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  • Author or Editor: Ryutaro Tao 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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Endodormancy release and the fulfillment of the chilling requirement (CR) are critical physiological processes that enable uniform blooming in fruit tree species, including apple (Malus ×domestica). However, the molecular mechanisms underlying these traits have not been fully characterized. The objective of this study was to identify potential master regulators of endodormancy release and the CR in apple. We conducted RNA-Sequencing (RNA-seq) analyses and narrowed down the number of candidates among the differentially expressed genes (DEGs) based on the following two strict screening criteria: 1) the gene must be differentially expressed between endodormant and ecodormant buds under different environmental conditions and 2) the gene must exhibit chill unit (CU)–correlated expression. The results of our cluster analysis suggested that global expression patterns varied between field-grown buds and continuously chilled buds, even though they were exposed to similar amounts of chilling and were expected to have a similar dormancy status. Consequently, our strict selection strategy resulted in narrowing down the number of possible candidates and identified the DEGs strongly associated with the transition between dormancy stages. The genes included four transcription factor genes, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), FLOWERING LOCUS C (FLC)-LIKE, APETALLA2 (AP2)/ETHYLENE-RESPONSIVE 113 (ERF113), and MYC2. Their expressions were upregulated during endodormancy release, and were correlated with the CU, suggesting that these transcription factors are closely associated with chilling-mediated endodormancy release in apple.

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Most commercial cultivars of japanese plum (Prunus salicina Lindl.) exhibit S-RNase-based gametophytic self-incompatibility (GSI), although some self-compatible (SC) cultivars exist. In this study, we characterized S-RNase and SFB, the pistil and pollen S determinants of the specificity of the GSI reaction, respectively, from four S-haplotypes, including a SC (Se ) and three SI (Sa , Sb , and Sc ) S-haplotypes of japanese plum. The genomic organization and structure of the SC Se-haplotype appear intact, because the relative transcriptional orientation of its S-RNase and SFB and their intergenetic distance are similar to those of the other three SI S-haplotypes of japanese plum and other Prunus L. species. Furthermore, there is no apparent defect in the DNA sequences of Se-RNase and SFBe . However, a series of transcriptional analyses, including real-time reverse transcriptase–polymerase chain reaction, showed that the Se-RNase transcript levels in the pistil are significantly lower than those of the Sa-, Sb-, and Sc-RNases, although transcripts of SFBa , SFBb , SFBc , and SFBe are present at similar levels in pollen. Furthermore, no Se-RNase spot was detected in two-dimensional polyacrylamide gel electrophoresis profiles of stylar extracts of the cultivars with the Se-haplotype. We discuss the possible molecular basis of SC observed with the Se -haplotype with special reference to the insufficient Se-RNase accumulation incited by the very low transcriptional level of Se-RNase in pistils.

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Most of the self-compatible (SC) cultivars of almond [Prunus dulcis (Mill.) D.A. Webb. syn. P. amygdalus Batsch] have the Sf haplotype. In this study, we cloned and characterized the S locus region of the Sf haplotype of SC ‘Lauranne’. The relative transcriptional orientation of SFBf and Sf-RNase and the physical distance between them are similar to those of other functional self-incompatible (SI) S haplotypes of Prunus, indicating that the genomic structure of the SC Sf haplotype appears to be intact. Although there is no apparent mutation in the coding sequence of SFBf , the Sf-RNase sequence in this study and previously reported Sf-RNase sequences show discrepancies. First, as opposed to previous indications, the ‘Lauranne’ Sf-RNase sequence encodes a histidine residue in place of a previously reported arginine residue in the conserved C2 region of Prunus S-RNase. Direct sequencing of the polymerase chain reaction products from the Sf-RNase of ‘Tuono’ confirmed that ‘Tuono’ Sf-RNase also encodes the histidine residue. We found another difference in the ‘Lauranne’ Sf-RNase sequence and other reported Sf-RNase sequences. Namely, ‘Lauranne’ Sf-RNase encodes a phenylalanine residue in place of a previously reported leucine residue in the conserved C5 region of Prunus S-RNase. This is also the case for ‘Tuono’ Sf-RNase. Expression analysis of Sf-RNase and SFBf by reverse transcriptase–polymerase chain reaction showed that Sf-RNase transcripts were barely detectable in pistil, whereas SFBf transcripts were accumulated at a similar level to the level that was observed with SFB of other functional SI S haplotypes of almond. We discuss the possible molecular mechanisms of SC observed with the Sf haplotype with special references to the expression of Sf-RNase.

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