# Morpho-anatomical adaptations to waterlogging by germplasm accessions in a tropical forage grass

Juan Andres Cardoso, Joisse Rincon, Juan de La Cruz Jimenez, Diana

Noguera, Idupulapati M. Rao

► To cite this version:

Juan Andres Cardoso, Joisse Rincon, Juan de La Cruz Jimenez, Diana Noguera, Idupulapati M. Rao. Morpho-anatomical adaptations to waterlogging by germplasm accessions in a tropical forage grass. AoB Plants, 2013, 5, plt047. 10.1093/aobpla/plt047. hal-01532154

HAL Id: hal-01532154

https://hal.sorbonne-universite.fr/hal-01532154

Submitted on 2 Jun 2017

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Research Article

# Morpho-anatomical adaptations to waterlogging by germplasm accessions in a tropical forage grass

Juan Andrés Cardoso<fnref n="1" />,<fnref n="2" />*, Joisse Rincón<fnref n="1" />, Juan de la Cruz Jiménez<fnref n="1" />, Diana Noguera<fnref n="3" />,<fnref n="4" />
and Idupulapati M. Rao<fnref n="1" />

**Received:** 16 July 2013; **Accepted:** 29 September 2013; **Published:** 21 October 2013

**Citation:** Cardoso JA, Rincón J, Jiménez JC, Noguera D, Rao IM. 2013. Morpho-anatomical adaptations to waterlogging by germplasm accessions in a tropical forage grass. *AoB PLANTS* 5: plt047; doi:10.1093/aobpla/plt047

**Abstract.** Soil waterlogging reduces gas exchange between the soil and the atmosphere, leading to oxygen deprivation in the rhizosphere. *Brachiaria* spp. are the most widely sown forage grasses in tropical America. Among commercial *Brachiaria* grasses, *B. humidicola* shows superior tolerance to waterlogged soils based on maintenance of growth and reduced leaf chlorophyll loss and senescence. However, little is known about the underlying traits of waterlogging tolerance in *B. humidicola* or their intraspecific variation. For this purpose, an outdoor study was conducted using 12 germplasm accessions of *B. humidicola* that were grown in soil cylinders under drained or waterlogged soil conditions for 21 days. Dry mass production and morpho-anatomical responses (aerenchyma in shoots and roots, root diameter, proportional area of stele in roots, number of nodal and lateral roots, and length of the longest root) were determined. All accessions showed shorter roots and reduced root dry mass under waterlogged soil conditions. All accessions showed aerenchyma in shoots and roots under drained conditions but were further increased under waterlogging. All accessions showed a reduction in the proportional area of stele of roots in response to waterlogging. The accession (CIAT 26570) that showed a higher proportion of aerenchyma in shoots and roots and an increased number of nodal roots (with higher diameter and a reduction in the number of lateral roots) showed longer roots, less reduction in root dry mass and increased shoot growth under waterlogged conditions. We conclude that superior growth of one accession (CIAT 26570) under waterlogged soil conditions is probably a result of morpho-anatomical traits acting together to enhance root aeration and shoot ventilation. Further research is needed to test the ability to recover from waterlogging in *B. humidicola* accessions.

**Keywords:** Aerenchyma; *Brachiaria humidicola*; dry mass production; oxygen deficiency; root penetration; root traits.

**Introduction**

Soil waterlogging (or flooding of the soil) occurs when soil is saturated with water. Owing to the slow diffusion of gases in water, gas exchange between the soil and the atmosphere is strongly hindered (Colmer 2003). Soil

waterlogging reduces plant growth as O₂ availability in the root zone decreases (Armstrong 1979; Jackson and Drew 1984). To cope with waterlogging, plants usually develop new roots with aerenchyma (Laan et al. 1989; Visser et al. 1996; Huber et al. 2009). Aerenchyma refers to tissue with air spaces that provide an internal

<footnote n="1">¹ Centro Internacional de Agricultura Tropical (CIAT), Apartado Aéreo 6713, Cali, Colombia</footnote>

<footnote n="2">² Programa de doctorado Biología Agraria y Acuicultura, Universidad de Granada, Avenida de Fuente Nueva s/n, Granada 18071, Spain</footnote>

<footnote n="3">³ BIO UPMC—Bioemco (UMR CNRS 7618), 32 Avenue Henri Varagnat, Bondy Cedex 93143, France</footnote>

<footnote n="4">⁴ Laboratori de Cartografia i SIG, Facultat de Geografia i Història, Universitat de Barcelona, Montalegre 6, Barcelona 08001, Spain</footnote>

<footnote>* Corresponding author's e-mail address: j.a.cardoso@cgiar.org</footnote>
pathway for oxygen diffusion in organs under water-
logged/submerged conditions (Armstrong 1979). Apart
from this, plants may display other adaptive strategies
that might act together to improve root aeration and
oxygen consumption within the root. These include
increased root diameter (Armstrong 1979; Visser et al.
1997, 2000; Aguilar et al. 2008), fewer lateral roots devel-
oping from the main root axis (Armstrong et al. 1983;
Sorrell et al. 2000; Aguilar et al. 2003) and narrower stele
(Armstrong 1979; Armstrong and Beckett 1987;
Armstrong et al. 2000; McDonald et al. 2002; Colmer
2003). Furthermore, to provide a continuum of gas
ventilation between shoots and roots, aerenchyma is
commonly present in shoots of plants that are well
adapted to waterlogging (e.g. wetland species; Jackson
and Armstrong 1999).

Soil waterlogging is a major limitation to pasture prod-
uctivity (Hare et al. 2004). Perennial *Brachiaria* grasses
(*Brachiaria* Trin. Griseb.) are the most widely sown forage
grasses in tropical America (Miles et al. 2004; Valle and
Pagliarini 2009). Among commercial *Brachiaria* grasses,
*B. humidicola* tolerates the conditions of temporary water-
logged soils better than others based on a lower reduction
in growth (Dias-Filho and Carvalho 2000; Silva et al. 2007;
Caetano and Dias-Filho 2008), maintenance of leaf chloro-
phyll content and reduced leaf senescence (Dias-Filho and
Carvalho 2000). *Brachiaria humidicola* is a highly stolon-
iferous grass that roots at each stolon node when in per-
manent contact with moist soil. This stoloniferous
growth habit facilitates the spreading of *B. humidicola*
over large areas (Canchila et al. 2011). However, a major
limitation of *B. humidicola* is its low forage production
and its moderate to low nutritional quality, which limits
animal performance (Argel and Keller-Grein 1996). Vari-
ation in morphological traits (e.g. leaf area, leaf-to-stem
ratio; Cruz 2009), forage production and protein concen-
tration (Juarez et al. 2011) has been found among
*B. humidicola* accessions. Furthermore, the discovery of a
*B. humidicola* accession with a sexual mode of reproduc-
tion (Valle and Glienke 1991) has opened the opportunity
to explore, through breeding, the genetic variation of
*B. humidicola* (e.g. retain waterlogging tolerance while
improving forage quality and yield).

