Study on Peroxidase Isozymes in Citrus and Fortunella

The acidic peroxidase isozymes in petals of 106 Citrus and Fortunella species and cultivars were studied using polyacrylamide gel electrophoresis. The results (Fig. 1; Tables 1 and 2) suggest that these isozymes be of the monomeric nature coded by a single locus. Five alleles were identified and designated as A, B, C, D and F respectively. No genetic difference in peroxidase isozymes was detected among the mandarins orignated from China, Japan and Indian. Man Ju may be a hybrid between Ponkan and Ougan. Ponkan may have not been involved in the origin of Jiao Gan. Zheng Jiang Jin Ju should be an infraspecific hybrid of C. reticulata. C. sinensis has a close relationship with some Chinese mandarins. Changshou Kumquat and Calamondin are shown to be intergeneric hybrids between Citrus andFortunella.     

伏令夏橙与宁波金柑属间体细胞杂种变异研究

伏令夏橙（Ｃｉｔｒｕｓｓｉｎｅｎｓｉｓ（Ｌ．）Ｏｓｂｅｃｋ）＋宁波金柑（ＦｏｒｔｕｎｅｌａｃｒａｓｉｆｏｌｉａＳｗｉｎｇｌｅ）属间体细胞杂种在田间生长６年,树势较弱,新梢经常枯死,树冠参差不齐。染色体检查发现,除了四倍体之外,还存在其它倍性的细胞,呈嵌合体状态。酯酶同工酶图谱上,多数单株出现一条双亲没有的新带。ＲＡＰＤ分析结果表明,部分植株丢失了亲本的标记带型,表现出遗传上的不稳定性     

Variation of Intergeneric Somatic Hybrids Between Citrus sinensis cv. Valencia and Fortunella crassifolia cv. Meiwa

Intergeneric somatic hybrid plants derived from protoplast fusion between Citrus sinensis (L.) Osbeck cv. Valencia and Fortunella crassifolia Swingle cv. Meiwa have been grown in the fields for 6 years. The plants grew less vigorously with uneven canopy size and some shoots frequently died. Chromosome count showed that the plants, containing non-tetraploid cells besides amphidiploid, were chimeras. Most somatic hybrid plants exhibited a new band of EST isozyme. RAPD analysis verified that several plants lacked some of the parents' markers. The results indicated that the intergeneric somatic hybrids were genetically unstable.     

The role of carotenoid cleavage dioxygenases in the regulation of carotenoid profiles during maturation in citrus fruit

To investigate the relationship between a carotenoid profile and gene expression for carotenoid cleavage dioxygenases, three citrus varieties that exhibit different 9-cis-violaxanthin levels in their juice sacs, Satsuma mandarin (Citrus unshiu Marc.; a variety accumulating a low level of 9-cis-violaxanthin), Valencia orange (Citrus sinensis Osbeck; variety accumulating a high level of 9-cis-violaxanthin), and Lisbon lemon (Citrus limon Burm.f.; a variety accumulating an undetectable level of 9-cis-violaxanthin) were used. Three cDNAs (CitCCD1, CitNCED2, and CitNCED3) were cloned. The recombinant CitCCD1 protein cleaved beta-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9-10 and 9'-10' positions and 9-cis-violaxanthin at the 9'-10' position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11-12 position to form xanthoxin, a precursor of abscisic acid (ABA). The gene expression of CitCCD1 increased in the flavedos and juice sacs of the three varieties during maturation. In Satsuma mandarin, the gene expression of CitNCED2 and CitNCED3 increased noticeably, accompanying a massive accumulation of ABA in the flavedo and juice sacs. In Valencia orange, the gene expression of CitNCED3 increased with a slight elevation of the ABA level in the flavedo, whereas neither the gene expression of CitNCED2 nor the ABA level increased noticeably in the juice sacs. In Lisbon lemon, the gene expression of CitNCED2 increased remarkably, accompanying increases in the ABA level in the flavedo and juice sacs. These results suggest that, in the juice sacs, the efficient cleavage reaction for ABA synthesis reduces the 9-cis-violaxanthin level in Satsuma mandarin and Lisbon lemon, whereas the low cleavage reaction maintains the predominant 9-cis-violaxanthin accumulation in Valencia orange.     

