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Overcoming the phenotyping gap - experiences with the LemnaTec-Scanalyzer 3D platform investigating drought tolerance in barley Kerstin Neumann, Nils Stein, Andreas Graner, Benjamin Kilian Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany LemnaTec HTS-Scanalyzer 3-D-platform NIR VIS Fluo
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Kerstin Neumann, Nils Stein, Andreas Graner, Benjamin Kilian
Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany
Overcoming the phenotyping gap - experiences with the LemnaTec-Scanalyzer 3D platform investigating
drought tolerance in barley
VIS
Fluo
NIR
LemnaTec HTS-Scanalyzer 3-D-platform
312 carriers with RFID-chip
Automated imaging and watering
Barley core set of spring barley varieties
Modern german cultivars: Old german cultivars
• Ack. Bavaria (1903)
• Apex (1983)
• Heils Franken (1895)
• Isaria (1909)
• Perun (1988)
• Pflugs Intensiv (1921)
• Sissy (1990)
• Trumpf (1973)
• Barke (1996)
• Beatrix (2004)
• Djamila (2003)
• Eunova (2000)
• Streif (2007)
• Ursa (2002)
• Victoriana (2007)
• Wiebke (?)
+ Barke and Morex (DH) from pre-experiments 18 cultivars
IPK involved in Crop.Sense.net project, Start 09/2010
Analysis of RGB Top and Side views with Image J Plugin HTPheno from DAS 22 to DAS 57
First Core-Set experiment
- 312 pots: 16 core Set lines (9+9) + Barke and Morex (DH) (6+6)
- pots on system since sowing
- Two treatments (well watered and drought stress)
- stress after 4 weeks of growth water stop for 2 weeks
- rewatering, recovery period for 2 weeks
- manual harvest at DAS 58: fresh + dry weight, plant height, tiller number
- image configuration: Top view, 2 Side views - 0° and 90°
Weigh in soil
First Core-Set experiment
Daily watering, imaging every 2nd day
Analysis
HTPheno Plugin for ImageJ (Hartmann et al. 2011)
Image
retrieval Morphology
Analysis
result Object
segmentation
Object
extraction Region
definition
Correlations within cultivars Correlations over all plants
dig
ital
bio
mas
s
Fresh weight in g
0.752
dig
ital
he
igh
t
Plant height in cm
0.922
First Core-Set experiment
dig
ital
bio
mas
s
Fresh weight in g
Dig.Bio = SQR (SideView0°*SideView90°*TopView)
New water tank
Open pots
Spoon
Adjusting basic equpiment
Watering system Pots
Rubber sleeve
Blue sand cover (aquaria) Soil surface
Cover to reduce background noise
Adjusting basic equpiment
Adjusting basic equpiment
Plant supports
greenhouse greenhouse
modified
Professional produced
3 Core-Set experiments after adaptation of experimental setup in 2011
•New plant supports, blue sand cover
•Longer stress phase (18 days instead of 14)
•More side view images (7: 0°-180°)
• First full experiment finished July, 25th
• Second finished September, 19th
• Third will be ready November, 13th
• sowing of next experiment 3 days before harvest of old one
Currently running experiment
Imaging growth over time
14 23 27 38 45 48 58
Apex – Contr.
Apex – Stress no watering
18 days stress period
rewatering
DAS 14 23 27 38 45 48 58
Apex – Contr.
Apex – Stress no watering
18 days stress period
rewatering
DAS
GS1 Kerstin Neumann, Benjamin Kilian
R2 = 0.95
Improved correlation
dig
ital
bio
mas
s Fresh weight in g
0.75
Experiment (old supports)
Fresh weight in g
dig
ital
bio
mas
s Dig.Bio: SQR(SV*SV*TV)
Minus 55% Minus 16%
Digital biomass Digital height
Influence of drought stress
Genotypic variation in Core-Set
Influence of drought stress
Growth under drought stress
New Image analysis pipeline for maize system ( poster)
Adaptation for barley in progress may even give better results
Fresh weight
Dig
ital
bio
mas
s (n
ew p
ipe
)
stress regrowth
Zoom fluctuation challenge calibration with blue markers
Plant height with fluctuation Plant height corrected
Example for blue marker correction
Outlook
Completing Core-Set testing
Statistical analysis of all three experiments with HTPheno and new developed pipeline of image analysis group for VIS
Analysing fluorescence images and later NIR
Increasing system from 312 pots to 520
Testing of association mapping panel
Acknowledgments
Ingo Mücke (technician for LemnaTec-systems)
Alexander Entzian and Christian Klukas (Image analysis group)