Showing papers in "Plant Molecular Biology Reporter in 1990"
TL;DR: By virtue of the diploid, self-fertilising and genetically homogeneous character of M. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.
Abstract: Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.
376 citations
TL;DR: The advantages of the described procedure are that it is fast, does not require expensive equipment (e.g., lyophilizer) and can be used in situations where large numbers of samples must be extracted.
Abstract: We describe an inexpensive method for dehydration of plant tissue and extraction of high molecular weight DNA. Tissue is dried for 12 to 24 hours in a food dehydrator and subsequently powdered for DNA extraction. Dicot tissue can be powdered in centrifuge tubesen masse using a commercial paint mixer and glass beads. With the use of the paint mixer, tissue never touches common surfaces that might lead to cross contamination, a potential benefit when the DNA is to be used for PCR reactions. The DNA is of a quality equal to that obtained from either lyophilized or fresh frozen tissue (commonly used in many labs). The advantages of the described procedure are that it is fast, does not require expensive equipment (e.g., lyophilizer) and can be used in situations where large numbers of samples must be extracted.
318 citations
TL;DR: DNA suitable for digestion with restriction endonucleases has been isolated from Theobroma cacao, a plant high in polyphenolic compounds, and several other plant taxa using the method presented in this paper.
Abstract: DNA suitable for digestion with restriction endonucleases has been isolated fromTheobroma cacao, a plant high in polyphenolic compounds, and several other plant taxa using the method presented in this paper. The method relies on concentrating nuclei away from most of the cytoplasmic components and inhibiting the formation of oxidized polyphenolic compounds and their interaction with the DNA fraction in the remaining steps.
126 citations
TL;DR: The primary purification process consists of washing the DNA with phenol while it is complexed with CTAB and dissolved in 1 M NaCl.
Abstract: Numerous DNA extraction methods failed to remove contaminants that interfere with restriction digests ofCuphea DNA. The method described here removes those contaminants and maintains relatively high DNA yields. The primary purification process consists of washing the DNA with phenol while it is complexed with CTAB and dissolved in 1 M NaCl.
121 citations
TL;DR: A modified protocol for theAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotina tabacum L.) leaf disks was developed for greater recovery of transgenic plants.
Abstract: A modified protocol for theAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotina tabacum L.) leaf disks was developed for greater recovery of transgenic plants. Modifications include transformation ofAgrobacterium by a freeze-thaw procedure, initial cocultivation of leaf disks andAgrobacterium under vacuum, subsequent growth with nurse cells for one week, rooting of shoots in medium lacking carbenicillin, longer, growth in rooting medium, and a shortened “hardening” step. By this procedure, an average of 1.3 kanamycin-resistant calli were obtained per leaf disk, and 38% of, the callus cultures used were regenerated to produce 133 independently transformed tobacco plants.
104 citations
TL;DR: A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-Radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase.
Abstract: A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.
63 citations
TL;DR: A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genus Abelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves.
Abstract: 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.
49 citations
45 citations
TL;DR: An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented and two-thirds of transgenic plants grown to maturity in the greenhouse bore viable seeds.
Abstract: An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented. In this protocol seed calli from the varietiesNipponbare andTaipei 309 were used to produce rice suspension cultures in General Medium. Protoplasts were isolated from suspension cells (8 × 106 protoplasts perg fresh weight), then were incubated with sterile DNA in the presence of MaMg solution, followed by addition of PEG to a final concentration of 25%. A hygromycin phosphotransferase (hph) gene under the plant transcriptional regulatory signals was used as a selectable marker gene. Hygromycin-resistant colonies were selected in the presence of 95 μM hygromycin B with apparent frequencies of 2×10−4 and 5×10−4 forNipponbare andTaipei 309, respectively. Plantlets were regenerated from resistant colonies in Murashige and Skoog plant regeneration medium. Among 628 transgenic plants grown to maturity in the greenhouse, two-thirds bore viable seeds.
44 citations
TL;DR: The strongest signalto-noise ratio and most reproducible localizations of nodulin transcripts were obtained with the RNA detection kit on chemically fixed, cryostat-sectioned nodules.
Abstract: A protocol for thein-situ localization of nodulin mRNAs using digoxigenin (Genius KitTM; Boehringer Mannheim) as a non-radioactive detection system was developed for alfalfa root-nodule tissue. The strongest signalto-noise ratio and most reproducible localizations of nodulin transcripts were obtained with the RNA detection kit on chemically fixed, cryostat-sectioned nodules. This technique should be applicable to a wide range of plant tissues.
35 citations
TL;DR: A simple procedure for the detection of rice RFLPs with non-radioactive probes is described, which showed that when digested total DNA was hybridized with the non- radioactive labeled DNA probes, RFLP for rice single-copy DNA could be successfully detected.
Abstract: A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes, RFLPs for rice single-copy DNA could be successfully detected.
TL;DR: A method is described which allows localization of plant virus in infected leaf tissue and should have broad applicability to identifiying the distribution and location of proteins expressed in plants.
