Production of potato intraspecific somatic hybrids with improved tolerance to PVY and Pythium aphanidermatum

Somatic hybridization can be an interesting alternative for the selection of heterozygous and vigorous potato plants through combination of dihaploid genomes. The resulting hybrids can harbour interesting characters and thus can be used in agriculture if they are in agreement with agronomic criteria.

In this report, we used an intraspecific somatic hybridization technique for the production of tetraploid potato lines. Two parental combinations were used in protoplast electrofusion procedure: Aminca-Cardinal and Cardinal-Nicola. The selection of somatic hybrids was based on in vitro plant vigour. Therefore, among the 75 regenerated plants obtained from Aminca-Cardinal fusion, 3 putative hybrids were retained and 2 plant lines were selected among the 54 regenerated from the Cardinal-Nicola fusion. Heterosis was observed in the larger hybrid tuber size compared to the parents’. Our results also showed a precocity in the in vitro tuberization for the hybrids. Moreover, all of the regenerated putative hybrids were tetraploid (2n=4x=48 chromosomes). Isocitrate dehydrogenase and malate dehydrogenase isoenzyme analyses confirmed the hybrid nature of these lines. A molecular characterization performed by PCR amplification of simple sequence repeats and inter-simple sequence repeats confirmed that all these lines were somatic hybrids.

The effect of potato virus Y infection on these hybrid lines was tested by mechanical inoculation of plants cultivated in a greenhouse. The majority displayed a reduction of infection rate associated with a delayed appearance of symptoms compared to the parents. Moreover, complete resistance was noted for one hybrid line (CN2). All hybrids also showed improved tolerance to Pythium aphanidermatum infection during tuber storage or after plant inoculation.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Foreign gene delivery into monocotyledonous species

Monocotyledonous plants are generally more recalcitrant to genetic transformation than dicotyledonous species. The absence of reliable Agrobacterium-mediated transformation methods and the difficulties associated with the culture of monocotyledonous tissues in vitro are mainly responsible for this situation. Until recently, the genetic transformation of monocotyledons was essentially performed by direct transfer of DNA into regenerable protoplasts or intact cells cultured in vitro, via polyethylene glycol treatment, electroporation or particle bombardment. Since 1990, the use of particle gun technology has revolutionized the genetic engineering of monocotyledonous species, allowing transformation to be more independent of the in vitro culture requirements. Today, at least one genotype of each major monocotyledonous crop species, including cereals, can be genetically transformed.     

Mapping oxidative DNA damage at nucleotide level

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the in vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50 mM H₂O₂, or purified genomic DNA was treated with 5 mM H₂O₂, 100 μM Ascorbate, and 50 μM, 100 μM, or 100 μM of Cu(II), Fe(III), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H₂O₂-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H₂O₂.     

A quantitative bacterial dot method for DNA-DNA hybridization and its correlation to the hydroxyapatite method

A filter hybridization method employing bacterial samples and [¹²⁵I]labeled chromosomal DNA as a probe was used for DNA-DNA hybridization. It was found that the hybrids had a thermal melting temperature very similar to that of duplexes formed by purified filterbound DNA. The difference in thermal denaturation midpoint between homologous and heterologous duplexes was determined for a number of strains ofAcinetobacter spp. andEnterobacter agglomerans. A comparison with the corresponding data obtained by the hydroxyapatite method showed good correlation between the two methods. The use of bacterial samples in filter hybridization omits the time-consuming DNA preparation procedure necessary for traditional DNA-DNA hybridization procedures. A simplified, two-step elution procedure is suggested for processing large numbers of strains.     

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.     

In vitro simulation and quantification of wear within the patellofemoral joint replacement

Quantification of the wear rate in vitro is now considered an essential step in the development of a new joint replacement prior to clinical trials. However, little research exists around in vitro simulation of wear in the patellofemoral joint (PFJ) despite over 200,000 being implanted annually within the European Union. A method to simulate wear in the laboratory using four input degrees of freedom within the PFJ of total knee replacement (TKR) has been developed. Wear simulation was validated through comparison of functional kinematics and patellar surface damage modes produced in vitro to clinical outcomes. The technique has been shown to replicate the prescribed in vivo kinematics in a reproducible and repeatable manner. The wear scar areas were similar to those found in vivo. However, geometrical measurements of wear were not reliable due to creep and geometry changes. As has been found previously with tibial inserts, geometrical determination of wear volume was not found to be an effective method of comparing wear from simulators and retrievals. Change in volume calculated gravimetrically was seen to be the most repeatable measure of patellar wear in vitro.     

