Orange Tissue Research Articles

Restraining Quiescence Release-Related Ageing in Plant Cells: A Case Study in Carrot.

The blackening of cut carrots causes substantial economic losses to the food industry. Blackening was not observed in carrots that had been stored underground for less than a year, but the susceptibility to blackening increased with the age of the carrots that were stored underground for longer periods. Samples of black, border, and orange tissues from processed carrot batons and slices, prepared under industry standard conditions, were analyzed to identify the molecular and metabolic mechanisms underpinning processing-induced blackening. The black tissues showed substantial molecular and metabolic rewiring and large changes in the cell wall structure, with a decreased abundance of xyloglucan, pectins (homogalacturonan, rhamnogalacturonan-I, galactan and arabinan), and higher levels of lignin and other phenolic compounds when compared to orange tissues. Metabolite profiling analysis showed that there was a major shift from primary to secondary metabolism in the black tissues, which were depleted in sugars, amino acids, and tricarboxylic acid (TCA) cycle intermediates but were rich in phenolic compounds. These findings suggest that processing triggers a release from quiescence. Transcripts encoding proteins associated with secondary metabolism were less abundant in the black tissues, but there were no increases in transcripts associated with oxidative stress responses, programmed cell death, or senescence. We conclude that restraining quiescence release alters cell wall metabolism and composition, particularly regarding pectin composition, in a manner that increases susceptibility to blackening upon processing.

Extracts of Ulva lactuca Induce Responses against Penicillium digitatum on Oranges

Penicillium digitatum causes severe damage to postharvest and stored citrus fruits, making it necessary to seek acceptable solutions limiting environmental and sanitary impacts. The seaweed liquid extracts (SLEs) obtained from the green macroalgae Ulva lactuca have previously shown anti-fungal activities towards postharvest green mold due to P. digitatum. Moreover, treatment with aqueous and ethanolic extracts had an influence on disease incidence and lesion severity on wounded oranges “Valentia late”. Therefore, we studied the abilities of the U. lactuca extracts and chlorophyll a (as a standard) to activate defense mechanisms in oranges. The analyses showed modifications in the protein concentrations and activities of β-1,3-glucanase and peroxidase in orange tissues, with a maximum activity level reached at 72 h after treatments. It appears that the SLEs were able to fight P. digitatum infections in the absence of direct contact with the pathogen, reflecting that the induction of host resistance is also a probable mode of action. Ulva lactuca fractions are rich in chlorophyll a, polyphenols, organic acids and ulvans, which might be the key molecules involved in the defense mechanism of oranges’ albedo. The use of these natural substances could be an alternative way to control postharvest citrus rot.

Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciens-mediated transformation for generation of marker-free transgenic plants from navel orange (Citrus sinensis Osbeck) mature stems using a Cre-loxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1–0.5 (1–2 weeks), > 0.5 cm (3–4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange (Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase (IPT) and an anti-bacterial peptide (PR1aCB). Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained. Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants (deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange (Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

Genome-Wide Characterization and Expression Analysis of the SBP-Box Gene Family in Sweet Orange (Citrus sinensis).

SBP-box is an important plant-specific transcription factor family and is involved in diverse biological processes. Here, we identified a total of 15 SBP-BOX genes in the important fruit crop sweet orange (Citrus sinensis) and characterized their gene structures, conserved domain and motif, chromosomal location, and cis-acting regulatory elements. SBP genes were classified into four subfamilies based on the amino acid sequence homology, and the classification is equally strongly supported by the gene and protein structures. Our analysis revealed that segmental duplication events were the main driving force in the evolution of CsSBP genes, and gene pairs might undergo extensive purifying selection. Further synteny analysis of the SBP members among sweet orange and other plant species provides valuable information for clarifying the CsSBP family evolutionary relationship. According to publicly available RNA-seq data and qRT-PCR analysis from various sweet orange tissues, CsSBP genes may be expressed in different tissues and developmental stages. Gene expression analysis showed variable expression profiles of CsSBP genes under various abiotic stresses, such as high and low-temperature, salt, and wound treatments, demonstrating the potential role of SBP members in sweet orange response to abiotic stress. Noticeably, all CsSBP genes were also downregulated in sweet orange upon the infection of an important fungal pathogen Diaporthe citri. Our results provide valuable information for exploring the role of SBP-Box in sweet orange.

