DNA isolation from dry and fresh samples of polysaccharide-rich plants

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion.     

Rapid extraction of genomic DNA from leaves and bracts of dove tree (Davidia involucrata)

Large amounts of polyphenolics in dove tree leaves make it difficult to obtain high-quality genomic DNA during extraction. A rapid DNA minipreparation method was developed for dove tree (Davidia involucrata) and yields 40–50 μg genomic DNA from 0.1 g fresh matured and young leaves and bracts. The yield and quality of the resulting DNA is satisfactory, and the protocol can be scaled up according to sample size. The obtained DNA is suitable for PCR and the restriction enzyme digestion needed for Southern blotting.     

Nuclear DNA content in species ofEleusine (Gramineae): A critical re-evaluation using laser flow cytometry

Interest in dinitroaniline herbicide resistant biotypes ofEleusine indica, and an as yet undetermined taxon ofEleusine, necessitated a revaluation of reported nuclear genome size estimates for available species in the genus. Laser flow cytometry showed that the nuclear DNA content of six of the seven species examined had 15 to 50% less DNA than reported previously. It was also determined that roots, as contrasted to leaves, possessed a large fraction of nuclei at the 4C or 8C DNA content level, in diploid or tetraploid species, respectively (i.e. the G2/M peak). Two major reasons for the previously reported overestimation may include sampling only of root tissues where endopolyploid and normal diploid nuclei both occur and the inappropriate choice of onion nuclei as an internal standard.     

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Homology-dependent DNA Transfer from Plants to a Soil Bacterium Under Laboratory Conditions: Implications in Evolution and Horizontal Gene Transfer

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.     

DNA Damage Measured by the Comet Assay in Eight Agronomic Plants

For most crops growing in polluted areas or treated with agricultural chemicals, no genotoxicity assays are available. We have studied the possibility of using the alkaline protocol of the plant-based molecular assay — the Single Cell Gel Electrophoresis (SCGE) assay (also called Comet assay) as a method for detecting induced DNA damage in 8 agronomic important plants (ordered according to the diameter of the nuclei): sugar beet, alfalfa, tobacco, lentil, maize, potato, hard wheat, and bread wheat. The monofunctional alkylating agent ethyl methanesulphonate (EMS) was applied as a model genotoxic agent on young excised leaves of the tested crops for 18 h at 26 °C in the dark. With increasing concentrations of 2 to 10 mM EMS, the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all crops studied. No correlation between the diameter of nuclei and sensitivity to EMS treatment was observed. The data obtained demonstrate the feasibility of using the Comet assay for detecting induced DNA damage in crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

Stratospheric Ozone Depletion: High Arctic Tundra Plant Growth on Svalbard is not Affected by Enhanced UV-B after 7 years of UV-B Supplementation in the Field

The response of tundra plants to enhanced UV-B radiation simulating 15 and 30% ozone depletion was studied at two high arctic sites (Isdammen and Adventdalen, 78° N, Svalbard).The set-up of the UV-B supplementation systems is described, consisting of large and small UV lamp arrays, installed in 1996 and 2002. After 7 years of exposure to enhanced UV-B radiation, plant cover, density, morphological (leaf fresh and dry weight, leaf thickness, leaf area, reproductive and ecophysiological parameters leaf UV-B absorbance, leaf phenolic content, leaf water content) were not affected by enhanced UV-B radiation. DNA damage in the leaves was not increased with enhanced UV-B in Salix polaris and Cassiope tetragona. DNA damage in Salix polaris leaves was higher than in leaves of C. tetragona. The length of male gametophyte moss plants of Polytrichum hyperboreum was reduced with elevated UV-B as well as the number of Pedicularis hirsuta plants per plot, but the inflorescence length of Bistorta vivipara was not significantly affected. We discuss the possible causes of tolerance of tundra plants to UV-B (absence of response to enhanced UV-B) in terms of methodology (supplementation versus exclusion), ecophysiological adaptations to UV-B and the biogeographical history of polar plants     

Rapid cytofluorimetric determination of leaf nuclear DNA content in the polyploid seriesRanunculus marsicus (R. auricomus agg.,Ranunculaceae)

A cytofluorimetric method for DNA amount determination on nuclei released from fixed leaves has been successfully tested on samples belonging to the polyploid series ofRanunculus marsicus Guss & Ten. (sect.Auricomus). The procedure was devised to screen rapidly for different ploidy levels within topodemes and progenies.     

