Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

Hybrid breeding for biomass yield in winter triticale: II. Combining ability and hybrid prediction

Accurate hybrid prediction and knowledge about the relative contribution of general (GCA) and specific combining ability (SCA) are of utmost importance for efficient hybrid breeding. We therefore evaluated 91 triticale single-cross hybrids in field trials at seven environments for plant height, heading time, fresh biomass, dry matter content and dry biomass. Fresh and dry biomass showed the highest proportion (23%) of variance due to SCA. Prediction accuracies based on GCA were slightly higher than based on mid-parent values. Utilizing parental kinship information yielded the highest prediction accuracies when both parental lines have been tested in other hybrid combinations, but still moderate-to-low prediction accuracies for two untested parents. Thus, hybrid prediction for biomass traits in triticale is currently promising based on mid-parent values as emphasized by our simulation study, but can be expected to shift to GCA-based prediction with an increasing importance of GCA due to selection in hybrid breeding. Moreover, the performance of potential hybrids between newly developed lines can be predicted with moderate accuracy using genomic relationship information.     

Diversity of root-knot nematodes in Moroccan olive nurseries and orchards: does <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> disperse according to invasion processes?

Background

Root-knot nematodes (RKN) are major pest of olive tree (Olea europaea ssp. europaea), especially in nurseries and high-density orchards. Soil samples were collected from main olive growing areas of Morocco, to characterize Meloidogyne species and to discuss the contribution of biotic and abiotic factors in their spatial distribution.

Results

RKN were found in 159 soil samples out of 305 from nurseries (52.1% occurrence) and in 11 out of 49 soil samples from orchards (23.2% occurrence). Biochemical and molecular characterisation (PAGE esterase and SCAR) revealed the dominance of M. javanica both in nurseries and orchards with minor presence of M. incognita only in nurseries, and M. arenaria in only one nursery. RKN were distributed on aggregated basis. Frequent presence of M. javanica in orchards might have come from nurseries. In contrast, the detection of M. incognita in nurseries alone suggests that this species could not reproduce in orchards because of either the competition with other plant-parasitic nematodes or unfit local habitats. The impact of environmental variables (climate, habitat origin and physicochemical characteristics of the substrates) on the distribution of Meloidogyne species is also discussed.

Conclusion

Olive nurseries in Morocco are not able to guarantee the safety of rooted plants. As a result, olive production systems are exposed to strong RKN invasion risks. Consequently, the use of healthy substrates in nurseries may prevent plant-parasitic nematode induction in orchards.

     

No effect of turkey herpesvirus infections on the outcome of avian pneumovirus infections in turkeys.

To determine whether turkey herpesvirus (HVT) impairs the aspecific and specific defense against an avian pneumovirus (APV) infection, specific-pathogen-free turkeys were inoculated at 7 days of age with HVT and 1, 5, or 7 wk later with APV. Clinical signs, APV replication, and development of antibodies against APV were evaluated. No differences were found between the birds that received both HVT and APV and those that received only APV.     

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.     

A Tremor Syndrome With a Central Axonopathy in Scottish Terriers

A central axonopathy in 2 male and 1 female Scottish Terrier puppies from 3 different but related litters is reported. Clinical signs consisting of severe whole-body tremors and ataxia were first detected at the age of 10 to 12 weeks. They worsened with activity and excitement and diminished during rest or sleep. Two dogs also had paraparesis. In 1 dog the neurological deficits progressed over several months. Neuropathological examination revealed widespread axonal changes, vacuolation, and gliosis in the white matter of the central nervous system.     

Particle Size Distribution in Two Lipid Emulsions Used for the Enrichment of Artemia nauplii as a Function of Their Preparation Method and Storage Time

The present study examined the distribution of particle sizes in two experimental standardized lipid emulsions (distributed by ICES, International Council for the Exploration of the Sea) as a function of the preparation method (hand shaking or ultrasonic blending) and as a function of storage time. A 24-h enrichment trial compared the incorporated HUFA levels in the nauplii of Artemia franciscana .
The emulsion droplets in the 50% HUFA emulsion (ICES 50, ethyl ester based) were much smaller than in the 30% HUFA emulsion (ICES 30, triacylglycerol-based) in which 90% of the droplets had a diameter below 12.3 μm as compared to 1.3 pn in ICES 50. The blending method highly affected particle sizes. High-shear blending instead of the classical hand shaking of the emulsion of both ICES 30 and 50 reduced the mean particle size from 5.06 to 1.07 μm and from 0.38 to 0.11 μm, respectively. The particle size distributions remained stable during the 1-wk storage, suggesting the absence of agglomeration or bacterial development. The fatty acid composition of 24-h enriched Artemia reflected differences in the HUFA profile of the emulsions, but was independent of observed differences in the size of the particles.     

Blastocyst Evaluation by Means of Differential Staining: a Practical Approach

Techniques for in vitro production of embryos have been developed world-wide in different species, with promising results in human and ruminants. Thousands of human IVF-babies have been born during the last 20 years and thousands of in vitro -produced calves have been born since the late 1980s. With current methods for bovine in vitro fertilization, about 30–40% of in vitro -fertilized bovine oocytes develop further to the blastocyst stage and can be used for transfer. A proper evaluation of blastocyst quality remains however, an important challenge for every researcher involved in embryology and for every clinician who wants to select the best embryos for transfer. This review attempts to summarize the different methods available for estimation of blastocyst quality with a special emphasis upon differential staining.     

Application of Airway Pressure Therapy in Veterinary Critical Care: Part I: Respiratory Mechanics and Hypoxemia

Over the past several decades, recognition of acute respiratory failure as the cause of death in patients suffering from various clinical conditions has prompted aggressiv investigation into the area of respiratory physiology and supportive respiratory care. With the evolution of emergency medicine and critical care services in both human and veterinary medicine, many patients previously considered unsalvageable due to the severity of their underlying disease are now being resuscitated and successfully supported, creating a new population of critically ill patients. Where only a decade ago these patients would have succumbed to their underlying disease, they now survive long enough to manifest the complications of shock and tissue injury in the form of acute respiratory failure. Investigation into the pathophysiology and treatment of this acute respiratory distress syndrom (ARDS) has facilitated increased clinical application of respiratory theerapy and machanical ventilation.¹ The purpose of this paper is to provide a basic review of respiratory mechanics and the pathophysiology of hypoxemia as they relate to airway pressure therapy in veterinary patients and to review the use of airway pressure therapy in veterinary patients This paper is divided into two parts; part I reviews respiratory mechanics and hypoxemia as they apply to respiratory therapy, while part II deals specifically with airway pressure therapy andits use in clinical cases.