Non-destructive determination of floral staging in cereals using X-ray micro computed tomography (µCT)
© The Author(s) 2017
Received: 20 August 2016
Accepted: 23 February 2017
Published: 28 February 2017
Accurate 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.
This 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.
The 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.
KeywordsArabidopsis thaliana Barley Floral staging Flower development Hordeum vulgare L. Image analysis X-ray computed tomography
There is an increasing awareness of the importance of food security, with an estimated requirement of 50% more food by 2030 . Male fertility in plants is highly sensitive to environmental change such as heat stress, thus fluctuations in global temperature may result in dramatic changes in yield and productivity . To understand the effect of heat stress on pollen development and therefore plant productivity, the analysis of morphological, genetic and biochemical information is required to determine the normal pollen developmental processes. This information can then be used for future modelling and plant breeding to identify strategies to overcome environmental changes . 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 monocotyledons (grass-like), flower development occurs internally and therefore there have been limited options available to study the very early stages of inflorescence development, since materials cannot be identified easily without dissection. Dissection is time-consuming and leads to the ultimate destruction of the floral tissue. The ability to accurately stage floral development by a non-destructive technique would be beneficial for a wide range of research in plant reproductive development. In the past experimental plants were characterised by age rather than precise growth stage ; such staging is not precise and therefore not suitable for detailed molecular analyses.
In Barley (Hordeum vulgare L.), florets begin their development within the pseudostem shoot (a false stem composed of blades and sheaths that surround it), meaning that floral developmental stages cannot be observed or checked without destruction of the floral tissue. Recently Gomez and Wilson  produced a non-destructive staging guide based on external morphological features for barley (variety Optic) to allow correct stages to be recognised by external vegetative features. As part of this study they observed that specific anther and pollen stages were tightly linked to spike size . This supported previous observations that spike size was linked to pollen development, which were first reported in the 1980s [6, 7]. While the staging suggested by Gomez and Wilson  goes a long way towards selecting appropriately staged material for detailed reproductive studies it has limitations, such as a requirement for controlled growth conditions, variation between genotypes and a high level of developmental variation between tillers . This means that only tillers immediately following the main stem could be used for accurate staging. This staging is also variety specific, with further analysis needed to apply this approach to other varieties, or different growth conditions . Using non-destructive methods that enabled accurate staging of floral development across all tillers, varieties, mutants and in all conditions, would therefore offer a unique insight into plant developmental stages.
X-ray micro computed tomography (μCT) has been used extensively in medical sciences since the 1970s to allow quantification and visualisation of internal structures in 3D in a non-invasive and non-destructive way [9, 10]. The use of this technology has been adopted successfully in many other areas of Science and Engineering. For example in archaeology , engineering , food science  and biology . In the last twenty years it has been widely used in the soil and plant sciences to visualise soil properties , the influence of soil microbes on soil structure [16, 17] and plant root development and architecture in 4D imaging .
There have been comparatively few studies to visualise and quantify above-ground structure of plants; nevertheless there has been an increased interest in this over the last 6 years [19–26]. One of the main problems for this analysis is the low X-ray attenuation of plant tissue which can result in low image contrast and high levels of image noise. To overcome this problem many studies have used fixing and then infiltration/stained material to provide contrast to enable tissue and cellular differentiation . Through fixation, dehydration and iodine staining high levels of resolution (13.8 and 0.8 μm) were achievable with Arabidopsis thaliana (Arabidopsis) flower and shoots respectively . Staedler et al.  did an in-depth study on Arabidopsis and Marcgravia caudata flowers using a number of different compounds used previously in transmission electron microscopy (TEM) experimentation and a number of different mounting techniques providing high resolution (1.5 μm) for the tissue. This work highlights the importance of effective sample mounting techniques to maximise image quality at high resolution as even a small movement e.g. due to moisture loss from the tissue, can cause blurred images.
