A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants

Protocols for genomic DNA extraction from plants are generally lengthily, since they require that tissues be ground in liquid nitrogen, followed by a precipitation step, washing and drying of the DNA pellet, etc. This represents a major challenge especially when several hundred samples must be screened/analyzed within a working day. There is therefore a need for a rapid and simple procedure, which will produce DNA quality suitable for various analyses. Here, we describe a time and cost efficient protocol for genomic DNA isolation from plants suitable for all routine genetic screening/analyses. The protocol is free from hazardous reagents and therefore safe to be executed by non-specialists. With this protocol more than 100 genomic DNA samples could manually be extracted within a working day, making it a promising alternative in genetic studies of large-scale genomic screening projects.     

Chromatin-bound DNA polymerase from higher plants

Chromatin-bound DNA polymerase has been extracted from pea (Pisum sativum L.) seedlings, and partially purified by solubilization from chromatin followed by chromatography on columns of either DEAE-cellulose or DEAE-Sephadex. The enzyme elutes from DEAE-cellulose as a single peak, but is fractionated into two peaks, CI and CII, by DEAE-Sephadex chromatography. If the enzyme is stored at-15°C for several days prior to chromatography, a third peak, CIII, derived from CII, is obtained. The polymerase is devoid of nuclease activity, and is relatively insensitive to N-ethyl-maleimide. These features, taken with the ion requirements and with data obtained from other plant species, lead to the suggestion that the chromatin-bound DNA polymerase of higher plants is similar to the DNA polymerase- from vertebrates.     

Terbutryn resistance in Vaucheria dichotoma

Infestations of Vaucheria dichotoma have been reported to become resistant to the herbicide terbutryn after successful control by annual or biennial applications for 11 years. V. dichotoma was isolated from a site where terbutryn treatment gave good control and from a site where terbutryn treatment had failed. The isolate from the latter was resistant to terbutryn in laboratory toxicity tests. The resistant strain was not killed until the dose of terbutryn was increased to more than double the concentration approved for use in British waters. The shape of the resistance curve indicated that the resistance mechanism was not by modification of the protein binding site, suggesting that differential uptake, translocation and/or degradation is involved.     

Nucleotide sequence of the chloroplast gene responsible for triazine resistance in canola

Summary The nucleotide sequence for the psbA gene from a triazine resistant cultivar of B. napus (cv Triton) has been determined. This gene encodes an open reading frame of 353 amino acids that is highly homologous to other higher plant psbA genes at both the nucleotide and amino acid levels. As has been found for other triazine resistant psbA genes, the Triton psbA contains an A to G nucleotide change which results in a serine to glycine amino acid substitution at position 264. The B. napus psbA gene also has a G insertion at position –9 resulting in a ribosome binding site sequence (AGGA) just before the initial methionine and suggesting that the entire open reading frame is translated. A large (72 bp) insertion is also found upstream of the B. napus psbA gene which resembles a similar insertion in the mustard psbA. The uncloneable nature of the entire gene is further investigated through reconstruction experiments and the implications discussed.     

A rapid DNA preparation for PCR fromChlamydomonas reinhardtii andArabidopsis thaliana

A method for preparing DNA for PCR has been adapted from the forensic work of Walsh et al. (Biotechniques 10:506–513) for use withChlamydomonas reinhardtii andArabidopsis thaliana. The method consists of a short incubation of cells or tissue in ethanol, followed by addition of Chelex-100 and heat treatment. Following centrifugation, the supernatant is added directly to the PCR reaction; forChlamydomonas, amplification product is visible over a range of four orders of magnitude of starting cells. Using this method, DNA suitable for PCR template can also be obtained fromArabidopsis leaf tissue without grinding, organic extraction or precipitation steps. This method may prove to be useful for other plant and algal species.     

Cereal DNA: A rapid high-throughput extraction method for marker assisted selection

A rapid, automated and novel method is presented to extract DNA suitable for polymerase chain reaction (PCR) from small amounts of cereal leaf tissue in a high-throughput cost-effective way. The method uses a 96-well plate in which leaf samples are frozen, mechanically crushed using a matrix mill, macerated in alkali and subsequently neutralized. The method was used routinely with barley and wheat leaf samples and the extracted material was used to screen for specific traits of interests, including barley yellow dwarf virus resistance and β-amylase activity in barley and stem rust resistance in wheat. This system allows complete PCR analysis of 384 seedlings or more by one person in a day.     

A simple,nondestructive spraying assay for the detection of an active kanamycin resistance gene in transgenic tomato plants

Summary A simple, nondestructive kanamycin spraying assay for detecting neomycin phosphotransferase II activity in tomato has been developed. This assay does not require the use of tissue culture or biochemical methods, but allows transgenic and non-transgenic tomato plants to be distinguished directly at the plant level in the green-house. Its potential applications in large-scale genetic analyses are discussed.     

