Nutzung phytobakterieller Gene zur Beeinflussung der pflanzlichen Apikaldominanz - Untersuchungen in transienten und induzierbaren transgenen Expressionssystemen

Abstract

In this study, different transgenic expression systems were investigated for their influence on auxin/cytokinin balance in plants in order to break apical dominance and promote lateral shoot growth in Nicotiana species and Petunia hydrida. In the first part of the work, different phytohormone genes (rolA, rolC, iaaH, iaaL und ipt) encoded by phytopathogenic bacteria A. rhizogenes, A. tumefaciens and P. syringae, known for its influence on phytohormonal balance in plants, where used to modify the development of plant shape. In the second part of the study it was analysed, if RNA interference (RNAi), induced by virus induced gene silencing (VIGS), can be used to suppress expression of genes from the plants cytokinin metabolism in order to induce phenotypic changes of plant shape. Initially the general potential of phytobacterial genes to induce plant shape modifications in N. tabacum, N. benthamiana and P. hybrida was characterized by applying a transient expression assay. Plant viral expression vectors based on Potato virus X (PVX) and Tobacco rattle virus (TRV) were used for expression of different phytohormone genes. Very young seedlings and young plants were infected with the TRV and PVX derived expression vectors and in case of ipt expression typical phenotypes of suboptimal increased cytokinin content were induced. In contrast, viral expression of iaaH induced strong phenotypes of high auxin supply, i.e. epinastic leaves and formation of root primordia at the scion. In addition to the aforementioned transient expression assay, experiments were carried out to express the phytohormone genes in stable transformed plants. For this purpose, the phytobacterial genes were placed under transcriptional control of an ethanol inducible expression system (alc system). In addition, the expression cassette of the binary plant transformation plasmid was supplied with gfp reporter gene (mgfp4) under control of an auxin inducible promotor (DR5). Besides the inducible changes of plant form, the auxin inducible GFP expression should enable the tissue specific analysis of changes in free auxin, depending on the ethanol induced expression of phytohormone genes. The obtained transgenic plant lines (T0 and T1) were characterized for the effects of induced and reversible controlled expression of genes on phenotypes of modified plant phytohormonal balance. Primary ethanol induction experiments with these lines revealed the suitability of the inducible expression system for the regulation of phytohormonal gene expression. Several in vitro as well as ex vitro expression experiments using plants of different developmental stages resulted in reproducible alterations of plant shape. An additional expression assay carried out, aimed to apply VIGS for the induction of transient RNAi in order to reduce the expression level of cytokinin metabolism genes in Petunia plants. Therefore Petunia gene fragments displaying high homology to isopentenyltransferases and cytokinin oxidases/dehydrogenases of different plants, were cloned and inserted into a VIGS vector. N. benthamiana and P. hybrida plants were inoculated with these different constructs but no visible changes of morphological traits could be observed. In conclusion, a selective influence on apical dominance was demonstrated using different strategies of manipulating the phytohormone metabolism in Nicotiana spec. and P. hybrida. For the first time, these results were achieved using plant viral expression vectors. In addition, the ethanol-inducible expression system was successfully used for modulation of auxin/cytokinin balance in transgenic tobacco plants.