Citrus Fruit Development Research Articles

Network Analysis of Differentially Expressed Genes across Four Sweet Orange Varieties Reveals a Conserved Role of Gibberellin and Ethylene Responses and Transcriptional Regulation in Expanding Citrus Fruits

Citrus represents the most important non-climacteric fruits and thus understanding transcriptional control during fruit development is important for improving fruit yield and quality. Compared to relatively intensive transcriptomic studies of ripening citrus fruits, much less is known regarding expanding fruits. To provide a systems view of hormone response and transcriptional regulation in citrus fruit development from Stage I (slow fruit growth) to Stage II (rapid growth), we re-analyzed the fruit transcriptomes which we previously collected from the sweet orange varieties, Newhall, Xinhui, Bingtang, and Succari (Citrus sinensis L. Osbeck). A total of 3145 genes were differentially expressed across all four varieties, indicating that they likely have conserved functions in orange fruit development. Using a gene coexpression network-based systems approach, we constructed the subnetworks respectively for gibberellin response, ethylene response, transcription factors and chromatin modifications. Analysis of these subnetworks has led to the identification of more than a dozen major hub genes, such as EXPA1, GASA1/14, ERF13, HB22, ATK1, and TOPII, which represent the most promising candidates for future functional characterization.

Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants

Identification of genes with differential transcript abundance (GDTA) in seedless mutants may enhance understanding of seedless citrus development. Transcriptome analysis was conducted at three time points during early fruit development (Phase 1) of three seedy citrus genotypes: Fallglo (Bower citrus hybrid (Citrus reticulata×C. reticulata×C. paradisi)×Temple (C. reticulata×C. sinensis)), grapefruit (C. paradisi), Pineapple sweet orange (C. sinensis), and their seedless mutants. Seed abortion in seedless mutants was observed at 26 days post anthesis (Time point 2). Affymetrix transcriptomic analysis revealed 359 to 1077 probe sets with differential transcript abundance in the comparison of seedless versus seedy fruits for each citrus genotypes and time points. The GDTA identified by 18 microarray probe sets were validated by qPCR. Hierarchical clustering analysis revealed a range of GDTA associated with development, hormone and protein metabolism, all of which may reflect genes associated with seedless fruit development. There were 14, 9 and 12 genes found exhibiting similar abundance ratios in all three seedless versus seedy genotype comparisons at time point 1, 2 and 3, respectively. Among those genes were genes coding for an aspartic protease and a cysteine protease, which may play important roles in seedless fruit development. New insights into seedless citrus fruit development may contribute to biotech approaches to create seedless cultivars.

Comparative Analysis of miRNAs and Their Target Transcripts between a Spontaneous Late-Ripening Sweet Orange Mutant and Its Wild-Type Using Small RNA and Degradome Sequencing.

Fruit ripening in citrus is not well-understood at the molecular level. Knowledge of the regulatory mechanism of citrus fruit ripening at the post-transcriptional level in particular is lacking. Here, we comparatively analyzed the miRNAs and their target genes in a spontaneous late-ripening mutant, “Fengwan” sweet orange (MT) (Citrus sinensis L. Osbeck), and its wild-type counterpart (“Fengjie 72-1,” WT). Using high-throughput sequencing of small RNAs and RNA degradome tags, we identified 107 known and 21 novel miRNAs, as well as 225 target genes. A total of 24 miRNAs (16 known miRNAs and 8 novel miRNAs) were shown to be differentially expressed between MT and WT. The expression pattern of several key miRNAs and their target genes during citrus fruit development and ripening stages was examined. Csi-miR156k, csi-miR159, and csi-miR166d suppressed specific transcription factors (GAMYBs, SPLs, and ATHBs) that are supposed to be important regulators involved in citrus fruit development and ripening. In the present study, miRNA-mediated silencing of target genes was found under complicated and sensitive regulation in citrus fruit. The identification of miRNAs and their target genes provide new clues for future investigation of mechanisms that regulate citrus fruit ripening.

