12 Root Growth: Methods of Measurement David Atkinson Scottish Agricultural College, Edinburgh, Scotland Lorna Anne Dawson Macaulay Land Use Research Institute, Aberdeen, Scotland I. INTRODUCTION The morphology of a plant root system is a function of its genetics and the envi- ronment in which it grows (Smucker, 1993; Aiken and Smucker, 1996). Mor- phology is also affected by interaction with soil microorganisms, e.g., arbuscular mycorrhizal fungi (Hooker et al., 1992). Both individual plant roots and whole root systems can and do show substantial variation within the potential range of their characteristics. Soil physical factors, particularly temperature, aeration, wa- ter potential, and mechanical impedance, are frequently the cause of limits to the expression of genetic potential. The morphology of the root system can thus be regarded as representing the integrated effects of three factors. This chapter first reviews those root properties that are most likely to be influenced by soil physical conditions and then describes methods that can be used in the field or laboratory to describe particular attributes of root systems. It illustrates some of the uses to which particular methods have been put and some of the limitations of their use. Bohm (1979) has given a more complete description of methods for measuring roots, and Atkinson (1981) has reviewed those methods relevant to tree crops. The impact of soil biological and chemical factors, and of the growth of the aerial parts of the plant, on root growth have been reviewed in general terms by Russell (1977). In both field and laboratory, many of the methods give information on a range of parameters. For example, when a root system is observed directly (e.g., through an observation panel), measurements can be made of length, diameter, Copyright © 2000 Marcel Dekker, Inc. longevity, and branching. It therefore seems more logical to divide studies on root systems by type of method rather than by root system property. Consequently, in this chapter, the major groups of available methods and the significance of the measurements they facilitate are discussed together, but in the context of the need to determine how plant function can be influenced by soil physical conditions. A. Root System Properties Root systems are branched structures composed of a number of individual roots with normally up to four orders of branching. Individual roots are themselves made up of large numbers of cells. The processes relating to root development have been characterized at both cellular (e.g., Scheres et al., 1996) and molecular levels (e.g., Chriqui et al., 1996). The size, shape, and form of these cells, the numbers in a particular tissue (e.g., xylem or cortex), and their function (Smucker 1993) may be altered by the growing environment. Major soil physical factors, such as soil water potential and soil mechanical resistance, can affect root prop- erties such as cell wall extensibility and wall pressure in a number of ways. Cell wall pressure is closely related to the rate of root growth, while osmoregulation is closely related to changes in soil water potential but less completely related to mechanical resistance. As a consequence of these effects, the length of individual roots, their rate of extension, and increases in root diameter can be changed by soil physical conditions, thus affecting the overall volume of soil exploited by roots, via effects on horizontal spread and the depth of penetration, which in turn influence the resources of water and nutrients available to the plant. Other pa- rameters that can vary include the angle at which roots grow through soil (e.g., their susceptibility to geotropism). The longevity of roots varies between species (Atkinson, 1985) and between root types in a species (Hooker et al., 1995). The rate of production, and the lon- gevity, determine the total root length and average root length density, i.e., the length of root (LA) under an area of soil surface or the length (LV) in a volume of soil. These factors are important to the ability of the root system to obtain nutri- ents for plant growth. In addition to possible effects of soil factors on morphology, root function (e.g., nutrient uptake per unit root length, surface area, or volume) may be altered as a consequence of effects on the types and ages of root present. However, the exact effects of physical conditions on the above parameters are incompletely described or understood, and considerable plasticity clearly exists in respect of most properties (e.g., Reynolds and D’Antonio, 1996). Roots are nor- mally considered in relation to their ability to supply water and nutrients to the plant, but they are also required to anchor the plant (Coutts, 1983) and to produce hormones, which may regulate the growth and performance of both root and shoot (Aiken and Smucker, 1996). The root system of most plants exists in nature in a symbiotic association with fungi (mycorrhizas), and so assessments of effects of 436 Atkinson and Dawson Copyright © 2000 Marcel Dekker, Inc. soil conditions on roots should also consider effects on mycorrhizas. Effects of physical factors on root characteristics are summarized in Table 1. B. Potential Effects of Physical Properties Many root system parameters can be influenced by a change in soil physical con- ditions (Table 1). Root effects depend upon many factors, including the nature of the changed soil variable, the species under investigation, and conditions in other parts of the soil. General principles were reviewed by Greacen and Oh (1972). 