The Effect of Vitrification and in vitro Culture on the Adenosine Triphosphate Content and Mitochondrial Distribution of Mouse Pre-Implantation Embryos

Background: The mitochondria are an important source of adenosine triphosphate (ATP) production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. Methods: The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. Results: The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively (P>0.05). The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups (P>0.05). Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Conclusions: Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown.Key Words: Mitochondria, Vitrification, Adenosine triphosphate (ATP)     

Genetic diversity and pathogenicity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolated from wilted Welsh onion in Japan

Thirty isolates of Fusarium oxysporum from wilted Welsh onion plants were examined for their diversity in nucleotide sequences of the ribosomal DNA (rDNA) intergenic spacer (IGS) region and for pathogenicity with regard to five Welsh onion cultivars. Phylogenetic analysis based on the IGS sequences revealed polyphyletic origins of the isolates and a relationship between phylogeny and pathogenicity; low virulence isolates differed genetically from those with high and moderate virulence. Mating type analysis revealed that all F. oxysporum isolates were MAT1-1 idiomorphs, suggesting that the pathogens may be clonal in the fields examined.     

Pathogenic variation and molecular characterization of <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from wilted Welsh onion in Japan

Thirty-two isolates of Fusarium species were obtained from wilted Welsh onion (Allium fistulosum) grown on nine farms from six regions in Japan and identified as F. oxysporum (18 isolates), F. verticillioides (7 isolates), and F. solani (7 isolates). The pathogenicity of 32 isolates was tested on five commercial cultivars of Welsh onion and two cultivars of bulb onion in a seedling assay in a greenhouse. The Fusarium isolates varied in the degree of disease severity on the cultivars. Five F. oxysporum isolates (08, 15, 17, 22, and 30) had a higher virulence on the cultivars than the other isolates. The host range of these five isolates was limited to Allium species. Molecular characterization of Fusarium isolates was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA. The 32 isolates were grouped into eight types (four types for F. oxysporum, one for F. verticillioides, and three for F. solani). Restriction patterns of the ITS region were not related to pathogenicity. However, the haplotypes obtained with five enzymes (RsaI, HinfI, HaeIII, ScrFI, and MspI) and the phylogenetic analysis permitted the discernment of the three Fusarium species. The PCR-RFLP analysis should provide a rapid, simple method for differentiating Fusaruim species isolated from wilted Welsh onion in Japan.     

Pathogenic and molecularVariability of Colletotrichum falcatum Went. Isolates from sugarcane with red rot disease symptoms

Pathogenic variability of different isolates of Colletotrichum falcatum Went. collected from six varieties: Co 419, CoC 671 (from Andhra Pradesh), Co 86032, Co87044, 89V74, and a local variety in Bhadrak (from Orissa) were established. Six red rot isolates were differentiated into four distinct pathotypes, viz., pathotype A - Cf 419 and Cf Bdkh; pathotype B - Cf 671; pathotype C - Cf 89V74 and pathotype D - Cf 86032, and Cf 87044. Pathotype A showed a susceptible reaction to Khakai, CoC 671, CoJ 64, and Co 419. Also pathotype B showed a susceptible reaction to Baragua, CoC 671, Co 997, CoJ 64, Co 1148, and Co 975, whereas pathotype C showed a susceptible reaction to CoC 671, Co 7717, Co 997, CoJ 64, Co 975, and Co 419. But pathotype D showed a susceptible reaction to CoC 671 and Co 997. Polymerase chain reaction (PCR) amplification and subsequent restricted fragment length polymorphism (RFLP) analysis of internal transcribed spacer region of ribosomal DNA (rDNA) of Cf 89V74, Cf 671 and Cf Vittal (C. falcatum isolate from Vittal, Karnatak) by four restriction enzymes, Alu I, Msp I, Rsa I, and Pvu II revealed two distinct groups viz.Group 1- Cf 89V74 and Cf Vittal; Group 2-Cf 671. Restriction enzymes Alu I, Msp I, Rsa 1, and Pvu II produced fragment lengths of 380, 520, 550, and 580 bp for Cf Vittal and Cf 89V74, whereas fragment lengths of 420, 370, 440, and 460 bp were observed in Cf 671, respectively. The present investigation with the red rot infected sugarcane samples clearly indicated the existence of two pathotypes viz., pathotype A and pathotype B in Andhra Pradesh and three pathotypes viz., pathotype A, pathotype C, and pathotype D in Orissa. Pathotype C (Cf 89V74) showed similar ITS-RFLP pattern in Cf Vittal which was isolated from Karnataka.     

(−)-Loliolide Isolated from Sargassum horneri Abate UVB-Induced Oxidative Damage in Human Dermal Fibroblasts and Subside ECM Degradation

Ultraviolet (UV) B exposure is a prominent cause of skin aging and a contemporary subject of interest. The effects are progressing through the generation of reactive oxygen species (ROS) that alter cell signaling pathways related to inflammatory responses. The present study evaluates the protective effects of (7aR)-6-hydroxy-4,4,7a-trimethyl-6,7-dihydro-5H-1-benzofuran-2-one (HTT) isolated from the edible brown algae Sargassum horneri against UVB protective effects in human dermal fibroblasts (HDFs). HTT treatment dose-dependently suppressed intracellular ROS generation in HDFs with an IC₅₀ of 62.43 ± 3.22 µM. HTT abated UVB-induced mitochondrial hyperpolarization and apoptotic body formation. Furthermore, UVB-induced activation of key nuclear factor (NF)-κB and mitogen-activated protein kinase signaling proteins were suppressed in HTT treated cells while downregulating pro-inflammatory cytokines (interleukin-1β, 6, 8, 33 and tumor necrosis factor-α). Moreover, HTT treatment downregulated matrix metalloproteinase1, 2, 3, 8, 9 and 13 that was further confirmed by the inhibition of collagenase and elastase activity. The evidence implies that HTT delivers protective effects against premature skin aging caused by UVB exposure via suppressing inflammatory responses and degradation of extracellular matrix (ECM) components. Extensive research in this regard will raise perspectives for using HTT as an ingredient in UV protective ointments.     

