Dataset Information

An Updated Protocol for High Throughput Plant Tissue Sectioning.

ABSTRACT: Quantification of the tissue and cellular structure of plant material is essential for the study of a variety of plant sciences applications. Currently, many methods for sectioning plant material are either low throughput or involve free-hand sectioning which requires a significant amount of practice. Here, we present an updated method to provide rapid and high-quality cross sections, primarily of root tissue but which can also be readily applied to other tissues such as leaves or stems. To increase the throughput of traditional agarose embedding and sectioning, custom designed 3D printed molds were utilized to embed 5-15 roots in a block for sectioning in a single cut. A single fluorescent stain in combination with laser scanning confocal microscopy was used to obtain high quality images of thick sections. The provided CAD files allow production of the embedding molds described here from a number of online 3D printing services. Although originally developed for roots, this method provides rapid, high quality cross sections of many plant tissue types, making it suitable for use in forward genetic screens for differences in specific cell structures or developmental changes. To demonstrate the utility of the technique, the two parent lines of the wheat (Triticum aestivum) Chinese Spring × Paragon doubled haploid mapping population were phenotyped for root anatomical differences. Significant differences in adventitious cross section area, stele area, xylem, phloem, metaxylem, and cortical cell file count were found.

SUBMITTER: Atkinson JA

PROVIDER: S-EPMC5632646 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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An Updated Protocol for High Throughput Plant Tissue Sectioning.

Atkinson Jonathan A JA Wells Darren M DM

Frontiers in plant science 20171004

Quantification of the tissue and cellular structure of plant material is essential for the study of a variety of plant sciences applications. Currently, many methods for sectioning plant material are either low throughput or involve free-hand sectioning which requires a significant amount of practice. Here, we present an updated method to provide rapid and high-quality cross sections, primarily of root tissue but which can also be readily applied to other tissues such as leaves or stems. To incr ...[more]

PMID: 29046689

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Project description:Confocal imaging is a well-established method for investigating plant phenotypes on the tissue and organ level. However, many differences are difficult to assess by visual inspection and researchers rely extensively on ad hoc manual quantification techniques and qualitative assessment. Here we present a method for quantitatively phenotyping large samples of plant tissue morphologies using triangulated isosurfaces. We successfully demonstrate the applicability of the approach using confocal imaging of aerial organs in Arabidopsis thaliana. Automatic identification of flower primordia using the surface curvature as an indication of outgrowth allows for high-throughput quantification of divergence angles and further analysis of individual flowers. We demonstrate the throughput of our method by quantifying geometric features of 1065 flower primordia from 172 plants, comparing auxin transport mutants to wild type. Additionally, we find that a paraboloid provides a simple geometric parameterisation of the shoot inflorescence domain with few parameters. We utilise parameterisation methods to provide a computational comparison of the shoot apex defined by a fluorescent reporter of the central zone marker gene CLAVATA3 with the apex defined by the paraboloid. Finally, we analyse the impact of mutations which alter mechanical properties on inflorescence dome curvature and compare the results with auxin transport mutants. Our results suggest that region-specific expression domains of genes regulating cell wall biosynthesis and local auxin transport can be important in maintaining the wildtype tissue shape. Altogether, our results indicate a general approach to parameterise and quantify plant development in 3D, which is applicable also in cases where data resolution is limited, and cell segmentation not possible. This enables researchers to address fundamental questions of plant development by quantitative phenotyping with high throughput, consistency and reproducibility.

Project description:BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or "orphan" crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1-13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Dataset Information

An Updated Protocol for High Throughput Plant Tissue Sectioning.

Publications

An Updated Protocol for High Throughput Plant Tissue Sectioning.

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