Types, structure and potential for axial water flow in the deepest roots of field-grown cereals

Authors

Errata

This article is corrected by:

  1. Errata: Corrigendum Volume 178, Issue 3, 690, Article first published online: 17 April 2008

Author for correspondence:
Michelle Watt
Tel:+61 26246 4902
Fax:+61 26246 5399
Email: michelle.watt@csiro.au

Summary

  • • Deep root systems that extend into moist soil can significantly increase plant productivity. Here, the components of soil-grown root systems of wheat, barley and triticale are characterized, and types and water conducting potential of deep roots in the field are assessed.
  • • Root system components were characterized in plants grown in soil in PVC tubes, based on their origin and number and the arrangement of xylem tracheary elements (XTE) viewed using fluorescence microscopy. A new nomenclature is proposed. Deep roots were harvested in the field, and root types of the current crop and remnant roots from previous crops were identified by fluorescence and cryo-scanning electron microscopy.
  • • Four types of axile (framework) and five types of branch root were distinguished in the three cereals. Six per cent of deep roots were axile roots that originated from the base of the embryo; 94% were branch roots, of which 48% had only two XTE (10 µm diameter), and thus potentially low axial flow. Only 30% of roots in the cores were from the current crop, the remainder being remnants.
  • • Selection for more deep-penetrating axile roots and increased vascular capacity of deep branches is of potential benefit. Conventional root-length density measurements should be interpreted and applied cautiously.

Introduction

In agricultural systems, deep roots in moist soil layers contribute substantially to growth and reproduction (Gregory et al., 1978b; Passioura, 1983; King et al., 2003; Manschadi et al., 2006; Kirkegaard et al., 2007). Kirkegaard et al. (2007) demonstrated in the field that an extra 10 mm of deep soil water, can contribute at extra 0.6 tonnes of grain ha−1. Analysis of 100 yr of rainfall records showed that deep roots in moist soil are valuable not only when seasonal rainfall is low, but especially so in years when seasonal rainfall has been high (Lilley & Kirkegaard, 2007). Deep crop roots, however, appear limited in their uptake of water (Passioura, 1983). Proposed explanations are clumping of roots in soil spaces and poor root–soil contact (Passioura, 1988), low root lengths (Li et al., 2002), time (Passioura, 1983) and low axial water flow in the deepest, immature apical regions of axile roots (Garrigues et al., 2006; Pierret et al., 2006).

Despite the obvious importance of deep roots, little is known of their types, structure and function in field-grown cereals, not only because of difficulties of access, identification and quantification, but also because of inconsistent nomenclature and lack of anatomical detail of the components of the systems. The nomenclature of the axile (framework) roots of grasses has long been in dispute (Percival, 1921; Esau, 1977; Klepper et al., 1984), in particular that of the ‘seminal roots’ originating from within the seeds. There is uncertainty about the origins of those roots from the seed, and a paucity of anatomical data on the axile and branch roots. These seminal roots are probably important for accessing deep soil resources in small-seeded cereals such as wheat; Weaver (1926) dug 3-m-deep pits into silt–loam soil in the field and followed the growth of spring and winter wheat root systems, and reported that what he termed the ‘nodal system’ penetrated only to 1 m while the ‘primary system’ reached 2 m.

Accurate quantification of roots is critical for modelling roots and for identifying root architectures suitable to agricultural or natural systems (Wang & Smith, 2004; Wu et al., 2005). Here we use the number and arrangement of xylem tracheary elements (XTE) to identify the deepest roots of field-grown, mature wheat, barley and triticale. The numbers and diameters of the XTE are also used to estimate the potential axial water flow of the root types using the Hagen–Poiseulle law (Varney et al., 1991). First we grew the cereals in soil in PVC tubes under controlled conditions, to determine the origins and xylem structure of the root types comprising the root systems of each species. Then we grew the same species in the field and, based on the classifications of root type identified under controlled conditions, we identified the cereal root types present at depth. We also distinguished the roots of the current crop from those that were remnants from previous crops, to evaluate the accuracy of conventional root-length density measurements.

Materials and Methods

Classification of root system components

Wheat (Triticum aestivum L. cv. Janz), barley (Hordeum vulgare L. cv. Beecher) and triticale (Triticale hexaploide Lart. cv. Currency) were grown in a glasshouse from July 2005 to February 2006 (day lengths 13–15 h; irradiance approx. 700–1700 µmol m−2 s−1), in a mix of 50% river sand and 50% recycled potting soil (organic matter, loam and sand supplemented with lime, 14.4% N, 6.6% P, 5% K) that drains well and washes from roots easily. The soil mix was loosely packed into PVC tubes (0.085 m diameter, 0.5 m high) and the bottom was sealed with a Petri dish, which had holes for drainage. Two seeds were sown per tube, and plants were harvested when they had one to two, three, five, and six leaves along the main stem, and when they were mature and filling grain. Two tubes with two plants were sampled per sampling age. Roots were gently washed from soil to avoid breakage of branch roots and stored in 1 : 1 ethanol : water at 4°C until analysed. For all harvest ages, the root systems of each plant were carefully spread out in a large tray of water, the origin (from seed, stem, axile or first-order branch root) of their component roots was noted, and the lengths of the axile (framework) roots were measured (total of 48 plants examined per cereal). To establish the anatomy of the component roots, plants harvested at one to two, five, and six leaves were used, in addition to a repeat experiment of plants grown to six leaves (total of 16 plants per cereal examined). Pieces approx. 2 cm long were excised from approx. 3–5 cm from the base of the axile and first-order branch roots, and placed in individual small vials with 1 : 1 ethanol : water. Short first- and second-order branch roots were preserved whole. Ropes of individual component root types were made as described below for field roots, frozen in liquid N2, stored until sectioned with a cryostat, and their anatomy determined by fluorescence microscopy as described below. At least one of each component root type was examined per plant.

