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Blood oranges have been cultivated in Italy since the early 19th century. Growers and breeders were able to identify and collect many somatic mutants, which differed in their major pomological traits, such as fruit size and firmness, pulp and peel pigmentation, and ripening period. Recently, in the framework of a genetic resource conservation program, most of the accessions collected at CREA in recent decades were planted in a single block to evaluate their characteristics. The presence of many selections in the same block, of the same age and grafted on the same rootstock is extremely important for a reliable characterization of the pomological characters, particularly pigmentation, which is strongly influenced by both the environment and the rootstock. The collection consists of 88 genotypes, including old lines and nucellar selections. Some commercial Tarocco, Moro and Sanguinello varieties were also included in the collection to facilitate comparison among clones. Moreover, the pomological features of 20 elite Tarocco clones cultivated nearby in the same experimental station were used as a further reference. The pomological characterization, based on 13 traits and performed in two consecutive years, revealed significant variability. Some genotypes showed strong year-to-year variability, mainly in fruit pigmentation, probably due to different climatic conditions during winter. Similarly, other traits such as fruit size, total soluble solids and acidity also varied among and within the varietal groups. These findings have a potential utility in identifying promising clones, which might fulfil the growing market needs such as higher anthocyanin content and longer shelf life.

International audience; In citrus, the production of anthocyanin pigments requires the activity of the transcriptional activator Ruby. Consequently, loss-of-function mutations in Ruby result in an anthocyaninless phenotype [1]. Several citrus accessions, however, have lost the ability to produce these pigments despite the presence of wild-type Ruby alleles. These specific mutants have captivated the interest of botanists and breeders for centuries because the lack of anthocyanins in young leaves and flowers is also associated with a lack of proanthocyanidins in seeds and, most notably, with an extreme reduction in fruit acidity (involving about a three-unit change in pH). These mutants have been defined collectively as "acidless" [24]. We have identified Noemi, which encodes a basic helix-loop-helix (bHLH) transcription factor and which controls these apparently unrelated processes. In accessions of Citron, limetta, sweet lime, lemon, and sweet orange, acidless phenotypes are associated with large deletions or insertions of retro- transposons in the Noemi gene. In two accessions of limetta, a change in the core promoter region of Noemi is associated with reduced expression and increased pH of juice, indicating that Noemi is a major determinant of fruit acidity. The characterization of the Noemi locus in a number of varieties of Citron indicates that one specific mutation is ancient. The presence of this allele in Chinese fingered Citrons and in those used in the Sukkot Jewish ritual [5] illuminates the path of domestication of Citron, the first citrus species to be cultivated in the Mediterranean. This allele has been inherited in Citron-derived hybrids with long histories of cultivation.

Citrus rootstocks are highly polyembryonic. Seeds usually produce many nucellar seedlings that are identical to the mother plant and sometimes one zygotic plant. Nucellar seedlings hamper the identification of hybrids if the morphological markers that distinguish sexual and nucellar plants are missing, and competition among embryos might impair zygotic embryo viability. Accurate morphological and/or molecular screening methods are needed to identify and discard the nucellar plants, which is labor-intensive and increases the costs of rootstock breeding programs. This study describes an affordable and reliable method for an early identification of hybrids in progenies derived from highly polyembryonic female parents based on DNA isolation from pooled leaf samples (2 to 4 plants per sample) coupled with a high resolution melting (HRM) analysis at two SNP loci. The method was validated on more than one thousand progeny derived from the crosses alemow ( Citrus macrophylla Wester) × Swingle citrumelo [ Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.] (A × S), and Cleopatra mandarin ( Citrus reshni Hort. ex Tan) × Swingle citrumelo (C × S). Bulking reduced the number of DNA isolations for population genotyping, while HRM unequivocally identified, in a single step, the bulks containing the unifoliate hybrids. A consistent difference was found between A × S and C × S populations in the frequency of uni- and multifoliate hybrids (69% unifoliate in A × S vs. 10% in C × S), suggesting that the combination of parents, rather than just the male parent carrying the trifoliate character, influenced the early multifoliate leaf expression. Moreover, several multifoliate nucellar seedlings were found among the C × S progenies, making the multifoliate leaf character inappropriate for the early identification of all hybrids.

Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus 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 by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

Sweet orange [Citrus sinensis (L.) Osbeck] represents the most important Citrus species, followed by clementine (C. clementina Hort. ex Tan.). Citrus species and genotypes are difficult to recognize as they have a moderate level of diversity due to nucellar selection, vegetative propagation and origin by single spontaneous mutation. Despite the large number of available sequences and the existence of a draft assembly of sweet orange and clementine, there are currently no single nucleotide polymorphism (SNP) databases for Citrus species. For this purpose, the QualitySNP software was used to discover SNPs in 19 Citrus species starting from 540,000 expressed sequence tags (ESTs) assembled in 52,000 contigs. The vast majority of ESTs, contigs and SNPs were found in C. clementina and C. sinensis: 4,400 out of 16,000 contigs (27 %) of C. clementina and 4,100 out of 17,000 contigs (24 %) of C. sinensis contained putative SNPs. A total of 3,634 sequences were associated with enzymes belonging to 121 metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, among which the secondary metabolite pathway was the most represented. A total of 163 SNPs from 52 contigs and genes of specific functional categories were validated and 81 polymorphic sites were found. Thirty-seven selected SNPs, validated by Sanger sequencing, confirmed that polymorphisms were mainly between species, while poor within-species variability was discovered. This work provides a collection of 15,879 putative SNP markers that could be exploited by the Citrus community. Furthermore, the validated SNPs associated with specific genes could be used for functional genetic studies in germplasm diversity analysis, mapping and breeding.

Ruby, a regulatory gene encoding a MYB transcription factor, is essential for anthocyanin production, and differences in its activity determine most of the natural variation in pigmentation in Citrus and related genera.

Blood orange juice (OJ) is an important source of anthocyanins (ACN). The latter molecules are endowed with antioxidant activity and might thus modulate different cell function. Our aim was to investigate ACN absorption following a 1-month daily supplementation of blood OJ and their potential effects on cell markers of platelet and leukocyte activation and interaction.Eighteen healthy subjects (10 men and 8 women) were supplemented for 4 weeks with 1 L/day of either blood OJ or blond OJ (that contains no ACN), following a cross-over design. Blood samples were obtained from fasting participants both at baseline and after each week of treatment to measure plasma ACN concentration. At the same time-intervals, 24-h urinary excretion of these molecules was also measured. At the beginning and the end of each 4-week intervention period, platelet and leukocyte markers and mixed cell conjugates were assessed both in basal condition and upon in vitro collagen/ADP activation.After 1 week supplementation with blood OJ, 24-h urinary excretion of ACN reached average levels of 11.47 ± 5.63 nmol that significantly differed from baseline and remained substantially unchanged until the end of treatment. No plasma accumulation of ACN following blood OJ supplementation was observed. Cellular markers were not significantly affected by either OJ after 4-week supplementation.Following supplementation of healthy volunteers with 1 L/day of blood OJ for 4 weeks, the ACN plasma levels reached were insufficient to significantly modify cell markers of platelet and leukocyte activation and interaction.

The productivity and the geographical distribution of most commercially important Citrus varieties are markedly affected by environmental low temperatures. As gene engineering has been shown to be a favourable alternative to produce germplasm with improved cold tolerance, a broad group of cold regulated genes have been previously identified from several Citrus spp. By contrast, little information regarding the cold stress response of pigmented sweet orange varieties is available although they might provide a pivotal contribution to define the whole events occurring in cold exposed Citrus fruits. In our work, the transcriptome analysis based on subtractive hybridisation was performed in order to emphasise the overall induction in gene expression after the exposure of blood oranges [(Citrus sinensis) L. Osbeck Tarocco Sciara] to low temperature. The cold induction of several gene expressions was then validated by real-time RT-PCR. Overall, we observed the enhancement of transcripts involved in the defence mechanisms against oxidative damage, osmoregulating processes, lipid desaturation as well as many ESTs implicated in the primary and secondary metabolisms. In particular, the results show that cold stress induces transcriptomic modifications directed towards the increase of flavonoid biosynthesis, including those reactions involved in anthocyanin biosynthesis, as well as of the metabolic pathways supplying it. By comparing the blood orange response to cold stress with those of other plant sources, such as grapefruit, it seems to be similar to that of the chilling acclimated species. Interestingly, among the genes encoding for the regulatory proteins, several transcription factors have been identified for the first time as cold responsive genes in plants, indicating novel investigation lanes which should receive special attention in the future.

