Pieter Badenhorst

The extraction of automated plant phenomics from digital images has advanced in recent years. However, the accuracy of extracted phenomics, especially for individual plants in a field environment, requires improvement. In this paper, a new and efficient method of extracting individual plant areas and their mean normalized difference vegetation index from high-resolution digital images is proposed. The algorithm was applied on perennial ryegrass row field data multispectral images taken from the top view. First, the center points of individual plants from digital images were located to exclude plant positions without plants. Second, the accurate area of each plant was extracted using its center point and radius. Third, the accurate mean normalized difference vegetation index of each plant was extracted and adjusted for overlapping plants. The correlation between the extracted individual plant phenomics and fresh weight ranged between 0.63 and 0.75 across four time points. The methods proposed are applicable to other crops where individual plant phenotypes are of interest.

Perennial ryegrass biomass yield is an important driver of profitability for Australian dairy farmers, making it a primary goal for plant breeders. However, measuring and selecting cultivars for higher biomass yield is a major bottleneck in breeding, requiring conventional methods that may be imprecise, laborious, and/or destructive. For forage breeding programs to adopt phenomic technologies for biomass estimation, there exists the need to develop, integrate, and validate sensor-based data collection that is aligned with the growth characteristics of plants, plot design and size, and repeated measurements across the growing season to reduce the time and cost associated with the labor involved in data collection. A fully automated phenotyping platform (DairyBioBot) utilizing an unmanned ground vehicle (UGV) equipped with a ground-based Light Detection and Ranging (LiDAR) sensor and Real-Time Kinematic (RTK) positioning system was developed for the accurate and efficient measurement of plant volume as a proxy for biomass in large-scale perennial ryegrass field trials. The field data were collected from a perennial ryegrass row trial of 18 experimental varieties in 160 plots (three rows per plot). DairyBioBot utilized mission planning software to autonomously capture high-resolution LiDAR data and Global Positioning System (GPS) recordings. A custom developed data processing pipeline was used to generate a plant volume estimate from LiDAR data connected to GPS coordinates. A high correlation between LiDAR plant volume and biomass on a Fresh Mass (FM) basis was observed with the coefficient of determination of R² = 0.71 at the row level and R² = 0.73 at the plot level. This indicated that LiDAR plant volume is strongly correlated with biomass and therefore the DairyBioBot demonstrates the utility of an autonomous platform to estimate in-field biomass for perennial ryegrass. It is likely that no single platform will be optimal to measure plant biomass from landscape to plant scales; the development and application of autonomous ground-based platforms is of greatest benefit to forage breeding programs.

Perennial ryegrass (Lolium perenne L.) is one of the most important forage grass species in temperate regions of Australia and New Zealand. However, it can have poor persistence due to a low tolerance to both abiotic and biotic stresses. A major challenge in measuring persistence in pasture breeding is that the assessment of pasture survival depends on ranking populations based on manual ground cover estimation. Ground cover measurements may include senescent and living tissues and can be measured as percentages or fractional units. The amount of senescent pasture present in a sward may indicate changes in plant growth, development, and resistance to abiotic and biotic stresses. The existing tools to estimate perennial ryegrass ground cover are not sensitive enough to discriminate senescent ryegrass from soil. This study aimed to develop a more precise sensor-based phenomic method to discriminate senescent pasture from soil. Ground-based RGB images, airborne multispectral images, ground-based hyperspectral data, and ground truth samples were taken from 54 perennial ryegrass plots three years after sowing. Software packages and machine learning scripts were used to develop a pipeline for high-throughput data extraction from sensor-based platforms. Estimates from the high-throughput pipeline were positively correlated with the ground truth data (p < 0.05). Based on the findings of this study, we conclude that the RGB-based high-throughput approach offers a precision tool to assess perennial ryegrass persistence in pasture breeding programs. Improvements in the spatial resolution of hyperspectral and multispectral techniques would then be used for persistence estimation in mixed swards and other monocultures.

