Plastid Marker Gene Excision in the Tobacco Shoot Apex by Agrobacterium-Delivered Cre Recombinase.

Abstract

Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. The example of the marker gene is the barau gene flanked by loxP sites in the plastid genome. For marker excision Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the barau gene, confers a visual aurea leaf phenotype, thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The success of in planta plastid marker excision proves that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA and the availability of visual marker genes.