Start Date: Jun 01, 2008
End Date: May 31, 2013
This research will primarily be focused on development of tissue culture regeneration & transformation technology for sunflower, to be used in the near future as a tool for studying effects of introduced transgenes on modulation of Sclerotinia resistance. Funding will be requested for one year, with the development of the technology occurring during that year. Germplasm generated within that year will subsequently be provided to colleagues, for evaluation of altered phenotypes. The proposed research is based on some recent results obtained in the PI’s laboratory on tissue culture responsiveness of sunflower germplasm that has not previously been evaluated. Based on collaborative efforts between John Finer (OSU) & Steve Knapp (University of Georgia), a confectionary sunflower line was identified, which appears to be remarkably suitable to tissue culture regeneration. This sunflower line is coincidently also one of the parents used in the sunflower breeding program at the University of Georgia, for the development of genetic markers. Based on our preliminary results, additional efforts were placed to develop consistency of shoot regeneration from the explants. Although shoot regeneration has already been repeated a number of times in the Finer Laboratory, additional efforts will still be required to increase efficiencies of shoot regeneration & integrate Agrobacterium-mediated transformation into the sunflower shoot regeneration system. These improvements entail standard tissue culture optimizations of shoot regeneration using different media manipulations, environmental culture conditions & most importantly, physiological status of the explant. Preliminary results suggest that the majority of improvements with this system will result not from media manipulations, but from varying preculture conditions of the explant. And since the sunflower line identified in the preliminary research was selected from ten diverse sunflower lines, including 8 Helianthus annuus genotypes & 2 additional species (H. argophyllus & H. tuberosus), genetic makeup of the most suitable line was obviously important. Populations have been developed (University of Georgia) which can be easily screened for regeneration potential to identify regions of the genome which contribute to regenerability in sunflower. Integration of Agrobacterium-mediated transformation with sunflower regeneration is not expected to be problematic as sunflower remains quite receptive to transformation using this biological vector. The Green Fluorescent Protein (GFP) will be used as a marker for optimization of transformation efficiency with antibiotic resistance genes as selectable markers. The PI has extensive experience with GFP & use of antibiotic resistance as a selection tool. In addition to the marker genes, 2 main “genes of interest” will be introduced into sunflower during the course of this research. Oxalate oxidase from wheat is already available in the PI’s laboratory & paperwork is well underway to obtain the Tnt transposon from tobacco from Marie-Angèle Grandbastien at INRA in France. These 2 genes represent two different approaches to understanding Sclerotinia resistance in sunflower.