| Centaurea montana is a source of major flavonoids, acetylenes and indoles which show significant pharmaco-medicinal and in vitro anticancer activity. For the first time the conditions for a robust and rapid regeneration of C. montana through direct organogenesis coupled to an efficient Agrobacterium-mediated transformation were described. Large numbers of regenerated shoots (62+/-15 shoots/explant) were obtained from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mgl-1 (0.5 muM) indole-3-acetic acid (IAA) and 2.0 mgl-1 (8.8 muM) 6-benzylaminopurine (BAP). Shoots up to 80 mm in length were elongated on MS medium with gibberellic acid (GA3) within 2 weeks. Elongated shoots were rooted on MS medium supplemented with 0.5 mgl-1 (2.4 muM) indole-3-butyric acid (IBA). Overall, C. montana plantlets regenerated in vitro were transferred to the green house in less than three months. Transformation was obtained by co-cultivation of leaf explants and Agrobacterium tumefaciens strain AGL1 carrying plasmid harboring the isopentyl phosphotransferase (IPT) gene under the control of the developmentally regulated Atmyb32 promoter. Molecular analyses, including PCR, Southern blotting, and RT-PCR were used to confirm chromosomic integration and expression of the transgene in phenotypically normal transgenic plants displaying the delayed senescence phenotype. We have obtained 18 transgenic plants from 990 leaf explants initially transformed, resulting in an overall transformation efficiency of 1.8%. In parallel, Nicotiana tabacum IPT-transgenic plants were successfully produced in order to study the senescence phenotype side by side with the Centaurea transgenic plants. Similarly, an increase of physiologically-active and translocation cytokinins lead to a marked delay in leaf senescence as well as to the production of healthy mature transgenic plants with no deleterious effects on growth and development. Moreover, conditions for the establishment and proliferation of developmentally stable, fine embryogenic cell suspension cultures from leaf-derived friable callus were described. Plant cell suspension cultures have a huge promising potential as alternative sources for the mass production of secondary metabolites of high industrial importance and value. Leaf explants formed embryogenic, fluffy calluses at a frequency of 75% when cultured on MS medium supplemented with 1 mgl-1 (4.5 muM) 2,4-Dichlorophenoxyacetic acid (2,4-D). Cell suspension cultures were established on liquid MS medium with varying concentrations of 2,4-D. We report here also the induction of in vitro flowers in Centaurea following hormonal manipulation in the culture medium. The induction of in vitro flowers in Centaurea is of utmost importance for the continuous supply of flower and seed material all year round. Moreover, the ability of flower buds to open and bloom inside the test tube makes it possible to shorten the life cycle of the plant and obtain pathogen-free seeds in vitro. This study offers an attractive system for the exploration of this plant species. Finally, a family of fifteen FBL genes were isolated and characterized from Arabidopsis thaliana plants. An evidence for their possible involvement in disease resistance signaling pathways was provided through a detailed analysis of AtFBL5 transcriptional expression in response to treatments with the disease resistance-related signaling molecules salicylic acid (SA), methyl jasmonate (JA) and the ethylene precursor 1-amino cyclopropane-1-carboxylic acid (ACC). |
