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Polyploidy is one of the main forces that drives the evolution of plants and provides great advantages for breeding. Somatic hybridization by protoplast fusion is used in citrus breeding programs. This method allows combining the whole... more
Polyploidy is one of the main forces that drives the evolution of plants and provides great advantages for breeding. Somatic hybridization by protoplast fusion is used in citrus breeding programs. This method allows combining the whole parental genomes in a single genotype, adding complementary dominant characters, regardless of parental heterozygosity. It also contributes to surpass limitations imposed by reproductive biology and quickly generates progenies that combine the required traits. Two allotetraploid somatic hybrids recovered from the citrus rootstocks- (CM) and Carrizo citrange (CC)-were characterized for morphology, genome composition using molecular markers (SNP, SSR, and InDel), and their tolerance to iron chlorosis, salinity, and (CTV). Both hybrids combine the whole parental genomes even though the loss of parental alleles was detected in most linkage groups. Mitochondrial genome was inherited from CM in both the hybrids, whereas recombination was observed for chloro...
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Mandarin (C. reticulata), citron (C. medica) and pummelo (C. maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of citrus: sweet orange, sour... more
Mandarin (C. reticulata), citron (C. medica) and pummelo (C. maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of citrus: sweet orange, sour orange, clementine, lemon, lime and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus. Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused b...
Research Interests: Pigmentation, Transcription Factors, Biological Sciences, Phylogeny, Plant Physiology, and 13 moreMutation, Flowers, Domestication, Citrus, Anthocyanins, Genotype, Species Specificity, Base Sequence, PLANT PROTEINS, Plant Leaves, alleles, reverse transcriptase polymerase chain reaction, and Retroelements
Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group... more
Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group within a 3.3 Mb genome region near the centromere in chromosome III. This work addresses fine-linkage mapping of this region for identifying candidate resistance genes and develops new molecular markers for ABS-resistance effective marker-assisted selection (MAS). Markers closely linked to ABSr locus were used for fine mapping using a 268-segregating diploid progeny derived from a heterozygous susceptible × resistant cross. Fine mapping limited the genomic region containing the ABSr resistance gene to 366 kb, flanked by markers at 0.4 and 0.7 cM. This region contains nine genes related to pathogen resistance. Among them, eight are resistance (R) gene homologs, with two of them harboring a serine/threonine protein kinase domain. These two genes along wi...
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Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in... more
Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exoder...
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Citrus triploid hybrids can be recovered by 2x X 2x hybridisations as a consequence of the formation of 2n gametes or by interploid hybridisation. Most of the 4x parents used in our program are doubled-diploid (DD), also known as... more
Citrus triploid hybrids can be recovered by 2x X 2x hybridisations as a consequence of the formation of 2n gametes or by interploid hybridisation. Most of the 4x parents used in our program are doubled-diploid (DD), also known as autotetraploid. The genetic structure of diploid gametes and particularly the rate of parental heterozygosity restitution (PHR) depend on the meiotic process by which they were originated. Second-division restitution (SDR) is the 2n gamete formation mechanism involved in 'Fortune' and clementines. With SDR, PHR is positively linked with the distance of each locus to the centromere. With DD parents, PHR depends on the rate of preferential pairing and thus the proportion of disomic versus tetrasomic segregations. We have compared the genetic structure of two populations of diploid gametes of clementine (one population of 2n gametes and one of 2x gametes produced by DD) with SNP and SSR markers covering the 9 citrus chromosomes. The DD displays mostly tetrasomic segregation; however three linkage groups present intermediate segregation and one displays a tendency for disomy. The PHR in the 2n gametes is 2/3 of that obtained in 2x gametes produced by DD. The two methods of triploid production appear complementary in terms of genotypic variability. 4x X 2x hybridizations are more efficient than 2x X 2x for developing new cultivars phenotypically closer to the diploid parent of the DD. Conversely, 2x X 2x hybridisations provides a greater opportunity to select innovative products. (Resume)
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Background / Purpose: The phylogenetic relationships between Citrus and its sexually compatible relatives are still unclear. Sequencing of nuclear genes and the use of mitochodrial markers will help to solve its organization. Main... more
Background / Purpose: The phylogenetic relationships between Citrus and its sexually compatible relatives are still unclear. Sequencing of nuclear genes and the use of mitochodrial markers will help to solve its organization. Main conclusion: C. reticulata and Fortunella form an independent clade, differentiated from the other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha).
