The main objectives are as follows:
* To identify Chinese wheat cultivars and/or synthesise new breeding lines which will be highly amenable to tissue culture and regeneration from protoplasts.
* To evaluate the most promising cereal transformation to determine the best for use with Chinese wheat cultivars.
* To assemble information on the molecular variation amongst isolates of both wheat spindle streak mosaic bymovirus (WSSMV) and soil-borne wheat mosaic furovirus (SBWMV) from different sites in Europe and China, and to design a pathogen-encoded antiviral gene strategy.
* To develop a suitable expression vector for wheat transformation and use constructs with these virus products to produce genetically-stable transformed wheat.
The work carried out in this project should produce transgenic wheat plants with virus resistance that will be available for use in breeding programmes in China. More importantly, reliable routine strategies for transformation of selected Chinese wheat cultivars should be established that can later be adapted by plant breeders to introduce a much wider range of desirable characteristics.
The key activities envisaged are:
* Identification of Chinese wheat genotypes with high regeneration capacity. These will then be used in evaluating the most promising transformation strategies: (i) PEG-mediated transformation of protoplasts; (ii) pollen-tube-pathway; (iii) biolistics; (iv) Agrobacterium tumefaciens-mediated DNA transfer.
* Molecular characterisation of WSSMV and SBWMV from different sites in Europe and China using RT-PCR and nucleotide sequencing. Viral transgene cassettes will then be designed which will express the most conserved components of selected genes (capsid protein, replicase, fungus transmission factor) either as RNA or as protein and for both viruses.
* Transformation of the selected wheat genotype(s) with two kinds of plasmid, one containing the selectable marker and the other the virus-derived gene of choice, under the control of a root-specific or pathogen-induced promoter.
* Tests of the transgenic plants for gene expression (by in situ hybridisation, sap analysis and by immunogold localisation of the virus gene products in the electron microscope) and virus resistance (by growing in infested soil under controlled conditions or by zoospore inoculation using sand cultures of the fungus vector). Another culture and crossing will be done to ensure that the introduced resistance(s) are stably integrated into the wheat genome and to check the purity of transgenic plants.
Funding SchemeCSC - Cost-sharing contracts
DD2 5DA Dundee