3.5 Serial transformation is achievable using CpPosNeg
To demonstrate that the CpPosNeg method could be repeated to create marker-free strains carrying multiple transgenes, we tested whether a second reporter, mVenCP could be introduced into the Luc2.R2 cell line that has lucCP at the psbH downstream locus. As shown in Supplementary Figure 1 , plasmid pVen1 was designed to targetmVenCP to the psbA downstream locus, again using thecodA-aadA marker. However, to avoid the possibility of recombinational interchange between the lucCP and mVenCPcassettes due to both having the same 5’ and 3’ elements, the 3’UTR used to create the direct repeat was changed from the 258 bp rbcL UTR to an identical sized UTR from atpB . As before, transformants were initially selected on Spc to achieve homoplasmy at the R1 stage, and subsequently re-streaked on 5-FC to select for loss of the marker. All transformant lines (termed Luc2:Ven1) were confirmed by PCR to have reached homoplasmy at the R2 stage (Figure 5a,b ). Phenotypic tests carried out on Luc2:Ven1 confirmed sensitivity to Spc and resistance to 5-FC following the loss of the marker (Figure 5c ). A representative Luc2:Ven1 line was maintained on medium without selection thereafter and displayed stable expression of themVenCP (Figure 5d ) and lucCP (Figure 5e ) reporters. Relative to the Luc2 parental line, there appeared to be a small reduction in the luciferase activity of Luc2:Ven1, although this was not statistically significant (p = 0.12; Student’st -test). These results show that the CpPosNeg strategy can be used to serially introduced multiple transgenes into the plastome with the codA-aadA dual marker efficiently removed at the end of each transformation cycle.