Location: Sunflower and Plant Biology Research2009 Annual Report
1a. Objectives (from AD-416)
Plant biotechnology has had a tremendous impact on crop production in the U.S., as well as world-wide. The percent of transgenic acreage of major crops in the U.S. is very high, with transgenics contributing 52% of maize, 79% of cotton, 82% of canola and 93% of soybeans. This shows remarkable acceptance of this technology, considering the first introduction of transgenics occurred in 1994. This success in plant biotechnology resulted from the ability to introduce genes into plants, which could not be introgressed using conventional breeding techniques. Breeding is still integral to continual improvements in crop genetics but transgenics provides an additional tool, which can accelerate advancements in crop production. Most of the transgenic plants, which are grown in the field, contain genes for increased resistance to specific insects and herbicides. Other plants in various stages of approval for commercialization contain genes for virus resistance, fungal resistance and both modified oil and protein. Advances in sunflower biotechnology have not kept pace with the other major crops for a variety of reasons. First and most importantly, commercialization of transgenic cultivated sunflower would present new challenges as it is sexually compatible with common wild sunflower in the U.S. Unless outcrossing restrictions are relaxed or outcrossing itself is completely controlled, commercialization of transgenic sunflower may be problematic. Even if commercialization of transgenic sunflower does not take place in the near future, gene introduction in this crop remains useful as a basic research tool. Transgenic approaches can be used to either evaluate the effects of transgenes directly or knock out native genes through RNAi approaches or generation of insertion mutants. All of these approaches require the development of consistent and reliable transformation approaches for sunflower, which unfortunately do not currently exist. The absence of efficient transformation methodologies for sunflower are surprising, considering that this crop was one of the first plants to be placed in tissue culture and one of the most responsive to Agrobacteriummediated transformation. The challenge in developing transformation systems is the coordination of transformation and regeneration; being able to target cells/tissues for DNA introduction into tissues that are also capable of regenerating into whole plants. Although there are reports of “efficient” transformation systems for sunflower, these methods are largely not effective and production of transgenic sunflower remains extremely difficult. The main objective of this research is to develop tissue culture regeneration and transformation technology for sunflower which can be used for introduction of both transgenes of interest and retrotransposons, to generate insertion mutants for reverse genetics approaches.
1b. Approach (from AD-416)
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.
3. Progress Report
This project was initiated on June 1, 2008, research is ongoing, and the overall objective is to develop tissue culture regeneration and transformation technology for sunflower which can be used for introduction of both transgenes of interest and retrotransposons, to generate insertion mutants for reverse genetics approaches. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. Transformation system will speed improved Sclerotinia resistance in sunflower: Plant biotechnology has had a tremendous impact on crop production in the U.S., as well as world-wide. The main objective of this research is the development a consistent and reliable transformation method for sunflower, using a line, recently identified by the PI as exceptionally responsive to tissue culture manipulation. Successful completion of this objective will lead to the production of transgenic sunflower with altered susceptibility to Sclerotinia. Introduction of an oxalate oxidase gene may provide enhanced resistance directly while introduction of a Tnt retrotransposon will allow mapping and rapid identification of native genes which provide base levels of resistance. We have been able to successfully demonstrate high efficiency shoot production from sunflower cotyledonary tissues using a confection sunflower line, RHA280. The variability in response that we initially observed has been largely reduced by using cotyledon pieces from freshly-harvested seeds of greenhouse-grown plants. Seeds are stored at -20C after harvest. Response of the explant is consistently about 75% of the explants forming multiple shoots. We have further demonstrated that an explant size of 2 mm is optimal for shoot production. Using optimized regeneration and explant preparation, we have also observed that a previously unresponsive sunflower line (RHA810) remains unresponsive. A combination of use of a responsive cultivar with optimized plant regeneration is apparently needed. Plants regenerated from these RHA 280 shoots flower prematurely and are tremendously reduced in height but plant regeneration has been shown. Using Agrobacterium-mediated transformation, we have also recovered transformed tissue (not plants), demonstrating that the sunflower line is responsive to Agrobacterium. Sonication treatments increase Agrobacterium-mediated gene introduction in target tissues but reduce shoot production. Inclusion of antioxidants during Agrobacterium co-culture have not reduced tissue browning or led to recovery of transgenic shoots. A sunflower polyubiquitin promoter has been recovered from sunflower genomic DNA and will be evaluated for regulation of gene expression. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.