Engineering genetic resistance against insects in Japanese persimmon using the cryIA(c) gene of Bacillus thuringiensis.

摘要

Japanese persimmon (Diospyros kaki) 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 beta-glucuronidase gene (uidA), and a synthetic reconstruct of cryIA(c) encoding the insecticidal crystal protein fragment of B. thuringiensis subsp. kurstaki HD-73. Leaf discs made from leaves of shoot cultures were cocultivated with A. tumefaciens and cultured on a callus-induction medium containing kanamycin and cefotaxime. Of 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 mediumcontaining 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 in fluorometric assay, and weresubjected to PCR and Southern analyses. Except for 2 lines, all results were consistent with a stable integration of T-DNA into the D. kaki 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 and Monema flavescens, when compared with non-transformed controls.