Genome-wide identification of sweet orange (Citrus sinensis) histone modification gene families and their expression analysis during the fruit development and fruit-blue mold infection process.

In eukaryotes, histone acetylation and methylation have been known to be involved in regulating diverse developmental processes and plant defense. These histone modification events are controlled by a series of histone modification gene families. To date, there is no study regarding genome-wide characterization of histone modification related genes in citrus species. Based on the two recent sequenced sweet orange genome databases, a total of 136 CsHMs (Citrus sinensis histone modification genes), including 47 CsHMTs (histone methyltransferase genes), 23 CsHDMs (histone demethylase genes), 50 CsHATs (histone acetyltransferase genes), and 16 CsHDACs (histone deacetylase genes) were identified. These genes were categorized to 11 gene families. A comprehensive analysis of these 11 gene families was performed with chromosome locations, phylogenetic comparison, gene structures, and conserved domain compositions of proteins. In order to gain an insight into the potential roles of these genes in citrus fruit development, 42 CsHMs with high mRNA abundance in fruit tissues were selected to further analyze their expression profiles at six stages of fruit development. Interestingly, a numbers of genes were expressed highly in flesh of ripening fruit and some of them showed the increasing expression levels along with the fruit development. Furthermore, we analyzed the expression patterns of all 136 CsHMs response to the infection of blue mold (Penicillium digitatum), which is the most devastating pathogen in citrus post-harvest process. The results indicated that 20 of them showed the strong alterations of their expression levels during the fruit-pathogen infection. In conclusion, this study presents a comprehensive analysis of the histone modification gene families in sweet orange and further elucidates their behaviors during the fruit development and the blue mold infection responses.

Monthly metabolic changes and PLS prediction of carotenoid content of citrus fruit by combined Fourier transform infrared spectroscopy and quantitative HPLC analysis

This work examined the potential of Fourier transform infrared spectroscopy (FT-IR) spectroscopy and high-performance liquid chromatography (HPLC) analysis in evaluating metabolic changes during ripening of citrus fruit. Further, the feasibility of prediction modeling of carotenoid content by multivariate statistical analysis combined with FT-IR spectral and HPLC data was examined without additional HPLC analysis. FT-IR spectra of citrus (Citrus unshiu Marc. cv. Miyagawa) fruit peels and flesh were measured at monthly intervals of fruit development. Quantification of carotenoids from the fruit was confirmed by quantitative HPLC analysis. The most remarkable evolution of FT-IR spectral decrease during ripening of fruit was found in the amide region (1500–1700 cm−1), whereas there was an increase in the carbohydrate region (1000–1200 cm−1). The evolution of different FT-IR spectral bands was related to fruit constituents, including organic acids, carbohydrates, carotenoids, and phenolic compounds. Significant qualitative changes in the carotenoid pattern included an increase in β-cryptoxanthin and decrease in lutein content during citrus fruit development. The content of antheraxanthin (R 2 = 0.9117), β-carotene (R 2 = 0.8816), β-cryptoxanthin (R 2 = 0.8856), and violaxanthin (R 2 = 0.7305) from peels of citrus fruit could be predicted from FT-IR spectral data using partial least square (PLS) regression modeling. Considering the results of PLS-discriminant analysis (PLS-DA) of FT-IR spectral data and PLS regression modeling of carotenoid content, FT-IR in combination with multivariate analysis enables not only discrimination of metabolic variation during fruit development, but also prediction of carotenoid content from citrus fruit.

