Effect of physical desiccation on plant regeneration efficiency in rice (Oryza sativa L.) variety super basmati

This experiment assessed the effect of partial physical desiccation on plant regeneration efficiency in scutellum-derived embryogenic calluses of rice (Oryza sativa L.) variety Super basmati. A number of callusing cultures were developed, and efficient callus induction was observed on MS (Murashige and Skoog) basal medium supplemented with 2.0 mg/L 2,4-dichlorophenoxy acetic acid. The calluses were proliferated on the same medium for 3 weeks and then shifted to dehydration desiccation treatment for 72 h. The desiccated calluses were cultured on different media for somatic embryogenesis and plant regeneration. A medium with 2.0 mg/L α-napthaleneacetic acid, 10.0 mg/L abscisic acid , 2.0 mg/L kinetin was best for somatic embryogenesis only, but not for further plant development. After 10 d, differentiated calluses were sub-cultured on medium with various concentrations and types of carbohydrates (carbon source) in ¹MS_2j medium. A large number of plantlets (14.51±2.81 and 8.56±2.90 plants/callus) were regenerated via chemical desiccation, on MS with 3% maltose+3% sorbitol and 6% sucrose, respectively. Under dehydration on only simple MS (3% sucrose), 11.23±3.22 plants/callus were developed. Under conditions of dehydration and chemical desiccation, plant regeneration rates were higher than the calluses cultured on simple MS medium in the presence of plant growth regulator. After somatic embryogenesis, >25% plants were sterile. The protocol used here may allow maximum regeneration of normal and fertile plantlets of super basmati rice within 3 months.     

Scarless and site-directed mutagenesis in <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>chromosome

Background  

A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.     

1,2,3-Triazole Fused Quinoline-Peptidomimetics: Studies on Synthesis, DNA Binding and Photonuclease Activity

Abstract  

We present, simple approach for the accession of 1,2,3-triazole fused quinoline peptide analogues from 3-(azidomethyl)-2-chloroquinoline in a three-step mechanistic pathway. The UV–Visible absorbance plot shows dynamic interaction of parent triazole derivative with CT DNA as efficient DNA intercalator (K _b = 4.6 × 10⁻⁴ M⁻¹). Finally, the efficient DNA damage was observed on photo-irradiation at 360 nm in the presence of 2-(9H-Fluoren-9-ylmethoxycarbonylamino)-propionic acid 1-(2-chloro-quinolin-3-ylmethyl)-1H-[1,2,3]triazole-4-ylmethyl ester (6a).     

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.     

Statistical Parameters Reflecting Morphogenetic Capacity of Soft Spring Wheat Calluses

Callus cultures of soft spring wheat were subcultured without separation into explants to follow the line one excised embryo–one callus. This approach revealed the following statistical correlations. Within every cultivar of Triticum aestivum L. and within a row of cultivars arranged in ascending order according to the frequency of embryogenic callus formation, positive correlations (at P = 95) were found between the proliferative activity of callus cells and the frequency of embryogenic callus formation. A reliable intraspecies correlation (significant at P = 95) between multiple regenerations of plants from calluses and the tillering trait (bushiness) of donor plants was also found. We assessed the importance of various statistical parameters of callus cultures for preliminary estimation of morphogenesis efficiency at early stages of culturing. Frequencies of callusogenesis and the growth curves for randomly selected calluses turned out to be noninformative characteristics, unless the morphogenetic activity of calluses was taken into account. The following statistical parameters were found to correlate with the morphogenetic capacity of wheat calluses: gradually increasing coefficients of variation in fresh weight of primary calluses, a larger callus size, and higher fresh weight gain in potentially morphogenetic calluses.     

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

Background

Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.

Methodology/Principal Findings

We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.

Conclusions/Significance

We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.     

Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin

Background

Although genomic DNA isolation using the Chelex 100 resin is rapid and inexpensive, the DNA obtained by this method has a low concentration in solution and contains suspended impurities. The presence of debris in the DNA solution may result in degradation of DNA on long term storage and inhibition of the polymerase chain reaction. In order to remove impurities and concentrate the DNA in solution, we have introduced modifications in the existing DNA isolation protocol using Chelex-100. We used ammonium acetate to precipitate proteins and a sodium acetate- isopropanol mixture to pellet out DNA which was washed with ethanol.

Results

A pure DNA pellet that can be dissolved in water or Tris-EDTA buffer and stored for a long time at ??80 °C was obtained. We also observed a 20-fold change in the DNA concentration following precipitation and re-dissolution.

Conclusion

Our method is different from other extraction methods since it uses non-toxic, easily available and inexpensive reagents as well as minimal amounts of blood or tissue samples for the DNA extraction process. Besides its use in sex determination and genotyping in lab animals as described in this paper, it may also have applications in forensic science and diagnostics such as the easy detection of pathogenic DNA in blood.

     

DNA microarray data and contextual analysis of correlation graphs

Background  

DNA microarrays are used to produce large sets of expression measurements from which specific biological information is sought. Their analysis requires efficient and reliable algorithms for dimensional reduction, classification and annotation.     

