Plant Physiology Reports

https://doi.org/10.1007/s40502-024-00789-1

ORIGINAL ARTICLE

WirPheno: an affordable medium throughput root phenotyping

protocol for assessment of waterlogging induced roots in cowpea
H. G. K. Chanumolu1,2 · P. S. Basavaraj1 · Vinay Hegde1,5 · Mahesh Kumar4 · Sudhir Kumar4 · J. Rane1,3

Received: 6 January 2024 / Accepted: 15 January 2024

© Indian Society for Plant Physiology 2024

Abstract
Implementing trait-based phenotyping for waterlogging stress in crop improvement has become imperative due to the
limitations of traditional methods for assessing abiotic stress tolerance. Therefore, there is a crucial need for efficient
phenotyping tools and protocols to non-invasively evaluate genotypes for advantageous traits associated with waterlogging
tolerance. In this context, the study was carried out to optimize an affordable phenotyping protocol to assess one of such
traits, namely waterlogging-induced roots (WIR) in cowpea genotypes. The data generated from optimized protocol for
stress imposition, image acquisition, and image analysis demonstrated effectively that WIR image features significantly
differentiated cowpea genotypes when they were subjected to waterlogging stress as evidenced by PCA and K-cluster analysis.
The study also revealed significant variation among genotypes in terms of WIR architecture based on image features such
as total root length (TRL), network area (NA), convex area (CA), volume (Vol) and Median number of roots (MeN) etc.
Efficacy of these traits in differentiating the waterlogging tolerant and intolerant genotypes of cowpea could be validated with
conventional parameters. A strong positive correlation between conventional and WIR image features indicated that WIR,
playing a role in waterlogging tolerance, can be reliably measured noninvasively. Furthermore, the phenotyping protocol
developed in this study together with growth parameters could help in identification of waterlogging tolerant genotypes
CG121 and CG221 that had enhanced WIR over other genotypes under waterlogging conditions. The affordable phenotyping
protocol developed in this study promises to serve as an effective phenotyping tool for assessing waterlogging-induced roots
in cowpea and promising genotypes like CG121 and CG221 may serve as donors for waterlogging tolerance.

Keywords Affordable root phenotyping · WirPheno · Waterlogging induced roots (WIR) · ImageJ · RhizoVision explorer ·
Image features

Introduction

The enhancement of crop performance through genetic

improvement, in pursuit of higher productivity and climate
resilience, relies significantly on the accurate and precise
* J. Rane assessment of desired traits. This necessity has driven sig-
jagadish.rane@icar.gov.in nificant advancements in the realm of plant phenomics in
recent times. Plant phenomics stands as a multidisciplinary
1
ICAR – National Institute of Abiotic Stress Management, approach (Demidchik et al., 2020; Negrão & Julkowska,
Baramati, Pune, Maharashtra, India
2020; Pieruschka & Schurr, 2019) with the primary objec-
2
The Graduate School, ICAR – Indian Agricultural Research tive of phenotyping crop plants for desired phenes which
Institute, New Delhi, India
refers to traits. Plant phenotyping has been a pivotal com-
3
ICAR – Central Institute for Arid Horticulture, Bikaner, ponent of crop and variety selection since the early days of
Rajasthan, India
agriculture when humans began domesticating crops (Kumar
4
ICAR – Indian Agricultural Research Institute, New Delhi, et al., 2015). However, characterisation of plant responses
India
was largely carried out with conventional emphasis on
5
Panjabrao Deshmukh Krishi Vidyapeeth, Akola, growth, yield and its components both under favourable
Maharashtra, India

