SummaryNovel 'super-binary' vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drugresistance, selection-marker gene and the other contained a gene for 13-glucuronidase (GUS), A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tumefaciens LBA4404 that carried the vectors. Frequency of cotransformation with the two T-DNAs was greater than 47%. GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of cotransformation was relatively low.

SummaryA rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identi®ed by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the ®rst restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present ®ndings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.

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We examined the effect of light on the mRNA levels of 11 genes (pral-pra9A, pra9B, and pra9C) encoding the small GTP-binding proteins that belong to the ras superfamily in Pisum sativum. When the dark-grown seedlings were exposed to continuous white light for 24 hr, the levels of several pra mRNAs in the pea buds decreased: pra2 and pra3 mRNAs decreased markedly; pra4, pra6, and pra9A mRNAs decreased slightly; the other 6 pra mRNAs did not decrease. We studied the kinetics of mRNA accumulation for pra2, pra3, and pra9B in detail during white light illumination and compared them with those of the phytochrome gene and the small subunit gene of ribulose bisphosphate carboxylase: mRNA levels ofpra2 and pra3 decreased in a manner similar to that of phytochrome while that of the small subunit increased as was expected. The decreases were triggered by a 2-min monochromatic red light (660 nm) irradiation. The effect of red light was reversed by subsequent exposure to far-red light, indicating an involvement of phytochrome as a photoreceptor in this light-regulated event. This work reports negative regulation of mRNA levels of small GTP-binding proteins by light, mediated by phytochrome.