Figure 4. Characterization of MT1 (128/Xyl, cf. Fig. S1e) transformants.

(A) PCR analysis of 13 randomly chosen doubled-haploid T₁ seedlings of primary transformant MT1 B4. Lanes: 1 = 1 kb ladder; 2 =  plasmid DNA; 3 =  wild type wheat DNA; 4–16 = T₁ seeds. (B) Southern blot analysis of homozygous MT1 transformants from T₁ seedlings with the xylanase gene. Lanes: C1, C2, C4 respectively showing 2, 4 and 8 copies of 837 bp probe; M =  DNA ladder; lanes 1–10 = T₁ doubled-haploid seedlings of 6 different T₀ transformants (1 and 2 = MT1-1, 3 and 4 = MT1-2, 5 and 6 = MT1-3, 7 = MT1B5, 8 and 9 = MT1-6, and 10 = MT1-7); lanes 11–13 = T₁ doubled-haploid seedlings of MT1-B4; lanes 14–15 =  wild type DNA. (C) Zymogram assay for identification of transgenic wheat grains synthesizing recombinant 1,4-β-xylanase. Transgenic wheat grains (T₂ of MT1-B4) secrete the enzyme into the medium containing oat-spelt xylan that is stainable with Congo Red. De-polymerization of the xylan by the enzyme results in an unstained yellow ring around the seed. Wild type wheat grains lack the yellow ring (arrows).