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Kristi K. Barckley, Sandra L. Uratsu, Thomas M. Gradziel and Abhaya M. Dandekar

The California almond industry is the largest supplier of almonds [Prunus dulcis (Miller) D.A. Webb] in the United States and throughout the world. Self-incompatibility is a major issue in almond production as it greatly affects nut set. In this study, we determined full-length sequences for alleles Sa - Si, determined the genotypes of 44 California cultivars, and assigned the cultivars to cross-incompatibility groups (CIGs). Newly identified S-alleles led to an increase in the number of CIGs. A pairwise distance tree was constructed using the aligned amino acid sequences showing their similarity. Four pairs of alleles (Sc and Se, Sg and Sh, Sd and Sj, and Sb and Sf) showed high sequence similarity. Because of its simplicity, reproducibility, and ease of analysis, PCR is the preferred method for genotyping S-alleles.

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Sandra L. Uratsu, Hamid Ahmadi, Royce S. Bringhurst and Abhaya M. Dandekar

Several strains of Agrobacterium tumefaciens and A. rhizogenes were shown to form tumors on runners of the diploid strawberry species Fragaria vesca L. Tumors, weighing from 0.1 to 8.3 mg, appeared from 2 to 4.5 weeks after infection. The majority of tumors tested for opine synthesis by high-voltage paper electrophoresis analysis showed positive results. These results demonstrate that diploid strawberry plants are susceptible to infection with Agrobacterium and that there are differences in the relative virulence of Agrobacterium strains.

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Matthew A. Escobar, Andrew Shilling, Pine Higgins, Sandra L. Uratsu and Abhaya M. Dandekar

The enzyme polyphenol oxidase (PPO) is nearly ubiquitous in Kingdom Plantae and catalyzes the oxidation of phenolic compounds into highly reactive quinones. Although the functional importance of PPO in plants remains uncertain, a putative antipathogen role for walnut (Juglans regia) PPO was posited as early as 1911. However, despite the rich diversity of phenolics present in walnut leaves and hulls, walnut PPO has been little studied since the early 1900s. We cloned a PPO-encoding gene from a walnut pistillate flower cDNA library and designated the gene jrPPO1. Genomic Southern analysis demonstrated that jrPPO1 is the sole PPO gene in walnut. Transgenic tobacco (Nicotiana tabacum) plants expressing jrPPO1 display greater than 10-fold increases in leaf PPO activity compared with wild-type tobacco, demonstrating that jrPPO1 encodes a functional enzyme. The jrPPO1 protein is expressed primarily in the leaves, hulls, and flowers of walnut trees and is not regulated by wounding or methyl jasmonate. To examine whether walnut PPO could affect pathogen resistance, tobacco plants expressing jrPPO1 were challenged with Pseudomonas syringae pv. tabaci. Based on both symptom development and quantitative analyses of bacterial growth in planta, the PPO-expressing plants did not display increased resistance to this pathogen. Leaf extract browning assays indicated that tobacco leaves lack the endogenous phenolic substrates required for significant jrPPO1 activity and quinone production in planta.

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Ryutaro Tao, Abhaya M. Dandekar, Sandra L. Uratsu, Patrick V. Vail and J. Steven Tebbets

Japanese persimmon (Diospyros kaki L. `Jiro') was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA101, carrying the binary plasmid vector, pDU92.710. The T-DNA region of pDU92.710 contained the kanamycin resistance gene (nptII), the β-glucuronidase gene (uidA), and a synthetic reconstruct of cryIA(c) encoding the insecticidal crystal protein fragment of Bacillus thuringiensis subsp. kurstaki HD-73. Leaf discs made from leaves of shoot cultures were cocultivated with Agrobacterium and cultured on a callus-induction medium containing kanamycin and cefotaxime. Among 720 infected leaf discs, 17 putative transformed callus lines showing kanamycin resistance were obtained after 8 weeks of culture. When these were cultured on a regeneration medium containing kanamycin, 15 formed adventitious buds. Of the 15 shoot lines, 11 grew well on a shoot-proliferation medium containing kanamycin, while 4 lines did not grow well. Of the 11 shoot lines, 10 showed GUS activities by fluorometric assay and were subjected to polymerase chain reaction (PCR) and Southern analyses. Except for two lines, all results were consistent with a stable integration of T-DNA into the persimmon genome. The production of CryIA(c) protein in transformed shoot lines was confirmed with Western analysis using anti-CryIA(c) serum. Insect bioassays were conducted with 10 lines showing GUS activity. Many of these lines showed high significant mortality of the test insects, Plodia interpunctella Hüber and Monema flavescens Walker, when compared to nontransformed controls.

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Abhava M. Dandekar, Gale H McGranahan, Sandra L. Uratsu, Charles Leslie, J. Steven Tebbets and Patrick V. Vail

Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis (Bt) encoded by cryIA(c) gene were shown in diet incorporation studies to be lethal to codling moth (CM; Cydia pomonella) the key insect pest for walnut. However transformed walnut tissues expressing cryIA(c) with Bt codon usage patterns and native DNA sequence revealed very low levels of expression in planta. To correct this problem synthetic versions of one of these genes, cryIA(c) was used to transform walnut tissue. A total of 61 individual transgenic embryo lines were obtained. 34% of these lines (21/61) were high expressors (“class A”) demonstrating 80 to 100% mortality of first in star CM larvae and displaying no further larval development. Twelve clones (20%) were designated “class B” and these showed a marked retardation of larval development and a mortality between 40 to 79%. Embryos from the remaining 28 lines designated “class C” (46%). although transformed, were indistinguishable from the control (untransformed embryos) and showed a mortality of 0 to 39%.