The complete mitochondrial DNA sequence of the cestode Echinococcus multilocularis (Cyclophyllidea: Taeniidae)

The 13,738 bp mitochondrial DNA from the cestode Echinococcus multilocularis has been sequenced. It contains two major noncoding regions and 36 genes (12 for proteins involved in oxidative phosphorylation, two for rRNAs and 22 for tRNAs) but a gene for ATPase subunit 8 is missing. All genes are transcribed in the same direction. Putative secondary structures of tRNAs indicate that most of them are conventional clover leaves but the dihydrouridine arm is unpaired in tRNA(Ser(AGN)), tRNA(Ser(UCN)), tRNA(Arg) and tRNA(Cys). The base composition at the wobble positions of fourfold degenerate codon families is highly biased toward U and against C.     

Analysis of DNA size,content and cell cycle in leaves of Napier grass (Pennisetum purpureum Schum.)

Summary Mesophyll cell nuclei isolated from leaves of Pennisetum purpureum were analysed by flow cytometry to determine the nuclear DNA content and the percentage of cells in different phases of the cell cycle. Samples taken from base, middle and tip regions of leaves 2 to 8 (leaf 1, which was adjacent to the meristem, was too small to sample) showed no significant differences in the amount of DNA per G₁ nucleus due to either age or position. The average amount of DNA per G₁ nucleus was 5.78 pg. Although the majority of cells for each sample were in G₁, samples taken from older leaves had higher percentages of cells in G₂ and S phases. More specifically, base and middle regions of older leaves had a higher percentage of cells in G₂ than all three positions in younger leaves. Electrophoretic analysis of nuclear DNA from leaves 2 to 7 showed no evidence of degradation or difference in fragment size for any sample or position. This study was compared to previous work on the relationship between leaf age and embryogenic competence in Pennisetum purpureum. The results suggest that changes in the cell cycle, and/or a loss or fragmentation of the nuclear DNA, are not responsible for loss of embryogenic competence in mature leaf tissue.     

Three new species ofBullera isolated from leaves in the Ogasawara Islands

Thirteen strains of ballistoconidium-forming yeasts were isolated from leaves collected in the Ogasawara Islands, Japan. They represent three different species in the genusBullera on the basis of morphological, physiological, and biochemical characteristics, analyses of the sequences of internal transcribed spacer regions and small subunit ribosomal DNA, and a nuclear DNA-DNA hybridization study. Three new species,Bullera boninensis (five strains),B. waltii (seven strains), andB. schimicola (one strain), are proposed for these 13 strains.     

An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel

An efficient and convenient method is presented for the isolation of nuclear DNA from leaves of wildVitis species that have been dried with silica gel. The nuclear DNA obtained with this method is suitable for both PCR amplification and digestion with restriction endonucleases.     

Female's preference for oviposition site and larval performance in the water-lily beetle,Galerucella nymphaeae (Coleoptera: Chrysomelidae)

Water-lily beetles prefer younger rather than older water-lily leaves as oviposition sites. By the time of hatching, however, young leaves have aged consieerably. Larval performance of the water-lily beetle was measured on different types of leaves of the yellow water-lily and compared with oviposition preference of females. The leaf types used in the experiments were categorized as (i) young, (ii) natal (medium-aged) and (iii) old. The natal leaves were the ones on which larvae from a particular egg-batch had hatched. There were two sets of experiments. First, larvae were raised from eggs to pupae on young and on old leaves. Second, the growth of the 1st-instar larvae was measured on young, natal, and old leaves. The development time from egg to pupa did not differ between young and old leaves, but larvae growing on young leaves attained a higher pupal weight. In the second experiment the 1st-instar larvae grew fastest on their natal leaves, but there was also variation in the growth rate of progeny from different egg-batches. Larval growth on young and old leaves did not differ significantly. Larvae tried to emigrate much less from natal than other types of leaves. Females tended to lay eggs on leaves where larval growth was fastest. It seems that medium-aged leaves are best for larval growth, but the leaf characteristics responsible for this remain unresolved.     

Construction of a genomic library using high-molecular-weight DNA prepared from peach leaves

A method for constructing a total genomic library of peach DNA is presented. DNA of 200 kb or more is efficiently isolated from relatively large amounts of peach leaves (15 to at least 50 g) and used to construct the library in bacteriophage λ.     

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls – Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2–4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose–response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.