Using WINRhizo and Photoshop to determine root length, diameter and branching
Contributors :Kathy Schneebeli Bas de Regt
This protocol provides a detailed description for the use of WINRhizo root scanning software with Photoshop software to quantify the total length, diameters and lengths of different root types within root systems that have been washed from soil.
We have used this protocol on cereals for both field and controlled environment grown plants ranging from seedlings to adult plant stages. It is applicable to any washed root samples. Some samples can be very large- more than 25 meters total root length- and need to be separated. For field sampling, we used shovels or cores pushed into the ground by hand or a hydraulic corer mounted on a tractor, pushed to 2 m deep. Most field root samples need to be “cleaned” if you are interested in roots of a particular plant at a given time, by manually removing dead root remnants or living roots of other plants under a dissecting microscope. This requires preliminary work to develop visual criteria to recognise the wanted and unwanted roots (see Watt et al., 2008).
- 'WINRhizo' software (see reference at bottom of page)
- Roots (gently wash soil from roots by soaking the pot of core first, and slowly washing away soil with a gentle spray while supporting roots with hands and a mesh; when you have the washed solution, remove roots of unwanted plants if from the field; preserve roots in 50% ethanol and water in the cold if not stained and scanned immediately)
- Toluidine blue (0.05% in pH 4.4)
- Beakers of water for root washing
- Scanning tray and flatbed scanner with top and bottom illumination
Units, terms, definitions
- Starting the computer and scanner
- Turn scanner on and set up according to manufacturers instructions
- Setting up WinRhizo
- Start the WinRhizo software
- Select the Epson Twain option
- Once WinRhizo loads, the large square button at the top left should show a grey scanner. If it shows a blue disk the scanner was not ready. Quit WinRhizo, wait until the scanner is ready and restart.
- Go to Image, Acquisition parameters and make sure "Use TWAIN interface" is ticked
- If you want to open an image from disk, go to Image -> Origin and check the "from disk" button.
- Root preparation and scanning
- Shake or vortex roots in solution to remove excess soil. Remove dead root remnants and other unwanted roots from sample under a dissecting microscope.
- Unless exceedingly dark, roots need to be stained. Toluidine blue works well, stain for 3 minutes. It is simple to use a cup to hold the roots with a mesh on the bottom, first submersed into the jar of stain, and then transferred into several rinsed of water.
- Rinse well after staining until rinsing water no longer turns blue.
- Put some water in the tray and spread the roots out so that if possible they are not touching. The root system may need to be divided. If roots are very soily and more pieces of stuff float off while the roots are being arranged, it is good to rinse the roots and the tray and re-spread. WinRhizo will classify small piece of grit as root length and this can make a massive difference to total length.
- Shut the scanner and click the acquire button (picture of scanner at top left). Your scanner may have different software, but you can still get an idea of the sequence of events here). A new interface will load.
- Select the following settings:
- Document source: TPU for Pos. Film
- Image Type: 8-bit Gray(Std)
- Destination: Epson Stylus Printer (or your printer)
You will need to choose your own resolution, see Note below.
- Click "preview". This will do a quick scan and bring up a preview of roots. Check that they are all in the picture and not overlapping, and adjust the marquee to the required size.
- Click Scan. After scanning (which takes a fair while) WinRhizo will prompt you to save the image.
- The image is now ready for analysis. You can zoom in or out to view the image by clicking the small and large Z buttons at the bottom left of the screen.
- Go to Analysis -> Filters and check that no filters are active. It is best to start with no filters and add the ones you need once you have optimised (e.g. Set Image smoothing to on, and exclude anything smaller than 0.001)
- Go to Analysis -> Root/Background & Color Analysis and check that Color Analysis is not ticked.
- Double click the x axis of the chart at the top of the screen (you can show or hide this graph by the clicking the little picture of a graph on the bottom left of screen) and set the root diameter classification classes. This will probably take some optimising. See Note below.
- Once you have set up all these parameters, go to Misc -> Save Configuration and save you configuration settings. Next time just use Misc -> Load configuration.
- To analyse, simply click on the image. A window will appear asking for identifier and operator; it is not vital to include these but very useful later.
- Click OK and the image will be analysed. You will now be prompted to Open or Create a data file, or Save Nothing. WinRhizo saves data to a text file. Click create, and enter a name. A colour skeleton should appear over the roots and the equivalent lengths will be shown on the graph.
- If you make a mistake, click Restart Analysis and start again. The old data will be replaced with new data.
- The image does not by default save with the colour skeleton, but it is possible to do. See sections 9.3.5 SAVE ORIGINAL IMAGE (WITH ANALYSIS) and 9.3.6 SAVE IMAGE REGION on page 90 of the manual (version 2009). Viewing a saved (in batch mode) analysed image including an overlay of the skeleton is only possible in WinRHIZO.
- Click the black/white or red/black buttons on the left of screen to see what pixels were counted as length, and then the colour skeleton without the image.
Other tools in analysis: Regions and Filters are both very useful. See pages 53-54 and 64 of the manual. Thresholding may be necessary if WinRhizo is missing part of your root system or including things which are too pale to be roots. See the manual.
Notes and troubleshooting tips
A Note about Resolution and Root Diameter Class Size The optimal resolution is the usual conflict between time to acquire and file size, and level of information required in the image. A picture scanned at higher resolution will take longer to scan and create a bigger file. As a guide, the A4 sized tray scanned at 400dpi creates a file of about 12.5 Mb. However it is also important to partially base the root diameter class sizes on the resolution of the image. WinRhizo uses a "number of pixels" method to calculate the root diameter. This means that class sizes smaller than a single pixel will be of no use. A resolution of 400dpi gives a pixel length of 0.063mm. Hence classifying roots in diameter classes 0.06mm wide (0-0.06mm, 0.06-0.12mm etc) will not be accurate and some classes will be empty. To accurately get the length of different root types within the root system (eg. Branch roots versus axile, parent roots), import the scanned TIF image into Photoshop and use the “STAMP” tool or other tool to selectively erase root types and regions, and then rescan the image in WINRhizo. Subtract the length of the second scan from the total original scan length to give the length of the roots of interest. Note that WINRhizo diameter classes alone are not an accurate indication of length of different root type and only indicate total lengths of regions with similar pixels. Roots have different diameters along their length (they thin and thicken as they progress through the soil), especially those grown in the field.
Toluidine Blue 0.05% Stain recipe
290 mg sodium benzoate
250 mg benzoic acid
200 mL deionised water
100 mg toluidine blue
Add sodium benzoate and benzoic acid to 200 mL water. Place on a stirring plate and stir on medium speed until dissolved. This may take several hours. Add toluidine blue powder and mix until toluidine blue is evenly dispersed.
Links to resources and suppliers
Watt, M., Kirkegaard, J. and Rebetzke, G. (2005) A wheat genotype developed for rapid leaf growth copes well with the physical and biological constraints of unploughed soil, Functional Plant Biology 32, 695-706. (This paper used this protocol). Watt M, Magee L, McCully ME (2008) Types, structure and potential for axial water flow in the deepest roots of field-grown cereals. New Phytologist 178: 135-146
Health, safety & hazardous waste disposal considerations
Toluidine blue may cause skin irritation and has previously been evaluated as a suspected carcinogen. Use gloves when using toluidine blue. Concentrated washing solutions should not be put down the sink, and should be disposed through a chemical waste safe disposal system.