FOLLOWING UP THE DEVELOPMENT OF WHEAT KERNEL BY MULTISPECTRAL IMAGING B. JAILLAIS1 AND D. BERTRAND2 1. INRA
BIA, NANTES, FRANCE. 2. DATA_FRAME, NANTES, FRANCE.
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INRA AND DATA_FRAME INRA, CEPIA division BIA Unit
BIBS Platform
MS
Microscopy
CHEMOTYPING Analysis of polysaccharides Sophie Le Gall, Poster n째9
NMR
PHENO CHEM
PHENOTYPING Analysis of internal structure of seeds Phenodays 2012
Data_frame Consulting in Chemometrics 2
WHEN HARVESTING? • Wheat is mature • Kernel is well filled • Yellow color of kernel At the right time! • Depends of area of cultivation, climate, cultivar… New tool to characterise changes in wheat kernel! Phenodays 2012
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GREEN TO YELLOW SPIKE? Changes : •Grain growth •Molecular and morphological changes inside •Grain filling •Change of colour about 20 DAA •Accumulation of carotenoids •Far-red fluorescence from chlorophyll decreasing •Blue-green fluorescence from hydroxycinnamic acids decreasing
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ENDOSPERM DEVELOPMENT Phase 1 : Formation of the first endosperm nucleus Phase 2 : Cellularization of the layer of endosperm cells lining the embryo sac Phase 3 : Completion of meristemic activity Phase 4 : Attainment of maximum dry weight Phase 5 : Harvest ripeness Phase 1
Phase 2
Phase 3
Phase 4
Phase 5
Adapted from Evers (1970)
Embryo and endosperm increase in mass and volume (all phases) Parenchymatous pericarp degenerates from center to the outside (P2, P3) Endosperm enlargement (P2, P3) Accumulation of storage components (P4) Drying (P5) Phenodays 2012
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OBJECTIVE • Application of multivariate imaging to study the state of physiological maturity of a wheat kernel HOW? • By using 8 wavelengths to characterise tissues • By obtaining RGB images for each wavelength • By merging all the RGB images for one sample • By processing pixel-vectors of spectral responses
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MATERIALS and METHODS
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SAMPLE COLLECTION • Cultivar : Récital • Grown under controlled conditions (INRA Rennes)
• 10 kernels per each development time • 14 Development times (in DAA) : 4
5
6
8
11
14
15
16
17
18
21
24
25
28
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SAMPLE PREPARATION Estimated time : 10 s
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MUWI (MUltiWay Imager) NIR 950 nm
CCD colour camera (+ cooling device)
NIR 875 nm
microscope
Red Green
Black box
Blue
4x 8-leds Blocks
UV 400 nm UV 370 nm
Sample
UV 360 nm
8 lightning conditions x 3 RGB channels = 24 image-plans for each sample
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EXPOSURE TIME VS WAVELENGTH • Exposure time required to obtain well-exposed images varies with wavelength • Imaging device requires the possibility to address different values of time • For the same aperture, needed time is : • About 7s for UV lights • Between 0.02 and 0.1 s for Visible lights • About 0.02s for infrared lights Estimated time : 30 s Phenodays 2012
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IMAGES IN FALSE COLOURS IR875red
IR875gre
IR875blu
UV360re d
UV360gre
UV360blu
gre enred
gree ngre
gree nblu
re d re d
red gre
re d blu
UV400re d
UV400gre
UV400blu
IR950red
IR950gre
IR950blu
blue re d
blue gre
blue blu
UV370re d
UV370gre
UV370blu
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DATA PREPROCESSING Unfolded Image Cube
Image 位
z=24
p
IR 950 n
IR 875 Red
z
Green Blue p
UV 360 UV 370
Image Cube
n
UV 400 Unfolding of multivariate image : Each pixel is an individual caracterised by a vector of Phenodays 2012 24 intensities (i.e. spectral responses)
nxp
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IMAGE PCA Unfolded Image Cube
Scores
z=24
Refolded Scores z=10
p PC1 n PC2
PCA
Refolding
PC3 PC4 PC5 PC6 PC7 PC8
nxp
nxp
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IMAGES FROM PCA Channe l #1
Channe l #2
50 100
50
PC1
100
150
150
200
200
250
250
300
300
350
350
400
400
450
450
500
500
100
200
300 400 Channe l #3
500
600
700
50 100
PC2
100
200
300 400 Channe l #4
500
600
700
200
300
500
600
700
50
PC3
100
150
150
200
200
250
250
300
300
350
350
400
400
450
450
500
500 100
200
300
400
500
600
700
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PC4
100
400
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SEGMENTATION s e uil > 0
x 10
-4
4 X: 217 Y: 199 Inde x: 0.0002628 RGB: 1, 0.0625, 0
X: 530 Y: 204 Inde x: -7.801e -005 RGB: 1, 1, 0
2 0 -2 -4 -6
X: 300 Y: 430 Inde x: 0.0003858 RGB: 0.75, 0, 0
-8 -10
s euil > 0.0002
> « Red » pixels Estimated time : 2 mn
> « Yellow » pixels s euil > 0.0003
> « Dark red » pixels Phenodays 2012
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RESULTS
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SEQUENCE of RGB IMAGES UV 360nm
5 DAA
6 DAA
8 DAA
15 DAA
18 DAA
28 DAA
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SEQUENCE of RGB IMAGES UV 400nm
5 DAA
6 DAA
8 DAA
15 DAA
18 DAA
28 DAA
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EVOLUTION OF PC1 SCORE-IMAGE
4 DAA
6 DAA
11 DAA
14 DAA
16 DAA
18 DAA
25 DAA
28 DAA
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THEORY/EXPERIMENT
Grain growth
Grain filling
Anthesis
Grain drying Days
4 DAA
6 DAA
11 DAA Phenodays 2012
21 DAA
28 DAA 21
EVOLUTION OF NUMBER OF PIXELS
Each point is the mean of the 10 kernels at the same time
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CONCLUSIONS
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CONCLUSIONS (1)
The MUWI system makes it possible to quantify the degree of maturity of wheat kernels.
4 DAA
6 DAA
11 DAA
14 DAA
16 DAA
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18 DAA
25 DAA
28 DAA
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CONCLUSIONS (2) • Rapid (about 3 mn/sample), simple, robust, low-cost method • No need to human inspection • No limit for image processing only for sample presentation : bottleneck of phenotyping • It’s possible to increase the throughput of analysis by development of robot • Extraction of parameters (shape, colour, texture) from multispectral images is easy chemometrics processing Phenodays 2012
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THANK YOU FOR YOUR ATTENTION !
AUTOMATE PHOTO
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