1. Case Study In a study of the effect of zones of contrasting bulk density on root system devel- opment in oats, the effect of a given value of bulk density varied according to its relation to the density of other areas in the soil column (Schuurman, 1965). Com- paction did not reduce branching, although it did influence root survival. The length of branch roots, which was normally highest in the surface, was affected (Fig. 1). Where the elongation of the main axis was reduced by a dense subsoil, its diameter increased and branching was stimulated. 2. Types of Response In addition to changes in overall root system length, mass, or volume, there can be alterations in the partitioning of dry matter within the root system (e.g., by increasing root branching or root number: Goss, 1977). In soil, root elongation was reduced by 60% by a mechanical resistance of 1– 8 MPa in ryegrass and by 2–6 MPa in pea (Gooderham, 1977). Goss (1977), using a ballotini (glass sphere) system, showed that the effect of increasing pressure on root growth inhibition varied between species, with barley being the most sensitive of the species tested. Root Growth: Methods of Measurement 437 Table 1 Root System Characteristics That Can Be Affected by Soil Physical Conditions Characteristic Parameter Anatomy Cell size, cortex width, balance of xylem cell types, epidermal wall form, root shape Individual root features Diameter, growth rate, angle, length, mass, longevity, root hair length and density, penetration pressure Branching pattern Amount, density, number of orders, position, distance between branches Whole root system properties Horizontal distribution, vertical distribution, length, mass, absolute and relative distribution Function Absorption of nutrients and water, anchorage, production of bio- logically active molecules (e.g., enzymes, phenolics) Copyright © 2000 Marcel Dekker, Inc. When roots are prevented from elongation, a resultant increase in the diameter is found, mainly due to an increase in the cross-sectional area of the cortex (Wier- sum, 1957). Goss showed that even when root system mass is unchanged, length can be reduced by 65% by mechanical impedance, while Logsdon et al. (1987) demonstrated that an increase in root diameter can compensate, in part, for a re- duction in total length. Appropriate measurements are clearly needed to establish such effects, although their physiological significance is still poorly understood. The use of penetrometers in such studies has been discussed by Bengough et al. (1994). An increasing concentration of roots at the ‘‘soil surface’’ in the laboratory had no effect on nutrient supply (Goss, 1977), but in the field, in the absence of irrigation, it might be expected to have adverse consequences because the surface would quickly dry out. This exemplifies why results cannot be directly transferred from laboratory to field. There can also be changes in the internal root turgor pressure, which has been primarily studied in relation to water potential effects. A novel approach, using a force transducer, allows the turgor in an impeded root to be measured without the need to remove the root from the impeding environ- ment (Clark et al., 1996). C. Purpose of Measurements The optimum method for the assessment of any root system will depend both on the characteristics of the root system itself and on the reason for making the mea- surement. The following are among the commonest reasons for measuring roots: 1. To assess the significance of changes in soil physical conditions on the plant 438 Atkinson and Dawson Fig. 1 Effect of density of the topsoil (0 –25 cm) and subsoil (Ͼ25 cm depth) on devel- opment of oat root systems. Bulk densities above and below the boundary are given in megagrams per cubic meter. (From Schuurman, 1965.) Copyright © 2000 Marcel Dekker, Inc. 2. To help to interpret a plant response to a particular soil treatment by understanding effects on water or nutrient supply 3. To improve the use of inputs (e.g., irrigation water or fertilizers) or to optimize the effects of tillage and other soil management practices 4. To allow the development of better plant root systems by conventional breeding or genetic enhancement D. Significance of Root Features Criteria for the selection of methods of assessing root performance are limited by our current understanding of the significance of particular root characteristics and of the consequences of changes to them. Information exists on the importance of some characteristics, and so we can identify circumstances in which particular measurements will be useful. 1. Characteristics Influencing Water Supply Soil water flux is the product of the hydraulic potential gradient between the soil and the root and the unsaturated hydraulic conductivity. Typical maximum flux rates seem to be around 2.5 mm d Ϫ1 (Russell, 1977). This should allow a rate of root water uptake of around 160 mLd Ϫ1 cm Ϫ1 . On this basis a root density of 2 cm of root per cm 2 of soil surface area should be able to supply transpira- tional needs in Northern Europe. The uniformity of root distribution and the mean distance between roots both influence water supply. Thus if an average root length density is clustered in one soil region, the flux to the root surface is likely to be inadequate. As soil water content and consequently unsaturated conductivity decrease, a greater root density will be needed to supply the same total flux to the shoot. The total volume of soil exploited by the root system directly affects the total amount of water and nutrients available to that plant. This