Detection of transgenic and endogenous plant DNA in digesta and tissues of sheep and pigs fed Roundup Ready canola meal

The persistence of plant-derived recombinant DNA in sheep and pigs fed genetically modified (Roundup Ready) canola was assessed by PCR and Southern hybridization analysis of DNA extracted from digesta, gastrointestinal (GI) tract tissues, and visceral organs. Sheep (n = 11) and pigs (n = 36) were fed to slaughter on diets containing 6.5 or 15% Roundup Ready canola. Native plant DNA (high- and low-copy-number gene fragments) and the cp4 epsps transgene that encodes 5-enolpyruvyl shikimate-3-phosphate synthase were tracked in ruminal, abomasal, and large intestinal digesta and in tissue from the esophagus, rumen, abomasum, small and large intestine, liver, and kidney of sheep and in cecal content and tissue from the duodenum, cecum, liver, spleen, and kidney of pigs. High-copy chloroplast-specific DNA (a 520-bp fragment) was detected in all digesta samples, the majority (89-100%) of intestinal tissues, and at least one of each visceral organ sample (frequencies of 3-27%) from sheep and swine. Low-copy rubisco fragments (186- and 540-bp sequences from the small subunit) were present at slightly lower, variable frequencies in digesta (18-82%) and intestinal tissues (9-27% of ovine and 17-25% of porcine samples) and infrequently in visceral organs (1 of 88 ovine samples; 3 of 216 porcine samples). Each of the five cp4 epsps transgene fragments (179-527 bp) surveyed was present in at least 27% of ovine large intestinal content samples (maximum = 64%) and at least 33% of porcine cecal content samples (maximum = 75%). In sheep, transgene fragments were more common in intestinal digesta than in ruminal or abomasal content. Transgene fragments were detected in 0 (esophagus) to 3 (large intestine) GI tract tissues from the 11 sheep and in 0-10 of the duodenal and cecal tissues collected from 36 pigs. The feed-ingested recombinant DNA was not detected in visceral tissues (liver, kidney) of lambs or in the spleen from pigs. Of note, however, one liver and one kidney sample from the pigs (different animals) were positive for a 278-bp fragment of the transgenic cp4 epsps (denoted F3). Examination of genomic libraries from these tissues yielded no conclusive information regarding integration of the fragment into porcine DNA. This study confirms that feed-ingested DNA fragments (endogenous and transgenic) do survive to the terminal GI tract and that uptake into gut epithelial tissues does occur. A very low frequency of transmittance to visceral tissue was confirmed in pigs, but not in sheep. It is recognized that the low copy number of transgenes in GM feeds is a challenge to their detection in tissues, but there was no evidence to suggest that recombinant DNA would be processed in the gut in any manner different from endogenous feed-ingested genetic material.     

Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.     

Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis

Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 degrees C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 degrees C).     

Determination of ammonium,amides, and soluble carboxylates in plant tissues

Methods for extraction and determination of ammonium, amides and soluble carboxylates in plant tissues are compared and discussed. The procedure recommended involves extraction of finely ground plant tissues with 0.2 M formic acid, determination of ammonium and amides in the extract by overnight alkaline hydrolysis and distillation in Conway microdiffusion dishes, measurement of extracted ammonium using Carlson's rapid diffusion‐conductivity instrument, and estimation of soluble organic acids by titration after separation and purification with ion exchange resins. The method described is reasonably rapid, and gives complete recovery and highly reproducible results. The final solution of organic acids is suitable for direct chromatographic analysis.     