A flesh-specific oligonucleotide custom array was designed to study gene expression during blood orange ripening. The array included 301 probes derived from a subtracted SSH library, a cDNA-AFLP collection, and a set of regulatory genes from the Harvest citrus database. The custom array was hybridized using samples of Moro, a pigmented cultivar, and Cadenera, a common cultivar, at three different ripening stages: the immature phase, the halfway point of maturation (corresponding to the start of Moro pigmentation) and the full ripening. Of the 301 probes, 27 in total, corresponding to 20 different transcripts, indicated differential expression in stage-to-stage and/or cultivar-to-cultivar comparisons. Transcripts encoding for anthocyanin biosynthesis represented most of the total over-expressed probes. The remaining differentially expressed transcripts were functionally associated with primary metabolism as flavor biosynthesis, defense and signal transduction. The expressed products associated with probes indicating differential expression were confirmed by qRT-PCR. The microarray was designed considering a small collection of sequences useful for monitoring specific pathways and regulatory genes related to fruit ripening and anthocyanin pigmentation. The main novelty of this customization is the use of expressed sequences specifically derived from blood orange flesh to study different cultivars and ripening stages, and the provision of further information about processes related to anthocyanin pigmentation in citrus fruit flesh.

In 1968, the CRA-Research Center for Citriculture and Mediterranean Crops (CRA-ACM) started a research program aimed at breeding citrus rootstocks. The monoembryonic species C. latipes (Swing.) Tan. was used as the female parent; trifoliate orange [Poncirus trifoliata (L.) Raf.], sour orange, and volkamer lemon (C. volkameriana Pasq.) were used as male parents. The behavior of some of these hybrids tested with other standard rootstocks in Sicily and Sardinia was evaluated. The cultivars under comparison included ‘Washington’ navel orange and ‘SRA 92’ clementine in Sardinia and ‘Tarocco’ orange in Sicily. Our results showed the dramatic influence of rootstock on plant growth and yield; only minor effects on fruit quality were observed. Among the standard rootstocks tested, Swingle citrumelo provided the highest yield. Some of the tested hybrids (F5 P12, F6 P12, and F6 P13) may improve plant yield, thus maintaining good fruit quality. Encouraging data obtained with these hybrids may justify the use of monoembryonic species of the Papeda subgenus for breeding citrus rootstocks.

Citrus tristeza virus (CTV) is the causal agent of various diseases with dramatic effects on citrus crops worldwide. Most Citrus species, grown on their own roots, are symptomless hosts for many CTV isolates. However, depending on different scion-rootstock combination, CTV infection should result in distinct syndromes, being 'tristeza' the more severe one, leading to a complete decline of the susceptible plants in a few weeks. Transcriptomic analyses revealed several genes involved either in defense response, or systemic acquired resistance, as well as transcription factors and components of the phosphorylation cascades, to be differentially regulated during CTV infection in Citrus aurantifolia species. To date little is known about the molecular mechanism of this host-pathogen interaction, and about the rootstock effect on citrus response to CTV infection. In this work, the response to CTV infection has been investigated in tolerant and susceptible scion-rootstock combinations by two-dimensional gel electrophoresis (2DE). A total of 125 protein spots have been found to be differently accumulated and/or phosphorylated between the two rootstock combinations. Downregulation in tolerant plants upon CTV infection was detected for proteins involved in reactive oxygen species (ROS) scavenging and defense response, suggesting a probable acclimation response able to minimize the systemic effects of virus infection. Some of these proteins resulted to be modulated also in absence of virus infection, revealing a rootstock effect on scion proteome modulation. Moreover, the phospho-modulation of proteins involved in ROS scavenging and defense response, further supports their involvement either in scion-rootstock crosstalk or in the establishment of tolerance/susceptibility to CTV infection.