Little is known of the underlying traits of waterlogging
tolerance in *B. humidicola* or their intraspecific variation.
Empirical observations in tropical forage grasses have asso-
ciated waterlogging tolerance with a stoloniferous growth
habit and the presence of hollow stolons (Baruch 1994).
Preliminary research has shown that *B. humidicola* devel-
ops aerenchyma in roots under drained conditions and
the trait is further increased under waterlogged conditions
(Cardoso et al. 2010). Therefore, the main objective was to
study in further detail some responses of *B. humidicola*

accessions under waterlogged soil conditions. For this
purpose we used a subset of 12 *B. humidicola* germplasm
accessions from a total set of 66 accessions held in the
gene bank of the International Center for Tropical Agricul-
ture (CIAT) that in a preliminary study showed less
damage in terms of leaf senescence and leaf chlorophyll
content under waterlogged conditions (Rincón et al.
2010). We report differences in dry mass production and
morpho-anatomical responses of shoots (aerenchyma in
leaf sheaths and stolon internodes) and nodal roots
(number of nodal roots, number of first-order lateral roots
developed from nodal roots, root diameter, proportional
areas of aerenchyma and stele, and length of the longest
root) under drained or waterlogged soil conditions. These
data will help to identify adaptive traits related to water-
logging tolerance in *B. humidicola*, and their variation,
and therefore contribute to *B. humidicola* breeding.

Methods

Accessions and growing conditions

The genotypes used in this study were 12 germplasm accessions of *B. humidicola* (CIAT 679, CIAT 6013, CIAT 6133, CIAT 6707, CIAT 16182, CIAT 16866, CIAT 16886, CIAT 16888, CIAT 26152, CIAT 26181, CIAT 26416 and CIAT 26570). Three *Brachiaria* grasses with poor adaptation to waterlogged soil conditions (*B. brizantha* cv. Toledo, *B. ruziziensis* Br 44-02 and a *B. hybrid* cv. Mulato II) were included for reference (checks) but were excluded from the analysis.

The soil used in this study was an Oxisol collected from Santander de Quilichao, Department of Cauca in Colombia (latitude 3°60'N; longitude 76°310'W; altitude 990 m), 0–20 cm from the soil surface. All genotypes were grown from vegetative propagules. Propagules were harvested from 60-day-old plants growing in pots filled with 4 kg of a mixture of soil and sand (2 : 1) under field capacity and fertilized soil conditions (milligrams of element per kilogram of soil–sand mixture: N 21, P 26, K 52, Ca 56, Mg 15, S 10, Zn 1.0, Cu 1.0, B 0.05, Mo 0.05). Harvested propagules were then washed for 1 min in 0.1 % commercial sodium hypochlorite before planting. A 1 : 1 mixture of soil and sand was used to ensure better root growth and to facilitate root separation for analysis. Before planting, the soil mixture was thoroughly mixed with fertilizers. The rate of nutrient application (milligrams of element per kilogram of soil–sand mixture: N 40, P 50, K 100, Ca 101, Mg 28, S 20, Zn 2.0, Cu 2.0, B 0.1, Mo 0.1) represented the recommended fertility level for crop-pasture establishment (Rao et al. 1992). The soil mixture (4.5 kg) was packed in transparent plastic tubes (80 cm high × 7.5 cm diameter) inserted into opaque polyvinyl chloride (PVC) pipes. Three propagules of similar size (~6 cm
length) were planted 2 cm below the soil surface in each soil cylinder and thinned to one after rooting (5 days). Plants were allowed to grow for 21 days. Data on the number of fully expanded leaves, leaf greenness and maximum rooting depth (cm) were collected before the start of the experiment. Leaf greenness was measured in soil plant analysis development (SPAD) units in two fully expanded young leaves using a hand-held chlorophyll meter (SPAD-502, Konika Minolta, Japan). A factorial combination of 15 genotypes by two drainage conditions (drained or waterlogged) was established in a six-replicate randomized complete block. Waterlogging treatment was imposed by sealing the lower end of the PVC pipes with a cap and maintaining a water level of 3 cm above the soil surface. The soil of plants growing under drained soil conditions was kept at field capacity.

The experiment was conducted in an open area at the CIAT (Palmira, Colombia, latitude 3°31′N; longitude 76°19′W; altitude 965 m). During the experiment, temperature ran at an average of 30.5/23.8 °C (day/night), with a relative air humidity of 40.5/59.9 % (day/night) and a maximum photosynthetic photon flux density of 1800 µmol m⁻² s⁻¹. Redox potentials were monitored in four soil cylinders without plants during the experiment (two for drained soil, two for waterlogged soil) using a platinum electrode and a calomel reference electrode connected to a micro-voltmeter (ODR meter, Eijkelkamp, The Netherlands). Redox potentials of drained soil remained stable around 450 mV. Soil redox potential decreased to values below 330 mV after 7 days of waterlogging treatment and stabilized at around 100 mV after 14 days.

## Harvest

Prior to harvesting, leaf greenness (SPAD) and maximum rooting depth (cm) for each experimental unit (plant) were assessed. SPAD values were recorded in the same leaves used for greenness determination before the start of the experiment. Maximum rooting depth was estimated in roots that looked white and healthy and that were growing next to the wall of the transparent cylinders. Plants were harvested after 21 days of growth under drained or waterlogged soil conditions. Shoots were cut at 3 cm above the soil and gently washed with running water. Leaves, stolons (runners) and tillers (erect culms) (these three representing the shoot) were separated and oven dried for 72 h at 60 °C for determination of dry mass. Fully expanded leaves were counted before dry mass determination.

The remaining parts (underground parts and 3 cm above the soil) were washed from soil with running water and placed in a container with a few drops of low-foaming detergent (polysorbate 20) for 10–15 min and rinsed again with tap water to clean up loosened soil.

After washing, parts were kept in 50 % ethanol solution and stored at 4 °C for later separation and analysis.

## Morphology and anatomy of nodal roots

Parts kept in 50 % ethanol solution were further separated. Roots were separated from shoots. Under a dissecting microscope, roots that appeared damaged were sorted out and discarded. After that, the number of nodal roots was counted. To confirm the maximum rooting depth in soil, the length of the longest nodal root (waterlogged only) for each plant was recorded. After that and for each plant, a sample of four nodal roots of 11–14 cm length was taken. These roots were placed in an acrylic tray filled with water and carefully arranged so that there was no overlapping of lateral roots. These roots were scanned to record grey images at 600 dpi with a dual scanner (EPSON Expression 1680, Japan). Scanned images were then used for manual determination of the number of lateral roots (first order only) developed from the main axis of nodal roots.

Roots employed for determination of the number of lateral roots were used in the measurement of the diameter and areas of aerenchyma and stele of nodal roots. The diameter and areas of aerenchyma and stele were measured in cross-sections taken at 2.0, 5.0, 10 and 11–14 cm behind the root tip. Sections of roots were taken using a hand-held razor blade and examined under a light microscope that was equipped with a digital camera (Nikon Coolpix 4500, Japan). An eyepiece reticle was used to record root diameters. The areas of aerenchyma and stele (expressed as a percentage of total cross-sectional area) were determined in each digital picture using ImageJ software (v. 1.38, National Institutes of Health, USA). Cross-sections of first-order lateral roots were also taken when possible.