Immunohistochemical localization of metallothionein in plant tissues

The localization of metallothionein ( MT ) in the seeds and roots of soybean was investigated by immunohistochemistry. The germinating seeds at 2 hr, 1, 2, 3, 4 and 6 d including 1-mo root tips of soybean ( c.v. Toyosuzu ) with and without heavy metals ( Cu 400 μg Lor Zn 3 μg ml^?1) treatment were used to demonstrate the localization of MT by the indirect immunoperoxidase technique using polyclonal rabbit antirat MT conjugated to ascaris as a primary antibody. Metallothionein was localized in the proliferating regions such as the embryo in seeds, and root and shoot apices of both the control and heavy metals-treated plants. The intensity of MT staining in the proliferating regions generally increased as the soybean seeds germinate. Starting at about 1 day after germination, MT was found in the veins and vascular bundles suggesting its translocation to other organs. Similar observation hold true in the case of plants treated with heavy metals. This means that heavy metals treatment had no effect on MT localization. However, the heavy metals-treated plants showed higher concentration of MT over the control with respect to the growth stage of soybean seeds. These indicate that MT found in soybean plays a physiological role in heavy metal transport, detoxification and cell division in a similar manner to mammalian MT.

     

用核酸探针检测鸡脏器及羽毛囊中禽呼肠孤病毒

摘要：用地高辛标记禽呼肠孤病毒（ARV）S1基因中编码σ3蛋白的基因片段作为核酸探针，在斑点分子杂交中可检测到1.6pg的ARV RNA。利用该核酸探针，通过检测鸡羽毛囊及体内病毒繁殖情况，比较研究了 ARV感染后在鸡体内及鸡群中的传播动态。结果显示，ARV感染后在24小时时就可侵染大部分器官，并且很快传播到同群未攻毒的鸡中。羽毛囊中的病毒检出率与鸡内脏器官中病毒的检出率一致。该研究证明用核酸探针检测鸡羽毛囊中ARV的方法检测ARV的感染与流行情况，具有灵敏、特异性高，操作方便的特点，尤其适用于大量样品的同时检测。     

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.     

Natural abundance of 15N in nitrate,ureides, and amino acids from plant tissues

The natural ¹⁵N abundances (δ¹⁵N values) were measured for nitrate and free and bound amino acids from the leaves of field-grown spinach (Spinacia oleracea L.) and komatsuna (Brassica campestris L.), as well as ureides and free and bound amino acids in the leaves and roots of hydroponically grown soybean (Glycine max L.) totally depending on dinitrogen. Nitrate from the spinach and komatsuna leaves and ureides from leaves and roots of soybean showed higher δ¹⁵N values than the total tissue N and N in free or bound amino acid fractions. The δ¹⁵N values of individual free and bound amino acids, determined by GC/C/MS using their acetylpropyl derivatives, were similar in leaf tissues except for proline but varied in soybean root tissues. The order of ¹⁵N enrichment was similar in the four samples: aspartic acid > glutamic acid > threonine, proline, valine > glycine + alanine +serine, γ-amino butyric acid, and phenylalanine.     

Extraction, gas-liquid chromatographic detection, and quantitation of hexachlorophene residues from plant tissues high in lipid content

A method has been developed for the extraction, cleanup, derivatization, detection, and quantitation of hexachlorophene (HCP) residues from 2 types of plant storage tissue high in lipid content. Wet soybean or peanut tissue was homogenized and extracted with ethyl ether and chromatographed on silica gel to remove the neutral lipids. The cleaned up sample was methylated with diazomethane and the dimethoxyhexachlorophene was eluted from a second silical gel column and chromatographed on a 6' glass column packed with 3% OV-1 or 3% SE-30 on Gas-Chrom Q. The instrument detection limit for the 63Ni electron capture detector was less than 0.1 ng for dimethoxyhexachlorophene and about 1 ppb HCP residue in plant issue. Recovery of 10-420 ppb HCP added to tissue averaged 90.9 +/- 5.7%. Interfering substances were removed, column life was increased, peak sharpness was increased, and tailing of the parent compound was decreased by using appropriate column chromatography.     

