Protocol

Authors

Liesje Mommer^1,2, Eric Visser²

Author affiliations

¹Nature Conservation and Plant Ecology group, Wageningen UR, P.O. box 47, 6700 AA, Wageningen, The Netherlands

²Radboud University Nijmegen, Institute for Water and Wetland Research, Experimental Plant Ecology, P.O. box 9010, 6500 GL, Nijmegen, The Netherlands

Overview

Root distributions in soils can be quantified using destructive analyses, after taking soil cores from any soil occupied by plant roots (mesocosms or fields) and careful root washing before further analysis of biomass or root length. We also discuss options to reduce work, as root washing may be a very time consuming activity.

Also see related protocols:

Root distribution in soils II. Non-destructive measurements by minirhizotron image analysis

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 some ecosystems (Jackson et al., 1996). Roots are thus with good reason referred to as the hidden half. They are out of sight living in the soil and it is very laborious to get them actually in hands. And if so, it is almost impossible to determine to species level by visual inspection, particularly when the root sample originates from high diverse plant communities.

Despite these methodological challenges, roots are being studied. Root responses for 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 getting more and more interest. This protocol helps in setting up a good sampling scheme for roots when plants are growing in pots, mesocosms (i.e., larger containers occupied by multiple plants typically been grown for longer periods of time) or the natural or agricultural field. Roots from these systems can be studied destructively by washing or nondestructively by taking root images from small, transparent, so-called minirhizotron tubes (for the latter, see protocol Root distribution in soils II. Non-destructive measurements by minirhizotron image analysis ).

Materials/Equipment

Root sampling and washing:

• Root corers or root augers- e.g. Eijkelkamp, the Netherlands, but also custom made – see Fig. 1, 2.
• Spatula, spoon, or alike
• Sharp knife
• Metal blades (only for collecting roots from quadrants of pots – see Fig. 1, 2)
• Plastic bags to keep the samples moist and labels to mark the samples
• Sieves of several sizes (e.g., diameter 16 cm; mesh size < 2 mm, but also plastic tea strainers)
• Pairs of tweezers
• Plastic trays – white or dark colored, contrasting with the color of the roots
• Flowing water (slightly warm might be comfortable)
• Root washing table is convenient from a health perspective when many samples have to be processed – see Fig 3.

Determining root length and diameter from soil cores or pots:

• plastic pots (20 ml) to store roots
• staining and preservation solution of mercury chloride (HgCl₂) and neutral red in citrate buffer (see below)
• pairs of tweezers
• tea strainer
• disposal container for the staining solution
• flatbed scanner which has double-size illumination (e.g., A3-sized Epson Expression 10000 XL, custom made changes by Regent Instruments Inc, Canada)
• WinRhizo software (Regent Instruments Inc, Canada)

Units, terms, definitions

• Root length density is the total root length per volume of soil – (m dm^-3)
• Specific root length is the total root length per g dry root mass (m g^-1)
• 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)

Procedure

• Root coring: the exact way to take a root core depends on the research question and experimental setup, but below two different approaches are explained.
  • In the case of single plants growing in pots for a relatively short time, the easiest way of taking the part of interest is using a relatively sharp knife. For example: Remove the pot and cut the whole soil volume including the roots in different depth slices or different quadrants. When interested in quadrants (e.g. in case of root foraging responses – see Visser et al. 2008) we use sharp custom made metal blades that can be hammered into the pots. In the case of pots we prefer to NOT use the root augers, as root density always tend to increase towards the sides of the pots. With root augers one normally does not core close to the sides of the pot and thus total root investment would be underestimated.

Figure 1: Custom-made device to take soil quadrants from a pot. Left the metal blades plain, right when it is hammered in a pot.

• Taking root samples in mesocosms, common garden experiments or from the field requires the use of root augers and two persons. A typical grassland profile would be: 0-5; 5-10; 10-20; 20-30; 30-45; 45-60; 60-80 cm and should be taken in as many steps as different layers are to be obtained. If the soil core up to 80 cm would be taken at once, the core will be compressed and the relationship with depth is lost as compaction is not necessarily linear over the soil profile. The desired profile should be marked on the root auger (best by marks that are engraved), such that the person who is actually coring is able to see the mark and can indicate when to stop. Coring requires some power, but more importantly careful turning the root auger into the soil in order to cut the roots. The accompanying person – on knees – gets the soil and roots out of the root auger into the plastic bag (including a label!!) with the spoon or spatula.

Figure 2: Root coring in action. Taking root cores in a field site – one person is drilling, another person sits down and carefully takes soil and roots out of the auger.

Root washing: critical choices to be made

Root washing is always a very time-consuming step. The procedure will be a compromise between precision and available time and thus, there will a tradeoff between time to spend on a sample and number of samples that can be processed. A few considerations to decrease the time needed per sample:

• Fragmentation of the root system: Washing is much easier if the roots are still attached to the plant. However, due to the harvesting method, this is often not feasible.
• Dead roots or other organic material (i.e. potting soil!) in the sample increases time to spend per sample significantly.
• Size of the sample – the smaller the sample, less soil needs to be washed away.
• Plant species diversity of the sample – more diverse root systems are more difficult to wash, as one cannot focus on one particular type of morphology. Particular if the samples are needed for DNA analysis (Mommer et al. 2010) it is important to obtain all small fragments of the various species. This can be accomplished by using a sieve with a sufficiently small mesh, and careful inspection of the content of the sieve holding it submerged against a black (or white, depending on the colour of the roots – see below) background.

Procedure for root washing

Put the soil sample in a sieve within a plastic tray in order to wash away most soil first. Throw away the bulk soil, and start washing the roots more carefully. This can be done by putting the roots in a tray filled with water, in which they can be gently rinsed; roots will float and moist soil particles will sink. Taking a tray of different colour sometimes helps to better find the roots. Use tweezers to take the roots and collect them in a small pot with water before taking sub samples.

