ISHS Acta Horticulturae 738: International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species
A GFP-CONTAINING BINARY VECTOR FOR AGROBACTERIUM TUMEFACIENS-MEDIATED PLANT TRANSFORMATION |
| Authors: | V. Orbović, G. Pasquali, J.W. Grosser |
| Keywords: | citrus, Valencia, Marsh, Carizzo, genetic transformation |
| Abstract:
A new binary vector (pT-LAB21) that contains a modification of the Aequorea victoria green fluorescent protein-GFP (erGFP7INT) as a reporter gene was constructed for the use in citrus transformation experiments. The gfp gene was excised from pLMNC95 plasmid and cloned into the pPZP212 binary vector containing an nptII gene for selection of putatively transformed shoots and the aadA gene for bacterial selection. pT-LAB21 was mobilized into EHA101, a hypervirulent disarmed strain of A. tumefaciens, by the freeze-thaw method. In order to test the plasmid, we performed transformation experiments on Carrizo citrange, ‘Marsh’ grapefruit, and ‘Valencia’ sweet orange by stem segment infection. Transformed plants of all three citrus cultivars were easily obtained, despite varying transformation efficiencies that were observed in these experiments. Highest efficiency of transformation was recorded for Carrizo citrange and the lowest for ‘Valencia’ orange. The possibility of detecting GFP fluorescence in seedlings by observing them under the microscope at an early stage and without doing any destructive assay has facilitated early screening of transgenic plants while maintaining their maximum vigor. To confirm the presence of foreign DNA in genomes of transformed citrus seedlings, we performed PCR and Southern analysis. This new binary vector is available for the transformation of citrus and other plants. | |
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