Programmed cell death induced by high levels of cytokinin in Arabidopsis cultured cells is mediated by the cytokinin receptor CRE1/AHK4

High levels of cytokinins (CKs) induce programmed cell death (PCD) both in animals and plant cells. High levels of the CK benzylaminopurine (BA) induce PCD in cultured cells of Arabidopsis thaliana by accelerating a senescence process characterized by DNA laddering and expression of a specific senescence marker. In this report, the question has been addressed whether members of the small family of Arabidopsis CK receptors (AHK2, AHK3, CRE1/AHK4) are required for BA-induced PCD. In this respect, suspension cell cultures were produced from selected receptor mutants. Cell growth and proliferation of all receptor mutant and wild-type cell cultures were similar, showing that the CK receptors are not required for these processes in cultured cells. The analysis of CK metabolites instead revealed differences between wild-type and receptor mutant lines, and indicated that all three receptors are redundantly involved in the regulation of the steady-state levels of isopentenyladenine- and trans-zeatin-type CKs. By contrast, the levels of cis-zeatin-type CKs were controlled mainly by AHK2 and AHK3. To study the role of CK receptors in the BA-induced PCD pathway, cultured cells were analysed for their behaviour in the presence of high levels of BA. The results show that CRE1/AHK4, the strongest expressed CK receptor gene of this family in cultured cells, is required for PCD, thus linking this process to the known CK signalling pathway

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T0 generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T1 seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T1 generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use

Phenotype and hormonal status of transgenic tobacco plants overexpressing the rolA gene of Agrobacterium rhizogenes T-DNA

The rolA gene of the TL-DNA of Agrobacterium rhizogenes Ri-plasmid plays a major role in establishing the hairy root syndrome in transgenic plants. Transgenic tobacco plants (Nicotiana tabacum L.) expressing constitutively the rolA gene under the transcriptional control of the 35S RNA promoter show pronounced phenotypical alterations. P35S-rolA transgenic tobacco plants are characterized by stunted growth, dark green wrinkled leaves with an altered length-to-width ratio, condensed influorescences, retarded onset of flowering, a reduced number of flowers and shortened styles. To investigate whether the pleiotropic alterations of growth and development are linked to an altered hormonal status we have compared the immunoreactive content of indole-3-acetic acid, cytokinins, abscisic acid, gibberellin and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) of seedlings and different tissues of P35S-rolA transgenic plants, transgenic plants expressing the rolA gene under control of its own phloem-specific promoter and wild-type plants. Multiple tissue-specific alterations of phytohormone concentrations are the consequence of rolA gene activity. Changes of phytohormonal content can explain part of the rolA-induced phenotypic alterations. Most strikingly, in young and fully developed leaves of rolA and P35S-rolA transgenic clones a 40-60% reduction of immunoreactive gibberellin A1 was found, as compared to wild-type leaves. Treatment of wild-type tobacco plants with inhibitors of gibberellin biosynthesis phenotypic alterations similar to those of rolA transgenic plants. This suggests that the reduction of gibberellic acid content is indirectly but causally involved in rolA-induced alterations of stem elongation and planar leaf blade growth