We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.

This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors. Oshox1 maps to chromosome 10 and belongs to a family of related rice genes. Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein. This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms. Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants. Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator. In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor. Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression. Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain.

The molecular mechanisms through which the complex patterns of plant vascular tissues are established are largely unknown. The highly ordered, yet simple, striate array of veins of rice leaves represents an attractive system to study the dynamics underlying pattern formation. Here we show that mutation in the RADICLELESS1 (RAL1) gene results in distinctive vascular pattern defects. In ral1 embryonic scutella, secondary veins are absent and in the prematurely aborted and discontinuous primary veins,cells are misaligned to each other. In ral1 leaves, longitudinal and commissural (transverse) veins display altered spacing and the commissural veins additionally show atypical branching and interruptions in their continuity. The vascular pattern alterations of ral1 occur in the context of normally shaped leaf primordia. Anatomical inspection and analysis of the expression of the procambium specification marker Oshox1-GUS and of the auxin-inducible reporter DR5-GUS demonstrates that all the vascular patterning aberrations of ral1 originate from defects in the procambium, which represents the earliest identifiable stage of vascular development. Furthermore, the ral1 mutant is unique in that procambium formation in leaf primordium development is delayed. Finally, the ral1 vascular patterning distortions are associated with a defective response to auxin and with an enhanced sensitivity to cytokinin. ral1 is the first mutant impaired in both procambium development and vascular patterning to be isolated in a monocot species.

To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.

A single copy gene has been isolated, termed GOS2, from rice. Sequence comparison revealed highly similar genes in mammals and yeast, indicating that GOS2 encodes an evolutionary conserved protein. GOS2 mRNA was detected in all tissues examined. When the upstream region was translationally fused to the reporter gene gusA it was found to drive expression in a variety of rice tissues and in cell suspensions of other monocot species following introduction by particle bombardment. Therefore, the GOS2 promoter is potentially useful for genetic engineering of monocots. A DNA-binding activity from rice, termed rice ASF-1, with similar binding specificity as the cloned tobacco transcription factor TGA-1a, was found to bind to a TGACG sequence motif in the GOS2 promoter. Possible roles for rice ASF-1 in the transcriptional activation of the GOS2 promoter are discussed.