Detection of transgenes in apple rootstocks using anchored PCR

In this study, the presence of transgenes was detected in apple rootstocks A2 and M26 by anchored PCR. Two rolA transformed clones of A2 (LA1 and LA2) and two rolB transformed clones of M26 (C and F), obtained by Agrobacterium-mediated gene transfer, were used as materials. Two left border specific primers and two right border specific primers were chosen as anchor primers for amplifying T-DNA/plant DNA junctions in the rolA and rolB transgenic plants. In addition, the rolB specific anchor primer was used for amplifying the rolB internal fragment in the rolB transformed plants. The results showed that, for the rolA transgenic plants, four T-DNA inserts were obtained when using the left border anchor primers and two to four T-DNA inserts for the right border anchor primers in clone LA1. For clone LA2, one T-DNA insert was detected using the right border primer RB1-A, while no inserts were observed when using the other anchor primers. The vector backbone was found for the two rolA transformed clones at the right border region. For the rolB transformed plants, three T-DNA inserts were detected with the left border anchor primer LB1-B and the right border anchor primer RB2-B in clone F. One T-DNA insert was observed when using the left border anchor primers and the right border anchor primer RB2-B in clone C. The rolB internal fragment was detected in the rolB transformed plants when using the rolB anchor primer.