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Industrial Crops & Products 146 (2020) 112162

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Industrial Crops & Products

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Quality classification of Jatropha curcas seeds using radiographic images and T

machine learning
André Dantas de Medeiros*, Daniel Teixeira Pinheiro, Wanderson Andrade Xavier,
Laércio Junio da Silva, Denise Cunha Fernandes dos Santos Dias
Department of Agronomy, Federal University of Viçosa, CEP 36570-900, Viçosa, MG, Brazil


Keywords: Efficient seed quality assessment methodologies are important for the seed industry. Advanced seed technology
High throughput analysis research requires the use of high productivity methods that provide detailed information on seed structural
Tissue density integrity and predict its physiological potential quickly and accurately. The aim of this study was to propose a
X-ray imaging method for predicting germination capacity and discriminate Jatropha curcas L. seeds regarding germination
Prediction of seed quality
speed and seedling vigor by combining automatic X-ray analysis and machine learning model. The study was
performed using automated analysis of radiographic images of seeds, obtaining a series of morphological and
tissue integrity descriptors. After the X-ray test, the seeds were submitted to physiological assessments. Based on
all individual seed descriptors, quality classes were created and LDA models were applied. Prediction of seed
viability, germination speed and seedling vigor resulted in an average of 94.36, 83.72 and 89.72% of correctly
classified seeds, respectively. High throughput X-ray image analysis can provide information needed to dis-
criminate individual Jatropha curcas seeds into different classes of quality, i.e., germination capacity, germi-
nation speed and seedling vigor. The methodology proposed can be used to discriminate between seed classes
quickly and robustly.

1. Introduction traits (Huang et al., 2015; Rahman and Cho, 2016; Xia et al., 2019).
Recent studies have recommended the use of the 2D X-ray technique
Jatropha curcas L. is an oilseed plant that stands out in the scenario combined with software analysis to assess seed physical characteristics
of growing demand for non-petroleum alternative fuels whose pro- of interest more accurately and faster (Leão-Araújo et al., 2019;
duction does not conflict with global food security (Steinbrück et al., Medeiros et al., 2018, 2019).
2019). Recently, this specie has also been studied for medicinal pur- However, one of the biggest challenges today is the development of
poses, presenting potential for the treatment of bacterial infections affordable tools that can determine in advance which seeds are able to
(Haq et al., 2019). In addition, J. curcas is resistant to various abiotic germinate and produce vigorous seedlings, providing fast, non-de-
stresses and can be grown on low fertility soils, which are unsuitable for structive and less subjective diagnosis of individual seeds. Recent ad-
conventional agriculture (Lama et al., 2018). vances in the field of digital image processing and artificial intelligence
Seed propagation is more common in this specie (Silva et al., 2018). have made this kind of approach possible.
Therefore, the selection of high-quality seeds for the establishment of Public domain software provides promising perspectives for ad-
crop fields is a fundamental part of the production process. The concept dressing this purpose by enabling the automation of seed radiographic
of seed quality is composed of several attributes, including genetic analysis (Medeiros et al., 2019). In addition, machine learning algo-
purity, health, viability, germination capacity and vigor (ISTA, 2019). rithms, especially Linear Discriminant Analysis (LDA) models, have
Some of this information related to seed viability, germination, and shown good results for seed classification when combined with imaging
vigor is difficult to obtain directly due to time and technological con- techniques (ElMasry et al., 2019; Sarigu et al., 2019, 2017).
straints (ElMasry et al., 2019). In this sense, the use of automatic analysis of radiographic images
Noninvasive optical technologies, such as X-ray testing, have been associated with machine learning models may offer a new perspective
successfully applied for non-destructive assessment of non-visible seed for classification of J. curcas seed performance. Thus, the method could

