Plant phenotyping is an emerging discipline in plant biology. Quantitative measurements of functional and structural traits help to better understand gene–environment interactions and support breeding for improved resource use efficiency of important crops such as bean (Phaseolus vulgaris L.). Here we provide an overview of state-of-the-art phenotyping approaches addressing three aspects of resource use efficiency in plants: belowground roots, aboveground shoots and transport/allocation processes. We demonstrate the capacity of high-precision methods to measure plant function or structural traits non-invasively, stating examples wherever possible. Ideally, high-precision methods are complemented by fast and high-throughput technologies. High-throughput phenotyping can be applied in the laboratory using automated data acquisition, as well as in the field, where imaging spectroscopy opens a new path to understand plant function non-invasively. For example, we demonstrate how magnetic resonance imaging (MRI) can resolve root structure and separate root systems under resource competition, how automated fluorescence imaging (PAM fluorometry) in combination with automated shape detection allows for high-throughput screening of photosynthetic traits and how imaging spectrometers can be used to quantify pigment concentration, sun-induced fluorescence and potentially photosynthetic quantum yield. We propose that these phenotyping techniques, combined with mechanistic knowledge on plant structure–function relationships, will open new research directions in whole-plant ecophysiology and may assist breeding for varieties with enhanced resource use efficiency varieties.

Three-dimensional canopies form complex architectures with temporally and spatially changing leaf orientations. Variations in canopy structure are linked to canopy function and they occur within the scope of genetic variability as well as a reaction to environmental factors like light, water and nutrient supply, and stress. An important key measure to characterize these structural properties is the leaf angle distribution, which in turn requires knowledge on the 3-dimensional single leaf surface. Despite a large number of 3-d sensors and methods only a few systems are applicable for fast and routine measurements in plants and natural canopies. A suitable approach is stereo imaging, which combines depth and color information that allows for easy segmentation of green leaf material and the extraction of plant traits, such as leaf angle distribution. We developed a software package, which provides tools for the quantification of leaf surface properties within natural canopies via 3-d reconstruction from stereo images. Our approach includes a semi-automatic selection process of single leaves and different modes of surface characterization via polygon smoothing or surface model fitting. Based on the resulting surface meshes leaf angle statistics are computed on the whole-leaf level or from local derivations. We include a case study to demonstrate the functionality of our software. 48 images of small sugar beet populations (4 varieties) have been analyzed on the base of their leaf angle distribution in order to investigate seasonal, genotypic and fertilization effects on leaf angle distributions. We could show that leaf angle distributions change during the course of the season with all varieties having a comparable development. Additionally, different varieties had different leaf angle orientation that could be separated in principle component analysis. In contrast nitrogen treatment had no effect on leaf angles. We show that a stereo imaging setup together with the appropriate image processing tools is capable of retrieving the geometric leaf surface properties of plants and canopies. Our software package provides whole-leaf statistics but also a local estimation of leaf angles, which may have great potential to better understand and quantify structural canopy traits for guided breeding and optimized crop management.

The effect that the canopy structure and the viewing geometry have on the intensity and the spatial distribution of passively measured sun-induced chlorophyll fluorescence at canopy scale is still not well understood. These uncertainties constrain the potential use of fluorescence to quantify photosynthesis at this level. Using a novel technique, we evaluated the diurnal changes in the spatial distribution of sun-induced fluorescence at 760 nm (F₇₆₀) within the canopy as a consequence of the spatial disposition of the leaves and the viewing angle of the sensor. High resolution spectral and stereo images of a full sugar beet canopy were recorded simultaneously in the field to estimate maps of F₇₆₀ and the surface angle distribution, respectively. A dedicated algorithm was used to align both maps in the post-processing and its accuracy was evaluated using a sensitivity test. The relative angle between sun and the leaf surfaces primarily determined the amount of incident Photosynthetic Active Radiation (PAR), which in turn was reflected in different values of F₇₆₀, with the highest values occurring in leaf surfaces that are perpendicularly oriented to the sun. The viewing angle of the sensor also had an impact in the intensity of the recorded F₇₆₀. Higher viewing angles generally resulted in higher values of F₇₆₀. We attribute these changes to a direct effect of the vegetation directional reflectance response on fluorescence retrieval. Consequently, at leaf surface level, the spatio-temporal variations of F₇₆₀ were mainly explained by the sun–leaf–sensor geometry rather than directionality of the fluorescence emission. At canopy scale, the diurnal patterns of F₇₆₀ observed on the top-of-canopy were attributed to the complex interplay between the light penetration into the canopy as a function of the display of the various leaves and the fluorescence emission of each leaf which is modulated by the exposure of the individual leaf patch to the incoming light and the functional status of photosynthesis. We expect that forward modeling can help derive analytical simplified skeleton assumptions to scale canopy measurements to the leaf functional properties.

The development of leaf area is one of the fundamental variables to quantify plant growth and physiological function and is therefore widely used to characterize genotypes and their interaction with the environment. To date, analysis of leaf area often requires elaborate and destructive measurements or imaging-based methods accompanied by automation that may result in costly solutions. Consequently in recent years there is an increasing trend towards simple and affordable sensor solutions and methodologies. A major focus is currently on harnessing the potential of applications developed for smartphones that provide access to analysis tools to a wide user basis. However, most existing applications entail significant manual effort during data acquisition and analysis. With the development of Plant Screen Mobile we provide a suitable smartphone solution for estimating digital proxies of leaf area and biomass in various imaging scenarios in the lab, greenhouse and in the field. To distinguish between plant tissue and background the core of the application comprises different classification approaches that can be parametrized by users delivering results on-the-fly. We demonstrate the practical applications of computing projected leaf area based on two case studies with Eragrostis and Musa plants. These studies showed highly significant correlations with destructive measurements of leaf area and biomass from both ground truth measurements and estimations from well-established screening systems. We show that a smartphone together with our analysis tool Plant Screen Mobile is a suitable platform for rapid quantification of leaf and shoot development of various plant architectures. Beyond the estimation of projected leaf area the app can also be used to quantify color and shape parameters of other plant material including seeds and flowers.

Breeding for Cercospora resistant sugar beet cultivars requires field experiments for testing resistance levels of candidate genotypes in conditions that are close to agricultural cultivation. Non-invasive spectral phenotyping methods can support and accelerate resistance rating and thereby speed up breeding process. In a case study, experimental field plots with strongly infected beet genotypes of different resistance levels were measured with two different spectrometers. Vegetation indices were calculated from measured wavelength signature to determine leaf physiological status, e.g., greenness with the Normalized Differenced Vegetation Index (NDVI), leaf water content with the Leaf Water Index (LWI) and Cercospora disease severity with the Cercospora Leaf Spot Index (CLSI). Indices values correlated significantly with visually scored disease severity, thus connecting the classical breeders’ scoring approach with advanced non-invasive technology.