Non-destructive determination of floral staging in cereals using X-ray micro computed tomography (µCT)

Abstract

BackgroundAccurate floral staging is required to aid research into pollen and flower development, in particular male development. Pollen development is highly sensitive to stress and is critical for crop yields. Research into male development under environmental change is important to help target increased yields. This is hindered in monocots as the flower develops internally in the pseudostem. Floral staging studies therefore typically rely on destructive analysis, such as removal from the plant, fixation, staining and sectioning. This time-consuming analysis therefore prevents follow up studies and analysis past the point of the floral staging.ResultsThis study focuses on using X-ray µCT scanning to allow quick and detailed non-destructive internal 3D phenotypic information to allow accurate staging of Arabidopsis thaliana L. and Barley (Hordeum vulgare L.) flowers. X-ray µCT has previously relied on fixation methods for above ground tissue, therefore two contrast agents (Lugol’s iodine and Bismuth) were observed in Arabidopsis and Barley in planta to circumvent this step. 3D models and 2D slices were generated from the X-ray µCT images providing insightful information normally only available through destructive time-consuming processes such as sectioning and microscopy. Barley growth and development was also monitored over three weeks by X-ray µCT to observe flower development in situ. By measuring spike size in the developing tillers accurate non-destructive staging at the flower and anther stages could be performed; this staging was confirmed using traditional destructive microscopic analysis.ConclusionThe use of X-ray micro computed tomography (µCT) scanning of living plant tissue offers immense benefits for plant phenotyping, for successive developmental measurements and for accurate developmental timing for scientific measurements. Nevertheless, X-ray µCT remains underused in plant sciences, especially in above-ground organs, despite its unique potential in delivering detailed non-destructive internal 3D phenotypic information. This work represents a novel application of X-ray µCT that could enhance research undertaken in monocot species to enable effective non-destructive staging and developmental analysis for molecular genetic studies and to determine effects of stresses at particular growth stages.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0162-x) contains supplementary material, which is available to authorized users.

Anther Morphological Development and Stage Determination in Triticum aestivum

“…X-ray micro computed tomography has been employed to measure spike and spikelet lengths in a non-destructive manner (Tracy et al, 2017 ). Incorporation of this technology with developmental staging method could allow for a high-throughput plant phenotyping system to be utilized for rapid, non-destructive staging of anther development.…”

Non-destructive, high-content analysis of wheat grain traits using X-ray micro computed tomography

“…μCT has traditionally been used, to great effect, in medical imaging, and its applications in plant science have increased over the last few years [ 17 – 25 ]. Methodologies developed in the medical field have been applied to wider biological studies, for example techniques used to model regions of the human heart [ 42 , 43 ] have more recently been used to examine seed anatomy [ 18 ] and methods used to study metamorphosis in insects [ 44 ], modified to track root development in soil and non-destructive floral staging [ 19 , 45 ].…”

“…For a wheat spike this can take several hours using typical hardware. To address this, the scans were performed at the lower resolution of 0.2 megapixels (512 × 512) rather than much higher resolutions used in previous studies, for example 5 megapixels (2048 × 2048) and larger is often used [ 19 ]. This also reduced the output file size on average by a factor of 16.…”

“…During our initial attempts to analyse the data produced through μCT we discovered that there was a lack of software that could handle the volume of the data and implement modern computer vision algorithms easily and was well-suited to high-throughput automation. VGStudio Max, a commercially licensed software package, and BoneJ, a free and open source software package, are often used in biological and medical science for image analysis and visualisation [ 19 , 46 , 47 ]. However they require human interaction on a per image basis.…”

Evaluation of X‐ray tomography contrast agents: A review of production, protocols, and biological applications

“…Various methods were compared in the paper, where iodine-based staining for CT investigation was found successful to evaluate the structure of the plant. To investigate FIGURE 1 Major medical purpose iodine-based contrast agents (iodixanol, iohexol, iopamidol, iopromide, and ioxaglate) the pollen and floral development of Arabidopsis thaliana, Tracy, Gómez, Sturrock, Wilson, and Ferguson (2017) used X-ray CT imaging techniques as well. The group used Lugol's solution and compared the result with another stain.…”