Corresponding author at: Department of Agronomy, Federal University of Viçosa, Av. P H Rolfs, s/n, Viçosa, 36570-900, MG, Brazil.
E-mail address: (A.D.d. Medeiros).
Received 18 September 2019; Received in revised form 20 January 2020; Accepted 22 January 2020
0926-6690/ © 2020 Elsevier B.V. All rights reserved.
A.D.d. Medeiros, et al. Industrial Crops & Products 146 (2020) 112162

be included in the production chain of this oilseed, saving time and TXT format.
resources, especially in the seed production process.
Thus, the main objective of this study was to propose a method to 2.3. Physiological analysis
predict J. curcas seed germination capacity and discriminate seeds re-
garding germination speed and seedling vigor by combining automatic After obtaining the radiographic images, the seeds were evaluated
X-ray analysis and machine learning model. for their physiological quality. For this, the germination test was con-
ducted on germination paper rolls (Germitest®) moistened with water
2. Materials and methods equivalent to 2.7 times the dry paper mass and kept in a germinator
(Mangelsdorf) at 25 °C for 12 days (Oliveira et al., 2014). The 800 seeds
2.1. Local and plant material were analyzed individually and daily for germination. Germinated
seeds were considered those with radicle greater than 2 mm. In the last
The plant material consisted of Jatropha curcas seeds harvested in count, the seeds were classified into germinated seeds and non-germi-
the 2018/2019 crop season in an experimental field located in Viçosa, nated seeds (those without root protrusion up to 12 days of assessment
Minas Gerais State, Brazil (20° 46′01.7″S 42° 52′05.6″ W). At least 20 and / or not viable). The seedlings generated were classified as normal
adult plants were randomly selected, and their fruits were harvested. (those that presented all well-developed structures) and abnormal
The seeds were extracted manually from the fruits and placed to dry (seedlings poorly developed or defective).
naturally in an uncontrolled environment (25 °C and 40 RH%) until
reaching the equilibrium moisture content (approximately 8%). 2.4. Seed classes

2.2. Digital X-ray acquisition and analysis According to the data obtained from the germination test, J. curcas
seeds were classified into different categories based on: i) germination
The seeds were submitted to internal morphology analysis by X-ray capacity; ii) germination speed and viability; and iii) seedling vigor.
technique. In total, 800 seeds were fixed on adhesive paper, in groups of Based on germination data, seeds were classified into two groups:
six seeds, in an orderly and equidistant manner. This procedure was Class 1 – germinated seeds; and Class 2 – non-germinated seeds. In addi-
performed to allow individual identification of each seed in subsequent tion, in a second classification, the germinated seeds were divided ac-
analyses, and each seed was analyzed individually. cording to the germination speed (time required to initiate root pro-
Radiographic images were generated by a Faxitron MX-20 model trusion). Then, three classes were tested: Class 1: rapid germination –
(Faxitron X-ray Corp. Wheeling, IL, EUA). The equipment was adjusted root protrusion up to 96 h after initiation of imbibition; Class 2: slow
to a voltage of 23 kV and the seeds were exposed to radiation for 10 s at germination - root protrusion after 96 h after initiation of imbibition;
a focal length of 41.6 cm. Image contrast has been calibrated to 16383 and Class 3: non-germinated seeds. The period of 96 h was defined based
(width) x 3124 (center). The images were saved in Tagged Image File on the accumulated germination curve for this experiment. At this time,
Format (TIFF) files and then analyzed. approximately 80% of the seeds were geminated. Finally, according to
Automated image analysis was performed using the PhenoXray seedling vigor, the germinated seeds were classified into two classes:
macro (Medeiros et al., 2019) developed for the software ImageJ® Class 1: normal – seeds that generated well-developed seedlings; and
( (Schindelin et al., 2015). Class 2: abnormal – seeds that generated seedlings showing absence,
The sequential analysis performed by the macro consisted of scale ca- underdevelopment or deformation of some essential structure (cotyle-
libration (39,6003 pixels / mm), segmentation and extraction of dif- dons, primordial leaves, roots).
ferent morphometric and tissue integrity descriptors of each seed con- For the analyses, the data set (800 seeds) was divided into two
tained in each image. subsets: a training set (n = 635 seeds), used to develop the dis-
In more detail, each opened image was duplicated and segmentation crimination models, and a validation set (n = 165 seeds), for validate
was done on the image copy. ImageJ® Threshold mode was used and the models developed (Table 2).
the Yen's automatic multilevel thresholding method was selected (Yen
et al., 1995) for segmentation. Then the selections in the binary image 2.5. Multivariate data analysis and machine learning
were redirected to the original image. Finally, the area of interest
(seeds) was analyzed using the “analyze particles” command, gen- Principal Component Analysis (PCA) was applied as a multivariate
erating 15 descriptors listed in Table 1. After processing, the analysis exploratory analysis technique to identify the hidden patterns in the
results were stored in a pivot table and automatically saved to a file in data obtained from X-ray analysis. Thus, the data obtained from

