Rapid and accurate early-stage detection of T-DNA/plant flanking sequences of resistant kumquats

Abstract

Agrobacterium-mediated genetic transformation is a widely applied tool in plant biotechnology. In this process, genes of interest are integrated into plant genomes via T-DNAs present on plasmids in Agrobacteria. Classical and standard methods for screening transformants, such as Southern blot, are inconvenient for most woodland plants because of extremely low transformation efficiency. For the purpose of identifying transgenic woody lines at early selection stages, a right-border T-DNA/plant conjunction sequence analysis was carried out. By analyzing these sequences, 15 out of 17 kanamycin-resistant kumquats were found to be integrated with foreign genes, and two or more copies were present in 33.3% of the transgenic lines, which is completely concordant with Southern blots. Moreover, T-DNA integration into plant nuclear DNA was random without any sequence hotspots, and cleavage sites are any base of the sequence ‘TGAC’. These results showed that this screening method could not only detect resistant woodland plants rapidly at the early selection stage, but unequivocally detect copy numbers. Compared with other screening technique, this method could save time and effort for conducting genetic transformation in woody plants, and also provides accurate integration information for transgenic plants.