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Yongjian Chang and Barbara M. Reed

Cold hardiness and cryogenic survival of micropropagated pear (Pyrus cordata Desv.) shoots were evaluated after pretreatments with ABA and sucrose. Shoot cold hardiness increased by 3 °C, and cryopreserved shoot tip growth increased by 17% after a 4-week 150 μm ABA pretreatment. Low temperature (LT) pretreatments improved the recovery of cryopreserved P. cordata shoot tips. Six to 10 weeks of LT were required for reaching high cryopreservation recovery. ABA and LT treatments produced significant synergistic effects on both cold hardiness and cryopreservation recovery. ABA shortened the LT requirement for high cryopreservation growth from 10 to 2 weeks. The optimal treatment for recovery of cryopreserved shoot tips was a 3 week culture on 50 μm ABA followed by 2 weeks of LT, while the maximum cold hardiness (-22.5 °C) was obtained with 150 μm ABA and 2-week LT. A 4 week culture on 150 μm ABA at 25 °C induced dormancy in 74% of shoot tips, but had little effect on cryopreservation growth unless combined with LT. Control and ABA-treated shoot tips, lateral buds, and leaves had similar cold hardiness (-10 to -12 °C), but LT and LT+ABA-treated shoot tips survived the lowest temperatures (-17 to -23 °C), lateral buds next (-15 to -20 °C), and finally leaves (-14 to -18 °C). An increase in the preculture-medium sucrose concentration from 2% to 7% combined with 2-week LT significantly increased cryopreserved shoot tip growth (0% to 75%) and decreased the LT50 from -7.8 to -12.4 °C. The optimal shoot pretreatment for successful recovery of cryopreserved P. cordata shoot tips was a 3 week culture on either 50 μm ABA or 5% to 7% sucrose medium followed by 2 weeks of LT, and increased shoot tip growth from zero to >70%. Chemical name used: abscisic acid (ABA).

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Xiang Wang, Rajeev Arora, Harry T. Horner and Stephen L. Krebs

early fall and winter ( Sakai and Larcher, 1987 ; Weiser, 1970 ). Evergreen rhododendrons are broad-leaved woody plants and the freezing tolerance of fully cold-acclimated leaves can be as low as –50 to –60 °C ( Sakai et al., 1986 ; Wei et al., 2005

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Lisa J. Rowland, Elizabeth L. Ogden, Mark K. Ehlenfeldt and Bryan Vinyard

Deacclimation response is an important part of reproductive success in woody perennials because late winter or early spring thaws followed by hard freezes can cause severe injury to dehardened flower buds. There is a need to develop more spring-frost tolerant cultivars for the blueberry (Vaccinium L.) industry. The identification of later or slower deacclimating genotypes could be useful in breeding for more spring-frost tolerant cultivars. This study was undertaken to investigate cold hardiness and deacclimation kinetics under field conditions for 12 Vaccinium (section Cyanococcus A. Gray) genotypes (the cultivars Bluecrop, Duke, Legacy, Little Giant, Magnolia, Northcountry, Northsky, Ozarkblue, Pearl River, Tifblue, and Weymouth; and a population of V. constablaei Gray) with different germplasm compositions and expected mid-winter bud hardiness levels. Examination of bud cold hardiness (BCH) vs. weeks of deacclimation over a 7-week period in 2 consecutive years (2002 and 2003) revealed clear genotypic differences in cold hardiness and timing and rate of deacclimation. Among cultivars, `Legacy' was the least cold hardy at initial evaluation, even less so than `Tifblue'. Regarding deacclimation kinetics, the weekly intervals with the largest losses (i.e., high rates of deacclimation) also varied among genotypes. For `Duke', the largest losses in BCH were detected at weeks 2 and 3, making it the earliest deacclimator. For `Bluecrop', `Ozarkblue', `Weymouth', `Tifblue', and `Legacy', the greatest losses in BCH were observed at weeks 3 and 4. For `Little Giant', `Magnolia', `Northcountry', `Northsky', and `Pearl River', losses in BCH were greatest at weeks 4 and 5, while for V. constablaei, losses were greatest at weeks 6 and 7, making it the latest deacclimator. Deacclimation kinetics were not correlated with mid-winter hardiness or chilling requirements in any fixed pattern. On the other hand, a strong positive correlation was found between BCH and stage of bud opening (r = 0.84). A comparison of timing of deacclimation with germplasm composition indicated that V. constablaei was particularly late to deacclimate. `Little Giant', a 50:50 hybrid of V. constablaei and V. ashei Reade, was nearly as late to deacclimate as the 100% V. constablaei selections. Thus, V. constablaei may be useful in breeding programs to contribute genes for late deacclimation, which should translate into greater spring frost tolerance, in addition to genes for mid-winter hardiness.

