Overcoming the phenotyping gap - experiences with the LemnaTec-Scanalyzer 3D platform investigating

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)