FLP-mediated conditional loss of an essential gene to facilitate complementation assays Page: 87
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mg/L glufosinate ammonium and 100 ug/L R7402 did not yield any resistant seedlings,
suggesting inefficient transformation or unsuccessful excision of the P450-ecSUC2 cassette in
between the FRT sites by the FLP recombinase. Selection of transformed seedlings on 1% MS
agar containing gentamycin showed no positive transgenic seedling. Molecular analysis of the
only "resistant" seedling obtained using PCR and primers designed specifically for the
gentamycin gene suggested a false-positive transgenic seedling.
In conclusion, although no putative transgenic seedling harboring the T-DNA of the
second vector series pCAM-Gent-TSpSUC2-EASE-FLP has been obtained, the two vectors of
the two-component system were constructed successfully (Refer to Appendix for complete
sequence analysis data). Floral-dips have been repeated on independent lines (SG 22-7, SG 22-6,
SG 32-22, SG 32-23, SG 32-11, and SG 32-13) to generate more putative transgenic seedlings.
The selection for transgenics was tried with all three plant selection markers in the
system, (i) Bar gene, conferring resistance to the herbicide glufosinate ammonium, (ii) P450
gene, conferring sensitivity to the pro-herbicide R7402, and (iii) aacC1 gene conferring
resistance to the antibiotic gentamycin. False-positive results were obtained with glufosinate
ammonium treatment at 50 mg/L probably due to leaky expression of the Bar gene, but
increasing the glufosinate ammonium concentration to 100 mg/L was reliable. The glufosinate
ammonium resistant control plants showed healthy growth and development even after treatment
with 100 mg/L herbicide, suggesting that the high concentration of herbicide did not have any
inhibitory effects.
The R7402 was used as a counter selection marker in the system, to eliminate possibility
of any sectoring. For instance, sectors of plant tissue which do not express the Bar gene may
survive glufosinate ammonium treatment due to effect of Bar gene expression in surrounding87
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Ganesan, Savita. FLP-mediated conditional loss of an essential gene to facilitate complementation assays, thesis, December 2007; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc5180/m1/97/: accessed April 17, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .