Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis> system

An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2Dwrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes’ integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T₁ progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.     

Relationships of spectral traits with yield and nutritional quality of snap beans (Phaseolus vulgaris L.) in dry seasons

Four snap bean varieties (green and yellow-podded) were grown in three water supplies (regularly irrigated, deficit-irrigated and non-irrigated) in order to examine the spectral reflectance at leaf and canopy levels, leaf area index (LAI) and their relationships with the yield and nutritional quality at the University of Debrecen, Debrecen, Hungary from 2011 to 2013. Under non-irrigated condition, the plant height decreased, SPAD values were high but LAI and normalized difference vegetation index (NDVI) were low. The difference in SPAD values was higher between the varieties during flowering under non-irrigated condition and it was more pronounced during pod development using deficit irrigation. In dry years, non-irrigated condition SPAD did not correlate with the yield but during pod development it was correlated with the dry matter content of the pods. The relationship of SPAD with protein and crude fiber content of pods was strongly dependent on the cropping years. Under deficit-irrigated condition, NDVI measured during flowering and pod development correlated closely with the yield where a higher NDVI (0.85) predicted either 6 or 9.5 t ha^?1 of yield depending on the variety. Under this condition Serengeti variety with green pods was prominent based on the studied traits among the varieties.     

Effect of Breeding Activity on the Microflora of the External Genitalia and in the Semen of Stallions,and the Relationship Between Micro‐organisms on the Skin and on the External Genitalia

A possible role of breeding activities in the composition of the microbial population in stallions' external genitalia (EG) and the relationship between micro‐organisms colonizing the skin of the abdomen and the ones colonizing the EG have not been studied. In experiment 1, EG microbiological samples were collected from 41 stallions used for both natural cover and semen collection (BST) and from 18 non‐breeding stallions (NBST). A higher (p < 0.05) frequency of isolation of potentially pathogenic species was found for BST. Age did not influence number of micro‐organism species isolated both in BST and NBST. In experiment 2, the microbial content of the EG and semen was compared in 23 BST. Most micro‐organisms isolated from the EG were present in semen, albeit with a numerically lower prevalence. In 7 stallions, six microbial species isolated from semen were absent from the EG cultures, suggesting contamination by the operator. In experiment 3, a numerically higher number of micro‐organism species was isolated from the EG of 31 stallions, than from their skin of the ventral abdomen in contact with the penis or from the skin of the thorax. With the sole exception of Escherichia coli, potentially pathogenic bacteria were only isolated from the EG but not from the skin. Results suggest that breeding activity increased the number of species colonizing the EG; most species isolated from the EG were also found in semen even if with a lower frequency, and additional semen contamination seemed to occur during its manipulation. Many micro‐organism species of the skin were also isolated from the penis, but independently of being or not in contact with the penis, skin did not seem to provide an adequate environment for the growth of potentially pathogenic bacteria that were isolated from EG, with the sole exception for E. coli.     

Defining the validity of a biochemical index of soil quality

 The native soils of Galicia (NW Spain) exhibit a biochemical equilibrium such that total soil N is a function of five biochemical and microbiological parameters: microbial biomass C, mineralized N, phosphomonoesterase, β-glucosidase and urease activities. To investigate whether the ratio of the total N calculated from biochemical soil properties (Nc) and the total N as measured by the Kjeldahl method (Nk; Nc/Nk) can be used as an index of soil quality, we determined these variables and consequently the ratio in three kinds of disturbed soils: an artificially Cu-contaminated soil, two lignite mine soils, and a number of arable soils. In none of the studied soils did the individual biochemical parameters respond consistently to the factors influencing soil quality, but in all cases soil degradation was reflected by the Nc/Nk value, which differed more or less markedly from 100%. Nc/Nk can therefore be used for the rapid evaluation of soil degradation, since it distinguishes among biochemically balanced soils, soils in a transient state of high microbiological and biochemical activity and degraded soils. It can also serve as a reliable basis for the rapid calculation of the "ecological dose" (ED₅₀) of soil pollutants. The use of Nc/Nk as an objective index of the biochemical quality of soils is recommended. Received: 20 December 1998     

Lack of haplotype structuring for two candidate genes for trypanotolerance in cattle

Bovine trypanotolerance is a heritable trait associated to the ability of the individuals to control parasitaemia and anaemia. The INHBA (BTA4) and TICAM1 (BTA7) genes are strong candidates for trypanotolerance‐related traits. The coding sequence of both genes (3951 bp in total) were analysed in a panel including 79 Asian, African and European cattle (Bos taurus and B. indicus) to identify naturally occurring polymorphisms on both genes. In general, the genetic diversity was low. Nineteen of the 33 mutations identified were found just one time. Seventeen different haplotypes were defined for the TICAM1 gene, and 9 and 12 were defined for the exon 1 and the exon 2 of the INHBA gene, respectively. There was no clear separation between cattle groups. The most frequent haplotypes identified in West African taurine samples were also identified in other cattle groups including Asian zebu and European cattle. Phylogenetic trees and principal component analysis confirmed that divergence among the cattle groups analysed was poor, particularly for the INHBA sequences. The European cattle subset had the lowest values of haplotype diversity for both the exon1 (monomorphic) and the exon2 (0.077 ± 0.066) of the INHBA gene. Neutrality tests, in general, did not suggest that the analysed genes were under positive selection. The assessed scenario would be consistent with the identification of recent mutations in evolutionary terms.     

Morphological development and allometric growth of yellowtail kingfish Seriola lalandi V. larvae under culture conditions

Morphological development and allometric growth patterns of Seriola lalandi larvae were assessed to characterize normal growth patterns under culture conditions. Early ontogenetic stages of yellowtail kingfish exhibited an exponential growth in terms of standard length as a function of age. Five development stages were characterized from hatching to the juvenile stage: larval stage I (0–2 days post hatch, dph) with endogenous feeding, characterized by a small yolk sac, unpigmented eyes, primordial finfold surrounding the body and a closed mouth; larval stage II (2–15 dph) characterized by mouth opening, complete pigmentation of eyes and the beginning of the exogenous feeding; subsequently, in the larval stage III (15–25 dph) the posterior tip of notochord of the larvae bended upward and the first rays appeared in fins, concomitant with a change in swimming behaviour; thereafter, larval stage IV (post‐flexion stage; 25–30 dph) began when larvae resembled in morphology to a juvenile organism; however, caudal and dorsal fins were not completely development. Lastly, the juvenile stage was reached 30 dph characterized by a morphology and fin structures similar to those of the adults. Growth and development of structures and organs associated with vital functions such as feeding, sensorial and breathing systems seemed to be more critical previous to 23 dph, which was reflected with a positive allometric growth of head and eyes during this period. The results from this study can be used as a tool‐guide to assess normal development in larval research with S. lalandi to improve existing rearing protocols in hatchery production.     

Competitive and allelopathic interference between soybean crop and annual wormwood (Artemisia annua L.) under field conditions

Annual wormwood interference on soybean crop growth and yield may result from competition and allelopathy, which are modulated by crop management. Allelochemicals released by annual wormwood (e.g. artemisinin) may affect the crop directly or indirectly through the effect on the nitrogen fixing symbiont, Bradyrhizobium japonicum. The objectives were (i) to quantify the crop response (i.e. biomass production, nodulation and yield) to weed interference and (ii) to determinate the relative change of competition and allelopathy interferences, when a sublethal dose of herbicide is applied. Two split plot field experiments with three replications were used. The experiment involved a factorial combination of five weed–crop density (soybean/annual wormwood, plants m^?2) levels: D1, pure soybean, 40/0 plants m^?2; D2, 40/2 plants m^?2; D3, 40/4 plants m^?2 and D4, 40/8 plants m^?2, and D5, pure annual wormwood, 0/8 plants m^?2, two activated carbon (allelopathy) levels: C?, with activated carbon (reduced allelopathy) and C+, non activated carbon applied (with allelopathy) and two herbicide levels: H?, untreated and H+, treated with a sub-lethal dose of glyphosate. Activated carbon to adsorb allelochemicals (with and without activated carbon) and glyphosate application (with and no herbicide) were assigned to sub-plots. Increasing weed density did not affect crop biomass at flowering, but changed nodule number and soybean yield with a different pattern depending on carbon and herbicide treatment. Relative crop yield decreased with increasing relative weed biomass. This decrease was particularly drastic when allelopathy was reduced by activated carbon and without herbicide application. The maximum yield losses of 33% in 2006 and 17% in 2007 were observed with the highest weed density (8 plants m^?2). In contrast, without carbon (high allelopathy level), soybean yield remained stable within the explored range of annual wormwood biomass, despite the fact that weed biomass at high densities (D4) was high enough to generate competition. The lack of response to increasing weed density could be related to the indirect effect of allelochemicals interacting with soil microorganisms (i.e. B. japonicum) that positively affected the nodulation (e.g. larger nodules in 2006 and increased nodules biomass due to higher number of roots in 2007 at high densities). With herbicide application, soybean yield of both carbon treatments remained stable when biomass of annual wormwood increased. This research provided strong evidence in support of the existence of positive effect of allelopathic and competitive interactions between annual wormwood and soybean crop under field conditions that may be overridden under herbicide application.     

