FLP-mediated conditional loss of an essential gene to facilitate complementation assays Page: 78

                                
secondary transformant lines were sensitive to treatment with glufosinate ammonium (100 mg/L)
as well as R7402 (100 [g/L). This could be because they either did not carry the T-DNA of the
second vector pCAM-Gent-TSpSUC2-EASE-FLP as would be the case if transformation
efficiency was very low or the DNA cassette between the FRT sites of the first vector in these
seedlings did not get excised, thus making them sensitive to both glufosinate ammonium and
R7402. Thus far, no putative transgenics have been obtained with glufosinate ammonium and
R7402 treatments for any of the secondary transformant lines.
A single, putatively resistant seedling was obtained from the seeds put out on 1% MS
media with gentamycin selection. The putative transgenic seedling was transferred to soil and
analyzed for excision using PCR. The primers 35SpFwd and Bar3-FRTRev were designed to
give a band of 600 bp indicative of excision of the DNA cassette between the FRT sites of the
first vector, whereas the primers SUC2FRTFwd and Bar3-FRTRev were designed to give a band
of size 1089 bp indicative of no excision. The PCR analysis with the designed primers gave a
1089 bp product, which indicated that no excision had taken place (Fig. 34a). The seedling was
also analyzed for presence of the gentamycin gene with PCR using designed primers Gent35Slox
and GentLBNar (Fig. 34b). The putative transgenic seedling did not give the expected 1.4 kb size
band. The PCR analysis with the gentamycin primers confirmed that the seedling did not harbor
the T-DNA of the second vector and hence there was no excision of the DNA cassette between
the FRT sites of the first vector. The above putative seedling was also treated with glufosinate
ammonium (100 mg/L) for 5 consecutive days and it was observed that the seedling was
sensitive to the treatment. This suggests non-expression of the Bar gene from the 35S promoter
due to absence of the T-DNA of the second vector and hence non-excision of the DNA cassette

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