Location: Sunflower and Plant Biology Research2012 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. The main objective of the proposed project is to develop a reliable transformation approach for sunflower using a confection line identified by the PI that has an unusually high shoot production response with cotyledonary tissues. The completion of the project will lead to the production of transgenic sunflower with altered susceptibility to Sclerotinia disease. Introduction of an oxalate oxidase will develop sunflower line resistant to Sclerotinia disease. What’s more, with higher efficiency of transformation, Tntretrotransponson can be introduced into sunflower to mutate native genes conferring basal resistance to Sclerotinia disease. Mapping and identification of new genes conferring basal resistance to the disease will follow. We previously reported that the RHA280 sunflower line showed highly efficient shoot induction from cotyledon explants. During the reporting period, we observed a great variation in regeneration response among different lots of harvested seeds. Part of this variation was reduced when seed-producing plants were transferred from temporary to permanent greenhouses, following the completion of greenhouse renovations after a tornado severely damaged these structures in Sept, 2010. To reduce the remaining variation in shoot induction response to acceptable levels, much effort has been place into improving the quality of harvested seed and maintaining that quality during seed storage. Although cotyledons from freshly harvested seed usually responded with over 90% forming shoots, this response declined to 15% following storage for as little as 1 month at freezer or refrigerator temperatures. Recent results over this reporting period suggest that seed quality (for shoot induction response) can be maintained when seed moisture content at harvest is 4-9%. Seed that is harvested at higher or lower moisture content either does not respond well to shoot induction or does not store well for consistent shoot induction. Seed germination was not correlated with seed moisture content or shoot induction response. In addition to shoot induction from cotyledon explants, shoot induction from the primary leaves of 7-day-old seedlings was observed for the first time. Shoot number and efficiency of induction increased when benzylaminopurine (BA), was added to the seed germination medium. Shoot induction occurred on 65-95% of leaf explants, with 3-18 shoots per leaf. This new regeneration system using primary leaves of seedlings will provide an alternative target tissue for transformation, if the cotyledon explants do not work out. For whole plant recovery from regenerating shoots, efforts have been placed in developing micro-grafting techniques. Generation of roots directly from shoots has been problematic. Success of micro-grafting, where shoots are grafted onto germinated seedling in vitro, has improved with elongation of shoots (sometimes ten out of ten shoots elongated), but the survival after transfer to soil is still marginal with less than two of ten grafted plants surviving. But, this frequency is still much higher than root induction from elongated shoots. Precocious flowering of regenerants is common and plants were fertile. Various gene introduction approaches, including explant pre-culture, particle bombardment, sonication, and vortexing of tissue with glass beads, were evaluated to enhance transformation efficiency and to target gene delivery to the area where induced shoots emerge from the cotyledons. Transformed shoots have been inconsistently recovered and the response has been highly variable. We are hopeful that the more consistent shoot regeneration that we have recently obtained, will lead to consistency in transformed shoot recovery. Vacuum infiltration of shoot-producing primary leaves yields high efficiency gene delivery but transformed shoots have not yet been recovered. Unfortunately, recovery of whole transgenic sunflower plants, which is one of the main objectives of this research, has not yet been obtained. To finish the efforts to obtain more consistent shoot recovery from seed materials, biochemical and molecular markers will be evaluated before and after seed storage, to determine if any markers exist for consistent regeneration from seedling cotyledons. Efforts will continue to focus on consistent shoot production from seed materials, increasing transgenic event recovery per explant; optimization of transgenic shoot selection and recovery of whole plants from transgenic shoots.