Effect of resistant starch on the intestinal health of old dogs: fermentation products and histological features of the intestinal mucosa

The effects of resistant starch (RS) intake on nutrient digestibility, microbial fermentation products, faecal IgA, faecal pH, and histological features of the intestinal mucosa of old dogs were evaluated. The same formulation was extruded in two different conditions: one to obtain elevated starch cooking degree with low RS content (0.21%) and the other lower starch cooking with high RS content (1.46%). Eight geriatric Beagles (11.5 ± 0.38 years old) were fed each diet for 61 days in a crossover design. Food intake, nutrient digestibility, fermentation products, faecal pH, and faecal IgA were examined via variance analysis. Histological results of intestinal biopsies were assessed via Wilcoxon test for paired data. The morphometric characteristics of large intestine crypts were evaluated via paired t tests (p < .05). Protein, fat, and energy digestibilities were higher for the low‐RS diet (p < .05). Dogs receiving the high‐RS diet had lower faecal pH and higher values for propionate, butyrate, total volatile fatty acids, and lactate (p < .05). No differences between diets were found in the histological parameters of the gut mucosa, and only a tendency for deeper crypts in the descending colon was observed for dogs fed the high‐RS diet (p = .083). The intake of a corn‐based kibble diet manufactured with coarse ground raw material and low starch gelatinization to obtain 1.4% of RS affected microbial fermentation products and faecal pH and tended to increase crypt depth in the descending colon of old dogs.     

Effect of age and gender in the pharmacokinetics of albendazole and albendazole sulphoxide enantiomers in goats

The kinetics of albendazole metabolites and albendazole sulphoxide enantiomers were studied in 2- and 14-month-old female and male goats, after a single oral dose administration (10 mg/kg) of an albendazole formulation. Blood samples from the jugular vein were collected at 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42, 48 and 54 h post-treatment and analyzed using a high performance liquid chromatography method. In all groups the area under the plasma concentration-time curve (AUC) and peak concentration (C_max) values of (+)-ABZSO were significantly higher than those of (−)-ABZSO. The AUC and C_max values obtained for (+)-ABZSO and (−)-ABZSO in adult animals were higher compared to the results in young animals, showing significant differences except for (+)-ABZSO in female animals. In young animals, independently of gender, the C_max appeared earlier compared to adult animals. The mean residence time (MRT) values were shorter in young animals compared to adult animals for all compounds analyzed. No sex-related differences were found for any of the parameters calculated except for the (+)-ABZSO from adult animals.     

Optimization of the extraction of antioxidants from Dunaliella salina microalga by pressurized liquids

In this work, extraction of antioxidant compounds from Dunaliella salina microalga is optimized by combining pressurized liquid extraction (PLE) and experimental design (three-level factorial design) with three different solvents (hexane, ethanol, and water). Two main factors were considered, the extraction temperature (40, 100, and 160 degrees C) and the extraction time (5, 17.5, and 30 min). As response variables, the extraction yield (percent dry weight/initial weight) and the antioxidant activity of the extracts (determined using the TEAC method) were used. The parameters of the model were estimated by multiple linear regression. Results showed that the extraction temperature was the factor having the strongest influence (positive) on the two response variables. The best yields were obtained with ethanol at the higher extraction temperature and time tested. Besides, although hexane extracts provided the best antioxidant activity, ethanol extracts were also very active. The chemical characterization of ethanol extracts was carried out using HPLC-DAD, and attempts have been made to correlate their chemical composition with the antioxidant activity measured. Results pointed out that the extracts contained, besides all-trans-beta-carotene and isomers, several different minor carotenoids that seemed to make a contribution to the antioxidant activity of the extracts.