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K.M. Kelley, S.A. Weinbaum, P.B. Catlin, and T.T. Muraoka

Nitrogen (N) deficiency reduced biomass and altered N allocation within large walnut tree canopies (Juglans regia L. cv Serr). N-fertilized control trees contained 2.5 times more N in current year spurs, leaves and fruit than did those of N-deficient trees. The N content and biomass allocated to kernels was reduced in N-deficient canopies to a greater extent than was al location to current year shoots and foliage. N removal in abscised leaves and fruit was 3 times greater in canopies of fertilized trees than in N-deficient trees.

A non-destructive method is described to calculate total spur, leaflet and fruit numbers. Calculations were based on ratios of fruit counts on selected scaffold limbs to total fruit number per tree. Dry weight and N content of representative spurs, leaflets and fruit permitted estimation of whole canopy biomass and N content in these organs. N contained in current year spurs and the N lost from the tree in fruit and leaf litter were calculated for both N-fertilized control and N-deficient trees.

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D.J. Gray, E. Hiebert, C.M. Lin, K.T. Kelley, M.E. Compton, and V.P. Gaba

Agrobacterium-mediated transformation (AMT) was compared with a particle bombardment (PB) for stable transformation of `Eden Gem', a melon with high embryogenic potential. Pretreated cotyledonary explants were either wounded via particle bombardment prior to Agrobacterium infection or were bombarded with plasmid-coated gold microparticles, using a modified particle inflow-type gun. Although similar numbers of embryos initially were obtained with each method, most produced via AMT became abnormal, possibly due to the growth-regulatory effects of the antibiotic mefoxine, which was used to inhibit Agrobacterium. Stably transformed plants and progeny were obtained only with PB, as determined by detection of the NPTII gene in R0 plant by Southern hybridization and, in progeny, by PCR amplification.

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D. J. Gray, Z. T. Li, D. L. Hopkins, M. Dutt, S. A. Dhekney, M. M. Van Aman, J. Tattersall, and K. T. Kelley

Pierce's disease (PD), caused by the xylem-limited bacterium Xylella fastidiosa, is endemic to the coastal plain of the southeastern United States. Although native southern grapevines are tolerant to X. fastidiosa, all varieties of Vitisvinifera grown in the region will succumb to PD. Genetic transformation to add disease resistance genes, while not disturbing desirable phenotypic characters, holds promise for expanding the southeastern U.S. grape industry by allowing use of established fruit and wine varieties. We utilize embryogenic cell cultures and Agrobacterium strain EHA105 to refine transformation systems for Vitis species and hybrids. V. vinifera`Thompson Seedless' is employed as a model variety to test various transgenes for disease resistance, since as many as 150 independent transgenic plant lines routinely are produced from 1 g of embryogenic culture material. Transgenic plants are stringently screened for PD resistance in greenhouses by mechanical inoculation with X. fastidiosa. Transgenic plants are compared with both susceptible and resistant control plants by assessing typical PD symptom development and by assaying bacterial populations in xylem sap over time. Using these procedures, nine putative PD resistance genes have been inserted into grapevine and over 900 unique transgenic lines have been evaluated. A range of susceptible-to-resistant responses has been catalogued. Thus far, the best construct for PD resistance contains a grape codon-optimized hybrid lytic peptide gene in a high-performance bi-directional 35S promoter complex. Certain transgenic plant lines containing this construct exhibit better resistance than that of resistant control vines.