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Despite potential benefits granted by genetically modified (GM) citrus trees, their release and commercialization raises concerns about their potential environmental impact. The transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. In this work, 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 much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations, indicating that pollen competition strongly contributed to transgene confinement. This is the first study on transgene flow in citrus. It provides crucial information on the safety and field performance of GM citrus that can serve as a basis for further field trials and as a guide for (case-by-case) regulatory policies.

Citrus tristeza virus (CTV) causes one of the most devastating diseases of citrus worldwide. It induces the death of sweet orange, mandarin, lime and grapefruit trees budded on sour orange which has caused its replacement by other CTV-tolerant rootstocks. The availability of a CTV-resistant rootstock with the sour orange attributes of productivity, fruit quality and tolerance to abiotic stresses would be a major benefit to the citrus industry worldwide. The objective of this work was to evaluate the response to CTV of 10 sour orange (Citrus aurantium) transgenic lines carrying CTV-derived sequences. They were obtained in the laboratories of IVIA, Spain and planted at INTA Experiment Station in Concordia, Argentina, where CTV is endemic and efficiently transmitted by the brown citrus aphid (Toxoptera citricida). Trees were obtained by budding rooted cuttings of transgenic sour orange lines with non-transgenic and virus-free Valencia late sweet orange (C. sinensis). Valencia trees budded on tolerant rootstocks as well as non-transgenic sour orange were planted as controls. Trees were planted in a complete randomized design with two trees per plot and 5 replications. Every six months imprints were taken to determine the progress of CTV infection in each tree. Four years after planting, almost 100% of the trees are CTV infected, showing stunting growth and yellowing of foliage.

Volatile organic compounds, most of them being terpenes, are signals emitted by plants for communication with the environment. Orange fruit accumulates mainly terpenes in mature peel oil glands with D-limonene accounting for approximately 97% of terpene content. We have generated transgenic orange plants carrying a D-limonene synthase gene in antisense (AS) configuration. Transgenic expression caused a dramatic decrease in the accumulation of D-limonene in fruit peels, being about 80-100 times lower in AS samples than in empty vector (EV) transgenic ones. This affected the interaction of fruits with their biotic environment because they resulted in resistance to different specialized pathogens. When antisense fruits were challenged with the fungus Penicillium digitatum, they showed marked resistance against this pathogen. A gene expression analysis of these fruits linked the decrease of D-limonene to upregulation of genes involved in the innate immunity response. High D-limonene content in mature orange peels may be a signal for attractiveness of microorganisms which might be likely involved in facilitating the access to the pulp of seed dispersal frugivores.

[EN] Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded., We thank J.E. Peris for his excellent technical assistance and Dr. W.O. Dawson (University of Florida, C.R.E.C., Lake Alfred, FL, USA) for providing the GFP-tagged CTV strain CTV947R-GFP. N.S. was supported by a PhD fellowship from the IVIA. C.F. is recipient of a postdoctoral Ramon y Cajal contract from the Ministerio de Ciencia e Innovacion (MICINN). This research was supported by grants AGL2009-08052, co-financed by Fondo Europeo de Desarrollo Regional-MICINN, and Prometeo/2008/121 from the Generalitat Valenciana.

[EN] Citrus tristeza virus (CTV), a phloem-restricted closterovirus infecting citrus, encodes three different silencing suppressors (p25, p20 and p23), one of which (p23) is a pathogenicity determinant that induces aberrations resembling CTV symptoms when expressed ectopically in transgenic citrus hosts. In this article, the effect of p23 ectopic expression on virus infection was examined in sweet orange (SwO), a highly susceptible host, and sour orange (SO), which severely restricts CTV cell-to-cell movement. Transgenic plants of both species ectopically expressing p23, or transformed with an empty vector, were graft inoculated with the mild CTV isolate T385 or with CTV-BC1/GFP, a clonal strain derived from the severe isolate T36 carrying the gene for the green fluorescent protein (GFP). CTV distribution in infected tissues was assessed by direct tissue blot immunoassay and fluorescence emission, and virus accumulation was estimated by quantitative real-time reverse transcriptase-polymerase chain reaction. CTV accumulation in p23-expressing and control SwO plants was similar, whereas the viral load in transgenic SO expressing p23 was 10-10(5) times higher than in the cognate control plants. Although few infection foci composed of a single cell were observed in the phloem of CTV-infected control SO, the number of foci in p23-expressing plants was higher and usually comprised two to six cells, indicating viral cell-to-cell movement. CTV was detected in mesophyll protoplasts and cells from infected SO and SwO expressing p23, but not in similar protoplasts and cells from infected control plants. Our results show that the ectopic expression of p23 enables CTV to escape from the phloem and, in addition, facilitates systemic infection of the resistant SO host. This is the first report of a viral-encoded protein that enhances virus accumulation and distribution in woody hosts., We thank J. E. Peris and J. Juarez for excellent technical assistance, Dr E. Marco-Noales for help with confocal microscopy and Dr W. O. Dawson (University of Florida) for providing the GFP-tagged T36 strain CTV-BC1/GFP. CF is a recipient of a post-doctoral Ramon y Cajal contract from the Ministerio de Ciencia e Innovacion (MICINN). This research was supported by grants AGL2009-08052, co-financed by Fondo Europeo de Desarrollo Regional (FEDER) and MICINN, and Prometeo/2008/121 from the Generalitat Valenciana.