Genes encoding chalcone synthase (CHS), anthocyanidin synthase (ANS), and UDP-glucose-fl avonoid 3-O- glucosyltransferase (UFGT), some of the enzymes of anthocyanin biosynthetic pathway, were assayed in two different experiments using quantitative real-time reverse transcriptase (RT)-PCR, in order to test their transcription levels in the fl esh of blood and common orange (Citrus sinensis (L.) Osbeck) fruit, and to investigate their role in anthocyanin accumulation in the same tissue. The fi rst experiment compared a blood orange and a common orange cultivar during seven different fruit maturation stages. This was followed by the test of 11 different genotypes at the end of the winter season. Data collected from the fi rst experiment, over the blood orange cultivar, were statistically analyzed using the Pearson correlation coeffi cient. Results show that CHS, ANS, and UFGT mRNA transcripts are up- and co-regulated in the blood orange cultivar, whereas they are down-regulated in the common orange cultivar. There is evidence of correspondence between the target genes expression level and the content of the pigment assessed. The second test confi rms this correlation and shows that enzyme synthesis levels and pigment accumulation, in plants grown under the same environmental conditions, are dependent on the differences occurring among the genotypes tested. These results suggest that the absence of pigment in the common orange cultivars may be caused by the lack of induction on the structural genes expression. This is the fi rst report on the characterization of the relationships between biosynthetic genes expression and fruit fl esh anthocyanin content in blood oranges. The blood orange is the typical product of Sicilian citriculture and its red to purple pigmented rind and fl esh makes the fruit more attractive. Red color in blood oranges is due to the pres- ence of anthocyanins, the largest class of pigments among the fl avonoids. They are secondary metabolites, widely spread in higher plants and produced in different organs, in response to a variety of developmental, environmental, and genetic cues (Mol et al., 1996). They serve various reproductive functions (e.g., in fl ower petals to attract pollinators, and in seeds and fruit to aid in seed dispersal (Holton and Cornish, 1995)) and they protect plant tissues against pathogens and damage from environmental conditions (e.g., ultraviolet (UV) irradiation, cold temperature (Dixon and Paiva, 1995) and water stress (Chalker-Scott, 1999)). This class of pigmented molecules also possesses some important therapeutic properties for human health (Saija, 1994), function- ing as antioxidants, anti-infl ammatories, in epithelium repair and protection, and in modulation of capillary permeability. In conferring these properties to blood orange fruit, anthocyanins contribute to their commercial popularity and value, therefore research has been focusing on understanding the biosynthetic pathway for anthocyanins in oranges (Lo Piero et al., 2005

Background Glutathione S-transferases (GSTs) represent a ubiquitous gene family encoding detoxification enzymes able to recognize reactive electrophilic xenobiotic molecules as well as compounds of endogenous origin. Anthocyanin pigments require GSTs for their transport into the vacuole since their cytoplasmic retention is toxic to the cell. Anthocyanin accumulation in Citrus sinensis (L.) Osbeck fruit flesh determines different phenotypes affecting the typical pigmentation of Sicilian blood oranges. In this paper we describe: i) the characterization of the GST gene family in C. sinensis through a systematic EST analysis; ii) the validation of the EST assembly by exploiting the genome sequences of C. sinensis and C. clementina and their genome annotations; iii) GST gene expression profiling in six tissues/organs and in two different sweet orange cultivars, Cadenera (common) and Moro (pigmented). Results We identified 61 GST transcripts, described the full- or partial-length nature of the sequences and assigned to each sequence the GST class membership exploiting a comparative approach and the classification scheme proposed for plant species. A total of 23 full-length sequences were defined. Fifty-four of the 61 transcripts were successfully aligned to the C. sinensis and C. clementina genomes. Tissue specific expression profiling demonstrated that the expression of some GST transcripts was 'tissue-affected' and cultivar specific. A comparative analysis of C. sinensis GSTs with those from other plant species was also considered. Data from the current analysis are accessible at http://biosrv.cab.unina.it/citrusGST/, with the aim to provide a reference resource for C. sinensis GSTs. Conclusions This study aimed at the characterization of the GST gene family in C. sinensis. Based on expression patterns from two different cultivars and on sequence-comparative analyses, we also highlighted that two sequences, a Phi class GST and a Mapeg class GST, could be involved in the conjugation of anthocyanin pigments and in their transport into the vacuole, specifically in fruit flesh of the pigmented cultivar.