Vol.:(0123456789)
 Plant Physiology Reports

and abiotic stress environments as many of the traits pro- 2020). Traditionally, plants were uprooted, and their root
posed did not allow large scale screening because of lack architecture was analysed using various methods such as
of robustness. Modern technologies at present, attempt to soil coring, root excavations, trenching (Weaver, 1926;
bridge this gap in phenotyping by engaging non-invasive Weaver et al., 1922), and shovelomics (Burridge et al., 2016;
tools and automation in a variety of plant phenomics plat- Trachsel et al., 2010). These conventional approaches have
form (Houle et al., 2010) with optimistic hope that external limitations and are often labour-intensive, making them less
features of shoot and root can help in interpretation of inter- suitable for high throughput phenotyping efforts.
nal processes in plants (Zhao et al., 2019) when exposed to Phenotyping platforms that have emerged for the non-
natural or imposed abiotic stresses. While ample attempts destructive quantification of root system architecture (RSA)
have been made to optimise phenomics protocol for stress engage technologies such as electromagnetic inductance as
tolerance like drought in legumes such as mungbean (Rane demonstrated in wheat (Whalley et al., 2017), computed
et al., 2021) and salinity in cereals (Al-Tamimi et al., 2016), tomography (CT) as demonstrated in wheat (Pfeifer et al.,
few information is available on waterlogging stress and 2015) and in rice (Teramoto et al., 2020). Further, magnetic
plant’s response in legume crops like cowpea. resonance imaging (Van Dusschoten et al., 2016) was used
The characterization of crop germplasm for waterlogging in crops like barley and maize for 3D quantification of RSA
tolerance remains a formidable task (Langan et al., 2022) and its activity in response to various edaphic and stress
despite an access to information on morpho-physiological factors. Since these technologies are highly expensive,
responses of plants to waterlogging. This challenge researchers have introduced affordable root imaging
primarily stems from the wide variability in waterlogging platforms to facilitate phenotyping for root traits (Bontpart
stress conditions and the multitude of additional factors that et al., 2020; Rinehart et al., 2022). A diverse array of tools
influence plant responses (Langan et al., 2022). Innovative for root phenotyping exists, including commercial software
setups for inducing waterlogging have been demonstrated, such as WinRHIZO, WinRhizoTRON, and RootSnap, open-
including the use of pots placed in buckets to evaluate shoot source and freeware options like ImageJ, SmartRoot, Root
responses as in lentil (Lake et al., 2021), hydroponics in System Analyzer, DIRT, RootTrace, IJ_Rhizo, DART,
soybean (Harrison et al., 2022). Waterlogging responses RootNav, EZ-Rhizo, Toporoot, and RhizoVision Explorer,
of plants were assessed by employing digital imaging and as well as on-demand platforms like Growth Explorer and
thermal cameras as in lentil (Lake et al., 2021), by UAV RootReader 3D (Li et al., 2022; Takahashi & Pradal, 2021).
based imagery systems (Zhou et al., 2021) and cost-effective These imaging software tools can be further categorized
3D imaging platforms (Cao et al., 2019) in soybean. into either 2D (such as WinRHIZO, GiA Roots, SmartRoot,
Furthermore, a smartphone-based phenomics tool has been and DIRT) or 3D (including RootTrak, RootReader3D, and
devised for evaluating drought responses in pulse crops like RSAtrace3D) based on the dimensions they analyse (Li
green gram, cowpea, chickpea, and black gram (Tamilselvan, et al., 2022).
2022). However, despite these advancements, there remains Legume crops such as cowpea are highly sensitive to
a dearth of research focusing on the identification of waterlogging and there is no standardized phenotyping
desirable waterlogging-tolerance traits, such as adventitious methodology available to comprehensively study various
roots, by refining non-invasive phenotyping techniques aspects of root system architecture under waterlogged
for screening. It has been evidenced that development of conditions (Langan et al., 2022). Hence, the current study
adventitious roots from stem region, which replace the was focused on optimising an affordable phenotyping
deteriorated main root system is one of the adaptations in protocol and to demonstrate its use in assessing waterlogging
cowpea (Hong et al., 1977; Olorunwa et al., 2022) and other induced roots (WIR) in cowpea genotypes.
legumes like pigeon pea (Hingane et al., 2015) in response
to waterlogging. Developing robust methods to identify and
evaluate such trait can significantly contribute to breeding Materials and methods
for waterlogging tolerance in legume crops.
Root phenotyping has emerged as a pivotal technique Experiment details
within plant phenomics, aiming to characterize various root
phenes or traits encompassing morphological, geometrical, The experiment was carried out at Plant Phenomics
topological, and dynamic aspects (York & Lobet, 2017). Facility of ICAR- National Institute of Abiotic Stress
This technique enables the exploration of how roots respond Management, Baramati (ICAR-NIASM) located at 18°
to both regular and extreme environmental conditions. 09′ 30.62′′ N latitude, 74° 30′ 03.08′′ E longitude and the
However, root phenotyping poses unique challenges due to altitude of 550 m from MSL in Baramati taluka, Pune,
the opaque nature of soil and the requirement for advanced Maharashtra, India in 2022–23. The seed of 20 cowpea
facilities to accurately assess these traits (Bontpart et al., genotypes including two local cultivars were collected
Plant Physiology Reports

