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United States Department of Agriculture

Agricultural Research Service

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Research Project: Use of a Transformation System in Sunflower for Sclerotinia Resistance Studies

Location: Sunflower Research

Project Number: 5442-21220-028-22
Project Type: Specific Cooperative Agreement

Start Date: Jun 01, 2013
End Date: May 31, 2015

Objective:
Since production of the first transgenic plants in the mid 1980s and the subsequent commercialization of the first transgenics in the early 1990s, plant biotechnology continues to have a tremendous impact on both basic plant biology research and crop production. The use of molecular markers has become an integral part of virtually all large-scale breeding programs and the contribution of transgenics to this research area continues to grow. Commercial production of transgenics for many of the major crops in the US is very high, with transgenics contributing to well over 75% of the maize, cotton, canola and soybean acreage. The US has led this adoption effort but there continues to be some resistance to commercialization of certain transgenics in parts of Europe. Transgenic technology is based on the introduction of genes into plants, which could not be introgressed using conventional breeding techniques. Therefore, transgenics provide an additional tool for breeding and crop improvment, which can accelerate advances in crop production. Transgenic plants, grown in the field, contain genes for increased resistance to virus diseases, specific insects and herbicides, as well as fungal resistance and modified oil and protein content. In addition to use of transgenics for commercial applications, plant gene transfer and biotechnology is revolutionizing all aspects of basic plant sciences. The availability of plant genome sequences combined with the tools for modulating gene expression using transgenic approaches has led to tremendous advances in our basic understanding of gene expression and gene function. By overexpressing and/or silencing native genes thought gene transfer technology, any recoverable phenotype will provide information on the role of genes in combating biotic and abiotic stresses as well as increase out understanding of the contribution of a gene to plant growth and development. Although transgenic sunflower has not yet been commercialized, it was one of the first plants to be placed in tissue culture and also one of the first shown to be responsive to transformation. These "firsts" were unusually short-lived as this crop continues to confound breeding and biotechnology laboratories with low rates of transformation and transgenic plant recovery. Transgenic sunflowers have been generated and are available but procedures are not very robust or user-friendly. The main challenge in developing a transformation system for sunflower is to coordinate the transformation and regeneration processes; ie targeting DNA introduction into cells/tissues which are also capable of regenerating into whole plants. We have previously identified a novel target genotype for sunflower regeneration that gives high efficiency of shoot regeneration from cotyledons. In addition, we have very recently discovered procedures that appear to give rise to high numbers of transgenic shoots for the first time. We are hopeful that some additional work in this area will be highly productive and lead to the consistent recovery of sunflower transgenics.

Approach:
This research will continue to be primarily focused on development of tissue culture regeneration and transformation technology for sunflower, which will be used to study the effects of introduced transgenes on modulation of Sclerotinia resistance. Funding has been previously awarded to the Finer Laboratory and exciting progress has recently been made on this project. The original funded research was based on early results obtained in the Finer Laboratory on tissue culture responsiveness of sunflower germplasm that had not previously been evaluated. In that work, a confection sunflower line (RHA280) was identified, which appeared to be remarkably suitable to tissue culture regeneration. RHA280 is also a commonly-used parent of sunflower breeding populations. Efforts have continued with this exceptional line. Since the project was initiated, we have demonstrated good improvements in shoot regeneration efficiency, using standard tools and some new approaches, which we have combined in unique ways. This project is moving to completion and we believe that we have finally discovered the approach needed to generate more consistent transformation results. Our approach of using a high shoot regeneration target tissue for Agrobacterium-mediated transformation appears to be justified. In our recent transformation experiments, transgenic shoot initials were obtained with multiple shoots buds expressing the introduced marker gene. These recent positive transformation results have utilized a combination of approaches that have not been previously used together in our sunflower work. And, in hindsight, these approaches seem to be so simple and straightforward that it may be unclear why so much time was needed to obtain these results. A large effort has been placed in this project and it was necessary to evaluate many variables before a working combination was discovered. These results will be disclosed later in this pre-plan and it is suggested that these results should be considered as PRELIMINARY and CONFIDENTIAL.

Last Modified: 4/17/2014
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