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Bjorn H. Karlsson, Jiwan P. Palta, Laurie S. Weiss, James F. Harbage and John B. Bamber

Cold-induced changes in gene expression have been demonstrated in a number of species that vary in freezing tolerance and acclimation capacity. Relative freezing tolerance was measured based on ion leakage for both nonacclimated and acclimated S. commersonii and S. cardiophyllum parents, F1 and backcross progeny segregating for cold tolerance and acclimation capacity. Western blot analyses showed increase in a dehydrin band (47 kD)(antisera courtesy of T. Close) following cold acclimation of cold tolerant S. commersonii, and a slight increase in cold sensitive S. cardiophyllum. Expression of 47 kD cosegregated with non acclimated freezing tolerance but not with acclimated freezing tolerance. Our results show that (i) expression of dehydrins is a heritable trait in the Solanum diploid population, (ii) there is no direct relationship between relative freezing tolerance and the presence or absence of dehydrm protein following cold acclimation and (iii) based on assays measuring the residual activity of the lactate dehydrogenase (LDH) enzyme following freezing, the cryoprotective influence of `boiling stable' proteins was species dependent and is related to the freezing tolerance of the species. Supported by USDA/NRI grant 91-3700-6636 to J.P.P. and J.B.B..

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Rita A. Teutonico, Jiwan P. Palta and Tom C. Osborn

Identification of the genes involved in freezing tolerance in oilseed Erussica could lead to genetic improvement of winter survival of this crop and other species, as well as provide greater understanding of the basis of cold stress tolerance in plants. We developed a genetic linkage map for B. rapa using restriction fragment length polymorphisms (RFLPs) and identified molecular markers which are linked to genes controlling vernalization requirement and freezing tolerance. We mapped the location of a group of cold-regulated (`cor') genes from Arabidopsis thaliana in this population and determined their association with freezing tolerance and vernalization requirement. We developed genetically fixed, recombinant inbred lines of B. rapa to assay the physiological processes involved in these cold responses. Specifically, we measured the differences in lipid composition of the plasma membranes of acclimated and nonacclimated plants of a subset of this population. We will determine if the genes involved in the physiological responses to cold temperature are also associated with the acquisition of freezing tolerance.

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Sandra E. Vega, Jiwan P. Palta and John B. Bamberg

Two major components of frost resistance are freezing tolerance in the nonacclimated state (growing in normal condition) and capacity to cold acclimate (increase in freezing tolerance upon exposure to chilling temperatures). In addition to these two major components, numerous factors contribute to frost survival. Although the rate of cold acclimation and deacclimation have been recognized as important factors contributing to frost survival, very little information about them is available. Our objective was to determine if there is variability in the rate of cold acclimation and deacclimation among tuber-bearing wild potato species: S. acaule Bitter, S. commersonii Dunal, S. megistacrolobum Bitter, S. multidissectum Hawkes, S. polytrichon Rydb., S. sanctae-rosae Hawkes, and S. megistacrolobum subsp. toralapanum (Cárdenas & Hawkes) Giannattasio&Spooner. Relative freezing tolerance of these species was measured after 0, 3, 6, 9 and 12 days of cold acclimation and after 12 and 24 hours deacclimation. Our results showed there were differences in the rates of cold acclimation and deacclimation among these species. With respect to the rate of acclimation we found these species can be divided into four groups: (i) early; (ii) late acclimators; (iii) progressive acclimators, and (iv) nonacclimators. Likewise, a wide range of cold deacclimation behavior was found. Some species showed as low a loss of 20% of their freezing tolerance, others showed as much as >60% loss after 12 hours of deacclimation. Significant deacclimation was observed in all cold acclimating species after 1 day. These results demonstrate that the rates of cold acclimation and deacclimation were not necessarily related to the cold acclimation capacity of a species. Rapid acclimation in response to low temperatures preceding a frost episode and slow deacclimation in response to unseasonably warm daytime temperatures could be advantageous for plants to survive frost events. Thus, in addition to nonacclimated freezing tolerance and acclimation capacity, it would be very desirable to be able to select for rapid acclimation and slow deacclimation abilities. Results demonstrate that variability for these two traits exists in Solanum L. (potato) species.

