In an attempt to generate soybean plants resistant to soybean dwarf virus (SbDV), we transformed a construct containing inverted repeat-SbDV coat protein (CP) genes spaced by beta-glucuronidase (GUS) sequences into soybean somatic embryos via microprojectile bombardment. Three T(0) plants with an introduced CP gene were obtained, and one generated T(1) seeds. The presence of the transgene in T(1) plants was confirmed by PCR and Southern blot hybridization analysis, but expression of CP was not detected by northern blot hybridization analysis. Two months after inoculation of SbDV by aphid, T(2) plants contained little SbDV-specific RNA and remained symptomless. These plants contained SbDV-CP-specific siRNA. These results suggest that the T(2) plants achieved resistance to SbDV by an RNA-silencing-mediated process.

Somatic embryos of Glycine max (L.) Merrill cultivar 'Jack' were co-transformed with coat protein (CP) gene of attenuated isolates of soybean mosaic virus (SMV) and hygromycin phosphotransferase (hpt) gene by means of microprojectile bombardment. These transformed embryogenic tissues were selected in hygromycincontaining liquid medium. The hygromycin-resistant embryogenic tissues obtained through the selection were regenerated, and CP gene was detected in the 11 transgenic plants out of them. In order to assess their resistance to SMV, mechanical inoculation was performed in T 1 generation. The disease symptom was examined visually and confirmed by the enzyme-linked immunosorbent assay (ELISA). Finally we obtained three independent lines highly resistant to SMV. This is the first report of the soybean plants that were conferred a high resistance to SMV by the transformation with CP gene of the SMV attenuated isolates. In these three lines, the presence of transgene transcript was confirmed by Northern blot analysis, and the transgene product was detected in two of them by Western blot analysis.

Somatic embryos of Glycine max (L.) Merrill cultivar 'Jack' were co-transformed with the coat protein (CP) gene of attenuated isolates of Soybean mosaic virus (SMV) and hygromycin phosphotransferase gene by microprojectile bombardment. CP gene was detected in eleven transgenic plants, and three independent lines highly resistant to SMV were obtained in a previous study. One of these lines, line No. 55, which was assumed to have acquired RNA-mediated resistance, was selected for further gene expression analysis of T 4 and T 5 plants in relation to their viral resistance. The resistant plants contained a lower level of transgenederived RNA than the susceptible ones. On the other hand, based on RNA analysis after mechanical inoculation, SMV-specific RNAs were detected faintly in the resistant plants, while large amounts of RNAs were found in the susceptible ones. During the development of the resistant lines, SMV CP sequence-specific small interfering RNAs (siRNAs) appeared initially in the leaves of the first leaf stage but not in those of the primary stage, and were detected thereafter in the leaves of later stages constantly. The presence of the siRNAs before SMV inoculation was strongly correlated with the resistant phenotype of the lines tested.

We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.

Agrobacterium rhizogenes was used for efficient transformation of chrysanthemum. Two types of Agrobacterium, A. rhizogenes (A-13) and A. tumefaciens (LBA4404), which harbor pIG121-Hm, were employed for infection. In the A. rhizogenes-infected explants, hairy roots were not observed on any tested medium or culture condition. When explants were cultured on shoot induction medium, calli were formed at the cutting edge within 4-6 weeks of culture, and shoot primordia were observed on the callus surface after 2 weeks of callus formation. Consequently, with gus introduction, a significantly higher transformation rate was observed for A. rhizogenes (6.0%) compared with A. tumefaciens (3.3%). However, only 0.6% of the frequency of rol insertion was exhibited in A. rhizogenes mediation. These results indicate that A. rhizogenes effectively introduces T-DNA of the binary plasmid into the chrysanthemum genome by introducing Ri T-DNA at a low frequency. It also indicates that the system is a useful alternative for the transformation of chrysanthemum.Abbreviations: CTAB -cetyltrimethylammonium bromide; GA 3 -gibberellic acid; GUS -b-glucuronidase; MS -Murashige and Skoog medium