woody perennials. Previously the authors determined the midwinter hardiness, dehardening resistance, and rehardening capacities in deciduous azalea (Rhododendron L.) floral buds in early winter. The purpose of this study was to investigate how these parameters changed as winter progressed and to compare rehardening response at three treatment temperatures. Experiments were also conducted to measure bud water content during dehardening and chilling accumulation of 10 azalea genotypes. Buds of R. arborescens (Pursh) Torr., R. canadense (L.) Torr., R. canescens (Michx.) Sweet, and R. viscosum (L.) Torr. var. montanum Rehd. were acclimated in the field and were dehardened in the laboratory at controlled warm temperatures for various durations. Dehardened buds were rehardened for 24 hours at 2 to 4 °C, 0 °C, or –2 °C. Bud hardiness (LT50) was determined from visual estimates of freeze injury during a controlled freeze–thaw regime. The midwinter bud hardiness in the current study was ≈4 to 8 °C greater than in early winter. R. canadense and R. viscosum var. montanum dehardened to a larger extent in late winter than in the early winter study whereas R. arborescens and R. canescens did not. The rehardening capacities were larger in early than in late winter. Even though rehardening occurred throughout the first 8 days of dehardening (DOD) in early winter in the previous study, in the current study it was only observed after 10 DOD (R. viscosum var. montanum) or 15 DOD (R. arborescens). There was no difference among the rehardening capacities at the three rehardening temperatures for any genotype. Water content decreased throughout dehardening in all four genotypes examined. R. canadense had the lowest chilling requirement (CR) [450 chilling units (CU)], followed by R. atlanticum (Ashe) Rehd., R. austrinum (Small) Rehd., R. canescens, and R. calendulaceum (Michx.) Torr. with intermediate CR [820, 830, 830, and 1000 CU respectively). The CR of R. arborescens, R. prinophyllum (Small) Millais, R. prunifolium (Small) Millais, R. viscosum var. montanum, and R. viscosum var. serrulatum (Small) Millais exceeded 1180 CU. Results of this study indicate that the dehardening kinetics (magnitude and rate) and the rehardening capacity of azalea buds are influenced by the progression of winter and the depth of endodormancy.
Styrax americanus Lam. (American snowbell) is a deciduous shrub or small tree seldom produced in nurseries. This species is distributed in patchy populations found mainly from Florida to southern Illinois, although a small, disjunct population exists in northern Illinois. The winter-hardiness and loss of hardiness during a period of increased temperature (deacclimation) of plants from this disjunct population may differ from those of S. americanus elsewhere. We examined cold-hardiness and deacclimation of stems of plants from the disjunct population, from southern Illinois, and from Florida. Segments of stems removed from plants grown outdoors in Ames, IA, were exposed to low-temperature ramping, and the temperature at which stems showed 50% damage (LT50) was determined by using the tissue-discoloration method. To assess deacclimation, stem segments were collected from cold-acclimated plants during winter in a minimally heated greenhouse and exposed to controlled warm temperatures for various time intervals followed by low-temperature ramping. Plants from Illinois were ≈15 °C more cold-hardy than plants from Florida in Feb. 2008. Plants from the disjunct population in northern Illinois showed less stem tip injury than did plants from southern Illinois. Deacclimation patterns were similar between plants from both Illinois populations. Plants sampled in Apr. 2009 from Florida deacclimated more rapidly than corresponding samples from Illinois, and the chilling required to overcome endodormancy increased with increasing latitude of plant origin. This research suggests that germplasm from the Illinois populations should be used in regions where the poorer hardiness and deacclimation resistance of most S. americanus would not permit survival.
A seasonal study was conducted to assess the freezing injury of `Boskoop Giant' black currant (Ribes nigrum L.) samples from Oct. 1991 through Mar. 1992. Buds were subjected to either differential thermal analysis (DTA) or one of a series of temperatures (0 to -36C). Freeze injury was then assessed either visually or with TTC. Results indicated that black currant floral buds have multiple low-temperature exotherms (LTE). Freeze injury in intact buds could not be visually quantified because of the lack of visible browning, nor assayed with TTC reduction. Excised floral primordia incubated in TTC, however, developed colored formazan following exposure to nonfreezing and sublethal freezing temperatures, but remained colorless when exposed to lethal temperatures. The percentage of floral primordia that were colored and colorless were tabulated and a modified Spearman-Karber equation was used to calculate the temperature at which 50% of floral primordia were killed (T50 The T50 temperature was correlated with the temperature at which the lowest LTE was detected (R2 = 0.62). TTC reduction assay using excised floral bud primordia was a good indicator of viability in frozen blackcurrant buds. Chemical name used: 2,3,5-triphenyltetrazolium chloride (TTC).
Mode of inheritance of cold hardiness (CH) in woody perennials is not wellunderstood. This study was undertaken to determine the mode of inheritance and gene action of CH in blueberry (Vaccinium section Cyanococcus). Two testcross populations (segregating for CH) derived from interspecific hybrids of V. darrowi (drw) × V. caesariense (csr) were used. Plants were cold-acclimated by a 4-week exposure to 4°C. Bud CH (LT50) was defined as the temperature causing 50% injury (visual) when subjected to controlled freeze–thaw. Results show that the drw and csr parents had an LT50 of –13° and –20°C, respectively. The F1 population exhibited mean LT50 of –14.7°C. The csr and drw testcross populations had a mean LT50 of –18° (39 individuals) and –14°C (33 individuals), respectively. Individuals of each population were distributed between parental values with center of distribution skewed toward the testcross parent. Since individuals having LT50s as same as the recurrent parents were present in each population of only 33–39 plants, data suggest that CH is determined by relatively few genes. To determine gene action, the estimates for various genetic parameters (calculated from joint scaling test) were used in generation means analysis to test various models. Results indicate that CH in blueberry can be best explained by simple-additive dominance model, whereas models including epistatic components did not satisfactorily explain the data.