## Areas of aerenchyma in leaf sheaths and internodes of stolons

Stolon pieces (3–5 cm length) kept in 50 % ethanol solution were used for determination of aerenchyma in leaf sheaths and internodes of stolons. The greenest stolon per plant was visually selected, and two to five cross-sections of leaf sheaths and internodes were taken at 1 cm from the first observable node from the root-shoot junction. The areas of aerenchyma in each cross-section were analysed as described above.

Once morpho-anatomical traits had been recorded, the remaining parts kept in 50 % ethanol were used for determination of dry weight. As cross-sections used for microscopy studies did not exceed 3 % of fresh weight, the remaining non-cross-sectioned parts were also taken into account for dry weight determination. The
remaining shoots and roots were oven dried at 60 °C for
72 h for dry mass determination.

Statistical analysis

Data were analysed to generate mean values, standard
deviation and analysis of variance (ANOVA) using R soft-
ware (v. 2.15.2) (R Development Core Team, 2012). Log
transformation was carried out to ensure normality of
data. Differences between accessions were analysed and
reported with the least significant difference (LSD) at
α = 0.05.

Results

Effect of waterlogging on leaf number, leaf greenness and dry mass production

Prior to the beginning of the experiment, all accessions
showed a similar number of leaves per plant (around 14
leaves). Leaf production increased in all accessions when
grown under drained or waterlogged soil conditions (P <
0.05), but CIAT 6013 produced more leaves than the rest
of the accessions tested (Table 1). There were no signifi-
cant differences in leaf production among plants grown
under waterlogging when compared with plants grown
under drained soil conditions (Table 1). SPAD values
decreased in all accessions of B. humidicola from the be-
ginning of the experiment under drained or waterlogged
soil conditions (P < 0.05), but these values were not
further reduced under waterlogging [see Supporting
Information]. When compared with the drained treat-
ment, only CIAT 26570, CIAT 679 and CIAT 6133 increased
their shoot dry mass, whereas CIAT 16866 and CIAT 16888
showed a reduction of shoot dry mass under waterlogged
conditions (Fig. 1). Waterlogging treatment significantly
reduced root dry mass in all accessions (Fig. 1). Data
shown for shoot dry mass are the sum of leaves, stolons
and tillers, but there was a trend to allocate more
biomass to stolons [see Supporting Information].

Effect of waterlogging on root morphology and anatomy

Waterlogging increased the number of nodal roots per
plant in six accessions (CIAT 16888, CIAT 16886, CIAT
6133, CIAT 6707, CIAT 679 and CIAT 26570; Table 2).
Nodal roots formed under waterlogged conditions were
almost invariably whiter than roots of plants grown
under drained conditions (visual observation). Most of
the nodal roots developed fewer lateral roots (first order)
when grown under waterlogged conditions; however,
this was only statistically significant for seven accessions
(CIAT 16888, CIAT 26416, CIAT 6013, CIAT 6133, CIAT
6707, CIAT 679 and CIAT 26570; Table 2). Prior to the begin-
ning of the experiment, the maximum rooting depth of all

accessions was around 25 cm below the soil surface [**see Supporting Information**]. When grown under drained soil conditions, nodal roots of all accessions reached the bottom of the cylinders (77 cm) prior to the completion of the experiment. Under waterlogged conditions, the maximum rooting depth of all accessions did not appear to grow deeper than 30 cm below the soil surface. This was confirmed at harvest with measurements of the length of the longest nodal root (Table 2). Among *B. humidicola* accessions, CIAT 26152 showed the shortest nodal roots (15.5 cm) whereas roots of CIAT 26570 were the longest (26.4 cm) under waterlogged soil conditions (Table 2).

Nodal roots grown under waterlogged soil tended to be of greater diameter, but only seven accessions (CIAT 16888, CIAT 26181, CIAT 26416, CIAT 6133, CIAT 6707, CIAT 679 and CIAT 26570) showed significant increases when compared with nodal roots of drained plants (Table 3). Almost invariably, nodal roots of waterlogged plants lacked root hairs (visual observation; Fig. 2). Aerenchyma developed in nodal roots of all accessions of *B. humidicola* grown under drained soil conditions (Fig. 2). The extent of aerenchyma development under drained conditions varied among accessions (Figs 2 and 3, Table 3). Growth in waterlogged soil increased aerenchyma development and decreased the relative proportion of stele in cross-sections of nodal roots in all accessions tested (Fig. 2, Table 3). For all accessions, the relative proportions of aerenchyma and stele along the root length decreased from the root base to the root tip (Fig. 3; data not shown for stele). Aerenchyma development in nodal roots was absent at sites of the presence and/or emergence of lateral roots (visual observation) [**see Supporting Information**]. Lateral roots showed negligible aerenchyma and their diameters were 10–30 times smaller than those of nodal roots (visual observation).

Effect of waterlogging on proportional areas
of aerenchyma in leaf sheaths and internodes
of stolons

In all *B. humidicola* accessions, aerenchyma developed in
leaf sheaths, the pith cavity and in the cortex of stolon
internodes even under drained conditions (Figs 4 and 5,
Table 4). Leaf sheaths and stolon internodes in all
accessions showed increased aerenchyma development
under waterlogged conditions (Figs 4 and 5, Table 4).
Aerenchyma was absent in nodes.