ARTP诱变猴头菌株的发酵菌丝体多糖理化性质及体外免疫活性

为探讨常压室温等离子体诱变的3株高产多糖猴头菌和出发菌株的多糖组分差异,通过液体发酵获得的菌丝体经水提、分级醇沉获得8个胞内多糖组分,对它们的理化性质、结构特征及体外免疫活性进行了研究。结果表明,3株ARTP诱变菌株414、321、236菌丝体多糖含量较出发菌株有较明显提升;ARTP诱变的猴头菌20%醇沉多糖组分较出发菌株分子量大,所占比例增加;诱变菌株60%醇沉多糖组分的分子量略大于出发菌株,所占比例相近。20%醇沉多糖主要由半乳糖、葡萄糖、甘露糖构成,诱变菌株该多糖组分中葡萄糖和甘露糖的比例较出发菌株均有明显提升,60%醇沉多糖组分单糖组成无明显差异;8个多糖组分均具有体外刺激巨噬细胞释放NO的活性,其中20%醇沉多糖的活性优于60%醇沉多糖,诱变菌株的生物活性优于出发菌株。本研究探讨了ARTP诱变对猴头菌胞内多糖结构及活性的影响,为猴头菌相关产品的开发提供了优质资源。     

Enhancement of myeloid cell growth by benzene metabolites via the production of active oxygen species

In low concentrations, benzene and its metabolite hydroquinone are known to have diverse biological effects on cells, including the synergistic stimulation with GM-CSF of hematopoietic colony formation in vitro, stimulation of granulocytic differentiation in vitro and in vivo, and general suppression of hematopoiesis in vivo. These chemicals are also known to be active in the induction of active oxygen species. We used several assays to determine the effects of benzene metabolites (hydroquinone, benzenetriol, benzoquinone) and active oxygen species (xanthine/xanthine oxidase) on cell growth and cell cycle kinetics of the human myeloid cell line HL-60. HL-60 cells treated with these chemicals for 2 h in PBS showed increased growth over untreated controls in a subsequent 18h growth period in complete media. Incorporation of ³H-thymidine was also increased proportionately by these treatments. Catalase treatment abrogated the increased cell growth of all chemicals, suggesting an oxidative mechanism for the effect of all treatments alike. Cell cycle kinetics assays showed that the growth increase was caused by an increased recruitment of cells from G₀/G₁ to S-phase for both hydroquinone and active oxygen, rather than a decrease in the length of the cell cycle. Benzene metabolite's enhancement of growth of myeloid cells through an active oxygen mechanism may be involved in a number of aspects of benzene toxicity, including enhanced granulocytic growth and differentiation, stimulation of GM-CSF-induced colony formation, apoptosis inhibition, and stimulation of progenitor cell mitogenesis in the bone marrow. These effects in sum may be involved in the benzene-induced “promotion” of a clonal cell population to the fully leukemic state.     

Activity of dehydroabietic acid derivatives against wood contaminant fungi

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and different substituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks).

The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. The Relative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparison with those of control cultures; the results are expressed as EC₅₀.

The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group of compounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means to investigate the preservative activities of these antifungal compounds under actual conditions of use.

The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and in situ.

The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a good level of protection of the wood against the organisms tested was achieved.

The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative to conventional fungicides.     

