Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica).

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid 'OLL8' sweet orange or 'Page' tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the 'OLL8' + FL fusion, while three putative cybrids were regenerated from the 'Page' + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

A comparison of the ultrastructure and composition of fruits' cuticular wax from the wild-type 'Newhall' navel orange (Citrus sinensis [L.] Osbeck cv. Newhall) and its glossy mutant.

KEY MESSAGE: The altered ultrastructure and composition of cuticular wax from 'glossy Newhall' (MT) fruits lead to its glossy phenotype. A novel mutant derived from the wild-type (WT) 'Newhall' navel orange (Citrus sinensis [L.] Osbeck cv. Newhall), named 'glossy Newhall' (MT), which produced much more glossy fruits that were easily distinguishable from the WT fruits was characterized in this report. The total wax loads of both WT and MT fruits varied considerably during the fruit development. The most abundant wax fraction of WT mature fruits was triterpenoids, followed by aldehydes, alkanes, fatty acids, primary alcohol and cholesterol. The total wax load in MT mature fruits was reduced by 44.2 % compared with WT. Except for the minor wax components of primary alcohol and cholesterol, the amounts of all major wax fractions in MT mature fruits were decreased in varying degrees. The major reduction occurred in aldehydes that decreased 96.4 % and alkanes that decreased 81.9 %, which was consistent with scanning electron micrographs of MT mature fruit surfaces that showed a severe loss of wax crystals. Hence, aldehydes and alkanes were suggested to be required for wax crystal formation in 'Newhall' navel orange fruits.

Structural analysis and involvement in plant innate immunity of Xanthomonas axonopodis pv. citri lipopolysaccharide.

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.

The antioxidant activity and polyphenolic contents of different plant seeds extracts.

Different plant seeds extracts of Citrus sinensis, Hordeum sativum, Triticum sativum, Canna indica, Citrullus vulgaris and Capsicum annuum were evaluated for their antioxidant activity by the following methods: 2,2-diphenyl-1-pycril-hydrazyl (DPPH) radical scavenging, reducing power, RBCs hemolysis and linoleic acid oxidation, a long with the determination of total phenolic and flavonoids contents. All the methanolic extracts showed high antioxidant activity and have high contents of phenolic and flavonoid. The Canna indica extract exhibited strong antioxidant as a reducing power and as DPPH radical-scavenging (3.61 absorbance, 87.12%, respectively), while the Hordeum sativum extract exhibited highest inhibitory effect on RBCs hemolysis (59.55%) and the Capsicum annuum extract has highest inhibitory effect on linoleic acid peroxidation (65.06%).

Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange (Citrus sinensis) red-flesh mutant.

BACKGROUND: Interest in lycopene metabolism and regulation is growing rapidly because accumulative studies have suggested an important role for lycopene in human health promotion. However, little is known about the molecular processes regulating lycopene accumulation in fruits other than tomato so far. RESULTS: On a spontaneous sweet orange bud mutant with abnormal lycopene accumulation in fruits and its wild type, comparative transcripts profiling was performed using Massively Parallel Signature Sequencing (MPSS). A total of 6,877,027 and 6,275,309 reliable signatures were obtained for the wild type (WT) and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed gene in MT is 18,106, larger than that in WT 17,670, suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts abundance between MT and WT revealed that 3,738 genes show more than two fold expression difference, and 582 genes are up- or down-regulated at 0.05% significance level by more than three fold difference. Functional assignments of the differentially expressed genes indicated that 26 reliable metabolic pathways are altered in the mutant; the most noticeable ones are carotenoid biosynthesis, photosynthesis, and citrate cycle. These data suggest that enhanced photosynthesis and partial impairment of lycopene downstream flux are critical for the formation of lycopene accumulation trait in the mutant. CONCLUSION: This study provided a global picture of the gene expression changes in a sweet orange red-flesh mutant as compared to the wild type. Interpretation of the differentially expressed genes revealed new insight into the molecular processes regulating lycopene accumulation in the sweet orange red-flesh mutant.

Removal of direct blue-86 from aqueous solution by new activated carbon developed from orange peel.

The use of low-cost, easy obtained, high efficiency and eco-friendly adsorbents has been investigated as an ideal alternative to the current expensive methods of removing dyes from wastewater. This study investigates the potential use of activated carbon prepared from orange peel for the removal of direct blue-86 (DB-86) (Direct Fast Turquoise Blue GL) dye from simulated wastewater. The effects of different system variables, adsorbent dosage, initial dye concentration, pH and contact time were studied. The results showed that as the amount of the adsorbent increased, the percentage of dye removal increased accordingly. Optimum pH value for dye adsorption was determined as approximately 2.0. Maximum dye was sequestered within 30min after the beginning for every experiment. The adsorption of direct blue-86 followed a pseudo-second-order rate equation and fit well Langmuir, Tempkin and Dubinin-Radushkevich (D-R) equations better than Freundlich and Redlich-Peterson equations. The maximum removal of direct blue-86 was obtained at pH 2 as 92% for adsorbent dose of 6gL(-1) and 100mgL(-1) initial dye concentration at room temperature. The maximum adsorption capacity obtained from Langmuir equation was 33.78mgg(-1). Furthermore, adsorption kinetics of DB-86 was studied and the rate of adsorption was found to conform to pseudo-second-order kinetics with a good correlation (R2>0.99) with intraparticle diffusion as one of the rate determining steps. Activated carbon developed from orange peel can be attractive options for dye removal from diluted industrial effluents since test reaction made on simulated dyeing wastewater show better removal percentage of DB-86.