Increasing dry matter yield (DMY) is the most important objective in perennial ryegrass breeding programs. Current yield assessment methods like cutting are time-consuming and destructive, non-destructive measures such as scoring yield on single plants by visual inspection may be subjective. These assessments involve multiple measurements and selection procedures across seasons and years to evaluate biomass yield repeatedly. This contributes to the slow process of new cultivar development and commercialisation. This study developed and validated a computational phenotyping workflow for image acquisition, processing and analysis of spaced planted ryegrass and investigated sensor-based DMY yield estimation of individual plants through normalized difference vegetative index (NDVI) and ultrasonic plant height data extraction. The DMY of 48,000 individual plants representing 50 advanced breeding lines and commercial cultivars was accurately estimated at multiple harvests across the growing season. NDVI, plant height and predicted DMY obtained from aerial and ground-based sensors illustrated the variation within and between cultivars across different seasons. Combining NDVI and plant height of individual plants was a robust method to enable high-throughput phenotyping of biomass yield in ryegrass breeding. Similarly, the plot-level model indicated good to high-coefficients of determination (R²) between the predicted and measured DMY across three seasons with R² between 0.19 and 0.81 and root mean square errors (RMSE) values ranging from 0.09 to 0.21 kg/plot. The model was further validated using a combined regression of the three seasons harvests. This study further sets a foundation for the application of sensor technologies combined with genomic studies that lead to greater rates of genetic gain in perennial ryegrass biomass yield.

Nutritive value (NV) of forage is too time consuming and expensive to measure routinely in targeted breeding programs. Non-destructive spectroscopy has the potential to quickly and cheaply measure NV but requires an intermediate modelling step to interpret the spectral data. A novel machine learning technique for forage analysis, Cubist, was used to analyse canopy spectra to predict seven NV parameters, including dry matter (DM), acid detergent fibre (ADF), ash, neutral detergent fibre (NDF), in vivo dry matter digestibility (IVDMD), water soluble carbohydrates (WSC), and crude protein (CP). Perennial ryegrass (Lolium perenne) was used as the test crop. Independent validation of the developed models revealed prediction capabilities with R2 values and Lin’s concordance values reported between 0.49 and 0.82, and 0.68 and 0.89, respectively. Informative wavelengths for the creation of predictive models were identified for the seven NV parameters. These wavelengths included regions of the electromagnetic spectrum that are usually excluded due to high background variation, however, they contain important information and utilising them to obtain meaningful signals within the background variation is an advantage for accurate models. Non-destructive field spectroscopy along with the predictive models was deployed infield to measure NV of individual ryegrass plants. A significant reduction in labour was observed. The associated increase in speed and reduction of cost makes targeting NV in commercial breeding programs now feasible.

Increasing herbage biomass is the predominant objective for pasture plant breeding programs. Three types of field trials are commonly involved during forage plant breeding, i.e., individually spaced plants, row plot, and sward trials. Assessments of biomass production at individual plant, row plot, and sward plot levels are through visual scoring and/or cutting of biomass manually or mechanically. Both visual scoring and cutting of plants are laborious, time consuming, and costly. The development of sensor technology such as multispectral sensors and unmanned aircraft systems (UAS) provide the opportunity to accelerate the process of biomass evaluation and to increase throughput, improve resolution, and reduce time and cost. We tested either the handheld Trimble GreenSeeker^® or Parrot Sequoia multispectral sensors attached to a 3DR Solo Quadcopter to assess biomass in perennial ryegrass field trials sown as spaced individual plants, row plots, and simulated sward plots. Significant correlations were observed between visual score and normalized difference vegetation index (NDVI) in a spaced plant field trial and between biomass yield and NDVI in row plot and sward trials (r = 0.12 ~ 0.93). NDVI obtained from multispectral sensors and UAS can replace visual scoring in spaced plant trials. It was also a valuable proxy for yield estimation in row plot and sward trials. These technologies will assist in transition for the forage grass breeding from pen and notepad to digital and data era.

Sensor-based phenotyping technologies may offer a non-destructive, high-throughput and efficient assessment of herbage yield (HY) to replace current inefficient phenotyping methods. This paper assesses the feasibility of combining normalised difference vegetative index (NDVI) from multispectral imaging and ultrasonic sonar estimates of plant height to estimate HY of single plants in a large perennial ryegrass breeding program. For sensor calibration, fresh HY (FHY) and dry HY (DHY) were acquired destructively, and plant height was measured at four dates each in 2017 and 2018 from a selected subset of 480 plants. Global multiple linear regression models based on K-fold and random split cross-validation methods were used to evaluate the relationship between observed vs. predicted HY. The coefficient of determination (R²) = 0.67–0.68 and a root mean square error (RMSE) between 5.43–7.60 g was obtained for the validation of predicted vs. observed DHY. The mean absolute error (MAE) and mean percentage error (MPE) ranged between 3.59–5.44 g and 22–28%, respectively. For the FHY, R² values ranged from 0.63 to 0.70, with an RMSE between 23.50 and 33 g, MAE between 15.11 and 24.34 g and MPE between ~22% and 31%. Combining NDVI and plant height is a robust method to enable high-throughput phenotyping of herbage yield in perennial ryegrass breeding programs.