Traditionally, Sicilian blood oranges (Citrus sinensis) have been associated with cardiovascular health, and consumption has been shown to prevent obesity in mice fed a high-fat diet. Despite increasing consumer interest in these health-promoting attributes, production of blood oranges remains unreliable due largely to a dependency on cold for full color formation. We show that Sicilian blood orange arose by insertion of a Copia-like retrotransposon adjacent to a gene encoding Ruby, a MYB transcriptional activator of anthocyanin production. The retrotransposon controls Ruby expression, and cold dependency reflects the induction of the retroelement by stress. A blood orange of Chinese origin results from an independent insertion of a similar retrotransposon, and color formation in its fruit is also cold dependent. Our results suggest that transposition and recombination of retroelements are likely important sources of variation in Citrus.

Oxygen deficit, which occurs in flooded or poorly drained soils, can limit plant growth and development. Low-oxygen environmental conditions also limit the distribution of many woody plants, such as citrus trees, which are considered flood-sensitive crops, although tolerance to this stress varies among genotypes and rootstocks. In this study, the rice transcription factor Mybleu was inserted into the pGA470 plant cloning vector and transferred into the epicotyl explants of the Carrizo citrange rootstock (Citrus sinensis × Poncirus trifoliata) using Agrobacterium tumefaciens-mediated transformation. The transgenic lines were confirmed for the presence and expression of the transgene, and physiological, biochemical and molecular parameters were evaluated for adaptation to hypoxic and anoxic stress conditions. The ectopic expression of Mybleu increased tolerance to oxygen deprivation in the transgenic lines, contributing to increased viability under this stress condition. This improved tolerance correlates with, and may depend on, the induction of genes and the activation of enzymes from various fermentation and carbohydrate metabolic pathways, antioxidant systems and nonsymbiotic haemoglobin-nitric oxide homeostasis mechanisms. Together, our data suggest a key role for Mybleu in coordinating the multifaceted plant response to low oxygen stress and the conservation of Mybleu-regulated pathways among species.

Members of the MYB and MYC family regulate the biosynthesis of phenylpropanoids in several plant species. Two sequences, called CsMYB8 and CsMYC2, were identified from Citrus sinensis, and both the cDNA and the genomic clones were isolated and characterized from the flesh of common and blood oranges. Analysis by real-time polymerase chain reaction showed that the expression pattern of CsMYC2 is generally higher in rind than in flesh and in blood oranges than in common ones. In contrast, no significant difference in expression was observed for CsMYB8. The expression pattern of the structural genes chalcone synthase, anthocyanidin synthase, and UDP-glucose-flavonoid 3-O-glucosyltransferase, which code for three enzymes involved in the anthocyanin biosynthetic pathway, was also analyzed and correlated with CsMYC2, in flesh, rind, and leaf of the common and blood oranges, and in leaf of Citrus limon cultivars (characterized by anthocyanin absence or variable content). Surprisingly, CsMYC2 is highly expressed in the leaf and expression is correlated with UFGT expression in this organ. These results suggest that CsMYC2 is involved in the regulation of the flavonoid biosynthetic pathway in Citrus.