ABSCISSION STUDY DURING CITRUS FRUIT MATURATION IN CORSICA: UNFAVORABLE ENVIRONMENTAL CONDITIONS FOR FRUIT SHEDDING

Citrus fruit development and ripening are complex processes involving physiological and biochemical changes that are under hormonal, nutritional and environmental control. One of the most evident phenomena in late maturation is shedding of ripe fruit. A previous study on sweet orange abscission supposed that fruit shedding was related to the increase of sugars content of pulp juice. To investigate this potential relationship between fruit abscission and internal maturity parameters we investigated the fruit maturity process of 10 mandarin x clementine hybrids and 9 commercial sweet orange varieties with different maturity time. Morphological and biochemical analyses (acidity, total soluble solids, fruit weight thickness of the flavedo, thickness of the peduncle and external color) were carried out on fruit sets of the different varieties during the maturation process, from December to June. The abscission initiation was evaluated by measuring the force required to detach the fruit of the peduncle. Analysis showed that under our local conditions, abscission was generally not expressed even though fruit maturation was evolving. Therefore, we suppose that even though the local conditions have an important effect, abscission of citrus fruit also results from environmental-genotype interactions. Moreover, evolution of abscission and fruit parameters are not correlated, suggesting the independence in the processing of maturation and abscission. (Resume d'auteur)

Chlorophyll a/b binding protein plays a key role in natural and ethylene-induced degreening of Ponkan (Citrus reticulata Blanco)

Chlorophyll content in peel gradually declines during citrus fruit development, and this can be accelerated by applying ethylene. In order to understand the molecular regulation of chlorophyll loss, the expression of several chlorophyll-related genes was determined in Ponkan (Citrus reticulata Blanco) peel during fruit maturation and ethylene-induced degreening. During fruit development, the transcript level of pheophorbide a oxygenase (CitPaO) and stay-green protein (CitSGR) was stable, and no obvious change of chlorophyll b reductase (CitNYC) mRNA was found. In addition, chlorophyllase (CitChlase) mRNA was decreased, indicating the decline of chlorophyll degradation capacity in this process. Only the reduced expression of Mg-chelatase (CitCHLH) and chlorophyll a/b binding protein (CitCAB1, 2) was found to be correlated with the reduction in chlorophyll content. Chlorophyll loss was greatly accelerated by postharvest ethylene fumigation. In this process, the expression of CitCHLH, CitPaO and CitSGR was not affected. However, it greatly increased the expression of CitNYC and CitChlase, and accelerated the decline in CitCAB expression. Taken together, these results indicate that the decrease in expression of CitCAB was highly associated with chlorophyll loss, no matter whether during natural or ethylene-induced degreening. However, the increase in CitChlase and CitNYC transcript abundance was only related to accelerated chlorophyll degradation in ethylene-induced degreening. In conclusion, the higher availability of free chlorophylls for degradation, resulting from down regulated expression of CitCABs, is likely to be a main reason for chlorophyll reduction during natural and ethylene-induced degreening.

Citrus sinensis L. Osbeck orthologs of FRUITFULL and SHATTERPROOF are differentially expressed during fruit development

Several regulatory steps and genes involved in fruit development were identified and characterized in Arabidopsis thaliana. FRUITFULL (FUL) and SHATTERPROOF (SHP), which belong to the MADS-box family of transcription factors, act together to promote the differentiation of the dehiscence zone and thus control the process of pod shattering in Arabidopsis. Homologs to these genes have been described in fleshy fruit species, but the specific nature of the regulatory hierarchy and interactions among these key regulators remains elusive in most plant species. Here, Citrus sinensis putative orthologs to FUL and SHP, named CsFUL and CsSHP respectively, were characterized. Phylogenetic comparisons indicated that CsFUL belongs to FRUITFUL sub-clade within the AP1/SQUA major clade while CsSHP falls into PLENA sub-clade from the AG/PLE clade. CsFUL and CsSHP protein sequences possess all of the characteristic conserved domains commonly found in A- and C-lineages of MIKC MADS-box proteins, respectively. Semi-quantitative RT-PCR showed preferential expression of both genes in developing fruits. In situ hybridization and a detailed analysis of Citrus fruit development using scanning electron microscopy allowed further characterization of these genes during C. sinensis fruit development. CsFUL and CsSHP are differentially expressed in exocarp, mesocarp and endocarp tissues in early stages of fruit development but their expression diminishes with fruit maturation. Moreover, the co-localization of CsFUL and CsSHP mRNA during oil glands and juice vesicle formation suggests a potential role in the development of such structures. Altogether, there results might contribute to a better understanding of the molecular mechanisms involved in Citrus fruit development.

Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development.

