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Abstract
In the present study, we analysed the effect of integration of single Agrobacterium rhizogenes rol genes or their combinations in the rol-containing Nicotiana glauca, in the non-rol Nicotiana langsdorffii, in their tumorous hybrid N. glauca×N. langsdorffii and in a non-tumorous mutant of it. We investigated whether the integration of transgenes could change the genotype's hormonal balance, thus inducing different in vitro morpho-physiological patterns. In the mutant, the presence and expression of endogenous rol genes and the effect of their induction were also studied. Transformation of N. langsdorffii with the whole A. rhizogenes complex and the integration of rolB and rolC genes in the mutant induced the production of autotrophic calluses exhibiting a tumorous morphology. Polymerase chain reaction (PCR) analysis of untransformed plants confirmed the presence of rolB and rolC in N. glauca and hybrids, whereas both genes were absent in N. langsdorffii. However, reverse-transcriptase PCR showed the loss of expression of both genes in the mutant. Treatment of the non-tumorous mutant plants with the demethylating agent 5-azacytidine revived rolB and rolC expression and induced the acquisition of hormone autotrophy.
Acknowledgements
This study was partially funded by grants from the Italian Ministry of University and Research (MIUR) within the framework of the PRIN-2007 project. The authors thank Prof. P. Costantino, Department of Genetics and Molecular Biology, University of Rome “La Sapienza”, for providing all the Agrobacterium strains. This work is dedicated to the memory of Dr. Domenico Mariotti, our friend and colleague.