from ICAR- NBPGR, New Delhi (Table 1) and the cowpea Imposition of waterlogging stress
plants were raised in pots filled with black soil devoid of
small stones and crop residues on top layers of soil up to The initial stage of WirPheno, stress imposition, a modi-
15 days after sowing (DAS) in shade net. Then the plants fied “pot-in-pot” method, which is based on the pot in a
sifted to greenhouse and arranged in CRBD design having bucket method described by Lake et al. (2021) was used to
2 treatments and 3 replications to assess the waterlogging impose waterlogging stress to the plants. In this modified
induced roots (WIR) of cowpea germplasm at seedling method, two pots of different sizes and heights were selected
stage with the help of an affordable root phenotyping where the small and big pots were named as pot-A and pot-B
system. respectively. Pot-A, having 20 cm height and 20 cm diameter
at top surface and holes at bottom, was used to grow the
cowpea plants and made a few more extra holes at sides of
Framework of WirPheno (waterlogging induced root pot A to oversaturate the soil within short time during stress.
phenotyping) Pot-B, dimensions of 31 cm in diameter at the top surface,
20 cm in diameter at the bottom surface, and a height of
The optimised WirPheno includes three stages namely 29 cm and devoid of holes, was used for imposing stress.
stress imposition, image acquisition, and image analysis. The pot A was kept in pot B. After transiently replacing the
The stress imposition was given in “pot-in-pot” method air in the soil pore spaces with water by adding water to half
followed by image acquisition with the help of smartphone of pot B, stress was applied (15 DAS) by raising the water
followed by image analysis with the help of amalgamation level by 4 cm from the soil in pot A. All 20 genotype pots
of ImageJ and RVE (RhizoVision Explorer) to extract the were treated with the same manner. The stress was allowed
data from images. for 10 days (Fig. 1).

Table 1  List of cowpea Gene ID Accession name Seed lot collection Gene ID Accession name Seed lot collection
genotypes collected for research
CG 035 EC 240861 ICAR- NBPGR CG 151 IC 488195 ICAR- NBPGR
CG 038 EC 240875 ICAR- NBPGR CG 162 IC 598466 ICAR- NBPGR
CG 041 EC 240801 ICAR- NBPGR CG 165 IC 560928 ICAR- NBPGR
CG 060 IC 488085 ICAR- NBPGR CG 203 IC 488067 ICAR- NBPGR
CG 080 EC 723735-B ICAR- NBPGR CG 212 IC 560916 ICAR- NBPGR
CG 082 IC 488270 ICAR- NBPGR CG 214 IC 554414 ICAR- NBPGR
CG 087 IC 488239 ICAR- NBPGR CG 221 EC 724791 ICAR- NBPGR
CG 091 EC 244175 ICAR- NBPGR CG 236 EC 724805 ICAR- NBPGR
CG 121 EC 240966-A ICAR- NBPGR DC 015 Local cultivar (Dharwad)
CG 133 IC 402125 ICAR- NBPGR RC 101 Local cultivar (Rajasthan)
X cm

Pot Pot B
Y cm

A
Providing extra holes to
get soil oversaturated
transiently

Adjusting the height

Keeping the small Levelling the Placing the pot Giving stress to Stress
after keeping pebbles
pebbles in pot B pebbles in pot B A in pot B plant imposed plant
to place pot A

Fig. 1  Schematic diagram of optimized waterlogging stress imposing protocol to assess cowpea WIR
 Plant Physiology Reports

Optimisation of affordable image acquisition system involves two steps namely, image segmentation and feature
extraction (Fig. 3). The image segmentation was done using
Image acquisition, a crucial stage in the WirPheno involves ImageJ followed by feature extraction using RVE. The rea-
the capturing of images by using an affordable root imag- sons for use of this amalgamation were explained in results
ing platform for assessing waterlogging induced roots. The and discussion part. In image segmentation stage, an image
affordable root imaging setup consists of four components was uploaded onto ImageJ where ROI (Region of Interest)
namely, smart phone and laptop, python configured software, selection with selection tool (oval), clearing the noise by
IP web cam application, hardware equipment. The smart making colour threshold followed by segmenting and con-
phone and laptop are used to capture and saving the images verting binary were involved in sequential manner. Based
respectively. The software, developed in ICAR-NIASM lab, on single image, made an ImageJ macro for batch segmenta-
allows real-time view of the images on the laptop screen tion of images. Then, the binarized images were loaded onto
as they are being captured. The IP webcam application RVE (Rhizo Vision Explorer) where image pre-processing,
allows the smart phone to access it remotely by laptop. The feature extraction and image output display customised set-
setup was made in such a way that the smartphone (Nokia, tings were made for extracting the data from those bina-
Android 10), act as a camera for capturing images, remotely rized images (Table 2). Nearly 600 images were segmented,
controlled by the laptop (Lenovo IdeaPad Slim 5 Pro) with binarized by ImageJ followed by processed through RVE
the help of IP Webcam Application and python configured for feature extraction (See SI Fig. 1, 2 in supplementary
software. An overhead tabletop mobile stand, was used to information).
provide support and ensure stability during the image acqui-
sition process for WIR architecture (Fig. 2). In acquisition Growth traits
process, after removing from pot B (25 DAS), the A pots
were kept manually on the system where the roots are being After exposing the 15-day old plants to a waterlogging
captured. The distance between plants and smartphone kept treatment for a period of 10 days (25 DAS), the
constant for all plants. The images were taken in three angles morphological attributes such as leaf number (LN), leaf
(0°, 90° and 180°). Since there was an opportunity for the area (LA), Total Fresh Weight (TFW) and Total Dry Weight
visibility of roots above the soil, it was possible to capture (TDW) were measured. The number of leaves of each
the WIR images. cowpea plant was measured manually. Leaf area of each
cowpea plant was measured destructively using LI-3100
Use of open‑source software for image analysis leaf area meter (LI-COR, Nebraska, USA). Finally, the total
fresh biomass followed dry biomass of cowpea plant after
Image analysis, also an important stage in WirPheno has drying it in a hot air oven at 65 °C for 2 days (Olorunwa
the use of amalgamated software i.e. ImageJ (freeware) et al., 2022) was recorded using analytical balance (ATX224
and RVE (Seethepalli & York, 2021). The image analysis Model, Shimadzu).