Phytohormone Changes and Carbohydrate Status in Sweet Orange Fruit from Huanglongbing-infected Trees

Huanglongbing (HLB) infection alters citrus fruit growth and development, resulting in small, misshapen, and poorly colored fruit containing aborted or partially developed seeds. Typically, symptomatic fruit have delayed maturation and abscise prematurely. We studied carbohy- drate and phytohormone changes in HLB-affected fruit to explain symptom development because (1) carbohydrate shortage has been linked to fruit growth arrest and eventually abscission and (2) hormonal signals regulate, at least par- tially, fruit set and development. Symptomatic fruit (S), asymptomatic fruit (AS) from symptomatic trees, and healthy fruit (H) from asymptomatic trees were harvested from 'Valencia' sweet orange trees (Citrus sinensis (L.) Osbeck) infected with the HLB pathogen or not, as verified by PCR. Mature S weighed less, had lower Brix, were smaller, had more aborted seeds, and were greener than AS or H. Starch and sucrose contents were lower in mature S flavedo compared with that of H and AS. S and AS harvested 7 and 12 months after full bloom produced significantly less ethylene than H. Indole-3-acetic acid (IAA) and abscisic acid (ABA) contents in flavedo removed from the stylar end, middle section, or stem end of fruit generally were higher in S flavedo than in AS and H. ABA content was fourfold higher in flavedo from the middle section of S than in AS and H. Flavedo excised from the large shoulder of misshapen S had significantly higher IAA content when compared with the normal-sized area of the same fruit on the opposite side. This increase corresponded to an increase in hypodermal cell area in S flavedo. Overall, these data reveal an imbalance of carbohydrate and phytohormone status in fruit from HLB- infected trees and suggest a role of such changes in fruit symptom development.

Rootstocks influence fruit oleocellosis in ‘Hamlin’ sweet orange ( Citrus sinensis L. Osbeck)

Oil spotting or oleocellosis, is a major problem in citrus crops. As the rootstock and fertilization play important roles in citrus growth and fruit development, we investigated the influence of different rootstocks on the growth, mineral nutrition metabolism, water relations, and fruit oleocellosis of eight-year-old field ‘Hamlin’ sweet orange trees. Trees grafted onto Lichi16-6 trifoliata ( Poncircus trifoliate) had the greatest rate of oleocellosis (RO), and trees grafted onto Goutou orange ( Citrus aurantium) had the greatest degree of oleocellosis (DO). In contrast, trees grafted onto Rangpur lime ( Citrus limonia Osbeck) had the lowest RO and DO. Trees were the most vigorous on Rangpur lime rootstocks, followed by Lichi16-6 trifoliata, and then Goutou orange. In addition, because the scion/stock girth ratio showed significant correlations with the RO and DO, oleocellosis parameters can be a good indicator of scion/stock affinity. The total N, total P, Ca 2+, and Mg 2+ in leaves from trees on Rangpur lime were significantly lower than in leaves from trees on Goutou orange or Lichi16-6 trifoliata. In addition, the RO showed a significant correlation with the leaf Ca 2+ and S concentrations and with the peel Mg 2+ concentration. The DO was significantly correlated with the total peel N and S concentrations. In addition, the RO showed a significant correlation with the net assimilation of CO 2 (ACO 2), stomatal conductance (GS), transpiration rate (ET), and water-use efficiency (WUE). However, the DO showed a significant correlation with the GS, ACO 2, and WUE. Taken together, these results indicate that rootstocks affect the development of oleocellosis in ‘Hamlin’ sweet orange due to their effects on the mineral nutrition balance and water relations.

