Machine learning-enabled computer vision for plant phenotyping: a primer on AI/ML and case study on stomatal patterning.

Artificial intelligence and machine learning (AI/ML) can be used to automatically analyze large image datasets. One valuable application of this approach is estimation of plant trait data contained within images. Here we review 39 papers that describe the development and/or application of such models for estimation of stomatal traits from epidermal micrographs. In doing so, we hope to provide plant biologists with a foundational understanding of AI/ML and summarize the current capabilities and limitations of published tools. While most models show human-level performance for stomatal density (SD) quantification at superhuman speed, they are often likely to be limited in how broadly they can be applied across phenotypic diversity associated with genetic, environmental or developmental variation. Other models can make predictions across greater phenotypic diversity and/or additional stomatal/epidermal traits, but require significantly greater time investment to generate ground-truth data. We discuss the challenges and opportunities presented by AI/ML-enabled computer vision analysis, and make recommendations for future work to advance accelerated stomatal phenotyping.

An unmanned ground vehicle phenotyping-based method to generate three-dimensional multispectral point clouds for deciphering spatial heterogeneity in plant traits.

Fusing three-dimensional (3D) and multispectral (MS) imaging data holds promise for high-throughput and comprehensive plant phenotyping to decipher genome-to-phenome knowledge. Acquiring high-quality 3D MS point clouds (3DMPCs) of plants remains challenging because of poor 3D data quality and limited radiometric calibration methods for plants with a complex canopy structure. Here, we present a novel 3D spatial-spectral data fusion approach to collect high-quality 3DMPCs of plants by integrating the next-best-view planning for adaptive data acquisition and neural reference field (NeREF) for radiometric calibration. This approach was used to acquire 3DMPCs of perilla, tomato, and rapeseed plants with diverse plant architecture and leaf morphological features evaluated by the accuracy of chlorophyll content and equivalent water thickness (EWT) estimation. The results showed that the completeness of plant point clouds collected by this approach was improved by an average of 23.6% compared with the fixed viewpoints alone. The NeREF-based radiometric calibration with the hemispherical reference outperformed the conventional calibration method by reducing the root mean square error (RMSE) of 58.93% for extracted reflectance spectra. The RMSE for chlorophyll content and EWT predictions decreased by 21.25% and 14.13% using partial least squares regression with the generated 3DMPCs. Collectively, our study provides an effective and efficient way to collect high-quality 3DMPCs of plants under natural light conditions, which improves the accuracy and comprehensiveness of phenotyping plant morphological and physiological traits, and thus will facilitate plant biology and genetic studies as well as crop breeding.

Overview of transcriptome changes and phenomic profile of sanitized artichoke vis-à-vis non-sanitized plants.

Plant tissue in vitro culture is increasingly used in agriculture to improve crop production, nutritional quality, and commercial value. In plant virology, the technique is used as sanitation protocol to produce virus-free plants. Sanitized (S) artichokes show increased vigour compared to their non-sanitized (NS) counterparts, because viral infections lead to a decline of growth and development. To investigate mechanisms that control the complex traits related to morphology, growth, and yield in S artichokes compared to NS plants, RNAseq analysis and phenotyping by imaging were used. The role of peroxidases (POD) was also investigated to understand their involvement in sanitized plant development. Results showed that virus infection affected regulation of cell cycle, gene expression and signal transduction modulating cellular response to stimulus/stress. Moreover, primary metabolism and photosynthesis were also influenced, contributing to explain the main morphological differences observed between S and NS artichokes. Sanitized artichokes are also characterized by higher POD activity, probably associated with increased plant growth, rather than strengthening of cell walls. Overall, results show that the differences in development of S artichokes may be derived from the in vitro culture stressor, as well as through pathogen elimination, which, in turn, improve qualitative and quantitative artichoke production.

A 3D printed plant model for accurate and reliable 3D plant phenotyping.

