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 Nicotana stems, leafpieces, and cucumber seedlings by A. tumefaciens infection. Shoots were observed in the infection site on all responding genotypes of Nicotana 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-4pt) or the unmodified ipt gene. Zeatin and zeatinriboside concentrations averaged 23 times greater in the 35Spt 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.Patternsiof differentiation in plants can be affected by several phytohormones including two major classes, the cytokinins and auxins. The relative levels of cytokinins and auxins in plants appear to be critically important. When cultured in vitro, cells of most plants require exogenous cytokinin and auxin in the medium for cell division to occur, presumably because of low endogenous pools of these phytohormones. Moreover, regeneration of shoots from cultured cells can be achieved with many plant species by increasing the cytokinin-to-auxin ratio in the medium (1, 2). However, failure of some species, among them important crop species, to respond in a morphogenic fashion to such treatment may reflect problems in hormone uptake, compartmentalization, or metabolism. The availability of phytohormone-specifying genes with plant regulatory sequences from Agrobacterium tumefaciens provides an alternate genetic approach to the study of morphogenesis through the in vivo manipulation of hormone ratios (3).A widely studied method of gene transfer to dicotyledonous plants is based on the tumor-inducing (Ti) plasmid of A.

Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.)

“…Classical tissue culture experiments have demonstrated that exogenous application of a low ratio of auxin to cytokinin promotes shoot regeneration from tobacco callus, while a high auxin to cytokinin ratio stimulates root formation (Skoog and Miller 1957). These observations are supported and extended by numerous studies in which heterologous expression of bacterial cytokinin-(isopentenyltransferase; ipt) and auxin biosynthesis enzymes (tryptophan monooxygenease, iaaM; indoleacetamide hydrolase; iaaH) has been used to alter endogenous levels of these hormones (Smigocki and Owens 1988;Sitbon et al 1992;Hewelt et al 1994;Eklöf et al 2000). BBM over-expression transgenics show elements of both cytokinin and auxin over-production phenotypes including ectopic shoot production and leaf wrinkling (cytokinin) and ectopic root production (auxin).…”

Cytokinin-to-Auxin Ratios and Morphology of Shoots and Tissues Transformed by a Chimeric Isopentenyl Transferase Gene

“…By replacing the native promoter on this gene with the 35S promoter from cauliflower mosaic virus, the level of ipt gene transcription was increased more than 10-fold in transformed tobacco and cucumber tissues. As we have reported, this correlated with more than a 100-fold increase in cytokinin content and an enhanced ability of the cells to undergo shoot organogenesis presumably due to increased cytokinin-to-auxin ratios (17). We report here the auxin levels for these transformed tissues and both cytokinin and auxin contents of additional samples not previously analyzed in order to study the effects of in vivo phytohormone ratios on the cells' morphogenic potential.…”

“…Strains of Agrobacterium tumefaciens used have been described previously ( 17). Briefly, in one strain, a truncated ipt gene cloned from pTiB6S3 was placed under the control of the 35S promoter from cauliflower mosaic virus, and the chimeric gene construct, termed 35S-ipt, was inserted into a binary plasmid, pBin 19.…”

“…To eliminate possible effects of other T-DNA genes, we cloned the gene encoding the first enzyme in the cytokinin biosynthetic pathway, isopentenyl transferase (ipt), from the T-DNA region (17). By replacing the native promoter on this gene with the 35S promoter from cauliflower mosaic virus, the level of ipt gene transcription was increased more than 10-fold in transformed tobacco and cucumber tissues.…”

Hormonal Characterization of Transgenic Tobacco Plants Expressing the rolC Gene of Agrobacterium rhizogenes TL-DNA

“…Plants expressing iaaL showed, as a result of increasing the cytokinin-to-IAA ratio, cytokinin-like effects, with reduced apical dominance and reduced root growth, which were correlated to reduced levels of free IAA and increased levels of IAA conjugates (Romano et al, 1991;Spena et al, 1991). Plants expressing the ipt gene failed to develop any further than small shoots when expressing a 35s-ipt construct (Smigocki et al, 1988(Smigocki et al, , 1989, but when the ipt gene was fused to a heat-shock promoter, plants expressing the gene displayed cytokinin effects in that they were dark green with reduced apical dominance and poor root formation. Accordingly, increased levels of a number of cytokinins could be detected in these plants (Medford et al, 1989;Smart et al, 1991;Smigocki, 1991).…”