ORHis, a Natural Variant of OR, Specifically Interacts with Plastid Division Factor ARC3 to Regulate Chromoplast Number and Carotenoid Accumulation

Chromoplasts are colored plastids that synthesize and store massive amounts of carotenoids. Chromoplast number and size define the sink strength for carotenoid accumulation in plants. However, nothing is known about the mechanisms controlling chromoplast number. Previously, a natural allele of Orange (OR), ORHis, was found to promote carotenoid accumulation by activating chromoplast differentiation and increasing carotenoid biosynthesis, but cells in orange tissues in melon fruit and cauliflower OR mutant have only one or two enlarged chromoplasts. In this study, we investigated an ORHis variant of Arabidopsis OR, genetically mimicking the melon ORHis allele, and found that it also constrains chromoplast number in Arabidopsis calli. Both in vitro and in vivo experiments demonstrate that ORHis specifically interacts with the Membrane Occupation and Recognition Nexus domain of ACCUMULATION AND REPLICATION OF CHLOROPLASTS 3 (ARC3), a crucial regulator of chloroplast division. We further showed that ORHis interferes with the interaction between ARC3 and PARALOG OF ARC6 (PARC6), another key regulator of chloroplast division, suggesting a role of ORHis in competing with PARC6 for binding to ARC3 to restrict chromoplast number. Overexpression or knockout of ARC3 in Arabidopsis ORHis plants significantly alters total carotenoid levels. Moreover, overexpression of the plastid division factor PLASTID DIVISION 1 greatly enhances carotenoid accumulation. These division factors likely alter carotenoid levels via their influence on chromoplast number and/or size. Taken together, our findings provide novel mechanistic insights into the machinery controlling chromoplast number and highlight a potential new strategy for enhancing carotenoid accumulation and nutritional value in food crops.

Improved protocol for the transformation of adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues

The production of transgenic citrus plants from adult tissues is difficult because of low regeneration and transformation rates. To increase the transformation efficiency of adult citrus tissues, an improved protocol involving adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues was developed. Explants were pre-incubated in a liquid medium prior to infection by Agrobacterium tumefaciens. Plant materials were also incubated on callus-induction medium supplemented with various combinations of cytokinin (Cyt) and kanamycin (Kan). An appropriate pre-incubation of the explants increased the transformation efficiency of adult tissues. During the callus-induction period, the Cyt type and Kan concentration had the largest and smallest effects on the transformation efficiency, respectively. The most effective combination of plant growth regulator and Kan for the transformation of ‘Tarocco’ blood orange tissues was 2 mg L−1 2-isopentenyl adenine and 50 mg L−1 Kan. The transformation efficiency under the optimized conditions was 11.7%. A Southern blot analysis confirmed the integration of the transgene. These results indicated that the transformation efficiency of adult citrus tissues can be enhanced by optimizing the transformation conditions.

Relationship of the orange tissue morphotype with shell and pearl colouration in the mollusc Pinctada margaritifera

Molluscs display a vast range of shell colours both between and within species. However, only a few species show colour variation in their soft tissues. In French Polynesia, the pearl oyster Pinctada margaritifera has three tissue morphotypes: the black wild-type and two rare mutations: white albino and orange mantle. Phenotypic transmission is known to occur from these phenotypes when they are used as graft donors for pearl production, leading to multicoloured and white pearls from black and albino mantle grafts, respectively. The present study furthers this knowledge by examining the phenotypic association between the orange mantle tissue morphotype and hard tissues: shells and cultured pearls. Based on a large experimental graft, shell colour quantification and pearl qualification showed that the orange morphotype is associated with light-coloured shells and pearls. Expression analysis of some candidate genes previously identified in the white mantle mutant, tested here on both graft and pearl sac tissues from orange mantle donors, confirmed the involvement of genes associated with shell matrix protein (shem4) and the melanin biosynthesis pathway (zinc). This study provides fundamental information on the mechanism behind mantle tissue colour in P. margaritifera and its association with biomineralisation and pigmentation processes that will be potentially valuable in future selection programs.