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R.A. Teutonico, T.C. Osborn and J.P. Palta

Identification of the genes involved in low temperature responses in oilseed Brassica could lead to genetic improvement of this crop and other species. We developed a genetic linkage map for B. rapa using restriction fragment length polymorphisms (RFLPs) and identified molecular markers which are linked to genes controlling vernalization requirement and freezing tolerance. We mapped the location of a group of cold-regulated (`cor') genes from Arabidopsis thaliana in this population and determined their association with these cold responses. We developed genetically fixed, recombinant inbred lines of B. rapa to assay the physiological processes involved in these cold responses. Specifically, we measured the differences in lipid composition of the plasma membranes of acclimated and nonacclimated plants of a subset of this population. We will determine if the genes involved in the physiological responses to low temperature are also associated with the acquisition of freezing tolerance.

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L. V. Gusta

Plants acclimate to abiotic stresses, e.g. heat, freezing drought and salinity, in response to environmental cues such as temperature, daylength and water. Plants can respond within minutes to the cue e.g. heat tolerance or within hours or days, e.g. drought and freezing tolerance. Heat shock proteins are measurable within 20 to 30 minutes of a heat stress and the plants aclimate almost immediately. In contrast, proteins related to freezing tolerance are measurable within hours but days are required before a measurable increase in freezing tolerance can be detected. In almost all stresses it appears that the environmental cue effects the water status of the plant which in turn affects the level of endogenous abscisic acid (ABA). ABA has been implicated to ameliorate the stress by inducing genes to produce stress proteins. There is a certain degree of commodity between stresses in ragards to stress proteins, however each stress has their own unique set of stress proteins. For example heat shock proteins did not confer stress tolerance. Proteins involved in water and osmotic stress tolerance share a high degree of commonality. I” all stresses a unique class of proteins are synthesized which are classified as heat or boiling stable (do not coagulate at 100°). These proteins are suggested to be involved in the stress response. Many of these heat stable proteins are induced by ABA alone or in combination with jasmonic acid (JA). Analogs of ABA which are either slowly converted to ABA or are degraded slowly or taken up at a faster rate than ABA have been tested for the efficacy in inducing the stress responses. Analogs have also been identified which inhibit the ABA induced response. How these analogs may have practical significance will be discussed.

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Christopher L. Owens, J.F. Hancock and A.F. Iezzoni

Sour cherry and strawberry are examples of two Rosaceous species that often suffer crop reductions due to spring freezes. Breeding for improved floral freezing tolerance has the potential to mitigate the susceptibility of these plants to spring frosts. In model plant systems, researchers have been able to identify genes that play a role in freezing tolerance by initially searching for mRNAs regulated in response to cold temperatures. To search for cold-responsive freezing-tolerance genes in strawberry and sour cherry, it is necessary to first define their cold acclimation response. To test the hypothesis that sour cherry and strawberry flowers have the ability to cold acclimate, blooming plants were exposed to 4 °C and 16 h light for 14 days. Sour cherry styles and strawberry receptacles from open, fully developed flowers were excised, and electrolyte leakage curves were generated over a range of subzero temperatures. The temperature at which 50% electrolyte leakage (EL50) occurred was used to compare treatments. The flowers of two strawberry cultivars were tested for the ability to cold acclimate. Non-acclimated `Chandler' receptacles had an EL50 of -2.9 °C, while non-acclimated `Honeoye' had an EL50 of -3.4 °C. Conversely, acclimated `Chandler' receptacles had an EL50 of -7.7 and acclimated `Honeoye' receptacles had an EL50 of -8.7 °C, both are significantly different from non-acclimated values (P ≤ 0.01). Additionally, sour cherry styles were collected from the field at full bloom from a mapping population of 86 individuals from the cross `Rheinische Schattenmorelle' × `Erdi Botermo' and acclimated as previously described. The EL50 of the 86 progeny ranged from approximately -2.0 to -6.0 °C.

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Fadi H. Karam and J. Alan Sullivan

Distinct differences in freezing tolerance among a cold-hardy wild rose species Rosa fedtschenkoana Regel., a garden rose, `Jack Frost', and their hybrid could be detected under laboratory conditions using 2-cm-long shoot segments with buds. The garden rose did not survive - SC, but the cold-hardy species survived freezing to -10C and the hybrid to –5C. One week of acclimation at 4C was adequate for R. fedtschenkoana; longer periods did not improve the rate of survival. Immersing tissue in 5%, 10%, or 20% sucrose during acclimation improved the rate of survival of R. fedtschenkoana but not of `Jack Frost'. Applications to rose breeding are discussed.