Validation of the Site Directed Recombination System “Flp-Frt” in Transgenic Sugarcane.

Sugarcane is a highly productive C4 crop, used for production of sugar and biofuel. Genetic transformation has tremendous potential for sugarcane improvement. Recently genetic transformation strategies often involve gene stacking. Site directed recombination technology like Flp-FRT or others can support gene stacking by site directed integration into pre-characterized sites or by excision of selectable marker genes. The latter is of importance since a very limited number of effective selectable markers are available if several transformation cycles are required for gene stacking. Sugarcane is vegetatively propagated which eliminates the possibility of marker removal by Mendelian segregation. Flp-FRT recombination is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase (Flp) derived from the plasmid of baker's yeast.

In this study, we are testing the efficacy of Flp-FRT system in sugarcane. So far we have stably integrated and expressed in sugarcane callus a constitutive expression cassette in which the selectable nptII gene is placed 5’ of the uidA ORF and flanked by FRT sites. We also generated a constitutive expression cassettes of Flp and FLPe, codon optimized for sugarcane (Flp-eco) to compare their effect on activation of the uidA reporter gene by FLP activated excision of the 5’ located nptII. Data describing Gus activity following Flp or Flp-eco bombardment will be presented.