De novo Transcriptome Assembly of the Clown Anemonefish (Amphiprion percula): A New Resource to Study the Evolution of Fish Color

A fundamental question of evolutionary biology is, why are some animals conspicuously colored? This question may be addressed from both a proximate (genetic and ontogenetic) and ultimate (adaptive value and evolutionary origins) perspective, and integrating these perspectives can provide further insights. Over the last few decades we have made great advances in understanding the causes of conspicuous coloration in terrestrial systems, e.g., birds and butterflies, but we still know relatively little about the causes of conspicuous, ‘poster’ coloration in coral reef fishes. Of all coral reef fishes, the clownfish Amphiprion percula, is perhaps the most conspicuously colored, possessing a bright orange body with three iridescent white bars bordered with pitch black. Here, we review what is known about the proximate and ultimate causes of the conspicuous coloration of clownfishes Amphiprion sp.: coloration has a heritable genetic basis; coloration is influenced by development and environment; coloration has multiple plausible signaling functions; there is a phylogenetic component to coloration. Subsequently, to provide new insights into the genetic mechanisms and potential functions of A. percula coloration we i) generate the first de novo transcriptome for this species, ii) conduct differential gene expression analyses across different colored epidermal tissues, and iii) conduct gene ontology (GO) enrichment analyses to characterize function of these differentially expressed genes. BUSCO indicated that transcriptome assembly was successful and many genes were found to be differentially expressed between epidermal tissues of different colors. In orange tissue, relative to white and black, many GO terms associated with muscle were over-represented. In white tissue, relative to orange and black tissue, there were very few over- or under-represented GO terms. In black tissue, relative to orange and white, many GO terms related to immune function were over-represented, supporting the hypothesis that black (melanin) coloration may serve a protective function. Overall, this study presents the assembly of the A. percula transcriptome, and represents a first step in an integrative investigation of the proximate and ultimate causes of conspicuous coloration of this iconic coral reef fish.

Investigation of morphological and anatomical underground parts structure of genus Primula L. Cultivated species

Investigation of morphological and anatomical underground parts structure of genus Primula L. Cultivated species

Multifocal spectral-domain optical coherence tomography based on Bessel beam for extended imaging depth.

To advance the practical application of optical coherence tomography (OCT) in the field of biomedical imaging, the imaging depth must be extended without sacrificing resolution while maintaining sufficient sensitivity. However, there is an inherent trade-off between lateral resolution and depth of field (DOF) in OCT. To address this shortcoming, this article proposes a multifocal Bessel beam spectral-domain optical coherence tomography (MBSDOCT) capable of increasing the DOF with unchanged lateral resolution and a high signal-to-noise ratio. The proposed technique is demonstrated by simulation and experiment. A three-focal MBSDOCT with an axicon lens theoretically achieved a DOF of ∼6 mm with a lateral resolution of ∼13 μm. In imaging experiments performed on the acinar cells of orange tissue, a measured DOF of ∼4 mm was demonstrated with a sensitivity penalty of ∼18.1 dB, relative to the Gaussian beam spectral-domain OCT, with a 9-mW light source.

Enhanced resistance to citrus canker in transgenic sweet orange expressing the sarcotoxin IA gene

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic “Pera” sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacterium-mediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm2) than in the transgenic plants (<5 lesions/cm2) after injection infiltration or spraying with Xcc inoculum. Accumulation of sarcotoxin IA peptide in sweet orange tissue did not cause any deleterious effects on the growth and development of the transgenic plants, indicating this approach is suitable to provide resistance to citrus canker.

Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation

Tissue culture is the best option to produce disease free seedling of the fruit crop rapidly. Micro-propagation and use of the in-vitro grafting (micro-grafting) is very helpful for production of virus free planting materials in mandarin. Different levels of the in-vitro hormone affect the success of callusing, shooting and plant regeneration in mandarin. Shoot bud, flower bud and in-vitro seedling epicotyl was used as explants to study the hormonal effect on mandarin micro-propagation. Similarly, 10 levels of BAP and IAA combination on MS media for mandarin tissue culture were used. Observation was done for 100 test tubes per treatment combination after 4, 8 and 12 weeks of culture. Data was arc sine transformed for analysis. Shooting from explants was significantly higher (71.72%) on medium level of the BAP (0.5 mg/L) and IAA (0.2 mg/L) using in-vitro seedling stem as explant, however, it was 27.91% for stem bud as explant. Stem bud showed higher level of callusing (6.15%, p&lt;0.001) in mandarin orange. However, flower bud didn’t develop shoot in mandarin tissue culture. Increment of the in-vitro regeneration of the shooting and callusing was observed by the increment of the in-vitro incubation duration in mandarin orange tissue culture.