Collection of deep, field-grown roots

The same cultivars as were used in the controlled environment study were sown in an agricultural field east of Canberra, Australia (147°47′ E, 34°32′ S) in May 2004, at a density of 200 plants m−2, in four randomized blocks of plots 6 × 2 m. Phosphorus, nitrogen and potassium were applied in granules with the seeds. The soil is a red-brown earth (type Kandosol) and below 50 cm is dense (approx. 1.6 g cm−3) with a pH (CaCl2) from 5.0–5.8. Crops grown previously in the experimental area were canola (2003), wheat (2002), and canola (2001). Roots were harvested in October, when the cereals were starting to flower, with steel tubes (0.041 × 2 m) pushed into the soil directly over a plant (two cores per plot) using a hydraulic press mounted onto a tractor. Extracted cores of soil were pushed carefully out of the steel tube onto a cradle, and the maximum depth of roots was determined by the ‘core-break’ method (Böhm, 1979). Cores were gently broken at intervals beginning at the top of the soil profile and following the root system down to where the root system ended (judged by eye or hand lens by the presence of whitish roots and fresh root tips). The root systems extended to 87 ± 22 cm from the soil surface for barley, 70 ± 9 cm for triticale, and 85 ± 16 cm for wheat. The bottom third of the root system with associated core soil was sealed in a plastic bag, returned to the lab, and maintained up to 17 d in a 4°C cold room until washed in a Hydropneumatic Root Elutriation System (modified from that of Smucker et al., 1982). This washer is specialized to gently remove soil from roots and minimize loss of fine roots by trapping them in cups lined with fine mesh (0.35- or 0.6-mm-diameter pores), then stored in 1 : 1 ethanol : water at 4°C until they were analysed for length and anatomy. Two cores were harvested per plot (n = eight cores per cereal).

Separation and measurement of field roots

Washed samples were transferred to a film of water in a clear Petri dish and observed on a white background with a magnifying lamp (×2). They were a mixture of roots of the current cereal crop (‘current roots’) and remnant roots from previous crops and weeds (‘remnant roots’). An attempt was made to separate the current and remnant roots based on the features in Table 1. Some samples were reclassified under a dissecting microscope (×10), where the features in Table 1 were more readily observed. It took approx. 40 min to separate a sample (approx. 745 cm of total root, 30 min under the dissecting lamp, 10 min under the dissecting microscope). Current roots were stained for 3 min with 0.05% Toluidine blue O, pH 4.4, rinsed in water, spread out in a thin film of water, scanned using an Epson 1680 flatbed scanner at 400 dpi, and analysed for total length and mean diameter with WINRhizo software (Regent Instruments, Quebec, Canada). Remnant roots were scanned and analysed with the ‘pale root’ function of WINRhizo. They were not stained because they contrasted sufficiently with the background to provide lengths, and we were interested only in lengths of the remnants. Current crop roots and remnant roots were preserved in 1 : 1 ethanol : water at 4°C for anatomical studies.

Table 1.  Colour and morphological features used to separate roots of current cereal crops from roots remnant from previous crops and weeds, in samples harvested from the field, washed and viewed at low magnification (×2 to ×10)
FeatureCurrent rootsRemnant roots
ColourPaler/whiterDarker
ShapeDistortions along roots with rounded edgesStick-like; branch roots emerge from parent root at right angles
Root hairsPresent and visible at low magnificationNo obvious root hairs; very difficult to see at low magnification
Ratio of stele diameter to cortex diameterLow; stele and cortex visible, with stele more opaque than cortexHigh; almost entirely opaque stele with little cortex
Diameter and lengthTend to be thicker and longer than remnant rootsTend to be finer and shorter than current roots, but not always

All cores were separated into current and remnant roots and scanned as described above. Then, for one core per plot, remnant and current roots were recombined and analysed by cryo-scanning electron microscopy (cryo-SEM) to quantify the proportion of current and remnant roots (described below). For the other core per plot, remnants and current roots were analysed separately by microscopy to quantify the accuracy of the separation criteria in Table 1.

Fluorescence and cryo-SEM

From the field material, separated samples of current crop and remnant roots, and mixed samples, were transferred through 25% ethanol to water. Groups of roots were aligned longitudinally by gently bunching them together with forceps, then dipping them in and out of water in a deep beaker several times to form a ‘rope’ of roots. This rope was immediately frozen in liquid nitrogen (LN2), and maintained frozen in a cryostorage unit until they were sectioned and observed with fluorescence microscopy or cryo-SEM. Bunching roots into ropes allowed observation of many roots in a single mounted sample.

