Effects of storage conditions on the composition of Citrus tamurana Hort. ex Tanaka (hyuganatsu) essential oil

Compositional changes of Citrus tamurana Hort. ex Tanaka (hyuganatsu) essential oil were analyzed after storage for 1, 3, 6, and 9 weeks at -21, 5, 20, and 30 degrees C. The total amount of oxides increased greatly. The contents of monoterpene alcohols and ketones also increased. The total amount of monoterpene hydrocarbons was unchanged up to 30 degrees C. However, the contents of myrcene, gamma-terpinene, and terpinolene decreased, while there was a considerable increase in p-cymene. Among the sesquiterpene hydrocarbons, considerable increases in the (-)-cedrene, gamma-elemene, and alpha-humullene contents were noted in samples stored at 30 degrees C, and the trans-beta-farnesene content decreased during storage. The total content of monoterpene alcohols was increased slightly. The content of trans-carveol increased during storage. p-Cymene, trans-carveol, isopiperitone, and limonene oxide contents increased, while gamma-terpinene, terpinolene, and citronellal contents decreased significantly during storage. The changes of these compounds during storage can serve as a quality index for hyuganatsu essential oil.     

Hyuganatsu orange (Citrus tamurana Hort. Ex Tanaka) contains a water soluble substance that suppresses bone loss in ovariectomized rats

We investigated whether Hyuganatsu orange (Citrus tamurana Hort. ex Tanaka) contains water and acetic-acid soluble substances that increase bone mineral density (BMD) in ovariectomized rats. In in vivo study, femoral BMD can significantly increased. In in vitro study, tartrate-resistant acid phosphatase (TRAP) positive cells significantly decreased. We speculate that Hyuganatsu orange contains biologically active substances other than hesperidin that increase BMD.     

Identification and characterization of volatile components of the Japanese sour citrus fruit Citrus nagato-yuzukichi Tanaka

A total of 39 aroma compounds were detected in the essential oil of Citrus nagato-yuzukichi Tanaka (nagato-yuzukichi) by gas chromatography-mass spectrometry (GC-MS). The essential oil was characterized by a high percentage of monoterpene hydrocarbons (12 components, 90.52%). The composition pattern of essential oil in C. nagato-yuzukichi was fairly similar to that of Citrus sudachi Hort. ex Shirai (Sudachi). Principal component analysis (PCA) of data obtained with an electronic nose indicated a variation of each oil along PC1. The oils of Citrus junos Tanaka (Yuzu) and Citrus sphaerocarpa Tanaka (Kabosu) showed a clear upward displacement as compared with those of C. nagato-yuzukichi and C. sudachi. However, in PC2, the oils of C. nagato-yuzukichi and C. sudachi showed a displacement in a negative direction and a positive one respectively.     

Comparative analysis of the volatile fraction of fruit juice from different Citrus species

The volatile composition of fruit from four Citrus varieties (Powell Navel orange, Clemenules mandarine, and Fortune mandarine and Chandler pummelo) covering four different species has been studied. Over one hundred compounds were profiled after HS-SPME-GC-MS analysis, including 27 esters, 23 aldehydes, 21 alcohols, 13 monoterpene hydrocarbons, 10 ketones, 5 sesquiterpene hydrocarbons, 4 monoterpene cyclic ethers, 4 furans, and 2 aromatic hydrocarbons, which were all confirmed with standards. The differences in the volatile profile among juices of these varieties were essentially quantitative and only a few compounds were found exclusively in a single variety, mainly in Chandler. The volatile profile however was able to differentiate all four varieties and revealed complex interactions between them including the participation in the same biosynthetic pathway. Some compounds (6 esters, 2 ketones, 1 furan and 2 aromatic hydrocarbons) had never been reported earlier in Citrus juices. This volatile profiling platform for Citrus juice by HS-SPME-GC-MS and the interrelationship detected among the volatiles can be used as a roadmap for future breeding or biotechnological applications.     

Composition of Peel Oil from Citrus Unshu

Constituents of aroma of peel oil (both oleoresin and cold-pressed oil) from Citrus Unshu were isolated by column chromatography and gas chromatography (GLC). and identified mostly by infrared (IR), partially by mass (MS) and nuclear magnetic resonance (NMR) spectroscopy.

One hundred and seven compounds were found in the oil and among them, four sesquiterpene hydrocarbons, five acetates, five carbonyls and five alcohols were identified as a main constituents which contributed to difference in flavor from other citrus peel oils. Characteristics of aroma of C. Unshu was discussed on the basis of quantitative analysis.     