Leucobacter chromiireducens sp. nov, and Leucobacter aridicollis sp. nov., two new species isolated from a chromium contaminated environment

Two strains designated strains L-1^T and L-9^T were isolated from activated sludge of a treatment plant that receives wastewater from the tannery industry contaminated with chromium. Phylogenetic analysis showed that the organisms represented two new species of the genus Leucobacter. Strains L-1^T and L-9^T could be distinguished from the type strain of L. komagatae and from the type strain of “L. albus” by the B-type peptidoglycan composition, fatty acid composition, several phenotypic and physiological characteristics. The major fatty acids of the organisms were iso- and anteiso-branched C15:0 and C17:0, straight-chain C16:0 was also found in relatively high proportions. The organisms were halotolerant, grew in medium containing 9% NaCl, and all strains, including the type strain of L. komagatae grew in medium containing 5 mM Cr(VI). On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, percentage of DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain L-1^T represents a new species of the genus Leucobacter for which we propose the name Leucobacter chromiireducens and that strain L-9^T represents an additional new species of the same genus for which we propose the name Leucobacter aridicollis.     

Quantitative microarray-based DNA-DNA hybridization assay for measuring genetic distances among bacterial species and its application to the identification of family Enterobacteriaceae

Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

Potential for control of seedling blight of wheat caused by Fusarium graminearum and related species using the bacterial endophyte Bacillus mojavensis

Fusarium-infected wheat seed decreases germination, seedling emergence, and causes post emergence seedling death, and can contribute to wheat scab and ear rot of maize, with consequent production of mycotoxins such as deoxynivalenol and zearalenone. Current seed treatments have proved ineffective in controlling seedling blight and scab. A patented endophytic bacterial strain, Bacillus mojavensis RRC 101, and several other strains of this species were studied to determine in vitro antagonism to some Fusarium species and to assess the potential of this bacterium to serve as an endophytic biocontrol for seedling blight of wheat produced by species within the F. graminearum complex, as well as other species of Fusarium. Seedling emergence and seed germination were two tests used as indicators of seedling blight. These tests were conducted in growth rooms with two wheat cultivars highly susceptible to scab, Norm and Pioneer 2552, and other cultivars with varying resistance to scab. The results indicated that all strains of this bacterium were antagonistic in vitro to the strains of F. graminearum and its seven related species, as well as four strains of F. pseudograminearum and the two strains of F. verticillioides. Germination of the highly scab susceptible cultivar 2552 was increased from 77 to 97% when planted in soil containing a mixed inoculum of F. graminearum and related species. Seedling emergence in the very susceptible wheat cultivar Norm increased from 20 to 82% when treated with the bacterium. The data indicated that inoculating wheat kernels with B. mojavensis reduced seedling blight of wheat produced by F. graminearum and related Fusarium species indicating the potential for this bacterium as a biocontrol under field condition.     

Specificity of the action of parathyroid hormone upon mitochondrial metabolism: Cyanogen bromide-derived parathyroid-hormone peptides

The mitochondrial response to cyanogen bromide-treated parathyroid hormone was studied as a means of testing further the relationship between the structure and the effects in vitro of this hormone. The treated hormone and appropriate control hormone were tested in a standard bioassay and in a mitochondrial assay system in vitro.

Reaction of more than 90 % of the methionine residues in the hormone resulted in total inactivation of the hormone both in vivo and in vitro. This result disagrees with previously published data.     

Glutamate in the glomus cells of the cat carotid body: Immunocytochemistry and in vitro release

The identity of the postulated excitatory transmitter released by glomus cells is not known. Since our preliminary work on paraffin sections of the cat carotid body indicated that most glomus cells were intensely immunoreactive to glutamate, we decided to investigate whether glutamate might be such a transmitter, using two approaches. One approach was to make a quantitative immunogold analysis of ultrathin sections to assess the level of glutamate immunoreactivity of glomus cells relative to glia and to afferent axon terminals. The other approach was to measure the potassium-induced release of glutamate from carotid bodies superfused in vitro. We consistently found that glomus cell profiles had 50% more immunogold particles per unit of area than glial cell or axonal profiles. However, the levels of glutamate immunoreactivity of glomus cells were lower than those expected for glutamatergic terminals. We also found that glutamate was not released from in vitro carotid bodies stimulated with high concentrations of potassium. These findings indicate that the oxygen-sensitive glomus cells have a high concentration of glutamate, which is not released by superfusion with high potassium. Thus, glutamate is not the excitatory transmitter released by glomus cells. We speculate that the high concentrations of glutamate might instead be related to the known dependence of the “in vitro” chemosensory activity on metabolic substrates.     

