SummarySeveral approaches have recently been adopted to improve Agrobacterium-mediated transformation of rice, both to generate the large number of T-DNA insertion plants needed for functional analysis of the rice genome, and for production of rice with additional agronomical value. However, about 3 months of in vitro culture is still required for isolation of transgenic rice plants. Here, we report the competency of scutellum tissue from 1-day pre-cultured seeds for Agrobacterium-mediated transformation. Furthermore, early infection of rice seeds with Agrobacterium enhanced efficient selection of transformed calli. Using our system, we successfully regenerated transgenic rice plantlets within a month of the start of the aseptic culture of mature seeds. Our new system should reduce the somaclonal variation accompanying prolonged culture of rice cells in the dedifferentiated state and facilitate the molecular breeding of rice.

Three random amplified polymorphic DNAs (RAPDs) closely linked to the vrs1 (formerly v) locus were sequenced and converted to sequence-tagged sites (STSs). Of the three STSs, two retained the RAPD polymorphism as dominant-recessive markers between 'Kanto Nakate Gold' (KNG; a two-rowed barley) and 'Azumamugi' (AZ; a six-rowed barley), while the other was co-dominant after digestion with restriction enzymes. Six restriction fragment length polymorphisms (RFLPs) linked to vrs1 were converted to six STSs. All six STSs were co-dominant between the two cultivars after digestion with restriction enzymes. A reliable protocol for small-scale DNA isolation from leaf tissue was developed. The STS markers and the small-scale DNA isolation protocol developed in this study are useful tools for mapping the vrs1 locus of barley.Key words: vrs1 locus (two-row vs. six-row), STSs, co-dominance, Hordeum vulgare.

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A quantitative trait locus (QTL) controlling shoot differentiation from immature embryo callus was identified by linkage analysis with morphological and isozyme markers in barley, Hordeum vulgare L. Immature embryos were isolated from cvs 'Azumamugi' (difficult to differentiate), 'Kanto Nakate Gold' (easy to differentiate), their hybrids (F1) and a backcross population derived from a cross 'Azumamugi' x F1. The embryos were cultured in vitro for callus initiation and subsequent shoot differentiation. The shoot differentiation rate was closely associated with ear type (v locus), isocitrate dehydrogenase isozyme (Idh-2), and esterase isozyme (Est-11). These markers were found to reside in a chromosome segment of approximately 30cM on chromosome 2. Recombination frequency was 9.9% between v and a proposed QTL named "Shd1" (shoot differentiation), 11.5% between Idh-2 and Shd1, and 21.3% between Est-11 and Shd1. All data showed the Idh-2, v, Shdl and Est-11 loci to be arranged in this order from proximal to distal on the long arm of chromosome 2.

A PCR-amplified DNA, cMWG699, is linked to the vrs1 (formerly v) locus controlling 2- and 6-rowed spikelets. Restriction analysis of the amplified DNA of 65 varieties from Europe, America, and East Asia revealed 3 alleles, named types K, A and D. Two-rowed varieties were mostly of type K allele, and 6-rowed varieties were mostly of type A allele. The type D allele was found only in three 6-rowed varieties. Sequence comparison of these alleles revealed that the type A allele is more closely related to the type K allele than to the type D allele. The sequence analysis also suggested that the types A and D alleles diverged earlier than when 6-rowed barley arose. On the assumption that 2-rowed barleys were the ancestors of 6-rowed barley, 6-rowed barleys with types A and D alleles probably differentiated from 2-rowed barleys with type A and D alleles, respectively, by independent mutations on the vrs1 locus.