Seminar presentation entitled 'Advances and limitations of a non-invasive methods for the phenotyping of plant root systems'

1. Advances and limitations of
a non-invasive methods for the
phenotyping of plant root systems
Słota Michał
Department of Genetics, University of Silesia
Phot.M.Slota

2. Słota M., 2015INTRODUCTION
Slota et al. 2015 (in review)

3. Słota M., 2015
Fiorani et al. 2012
RGB
imaging
NIR
imaging
INTRODUCTION
Thermal
imaging
1 2 3

4. Słota M., 2015
Techniques for non-invasive imaging of plants in soil
Phenotypic data can be obtained by detecting reflected, transmitted or emitted
electromagnetic radiation, or magnetic resonance properties of nuclei.
3D structural as well as physiological traits can be acquired non-invasively by
positron emission tomography (PET), magnetic resonance imaging (MRI) and
Micro Computed Tomography (MicroCT).
Method Description Resolution Application Studies
MRI
(Magnetic Resonance
Imaging)
Water (1H)
mapping
200-500 µm
1-600 s
Morphometric
parameters in 3D,
water content
B. vulgaris,
H. spontaneum,
bean, sugar beet
PET
(Positron Emission
Tomography)
Radiotracer
mapping
1-2 mm,
10 s, 20 min
Transport
partitioning,
flow velocty
H. spontaneum,
B. vulgaris
MicroCT
(Micro Computed
Tomography)
X-ray digital
radiography
<100 µm
hours
Morphometric
parameters in 3D,
grain quality
O. sativa,
T. aestivum
INTRODUCTION
(Li et al. 2014)

5. Słota M., 2015
Magnetic Resonance Imaging (MRI)/ Nuclear Magnetic Resonance (NMR)
▪ imaging technique used in radiology to investigate the anatomy and
physiology of the living organisms,
▪ MRI scanner is actually a long coil of superconducting wire (magnet) and RF
(Radio frequency) transmitters that create localized electromagnetic fields,
which push some of these protons out of alignment. When the radio
transmitters are turned off the displaced protons flip back into alignment,
generating a radio signal that is picked up.
IMAGING TECHNIQUES
An image from Damadian's MRI Patent (1974).
1) The magnetic field is used to
align hydrogen protons in the body.
2) Radio frequency waves are
absorbed by the protons and then
emitted as a signal.
3) A radio frequency coil
(transceiver) picks up the signal
and transmits it to the computer.
The computer processes the data
and an image is generated.
[http://www.bangscience.org/]

6. Słota M., 2015
x-ray Tomography/ Micro–Computed Tomography (microCT)
▪ non-destructive imaging technique that uses x-rays to provide three-
dimensional images of the internal structure of an object,
▪ There are two types of imaging setups:
1) X-ray source and detector can be stationary during the scan while the
sample/animal rotates.
2) clinical CT scanner, where the sample is stationary in space while the X-ray
tube and detector rotate around.
▪ The gray levels in a CT slice image correspond to X-ray attenuation, which
reflects the proportion of X-rays scattered or absorbed as they pass through
each voxel (volume element).
IMAGING TECHNIQUES
[www.mayo.edu/]
« Schematic diagram of
a micro-CT scanner. The
specimen is mounted on
a rotating stage and is
illuminated by a X-ray
beam.

7. Słota M., 2015
Positron Emission Tomography (PET)
▪ functional imaging technique that produces a 3D image of functional
processes in the living organism,
▪ the system detects pairs of gamma rays emitted indirectly by a positron-
emitting radionuclide (tracer),
▪ tracer with a short-lived positron-emitting radionuclide of carbon, oxygen,
nitrogen, or fluorine is injected into the living organism. The activity of such a
compound is measured throughout the target organs by detectors.
IMAGING TECHNIQUES
[www.medchem.web.ne.kr/]
« Schematic illustration
of an annihilation
reaction (A) and the
subsequent coincidence
detection (B).

8. Słota M., 2015
Positron Emission Tomography (PET)
IMAGING TECHNIQUES
(Hubeau and Steppe 2015)
Overview of the most commonly used
short-lived positron-emitting
radionuclides in biological research.
a- Half-life of the radionuclides.
b- Distance which the emitted positron travels on
average in water before annihilating with an
electron. This is an important factor in
determining the resolution of PET.
Radio-
nuclide
t1/2 (min)a
Mean
travel
distance
in water
(mm)b
11C 20.4 1.1
13N 9.96 1.5
15O 2.03 2.5
18F 109.8 0.6

9. Słota M., 2015
(a) NMR image of a Hordeum vulgare plant grown in a pot with a volume of 1.3 L for 44
days. Roots in the inner 50% of the soil volume are colour-coded yellow, roots in the
outer 50% blue (b) Idem for a Beta vulgaris plant 48 days after sowing. The developing
storage root is colour-coded red. (Poorter et al. 2012)
APPLICATION IN RESEARCH
NMR: ’Pot size matters’
NMR image

10. Słota M., 2015
(Pietruschka el al. 2012)
APPLICATION IN RESEARCH
Combining MRI and PET data
Coregistration of MRI and PET data workflow.