Dehydrotomatine and alpha-tomatine content in tomato fruits and vegetative plant tissues

Tomato plants (Lycopersicon esculentum) synthesize the glycoalkaloids dehydrotomatine and alpha-tomatine, possibly as a defense against bacteria, fungi, viruses, and insects. We used a high-performance liquid chromatography method with UV detection at 208 nm for the analysis of these compounds in various tissues. An Inertsil ODS-2 column with a mobile phase of acetonitrile/20 mM KH2PO4 (24/76, v/v) afforded good separation of the two glycoalkaloids in mini-tomato extracts, fruit harvested at different stages of maturity, and calyxes, flowers, leaves, roots, and stems. The two peaks appeared at approximately 17 and approximately 21 min. Recoveries from tomato fruit extracts spiked with dehydrotomatine and alpha-tomatine were 87.7 +/- 6.8 and 89.8 +/- 3.4% (n = 5), respectively. The detection limit is estimated to be 0.39 microg for dehydrotomatine and 0.94 microg for alpha-tomatine. The dehydrotomatine and alpha-tomatine content of tomatoes varied from 42 to 1498 and 521 to 16 285 microg/g of fresh weight, respectively. The ratio of alpha-tomatine to dehydrotomatine ranged from 10.9 to 12.5 in tomatoes and from 2.3 to 7.8 in the other plant tissues. These results suggest that the biosynthesis of the glycoalkaloids is under separate genetic control in each plant part. Degradation of both glycoalkaloids occurred at approximately the same rate during maturation of the tomatoes on the vine. An Inertsil NH2 column, with acetonitrile/1 mM KH2PO4 (96/4, v/v) as the eluent, enabled the fractionation of commercial tomatidine into tomatidenol and tomatidine, the aglycons of dehydrotomatine and alpha-tomatine, respectively. The information should be useful for evaluating tomatoes and vegetative tissues for dehydrotomatine/alpha-tomatine content during fruit development and their respective roles in host-plant resistance and the diet.     

Gas chromatographic determination of avilamycin total residues in pig tissues, fat, blood, feces, and urine

A gas chromatographic (GC) procedure is presented for the determination of residues of avilamycin and all its metabolites/conjugates which can be converted to the common moiety dichloroisoeverninic acid (DIA). The method involves alkaline hydrolysis to DIA, cleanup by partitioning with chloroform, acidification of the aqueous phase, and partitioning of DIA into methylene chloride. After methylation of DIA, the product, 3,5-dichloro-4,6-dimethoxy-2-methylbenzoic acid methyl ester, is cleaned up on a silica gel column prior to the final determination by electron capture GC. The method is sensitive to 0.1 mg/kg avilamycin equivalent. Overall average recoveries were 85.4%, with a standard deviation of 9.1% for n = 20. Analyses of feces, urine, tissues, and fat of pigs treated with avilamycin demonstrated that 93% of the administered substance is excreted in feces and urine, within 72 h after treatment, and that no residues (less than 0.01 mg/kg) can be found in the tissues and fat of the animals at any time between 0 and 7 days after treatment with medicated feed.     

Antifungal and peroxidative activities of nonheme chloroperoxidase in relation to transgenic plant protection

Nonheme chloroperoxidase (CPO-P) of Pseudomonas pyrrocinia catalyzes the oxidation of alkyl acids to peracids by hydrogen peroxide. Alkyl peracids possess potent antifungal activity as found with peracetate: 50% killing (LD(50)) of Aspergillus flavus occurred at 25 microM compared to 3.0 mM for the hydrogen peroxide substrate. To evaluate whether CPO-P could protect plants from fungal infection, tobacco was transformed with a gene for CPO-P from P. pyrrocinia and assayed for antifungal activity. Leaf extracts from transformed plants inhibited growth of A. flavus by up to 100%, and levels of inhibition were quantitatively correlated to the amounts of CPO-P activity expressed in leaves. To clarify if the peroxidative activity of CPO-P could be the basis for the increased resistance, the antifungal activity of the purified enzyme was investigated. The LD(50) of hydrogen peroxide combined with CPO-P occurred at 2.0 mM against A. flavus. Because this value was too small to account for the enhanced antifungal activity of transgenic plants, the kinetics of the enzyme reaction was examined and it was found that the concentration of hydrogen peroxide needed for enzyme saturation (K(m) = 5.9 mM) was already lethal. Thus, the peroxidative activity of CPO-P is not the basis for antifungal activity or enhanced resistance in transgenic plants expressing the gene.     

