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Elsayed, Ashraf (2011): Analysis of paternal plastid inheritance in Arabidopsis thaliana and its impact on biosafety of gene-modified plants. Dissertation, LMU München: Faculty of Biology
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Abstract

Genetically modified plants for the use of transgene containment are a central concern. Nuclear gene flow is one of the most discussed topics in our days; therefore, plastid genetic engineering is a promising tool to reduce the risk of transgene flow, because in most angiosperm species plastids are inherited maternally. In addition, plastid transformation has the advantage that the site of gene insertion can be controlled, high rates of transgene expression and protein accumulation can be achieved and epigenetic effects are absent. In Arabidopsis pollen, plastids are inherited also maternally and not created de novo, but arise from pre-existing plastids by fission. The aim of this study was to assess the frequency of plastid transfer from atrazin-resistant ElyF3BC4 Arabidopsis thaliana plants bearing a point mutation in the plastid psbA gene to male sterile N75 plants by spontaneous crossing under field conditions. Also the plastid transfer from atrazin-resistant, EMS-mutagenized M2ElyF3BC4 plants to wild-type A. thaliana plants by manual crossings under green house conditions was estimated. It was found that plastid-encoded atrazin resistance could not be transmitted via pollen, neither by manual pollination among 65,000 hybrid seeds nor by spontaneous pollination among 2,444,465 hybrid seeds in A. thaliana. Although various random nuclear mutations were screened for their potential to allow the transfer of paternal plastids into the egg-cells of the recipient plant, a corresponding mutant line could not be isolated. Explanation for this could be duplication or redundancy of nuclear genes mediating maternal inheritance and suppressing paternal leakage in Arabidopsis in such a way that the defect in one gene is compensated for by the function of its homologue. Therefore, a double mutant of two genes, atg4a and atg4b, which are involved in autophagy, were studied to test this hypothesis. However, the frequency of paternal plastid transfer was not increased. Taken together, in this study paternal leakage of Arabidopsis plastids could not be induced by mutations. To be able to follow plastid fate in developing pollen tubes, the colorless plastids in Arabidopsis pollen were visualized by the expression of a GFP fusion protein under the control of a pollen specific promoter. However, the affiliation of the GFP labeled plastids to either the vegetative or the generative cells was not clear. Placing particular emphasis on plastid behavior during specification of sperm cells in pollen of Arabidopsis might shed some light on this very strict process of maternal inheritance in the future work.