EXMOTIF: efficient structured motif extraction

Background  

Extracting motifs from sequences is a mainstay of bioinformatics. We look at the problem of mining structured motifs, which allow variable length gaps between simple motif components. We propose an efficient algorithm, called EXMOTIF, that given some sequence(s), and a structured motif template, extracts all frequent structured motifs that have quorum q. Potential applications of our method include the extraction of single/composite regulatory binding sites in DNA sequences.     

An optimized chloroplast DNA extraction protocol for grasses (Poaceae) proves suitable for whole plastid genome sequencing and SNP detection

Background

Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering) and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses.

Methodology/Principal Findings

We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge.

Conclusions/Significance

The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus×giganteus, Panicoideae). The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.     

An expressed sequence tag (EST) library from developing fruits of an Hawaiian endemic mint (Stenogyne rugosa, Lamiaceae): characterization and microsatellite markers

Background  

The endemic Hawaiian mints represent a major island radiation that likely originated from hybridization between two North American polyploid lineages. In contrast with the extensive morphological and ecological diversity among taxa, ribosomal DNA sequence variation has been found to be remarkably low. In the past few years, expressed sequence tag (EST) projects on plant species have generated a vast amount of publicly available sequence data that can be mined for simple sequence repeats (SSRs). However, these EST projects have largely focused on crop or otherwise economically important plants, and so far only few studies have been published on the use of intragenic SSRs in natural plant populations. We constructed an EST library from developing fleshy nutlets of Stenogyne rugosa principally to identify genetic markers for the Hawaiian endemic mints.     

Microarray-based method for detection of unknown genetic modifications

Background  

Due to the increased use of genetic modifications in crop improvement, there is a need to develop effective methods for the detection of both known and unknown transgene constructs in plants. We have developed a strategy for detection and characterization of unknown genetic modifications and we present a proof of concept for this method using Arabidopsis thaliana and Oryza sativa (rice). The approach relies on direct hybridization of total genomic DNA to high density microarrays designed to have probes tiled throughout a set of reference sequences.     

SMOTIF: efficient structured pattern and profile motif search

Background  

A structured motif allows variable length gaps between several components, where each component is a simple motif, which allows either no gaps or only fixed length gaps. The motif can either be represented as a pattern or a profile (also called positional weight matrix). We propose an efficient algorithm, called SMOTIF, to solve the structured motif search problem, i.e., given one or more sequences and a structured motif, SMOTIF searches the sequences for all occurrences of the motif. Potential applications include searching for long terminal repeat (LTR) retrotransposons and composite regulatory binding sites in DNA sequences.     

Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle,sheep, goat,roe buck and deer by PCR-RFLP

Background  

As an alternative to direct DNA sequencing of PCR products, random PCR-RFLP is an efficient technique to discriminate between species. The PCR-RFLP-method is an inexpensive tool in forensic science, even if the template is degraded or contains only traces of DNA from various species.     

Plant regeneration from leaf petioles in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Summary Camptothecin, produced by Camptotheca acuminata, is a pharmaceutically important compound. Transgenic technology has potential uses for the enhancement of camptothecin production; however, an efficient plant regeneration protocol for C. acuminata is not currently available. Factors that affected successful seedling germination were evaluated. The regeneration potential of various parts of seedlings was tested. Camptothecin production in regenerated plants was compared to its production in calluses and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival rate after germination. The best shoot induction medium was found to be Gamborg's B5 medium+8.9 μM benzyladenine. Among the calluses induced from various parts of seedlings, leaf petiole calluses, leaf dise calluses, and cotyledon calluses regenerated shoots, but internode calluses did not. Furthermore, leaf petiole calluses and leaf dise calluses regenerated normal shoots, while cotyledon calluses regenerated hyperhydric shoots. Moreover, leaf petiole calluses had a higher shoot regeneration rate, 50% versus 9%, and a higher shoot number, 6.2±0.5 versus 2.0±0.3, than did leaf dise calluses on the best shoot induction medium. It took 4–6 wk to regenerate shoots after transfer into shoot induction media. Camptothecin concentration in the regenerated plants was significantly higher than that in the calluses and similar to that in the original seedlings. In conclusion, leaf petioles provide efficient plant regeneration of C. acuminata.     

Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis

Purpose of work  

A simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities of DNA are usually obtained, and with large scale analysis multiple isolations are required.     

DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks

Background  

DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability.     

CoreGenes: A computational tool for identifying and cataloging "core" genes in a set of small genomes

Background  

Improvements in DNA sequencing technology and methodology have led to the rapid expansion of databases comprising DNA sequence, gene and genome data. Lower operational costs and heightened interest resulting from initial intriguing novel discoveries from genomics are also contributing to the accumulation of these data sets. A major challenge is to analyze and to mine data from these databases, especially whole genomes. There is a need for computational tools that look globally at genomes for data mining.     

An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents

Background

Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found.

Methodology/Principal Findings

We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species.

Conclusions/Significance

Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.