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Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes.... more
Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes. Their formation mechanism, i.e., First-Division Restitution (FDR) or Second-Division Restitution (SDR), greatly impacts the gametic and population structures and, therefore, the breeding efficiency. Previous methods to identify the underlying mechanism required the analysis of a large set of markers over large progeny. This work develops a new maximum-likelihood method to identify the unreduced gamete formation mechanism both at the population and individual levels using independent centromeric markers. Knowledge of marker-centromere distances greatly improves the statistical power of the comparison between the SDR and FDR hypotheses. Simulating data demonstrated the importance of selecting markers very close to the centromere to obtain significant...
Research Interests: Biology, Plant Breeding, Medicine, Polyploidy, Meiosis, and 5 moreCitrus, Genotype, Likelihood Functions, centromere, and alleles
Citrus are most important fruit crop worldwide. Seed1essness is a key characteristic for the fresh fruit market and the development of triploid hybrids is one strategy developed by several groups over the world. Indeed, triploid hybrids... more
Citrus are most important fruit crop worldwide. Seed1essness is a key characteristic for the fresh fruit market and the development of triploid hybrids is one strategy developed by several groups over the world. Indeed, triploid hybrids are generally sterile and produce seedless fruits and do not pollinate other varieties. Triploid citlllS hybrids can be obtained by several strategies, including hybridization betwe6n diploid parents. Mechanism of 2n gamete formation and its implication on parental heterozygosity restitution is a main parameter determining the genetic and phenotypic structure of the triploid population. In the case of Citrus it has been shown that the 2n gametes are of maternaI origin. It has been proposed that the origin of 2n gametes is from the second division restitution (SDR) in Clementines and from the first division restitution (FDR) in sweet oranges. No data is available for other genotypes and particularly 'Fortune', a mandarin hybrid producing very high rate of triploids in 2x x 2x crosses and massively used to create triploid progenies. The aim of this work was to analyse the mechanism of 2n gamete formation in 'Fortune' mandarin genotype. One hundred and five triploid hybrids from the crosses between 'Fortune' as female diploid parent and 'Murcott' or 'Mandarino ComUIl' as male diploid parents were genotyped for twenty- four codominant molecular SSRs (Simple Sequence Repeat) markers using a capillary genetic fragment analyzer. Estimation of ailelic doses from relative peaks area ailowed inferring the female and male gamete structures and thus the heterozygosity restitution in the 2n gametes.; this demonstrated that ail triploid arise from 2n megaspores. The unimodal distribution of heterozygosity restitution in the 2n megaspores among the analyzed genotypes suggests that an these 2n gametes arise from a same mechanism. Restitution of maternaI heterozygosity for the used rnarkers makes suppose that underlying mechanism in the 2n gamete formation is SDR. Indeed there are six markers with less than 50% of maternaI heterozygosity restitution, which is incompatible with FDR hypothesis. SDR hypothesis is coherent with the results published in case of the clementine, which is one of the parents of the 'Fortune' variety. Under this hypothesis, the relatively high global heterozygosity restitution level (60,95%) should indicate that a majority of the analyzed markers are far frorn the centromeres. This genetic struc turation will soon be confronted with phenotypic variability and compared with structuration obtained with other triploid creation strategies such as 2x x 4x hybridization. (Texte integral)
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Virus induced gene silencing (VIGS) is an effective technology for gene function analysis in plants. We assessed the VIGS effectiveness in Nicotiana benthamiana and citrus plants of different Citrus leaf blotch virus (CLBV)-based vectors,... more
Virus induced gene silencing (VIGS) is an effective technology for gene function analysis in plants. We assessed the VIGS effectiveness in Nicotiana benthamiana and citrus plants of different Citrus leaf blotch virus (CLBV)-based vectors, using insets of the phytoene desaturase (pds) gene. While in N. benthamiana the silencing phenotype was induced only by the construct carrying a 58-nt pds hairpin, in citrus plants all the constructs induced the silencing phenotype. Differences in the generation of secondary small interfering RNAs in both species are believed to be responsible for differential host-species effects. The ability of CLBV-based vectors to silence different endogenous citrus genes was further confirmed. Since CLBV-based vectors are known to be stable and induce VIGS in successive flushes for several months, these vectors provide an important genomic tool and it is expected that they will be useful to analyze gene function by reverse genetics in the long-lived citrus pla...
