GFP transient expression and silencing in fragaria x ananassa

Abstract

Background: Stable transformation, transient expression, and post-transcriptional gene silencing (PTGS) are powerful methodologies that allow exploration of gene function. Objective: We aimed to apply these methodologies to strawberry leaves. Methods: the binary vectors pBIN19-sgfp, pBICds- GFP and pBIN61-P19 were transferred into A. tumefaciens EHA105 supervirulent strain by electroporation. The sgfp gene silencing was carried out in stably transformed GFP (green fluorescent protein) F. x ananassa Duch. cultivar ‘P´ajaro’ strawberry plants by agroinfiltration. GFP-fluorescence was observed using a stereomicroscope (507 nm). Results: We attained a GFP transgenic F. x ananassa plant that expresses the functional protein in all the tissues during a complete and normal life cycle. In planta sgfp transient expression and silencing have also been achieved in F. x ananassa cv. ‘P´ajaro’ leaves of wild type and GFP transgenic plants, respectively. Agrobacterium-mediated transient expression was visualized as high intensity green fluorescent spots as early as 7 days post-agroinfiltration (dpa), peaking between 10 and 14 dpa and persisting as long as 24 dpa. A knockdown GFP phenotype was achieved by silencing using a dsGFP hairpin. Conclusion: This work contributes significantly to the reverse genetics field in strawberry, might help to gain knowledge in the analysis of functional promoters and thereby allow protein expression and silencing of genes. This will help to develop resistant plants expressing plant defense elicitors or silencing pathogen receptors and/or negative regulators of plant defense.