Targeted deletion of the plastid <i>trnL-CAA</i> gene.

<p>(A) Physical map of the region in the tobacco plastid genome containing the gene for <i>trnL-CAA</i>. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning and RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in Δ<i>trnL-CAA</i> transplastomic plants. The <i>aadA</i> cassette replacing <i>trnL-CAA</i> is shown as grey box. (C) RFLP analysis of Δ<i>trnL-CAA</i> plastid transformants. All lines are homoplasmic and show exclusively the 3.1-kb band diagnostic of the transplastome. Wt: wild type. (D) tRNA-Leu(CAA) accumulation in the wild type and Δ<i>trnL-CAA</i> lines assessed by northern blotting. Hybridization to a plastid <i>trnL-CAA</i> probe confirms complete absence of the tRNA from homoplasmic knock-out lines. The ethidium bromide-stained agarose gel prior to blotting is also shown. (E) Confirmation of the homoplasmic state of the Δ<i>trnL-CAA</i> lines by inheritance assays. Germination of seeds from transplastomic plants on spectinomycin-containing medium results in a homogeneous population of green antibiotic-resistant seedlings. (F) Wild-type seedlings are sensitive to spectinomycin and bleach out in the presence of the antibiotic.