Fig. 2  Schematic representation

of optimized image acquisition
for WIR
Plant Physiology Reports

Fig. 3  Schematic representation of optimized image analysis to assess WIR in cowpea

Statistical analysis affordable tools as described in material and methods.

However, during the subsequent image analysis phase
The data retrieved from images was analysed using R using ImageJ, it became evident that most of the captured
programming. To classify genotypes based on Waterlogging- images containing region of interest (ROI) i.e., WIR had
Induced Roots (WIR) image features, Principal Component background noise, making it challenging to segment ROI
Analysis (PCA) biplot analysis was employed, with R from background (Fig. 4). This could be solved through
packages such as ‘factoextra,’ ‘FactoMineR,’ and ‘ggbiplot.’ subsequent experiments with utmost precautions to clear
To validate the PCA biplot analysis and determine the optimal of noise due to pebbles and algae. These efforts enable
number of clusters, K cluster analysis was conducted, utilizing segmentation of WIR with greater clarity (Fig. 5). Binary
R packages like ‘tidyverse,’ ‘parameters,’ ‘factoextra,’ and images obtained from ImageJ were further used for extrac-
‘cluster’. Furthermore, Pearson’s correlation analysis was tion of their features to explain the waterlogging responses
performed to establish correlations between conventional of cowpea genotypes by employing RhizoVision Explorer
parameters (e.g., fresh weight, dry weight, leaf number and (RVE), a software that allow a more comprehensive analy-
leaf area) and image features (including Total Root Length, sis. This process could provide more than 20 image fea-
Network Area, Convex Area, Perimeter, Branching Points, tures for each of the images.
Root Tips and Width, etc.). This correlation analysis was The graph (Fig. 6) shows a close association between
facilitated using R packages like ‘corrplot’ and ‘caret.’ To WIR-image features derived from RVE and those from Lem-
assess genetic variation among genotypes based on image naTec Grid (LT). For example, there was highly significant
features, the Duncan Multiple Range Test (DMRT) was positive correlation between the root area (NA) extracted
employed. For factorial randomized block design (RBD) from RVE and area obtained from Lemna Grid (r = 0.97).
analysis, Analysis of Variance (ANOVA) was carried out with Similarly convex area (CA) obtained from RVE and same
the assistance of the ‘Agricolae’ R package. derived from LT perfectly corelated (r = 1.00). The strong
correlation observed between the features extracted from
RVE and those obtained from Lemna Grid provides com-
Results pelling evidence for the accuracy and reliability of the data
derived from RVE (RhizoVision Explorer). This correlation
Optimisation of protocol shows the effectiveness of the method that was optimized
and suggests that it can be confidently applied in future
In the preliminary trial, experiments were aimed to design experiments and analyses.
image acquisition platform for phenotyping WIR with
 Plant Physiology Reports

Table 2  Root image features derived from ImageJ and RhizoVision explorer
S. No Root features from image Abbreviation Unitsa Description

1 No. of Median and Maximum Roots MeN and MaN n Each row of the segmented image is scanned horizontally
from the left to the right to ascertain the presence of roots.
On the right side of each line being scanned, when the
current pixel value transitions from 0 to 1, it indicates
the presence of a root in the image. Root counts are
determined for each row of the segmented image, and
subsequently, the average and maximum root counts are
calculated
2 Number of Root Tips RT n Calculated by counting all of the tip pixels in the
topologically skeletonized image
3 Total Root Length TRL px The calculation involves the total sum of all the pixels in the
skeletonized image. For diagonal pixels, a square root of 2
is applied to account for their diagonal distance
4 Network Area/Root area NA px2 The network area is constituted by the total number of pixels
in the segmented image
5 Perimeter Peri px It is the pixel count represents the total number of pixels in
the perimeter image
6 Convex Area and Solidity CA & solidity px2 & no units A smallest possible convex polygon in geometric form is
called the convex hull. A measure of solidity is the ratio of
network area to convex area
7 Average, median, and maximum diameter AvgD, px The distance from each pixel in the skeletonized image to
MeD & the closest non-root pixel is calculated, and a circle is
MaD fitted using this value as its radius. Each pixel’s diameter
is identified as the diameter of the circle to it. The average,
median, and maximum diameters are calculated using the
list of diameters from every pixel along the medial axis
8 Average Root Orientation ARO degree The mean orientation of medial axis pixels within a locality
of 40 × 40 pixels is used to calculate the orientation at each
pixel for each medial axis pixel. The term “average root
orientation” refers to the average of all these orientations
9 Branch Points BP n Calculated by counting all of the branch pixels in the
topologically skeletonized image
10 Volume and Surface Area Vol & SA px3& px2 The sum of the cross-sectional areas across all medial axis
pixels is indicated as volume, and the sum of the perimeter
across all medial axis pixels is noted as surface area, using
the radii previously computed. Calculated by counting
all of the branch pixels in the topologically skeletonized
picture
a
n: number and px: pixel

