硫酸锰矫治红壤改良橙（Citrus Sinensis Osbeck）缺锰的效应

对酸性红壤改良橙缺锰植株土施及叶面喷布硫酸锰均可明显地提高植株锰含量，促进枝梢生长与结果。土壤施锰可明显地提高土壤代换态锰及易还原态锰含量，有效地改善缺锰红壤柑桔园的土壤与树体锰素水平。可在盛果期每株年施硫酸锰０．２５ｋｇ，亦可在春夏季叶面喷布０．２％～０．４％硫酸锰。

两类柑桔杂种砧木对脐橙(Citrus sinensis Osbeck)幼树生长和叶片糖营养含量年变化的影响

以体细胞杂种红桔+枳和红桔+粗柠檬、有性杂种Troyer枳橙和Swingle枳柚作砧木的耐湿脐橙(CitrussinensisOsbeck)2年生嫁接苗为试材,通过盆栽试验研究了砧木对苗木生长和叶片糖营养含量年变化动态的影响。结果表明:红桔+枳的生长势和花量明显地强于和大于其它砧木,红桔+粗柠檬的生长和花量表现介于2种有性杂种之间。叶绿素a/b和类胡萝卜素含量在不同砧木间无显著差异,而不同砧木显著或极显著地影响叶绿素含量各指标。砧木影响叶片可溶性糖和淀粉含量年变化动态的基本特征。在休眠期,红桔+枳的可溶性糖和淀粉含量都高于2种有性杂种砧木,而红桔+粗柠檬则反之;在生长期,红桔+粗柠檬的可溶性糖和淀粉含量表现出高于有性杂种砧木的趋势,而红桔+枳则介于2种有性杂种砧木之间。同时还重点讨论了2种体细胞杂种砧木的利用价值。

In vitro Plant Regeneration from the Mature Tissue of Navel Orange (Citrus sinensis L. Osbeck) by Direct Organogenesis

An efficient in vitro regeneration system by direct organogenesis from mature nodal and internodal stem segments ofNewhall navel orange (Citrus sinensis L. Osbeck) was developed. Illuminating conditions together with plant growthregulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation isbeneficial for the adventitious bud regeneration. The highest regeneration frequency (85.2%) and bud formationefficiency (3.7 per responsive internodal stem segment) were obtained in the media supplemented with 1.0 mg L-1 BAPand 0.5 mg L-1 NAA. ABA at 0.2 mg L-1 positively affected the bud formation efficiency, which amounted to 8.5 buds perinternodal segment in the presence of BAP at 1.0 mg L-1. The adventitious shoots successfully rooted and weretransferred to the soil.

Phytoene Synthase Gene Cloning from Citrus sinensis Osbeck cv. Cara Cara and Its Prokaryotic Expression

Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.

<i>In-Vitro</i>Regeneration of <i>Citrus sinensis</i>(L.) Osbeck from Mature Seed Derived Embryogenic Callus on Different Solid Basal Media

In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbeck. Observations of the effect of PGRs were carried out using different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D),1-naphthalene acetic acid (NAA) and combinations of 2,4-D and NAA using different basal media. This study found Citrus sinensis (L.) Osbeck exhibited a high frequency of callus induction on MS medium supplemented with 3 mg/L 2,4-D and callus induction frequency was 86.7% ± 3.4% whereas N6 and B5 showed lower callus induction frequency of 83.3% ± 8.8% and 82.2% ± 1.9% respectively compared to that of MS media with supplementation of the same hormone. Among the induced calli, the morphological analysis showed only 40% - 50% was embryogenic calli. Regeneration of plantlets from calli was done using different concentrations and combinations of auxin and cytokinin. The study showed that 3 mg/L 6-benzylaminopurine (BAP) supplemented medium has the maximum potential to promote regeneration of Citrus sinensis (L.) Osbeck from embryogenic calli with the frequency of 89.3% ± 8.8% but no regeneration occurred from the non-embryogenic calli. The regenerated plantlets were rooted on MS medium with supplementation of 5 mg/l NAA. These observations in Citrus sinensis (L.) Osbeck regeneration will be helpful for genetic improvement with desired traits.

