Pyramiding the antimicrobial PR1aCB and AATCB genes in ‘Tarocco’ blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major bacterial disease responsible for substantial economic losses in citrus-producing areas. To breed transgenic citrus plants with enhanced resistance to citrus canker, two antimicrobial peptide genes, PR1aCB and AATCB, were incorporated into ‘Tarocco’ blood orange (Citrus sinensis Osbeck) plants via co-transformation and sequential re-transformation. The presence of PR1aCB and AATCB in double transgenic plants was confirmed by PCR. The expression of PR1aCB and AATCB in double transformants was demonstrated by quantitative real-time PCR. An in vivo disease resistance assay involving the injection of Xcc revealed that the double transformants were more resistant to citrus canker than the single gene transformants and wild-type plants. An analysis of the bacterial population indicated that the enhanced citrus canker resistance of the double transformants was due to inhibited Xcc growth. These results proved that the pyramiding of multiple genes is a more effective strategy for increasing the disease resistance of transgenic citrus plants than single gene transformations.