Comparative transcriptional profiling of orange fruit in response to the biocontrol yeast Kloeckera apiculata and its active compounds.

BackgroundThe yeast Kloeckera apiculata strain 34–9 is an antagonist that shows biological control activity against the postharvest fungal pathogens of citrus. An antifungal compound, 2-phenylethanol (PEA), has been identified from the extract of K. apiculata. To better understand the molecular processes underlying the response of citrus fruit tissue to K. apiculata, the extract and PEA, microarray analyses were performed on navel oranges using an Affymetrix Citrus GeneChip.ResultsAs many as 801, 339 and 608 differentially expressed genes (DEGs) were identified after the application of K. apiculata, the extract and PEA, respectively. In general, K. apiculata induced the expression of defence-related genes. In addition to chitinase and β-1,3-glucanase, genes involved in ethylene (ET), jasmonic acid (JA), calcium signalling, MAPK signalling and phenylalanine metabolism were induced. In contrast, monodehydroascorbate reductase, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and carotenoid biosynthesis genes were down-regulated. The expression profiles for the extract- and PEA-treated samples were similar to that found for yeast (sharing 57.4 % DEGs), with a significant increase in the transcript levels of defence-related genes.ConclusionThis study provides a global picture of the gene expression changes in navel oranges after the application of the antagonist yeast K. apiculata, its extract and PEA. The interpretation of the DEGs revealed new insight into the molecular processes that regulate the defence responses in orange tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2333-3) contains supplementary material, which is available to authorized users.

Prediction and functional analysis of the sweet orange protein-protein interaction network.

BackgroundSweet orange (Citrus sinensis) is one of the most important fruits world-wide. Because it is a woody plant with a long growth cycle, genetic studies of sweet orange are lagging behind those of other species.ResultsIn this analysis, we employed ortholog identification and domain combination methods to predict the protein-protein interaction (PPI) network for sweet orange. The K-nearest neighbors (KNN) classification method was used to verify and filter the network. The final predicted PPI network, CitrusNet, contained 8,195 proteins with 124,491 interactions. The quality of CitrusNet was evaluated using gene ontology (GO) and Mapman annotations, which confirmed the reliability of the network. In addition, we calculated the expression difference of interacting genes (EDI) in CitrusNet using RNA-seq data from four sweet orange tissues, and also analyzed the EDI distribution and variation in different sub-networks.ConclusionsGene expression in CitrusNet has significant modular features. Target of rapamycin (TOR) protein served as the central node of the hormone-signaling sub-network. All evidence supported the idea that TOR can integrate various hormone signals and affect plant growth. CitrusNet provides valuable resources for the study of biological functions in sweet orange.

Acidification of apple and orange hosts by Penicillium digitatum and Penicillium expansum

New information about virulence mechanisms of Penicillium digitatum and Penicillium expansum could be an important avenue to control fungal diseases. In this study, the ability of P. digitatum and P. expansum to enhance their virulence by locally modulating the pH of oranges and apples was evaluated. For each host, pH changes with a compatible pathogen and a non-host pathogen were recorded, and the levels of different organic acids were evaluated to establish possible relationships with host pH modifications. Moreover, fruits were harvested at three maturity stages to determine whether fruit maturity could affect the pathogens' virulence. The pH of oranges and apples decreased when the compatible pathogens (P. digitatum and P. expansum, respectively) decayed the fruit. The main organic acid detected in P. digitatum-decayed oranges was galacturonic acid produced as a consequence of host maceration in the rot development process. However, the obtained results showed that this acid was not responsible for the pH decrease in decayed orange tissue. The mixture of malic and citric acids could at least contribute to the acidification of P. digitatum-decayed oranges. The pH decrease in P. expansum decayed apples is related to the accumulation of gluconic and fumaric acids. The pH of oranges and apples was not affected when the non-host pathogen was not able to macerate the tissues. However, different organic acid contents were detected in comparison to healthy tissues. The main organic acids detected in P. expansum–oranges were oxalic and gluconic and in P. digitatum–apples were citric, gluconic and galacturonic. Further research is needed to identify the pathogenicity factors of both fungi because the contribution of organic acids has profound implications.