Identification of Genetic Variation in Citrus sinensis from Hunan Based on Start Codon Targeted Polymorphism

[Objective] The aim was to identify genetic variation in Citrus sinensis （sweet orange） germplasm from Hunan Province according to the Start Codon Targeted （SCoT） Polymorphism. [Method] The reaction system for SCoT amplification from sweet orange was first optimized, and then the SCoT fragments were amplified from 24 sweet orange cultivars collected in Hunan Province and sequenced for genetic variation analysis. [Result] The optimum reaction system for SCoT markers amplification was 2.0 μl containing 80 ng of template DNA, 0.3 mmol/L dNTPs, 0.2 μmol/L primer, 1.6 mmol/L Mg2＋, 1.6 U of Taq DNA polymerase and 10×PCR buffer. By using this reaction system, the PCR products from the sweet orange cultivars produced clear and reproducible bands at 100-2 000 bp through electrophoresis. The SCoT fragments of the 24 sweet orange cultivars were 1 090-1 091 bp, with the homology of 99.84% and nucleotide deletion and substitution. After being sequenced, the SCoT polymorphisms could distinguish 12 sweet orange cultivars. In addition, the BLAST result showed that part of the SCoT fragments coding region shared high homology with ribosomal protein S3 N superfamily. [Conclusion] This study will provide a theoretical basis for breeding sweet orange cultivars.

甜橙(Citrus Sinensis)皮提取物清除超氧自由基作用的研究

本文以邻苯三酚所致红细胞膜氧化损伤为实验模型,发现甜橙(Citrus sinensis)皮提取物具有较强的清除超氧自由基(O_2^-)能力:能明显地抑制膜脂过氧化和膜蛋白氧化交联作用.该提取物是一种有应用前景的新型超氧自由基清除剂.

柚子(Citrus grandis Osbeck)S-like核酸酶基因CgSL1的分离与特征分析

从柚子 (Citrus grandis Osbeck) 2个自交不亲和品种溪蜜柚和度尾蜜柚的花柱中克隆出一个类似 S核酸酶基因 Cg SL1(C.grandis S- like RNase) ,它的 c DNA序列全长 10 74 bp,编码 2 97个氨基酸。通过与其它植物S- like核酸酶和 S核酸酶氨基酸序列进行比较 ,发现 Cg SL1类似于 S- like核酸酶 ,与拟南芥中的 RNS2一致性为6 2 .5 %。对 Cg SL1的表达分析表明该基因在花柱、花药、叶片不同器官以及花柱的不同发育阶段均有表达 ,且在花柱中的表达随衰老增强 ,由此推测它可能与衰老有关。

红肉脐橙(Citrus sinensis L.)果肉中特征色素提取方法探索

红肉脐橙是目前为止惟一果肉呈粉红色或红色的脐橙,其呈色色素尚未见报道。为深入研究其果肉中特征色素的构成及在果实生长发育和贮存过程中色素含量的变化,本研究在对该色素初步定性的基础上对提取方法进行了探索。在分析红肉脐橙果肉中色素萃取液的紫外可见吸收光谱时发现,该色素有类胡萝卜素的特征吸收峰,将萃取液进行薄层层析则进一步证实其内含有番茄红素。在色素提取的前处理方式上,本研究认为冷冻干燥样品的浸提效果较真空干燥的样品稳定(变异系数小),且浸提率相差无几,所以选择冷冻干燥作为合适的样品前处理方式。色素浸提条件的正交实验结果表明,最佳浸提条件为50℃、1.5h内浸提4次,浸提的料液比为1:60。此外,浸提温度、浸提时间、浸提料液比和浸提次数对浸提率都有显著影响,影响最大的因素是浸提温度,最佳温度为50℃,随温度降低浸提率显著降低。其次是侵提时间的长短,最佳浸提时间为1.5h,延长或缩短0.5h都会使浸提率下降。浸提次数对浸提率的影响是最小的。

