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Mark Ehlenfeldt and Lisa Rowland

In recent years, the U.S. Department of Agriculture has generated V. ashei and V. constablaei derivatives to provide late-flowering, earlyripening rabbiteye germplasm for the southern U.S.; however, this germplasm has also performed well in New Jersey, and could potentially provide northern-adapted rabbiteye types. When combined, these germplasms complement each other in many respects. Crosses with varying percentages of V. constablaei germplasm ranging from 100% to 0%, in 25% increments, were evaluated for mid-winter cold-hardiness using a detached twig assay. Hybrids with 50% or more V. constablaei germplasm were hardy to –25° C or lower. Further backcrosses to V. ashei resulted in dramatic segregation for hardiness, but still yielded some progeny as hardy as typical northern highbush. Assays of rabbiteye cultivars have been recently completed to enhance the ability to generate cold-hardy hybrids. Recombination and selection have begun to yield hybrids improved for critical commercial characteristics, and further rapid progress is expected.

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Rajeev Arora and Lisa J. Rowland

To survive winters, woody perennials of temperate zone must enter into endodormancy. Resuming spring growth requires sufficient exposure to low temperature or chill units (CUs) in winter, referred to as chilling requirement (CR), which also plays a role in the development of freezing tolerance (cold acclimation; CA). Physiological studies on the breaking of dormancy have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of CU accumulation. However, whether these changes are associated with breaking dormancy or CA is not clear. We conducted a study, using greenhouse blueberry (Vaccinium section Cyanococcus) plants, to address this question Three blueberry cultivars (`Bluecrop', `Tifblue', and `Gulfcoast'), having CRs of ≈1200, 600, and 400 CUs, respectively, first were exposed to 4° for long enough to provide CUs equivalent to one-half of their respective CRs. This treatment resulted in CA. Plants were then transferred to 15C for 2 weeks (a treatment which should not negate CU accumulation but did result in deacclimation). Before and after each treatment cold hardiness (using a controlled freezing bath) and dormancy status (observe budbreak after placing shoots in water at 20C for 2 to 3 weeks) of floral buds were determined. Proteins were extracted from buds collected, simultaneously and separated by SDS-PAGE. To determine the association of dehydrin-like proteins with dormancy or CA, electroblots were probed with anti-dehydrin antibody. The relationship of protein and western blots data to cold acclimation and dormancy are presented.

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Rajeev Arora and Lisa J. Rowland

Freezing is a major environmental stress during an annual cycle of overwintering, temperate-zone perennials. The timing and extent of seasonal cold acclimation (development of freezing tolerance in the fall) and deacclimation (loss of acquired freezing tolerance in response to warm temperatures) are of critical importance for winter survival, particularly in view of the climate change, i.e., unpredictable extreme weather occurrences. For example, plants may acclimate inadequately if exposed to a milder fall climate and may be damaged by sudden frosts. Alternatively, they may deacclimate prematurely as a result of unseasonable, midwinter warm spells and be injured by the cold that follows. Efficient cold acclimation ability, high deacclimation resistance, and efficient reacclimation capacity are, therefore, important components of winter survival in overwintering perennials. These components should be evaluated separately for a successful breeding program focused on improving winter-hardiness. Another layer of complexity that should be carefully considered is that endodormant status (shallow versus deep) of the reproductive/vegetative apices can significantly impact these components of winter-hardiness. Winter survival, especially by woody evergreens, requires tolerance of light stress, which can result in photo-oxidative damage at cold temperatures when biochemistry of photosynthesis is somewhat compromised but light harvesting is unaffected. Accumulation of Elips (early light-induced proteins) in overwintering evergreens during winter represents a relatively novel strategy to cope with such light stress, and investigations on the precise cellular mechanism and genetic control of this strategy deserve research in the future. Investigations into the mechanisms for cold acclimation use laboratory-based, artificial acclimation protocols that often do not closely approximate conditions that plants are typically exposed to in nature. To draw meaningful conclusions about the biology of cold acclimation and ultimately improve freeze resistance under field conditions, one should also include in cold acclimation regimens parameters such as exposure to subfreezing temperatures and realistic diurnal temperature fluctuations and light levels to simulate natural conditions. One of the main objectives of this article is to highlight two areas of research that we believe are important in the context of plant cold-hardiness but, so far, have not received much attention. These are: 1) to understand the biology of deacclimation resistance and reacclimation capacity, two important components of freeze-stress resistance (winter-hardiness) in woody perennials; and 2) to investigate the cellular basis for various strategies used by broad-leaved evergreens for photoprotection during winter. Our emphasis, in this context, is on a family of proteins, called Elips. The second objective of this article is to draw attention of the cold-hardiness research community to the importance of using realistic cold acclimation protocols in controlled environments that will approximate natural/field conditions to be better able to draw meaningful conclusions about the biology of cold acclimation and ultimately improve freeze resistance. Results from our work with Rhododendron (deciduous azaleas and broad-leaved evergreens), blueberry, and that of other researchers are discussed to support these objectives.

