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

Cold injury to plants can occur by early fall freezes before cold acclimation, by severe midwinter freezes that exceed the limits of the plant's tolerance, or by hard freezes in late winter or early spring after partial or complete deacclimation. Ideally, blueberry (Vaccinium L.) cultivars for temperate regions should acclimate to cold quickly in the fall, have a high midwinter-hardiness, and deacclimate late and/or slowly during spring or during unseasonably warm spells in winter, and do all of this without adversely delaying time of fruiting. Until recently, only limited evaluations have been done on the acclimation and deacclimation process in blueberry, although it is an integral part of flower bud survival and, thus, is directly related to potential yield. In this study, we have measured the timing and rate of acclimation and deacclimation in seven blueberry genotypes with different amounts of diverse species germplasm in their backgrounds. Primary differences observed among the seven genotypes were differences in maximum hardiness levels and the date at which they were reached, and differences in the date at which maximum acclimation levels were no longer sustained and deacclimation started. Highbush cultivars Bluecrop and Legacy (V. corymbosum L.), rabbiteye cultivar Tifblue [V. ashei Reade (= V. virgatum Aiton)], and two rabbiteye hybrid derivatives (US 1043 and US 1056) all reached maximum or near maximum cold-hardiness by late December with temperatures causing 50% lethality (LT50) in a range from –22 to –27 °C. The half-high, ‘Northsky’, and a hybrid of V. constablaei Gray × V. ashei ‘Little Giant’ both achieved cold acclimation of –28 °C or below (the lowest value we could measure) by the end of November. After reaching their maximum hardiness in late December, ‘Legacy’, ‘Tifblue’, and US 1043 began a sustained and relatively linear deacclimation, whereas US 1056, ‘Bluecrop’, ‘Northsky’, and ‘Little Giant’ sustained their acclimation for longer intervals. ‘Bluecrop’ and US 1056 did not begin to deacclimate until early March, and ‘Little Giant’ and ‘Northsky’ had no LT50 values higher (warmer) than –25 °C until late March. As concerns about climate change increase, knowledge of the ability of breeding germplasm to tolerate greater temperature extremes and fluctuations will prove increasingly valuable.

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Stan C. Hokanson, Kelvin G. Grant, Elizabeth L. Ogden and Lisa J. Rowland

Commercial strawberry plantings in the mid-Atlantic region are often quickly infected with one or more aphid-transmitted viruses, resulting in the loss of plant vigor, stunting, lowered yields, etc. To produce virus-free plant material for the strawberry industry and for cultivar development programs, heat therapy and/or meristem tip culture protocols are generally employed. One of the problems associated with meristem culturing is the potential for somaclonal mutations to occur in the meristem or surrounding proliferating tissue. As a result, distinct “bud lines” displaying functionally insignificant to distressingly high levels of phenotypic variation can arise from individual meristems. It would be desirable to differentiate these off-types by genetic fingerprinting to maintain trueness-to-type. Randomly amplified polymorphic DNA (RAPD) markers were evaluated for the potential to differentiate six pairs of strawberry bud lines that exhibit slight to fairly extreme levels of phenotypic variation. Reproducible RAPD marker profiles were generated using 10 primers in amplification reactions with genomic DNA obtained from multiple extractions. While five of the bud line pairs remained indistinguishable, three primers distinguished two variants of the Mohawk cultivar that are now in existence in the strawberry industry. Results suggest that typical somaclonal variation produced in the meristem culture process is of a magnitude that is not readily detectable with the RAPD protocol. The two Mohawk lines were probably produced by a higher magnitude mutation event than generally occurs or a cultivar mix-up.

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Lisa J. Rowland, Elizabeth L. Ogden, Mark K. Ehlenfeldt and Rajeev Arora

Cold hardiness in woody perennials is determined by complex interacting factors: the timing and rate of cold acclimation; the maximum level of cold tolerance attained; the maintenance of cold tolerance during the winter; and the rate of loss of cold tolerance or deacclimation on resumption of spring growth. For highbush blueberry, the degree of winter freezing tolerance and susceptibility to spring frosts have been identified as the most important genetic limitations of current cultivars. Depending on the winter and the location, both winter freezes and spring frosts can cause damage to floral buds or flowers resulting in substantial losses in yield. To identify genotypes that are particularly slow or late to deacclimate and thus may be useful in breeding for spring frost-tolerant cultivars, we compared deacclimation kinetics under controlled laboratory and field conditions among several blueberry genotypes with diverse genetic backgrounds. Clear genotypic differences in timing and rate of deacclimation were found. In the field study, the species Vaccinium constablaei Gray was identified as particularly late to deacclimate, and ‘Little Giant’ (50:50 hybrid of V. constablaei and V. ashei Reade) was nearly as late to deacclimate as 100% V. constablaei. Recently, we extended our cold tolerance measurements from October through midwinter comparing acclimation kinetics and maximum cold tolerance levels among genotypes. Although all genotypes appeared to reach maximum cold tolerance about mid-December under the study conditions, genotypic differences were detected in other aspects, including initial cold tolerance, rate of acclimation, maximum cold tolerance, and length of the plateau. ‘Little Giant’ and ‘Northsky’ (75:25 hybrid of V. corymbosum L. and V. angustifolium Ait.) were very early to acclimate and were hardier than the other genotypes both initially and when maximum cold tolerance was reached. Understanding how cold tolerance levels change throughout the dormant period should help us to develop cultivars better suited to their environments.