Table 1
List of descriptors generated by the analysis of radiographic images of seeds.
Abreviation Name descriptor Description

Area Area (mm2) Area inside the polygon defined by the perimeter.
Mean Mean Gray (gray·pixel−1) Average gray value within the selection.
StdDev Standard Deviation Standard deviation of the gray values.
Perim. Perimeter (mm) Length in millimeters of the outer limit of the selection.
Width Width (mm) Width of the smallest rectangle that encompasses the selection.
Height Heigth (mm) Height of the smallest rectangle that encompasses the selection.
Circ. Circularity Circularity = 4·π·Area/Perimeter2
IntDen Integrated density (gray·mm·pixel−1) The sum of the greyscale values in the particle
Median Median Median greyscale
Skew Skewness The third order moment about the mean
Kurt Kurtosis The fourth order moment about the mean
%Area Seed filling (%) Determined by dividing the area effectively filled with high-density tissue (grey levels above the initially defined threshold)
by the total area of each seed
AR Aspect ratio Aspect ratio = major axis/minor axis
Round Roundness Round = 4·Area/(π·major axis2)
Solidity Solidity Solidity = Area/ConvexArea

A.D.d. Medeiros, et al. Industrial Crops & Products 146 (2020) 112162

Table 2 and gray level of the images (e.g. Mean, IntDen, Median, %Area, Skew,
Final number of seeds for each class used in the training and validation data Kurt). Except for the asymmetry, the other descriptors related to the
sets. gray level of the radiographs tended to have the highest averages as-
Classification type Training set Validation set sociated with Class 1 (Germinated) seeds and the lowest averages as-
sociated with Class 2 (Non-germinated) seeds. This trend was main-
Class 1 Class 2 Class 3 Class 1 Class 2 Class 3 tained in the classifications based on germination speed and seedling
vigor, where seeds with better performance, i.e., Class 1 seeds, had
Germination 468 167 – 129 36 –
Speed of germination and 377 91 167 91 38 36 higher averages for the descriptors.
Seedling Vigor 414 221 – 108 57 –
3.2. Viable and non-viable Jatropha curcas seeds overview

radiographic analysis for all seeds were arranged in an X matrix The morphometric and tissue integrity data, obtained from the
(800 × 15), where the rows represented the observations (seeds) and analysis of radiographic images of the seeds, were submitted to PCA
the columns represented the variables. analysis (Fig. 1). The first three main components explained 79.88% of
Then, descriptors obtained from seed radiographs were used to the original variation between seed descriptors with 45.36, 26.51 and
develop multivariate discrimination models for classifying seeds into 8.01% for PC1, PC2 and PC3, respectively. As presented in the PCA
different categories based on their germination and seedling data. The scores, there was seed separation according to germination in the ger-
seeds were divided into training and validation sets (Table 2), and the minated (yellow) and non-germinated (blue) classes. These data show
Linear Discriminant Analysis (LDA) classification models were devel- that, from the assessment of the internal morphology of the seeds, it is
oped using the training set and the obtained models were validated possible to infer about their germination capacity. The parameters that
using the independent validation set, which was not used during the most contributed to the constitution of the first dimensions of the
construction of the models. For the development of the models using components were seed filling and tissue integrity (e.g. seed filling, gray
the LDA method, the data obtained from the radiographic images were median, gray mean, skewness – supplementary material), which de-
arranged in an X matrix (the predictors) and the data resulting from the monstrates the relationship of these characteristics with the physiolo-
Y vector germination test (response variable). In this study, the accu- gical quality of seeds. However, some non-germinated seeds were
racy of the LDA models was assessed by the percentage of correctly grouped with germinated seeds and vice versa (Fig. 1).
classified seeds. All statistical analyses were performed using the soft-
ware R 3.6.1 (R Core Team, 2019).
3.3. Discrimination models for seed classification
3. Results
Three different models were developed based on Linear
3.1. Seed internal morphology and physiological data Discriminant Analysis (LDA) aiming to classify seeds into different
classes, based on germination capacity, germination speed and viability
The averages of the morpho-anatomical parameters obtained and seedling vigor. The classifications were stored in the Y vector, and
through the automated analysis of the seed radiographs for each clas- the data obtained from the automatic analysis of digital seed radio-
sification type are presented in Table 3. For germination-based classi- graphs were stored in the matrix X.
fication, major differences in the averages between classes were ob-
served for most descriptors, especially those related to the distribution