柚(Citrus grandis Osbeck cv.)MADS box基因的表达分析

以De novo转录组学方法分析柚(Citrus grandis Osbeck cv.)MADS box基因家族的基因.以特早熟龙柚(变异型)和琯溪蜜柚(参照型)嫩果(不超过5天)和新叶为材料,经过RNA提取、反转录、测序、组装得到Unigene序列.Unigene序列在Nr、Swiss-Prot、KEGG和COG四大数据库中进行比对,获得MADS box转录因子家族基因注释结果.采用数字基因表达标签(digital gene expression tag,DGE)技术,对样本中基因转录丰度以RPKM值表示,比较处理组和对照组的RPKM值,以倍性变化≥2确定差异表达基因(differentially expressed genes,DEGs).从特早熟龙柚和琯溪蜜柚嫩果和叶片中,总共注释到28个MADS box基因家族基因.在龙柚嫩果(FE)和蜜柚嫩果(FL)中表达的有25个,其中有6个属于差异表达基因.在龙柚叶片(LE)和蜜柚叶片(LL)中表达的有27个,其中有7个属于差异表达基因.注释到的28个MADS box基因家族基因在克莱门特柚(Citrus clementina)和中国甜橙(Citrus sinensis)报道过的分别有18个和20个,其中有5个基因既在中国甜橙和克莱门特柚都没有报道过,也没有发现在其它Citrus物种报道过.

Larvicidal and irritant activities of hexane leaf extracts of Citrus sinensis against dengue vector Aedes aegypti L.

Objective:To assess the larvicidal and irritant activities of the hexane extracts of leaves of Citrus sinensis(C.sinensis)against the early fourth instars and female adults of Aedes aegypti(Ae.aegypti).Methods:The larvicidal potential of the prepared leaf extract was evaluated against early fourth instar larvae of Ae.aegypli using WHO protocol.The mortality counts were made after 24 h and LC_(50)and LG_(50)values were calculated.The efficacy of extract as mosquito irritant was assessed by contact irritancy assays.Extract-impregnated paper was placed on a glass plate over which a perspex funnel with a hole on the top was kept inverted.Single female adult,3-day old unfed/blood-fed,was released inside the funnel.After 3 min of acclimatization time,the time taken for the first take-off and total number of flights undertaken during 15 min were scored.Results:The citrus leaf extracts from hexane possessed moderate larvicidal efficiency against dengue vector.The bioassays resulted in an LC_(50)and LC_(90)value of 446.84 and 1370.96 ppm,respectively after 24 h of exposure.However,the extracts were proved to be remarkable irritant against adults Ae.aegypti,more pronounced effects being observed on blood-fed females than unfed females.The extract-impregnated paper was thus proved to be 7-11 times more irritable as compared with the control paper.Conclusions:The hexane extracts from C.sinensis leaves are proved to be reasonably larvicidal But remarkably irritant against dengue vector.Further studies are needed to identify the possible role of extract as adulticide,oviposition deterrent and ovicidal agent.The isolation of active ingredient from the extract could help in formulating strategies for mosquito control.

CsPrx25,a class III peroxidase in Citrus sinensis,confers resistance to citrus bacterial canker through the maintenance of ROS homeostasis and cell wall lignification

Citrus bacterial canker(CBC)results from Xanthomonas citri subsp.citri(Xcc)infection and poses a grave threat to citrus production.Class III peroxidases(CIII Prxs)are key proteins to the environmental adaptation of citrus plants to a range of exogenous pathogens,but the role of CIII Prxs during plant resistance to CBC is poorly defined.Herein,we explored the role of CsPrx25 and its contribution to plant defenses in molecular detail.Based on the expression analysis,CsPrx25 was identified as an apoplast-localized protein that is differentially regulated by Xcc infection,salicylic acid,and methyl jasmone acid in the CBC-susceptible variety Wanjincheng(C.sinensis)and the CBC-resistant variety Calamondin(C.madurensis).Transgenic Wanjincheng plants overexpressing CsPrx25 were generated,and these transgenic plants exhibited significantly increased CBC resistance compared with the WT plants.In addition,the CsPrx25-overexpressing plants displayed altered reactive oxygen species(ROS)homeostasis accompanied by enhanced H_(2)O_(2) levels,which led to stronger hypersensitivity responses during Xcc infection.Moreover,the overexpression of CsPrx25 enhanced lignification as an apoplastic barrier for Xcc infection.Taken together,the results highlight how CsPrx25-mediated ROS homeostasis reconstruction and cell wall lignification can enhance the resistance of sweet orange to CBC.