Perennial ryegrass (Lolium perenne L.) is one of the most important forage grass species in temperate regions of the world, but it is prone to having poor persistence due to the incidence of abiotic and biotic stresses. This creates a challenge for livestock producers to use their agricultural lands more productively and intensively within sustainable limits. Breeding perennial ryegrass cultivars that are both productive and persistent is a target of forage breeding programs and will allow farmers to select appropriate cultivars to deliver the highest profitability over the lifetime of a sward. Conventional methods for the estimation of pasture persistence depend on manual ground cover estimation or counting the number of surviving plants or tillers in a given area. Those methods are subjective, time-consuming and/or labour intensive. This study aimed to develop a phenomic method to evaluate the persistence of perennial ryegrass cultivars in field plots. Data acquisition was conducted three years after sowing to estimate the persistence of perennial ryegrass using high-resolution aerial-based multispectral and ground-based red, green and blue(RGB) sensors, and subsequent image analysis. There was a strong positive relationship between manual ground cover and sensor-based ground cover estimates (p < 0.001). Although the manual plant count was positively correlated with sensor-based ground cover (p < 0.001) intra-plot plant size variation influenced the strength of this relationship. We conclude that object-based ground cover estimation is most suitable for use in large-scale breeding programs due to its higher accuracy, efficiency and repeatability. With further development, this technique could be used to assess temporal changes of perennial ryegrass persistence in experimental studies and on a farm scale.

The nutritive value (NV) of perennial ryegrass is an important driver of productivity for grazing stock; therefore, improving NV parameters would be beneficial to meat and dairy producers. NV is not actively targeted by most breeding programs due to NV measurement being prohibitively slow and expensive. Nondestructive spectroscopy has the potential to reduce the time and cost required to screen for NV parameters to make targeted breeding of NV practical. The application of a field spectrometer was trialed to gather canopy spectra of individual ryegrass plants to develop predictive models for eight NV parameters for breeding programs. The targeted NV parameters included acid detergent fibre, ash, crude protein, dry matter, in vivo dry matter digestibility, in vivo organic matter digestibility, neutral detergent fibre, and water-soluble carbohydrates. The models were developed with partial least square regression. Model predicted ranking of plants had R² between (0.87 and 0.39) and lab rankings of highest preforming plants. The highest ranked plants, which are generally the selection target for breeding programs, were accurately identified with the canopy-based model at a speed, cost and accuracy that is promising for NV breeding programs.

Increasing the yield of perennial forage crops remains a crucial factor underpinning the profitability of grazing industries, and therefore is a priority for breeding programs. Breeding for high dry matter yield (DMY) in forage crops is likely to be enhanced with the development of genomic selection (GS) strategies. However, realising the full potential of GS will require an increase in the amount of phenotypic data and the rate at which it is collected. Therefore, phenotyping remains a critical bottleneck in the implementation of GS in forage species. Assessments of DMY in forage crop breeding include visual scores, sample clipping and mowing of plots, which are often costly and time-consuming. New ground- and aerial-based platforms equipped with advanced sensors offer opportunities for fast, nondestructive and low-cost, high-throughput phenotyping (HTP) of plant growth, development and yield in a field environment. The workflow of image acquisition, processing and analysis are reviewed. The “big data” challenges, proposed storage and management techniques, development of advanced statistical tools and methods for incorporating the HTP into forage breeding systems are also reviewed. Initial results where these techniques have been applied to forages have been promising but further research and development is required to adapt them to forage breeding situations, particularly with respect to the management of large data sets and the integration of information from spaced plants to sward plots. However, realizing the potential of sensor technologies combined with GS leads to greater rates of genetic gain in forages.