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Jay Young, Charles Heuser and E.J. Holcomb

The use of spent mushroom compost (SMC) as a media amendment for containerized greenhouse and nursery crop production is a promising alternative to disposal of this by-product of mushroom production. Fresh SMC is the compost that is removed from the mushroom house and used without further weathering. The objectives of this study include first, identification of key factors involved its successful use and second demonstration of the effective use of SMC by nurserymen. The plant material used includes both bedding plants and woody perennial species. Results demonstrate that the key factor in the use of SMC for plant production is high soluble salts. Leaching can reduce the high soluble salts. In addition, special consideration should be given to the reduction in potted media volume over time due to composting that continues after the material is removed from mushroom production. SMC as the sole growing media was not as effective as when SMC was amended with a commercial nursery growing mix. Several species were grown in 0%, 25%, 50%, 75%, and 100% mixtures of SMC and a commercial nursery mix. All species grew well in 50% SMC/50% nursery mix.

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Laurie E. Boyden and Peter S. Cousins

Breeders of woody perennials seek to shorten the time from propagation to flowering and the turnover time between generations. Grapevines usually flower and fruit no earlier than their third season. Onset of flowering occurs when anlagen, undifferentiated primordia arising from axillary and terminal bud meristems, begin to develop into inflorescences as well as tendrils. This occurs in response to hormonal stimuli; high levels of gibberellins in juvenile tissue favor vegetative growth, whereas increased cytokinin levels in physiologically mature tissue favor reproductive growth. We modified a method developed for Vitis vinifera for use on grape rootstock seedlings. Exogenous applications of chlormequat and N-benzyl-9-(2-tetrahydropyranyl) adenine (PBA, a cytokinin) were used induce precocious flowering by increasing the cytokinin:gibberellin ratio, triggering anlagen to develop into inflorescences on physiologically juvenile vines. The optimum treatment was a single application of 3000 micromolar chlormequat and 250 micromolar PBA, followed by 10 subsequent daily applications of PBA alone. Lower concentrations of treatments resulted in a loss of efficacy, and higher treatment concentrations and/or longer durations resulted in phytotoxicity. Abnormalities in flower and leaf morphology were observed with all treatments. Grape rootstock seedlings are dioecious and staminate vines were more responsive to the treatments than pistillate vines. We did not observe production of hermaphroditic flowers on staminate vines. Pollen collected from flowering staminate seedlings was successfully used in crosses that produced fruit and viable seeds. The ability to induce precocious flowering in juvenile grape seedlings has many applications in grape breeding and genetic research.

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Douglas G. Bielenberg, Ying Wang, Gregory L. Reighard and Albert G. Abbott

`Evergrowing' (evg) peach is a naturally occurring mutant unable to enter winter dormancy in response to dormancy inducing conditions. The evg mutant is one of only two described mutants affecting winter dormancy in woody perennial trees. The evg mutation segregates as a single recessive gene and previous work by our group has fine mapped the trait between flanking markers separated by 3.3 centiMorgans. This region was physically mapped using a bacterial artificial chromosome (BAC) library from and a contig of overlapping genomic fragments identified. We have utilized several approaches to complete the sequencing of a 132 kilobase region of the peach genome derived from three overlapping BACs that encompass the complete EVG gene containing region. We present here our analysis and annotation of the genomic region, including putative and experimentally verified gene coding sequences. A primary feature of the region is a large tandem duplication of a region containing a MADS-box type DNA binding transcription factor resulting in six similar copies of the gene, all of which appear to be expressed at the mRNA level in wild-type germplasm. Hybridization analysis revealed the presence of a large deletion in the mutant genome. Five of the identified genes fall within the evg mutation and represent new candidates for the control of entrance into winter dormancy.