Sodium carbonate and bicarbonate treatments induce resistance to postharvest green mould on citrus fruit

The aim of this study was to investigate the ability of two salts, sodium carbonate and bicarbonate, to activate defence mechanisms in citrus fruit against postharvest green mould caused by Penicillium digitatum. In particular, once there was confirmed salt antifungal activity in the absence of direct contact with the pathogen, changes in enzyme activity and expression levels of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase (PAL), and phytoalexin (scoparone, scopoletin, umbelliferone) and sugar (glucose, fructose, sucrose) contents in treated oranges were analyzed. Overall, sodium carbonate and bicarbonate increases the activity of β-1,3-glucanase, peroxidase, and PAL enzymes in orange tissues. Gene expression analyses confirmed PAL up-regulation particularly 12h after treatment application. HPLC analyses of peel extracts showed increased amounts of the sugars and phytoalexins, compared to control tissues, with sucrose and scoparone being the most represented. The results suggest that, although salts exert a direct antifungal effect on P. digitatum, they are also able to induce citrus fruit defence mechanisms to postharvest decay. The defence response seems correlated with the up-regulation of the phenylpropanoid pathway, which has a role in the adaptation to various stresses. This response could result in natural reaction to wounding and pathogen attack in citrus, enhancing its protective effect. As a consequence, the fruit might have a better chance of successful defence against the decay.

STUDY OF RELATIVE EXPRESSION OF DEFENCE-RELATED GENES IN ORANGE FRUIT IN RESPONSE TO ANTAGONIST-PATHOGEN INTERACTION

Pseudomonas spp. produce a wide spectrum of phytotoxic compounds, some of which correlated with post-harvest disease control. Additionally, biocontrol agents can induce a range of defence mechanisms of resistance in citrus tissue that result in a broad spectrum of metabolic modifications such systemic acquired resistance, induced systemic resistance and reactive oxygen species. In the present study we investigated the expression level of genes that encode chitinase (CHI1), gluthatione peroxidase (GPX), phenylalanine-ammonia lyase (PAL), and allene oxide synthase (AOS) in ‘Tarocco’ sweet orange [Citrus sinensis (L.) Osbeck] treated with P. syringae biocontrol strain and the green mold pathogen Penicillium digitatum. Quantitative reverse transcriptase PCR (qRT-PCR) showed that antagonistic P. syringae cells and P. digitatum applied singly increases the transcription of chitinase (CHI1) in the flavedo relative to the untreated control. Interestingly, the transcription of CHI1 in the flavedo was markedly induced by application of P. syringae challenged by P. digitatum, and it was pronounced 24 and 48 h after inoculation. P. syringae inoculation, alone or followed by challenge inoculation with P. digitatum, was weakly effective in enhancing AOS, GPX1, and PAL1 gene expression, whereas P. digitatum alone strongly enhanced the expression of AOS and GPX1 (24 h after inoculation) and PAL1 (48 h after inoculation) in orange tissues. Overall, we assume that CHI1 gene is most likely to be part of the molecular mechanisms involved in pathogen defence responses in citrus fruit.

Anti-oxidant activity of oranges after infection with the pathogen Penicillium digitatum or treatment with the biocontrol agent Pantoea agglomerans CPA-2

