A functional cDNA for the resistance gene N

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the beta-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants.

SUMMARY In clubroot disease, gall formation is induced by infection with the obligate biotroph Plasmodiophora brassicae due to increased levels of auxins and cytokinins. Because aldehyde oxidase (AO) may be involved in auxin biosynthesis in plants, we isolated two AO genes (BrAO1 and BrAO2) from Chinese cabbage (Brassica rapa ssp. pekinensis cv. Muso), which are the most similar to AAO1 among Arabidopsis AO genes, and examined their expressions during clubroot development. The expression of BrAO1 was enhanced in inoculated roots from 15 days post-inoculation (dpi) when visible clubroots were still undetectable. Thereafter, BrAO1 expression increased with clubroot development compared with uninoculated roots, although BrAO2 expression was repressed. In situ hybridization revealed that BrAO1 was strongly expressed in tissues that were invaded by immature plasmodia at 35 dpi, suggesting that BrAO1 expression was enhanced by the pathogen in order to establish its pathogenesis. In addition, we detected AO activity, as evidenced by the occurrence of at least six bands (BrAO-a to BrAO-f) in the roots of Chinese cabbage using an active staining method with benzaldehyde and indlole-3-aldehyde as the substrate. Coincidental with BrAO1 expression, the signals of BrAO-a and BrAO-d increased with inoculation by P. brassicae during clubroot development compared with healthy roots, resulting in an increase in total AO activity. By contrast, the band BrAO-b decreased post-inoculation, in parallel with the expression of BrAO2. The other bands of activity were not clearly influenced by the infection. Based on these results, we discuss the involvement of AO in auxin-overproduction during clubroot development in Chinese cabbage.

Heading Chinese cabbage (Brassica rapa ssp. pekinensis) has been recalcitrant to regeneration and transformation. In particular, there are few reports concerning Japanese cultivars. We evaluated the factors that inhibit Agrobacterium-mediated transformation in heading Chinese cabbage. Then, we investigated the effects of selectable markers using heading Chinese cabbage cv. Chihiri 70 and compared the effects with those in broccoli cv. Ryokurei (B. oleracea var. italica) as a control for an easily transformable Brassica species. To utilize a selectable marker derived from a host plant cisgene, we cloned a genomic DNA fragment containing regulatory and coding sequences of the acetolactate synthase (ALS) gene from Chinese cabbage and mutagenized it to a herbicide resistant form. After transformation of Chinese cabbage and broccoli with this construct, transgenic plants were efficiently selected with the herbicide bispyribac sodium salt and screened by DNA gel blot analysis. The average transformation frequency of Chinese cabbage was 1.2±0.2%, which was similar to those in reports using antibiotic selectable markers and was lower than for broccoli (13.9±2.0%). Furthermore, the escape rate was restricted at a low level (about 35-50% lower than hygromycin selection), which is an advantage on practical transformation. We confirmed transgene inheritance and herbicide resistance of potted plants in the T 1 generation. This report is the first to describe a selection system for the transformation of a Brassica crop that uses a herbicide-tolerant selectable marker derived from a cisgene.

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