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Scott Kalberer, Norma Leyva-Estrada, Stephen Krebs and Rajeev Arora

Winter survival of temperate-zone woody perennials requires them to resist loss of frost hardiness (deacclimation) during winter and early spring thaws. However, little is known about deacclimation response in woody landscape plants. Moreover, what impact, if any, the degree of deacclimation has on reacclimation capacity has not been systematically studied. We used nine genotypes of deciduous azaleas (Rhododendron subgenus Pentanthera) to investigate effects of deacclimating conditions on bud cold hardiness and reacclimation ability. Dormant floral buds, with 3–5 cm stem attached, were collected in late December from field-grown plants, and placed in constant warm [22 °C 15 °C (D/N)] and humid conditions for increasing durations (0-day to 14-day) to stimulate deacclimation. Bud cold hardiness (lt 50) was determined (using logistic regressions) by evaluating immature flower survival at subfreezing treatment temperatures. Results indicated that azalea genotypes from colder provenances showed greater initial frost hardiness. Typically northern genotypes had slow to intermediate deacclimation rates, while rates of southern genotypes were intermediate to rapid. High initial frost hardiness was frequently associated with slow deacclimation. Buds retained the capacity to reacclimate upon cold exposure [2 °C/–2 °C; (12 h/12 h)] even after 8 days of deacclimation. Distinct differences were observed between the two latitudinal ecotypes of R. viscosum with respect to their initial bud hardiness, deacclimation rates, and reacclimation capacities. We suggest that the three attributes, i.e., high initial hardiness, slow deacclimation, and high reacclimation capacity, together may be important for winter-survival of azalea buds.

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Hongwen Huang, Desmond R. Layne and R. Neal Peterson

The utility of isozyme phenotypes for identifying and determining genetic variation in pawpaw cultivars was studied using isoelectric focusing in thin-layer polyacrylamide gels. Based on a sample of 32 clones (cultivars and advanced selections) and 23 enzyme systems, 7 enzymes were found to be polymorphic, involving 9 polymorphic loci [acid phosphatase (ACP), dihydrolipoamide dehydrogenase (DDH), malic enzyme (ME), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), peroxidase (PRX), and shikimate dehydrogenase (SKD)]. Altogether these 9 loci and 32 clones yielded 28 multi-locus isozymic phenotypes useful for cultivar identification; 24 of the 32 clones were uniquely identified. The allozyme variation in these clones has the average of other long-lived woody perennials of widespread geographic range in temperate regions with insect-pollinated outcrossing breeding systems, secondary asexual reproduction, and animal-dispersed seed. Genetic differentiation among these pawpaw clones, measured by Nei's distance, D, was substantial: 496 pairwise comparisons of genetic distance among the 32 clones indicated that they differed on average of D = 0.068 ± 0.04 and ranged from 0 to 0.188. Cluster analysis (UPGMA) produced a most likely division of the 32 clones into 7 groups; however, these groups did not conform to known pedigree relations. Additional polymorphic enzymes are needed for accurate allozyme-based genetic discrimination.

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Thomas E. Marler and Yasmina Zozor

Leaf gas exchange, chlorophyll fluorescence, water relations, and mineral nutrient relations responses of Annona squamosa seedlings to mild salinity were studied in sand culture in five experiments during 1990, 1991, and 1993. Trees were irrigated with a complete nutrient solution (control) or with this solution amended to 3 or 6 dS·m-1 with sea salt. Inhibition of net CO2 assimilation, stomatal conductance of CO2, and transpiration was apparent within 2 weeks of initiating salinity treatments, and gas exchange continued to decline until day 30 to 35. The diurnal pattern of leaf gas exchange was not altered by increased salinity. Salinity reduced CO2, light energy, and water-use efficiencies. Salinity sometimes reduced the ratio of variable to maximum fluorescence below that of the control, and this response was highly dependent on the ambient light conditions that preceded the measurements. Dark respiration was unaffected by salinity stress. Root zone salinity of 3 dS·m-1 administered for 52 days did not influence foliar sodium concentration or the ratio of sodium to potassium, but increased chloride concentration and decreased nitrogen concentration. The sodium response indicated that some form of exclusion or compartmentation occurred. Salinity reduced osmotic potential of root tissue but did not influence foliar osmotic or predawn xylem potential. These results indicate that A. squamosa is sensitive to salinity stress, and that the responses to salinity are consistent with other salt-sensitive woody perennial species.