Electrothermal atomic absorption spectroscopic determination of chromium in plant tissues

This method involves sample digestion in nitric, perchloric, and sulfuric acids. Chromium is concentrated by coprecipitation with ferric hydroxide. Redissolved iron is removed by liquid-liquid extraction, remaining silica is dissolved with hydrofluoric acid, and chromium is determined by electrothermal atomic absorption spectrometry. The sensitivity and detection limits for chromium approach those given by the manufacturers of the various instruments. Recovery studies and analysis of standard materials show that this method is reliable.     

Silicon stimulates initial growth and chlorophyll a/b ratio in rice seedlings,and alters the concentrations of Ca,B, and Zn in plant tissues

Abstract

Silicon (Si) is considered a beneficial element for plants due to the far-reaching benefits it confers, including enhanced growth, yield, and crop quality, as well as stress resistance. In this study, we evaluated the effect of Si during germination and initial growth (0.0, 0.5, 1.0, or 1.5?mM Si) and during vegetative growth (0, 1, 2, or 3?mM Si) in rice (Oryza sativa) cv. Morelos A-98. Si did not affect germination but stimulated seedling height, root length, number of roots, as well as fresh and dry biomass weight of shoots and roots during initial growth. During vegetative growth, the application of 3?mM Si significantly increased the chlorophyll a/chlorophyll b ratio, but no major changes were observed either in growth or in concentrations of most nutrients, with the exception of Ca (which increased with 3?mM Si), and B and Zn (which decreased in the presence of Si). In conclusion, applying Si had positive effects during the initial stage of growth, increasing seedling height, root length, root number, and fresh and dry biomass weight. Under our experimental conditions, Si did not affect germination and vegetative growth, but increased Ca concentrations and decreased B and Zn concentrations.     

Detection of GFP-labeled Paenibacillus polymyxa in autofluorescing pine seedling tissues

Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedling stem tissue and fixes nitrogen (N) in association with pine and western red cedar (Thuja plicata Donn.). To evaluate endophytic colonization by this microorganism, we generated P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, and grew pine seedlings that were inoculated with the marked strain in a N-limited soil. Tissue disintegration during sample preparation precluded examination of needles for the GFP-labeled endophyte but GFP was detected on roots and in stems of 2- to 14-week-old pine seedlings using confocal laser scanning microscopy. Due to excessive autofluorescence of seedling tissues, labeled bacteria were clearly discernible only in stem tissues of 4- and 6-week-old seedlings. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly. Some labeled bacteria appeared to contain endospores and none were detected in vascular tissues. We conclude that P. polymyxa P2b-2R is capable of endophytic colonization of pine seedlings with specific colonization sites that include the stem cortex but that GFP labeling is of limited value for localization of endophytic bacteria in pine seedling tissues.     

Levels of organochlorine pesticides in soils and rye plant tissues in a field study

The organochlorine pesticides are lipophilic and persistent and tend to accumulate in soils and growing plants. The contamination of growing plants occurs by adhesion of volatile substances from the air to the plant surface and by the migration of contaminants through xylem in inner ascendant transport. Persistent organochlorine pesticides (HCB, alpha,gamma-HCH, pp'DDE, op'DDT, pp'DDT) levels were determined in soils and rye plants. The aims of the study were the monitoring of organochlorine pesticide concentrations and the comparison of these levels among soil, rye straw, and rye grains. Fifty soil samples and 50 rye plant (50 straw and 50 grains) samples were taken. The GLC-ECD chromatographic results indicated the following contamination levels distributed among soil, straw, and grains: HCB (0.7-1.2-0.7 microg.kg(-1)), alpha-HCH (0.6-3.4-1.2 microg.kg(-1)), gamma-HCH (1.8-27.3-4.4 microg.kg(-1)), Sigma-HCH (2.5-30.7-5.6 microg.kg(-1)), pp'DDE (1.0-7.8-5.5 microg.kg(-1)), op'DDT (16.1-20.4-17.0 microg.kg(-1)), pp'DDT (38.0-41.7-49.6 microg.kg(-1)), and Sigma-DDT (54.2-63.2-72.1 microg.kg(-1)). The study verified the presence of organochlorine pesticides in the Mexican agricultural environment and their migration from soil to the growing rye plants. However, DDT has been banned since 1999 for sanitary reasons, and Lindane is applied only in some cases as a seed dresser. The determined organochlorine pesticide levels in rye plants are low, at residual levels that are below Codex Alimentarius Commission maximum residue limits.     