Flavonoid composition of fruit tissues of citrus species

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.     

Quantification of multi-residue levels in peach juices, pulps and peels using dispersive liquid-liquid microextraction based on floating organic droplet coupled with gas chromatography-electron capture detection

In this paper, polychlorinated biphenyl (PCB), organochlorine pesticide (OCP) and pyrethroid pesticides in peach was investigated by comparing their residual level in peach juice, pulps and peels using dispersive liquid-liquid microextraction based on solidification of floating organic droplet (DLLME-SFO) combined with gas chromatography-electron capture detection (GC-ECD). Extraction conditions such as the type of extractant, volume of extractant and dispersant, salt effect and extraction time were optimized. For juice samples, the linearity of the method was obtained in the range of 10-2000 ng L(-1),with determination coefficients>0.99. The limits of detection (LOD) of the method were ranged between 2.8 and 18.5 ng L(-1). For pulp and peel samples, the developed method is linear over the range assayed, 1-20 μg kg(-1),with coefficients also >0.99. The relative recoveries of compounds analyzed from juice, pulp and peel samples were in the range of 73-106% with a relative standard deviation between 2.6 and 11.8%. The proposed method was applied to the simultaneous analysis of residues in real peach juice, pulp and peel samples. As a result, there were no target analytes found in peach juices and pulps while 3.3 μg kg(-1) cyhalothrin and 3.5 μg kg(-1) fenvalerate were found in peels. The experiment results revealed that the pyrethroid residues just deposited on the peels of the fruits, but did not move into pulps and juices.     

The Transport of Sugars in Developing Fruits of Satsuma Mandarin

Transport of sugars to the juice sacs of developing satsumamandarin (Citrus unshui Marc) has been studied in attached fruitsand in isolated fruit pieces. ¹⁴CO₂ fed to the leaves resultedin [¹⁴C]sugar accumulation in the juice sacs, mainly as [¹⁴C]sucrose.Uptake of sucrose and glucose by the excised fruit pieces proceededlinearly with time. Sucrose uptake was linearly related to sucroseconcentration over the range 25–300 mM, with no indicationof saturation. This uptake was insensitive to pH (5, 7 or 9),Ca²⁺(3 mM), PCMBS (2.5 mM), DNP (1 mM) or vanadate (0.1 mM)but was slightly reduced by erythrosin (21 % by 0–1 mM;27 % by 1 mM). No competitive effect of glucose (up to 100 mM)was detectable on sucrose uptake from 100 mM solution. Mostof the [¹⁴C]sucrose uptake observed was reversible, althoughconsiderable hydrolysis and metabolic conversion were evidenced.A vanadate-sensitive ATPase was demonstrated by EM localizationon the plasma membrane of the juice sac cells. These resultsare interpreted in relation to the accumulation of assimilatesby the developing fruit. Transport: sugar, satsuma mandarin, juice sacs     

Management of Citrus sinensis peels for protection and treatment against gastric ulcer induced by ethanol in rats

Abstract

Introduction: Stomach ulcer is one of the most prevalent disorders worldwide. The study was aimed to isolate and characterize the major polymethoxylated flavonoids in Citrus sinensis peels petroleum ether extract and investigate its protective and curative effect on gastric ulcer.

Material and methods: Some spectral analyses were used for identification of the isolated compounds from the petroleum ether extract of Citrus sinensis peels. One oral dose (0.5?ml/100?g b.wt.) of absolute ethanol was orally given to rats after starvation for 24?h to induce gastric ulcer. To explore the protective and curative role of the plant extract, it was orally (250?mg/kg b.wt.) given for 1 week either before or post-ulcer induction. A reference drug, ranitidine (100?mg/kg b.wt.), was also evaluated. Stomach acidity, gastric volume, lesion counts, glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), acid phosphatase (AP), interlukin-10 (IL-10) and prostaglandin E2 (PGE2) were estimated. Stomach histopathological features were monitored.