The suppression or generation of hydrogen peroxide (H 2O 2) in fruit as well as the changes in the superoxide dismutase (SOD) and catalase (CAT) activities due to Penicillium digitatum infection were evaluated on oranges. These biochemical parameters were also determined when oranges were treated with the biocontrol agent Pantoea agglomerans CPA-2. After harvest, oranges were divided in two batches of fruit: one was used immediately and another stored at 4 °C for 2 months. In both cases, H 2O 2 content and changes in enzyme activities were determined at specific time intervals in oranges inoculated with P. digitatum or treated with P. agglomerans. Similar levels of H 2O 2 were observed during the first 3 days after inoculation in all treatment (control, infected and treated fruit). A short time after, CPA-2 treated fruit showed an accumulation of H 2O 2 while infected fruit exhibited a sharp decrease in H 2O 2 levels. An increase of SOD and CAT activities was also observed in CPA-2 treated oranges; in contrast, infected fruit showed a significant reduction of both enzymatic activities. This biochemical approach suggests that P. digitatum suppresses H 2O 2 production as well as SOD and CAT activities in orange tissue as response to infection process. In contrast, the biocontrol agent P. agglomerans CPA-2 triggers H 2O 2 production and both enzymatic activities probably as a mechanism of action to prevent citrus fruit against future infections of green mold. Our data suggest that P. agglomerans CPA-2 may act as antagonist against green mold not only by competition for nutrients and space but also by inducing oxidative response in orange tissue.

221 EFFECT OF STAGE OF CORPUS LUTEUM DEVELOPMENT ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

The objective of this study was to investigate the effect of stage of corpus luteum (CL) development on the in vitro production of bovine embryos. Ovaries were classified according to the expected day of the oestrous cycle based on the morphology of the ovaries. Ovaries with a corpus hemorrhagicum and the remnant of the follicular lumen filled with blood were considered the early luteal stage (Days 2 to 4; Day 0 = day of ovulation, n = 46). Ovaries with a large mass of orange tissue in the CL were classified as the midluteal stage (Days 7 to 10, n = 42). Cumulus–oocyte complexes (COC) were collected by aspiration of 2- to 6-mm follicles. The COC were classified into the following grades: COC with &gt;3 compact layers of cumulus cells and evenly granulated cytoplasm were classified into Grade 1; COC with &gt;3 layers cumulus cells and evenly granulated cytoplasm were classified into Grade 2; COC with partially remaining cumulus cells and abnormal cytoplasm were classified into Grade 3; COC without cumulus cells or those with expanded cumulus cells were classified into Grades 4 and 5, respectively. Grades 1 and 2 COC were in vitro matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 mg mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air. Matured COC were inseminated with 5 × 106 sperm for 18 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). The mean number of COC and the proportion of COC classified as Grades 1 and 2 were analysed by ANOVA. Cleavage rates on Day 3 and blastocyst rates on Days 7 to 9 were analysed by a chi-square test. The mean number of recovered oocytes in the early luteal stage (18.7 ± 9.5) was significantly higher (P &lt; 0.05) than the number in the midluteal stage (12.2 ± 5.7). The proportion of Grades 1 and 2 oocytes in the early luteal stage [66.7% (531/789)] was significantly higher (P &lt; 0.01) than that in the midluteal stage [51.6% (252/484)]. The cleavage and blastocyst rates in the early luteal stage [60.9% (181/297) and 32.7% (97/297), respectively] were significantly higher (P &lt; 0.05) than those in the midluteal stage [50.7% (76/150) and 20.7% (31/150) respectively].The present study suggests that the stage of development of the CL in bovine ovaries influences the number of recovered oocytes per ovary and the development of in vitro production of bovine embryos.

Development of &lt;I&gt;Epiphyas postvittana&lt;/I&gt; (Lepidoptera: Tortricidae) on Leaves and Fruit of Orange Trees

Development of Epipyas postvittna (Walker) (Lepidoptera: Tortricidae), on leaves and fruit of 'Valencia', 'Washington navel', and 'Navelina' orange trees was studied under constant and fluctuating temperatures. E. postvittna was able to complete its life cycle feeding exclusively on leaves or fruit of orange trees. However, larval survival rate was very low (< 20%) on orange tissues compared with that on noncitrus hosts. Among the four types of orange tissues, young orange leaves and fruit afforded larvae higher survival rates than mature orange leaves and fruit. Fruit (young or mature) produced heavier pupae than leaves (young or mature). Larvae developed more slowly on mature orange fruit than on other orange materials and more slowly on orange leaves than on leaves of most noncitrus hosts. Degree-day accumulations based on the fastest developmental rates obtained in this study suggested that E. postvittna is capable of completing 4.4-4.7 generations per year in orange orchards in the Riverina region of New South Wales, Australia. Implications of the results in the management of the insect in citrus are discussed.