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     

Effects of elevated CO2 concentration on bulbil germination and early seedling growth in Chinese yam under different air temperatures

The present study investigated the effects of elevated carbon dioxide concentration ([CO₂]) and air temperature on the germination of seed bulbils and the seedling vigour of two Chinese yam lines. Plants were grown under two [CO₂] levels, ambient and elevated (ambient + 200 μmol mol^?1), and two mean air temperature regimes, 22.2 °C (ambient + 1.4 °C) and 25.6 °C (ambient + 5.2 °C). Elevated [CO₂] did not affect bulbil germination under both air temperature regimes. During the early growth stage, the dry weight (DW) of leaves, vines, shoots, roots, belowground parts (roots + tubers) and whole plants were higher under elevated [CO₂] than ambient [CO₂] for both lines under the low- and high-temperature regimes. The values of vigour indexes (index I = germination % × seedling length and index II = germination % × seedling DW) were also higher under elevated [CO₂] than ambient [CO₂] for both lines. These results indicated that Chinese yam seedlings respond positively to elevated [CO₂] during the early growth stage. The above:belowground DW ratios were lower under elevated [CO₂] than ambient [CO₂] in seedlings with very small new tubers for both yam lines, indicating that elevated [CO₂] strongly affected the root growth in the early growth stage. The DWs of post-treatment seed bulbils were higher in the elevated [CO₂] under both air temperature regimes. The results showed that Chinese yam used a smaller amount of the reserves in seed bulbils under elevated [CO₂] than under ambient [CO₂].     

Bovine viral diarrhea virus subgenotype 1b in water buffalos (Bubalus bubalis) from Brazil

Tropical Animal Health and Production - Serological studies have characterized the presence of the bovine viral diarrhea virus (BVDV) infection in water buffalo herds worldwide. However, the...     

Inclusion levels and modes of whole grain incorporation into wheat-based rations differentially influence the performance of broiler chickens

1. The objective was to compare three whole grain (WG) inclusion levels (7.5, 15 and 30%) offered to broiler chickens by three modes of WG incorporation: (i) pre-pellet WG inclusion, (ii) post-pellet WG inclusion as a blend of WG and pelleted concentrate and (iii) post-pellet WG inclusion where WG and pelleted concentrate were provided in separate feed trays against a ground-grain, wheat-based control diet.

2. Ten dietary treatments were offered to 6 replicate cages (6 birds per cage) of male Ross 308 chickens from 7 to 28 d post-hatch. The effects of treatment on relative gizzard weights, gizzard contents, pancreatic weights and pH of gizzard digesta were monitored. Parameters of growth performance, nutrient utilisation (apparent metabolisable energy [AME], metabolisable to gross energy [ME:GE] ratios, nitrogen [N] retention and N-corrected AME [AMEn]), apparent starch and protein (N) digestibility coefficients and disappearance rates in for small intestinal segments and concentrations of free amino acids in plasma taken from the anterior mesenteric vein were determined.

3. Whole grain feeding (WGF) did not influence weight gain, but 30% post-pellet blended and 15 and 30% post-pellet separated treatments significantly depressed (P < 0.05) feed intakes while the 30% post-pellet separated treatment improved (P < 0.01) feed conversion ratios (FCR). WGF regimes significantly increased relative gizzard weights.

4. Overall, WGF generated profound responses in AME, ME:GE ratios, N retention and AMEn that were highly correlated with relative gizzard weights. In general, WGF improved starch and protein (N) digestibilities and again there were some correlations with these outcomes and relative gizzard weights.

5. Post-pellet WG inclusions where WG and pelleted concentrate were offered separately provided chickens with the opportunity to choice feed. Birds showed a preference for the relatively high-protein pelleted concentrate and at 30% WG, this resulted in an improvement in FCR of 7.69% (1.260 versus 1.365; P < 0.001) relative to the ground-grain control diet.     

Effects of Fermentation Conditions Using Lactobacillus plantarum on the Charantin,Stigmasterol Glucoside and β-sitosterol Glucoside Contents of Bitter Gourd (Momordica charantia L.) Juice

Plant Foods for Human Nutrition - Bitter gourd fruits contain high amounts of charantin, stigmasterol glucoside and β-sitosterol glucoside, which have been shown to provide health benefits for...     

