Seminar: Novel methods and challanges in plant phenotyping

1. NOVEL METHODS AND CHALLANGES
IN PLANT PHENOTYPING
AUTHOR: MICHAŁ SŁOTA

2. PHENOTYPIC VS. GENOTYPIC DATA ACQUISITION
Słota M., 2012 1.
High throughput techniques:
✓ next generation sequencing,
✓ microarrays,
✓ bioinformatics.
27 000 genes Phenome = gene x
environment
34 000 proteins Possible interactions = ?
[Integr8 - A.thaliana Genome Statistics]
PHENOTYPING = HIGH THROUGHPUT PLANT PHYSIOLOGY (?)
✓ automated image acqusition
✓ robotics/ automated sensoring
✓ bioinformatics/ image analysis
(Furbank and Tester, 2011)

3. PHENOMICS
GENOMICS
PROTEOMICS
METABOL
-OMICS
TRANSCRIPT
-OMICS
Słota M., 2012 2.

4. High throughput phenotyping is essential for:
▪ functional analysis of specific genes;
▪ forward and reverse genetic analyses;
▪ production of new plants with beneficial characteristics;
▪ screening for the desirable lines:
✓ mutant populations,
✓ mapping populations,
✓ breeding populations,
✓ germplasm collections.
Plant phenotyping - the comprehensive assessment
of plant complex traits such as growth, development,
tolerance, resistance, architecture, physiology, ecology, yield
and the basic measurement of individual quantitative
parameters that form the basis for the more complex traits.
(Furbank and Tester, 2011)
Słota M., 2012 3.

5. [www.arabidopsis.org]Słota M., 2012 4.

6. Cumulative growth in the number of sequences deposited in International Nucleotide
Sequence Database Collaboration database (Karsch-Mizrachi et al., 2012).
Słota M., 2012 5.

7. Cumulative growth in the genome’s number deposited in International Nucleotide
Sequence Database Collaboration database (Karsch-Mizrachi et al., 2012).
Słota M., 2012 6.

8. Installation Maximal capacity/ month Plants
ShootTraits
ABER IPC 850 ●
FZJ SCREEN Chamber 2500 ○
FZJ SCREEN House 250 ●
HAS SSDS 150 ●
HMGU expoScreen 100 ●
HMGU sunScreen 50 ○
INRA Phenodyn/Phenoarch 1680 ●
INRA Phenopsis 1510 ○
INRA PPHD 1800 ●
IPK APPP 396 (●)/ 4608 (○) -
RootTraits
FZJ SCREEN Root LP 72 ●
FZJ SCREEN Root SP 1800 ○
HAS RSDS 100 ●
UNOTT MicroCT ~10 ○
UNOTT Root Trace 400 ○
UNOTT SCREEN Glasshouse 250 ●
UNOTT Vertical Confocal ~10 ●/○
Metabol.
ABER FTIR/NMR 4400 ●/○
ABER Micro Raman ~60 ●/○
ABER TGA-py GC/MS 150 ●/○
IPK MP 768 ●/○
Field
INRA Diaphen 100 plots ●
UNOTT SCREEN Field 20 ha ●
Total month capacity:
5648 large plants (●)
10868 small plants (○)
Słota M., 2012 7.[www.plant-phenotyping-network.eu]

9. a) Mendel’s garden (Augustinian Abbey, Brno),
b) Mendel’s phenotyping instrument: microscope
Gregor Mendel (1822-1884) Pisum sativum
[www.zlgc.seu.edu.cn;www.mendel-museum.com]
Time period: 1856- 1863’ (7 years)
Plant material: 29 000 pea plants (Pisum sativum)
Plot: 2 ha monastery garden
Traits tested: 7 traits (7 different loci, each possesing 2 alleles)
(color and seed smoothness, color of the cotyledons, color of the
flowers, shape of the pods, color of the unripe pods, position of
flowers and pods, height of the plants).(Butler, 2009)
Słota M., 2012 8.

10. 1900’
2011’
Gartons Limited
'recording the action
of the reproductive
organs on the living
plant by the aid of
the camera and the
microscope'
LemnaTec
ScanalyzerField
Discovery Platform
'automated field
phentoyping systems
allowing digital
phenotyping of a
multitude of crops
and small trees'
Słota M., 2012 9.
[www.lemnatec.com][www.commons.wikimedia.org]

11. Słota M., 2012 [http://www.iupui.edu]
Bunsen’s spectroscope (1859’) AAS spectrophotometer,
Perkin-Elmer Model 403 (1955’)
NIR absorption spectroscopy (1985’)
0
10
20
30
40
1870s 1930s 1980s
Spinach Fe content [mg per 100g]
(Fullerton-Smith, 2007)
[www.commons.wikimedia.org]
10.

