Isolation of precise plastid deletion mutants by homology‐based excision: a resource for site‐directed mutagenesis, multi‐gene changes and high‐throughput plastid transformation

Summary We describe a simple and efficient homology‐based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aad A‐based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atp B promoter region. Efficient recombination between atp B repeats deletes the intervening foreign genes and 1984 bp of plastid DNA (co‐ordinates 57 424–59 317) containing the rbc L gene. Only five foreign bases are present in Δ rbc L plants illustrating the precision of homology‐based excision. Sequence analysis of non‐functional rbc L‐related sequences in Δ rbc L plants indicated an extra‐plastidic origin. Mutant Δ rbc L plants were heterotrophic, pale‐green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aad A‐based transformation with the wild‐type rbc L gene ruled out mutations in other genes. Excision and re‐use of aadA shows that, despite the multiplicity of plastid genomes, homology‐based excision ensures complete removal of functional aad A genes. Rescue of the Δ rbc L mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbc L deletion mutant isolated here is readily transformed with the efficient aad A marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site‐directed mutant alleles and is not easily achieved using other methods.