For fluorescence microscopy, a piece of frozen rope of roots (approx. 1 cm long) was positioned perpendicularly in low-temperature mounting medium (TBS, Durham, NC, USA) on the sectioning block in the chamber of a cryostat maintained at −25°C (International Cryostat Model CTI, International Equipment Company, Needham Heights, MA, USA). Transverse sections (8 µm) of ropes or individual root were cut with a single-edged carbon razor, placed on Poly/sine‘ pre-cleaned slides (Biolab Scientific, Clayton, Australia), and dried on a hotplate at 60°C for 1 h. The sections were flooded with rhodamine B (British Drug Houses Ltd, 0.0001% w/w in water) for 5 min, drained of excess stain, redried (5 min at 60°C on a hotplate), mounted in water under a coverslip, and observed with UV fluorescence optics (EX 360/40 nm, EM 470/40 nm) with a Leica DMR compound microscope. Some sections were observed without stain with UV fluorescence optics, which also showed XTE clearly.

For cryo-SEM, pieces of frozen root ropes (approx. 0.5 cm long) were quickly positioned in stubs in low-temperature mounting medium (TBS), refrozen in LN2, transferred to a cryomicrotome (Reichert-Jung Leica Ultracut E, Nussloch, Germany) and planed transversely with a diamond knife at −100°C. Stubs with planed samples were transferred frozen to the cryostage of a cryo-SEM (JEOL 6400, Jeol Ltd, Tokyo, Japan) and sublimed at −80°C for approx. 4 min, when cell walls extended above the frozen face and outlines of cells could be easily observed. The specimen was coated with gold and then observed at 10 kV (Refshauge et al., 2006).

Photography and image analysis

Cryo-SEM images were captured digitally with SpectrumMono ver. 2.01 (Dindima Group Pty Ltd, Ringwood, Victoria, Australia; resolution 0.4–1.2 µm per pixel). Fluorescence microscopy images were captured with a Leica DC500 digital camera and Adobe photoshop ver. 7.0 (resolution 0.2–0.7 µm per pixel). Anatomical measurements were made using AnalySIS ver. 3.2 (Soft Imaging Systems, Muenster, Germany). Images were first calibrated using a scale bar on the cryo-SEM images or known widths of fields of view on the fluorescence images, and the AnalySIS commands ‘image’ and ‘calibrate image’. Then diameters were determined using the ‘graph’, ‘measure’ and ‘arbitrary distance’ commands. The stele was taken to be the distance between the outer walls of the endodermis. For the stele and root diameters, two measurements, at the widest and narrowest points, were taken and averaged per section. Diameters of central and peripheral XTE were measured at their widest points for each section. The diameters were halved for radii and the areas of the root and stele were determined using the formula area = πr2.

Statistics

Least significant differences (LSD) of means were determined with a one-way anova using GenStat ver. 10.1 (VSN International Ltd, Hemel Hempstead, UK).

Results

Classification of components of cereal root systems

The component roots of the root systems of each cereal grown in the controlled environment fitted into nine categories, based on the tissues from which they originated and their structure, principally the number and arrangement of XTE in their mature regions (Table 2; Figs 1–3). There were primary axile roots, three types of nodal axile root, and five types of branch root.

Table 2.  Classification of component roots of wheat (Triticum aestivum), barley (Hordeum vulgare) and triticale (Triticale hexaploide) grown in a controlled environment in sandy soil, based on origin and structurea
Root classificationOriginbStructure: arrangement of xylem tracheary elements (XTE)c
  • a

    See Figs 1 and 2.

  • b

    n = 48 plants per cereal, aged 1.5 leaves to flowering.

  • c

    n = 170 axile roots, n = 246 first-order branch roots, n = 100 second-order branch roots from 16 plants per cereal, aged 1.5 to six leaves. XTE, xylem tracheary element, either in centre of root as for primary axile roots, inner surrounding a central pith, or peripheral, surrounding inner XTE. See Table 3 for numbers for cereals.

  • d

    Br1 to Br5 were first- or second-order roots; no third-order roots were observed.

Primary axileBottom of embryo within the seedCentral and multiple peripheral XTE
Scutellar node axileScutellum within the seedCentral pith surrounded by inner and peripheral XTE
Coleoptile node axileStem just above the seedCentral pith surrounded by inner and peripheral XTE
Leaf node axileStem at leaf nodesCentral pith surrounded by inner and peripheral XTE
Branch rootd (Br1)Axile root or first-order branch rootTwo peripheral XTE
Branch root (Br2)Axile root or first-order branch rootThree peripheral XTE
Branch root (Br3)Axile root or first-order branch rootOne central and three peripheral XTE
Branch root (Br4)Axile root or first-order branch rootOne central and four peripheral XTE
Branch root (Br5)Axile root or first-order branch rootOne central and five peripheral XTE
Figure 1.

(a) Classification of the axile roots in wheat (Triticum aestivum), barley (Hordeum vulgare) and triticale (Triticale hexaploide), their positions of emergence, and their order of emergence in parentheses. Solid lines, primary axile roots, anatomy shown in Fig. 3a; dashed lines, nodal axile roots, anatomy shown in Fig. 3b–d. The primary axile roots emerge first, from the root end of the embryo within the seed; the scutellar node axile roots from the scutellar node midway along the embryo axis; the coleoptile node axile roots from the coleoptile node just above the seed along the stem; the leaf node axile roots from leaf nodes along the stems. (b) Diagram of an axile root with first- and second-order branch roots with subtending axile root. No third-order branch roots were observed. The branch roots could be any one of the five anatomical classes (Fig. 3e–i).