A label-free differential quantitative mass spectrometry method for the characterization and identification of protein changes during citrus fruit development

BackgroundCitrus is one of the most important and widely grown commodity fruit crops. In this study a label-free LC-MS/MS based shot-gun proteomics approach was taken to explore three main stages of citrus fruit development. These approaches were used to identify and evaluate changes occurring in juice sac cells in various metabolic pathways affecting citrus fruit development and quality.ResultsProtein changes in citrus juice sac cells were identified and quantified using label-free shotgun methodologies. Two alternative methods, differential mass-spectrometry (dMS) and spectral counting (SC) were used to analyze protein changes occurring during earlier and late stages of fruit development. Both methods were compared in order to develop a proteomics workflow that could be used in a non-model plant lacking a sequenced genome. In order to resolve the bioinformatics limitations of EST databases from species that lack a full sequenced genome, we established iCitrus. iCitrus is a comprehensive sequence database created by merging three major sources of sequences (HarvEST:citrus, NCBI/citrus/unigenes, NCBI/citrus/proteins) and improving the annotation of existing unigenes. iCitrus provided a useful bioinformatics tool for the high-throughput identification of citrus proteins. We have identified approximately 1500 citrus proteins expressed in fruit juice sac cells and quantified the changes of their expression during fruit development. Our results showed that both dMS and SC provided significant information on protein changes, with dMS providing a higher accuracy.ConclusionOur data supports the notion of the complementary use of dMS and SC for label-free comparative proteomics, broadening the identification spectrum and strengthening the identification of trends in protein expression changes during the particular processes being compared.

Preharvest Foliar Sprays of Prohexadione–calcium, a Gibberellin-biosynthesis Inhibitor, Induce Chlorophyll Degradation and Carotenoid Synthesis in Citrus Rinds

Rind color is an important cosmetic preference of consumers when purchasing citrus fruit. As citrus fruit mature, a decrease in chlorophyll concentration unmasks the presence of carotenoid pigments followed by further synthesis of carotenoids, resulting in the first appearance of the characteristic orange color of mandarins and sweet oranges. Factors contributing to invigorating growing conditions are antagonistic to optimal rind color development and tree vegetative vigor as well as high gibberellin and cytokinin levels are also thought to adversely affect rind color. Thus, a method to increase preharvest rind color by moderating vegetative vigor using a growth retardant was investigated. Prohexadione–calcium (ProCa; Regalis®), a gibberellin-biosynthesis inhibitor with growth retardant activity, was applied to ‘Nules Clementine’ mandarin (Citrus reticulata Blanco), ‘Navelina Navel’ orange [C. sinensis (L.) Osbeck], and ‘Eureka’ lemon [C. limon (L.) Burm. f.] during the 2005 and 2006 seasons at 200 and 400 mg·L−1 a.i. Rind color rating, colorimeter measurements, and pigment analyses were conducted directly after harvest, after ethylene degreening, and 3 weeks after cold storage. In the 2005 season, ProCa significantly increased rind color of ‘Nules Clementine’ mandarin and ‘Navelina Navel’ orange directly after harvest and after ethylene degreening by decreasing chlorophyll and increasing carotenoid concentrations in the flavedo of fruit but did not affect the pigment concentration of ‘Eureka’ lemon despite an improvement in rind color rating. After cold storage, however, rind color was not significantly different among treatments. In the 2006 season, rind color was significantly increased directly after harvest, and chlorophyll degradation together with carotenoid synthesis of all Citrus spp. tested were stimulated by the late 400 mg·L−1 ProCa application. Therefore, foliar spray application of ProCa at a concentration of 400 mg·L−1 applied 6 plus 3 weeks before anticipated harvest has the potential to increase preharvest rind color of early-maturing citrus cultivars as a result of increased carotenoid-to-chlorophyll ratio. This treatment provides a novel approach to manipulate chlorophyll degradation and carotenoid synthesis in citrus fruit, and these results support the hypothesis that there may be an inverse relationship between vegetative vigor and rind color development of citrus fruit. Therefore, by moderating vegetative vigor through the use of growth retardants, rind color of citrus fruit can be enhanced.