Results: Nine polymethoxy flavonoids were identified from the extract. Treatment with C. sinensis peels extract recorded amelioration in all parameters.

Conclusion: Citrus sinensis petroleum ether peels extract had protective and curative effects against gastric ulcer. Therefore, the extract recorded anti-secretory, anti-ulcerative, anti-inflammatory, and antioxidant effects. Its healing action exceeded its protective role due to its richness in polymethoxylated flavonoids     

A Numerical Taxonomic Study of Citrus and Its Close Relatives

Fifty nine taxa of Citrus, Fortunella and Poncirus were studied by hierarchical agglomerative clustering analysis and quantas type 3 analysis using 86 morphological characters. Five affinity groups were obtained in Citrus. The first group includes C. medica, C. limon, C. limonia, C. aurantifolia and C. iambhiri; the second includes C. grandis, C. aurantium, C. sinensis and C. paradisi; the third is mandarin (C. reticulata); the forth is C. ichangensis; The last is C. hongheensis. Mandarin oranges show two subgroups: one comprise satsuma, King, Shagan, Tankan and Bendiguangju, and the other consists of the remaining typical mandrins. The two papeda oranges, honghe papeda and ichang papeda, could not be clustered into the same group, indicating that they might have evolved from two compeletly different ancestors. Pumelos(C. grandis) show some relationships to honghe papeda orange. C. limonia is assumed to be a hybrid between rough lemon and mandarin. The sweet orange(C. sinensis) is considered to be the offspring of pomelo and mandarin. Zhekiang jinju a small mandarin generally considered similar to calamondin in China, should be a true mandarin orange based on our results. Poncirus was well distinguished from the other two genera, Citrus and Fortunella, by the numerical methods used in this study. Our results show that citron (C. medica), mandarin (C. reticulata) and pumelo (C. grandis) are the original species of the subgenus Citrus, which was congruent with the re-sults obtained by Barrett and Soost (1976), Potvin (1983) and Handa (1985).     

A Study on Glutamate Oxaloacetate Transaminase Isozymes of Citrus Germplasm Resources

The glutamate oxaloacetate transaminase isozymes (GOT) extracted from 125 biotypes of Citrus and its relatives, Fortunella, Poncirus and Microcitrus were analyzed using polyacrylamide gel electrophoresis to investigate the taxonomic relationships among citrus plants. Besides all the isozymes reported before, two new bands were detected and designated their putative alleles B and C in GOT-l. Among pummelo cultivars wide variations were found. Most of the mandarins were identical, having SS for GOI-1 and MM for GOT-2 except for Zou-Pi-Gan and Yao-Gan, which both had FS at GOT-1 and might be hybrids. This finding suggests that all of the mandarins may have originated from a common ancestor. Sour orange biotypes showed a considerable variation in GOT isozymes. Most of the sour oranges in China were assumed to be hybrids between pummelo and mandarin based on GOT isozyme patterns, but two biotypes, Xiao-Hong-Cheng and Zhu-lan, had FS at GOT-1 and MB at GOT-2, which strongly suggests that they be hybrids of pummelo and Ichang papeda because B allele of GOT-2 occurs only in Ichang papeda and its close relatives Yuzu and Ichang lemon. From this study Yuzu is assumed to be derived from hybridizationof Ichan, papeda and mandarin.     

