Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

This method can answer key questions in the growth and morphology of stolons and rhizomes of different crops such as turfgrasses, meadows, and pastures. The main advantage of this technique is that stolon and rhizome length can be measured in samples with a large amount of biomass for which weight is the only parameter currently used. Collect samples using a soil core sampler.

Include the above ground biomass and a soil layer with an appropriate depth, depending on species to ensure collection of both stolons and rhizomes. Alternatively, define the surface area to collect with a frame and collect the samples with a spade. Label each sample with laboratory tape.

To clean the biomass samples, place the sample in a large sieve with 0.5 to 1.5 millimeter openings, depending on stolon or rhizome size. The openings should be small enough to retain all stolons and rhizomes, but large enough to allow the soil particles to be removed. Clean the samples with a stream of water with enough power to remove soil particles without damaging the plants.

Retrieve the clean samples and place them in a tray with paper towels, taking care to label the trays appropriately. Further clean the samples by removing roots and leaves with scissors. During this process, separate the stolons and rhizomes if needed and record additional information, such as the number of plants, tillers, and stolons per plant.

Place the stolons and rhizomes in paper labeled bags. Place the sample on a transparent plastic tray of the WinRHIZO standard scanning equipment. Manually place the stolons and rhizomes using laboratory forceps to minimize overlapping.

Large samples may need to be split into sub-samples. Place the tray on the scanner surface, then turn on the scanner and start running the program. For further control in a saved image, check the image dpi in the Image menu by selection the command Image Acquisition Parameter.

For good classification of pixels belonging to the scanned organs, check the threshold and analysis by selecting the command Root Background Distinction. Check that the whole tray surface will be scanned in the Image menu by selecting the command Image Acquisition Parameter. Now, check the diameter class displayed for organs distribution per diameter in the graphic area above the scanned image.

Select 20 equal width classes with 0.1 millimeter intervals by clicking on horizontal access of the graph. This function allows the exclusion of data belonging to roots or small organs when stolons or rhizomes were not perfectly cleaned. The literature reports that most roots of turf species have diameter lower than 0.2 millimeters.

Run the first sample scanning and check that the edit allows for good analysis. Follow the software instructions for saving the image. Finally, process the analysis following software option and label the image and analysis with the sample label.

Proceed with the scanning of all samples. To perform measurement of dry weight, place the scanned samples in a tared aluminum tray on a precise electronic balance and record their tare. Repeat this step for all scanned samples.

Insert all the samples into an often set to 105 degrees Celsius and dry them for 24 hours. The next day, remove the samples and wait until the tissue weight has stabilized. Then, weigh all the samples with their tare.

Subtract the tare from the recorded weight to obtain the net weight of each sample. Example results from a field trial comparing the stolon and rhizome length density of four turf-type Bermudagrass cultivars are shown. There is a significant different in the stolon length density between the vegetative cultivars Patriot and Tifway, and the seeded cultivars La Paloma and Yukon in samples collected in July 2014.

For the rhizomes, Patriot displays the highest length density, followed by Tifway, and the seeded cultivars. The development of stolon length density of cultivar Patriot throughout the study period shows an increase from March 2014 to July 2014 and it did not vary from July 2014 to July 2015. Few rhizomes were found in samples collected in October 2013 and March 2014.

Rhizome length density increases in July 2014, reaching highest values. But it decreases again in October 2014. When attempting this procedure, it's important to remember to select representative samples, to meticulously clean stolons and rhizomes before analysis, and to select the width of diameter classes using software options to include only stolons and rhizomes in the analysis.