Transfer of citrus tristeza virus (CTV)-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium-mediated genetic transformation

Transgenic plants of grapefruit (Citrus paradisi Macf.) cvs. 'Duncan', 'Flame', 'Marsh', and 'Ruby Red' were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Two citrus tristeza virus (CTV)-derived candidate resistance genes: '392' (3' region of the p23 ORF plus 3' untranslated region-UTR) and p23 hairpin' (sense-p23 ORF plus UTR plus antisense-p23 ORF) were introduced into grapefruit using Agrobacterium strains EHA105 and EHA101, respectively. Epicotyl explants from 1-mo.-old in vitro etiolated seedlings were incubated in bacterial suspension. Green shoots that formed on explants after 4-5 wk after bacterial incubation were tested for the presence of the GUS gene by histochemical analysis. The percentage of GUS-positive shoots and transformation efficiency was 30.3 +/- 3.3% and 3.5% for treatment with EHA101 and 15.3 +/- 1.7% and 1.3% for treatment with EHA105. GUS-positive shoots were micrografted onto Carrizo citrange (Citrus sinensis L. Osbeck x Poincirus trifoliata L. Raf.) seedling rootstocks, and the presence of transgene sequences in these plants was confirmed by polymerase chain reaction (PCR), Southern blot, and reverse transcription PCR analyses. Resulting transgenic grapefruit plants were challenged with CTV and tobacco mosaic virus using a protoplast challenge assay as an initial screen to determine the effects of the transgenes on virus replication. Although complete RNA-mediated resistance was not achieved, preliminary results showed that 5.2% of the recovered transgenic plants containing the '392' CTV-derived sequence repeatedly exhibited reduced CTV replication in protoplasts. These plants are being further evaluated using the traditional method of virus inoculation followed by enzyme-linked immunosorbent assay.