Discussion

In the present study, an increase in the number of leaves in
all *B. humidicola* accessions was an indication that plants
kept growing under waterlogging, even if some accessions
showed reductions of shoot dry mass under this condition
<table><caption>**Table 1.** Number of leaves of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) before the beginning of the experiment and after 21 days of growth under drained or waterlogged soil. Data shown are means of six replicates ± SD. An asterisk represents significant differences between treatments for each accession (statistical significance at the \*0.05, \*\*0.01 and \*\*\*0.001 probability levels). All accessions showed an increase in the number of leaves under both treatments when compared with initial values (*P* < 0.05). NS: not significant. *P*<sub>anova</sub> and LSD values exclude checks.</caption><thead><tr><th rowspan="3">Accession</th><th colspan="4">Number of leaves (plant<sup>-1</sup>)</th></tr><tr><th colspan="2">0 days of treatment</th><th colspan="2">21 days of treatment</th></tr><tr><th>Drained</th><th>Waterlogged</th><th>Drained</th><th>Waterlogged</th></tr></thead><tbody><tr><td>CIAT 26570</td><td>13.7 ± 5.1</td><td>14.3 ± 5.7</td><td>56.5 ± 20.1</td><td>72.0 ± 23.2</td></tr><tr><td>CIAT 679</td><td>13.8 ± 6.0</td><td>12.7 ± 4.8</td><td>39.5 ± 16.8</td><td>40.0 ± 16.2</td></tr><tr><td>CIAT 6133</td><td>13.5 ± 4.6</td><td>14.3 ± 5.6</td><td>33.8 ± 11.5</td><td>38.0 ± 11.6</td></tr><tr><td>CIAT 16182</td><td>15.8 ± 7.8</td><td>14.8 ± 9.4</td><td>47.2 ± 19.6</td><td>38.5 ± 16.9</td></tr><tr><td>CIAT 6707</td><td>14.0 ± 2.8</td><td>12.7 ± 5.1</td><td>39.0 ± 11.0</td><td>38.3 ± 12.0</td></tr><tr><td>CIAT 16886</td><td>10.3 ± 4.3</td><td>14.3 ± 3.9</td><td>32.5 ± 17.8</td><td>51.2 ± 30.2</td></tr><tr><td>CIAT 26152</td><td>17.0 ± 5.2</td><td>14.0 ± 4.0</td><td>76.3 ± 31.8</td><td>52.0 ± 16.1</td></tr><tr><td>CIAT 6013</td><td>16.7 ± 5.9</td><td>18.5 ± 5.0</td><td>122.7 ± 59.2</td><td>82.5 ± 30.7</td></tr><tr><td>CIAT 26416</td><td>12.5 ± 7.2</td><td>12.0 ± 4.0</td><td>49.0 ± 9.9</td><td>39.8 ± 14.5</td></tr><tr><td>CIAT 26181</td><td>12.2 ± 3.8</td><td>10.3 ± 5.3</td><td>45.5 ± 28.1</td><td>25.0 ± 14.8</td></tr><tr><td>CIAT 16866</td><td>16.3 ± 8.1</td><td>14.7 ± 4.1</td><td>46.8 ± 27.6</td><td>37.3 ± 9.2</td></tr><tr><td>CIAT 16888</td><td>11.0 ± 3.1</td><td>12.0 ± 4.1</td><td>56.3 ± 31.4</td><td>28.0 ± 5.4</td></tr><tr><td>P<sub>anova</sub></td><td colspan="2">0.5159</td><td colspan="2">0.0000</td></tr><tr><td>LSD<sub>0.05</sub></td><td colspan="2">NS</td><td colspan="2">51.4</td></tr><tr><td>Checks</td><td colspan="4"></td></tr><tr><td>B. brizantha</td><td>11.2 ± 3.4</td><td>9.8 ± 1.9</td><td>21.3 ± 8.5</td><td>7.0 ± 2.4**</td></tr><tr><td>B. ruziziensis</td><td>16.0 ± 5.7</td><td>15.0 ± 5.9</td><td>42.2 ± 5.8</td><td>10.0 ± 3.5**</td></tr><tr><td>B. hybrid</td><td>9.0 ± 3.5</td><td>9.8 ± 3.0</td><td>33.7 ± 5.3</td><td>7.0 ± 1.7**</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Shoot and root dry mass of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained (white column) or waterlogged soil (grey column) conditions for 21 days. Columns represent means and error bars their standard deviation (n = 6). An asterisk represents significant differences between treatments for each accession (statistical significance at the \*0.05, \*\*0.01 and \*\*\*0.001 probability levels). LSD values exclude checks.</figcaption></figure>
<table><caption>**Table 2.** Number of nodal and lateral roots and length of the longest nodal root (LLN) of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained or waterlogged soil for 21 days. The number of lateral roots (first order only) was determined from nodal roots of 11–14 cm length. Data shown are means of six replicates ± SD. An asterisk represents significant differences between treatments for each accession (statistical significance at the *0.05*, **0.01 and ***0.001 probability levels). *P*<sub>anova</sub> and LSD values exclude checks.</caption><thead><tr><th rowspan="2">Accession</th><th colspan="2">Number of nodal roots (plant<sup>-1</sup>)</th><th colspan="2">Number of lateral roots</th><th>LLN (cm plant<sup>-1</sup>)</th></tr><tr><th>Drained</th><th>Waterlogged</th><th>Drained</th><th>Waterlogged</th><th>Waterlogged</th></tr></thead><tbody><tr><td>CIAT 26570</td><td>21.2 ± 3.5</td><td>26.5 ± 4.1*</td><td>10.5 ± 0.8</td><td>7.0 ± 0.7***</td><td>26.4 ± 5.6</td></tr><tr><td>CIAT 679</td><td>17.0 ± 3.9</td><td>36.0 ± 8.0***</td><td>10.8 ± 1.4</td><td>5.6 ± 0.5***</td><td>21.0 ± 3.7</td></tr><tr><td>CIAT 6133</td><td>20.0 ± 4.7</td><td>31.3 ± 7.3**</td><td>9.2 ± 1.1</td><td>7.8 ± 1.0*</td><td>23.2 ± 3.2</td></tr><tr><td>CIAT 16182</td><td>22.8 ± 6.9</td><td>28.8 ± 7.4</td><td>10.0 ± 1.5</td><td>9.6 ± 3.0</td><td>19.3 ± 3.0</td></tr><tr><td>CIAT 6707</td><td>20.0 ± 2.6</td><td>27.7 ± 6.3*</td><td>10.1 ± 1.4</td><td>6.2 ± 0.9***</td><td>22.2 ± 4.8</td></tr><tr><td>CIAT 16886</td><td>20.5 ± 8.5</td><td>37.3 ± 3.1**</td><td>10.3 ± 1.6</td><td>9.0 ± 2.3</td><td>17.2 ± 4.4</td></tr><tr><td>CIAT 26152</td><td>37.0 ± 10.8</td><td>39.3 ± 13.6</td><td>8.8 ± 1.2</td><td>8.0 ± 1.7</td><td>15.5 ± 2.6</td></tr><tr><td>CIAT 6013</td><td>42.7 ± 10.7</td><td>31.8 ± 9.4</td><td>10.2 ± 1.0</td><td>8.4 ± 1.5*</td><td>21.8 ± 4.8</td></tr><tr><td>CIAT 26416</td><td>41.5 ± 9.3</td><td>34.0 ± 4.5</td><td>11.9 ± 1.3</td><td>6.4 ± 1.7***</td><td>20.0 ± 3.3</td></tr><tr><td>CIAT 26181</td><td>26.7 ± 8.1</td><td>34.0 ± 5.2</td><td>6.8 ± 0.6</td><td>7.6 ± 1.5</td><td>18.0 ± 1.8</td></tr><tr><td>CIAT 16866</td><td>26.5 ± 4.1</td><td>34.8 ± 8.9</td><td>7.1 ± 1.1</td><td>7.8 ± 1.7</td><td>19.7 ± 4.4</td></tr><tr><td>CIAT 16888</td><td>42.7 ± 3.1</td><td>53.7 ± 10.7*</td><td>9.1 ± 1.1</td><td>6.3 ± 0.7***</td><td>17.0 ± 2.4</td></tr><tr><td>P<sub>anova</sub></td><td colspan="2">0.0000</td><td>0.0000</td><td>0.001</td><td>0.000</td></tr><tr><td>LSD<sub>0.05</sub></td><td colspan="2">13.3</td><td>2.3</td><td>3.0</td><td>7.3</td></tr><tr><td>Checks</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B. brizantha</td><td>33.2 ± 4.6</td><td>30.3 ± 3.1</td><td>6.6 ± 1.4</td><td>5.5 ± 1.2</td><td>12.5 ± 2.7</td></tr><tr><td>B. ruziziensis</td><td>49.2 ± 2.3</td><td>48.8 ± 9.1</td><td>9.0 ± 1.0</td><td>8.5 ± 2.4</td><td>12.5 ± 1.9</td></tr><tr><td>B. hybrid</td><td>46.3 ± 8.9</td><td>34.0 ± 4.1*</td><td>5.3 ± 1.2</td><td>4.4 ± 1.3</td><td>9.3 ± 2.0</td></tr></tbody></table>

when compared with the drained treatment (Fig. 1). It was noteworthy that one accession, CIAT 6013, was very productive under both drained and waterlogged soil conditions (Fig. 1). The high production of leaves from the start of the experiment together with the higher production of shoot dry mass of CIAT 6013 suggest a faster growth rate than the rest of the tested accessions. From an agro-nomic point of view, CIAT 6013 shows promise for use in zones with a risk of waterlogging events. However, the inherent high productivity of CIAT 6013 may not be in itself causally related to waterlogging tolerance since it has been shown in other grasses (e.g. wheat, barley and oats) that vigour and tolerance are not necessarily positively linked ( Setter and Waters 2003; Pang et al. 2004).

