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.
Highlights
Accurate floral staging is required to aid research into pollen and flower development, in particular male development
Using the scanning conditions (Table 1) for Arabidopsis within the Nanotom, samples were detected at the organ level, and to some respect the tissue level for pollen and ovules giving a potential level of resolution of 2.25 μm, and actual spatial resolution of 10 μm (Fig. 2; Additional file 2: Video S1)
Despite movement within the oldest tissue causing blurred images, the different organs were identifiable and released pollen could be detected (Fig. 2e; Additional file 3: Video S2). This shows that while the micro computed tomography (μCT) scanning technique is able to produce sliced images of Arabidopsis without the requirement for fixing and sectioning, it is unable to provide detailed information to allow for specific Anther or Pollen stages [31], flower stages were identifiable [30]
Summary
Accurate floral staging is required to aid research into pollen and flower development, in particular male development. 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 typically rely on destructive analysis, such as removal from the plant, fixation, staining and sectioning. To conduct molecular research of flower development and the stage-specific effects of heat stress accurate staging of flower development is required. This is critical so that traits can be analysed correctly for stress treatments, or for precise collection of materials for molecular, genetic and comparative studies. In the past experimental plants were characterised by age rather than precise growth stage [4]; such staging is not precise and not suitable for detailed molecular analyses
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