ISHS Acta Horticulturae 632: XXVI International Horticultural Congress: Citrus and Other Subtropical and Tropical Fruit Crops: Issues, Advances and Opportunities
TRANSFER OF A POTENTIAL CANKER RESISTANCE GENE INTO CITRUS PROTOPLASTS USING GFP AS THE SELECTABLE MARKER |
| Authors: | W.W. Guo, J.W. Grosser |
| Keywords: | sweet orange, green fluorescent protein, Xa21 gene, co-transformation, in vitro grafting |
| Abstract:
Citrus canker disease caused by the bacterial pathogen Xanthomonas axonopodis pv. citri is endemic in several citrus growing areas, and many citrus species including sweet orange, grapefruit, and Mexican lime are highly susceptible. Introduction of resistance genes in susceptible cultivars is potentially the best procedure to control this disease. Protoplasts isolated from embryogenic callus of Newhall navel orange, one of the leading commercial cultivars in China because of its seedlessness and other good qualities, ‘Early Gold’ sweet orange and ‘Murcott’ tangor were used for transformation. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene (p524EGFP.1) and the plasmid DNA of a potential canker resistance gene (pC822) from the Xa21 gene family of rice (which provides broad spectrum Xanthomonas resistance in rice) were PEG-mediated co-transformed into protoplasts. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of GFP, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic embryoids expressing GFP were recovered. Shoots were regenerated from the three cultivars, and their growth was expedited by in vitro grafting. PCR analysis revealed that the Xa21 gene was present in all of the six analyzed shoots from ‘Early Gold’ sweet orange, and in none of the nineteen analyzed samples of Newhall navel orange. Southern blot analysis is underway, and any plants containing the Xa21 gene will be evaluated for canker resistance. | |
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