Figure 2.

Root system of triticale (Triticale hexaploide), approximately at the five-leaf stage of development, with primary and nodal axile roots indicated.

Figure 3.

Representative cross-sections of component roots of the root systems of wheat (Triticum aestivum), barley (Hordeum vulgare) and triticale (Triticale hexaploide), grown in a controlled environment in a sandy potting soil, stained with rhodamine B and viewed with UV fluorescence optics. The anatomy of each component root did not vary among cereals (Table 3); here all images except (c) are from wheat cv. Janz; (c) is from barley cv. Beecher. (a) Primary axile root, bar, 100 µm ; (b) scutellar node axile root, bar, 100 µm; (c) coleoptile node axile root, bar, 100 µm; (d) leaf node axile root, bar, 100 µm; (e) Br5, branch root with six xylem tracheary elements (XTE), bar, 10 µm. (f) Br4, branch root with five XTE, bar, 10 µm; (g) Br3, branch root with four XTE, bar, 10 µm; (h) Br2, branch root with three XTE, bar, 10 µm; (i) Br1, branch root with two XTE, bar, 10 µm; Arrowheads, central or inner XTE; arrows, peripheral XTE; double arrowhead, root hair; c, cortex. See Fig. 1 for diagrams of the origins of roots.

The first roots that emerge from the seed are termed primary axile roots. These originate from the root end of the embryo. Most sections of these roots had one central XTE surrounded by six to nine peripheral XTE (Fig. 3a; Table 3), and no central pith parenchyma cells. A few wheat and triticale primary axile roots had two or three central XTE separated by one, or very rarely two or three, layers of parenchyma cells. Wheat produced two to five primary axile roots, barley three to six, and triticale four to six. For the three cereals, these were emerged and approx. 15 cm long by the time one to two leaves had developed. When the cereals had finished flower development, all primary axile roots had extended > 50 cm.

Table 3.  Central and peripheral xylem tracheary elements (XTE) and branch classes of axile roots of wheat (Triticum aestivum), barley (Hordeum vulgare) and triticale (Triticale hexaploide), grown in PVC tubes in a controlled environmenta
Axile root type WheatBarleyTriticale
  • a

    See Table 2; Figs 1 and 2 for description of root types. n = 170 axile roots, n = 246 first-order branch roots, n = 100 second-order branch roots, taken from 16 plants per cereal, aged 1.5 to six leaves.

PrimaryAxile1–3 central1 central1–3 central
+7 peripheral+7 to 9 peripheral+6 to 8 peripheral
First-order branch rootsBr1 to Br4Br1 to Br4Br3 to Br4
Second-order branch rootsBr1Br1 to Br3Br1
Scutellar nodeAxile4–5 central2–6 central2–4 central
+10 to 11 peripheral+8 to 10 peripheral+8 to 9 peripheral
First-order branch rootsBr2Br1, Br2, Br4Br1, Br2
Second-order branch rootsBr1Br1, Br2Br1
Coleoptile nodeAxile4 central4–5 central4 central
+9 to 12 peripheral+10 to 14 peripheral+8 to 11 peripheral
First-order branch rootsBr1 to Br3Br1 to Br5Br1 to Br3
Second-order branch rootsBr1, Br2Br1, Br2, Br4None
Leaf nodeAxile5–7 central3–6 central4–6 central
+10 to 14 peripheral+12 to 15 peripheral+11 to 14 peripheral
First-order branch rootsBr1 to Br4Br1, Br2, Br4Br1, Br2, Br4
Second-order branch rootsBr1Br2, Br3, Br4Br1

The nodal axile root types originate from embryonic or stem tissues (Figs 1a, 2). The first of these to emerge develops midway along the embryonic axis within the seed, approximately at the scutellar node. We term this type a scutellum node axile root. A third axile root type develops next at the coleoptile node just above the seed, and this is termed a coleoptile node axile root. Leaf node axile roots subsequently develop at foliar leaf nodes. All the nodal axile root types have a central pith of parenchyma cells surrounded by two to seven central XTE and eight to 14 peripheral XTE, depending on tissue origin and cereal species (Fig. 3b–d; Table 3). The scutellum node axile roots emerged after the primary axile roots, when the third leaf was expanding. Although they emerged very close to the primary axile roots, they were possible to distinguish because they were thicker and emerged from the seed at a different angle. Generally one developed per seed; occasionally none, and very rarely two. By the time the cereals were flowering, the scutellar node axile roots had extended 40–50 cm. The coleoptile node axile emerged close to the seed, had grown approx. 9–25 cm long when the fourth leaf was expanding, and by flowering had extended 8–66 cm. Two emerged per plant. The leaf node axile roots also had emerged and had extended 3–14 cm long as the fourth leaf was expanding. At flowering, a total of nine to 19 nodal axile roots were counted, extending 10–68 cm long. The nodal axile roots emerged and grew at similar stages across the three cereals.