The reduction in growth of root mass in water-saturated soils is common in flood-tolerant grasses (Naidoo and Naidoo 1992; Baruch 1994; Loreti and Oesterheld 1996). Waterlogging-induced new root formation is a well-documented response that presumably allows the plant to compensate for the decay of the original root system brought about by a lack of oxygen (Jackson and Drew 1984; Vartapetian and Jackson 1997). Therefore,

increasing the number of nodal roots is considered of high importance for survival under waterlogged conditions (Visser et al. 1996). In the present study, six accessions showed an increase in the number of nodal roots under waterlogged conditions (Table 2). However, none of the remaining accessions showed a reduction in the number of nodal roots under waterlogged conditions when compared with plants grown under drained soil (Table 2). Therefore, it is likely that a reduction of root dry mass in all accessions was mainly a reflection of the restriction of root penetration below 30 cm soil depth.

Roots can display various adaptations to waterlogged soils; among them, the presence of aerenchyma is the most obviously adaptive one (Laan et al. 1989). All accessions of *B. humidicola* developed aerenchyma in nodal roots even under well-drained soil, suggesting that this is a constitutive trait in this species (Figs 2 and 3, Table 3). Constitutive root aerenchyma is common in plants adapted to waterlogged conditions (Justin and Armstrong 1987; Jackson and Armstrong 1999; Visser et al. 2000; Mano et al. 2006; Abiko et al. 2012). As development of aerenchyma takes time, constitutive root aerenchyma would
<table><caption>**Table 3.** Average diameter, aerenchyma and stele of nodal roots of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained or waterlogged soil for 21 days. Data shown are means of six replicates ± SD. An asterisk represents significant differences between treatments for each accession (statistical significance at the *0.05*, **0.01 and ***0.001 probability levels). *P*<sub>anova</sub> and LSD values exclude checks.</caption><thead><tr><th rowspan="2">Accession</th><th colspan="2">Diameter (mm)</th><th colspan="2">% aerenchyma</th><th colspan="2">% stele</th></tr><tr><th>Drained</th><th>Waterlogged</th><th>Drained</th><th>Waterlogged</th><th>Drained</th><th>Waterlogged</th></tr></thead><tbody><tr><td>CIAT 26570</td><td>1.5 ± 0.2</td><td>1.8 ± 0.1**</td><td>14.5 ± 1.7</td><td>34.4 ± 3.8***</td><td>15.0 ± 1.1</td><td>6.4 ± 0.8***</td></tr><tr><td>CIAT 679</td><td>1.4 ± 0.1</td><td>1.7 ± 0.1***</td><td>13.5 ± 1.2</td><td>29.3 ± 1.6***</td><td>16.9 ± 2.9</td><td>8.1 ± 0.8***</td></tr><tr><td>CIAT 6133</td><td>1.5 ± 0.0</td><td>1.7 ± 0.0***</td><td>13.3 ± 1.5</td><td>28.3 ± 2.0***</td><td>16.6 ± 1.9</td><td>8.2 ± 0.9***</td></tr><tr><td>CIAT 16182</td><td>1.1 ± 0.1</td><td>1.2 ± 0.3</td><td>13.2 ± 1.5</td><td>29.6 ± 3.5***</td><td>11.3 ± 0.9</td><td>6.9 ± 0.8***</td></tr><tr><td>CIAT 6707</td><td>1.0 ± 0.2</td><td>1.4 ± 0.1**</td><td>10.4 ± 1.0</td><td>28.9 ± 2.2***</td><td>14.8 ± 2.2</td><td>6.7 ± 0.2***</td></tr><tr><td>CIAT 16886</td><td>1.2 ± 0.1</td><td>1.1 ± 0.3</td><td>11.0 ± 1.9</td><td>31.5 ± 2.1***</td><td>15.0 ± 2.7</td><td>7.6 ± 0.3***</td></tr><tr><td>CIAT 26152</td><td>1.4 ± 0.1</td><td>1.5 ± 0.3</td><td>11.8 ± 1.4</td><td>29.8 ± 2.1***</td><td>12.5 ± 1.2</td><td>7.4 ± 0.7***</td></tr><tr><td>CIAT 6013</td><td>1.6 ± 0.3</td><td>1.9 ± 0.3</td><td>15.3 ± 1.7</td><td>31.2 ± 3.8***</td><td>15.2 ± 2.2</td><td>9.2 ± 1.4***</td></tr><tr><td>CIAT 26416</td><td>1.1 ± 0.1</td><td>1.5 ± 0.1***</td><td>9.5 ± 1.7</td><td>29.7 ± 3.0***</td><td>23.8 ± 1.9</td><td>9.5 ± 1.5***</td></tr><tr><td>CIAT 26181</td><td>1.2 ± 0.2</td><td>1.7 ± 0.1***</td><td>9.6 ± 2.5</td><td>28.8 ± 1.9***</td><td>12.5 ± 3.0</td><td>7.3 ± 1.6**</td></tr><tr><td>CIAT 16866</td><td>1.2 ± 0.1</td><td>1.3 ± 0.3</td><td>14.0 ± 2.4</td><td>28.3 ± 1.3***</td><td>14.2 ± 1.9</td><td>7.0 ± 0.6***</td></tr><tr><td>CIAT 16888</td><td>1.5 ± 0.0</td><td>1.6 ± 0.1*</td><td>12.8 ± 1.7</td><td>31.5 ± 2.1***</td><td>12.6 ± 0.2</td><td>7.4 ± 1.5***</td></tr><tr><td>P<sub>anova</sub></td><td>0.0000</td><td>0.0000</td><td>0.0000</td><td>0.01</td><td>0.0000</td><td>0.0000</td></tr><tr><td>LSD<sub>0.05</sub></td><td>0.3</td><td>0.4</td><td>3.3</td><td>4.9</td><td>3.8</td><td>2.0</td></tr><tr><td>Checks</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B. brizantha</td><td>1.9 ± 0.1</td><td>2.0 ± 0.1</td><td>-</td><td>15.6 ± 1.4</td><td>17.8 ± 1.7</td><td>14.9 ± 2.8</td></tr><tr><td>B. ruziziensis</td><td>1.1 ± 0.1</td><td>1.3 ± 0.2</td><td>-</td><td>18.3 ± 1.3</td><td>19.2 ± 2.5</td><td>17.1 ± 1.1</td></tr><tr><td>B. hybrid</td><td>2.0 ± 0.1</td><td>1.7 ± 0.1***</td><td>-</td><td>16.8 ± 1.5</td><td>19.1 ± 1.7</td><td>16.2 ± 1.1**</td></tr></tbody></table>