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

From 2004 to 2006, cold hardiness assays were performed to evaluate the relative winterhardiness of flower buds in selections of pure Vaccinium ashei Reade and V. constablaei Gray as well as in selections/families composed of various combinations of V. ashei and V. constablaei germplasm. Significant differences were observed among entries with LT50 values ranging from −17.2 to −28.4 °C. An analysis of LT50 versus percent V. constablaei yielded a regression of LT50 (°C) = (−0.08 × V. constablaei percentage) – 21.57. Families or selections with 50% (or greater) V. constablaei and some with 25% V. constablaei had LT50 values equivalent to or better than ‘Bluecrop’. Based on this information, a 25% V. constablaei constitution appears suitable to develop northern-adapted rabbiteye types if proper parents are selected and if sufficient selection pressure for winterhardiness is exercised.

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

The midwinter cold hardiness of 25 rabbiteye (V. ashei) blueberry cultivars was assayed across 2 years using a shoot freezing assay. LT50values (i.e. temperature at which 50% of buds are damaged) for the cultivars ranged from –24.9 °C for `Pearl River' (a 50% V. ashei derivative) to –13.7 °C for `Chaucer'. Under New Jersey conditions, numerous cultivars were observed to exhibit dimorphism for dormant floral bud size. Comparisons of bud dimorphism with LT50 values, found dimorphism more common in cultivars with lower floral bud hardiness. LT50 values generally supported empirical observations of winter hardiness, but exceptions suggest that additional factors contribute to observed winter hardiness under field conditions.

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Rajeev Arora, Lisa J. Rowland, Elizabeth L. Ogden, Anik L. Dhanaraj, Calin O. Marian, Mark K. Ehlenfeldt and Bryan Vinyard

Loss of freeze tolerance, or deacclimation, is an integral part of winter survival in woody perennials because untimely mid-winter or spring thaws followed by a hard freeze can cause severe injury to dehardened tissues. This study was undertaken to investigate deacclimation kinetics, particularly the timing and speed, of five blueberry (Vaccinium L.) cultivars (`Bluecrop', `Weymouth', `Ozarkblue', `Tifblue', and `Legacy'), with different germplasm compositions and mid-winter bud hardiness levels, in response to an environmentally controlled temperature regime. Based upon bud cold hardiness evaluations in 2000 and 2001, `Tifblue', a Vaccinium ashei Reade cultivar, was one of the least hardy and the fastest to deacclimate; `Bluecrop', a predominantly V. corymbosum L. cultivar, was the most hardy and the slowest to deacclimate; and `Ozarkblue', a predominantly V. corymbosum cultivar but including southern species V. darrowi Camp. and V. ashei, was intermediate in speed of deacclimation. `Weymouth' (predominantly V. corymbosum) and `Legacy' (73.4% V. corymbosum and 25% V. darrowi) were slow to intermediate deacclimators. Deacclimation rates did not correlate strictly with mid-winter bud hardiness. Data suggest that the southern germplasm component V. ashei may be responsible for the observed faster deacclimation whereas both southern species, V. darrowi and V. ashei, may contribute genes for cold sensitivity. Strong positive correlations between stage of bud opening and bud cold hardiness existed in both years (r = 0.90 and 0.82 in 2000 and 2001 study, respectively). Previously identified major blueberry dehydrins, 65-, 60-, and 14-kDa, progressively decreased in their abundance during incremental dehardening in `Bluecrop', `Weymouth', and `Tifblue'. However, down-regulation of the 14-kDa dehydrin most closely mirrored the loss in cold hardiness during deacclimation, and, therefore, may be involved in regulation of bud dehardening. Because differences in deacclimation rate were clearly evident among the genotypes studied, rate of deacclimation of the flower buds of blueberry should be an important consideration in breeding to improve winter survival.

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Lisa J. Rowland, Smriti Mehra, Anik L. Dhanaraj, Elizabeth L. Ogden, Janet P. Slovin and Mark K. Ehlenfeldt

Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (Vaccinium spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry. Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5' and 3' ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and were tested in amplification reactions with genomic DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.

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Lisa J. Rowland, Elizabeth L. Ogden, Fumiomi Takeda, David Michael Glenn, Mark K. Ehlenfeldt and Bryan T. Vinyard

Injury of open flowers often occurs in fruit crops by late winter or early spring frosts and can result in significant reduction in yield. In this study, freezing tolerance of open flowers of five highbush blueberry cultivars, Bluecrop, Elliott, Hannah’s Choice, Murphy, and Weymouth, was determined using two freezing methods. Methods involved either placing whole plants in a radiation frost chamber or detached shoots in a glycol-freezing bath. In both methods, plants (or excised shoots) with opening flowers were exposed to temperatures ranging from –2 to –10 °C. After freeze treatments, several flower parts were evaluated for damage and the lethal temperature50 (LT50) determined. In order, from the most sensitive flower part to the least sensitive on average, were the corolla, filament, anther, style, exterior ovary, stigma, ovules, interior ovary, and placenta. A two-way analysis of variance (ANOVA) found no significant effect of the freezing method on the calculated freeze damage to most of the various flower parts. However, a significant genotype effect was found on freeze damage to the style, filament, anthers, and exterior ovary. Overall, ‘Bluecrop’ was the most sensitive to freezing, whereas ‘Hannah’s Choice’ and ‘Murphy’ were the most freezing-tolerant. In conclusion, genotypic variability in frost tolerance of open highbush blueberry flowers was detected, which can be exploited in breeding for more frost-tolerant cultivars.