The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium-mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

The purpose of this study was to characterize the T-DNAs introduced into the transgenic OsCK rice, as part of a biosafety evaluation. Choline Kinase (CK) gene is upregulated in the transgenic OsCK rice. We identified the insertion sites, flanking sequences, structures and sequences of the inserted T-DNAs. Based on the adaptor-ligation PCR, we found that the right border of the T-DNA was inserted at position no. 129971, on BAC clone OSJNBa0014J14 of chromosome 10. The flanking sequences of the left border region of the T-DNA (which was later identified as a region harboring a 1-kb long deleted sequence), could not be identified by various PCR-based trials. However, it was finally identified with whole-genome shotgun sequencing, using an Illumina sequencer. The result indicated that one of the T-DNAs was inserted into the CaMV 35S promoter region, whereas the other T-DNA was introduced at position 128947 on OSJNBa0014J14 clone, with an inverse orientation. During the insertion process, a 1024-bp-long chromosome sequences flanked by the right border of the T-DNA region was deleted. A 370-bp long left border region and 199-bp long right border region corresponding to the matrix attachment region (MAR) sequences of the T-DNA were also deleted. Collectively, these results indicate that whole-genome shotgun sequencing is a useful tool to reveal the detailed sequences and structures of the introduced T-DNAs, especially in the case of multiple T-DNA insertions. The expenses incurred on genome sequencing can be easily compensated by minimizing the time and efforts invested in conventional molecular analyses.