Biochemical Properties of α-Amylase from Peel of Citrus sinensis cv. Abosora

alpha-Amylase activity was screened in the peel, as waste fruit, of 13 species and cultivars of Egyptian citrus. The species Citrus sinensis cv. Abosora had the highest activity. alpha-Amylase AI from Abosora peel was purified to homogeneity using anion and cation-exchange, and gel filtration chromatographies. Molecular weight of alpha-amylase AI was found to be 42 kDa. The hydrolysis properties of alpha-amylase AI toward different substrates indicated that corn starch is the best substrate. The alpha-amylase had the highest activity toward glycogen compared with amylopectin and dextrin. Potato starch had low affinity toward alpha-amylase AI but it did not hydrolyze beta-cyclodextrin and dextran. Apparent Km for alpha-amylase AI was 5 mg (0.5%) starch/ml. alpha-Amylase AI showed optimum activity at pH 5.6 and 40 degrees C. The enzyme was thermally stable up to 40 degrees C and inactivated at 70 degrees C. The effect of mono and divalent metal ions were tested for the alpha-amylase AI. Ba2+ was found to have activating effect, where as Li+ had negligible effect on activity. The other metals caused inhibition effect. Activity of the alpha-amylase AI was increased one and half in the presence of 4 mM Ca2+ and was found to be partially inactivated at 10 mM Ca2+. The reduction of starch viscosity indicated that the enzyme is endoamylase. The results suggested that, in addition to citrus peel is a rich source of pectins and flavanoids, alpha-amylase AI from orange peel could be involved in the development and ripening of citrus fruit and may be used for juice processing.

2A1-B12 カンキツ果実用移動型選果ロボットの試作(農業用ロボット・メカトロニクス)

Recently, social problems about "Food" are frequently reported such as BSE and camouflage-agricultural product, chemical residues and so on. Accurate traceability information on agricultural production is very important and necessary for solving the problems. To establish the traceability system, sensors collecting information of agricultural products, equipments of network and database are essential. In other words, it is necessary for informatization-agriculture to introduce innovative mechanical systems with a large scale memory function of PC. In this research, development of a mobile citrus fruit grading grading robot is one of objectives. This precision agriculture oriented robot brings labor reduction, farming guidance, quality improvement, environment conservation, and security and safety information. In this paper, constitution of the mobile grading robot and its monitoring camera are described.

Changes in Organic Acids and Sugars during Early Stages of Development of Acidic and Acidless Citrus Fruit

Most of the studies on organic acids and sugars in citrus were performed during fruit maturation, and less is known before this stage of development. The aim of our study was to investigate acids and sugars in lemon, lime, and orange from fruit-set toward development. We chose to compare organic acid and sugar accumulation among acidic and acidless varieties within three species. We estimated the acidity by titrimetry and quantified the concentrations of seven organic acids and three sugars by reverse HPLC. During the first 50 days of development, quinic acid was the major organic acid whatever the variety. Afterward, citric acid predominated in acidic varieties, while in acidless, malic acid exceeded it. Fructose substituted citric acid in acidless and could be synthesized either from citric acid or directly from glucose. Our results provided the first complete report on sugar and organic acid accumulation during the early stages of fruit development in several citrus varieties.

Global Analysis of Gene Expression During Development and Ripening of Citrus Fruit Flesh. A Proposed Mechanism for Citric Acid Utilization

Microarrays of cDNA have been used to examine expression changes of 7000 genes during development and ripening of the fruit flesh of self-incompatible Citrus clementina, a non-climateric species. The data indicated that 2243 putative unigenes showed significant expression changes. Functional classification revealed that genes encoding for regulatory proteins were significantly overrepresented in the up-regulated gene clusters. The transcriptomic study together with the analyses of selected metabolites highlighted key physiological processes occurring during citrus fruit development and ripening such as water accumulation, carbohydrate build-up, acid reduction, pigment substitutions (carotenoid accumulation and chlorophyll decreases) and ascorbic acid diminution. Often, the combined analyses strongly suggested prevalence of specific metabolic alternatives. This observation has been exemplified with the proposal for a mechanism for citrate utilization, a process of much importance in citrus industry. Microarray data validated by real-time RT-PCR suggested that citrate was sequentially metabolyzed to isocitrate, 2-oxoglutarate and glutamate. Thereafter, glutamate was both utilized for glutamine production and catabolyzed through the gamma-aminobutirate (GABA) shunt (GABA --> succinate semialdehyde --> succinate). This last observation appears to be of special relevance since it links the proton consuming reaction glutamate + H(+)--> GABA + CO(2) with high acid levels. GG-MS determinations showed that glutamate was constant while GABA levels decreased at ripening in agreement with a feasible activation of the GABA shunt during acid catabolism. This suggestion provides a convincing explanation for the strong reduction of both citrate and cytoplasmatic acidity that takes place in citrus fruit flesh during development and ripening.