Characterization of the soil organic matter and plant tissues in an initial stage of the plant succession and soil development by means of curie-point pyrolysis coupled with GC-MS

The composition of the soil organic matter in soil developing under the influence of vegetation during the primary succession in the poor sandy area was investigated. The Curie-point pyrolysis method coupled with gas chromatographic separation and mass spectrometric identification of pyrolysates was applied during the investigation. A comparison of occurrence and diversity in composition of organic compounds in plant tissues and humus horizon of soils under the communities of coniferous forest series in the initial stages and phases was carried out. A large diversity of organic compounds under Algae-Cyanophyta communities, biological soil crusts and Polytrichum piliferum was noticed. A clear differentiation in the composition of the soil organic matter at different phases of succession under predominating communities with cryptogamous and vascular plants was observed. The analysis of organic compounds in plant tissues was found to facilitate the determination of origin of various groups of organic compounds in the soil. The results obtained from the Curie point pyrograms in the humus horizon (A) under grasses (Corynephorus canescens, Koeleria glauca) differed from the pyrolysates obtained under Algae-Cyanophyta communities. The polysaccharide derivates are more frequent in the pyrolysis products under algae, grasses (Corynephorus canescens, Koeleria glauca) and mosses than under Pinus sylvestris. In the beginning of the terminal stage of succession, lignin, phenols and aliphatic substances, prevailed over the polysaccharide derivates in the humus horizon.     

Comparison of the reflectances of black plant fragments in melanic Andisols with those of fresh charcoal from modern fires

Abstract

We investigated the reflectances of black plant fragments in melanic Andisols and of fresh charcoal produced during modern grassland and forest fires with the aim of clarifying the origin of fragments in melanic Andisols. The reflectance values of most Andisol plant fragments were less than 2%; all values were below 3%. These values are considered to have formed at temperatures of at least 200°C, indicating that these fragments are charcoal fragments. The reflectances of the fragments in melanic Andisols were similar to those of grass charcoal from grassland fires rather than wood charcoal produced during forest fires. This finding indicates that most charcoal fragments in melanic Andisols are the product of grassland fires. The small difference between the reflectance values of the charcoal fragments in the Andisols and the grass charcoal is possibly derived from the fact that charcoal fragments with lower reflectance are more likely to be retained in the soil than fragments with higher reflectance values.     

Comparison of the reflectances of black plant fragments in melanic Andisols with those of fresh charcoal from modern fires

We investigated the reflectances of black plant fragments in melanic Andisols and of fresh charcoal produced during modern grassland and forest fires with the aim of clarifying the origin of fragments in melanic Andisols. The reflectance values of most Andisol plant fragments were less than 2%; all values were below 3%. These values are considered to have formed at temperatures of at least 200°C, indicating that these fragments are charcoal fragments. The reflectances of the fragments in melanic Andisols were similar to those of grass charcoal from grassland fires rather than wood charcoal produced during forest fires. This finding indicates that most charcoal fragments in melanic Andisols are the product of grassland fires. The small difference between the reflectance values of the charcoal fragments in the Andisols and the grass charcoal is possibly derived from the fact that charcoal fragments with lower reflectance are more likely to be retained in the soil than fragments with higher reflectance values.