BACKGROUND: This study addresses the importance of precise referencing in 3-dimensional (3D) plant phenotyping, which is crucial for advancing plant breeding and improving crop production. Traditionally, reference data in plant phenotyping rely on invasive methods. Recent advancements in 3D sensing technologies offer the possibility to collect parameters that cannot be referenced by manual measurements. This work focuses on evaluating a 3D printed sugar beet plant model as a referencing tool. RESULTS: Fused deposition modeling has turned out to be a suitable 3D printing technique for creating reference objects in 3D plant phenotyping. Production deviations of the created reference model were in a low and acceptable range. We were able to achieve deviations ranging from -10 mm to +5 mm. In parallel, we demonstrated a high-dimensional stability of the reference model, reaching only ±4 mm deformation over the course of 1 year. Detailed print files, assembly descriptions, and benchmark parameters are provided, facilitating replication and benefiting the research community. CONCLUSION: Consumer-grade 3D printing was utilized to create a stable and reproducible 3D reference model of a sugar beet plant, addressing challenges in referencing morphological parameters in 3D plant phenotyping. The reference model is applicable in 3 demonstrated use cases: evaluating and comparing 3D sensor systems, investigating the potential accuracy of parameter extraction algorithms, and continuously monitoring these algorithms in practical experiments in greenhouse and field experiments. Using this approach, it is possible to monitor the extraction of a nonverifiable parameter and create reference data. The process serves as a model for developing reference models for other agricultural crops.

Bagging Improves the Performance of Deep Learning-Based Semantic Segmentation with Limited Labeled Images: A Case Study of Crop Segmentation for High-Throughput Plant Phenotyping.

Advancements in imaging, computer vision, and automation have revolutionized various fields, including field-based high-throughput plant phenotyping (FHTPP). This integration allows for the rapid and accurate measurement of plant traits. Deep Convolutional Neural Networks (DCNNs) have emerged as a powerful tool in FHTPP, particularly in crop segmentation-identifying crops from the background-crucial for trait analysis. However, the effectiveness of DCNNs often hinges on the availability of large, labeled datasets, which poses a challenge due to the high cost of labeling. In this study, a deep learning with bagging approach is introduced to enhance crop segmentation using high-resolution RGB images, tested on the NU-Spidercam dataset from maize plots. The proposed method outperforms traditional machine learning and deep learning models in prediction accuracy and speed. Remarkably, it achieves up to 40% higher Intersection-over-Union (IoU) than the threshold method and 11% over conventional machine learning, with significantly faster prediction times and manageable training duration. Crucially, it demonstrates that even small labeled datasets can yield high accuracy in semantic segmentation. This approach not only proves effective for FHTPP but also suggests potential for broader application in remote sensing, offering a scalable solution to semantic segmentation challenges. This paper is accompanied by publicly available source code.

Stress phenotyping analysis leveraging autofluorescence image sequences with machine learning.

Background: Autofluorescence-based imaging has the potential to non-destructively characterize the biochemical and physiological properties of plants regulated by genotypes using optical properties of the tissue. A comparative study of stress tolerant and stress susceptible genotypes of Brassica rapa with respect to newly introduced stress-based phenotypes using machine learning techniques will contribute to the significant advancement of autofluorescence-based plant phenotyping research. Methods: Autofluorescence spectral images have been used to design a stress detection classifier with two classes, stressed and non-stressed, using machine learning algorithms. The benchmark dataset consisted of time-series image sequences from three Brassica rapa genotypes (CC, R500, and VT), extreme in their morphological and physiological traits captured at the high-throughput plant phenotyping facility at the University of Nebraska-Lincoln, USA. We developed a set of machine learning-based classification models to detect the percentage of stressed tissue derived from plant images and identified the best classifier. From the analysis of the autofluorescence images, two novel stress-based image phenotypes were computed to determine the temporal variation in stressed tissue under progressive drought across different genotypes, i.e., the average percentage stress and the moving average percentage stress. Results: The study demonstrated that both the computed phenotypes consistently discriminated against stressed versus non-stressed tissue, with oilseed type (R500) being less prone to drought stress relative to the other two Brassica rapa genotypes (CC and VT). Conclusion: Autofluorescence signals from the 365/400 nm excitation/emission combination were able to segregate genotypic variation during a progressive drought treatment under a controlled greenhouse environment, allowing for the exploration of other meaningful phenotypes using autofluorescence image sequences with significance in the context of plant science.

Unlocking the genetic diversity and population structure of the newly introduced two-row spring European HerItage Barley collecTion (ExHIBiT).

