Engineering the chloroplast genome is key to improving the efficiency of photosynthesis, the process that converts sunlight energy into biomass in crops. In most plants, chloroplast engineering is very inefficient. However, a recent breakthrough has revealed the genetic bottleneck for this process, and this project will leverage the new information to develop methods for efficient engineering of chloroplasts in the model plant, Arabidopsis thaliana. The experimental design will be directly applicable to crops in the evolutionary related Brassicaceae family, broccoli, an important vegetable, and oilseed rape, an important source of edible oil. The ability to engineer chloroplasts in these crops could lead to significant production improvements. The project will also have educational impact by providing research training for undergraduates, including women and underrepresented minorities. Undergraduates will be recruited from Rutgers University through The Douglass Project for Rutgers Women in Math, Science and Engineering, and The Rutgers Aresty Research Center for Undergraduates. Under-represented minorities, low-income, and first-generation college students will also be recruited from the Collegiate Science and Technology Entry Program or the First in the World Grant programs at Farmingdale State College, Farmingdale, NY. A summer research experience in chloroplast biotechnology will be offered to an undergraduate student from the University of Sao Paulo (USP), Brazil as part of a plan to recruit students for the joint Rutgers/USP PhD program. Transformation of the plastid genome is routine in tobacco, but 100-fold less frequent in the model plant, Arabidopsis thaliana, preventing its widespread use for studying questions in plastid biology. Recent data have revealed that plastid transformation efficiency is 100-fold enhanced in Arabidopsis plants with a defective ACC2 gene. This mutant line is hypersensitive to spectinomycin, the selective agent used for plastid transformation. Proof of principle for increased plastid transformation frequency was obtained in the Columbia background, because ACC2 T-DNA knockout lines are available in this accession. However, the Columbia accession is recalcitrant to plant regeneration from cultured cells where plastid transformation is carried out. The goal of this research is to provide Arabidopsis plastid transformation research tools for the plant community. This will be achieved through (1) obtaining ACC2 knockout lines in the regenerable RLD and Ws accessions; (2) developing plant regeneration protocols from transplastomic cells to obtain fertile transplastomic plants; (3) obtaining regenerable forms of the Columbia ecotype; and (4) constructing plastid transformation vectors that are suitable to achieve these goals. The results will accelerate studies of plastid function, thereby allowing better understanding of the 'rules' that govern interactions between this important organelle and the nucleus and contribute to essential plastid functions.This award is co-funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences and by the Plant Genome Research Program in the Division of Integrative Organismal Systems; both programs are in the Directorate for Biological Sciences.
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