Efficient induction of microspore embryogenesis using abscisic acid,jasmonic acid and salicylic acid in Brassica napus L

The stress hormones abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) play an important role in the regulation of physiological processes and are often used in tissue culture to promote somatic embryogenesis and to enhance the quality of somatic embryos. Despite many studies on Brassica napus microspore culture, the effects of stress hormones (ABA, JA and SA) on microspore embryogenesis are not well explored. In this study, the effects of three incubation periods (6, 12 and 24 h) at different levels of ABA, JA and SA (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1) on microspore embryogenesis of rapeseed (B. napus L.) cv. ‘Regent’ were investigated. ABA (0.5 mg l^?1 for 12 h) enhanced microspore embryogenesis by about threefold compared with untreated cultures and increased normal plantlet regeneration by 68 %. ABA treatment also effectively reduced secondary embryo formation at all concentrations tested but enhanced callusing at high levels, for example 67 % at 1.0 mg l^?1 for 24 h. Highest embryo yield (286.0 embryos Petri dish^?1) was achieved using 1.0 mg l^?1 JA for 24 h and highest normal plantlet regeneration (54 %) was observed in cultures exposed to 0.5 mg l^?1 JA for 12 h. JA (5.0 mg l^?1 for 24 h) also reduced the germination of microspore-derived embryos on regeneration medium by 21 %. SA at 0.2 and 0.5 mg l^?1 for 6 h increased microspore embryogenesis (184.0 and 193.4 embryos Petri dish^?1) relative to the control (136.2 embryos Petri dish^?1). However, SA did not improve normal regeneration, secondary embryo formation or callusing. Microspore embryogenesis and plant regeneration could be improved by ABA, JA as well as SA when the appropriate level and duration of incubation were selected.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

Expression and hypoxia-responsiveness of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 in mammary gland malignant cell lines

Recently, we have shown that PFKFB4 gene which encodes the testis isoenzyme of PFKFB is also expressed in the prostate and hepatoma cancer cell lines. Here we have studied expression and hypoxic regulation of the testis isoenzyme of PFKFB4 in several malignant cell lines from a female organ--the mammary gland. Our studies clearly demonstrated that PFKFB4 mRNA is also expressed in mammary gland malignant cells (MCF-7 and T47D cell lines) in normoxic conditions and that hypoxia strongly induces it expression. To better understand the mechanism of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, a specific inhibitor of HIF-1alpha hydroxylase enzymes, which strongly increases HIF-1alpha levels and mimics the effect of hypoxia. It was observed that PFKFB4 expression in the MCF7 and T47D cell lines was highly responsive to dimethyloxalylglycine, suggesting that the hypoxia responsiveness of PFKFB4 gene in these cell lines is regulated by HIF-1 proteins. Moreover, desferrioxamine and cobalt chloride, which mimic the effect of hypoxia by chelating or substituting for iron, had a similar stimulatory effect on the expression of PFKFB mRNA. In other mammary gland malignant cell lines (BT549, MDA-MB-468, and SKBR-3) hypoxia and hypoxia mimics also induced PFKFB4 mRNA, but to variable degrees. The hypoxic induction of PFKFB4 mRNA was equivalent to the expression of PFKFB3, Glut1, and VEGF, which are known HIF-1-dependent genes. Hypoxia and dimethyloxalylglycine increased the PFKFB4 protein levels in all cell lines studied except MDA-MB-468. Through site-specific mutagenesis in the 5'-flanking region of PFKFB4 gene the hypoxia response could be limited. Thus, this study provides evidence that PFKFB4 gene is also expressed in mammary gland cancer cells and strongly responds to hypoxia via an HIF-1alpha dependent mechanism. Moreover, the PFKFB4 and PFKFB3 gene expression in mammary gland cancer cells has also a significant role in the Warburg effect which is found in all malignant cells.     

Developmental Expression Patterns of Arabidopsis XTH Genes Reported by Transgenes and Genevestigator

The plant cell wall is the structural basis of cellular form and thus forms a foundation on which morphogenesis builds organs and tissues. Enzymes capable of modifying major wall components are prominent candidates for regulating wall form and function. Xyloglucan endotransglucosylases/hydrolases (XTHs) are predicted to participate in xyloglucan integration and/or restructuring. XTHs are encoded by large gene families in plants; the Arabidopsis genome encodes 33 XTHs. To gain insight into the potential physiological relevance of the distinct members of this family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data reveal that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs show XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes. These data suggest that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Degree of Hybridization in Seed Stands of Pinus engelmannii Carr. In the Sierra Madre Occidental,Durango, Mexico

Hybridization is an important evolutionary force, because interspecific gene transfer can introduce more new genetic material than is directly generated by mutations. Pinus engelmannii Carr. is one of the nine most common pine species in the pine-oak forest ecoregion in the state of Durango, Mexico. This species is widely harvested for lumber and is also used in reforestation programmes. Interspecific hybrids between P.engelmannii and Pinus arizonica Engelm. have been detected by morphological analysis. The presence of hybrids in P. engelmannii seed stands may affect seed quality and reforestation success. Therefore, the goals of this research were to identify introgressive hybridization between P. engelmannii and other pine species in eight seed stands of this species in Durango, Mexico, and to examine how hybrid proportion is related to mean genetic dissimilarity between trees in these stands, using Amplified Fragment Length Polymorphism (AFLP) markers and morphological traits. Differences in the average current annual increment of putative hybrids and pure trees were also tested for statistical significance. Morphological and genetic analyses of 280 adult trees were carried out. Putative hybrids were found in all the seed stands studied. The hybrids did not differ from the pure trees in vigour or robustness. All stands with putative P. engelmannii hybrids detected by both AFLPs and morphological traits showed the highest average values of the Tanimoto distance, which indicates: i) more heterogeneous genetic material, ii) higher genetic variation and therefore iii) the higher evolutionary potential of these stands, and iv) that the morphological differentiation (hybrid/not hybrid) is strongly associated with the Tanimoto distance per stand. We conclude that natural pairwise hybrids are very common in the studied stands. Both morphological and molecular approaches are necessary to confirm the genetic identity of forest reproductive material.     

