1. We will use high-efficiency Arabidopsis whole-plant DNA transformation as a tool for gene rescue. By a """"""""successive approximation"""""""" method, we will conduct large-scale, whole-plant transformation with a matrix- organized DNA library so as to be able to infer individual clones (library members) giving rise to transformants. 2. We will use the transformation-competent Arabidopsis genomic library in Agrobacterium as donor DNA to rescue: (a) the ahs1-1 gene conferring resistance to the sulfonylurea herbicide chlorsulfuron, (b) the DET3 gene which controls light regulated growth and development, and will collaborate on the rescue of TTG, er, bp and yi genes, all from the same, large-scale transformation experiment. 3. We will define a simple procedure for gene-targeting in Arabidopsis plants. To do so, we will screen phenotypically rescued mutants for perfect DNA replacements resulting from homologous, double-recombination events. 4. We will YAC-sort the transformation competent Arabidopsis genomic library so as to facilitate map-based gene cloning. This will cross- reference our Arabidopsis cosmid library with the Arabidopsis YAC libraries, whose clones are anchored to the genomic physical map. Gene-rescue will allow the expeditious isolation of Arabidopsis genes central to the control of plant growth and development. It will also allow efficient integration of Arabidopsis physical-genome and genetic maps.