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Julie P. Newman, Joseph P. Albano, Donald J. Merhaut and Eugene K. Blythe

Release characteristics of four different polymer-coated fertilizers (Multicote, Nutricote, Osmocote, and Polyon) were studied over a 47-week period in a simulated outdoor, containerized plant production system. The 2.4-L containers, filled with high-fertility, neutral-pH substrate, were placed on benches outdoors to simulate the environmental conditions often used for sun-tolerant, woody perennials grown in the southwestern United States. Container leachates were collected weekly and monitored for electrical conductivity, pH, and concentrations of NH4 +N, NO3 N, total P, and total K. Concentrations of most nutrients in leachates were relatively high, but fluctuated frequently during the first third of the study period, and then gradually decreased and stabilized during the last 27 weeks. Osmocote often resulted in greater NH4 + and total inorganic N concentrations in leachates than other fertilizers during weeks 1 through 5, whereas Multicote produced higher NH4 + in leachates than most of the other fertilizer types during weeks 9 through 12. Overall, total P concentrations were greater with Multicote during a third of the experimental period, especially when compared with Osmocote and Polyon. Differences were also observed among treatments for leachate concentrations of K, with Polyon and Multicote fertilizers producing greater K concentrations in leachates compared with Osmocote during several weeks throughout the experimental period. Leachate concentrations of NO3 N and P from all fertilizer types were usually high, especially from week 5 through week 30.

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Anik L. Dhanaraj*, Janet P. Slovin and Lisa J. Rowland

To gain a better understanding of changes in gene expression associated with cold stress in the woody perennial blueberry (Vaccinium spp.), a genomics approach based on the analysis of expressed sequence tags (ESTs) was undertaken. Two cDNA libraries were constructed using RNA from cold acclimated (mid winter conditions when the plants are cold stressed) and non-acclimated (before they received any chilling) floral buds of the blueberry cultivar Bluecrop. About 600 5'-end ESTs were generated from each of the libraries. Putative functions were assigned to 57% of the cDNAs that yielded high quality sequences based on homology to other genes/ESTs from Genbank, and these were classified into 14 functional categories. From a contig analysis, which clustered sequences derived from the same or very similar genes, 430 and 483 unique transcripts were identified from the cold acclimated and non-acclimated libraries, respectively. Of the total unique transcripts, only 4.3% were shared between the libraries, suggesting marked differences in the genes expressed under the two conditions. The most highly abundant cDNAs that were picked many more times from one library than from the other were identified as representing potentially differentially expressed transcripts. Northern analyses were performed to examine expression of eight selected transcripts and seven of these were confirmed to be preferentially expressed under either cold acclimating or non-acclimating conditions. Only one of the seven transcripts, encoding a dehydrin, had been found previously to be up-regulated during cold stress of blueberry. This study demonstrates that analysis of ESTs is an effective strategy to identify candidate cold-responsive transcripts in blueberry.

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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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Lynn Jo Pillitteri, Carol J. Lovatt and Linda L. Walling

Homologues of the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) were isolated from the hybrid perennial tree crop `Washington' navel orange (Citrus sinensis) and designated CsLFY and CsAP1, respectively. Citrus has an extended juvenile period unlike herbaceous plants and responds to different floral stimuli than herbaceous plants or deciduous tree species. Despite these differences, the deduced amino acid sequences of CsLFY and CsAP1 genes are at least 65% identical with their Arabidopsis thaliana L. Heynh counterparts and share even greater sequence similarity to LFY and AP1 from the deciduous woody perennials, Populus balsamifera Bradshaw and Populus tremuloides Michaux, respectively. Like A. thaliana LFY (AtLFY) and AP1 (AtAP1), CsLFY and CsAP1 expression was restricted almost exclusively to reproductive tissues, but observed expression of CsAP1 in the fourth whorl carpel tissue of mature flowers was distinct from other plant AP1 genes. Transgenic A. thaliana plants ectopically expressing CsLFY or CsAP1 showed early-flowering phenotypes similar to those described for overexpression of AtLFY and AtAP1. In addition, the 35S:CsLFY and 35S:CsAP1 transgenes partially complemented the lfy-10 and ap1-3 mutants, respectively. The severity of the overexpression phenotypes correlated with the accumulation of CsLFY or CsAP1 transcripts. LFY is a single-copy gene in flowering plants but consistent with its hybrid origin, the genome of C. sinensis `Washington' has two easily distinguishable CsLFY and CsAP1 alleles derived from it's parental genotypes, C. maxima L. Osbeck (pummelo) and C. reticulata Blanco (mandarin). Allelic polymorphism at both the CsLFY and CsAP1 loci was restricted to the 5′- and 3′-flanking regions.