In the last century, breeding programs have traditionally favoured yield-related traits, grown under high-input conditions, resulting in a loss of genetic diversity and an increased susceptibility to stresses in crops. Thus, exploiting understudied genetic resources, that potentially harbour tolerance genes, is vital for sustainable agriculture. Northern European barley germplasm has been relatively understudied despite its key role within the malting industry. The European Heritage Barley collection (ExHIBiT) was assembled to explore the genetic diversity in European barley focusing on Northern European accessions and further address environmental pressures. ExHIBiT consists of 363 spring-barley accessions, focusing on two-row type. The collection consists of landraces (~14%), old cultivars (~18%), elite cultivars (~67%) and accessions with unknown breeding history (~1%), with 70% of the collection from Northern Europe. The population structure of the ExHIBiT collection was subdivided into three main clusters primarily based on the accession's year of release using 26,585 informative SNPs based on 50k iSelect single nucleotide polymorphism (SNP) array data. Power analysis established a representative core collection of 230 genotypically and phenotypically diverse accessions. The effectiveness of this core collection for conducting statistical and association analysis was explored by undertaking genome-wide association studies (GWAS) using 24,876 SNPs for nine phenotypic traits, four of which were associated with SNPs. Genomic regions overlapping with previously characterised flowering genes (HvZTLb) were identified, demonstrating the utility of the ExHIBiT core collection for locating genetic regions that determine important traits. Overall, the ExHIBiT core collection represents the high level of untapped diversity within Northern European barley, providing a powerful resource for researchers and breeders to address future climate scenarios.

Deep learning for automated segmentation and counting of hypocotyl and cotyledon regions in mature Pinus radiata D. Don. somatic embryo images.

In commercial forestry and large-scale plant propagation, the utilization of artificial intelligence techniques for automated somatic embryo analysis has emerged as a highly valuable tool. Notably, image segmentation plays a key role in the automated assessment of mature somatic embryos. However, to date, the application of Convolutional Neural Networks (CNNs) for segmentation of mature somatic embryos remains unexplored. In this study, we present a novel application of CNNs for delineating mature somatic conifer embryos from background and residual proliferating embryogenic tissue and differentiating various morphological regions within the embryos. A semantic segmentation CNN was trained to assign pixels to cotyledon, hypocotyl, and background regions, while an instance segmentation network was trained to detect individual cotyledons for automated counting. The main dataset comprised 275 high-resolution microscopic images of mature Pinus radiata somatic embryos, with 42 images reserved for testing and validation sets. The evaluation of different segmentation methods revealed that semantic segmentation achieved the highest performance averaged across classes, achieving F1 scores of 0.929 and 0.932, with IoU scores of 0.867 and 0.872 for the cotyledon and hypocotyl regions respectively. The instance segmentation approach demonstrated proficiency in accurate detection and counting of the number of cotyledons, as indicated by a mean squared error (MSE) of 0.79 and mean absolute error (MAE) of 0.60. The findings highlight the efficacy of neural network-based methods in accurately segmenting somatic embryos and delineating individual morphological parts, providing additional information compared to previous segmentation techniques. This opens avenues for further analysis, including quantification of morphological characteristics in each region, enabling the identification of features of desirable embryos in large-scale production systems. These advancements contribute to the improvement of automated somatic embryogenesis systems, facilitating efficient and reliable plant propagation for commercial forestry applications.

Non-destructive, whole-plant phenotyping reveals dynamic changes in water use efficiency, photosynthesis, and rhizosphere acidification of sorghum accessions under osmotic stress.

Noninvasive phenotyping can quantify dynamic plant growth processes at higher temporal resolution than destructive phenotyping and can reveal phenomena that would be missed by end-point analysis alone. Additionally, whole-plant phenotyping can identify growth conditions that are optimal for both above- and below-ground tissues. However, noninvasive, whole-plant phenotyping approaches available today are generally expensive, complex, and non-modular. We developed a low-cost and versatile approach to noninvasively measure whole-plant physiology over time by growing plants in isolated hydroponic chambers. We demonstrate the versatility of our approach by measuring whole-plant biomass accumulation, water use, and water use efficiency every two days on unstressed and osmotically stressed sorghum accessions. We identified relationships between root zone acidification and photosynthesis on whole-plant water use efficiency over time. Our system can be implemented using cheap, basic components, requires no specific technical expertise, and should be suitable for any non-aquatic vascular plant species.

The dosage- and size-dependent effects of micro- and nanoplastics in lettuce roots and leaves at the growth, photosynthetic, and metabolomics levels.