First- and second-order branch roots developed from the primary and nodal axile roots of all three cereals, except for the coleoptile node axile roots of triticale, which developed only first-order branch roots (Table 3). Third-order branch roots were never observed. The branch roots fell into five anatomical classes based on numbers of XTE, which totalled two to six (Table 2; Fig. 3e–i). For wheat and triticale, first-order branch roots ranged from Br4 with five (Fig. 3e) to the lowest anatomical class, Br1 with two small XTE (Fig. 3i). Second-order branch roots were almost always the Br1 class for these two cereals, except for those on the coleoptile node axile roots of wheat (Table 4), which occasionally were class Br2 (Fig. 3h). Barley branch roots generally had more XTE than wheat or triticale. For example, classes Br4 (Fig. 3f) and Br5 (Fig. 3e) first-order branches developed from the primary and coleoptile node axile roots of barley, but not wheat and triticale. Further, the second-order branches of barley fell into classes Br1 to Br4 on all axes, while those of wheat and triticale fell into class Br1 or Br 2 (Table 3).

Table 4.  Root-length densities of current crop roots and remnant roots from previous crops and weeds, harvested from the field, washed and separated by eye using the features listed in Table 1
CerealRoot-length density (cm root cm−3 soil)aCurrent root length per total root length (%)
Total root length: current roots plus remnant rootsCurrent rootsRemnant roots
  • a

    Means, max. and min. in parentheses. LSDs and probabilities of differences between the means of the cereals determined by one-way anova; no significant differences were found. n = 8 cores from four plots per cereal. Root lengths measured using a scanner and WINRhizo as described in the text.

Wheat (Triticum aestivum)2.40.81.627
(0.4 to 5.1)(0.03–1.7)(0.4–3.5) 
Barley (Hordeum vulgare)2.20.71.436
(0.7 to 4.5)(0.2–1.5)(0.04–3.07) 
Triticale (Triticale hexaploide)2.40.91.533
(0.4 to 6.0)(0.1–3.4)(0.3–2.6) 
LSD1.70.81.020
P0.940.890.930.62

Field roots

The deep roots washed out of the cores were dominated by dead roots that were remnants from previous crops and weeds (Table 4; Fig. 4a). When separated by eye using colour and the morphological features listed in Table 1, roots of the current cereal crops appeared to comprise only approx. 32% of the total root length, the remaining length being remnant roots (Table 4). The lengths of wheat, barley and triticale crop roots did not differ.

Figure 4.

Roots harvested from deep soil layers in the field, frozen in liquid nitrogen, planed for cross-sectional views, etched to emphasise cell walls, and observed with a cryo-scanning electron microscope. (a) Mixture of cereal roots (double black arrowheads) and roots remnant from previous crops and weeds (arrows), wicked together into a rope before sectioning and viewing (see text for details). Bar, 1 mm. (b) Remnant branch root likely from the cereal crop planted 2 yr before the experiment. The cortical cells and inner walls of the epidermal cells are completely degraded (s, spaces). The outer walls of the epidermal cells, including root hairs, are intact, as are the walls of the xylem tracheary elements (XTE) in the stele (white arrow). Bar, 10 µm. (c) Remnant dicotyledon root. The secondary xylem development (x) reveals that the root is not from a monocotyledon. The phloem tissue appears to be degraded (s, space), while the cortical cells walls are intact and have phi thickenings (black arrow). Bar, 100 µm. (d) Remnant of xylem vessel (x) walls, from a dicotyledon. Bar, 10 µm. (e–g) Current cereal crop roots with intact stele and cortical cell walls. (e) A primary axile root with one central XTE and six surrounding XTE. The epidermis and outer cortical cells have collapsed. Bar, 100 µm. (f) A branch root, probably type Br4, with five XTE. Bar, 100 µm. (g) A branch root, probably type Br1, with two XTE, three layers of intact cortical cells and an intact epidermis with root hairs. Bar, 100 µm. (b–g): s, space; arrowhead, central or inner XTE; arrow, peripheral XTE; c, cortex; double arrowhead, root hair.

Current cereal roots and remnant roots were distinguished in cryo-SEM images of transverse faces of the ropes of roots obtained from cores (Fig. 4). The remnant roots consisted of degraded cereal roots (Fig. 4b) and dicotyledon roots, which had characteristic xylem development (Fig. 4c,d). Occasionally the only remnant was a chain of primary xylem vessels (Fig. 4d). Remnant cereal roots (Fig. 4b) often had degraded cortices but intact outer epidermal cell walls, including the walls of root hairs, and dicotyledon remnant roots occasionally had phi thickenings on cortical and endodermal cells (Fig. 4c). Analysis of ropes such as that in Fig. 4a indicated that there was no significant difference between the cereals in numbers of crop roots per total roots (data not shown). In these field samples, 21 ± 12% of the total roots were current cereal root, 68 ± 12% were remnants, and 11 ± 12% were not identifiable as a root (mean ± SD, 12 cores from four plots per cereal; P > 0.001, LSD = 12). These mean values were approx. 10% lower than, but within the same range as, those obtained by separating roots and measuring their lengths (Table 4). We validated the separation features in Table 1 by observing ropes of separated crop roots and ropes of separated remnant roots with the cryo-SEM. Ropes thought to be entirely cereal crop roots were found to be 73 ± 23% cereal crop roots, 19 ± 23% remnant roots and 8 ± 7% unidentified (mean ± SD; P > 0.001, LSD = 17), suggesting that selection by eye based on the features in Table 1 was 27% inaccurate. Ropes selected to be entirely remnant roots were shown with the cryo-SEM to be 7 ± 7% cereal crop roots, 80 ± 11% remnant roots and 12 ± 9% unidentified (mean ± SD; P > 0.001, LSD = 8). Unidentifiable remnants were groups of root cells that could not be classed as dicotyledon or monocotyledon because their vascular structures were degraded.

