High-Throughput Field Imaging and Basic Image Analysis in a Wheat Breeding Programme.

Visual assessment of colour-based traits plays a key role within field-crop breeding programmes, though the process is subjective and time-consuming. Digital image analysis has previously been investigated as an objective alternative to visual assessment for a limited number of traits, showing suitability and slight improvement to throughput over visual assessment. However, easily adoptable, field-based high-throughput methods are still lacking. The aim of the current study was to produce a high-throughput digital imaging and analysis pipeline for the assessment of colour-based traits within a wheat breeding programme. This was achieved through the steps of (i) a proof-of-concept study demonstrating basic image analysis methods in a greenhouse, (ii) application of these methods to field trials using hand-held imaging, and (iii) developing a field-based high-throughput imaging infrastructure for data collection. The proof of concept study showed a strong correlation (r = 0.95) between visual and digital assessments of wheat physiological yellowing (PY) in a greenhouse environment, with both scores having similar heritability (H2 = 0.85 and 0.76, respectively). Digital assessment of hand-held field images showed strong correlations to visual scores for PY (r = 0.61 and 0.78), senescence (r = 0.74 and 0.75) and Septoria tritici blotch (STB; r = 0.76), with greater heritability of digital scores, excluding STB. Development of the high-throughput imaging infrastructure allowed for images of field plots to be collected at a rate of 7,400 plots per hour. Images of an advanced breeding trial collected with this system were analysed for canopy cover at two time-points, with digital scores correlating strongly to visual scores (r = 0.88 and 0.86) and having similar or greater heritability. This study details how high-throughput digital phenotyping can be applied to colour-based traits within field trials of a wheat breeding programme. It discusses the logistics of implementing such systems with minimal disruption to the programme, provides a detailed methodology for the basic image analysis methods utilized, and has potential for application to other field-crop breeding or research programmes.

Backprojection Wiener deconvolution for computed tomographic reconstruction.

Analytical CT reconstruction is popular in practice because of its computational efficiency, but it suffers from low reconstruction quality when an insufficient number of projections are used. To address this issue, this paper presents a new analytical method of backprojection Wiener deconvolution (BPWD). BPWD executes backprojection first, and then applies a Wiener deconvolution to the whole backprojected image. The Wiener filter is derived from a ramp filter, enabling the proposed approach to perform reconstruction and denoising simultaneously. The use of a filter after backprojection does not differentiate between real sampled projections and interpolated ones, introducing reconstruction errors. Therefore a weighted ramp filter was applied to increase the contribution of real sampled projections in the reconstruction, thus improving reconstruction quality. Experiments on synthetic data and real phase-contrast x-ray images showed that the proposed approach yields better reconstruction quality compared to the classical filtered backprojection (FBP) method, with comparable reconstruction speed.

Land-based crop phenotyping by image analysis: Accurate estimation of canopy height distributions using stereo images.

In this paper we report on an automated procedure to capture and characterize the detailed structure of a crop canopy by means of stereo imaging. We focus attention specifically on the detailed characteristic of canopy height distribution-canopy shoot area as a function of height-which can provide an elaborate picture of canopy growth and health under a given set of conditions. We apply the method to a wheat field trial involving ten Australian wheat varieties that were subjected to two different fertilizer treatments. A novel camera self-calibration approach is proposed which allows the determination of quantitative plant canopy height data (as well as other valuable phenotypic information) by stereo matching. Utilizing the canopy height distribution to provide a measure of canopy height, the results compare favourably with manual measurements of canopy height (resulting in an R2 value of 0.92), and are indeed shown to be more consistent. By comparing canopy height distributions of different varieties and different treatments, the methodology shows that different varieties subjected to the same treatment, and the same variety subjected to different treatments can respond in much more distinctive and quantifiable ways within their respective canopies than can be captured by a simple trait measure such as overall canopy height.

Detecting spikes of wheat plants using neural networks with Laws texture energy.

BACKGROUND: The spike of a cereal plant is the grain-bearing organ whose physical characteristics are proxy measures of grain yield. The ability to detect and characterise spikes from 2D images of cereal plants, such as wheat, therefore provides vital information on tiller number and yield potential. RESULTS: We have developed a novel spike detection method for wheat plants involving, firstly, an improved colour index method for plant segmentation and, secondly, a neural network-based method using Laws texture energy for spike detection. The spike detection step was further improved by removing noise using an area and height threshold. The evaluation results showed an accuracy of over 80% in identification of spikes. In the proposed method we also measure the area of individual spikes as well as all spikes of individual plants under different experimental conditions. The correlation between the final average grain yield and spike area is also discussed in this paper. CONCLUSIONS: Our highly accurate yield trait phenotyping method for spike number counting and spike area estimation, is useful and reliable not only for grain yield estimation but also for detecting and quantifying subtle phenotypic variations arising from genetic or environmental differences.

Phenotyping of plants in competitive but controlled environments: a study of drought response in transgenic wheat.