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Research Interests: Plant Biology, Biology, Ecology, Medicine, Competence, and 3 moreTree Physiology, Tree, and Forestry Sciences
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Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in... more
Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necrotic-inducing ability of p23 when launched in N. benthamiana from Potato virus ...
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Ectopic expression of the p23 gene from a severe (T36) strain of Citrus tristeza virus (CTV) induces viral-like symptoms in Mexican lime. Here, we report that expressing the same gene from a mild strain induced similar symptoms that... more
Ectopic expression of the p23 gene from a severe (T36) strain of Citrus tristeza virus (CTV) induces viral-like symptoms in Mexican lime. Here, we report that expressing the same gene from a mild strain induced similar symptoms that correlated with accumulation of p23 protein irrespective of the source strain. CTV inoculation of transgenic limes showing CTV-like leaf symptoms and high p23 accumulation did not modify symptoms initially, with the virus titer being as in inoculated nontransgenic controls; however, at later stages, symptoms became attenuated. Transformation with p23-T36 of CTV-susceptible sweet and sour orange and CTV-resistant trifoliate orange also led to CTV-like leaf symptoms that did not develop when plants were transformed with a truncated p23 version. In transgenic citrus species and relatives other than Mexican lime, p23 was barely detectable, although symptom intensity correlated with levels of p23 transcripts. The lower accumulation of p23 in sweet and sour or...
Research Interests: Genetics, Microbiology, Plant Biology, Biology, Tobacco, and 12 morePlant Molecular Biology, Medicine, Gene expression, Plant diseases, Molecular Plant Microbe Interactions, Citrus, Citrus Tristeza Virus, Species Specificity, Amino Acid Sequence, Closterovirus, Plant Leaves, and Molecular Sequence Data
Viral vectors have been used to express foreign proteins in plants or to silence endogenous genes. This methodology could be appropriate for citrus plants that have long juvenile periods and adult plants that are difficult to transform.... more
Viral vectors have been used to express foreign proteins in plants or to silence endogenous genes. This methodology could be appropriate for citrus plants that have long juvenile periods and adult plants that are difficult to transform. We developed viral vectors based on Citrus leaf blotch virus (CLBV) by duplicating a minimum promoter (92 bp) either at the 3′ untranslated region (clbv3′pr vector) or at the intergenic region between the movement and coat protein (CP) genes (clbvINpr vector). The duplicated fragment (–42/+50) around the transcription start site of the CP subgenomic RNA (sgRNA) had the full promoter activity and induced synthesis of a new sgRNA in infected plants. Agroinoculation with these vectors resulted in systemic infection of Nicotiana benthamiana and the resulting virions systemically infected citrus plants. A clbvINpr vector carrying the green fluorescent protein (GFP) gene expressed GFP in citrus plants and triggered gfp silencing in gfp-transgenic citrus pl...
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A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times... more
A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times more sensitive than detection by conventional RT-PCR. An external standard curve using in vitro synthesized RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range (seven logarithmic units of concentration) and very low variation coefficient values. This protocol enabled detection of as little as 100 copies of CLBV RNA in various tissues and citrus varieties infected with CLBV sources from different geographical origins. The new assay greatly improves current detection methods for CLBV and it has been most helpful for the Spanish citrus sanitation, quarantine and certification programs, and fitness evaluation of infectious cDNA clones of CLBV, useful potentially as viral vectors for citrus.