All these methods require lengthy preparation, removal from the plant and results in the destruction of the tissue with the dehydration and fixation stages. More recent analysis has moved towards living tissue, as both the contrast and speed of the µCT scanners has increased, allowing external and internal structures to be visualised without fixing and staining. For example, studies of rice panicle development over time . The main limitation of in vivo analysis is that there is a resolution versus sample size trade off, where fine detail scans require lengthy scanning and processing times, whilst larger samples adversely affect spatial resolution and the ability to differentiate tissue/cells.
To take advantage of the staining for individual tissue differentiation as observed by Staedler et al. , we adapted their study to look at living tissue and focused on small molecular stains which would have limited/no impact on plant viability. Another important factor to take into account is the ionizing effect of the X-rays themselves, previous studies by  showed that consecutive daily in vivo scanning (10–20 min) for a fortnight resulted in temporal growth inhibition in Arabidopsis seedlings. Therefore we aimed to keep our scans to a minimum duration to minimise the X-ray dose received by each sample.
The model plant Arabidopsis was used to optimise the X-ray µCT scanning technique and to enhance image quality different contrast agents were explored. This knowledge was then transferred to the crop Barley to optimise monocot imaging. To date there have been no previous studies of aerial parts of Barley using X-ray µCT scanning, this is therefore the first report of analysis of dissected Barley spikes and aerial parts from pot grown Barley plants. This novel method benefits from utilising the non-invasive technique so that we can obtain a 3D visualisation of floral development in the same flower over a growth period, and allows for non-destructive, quick and accurate staging of material.
The external whole inflorescence and single floral buds of Arabidopsis thaliana [wildtype (wt) ecotype Col-0] were used as it is a widely studied model plant; it comprises compact, complex organs and has been used previously in X-ray µCT studies . Arabidopsis was grown in Levington M3 (The Scotts Company Ltd, UK) compost supplemented with Met52 (Novozymes, France) and nematodes (Sygenta Bioline, UK) under controlled conditions of 22/2 h photoperiod at 21/17 °C light/dark cycle. Flower development was based on the flower stages by Smyth et al.  and further expanded by Sanders et al. . Whole plant and internal spikes of Barley (Horedum vulgare cv. Optic) were scanned to assess the penetration of the X-rays into thicker and denser tissue barriers. Spring barley, Optic, was sown 3 plants to a pot in John Innes number 3 (John Innes, UK) compost and then transferred to Levington C2 compost after 3 weeks; they were grown under controlled conditions of 16/8 h photoperiod 15/12 °C light/dark cycle (80% relative humidity). Barley was observed after the onset of elongation stages to heading to allow the reproductive development to be followed. Flower development was based on flower stages as described by . Spike size was measured from the base of the most basal floret to the tip of the most apical floret.
To enhance visualisation of plant tissue contrast agents were used. Iodine (Lugol’s solution) and bismuth acetate were selected based on existing studies as least toxic to a developing plant and small enough to be taken up by the root system. Plants were treated with the contrast agents by watering two days prior to scanning (Arabidopsis) or one week prior to scanning (Barley). Treatments included 1% Lugol’s solution, 5 mM bismuth solution, and water (non-treated control).
After infiltration treatments of 2 days, Arabidopsis whole inflorescence were manually removed from the plant and placed into 0.5 ml Eppendorf tubes to secure the sample and prevent movement throughout the scanning. The tubes were then mounted onto a glass cylinder and left for 20 min to equilibrate before scanning.
After infiltration treatments of 1 week, Barley spikes were dissected from the main tiller and scanned individually. These were either kept within the pseudostem (cut above and below the spike) to provide structure and prevent movement within the scan time, or the spike was manually removed from the pseudostem. These were then either mounted directly into the machine or placed within a 30 ml tube respectively; polystyrene was used to secure the spike in a vertical position within the tube and prevent movement, the tube was then mounted directly into the machine for the scan.
Material, treatment, scanner and scanning parameters for each scan performed
2 × 2
3 × 0
1% Lugols solution/5 mM bismuth/water
2 × 2
3 × 0
5 mM bismuth/water
1 × 1
Barley whole plant
1 × 1
Histological analysis of anther development
Florets from the middle zone of the spike were collected from different-sized spikes and fixed in paraformalehyde and then embedded in technovit resin in order to determine reproductive development stage. Fixation and staging occurred following the protocol of , sections were obtained using the Bright 5040 microtome (Bright Instruments, UK). Slides were stained with 0.05% (w/v) toludine blue prior to imaging.