Captured image Segmented image Binary image

Fig. 4  Attempts to capture the images of WIR in preliminary experiment

Classification of genotypes by WIR image features genotypes and image features were represented by dots (.)
and vectors ( →) respectively. The analysis could clearly
The first two principal components obtained from PCA differentiate the genotypes with higher values from those
biplot analysis represented by dimension 1 and dimension exhibiting lower values of image features. Furthermore,
2 (Fig. 7) together could explain nearly 89% of variability the alignment of genotypes with vectors signifies the sig-
in the features extracted from image analysis of WIRs. The nificance of contribution of respective image features in the
Plant Physiology Reports

Captured image Segmented image Binary image

Fig. 5  Captured the images of WIR in main experiment

vectors. When the angle between a pair of vectors is narrow,

it signifies a high degree of correlation. To exemplify,
consider the case of roots with a slender structure, such
as D1 root length, surface area, volume, and projected
area, which are represented by RLD1, SAD1, VD1, and
PAD1 respectively. These parameters largely overlap, with
the angles between their corresponding vectors nearly
approaching zero. This suggests strong correlations,
implying the potential derivation of these parameters from
a common source. Similar scenario was observed in case of
the surface area of both the thickest (D3) and moderately
Fig. 6  Validation of RVE image analysis with LemnaTec (sym- thick (D2) roots.
bols = “***”, “**”, “*” indicate significance at p < 0.001, 0.01, 0.05
respectively) k‑means clustering of genotypes from WIR Image
features
corresponding genotypes. In comparison to other genotypes,
the genotype CG121 was characterized by significantly Prior to clustering by k-mean analysis the ideal cluster count
higher values of traits such as MeN, BP, TRL, Peri. was performed with gap statistic approach of R program.
Virtually, all image features align consistently with The Fig. 8a suggests that the optimal cluster number that
their respective vectors. This observation further uncovers could be generate were only two. Subsequently, the gap
those numerous parameters extracted from images exhibit statistic approach validates the existence of two distinct
correlations amongst themselves, as demonstrated by the clusters, represented by a dotted line. Furthermore, the gap

