Cytokinin gene fused with a strong promoter enhances shoot organogenesis and zeatin levels in transformed plant cells

Abstract

The isopentenyltransferase (ipt) gene associated with cytokinin biosynthesis in plants was cloned from a tumor-inducing plasmid carried by Agrobacterium tumefaciens and placed under the control of promoters of differing activities, the cauliflower mosaic virus 35S promoter and the nopaline synthase promoter. These promoter-gene constructs were introduced into wounded Nicotiana stems, leaf pieces, and cucumber seedlings by A. tumefaciens infection. Shoots were observed in the infection site on all responding genotypes of Nicotiana plants infected with the 35S promoter construct (35S-ipt), whereas only 41% responded similarly to infection with the unmodified gene. Furthermore, shoots were observed 19 days after infection with the 35S-ipt gene but not until 28 to 45 days with the unaltered ipt gene. Shoots were more numerous (>40) on galls incited by 35S-ipt and were up to 6 times taller than shoots induced by the native gene. On Cucumis (cucumber), shoots were observed only on galls incited by the 35S-ipt construct. These galls were on the average 7.5 times larger than those incited by the nopaline synthase promoter construct (NOS-ipt) or the unmodified ipt gene. Zeatin and zeatinriboside concentrations averaged 23 times greater in the 35S-ipt transformed shoots than in ones transformed with the native ipt gene. These results suggest that a more active promoter on the ipt gene can enhance or change the morphogenic potential of transformed plant cells by increasing their endogenous cytokinin levels.