Whole-tree level water balance and its implications on stomatal oscillations in orange trees [Citrus sinensis (L.) Osbeck] under natural climatic conditions.

Sustained cyclic oscillations in stomatal conductance, leaf water potential, and sap flow were observed in young orange trees growing under natural conditions. The oscillations had an average period of approximately 70 min. Water uptake by the roots and loss by the leaves was characterized by large time lags which led to imbalances between water supply and demand in the leaves. The bulk of the lag in response between stomatal movements and the upstream water balance resided downstream of the branch, with branch level sap flow lagging behind the stomatal conductance by approximately 20 min while the stem sap flow had a much shorter time lag of only 5 min behind the branch sap flow. This imbalance between water uptake and loss caused transient changes in internal water deficits which were closely correlated to the dynamics of the leaf water potential. The hydraulic resistance of the whole tree fluctuated throughout the day, suggesting transient changes in the efficiency of water supply to the leaves. A simple whole-tree water balance model was applied to describe the dynamics of water transport in the young orange trees, and typical values of the hydraulic parameters of the transpiration stream were estimated. In addition to the hydro-passive stomatal movements, whole-tree water balance appears to be an important factor in the generation of stomatal oscillations.

Hydraulic characteristics and water relations in pigment-less mutant shoots of an orange tree.

Phenotypes more or less deficient in photosynthetic pigments show reduced productivity. Not much is known, however, about the influence of pigment-less twigs on the water balance of whole trees. We studied the water relations and hydraulic properties of normal and pigment-less (white) and 1-year-old shoots of 12-year-old Citrus sinensis L. trees. Compared with green leaves, white leaves showed a pronounced deficiency of pigments, higher stomatal density, the absence of chloroplasts in the guard cells and a different organization of leaf parenchyma. Stomatal conductance (gL) and transpiration rate (EL) were higher in white leaves than in green leaves during the hottest hours of the day, especially in July and September. The absence of chloroplasts in the stomatal guard cells seemed to be one of the factors causing insufficient stomatal control. Hydraulic conductance (KL) was higher in white leaves than in green leaves (16.96+/-2.24x10(-5) versus 11.26+/-0.66x10(-5) kg s-1 m-2 MPa). The ratio between the sum of the fourth power of xylem conduit radius (Sigmar4) (which determines theoretical conductance) and the total leaf area supplied by petioles and midribs was higher in white leaves than in green leaves. This was because of a smaller leaf area in white leaves and a statistically different distribution of lumen diameters of the conduits in midribs and petioles. The hydraulic properties of white twigs profoundly disturbed the water balance and physiology of the whole tree.

Calibration of sap flow estimated by the compensation heat pulse method in olive, plum and orange trees: relationships with xylem anatomy.

The compensation heat pulse method is widely used to estimate sap flow in conducting organs of woody plants. Being an invasive technique, calibration is crucial to derive correction factors for accurately estimating the sap flow value from the measured heat pulse velocity. We compared the results of excision and perfusion calibration experiments made with mature olive (Olea europaea L. 'Manzanilla de Sevilla'), plum (Prunus domestica L. 'Songal') and orange (Citrus sinensis (L.) Osbeck. 'Cadenero') trees. The calibration experiments were designed according to current knowledge on the application of the technique and the analysis of measured heat pulse velocities. Data on xylem characteristics were obtained from the experimental trees and related to the results of the calibration experiments. The most accurate sap flow values were obtained by assuming a wound width of 2.0 mm for olive and 2.4 mm for plum and orange. Although the three possible methods of integrating the sap velocity profiles produced similar results for all three species, the best results were obtained by calculating sap flow as the weighted sum of the product of sap velocity and the associated sapwood area across the four sensors of the heat-pulse-velocity probes. Anatomical observations showed that the xylem of the studied species can be considered thermally homogeneous. Vessel lumen diameter in orange trees was about twice that in the olive and plum, but vessel density was less than half. Total vessel lumen area per transverse section of xylem tissue was greater in plum than in the other species. These and other anatomical and hydraulic differences may account for the different calibration results obtained for each species.

Spiroplasmas are the causal agents of citrus little-leaf disease.

A spiroplasma isolated from citrus with little-leaf disease was grown in a cell-free medium and injected into leafhoppers (Euscelis plebejus). Injected leafhoppers, but not those fed on infected plants, transmitted the spiroplasma to white clover (Trifolium repens cv. S100) and sweet orange (Citrus sinensis cv. Valencia). Infected clover plants were severely stunted; infected sweet orange plants showed typical symptoms of citrus little-leaf disease. The spiroplasma was detected in clover and sweet orange plants by electron microscopy; the helical morphology of the organisms was most easily recognizable in sections 150-200 nm thick. The organism was re-isolated in cell-free media both from infected plants and from injected E. plebejus. The original isolate and those re-isolated from experimentally infected clover and sweet orange appeared by morphological, cultural, biochemical and serological criteria to be identical to each other and to the R8-A2 (type) and C-189 strains of Spiroplasma citri. Serological tests and electrophoretic analysis of protein preparations indicated no relationship to Acholeplasma laidlawii, although this organism survived for at least 10 wk after injection into E. plebejus. Our results show that the causal agent of little-leaf disease is related to S. citri.