volume can be represented by the horizontal spread times the maximum rooting depth (or the depth containing 95% of roots), although the effective volume will be increased by capillary rise. Average root length density and root distribution within the soil volume exploited are both important. This, however, gives only a static picture. In the field, the transpiration rate will change during the season, as a consequence of weather and plant needs. It is thus important to be able to assess the root system as it grows, and to characterize factors such as root death; root longevity in some species is of the order of days, not weeks (Atkinson, 1985). E. Commonly Measured Characteristics Despite the long list of root parameters that might be measured (Table 1), re- searchers have commonly determined a smaller set. They are summarized in Table 2. Root Growth: Methods of Measurement 439 Copyright © 2000 Marcel Dekker, Inc. Table 2 Functional Significance of Major Root Systems Parameters and the Principal Means of their Estimation Root parameter Functional significance Method Length Total system size Potential for the absorption of nutrients or water Soil microbial activity (Temporal variation: frequent estima- tion needed) Monolith methods (soil coring, etc.) Rhizotron/minirhizotron meth- ods (with assumptions) Profile wall methods (with assumptions and where limited precision is needed) Mass Total root system size Monolith methods (especially soil coring) Excavation (woody perennials) Number Growth regulator production Counts on soil cores Profile walls Rhizotron/minirhizotron Root: shoot ratio Relative allocation strategy Calculation from root and shoot weights Length density Limitations to soil nutrient and water exploitation Calculation from root length and soil volume Specific length Within-root-system allocation strategy Relative importance of soil exploitation Calculation from root length and weight Diameter Potential for mycorrhizal development Regulation of water stress Potential for apoplast/symplast exchange Growth potential Response to soil physical conditions Direct measurement or calcula- tion from root length and volume Amount of secondary thickened root Investment in system infrastructure Measurement of weight or length Branching pattern Intensity of soil exploitation Measurement of lengths or num- ber of roots of different orders Longevity Potential for rapid adjustment to root length Plasticity Flux of carbon to rhizosphere Cohort analysis of rhizotron or minirhizotron images Production Overall potential for soil exploitation Ability to increase system length Sequential soil core estimates or rhizotron/minirhizotron measurements Mycorrhizal infection Carbon allocation strategy Surface for nutrient uptake Estimation from stained root samples Vertical distribution Physical stability Depth of soil exploited Potential for resource use Soil core sampling Profile walls Horizontal distribution Stability Interaction with other species Profile walls Excavation Copyright © 2000 Marcel Dekker, Inc. II. METHODS OF STUDYING ROOT SYSTEMS IN THE LABORATORY Laboratory studies are normally concerned with assessing the effect of either a single or a limited range of soil physical properties on plant and root performance. Single-factor studies usually relate to soil temperature, water potential, osmotic potential, or aeration, whereas studies of the effect of soil impedance often involve simultaneous changes in other factors. For example, when bulk density was changed from 1.24 to 1.52 Mg m Ϫ3 (Fig. 1), there was also a reduction in the vol- ume of pores that were filled with air at field capacity and an increase in the volume of pores from which water would be unavailable to plants (Schuurman, 1965). Because the results of such experiments are likely to be influenced by the types of containers and media used, these are also briefly reviewed. A. Containers 1. General Factors Container methods permit the isolation of individual environmental factors that will normally interact with other characteristics and influence root growth in the field. Replication and ‘‘management’’ are easier than in the field, although con- tainer effects on root growth may be unnatural because of the restricted space and absence of soil organisms (bacteria, fungi, soil arthropods, etc.). Container meth- ods are best suited for studying plants with small root systems or for investigating the early stages of plant development. 2. Container Types The size of a container determines the total volume of soil available to a plant. Conventional plastic or clay plant pots, Mitscherlich pots, glass pots, petri dishes, tubes made from glass or plastic, and cardboard cartons have all been successfully used as containers, but their limited volume frequently results in roots concentrat- ing near the walls of the vessel and around its bottom. As a consequence of mois- ture and temperature differentials, the concentration of roots between the wall of a pot and the soil tends to be greater in a porous clay pot than in a plastic pot. In container experiments designed principally to study root growth and dis- tribution, the depth of the container needs to be large because of the root’s ten- dency to grow downward when restricted by the walls of a container (Bohm, 1979). Boxes 80 cm high, made from metal, wood, or plastic, have been used in this type of study, but cylindrical tubes are more common. Iron, clay, asbestos, plastic, acrylic, and glass have all been used. Where tubes are of a transparent material, or where boxes have glass windows set into their