Figure 4.

Southern blot analysis results. The [³²P]dCTP-labeled 660-bp DNA fragment from the N-terminal end of the gene was used as the probe for Southern blot analysis. Digested or undigested genomic DNA from each sample was fractionated through an 0.8% agarose gel and blotted to Hybond-N⁺ nylon membrane (Amersham). After hybridization, the membrane was exposed to Kodak X- Omat film at −70°C. B, BamHI; C, untransformed plant; Ev, EcoRV; U, undigested. (A) Genomic DNA (6 μg per lane) from T₀ plants was digested with EcoRV (which has no restriction site in the transforming DNA). Six independent T₀ transgenic plants are shown in lanes 1–6, events 3112, 3421, 4017, 756, 1321, and 1804, respectively. (B) Undigested genomic DNA (4 μg per lane) of T₀ event 4017 (lane 1) and its five T₁ progenies (lanes 2–6). (C) Genomic DNA (6 μg per lane) of the same plants as in B digested with BamHI and with EcoRV. Lanes: 1, T₀ event 4017; 2–6, T₁ progenies, events 4017-62, 4017-14, 4017-23, 4017-51, and 4017-39, respectively. The EcoRV fragments indicate that event 4017 has two linked integration sites. The fainter high molecular weight band probably represents a single copy, whereas the 9.4-kb band represents multiple copies of the gene in a single site. (D) Genomic DNA (6 μg per lane) of T₀ event 3112 (lane 1) and four T₁ progenies (lanes 2–5, events 3112-20, 3112-27, 3112-38, and 3112-42, respectively) were digested with EcoRV or not digested. Among the transgenic lines, the presence of a single band in the EcoRV-digested lanes is evident.