Fig. 7  Principal component analysis (PCA) of WIR surrogate traits

 Plant Physiology Reports

statistic method is reinforced by over 20 other techniques for genotypes had notably lower NA values (9336.8, 9138.6 p­ x2
determining the optimal cluster number. The Fig. 8b depicts respectively). In case of CA image feature, CG121, CG236,
the k-means clustering of genotypes on WIR image features and CG221 genotypes exhibited significantly larger convex
basis. This analysis could validate the results obtained from areas (610,840.8, 564,517.7 and 556,167.4 px2 respectively),
PCA biplot analysis by grouping the genotypes into two dif- in contrast to other genotypes. The genotypes CG121,
ferent clusters which shares common values of 27 image CG041, and CG221 exhibited significantly larger perimeters
features. The first two principal components represented by (21,765.25, 20,409.404 and 17,758.672 px respectively),
Dim1 and Dim2 together could explain more than 87% of in contrast to the other genotypes. In case of Vol image
the variations in existed in the data set. feature, the genotypes CG121, CG060, CG236, and CG221
exhibited significantly greater root volumes (328,402.52,
Genetic variation by WIR image features 319,900.82, 313,945.37 and 219,059.29 px3 respectively),
compared to the other genotypes. ANOVA revealed highly
The assessment of genetic variation in the genotypic significant effect (p < 0.001) of genotype on image features
responses to waterlogging with respect to waterlogging (See SI 1 to 4 in Supplementary Information).
induced roots (WIR) was carried out by image features
extracted from WIR images of 20 genotypes such as MeN, Association between growth traits and image
MaN, RT, BP, TRL, Width, NA, CA, Peri, Vol, SA, Holes, derived parameters
Solidity, AvgD and other derived parameters based on the
diameter ranges of root such as Root Length (RLD), Pro- The relation among growth parameters and image derived
jected Area (PAD), Volume (VD) of Diameter Ranges 1(0–2 WIR parameters of 20 genotypes was evaluated by using
px), 2 (2–5px) and 2 (> 5px). Moreover, among the diverse Pearson’s correlation analysis. Here, correlation was
image features, priority was assigned to those that could depicted in a Fig. 10. Four growth parameters viz., total
effectively distinguish genotypes. In case of MeN, CG060, fresh weight (TFW), total dry weight (TDW), Leaf Num-
CG121, and CG041 genotypes exhibited more than twice ber and leaf area and 12 image derived WIR parameters
the values (6.83, 6.5, and 6.25, respectively) compared to i.e., total root length (TRL), Network Area (NA), Branch-
CG091 and CG080 genotypes, which had observed val- ing points (BP), Surface Area (SA), Perimeter (Peri), Total
ues of 2.4 and 2.25, respectively. The genotypes CG121, width (Width), Median Number of Roots (MeN), Maximum
CG041, and CG236 exhibited significantly higher TRL val- Number of Roots (MaN), Root Tips (RT), Convex Area
ues (13,519.933, 11,320.253, 10,871.558 px respectively), (CA), Surface Area of Diameter 2 (SAD2) and Volume
whereas the genotypes CG151 and CG091 had notably lower (Vol) were used to evaluate the correlation among them.
TRL values (3041.982, 2780.906 px respectively) (Fig. 9). In Fig. 10, the total fresh weight (TFW) was positively cor-
The genotypes CG121, CG236, and CG221 displayed related with TRL (r = 0.86, ***), NA (r = 0.88, ***), BP
significantly higher NA values (49,273.83, 45,749.33, (r = 0.84, ***), Peri (r = 0.84, ***), Width (r = 0.79, ***),
37,639.6 px 2 respectively), while CG165 and CG091 SA (r = 0.85, ***), MeN (r = 0.69, ***), MaN (r = 0.73, ***),

Fig. 8  a Optimal number of clusters, b k-means cluster analysis

Plant Physiology Reports

Fig. 9  Genetic variation from image features of WIR

Fig. 10  The correlation between conventional parameters and image-derived WIR traits using Pearson’s correlation (symbols = “***”, “**”, “*”
indicate significance at p < 0.001, 0.01, 0.05 respectively)

RT (r = 0.79, ***), CA (r = 0.87, ***), SAD2 (r = 0.86, ***) and moderately with BP (r = 0.68, **),Width (r = 0.61, **),
and Vol (r = 0.83, ***). Similarly, the other conventional MeN (r = 0.63, **), MaN (r = 0.56, **), RT (r = 0.63, **).
trait, total dry weight (TDW) was also strongly correlated Further, the leaf area also strongly correlated with TRL
with WIR image features such as TRL (r = 0.70, ***), NA (r = 0.80, ***), NA (r = 0.77, ***), BP (r = 0.80, ***), Peri
(r = 0.74, ***), Peri (r = 0.68, ***), SA (r = 0.71, ***) CA (r = 0.77, ***), MeN (r = 0.76, ***), RT (r = 0.77, ***),
(r = 0.76, ***), SAD2 (r = 0.74, ***) and Vol (r = 0.71, ***), SA (r = 0.74, ***) CA (r = 0.73, ***), SAD2 (r = 0.70,
***) and Vol (r = 0.70, ***) and moderately with MaN
 Plant Physiology Reports