The diameters of the different classes of deep cereal roots were not different from each other, and did not differ among cereals (Fig. 5). It is worth noting that the mean diameter of remnant roots was 267 µm (n = 24 samples from the three cereals and eight cores per cereal), but ranged from < 130 to > 1170 µm and fell within the ranges observed for the crop roots (Fig. 5), highlighting that diameter is not a predictor of root class and age. The diameters of the steles were also highly variable, and overlapped across the primary axile roots and branch classes of the three cereals (Table 5). The mean cross-sectional area that was cortex, across the three cereals, was 86% for the primary axile roots, 89% for Br5 and 4, and 92% for Br3, 2 and 1, but these differences were not significant between root classes and cereals (data not shown). Although the diameters of XTE varied within root types, generally the diameters of the central XTE decreased in the following order: primary axile roots > Br5 > Br4 > Br3. The diameters of XTE were similar among cereals (Table 5).

Figure 5.

Mean diameter of roots of wheat (Triticum aestivum), barley (Hordeum vulgare) and triticale (Triticale hexaploide), harvested from the bottom third of the root systems, from the field. Cereals are combined because they did not differ significantly from each other. Bars indicate minimum and maximum values. Mean of remnant roots presented without range because minimum diameter was < 130 µm and maximum diameter was > 1170 µm. n > 20 samples of roots from four plots for each cereal.

Table 5.  Diameters of stele and numbers and diameter of xylem tracheary elements (XTE) of the deepest roots of field-grown wheat (Triticum aestivum), barley (Hordeum vulgare) and triticale (Triticale hexaploide)a
Root typeCerealStele diameter (µm)Central XTEPeripheral XTE
NumberDiameter (µm)NumberDiameter (µm)
  • a

    Cereals grown to time of flowering, roots harvested by coring and anatomy determined by fluorescence microscopy or cryo-SEM. Mean and absolute range of all sections observed per root type and cereal presented in parentheses. Roots from four plots per cereal. For the three cereals, n = 27 primary axile roots; 28 Br5; 42 Br4; 21 Br3; 43 Br2; 95 Br1 roots. np, not present.

Primary axile rootWheat136 (81–228)155 (46–87)714 (7–19)
Barley149 (109–195)157 (23–80)714 (8–21)
Triticale114 (95–133)144 (31–69)712 (7–23)
Branch 5Wheat 96 (51–151)139 (16–50)512 (5–18)
Barley112 (72–187)137 (7–69)512 (6–23)
Triticale 88 (37–146)134 (22–60)512 (7–20)
Branch 4Wheat 99 (63–142)130 (13–46)412 (5–20)
Barley 82 (54–128)124 (9–57)410 (5–17)
Triticale 91 (40–136)125 (11–49)410 (5–21)
Branch 3Wheat 68 (45–92)113 (5–27)3 8 (4–12)
Barley 67 (49–100)112 (8–18)3 7 (3 to 13)
Triticale 76 (44–103)116 (10–27)311 (8–18)
Branch 2Wheat 62 (42–85)0np3 8 (4–15)
Barley 61 (42–105)0np3 8 (4–15)
Triticale 70 (51–112)0np311 (6–19)
Branch 1Wheat 55 (27–164)0np2 8 (5–13)
Barley 50 (30–70)0np2 8 (3–14)
Triticale 50 (34–98)0np211 (3–17)

The current crop roots at depth were either primary axile roots or one of the five branch classes observed in the controlled environment studies (Fig. 6a; cf. Fig. 3). The cereals had similar allocation to the different root classes (Fig. 6a). No nodal axile roots were observed in the field-grown deep roots. The primary axile roots comprised < 10% of the cereal roots. Approximately half of all roots were the smallest class, Br1, with two small XTE approx. 10 µm in diameter (Fig. 3i; Table 5). According to the Hagen–Poiseulle law, which states that flow in tube varies with the radius of that tube to the fourth power, these Br1 branches could have potentially 1000-fold less axial water flow compared with the primary axile roots (Fig. 6b).

Figure 6.

(a) Roots of the bottom third of the root systems of field-grown wheat (Triticum aestivum, grey), barley (Hordeum vulgare, black) and triticale (Triticale hexaploide, striped) harvested at the time of flowering, expressed as a percentage of all cereal crop roots observed. Root types classified based on their origins and structure, mainly the number and arrangement of their xylem tracheary elements determined using cryo-scanning and fluorescence microscopy (Fig. 2). Mean and LSD within each root type are presented. n > 8 sections from 16 plants from four plots in the field, per cereal. (b) Estimation of the potential for axial water flow of the different root types, calculated using the Hagen–Poiseulle law and presented as log10. For each root type, estimated potential axial flow = sum (radius4 of xylem tracheary elements). See Table 5 for numbers and diameters of xylem tracheary elements of each root type.