Immature embryo rescue, culture and seedling development of acid citrus fruit derived from interploid hybridization

Interploid sexual hybridizations were completed in 2001 and 2002 between seven lemon (Citrus limon(L.) Burm. f.) varieties, ‘Key’ lime (C. aurantifolia (Cristm.) Swing), Palestine sweet lime (C. imettioides Tan.), ‘Lakeland’ limequat (C. aurantifolia x Fortunella japonica (Thumb.) Swing.), and ‘Etrog’ citron (C. medica L.) as diploid progenitors and four allotetraploid somatic hybrids (‘Key’ lime + ‘Valencia’ orange, ‘Hamlin’ orange + ‘Femminello’ lemon, ‘Valencia’ orange + Rough lemon, and ‘Valencia’ orange+ ‘Femminello’ lemon) in efforts to generate improved seedless triploid acid fruit hybrids. Efficient recovery of triploid progeny from such crosses requires embryo rescue to avoid embryo abortion due to endosperm failure. Germination of rescued genetically diverse immature embryos was induced on two culture media (EME and Gamborg’s B5), with two sucrose concentrations (50 or 70 g l−1). All media contained 0.5 g l−1 malt extract and 4.50 μM GA3. Germination of globular, heart and torpedo shaped embryos (defined as small embryos) was significantly (p < 0.05) affected by medium and genotype. Gamborg’s medium induced 82.89% germination. Of germinated embryos, 11–65% developed into normal plants with differences among crosses. Cotyledonary embryos (defined as immature embryos with fully developed cotyledons) germinated and developed into normal plants at higher rates than less-developed embryos. In efforts to improve the efficiency of plant recovery, small embryos from ‘Todo el ano’ × HF and ‘Lisbon’ × HF crosses conducted during 2002 were rescued and cultured on three media (MS, Gamborg’s, and RMA) for comparison. Media did not significantly affect the proportion of normal plant recovery.

Toward comprehensive expression profiling by microarray analysis in citrus: monitoring the expression profiles of 2213 genes during fruit development

A citrus cDNA microarray containing 2213 independent genes was developed and utilized for a gene-expression profile of fruit development from ovary to mature fruit of Satsuma mandarin ( Citrus unshiu Marc.). A principal component analysis indicated that there were two major patterns bordering on tissue division and specialization. The expression profile of genes responding to fruit development was quite different in each fruit tissue, including the juice sac, albedo and flavedo, suggesting the way in which the unique genetic regulatory networks might be organized during citrus fruit development. The cDNA microarray analysis could efficiently provide an individual gene-expression profile and a new insight to elucidate the biological mechanisms responsible for fruit development.

Flavonol synthase gene expression during citrus fruit development.

We isolated a cDNA clone (CitFLS) encoding flavonol synthase (FLS) from the satsuma mandarin (Citrus unshiu Marc.) fruit and investigated the steady state of CitFLS RNA expression during the fruit development. The CitFLS was 1274 bp long, encoded 335 amino acid residues, and belonged to a family of 2-oxoglutarate-dependent dioxygenases. The level of CitFLS transcript was higher in the young leaves than in the old leaves, and it was high at the early developmental stage and low at the mature stage in the juice sacs/segment epidermis (edible part). On the other hand, the CitFLS transcript increased in the peel during fruit maturation. These results indicated that the satsuma mandarin CitFLS was differentially regulated in the developmental stage and in a tissue-specific manner. Additionally, satsuma mandarin peel tissues produced rutin (a flavonol glycoside) from an exogenous dihydroquercetin (taxifolin), indicating the ability of these tissues to produce flavonols.