Influence of tuber weight and cutting on growth and yield of safed musli (Chlorophytum borivilianum)

The present study was aimed to investigate the effect of different weight of planting material and cutting on safed musli cultivation. Tubers were divided into three weight categories (3, 6 and 9 g bulk^?1) and were either planted as whole or cut tubers. The plant growth and development were recorded at 60, 90 and 120 days after sprouting (DAS). The results showed that both factors had no interaction for all parameters measured. The sprouting percentage was not significantly different in all the treatments. The weight of planting materials used in the treatments had no influence on the overall plant growth and development. However, there was a significant difference observed between whole tuber and cut tuber treatments. At 90 DAS, tubers subjected to cutting encouraged the plants to produce the higher number of leaves (22.7) with improved total leaf area (642 cm²), fibrous root length (2795 cm) and tuber dry weight (7.8 g). The same pattern was observed at 120 DAS where the cut tubers produced the higher number of tubers (20.7) with increased length (8.6 cm) and diameter (6.6 mm). Consequently, these factors resulted in higher yield, 15.7 as compared to 13.6 g bulk^?1 in non-cut treatment.     

Portable acoustic device for detection of coconut palms infested by Rynchophorus ferrugineus (Coleoptera: Curculionidae)

Acoustic methods have been developed and used to detect insects in concealed habitats. The larvae of red palm weevil, Rynchophorus ferrugineus (Olivier), a serious pest of the coconut palm, Cocos nucifera L. feed on the soft tissues inside the stem and bud region. Detection of infested coconut palms in the early stages by the conventional method of checking for external symptoms is time consuming, labour intensive and costly. This paper describes the development of a portable and efficient acoustic device and its potential in detection of infested palms in the field. The device comprises a sensor to mount on the palm and to acquire the sounds of red palm weevil larvae, an electronic unit that processes the acquired sounds and a set of headphones to receive the output sound by the listener. It is light weight, user-friendly and powered by batteries. The highest accuracy of identifying infested palms was obtained when the palm was checked at four positions; either side of palm base and bases of the two lowermost leaves. The infested palms were detected with over 97% accuracy, while the probability of not detecting uninfested palms was over 92%. A second check increased the rate of accuracy. Many difficulties encountered with conventional methods could be overcome by the use of this device.     

Lower soil nitrous oxide emissions by a high lipid genetically modified perennial ryegrass line compared to its wild type

We describe an experiment where cattle urine was applied at a rate of 420 kg N ha⁻¹, equivalent to 10 L m⁻², to mesocosm swards of a high lipid genetically modified perennial ryegrass line (HME) and its wild type (WT). We measured N pools and fluxes in the plant and soil, soil microbial populations and N₂O production. HME plants produced 21% greater biomass than WT (p = .02), resulting in greater N uptake (27% higher in HME, p = .05). Urine N recovery in total plant biomass during the experiment in HME and WT swards were 54.7% and 33.9% respectively. The nitrification potential of soil was significantly lower (p = .01) in HME than WT. Partial least square-discriminant analysis using microbial gene abundance data indicated that HME and WT plant growth induced distinct microbial populations in the soil. These differences in plant soil microbial interactions between HME and WT swards resulted in significantly lower N₂O emissions from the HME sward. Total N₂O emissions over the 4 weeks after urine application was 38% lower (p < .03) in HME swards than in WT swards. The next step in this work is to identify the specific changes in HME traits that drive the reduction in N₂O.     

Adsorption of deoxyribonucleic acid (DNA) by willow wood biochars produced at different pyrolysis temperatures

Adsorption of DNA by biochars was investigated in the present study. Biochars were produced from air-dried willow wood chips at 300, 400, 500, and 600 °C under limited oxygen supply. The resulting products, referred to as BC300, BC400, BC500, and BC600, respectively, were characterized for their elemental composition, cation exchange capacity (CEC), specific surface areas (SSA), and microporosity. According to a Langmuir isotherm, maximum DNA adsorption capacity of biochars was ranked as BC500?>?BC600?>?BC400?>?BC300. Increasing solution pH (from 4.0 to 9.0) faintly decreased DNA adsorption onto biochars. The addition of Na⁺, Mg²⁺, and Ca²⁺ slightly increased the adsorption of DNA, and the effect decreased by increasing the pyrolysis temperature of biochars, indicating that electrostatic interaction was not the main driving force for DNA adsorption onto those biochars. Correlation analysis showed that SSA and micropore surface area were the main factors influencing DNA adsorption on biochars.     

白花草木樨半同胞家系的生物固氮性状评价

以不同的白花草木樨(Melilotus albus)半同胞家系为试验材料,分别种植于甘肃临泽和榆中,测定其根干重、根瘤数、根瘤重、叶全氮、叶全磷和生物固氮百分率,以此评价其生物固氮能力。结果表明,白花草木樨在两个地区表现出的各项固氮指标变异系数均大于15%,主成分分析后,可将半同胞家系划分为4组,组1白花草木樨家系的根干重、根瘤数、根瘤重、叶全氮、叶全磷和生物固氮百分率分别高出对照当地白花草木樨品种(RX-02) 4%、26%、10%、17%、24%、11%,其中生物固氮百分率的最大值和最小值分别为58%和43%。本研究筛选出的优良生物固氮性状的后代家系,可为进一步选育生物固氮能力强的新品种奠定基础。