Numerical Chemotaxonomical Study on Citrus Plants Cultivated in China

Soluble proteins were extracted from mature leaves of 83 citrus biotypes. Their protein patterns were obtained by acrylamide gel electrophoresis. The similarity of the protein patterns among the samples was calculated by computer according to the principle and methods of numerical taxonomy. The similarity comparison was made between different species and varieties included in the Swingl’s system. The distance coefficients of similarity (DCS) is 2.13 between species in the genus Citrus, 0.927 in Poncirus and 0.617 among Fortunella species. The DCS of biotypes in Citrus limon, C. paradisi and C. sinensis are zero, and those in C. grandis and C. reticulata are 0.656 and 0.863 respectively. There is no difference among satsuma mandarin, Tankan and King mandarin, which are considered respectively as a good species in Tanaka’s system. Ancestors of some biotypes. whose origin was unknown berore, are postulated in this work. Jiang Bai Shang Cheng is probably a hybrid between C. junos Tanaka and mandarin, rough lemon may be a hybrid of mandarin with citron or lime, but not with lemon. Some so-called lemon biotypes are not in the same cluster as true lemon (Eureka or Lishon). The biotypes. of sour orange, all of which are scattered in the phenetic tree of cluster analysis (PTCA), might have been derived from different ancestors. In the PTCA the evolutionary tendency from Poncirus to Macroacrumen in the citrus plants may be found on the basis of the DCS of different genera, sections and subsections respectively with the subsection Macroacrumen. The present author considers that if Fortunella is recognized as true genus, Cephalocitrus should also be an acceptable genus. The position of Fortunella in the PTCA is between the sections Cephalocitrus and Aurantium, but it does not represent its position in the phylogeny. Taking comprehensive data into consideration it appear to be more reasonable to place C. ichangensis, as member of Papeda originated in the subtropics, in the section Papeda.     

Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids

Organelle DNA inheritance of four 10-year-old somatic hybrid trees between Valencia orange [Citrus sinensis (L.) Osbeck] and Meiwa kumquat (Fortunella crassifolia Swingle) was analyzed by cleaved amplified polymorphic sequence (CAPS) and restriction fragment length polymorphisms (RFLPs). Five chloroplast (cp) and three mitochondrial (mt) universal primer pairs were amplified, but no polymorphisms were detected. When the polymerase chain reaction products were digested by 15 restriction enzymes, four polymorphic cpDNA-CAPS and two mtDNA-CAPS markers were found. Both the cpDNA and mtDNA in the somatic hybrids were derived from Valencia orange (the embryogenic suspension parent). Genomic DNA of the somatic hybrids and corresponding parents was digested by five restriction endonucleases and hybridized with one chloroplast probe (RbcL- RbcL) and nine mitochondrial probes (coxI, coxII, c oxIII, c ob, atpA, tyr, proI, atp6 and atp9). The results indicated that three hybrid plants shared one strong cpDNA band with both parents and that the remaining one plant had two additional novel bands besides the shared band, while their mtDNA was identical to that of Valencia orange plus non-parental bands. When data on the mtDNA banding patterns were combined with observations on phenotypic performance in the field, it was found that the more complex mtDNA banding pattern coincided with increased vigor of the plant. The stability of the organelle genomes was studied by extracting the genomic DNA of one hybrid plant at monthly intervals for 1 year and then analyzing it using RFLPs. Before the dieback of the shoots, two fragments of the mtDNA were lost while the cpDNAs remained stable. Ploidy analysis by flow cytometry showed that all of the hybrids were stable tetraploids. Four simple sequence repeat primer pairs were applied to detect microsatellite alleles of the four hybrid plants, both parents and the 12 DNA samples from one plant. The results showed that all hybrids had biparental bands uniformly, which indicated that they had the same nuclear background. These results suggest that the mtDNA pattern is correlated with the phenotypic abnormality of Valencia and kumquat somatic hybrid plants and that nuclear-cytoplasm incompatibility may be the cause of dieback.     

Postphloem, nonvascular transfer in citrus: kinetics, metabolism, and sugar gradients

