Towards the Development of Broad-Spectrum Disease Resistance in Citrus

Transgenic plants expressing Cyclic nucleotide-gated channel (CNGC) and Bcell lymphoma 2 (bcl-2) transgenes have been shown to be resistant to fungal and viral pathogens. A PCR amplification product comprising CNGCcit open reading frame (ORF) with Xba I and Eco RI ends was generated, inserted into pRTL22 plasmid, transformed into E. coli, and sequenced. The 3.2 kb Hind III fragment of pRTL22/CNGCcit containing the CaMV 35S promoter with dual enhancer, CNGCcit, and CaMV 35S terminator was inserted into the Hind III site of pBin 34SGUS to generate pBin35SCNGCcit construct, and transformed into E. coli. The cDNA clone of bcl-2 in the pPTN binary vector was digested with Hind III to release a fragment consisting of CaMV 35S promoter, bcl-2, and CaMV 35S terminator and inserted into the Hind III site of pBin 34SGUS to generate pBin35SBcl-2 construct. These constructs along with pPTN334Bcl-2 were used in Agrobacterium tumefaciens-mediated transformation of sour orange rootstock, ?Rio Red?, ?Ruby Red? and ?Duncan? grapefruit cultivars. The presence and expression of CNGCcit and bcl-2 in the transgenic plants was verified by b-glucuronidase histochemical assay, nptII enzyme-linked immunosorbent assay, polymerase chain reaction, Southern blot, northern blot, relative quantitative reverse transcription PCR , and quantitative real-time PCR. In this study, two transgenic ?Ruby Red? plants were successfully produced with an incorporated CNGCcit gene that gave a positive result with all the analyses. None of the putative transgenic plants was transgenic for bcl-2 gene based on Southern and northern analyses. Detached leaf assays of the transgenic ?Ruby Red? plants showed an enhanced resistance to Xanthomonas axonopodis pv. citri, Alternaria alternata, and Phytophthora nicotianae. Also, the leaves did not show any sensitive response to tentoxin. The citrus cytosolic ascorbate peroxidase (cAPXcit) genomic clone was isolated and characterized. Expression of cAPXcit in ?Duncan?, non-transgenic control ?Ruby Red?, and ?Rio Red? was suppressed due to A. alternata and P. nicotianae inoculations. However, an increased expression of cAPXcit was observed in the inoculated transgenic ?Ruby Red? leaves