Analyses of T
plants with transgene autoexcision in pollen. (a) Determination of the copy number of the pMBXS824 T-DNA (Figure 3a) by Southern blot hybridization of genomic DNA isolated from leaves of transgenic and control plants. Lanes: WT, a non-transgenic plant; MB, individual transgenic plants from lines MB1, 2, 5, 6, 17a, 17b, 18, and 24. (b) PCR analysis for detection of Bxb1-mediated autoexcision in pollen from select transgenic lines. Genomic DNA was isolated from mature pollen grains collected from perfect florets at anthesis. PCR reactions: designated lane e, detection of autoexcision (primers e3&e4, PCR product size 0.51 kb); b, detection of the marker gene bar (primers b1&b2, 0.41 kb); r, detection of the recombinase gene bxbNom (primers r3&r4, 0.68 kb); c, detection of the excised DNA fragment (primers c1&c2, 0.57 kb). (c) Detection of GUS activity in mature pollen (magnification 63×). (d) RT-PCR for detection of the bxbNom and gusA transcripts in pollen. The following amounts of total RNA per reaction were used: 10 ng for the bxbNom gene, 25 ng for the gusA gene, and 2 ng for actin as a loading control. The plant samples are in the same order as in (b).