aid root aeration during the initial stages of waterlogging (Mano et al. 2006; Colmer and Voesenek 2009; Abiko et al. 2012). Overall aerenchyma in cross-sections of nodal roots under drained conditions in *B. humidicola* accessions (14–24 %) was similar to values found in other grass species that are adapted to waterlogging such as *Oryza sativa* (Armstrong 1971; Colmer et al. 1998), *Glyceria maxima* (Smirnoff and Crawford 1983), *Brachiaria mutica* and *Echinochloa polystachya* (Baruch and Merida 1995), which typically range from 20 to 25 % under O₂-sufficient conditions (McDonald et al. 2001). The area of aerenchyma in roots of *B. humidicola* accessions increased over 2-fold from constitutive levels when grown in waterlogged soil (Table 3). This finding is similar to the 1.5- to 3.0-fold increase reported for several wetland grasses grown in flooded compared with drained sand (Smirnoff and Crawford 1983). In the present study, the accessions that showed greater levels of root aerenchyma under drained conditions did not necessarily show greater aerenchymatous tissue under waterlogged soils, as was shown in other species (e.g. *Zea nicaraguensis*; Mano and Omori 2013). This indicates that high constitutive aerenchyma

alone may not be a good indicator of the adaptation ability to transient waterlogging among *B. humidicola* accessions and, therefore, evaluation of aerenchyma development under this soil condition should be taken into account.

An increase in root thickness will allow the plant to cope better with waterlogged soils as it contributes to an increase in O₂ flow to the root tip (Armstrong 1979; Aguilar et al. 2008). Seven accessions showed this morphological acclimation (Table 3). An increase in root diameter under waterlogged conditions was previously reported in other tropical grasses (Baruch and Merida 1995; Imaz et al. 2012). The development of nodal roots with a larger diameter may increase the cortex area in which aerenchyma can develop and would also help in ventilating gases such as ethylene and carbon dioxide, which typically increases under waterlogging conditions (Visser et al. 1997, 2000).

The proportional area of stele in *B. humidicola* accessions decreased by 39–60 % under waterlogging (Table 3). This morphological acclimation was also documented in other species such as *Hyparrhenia ruffa* (34 % reduction; Baruch
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Aerenchyma development in nodal roots of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained (D) or waterlogged (W) soil conditions for 21 days. Cross-sections taken at 10 cm from the root tip. The arrow indicates an air space in the cortex. Scale bars = 250 µm.</figcaption></figure>

and Merida 1995) and *Hordeum vulgare* (20% reduction; Pang et al. 2004). Studies on *Zea mays* (Armstrong 1994) and *Musa* sp. (Aguilar et al. 2003) found that the respiratory demands of stele are about four to six times greater than those found in the cortex; thus a useful adaptation to restricted O₂ supply might be a narrower stele (Armstrong et al. 2000). A narrower stele would allow less consumption of O₂ per unit length of the diffusion path of the aerenchyma that leads to the root tip (Armstrong et al. 2000) and would also allow a greater cortex where aerenchyma can be formed (Justin and Armstrong 1987; McDonald et al. 2002).

A reduction in the number of lateral roots under oxygen-deficient conditions might be beneficial as each lateral root demands oxygen for growth and maintenance, thereby reducing transport of O₂ along the root where they develop and therefore to the root tip (Armstrong et al. 1983; Aguilar et al. 2003). The reduction in the number of lateral roots under waterlogging, as shown by seven accessions (Table 2), was also recorded for other species such as *Pisum sativum* (Armstrong et al. 1983) and *Musa* sp. (Aguilar et al. 2003). Furthermore, development of lateral roots seemed to locally restrict

aerenchyma development in the nodal roots of *B. humidicola* [see Supporting Information]. Lack of aerenchyma adjacent to lateral root emergence was also noted by other authors (Smirnoff and Crawford 1983; Justin and Armstrong 1991; Enstone and Peterson 2005). As lateral roots develop from the pericycle, their presence occupies space that would otherwise be aerenchymatous (Justin and Armstrong 1991).

Reduction of root penetration from the beginning of waterlogging coincides with depletion of O₂ as shown by redox potential values <330 mV (where molecular oxygen is undetectable; Ponnamperuma 1972) after 7 days. Roots stop growing if the root tip becomes anoxic (Armstrong and Drew 2002). As root penetration into anaerobic soil relies greatly upon the capacity for internal O₂ transport to the root tip (for a review see Colmer 2003; Colmer and Greenway 2005), greater aerenchyma formation, as shown by CIAT 26570, would facilitate deeper penetration of roots into waterlogged soil.

Aerenchyma in the shoot provides a ventilation channel between shoots and submerged roots (Sorrell et al. 1994; Colmer 2003; Afreen et al. 2007; Armstrong et al. 2009).
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Percentage of aerenchyma along nodal roots of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained (open circles) or waterlogged soil (closed circles) conditions for 21 days. Circles represent means and error bars their standard deviation (n = 6). Significant differences at *P* < 0.01 for treatments at each root distance were found.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Aerenchyma development in leaf sheaths of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained (D) or waterlogged (W) soil conditions for 21 days. Cross-sections taken at 1 cm from the first observable node from the root-shoot junction. An arrow indicates air spaces in the leaf sheath. Scale bars = 500 µm.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Aerenchyma development in stolon internodes of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained (D) or waterlogged (W) soil conditions for 21 days. Cross-sections taken at 1 cm from the first observable node from the root-shoot junction. The arrow indicates an air space in the cortex of stolons. All stolons of *B. humidicola* grown either under drained or waterlogged conditions showed a hollowed pith. Scale bars = 500 µm.</figcaption></figure>

Although aerenchyma was absent in the nodes of *B. humidicola*, it is possible that the continuum of the ventilation path between shoots and roots was restricted (rather than impeded) to intercellular spaces in the nodes, as was suggested in rice (Butterbach-Bahl *et al.* 1997; Steffens *et al.* 2011). In this sense, the presence of a ventilation channel in leaf sheaths and stolon internodes (Figs 4 and 5, Table 4) might also contribute to the good adaptation of *B. humidicola* to waterlogged soils by enabling O₂ transport from shoot to root, but also by facilitating the escape of gases (such as both carbon dioxide and ethylene which usually accumulate in the roots under waterlogged soil conditions; Visser *et al.* 1997). Very interestingly, CIAT 26570 also showed more aerenchyma in leaf sheaths and internodes than the rest of the accessions tested (Figs 4 and 5, Table 4).

