The document describes a flood-and-drain based hydroponics system for root phenotyping. The system uses acrylic tubes filled with glass beads as the rooting substrate and an automated watering system to flood and drain the tubes on a set schedule. It measures various root system parameters such as length, surface area, diameter and architecture. The system is inexpensive at 7150 PLN to analyze 48 plants simultaneously and takes approximately 14 days to complete an experiment, providing a low-cost, high-throughput method for root phenotyping.

1. The installation and experiment
guidelines of a flood-and-drain
based hydroponics system
Słota Michał, Tkaczyk Oliver
Department of Genetics, University of Silesia
Phot.M.Slota

2. Flood-and-drain based system (Slota et al. 2016)
Slota M., 2017
▪ root architecture phenotyping method adequate for accurate, efficient and
cost-effective analysis of root system,
▪ automated water supply system,
▪ non-invasive root growth observation,
▪ dedicated for subsequent root image acquisition with a reduced background
noise.
Flood-and-drain based system

3. Slota M., 2017Flood-and-drain based system
Publication in the Bulletin of Public Information (BIP)Slota M, et al. Plant Methods 2016

4. 1
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1. Air pumps.
2. Transformer with programmable timer.
3. Afferent pump placed in the tank with
the culture medium.
4. Distributors of 12 outlets.
5. Distribution pipes
6. Acrylic tubes filled with substrate.
7. Efferent pump.
8. Draining system.
9. PLC controller
Prototype of the system
The layout of the sub-assemblies:
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5
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Slota M., 2017Flood-and-drain based system

5. Slota M., 2017
Cover tubes
Bulk container
for 24 tubes
Distribution
pipes
Water
distributors
Transformers for
water pumps
Air pump
Medium
drainage pipes
UV lamp
Air pipes
Box with wiring
Power strip
Supply pipes
Tank with a
medium
1 2 3 4
Flood-and-drain based system
Example of the implementation of the system: Plant Breeding and Acclimatization Institute (IHAR), Radzików, Poland.

6. Slota M., 2017Flood-and-drain based system
A non-invasive (A) and invasive (B) visualization of root system of a 14-day-old barley seedling.
Transparent acrylic
tubes filled with
glass beads
Up to 48 plants per
one module
Automated
irrigation system
Continuous sensing
of medium parameters
(pH, temperature, ion
concentration)
Regulation of medium
composition, flow rate
A B

7. Experiment maintenance
Germination of the seeds
▪ Seeds are sterilized in 5% solution of sodium hypochlorite (Sigma, Art. no 71696) for 15
min and washed with sterile water, 3 times for 5 min.
▪ Seeds are planted on sterile square plastic 120x120 mm Petri plates (Gosselin, Art. no
BP124-05) filled to the middle with sterile moist filter paper.
▪ Seeds are incubated in 4°C for 24h and then the plates are transferred for next 48h to
incubator to 24-25°C.
(Phot. M. Slota)
Slota M., 2017

8. Experiment maintenance
Implantation of the seedlings
▪ The appropriate amount of soda-lime glass beads (900 g of beads per one tube), 24
acrylic tubes for one module, and other necessary laboratory glassware should be
autoclaved in advance .
▪ Acrylic tubes are filled with soda-lime glass beads leaving a top space of approx. 5 cm.
▪ Seedlings are implanted into the acrylic tubes and replenished with additional portion
of soda-lime glass beads leaving a top space of approximately 1 cm.
(Phot. M. Slota)
Slota M., 2017

9. Preparation of the tubes
▪ Acrylic tubes should be properly labeled with the genotype indication.
▪ Seedlings in tubes should be kept in high moinsture conditions and moderate heat for
first 12h.
▪ Tubes are inserted into opaque cover tubes and settled within previously prepared bulk
container. Bulk container should be placed above the medium container and draining
system for effective work.
(Phot. M. Slota)
Slota M., 2017Experiment maintenance

10. Assembling of the system
▪ Distributor pipes (1) are attached to pipe pegs (2) attached to the sides of the cover
tube (3).
▪ Each distributor pipe is joined with supply pipe (4) topped with adequate distributor (60
ml/min) of 12 outlets (5).
▪ Two supply pipes are attached to two of the three water connections of the afferent
pump and screw sealed with the sleeve.
1
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3
4
(Phot. M. Slota)
Slota M., 2017Experiment maintenance

11. Course of the experiment
▪ Afferent pump is placed on the bottom of opaque 30 liter tank filled with culture
medium ad connected to atransformer controlled by a control timer.
▪ Efferent pump is placed on the bottom of bulk container.
▪ All the tubing connections should be checked and afferent and efferent transformers
schould be pluged to a power source.
▪ System is placed in the greenhouse under controlled conditions.
(Phot.M.Slota)
Slota M., 2017Experiment maintenance

12. 4rd day of barley plants growth after seedlings transplantation
(Phot. M. Slota)
Slota M., 2017Experiment maintenance

13. 14th day of barley plants growth after seedlings transplantation
(Phot.M.Slota)
Slota M., 2017Experiment maintenance

14. Termination of the experiment
▪ After 14 days of growth experiment is terminated (roots reach the length of 50 cm).
▪ Distriburtor pipes are carrefully disconnected and acrylic tubes are removed.
▪ Plants are extracted from acrylic tubes after moistening the substrate and pouring the
water through bottom drainage opening with a syringe (100 ml) or a beaker.
(Phot.M.Slota)
Slota M., 2017Experiment maintenance