The occurrence of microplastics (MPs) and nanoplastics (NPs) in soils potentially induce morphological, physiological, and biochemical alterations in plants. The present study investigated the effects of MPs/NPs on lettuce (Lactuca sativa L. var. capitata) plants by focusing on (i) four different particle sizes of polyethylene micro- and nanoplastics, at (ii) four concentrations. Photosynthetic activity, morphological changes in plants, and metabolomic shifts in roots and leaves were investigated. Our findings revealed that particle size plays a pivotal role in influencing various growth traits of lettuce (biomass, color segmentation, greening index, leaf area, and photosynthetic activity), physiological parameters (including maximum quantum yield - Fv/Fmmax, or quantum yield in the steady-state Fv/FmLss, NPQLss, RfdLss, FtLss, FqLss), and metabolomic signatures. Smaller plastic sizes demonstrated a dose-dependent impact on aboveground plant structures, resulting in an overall elicitation of biosynthetic processes. Conversely, larger plastic size had a major impact on root metabolomics, leading to a negative modulation of biosynthetic processes. Specifically, the biosynthesis of secondary metabolites, phytohormone crosstalk, and the metabolism of lipids and fatty acids were among the most affected processes. In addition, nitrogen-containing compounds accumulated following plastic treatments. Our results highlighted a tight correlation between the qPCR analysis of genes associated with the soil nitrogen cycle (such as NifH, NirK, and NosZ), available nitrogen pools in soil (including NO3- and NH4), N-containing metabolites and morpho-physiological parameters of lettuce plants subjected to MPs/NPs. These findings underscore the intricate relationship between specific plastic contaminations, nitrogen dynamics, and plant performance.

The improved stratified transformer for organ segmentation of Arabidopsis.

Segmenting plant organs is a crucial step in extracting plant phenotypes. Despite the advancements in point-based neural networks, the field of plant point cloud segmentation suffers from a lack of adequate datasets. In this study, we addressed this issue by generating Arabidopsis models using L-system and proposing the surface-weighted sampling method. This approach enables automated point sampling and annotation, resulting in fully annotated point clouds. To create the Arabidopsis dataset, we employed Voxel Centroid Sampling and Random Sampling as point cloud downsampling methods, effectively reducing the number of points. To enhance the efficiency of semantic segmentation in plant point clouds, we introduced the Plant Stratified Transformer. This network is an improved version of the Stratified Transformer, incorporating the Fast Downsample Layer. Our improved network underwent training and testing on our dataset, and we compared its performance with PointNet++, PAConv, and the original Stratified Transformer network. For semantic segmentation, our improved network achieved mean Precision, Recall, F1-score and IoU of 84.20, 83.03, 83.61 and 73.11%, respectively. It outperformed PointNet++ and PAConv and performed similarly to the original network. Regarding efficiency, the training time and inference time were 714.3 and 597.9 ms, respectively, which were reduced by 320.9 and 271.8 ms, respectively, compared to the original network. The improved network significantly accelerated the speed of feeding point clouds into the network while maintaining segmentation performance. We demonstrated the potential of virtual plants and deep learning methods in rapidly extracting plant phenotypes, contributing to the advancement of plant phenotype research.

AgTC and AgETL: open-source tools to enhance data collection and management for plant science research.

Advancements in phenotyping technology have enabled plant science researchers to gather large volumes of information from their experiments, especially those that evaluate multiple genotypes. To fully leverage these complex and often heterogeneous data sets (i.e. those that differ in format and structure), scientists must invest considerable time in data processing, and data management has emerged as a considerable barrier for downstream application. Here, we propose a pipeline to enhance data collection, processing, and management from plant science studies comprising of two newly developed open-source programs. The first, called AgTC, is a series of programming functions that generates comma-separated values file templates to collect data in a standard format using either a lab-based computer or a mobile device. The second series of functions, AgETL, executes steps for an Extract-Transform-Load (ETL) data integration process where data are extracted from heterogeneously formatted files, transformed to meet standard criteria, and loaded into a database. There, data are stored and can be accessed for data analysis-related processes, including dynamic data visualization through web-based tools. Both AgTC and AgETL are flexible for application across plant science experiments without programming knowledge on the part of the domain scientist, and their functions are executed on Jupyter Notebook, a browser-based interactive development environment. Additionally, all parameters are easily customized from central configuration files written in the human-readable YAML format. Using three experiments from research laboratories in university and non-government organization (NGO) settings as test cases, we demonstrate the utility of AgTC and AgETL to streamline critical steps from data collection to analysis in the plant sciences.

Completing the picture of field-grown cereal crops: a new method for detailed leaf surface models in wheat.

