Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

Citrus tissue culture: stimulation of fruit explant cultures with orange juice

In vitro growth of explant (juice vesicle or albedo tissues) cultures from citron (Citrus medica), lemon (C. limon), grapefruit (C. paradisi), sweet orange (C. sinensis), and mandarin (C. reticulata) fruits was stimulated by addition of orange juice (10% v/v optimum) to a basal medium containing Murashige and Skoog salts, 50 grams per liter sucrose, 100 milligrams per liter myo-inositol, 5 milligrams per liter thiamine·HCl, 2 milligrams per liter 2,4-dichlorophenoxyacetic acid and 0.5 milligrams per liter kinetin. In analyzing this effect of orange juice on citron explant cultures, we failed to obtain increased yields by addition of appropriate concentrations of citric acid to the basal medium but obtained growth stimulation when the medium was supplemented with juice from an “acidless” orange variety (cv. Lima). These facts suggest that some component(s) other than citric acid is involved. Addition of the inorganic ash corresponding to 10% (v/v) orange juice to the basal medium had no effect on yields. Similarly, the stimulatory effect of orange juice could not be explained based on its content of sucrose or of organic growth factors already present in the basal medium.     

Citrus Tree Decline Caused by Pratylenchus coffeae

The pathogenic effects of Pratylenchus coffeae on growth and yield of tangelo (Citrus paradisi × C. reticulata) scions grafted on rough lemon (C jambhiri), sour orange (C. aurantium) and ''Cleopatra'' mandarin (C. reticulata) rootstocks were evaluated under field conditions for 4 years. Pratylenchus coffeae on inoculated trees increased to significantly damaging population densities on rough lemon rootstock the second year, on sour orange the third and on Cleopatra mandarin the fourth year after planting. Mean growth reduction of P. coffeae-infected trees after 4 years was 80, 77 and 49%, respectively, for the three rootstocks. Noninoculated trees on rough lemon and sour orange rootstocks yielded significantly more fruit than comparable inoculated trees. Natural migration of P. coffeae occurred horizontally on roots for a distance of 4.5 m.     

Molecular Cloning, Sequencing and Phylogenetic Analysis of Coat Protein Gene of a Biologically Distinct Citrus Tristeza Virus Isolate Occurring in Central India

Citrus tristeza virus (CTV) culture, collected from a fifteen-year-old wilted and declined mosambi sweet orange [Citrus sinensis (L) Osb] plant, was maintained under green house into acid lime [C. aurantifolia Swing] and mosambi seedlings. It gave positive reaction in ELISA, both with CTV specific polyclonal antibodies (G604) and monoclonal antibody MCA13, which specifically detects severe CTV strains. Coat protein gene (CPG) of the virus was amplified by RT PCR using CPG specific primers yielding an amplicon of 672 bp. Sequence analysis of the CPG amplicon showed 97% nucleotide sequence homology with Chinese isolate CTV-0002 (Acc. No. AJ518841) and isolate S4 (Acc. No. EF063109). In phylogenetic analysis, the present CTV isolate was displayed in different clade than other reported Indian CTV isolates, but it shared a separate clade with isolates from China, Israel, Jordan and New Zealand.     

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.     

Symptomatic expression of tristeza-infected citrus plants in Pakistan

Variable symptoms were recorded during a survey in the citrus trees infected or suspected to be infected with citrus tristeza virus based on ELISA tests. Sweet orange manifested main symptoms like bud union crease, pin holing, bark cracking, incompatibility, yellowing and dropping of leaves. Pin holing was particularly prevalent in Sweet orange (Citrus sinensis Osbeck), Kinnow (Citrus reticulata Blanco) and Grapefruit (Citrus paradisii Mcaf.), while incompatibility was only in Sweet orange and Grapefruit.     

Improved Transformation Efficiency in Citrus by Plasmolysis Treatment

Procedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of eight different citrus species [’Milam’ Rough lemon (Citrus jambhiri Lush), ‘Volkamer’ lemon (Citrus volkameriana L), Rangpur lime (Citrus limonia L), ‘Hamlin’ sweet orange (Citrus sinensis L Osbeck), ‘Duncan’ grapefruit (’Citrus paradisi’ Macf), Sour orange (Citrus aurantium L), ‘Cleopatra’ mandarin (Citrus reticulata Blanco) and Carrizo citrange (Citrus sinensis L Osbeck x Poncirus trifoliata L Raf) ] were plasmolyzed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v) ] prior to Agrobacterium inoculation. Plasmolyzed epicotyl explants were cocultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agl-1 (carrying pCAMBIA1303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and β-glucuronidase (GUS) genes. The binary vector, pCAMBIA1303 also contained a fused mGFP5 gene at the 3’ end of GUS gene as a reporter. Epicotyl explants of Rangpur lime, Rough and ‘Volkamer’ lemons plasmolyzed in 9–12 % maltose showed transient GUS gene expression comprising up to 95 % of the cut surface of explants, while Carrizo citrange showed 80 % expression when they were plasmolyzed in 6–10 % sucrose. On the other hand, epicotyl explants of ‘Hamlin’ sweet orange, Grapefruit, Sour orange and ‘Cleopatra’ mandarin showed transient GUS expession in 80–90 % of explants with 6–10 % sucrose. Basal portions of the regenerated putative transgenic shoots harvested from the cut surface of epicotyl explants within 2–3 months, were assayed for GUS, and apical portions were shoot-tip grafted in vivo for the production of whole plants. The transformation efficiencies in different species obtained are the highest so far reported for citrus.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Restoration of vigor and rooting competence in stem tissues of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro

Summary Repeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.     

