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In this study, we searched for anther-specific genes involved in male gametophyte development in apple (Malus x domestica Borkh. cv. Fuji) by differential display-PCR. Three full-length cDNAs were isolated, and the corresponding genomic sequences were determined by genome walking. The identified genes showed intronless 228- to 264-bp open reading frames and shared 82-90% nucleotide sequence. Sequence analysis identified that they encoded a putative arabinogalactan protein (AGP) and were designated MdAGP1, MdAGP2, and MdAGP3, respectively. RT (reverse transcriptase)-PCR revealed that the MdAGP genes were selectively expressed in the stamen. Promoter analysis confirmed that the MdAGP3 promoter was capable of directing anther- or pollen-specific expression of the GUS reporter in tobacco and apple. Furthermore, expression of ribosome-inactivating protein under the control of the MdAGP3 promoter induced complete sporophytic male sterility as we had expected.

To develop an efficient screening method for detection of the transgene in Chinese cabbage (Brassica rapa spp. pekinensis) utilizing Basta spray, optimal conditions for Basta application were examined in this study. Two transgenic Chinese cabbage lines were obtained through Agrobacterium-mediated transformation and used as transgenic positive controls in the Basta screening experiment. Differential concentrations of glufosinate-ammonium were sprayed into three different growth stages of 12 commercial Chinese cabbage cultivars. The results showed that no plants could survive higher than 0.05% glufosinateammonium, and plants at the 2-3 leaf stage were most vulnerable to glufosinate-ammonium. On the other hand, no damage was observed in the transgenic control plants. Reliability of the Basta spray method was proven by showing perfect co-segregation of the tolerance to glufosinate-ammonium and the presence of the bar gene in T 1 segregating populations of the transgenic lines, as revealed by both PCR and Southern blot analyses. Using the developed Basta screening method, we tried to investigate the transgene flow through pollen dispersal, but failed to detect any transgene-containing non-transgenic Chinese cabbages whose parents had been planted adjacent to transgenic Chinese cabbages in field conditions. However, the transgene was successfully detected using Basta spray from the non-transgenic plants bearing the transgene introduced by hand-pollination. Since the Basta spray method developed in this study is easy to apply and economical, it will be a valuable tool for understanding the mechanism of gene flow through pollen transfer and for establishing a biosafety test protocol for genetically modified (GM) Chinese cabbage cultivars.

A cluster of oligonucleotide repeat sequences was identiWed that is linked to the melon (Cucumis melo L.) andromonoecious (a) locus, which controls andromonoecy. Six diVerent repeat units, ranging from 11 to 49 base pairs, were clustered within a 0.6-kb intergenic region. The number of repeat units varied from two to six. After sequence analysis of diverse melon germplasms, four haplotypes of this cluster were identiWed. Length variations among the four haplotypes resulted from insertion or deletion of repeat sequences. Particularly, a tandem array of six repeats of 21 nucleotides was a hotspot for insertion/deletion mutations. A simple PCR-based marker was developed to identify haplotypes of this cluster based on the length polymorphism. In practice, this marker was successfully used in genetic purity tests of melon F 1 hybrid cultivars. Four selfpollinated contaminants, which were conWrmed by phenotypic examination in grow-out tests, were easily discriminated from 99 F 1 hybrid individuals. In addition, the genetic distance between this marker and the andromonoecious (a) locus was calculated as 7.9 cM, after analyzing melon F 2 populations originating from a cross between monoecious and andromonoecious parental lines. Therefore, this marker will be useful as a recombinant selection marker in marker-assisted backcrossing of monoecy in melon breeding programs.