10 pages, 6 figures, 3 tables, Blood orange cultivars of the sweet orange [Citrus sinensis (L.) Osbeck] differ from the common sweet orange group (Valencia Late, Washington navel, Navelina) by the presence in the flesh and sometimes in the rind, of red anthocyanin pigments. Among blood orange varieties, Tarocco is the most variable due to its particular characteristics. The presence of several Tarocco varieties, often characterized by similar maturation periods, necessitates accurate postharvest fruit evaluation, particularly appearance, since this is a primary criterion of consumer preference. In this work a total of 929 fruit belonging to 17 different Tarocco genotypes were analyzed. Optoelectronic techniques were used to discriminate among fruit shapes using elliptic Fourier analysis (EFA) to analyse fruit lateral shapes. Fruit shape for different genotypes was classified according to the IPGRI e Citrus Industry classification. The efficiency of these methods was tested by reclassifying fruit shape typologies by k-means analysis. We also computed the best number of k (4) by implementing a suited script in MatLab. Results were screened by multivariate classification techniques (i.e., PSLDA) in order to evaluate the efficiency of the group classifications. The combined EFA and k-means analysis increased the efficiency of genotype classification based on fruit shape in comparison with reported descriptive methods. For example, comparing the two models with 5 groups (Citrus Industry and k-means-5), the percentage of correct classification in the independent test dataset was higher in the k-means-5 model (respectively, 46.6% vs. 26.0% compared to a random probability of classification of 20%). EFA could measure single fruit shape allowing the comparison of their conformity within a standard of reference. The results set the basis for a shape description of different Tarocco varieties based on quantitative morphological statistics, a practice that, until now, has been carried out exclusively in a descriptive fashion. Consequently, our work represents the first discrimination of genetically different cultivars of the same species based on fruit shape. © 2009 Elsevier B.V. All rights reserved, This study was supported by a grant from the Ministero Italiano per le Politiche Agricole (Law 41/82) entitled “Ricerche avanzate in agrumicoltura e loro applicazioni” Paper No. 29. The authorswould like to thank Mrs. Iliana Niciarelli and Mr. Matteo Cegna for the support during the laboratory operation. Jacopo Aguzzi is a fellow of the Juan de la Cierva Postdoctoral Program (MEC, Spain)

A combination of 2-DE and LC-MSMS approaches was used to identify the differentially expressed proteome of a pigmented sweet orange (Citrus sinensis, cv. Moro) in comparison with a common cultivar (Cadenera) at ripening time. The comparison of the protein patterns of Moro and Cadenera showed 64 differential expressed protein spots. Fifty-five differentially expressed proteins were identified. Proteins were classified according to their putative function and known biosynthetic pathways. Most of the proteins related to sugar metabolism were overexpressed in Moro, while those related to stress responses were overexpressed in Cadenera. The abundance of proteins belonging to Unknown/Unnamed and Hypothetical classes could be associated to the incomplete data available on the Citrus genome. The relative abundance of Secondary metabolism and Oxidative process proteins substantiated the key role of the anthocyanin pathway in Moro, which is characterized by a strong pigmentation at ripening time. The potential role of protein differential expression between Moro and Cadenera fruits was discussed, and proteomic results were compared with the known variations of transcripts of the same fruits. The latter analyses highlighted many discrepancies, confirming the necessity to associate both proteomic and transcriptomic approaches in order to achieve a more complete characterization of the biological system.

Food health and quality are the keywords recurring both into the VII Framework Programme of EU, both in the PNR and development plans arranged by several Italian regions. Essentially, the increase and definition of quality of agri-food products represent necessary tools to give new impulse and renovation to this sector. The late developments of European and Italian laws show a clear trend towards the need to give consumer clear indications on different qualitative aspects of agri-food products, besides their microbiological security. The capacity to increase the quantity of secondary metabolites (vitamins and antioxidants) in crops is particularly interesting for the development of products with high health value (functional food). Breeding programmes have already obtained lines improved about these characteristics. The need to develop assessment strategies giving scientifically reliable information on traceability and health of food derived from genetically modified plants. Assessment of the risk coming from genetically modified plants is mainly based on the so-called “principle of substantial equivalence” or “of comparative safety”, claiming that a transgenic genotype shouldn’t show any substantial change with respect of the corresponding non-transgenic genotype. These changes are obviously considered healthy in case of the current commercial varieties. The new “-omics” disciplines such as genomics, transcriptomics, proteomics and metabolomics, born together with and subsequent to the complete sequencing of model genomes, can give an overview of first matters and food compositions, thus allowing the making of an overall assessment of their quality and safety. These new and sophisticated tools for molecular investigation can help and strengthen the commonly used methods, meeting the new requirements of traceability and authenticity certification.

