Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

Background  

Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.     

Proposal for field sampling of plants and processing in the lab for environmental metabolic fingerprinting

Background  

Samples for plant metabolic fingerprinting are prepared generally by metabolism quenching, grinding of plant material and extraction of metabolites in solvents. Further concentration and derivatisation steps follow in dependence of the sample nature and the available analytical platform. For plant material sampled in the field, several methods are not applicable, such as, e.g., collection in liquid nitrogen. Therefore, a protocol was established for sample pre-treatment, grinding, extraction and storage, which can be used for analysis of field-collected plant material, which is further processed in the laboratory. Ribwort plantain (Plantago lanceolata L., Plantaginaceae) was used as model plant. The quality criteria for method suitability were high reproducibility, extraction efficiency and handling comfort of each subsequent processing step.     

A simple and efficient method for the long-term preservation of plant cell suspension cultures

Background  

The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.     

A modified Gateway cloning strategy for overexpressing tagged proteins in plants

Background  

Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale.     

Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants

Background  

We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.     

A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta

Background  

Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems.     

A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context

Background  

In silico analyses based on sequence similarities with animal channels have identified a large number of plant genes likely to encode ion channels. The attempts made to characterise such putative plant channels at the functional level have most often relied on electrophysiological analyses in classical expression systems, such as Xenopus oocytes or mammalian cells. In a number of cases, these expression systems have failed so far to provide functional data and one can speculate that using a plant expression system instead of an animal one might provide a more efficient way towards functional characterisation of plant channels, and a more realistic context to investigate regulation of plant channels.     

High-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated barley transformation

Background  

Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.     

Computer aided data acquisition tool for high-throughput phenotyping of plant populations

Background  

The data generated during a course of a biological experiment/study can be sometimes be massive and its management becomes quite critical for the success of the investigation undertaken. The accumulation and analysis of such large datasets often becomes tedious for biologists and lab technicians. Most of the current phenotype data acquisition management systems do not cater to the specialized needs of large-scale data analysis. The successful application of genomic tools/strategies to introduce desired traits in plants requires extensive and precise phenotyping of plant populations or gene bank material, thus necessitating an efficient data acquisition system.     

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background  

Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.     

A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid

Background  

Salicylic acid (SA) is an important signalling molecule in plant defenses against biotrophic pathogens. It is also involved in several other processes such as heat production, flowering, and germination. SA exists in the plant as free SA and as an inert glucose conjugate (salicylic acid 2-O-β-D-glucoside or SAG). Recently, Huang et al. developed a bacterial biosensor that responds to free SA but not SAG, designated as Acinetobacter sp. ADPWH_lux. In this paper we describe an improved methodology for Acinetobacter sp. ADPWH_lux-based free SA quantification, enabling high-throughput analysis, and present an approach for the quantification of SAG from crude plant extracts.     

In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis

Background  

Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo.     

Development of a pooled probe method for locating small gene families in a physical map of soybean using stress related paralogues and a BAC minimum tile path

Background  

Genome analysis of soybean (Glycine max L.) has been complicated by its paleo-autopolyploid nature and conserved homeologous regions. Landmarks of expressed sequence tags (ESTs) located within a minimum tile path (MTP) of contiguous (contig) bacterial artificial chromosome (BAC) clones or radiation hybrid set can identify stress and defense related gene rich regions in the genome. A physical map of about 2,800 contigs and MTPs of 8,064 BAC clones encompass the soybean genome. That genome is being sequenced by whole genome shotgun methods so that reliable estimates of gene family size and gene locations will provide a useful tool for finishing. The aims here were to develop methods to anchor plant defense- and stress-related gene paralogues on the MTP derived from the soybean physical map, to identify gene rich regions and to correlate those with QTL for disease resistance.     

UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

Background  

Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids.     

Development of expression vectors based on pepino mosaic virus

Background  

Plant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV.     

High-throughput phenotyping of lateral expansion and regrowth of spaced <Emphasis Type="Italic">Lolium perenne</Emphasis> plants using on-field image analysis

Background

Genetic studies and breeding of agricultural crops frequently involve phenotypic characterization of large collections of genotypes grown in field conditions. These evaluations are typically based on visual observations and manual (destructive) measurements. Robust image capture and analysis procedures that allow phenotyping large collections of genotypes in time series during developmental phases represent a clear advantage as they allow non-destructive monitoring of plant growth and performance. A L. perenne germplasm panel including wild accessions, breeding material and commercial varieties has been used to develop a low-cost, high-throughput phenotyping tool for determining plant growth based on images of individual plants during two consecutive growing seasons. Further we have determined the correlation between image analysis-based estimates of the plant’s base area and the capacity to regrow after cutting, with manual counts of tiller number and measurements of leaf growth 2 weeks after cutting, respectively. When working with field-grown plants, image acquisition and image segmentation are particularly challenging as outdoor light conditions vary throughout the day and the season, and variable soil colours hamper the delineation of the object of interest in the image. Therefore we have used several segmentation methods including colour-, texture- and edge-based approaches, and factors derived after a fast Fourier transformation. The performance of the procedure developed has been analysed in terms of effectiveness across different environmental conditions and time points in the season.

