Root Length Determination

Root length and surface area can be controlling variables for water and nutrient uptake. As such, it is important to be able to quantify them. WinRhizo, a product of Regent Instruments, inc. is an image analysis software package designed to allow this type of analysis. WinRhizo allows the acquisition of images of washed roots, and the measurement of root length, root diameter, and root surface area. Root length distribution by diameter classes and root topology are also quantified. We regularly use WinRhizo to measure root length in plants grown in greenhouse experiments, in solution culture, in growth pouches, and soil cores taken from field experiments.

This is intended as a brief overview of the use of WinRhizo, more detailed and specific information is located at: http://www.regentinstruments.com/products/rhizo/Rhizo.html, or in the manuals included with WinRhizo.

Acquiring Washed Roots

The first step is acquiring washed roots. This can be the most difficult and laborious step in the experiment if plants are grown in a solid medium. This should be considered in design of experiments. Soil or growth media with a high sand content can greatly simplify this step, while high clay or organic matter content can make it extremely tedious. Harvesting of younger plants also can simplify this step. In many cases, we find it impractical to scan the entire roots system of a plant. In such cases, we take a representative sample that we scan and analyze before determining dry mass. We then use the length/dry mass ratio (Specific Root Length) of the sample to estimate root length of the whole root system based on dry mass. In studies of root architecture, each class of root or soil horizon could be sampled separately – the sampling design will depend on the research questions.



Washed bean roots poured into tray for scanning. These plants were grown in a sand/vermiculite mix to facilitate washing of roots.

Preparing Roots for Scanning

Prior to acquiring root images, roots may be stained . Staining can increase contrast in the image, which improves the accuracy of measurement, and can improve the determination of root diameter. However, it is an additional step, which requires more time and handling of samples. Staining may especially be problematic when plants are very small and the entire root is being scanned as staining could prevent further chemical analysis of tissue. We recommend a preliminary trial where a sample is scanned before and after staining to determine if staining is necessary or not. We have successfully used Neutral Red (0.162 g/L in water) for root staining.

Roots are floated in water in acrylic trays on the scanner. This allows the roots to be arranged to reduce overlap and crossing of roots. We use plastic forceps and very fine plastic pipettes as tools. This is delicate work; good lighting and steady hands are helpful.

Roots can be arranged to reduce overlap and crossing while floating in the trays.

Scanning Roots

For best results, WinRhizo is used with an approved scanner, which allows the roots to be lit from above and below while being scanned. This is an important feature (called "Dual Scan" in Regent's documentation), which reduces shadows on the root image. The Regent Positioning System allows the trays to be consistently placed, thus obviating the need to preview each scan.



? Root scanner ready for action. Note that the top has the secondary light source which allows the "Dual Scan" mode. Here the tray is placed using the Regent Positioning System, a series of spacers (red in this photo) which allow consistent tray placement.

? Screen image of the above root sample after scanning at 200 dpi

 

Optimum scanning resolution depends on the type of samples. Phaseolus vulgaris roots may be scanned at 200-400 dpi in 20x30 cm trays, while Arabidposis roots may be scanned at 600 dpi in 10x5 cm trays. Lower resolution can speed up scanning significantly, especially if the samples require the use of large trays. We recommend scanning a sample at a range of resolutions to determine if lower resolution leads to loss of precision in measurement. Root length analyses are carried out with grayscale images; saving images in grayscale reduces the image file size substantially.

 

 

The Right Threshold Value is Important

Analysis results can be sensitive to the threshold parameters used. WinRhizo can automatically set these, but you may want to manually tweak them from time to time. These analysed images are from the root image previously shown, but are a selection of a smaller region for clarity. The color traces on the root indicate where roots have been detected.


In this image, the majority of the fine roots have not been detected. Total Root length (whole sample) was 77.63 cm

Here, with the proper threshold setting, root detection is nearly complete, and Total Root Length was 332.43 cm.

 

Analyzing Scanned Images

  

To analyze the image, the user selects the region(s) of interest, and it is analyzed. When scanned images are analyzed, the software uses thresholding to determine what is root and what is not root (each pixel is classified as either root or not root based on its grayscale value; this is why shadows in images are problematic). Portions of the image can be excluded from analysis if necessary, and there are basic editing tools if  minor image  editing is required.

In this screen image, the color traces on top of the image show roots classified by diameter. The color histogram shows length by diameter class 



 

 

   

  WinRhizo will prompt the user to open or create a file for analysis results to be saved to. This is a text file that can easily be opened in standard spreadsheet programs. 

Belowis from an Excel file corresponding to the above image. Several columns were deleted, and the output was "stacked" to allow it to fit the screen. 

 

 

I Don't have Access to WinRhizo

There are other ways to determine root length also. The classical method is the line-intercept method, where root length is estimated on the basis of the number of intersections of roots with a grid which is superimposed on them. For a complete explanation, see:

A Test of a Modified Line Intersect Method of Estimating Root Length

D. Tennant, The Journal of Ecology, Vol. 63, No. 3. (Nov., 1975), pp. 995-1001.

Another approach might be to use the best scanner available and use Image J, a free image analysis program from the US NIH. This approach is presented in:

Root length and diameter measurement using NIH Image: application of the line-intercept principle for diameter estimation

K. Kimura and S. Yamasaki, Plant and Soil, Vol 234, No. 1. (July, 2001), pp. 37-46

We also find image J to be useful for analysis of microscope images and particle counting (eg, counting Arabidopsis seed)

Delta T scan is another image analysis program which we have used to estimate root length.

Other Uses for WinRhizo

WinRhizo also contains tools for color analysis. We have used WinRhizo to measure leaf area of individual plants or percent ground cover of stands of plants. In this case, images are acquired using a camera, and an object of known dimension should be included in each image for proper calibration of that image. The user creates color classes which can be assigned to groups, for example the group “leaves” may contain several shades of green, the group “soil” may contain shades of brown and black, and the group “litter” may contain shades of brown and yellow. WinRhizo documentation is helpful for learning this process.

Similar color analyses can be carried out in Photoshop or GIMP (Gnu Image Manipulation Program). Photoshop is the most standard photo manipulation program, but GIMP has the advantage of being freeware.