Biodiversity of sweetpotato (iPomoea batatas,Convolvulaceae) in Southern Mexico

Between 1987 and 1992 the phytogeographic region of southern Mexico was explored during three collecting trips made in search of cultivated and wild germplasm of the sweetpotato (Ipomoea batatas). The first trip was made in 1987, when we collected wild species in Ipomoea section Batatas found in the southeastern and southwestern regions of Mexico. A second trip was made in 1990, when we collected accessions of the cultivated species as well as wild species in the southeast, southwest and northeast. The third and final trip was oriented at identification, characterization and collecting seeds in the ecological niches ofI.tabascana andI.umbraticola. As a result of the three trips we collected 165 accessions of cultivated and wild germplasm with populations dispersed in 147 localities in 15 states of the Mexican region: Veracruz, Tabasco, Campeche, Chiapas, Oaxaca, Yucatán, Guerrero, Michoacán, San Luis Potosí, Hidalgo, Querétaro, Tamaulipas, Guanajuato, Puebla and México. Of the total accessions some 64 (38.3%) were of the cultivated species including nine accessions of feral material, and 103 accessions (61.7%) were of wild species made up of 59 accessions of seven species in the section Batatas, 37 of other species in the family Convolvulaceae, and seven yet to be determined. We have identified the largest genetic biodiversity in six localities of five states: Tabasco, Oaxaca, Michoacán, San Luis Potosí, and Puebla. Biodiversity maintenance in this region is associated with the day-of-the-dead festivities.     

Parthenolide sensitizes cells to X-ray-induced cell killing through inhibition of NF-kappaB and split-dose repair

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.     

DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: Evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41°C and 45°C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24h), or acute (1h) exposure to each treatment followed by incubation at 37°C over a period of 24h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF.     

Interaction of Photoprotective and Acclimation Mechanisms in Ulva rigida (Chlorophyta) in Response to Diurnal Changes in Solar Radiation in Southern Chile

Species of the genus Ulva (Chlorophyta) are regarded as opportunistic organisms, which efficiently adjust their metabolism to the prevailing environmental conditions. In this study, changes in chlorophyll‐a fluorescence‐based photoinhibition of photosynthesis, electron transport rates, photosynthetic pigments, lipid peroxidation, total phenolic compounds, and antioxidant metabolism were investigated during a diurnal cycle of natural solar radiation in summer (for 12 h) under two treatments: photosynthetically active radiation (PAR: 400–700 nm) and PAR+ ultraviolet (UV) radiation (280–700 nm). In the presence of PAR alone, Ulva rigida showed dynamic photoinhibition, and photosynthetic parameters and pigment concentrations decreased with the intensification of the radiation. On the other hand, under PAR+UV conditions a substantial decline up to 43% was detected and an incomplete fluorescence recovery, also, P‐I curve values remained low in relation to the initial condition. The phenolic compounds increased their concentration only in UV radiation treatments without showing a correlation with the antioxidant activity. The enzimatic activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased over 2‐fold respect at initial values during the onset of light intensity. In contrast, catalase (CAT) increased its activity rapidly in response to the radiation stress to reach maxima at 10 a.m. and decreasing during solar. The present study suggests that U. rigida is capable of acclimating to natural radiation stress relies on a concerted action of various physiological mechanisms that act at different times of the day and under different levels of environmental stress.     

Grazing effects on plant diversity in the endemic Erica mackayana heathland community of north-west Spain

Background: Large areas of heathland landscapes in Galicia, north-west Spain, have traditionally been extensively grazed by free-ranging cattle and wild ponies. Recently, a large reduction in the number of these larger herbivores has been observed, with unknown consequences for the habitat.

Aims: To evaluate the effects of grazing and herbivore density on plant diversity, community composition and vegetation structure of the endemic wet heathlands dominated by Erica mackayana in Galicia.

Methods: Field sampling of vascular plants, generalised linear models (GLMs), non-metric multidimensional distance scaling (NMDS).

Results: Grazed sites had significantly higher total and rare species richness and diversity than ungrazed sites. Higher densities of cattle resulted in lower numbers of rare species, while wild pony density had no effect on rare species richness. In grazed sites, vegetation was lower with greater variation in height, resulting in greater heterogeneity of the habitat. Precipitation and summer temperatures were related to plant diversity, mainly beta diversity. Soil organic matter negatively correlated with rare species.

Discussion: Grazing, mostly by wild ponies, was demonstrated to be positively related to plant diversity and vegetation structure. Lack of grazing or high cattle densities resulted in a negative effect on total and rare species richness and diversity. Future climate change may negatively affect heathland plant diversity. Galician wild ponies represent a unique case of sustainable management of a wild species and an invaluable cultural heritage. Moreover, they have a significant role in maintaining the endemic E. mackayana heathlands, what would justify specific conservation actions for these large herbivores.