A guide to choosing vectors for transformation of the plastid genome of higher plants.

Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3'rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3''-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3''-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.