The development of genetic markers for plastid transformation in higher plants

The four studies that comprise my dissertation were undertaken in an effort to develop new selectable marker systems and expression elements that could make chloroplast transformation possible in monocot crop species. For my first project, I developed a new selectable marker system for nuclear transformation. I identified seven putative lincosamide-tolerance genes from medical studies and modified them for expression in plants. I then tested them as selectable markers for Agrobacterium mediated transformation of tobacco and generated transgenic plants with high efficiency from four of them: lnuAN2s, lnuBs, lnuDs and lnuFs. These four genes were then tested in Arabidopsis and potato. The lnuAN2s gene yielded transgenic events under lincosamide selection with similar transformation efficiencies as kanamycin and gentamicin. The performance of the new lnu marker genes makes this lincomycin selection scheme a valuable new tool for plant biologists. For my second project, I sequenced the plastid genomes of three maize lines important to transformation and breeding. The sequence was used to construct a maize-specific plastid-targeting vector and a powerful chimeric promoter for high expression in non-green tissue. I also sequenced the plastid genomes of three cytoplasmic male sterile lines, and used this information for an evolutionary study confirming a single origin and maternal co-transmission of the mitochondrial cms-T trait. For my third project I tested an herbicidal selection scheme for plastid transformation in tobacco. The glyphosate acetyltransferase (gat) gene, in combination with the chimeric maize expression element, provided protection to transplastomic cells under glyphosate selection. Because glyphosate targets a pathway that is universal in plants, the gat gene may work as a plastid marker gene across a wider range of species. For my fourth project I tested lnu genes as primary selectable marker genes for plastid transformation in tobacco. I constructed plastid-targeting vectors carrying the four efficacious lnu genes identified in my earlier study. This set of experiments did not yield transplastomic events, but I obtained five lincosamide-resistant mutants under selection in tissue culture. I sequenced the plastid rrn23 region of each line to identify the mutation responsible for the resistant phenotype.Ph.D.Includes bibliographical referencesby Massimo Bosacch