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F.S. Cheng, S.K. Brown and N.F. Weeden

A DNA extraction protocol was developed for tissues from woody species. DNA was extracted successfully from 11 species and five different types of tissues and was suitable for RAPD and restriction analysis. Spermine precipitation was used to further purify DNA. The protocol can be used for large-scale analysis and mini-preparations.

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Minou Hemmat, Norman F. Weeden, Frank S. Cheng and S.K. Brown

The positions of over 50 SSR loci and other sequence tagged sites (STSs) have been located on the linkage maps of five apple cultivars (Rome Beauty, White Angel, Golden Delicious, Liberty, McIntosh) and two New York accessions. In most cases, the primers used produced single amplification products, permitting identification of homologous loci in the different cultivars and the precise alignment of the linkage maps generated for each. Based on this information, we present a general linkage map for apple with STS markers on each linkage group. The map consists of 17 linkage groups (equal to the haploid chromosome number for the species) with over 500 markers. The positions of several resistant gene analogues have been located on this linkage map. None of these sequences map near genes conferring resistance to scab or powdery mildew. SSR loci exhibited a tendency to cluster in certain regions of the linkage map. This clustering slightly reduces their effectiveness as genome markers for comparative mapping or germplasm diversity. However, the SSR markers definitely displayed a high level of polymorphism, making them particularly useful for genetic studies.

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D.S. Lawson, S.K. Brown, J.P. Nyrop and W.H. Reissig

A barrier system for pest control consisting of insect-exclusionary cages covered with three types of mesh material was placed over columnar apple (Malus domestica Borkh.) trees. This system has been shown to provide arthropod control equivalent to insecticides. Light intensity, evaporation, and air and soil temperature were reduced inside the cages. Shoot elongation of columnar apple trees grown inside insect-exclusionary cages was significantly greater than that of trees grown outside the cages. However, this increased shoot growth was not due to etiolation. Tree performance was unaffected by insect-exclusionary cages. Fruit set and fruit soluble solids concentration were not reduced by the cages; however, fruit color intensity was reduced as the degree of shading from the mesh increased. These findings, in conjunction with high levels of arthropod control by insect-exclusionary cages, may allow insect-exclusionary cages to be used for evaluating integrated pest management thresholds, predator-prey relationships, and apple production without insecticides.

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A.M.S. Nyomora, P.H. Brown, K. Pinney and V.S. Polito

The effect of boron (B) on in vivo and in vitro development of almond [Prunus dulcis (Mill.) D.A. Webb (syn. P. amygdalus Batsch)] pollen and pollen tubes and the resultant effect on fruit set was studied in mature trees. The cultivars Mono (pistil donor) and Butte (pollinizer) in an orchard with low soil B in Fresno, California were sprayed with B at 0, 0.8, 1.7, or 2.5 kg·ha-1 during Fall 1993. Pollen viability as indicated by the fluorescein diacetate method (FDA) was >85% and was not affected by field-applied B, however, in vivo pollen germination and tube growth were enhanced by foliar-applied B. More effect of applied B on in vivo growth appeared as pollen tubes progressed toward the ovary. For in vitro germination, foliar-applied B reduced bursting of tubes, and addition of B to the culture media significantly increased pollen germination and pollen tube growth.

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Hideyuki Takahashi, Christopher S. Brown, Thomas W. Dreschel and Tom K. Scott

Orientation of root growth on earth and under microgravity conditions can possibly be controlled by hydrotropism-growth toward a moisture source in the absence of or reduced gravitropism. A porous-tube water delivery system being used for plant growth studies is appropriate for testing this hypothesis since roots can be grown aeroponically in this system. When the roots of the agravitropic mutant pea ageotropum (Pisum sativum L.) were placed vertically in air of 91% relative humidity and 2 to 3 mm from the water-saturated porous tube placed horizontally, the roots responded hydrotropically and grew in a continuous arch along the circular surface of the tube. By contrast, normal gravitropic roots of `Alaska' pea initially showed a slight transient curvature toward the tube and then resumed vertical downward growth due to gravitropism. Thus, in microgravity, normal gravitropic roots could respond to a moisture gradient as strongly as the agravitropic roots used in this study. Hydrotropism should be considered a significant factor responsible for orientation of root growth in microgravity.