For some measurements (e.g., if DNA extraction or other destructive analysis will be done), subsamples may need to be taken. In order to know which proportion of the roots was used, the fresh mass of the roots should be determined. However, this requires very careful standardization, as stronger or longer blotting with dry tissue will result in varying fresh masses. Therefore, this procedure is preferably done by the same person.

Figure 3: Root washing table – overview of the facility (left) and detail (right) how roots are washed over sieves. This facility gives place to 4-6 persons washing roots simultaneously. The washed soil will be collected in the grey containers below the table. Most of the water flows out via the tube. These containers will be transported by a hand pallet truck to the place where they can be emptied.

Procedure for determining root length and diameter on root samples (Winrhizo)

Once the roots are washed and separated from the soil, roots are colored to make them better visible, and they are scanned, digitized and analysed for their length and diameter using WinRhizo software (see Watt’s protocol on WhinRhizo). After this procedure, roots can be dried at 70 C until constant weight to measure their dry mass, so dry mass per soil volume or layer or soil surface area can be determined.

First the roots of a (sub) sample are stained because unstained roots may give insufficient contrast on digital scans to result in accurate measurements of root length and diameter. Particularly diameter estimations are very sensitive to this contrast. Therefore, we advise to stain roots whenever possible, except maybe for the coarsest root systems or roots that are dark-coloured already.

• Place roots for at least 20 min in a neutral red solution (0.035 % neutral red in a 25 mM citrate buffer at pH 6; i.e., 5.25 g citric acid and 2.6 g NaOH in demiwater; a phosphate buffer can also be used, but not when after scanning P-content will be measured). Leaving the roots in the staining solution for 24 h is a better choice, but make sure all roots are in sufficient contact with the solution (not in a tight bunch). If longer storage of roots is needed (up to two weeks), HgCl₂(0.01 %) can be added, after which samples are stored in the refrigerator.

• Put a transparent tray on the scanner and fill it with tap water. Be careful! The scanner is not watertight; do not spill water on top of the scanner!!

• Rinse stained roots in tap water to remove any adhering particles. Spread the roots over the entire surface (Fig. 3), taking care that roots are not arrange parallel and touching each other, as the software will recognize this as one thicker root. Sample density on the scanner surface should not exceed 0.5 mm root length mm^-2; i.e. total root length per scan not exceeding 28 m. If samples are larger, either divide the roots over multiple scans, or take a subsample and calculate total root length based on the ratio dry mass of subsample to dry mass of entire sample. Keep roots about 0.5-1 cm from the edges of the tray.

Figure 4. Example of scanned root system of Arabidopsis. Typically, roots will need to be more spread out than in this particular case.

• Use WinRhizo software to scan the tray and analyze the scan. We typically use the following settings (but for alternative information on this, see Michelle Watt’s protocol). This protocol provides the total length and average diameter for the root sample. Also, length per diameter class can be determined – (Fig. 4).
  • Scan resolution 400 dpi (coarser roots) or 600 dpi (fine roots) in grey scale
  • Under Analysis/Measurements àMorphology and diameter interpolation: regular
  • Under Filters àImage Smoothing Low, and Remove objects typically at 0.01 cm and Length/Width ratio of 6
  • Pixel classification set to Automatic. The option -Very pale roots’ is not a good method for stained roots.
  • Diameter classes can be set by clicking on the bar chart at the upper part of the screen. Moving the cursor over the bars displays the length in each class.

Fig 5. Example of an analyzed root scan, with roots in different diameter classes indicated in different colours.

Notes and troubleshooting tips

Root washing is all about sample management. Ideally, roots that are cored should be washed the same day, or as soon as possible thereafter. By experience we found out that root washing is team work. Better do it one week with many people, than many weeks with only one or two people. If needed, samples could be stored for some time in the fridge (4^∘C). Putting samples in the freezer is also an option people might want to try, but we do not have experience with this.

Options to reduce amount of work

With regard to pots and interest in depths: Do first take quadrants, than cut a quadrant (or even 1/8 art) in different depths in order to reduce workload to ¼.

Related protocols:

Root distribution in soils II. Non-destructive measurements by minirhizotron image analysis

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

Links to resources and suppliers

Root corers: Eijkelkamp, The Netherlands, http://www.eijkelkamp.com

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

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

Literature references

Bouma TJ, Nielsen KL, Koutstaal B (2000) Sample preparation and scanning protocol for computerised analysis of root length and diameter. Plant and Soil 218: 185-196.

Hodge A (2004) The plastic plant: root responses to heterogeneous supplies of nutrients. New Phytologist 162: 9-24.

Hutchings MJ, de Kroon H (1994) Foraging in plants: the role of morphological plasticity in resource acquisition. Advances in Ecological Research 25: 160-238.

Jackson RB, Canadell J, Ehleringer JR, Mooney HA, Sala OE, Schulze ED (1996) A global analysis of root distributions for terrestrial biomes. Oecologia 108: 389-411.

Mommer L, van Ruijven J, de Caluwe H, Smit-Tiekstra AE, Wagemaker CAM, Ouborg NJ, Bogemann GM, van der Weerden GM, 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.

Visser EJW, Bögemann GM, Smeets M, de Bruin S, de Kroon H, Bouma TJ (2008) Evidence that ethylene signalling is not involved in selective root placement by tobacco plants in response to nutrient-rich soil patches. New Phytologist 177: 457-465.

Health, safety & hazardous waste disposal considerations

Neutral red solution should be disposed off in an appropriate way; please take particular care if you have added HgCl₂ to this solution. This compound is highly toxic and should be treated with extra precautions.