## Conclusions

The results obtained from this study help to identify adaptive traits associated with waterlogging tolerance in *B. humidicola*. Constitutive aerenchyma in nodal roots may allow *B. humidicola* accessions to tolerate oxygen

deficiency from the onset of waterlogging. During waterlogging, development of more and thicker nodal roots with a higher proportion of aerenchyma, smaller stele area and showing a reduction of lateral roots may act together for a more efficient system for O₂ transport to the elongation zone of the root (cf. Colmer 2003; cf. Colmer and Greenway 2005). The accession that showed this combination of root traits (CIAT 26570) showed larger roots (Table 3) and less reductions of root dry mass under waterlogged conditions (Table 1). Furthermore, the increase of aerenchyma in leaf sheaths and internodes above constitutive levels might improve the ventilation of gases between roots and shoots under waterlogged soil conditions. A more extensive root aeration system combined with a more prominent ventilation system in the shoot would probably allow CIAT 26570 to grow more quickly under waterlogged conditions than the other accessions we tested.

Evaluation of plants for their tolerance to waterlogging should also consider their ability to recover after the soil drains (Malik *et al.* 2002; Zhou 2010; Bramley *et al.* 2011; Striker 2012). For example, genotypes may differ in their responses to waterlogging but their variation after
<table><caption>**Table 4.** Percentage of aerenchyma in shoots (leaf sheaths and stolon internodes) of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained or waterlogged soil for 21 days. Data shown are means of six replicates ± SD. An asterisk represents significant differences between treatments for each accession (statistical significance at the *0.05*, **0.01** and ***0.001** probability levels). *P*<sub>anova</sub> and LSD values exclude checks.</caption><thead><tr><th rowspan="3">Accession</th><th colspan="6">% aerenchyma in shoot</th></tr><tr><th colspan="2">Leaf sheaths</th><th colspan="2">Pith (stolon internode)</th><th colspan="2">Cortex (stolon internode)</th></tr><tr><th>Drained</th><th>Waterlogged</th><th>Drained</th><th>Waterlogged</th><th>Drained</th><th>Waterlogged</th></tr></thead><tbody><tr><td>CIAT 26570</td><td>4.9 ± 0.7</td><td>10.3 ± 0.7***</td><td>3.3 ± 1.6</td><td>7.7 ± 1.2***</td><td>7.8 ± 0.7</td><td>18.4 ± 1.7***</td></tr><tr><td>CIAT 679</td><td>4.2 ± 0.8</td><td>8.8 ± 0.8***</td><td>5.1 ± 0.8</td><td>7.6 ± 1.3**</td><td>4.5 ± 1.5</td><td>9.2 ± 1.5***</td></tr><tr><td>CIAT 6133</td><td>4.0 ± 0.9</td><td>7.4 ± 0.7***</td><td>4.7 ± 1.0</td><td>7.1 ± 1.4**</td><td>1.3 ± 0.5</td><td>6.8 ± 0.7***</td></tr><tr><td>CIAT 16182</td><td>4.3 ± 0.5</td><td>7.7 ± 0.7***</td><td>2.8 ± 0.7</td><td>4.9 ± 0.9**</td><td>3.3 ± 0.9</td><td>7.1 ± 1.5***</td></tr><tr><td>CIAT 6707</td><td>3.6 ± 0.5</td><td>7.8 ± 0.8***</td><td>2.3 ± 0.6</td><td>5.2 ± 1.1***</td><td>1.3 ± 0.8</td><td>5.9 ± 0.8***</td></tr><tr><td>CIAT 16886</td><td>2.7 ± 0.5</td><td>5.6 ± 0.5***</td><td>7.2 ± 0.8</td><td>14.6 ± 1.4***</td><td>1.2 ± 0.6</td><td>5.9 ± 1.5***</td></tr><tr><td>CIAT 26152</td><td>3.5 ± 0.7</td><td>6.4 ± 0.8***</td><td>2.4 ± 0.6</td><td>4.5 ± 1.0**</td><td>3.2 ± 0.5</td><td>8.1 ± 0.7***</td></tr><tr><td>CIAT 6013</td><td>4.3 ± 0.7</td><td>10.2 ± 0.8***</td><td>4.4 ± 1.4</td><td>8.0 ± 1.3**</td><td>1.9 ± 0.4</td><td>10.7 ± 2.2***</td></tr><tr><td>CIAT 26416</td><td>3.8 ± 0.7</td><td>6.2 ± 0.6***</td><td>3.0 ± 0.8</td><td>6.1 ± 0.9***</td><td>3.4 ± 1.5</td><td>7.9 ± 1.3***</td></tr><tr><td>CIAT 26181</td><td>3.1 ± 0.5</td><td>6.9 ± 0.7***</td><td>5.2 ± 1.3</td><td>9.1 ± 1.8**</td><td>1.6 ± 0.5</td><td>7.8 ± 1.8***</td></tr><tr><td>CIAT 16866</td><td>3.8 ± 0.5</td><td>7.3 ± 1.0***</td><td>4.0 ± 0.8</td><td>7.5 ± 1.1***</td><td>2.7 ± 0.6</td><td>6.9 ± 1.5***</td></tr><tr><td>CIAT 16888</td><td>5.3 ± 0.3</td><td>8.5 ± 0.6***</td><td>5.3 ± 0.7</td><td>7.8 ± 1.2**</td><td>2.9 ± 1.2</td><td>5.9 ± 1.3***</td></tr><tr><td>P value</td><td colspan="2">0.0000</td><td colspan="2">0.0000</td><td colspan="2">0.0000</td></tr><tr><td>LSD<sub>0.05</sub></td><td colspan="2">1.2</td><td colspan="2">1.4</td><td colspan="2">2.7</td></tr><tr><td>Checks</td><td colspan="6"></td></tr><tr><td>B. brizantha</td><td>-</td><td>6.5 ± 0.6</td><td>-</td><td>5.2 ± 1.9</td><td>-</td><td>-</td></tr><tr><td>B. ruziziensis</td><td>-</td><td>7.0 ± 0.7</td><td>-</td><td>5.5 ± 1.3</td><td>-</td><td>-</td></tr><tr><td>B. hybrid</td><td>-</td><td>6.3 ± 0.9</td><td>-</td><td>4.6 ± 2.0</td><td>-</td><td>-</td></tr></tbody></table>

subsequent drainage might be even more marked (e.g. cool-season grain legumes; Solaiman et al. 2007). Morpho-anatomical changes of the root might be beneficial for growth under waterlogging but may bring up limitations for growth under non-waterlogged conditions (McDonald et al. 2002). For example, reduced stele size was associated with smaller meta-xylem vessels in *Alisma triviale*, which in turn might be responsible for reduced hydraulic conductivity (Ryser et al. 2011). Furthermore, waterlogging significantly reduces root penetration, and thereby resumption of deep root growth may be of great importance for exploration of soil nutrients and water after the waterlogged soil has been drained (Thomson et al. 1992). In this sense, aspects of root function and growth after waterlogging, and whether tolerance of *B. humidicola* accessions during waterlogging translates into the ability to grow after subsequent soil drainage, should be taken into consideration in future studies.

## Sources of Funding

This work was partially funded by Fontagro (USA) (project number: FTG-8060/08).

