Protocol

Author

This article is modified from Perez-Harguindeguy et al (2013). The “New handbook for standardised measurement of plant functional traits worldwide” is a product of and is hosted by Nucleo Diversus (with additional Spanish translation). For more on this and on its context as part of the entire trait handbook visit its primary site Nucleo DiverSus at http://www.nucleodiversus.org/?lang=en

Contributing Authors

Background

Specific root length (SRL), the ratio of root length to dry mass of fine roots, is the below-ground analogue of SLA, providing a ratio of a standard unit of acquisition (root length) to resource investment (mass). Plants with high SRL build more root length for a given dry-mass investment and are generally considered to have higher rates of nutrient and water uptake (per dry mass), shorter root lifespan and higher RGRs than for low-SRL plants. Yet, high SRL can result from having a low diameter or low tissue density, each of which is independently associated with different traits. For example, thin roots exert less penetrative force on soil and transport less water, whereas roots with low tissue density have lower longevity but greater rates of uptake under high nutrient conditions. As there is little operational difference in measuring just SRL or its two components, we recommend that both metrics be measured when measuring the functional traits of roots.

Materials/Equipment

• flatbed scanner with a resolution of 1600 dpi (a resolution of 15 μm) and preferably with a transparency adaptor that illuminates items on the scanner bed from above
• image-analysis software (see SLA for free software) or for a large number of samples or root length, the commercially available application, WinRhizo (Régent Instruments, Quebec, Canada), is recommended.

Units, terms, definitions

• RGR – Relative growth rate: the (exponential) increase in size relative to the size of the plant present at the start of a given time interval (measured on a dry-mass basis for the whole plant, including roots).
• SLA – Specific leaf area : the one-sided area of a fresh leaf, divided by its oven-dry mass (units: mm² mg^-1 or m² kg^-1; range of values: <1-300)
• SRL – Specific root length: the ratio of root length to dry mass of fine roots.

Procedure

What and how to collect

Roots are often measured in aggregate when comparing the live fine roots of plants, although individual fine roots or small numbers of fine roots can be enough to measure root diameter or branching order. Separating fine roots according to the timing or depth of sampling could be informative to answer particular questions. Roots measured in the field span a range of root age, whereas roots acquired from ingrowth cores or young plants would constrain age. The basis of comparison should be clear and root acquisition and preparation considered each time. Roots from the top 20 cm are often considered the standard basis of comparison; however, the actual depth sampled should be allowed to be as varied as the height above ground from which to collect leaves.

In mixed-species assemblages, fine roots should be traced back to shoots for positive species identification. This may not be necessary in uniform stands and roots can often be distinguished among a small number of species. For small plants, it is often most feasible to excavate the entire plant to be washed out later, aiding identification. A typical amount of root necessary for measurement generally fits in the palm of your hand. In general, it is better to have a small amount of root that is better prepared than a larger amount of less well prepared root. Preferably, atypically large or small individuals should be avoided.

Storing and processing

Unwashed roots can generally be stored under humid, chilled conditions for a week, with little degradation of structure. Washing techniques should be gentle for species with low-density roots, whereas more vigorous washing might be more suitable for high-density roots in soils with heavy clays or coarse organic matter that could compromise measurements. Washing roots from a sandy soil can require as little as 30 s under a hose, whereas clearing roots of organic matter from a tundra soil might require hours of painstaking plucking. In general, cleaning roots will require a combination of running water over a fine mesh sieve (0.2-1 mm) to remove fine heavy particles such as sand, rinsing in containers of water to remove coarser heavy particles such as pebbles, and plucking of debris with forceps to remove contaminants that are of a similar size and density as the roots of interest. Often roots have to be finger-massaged and individual roots separated to allow particles to be removed. If some fine particles such as clays are too difficult to remove, roots can be ashed at 650^∘C later and ash mass subtracted from gross root dry mass. Washed roots can be stored in a 50% ethanol solution for longer periods of time. A useful rule of thumb is to stop washing roots when it appears that you are losing as much of the fine roots as you are removing soil, or preferably slightly before.

Measuring

If necessary, under a dissecting microscope, sort apparently live, healthy roots from the recently washed sample. Live roots generally have a lighter, fully turgid appearance, compared with dead or dying roots of the same species which appear darker and floppy or deflated; however, note that live and dead roots may not be distinguishable by colour. It will help to observe a range of ages and colours of absorptive roots for each plant species before measurement, so as to properly identify healthy live roots. For woody species, roots are often divided by root (ramification) order, to better standardise comparisons across species.