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza <i>Closterovirus</i>

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards. The citrus species grafted on sour orange rootstock are affected by this disease. Predominantly, the sweet orange, grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline, vein clearing, pin holing, bark scaling and degeneration leading to variable symptoms. Symptomatic expression of Citrus tristeza virus (CTV) in different hosts has been attributed to virus isolates which are from severe to mild. Different serological and molecular assays have been deployed to differentiate the strains of CTV. Citrus tristeza virus is diversified towards its strains on the basis of biological, serological and molecular characterization. Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation. This review will give a brief idea about the different CTV isolates, their characterization based on nucleic acid and serological assays. Different methods along with salient features for strain characterization has also been reviewed. This review will also open the new aspects towards formulation of management strategies through different detection techniques.     

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

6,7-Dimethoxycoumarin in the peels of Citrus

6,7-Dimethoxycoumarin was isolated and identified from the peels of Citrus sinensis cv. Pineapple orange, of C. paradisi x C. paradisi x C. reticulata cv. Wekiwa and of C. mitis cv. Clamondin. The coumarin was tentatively identified in two other sweet oranges, C. sinensis cv. Mediterranean Sweet and Waite Parson Brown, and in C. aurantifolia cv. West Indian lime and in C. aurantium cv. Bitter Sweet orange.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

Somatic hybrid plants from sexually incompatible woody species: Citrus reticulata and Citropsis gilletiana

Summary Allotetraploid intergeneric somatic hybrid plants between Citrus reticulata Blanco cv. Cleopatra mandarin and Citropsis gilletiana Swing. & M. Kell. (common name Gillet's cherry orange) were regenerated following protoplast fusion. Cleopatra protoplasts were isolated from an ovule-derived embryogenic suspension culture and fused chemically with leaf-derived protoplasts of Citropsis gilletiana. Cleopatra mandarin and somatic hybrid plants were regenerated via somatic embryogenesis. Hybrid plant identification was based on differential leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose mutase (PGM) and phosphohexose isomerase (PHI) isozyme banding patterns. This is the first somatic hybrid within the Rutaceae reported that does not have Citrus sinensis (sweet orange) as a parent, and the first produced with a commercially important citrus rootstock and a complementary but sexually incompatible, related species.Abbreviations PGM phosphoglucose mutase - PHI phosphohexose isomerase - MES 2[N-morpholino] ethane sulfonic acid - BH3 protoplast culture medium (Grosser and Chandler, 1987) - PEG polyethylene glycol - MT Murashige and Tucker (1969) basal medium - NAA 1-naphthaleneacetic acid - GA3 gibberellic acid - H+H and EME citrus embryogenic cell culture media (Grosser and Gmitter, 1990b) - B embryo germination medium - RMAN rooting medium Florida Agricultural Experiment Station Journal Series No. R-00298.     

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).     

Evaluation of the Genetic Structure of Xylella fastidiosa Populations from Different Citrus sinensis Varieties

Xylella fastidiosa was isolated from sweet orange plants (Citrus sinensis) grown in two orchards in the northwest region of the Brazilian state of São Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of C. sinensis cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (H_T) values of X. fastidiosa were similar for both sets of strains; however, H_TRAPD values were substantially lower than H_TVNTR values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the X. fastidiosa strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of X. fastidiosa. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.     

Computerized Optimization of the Relative Growth of Callus Cultures of Orange Fruit Tissues7

The induction and growth of callus cultures from different partsof the fruit of Coorg mandarin orange (Citrus reticulata, Blanco)and the hormonal combinations that can maximize the growth witha basal MS medium, have been explored using Response SurfaceMethodology. The relative growth, protein, peroxidase pattern,and GC fingerprinting of volatiles have been studied. Calluscultures from juice vesicles were found to be more similar totheir explant counterpart than those from albedo, for all propertiesstudied. Key words: Callus culture, Citrus, albedo, juice vesicles, response surface methodology, growth optimization     

A field study investigating the insecticidal efficacy against Diaphorina citri Kuwayama on Kinnow mandarin,Citrus reticulata Blanco trees

Asian citrus psyllid is a most damaging insect pest of citrus. In this field study, the efficacy of seven insecticides (emamectin benzoate, bifenthrin, chlorfenapyr, fipronil, imidacloprid, pyriproxyfen and thiamethoxam) was evaluated against Diaphorina citri Kuwayama in the citrus orchard of Kinnow mandarin, Citrus reticulata Blanco. The insecticides revealed a differential and substantial relative efficacy against D. citri compared to the untreated plants. The insecticidal effect attributed as percent reduction in insect population was more prominent after three days of spray: highest reduction values were recorded with thiamethoxam (50.89%), imidacloprid (44.27%) and bifenthrin (42.94%) after first spray, and thiamethoxam (83.36%), imidacloprid (73.20%) and bifenthrin (72.66%) after second spray. Thus, neonicotinoids (thiamethoxam and imidacloprid) and pyrethroid (bifenthrin) resulted as highly effective against D. citri at three days after both sprays. At seven days, imidacloprid (63.53%) and fipronil (62.47%) presented relatively higher population reduction after first spray, and thiamethoxam (92.66%) and chlorfenapyr (89.59%) after second spray. At 12 days, the insecticidal effect on insect population became significantly at par after each spray except chlorfenapyr that reflected high population reduction (93.17%) only after second spray. It is also obvious from the data that there is need of regular monitoring to suppress the psyllids population below threshold level by timely application of the second insecticidal spray.