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  • Author or Editor: J. Steven Tebbets x
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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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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%.

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