Nucellar embryogenesis in undeveloped ovules in some navel orange cultivars. - Nucellar embryogenesis has been studied in some cultivars of navel orange, Citrus sinensis (L.) Osbeck. Histological observations have been carried out in undeveloped ovules excised 2, 4, 6, 8 months after anthesis. Ovules under study derived a) from flowers naturally pollinated or hand-pollinated with pollen of Poncirus trifoliata (L.) Raf.; b) from emasculated flowers, and c) from flowers deprived of stigma. In absence of pollination, as well as in absence of fertilization, the nucellar embryos reached the globular stage.

Characterization by SEM of the pollen of some species of Citrus and related genera at two ploidy levels (2x and 4x).—The pollen grain morphology of some Citrus species, of some related genera, and of some intergeneric hybrids is described. The study made with the SEM has shown: a) the more typical shape of Citrus pollen is spheroidal with four furrows; the sculptures of ektexine in all Citrus species can be an useful character for taxonomy; the ektexine can be pitted (especially in edible citrus fruits) or reticulated as (in some species valuable as rootstock); in two types of ektexine there are differences that can allow the individualization of some species; these preliminary observations do not allow to indicate useful elements to individualize the cultivars of the same species; b) pollen morphology is a valid diagnostic criterion for the ploidy levels 2x and 4x of the same species in the genera Citrus and Poncirus; pollen of tetraploid plants is larger and has a higher number of furrows than ...

In this study we isolated the partial cDNA clone of three enzymes active in the late steps of anthocyanins biosynthetic pathway such as DFR, ANS and UFGT from Citrus sinensis (L) Osbeck. The predicted aminoacids sequences shared very high identity values ranging from 77 % for DFR (Pyrus communis, Malus domestica), to 86 % for ANS (Vitis vinifera) with homologous available protein sequences. Only the UFGT sequence showed lower identity value (59 %), but this finding is in accordance with data obtained for other enzymes belonging to the plant glucosyl transferase superfamily. The expression of CHS, CHI, F3OH, DFR, ANS and UFGT was also investigated in both coloured and uncoloured oranges juice vesicles. Transcripts encoding the enzymes involved in the earlier reactions have been detected in either blood and blonde oranges by RT-PCR. Conversely, considerable difference emerged in the case of the later gene ufgt which was totally unexpressed in blonde oranges juice vesicles probably determining the lack of anthocyanins production in all blonde orange cultivars analysed.

Iron chlorosis is one of the most serious abiotic stresses affecting citrus (Citrus sp.) culture in the Mediterranean Basin. A trial was performed with potted tolerant and sensitive rootstocks that were grown in volcanic and calcareous soils. Microarray analysis allowed for the identification of differentially expressed genes putatively involved in iron (Fe) deficiency. Most of the differentially expressed genes isolated from the root tips were of unknown function; the remaining genes were related to the oxidative stress response (e.g., glutathione peroxidase), hormone metabolism and signaling (e.g., small auxin up RNA family protein genes), biological regulation, protein turnover, and the tricarboxylic acid cycle (e.g., aconitase). Additionally, the majority of the Fe stress-related genes expressed in the sensitive Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) and tolerant Carrizo citrange (Citrus sinensis × P. trifoliata) rootstocks identified using real-time reverse transcription–polymerase chain reaction (RT-PCR) were related to regulation, the oxidative stress response, and hormone metabolism and signaling, thereby confirming the array data. Furthermore, validation of the differentially expressed genes in seven tolerant and sensitive rootstocks grown in a field trial under chlorotic conditions was performed. In general, the gene expression profiles reflect the different responses of rootstocks, possibly as a result of the various genetic mechanisms involved in the response to Fe deficiency. Moreover, the expression of aconitase was analyzed in the roots and juice to evaluate the implication of the different aconitase isoforms (Aco), which are derived from specific cellular compartments, in the different tissues. The involvement of the mitochondrial isoform (Aco2) was directly correlated with the acidity of the juice, whereas the cytosolic one (Aco3), which corresponds to the aconitase isolated during the microarray analysis, was found specifically in the roots.