sides, roots can be directly observed and measured (Fig. 2). Containers of all dimensions can be Root Growth: Methods of Measurement 441 Copyright © 2000 Marcel Dekker, Inc. 442 Atkinson and Dawson Fig. 2 Use of acrylic cylinders to observe differences in root form, density, and distri- bution in spring barley. To calculate information, such as total or average root lengths, the relationship between length at the observation surface and in the whole soil volume must be known. (From Atkinson, 1987.) Copyright © 2000 Marcel Dekker, Inc. modified and constructed to have a viewing window or sides to permit repeated observations to be made of root growth (Mackie-Dawson et al., 1995a). Tubes may be buried in the soil or in insulated boxes to prevent the establishment of unrealistic temperature differentials (Mackie-Dawson et al., 1995a) and video re- cording equipment can be used to record root growth. Root boxes vary in size in relation to the type of plant being investigated. For studies of M.1 apple rootstocks, boxes 60 ϫ 17.5 ϫ 42.5 cm high were used (Rogers, 1939a), while for studies of maize, Walker and Barber (1961) used smaller boxes. Such boxes with windows may be used simply as a means of ob- serving the response of roots receiving particular treatments or as a means of as- sessing the uptake of radioisotopes incorporated into the soil adjacent to the soil- observation interface. The latter method has been used to observe the uptake of 86 Rb from the soil around individual roots (Walker and Barber, 1961). Such boxes have been used to assess the effect of soil moisture and soil temperature on the root growth of grass and clover species (Garwood, 1968). Us- ing the observation windows, it was possible to assess treatment effects on root system length, the elongation of individual roots, root diameter, and root number. All these parameters were affected by soil temperature. Where plants are grown in containers that allow observation of the root system, the possible effects of light on root growth must be considered, although few studies have addressed this issue. A comparison of the effect of a range of different light exposures, varying from total darkness to total light, on the growth of apple rootstocks showed that while continuous illumination severely checked growth, increased suberization, and reduced the development of lateral roots, the short periods of 3 ϫ 20 minutes per week or 2 hours per 2 weeks needed for observation had little obvious effect. Effects were greatest during periods of maxi- mum root growth, with length being more affected than root number (Rogers, 1939a). Given the paucity of data on the effects of light, it is prudent to reduce unnecessary exposure to a minimum. 3. Filling Containers Care must be taken when filling containers (both tubes and boxes), particularly when physical conditions (e.g., bulk density) are being controlled (Schuurman, 1965). Soil should be sieved when nearly air-dry, the appropriate fertilizer added, and the soil then moistened to a friable condition and mixed. It should then be put in the containers and compressed, layer by layer, several cm at a time, with the top few mm of each layer loosened before adding the next layer, to avoid stratifica- tion. All containers should normally be watered and allowed to stand for a period from a week to a month before the experiment begins, to allow the soil to settle. Where short-lived radioisotopes are being used, however, this may not be possible (Walker and Barber, 1961). These procedures are needed both for tubes and boxes. Root Growth: Methods of Measurement 443 Copyright © 2000 Marcel Dekker, Inc. Where an observation surface is to be viewed, it is essential that care be taken to prevent smearing and the formation of voids. B. Media For experimental purposes, plants can be grown in a solid, liquid, or gaseous root- ing medium; the type used depends on the scope and aims of the experiment. 1. Solid Media Soil. Soil is the most realistic growing medium for terrestrial plants and for long-term experiments. However, the extraction of whole root systems from soils other than very sandy ones is difficult, as is the complete removal of soil particles from recovered roots (Atkinson, 1987; McCully, 1987). Although exact nutrient compositions cannot be produced easily, soil temperature, water content, and compaction levels can all be manipulated. For example, containers of soil have been produced in which one layer is varied in bulk density, thickness, and depth from the surface (Baligar et al., 1981). Maintenance of a given matric potential is usually made using tensiometers or by weighing. A variation of this technique in which roots are grown in soil within porous membrane envelopes has been used successfully (Brown and ul Haq, 1984). Here the root system was confined within the ‘‘envelope,’’ and water and nutrients were able to move across the membrane. Undisturbed soil columns have the advantage that the structure, texture, and water availability, and also the complex structural and mycorrhizal network, remain relatively undisturbed. Columns allow the experimenter to control cer- tain soil conditions (e.g., water content) and plant growing conditions (e.g., tem- perature), but properties such as pore size distribution, structure, and bulk den- sity cannot be precisely determined before experimentation. Undisturbed columns may be very large, e.g., 1 m 3 (Belford, 1979), and can be collected by hand coring, power coring, or hydraulic sampling machines. The columns can be preserved with a coating of paraffin wax, plastic, foil, or