Protocol

Authors

Francisco M. Padilla¹, Eric Visser¹, Liesje Mommer^1,2

AUTHOR AFFILIATIONS

¹Radboud University Nijmegen, Institute for Water and Wetland Research, Experimental Plant Ecology, P.O. box 9010, 6500 GL, Nijmegen, The Netherlands

²Nature Conservation and Plant Ecology group, Wageningen UR, P.O. box 47, 6700 AA, Wageningen, The Netherlands

OVERVIEW

This protocol describes how root distributions of plants and vegetation can be measured non-destructively. Root production and loss can be monitored over time using below ground transparent minirhizotron tubes. Roots grow to the outer surface of such tubes, and images of those roots can be taken and analysed for the production and loss of roots, also in relation to the position in the soil.

Also see related protocols:

Root distribution in soils I. Root core sampling and destructive pot harvests

Using WINRhizo and Photoshop to determine root length, diameter and branching

BACKGROUND

In science, roots have received far less attention than their aboveground counterparts despite their key role in soil resource acquisition and anchorage in soils. Moreover, the biomass in roots may be similar or even exceed the biomass above-ground in several ecosystems (Jackson et al. 1996; Mokany et al. 2006). It is with good reason that roots are referred to as the hidden half. They are out of sight living in the soil and it is very laborious to get them in hand. Even then, it is rarely possible to determine them to species level by visual inspection.

Despite these methodological challenges, roots are being studied. Root responses to nutrients and water (Hutchings and de Kroon 1994; Hodge 2004) are exemplary for the plasticity of plants. Root interactions among plant species and soil biota are receiving greater and greater interest. This protocol helps in setting up a good root sampling scheme in mesocosms (i.e., larger containers occupied by multiple plants typically being grown for longer periods of time) or the field. Roots from these systems can be studied nondestructively by taking root images from small, transparent, so-called minirhizotron tubes or destructively by washing (for the latter, see protocol Root distribution in soils I. Root core sampling and destructive pot harvests).

MATERIALS/EQUIPMENT

• Minirhizotron tubes – e.g. ESACRIL (extruded acrylic tubes) with outer diameter 70 mm, inner diameter 64 mm, or follow instructions from root scanner manufacturer.
• Root scanner – CI-600 Root Scanner, CID Inc., Camas, WA, USA
• WinRhizoTron software (Regent Instruments Inc, Canada)
• Image processing software (e.g., Microsoft Paint, Adobe Photoshop, Corel Photo-Paint)

UNITS, TERMS, DEFINITIONS

Root length density is the total root length per volume of soil – (m dm^-3) or per observed area through minirhizotron tubes – (m m^-2) .

Root standing biomass is root mass per square metre of soil surface (g m^-2; alternatively, root length per square meter of soil surface can be used, m m^-2)

Root lifespan (days) is the time span between root birth and death. Take into account that roots, while still visible through minirhizotron tubes, might actually be dead.

Root lifespan (days) is the time span between root birth and its disappearance.

Root length production rate (m day^-1 m^-2) is length of new roots produced over two consecutive censi.

Root length loss rate (m day^-1 m^-2) is length of roots gone over two consecutive censi.

PROCEDURE OF MINIRHIZOTRON PLACING & WINRHIZOTRON ANALYSES

This procedure consists of three steps. (1) The installation of the minirhizotrons in the soil; (2) The use of a CI-600 scanner to make and save images of the roots growing around a minirhizotron tube; (3) The use of WinRhizo Software to analyse the root distribution around the minirhizotron tube, at a specific moment in time but also during longer term censi, thus following root system development and turn-over.

 

Setup of minirhizotron tubes.

• Set up minirhizotron tubes either horizontally or inclined in the soil. The angle can vary from 30^∘ to 60^∘, although vertical setup of tubes is also possible. In general, the research question to be addressed, the root system morphology and the soil physical characteristics will determine the length and inclination of the minirhizotron tubes.
• Avoid disturbing soil and roots as much as possible at minirhizotron setup. In mesocosm and potted research, place tubes before plantation. In nature, either use a soil auger (recommended) to insert the tubes or dig a deep trench (much more disturbing). In these latter case, allow time for soil stabilization and root colonization (up to 1 year, depending on the system).

Figure 1. Setup of minirhizotron tubes in mesocosms.

 

Scanning with the CI-600 scanner (follow manufacturer instructions).