Discussion

Validity of component root type classification

Traditionally, the axile or framework roots of cereal root systems have been classified into two types: seminal (those that develop early and originate from structures in or very close to the seed); and adventitious, nodal or crown (those that originate later from nodes of the stem) (Percival, 1921). In this classification, those roots we have termed primary axile, as well as those originating from the scutellar node region and the coleoptile node, have usually all been included in the seminal group (Klepper et al., 1984). The presence of a large central XTE in the seminal axile roots allows them to be distinguished clearly from nodal axile roots, in which a parenchyma core is surrounded by two peripheral rings of XTE (Percival, 1921; Wenzel et al., 1989). In the small-grain cereals, the scutellar node is the first node above the primary axile root, and is closely followed by the coleoptile node (Esau, 1977). Our finding that those roots that develop at the scutellar node and at the coleoptile node have the same distinctive xylem arrangement as the leaf node axile roots was a surprise, and has prompted the proposed nomenclature (Fig. 1). The occasional primary axile roots with two or three XTE in the centre may arise from tissue closer to the scutellar node in the short length of stem (hypocotyl) between the base of the first primary root and the scutellar node.

The anatomy of the branch roots of these small-grain cereals has, to our knowledge, been totally neglected. The finding of five distinct types of these branches, based on xylem anatomy and not on root diameter or branching order, has also been a surprise, and raises the question of their functional significance. Anatomy has been used to identify types and transport capacities of field-grown roots in previous studies (Varney et al., 1991; Pate et al., 1995; Eissenstat & Achor, 1999). Varney et al. (1991) divided the first-order branch roots of field-grown maize into four classes, also based on their XTE. These anatomical studies provide more insight into fine root function than diameter and density measurements alone (Zobel, 2003).

Components of root systems at depth

To identify the origins of deep roots in the field, it was necessary to classify the components of the wheat, barley and triticale root systems and establish an anatomical signature for each. This was achieved by combining knowledge of the originating tissue of a component root with the number and arrangement of its XTE in the stele (Table 2; Figs 1–3). This is possible in most monocotyledons, as they do not have a vascular cambium and secondary vascular tissues are not produced. The number and arrangement of XTE, past the apical 1–2 cm, changes much less along a length of monocotyledonous root than in dicotyledonous plants. In dicotyledenous plants, the vascular cambium produces secondary xylem and phloem cells, and the number and size of tracheary elements increases along the length of roots with age and diameter (Vercambre et al., 2002). There have been reports of anatomical changes in roots with depth. In dicotyledonous tree-root systems, deeper roots can have much larger mean vessel diameters than shallower roots (McElrone et al., 2004). In barley roots, Luxová (1989) found that the diameter of the central XTE of the seminal axile roots increased with depth and the number of central XTE in the nodal axile roots decreased with depth. However, the pattern of XTE remained clearly distinct between the nodal axile roots, which retained their central pith, and the seminal axile roots, which retained their central XTE. These studies were done in pots of uniformly packed soil. We found that the seminal axile roots at depth in the field all had one central XTE, which varied almost twofold in diameter depending on the root sampled, suggesting substantial phenotypic variation at depth depending on the soil conditions encountered by an individual root (Table 5).

The deep field roots were primary axile roots or one of five classes of branch root observed in the controlled environment studies; no new classes of root were observed in the field. Seminal axile roots are very valuable to cereals, as they not only form the deepest part of the root system, but are the first to emerge, and seedling survival is dependent on them. Wheat (Passioura, 1972) and maize (Shane & McCully, 1999) can grow to maturity and yield entirely on the central primary axile root and its branches.

No nodal axile roots were observed at depth, similar to the findings of (Weaver, 1926). They may have been restricted by low water and nutrients (Tennant, 1976; Gregory et al., 1978b), weak positive gravitropism, or insufficient time to reach the deeper soil. Low water and low nutrients specifically reduce the nodal system in wheat, with little effect on the primary system (Tennant, 1976). The leaf node axile roots would be necessary for anchorage where wheat is irrigated and surface soil is soft, for new axes for branch roots in surface soil where deep soil is dry, and where biotic factors such as Rhizoctonia sever primary axile roots and abiotic factors such as water logging severely restrict deeper seminal root function.

The deep crop roots were dominated by branch roots. We found large variation in diameter within the root classes (Fig. 5), possibly because the dense soil forced roots into cracks and pores, causing them to thicken and thin depending on space (cf. Watt et al., 2005). The stele diameters were also highly variable and independent of root type (Table 5). The distribution of roots and their diameters may be different in another soil type or field environment, and needs to be determined in future studies. For example, we found that in the controlled environment, barley tended to have more Br4 and Br5 first-order branch roots, and more Br3 and Br4 second-order branch roots, than wheat and triticale (Table 3); barley and triticale had more seminal axile roots than wheat. These differences were not observed in the field (Fig. 6a).

Management or genetic efforts to increase deep crop roots are worthy (Passioura, 1983). Our studies suggest that we should focus on the primary axile roots, the scutellar node and coleoptile node axile roots, and their branches. Genetic variation exists for number of primary axile roots (MacKey, 1973). Primary axile roots would need to double at least, as the difference between wheat (three primary axile roots), and barley and triticale (six) in the controlled environment plants, did not result in more deep axile roots in the field. The scutellar and coleoptile nodal axile roots emerged by the two- to three-leaf stage and extended to 68 cm in the controlled environment in sandy soil, suggesting that there may be opportunities to increase their number or rate of penetration, as these can have strong positive gravitropism similar to primary axile roots (Araki & Iijima, 2001). Faster-penetrating axile roots would have more time to develop branch roots (classes Br5, Br4, Br3; Fig. 3). Branches with greater vascular capacity would have more pericycle cells from which to develop second-order branch roots, and more phloem sieve tubes to transport carbohydrate for those new branches that can be severely restricted by low carbohydrate in wheat (Tennant, 1976).

