Fig. 4

PCR and Southern blot analysis of GT events in pigmented plants. a Maps of the WT ANT1 locus, the ANT1 locus with a precise insertion, and an ANT1 locus that has sustained a one-sided GT event. Primers used for PCR are indicated by numbered arrows. b PCR results from 26 purple plants recovered from four independently derived purple calli (events 1, 2, 10 and 11). PCR products of the expected size were obtained from all plants at the right junction. PCR products of the expected size of the left junction were obtained in all plants from events 2 and 10 and all plants from event 1 except for plant 1.10. Of the plants regenerated from event 11, only plant 11.3 proved positive for the left junction. Viral replicons were not detected in any of the mature plants. Primers used for detecting viral replicons were the same as in Fig. S4 in Additional file 1. M 2-Log DNA ladder (New England BioLabs), WT wild type plant, C positive control for virus circularization (genomic DNA from tissue 8 weeks after inoculation with the viral GT vector). Plants selected for Southern blot analysis are marked by asterisks. c Southern blot analysis of NsiI-digested genomic DNA from purple plants 1.9, 11.1 and 2.5. The 4.4-kb band in plants 1.9 and 2.5 is the size expected for precise insertion by HR. Plant 11.1 showed an approximately 6.3-kb band, indicative of a one-sided GT event. The 2.5-kb WT band was detected in all plants, demonstrating that they are heterozygous for the targeted insertion. No other bands were detected in any of the tested GT plants, suggesting that random integration of the T-DNA did not occur