In-depth analysis of Arabidopsis single buds
Arabidopsis and barley flower stage reference guide, showing flower stage in relation to anther/pollen development
Arabidopsis flower stage
Barley flower stage
Sporogenous cells and three anther cell layers
Spike 0–1 cm
Zadoks stage 31
Sporogenous cells and three anther cell layers
Four anther cell layers, pollen mother cell formed and undergo meiosis
Spike 1–2 cm
Zadoks stage 32
Four anther cell layers, pollen mother cells formed
Pollen mother cell meiosis completed, and single microspores released from the tetrad
Spike 2–3 cm
Zadoks stage 33
Pollen mother cell meiosis
Spike 3-4 cm
Zadoks stage 33.5
Released single microspores from tetrad
Single microspores and tapetum undergoes degeneration
Spikes 4–5 cm
Zadoks stage 34
Single microspores and middle layer undergoes crushing
Spikes 5–6 cm
Zadoks stage 35–37
Vacuolated microspores. Tapetum degenerating
Pollen mitosis I, and tapetum degeneration almost completed
Spikes 6–8 cm
Tapetum degeneration finished. Pollen mitosis I begins
Spikes ~9–10 cm
Pollen mitosis I completed
Binuclear pollen. Pollen mitosis II occurs, forming tri-nucleated pollen
Spikes ~9–10 cm
Binuclear pollen. Pollen mitosis II occurs.
Spikes ~9–10 cm
Tri-nucleated pollen and anthesis taking place
Flower stage 11 (Fig. 2a) were further analysed as it gave the highest resolution images, and a 3D model was created in VG StudioMAX© using image analysis techniques (segmentation using the Region Grower tool VG StudioMAX©), petals and sepals were easily virtually removed to give the internal flower structure. This shows the amount of detail at the organ level achievable by µCT scanning. Differences in organ sizes between different treatments or mutants could be measured in this way rather than by dissection.
Analysis of Arabidopsis inflorescence
Whole inflorescence of Arabidopsis was used to test the detection capabilities of the μCT scanner on living tissue using different contrast agents (Fig. 3a). Under the employed scanning conditions, samples and separate organs were detectable using all treatments. Greyscale intensity was used to observe the effect of different contrast agents; the histogram showed that both treatments had little effect on the overall contrast (Fig. 3b). These images were then further analysed to see if the contrast agents could generate differences between different organ structures in living tissue by using transverse sections of the Arabidopsis buds. Examining the average mean values across at least five anthers, carpels and sepals for each treatment showed that there were no statistical differences in the contrast between the different organs. Therefore there were no advantages of using a contrast agent compared to the water control for analysis of flower development in living tissue in our study.
Analysis of Barley spike
Barley spikes were manually removed and placed into a 30 ml tube to hold the sample static for imaging by the Nanotom (Fig. 1c, d). Figure 4c, d, f, g were kept sheathed with the Barley plant cut above and below the spike, while Fig. 3a, b, e were unsheathed before scanning leaving only the barley spike. Using the scanning conditions (Table 1) for Barley within the Nanotom, samples were easily detected at the organ level. No segregation at the tissue level could be achieved, with a potential resolution of 3.65 μm and actual resolution of 15 µm was achieved (Fig. 4). However, due to the denser tissue and thicker stem there was less movement during the thirty minute scan which resulted in sharper images. The µCT scanning technique was able to produce sliced images of Barley without the requirement for fixing and sectioning, but it was unable to provide detailed information to allow for specific anther or pollen stages and earlier flower stages (Additional file 4: Video S3). Similar to the Arabidopsis, no contrast improvements between the sheath, anther and carpel tissue were found (Additional file 1: Figure S1). However the Bismuth contrast agent gave a wider and shifted histogram pattern showing that it was taken up into the tissue, with an obvious peak for air (first peak) which was not detectable in the water control (Additional file 1: Figure S1).