The expression of engineered single-chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A-3 (six non-infected plants out of 10), 6K2-1 (17 out of 33) and NLS-11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non-structural NIb protein in different cell compartments during viral infection.

In any plant genetic transformation system, the use of a selectable marker gene is essential to promote the recovery of only transgenic plants. However, the presence of marker genes conferring antibiotic resistance in plants represents a serious obstacle for public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. Search of alternatives to nptII selection is not easy. An attractive possibility is offered by the MAT (Multi-Auto-Transformation) system, which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transgenic shoots for ipt gene lose apical dominance and show a characteristic phenotype. We are testing the efficiency of positive selection and excision by recombination with a MAT vector that comprises the ipt gene as selectable marker and the recombinase R gene, in citrus. Transformation with the MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. X Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly shown in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.

Plum pox virus (PPV) o virus de la sharka produce importantes daños y pérdidas económicas en frutales de hueso. En este trabajo se propone probar una nueva y prometedora estrategia de resistencia a la enfermedad de la sharka basada en el uso de anticuerpos recombinantes. La expresión constitutiva de genes o fragmentos de genes de anticuerpo (anticuerpos recombinantes o rAbs) específicos de proteínas virales se ha demostrado que puede inmunomodular o interferir con la infección viral (Tavladoraki y col, 1993). La eficacia de esta estrategia depende principalmente de la naturaleza del antígeno y/o epítopo reconocido por el fragmento de anticuerpo recombinante, de la correcta expresión y acumulación del anticuerpo recombinante y del compartimento celular al que vaya dirigido. El objetivo de este trabajo ha sido evaluar el efecto sobre la infección viral de la expresión de anticuerpos recombinantes específicos de proteínas del virus de la sharka en plantas transgénicas de una especie modelo (Nicotiana benthamiana) como paso previo a su uso en Prunus cuando estos puedan ser transformados y regenerados con éxito.

Desde hace 12 años, los laboratorios de Transformación Genética y Cultivo de Tejidos Vegetales del Departamento de Protección Vegetal y Biotecnología del IVIA, estamos trabajando en el desarrollo de la tecnología de transformación genética aplicada a cítricos y en investigar sus posibilidades de utilización para la mejora tanto de patrones como de variedades. Nuestra intención en este artículo es resumir las investigaciones llevadas a cabo y destacar los logros más importantes obtenidos hasta la fecha. En el contexto de la imperiosa necesidad de mejora de nuestros cítricos para poder seguir siendo competitivos en los mercados internacionales, es necesaria la mejora genética y, entre las tecnologías existentes, la transformación genética de plantas ofrece enormes posibilidades, ya que permite introducir caracteres únicos en genotipos de élite sin alterar su fondo genético. Por ejemplo, se podría incorporar a naranjo amargo un gen (o unos pocos genes) de resistencia a tristeza, de manera que obtuviésemos un naranjo amargo resistente a este virus. Además, los caracteres de mejora no tienen que proceder necesariamente de cítricos, sino que pueden ser aislados de otros organismos vivos. Así se podría conseguir resistencia a ciertas plagas utilizando genes procedentes de bacterias.