(r = 0.67, **) and Width (r = 0.67, **). Leaf Number, an adapted from pot in bucket method used for waterlogging
another conventional parameter positively correlated with tolerance in lentil by Lake et al. (2021), who assessed the
TRL (r = 0.83, ***), NA (r = 0.81, ***), BP (r = 0.80, ***), shoot responses of lentil genotypes to waterlogging using
Peri (r = 0.80, ***), MeN (r = 0.74, ***), RT (r = 0.76, high throughput phenotyping protocol as an alternative to
***), SA (r = 0.81, ***) CA (r = 0.79, ***), SAD2 (r = 0.79, traditional methods.
***), Width (r = 0.76, ***) and Vol (r = 0.79, ***) and While traditional methods for assessing plant responses
moderately with MaN (r = 0.65, **). This shows that the to waterlogging have shown effectiveness, their efficiency
WIR traits directly playing a key role in survival of plants declines when dealing with a large number of genotypes,
under waterlogging conditions. Moreover, it was observed particularly in terms of repeatability and time-course
that the image-derived waterlogging induced root (WIR) observations. Therefore, persistent efforts are being made
traits showed a strong positive correlation existed among to develop non-invasive phenomics techniques for the
them, as clearly illustrated in the PCA biplot analysis. precise phenotyping of plants with desirable traits, aiming to
advance crop improvement (Langan et al., 2022). However,
the primary goal of the current research was to establish a
Discussion phenotyping protocol for WIR which has not been attempted
so far. The non-invasive phenotyping protocol for WIR
The affordable medium throughput root phenotyping involved the development of an affordable root image
protocol for assessment of waterlogging induced roots in acquisition platform and image analysis techniques using
cowpea involved optimisation of protocol for inducing open-source software. The intention was to streamline the
waterlogging stress, optimisation of protocols for non- assessment of cowpea genotypes for their waterlogging
invasive measurement that involved image acquisition, tolerance, with a particular focus on WIR image features
analysis, feature extraction, data analysis and interpretation that could demonstrate meaningful correlations with
which have been discussed below. growth traits. The imaging tools such as cameras (Bates
The stress imposition protocol is a prerequisite for any & Lynch, 1996), industrial digital lenses (Liu et al., 2020)
plant phenotyping method to differentiate the responses of and monochrome vision cameras (Seethepalli et al., 2020)
plants to environmental stresses (Awlia et al., 2016; Junker have been frequently employed in in-situ root imaging
et al., 2015; Langan et al., 2022). Many of the previous platforms (Zhao et al., 2022). Nonetheless, these equipment
studies carried out for waterlogging tolerance focused on options come with a high cost and are not well-customised
in situ shoot responses rather than root responses taking for extensive, large-scale applications (Cao et al., 2019)
into consideration feasibility. The current study focused on in waterlogging responses of plants. The method we have
assessing traits associated with waterlogging tolerance and optimized for quantifying WIR consisted of two distinct
one of these desirable traits, also known as phenes, is the steps, as described below.
development of adventitious roots induced by waterlogging The first step involved image acquisition, which
(Kumar et al., 2013; Olorunwa et al., 2022; Sathi et al., incorporates a smart phone placed on an overhead stand and
2022), which has been specifically referred to in this thesis operated by a laptop running a python program. This setup
as “Waterlogging-Induced Roots” (WIR). The development ensured the acquisition of images with the desired quality, a
of Waterlogging-Induced Roots (WIR) is a complex process, critical factor for the segmentation of roots and the extraction
making it a quantitative trait influenced by various factors. of features necessary for interpreting the responses of WIR
These factors encompass species, genotype, growth stage, in cowpea plants. Cost-effective image acquisition platforms
water temperature, as well as the duration and level of have been demonstrated for characterizing shoot traits (Cao
waterlogging. During the seedling stage, the emergence et al., 2019) and root traits in previous studies (Bontpart
of adventitious roots is believed to play a crucial role in et al., 2020; Rinehart et al., 2022).
conferring waterlogging tolerance (Kyu et al., 2021; Zhang The imaging platforms discussed in the study by Bontpart
et al., 2015). et al. (2020) faced a limitation due to the soil’s opacity,
Hence, it was necessary to design the stress imposition which constrained topological analysis by hiding sections
protocol in a way to ensure visibility of WIR for optimisation of the root system, resulting in an incomplete representation
of phenomics. The review of literature and a couple of of root topology. Additionally, in the study by Rinehart et al.
trial-and-error methods finally led to selection of pot in (2022), a limitation was observed where the root imaging
pot method described in the framework of WirPheno in process was performed destructively, leading to the loss of
this paper for imposition of waterlogging stress in cowpea certain portions of the root structure. In our current study,
at seedling stage. This method was customised to allow the roots emerged on the soil surface due to waterlogging,
acquisition of both the architectural traits and growth making them easily visible and facilitating non-destructive
patterns of WIRs effectively. The pot-in-pot method was image capture. Additionally, the hardware used in our
Plant Physiology Reports

study was cost-effective, with an approximate cost of ₹ protocol for WIR and to identify potential cowpea genotypes
2000, which stands in stark contrast to the costs associated that could be valuable genetic resources for enhancing
with other affordable imaging platforms, such as $7400, waterlogging tolerance.
as reported in previous studies by Rinehart et al. (2022). Efforts made in this study aligns with a growing interest

current study was ₹ 12,000 excluding the cost of laptop.