Analysis of Barley plant for flowering stage floral inflorescence
Knowledge was gained regarding the visualisation of Barley and Arabidopsis aerial organs using X-ray µCT scanning technique. The model plant Arabidopsis thaliana was used to optimise scan settings, mounting techniques and contrast agents for internal organ identification and staging of flower development. Mounting techniques that held the samples secure during the scan to minimise movement presented the clearest images (Eppendorf tube and 50 ml tube for Arabidopsis and Barley respectively). These techniques seemed to have little impact on the resolution when compared to those that were mounted directly. We have shown that there can be problems caused by movement within the scan both by the mechanical movement whilst being scanned, but also due to dehydration and water loss during the scan. We have found that allowing the tissues to acclimatise once they have been removed from the plant for at least twenty minutes ameliorated this effect for the older bud tissue in Arabidopsis. Movement during the scan was less of an issue for monocotyledon plants such as Barley, since the tiller is sturdy and limits movement and dehydration of the spike.
Contrast agents considered were used by  however as their study destroyed the tissue by fixation and staining, it was important to select those compounds which had small molecular size so that they could be taken up by the root system and distributed within the plant. They also had to have a low level of known toxicity to prevent deleterious side effects. Lugol’s solution and Bismuth were chosen based on these criteria and Bismuth was shown in previous studies to give differential contrast to the filament, petals and to a lesser extent sepals and ovules . Both Iodine and Bismuth are used for µCT scanning for animal/human studies due to the contrast and low toxicity . In our study we have demonstrated that in Arabidopsis, both Bismuth and Lugol’s solution gave a similar profile to the water control and it is possible that these agents were not taken up successfully by the plant over the 2 days infiltration. Unsurprisingly for both of these treatments there was also no differentiation between different tissue layers. In Barley Bismuth was taken up successfully by the plants and provided an increase of X-ray attenuation of the tissue, although it did not lead to differentiation between tissues. Therefore under the conditions used the contrast agents did not enhance the differences between the living tissue flower organs which would allow for their automatic segmentation as observed by . The whole Barley plant growing in pots were therefore observed with no addition of contrast agents to determine floral staging by spike size.
To validate the imaging technique, florets were collected, sectioned and DAPI stained to enable detailed staging through cytological analysis. This allowed the determination of floral stage for the corresponding spike sizes, showing that a particular spike size is linked to flower development stages. Previous work by Gomez & Wilson  developed a modified Zadoks scale for barley reproductive development that was linked to vegetative features at different stages of flowering development, this showed that spike size is correlated to particular stages in development. The work presented here builds on this and allows accurate staging simply based on spike size measured by a quick 1 min scan and a simple measurement of the spike length by computational analysis. With 0–10 mm showing sporogenesis tissue with three cell layers, 10–20 mm showing four cell layers, 20–30 mm showing pollen mother cell meiosis, 30–40 mm showing microspore release and mitosis I and II in the 70 mm spikes (Fig. 6). The latter stages showing similar spike sizes and therefore external physiological features need to be used to differentiate between these stages, these are easily identifiable both visually and in the µCT scan as the spike has matured out of the pseudostem as described by Gomez and Wilson .
In this article we demonstrate the feasibility of using X-ray µCT for non-destructively visualising and quantifying the internal organ (spike) size of Barley to allow for accurate staging of floral tissue. The rapid 1 min scans require minimal preparation and computational analysis to enable accurate development staging. This has a major advantage over current staging methods that are destructive resulting in the death of the tiller for spike measurement and cytological analysis. Cytological analysis is also time consuming and requires a number of different steps, such as fixation, sectioning and staining before eventual microscope analysis of the stages. This means that accurate staging often occurs post-sample collection. Both of these current methods provide no opportunity for on-going developmental analysis, or subsequent sample collection if staging has been premature. The added benefit of this method is that the stage of all the spikes within the barley tillers can be established and therefore multiple stages can be observed in the same plant under particular treatments/mutants/cultivars. This is more consistent with what would happen in the field with environmental stresses affecting tillers differently depending on the current developmental stage of the flower.