12. Breeding
Agricultural
production
Biodiversity
assessment
Functional
genomics
Horticultural
production
Climate
change
IDENTIFICATION
OF HERITABLE TRAITS
FIELD ENVIRONMENTPLANT GROWTH STATUS
(Walter ta al., 2009)
Słota M., 2012 11.

13. PLANT CULTURE
(organic/soil, hydroponic, aeroponics etc.)
DATA COLLECTING
(digital camera images, high resolution
scanning, microscope imaging)
DATA ANALYSIS
(noise reduction, segmentation, filtering
counting/measuring, statistical analysis)
Słota M., 2012 12.(Furbank and Tester, 2011)

14. HIGH THROUGHPUT
HIGH RESOLUTION
University of Nottingham;
MicroCT (High Resolution X-ray
micro-Computed Tomograph). »
« National Institute for Agricultural
Research (INRA) in Montpellier;
High Throughput Plant Phenotyping
Platform (PPHD).
Słota M., 2012 13.

15. IN-VITRO CULTURES
SCREENING
Słota M., 2012 14.
GROWTH CHAMBER/
GREENHOUSE ASSAYS
FIELD EXPERIMENTS
✓ highly controlled
conditions,
✓ high throuput,
✓ high capacity,
✓ easy imaging,
- adapted only for
small plants.
✓ controlled
conditions,
✓ high throuput,
✓ adapted for
medium and
large plants,
- high costs,
- amount of data.
✓ high capacity,
✓ natural
conditions,
- high costs,
- complex imaging,
- extremely high
amount of data.

16. Quality
Trait
Analysis INSTALLATION SPECIFICITY
ROOT
Agar Soil
SHOOT
Small
plant
Large
plant
TISSUE/
METABOLITE
-
FIELD
EXPERIMENT
-
▪ root biomass,
▪ root growth,
▪ root structure.
▪ shoot biomass,
▪ growth,
▪ structure,
▪ photosynthesis,
▪ water relations.
Słota M., 2012 15.[www.plant-phenotyping-network.eu]

17. COLOUR IMAGES (5)
Plant area, volume, mass, structure,
phenology
Senescence, relative chlorophyll
content, pathogenic lesions
Seed yield, agronomic traits
NEAR IR IMAGING (1-4)
Tissue water content
Soil water content
FAR IR IMAGING
Canopy, leaf temperature,
water use, salt tolerance
Słota M., 2012 16.
HYPERSPECTRAL IMAGING (6)
Carbohydrates, pigments and protein
CARBON ISOTOPE RATIO
Transpiration efficiency,
photosynthetic pathway (TDL/MS)
FTIR IMAGING SPECTROSCOPY
Cellular localisation of metabolites
(sugars, protein, aromatics)
FLUORESCENCE IMAGING (7)
Physiological state of photosynthetic
machinery
(Fiorani et al., 2012)

18. SCREEN Root ’Root carousel’
Słota M., 2012 17.
Installation for: small plants (seedings)
Environmental monitoring: temperature, humidity, light, intensity/quality
Parameters: root growth, root structure, shoot growth,
shoot structure
Capacity: >1800 plants
Experiment duration: 4-6 weeks (Nagel et al., 2009)

19. Phenoarch, Lemnatec
Installation for: medium/large plants
Environmental monitoring: temperature, humidity, light, soil water content
Parameters: growth, transpiration, growth rate, leaf area
transpiration, biomass, 3D architecture
Capacity: 1680 plants
Experiment duration: 90 days
Słota M., 2012 18.(Neumann et al., 2012)

20. Phenoarch, Lemnatec
Słota M., 2012 19.
PHENOTYPING PLATFORM: LemnaTec Scanalyzer 3D
AIM OF STUDIES: drought tolerance in old and modern barley cultivars
PLANT MATERIAL:
Old german cultivars
• Ack. Bavaria (1903)
• Apex (1983)
• Heils Franken (1895)
• Isaria (1909)
• Perun (1988)
• Pflugs Intensiv (1921)
• Sissy (1990)
• Trumpf (1973)
+ Barke and Morex (DH) from pre-experiments, 18 cultivars
Modern german cultivars:
• Barke (1996)
• Beatrix (2004)
• Djamila (2003)
• Eunova (2000)
• Streif (2007)
• Ursa (2002)
• Victoriana (2007)
• Wiebke (?)
(Neumann et al., 2012)

