Table II. Analysis of the intactness of the GUS expression cassette in plants regenerated from triticale microspores.
The presence and intactness of the integrated DNA carrying the GUS expression cassette were analyzed by PCR on genomic DNA isolated from triticale plants regenerated from microspores treated with DNA or DNA/protein complexes in the presence or absence of the Tat2 CPP or from untreated (control) microspores. Primers and segments of the GUS expression cassette, which contain the primer annealing sites, are indicated (Fig. 1; Supplemental Fig. S2).
| Plants Containing the Amplified Regions of the Transgene Source DNA |
||||||||||
| Treatment | P1-RS p7-p8 | P1-Act p9-p10 | Act-GUS p11-p12 | Act-GUS p13-p3 | Act-GUS p5-p3 | GUS-nos p4-p6 | GUS-nos p4-p14 | GUS-nos p4-p15 | GUS-P2 p4-p16 | GUS-P2 p4-p17 |
| No treatment | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| (21 plants) | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% |
| No DNA + Tat2 | 0 | 0 | 0 | 0 | 1 | 2 | 2 | 2 | 0 | 0 |
| (26 plants) | 0% | 0% | 0% | 0% | 3.9% | 7.7% | 7.7% | 7.7% | 0% | 0% |
| pACT-1D/PstI + Tat2 | 0 | 0 | 12 | 14 | 14 | 14 | 14 | 14 | 14 | 14 |
| (63 plants) | 0% | 0% | 19.1% | 22.2% | 22.2% | 22.2% | 22.2% | 22.2% | 22.2% | 22.2% |
| dsT-DNA + Tat2 | 5 | 7 | 9 | 10 | 13 | 13 | 13 | 11 | 6 | 4 |
| (44 plants) | 11.4% | 15.9% | 20.5% | 22.7% | 29.6% | 29.6% | 29.6% | 25.0% | 13.6% | 9.1% |
| ssT-DNA + Tat2 | 1 | 1 | 1 | 1 | 2 | 2 | 2 | 2 | 1 | 1 |
| (27 plants) | 3.7% | 3.7% | 3.7% | 3.7% | 7.4% | 7.4% | 7.4% | 7.4% | 3.7% | 3.7% |
| ssT-DNA-RecA + Tat2 | 2 | 2 | 3 | 3 | 4 | 4 | 4 | 4 | 2 | 2 |
| (23 plants) | 8.7% | 8.7% | 13.0% | 13.0% | 17.4% | 17.4% | 17.4% | 17.4% | 8.7% | 8.7% |
| VirD2-ssT-DNA + Tat2 | 3 | 3 | 4 | 5 | 6 | 6 | 5 | 4 | 3 | 3 |
| (23 plants) | 13.0% | 13.0% | 17.4% | 21.7% | 26.1% | 26.1% | 21.7% | 17.4% | 13.0% | 13.0% |
| VirD2-ssT-DNA-RecA + Tat2 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 4 |
| (20 plants) | 25.0% | 25.0% | 25.0% | 25.0% | 25.0% | 25.0% | 25.0% | 25.0% | 25.0% | 20% |
| No DNA | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| (14 plants) | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% |
| pACT-1D/PstI | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 0 |
| (Nine plants) | 0% | 0% | 0% | 0% | 11.1% | 11.1% | 11.1% | 11.1% | 0% | 0% |
| dsT-DNA | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| (Three plants) | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% |
| VirD2-ssT-DNA-RecA | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| (10 plants) | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% | 0% |