Abstract: A method, termed press blotting, is described which allows localization of plant virus in infected leaf tissue. Press blotting should have broad applicability to identifiying the distribution and location of proteins expressed in plants.
TL;DR: A distinct pattern of GUS activity was found in radish roots infected with themas-uidA fusion indicating a specificity of expression in the metabolically active cambium and phloem parenchyma cells.
Abstract: A mannopine synthase—β-glucuronidase gene fusion,mas-uidA, was used to detect T-DNA transfer 48 hours afterA. tumefaciens infection of radish root disks. A detailed procedure for infection, tissue preparation and GUS histochemistry is given. A CaMV 35S promoter was shown to be unsuitable as it was highly expressed in the bacteria. A distinct pattern of GUS activity was found in radish roots infected with themas-uidA fusion indicating a specificity of expression in the metabolically active cambium and phloem parenchyma cells. This assay is useful for studying T-DNA transfer and host range differences amongA. tumefaciens strains.
TL;DR: Techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice are developed, statistical methods are used to determine which restriction endonucleases are rare-cutting, and pulsed-field gel electrophoresis (PFE) is used to separate large fragments of rice DNA.
Abstract: TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.
TL;DR: It is shown here that for preparing libraries in a λ replacement vector, the use of suitablemcr hosts andmcr packaging mixes can increase the efficiency of cloning of plant genomic DNAs by at least two orders of magnitude.
Abstract: It has recently become apparent that many strains ofE. coli contain nucleases encoded by themcrA andmcrB loci that, recognize the modified base 5-methylcytosine in DNA. Plant DNAs have particularly high levels of this modification and the activity of these 5-methylcytosine-specific nucleases is particularly relevant to cloning plant genomic DNAs. We show here that for preparing libraries in a λ replacement vector, the use of suitablemcr hosts andmcr packaging mixes can increase the efficiency of cloning of plant genomic DNAs by at least two orders of magnitude. We also provide evidence that the activity of themcr nucleases is probably a significant source of bias in the representation of sequences in plant genomic libraries.
TL;DR: The isolation of mitochondria from sugar beet and sunflower has been carried out using a buffered medium of high ionic strength (I=1.40 M) and tDNAs obtained were enriched for supercoiled molecules of low molecular weight.
Abstract: The isolation of mitochondria from sugar beet and sunflower has been carried outina buffered medium of high ionic strength (I=1.40 M). Them tDNAs obtained were enriched for supercoiled molecules of low molecular weight. Chloroplast and mitochondrial DNA were successively prepared and used from a single leaf preparation for further analysis of restriction fragment length polymorphism. The few available wild relatives of sugar beet or sunflower have also been analyzed by comparison of the restriction patterns of their chloroplast and mitochondrial DNAs.
TL;DR: This work describesin-situ CNBr cleavage of proteins electrophoretically separated in polyacrylamide gels and presents simple methods for the generation and separation of peptides suitable for automated amino acid sequencing using Edman chemistry.
Abstract: We present simple methods for the generation and separation of peptides suitable for automated amino acid sequencing using Edman chemistry. We describein-situ CNBr cleavage of proteins electrophoretically separated in polyacrylamide gels. The generated peptides are separated by gel electrophoresis and blotted onto polyvinylidene diflouride membrane. Membranebound peptides are detected by Coomassie Blue staining and directly applied to an automated sequencer.
TL;DR: The anin-vitro system consists of a wheat-germ chromatin extract, which serves as a source of RNA polymerase II and basic transcription factors, substrates, and exogenously added DNA templates for accurate transcription initiation of plant promoters.
Abstract: We describe here anin-vitro system for accurate transcription initiation of plant promoters. The system consists of a wheat-germ chromatin extract, which serves as a source of RNA polymerase II and basic transcription factors, substrates, and exogenously added DNA templates. Both linear and circular DNA are equally effective as templates, but optimal conditions for their transcription differ slightly. Specific initiation at the promoter of a linear template is determined by size analysis of the transcript on a polyacrylamide gel containing 8 M urea. In the case of circular DNA template, the initiation frequency is measured by the amount of specific RNA transcribed from a G-free sequence (Sawadogo and Roeder, 1985) in a reaction mixture without GTP. We believe that ourin-vitro system detailed here will be useful for the elucidation of transcription mechanisms in plants.
TL;DR: A procedure which avoids the use of phenol-chloroform and RNAase for the isolation of total DNA from A. tumefaciens is described and is probably applicable to other Gramnegative bacteria.
Abstract: A procedure which avoids the use of phenol-chloroform and RNAase for the isolation of total DNA fromA tumefaciens is described Specific precipitation of protein by 25 M ammonium acetate is employed and much of the RNA is removed by an isopropanol precipition step The procedure yields easily restrictable, good quality DNA and is probably applicable to other Gramnegative bacteria
TL;DR: A rapid and efficient procedure for the isolation of chloroplast DNA from walled cells of Chlamydomonas reinhardtii using a high-speed table-top ultracentrifuge is described.
