Diagrammatic representation of the processes for chloroplast genome transformation. a Basic design of a typical vector for transforming the plastid genome. Both the expression cassette and the selection cassette are placed between the two plastid regions. These flanking regions are taken from the wild-type plastid genome of a plant species whose plastome is to be manipulated, to allow a crossover event take place to integrate DNA sequences between them. Green arrows in the chloroplast expression vector represent promoters (P) and the direction of transcription, whereas terminators (T) are indicated by red rectangles. The untranslated regions are represented by white circles. The thin dotted lines with arrows indicate homologous recombination. b Delivery of transforming plasmids into chloroplasts in leaf cells using a particle delivery system. The plasmid DNA is coated on the surface of the microparticles of either gold or tungsten and then shot on to the abaxial surface of 4- to 6-week-old sterile leaves using a gene gun. The bombarded leaves are incubated for 48 h in the dark, cut into small discs and placed on regeneration medium supplemented with the appropriate antibiotic and hormones. Primary shoots generally arise within 2–3 months. c The process of recovering a stable homoplasmic transplastomic plant line. Initially, only a few copies of the plastome are transformed, and therefore the explant contains a mixture of both transformed as well as untransformed copies, a state known as heteroplasmy. The wild-type copies (indicated by light-coloured ovals) are sorted out gradually by repeating two or three regeneration cycles under selection to reach homoplasmy, a state where all copies of the plastome are transformed (indicated by dark grey ovals).