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

Abstract

Transgenic plants of grapefruit (Citrus paradisiMacf.) 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 antisensep23 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×Poncirus 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.

Citrus Transformation: Challenges and Prospects

“…Lines with normal phenotype (no symptoms) were further propagated and tested for CTV resistance and again the results were mixed with some plants completely immune to the virus while others from the same line had delayed symptom development and virus accumulation (Fagoaga et al, 2006;López et al, 2010). The use of the 3' region of the p23 and the contiguous 3'-untranslated region (UTR), either as a hairpin or as single copy, has also been transformed into 'Duncan', 'Flame', 'Marsh', and 'Ruby Red' grapefruit and alemow plants with similar results as described above in which some plants derived from a particular line were fully resistant and others were not (Ananthakrishnan et al, 2007;Batuman et al, 2006;Febres et al, 2008). Only in one line full resistance was observed (Febres et al, 2008).…”

Citrus biotechnology: Achievements, limitations and future directions

“…Several Citrus species have been genetically engineered in this direction to enable them to counter various biotic and abiotic stresses. Presently, production of transgenic plants resistant to viruses and bacteria is underway by the expression of coat protein and pathogen-related proteins, respectively, but adequate levels of resistance is yet to be achieved (Olivares-Fuster et al, 2003;Ananthakrishnan et al, 2007). In contrast, salinity tolerance has been achieved by introduction of HAL2 gene.…”

Grapefruit

“…In C. paradisi, some of the commercially important grapefruit cultivars are seedless (zero to five seeds per fruit), including "White Marsh", "Ruby Red", "Rio Red", and "Star Ruby" as well as some other commercial sweet oranges, which make it more difficult to obtain adequate nucellar seedling explants for Agrobacterium-mediated transformation (Fleming et al, 2000). The Agrobacterium-mediated transformation system is best suited for highly nucellar seedy citrus genotypes, but also works for lowseeded nucellar cultivars if abundant fruit is available, including the mentioned low-seeded grapefruit cultivars (Bond and Roose, 1998;Gonzalez-Ramos et al, 2005;Ananthakrishnan et al, 2007). This limitation in citrus transformation can also be overcome for some cultivars by using a protoplast transformation system with a nondestructive selection system (Niedz et al, 1995;Fleming et al, 2000).…”

“…Mirkov, unpublished data). A few transgenic "Marsh" grapefruit plants containing the 392-CTV sequence (containing part of p23 and the untranslated region (UTR) of CTV genome) showed altered replication in protoplasts with CTV virions based on Northern blot analysis (Ananthakrishnan et al, 2007), but it has yet to be determined if this result has any value at the whole-plant level. Information on transgenic grapefruit plants and the CTV-derived target genes is summarized in Table 2.…”