Differences in Ion Accumulation and Salt Tolerance among Glycine Accessions

Salinity reduces crop yield by limiting water uptake and causing ion‐specific stress. Soybean [Glycine max (L.) Merr.] is sensitive to soil salinity. However, there is variability among soybean genotypes and wild relatives for salt tolerance, suggesting that genetic improvement may be possible. The objective of this study was to identify differences in salt tolerance based on ion accumulation in leaves, stems and roots among accessions of four Glycine species. Four NaCl treatments, 0, 50, 75 and 100 mm , were imposed on G. max, G. soja, G. tomentella and G. argyrea accessions with different levels of salinity tolerance. Tolerant genotypes had less leaf scorch and a greater capacity to prevent Na⁺ and Cl^? transport from soil solution to stems and leaves than sensitive genotypes. Magnitude of leaf injury per unit increase in leaf Na⁺ or Cl^? concentrations was lower in tolerant than in susceptible accessions. Also, plant injury was associated more with Na⁺ rather than with Cl^? concentration in leaves. Salt‐tolerant accessions had greater leaf chlorophyll‐meter readings than sensitive genotypes at all NaCl concentrations. Glycine argyrea and G. tomentella accessions possessed higher salt tolerance than G. soja and G. max genotypes.     

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

The effects of maxillary nerve block,ethmoidal nerve block and their combination on cardiopulmonary responses to nasal stimulation in anesthetized Beagle dogs

ObjectiveTo describe an approach for ethmoidal nerve block (E_BLOCK) and to compare the effects of a maxillary nerve block (M_BLOCK), E_BLOCK and their combination (M-E_BLOCK) on heart rate (HR), systolic (SAP), mean (MAP), diastolic (DAP) arterial pressures and respiratory rate (f_R) during nasal stimulation in dogs.Study designProspective, blinded, randomized, crossover placebo-controlled study.AnimalsBeagle dogs (five cadavers, nine live dogs), with a median (interquartile range) weight of 10.5 (10.3–11.0) kg.MethodsThe accuracy of iohexol injections (each 1 mL) at the maxillary and ethmoidal foramina in cadavers was evaluated using computed tomography. Then, anesthetized dogs were administered four bilateral treatments separated by 1 week, saline or 2% lidocaine 1 mL per injection: injections of saline at the maxillary and ethmoidal foramina (Control), injections of lidocaine at the maxillary foramina and saline at the ethmoidal foramina (M_BLOCK), injections of saline at the maxillary foramina and lidocaine at the ethmoidal foramina (E_BLOCK) and injections of lidocaine at all foramina (M-E_BLOCK). The ventral nasal meatus was bilaterally stimulated using cotton swabs, and HR, SAP, MAP, DAP and f_R were continuously recorded. Values for each variable were compared before and after stimulation using Wilcoxon signed-rank test. Changes in variables among treatments were analyzed using Mann–Whitney U and Kruskal–Wallis tests (p ≤ 0.05).ResultsComputed tomography revealed iohexol distribution around the openings of the target foramina in all cadavers. In living dogs, HR, SAP, MAP, DAP and f_R significantly increased after stimulation within each treatment (p < 0.03). Physiologic responses were significantly attenuated, but not absent, in the M-E_BLOCK [HR (p = 0.019), SAP, MAP, DAP and f_R (all p ≤ 0.001)] compared with those in the Control.Conclusions and clinical relevanceConcurrent injections of lidocaine at the maxillary and ethmoidal foramina attenuated HR, arterial pressure and f_R responses to nasal stimulation in Beagle dogs.     

A morphological analysis of the skull size and shape of Kerivoulinae (Chiroptera: Vespertilionidae) from Vietnam

Southeast Asia is a region of high biodiversity, containing species of plants and animals that are yet to be discovered. In this region, bats of the subfamily Kerivoulinae are widespread and diverse with six species recorded in Vietnam. However, the taxonomy of the Kerivoulinae in Asia is complicated. In our study, we used diagnostic characters and multivariate analysis to determine morphological differences between the genera Kerivoula and Phoniscus. We showed that the two genera are distinguishable by the size of second upper incisors, the shape of skull, nasal sinus, canines, second upper and lower premolars. In addition, the two genera can be osteometrically separated by measurements of the braincase height, interorbital width and shape of anterior palatal emargination. Our data clearly revealed the morphological variations in the skull shape of Kerivoula hardwickii in Vietnam. This suggests a possible separation into three morphotypes, representing cryptic species supported by statistical differences with wide variation in skull shape, size and teeth. These results demonstrated Kerivoula hardwickii can be separated three subspecies, and the result will serve as the basis for the future assessment and classification of this group in Southeast Asia.