Development and validation of a method for the detection of altered resistance in transgenic plants against herbivore-pathogen-complexes

摘要

Enhanced or reduced uptake of viruses by vectors and changes in resistance level can be sensitive indicators for metabolic changes in transgenic plants caused by the new trait and not observed by conventional methods. In addition, the plant transformation process itself can lead to such changes. To be able to investigate this hypothesis in cereals we decided to use two highly important insect-transmitted viruses infecting them - _Barley yellow dwarf virus_ (BYDV) and _Wheat dwarf virus_ (WDV). Corresponding molecular tools for their quantification in plants as well as virus vectors had to be developed. Both viruses cause similar symptoms: dwarfing, stunting, leaf discoloration leading to yield losses or death of plant. BYDV (_Luteoviridae_) is one of the most important cereal-infecting viruses worldwide causing substantial yield losses. In Germany, the strain PAV is widespread. It is transmitted by the aphids _Rhopalosiphum padi_ and _Sitobion avenae_ in a persistent manner. WDV (_Geminiviridae_) is found in Germany since the early 1990s and has gained importance over the last years. It is transmitted by the leafhopper _Psammotettix alienus_ in a persistent manner. Real-time PCR is the state of the art method for specific detection of viruses even in minor quantities. It allows exact quantification of the virus content of plants and vectors and is hence suited to monitor changes of it. We developed qPCR assays for WDV and RT-qPCR assays for BYDV-PAV based on TaqMan probe technology as well as the DNA-binding dye SybrGreen. The qPCR assays proved to be more sensitive than DAS-ELISA. For example, WDV is detected even at dilutions of 1:10^8^, whereas the corresponding threshold for ELISA is about 1:10^4^. As nucleic acid extraction procedures proved to be time consuming and expensive, detection methods are adopted now for immunocapture procedures. These methods will be applied to the analysis of several transgenic wheat lines.