Endogenous free polyamines and their role in fruit set of low and high parthenocarpic ability citrus cultivars

Endogenous free polyamines (PAs), putrescine, spermidine and spermine, from developing fruitlets of Citrus species (Citrus unshiu Marc. and Citrus clementina Hort ex Tanaka) which differ in their parthenocarpic ability, and from uniflowered leafy and leafless inflorescences differing in their ability to set, have been determined by dansylation and separation of dansyl derivatives by HPLC. No significant differences in PAs content were observed between species or between leafy and leafless inflorescences which, nevertheless, significantly differed in fruit set. However, significant differences in their content were found in developing fruitlets, depending on the preceding flowering intensity of the tree and on the fruitlet load. These results suggest that, in Citrus, PAs may act as a nitrogen source rather than a regulator of fruit set.     

Changes in the Volatile Flavor Constituents of Citrus sinensis (L.) Osbeck During Storage

The methods of GC with fused silica capillary column, its Kovats retention index of GC and GC-MS-DS etc. We were used to study the aromatic compounds of the peel essential oil of Citrus sinensis (L.) Osbeck and aromatic concentrates of fruit flesh. 29 chemical constituents from the peels and 40 chemical constituents from the fruit flesh were identified. The trace constituents α-sinensal 0.01% and β-sinensal 0.01% are the special aroma ones for Citrus sinensis. At the same time, the changes flavor components in Citrus sinensis peels and fruit flesh during storage were also explored.     

Improvement of<Emphasis Type="Italic"> Citrus clementina</Emphasis> Hort. ex Tan. microspore-derived embryoid induction and regeneration

An improvement of the protocol for haploid induction through anther culture of Citrus clementina Hort. ex Tan. cv. Nules was achieved following the evaluation of a number of the factors affecting androgenesis. The influence of thidiazuron (TDZ) and three temperature pre-treatments (4°C, 25°C, 32°C) on the floral buds with respect to anther culture of C. clementina Hort. ex Tan., cv. Nules was investigated. An increased embryoid production was induced in the medium supplemented with TDZ. Pre-treatment temperatures of 4°C and 25°C were more favorable for embryo production than 32°C. Regeneration of androgenic haploid plantlets from cv. SRA 63 of C. clementina is reported here for the first time.Abbreviations 6-BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - KI Kinetin - NAA -Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea) - ZEA Zeatin Communicated by L. PeñaBoth authors have contributed equally to this article.     

Temperatures during flower bud development affect pollen germination,self‐incompatibility reaction and early fruit development of clementine (Citrus clementina Hort. ex Tan.)

• One of the key environmental factors affecting plant reproductive systems is temperature. Characterising such effects is especially relevant for some commercially important genera such as Citrus. In this genus, failure of fertilisation results in parthenocarpic fruit development and seedlessness, which is a much‐prized character. Here, we characterise the effects of temperature on flower and ovary development, and on pollen–pistil interactions in ‘Comune’ clementine (Citrus clementina Hort. ex Tan.).
• We examine flower bud development, in vitro pollen germination and pollen–pistil interaction at different temperatures (15, 20, 25 or 30 °C). These temperatures span the range from ‘cold’ to ‘hot’ weather during the flowering season in many citrus‐growing regions.
• Temperature had a strong effect on flower and ovary development, pollen germination, and pollen tube growth kinetics. In particular, parthenocarpic fruit development (indicated by juice vesicle growth) was initiated early if flowers were exposed to warmer temperatures during anthesis.
• Exposure to different temperatures during flower bud development also alters expression of the self‐incompatibility reaction. This affects the point in the pistil at which pollen tube growth is arrested and confirms the role of sub‐ and supra‐optimal temperatures in determining the numbers of pollen tubes reaching the ovary.

     

Temperature stress interferes with male reproductive system development in clementine (Citrus clementina Hort. ex. Tan.)

Male gametophyte development is a critical phase of the plant life cycle due to its high sensitivity to environmental stresses. The rise in the average global temperature, often accompanied by extreme fluctuations, has an important impact on biological processes. Among those, male gametophytes are particularly sensitive to temperature stress during flower bud development and anthesis. Male gametophyte development was extensively studied in several plant species, but little information is available about the effects of temperature stress on male gametophyte development in the genus Citrus. We evaluated the effects of cold and hot temperatures during microsporogenesis and microgametogenesis on one of the most economically valuable citrus species, the “Comune” clementine (Citrus clementina Hort. ex. Tan.). The effect of constant temperature on the androecium was evaluated by a time course histological analysis performed on the anthers and by monitoring in vitro pollen germination. The results revealed how suboptimal hot and cold temperatures induce drastic alterations on the morphology of the tapetal cells, microspores and mature pollen grains. Shifting from the optimal temperature affected the timing of starch depletion in the anther walls, such as epidermis, endothecium and middle layer, influencing the pollen germination rate and pollen tube growth. To the best of our knowledge this is the first study attempting to assess how temperature stress affects male reproductive development in citrus. A better understanding of the mechanisms underlining male sterility will provide novel insights to elucidate the physiology of this agronomical important quality trait.     