Table 3
Averages followed by the standard deviation obtained for morpho-anatomical descriptors of Jatropha curcas seeds in different classes, based on germination capacity,
germination speed and viability and seedling vigor.
Classification type Descriptor

Class Area Mean StdDev Perim. Width

Germination 1 153.5 ± 11.28 169.6 ± 17.57 70.4 ± 6.03 59.4 ± 12.27 10.9 ± 0.45
2 140.4 ± 14.07 99.3 ± 33.04 66.6 ± 9.69 59.6 ± 9.22 10.1 ± 0.73
Speed of germination and viability 1 155.1 ± 10.86 173.2 ± 13.76 70.6 ± 5.78 60.9 ± 13.06 10.9 ± 0.44
2 147.9 ± 11.01 156.8 ± 23.1 69.4 ± 6.79 53.7 ± 6.19 10.7 ± 0.45
3 140.4 ± 14.07 99.31 ± 33.04 66.6 ± 9.69 59.6 ± 9.22 10.1 ± 0.73
Seedling Vigor 1 154.3 ± 10.94 172.3 ± 14.54 70.4 ± 5.74 60.0 ± 12.58 10.9 ± 0.44
2 142.6 ± 14.04 113.2 ± 38.51 67.5 ± 9.31 58.3 ± 9.3 10.3 ± 0.74
Height Circ. IntDen Median Skew
Germination 1 17.7 ± 0.84 0.59 ± 0.15 26114 ± 3606 195.1 ± 21.8 −0.91 ± 0.21
2 18.2 ± 1.22 0.52 ± 0.12 14231 ± 5794 98.6 ± 46.27 0.21 ± 0.57
Speed of germination and viability 1 17.8 ± 0.83 0.57 ± 0.15 26881 ± 2915 199.3 ± 17.46 −0.94 ± 0.17
2 17.4 ± 0.81 0.66 ± 0.09 23330 ± 4423 180.1 ± 28.4 −0.79 ± 0.26
3 18.2 ± 1.22 0.52 ± 0.12 14231 ± 5794 98.6 ± 46.27 0.21 ± 0.57
Seedling Vigor 1 17.7 ± 0.82 0.58 ± 0.15 26626 ± 3130 198.2 ± 18.23 −0.93 ± 0.17
2 18.0 ± 1.19 0.55 ± 0.13 16474 ± 6615 118.9 ± 54.21 −0.04 ± 0.66
Kurt %Area AR Round Solidity
Germination 1 −0.29 ± 0.37 97.9 ± 2.12 1.63 ± 0.07 0.61 ± 0.03 0.99 ± 0
2 −0.69 ± 0.67 90.0 ± 6.95 1.79 ± 0.15 0.56 ± 0.05 0.98 ± 0.01
Speed of germination and viability 1 −0.24 ± 0.35 98.3 ± 1.25 1.63 ± 0.07 0.61 ± 0.03 0.99 ± 0
2 −0.45 ± 0.38 96.6 ± 3.61 1.63 ± 0.08 0.62 ± 0.03 0.99 ± 0
3 −0.69 ± 0.67 90.0 ± 6.95 1.79 ± 0.15 0.56 ± 0.05 0.98 ± 0.01
Seedling Vigor 1 −0.25 ± 0.35 98.2 ± 1.34 1.63 ± 0.07 0.61 ± 0.03 0.99 ± 0
2 −0.66 ± 0.61 91.5 ± 6.86 1.75 ± 0.15 0.58 ± 0.05 0.98 ± 0.01

A.D.d. Medeiros, et al. Industrial Crops & Products 146 (2020) 112162

Fig. 1. PCA biplot obtained for seed morphometric data (n = 800) of Jatropha curcas showing two seed classes (germinated and non-germinated).

