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1.  A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update 
In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system.
PMCID: PMC3698417  PMID: 23847598
CTV; infectious cDNA clones; agroinoculation; Agrobacterium tumefaciens; oncogenic strains; graft transmission; N. benthamiana protoplasts; RNA silencing suppressor
2.  Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics 
BMC Biotechnology  2012;12:41.
The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees.
The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability.
Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.
PMCID: PMC3462728  PMID: 22794278
Citrus; Transgenic trees; Selectable marker genes; uidA; nptII; Substantial equivalence; Pleiotropic effects; Long-term transgene stability; Phenotypic assessment; Field performance.
3.  The monoterpene limonene in orange peels attracts pests and microorganisms 
Plant Signaling & Behavior  2011;6(11):1820-1823.
Plant volatiles include terpenoids, which are generally involved in plant defense, repelling pests and pathogens and attracting insects for herbivore control, pollination and seed dispersal. Orange fruits accumulate the monoterpene limonene at high levels in the oil glands of their fruit peels. When limonene production was downregulated in orange fruits by the transgenic expression of a limonene synthase (CitMTSE1) in the antisense configuration, these fruits were resistant to the fungus Penicillium digitatum (Pers.) Sacc. and the bacterium Xanthomonas citri subsp. citri and were less attractive to the medfly pest Ceratitis capitata. These responses were reversed when the antisense transgenic orange fruits were treated with limonene. To gain more insight into the role of the limonene concentration in fruit responses to pests and pathogens, we attempted to overexpress CitMTSE1 in the sense configuration in transgenic orange fruits. Only slight increases in the amount of limonene were found in sense transgenic fruits, maybe due to the detrimental effect that excessive limonene accumulation would have on plant development. Collectively, these results suggest that when limonene reaches peak levels as the fruit develops, it becomes a signal for pest and pathogen attraction, which facilitate access to the fruit for pulp consumers and seed dispersers.
PMCID: PMC3329358  PMID: 22212123
co-evolution; defense; repellency; secondary metabolism; seed dispersers; trophic interaction; volatiles
4.  Pollen Competition as a Reproductive Isolation Barrier Represses Transgene Flow between Compatible and Co-Flowering Citrus Genotypes 
PLoS ONE  2011;6(10):e25810.
Despite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved.
Methodology/Principal Findings
Three different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17–2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations.
Pollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species.
PMCID: PMC3185051  PMID: 21991359
5.  Early Events in Agrobacterium‐mediated Genetic Transformation of Citrus Explants 
Annals of Botany  2004;94(1):67-74.
• Background and Aims Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so‐called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co‐cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co‐cultivation medium as possible enhancers of gene transfer was also studied.
• Methods A highly responsive citrus genotype and well‐established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co‐cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry.
• Key Results It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium‐inoculated and non‐inoculated explants. Furthermore, co‐ cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S‐phase, the stage in which cells are more prone to integrate foreign DNA.
• Conclusions Use of proper co‐cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.
PMCID: PMC4242373  PMID: 15145796
Agrobacterium tumefaciens; callus; cambium; cell cycle; citrange (Citrus sinensis × Poncirus trifoliata); citrus; competence; genetic transformation; sweet orange (C. sinensis); regeneration; S‐phase; transgenic

Results 1-5 (5)