BACKGROUND: The leaf angle distribution (LAD) is an important structural parameter of agricultural crops that influences light interception, radiation fluxes and consequently plant performance. Therefore, LAD and its parametrized form, the Beta distribution, is used in many photosynthesis models. However, in field cultivations, these parameters are difficult to assess and cereal crops in particular pose challenges since their leaves are thin, flexible, and often bent and twisted around their own axis. To our knowledge, there is only a very limited set of methods currently available to calculate LADs of field-grown cereal crops that explicitly takes these special morphological properties into account. RESULTS: In this study, a new processing pipeline is introduced that allows for the generation of realistic leaf surface models and the analysis of LADs of field-grown cereal crops from 3D point clouds. The data acquisition is based on a convenient stereo imaging setup. The approach was validated with different artificial targets and results on the accuracy of the 3D reconstruction, leaf surface modeling and calculated LAD are given. The mean error of the 3D reconstruction was below 1 mm for an inclination angle range between 0° and 75° and the leaf surface could be quantified with an average accuracy of 90%. The concordance correlation coefficient (CCC) of 99.6% (p-value = [Formula: see text]) indicated a high correlation between the reconstructed inclination angle and the identity line. The LADs for bent leaves were reconstructed with a mean error of 0.21° and a standard deviation of 1.55°. As an additional parameter, the insertion angle was reconstructed for the artificial leaf model with an average error < 5°. Finally, the method was tested with images of field-grown cereal crops and Beta functions were approximated from the calculated LADs. The mean CCC between reconstructed LAD and calculated Beta function was 0.66. According to Cohen, this indicates a high correlation. CONCLUSION: This study shows that our image processing pipeline can reconstruct the complex leaf shape of cereal crops from stereo images. The high accuracy of the approach was demonstrated with several validation experiments including artificial leaf targets. The derived leaf models were used to calculate LADs for artificial leaves and naturally grown cereal crops. This helps to better understand the influence of the canopy structure on light absorption and plant performance and allows for a more precise parametrization of photosynthesis models via the derived Beta distributions.

End-to-end multimodal 3D imaging and machine learning workflow for non-destructive phenotyping of grapevine trunk internal structure.

Quantifying healthy and degraded inner tissues in plants is of great interest in agronomy, for example, to assess plant health and quality and monitor physiological traits or diseases. However, detecting functional and degraded plant tissues in-vivo without harming the plant is extremely challenging. New solutions are needed in ligneous and perennial species, for which the sustainability of plantations is crucial. To tackle this challenge, we developed a novel approach based on multimodal 3D imaging and artificial intelligence-based image processing that allowed a non-destructive diagnosis of inner tissues in living plants. The method was successfully applied to the grapevine (Vitis vinifera L.). Vineyard's sustainability is threatened by trunk diseases, while the sanitary status of vines cannot be ascertained without injuring the plants. By combining MRI and X-ray CT 3D imaging with an automatic voxel classification, we could discriminate intact, degraded, and white rot tissues with a mean global accuracy of over 91%. Each imaging modality contribution to tissue detection was evaluated, and we identified quantitative structural and physiological markers characterizing wood degradation steps. The combined study of inner tissue distribution versus external foliar symptom history demonstrated that white rot and intact tissue contents are key-measurements in evaluating vines' sanitary status. We finally proposed a model for an accurate trunk disease diagnosis in grapevine. This work opens new routes for precision agriculture and in-situ monitoring of tissue quality and plant health across plant species.

Machine and Deep Learning: Artificial Intelligence Application in Biotic and Abiotic Stress Management in Plants.

Biotic and abiotic stresses significantly affect plant fitness, resulting in a serious loss in food production. Biotic and abiotic stresses predominantly affect metabolite biosynthesis, gene and protein expression, and genome variations. However, light doses of stress result in the production of positive attributes in crops, like tolerance to stress and biosynthesis of metabolites, called hormesis. Advancement in artificial intelligence (AI) has enabled the development of high-throughput gadgets such as high-resolution imagery sensors and robotic aerial vehicles, i.e., satellites and unmanned aerial vehicles (UAV), to overcome biotic and abiotic stresses. These High throughput (HTP) gadgets produce accurate but big amounts of data. Significant datasets such as transportable array for remotely sensed agriculture and phenotyping reference platform (TERRA-REF) have been developed to forecast abiotic stresses and early detection of biotic stresses. For accurately measuring the model plant stress, tools like Deep Learning (DL) and Machine Learning (ML) have enabled early detection of desirable traits in a large population of breeding material and mitigate plant stresses. In this review, advanced applications of ML and DL in plant biotic and abiotic stress management have been summarized.