Citrus are subject to many biotic and abiotic stresses and markets are continuously requiring fruits of higher quality. These pose important problems in most citrus growing areas that can only be solved with the establishment of citrus improvement programs to recover new and healthy genotypes to be used as rootstocks and varieties. Success of traditional citrus breeding strategies is limited by the peculiar genetic and reproductive characteristics of citrus. Biotechnology offers different approaches that can overcome many of these limitations. In this paper the following technologies used in Spain are reviewed: a) Shoot tip grafting in vitro. It is used worldwide to recover plants free of all known citrus pathogens, and already hundreds of millions of trees originated in this technique have been planted in the field; b) Embryo rescue. It is used in large programs to recover triploid seedless hybrid varieties from aborted seeds from 2n x 4n and 2n x 2n crosses; c) Protoplast fusion. It allows the production of allotetraploid hybrids among sexually compatible and incompatible parents to be used as rootstocks or as parentals for interploid breeding. Fusions between diploid and haploid cell lines are also used to produce triploid hybrids. d) Genetic transformation. Efficient protocols for transformation of several genotypes have been developed and introduction in citrus of genes of potential agronomical interest has been accomplished.

Actualmente, todos los nuevos patrones y variedades que se están produciendo en el mundo se someten a procesos de patente y su utilización está muchas veces restringida por intereses comerciales. Además, en diversos países como EE.UU., Japón, Israel, Francia e Italia se están realizando grandes esfuerzos en la aplicación de nuevas biotecnologías para la mejora de los cítricos y las tecnologías desarrolladas también serán objeto de patente.

En los últimos años, se está planteando en los países citrícolas más avanzados la utilización de diversas biotecnologías complementarias a la mejora convencional para tratar de conseguir genotipos más tolerantes o resistentes a patógenos de cítricos. Entre ellas, la transformación genética es especialmente prometedora, ya que permite la incorporación de caracteres específicos en genotipos de élite sin alterar su fondo genético. Nuestro grupo está trabajando en la incorporación de transgenes en cítricos para tratar de conseguir mayor tolerancia al oomiceto Phytophthora sp. y resistencia al virus de la tristeza de los cítricos (CTV).

The p25 coat protein (CP) gene of Citrus tristeza virus (CTV) was incorporated to Mexican lime plants and forty-two transgenic lines were produced, 25 containing the p25 CP gene of the severe CTV strain T-305 and 17 with that of the mild strain T-317. When plants propagated from each transgenic line were graft-inoculated with CTV T-305 or aphidinoculated with T-300, two types of response to viral challenge were observed: some lines developed CTV symptoms similar to those of non-transgenic controls, whereas others exhibited protection against the virus. This protection consisted of a proportion of plants, ranging from 10 to 33%, that were resistant to CTV, and the rest of them that showed a significant delay in virus accumulation and symptom onset. Protection was efficient against non-homologous CTV strains and was generally accompanied by high accumulation of p25 CP in the protected lines, which suggest a CP-mediated protection mechanism in most cases. This is the first report demonstrating pathogen-derived resistance in transgenic plants against a Closterovirus member in its natural host.

The 23 kDa protein (p23) coded by the 3'-terminal gene of Citrus tristeza virus (CTV), a member of the genus Closterovirus with the largest genome among plant RNA viruses, is an RNA-binding protein that contains a motif rich in cysteine and histidine residues in the core of a putative zinc-finger domain. On this basis, a regulatory role for CTV replication or gene expression has been suggested for p23. To explore whether over-expression of this protein in transgenic plants could affect the normal CTV infection process, transgenic Mexican lime plants were generated carrying the p23 transgene, or a truncated version thereof, under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Constitutive expression of p23 induced phenotypic aberrations that resembled symptoms incited by CTV in non-transgenic lime plants, whereas transgenic plants expressing the p23 truncated version were normal. The onset of CTV-like symptoms in p23 transgenic plants was associated with the expression of p23, and its accumulation level paralleled the intensity of the symptoms. This demonstrates that p23 is involved in symptom development and that it most likely plays a key role in CTV pathogenesis. This is the first case in which a protein encoded by a woody plant-infecting RNA virus has been identified as being directly involved in pathogenesis in its natural host. This finding also delimits a small region of the large CTV genome for the future mapping of specific pathogenic determinants.

Very active programs to produce new mandarin varieties are being carried out in several countries using different approaches and technologies (diploid and triploid breeding, irradiation, somatic hybridization, genetic transformation, etc.). The objective of this workshop was to discuss about the most important breeding methods, the main limitations of the different approaches and methodologies, which is the challenge of new mandarin varieties in the next future and also their relationship with the legislation related to essentially derived varieties that in some cases may preclude the protection of breeder’s rights of new varieties.