3.4. Discrimination between germinated and non-germinated seeds 76.00% accuracy, for training and validation data, respectively).
Based on the first two discriminant factors (LD1 and LD2), which
The discrimination model developed to classify seeds into germi- explained 95.52% and 4.48% of the total variance, respectively, the
nated and non-germinated was established to identify whether the in- seeds were grouped according to the three classes (Fig. 2A). Non-ger-
dividual seed had internal characteristics (assessed from images ob- minated seeds, which were located in the negative LD1 scores, were
tained in the X-ray spectral range) that affected its viability and mostly separated and well differentiated from the classes Fast germi-
germination capacity. The results of the prediction of seed participation nation and Slow germination. It is important to emphasize that the dif-
in the classes and the correctness general classification of the model are ference between seeds germinated rapidly and germinated slowly is
presented in Table 4 for the training data and the independent vali- very subtle, as these two classes comprises seeds that germinated. Thus,
dation. there were greater interactions between the classes Fast germination and
The LDA model obtained was highly accurate to distinguish ger- Slow germination due to the low variation between individual seeds in
minated and non-germinated seeds, with an accuracy of 94.8, 93.93% terms of their physical properties. Thus, the model is efficient to dis-
for training and independent validation data sets, respectively. tinguish seeds with germination potential from non-viable seeds, based
on the physical characteristics assessed by automatic analysis of
3.5. Seed discrimination based on germination speed and seed viability radiographic images.
The discriminant function included 15 descriptors. In Fig. 2C, the
Seeds were classified according to germination speed and seed first three columns show the averages of each descriptor by category.
viability into three groups: seeds that germinated up to 96 h (rapid Higher averages are shaded in blue and lower averages in red, with
germination); seeds that germinated after 96 h (slow germination); and significant values at the 5% level in bold. The R-Squared column shows
non-germinated seeds (not viable). the proportion of variation within each row that is explained by the
The LDA model obtained allowed to discriminate between these descriptor. In this measure, the five most discriminating descriptors
three classes with accuracy of 85.03 and 82.42%, for training and va- were mean (gray mean), median (gray median), skewness (asymmetry),
lidation data, respectively (Table 5). It was observed that the model IntDen (integrated density) and %Area (seed filling). Seeds with fast
accuracy was higher for distinguishing fast germinating seeds and non- germination, in general, presented higher averages in the dis-
germinated seeds compared to slow germinating seeds (61.02% and criminating factors listed, except Skewness, which presented negative

Table 4
LDA Confusion Matrices for participation in 'Germinated' and 'Un-germinated' Jatropha curcas seed classes in training and validation sets.
Feature Training set (n = 635) Validation set (n = 165)

Germinated Non-Germinated %Correct Germinated Non-Germinated %Correct

Germinated 463 28 94.30 124 5 96.12

Non-Germinated 5 139 96.53 5 31 86.11
Overall correct classification (%) 94.8 93.93

A.D.d. Medeiros, et al. Industrial Crops & Products 146 (2020) 112162

Table 5
LDA Confusion Matrices for participation in Jatropha curcas seed classes based on germination speed and viability in training and validation sets.
Feature Training set (n = 635) Validation set (n = 165)

Fast Slow Non-Germinated %Correct Fast Slow Non-Germinated %Correct

Fast 366 53 15 84.33 87 16 3 82.08

Slow 9 36 14 61.02 3 19 3 76.00
Non-Germinated 2 2 138 97.18 1 3 30 88.24
Overall correct classification (%) 85.03 82.42

Fig. 2. LDA graph obtained from Jatropha curcas seeds morpho-anatomical data and classification according to germination speed. LDA ordination diagram with the
arrangement of individuals in their respective groups (A). LDA biplot with predictors and the centroid of the inserted classes (B). Averages, contribution and
significance of the descriptors for class differentiation (C). The training data were used to draw the graph.