21. Scanalyzer Field
Installation for: multitude of crops, small trees
Environmental monitoring: CO2, humidity, wind, light, temperature
Parameters: plant heigh, coverage, leaf area index, N
content, transpiration (FIR)
Capacity: 10 x 40 m field
Experiment duration: up to 6 months
Słota M., 2012 20.
Field growth
monitoring
instalation in
Jülich.
(Sirault et al., 2009)

22. Scanalyzer Field
AIM OF STUDIES: Screening for osmotic component of salinity tolerance in
cereals using infrared thermography.
PLANT MATERIAL: durum wheat commercial varieties
PLATFORM: field trial (5m x 2m plots per genotype)
Imaging: ThermaCAM SC660 IR camera
Słota M., 2012 21.(Sirault et al., 2009)

23. EXPERIMENT REPRODUCIBILITY REQUIRES:
✓ data objectively described in a mathematical, easily digitized and searchable
format (using ontologies),
✓ information on how the experiment was carried out (plant material, growth
conditions) ,
✓ standardization of the employed phenotyping techniques
(Furbank and Tester 2011)
(Poorter et al., 2012)
Meta analysis of Leaf area of
Arabidopsis thaliana Col. plants
grown with the same protocol, the
same seed stock and the same soil in
growth rooms of nine different
laboratories. »
Słota M., 2012 22.

24. Słota M., 2012 23.
32 750 PLN

25. [www.lemnatec.com]Słota M., 2012 24.

26. Low-tech vehicle equipped with infrared temperature and reflectance sensors
/U.S. Arid Land Agricultural Research Center (ALARC), Arizona/
▪ built of a pair of used bicycles fixed to a large metal frame and platform,
▪ guided and position-recorded via global positioning systems (GPS),
▪ deploys sensors that can quantify few plant traits,
▪ works on time scale of a few seconds per plot.
[http://www.trivalleycentral.com]
Measured parameters:
✓ plant height,
✓ infrared temperature,
✓ water evaporation.
Słota M., 2012 25.
« ALARC phenotyping
low-tech vehicle .

27. Słota M., 2012 26.
▪ Butler, J.M. 2009. Fundamentals of Forensic DNA Typing, Elsevier Academic Press,
Burlington, p. 224
▪ Fiorani, F. , U. Rascher and S. Jahnke. 2012. Imaging plants dynamics in heterogenic
environments. Current opinion in biotechnology 23: 227-235
▪ Fullerton-Smith, J. 2007. The Truth About Food. Bloomsbury Publishing, London.
▪ Furbank, R.T. and M. Tester. 2011. Phenomics--technologies to relieve the phenotyping
bottleneck. Trends Plant Sci. 16(12): 635-44
▪ Karsch-Mizrachi, I., Y. Nakamura, G. Cochrane. 2012. The International Nucleotide
Sequence Database Collaboration. Nucleic Acids Res. 40: D33–D37
▪ Nagel, K.A., B. Kastenholz, S. Jahnke, D. van Dusschoten and T. Aach. 2009. Temperature
responses of roots: impact on growth, root system architecture and implications for
phenotyping. Functional Plant Biology 36: 947-959
▪ Neumann, K., N. Stein, A. Graner, C. Klukas, A. Entzian and B. Kilian. 2012. Non-destructive
phenotyping using the high-throughput LemnaTec-Scanalyzer 3D platform to investigate
drought tolerance in barley, European Cereals Genetics Co-operative Newsletter 158-160.
▪ Poorter, H., F. Fiorani, M. Stitt, U. Schurr, A. Finck, Y. Gibon, B. Usadel, R. Munns, O. Atkin,
F. Tardieu and T.L. Pons . 2012. The art of growing plants for experimental purposes: a
practical guide for the plant biologist. Funct. Plant Biol. 39(11) 821-838
▪ Sirault, X.R.R., R.A. James and R.T. Furbank. 2009. A new screening method for osmotic
component of salinity tolerance in cereals using infrared thermography. Functional Plant
Biology 36: 970-977

28. QUESTIONS & DISCUSSION