Optimization of recombinase-mediated marker excision in citrus protoplasts

Citrus is a perennial crop prone to several diseases and abiotic stress factors in its life cycle. A long term solution to these problems is production of disease and abiotic stress resistant transgenic plants. Transgenic plants still have limitations such as the presence of selectable molecular markers producing antibiotic or herbicide resistance which cause public concern. One method to produce marker-free transgenic plants is recombinase-mediated cassette exchange (RMCE). Our objective in this study is to determine the efficiency of the enzyme recombinase in excising molecular markers from transgenic plants using RMCE. The recombinase gene was driven by a promoter induced by chemicals dexamethasone or estradiol. Concentration of inducing chemicals and optimal temperature for recombinase activity was evaluated. We utilized six plasmids specially constructed in a way that the visual marker gene, mCherry, has a promoter split in two with one part upstream of the recombinase cassette (which include the markers) and the other downstream so that only when the recombinase cassette is excised, the mCherry gene is expressed. We developed a quick methodology for screening marker excision using electroporation of citrus protoplasts. Results after 7 days of induction demonstrated that excision efficiency of recombinase varies with type of construct and concentration of inducer applied