values (Table 3). The opposite was observed for non-viable seeds. et al., 2019). Optical technologies, able to non-destructively access
these changes, generate time and resource savings (Rahman and Cho,
3.6. Discrimination between normal and abnormal seedlings 2016). The 2D X-ray technique is well-established for seed analysis,
indicated by the International Seed Testing Association (ISTA, 2019),
The LDA model constructed to classify J. curcas seeds capable of and for its simplicity of execution and lower cost can be considered
producing normal seedlings and seeds that produced abnormal seed- attractive from a practical standpoint compared to other methods more
lings revealed high accuracy. The results demonstrate an overall clas- advanced, that is, magnetic resonance imaging, microtomography, high
sification accuracy of 89.76 and 89.69% in training and validation cost multispectral equipment.
datasets, respectively (Table 6). These results point to a direct re- However, when the analysis of 2D X-ray images is performed vi-
lationship in the internal morphology of the seeds and the ability to sually, the risks of error increase considerably due to the subjective
generate well developed seedlings. interpretations of analysts, in addition to the great time spent on the
From the data of the morpho-anatomical parameters of the seeds analysis (Xia et al., 2019). The high-performance x-ray analysis pro-
(Table 3), it can be observed that the seeds that originated normal posed in this paper is a valuable alternative to reduce these difficulties.
seedlings presented higher averages in the descriptors related to the Some descriptors generated from the automated analysis of radio-
gray level of the images, that is, the images of the seeds had higher graphic images proved to be important in the discrimination between
radiopacity. seed classes regarding physiological quality, such as mean and median
of gray, integrated density, asymmetry and filling. These parameters
4. Discussion were correlated with the physiological quality of seeds of other species,
such as Brachiaria ruziziensis (Medeiros et al., 2019) and Leucaena leu-
Changes in morpho-anatomical properties of Jatropha curcas seeds cocephala (Medeiros et al., 2018).
may occur due to several factors such as physiological disturbances The variations observed in the descriptors in the range of the X-ray
during maturation, insect predation, tissue deterioration, incidence of electromagnetic spectrum between individuals are attributed to
pathogens, damage caused by improper storage conditions, among changes in seed morphological and anatomical properties. Compared to
others (Moncaleano-Escandon et al., 2013; Silva et al., 2018; Suresh the higher quality seeds (germinated, fast germinating and well-formed

Table 6
LDA Confusion Matrices for partition into Jatropha curcas seeds classes that originated normal and abnormal seedlings.
Feature Training set (n = 635) Validation set (n = 165)

Normal Abnormal %Correct Normal Abnormal %Correct

Normal 164 8 95.35 45 5 90.00

Abnormal 57 406 87.69 12 103 89.57
Overall correct classification (%) 89.76 89.69