Postphloem, nonvascular assimilate transport occurs over an unusually long area in citrus fruit and thus facilitates investigation of this process relative to sugar entry into many sink structures. Labeled photosynthates moving into juice tissues of grapefruit (Citrus paradisi Macf.) slowed dramatically after entering the postphloem transport path (parenchyma cells, narrow portions of segment epidermis, and hair-like, parenchymatous stalks of juice sacs). Kinetic, metabolic, and compositional data indicated that transfer through the nonvascular area was delayed many hours by temporary storage and/or equilibration with sugars in compartments along the postphloem path. Labeled assimilates were generally recovered as sucrose throughout the path, and extent of hexose formation enroute bore no apparent relationship to the assimilate transfer process. Even after 24 hours, radiolabel was restricted to discrete, highly localized areas directly between vascular bundles and juice sacs. Postphloem transfer occurred against an ascending sucrose concentration gradient in young fruit, whereas a descending gradient (favoring diffusion/cytoplasmic streaming) developed only later in maturation. Involvement of a postphloem bulk flow is complicated in the present instance by the extremely limited water loss from juice sacs either via transpiration or fluid backflow. Nonetheless, tissue expansion can account for a collective water inflow of at least 1.0 milliliter per day throughout the majority of juice sac development, thus providing a modest, but potentially important means of nonvascular solution flow. Overall, data indicate postphloem transfer (a) can follow highly localized paths through sizable nonvascular areas (up to 3.0 centimeters total), (b) appears to involve temporary storage and/or equilibration with compartmentalized sugars enroute, (c) can occur either against an overall up-hill sugar gradient (young tissues) or along a descending gradient (near full expansion), and (d) appears to involve at least some contribution by nonvascular mass flow accommodated by tissue expansion.     

不同贮藏条件对沙田柚果实苦味物质含量的影响

沙田柚原产广西,营养丰富且适合高血压和糖尿病等患者食用,近年来在沙田柚产业中存在品质下降、有苦味、异味生成等问题。为了研究不同贮藏条件下沙田柚苦味物质的含量变化,该文利用高效液相色谱仪,对室温包薄膜袋(A)、室温不包薄膜袋(B)和4℃低温不包薄膜袋(C)三种不同贮藏条件下,沙田柚外果皮、中果皮、囊衣、汁胞和种子中柚皮苷、柠檬苦素和诺米林在贮藏过程中的含量变化进行了测定。结果表明：沙田柚果实苦味物质以柚皮苷为主,主要分布在中果皮和囊衣中,柠檬苦素主要分布在种子和外果皮中,诺米林主要分布在种子中。各处理果实在贮藏过程中苦味物质含量变化不同,其中柚皮苷含量略有上升,柠檬苦素和诺米林含量整体呈下降趋势,略有波动,而汁胞中柠檬苦素和诺米林含量先上升再下降。三种不同贮藏条件相比,4℃低温不包薄膜袋贮藏的果实汁胞中柚皮苷和柠檬苦素的含量最高,在贮藏第30天,A、B、C 三种不同贮藏条件果实汁胞中柚皮苷含量分别为(0.139±0.006)、(0.190±0.009)和(0.194±0.019) mg?g-1,柠檬苦素含量分别为(47.28±1.91)、(33.64±1.90)和(84.19±5.56)μg?g-1。与此相反,外果皮、中果皮、囊衣和种子中柚皮苷、柠檬苦素和诺米林含量最低。该研究结果为采后沙田柚贮藏保鲜方式的选择提供了理论指导。     

Studies on Taxonomy and Evolution of Citrus and Its Related Genera by Isozyme Analysis