This approach enables rapid and accurate staging of materials for collection for molecular analysis; this is critical given the specificity of gene expression over these developmental transitions and the requirement for accurate collection of materials, this is particularly the case when materials are limited, for example during transgenic line analysis. This technique would also be useful when looking at the effects of abiotic and biotic stress during plant flower development, as individual tillers could be monitored separately to see what effect the stress has over whole plant yield and yield during a particular flower stage. Temperature variability and extreme fluctuations (heat stress) have been shown to affect plant yield . Studies in high temperature (as well as other stresses) are essential to help identify germplasm that is more tolerant of abiotic stress and also the adaption of agricultural practices to mitigate the impact of such stresses on global food security, such as shifting sowing time, and changing cultivars to reflect the changing environmental conditions.
Furthermore, this approach could be developed into phenotyping platforms which combined with data such as plant height, chlorophyll content, root structure etc. would allow high-through put large-scale data to be correlated with fertility traits to give an overview of crop development and yield. Automated plant phenotyping is rapidly becoming mainstream, with buildings being designed specifically for automated phenotyping to measure a number of different aspects of plant growth, using imaging and different tools to record these . Phenotyping approaches that enable the collection of this information alongside other data on plant physiology and development offer a more holistic analysis of growth, thus enabling links between general plant growth, reproductive development and final yield to be made. This technique for staging flowers is also directly applicable to other monocot species where the flower develops internally, such as wheat and rice, to aid scientific research.
We have shown that while there is regulation of floral development between spike size and flower development, the progression of individual tillers is variable. This is likely to be based on the environment and other features such as tiller position, for example primary versus secondary, total number of tillers and where they are in development can effect flower development [33, 37]. This differential flower development and spike growth has been previously observed and is the reason why scientific observations are frequently limited to primary and secondary tillers for accurate development progression when observing traits. The use of the µCT scanning technique means that the particular stage of all tillers can be observed without relying on them developing at a certain rate under controlled conditions, giving an in-depth analysis of floral stage and final yield.
X-ray μCT can be used for visualisation of both Arabidopsis and Barley without any prior fixation or staining allowing living plants to be analysed progressively as they develop. As X-ray μCT scanners improve it will allow for rapid screening of plant tissue development in 3D over time. In X-ray μCT studies there will always be a trade-off between magnification and measured volume, however an advantage of using this technique for plant aerial parts is that because the parts are individually smaller they can be placed close to the X-ray tube to enable high resolution, but still be grown under field conditions or in field representative pots. This has been an issue for using μCT analysis for root and soil studies, as the whole diameter of the pot is usually included in the scan to extract the full root system architecture, so limits the achievable resolution.
X-ray μCT can be used for living plants to be analysed progressively as they develop, for 2D (sliced sections), 3D (structure) and 4D (over time) analysis. This study has shown that prior fixation or staining is not required for a model dicot and monocot plant species (Arabidopsis and Barley). X-ray μCT can be used to accurately stage flower development non-destructively which is especially important in the monocotyledon species where the flowers begin their development internally. Accurate staging based simply on spike size was measured by a quick 1 min scan. The rapid 1 min scans require minimal preparation and computational analysis to enable accurate development staging. This approach could be incorporated into high-throughput plant phenotyping platforms. Combination of this technique and method has the opportunity to support research into flower development and further understanding of plant developmental responses to biotic and abiotic stresses.
last flag elongation
transmission electron microscopy
micro computed tomography
ACF, SRT and ZAW received the funding; ACF designed the experiment; ACF, SRT and CJS performed the µCT scanning; ACF, SRT analysed the data; JFG performed the histological analysis; ACF, SRT wrote the manuscript. All authors edited the draft manuscript. All authors read and approved final manuscript.
All µCT scanning was conducted at The Hounsfield Facility, School of Biosciences, University of Nottingham which received funding from the European Research Council (Futureroots Project), Biotechnology and Biosciences Research Council, Engineering and Science Research Council and The Wolfson Foundation.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published article (and its additional information files).
This research was supported by the University of Nottingham Interdisciplinary Centre for Analytical Science (UNICAS) funding, and the Biotechnology and Biosciences Research Council (BB/J001295/1).
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