Functional significance of deep roots

The very small diameters of the XTE in the Br1 class suggest that many of the deepest cereal roots have low axial water flow (Figs 3i, 6a,b) compared with larger branch roots and primary axile roots with open XTE, based on the Hagen–Poiseulle law (Fig. 6b), which can overestimate axial conductance in roots by one to two orders of magnitude (Melchior & Steudle, 1993; Jeschke & Pate, 1995). Large axial resistances to water movement may also occur in apical regions of the primary axile roots with closed, immature XTE, and in narrow (10 µm diameter) xylem vessels at complex vascular junctions between roots (shown in maize, Wenzel et al., 1989; McCully & Mallett, 1993; Shane et al., 2000).

All the field branch roots, including the Br1 class, had intact cortices and epidermal cells with root hairs, suggesting they are involved in absorbing water and nutrients. They also all have fluorescent endodermal layers, suggesting suberization and specialization in that layer of cells. The small XTE in the numerous small branches may limit water flow within the deep roots of wheat, barley and triticale. This may be a mechanism by which plants grown in monoculture ‘slow down’ drying of surrounding soil, conserve the valuable deep water, and increase the time to absorb nutrients.

Significance of findings for assessing the amount of current roots at depth

Two-thirds of the total root length was remnant from previous crops that were at least 3 yr old. Inclusion of dead root length from our studies in models would underestimate crop root water and nutrient uptake by approx. 70% per total length. Accurate removal of remnant roots is an ongoing problem for quantification of field roots. Different methods include separation based on features (Gregory et al., 1978b), staining (Ward et al., 1978; Hamblin & Tennant, 1987), and image analysis of washed samples for shape and surface area (Benjamin & Nielsen, 2004). To our knowledge, these have not been validated with direct sampling. Cryo-SEM enabled us to observe and distinguish the cereal crop roots from dicotyledon roots with secondary xylem development, and highly decomposed cereals roots, to show that the five visual criteria in Table 1 were approx. 80% effective. It is possible that the highly decomposed cereal roots belonged to the current crop, but if that were so, we would have expected to see many more such highly decomposed roots with secondary walls broken down by microorganisms. Separation and accurate measurement of field root-length density is feasible for data for modelling or for comparing a few extreme genotypes, but is not suitable as a selection tool across a large number of lines, because variation within cereal type is high. Separation took 40 min per sample, and validation with microscopy requires specialized skills and equipment. The appearance of crop and remnant roots will vary with soil type and cropping practice. Selection criteria can be developed for each study by initially observing the remnant roots (core in an area without crop), then carefully observing mixed samples to identify unique features of the crop roots, and then using anatomy to validate. More recent techniques to identify species of mixed field roots include nucleic acid sequences (Jackson et al., 1999) and alkane (wax) composition (Dawson et al., 2000; Dove & Bolger, 2005). These do not distinguish roots within a species, and tissue development such as vascular capacity. One advantage of anatomy over nucleic acid analysis is that old and dead roots (with only cell wall remains) can be quantified and identified.

Root remnants influence the structure of the soil, opening spaces for roots, organisms and water, and are sources of nutrients in the subsoil. High amounts of remnant roots are not just a feature of Kandosol soils in south-eastern Australia (this study; Watt et al., 2005; Pierret et al., 2005), but are reported in a sandy loam over gravelly sand in the UK (Gregory et al., 1978a), a loamy sand, sandy clay and sodic red-brown clay in Western Australia (Hamblin & Tennant, 1987), and in several soils in central USA (Ward et al., 1978; Benjamin & Nielsen, 2004). In surface soil, 50% of the crop roots directly contact remnant root, and thus processes on remnants may be important for crop roots (Watt et al., 2005). The amount of remnant root would depend on the root growth in the preceding cropping practice, the presence of decomposing organisms, and the duration of conditions that favour decomposition. Although numerous studies have quantified root turnover carefully, measuring rates of root production and disappearance with mini-rhizotrons, processes regulating root senescence and decomposition remain poorly understood. Detailed studies of decomposition of soil organic matter traditionally remove particles > 2 mm, thus avoiding remnant root pieces. In future, anatomy can be combined with nucleic acid and lipid-based techniques to separate species and root types, and with stable isotope signatures to determine age and root type, as has been done with carbon isotopic signatures of microbial phospholipid fatty acids for ages of carbon pools consumed by bacteria or fungi (Kramer & Gleixner, 2006).

Acknowledgements

We are grateful to Bernie Mickelson and the staff at the CSIRO Ginninderra Experimental Research Station for managing the field experiments, Geoff Howe for help harvesting the field roots, Dr John Kirkegaard for the soil measurements and stimulating discussions, Dr Cheng Huang for performing the cryo-scanning electron microscopy and Dr Rosemary White for help with the cryostat sectioning at the CSIRO Microscopy Centre, Canberra. We thank the Australian National University, Canberra, for the Hydropneumatic Root Elutriation System. This study was funded by the Australian Grains Research and Development Corporation.

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