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Minou Hemmat, Norman F. Weeden, Herb S. Aldwinckle and Susan K. Brown

Bulked segregant analysis was used to identify RAPD markers that display tight linkage to the Vf gene in apple (Malus sp.) that confers resistance to five races of apple scab [Venturia inaequalis (Cke.) Wint.]. We identified several new RAPD markers linked to Vf. The most tightly linked marker in the test population, S52500, was cloned and sequenced. A linkage map of the Vf region was developed using these markers, RAPD markers previously described by other laboratories, and the isozyme locus Pgm-1. An assay was developed for Vf by multiplexing the two markers closely flanking the Vf locus. This assay has a theoretical `escape' value (discarding a resistant plant) of 3% and an error rate (selection of a susceptible plant) of 0.02%.

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Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle and Susan K. Brown

Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.

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Kisung Ko, Susan K. Brown, John L. Norelli and Herb S. Aldwinckle

Seven nptII and gus transgenic lines of the apple (Malus ×domestica Borkh.) rootstock Malling 7 (M.7) were examined by glucuronidase (GUS) histochemical testing and a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). These lines had different amounts of neomycin phosphotransferase II (NPTII). The amounts of NPTII among lines was positively correlated with the ability of the transgenic lines to regenerate in the presence of kanamycin, paromomycin, or geneticin. Regenerants derived from transgenic lines also varied greatly in GUS expression. The apical portion of regenerant stem tissues had stronger GUS staining than the basal portion of stem. All regenerated tissue of T1, a transgenic line originally classified as a uniform GUS staining line, showed non-GUS staining, while the regenerated tissues of chimeric transgenic lines showed nonstaining, chimeric staining, or uniform GUS staining, indicating the potential to select uniform GUS staining lines from chimeras. Polymerase chain reaction (PCR) indicated the gus gene was present in GUS negative (nonstaining) lines. Negative PCR results with primers derived from vir G of Agrobacterium tumefaciens, and failure to isolate A. tumefaciens from M.7 transgenics indicated that PCR and GUS staining results were not due to A. tumefaciens. A modified PCR methylation assay (MPMA) indicated that methylation of cytosines of the CCGG site in the gus gene, and in the border between the CaMV35S promoter and the gus gene, was positively correlated with complete gus gene silencing (nonstaining lines). However, the MPMA indicated that methylation was not always associated with variable GUS expression, suggesting that chimeric staining could be due to a mixture of transformed and nontransformed cells in some transgenic lines.

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S.J. Browning, T.P. Riordan, R.K. Johnson and J. Johnson-Cicalese

Buffalograss [Buchloë dactyloides (Nutt.) Engelm] is a drought-resistant, dioecious species, native to the Central Great Plains, which shows excellent potential as a low-maintenance turfgrass. Although buffalograss can be propagated vegetatively, there is a need for seeded turf-type cultivars. To assist in developing seeded cultivars, heritabilities of turf characteristics were estimated. Heritabilities from maternal half-sib analyses ranged from h2 = 0.04 ± 0.03 for the 1988 uniformity rating to h2 = 0.62 ± 0.26 for the 1989 spring color rating. Heritability estimates calculated from offspring-parent regression were also variable and generally lower than maternal half-sib analysis. The results suggest that some turf characteristics are highly heritable and that growing conditions markedly affect heritability estimates.

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Jyothi Prakash Bolar, Susan K. Brown, John L. Norelli and Herb S. Aldwinckle

The overall goal of our research is to develop an efficient transformation and regeneration system for `McIntosh' apple. The first objective was to determine the optimum combination of Gelrite (G) and agar (A) to maximize regeneration and minimize vitrification. Treatments included the following combinations of agar (in gliter–1) and Gelrite (in gliter–1): 1) 7 and 0; 2) 5.25 and 0.625; 3) 3.5 and 1.875; 4) 1.75 and 1.875; and 5) 0 and 2.5. There were 10 replications, and a single petri plate containing six leaf pieces was the unit of replication. Both 5.25(A) and 0.62(G) and 3.5(A) and 1.25(G) provided high regeneration of healthy, nonvitrified shoots. Since modification of media affects the concentration of antibiotics used in selection due to precipitation of antibiotics, the second objective was to determine the optimal concentration of antibiotic for the selection and regeneration of transformed `McIntosh' on gelrite–agar-based media. Kanamycin was tested at 0, 10, 25, 50, 75, and 100 μgml–1 and paromomycin was tested at 0, 50, 100, 150, 200, and 250 μgml–1. Antibiotic selection will be discussed relative to optimum concentration and efficiency of selection.