liquid plastic material. Care needs to be taken during transportation so that artificial voids are not created. Sand. Sand is often used in nutrient experiments because of its low buffering capacity and the ease with which it can be manipulated. It is also relatively easy to wash from the roots, although some may still firmly adhere (Atkinson, 1987). The physical properties of the medium can be altered by varying the particle size. Fine sand gives a higher water-holding capacity, while coarse sand is more freely draining and better aerated. Root systems obtained from experimental media of this type are generally similar to those grown in solution culture (Bohm, 1979). Other Solid Media. Perlite, which is composed of expanded volcanic rock frag- ments, is uniform and inert and so is suitable for studies of germination or seedling 444 Atkinson and Dawson Copyright © 2000 Marcel Dekker, Inc. [...]... composition, and so require more routine maintenance than is needed for soil- based systems Solution culture is only of limited use in soil physical studies The uniform medium, lack of physical resistance, and the absence of soil flora and fauna make it difficult to compare root growth in solution culture with that in soil However, this approach has applications in studies of impeded aeration, water stress, and. .. have used both 2-D and 3-D images and have been able to distinguish plant vasculature from surrounding parenchymal tissue (MacFall and Johnson, 1994) Images can now be measured for root surface area, volume, and orientation (MacFall and Johnson, 1996) However, there are still limits to the resolution at which it can operate X-Ray Computer-Aided Tomography This is a non-destructive x-ray technique that... a trench or pit is dug to expose a vertical soil profile from which records of partially exposed roots can be made Horizontal areas, at different soil depths, can be prepared in the same way Unlike pinboard and soil coring methods, this technique can be used on stony soils The method is, however, labor-intensive and time-consuming, and it leads to extensive soil disturbance; moreover it can be difficult... root system (soil monolith, soil cores, needleboards) are either removed from the soil or assessed in situ (profile wall) Observation Methods A viewing surface is inserted into the soil, either a small observation window (Asamoah, 1984), a minirhizotron (Hendrick and Pregitzer, 1992a), or a large walk-in facility (Rogers, 1939b, 1969; Rogers and Head, 1963; Fogel and Lussenhop, 1991) Indirect Methods The... study the soil pore network available to protozoa and roots Although the method is expensive and labor-intensive, it allows detailed examination of the soil- root interface Root soil interactions, at a more detailed level, can be assessed using the scanning electron microscope (Fig 3) Copyright © 2000 Marcel Dekker, Inc Root Growth: Methods of Measurement 447 Fig 3 A root of spring barley and the soil attached... intercept methods due to root overlap, and correction factors have been added to compensate for this (Sackville-Hamilton et al., 1991b; Kirchoff, 1992) Also, roots have to be well spread out to ensure random orientation Alternatively, pixel-count methods can be used on a root skeleton and have been shown to be more accurate and more precise than the line intercept method (Lebowitz, 1988; Ewing and Kaspar,... Inc 454 Atkinson and Dawson Fig 4 Root system of 26-year-old apple tree (Fortune/M9) excavated by the skeleton method (From Atkinson, 1972.) Total Excavation Methods A major study of apple root systems has involved the excavation of 26 mature apple trees grown on either a light sand, a sandy loam, or a heavy clay loam with about 1000 Mg of soil having to be moved by hand (Rogers and Vyvyan, 1934)... digitized methods indirect methods Root length can be calculated from counts of root numbers found in sectioned impregnated blocks of soil Formulas are available to convert root number to root length for both random (Melhuish and Lang, 1968) and strongly anisotropic (Lang and Melhuish, 1970; Melhuish and Lang, 1971) root distributions For the former, provided a reasonably large sample of randomly orientated... between plowing and direct drilling (Finney and Knight, 1973) Board sampling a volume 30 ϫ 5 ϫ 30 cm deep has been employed to study the cabbage root Copyright © 2000 Marcel Dekker, Inc Root Growth: Methods of Measurement 461 system (Goodman and Greenwood, 1976) In this study, roots and soil were removed from the board as 36 samples of 5 cm 3 each, and the roots were washed free of soil, and their length... mm diameter round glass tubes but with the soil adjacent to the tube illuminated by a bulb and a magnifying glass at the top of the observation tube (Fig 11b) He found it necessary to pack air-dried soil around the tube to get good contact between glass and soil Observations were made of the effects of cultivation on spring barley and oilseed rape Sanders and Brown (1978) used a medical duodenoscope . growing environment. Major soil physical factors, such as soil water potential and soil mechanical resistance, can affect root prop- erties such as cell wall extensibility and wall pressure in a. Calculation from root and shoot weights Length density Limitations to soil nutrient and water exploitation Calculation from root length and soil volume Specific length Within-root-system allocation. needed for soil- based systems. Solution culture is only of limited use in soil physical studies. The uniform medium, lack of physical resistance, and the absence of soil flora and fauna make it