Figure 4: (A ) Illustration of the plastome loci (i.e. downstream of psbH or psbA ) prior to transgene insertion (WT), as the intermediate recombinant loci (R1), and following loop-out of the codA-aadA marker (R2). The locations of the primers (P1 – P4) used in the four-primer PCR analysis of transformant lines are indicated together with the expected product sizes. (B ) PCR analysis of six transformant lines for each locus indicate that all are homoplasmic for the R1 plastome following three rounds of selection on Spc. The WT band is the product of P1+P2, transformants bands are the product of P3+P2. (C,D ) PCR analysis following two further rounds of restreaking on TAP or TAP+5-FC media demonstrates the stability of the R1 genotype in the absence of selection (P3+P2 product retained), and rapid loss of the codA-aadA cassette under 5-FC selection (new band corresponding to P4+P2 product). (E ) Luminescence analysis of mid-log cultures of Luc1.R1, Luc1.R2, Luc2.R1 and Luc2.R2 lines. Luminescence units were normalised to optical cell density (OD750) with values based on 3n replicates. (F ) Spot tests of mid-log cultures and 1:10 and 1:100 dilutions of WT and transformant lines C-A, Luc1.R2 and Luc1.R2 on TAP plates containing: no antibiotic, Spc or 5-FC.