Evaluation of an ELISA assay for rapid detection and quantification of neomycin phosphotransferase II in transgenic plants

Neomycin phosphotransferase II (neo) is a selectable marker gene used extensively in plant transformation experiments. Here we evaluate immunological detection of its gene product (NPTII) as an alternative to widely used radioactive assays. We have taken a commercially available non-radioactive NPTII Enzyme linked-Immunosorbant Assay (ELISA) kit, modified the protocol for application to plant tissues, and used it to quantify levels of NPTII protein in transformed plants. The ELISA proved safe, economical and convenient to reliably screen and quantify NPTII protein in large numbers of plant samples. The sensitivity of the ELISA for NPTII detection in tobacco plants is at least an order of magnitude greater than a widely used radioactive gel assay. Using three replicates per sample, standard errors are low and the assay is highly reproducibleover time for tissue-cultured tobacco. However, background readings varied with plant species, and also with plant age for untransformed glasshouse-grown tobacco. It is therefore essential to ensure that untransformed controls are closely matched to test plant.     

Identification of RAPD markers linked to a major rust resistance gene block in common bean

Rust in bean (Phaseolus vulgaris L.), caused byUromyces appendiculatus (Pers.) Unger var.appendiculatus [ =U. phaseoli (Reben) Wint.], is a major disease problem and production constraint in many parts of the world. The predominant form of genetic control of the pathogen is a series of major genes which necessitate the development of efficient selection strategies. Our objective was focused on the identification of RAPD (random amplified polymorphic DNA) markers linked to a major bean rust resistance gene block enabling marker-based selection and facilitating resistance gene pyramiding into susceptible bean germplasm. Using pooled DNA samples of genotyped individuals from two segregating populations, we identified two RAPD markers linked to the gene block of interest. One such RAPD, OF10₉₇₀ (generated by a 5-GGAAGCTTGG-3 decamer), was found to be closely linked (2.15±1.50 centi Morgans) in coupling with the resistance gene block. The other identified RAPD, OI19₄₆₀ (generated by a 5-AATGCGGGAG-3 decamer), was shown to be more tightly linked (also in coupling) than OF10₉₇₀ as no recombinants were detected among 97 BC₆F₂ segregating individuals in the mapping population. Analysis of a collection of resistant and susceptible cultivars and experimental lines, of both Mesoamerican and Andean origin, revealed that: (1) recombination between OF10₉₇₀ and the gene block has occurred as evidenced by the presence of the DNA fragment in several susceptible genotypes, (2) recombination between OI19₄₆₀ and the gene block has also occurred indicating that the marker is not located within the gene block itself, and (3) marker-facilitated selection using these RAPD markers, and another previously identified, will enable gene pyramiding in Andean germplasm and certain Mesoamerican bean races in which the resistance gene block does not traditionally exist. Observations of variable recombination among Mesoamerican bean races suggested suppression of recombination between introgressed segments and divergent recurrent backgrounds.Research supported by the Michigan Agricultural Research Station and the USDA-ARS. Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable     

Characterization of induced resistance in tomato plants

We have characterized, using several types of bioassays, the resistance induced in young tomato plants by feeding of the corn earworm, Helicoverpa zea. Beet armyworm larvae, Spodoptera exigua, and leafminers, Liriomyza trifolii, were used to assay the induced resistance. In whole-plant experiments, damage localized to a single leaflet of fourleaf tomato plants induced a systemic increase in resistance such that beet armyworm larvae confined to previously damaged (induced) plants grew at a rate about half that of larvae raised on control plants and consumed less leaf tissue from induced plants than from control plants. In experiments using excised leaves, beet armyworm larvae suffered increased mortality when reared on leaves from induced plants. The strength of this induced resistance varied spatially relative to the damaged position; moreover, the spatial distribution of induced resistance changed over a three-week period following damage. Other experiments demonstrated that the mechanisms of induced resistance in tomato foliage involves both a decrease in larval preference for and a decrease in the nutritional value of induced foliage. Induction also retarded the oviposition and/or early development of leafminers. Thus, induced resistance has relatively severe effects on the biology of subsequent herbivores. These data should allow us to begin to elucidate cause-effect relationships between induced resistance and induced chemistry in tomato plants.     

Quantitative assessment of heteroplasmy levels in <Emphasis Type="Italic">Senecio vulgaris</Emphasis> chloroplast DNA

Heteroplasmy in coding chloroplast DNA was only recently shown to occur and was so far not quantitatively assessed. We present a quantitative analysis of cpDNA heteroplasmy levels at a triazine-resistance determining site within and between individual Senecio vulgaris plants. Detectable levels of heteroplasmic haplotypes were observed in all tested plants. As expected, the levels of heteroplasmy vary greatly between plants. However, even within individual plants, the fraction of one haplotype may cover a range from below 1 to well over 90. Our results suggest that heteroplasmy may be a common phenomenon in S. vulgaris.Possible consequences for molecular diagnostics of chloroplast encoded traits as well as evolutionary consequences of chloroplast heteroplasmy are discussed.     

