Exogenous Phytohormone-Independent Growth and Regeneration of Tobacco Plants Transgenic for the 6b Gene of Agrobacterium tumefaciens AKE10

Abstract

The 6b gene of Agrobacterium tumefaciens AKElO (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium. We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A. fumefaciens AKEl O. Tobacco (Nicotiana tabacum) leaf discs infected with A. tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium. The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization. Some of these immature shoots spontaneously developed into mature shoots, of which severa1 leaves displayed morphological abnormalities. When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential. Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-66 expression is regulated in plants and essential for the early stages of regeneration. Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content.

The Protein Encoded by Oncogene 6b from Agrobacterium tumefaciens Interacts with a Nuclear Protein of Tobacco

“…Thus, the product of the 6b gene appears to be nondiffusible (Tinland et al, 1992). This conclusion is consistent with the hypothesis that the levels of cytokinin and auxin, which basically are diffusible within a plant, are not affected by expression of the 6b gene (Wabiko and Minemura, 1996). Some clues to the mode of action of the 6b protein might be found in its amino acid sequence.…”

“…It also has been proposed that the 6b gene might affect the concentration of endogenous plant hormones in transformed cells (Spanier et al, 1989). However, shoot-bearing calli of tobacco whose formation has been induced by the 6b gene contain normal levels of active cytokinins (Wabiko and Minemura, 1996). Moreover, when we consider the possible mechanisms of action of the 6b protein in plant cells, we must remember that, in chimeric plants that were generated by grafting the stem of a 6b -transformed plant onto that of a normal plant, the effect of 6b was found only in the 6b -transformed portion.…”

“…Various phenotypic effects associated with the expression of 6b have been reported, as follows: (1) formation of tumors on certain plants (Hooykaas et al, 1988;Spanier et al, 1989;Tinland et al, 1989Tinland et al, , 1992; (2) stimulation of the ipt -induced and iaaM / iaaH -induced division of cells (Tinland et al, 1989(Tinland et al, , 1990Wabiko and Minemura, 1996); (3) reduction in the formation of shoots on leaf discs that is normally induced by appropriate levels of exogenous or endogenous cytokinin (Spanier et al, 1989); (4) generation of shoot-bearing calli on leaf discs on phytohormone-free medium (Wabiko and Minemura, 1996); (5) inhibition of the growth of Rol -induced hairy roots via the induction of an undifferentiated state and the formation of calli (Tinland et al, 1990); and (6) alterations in the morphology of leaves of transgenic tobacco plants that express the 6b gene (Tinland et al, 1992;Wabiko and Minemura, 1996). Although there are some discrepancies among previously reported results (Leemans et al, 1982;Ream et al, 1983), it is accepted generally that the product of 6b stimulates the proliferation of plant cells and affects the development of 1 Current address: National Institute for Basic Biology, 38 Nishigounaka, Myo-daiji-cho, Okazaki 444-8585, Japan.…”

“…It has been suggested that the 6b gene might function in the activation and/or inactivation of cytokinin and auxin (Hooykaas et al, 1988;Spanier et al, 1989); it might increase and/or decrease the sensitivity of transformed cells to these plant hormones (Hooykaas et al, 1988;Tinland et al, 1989) by modifying putative hormone transport systems or reporters (Spanier et al, 1989); its product might interact with ipt and iaa genes and/or their products (Tinland et al, 1989) and/or it might stimulate signal perception systems (Wabiko and Minemura, 1996). It also has been proposed that the 6b gene might affect the concentration of endogenous plant hormones in transformed cells (Spanier et al, 1989).…”

Prussian Blue acts as a mediator in a reagentless cytokinin biosensor

“…fractionated measurements such as by gas chromatography [1,2], capillary electrophoresis [3], HPLC 46 [4], ELISA [5,6] and radioimmunoassay [7,8]. Moreover many require elaborate sample work-up, 47 radioactive chemicals and are time-consuming.…”