Delivery of an antisense ACC oxidase gene into Citrus reticulata Blanco. mediated by Agrobacterium tumefaciens

This study was carried out to optimize the conditions for plantlet regeneration and genetic transformation of Citrus reticulata Blanco. with an antisense ACC oxidase gene mediated by Agrobacterium tumefaciens. The highest percentage of plantlet regeneration of 100 was successfully achieved on the MS medium supplemented with 3 μM BA, while the half-strength MS medium amended with 8 μM NAA was suitable for root induction. Explants that had high potency of regeneration were shoots, nodes and internodes, respectively. The delivery of an antisense ACC oxidase gene into the species C. reticulata mediated by Agrobacterium tumefaciens strain EHA 105 was successful by cocultivating explants with the strain EHA105 for 15 min, following that by eliminating the bacterium with 200 mg/l cefotaxime, and subsequently selecting transformed embryoid with 20 mg/l hygromycin. Verified histochemically by GUS assay, putative transformants showed the percentage of gus gene expression of 100. Molecular analysis using PCR confirmed the integration of the antisense ACC oxidase gene into plant genome.