Genetic Transformation of <i>Citrus sinensis</i>L. with an <i>antisense ACC oxidase</i>Gene

This work was carried out to optimize the conditions for highly effective embryogenic callus induction from mature seeds, plantlet regeneration and genetic transformation of Citrus sinensis L. by Agrobacterium tumefaciens strain EHA105 (pCAMBIA 1305.1). Embryogenic calli could be successfully induced from mature seeds employing the MT medium supplemented with 500 mg/l malt extract. The percentage of embryogenic callus induction was 85. With the same medium, the high proliferation rate of embryogenic callus was achieved. The liquid MT medium containing 500 mg/l malt extract in combination with 50 mg/l lactose could be used as the embryoid development medium. Somatic embryos, however, could be regenerated with normal shoots and roots in the MS medium, with the regeneration percentage of 60. The delivery of an antisense ACC oxidase gene into the species C. sinensis mediated by Agrobacterium tumefaciens strain EHA 105 was successful by co-cultivating explants with the strain EHA 105 for 10 min, following that by eliminating the bacterium with 200 mg/l cefotaxime, and subsequently selecting transformed embryoid with 20 mg/l hygromycin. Verified histochemically by GUS assay, putative transformants showed the percentage of gus gene expression of 100. Molecular analysis using PCR confirmed the integration of the antisense ACC oxidase gene into plant genome.

Stable Silver Nanoparticles Synthesis by Citrus Sinensis(Orange) and Assessing Activity Against Food Poisoning Microbes

Silver nanoparticles are considerecl as good antimicrobial agent. AgNPs were synthesized by mixing silver nitrate solution with citrus sinesb extract for 2 h at 37 and analyzed by UV-visible spectra, SEM, XRD, and FTIR. AgNPs were tested against B. subtilis, Shigello, S. oureus, ond E. coli. Minimum inhibitory concentration of AgNPs was 20 I^g/mL for B. subtilis and Shigello and 30 I^g/mL for S. oureus and E. coll. Antibiofilm activity （80% to 90%） was observed at 25 IJg/mL. AgNPs were stable for five months with sustained an＇timicrobial activity. Biosynthesized AgNPs can bE： used to inhibit food poisoning microbial growth.

Incorporation of Essential Oils from Cymbopogon Citratus and Ceasalpinia Decapitala Leaves, Citrus Lemon and Citrus Sinensis Peels into HPC （HydroxyPropyl Cellulose） Films and Assessment of Their Antibacterial Activity

In this study, essential oils from Cymbopogon citratus and Ceasalpinia decapitala leaves, Citrus lemon and Citrus sinens＆ peels, were extracted, chemically characterized and evaluated on both Gram positive and Gram negative bacteria, Salmonella Typhimurium, Escherichia coli, Listeria innocua, Staphylococcus aureus, Klebsiella pneumonia and Streptococcus pyogenes. Essential oil of C. citratus was found to be rich in geranial （33.0%） and neral （49.7%）, that from C. decapitala in 13-pinene （8.4%）, ct-pinene （25.5%）, 13-ocymene （31.6%）, 15-phellandrene （8.4%）, caryophyllene （7.5%） and geranial （5.9%）. The essential oil from C. lemon and C. sinensis where mainly composed by limonene with percentages of 77.5% and 83.3% respectively, and 13-phellandrene with percentages of 8.1% and 10.8% for the essential oil of C. lemon and C. sinensis, respectively. The antibacterial activity assessment realized on different microorganisms contaminating food showed that essential oil from C. citratus was shah more effective against all tested bacteria while that from C. sinensis was the less active essential oil. The incorporation of essential oils in HPC （hydroxy-propyl cellulose） films showed that those essential oils, especially essential oils from C. citratus, maintained their antibacterial activities against targeted microorganisms and therefore can be applied in food packing.

Phytochemical Screening of Essential Oil of Citrus sinensis by Gas Chromatography-Flame Ionization Detector

Citrus sinensis commonly called sweet orange belongs to the family Rutaceae. Nutritionally, it is highly recommended due to its high content of micronutrients. However, the rejection of a large amount of epicarp in nature contributes to the emission of greenhouse gas and the development of leachate which contaminate surface water and groundwater. The aim of this work was to identify the essential oil components from Citrus sinensis epicarp, and then look after the biological activity of these components in order to underline the worth to reuse the Citrus sinensis epicarp as a gainful mean. The essential oil of 4,000 g of Citrus sinensis epicarp was done through the water steam distillation and 0.0287 g of essential oil was obtained; so a yield of 0.0007%. The essential oil was then submitted to gas chromatography-flame ionization detector （GC-F1D）. The result revealed that the essential oil was teemed with 28 volatile compounds, including terpene compounds （50%）, aldehydes （32%） and alcohols （18%） whose anti-inflammatory, anti-diabetic, larvicidal and antioxidant activities were underlined.