In recent years, the interest in new technologies for wheat improvement has increased greatly. To screen genetically modified germplasm in conditions more realistic for a field situation we developed a phenotyping platform where transgenic wheat and barley are grown in competition. In this study, we used the platform to (1) test selected promoter and gene combinations for their capacity to increase drought tolerance, (2) test the function and power of our platform to screen the performance of transgenic plants growing in competition, and (3) develop and test an imaging and analysis process as a means of obtaining additional, non-destructive data on plant growth throughout the whole growth cycle instead of relying solely on destructive sampling at the end of the season. The results showed that several transgenic lines under well watered conditions had higher biomass and/or grain weight than the wild-type control but the advantage was significant in one case only. None of the transgenics seemed to show any grain weight advantage under drought stress and only two lines had a substantially but not significantly higher biomass weight than the wild type. However, their evaluation under drought stress was disadvantaged by their delayed flowering date, which increased the drought stress they experienced in comparison to the wild type. Continuous imaging during the season provided additional and non-destructive phenotyping information on the canopy development of mini-plots in our phenotyping platform. A correlation analysis of daily canopy coverage data with harvest metrics showed that the best predictive value from canopy coverage data for harvest metrics was achieved with observations from around heading/flowering to early ripening whereas early season observations had only a limited diagnostic value. The result that the biomass/leaf development in the early growth phase has little correlation with biomass or grain yield data questions imaging approaches concentrating only on the early development stage.

Quantifying the Onset and Progression of Plant Senescence by Color Image Analysis for High Throughput Applications.

Leaf senescence, an indicator of plant age and ill health, is an important phenotypic trait for the assessment of a plant's response to stress. Manual inspection of senescence, however, is time consuming, inaccurate and subjective. In this paper we propose an objective evaluation of plant senescence by color image analysis for use in a high throughput plant phenotyping pipeline. As high throughput phenotyping platforms are designed to capture whole-of-plant features, camera lenses and camera settings are inappropriate for the capture of fine detail. Specifically, plant colors in images may not represent true plant colors, leading to errors in senescence estimation. Our algorithm features a color distortion correction and image restoration step prior to a senescence analysis. We apply our algorithm to two time series of images of wheat and chickpea plants to quantify the onset and progression of senescence. We compare our results with senescence scores resulting from manual inspection. We demonstrate that our procedure is able to process images in an automated way for an accurate estimation of plant senescence even from color distorted and blurred images obtained under high throughput conditions.

RootGraph: a graphic optimization tool for automated image analysis of plant roots.

This paper outlines a numerical scheme for accurate, detailed, and high-throughput image analysis of plant roots. In contrast to existing root image analysis tools that focus on root system-average traits, a novel, fully automated and robust approach for the detailed characterization of root traits, based on a graph optimization process is presented. The scheme, firstly, distinguishes primary roots from lateral roots and, secondly, quantifies a broad spectrum of root traits for each identified primary and lateral root. Thirdly, it associates lateral roots and their properties with the specific primary root from which the laterals emerge. The performance of this approach was evaluated through comparisons with other automated and semi-automated software solutions as well as against results based on manual measurements. The comparisons and subsequent application of the algorithm to an array of experimental data demonstrate that this method outperforms existing methods in terms of accuracy, robustness, and the ability to process root images under high-throughput conditions.

Genetic diversity for root plasticity and nitrogen uptake in wheat seedlings.

Enhancing nitrogen use efficiency (NUE) of wheat is a major focus for wheat breeding programs. NUE may be improved by identifying genotypes that are competitive for nitrogen (N) uptake in early vegetative stages of growth and are able to invest that N in grain. Breeders tend to select high yielding genotypes under conditions of medium to high N supply, but it is not known whether this influences the selection of root plasticity traits or whether, over time, breeders have selected genotypes with higher N uptake efficiency. To address this, genotypes were selected from CIMMYT (1966-1985) and Australian (1999-2007) breeding programs. Genotypes from both programs responded to low N supply by expanding their root surface area through increased total root number and/or length of lateral roots. Australian genotypes were N responsive (accumulated more N under high N than under low N) whereas CIMMYT genotypes were not very N responsive. This could not be explained by differences in N uptake capacity as shown by 15N flux analysis of two representative genotypes with contrasting N accumulation. Expression analysis of nitrate transporter genes revealed that the high-affinity transport system accounted for the majority of root nitrate uptake in wheat seedlings under both low and high N conditions.

A complete system for 3D reconstruction of roots for phenotypic analysis.

Here we present a complete system for 3D reconstruction of roots grown in a transparent gel medium or washed and suspended in water. The system is capable of being fully automated as it is self calibrating. The system starts with detection of root tips in root images from an image sequence generated by a turntable motion. Root tips are detected using the statistics of Zernike moments on image patches centred on high curvature points on root boundary and Bayes classification rule. The detected root tips are tracked in the image sequence using a multi-target tracking algorithm. Conics are fitted to the root tip trajectories using a novel ellipse fitting algorithm which weighs the data points by its eccentricity. The conics projected from the circular trajectory have a complex conjugate intersection which are image of the circular points. Circular points constraint the image of the absolute conics which are directly related to the internal parameters of the camera. The pose of the camera is computed from the image of the rotation axis and the horizon. The silhouettes of the roots and camera parameters are used to reconstruction the 3D voxel model of the roots. We show the results of real 3D reconstruction of roots which are detailed and realistic for phenotypic analysis.