11. Słota M., 2015APPLICATION IN RESEARCH
Combining the NMR and PET data
Roots of a maize plant growing in soil
visualized by co-registered MRI–PET
images consecutively obtained on days
16 and 17 after sowing, respectively
[b-c]. Data for a second maize plant
growing in sand [d-g]. Temporal
profiles of 11C radioactivity of the tip of
root in ROIs shown in (d) and (g) [f-h].
(Jahnke et al. 2009)

12. Słota M., 2015
Wheat cv. Gregory with even
supply of phosphate (left)
compared with a patch of
phosphate applied at 8
μM between the blue beads
(right). »
(Flavel et al. 2012)
Values followed by the same superscript letter within a column are not significantly
different (P > 0.05). LSD, least significant difference.
APPLICATION IN RESEARCH
MICROCT: P metabolism

13. Słota M., 2015
P treatment
Root length [mm]
Micro-CT (diameter ∼250 μm) WinRhizo (diameter >280 μm)
Control 2860a 3120a
Diffuse 2960a 4230a,b
Banded 3580a 4570b
LSD 1067 1152
Comparison of root-length measurements
of wheat plants obtained using micro-CT
and WinRhizo.»
Comparison of root-length measurements
of wheat plants obtained using micro-CT
and WinRhizo.
(Flavel et al. 2012)
Values followed by the same superscript letter within a column are not significantly
different (P > 0.05). LSD, least significant difference.
APPLICATION IN RESEARCH
MICROCT: P metabolism
»

14. Słota M., 2015APPLICATION IN RESEARCH
MICROCT: hydropatterning of root development
MicroCT-generated images of maize seed- lings grown through a macropore of air
(I and K) or a continuous volume of soil (J). Root tissue is false-colored in white,
and soil is false-colored in brown. (Bao et al. 2014)

15. Słota M., 2015
MRI and CT images of a bean root system in a
medium soil filled pot. The root system segmented
from the same CT image (b) as in on a voxel size of
56 × 56 × 56 μm3. Shows the same root system
imaged one day later with MRI (c) and a voxel size of
333 × 333 × 1000 μm3 with the last value representing
the vertical dimension. »
Root traits calculated from CT and MRI root
images of the volumes.
Small pots (I.D. 34 mm) Medium pots (I.D. 56) Large pots (I.D. 81)
Measurement modality CT MRI
Win-
RHIZO
CT MRI
Win-
RHIZO
CT MRI
Total root length [mm] 2775 2910 4012 2787 4602 4855 6632 10496
Percent root length of
WinRHIZO [%]
69 73 57 95
APPLICATION IN RESEARCH
MICROCT and MRI comparison
»

16. ▪ High costs ($$$).
▪ Low throughput.
▪ Labour-intensive (data analysis).
▪ It requires to establishspecialized
facilities
Słota M., 2015SUMMARY
What are the pros and cons?
▪ High precision (up to <10µm).
▪ Acquiring a 3D root architecture
of a soil-grown plant.
▪ Wide range of applications.
(2-Tesla MRI at Forschungszentrum Jülich)

17. Słota M., 2015
(Downie et al. 2014)
SUMMARY
What are the other applications?
Applicability of imaging techniques to root:rhizosphere interactions
(x, low usage to xxx, highly suitable)
Current research topics:
▪ Impact of compaction on roots.
▪ Visualization of water distribution in bulk and rhizosphere soils.
▪ Root interaction with fertilizer granules.
▪ Effect of intercroping on root system architectures.

18. Słota M., 2015
(Nagel el al. 2012, FZJ Jülich)
SUMMARY
Non-invasive imaging – is there a proxy?
Correlation of visible root length with root biomass.

19. Słota M., 2015SUMMARY
Non-invasive imaging – is there a proxy?
VS.
VolumeGraphics rendering WinRHIZO
Volume [mm2] 102,01 121,5
Area [mm2] 1148,67 1320,65
Convex Hull Volume [mm3] 45759,4 -
Depth [mm] 79,38 -
Width [mm] 50,1811 -

20. Slota M., 2015
LITERATURE
Bao, Y., Aggarwal, P., Robbins, N. E., Sturrock, C. J., Thompson, M. C., Tan, H. Q., &
Dinneny, J. R. (2014). Plant roots use a patterning mechanism to position lateral root
branches toward available water. Proceedings of the National Academy of Sciences,
111(25), 9319-9324.
Fiorani, F., Rascher, U., Jahnke, S., & Schurr, U. (2012). Imaging plants dynamics in
heterogenic environments. Current opinion in biotechnology, 23(2), 227-235.
Flavel, R. J., Guppy, C. N., Tighe, M., Watt, M., McNeill, A., & Young, I. M. (2012). Non-
destructive quantification of cereal roots in soil using high-resolution X-ray tomography.
Journal of Experimental Botany, err421.
Jahnke, S., Menzel, M. I., Van Dusschoten, D., Roeb, G. W., Bühler, J., Minwuyelet, S., &
Schurr, U. (2009). Combined MRI–PET dissects dynamic changes in plant structures
and functions. The Plant Journal, 59(4), 634-644.
Hubeau, M., & Steppe, K. (2015). Plant-PET Scans: In Vivo Mapping of Xylem and
Phloem Functioning. Trends in plant science, 20(10), 676-685
Li, L., Zhang, Q., & Huang, D. (2014). A review of imaging techniques for plant
phenotyping. Sensors, 14(11), 20078-20111
Nagel KA Putz A Gilmer F et al . 2012. GROWSCREEN-Rhizo is a novel phenotyping
robot enabling simultaneous measurements of root and shoot growth for plants grown
in soil-filled rhizotrons. Functional Plant Biology 39, 891–904.
Poorter, H., Bühler, J., van Dusschoten, D., Climent, J., & Postma, J. A. (2012). Pot size
matters: a meta-analysis of the effects of rooting volume on plant growth. Functional
Plant Biology, 39(11), 839-850.