Spain is the largest citrus producer within the European Union and the fifth worldwide with an average production of 5 million tons per season. Most of it is exported to third countries. Indeed, according to FAO, Spain is considered the first citrus exporter worldwide. Spanish citrus production is mainly destined to fresh market. Due to the high fruit quality standards demanded by this market, citrus pest management has to provide fruit with almost no pest presence or damage and at the same time, with minimal pesticide residues. International market demands place Spanish citrus industry in an unbeatable position to implement area-wide Integrate Pest Management (aw-IPM) programs. In the last years the citrus producing area that adopted IPM programs has dramatically increased. In 2012 more than 19% of the production already followed IPM regulations and that was a 5-fold increment relative to 2005. The current trend indicates that the number of growers adopting IPM systems will keep increasing over the coming years. The present article summarizes the current status of Spanish citrus IPM, how pests are hierarchized according to their biological control and which are the available IPM tools for growers. Large scale implementation of this management approach will permit Spanish citrus production to make a qualitative leap in a society that seeks for healthier eating habits and more environmental friendly production systems.

This research work presents the development of a protocol for the micropropagation of three allotetraploid somatic hybrid rootstocks, obtained at IVIA from two hybridizations: Citrus macrophylla + citrange Carrizo and C. macrophylla + C. taiwanica. We have determined the conditions for the in vitro introduction and the establishment of micropropagation lines in the laboratory, with a high efficient multiplications rate of 3 every two weeks. The environmental conditions for the successful acclimatization and growth in the greenhouse have also been established. Finally, we have accomplished the large-scale adaptation for efficient commercial propagation.

In the Mediterranean area, citrus fruits are primarily produced for the fresh fruit market and Spain is the principal producer of the area. Seedlessness is one of the most important characteristics for mandarin fresh fruits. Triploid hybrids allow implementing this trait in commercial cultivars. In this work we described 'Alborea' and 'Albir', two new triploid hybrids originated from the IVIA triploid breeding program, which will be soon released to growers. 'Alborea' was obtained from an open pollinated 'Fortune' mandarin. Fruits reach optimum maturity at the beginning of January, although they can be harvested from December until the end of January. Fruits are easy-to-peel, with a diameter of 50-60 mm, and rind is deep orange red in color. 'Albir' was obtained from a cross between 'Fortune' and 'Kara' mandarins. Fruits reach optimum maturity at the beginning of February, although they can be harvested from January until second half of February. Fruits are easy-to-peel with a diameter of 60-70 mm and fruit rind is orange red in color. This variety has the same origin as 'Safor', a previously released triploid hybrid. The fruits of the two varieties are somewhat similar, but 'Albir' matures one month earlier. With these cultivars, 'Albir' and 'Safor', it will be possible to supply similar fruits to the market from January to the end of March.

Citrus cultivars must meet two basic conditions for fresh consumption: i) they should cover the widest possible commercial calendar, and ii) allow continuous adaptation to consumer preferences. For citrus growers, this requires permanent adoption of new technology and changes in the cultivars grown, driven by income-risk criteria, as well as by technical and commercial experience. The duration of an investment depends on the agronomic performance of the cultivar or its commercial life, which may be shortened due to lack of consumer acceptance. The survival of a cultivar depends on many variables: technical, quality, and competitiveness of the cultivar. Spanish citrus production falls into five main groups of cultivars: lemons (10.8%), clementines (25%), mandarin hybrids (8.7%), navel oranges (35%) and common oranges (15%). The most productive cultivars are ‘Fino’ lemon, ‘Navelina’ navel, ‘Clemenules’, ‘Lane Late’ navel and ‘Valencia’ sweet orange. In relation to changes in varietal composition, it is of interest the introduction of new cultivars of the clemenules group (‘Orogrande’, ‘Nulessin’), navel (‘Powell’, ‘Rohde’, ‘Barnfield’, ‘Chislett’ and ‘Fukumoto’) and Valencia (‘Barberina’, ‘Midknight’, ‘Valencia Late’ Frost and ‘Delta Seedless’). There is constant pressure to supply new early clementines cultivars, but their survival is usually short. Currently, mandarin hybrids represent a group with a strong potential to expand.