The leaf or bark tissue extracts of 108 biotypes of Citrus and its five related genera were analyzed by polyacrylamide gel electrophoresis for isozymes of peroxidase, glutamate oxaloacetate transaminase, phosphoglucose isomerase, phosphoglucose mutase, superoxide dismutase, tetrazolium oxidase, NADP+- dependent malate dehydrogenase and esterase. The inter-or intra-generic differences of isozymograms were compared. Dissimilarities of the isozyme patterns among the samples were calculated by computer based on the UPGMA method to demonstrate the phylogenetic relationship of the biotypes. There are remarkable isozymogramatic differences among the six genera. Generally speaking, each genus possesses its own unique bands. Based on the cluster analysis, the total Citrus biotypes are classified into seven groups, i.e. Honghe papeda, Mauritius papeda, Pummelo, Ichang papeda, Citron, Lemon-lime and Mandarin-orange. Ichang papeda is suggested to be the third subgenus of the genus Citrus. The Fuming trifoliata orange is accepted as a new species of Poncirus-P. polyandra S. Q. Ding. It might be the evolutionary bridge between Poncirus and Citrus, and one of the “missing links” as suggested by W. T. Swingle. Microacrumen (small- fruited mandarin) is the primitive form of the mandarins. It seems better to consid er“ Horse-nose mandarin ”or “Xipigoushigan mandarin” as the typic mandarin originated in China. Euacrumen (large-fruited mandarin) might have arised through two ways. One is hybridization of Microacrumen and C. sinensis, and the other is just the variation of Microacrumen. Ancestors of some biotypes are inferred based on isozyme data. Fortunella obovata Tan. may be a hybrid of Fortunella and Citrus. C. unshiu Marc. arose from the nucellar mutation of“Huangyanbendiguangju mandarin”( C. nobilis Lour.) in Huangyan of Zhejiang Province. C. junos Sieb. came from hybridization of Ichang papeda and mandarin. C. grandis Osbeck and mandarin may have been involved in the origin of C. aurantium L.. C. limonia Osbeck may be a hybrid between C. medica L. and mandarin. The evolutionary trend of citrus fruit trees is proposed, anda tentative phylogenetic diagram is drawn.     

25个种和品种的柑桔果皮精油的化学成分

用双柱毛细管气相色谱保留时间法和GC-MS法分析了25个种和品种的柑桔果皮精油的化学成分,共鉴定出精油化学成分61个。所有精油的主要成分都是柠檬烯。依据精油中柠檬烯含量的不同,将被分析的25个植物种和品种分成三组:枸橼、柠檬类为一组,精油中柠檬烯含量为50％～70％;酸橙、甜橙和柚类为一组,精油中柠檬烯的含量大于92％;宽皮柑桔类为一组,精油中柠檬烯的含量为70％～90％(少数种达94.18％)。这一实验结果与近来有关栽培柑桔只有枸橼、柚和宽皮柑桔具有生物学种的价值的看法相类似。     

Isoflavones in the Rutaceae family: twenty selected representatives of the genera Citrus, Fortunella, Poncirus, Ruta and Severinia

Enzyme-linked immunosorbent assays in combination with semi-preparative high-performance liquid chromatography (HPLC) and analytical HPLC with mass spectroscopy in the selective ion monitoring mode were used for the determination of selected isoflavones, daidzein, genistein, biochanin A and their homologues, in 20 representatives of the Rutaceae family. Species belonging to five genera were studied, namely Citrus, Fortunella, Poncirus, Ruta and Severinia. The enzyme immunoassays used were based on polyclonal antibodies raised against isoflavonoid conjugates with bovine serum albumin (BSA), namely biochanin A-7-BSA, daidzein-7-BSA, daidzein-4'-BSA, genistein-7-BSA and genistein-4'-BSA. Aglycones as well as glycosides were detected, and methoxyisoflavones appeared to be more abundant than hydoxyisoflavones. The content of individual isoflavonoids ranged from 0 to 2.6 mg/kg (dry weight); the sum of all measured substances reached up to 5.9 mg.     

Saturated and mono-unsaturated long-chain hydrocarbon profiles from mandarin juice sacs

The long-chain saturated and mono-unsaturated hydrocarbon content of the juice sacs of five mandarin cultivars (Mediterranean, Honey, Wilking, Kinnow, King) were examined. Normal homologues accounted for more than 47% of the saturated and more than 75% of the monoene hydrocarbons. In the saturated fraction the major hydrocarbon was n-C₂₅ but in the monoene fraction n-C₂₅ predominated in Kinnow and King while C₂₉ predominated in Mediterranean, Honey and Wilking. All five cultivars could be differentiated from each other and from other citrus species by their hydrocarbon patterns. The noticeably high normal/iso ratios of saturated C₂₃ and C₂₅ hydrocarbons previously shown to be characteristic of mandarin species, Citrus unshiu and C. reticulata, were also found in C. nobilis and C. deliciosa.