• Connect the measuring rod to the scanner. Connect the USB cable to the scanner, but not to the computer yet, then use an Allen wrench to tighten the screws on the security bracket and install the measuring rod cap.
• Slide the scanner inside the calibration tube. It might be recommended to wrap the calibration tube with aluminum foil to prevent light inside the calibration tube.
• Insert the USB interface cable into de computer port and wait about ten seconds for the automatic calibration to complete its process. During the recognition or initialization period, the scanner may give an error message. If this happens, it will be necessary to disconnect the USB cable, remove the CI-600 scanner from the calibrating tube and carefully rotate the scanner to the other hard stop.
• Insert the scanner into the minirhizotron tube. Make sure to place the scanner in the minirhizotron tubes always in the same position. For this purpose, make a mark on the scanner and at the top of the minirhizotron tube and make them match every time you are scanning.
• Open the software CanoScan LiDE that comes with the CI-600 unit and scan images at color mode at 300 dpi. 400 or 600 dpi would be desired, but may result in large files.
• Save the image as TIFF or JPG file in the desired folder. The file name should follow this scheme:
  • Experiment_TXXX_LXXX_date_time_SSS_Gat.tif
  • with: experiment name of up to 25 characters without spaces, tube number of a 3 digit number (001, 002) preceded by the letter T, location number of a 3 digit number (001, 002) preceded by the letter L (it refers to location inside the tube, from the top to bottom), date of scanning (mmddyyyy), time of scanning (hhmmssAM/PM), session number of a 3 digit number (001, 002) used to differentiate images over time, and data gatherer (3 characters).
  • Example: Gradient_T001_L001_01122009_173045PM_001_FMP.jpg

 

WinRhizoTron Analysis.

• Start WinRhizoTron and go to Calibration » Method and select Intrinsic calibration.
• Go to Analysis » Parameters and make sure that the ICAP naming scheme is ticked. Select Load analysis of current image and leave the other options as default.
• Go to Image » Origin and select disk.
• Go to Image » Acquire and select the image you want to process.
• When asked about the result data file, always name it as done with the image (same name but different extension only; txt) and keep it in the same folder where the image is.

Figure 2. Example of a minirhizotron image before analysis.

 

• Select the interactive measurement mode and start digitizing root segments.

Figure 3. Former image after digitizing every single root segment.

 

• In subsequent analyses over time, to import root segments already digitized, go to Roots » Import traced roots, browse the folder where the previous image (session) is, and select the .PAT file corresponding to the previous image. Path files are created automatically every time you analyze a new image on WinRhizoTron, and keep records of the traces you have drawn.
• To mark a traced, living root as dead or gone in subsequent analyses, select the target root by clicking on it, go to Roots » Edit root properties, and activate Dead or Gone if the root segment you selected appears to be dead or gone.
• This method can provide the root length density at any moment in time, and root production and loss when the roots are monitored during subsequent periods in time.

Figure 4. Example of the kind of data obtained through minirhizotron observations over time.

NOTES AND TROUBLE SHOOTING TIPS

Processing of minirhizotron images is quite time-consuming. Sampling interval depends on the ecosystem and species involved. In general, a one-month period between consecutive scans seems appropriate for most systems in the growing season. In winter, two-month periods or even no scanning is recommended.

Sufficient replication of minirhizotron tubes in natural systems is needed if the soil environment is heterogeneous. Typically variability is relatively large, so choose a sufficient number of minirhizotron replicates. If time is limited, sufficient replication of short tubes should have priority over replication of deeper tubes as most of the fine root biomass will likely concentrate in the upper soil centimeters.

Options to reduce the amount of work

There is no need to analyze the whole minirhizotron image. To save time and work, crop all the images in a consistent manner (same location, size) and give results per analyzed surface area. Sub-images on the same image or a fringe corresponding to the top part of the minirhizotron tube can be cropped with image processing software.

Related protocols:

Root distribution in soils I. Root core sampling and destructive pot harvests

Using WINRhizo and Photoshop to determine root length, diameter and branching

LINKS TO RESOURCES AND SUPPLIERS

Minirhizotron tubes: Vink kunststoffen, Didam, The Netherlands

CID root scanner, http://www.cid-inc.com/ci-600.php

WinRhizotron software, http://www.regentinstruments.com/

Nijmegen Phytotron – https://www.ru.nl/science/plant/research/facilities/

LITERATURE REFERENCES

Bouma T.J., Nielsen K.L., Koutstaal B. 2000. Sample preparation and scanning protocol for computerised analysis of root length and diameter. Plant and Soil 218: 185-196.

Hendrick, R.L. and K.S. Pregitzer. 1996. Applications of minirhizotrons to understand root function in forests and other natural ecosystems. Plant and Soil 185: 293-304.

Jackson R.B., Canadell J., Ehleringer J.R., Mooney H.A., Sala O.E. and Schulze E.D. 1996. A global analysis of root distributions for terrestrial biomes. Oecologia 108:389-411.

Joslin, J.D. and M.H. Wolfe. 1999. Disturbances during minirhizotron installation can affect root observation data. Soil Science Society of America Journal 63: 218-221.

Johnson, M.G., et al. 2001. Advancing fine root research with minirhizotrons. Environmental and Experimental Botany 45: 263-289.

Metcalfe, D., P. Meir, and M. Williams. 2007. A comparison of methods for converting rhizotron root length measurements into estimates of root mass production per unit ground area. Plant and Soil 301: 279-288.

Mokany K., Raison R.J. and Rokushkin A.S. 2006. Critical analysis of root : shoot ratios in terrestrial biomes. Global Change Biology 12: 84-96.

Mommer L, van Ruijven J, de Caluwe H, Smit-Tiekstra A E, Wagemaker C A M, Ouborg N J, Bogemann G M, van der Weerden G M, Berendse F and de Kroon H 2010 Unveiling below-ground species abundance in a biodiversity experiment: a test of vertical niche differentiation among grassland species. Journal of Ecology 98, 1117-1127.