Results

The procedure developed was able to analyse correctly 77.2 % of the 24,048 top view images processed. High correlations were found between plant’s base area (image analysis-based) and tiller number (manual measurement) and between regrowth after cutting (image analysis-based) and leaf growth 2 weeks after cutting (manual measurement), with r values up to 0.792 and 0.824, respectively. Nevertheless, these relations depend on the origin of the plant material (forage breeding lines, current forage varieties, current turf varieties, and wild accessions) and the period in the season.

Conclusions

The image-derived parameters presented here deliver reliable, objective data, complementary to the breeders’ scores, and are useful for genetic studies. Furthermore, large variation was shown among genotypes for the parameters investigated.

     

Delivering high-resolution landmarks using inkjet micropatterning for spatial monitoring of leaf expansion

Background  

Inkjet micropatterning is a versatile deposition technique with broad applications in numerous fields. However, its application in plant science is largely unexplored. Leaf expansion is one of the most important parameters in the field of plant science and many methods have been developed to examine differential expansion rates of different parts of the leaf lamina. Among them, methods based on the tracking of natural landmarks through digital imaging require a complicated setup in which the leaf must remain fixed and under tension. Furthermore, the resolution is limited to that of the natural landmarks, which are often difficult to find, particularly in young leaves. To study the fine scale expansion dynamics of the leaf lamina using artificial landmarks it is necessary to place small, noninvasive marks on a leaf surface and then recover the location of those marks after a period of time.     

Growth response of Ficus benjamina to limited soil volume and soil dilution in a skeletal soil container study

The interactive effects of rooting volume and nutrient availability in a skeletal soil medium designed to meet street tree and pavement needs were observed in a containerized experiment. Benjamin fig (Ficus benjamina L.) was grown in a stone-soil blended skeletal soil material (CU-Soil™) and compared to a loam soil. The same topsoil used as the soil component in the skeletal soil material was used as the sole component in the comparison soil-only treatment.Plants grown in the skeletal soil material had reduced leaf tissue N content and depressed growth compared with plants grown in non-diluted soil. No other mineral deficiencies were found. Leaf number, chlorophyll concentration, shoot weight, and root characteristics were all affected.Reduced growth from soil dilution could be offset by the provision of an enlarged rooting volume for root development. Large containers of skeletal soil were observed to have smaller root systems compared to equivalent net volumes of loam soil at the first two harvest dates of the study. By the end of the study, the large containers of skeletal soil were observed to have developed larger root systems compared to equivalent net volumes of loam soil; resulting in comparable leaf N levels and total plant dry matter. Plants in skeletal soil had lower shoot: root ratios at the end of the study. Investing resources to further root growth in times of nutrient shortages is a probable plant reaction as evidenced by differences in specific root length between treatments. The study allowed a method for directly partitioning the containerization effect by having equivalent amounts of soil over two volumes.     

Fine-grained recognition of plants from images

Background

Fine-grained recognition of plants from images is a challenging computer vision task, due to the diverse appearance and complex structure of plants, high intra-class variability and small inter-class differences. We review the state-of-the-art and discuss plant recognition tasks, from identification of plants from specific plant organs to general plant recognition “in the wild”.

Results

We propose texture analysis and deep learning methods for different plant recognition tasks. The methods are evaluated and compared them to the state-of-the-art. Texture analysis is only applied to images with unambiguous segmentation (bark and leaf recognition), whereas CNNs are only applied when sufficiently large datasets are available. The results provide an insight in the complexity of different plant recognition tasks. The proposed methods outperform the state-of-the-art in leaf and bark classification and achieve very competitive results in plant recognition “in the wild”.

Conclusions

The results suggest that recognition of segmented leaves is practically a solved problem, when high volumes of training data are available. The generality and higher capacity of state-of-the-art CNNs makes them suitable for plant recognition “in the wild” where the views on plant organs or plants vary significantly and the difficulty is increased by occlusions and background clutter.