Insecticidal Activity of the Powders Obtained from the Bauxite Red Mud, Zyzygium aromaticum and Citrus sinensis Peel Extracts on the Conservation of Corn against Sitophilus zeamais: Physicochemical Properties

The remarkable push of technologies increasingly recommends materials for their constructions.Hence the extraction of alumina is for different uses.The exploitation of bauxite pollutes the surrounding areas in this case the discharge of mud wheel.Furthermore,the excessive use of dangerous conventional synthetic insecticides continues to be in circulation in Cameroon despite the known adverse effects.These problems lead us to formulate bio insecticides from rejection of the extraction of alumina and extracts of S.aromaticum and Citrus sinensis which respectively offer us essential oils eugenol and limonene having insecticidal properties against the main insects,pests of corn stocks,Sitophilus zeamais.From the kinetic point of view of the adsorption of the extracts of eugenol,limonene and their association by the red mud,the adsorption curve shows that the Eu/Li mixture(30/70)is better by 0.36 mg/g during the 24 h.The results showed a high toxicity of the Eu-Li binary mixture of ratio 30/70 with respective mortality rates of 95.88%after 8 days of exposure to the concentration of 5 mL/L on the adults of Sitophilus zeamais.The other composites induce an average rate of 80%.The results from the physicochemical analysis of corn before and after treatment with formulated insecticides showed a slight loss in nutritional quality of 14.90%in ash,12.5%in protein,16.14%in lipids and 13.63%in total sugars for 180 days.The powdery formulation from red mud and Eu-Li(30/70)mixture can therefore ensure the protection of corn stocks against Sitophilus zeamais in a farming environment.

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.

Integrated multi-omics analysis of developing‘Newhall’orange and its glossy mutant provide insights into citrus fragrance formation

Sesquiterpene valencene is dominant in flavedo tissues of sweet oranges and imparts a unique woody aroma.However,the interaction between the biosynthetic pathways of valencene and other nutritional compounds is less studied.Sesquiterpenoids were significantly accumulated in a previously reported glossy mutant of orange(MT)than the wild type(WT),especially valencene and caryophyllene.In addition,we identified several other pathways with variations at both the transcriptional and metabolic levels in MT.It’s interesting to found those upregulated metabolites in MT,such as eukaryotic lipids,kaempferol and proline also showed strong positive correlation with valencene along with fruit maturation while those down-regulated metabolites,such as phenylpropanoid coumarins and most of the modified flavonoids exhibited negative correlation.We then categorized these shifted pathways into the‘sesquitepenoid-identical shunt’and the sesquitepenoid-opposite shunt’and confirmed the classification result at transcriptional level.Our results provide important insights into the connections between various fruit quality-related properties.

Effect of Copper on Growth Characteristics and Disease Control of the Recently Introduced <i>Guignardia citricarpa</i>on Citrus in Florida

Guignardia citricarpa, the plant pathogenic fungus that causes citrus black spot, was recently introduced into the United States. The development of this disease in the presence of multiple applications of copper per year to manage citrus canker warrants an investigation into the effects of copper on growth of isolates of G. citricarpa from citrus in Florida. Guignardia citricarpa and G. mangiferae isolates, confirmed by internal transcribed spacer (ITS) sequencing of ribosomal DNA and DNA homology, were inoculated on non-amended media and media amended with 50 and 500 μg·ml-1 copper sulfate. Radial colony growth was assessed over a 26 to 59 day period. Copper reduced the growth of G. citricarpa isolates in media amended with 500 μg·ml-1 copper but had variable effects on radial growth in media amended with 50 μg·ml-1 copper. There was little effect of copper on the in vitro growth of G. mangiferae isolates. Field application of copper with and without an adjuvant for the control of citrus black spot was undertaken in a commercial grove in Florida in 2011. Spray applications were made on a 23.3 ± 4.7 day interval and fruit accessed between December 2011 and March 2012 for black spot symptoms. Copper failed to reduce the proportion of fruit exhibiting symptoms compared to that of the controls.