15. Cleaning of the root systems
▪ Plant roots are rinsed with tap water above the sieve to remove the remaining glass
beads.
▪ Extracted seedlings should be necessarily kept wet.
(Phot.M.Slota)
Slota M., 2017Experiment maintenance

16. Preparation for WinRHIZO analysis
▪ Plants are stored in properly marked 50 ml conicals, immersed in deionized water until
the time of the root system scanning using WinRHIZO system.
▪ Time preceding the root system analysis should be reduced to a minimum.
(Phot.M.Slota)
Slota M., 2017Experiment maintenance

17. Root system scaning using WinRHIZO system (Regent Instrument)
▪ Roots of single plant are cut using a sharp scissors to separate all embryonic roots.
▪ Roots (1-2 embryonic roots at once) are placed on Regent’s water-proof trays.
▪ Roots are positioned to avoid overlapping lateral roots and ensure equal distribution.
(Phot.M.Slota)
Slota M., 2017Experiment maintenance

18. (Phot.M.Slota)
Non-invasive analysis Invasive analysis
Slota M., 2017Experiment maintenance

19. Image processing
▪ Parameters generated by WinRHIZO software include: total length of the root system
[cm], root system surface [cm2], root system volume [cm3], root diameter [mm].
▪ Images can be saved in .jpeg or .tiff format and exported the data files in ASCII easily
readable by many programs.
Slota M., 2017Experiment maintenance

20. Course of the experiment
Slota M., 2017Experiment maintenance
Stages of the experiment Duration
Preparation
Seeds sterilization 30 min.
Preparation of tubes and substrate 2h
Medium preparation 2h
Plant growth
Seed incubation [4oC] 24h
Seed incubation [24oC] 72h
12- days of growth
Data acquisition
and analysis
Experiment termination 2h
Roots scaning (48 plants) 6h
WinRHIZO analysis 4h
Data analysis (WinRHIZO) 2h
Data analysis (SmartRoot) 8h

21. Slota M., 2017Experiment maintenance
Course of the experiment
Experiment timeline 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Seeds sterilization
Preparation of tubes and substrate
Medium preparation
Seed incubation [4oC]
Seed incubation [24oC]
12- days of growth
Experiment termination
Roots scaning (48 plants)
WinRHIZO analysis
Data analysis (WinRHIZO)
Week 1 Week 2 Week 3
END
45 min each 3rd day

22. Slota M., 2017Experiment maintenance
Data output Time-consumingBalance of costs
SUMMARY Flood-and-drain based system for root phenotyping
Measured parameters:
▪ length, surface area and
volume of a root system,
▪ number of lateral roots,
▪ surface area and volume of
lateral roots,
▪ location and density of the
lateral roots,
▪ diameter of embryonic and
lateral roots.
Modifable parameters:
▪ medium composition,
▪ medium flow,
▪ abiotic stress application.
System setup:
7150 PLN/ 1663 EUR
for 48 plants
(2 x 24 plants, 2 possible
medium combinations)
Consumables:
▪ medium components,
▪ water, electricity
(water pumps- 24 W x 4 x 6
min/day, air pumps- 4 W x 2 x
90 min/day),
▪ glass beads exchange.
Experiment setup (48 plants):
4,5h
Medium preparation and refill:
45 min each 3rd day
System check and maintenance:
15 min each day
Experiment termination:
2h
Roots scaning:
6h
WinRHIZO analysis:
4h
TOTAL experiment duration:
14 days

23. Slota M., 2017Summary
Flood-and-drain based system – protocols (Slota, Tkaczyk 2017)

24. Slota M., 2017Summary
The overview of the pipeline for the preparation of phenotyping experiments.

25. Summary
Salt stress
Dehydration (drought)
Temperature stress (high/low temp.)
Toxic elements (heavy metals, Al3+, B)
Mineral nutrition (e.g. nutrient deficiency tests)
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Possible applicatons
Slota M., 2017

26. References
▪ Hoagland DR, Arnon DI. The water-culture method for growing plants without soil. 2nd ed.
California: California Agricultural Experiment Station Circular; 1950. p. 347.
▪ Lobet G, Pagès L, Draye X. A novel image-analysis toolbox enabling quantitative analysis of root
system architecture. Plant Physiology 2011. 157: 29-39.
▪ Slota M, Maluszynski M, Szarejko I. An automated, cost-effective and scalable, flood-and-drain
based root phenotyping system for cereals. Plant Methods 2016:12(34); DOI: 10.1186/s13007-
016-0135-5.
▪ Slota M, Maluszynski M, Szarejko I. Root Phenotyping Pipeline for Cereal Plants. In: Jankowicz-
Cieslak J, Tai T, Kumlehn J, Till B J (Eds.): Biotechnologies for plant mutation breeding:
protocols. Springer International Publishing (2017); ISBN 978-3-319-45019-3.
▪ Wang MB, Zhang Q. Issues in using the WinRHIZO system to determine physical characteristics
of plant fine roots. Acta Ecologica Sinica 2009. 29: 136-138.
▪ Publication in Bulletin of Public Information (BIP): P.412973 (16.01.2017)
Slota M., 2017

27. Michał Słota
Department of Genetics, Faculty of Biology and Environmental Protection
University of Silesia, Jagiellonska 28, 40-032 Katowice(michal.slota@us.edu.pl)
Slota M., 2017