Application of the acetolactate synthase gene as a cisgenic selectable marker for Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa ssp pekinensis)

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.