Articular cartilage mechanical and biochemical property relations before and after in vitro growth

The aim of this study was to design in vitro growth protocols that can comprehensively quantify articular cartilage structure–function relations via measurement of mechanical and biochemical properties. Newborn bovine patellofemoral groove articular cartilage explants were tested sequentially in confined compression (CC), unconfined compression (UCC), and torsional shear before (D0, i.e. day zero) and after (D14, i.e. day 14) unstimulated in vitro growth. The contents of collagen (COL), collagen-specific pyridinoline (PYR) crosslinks, glycosaminoglycan, and DNA significantly decreased during in vitro growth; consequently, a wide range of biochemical properties existed for investigating structure–function relations when pooling the D0 and D14 groups. All D0 mechanical properties were independent of compression strain while only Poisson's ratios were dependent on direction (i.e. anisotropic). Select D0 and D14 group mechanical properties were correlated with biochemical measures; including (but not limited to) results that CC/UCC moduli and UCC Poisson's ratios were correlated with COL and PYR. COL network weakening during in vitro growth due to reduced COL and PYR was accompanied by reduced CC/UCC moduli and increased UCC Poisson's ratios.     

Phycomyces blakesleeanus car B mutants: Their use in assays of phytoene desaturase

Strains of car B (phytoene-accumulating) mutants of Phycomyces blakesleeanus have been characterized with respect to their carotene contents, in vitro formation of isoprenoids from [2-¹⁴C] mevalonic acid and their ability to produce [¹⁴C]phytoene in situ for use in coupled assays of phytoene desaturase activity. All strains produced predominantly (15-Z)-phytoene both in vivo and in vitro. Other isoprenoids were produced by cell extracts including squalene, sterols, prenyl diphosphates and prenyl alcohols. The addition of 1% Tween 60 to crude cell extracts of the mutants partially restored wild type carotenogenic activity and also altered the proportions of other isoprenoids formed. However, in a cytosolic fraction of the car B mutant, the addition of 1% Tween 60 did not result in the production of any carotenoid from phytoene. This fraction was the most effective source of [¹⁴C] phytoene for use in coupled assays of phytoene desaturase activity.     

PCR-RFLP and total DNA homology revealed three related genomic species among broad-host-range Frankia strains

Abstract: Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified nif D-K intergenic spacer (IGS) region was used to cluster 22 Frankia strains of the Elaeagnus host specificity group into seven genomic groups and to measure the degree of genetic similarity among them. This PCR-RFLP analysis could assign freshly isolated strains to described genomic species and revealed genomic groups not yet described among Frankia strains of the Elaeagnus specificity group. Six broad-host-range Frankia strains, infective on both Alnus and Elaeagnus , fell into three closely related PCR-RFLP clusters. DNA-DNA hybridization was then used to establish the correlations between PCR-RFLP clusters and total DNA relatedness groups. The three PCR-RFLP clusters agreed with two new and one reference genomic species, indicating that Frankia ability to nodulate with Alnus and Elaeagnus is a monophyletic trait shared by three genomic species.     

Application of a colorimetric DNA-DNA hybridization method for identification ofAeromonas genomic species,isolated from diarrheal stools

The colorimetric DNA-DNA hybridization method for the identification of 18 strains ofAeromonas spp. isolated from human stools was used. Bacterial isolates were also examined by phenotypic characteristics. On the basis of biochemical tests 13 strains were included in phenogroupA. caviœ and 5 strains inA. sobria. Identification to the species level was obtained by colorimetric hybridization method. DNA-DNA similarity values showed that isolates ofA. caviœ group belong to hybridization group (HG) 4 whereas isolates ofA. sobria belong to HG 8/10. DNA relatedness results obtained by the colorimetric method showed good agreement with values detected by the spectrophotometric method. The background in the colorimetric method is lower than in the spectrophotometric one. Results of this study indicate the usefulness of the colorimetric DNA-DNA hybridization in microplates method for the identification ofAeromonas genomic species, isolated from human diarrheal stools.