The phiC31 Phage Integrase for Plastid Engineering in Higher Plants

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Plastid transformation is inefficient in the model plant species Arabidopsis thaliana. Proposed research will test the hypothesis that inefficient plastid transformation in Arabidopsis is due to the inefficiency of the plastid's homologous recombination machinery to incorporate foreign DNA. Increased transformation efficiency by a system independent of homologous recombination, utilizing the phiC31 phage integrase (INT), will test this hypothesis. Plastid transformation by INT will depend on the availability of a recipient line in which an attB site has been incorporated in the plastid genome. Plastid transformation will involve insertion of an attP vector into the attB site by a nuclear-encoded, plastid-targeted INT. Cells with transformed plastid genomes will be selected by kanamycin or spectinomycin resistance carried in the attP plastid vector. Transformation of the Arabidopsis plastid genome on a routine basis will allow plastid transgene-based genetic screens for factors mediating communication and molecular interactions between the nuclear and organellar compartments. Methods developed for Arabidopsis are likely to be applicable to oilseed rape (Brassica napus) and vegetable brassicas (broccoli, cauliflower and cabbage; Brassica oleracea), species related to Arabidopsis with significant gene-flow problems in North America. Since plastids are not transmitted by pollen in Brassicaceae, the methods developed in Arabidopsis may also find agricultural applications to control transgene flow in the field.