Figure 3.

RFLP analyses as test for homoplasmy of ycf9 knockout plants. A, Restriction analysis of total cellular DNA with BamHI. Three independently generated transplastomic lines (designated 9-1, 9-2, and 9-3; capital letters mark individually regenerated plants from one and the same line) and two wild-type controls (WT) are shown. Digestion with BamHI and hybridization to a radio-labeled ycf9-specific probe yields a 4.5-kb fragment for the wild-type genome and a 5.6-kb fragment for the transplastome (see Fig. 1). Note presence of a weak band of wild-type size in all transplastomic lines in addition to the 5.6-kb band characteristic of transformed plastid genomes. B, Restriction analysis of purified chloroplast DNA. Digestion with BamHI and hybridization to the same probe as in A detects only the 5.6-kb fragment diagnostic of the transplastome. Absence of the weak band of wild-type size detected in the total DNA samples from all transplastomic lines indicates homoplasmy and suggests the presence of promiscuous ycf9 copies DNA in an extraplastidic cellular compartment.