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