The effect of light quality on anther culture of <Emphasis Type="Italic">Citrus clementina</Emphasis> Hort. ex Tan.

This preliminary research reports results on the influence of light quality on anther culture of Citrus clementina Hort. ex Tan., cultivar Nules. After one month of cultivation in darkness, four light qualities were tested: White, Red, Far-Red and Blue. Continuous Darkness and White light under photoperiod of 16 hrs were used as a control conditions. Gametic embryoids and embryogenic callus were obtained only under photoperiodic conditions of White light, suggesting that the alternation of light and dark can be used for the process of gametic embryogenesis in Citrus.     

Isotope Ratio by HRGC-MS of Citrus junos Tanaka (Yuzu) Essential Oils: m/z 137/136 of Terpene Hydrocarbons

The isotope ratios of monoterpene hydrocarbons in Citrus junos Tanaka (yuzu) essential oils from different origins were determined by ordinary high-resolution gas chromatography-mass spectrometry (HRGC-MS). Both intensities of the molecular mass peaks (m/z 136) and of the isotope peaks (m/z 137) of monoterpene hydrocarbons were measured by single-ion monitoring with an MS analysis. The isotope ratios (m/z 137/136) of the ten monoterpene hydrocarbons commonly contained in citrus essential oils, α-pinene, β-pinene, sabinene, myrcene, α-phellandrene, α-terpinene, limonene, γ-terpinene, β-phellandrene and terpinolene, were determined in yuzu samples of the highest commercial quality from 42 different production districts. Statistical treatment of these data by the t-test and sign test revealed significant differences of the isotope effects in each yuzu sample. It is suggested that this technique will be applicable for evaluating the quality, genuineness and origin of citrus fruits and their products. The isotope fingerprints were also demonstrated in several citrus fruits other than the yuzu samples.     

水分胁迫下丛枝菌根真菌对红橘叶片活性氧代谢的影响

研究了水分胁迫下接种地表球囊霉（Glomus versiforme (Karsten) Berch）对红橘(Citrus tangerine Hort. ex Tanaka)叶片活性氧代谢的影响.结果表明：水分胁迫显著抑制了地表球囊霉对红橘根系的侵染,抑制率为33％.在正常供水和水分胁迫下,接种地表球囊霉处理的红橘叶片磷含量显著增加,与未接种处理相比,分别增加了45％和27%,丙二醛（MDA）和H₂O₂含量分别降低了25％、21％和16％、16％.正常供水和水分胁迫下接种地表球囊霉增强了叶片超氧化物岐化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性;提高了可溶性蛋白质、还原型抗坏血酸（ASC）和总抗坏血酸(TASC)含量.水分胁迫下接种处理显著降低了叶片超氧阴离子自由基（O₂^-_·）含量,与正常供水相比降低了31％.表明菌根化红橘植株的抗旱性增强.     

Intervention thresholds for Aphis spiraecola (Hemiptera: Aphididae) on Citrus clementina

The production of clementine fruit was affected by varying densities of Aphis spiraecola Patch (Hemiptera: Aphididae) maintained on four groups of clementine, Citrus clementina Hort. ex Tan., trees individually isolated in mesh cages. A formula relating the number of aphids per square meter of canopy per group of trees to yield loss in that group of trees was obtained, permitting us to calculate the economic injury levels (EILs) and economic thresholds (ETs) for treatment against A. spiraecola, i.e., EIL, ET, environmental economic injury level (EEIL), and environmental economic threshold (EET). In an example case with current values, EIL = 370, ET = 322, EEIL = 614, and EET = 533 aphids per m2 of canopy. Formulae designed for easier use in the field were obtained to express each of these thresholds. The formulae are compared with those obtained for Aphis gossypii Glover in a previous study; the intervention thresholds for A. spiraecola are slightly higher than those found for A. gossypii.     

1-Aminocyclopropane-1-Carboxylic Acid Transported from Roots to Shoots Promotes Leaf Abscission in Cleopatra Mandarin (Citrus reshni Hort. ex Tan.) Seedlings Rehydrated after Water Stress

The effect of water stress and subsequent rehydration on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity, ethylene production, and leaf abscission was studied in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings. Leaf abscission occurred when drought-stressed plants were allowed to rehydrate, whereas no abscission was observed in plants under water stress conditions. In roots of water-stressed plants, a high ACC accumulation and an increase in ACC synthase activity were observed. Neither increase in ACC content nor significant ethylene production were detected in leaves of water-stressed plants. After rehydration, a sharp rise in ACC content and ethylene production was observed in leaves of water-stressed plants. Content of ACC in xylem fluid was 10-fold higher in plants rehydrated for 2 h after water stress than in nonstressed plants. Leaf abscission induced by rehydration after drought stress was inhibited when roots or shoots were treated before water stress with aminooxyacetic acid (AOA, inhibitor of ACC synthase) or cobalt ion (inhibitor of ethylene-forming enzyme), respectively. However, AOA treatments to shoots did not suppress leaf abscission. The data indicate that water stress promotes ACC synthesis in roots of Cleopatra mandarin seedlings. Rehydration of plants results in ACC transport to the shoots, where it is oxidized to ethylene. Subsequently, this ethylene induces leaf abscission.     

