A rapid Agrobacferium fumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. l h e explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the p-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants i n soil was

SummaryWe describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-speci®c plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green¯uorescent protein was used as a visual marker for identi®cation of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.

SummaryThe visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum in®ltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per in®ltrated plant were achieved. The results showed that the majority of the transgenic seeds from single in®ltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the in®ltrated plants and the microscopic analysis of GUS expression in ovules of in®ltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-in®ltration. In addition, all ovules from a single in®ltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3±5 days postin®ltration, whereas frequencies of up to 6% were detected with ovules of unopened¯owers 5±11 days post-in®ltration. Most importantly, transgenic seeds were obtained only from genetic crosses using in®ltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.

Rupestris stem pitting (RSP), a component of the rugose wood complex, is one of the most widespread graft-transmissible diseases of grapevines. Here we report on the consistent association of a high molecular mass dsRNA (ca. 8n7 kbp) with RSP. The dsRNA was reverse-transcribed and cDNAs generated were cloned into Lambda ZAP II. Sequence analysis of the cDNA clones showed that the dsRNA was of viral origin and the putative virus was designated rupestris stem pitting associated virus-1 (RSPaV-1). The genome of RSPaV-1 consists of

Summary Transgenic lettuce plants expressing the nucleocapsid (N) protein gene of the lettuce isolate of tomato spotted wilt virus (TSWV‐BL) were protected against TSWV isolates via transgenic N protein when the protein accumulated at high levels or via an N transgene silencing mechanism activated by its overexpression. In a transgenic lettuce line, post‐transcriptional gene silencing was activated at a relatively earlier developmental stage in homozygous than in hemizygous progenies. As a result, the homozygous progenies generally showed a uniform suppression of N protein accumulation and consequently high levels of virus resistance in all leaves of the silenced plants. in contrast, N protein accumulated at high levels in the lower leaves of the hemizygous progenies and at much reduced levels (due to transgene silencing) in the successive leaves, resulting in moderate levels of virus resistance. It was also observed that the timing of the N transgene silencing in both homozygous and hemizygous plants was affected by environmental factors.

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We generated transgenic tobacco plants expressing the sense or antisense untranslatable N coding sequence of the lettuce isolate of tomato spotted wilt virus (TSWV-BL) as well as transgenic plants containing the promoterless N gene of the virus. Both sense and antisense untranslatable N gene RNAs provided protection against homologous and closely related isolates but not against distantly related Tospoviruses. These RNA-mediated protections were most effective in plants that synthesized low levels of the respective RNA species and appears to be achieved through the inhibition of viral replication. Unlike the sense RNA-mediated protection, the level of the antisense RNA-mediated protection depended on the concentration of the inoculum and the size of the test plants. Comparisons with previous results in transgenic plants expressing the intact N gene suggest that resistance to homologous and closely related TSWV isolates in plants that express low levels of the translatable N gene is due to the presence of the N gene transcript and not the N protein. In contrast, resistance to distantly related Tospoviruses is due to accumulation of high levels of the N protein and not due to the presence of the N gene transcript.