FRET hybridization probes for the rapid detection of disease resistance alleles in plants: detection of corky root resistance in lettuce

We describe the development of a non-electrophoresis PCR-based assay for allele discrimination at a disease resistance locus. The assay is based on the emission of light by fluorescence resonance energy transfer (FRET) upon annealing of two hybridization probes. The analysis of melting curve profiles of the probes and templates allowed the detection of single nucleotide polymorphisms. The assay was applied to the detection of alleles at the cor locus in lettuce (Lactuca sativa) that confers recessive resistance to corky root disease. Probes and primers for the assay were designed after the characterization of a single nucleotide polymorphism between alleles of PCR products amplified using a linked marker. That polymorphism was validated in a collection of lettuce varieties representing different genetic backgrounds. The FRET hybridization probes approach provided fast and accurate genotyping of breeding material directly in a one-tube reaction. The absence of electrophoresis makes this approach suitable for applications that require automation and high-throughput genotyping analyses such as marker-assisted selection programs.     

A rapid visual method to identify transformed plants

Summary A rapid, efficient assay that is nondestructive and semiquantitative for identifying transgenic plants and progeny from Biolistic^? and protoplast transformations is described. Leaf sections of maize and wheat plants are placed on an indicator medium containing chlorophenol red and the selection agent. Changes in the color of the medium from red to yellow resulting from altered pH indicate transformed plants within 2-5 d. The method is particularly suited to use with phosphinothricin and could be used with other suitable selectable markers.     

A rapid and efficient method for the isolation of restrictable total DNA from plants of the genusAbelmoschus

A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genusAbelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves. This problem is resolved using cotyledons of dark-grown seedlings.     

A rapid HPLC assay for zearalenone in laboratory cultures ofFusarium graminearum

A high pressure liquid chromatographic (HPLC) method to determine zearalenone in corn contaminated withFusarium graminearum is described. After extraction with methanol-water and solvent partition, samples were cleaned up by applying the extract to a disposable silica cartridge and by eluting the toxin with a mixture of hexane/dry ethyl ether (5/5). Separation was achieved by a reverse phase Bondapak C₁₈ column followed by fluorescence detection using an excitation wavelength at 274 nm and an emission wavelength at 440 nm. Detection limit was about 5 ng. Recoveries ranging from 85.37 to 100.97%, in standard solutions range 30–0.5 µg/ml, were found.     

A role for managanese in the resistance of wheat plants to take-all

Summary The hypothesis that wheat plants deficient in managenese are predisposed to infection byGaeumannomyces graminis is outlined, and a test of the hypothesis in a soil system is reported. The results supported the hypothesis: wheat plants growing in managanese-deficient soil, although not showing foliar symptoms, were markedly more susceptible to infection; plant analysis confirmed the nutrient status of the plants. A review of the literature on take-all in wheat coupled with the results of our experiments suggests a reinterpretation of the etiology of this disease, since those edaphic factors which promote infection by this organism are those which also render managese unavailable to the host. Managenese nutrition is proposed as a common factor in many of the environmental conditions which influence the host-pathogen balance.     

A rapid,single leaf,nucleic acid assay for determining the cytoplasmic organelle complement of rapeseed and related Brassica species

Summary An assay is described whereby Eco RI restriction fragment length polymorphisms of mitochondrial and chloroplast DNAs can definitively identify cytoplasms of interest in Brassica crop development. Restrictable mitochondrial and chloroplast DNA is extracted from as little as 2–3 g and 0.5 g leaf tissue, respectively, and the donor plants are able to continue to develop in a normal manner. An unknown cytoplasm can be identified in three days, which is a considerable saving in time and labor compared to the several years required by traditional methods. The assay is very inexpensive and should be established as a routine procedure in laboratories involved in sexual or somatic Brassica hybrid production.     

The combination of polima cms and cytoplasmic triazine resistance in Brassica napus

Summary Protoplast fusion was used to combine cytoplasmic triazine resistance (ctr) and Polima type cytoplasmic male sterility (cms) in Brassica napus. The cybrids produced constitute the major biological input required for the production of commercial single-cross hybrid rapeseed bearing cytoplasmic triazine resistance. The results also indicate that Polima cms is associated with the mitochondrial genome.     

A rapid PCR based method to establish the potential for paternal inheritance of chloroplasts inPelargonium

A simple procedure for the amplification of chloroplast DNA from pollen is described. This allows the potential for biparental or paternal inheritance of chloroplasts in angiosperms to be established rapidly and reliably. Such information is important when chloroplast DNA, which is generally assumed to be inherited maternally, is used to study gene flow or reconstruct phylogenies. The technique could also prove valuable in assessing the risks of transgene escape via pollen of genetically modified plants, in which the chloroplast genome rather than the nucleus has been transformed.