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Lisa J. Rowland and Elizabeth L. Ogden

Conditions for improving the efficiency of shoot regeneration from leaf sections of highbush blueberry (Vaccinium corymbosum L.) were investigated. Effectiveness of tissue culture medium supplemented with the cytokinin conjugate zeatin riboside or the cytokinin zeatin at 10, 20, or 30 μm was compared with medium supplemented with the optimum 2iP concentration of 15 μm. Use of 20 μm zeatin riboside resulted in the most shoots per leaf section, » 6-fold higher than the number of shoots produced on 2iP medium. The number of shoots produced on medium supplemented with zeatin was not significantly higher than the number of shoots produced on 2iP medium. Consequently, we concluded that the cytokinin conjugate zeatin riboside was more effective than either of the free cytokinins, 2iP or zeatin, in promoting shoot regeneration from leaf sections of highbush blueberry. Chemical names used: 6-(y,y-dimethylallylamino)-purine (2iP); 6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin).

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Rajeev Arora, Lisa J. Rowland and Karen Tanino

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Lisa J. Rowland, M. Muthalif and Mubarack

There is evidence from several plant species that low, nonfreezing temperatures induce the accumulation of specific proteins in leaves and stems. Only recently, however, have attempts been made to identify changes in gene expression in dormant buds of woody plants in response to chilling. For our investigations, we have used dormant blueberry plants representing three different species, placed them in a room maintained at about 4C, and collected buds after 0, 500, 1000, and 1500 hours. Proteins were extracted from bud samples and analyzed by SDS-PAGE. Results indicated that various cultvars and species responded differently to chilling unit accumulation. The most dramatic changes were noted in the V. corymbosum cultivar `Bluecrop'. The concentration of about 6 polypeptides with molecular weights ranging from 60-115 kD increased by 500 hours. The concentration of a 17.5 kD polypeptide increased after 1000 hours and the concentration of a 114 kD polypeptide decreased throughout the treatment.

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Peter Boches, Nahla V. Bassil and Lisa Rowland

Sixty-nine accessions representing wild and domesticated highbush blueberry (Vaccinium corymbosum L.) germplasm were genotyped using 28 simple sequence repeats (SSRs). A total of 627 alleles was detected and unique fingerprints were generated for all accessions. Suspected duplicate accessions of `Coville' and `Ivanhoe' had DNA fingerprints that were identical to `Coville' and `Ivanhoe', respectively. Genetic similarity measures placed wild and cultivated blueberries in separate groups. Northern highbush blueberries grouped among ancestral clones that were used extensively in blueberry breeding such as `Rubel' and `Stanley'. Southern highbush blueberries formed a separate group from northern highbush blueberries. The microsatellite markers used here show excellent promise for further use in germplasm identification, in genetic studies of wild Vaccinium L. populations, and for constructing linkage maps.

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Rajeev Arora, Michael Wisniewski and Lisa J. Rowland

Seasonal changes in cold tolerance and proteins were studied in the leaves of sibling deciduous and evergreen peach [Prunus persica (L.) Batsch]. Freezing tolerance [defined as the subzero temperature at which 50% injury occurred (LT50)] was assessed using electrolyte leakage. Proteins were separated by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Electroblots were probed with anti-dehydrin and anti-19-kD peach bark storage protein (BSP) antibodies. Leaf LT50 decreased successively from -5.8 °C on 18 Aug. to -10.3 °C in the evergreen genotype and from -7.0 °C to -15.0 °C in the deciduous genotype by 14 Oct. Protein profiles and immunoblots indicated the accumulation of a 60- and 30-kD protein during cold acclimation in the leaves of deciduous trees; however, levels of these proteins did not change significantly in the evergreen trees. Immunoblots indicate that the 60-kD protein is a dehydrin-like protein. Gel-electrophoresis and immunoblots also indicated that the 19-kD BSP progressively disappeared from summer through fall in leaves of deciduous peach but accumulated to a high level in bark tissues. A similar inverse relationship was not evident in evergreen peach.

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Amnon Levi, Elizabeth Ogden and Lisa J. Rowland

Efforts are underway to develop genetic linkage maps for two interspecific blueberry populations (Vaccinium darrowi × V. elliottii and V. caesariense-derived populations). To date, 72 RAPD markers have been mapped, and another 200 markers have been identified as suitable for mapping in the V. darrowi × V. elliottii-derived population. Inheritance of 40 RAPD markers has been followed, and additional 40 RAPD markers have been identified as suitable for mapping in the V. darrowi × V. caesariense population. These two populations are comprised of individual plants that should have a wide range of chilling requirements. At a later date, plants will be classified according to their chilling requirements to identify RAPD markers that cosegregate with chilling requirements. Presently, a bulked-segregant analysis is being performed on a tetraploid breeding population (primarily V. corymbosum) to identify RAPD markers linked to chilling requirement genes.