Research Interests: Microbiology, Medical Microbiology, Biology, Virology, Medicine, and 14 moreQuantitative analysis, Real-time RT-PCR, Plant diseases, Method, Journal of Virological Methods, Citrus, Real Time, Quantitative Analysis, Sensitivity and Specificity, Standard Curve, cDNA cloning, reverse transcriptase polymerase chain reaction, Viral Vector, and Reverse transcription polymerase chain reaction
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Rapid flowering is crucial to perform functional genomic studies to investigate reproductive biology characteristics and fruit quality-related traits in fruit trees. However, long generation cycles of woody plants considerably delay this... more
Rapid flowering is crucial to perform functional genomic studies to investigate reproductive biology characteristics and fruit quality-related traits in fruit trees. However, long generation cycles of woody plants considerably delay this evaluation. Through genetic transformation, juvenile periods can be significantly shortened by overexpression of flower meristem-identity genes. Transgenic APETALA1 (AP1) citrus plants behave as rapid-cycling trees, since 1-year-old seedlings promptly show precocious flowering and fruiting. By transgene stacking into these short-generation AP1 and nptII/GUS-positive plants, expression of novel transgenes could theoretically be examined as quickly as 1 year after retransformation. Establishment of the selection and regeneration conditions for the production of retransformed individuals with marker genes is detailed in this communication. Hpt and bar genes were used as the second selectable marker genes. PCR and Southern blot analyses confirmed the recovery of retransformed shoots. AP1 transcript accumulation and GUS and GFP expression were assessed in leaves, and flowers and fruit organs of rapid-cycling retransformed lines, respectively, as early as 1 year after plant generation and during three consecutive years, demonstrating that the principle of stable transgene stacking on early-fruiting transgenic trees is feasible.
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Reintroducciones y reforzamientos poblacionales de especies amenazadas en la Comunidad Valenciana (pp. 11) La gestión in situ de la flora amenazada en el Parque Natural de las Sierras de Cazorla, Segura y Las Villas (pp. 13) El control de... more
Reintroducciones y reforzamientos poblacionales de especies amenazadas en la Comunidad Valenciana (pp. 11) La gestión in situ de la flora amenazada en el Parque Natural de las Sierras de Cazorla, Segura y Las Villas (pp. 13) El control de la explotación de las plantas ...
Research Interests: Biology and Conservación
ABSTRACT Citrus suffers severe losses from virus and similar diseases, which are mainly spread by grafts, stocks and scions. This planting material needs to be made virus-free throughout the Mediterreanean region. The standards published... more
ABSTRACT Citrus suffers severe losses from virus and similar diseases, which are mainly spread by grafts, stocks and scions. This planting material needs to be made virus-free throughout the Mediterreanean region. The standards published by EPPO in 1980 remain valid for this purpose: they provide techniques for elimination of the viruses and for protecting healthy material from reinfection. The actual situation in individual Mediterranean countries is reviewed, especially with regard to certification programmes (still not started in several cases). Healthy planting material of certain cultivars can be obtained on the international market, but this is not true of local cultivars which also need to be certified. In any case, the import of planting material carries the risk of introducing diseases not yet present in the Mediterranean region, so that strict quarantine procedures are needed.
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Research Interests: Microbiology, Plant Biology, Biology, Medicine, Plant Genome Project, and 3 moreCitrus, alleles, and Haploidy
Research Interests: Bioinformatics, Genetics, Molecular Biology, Genomics, Computational Biology, and 15 moreMolecular Evolution, Biology, Breeding, Molecular Genetics, Plant breeding and genetics, Medicine, Biological Sciences, Haplotypes, Molecular Phylogenetics and Evolution, Citrus, Genotype, Species Specificity, Genetic Markers, Synteny, and Medical and Health Sciences
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The first outbreak of citrus tristeza disease in Spain caused by Citrus tristeza virus (CTV) was recorded in 1957 in the Valencian Community (VC). In total c. 40 million trees, mainly of sweet orange and mandarin grafted on sour orange... more
The first outbreak of citrus tristeza disease in Spain caused by Citrus tristeza virus (CTV) was recorded in 1957 in the Valencian Community (VC). In total c. 40 million trees, mainly of sweet orange and mandarin grafted on sour orange rootstocks, declined due to CTV. ...