A Novel Correction Methodology to Improve the Performance of a Low-Cost Hyperspectral Portable Snapshot Camera.

The development of spectral sensors (SSs) capable of retrieving spectral information have opened new opportunities to improve several environmental and agricultural practices, e.g., crop breeding, plant phenotyping, land use monitoring, and crop classification. The SSs are classified as multispectral and hyperspectral (HS) based on the number of the spectral bands resolved and sampled during data acquisition. Large-scale applications of the HS remain limited due to the cost of this type of technology and the technical difficulties in hyperspectral data processing. Low-cost portable hyperspectral cameras (PHCs) have been progressively developed; however, critical aspects associated with data acquisition and processing, such as the presence of spectral discontinuities, signal jumps, and a high level of background noise, were reported. The aim of this work was to analyze and improve the hyperspectral output of a PHC Senop HSC-2 device by developing a general use methodology. Several signal gaps were identified as falls and jumps across the spectral signatures near 513, 650, and 930 nm, while the dark current signal magnitude and variability associated with instrumental noise showed an increasing trend over time. A data correction pipeline was successfully developed and tested, leading to 99% and 74% reductions in radiance signal jumps identified at 650 and 830 nm, respectively, while the impact of noise on the acquired signal was assessed to be in the range of 10% to 15%. The developed methodology can be effectively applied to other low-cost hyperspectral cameras.

A Point-Cloud Segmentation Network Based on SqueezeNet and Time Series for Plants.

The phenotyping of plant growth enriches our understanding of intricate genetic characteristics, paving the way for advancements in modern breeding and precision agriculture. Within the domain of phenotyping, segmenting 3D point clouds of plant organs is the basis of extracting plant phenotypic parameters. In this study, we introduce a novel method for point-cloud downsampling that adeptly mitigates the challenges posed by sample imbalances. In subsequent developments, we architect a deep learning framework founded on the principles of SqueezeNet for the segmentation of plant point clouds. In addition, we also use the time series as input variables, which effectively improves the segmentation accuracy of the network. Based on semantic segmentation, the MeanShift algorithm is employed to execute instance segmentation on the point-cloud data of crops. In semantic segmentation, the average Precision, Recall, F1-score, and IoU of maize reached 99.35%, 99.26%, 99.30%, and 98.61%, and the average Precision, Recall, F1-score, and IoU of tomato reached 97.98%, 97.92%, 97.95%, and 95.98%. In instance segmentation, the accuracy of maize and tomato reached 98.45% and 96.12%. This research holds the potential to advance the fields of plant phenotypic extraction, ideotype selection, and precision agriculture.

Deep incorporation of organic amendments into soils of a 'Calardis Musqué' vineyard: effects on greenhouse gas emissions, vine vigor, and grape quality.

Background: Traditional wine growing regions are increasingly endangered by climatic alterations. One promising approach to mitigate advancing climate change could be an increase of soil organic matter. Here, especially subsoils are of interest as they provide higher carbon storage potential than topsoils. In this context, vineyard subsoils could be particularly suitable since they are deeply cultivated once before planting and afterwards, left at rest for several decades due to the perennial nature of grapevines. Methods: For this purpose, a biochar compost substrate and greenwaste compost were incorporated in up to 0.6 m depth before planting a new experimental vineyard with the fungus-resistant grapevine cultivar 'Calardis Musqué'. The influence of this deep incorporation on greenhouse gas emissions and grapevine performance was evaluated and compared to a non-amended control using sensor-based analyses. Results: Increased CO2 emissions and lower N2O emissions were found for the incorporation treatments compared to the control, but these differences were not statistically significant due to high spatial variability. Only few plant traits like chlorophyll content or berry cuticle characteristics were significantly affected in some of the experimental years. Over the course of the study, annual climatic conditions had a much stronger influence on plant vigor and grape quality than the incorporated organic amendments. Discussion: In summary, organic soil amendments and their deep incorporation did not have any significant effect on greenhouse gas emissions and no measurable or only negligible effect on grapevine vigor, and grape quality parameters. Thus, according to our study the deposition of organic amendments in vineyard subsoils seems to be an option for viticulture to contribute to carbon storage in soils in order to mitigate climate change.