指標 (GUS) 遺伝子を有する<I>Agrobacterium temefaciens</I>と<I>A. rhizogenes</I>利用によるハナタバコおよびハボタンの形質転換

Abstract

Transformation of ornamental tobacco (Nicotiana × Sanderae Hort.) and kale (Brassica oleracea var. acephala DC) was studied using the Agrobacterium tumefaciens or A. rhizogenes -vector system. A plasmid, pBI 121 (β-glucuronidase (GUS) gene combined with kanamycin resistant gene, nptII) was transferred to Agrobacterium by tri-parental mating or freeze-thaw method.After leaf disks of ornamental disks were inoculated with a suspension of A. tumefaciens harboring pBI 121, they were cultured on Murashige and Skoog (MS) medium supplemented with growth regulators, kanamycin sulfate and carbenicillin sodium. Six calli appeared from edges of leaf disks from which shoots differentiated. Of the 10 shoots examined, nine showed positive reaction to 5 bromo-4-chloro-3-indolyl glucuronide (X-gluc) suggesting that GUS gene was transferred to plant cells. These nine shoots survived on MS medium containing kanamycin sulfate whereas shoots regenerated from non-infected leaf disks died. Based on these results, nine shoots were recognized to be transformants harboring pBI 121.Petioles of ornamental kale lelaves were inoculated with a suspension of A. rhizogenes harboring a plasmid, pBI 121. Ten roots developed, but only two roots showed positive reaction to X-gluc. Five shoots which were regenerated from these two roots on MS medium supplemented with growth regulators and carbenicillin sodium gave a positive reaction to X-gluc, and to 4-methyl umbelliferyl glucuronide (MUG). The shoots survived on MS medium containing kanamycin sulfate, indicating that the five regenerated shoots were transformed.