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The p25 coat protein (CP) gene of Citrustristezavirus (CTV) was incorporated to Mexican lime plants and forty-twotransgeniclines were produced, 25 containing the p25 CP gene of thesevere CTV strain T-305 and 17 with that of the mild... more
The p25 coat protein (CP) gene of Citrustristezavirus (CTV) was incorporated to Mexican lime plants and forty-twotransgeniclines were produced, 25 containing the p25 CP gene of thesevere CTV strain T-305 and 17 with that of the mild strain T-317. When ...
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Citrus leaf blotch virus (CLBV), a member of the family Flexiviridae, has a ~9-kb single-stranded, positive-sense genomic RNA encapsidated by a 41-kDa coat protein. CLBV isolates are associated with symptom production in citrus including... more
Citrus leaf blotch virus (CLBV), a member of the family Flexiviridae, has a ~9-kb single-stranded, positive-sense genomic RNA encapsidated by a 41-kDa coat protein. CLBV isolates are associated with symptom production in citrus including leaf blotching of ...
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Carmen Fagoaga, Ismael Rodrigo1, Vicente Conejero1, Concha Hinarejos, Juan José Tuset, Joaquın Arnau1, José Antonio Pina, Luis Navarro1 and Leandro Peña Dpto. Protección Vegetal y Biotecnologıa, Instituto Valenciano de Investigaciones... more
Carmen Fagoaga, Ismael Rodrigo1, Vicente Conejero1, Concha Hinarejos, Juan José Tuset, Joaquın Arnau1, José Antonio Pina, Luis Navarro1 and Leandro Peña Dpto. Protección Vegetal y Biotecnologıa, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado ...
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Summary In preliminary experiments on Agrobacterium-mediated transformation of citrus, we found transformation events occurring in callus formed from the cambium. Factors affecting Agrobacteriumsour orange (Citrus aurantium L.)... more
Summary In preliminary experiments on Agrobacterium-mediated transformation of citrus, we found transformation events occurring in callus formed from the cambium. Factors affecting Agrobacteriumsour orange (Citrus aurantium L.) interactions, such as culture medium, ...
Research Interests: Plant Biology, Biology, Ecology, Medicine, Competence, and 3 moreTree Physiology, Tree, and Forestry Sciences
A. Domínguez 7 J. Guerri 7 M. Cambra 7 L. Navarro P. Moreno 7 L. Pena (И) Dpto. Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, E-46113-Moncada, Valencia, Spain e-mail:... more
A. Domínguez 7 J. Guerri 7 M. Cambra 7 L. Navarro P. Moreno 7 L. Pena (И) Dpto. Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, E-46113-Moncada, Valencia, Spain e-mail: lpenya6ivia.es Tel: c34-96-1391000 ...
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The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which... more
The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expres...
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Previously, we have shown that most Mexican limes (Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus tristeza virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent... more
Previously, we have shown that most Mexican limes (Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus tristeza virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent transgenic lines having normal phenotype displayed characteristics typical of post-transcriptional gene silencing (PTGS): multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of p23-specific small interfering RNAs (siRNAs). When graft- or aphid-inoculated with CTV, some propagations of these silenced lines were immune: they neither expressed symptoms nor accumulated virions and viral RNA as estimated by DAS-ELISA and Northern blot hybridization, respectively. Other propagations were moderately resistant because they became infected later and showed attenuated symptoms compared to controls. The susceptible propagations, in addition to symptom expression and elevated virus titer, ac...
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Summary Genetic transformation of mature trees is difficult because adult tissues are recalcitrant to Agrobacterium tume-faciens infection and transformation and because transgenic mature events are less competent for regeneration. We... more
Summary Genetic transformation of mature trees is difficult because adult tissues are recalcitrant to Agrobacterium tume-faciens infection and transformation and because transgenic mature events are less competent for regeneration. We have shown that reinvigoration allows ...
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Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol... more
Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod) were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 µmol L-1; Explants ends had a longitudinal terminal incision (2-3 mm); co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm) with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C, in the absence of acetosyringone.
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The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic... more
The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New emb...