Once roots have been obtained and prepared, determining SRL, diameter and tissue density requires digitising the roots and measuring their length and diameter. Digitisation can be carried out with almost any low-end flatbed scanner. A scanner with a resolution of 1600 dpi provides a resolution of 15 μm, which is still half the width of the finest roots of any plant. Nevertheless, a scanner with lower resolution may also work. A scanner that has a transparency adaptor that illuminates items on the scanner bed from above, is recommended to provide crisp root images. Roots are best imaged while submerged in a small amount of water, which also aids in teasing individual roots apart. A clear plastic tray works well. There should be no need to stain most roots to image them. After scanning, scanned roots should be dried (48 h at 60^∘C) and weighed. These root samples can also be ground and analysed for nutrient concentrations.

When roots have been scanned, units of root length need to be traced and their diameter determined. For a small number of roots, this can be carried out with image-analysis software (see SLA for free software). For a large number of samples or root length, the commercially available application, WinRhizo (Régent Instruments, Quebec, Canada), is recommended. The commercial software will automatically determine the length, diameter and root volume distribution of a sample of root length, enabling easy calculations of SRL, average root diameter and root tissue density (root dry mass over volume, the latter being derived from length and radius). Although the software is expensive for occasional use, roots can be scanned independent of analysis software, saved in the JPEG format and analysed later by someone who owns the software. See Notes section for manual methods when none of the above facilities is available.

Notes and troubleshooting tips

(1) Root diameter and tissue density.

Not all roots of a given diameter and tissue density have similar cellular structure. Roots can vary in their relative proportions of cortex and stele (mainly phloem and xylem) as well as the construction of each. For these reasons, secondary to measuring gross morphology of fine roots, we also recommend cross-sectioning roots, so as to determine their cellular structure. For this purpose, multiple roots of each species are embedded in a polymer, cut on a microtome into 4-μm slices, stained with toluidine blue, which stains lignin blue-green and cellulose purple or red-violet, and then mounted on a glass slide. Digital images are made for each species using light microscopy at x100 magnification and the cross-sectional areas of the root, stele, endodermis and large xylem elements are determined by tracing each portion of the root manually in image-analysis software. With these data, cell diameters and amounts of different tissues can be calculated relative to one another and to total cross-sectional area.

Literature references

Craine JM (2009) -Resource strategies of wild plants.’ Princeton University Press: Princeton, NJ.

Eissenstat DM, Yanai RD (1997) The ecology of root lifespan. Advances in Ecological Research 27, 1-60. doi:10.1016/S0065-2504(08)60005-7

Paula S, Pausas JG (2011) Root traits explain different foraging strategies between resprouting life histories. Oecologia 165, 321-331. doi:10.1007/s00442-010-1806-y

Pregitzer KS, DeForest JL, Burton AJ, Allen MF, Ruess RW, Hendrick RL (2002) Fine root architecture of nine North American trees. Ecology 72, 293-309.

Roumet C, Urcelay C, Díaz S (2006) Suites of root traits differ between annual and perennial species growing in the field. New Phytologist 170, 357-368. doi:10.1111/j.1469-8137.2006.01667.x

Steudle E (2001) Water uptake by plant roots: an integration of views. In -Recent advances of plant root structure and function’. Eds O Gašparíková, M Ciamporová, I Mistrík, F Baluška, pp. 71-82. Kluwer Academic Publishers: Dordrecht, The Netherlands.

Wahl S, Ryser P (2000) Root tissue structure is linked to ecological strategies of grasses. New Phytologist 148, 459-471. doi:10.1046/j.1469-8137.2000.00775.x

More on methods:

Böhm W (1979) -Methods of studying root systems. Ecological studies 33.’ Springer: Berlin.

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. doi:10.1023/A:1014905104017

Craine JM (2009) -Resource strategies of wild plants.’ Princeton University Press: Princeton, NJ.

Craine JM, Tilman DG, Wedin DA, Chapin S III (2001) The relationships among root and leaf traits of 76 grassland species and relative abundance along fertility and disturbance gradients. Oikos 93, 274-285. doi:10.1034/j.1600-0706.2001.930210.x

Fitter A (1996) Characteristics and functions of root systems. In -Plant roots: the hidden half’. 2nd edn. Eds Y Waisel, A Eshel, U Kafkafi, pp. 1-20. Marcel Dekker: New York.

Newman EI (1966) A method of estimating the total root length of a sample. Journal of Applied Ecology 3, 139-145. doi:10.2307/2401670

Tennant D (1975) Atest of a modified line intersect method of estimating root length. Journal of Ecology 63, 995-1001. doi:10.2307/2258617