Plant segmentation and trait extraction for individual organs are two of the key challenges in high-throughput phenotyping (HTP) operations. To address this challenge, the Ag Alumni Seed Phenotyping Facility (AAPF) at Purdue University utilizes chlorophyll fluorescence images (CFIs) to enable consistent and efficient automatic segmentation of plants of different species, age, or color. A series of image analysis routines were also developed to facilitate the quantitative measurements of key corn plant traits. A proof-of-concept experiment was conducted to demonstrate the utility of the extracted traits in assessing drought stress reaction of corn plants. The image analysis routines successfully measured several corn morphological characteristics for different sizes such as plant height, area, top-node height and diameter, number of leaves, leaf area, and angle in relation to the stem. Data from the proof-of-concept experiment showed how corn plants behaved when treated with different water regiments or grown in pot of different sizes. High-throughput image segmentation and analysis basing on a plant’s fluorescence image was proved to be efficient and reliable. Extracted trait on the segmented stem and leaves of a corn plant demonstrated the importance and utility of this kind of trait data in evaluating the performance of corn plant under stress. Data collected from corn plants grown in pots of different volumes showed the importance of using pot of standard size when conducting and reporting plant phenotyping data in a controlled-environment facility.

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Background Breakthrough imaging technologies may challenge the plant phenotyping bottleneck regarding marker-assisted breeding and genetic mapping. In this context, X-Ray CT (computed tomography) technology can accurately obtain the digital twin of root system architecture (RSA) but computational methods to quantify RSA traits and analyze their changes over time are limited. RSA traits extremely affect agricultural productivity. We develop a spatial–temporal root architectural modeling method based on 4D data from X-ray CT. This novel approach is optimized for high-throughput phenotyping considering the cost-effective time to process the data and the accuracy and robustness of the results. Significant root architectural traits, including root elongation rate, number, length, growth angle, height, diameter, branching map, and volume of axial and lateral roots are extracted from the model based on the digital twin. Our pipeline is divided into two major steps: (i) first, we compute the curve-skeleton based on a constrained Laplacian smoothing algorithm. This skeletal structure determines the registration of the roots over time; (ii) subsequently, the RSA is robustly modeled by a cylindrical fitting to spatially quantify several traits. The experiment was carried out at the Ag Alumni Seed Phenotyping Facility (AAPF) from Purdue University in West Lafayette (IN, USA). Results Roots from three samples of tomato plants at two different times and three samples of corn plants at three different times were scanned. Regarding the first step, the PCA analysis of the skeleton is able to accurately and robustly register temporal roots. From the second step, several traits were computed. Two of them were accurately validated using the root digital twin as a ground truth against the cylindrical model: number of branches (RRMSE better than 9%) and volume, reaching a coefficient of determination (R2) of 0.84 and a P < 0.001. Conclusions The experimental results support the viability of the developed methodology, being able to provide scalability to a comprehensive analysis in order to perform high throughput root phenotyping.