Quantitative trait locus (QTL) analysis of fruit-quality traits for mandarin breeding in Japan

The improvement of fruit quality is an important objective in citrus breeding. Using an F₁ segregating population from a cross between citrus cultivars ‘Harehime’ (‘E647’—‘Kiyomi’ [Citrus unshiu Marcow. ‘Miyagawa Wase’ × Citrus sinensis (L.) Osbeck ‘Trovita’] × ‘Osceola’—a cultivar of clementine [Citrus clementina hort. ex Tanaka] × ‘Orland’ [Citrus paradisi Macfad. ‘Duncan’ × Citrus tangerina hort. ex Tanaka] × ‘Miyagawa Wase’) and ‘Yoshida’ ponkan (Citrus reticulata Blanco ‘Yoshida’), a SNP-based genetic linkage map was constructed and quantitative trait locus (QTL) mapping of four fruit-quality traits (fruit weight, sugar content, peel puffing, and water rot) was performed. The constructed genetic linkage map of ‘Harehime’ consisted of 442 single nucleotide polymorphisms (SNPs) on 9 linkage groups (LGs) and covered 635.8 cM of the genome, while that of ‘Yoshida’ ponkan consisted of 332 SNPs on 9 LGs and covered 892.9 cM of its genome. We identified four QTLs associated with fruit weight, one QTL associated with sugar content, three QTLs associated with peel puffing, and one QTL associated with water rot. For these QTL regions, we estimated the haplotypes of the crossed parents and verified the founding cultivars that these QTLs were originated from and their inheritance in descendant cultivars using pedigree information. QTLs identified in this study provide useful information for marker-assisted breeding of citrus in Japan.     

Studies on Leaf Oils of Citrus Species

Leaf oils from 6 domestic citrus species were analysed by gas chromatography and 32 compounds were identified as constituents of one or more oils. Most components present were common to all 6 citrus leaf oils, but the percent composition of some components, i.e. β-pinene, limonene, β-phellandrene, γ-terpinene, p-cymene, p-α-dimethylstyrene, citronellal, linalool and thymol methyl ether, differed considerably from species to species. For example, γ-terpinene is a main component (36.5%) of Shiikuwasha (Citrus depressa Hayata), but is a trace in leaf oils from Kawabata-mikan (C. aurea Tanaka) and Otaheite-orange (C. limonia Osbeck var. otaitensis Tanaka). Yuzu (C. Junos Sieb. ex Tanaka) is characterized by relatively high contents of β-phellandrene (11.2%) and p-α-dimethylstyrene (6.7%), which are minor constituents in the other 5 citrus leaf oils. Otaheite-orange is characterized by high contents of limonene (39.7%) and citronellal (10.0%).     

沙田柚配子体自交不亲和花柱消减文库的构建及分析

分别以沙田柚自交花柱cDNA为tester,异交花柱cDNA为driver,利用抑制性消减杂交技术构建了消减文库,文库的重组率高于95%,插入片段集中在100～500 bp之间,对文库部分克隆进行测序并与GenBank中的同源序列进行比较,发现了一些类似于SI、S9-RNase、激酶类等与自交不亲和相关的基因.     

Isoflavonoids in the Rutaceae family: 1. Fortunella obovata, Murraya paniculata and four Citrus species

Several types of compounds with immunoreactivity similar to isoflavonoids were detected in water: ethanol extracts of leaves of Fortunella obovata Hort. ex Tanaka, Murraya paniculata Jack. and four Citrus species, namely C. aurantium L, C. grandis Osbeck, C. limonia Osbeck., and C. sinensis Osbeck (Rutaceae). The chromatographic mobilities of the immunoreactive substances were compared with those of authentic standards, revealing a spectrum of isoflavonoid metabolites in all plants studied. Aglycones as well as glycosides were recognized, namely daidzin, genistin, daidzein, genistein, formononetin, biochanin A, prunetin, and several incompletely characterized isoflavonoids. A subsequent HPLC-MS study verified the identities of the main immunoreactive isoflavonoids and established the identities of several others, viz. glycitein, glycitin, ononin and sissotrin, including the malonylated and acetylated isoflavonoid glucosides. The estimated content of the individual immunoreactive entities ranged from a few microg to about 2 mg/kg (dry weight). It is concluded that the isoflavonoid metabolic pathway is present throughout the Rutaceae family.