The net cost involved in designing the imaging tool in the in the study of root phenomics, which is increasingly
recognized as a pivotal component of crop breeding
This affordability makes our imaging platform particularly strategies (Chen et al., 2020; Liu et al., 2021). Hypotheses
accessible and cost-effective in line with the economic tested in this study revolved around the notion that there
considerations of Indian living standards. exists genotypic variation in WIR architecture among
In the second step, image analysis was executed using cowpea genotypes under waterlogging stress conditions, and
a combination of two open-source software: ImageJ and that the extracted image features of WIR could be employed
RhizoVision Explorer (RVE). The user-friendly software- for genotype classification. To test these hypotheses, the
features for thresholding and batch processing embedded initial experimental phase focusing on WIR phenotyping
in ImageJ facilitated analysis of all the images of WIR was carried out. This study unveiled significant variations
acquired through affordable imaging platform that was in the waterlogging-induced root traits of the 20 cowpea
meticulously designed in this study. The macro developed genotypes examined. In our current study, several WIR
for this purpose could process 600 images of WIR within image features were measured, and it was evident that
3–5 min resulting similar number of binary images clearly certain traits, including Total Root Length (TRL), Network
segmented from the background, which otherwise leads Area (NA), Convex Area (CA), Perimeter (Peri), Branching
to over or under estimation of root features. However, the Points (BP), Surface Area (SA), Volume (Vol), Maximum
WIR feature extraction was the challenge to be addressed No of Roots (MaN), and Median No of Roots (MeN),
in the next step of image analysis with binary images. To exhibited significant variations among the tested genotypes.
complete this task, we preferred the user-friendly features This indicates the importance of these root traits as potential
of RhizoVision Explorer over ImageJ due to certain inherent surrogates for assessing waterlogging tolerance in cowpea
limitations in the latter. Thus, the amalgamation of these genotypes non-invasively. This is highly evident from the
two-software enabled the extraction of image features from efficacy of traits such as root length (TRL) and root area
WIR images. This approach was supported by prior research, (NA) in differentiating waterlogging tolerant (CG121,
where a compatible combination of software, such as CG236, CG221, and CG041) and waterlogging intolerant
ImageJ + GiA Roots in barely (Khodaeiaminjan et al., 2023), (CG151, CG165, and CG091) genotypes. Additionally, WIR
Adobe Photoshop + Weka + RhizoVision Explorer in maize image features such as the median and maximum number
(Rinehart et al., 2022) and adobe photoshop + RootReader2D of roots could effectively differentiate between tolerant and
in rice (Clark et al., 2012) were employed to evaluate root intolerant genotypes when exposed to waterlogging. This
architectural traits. The combination of software for image finding is consistent with research conducted on cowpea
analysis described by Rinehart et al. (2022) and Clark et al. under waterlogging stress (Olorunwa et al., 2022), as well
(2012) comes with the limitation of requiring multiple as studies involving mung bean and black gram crops (Kyu
software tools, including subscription-based products like et al., 2021), where manual counting of adventitious roots
Adobe. In contrast, in current study, we utilized open-source similarly supported the indications of enhanced waterlogging
and freely available software, which are easily accessible tolerance. These findings provide valuable insights into
without additional costs. the diversity of root traits among different genotypes and
In the current research, we validated the effectiveness highlight the potential relevance of these traits in the context
of image segmentation using ImageJ and feature extraction of waterlogging tolerance. An investigation involving
with RVE software by comparing the data obtained with soybean genotype screening focused on efficient nutrient
those obtained using the image analysis grid of LemnaTec, uptake revealed a similar scenario, highlighting genotypic
resulting in high correlation coefficients (r = 0.99 and 1.00 variability in root traits (Liu et al., 2021; Salim et al., 2021).
for root area and convex hull as illustrated in (Fig. 6). The Principal Component Analysis (PCA) is a valuable
use of customized LemnaTec grids for extracting shoot statistical technique for selecting superior lines in breeding
features of various crops has been reported in studies by programs (Debnath et al., 2022). This approach streamlines
Rane et al. (2021) and for root features, as elucidated by decision-making by concurrently evaluating multiple traits,
Cardinal (2021). The feature extraction process in the with the aim of enhancing crop performance and resilience
previous step resulted in generation of huge set of data to particular adverse conditions, such as waterlogging
related to 27 different parameters. Subsequently, this dataset tolerance. PCA biplot analysis provided evidence that WIR
underwent statistical analysis to enhance the understanding image features generated through the phenotyping protocol
of the effectiveness of the cost-effective phenotyping successfully segregated cowpea genotypes into two distinct
 Plant Physiology Reports

groups (Fig. 7). This separation was further validated by tolerance in cowpea. This can be achieved by expanding
k-cluster analysis (Fig. 8). These results underscore the the study to encompass a larger pool of diverse genotypes,
effectiveness of the phenotyping protocol in differentiating facilitating a more comprehensive analysis of genetic
cowpea genotypes based on their levels of tolerance to variations and their impact on waterlogging tolerance.
waterlogging.
Supplementary Information The online version contains supplemen-
This interpretation derives further support from Pearson tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 40502-0​ 24-0​ 0789-1.
correlation analysis that revealed significant positive
association between the conventional parameters and Funding The funding was provided by Indian Agricultural Research
image derived parameters at p < 0.001 and p < 0.01 levels Institute New Delhi.
of significance (Fig. 10). A comparable correlation between
image features and conventional parameters was also
reported in prior studies of crops like lentil (Dissanayake References
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