Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes

Abstract

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Citrus Transformation: Challenges and Prospects

“…genetically identical), 10 to 33% were resistant to CTV while the rest developed typical symptoms, despite a significant delay in virus accumulation (Domínguez et al, 2002). Similar results were obtained in 'Duncan' grapefruit when translatable and untranslatable versions of the major CP were transformed (Febres et al, 2003(Febres et al, , 2008. Various forms (full length, hairpins) of the p23 gene, located in the 3' end of the viral genome, have also been transformed into citrus genotypes.…”

“…This line is currently being evaluated in the field for its horticultural value and durability of the resistance under natural conditions. Other CTV genes have been used but either no transgenic plants were regenerated (p20 and minor CP/p27) or they did not show resistance (RdRp gene) (Febres et al, 2003(Febres et al, , 2008. Resistance to another important viral disease, Citrus psorosis virus (CPsV), has been reported in transgenic sweet orange plants transformed with intron-hairpin constructs (ihp) corresponding to the viral CP, the 54K or the 24K genes (Reyes et al, 2011).…”

The use of the PMI/mannose selection system to recover transgenic sweet orange plants ( Citrus sinensis L. Osbeck)

“…Most of these use epicotyl or internodal segments excised from seedlings germinated either in vitro or in the greenhouses and transformed via Agrobacterium tumefaciens with the nptII (neomycin phosphotransferase II) selection gene in combination with the antibiotic kanamycin as a selection agent. Important genes such as the coat protein (GutiØr-rez-E. et al 1997;Domínguez et al 2000Domínguez et al , 2002aYang et al 2000;Febres et al 2003) and the p23 (Ghorbel et al 2001) from the citrus tristeza virus, the HAL2 (Cervera et al 2000b), the LEAFY and APETALA1 and the PR-5 pathogenesis-related protein gene ) have been introduced into citrus genomes.…”

Detection and characterization of Citrus tristeza virus stem pitting isolates in Jamaica

“…Of note, these CTV-SP strains were MCA-13 reactive. The Jamaican isolates that induced grapefruit and sweet orange stem pitting clustered with the severe quick decline and stem pitting strains from Mexico and Florida (T36), but the Jamaican sour orange stem pitting strain was most similar to and grouped separately with the Venezuelan B249, a grapefruit and sweet orange stem pitting strain (Febres et al 2003). Other severe isolates formed two groups, 4 and 5; one with isolates from Eurasia, the US and Brazil in a related cluster, and those from Croatia in a distinct branch.…”