A.D.d. Medeiros, et al. Industrial Crops & Products 146 (2020) 112162

seedlings), the lower quality seeds had lower resistance to X-ray pas- for seed classification. In cowpea, using single-seed spectral signatures
sage generating images with lower gray levels (Table 3). The mean gray extracted from VideometerLab3® multispectral images, different linear
level of the seed is directly related to its tissue density (Medeiros et al., discriminant analysis (LDA) models classified seeds for germination
2018), higher gray means are related to seeds with higher amounts of with a precision percentage greater than 96% (ElMasry et al., 2019).
reserves, lower incidence of physical damage and higher physical in- Seeds with rapid germination speed and vigorous seedlings are ex-
tegrity of the tissue. In addition to the gray mean, other descriptors tremely important for the agricultural system. These seeds and seed-
studied as% Area and IntDen are closely related to the filling and lings are less subject to biotic and abiotic stresses, which ensures proper
density of tissue of reserves available in J. curcas seed. These char- establishment of the plant stand in the field (Finch-Savage and Bassel,
acteristics are physiologically important, linked to a good initial seed- 2016). Thus, finding a good relationship between easily measurable
ling establishment in the field (Finch-Savage and Bassel, 2016) and descriptors and complex attributes of physiological quality are valuable
related to other properties of interest to the industry, such as oil content findings for the advancement of seed science. As observed in the present
(Pinto et al., 2018). work, descriptors of J. curcas seed internal morphology were efficiently
It is important that the proposed method for seed quality assessment used in the elaboration of seed quality prediction models. Seed com-
is efficient in distinguishing high quality seed from low quality seed panies can use these descriptors to make decisions regarding the des-
(Huang et al., 2015; Kotwaliwale et al., 2014; Rahman and Cho, 2016). tination and disposal of seed lots, which saves time and resources
In this sense, the focus of the present work was to use the information during processing and quality control of seed production.
extracted from radiographic images of seeds to predict the capacity of Fig. 4 highlights the gray levels assessed in the seed radiographs as
these seeds to germinate and produce normal seedlings. The LDA being associated with physiological characteristics related to viability
models developed in this research for the different J. curcas seed classes and vigor of J. curcas seeds. It shows the original radiographic image,
showed a high degree of discrimination between them, as can be seen the seed gray histogram and the color 3D representation of the tissue
briefly in Fig. 3. density in gray per pixel. Warm colors are associated with high-density
The results revealed that the method is highly satisfactory to predict regions, while cool colors are associated with low-density regions.
germination capacity and to explore the possibility of discriminating Seeds of higher physiological quality (Fig. 4D) have higher gray values,
the vigor of individual seedlings. In general, the classical statistical which means higher tissue density, and give rise to better developed
approach of LDA achieves maximum class discrimination by calculating seedlings. On the other hand, seeds with lower gray values give rise to
the optimal transformation that minimizes distance within the class and abnormal seedlings (Figs. 4B and C) or do not have the ability to ger-
maximizes the distance between classes simultaneously (Maione and minate (Fig. 4A), as observed in this experiment. This representation
Barbosa, 2018). However, we inquired that other machine learning only visually reinforces what has been observed in previous results,
models (e.g. artificial neural networks) could be examined in future which point to a strong link between these characteristics.
research to increase overall classification accuracy, especially for ger- Thus, the present work presented promising results on the re-
mination rate related classes. It is important to emphasize that this is a lationship between seed physical characteristics and physiological
pioneering work, combining high yield analysis technique of radio- quality, but this relationship cannot always be easily visualized, since
graphic images with machine learning models to predict seed physio- seeds as biological objects are multi-parametric and multicomponent
logical quality. nonlinear complex systems (Arkhipov et al., 2019), which are influ-
Recent research using seed X-ray image analysis has found a good enced by other factors, mainly environmental. However, this work of-
correlation between the descriptors of seed filling and tissue integrity fers new perspectives for further study of the association of imaging
with seed germination and seedling length in Brachiaria ruziziensis techniques with physiological, biochemical and genetic processes in
(Medeiros et al., 2019), Campomanesia adamantium (Leão-Araújo et al., seeds.
2019), Leucena leucocephala (Medeiros et al., 2018), among other High-throughput analysis of J. curcas seed radiographs combined
works. In this sense, descriptors generated using the PhenoXray macro with LDA machine learning models proved to be a fast, non-destructive,
in digital radiographs were correlated with physiological attributes of non-subjective and highly accurate method for classifying seeds for
brachiaria seeds (Brachiaria ruziziensis). The results revealed high cor- viability and vigor.
relations (r > 0.94) between the physical descriptors of the radio-
graphic image with physiological quality aspects, and that seed lots
with lower physiological quality presented lower gray levels (called 5. Conclusion
relative density) than the good quality lots (germination > 60%)
(Medeiros et al., 2019). Other imaging techniques have also been ap- High throughput X-ray image analysis provides information needed
plied in previous work in combination with machine learning models to discriminate Jatropha curcas individual seeds into different quality
level classes quickly and robustly. LDA-based machine learning models
were adequate to discriminate seed classes with high accuracy.

CRediT authorship contribution statement

André Dantas de Medeiros: Methodology, Formal analysis,

Writing - original draft. Daniel Teixeira Pinheiro: Conceptualization,
Data curation. Wanderson Andrade Xavier: Data curation. Laércio
Junio da Silva: Supervision. Denise Cunha Fernandes dos Santos
Dias: Project administration.

Declaration of Competing Interest

Fig. 3. Accuracy of LDA models developed for different classification scenarios The authors declare that they have no known competing financial
of Jatropha curcas seeds in classes, based on germination and development interests or personal relationships that could have appeared to influ-
parameters of seedlings, using morpho-anatomical parameters of seeds. ence the work reported in this paper.

A.D.d. Medeiros, et al. Industrial Crops & Products 146 (2020) 112162

Fig. 4. Radiographs of Jatropha curcas seeds with their respective histograms and 3D color representation of the gray levels of each pixel. Non-germinated seed (A),
slow germinating seed and abnormal seedling (B), embryo-damaged seed and abnormal seedling (C) and fast germinating healthy seed and normal seedling (D).

Acknowledgment Leucaena leucocephala seeds. Ciência e Agrotecnologia 42, 643–652.

Medeiros, A.D., SILVA, L.J., Pereira, M.D., Oliveira, A.M.S., Dias, D.C.F.S., 2019. High-
The authors are grateful to the Coordenação de Aperfeiçoamento de throughput Phenotyping of Brachiaria Grass Seeds Using Free Access Tool for
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