Abstract: We describe a rapid and efficient procedure for the isolation of chloroplast (and nuclear) DNA from walled cells ofChlamydomonas reinhardtii. Total nucleic acids are prepared and separated by equilibrium centrifugation in CsCl-bisbenzimide gradients using a high-speed table-top ultracentrifuge. Chloroplast DNA sufficient for several restriction analysis is obtained from 1 liter of cells in one to two days.
TL;DR: TwoPetunia hybrida homeotic flower mutants, Blind and Green Petals, were characterized as a way to study floral morphogenesis and the influence of these homeotic mutations on the expression of the two genes encoding the flavonoid biosynthesis enzyme chalcone flavanone isomerase was investigated.
Abstract: TwoPetunia hybrida homeotic flower mutants,Blind (Bl) andGreen Petals (Gp), were characterized as a way to study floral morphogenesis. The influence of these homeotic mutations on the expression of the two genes encoding the flavonoid biosynthesis enzyme chalcone flavanone isomerase was investigated. TheBl mutation gives rise to highly modified flowers in which the limbs are transformed into antheroid limbs which develop on top of a normally formed tube. In wild type plants,chiA transcripts are detected in the flower limb. In antheroid limbs, however, expression of the anther-specificchiB gene was observed. This is in line with the homeotic nature of theBl mutation. TheGp mutation gives rise to mutated flowers in which the petals are transformed into sepaloid petals. An absence of CHI activity andchi transcripts in sepaloid petals was observed, which is in line with the homeotic nature of theGp gene.
TL;DR: A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen.
Abstract: A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.
TL;DR: A NATO Advanced Research Workshop was held 21--26 May 1989 in Biddinghuizen, The Netherland, on the subject of molecular signals involved in intercellular communication within plants as well as in microbe-plant interactions.
Abstract: A NATO Advanced Research Workshop was held 21--26 May 1989 in Biddinghuizen, The Netherland s, on the subject of molecular signals involved in intercellular communication within plants as well as in microbe-plant interactions. The Workshop covered methods required to assay, purify, and identify signals; the design of experiments to test putative roles of such molecules; and the perception and transduction of signals. Since signal molecules may function similarly in plant and animal systems, M.G. Hahn and C.A. West were invited to discuss mechanisms of signal perception and signal transduction in animal systems. K. Palme discussed genes involved in the mechanism of plant hormone activity and Ca 2. metabolism. P. Albersheim discussed plant metabolites, specifically cell wall-derived molecules which play a variety of roles, from flowering to hypersensitive reactions. In discussing the use of Arabidopsis thaliana as a model plant for studying plant-pathogen interactions, K.R. Davis presented an emerging picture of specific initial interactions dependent on the type of microorganism followed by later stages that may be similar or even identical. N.T. Keen's discussion on avirulence genes and K. Hahlbrock's discussion on plant-fungus interactions presented novel data. Three speakers reported on the host specificity with Rhizobium plant interactions. J.W. Kijne showed that plant lectin is a major determinant of host-specificity; Spaink showed that the bacterial gene nodE is also a major determinant of host-specificity; and T. Bisseling showed that in addition to the common genes, nodA, B and C, the correct origin of nodE is essential for the synthesis of the plant nodulin ENOD 12. The organizing committee included E. J. J. Lugtenberg (Leiden) as chairman, P. Albersheim (Athens, Georgia, USA), K. Hahlbrock (K61n), and R. A. Schilperoort (Leiden). The proceedings of the workshop will be published by SpringerVerlag. Partial support was provided by the ISPMB.
TL;DR: This report will mainly focus on nitrate metabolism of higher plants where progress has been made in understanding NR genes and their regulation through the application of molecular biology techniques and the analysis of NR mutants.
Abstract: T HE Third International Symposium on Nitrate Assimilation, organized by Michel Caboche and Frdd(~rique Pelsy of the Laboratoire de Biologie Cellulaire, INRA-Versailles, was held in Bombannes, France, 13 to 18 May 1990. About 130 scientists from 17 countries participated in the meeting. During 31 oral presentations and on more than 60 posters, new data were presented on nitrate metabolism in bacteria and thallophytes, nitrate transport, structure of nitrate reductase (NR), the molybdenum cofactor, electron transport in nitrate reductase, molecular biology of NR and nitrite reductase. This report will mainly focus on nitrate metabolism of higher plants where progress has been made in understanding NR genes and their regulation through the application of molecular biology techniques and the analysis of NR mutants.
TL;DR: Representative genomic libraries of the soft wheat variety carrying a translocated rye chromosome are 1RS were constructed in arecD-minusE strain to reduce recombinational loss.
Abstract: The large genome size and the great amount of DNA repeats make it rather diffiult to obtain a representative hexaploid wheat genomic library. The protocol is given with modifications to phage isolation and to purification of vector and plant DNAs by electrophoresis in low-concentration SeaKem agarose gels. Representative genomic libraries of the soft wheat variety carrying a translocated rye chromosome are 1RS were constructed in arecD-minusE. coli strain to reduce recombinational loss.