## Contributions by the Authors

J.A.C. was involved in designing the experiments, data collection and analysis, manuscript preparation and submission. J.R. contributed to the set-up of experiments. J.C.J. and D.N. provided technical assistance during experiments. I.M.R. was involved in designing and supervision of experiments, manuscript preparation and submission.

## Conflicts of Interest Statement

None declared.
**Acknowledgements**

We thank Dr M. Akutsu, JIRCAS, Japan; and E. Cuarán and H. Mina, CIAT, Colombia for their help during the course of the experiment. We thank the associate editor and two anonymous reviewers for useful comments that improved this manuscript.

**Supporting Information**

The following Supporting Information is available in the online version of this article –

**File 1.** Table. Changes in leaf greenness (SPAD units) of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained or waterlogged soil for 21 days. Data shown are means of six replicates ± SD. An asterisk represents significant differences between treatments for each genotype (statistical significance at the \*0.05, \*\*0.01 and \*\*\*0.001 probability levels). All accessions showed a decrease in SPAD values under both treatments when compared with initial values (*P* < 0.05). *P*<sub>anova</sub> and LSD values exclude checks.

**File 2.** Table. Relative allocation of the dry mass of leaves, stolons and tillers to total shoots of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) grown under drained or waterlogged soil for 21 days. Data shown are means of six replicates ± SD. An asterisk represents significant differences between treatments for each accession (statistical significance at the \*0.05, \*\*0.01 and \*\*\*0.001 probability levels). NS: not significant. *P*<sub>anova</sub> and LSD values exclude checks.

**File 3.** Table. Maximum rooting depth of 12 *B. humidicola* accessions (plus three checks: *B. brizantha*, *B. ruziziensis* and *B. hybrid*) before the beginning of the experiment. Data shown are means of six replicates ± SD. NS: not significant. *P*<sub>anova</sub> and LSD values exclude checks.

**File 4.** Figure. Local inhibition of aerenchyma development by lateral roots. (A) Sequential cross-sections taken at ~9.5–10.0 cm from the root tip of a nodal root of *B. humidicola* (CIAT 6707) grown under waterlogged conditions and (B) emergence of a lateral root from a nodal root (CIAT 16888) grown under waterlogged conditions. AE: air space; ST: stele; LR: lateral root. Scale bars = 250 µm.

**Literature Cited**

Abiko T, Kotula L, Shiono K, Malik AI, Colmer TD, Nakazono M. 2012. Enhanced formation of aerenchyma and induction of a barrier to radial oxygen loss in adventitious roots of *Zea nicaraguensis* contribute to its waterlogging tolerance as compared with maize (*Zea mays* ssp. *mays*). *Plant, Cell and Environment* **35**: 1618–1630.

Afreen F, Zobayer SMA, Armstrong J, Armstrong W. 2007. Pressure gradients along whole culms and leaf sheaths, and other aspects of humidity-induced gas transport in *Phragmites australis*. *Journal of Experimental Botany* **58**:1651–1662.

Aguilar EA, Turner DW, Gibbs DJ, Armstrong W, Sivasithamparam K. 2003. Oxygen distribution and movement, respiration and nutrient loading in banana roots (*Musa* sp. L) subjected to aerated and oxygen-depleted environments. *Plant and Soil* **253**:91–102.

Aguilar EA, Santos PJA, Tamisin LT Jr. 2008. Root characteristics of five local banana cultivars (*Musa* spp.) under waterlogged conditions. *The Philippine Journal of Crop Science* **33**:14–23.

Argel PJ, Keller-Grein G. 1996. Regional experience with *Brachiaria*: tropical America-humid lowlands. In: Miles JW, Mass BL, Valle CB, eds. *Brachiaria: biology, agronomy and improvement*. Cali: CIAT/Embrapa, 205–224.

Armstrong J, Keep R, Armstrong W. 2009. Effects of oil on internal gas transport, radial oxygen loss, gas films and bud growth in *Phragmites australis*. *Annals of Botany* **103**:333–340.

Armstrong W. 1971. Radial oxygen losses from intact rice roots as affected by distance from the apex, respiration and waterlogging. *Physiologia Plantarum* **25**:192–197.

Armstrong W. 1979. Aeration in higher plants. *Advances in Botanical Research* **7**:225–332.

Armstrong W. 1994. Polarographic oxygen electrodes and their use in plant aeration studies. *Proceedings of the Royal Society of Edinburgh* **102B**:511–528.

Armstrong W, Beckett PM. 1987. Internal aeration and the development of stellar anoxia in submerged roots: a multishelled mathematical model combining axial diffusion of oxygen in the cortex with radial losses to the stele, the wall layers and the rhizosphere. *New Phytologist* **105**:221–245.

Armstrong W, Drew MC. 2002. Root growth and metabolism under oxygen deficiency. In: Waisel Y, Eshel A, Kafkafi U, eds. *Plant roots: the hidden half*, 3rd edn. New York: Marcel Decker Inc., 729–761.

Armstrong W, Healy MT, Lythe S. 1983. Oxygen diffusion in pea. II. The oxygen status of the primary root as affected by growth, the production of laterals and radial oxygen loss. *New Phytologist* **94**: 549–559.

Armstrong W, Cousins D, Armstrong J, Turner DW, Beckett PM. 2000. Oxygen distribution in wetland plant roots and permeability barriers to gas-exchange with the rhizosphere: a microelectrode and modelling study with *Phragmites australis*. *Annals of Botany* **86**:687–703.

Baruch Z. 1994. Responses to drought and flooding in tropical forage grasses. I. Biomass allocation, leaf growth and mineral nutrients. *Plant and Soil* **164**:87–96.

Baruch Z, Merida T. 1995. Effects of drought and flooding on root anatomy of four tropical forage grasses. *International Journal of Plant Sciences* **156**:514–521.

Bramley H, Tyerman SD, Turner DW, Turner N. 2011. Root growth of lupins is more sensitive to waterlogging than wheat. *Functional Plant Biology* **38**:910–918.

Butterbach-Bahl K, Papen H, Rennenberg H. 1997. Impact of gas transport through rice cultivars on methane emission from rice paddy fields. *Plant, Cell and Environment* **20**:1175–1183.

Caetano LPS, Dias-Filho MB. 2008. Responses of six *Brachiaria* spp. accessions to root zone flooding. *Revista Brasileira de Zootecnia* **37**:795–801.
Canchila ER, Soca M, Wencomo HB, Ojeda F, Mateus H, Romero E, Argüello G, Ruiz R, Canchila N. 2011. Comportamiento agronómico de siete accesorias de *Brachiaria humidicola* durante la fase de establecimiento. *Pastos y Forrajes* **34**:155-166.

Cardoso JA, Jiménez JC, Rincón JD, Rao IM. 2010. *Variation for root aerenchyma formation in accessions of B. humidicola under waterlogged and drained soil conditions*. *Tropical Forages Annual Report*. Cali: CIAT, 48-52.

Colmer TD. 2003. Long-distance transport of gases in plants: a perspective on internal aeration and radial oxygen loss from roots. *Plant, Cell and Environment* **26**:17-36.

Colmer TD, Greenway H. 2005. Oxygen transport, respiration, and anaerobic carbohydrate catabolism in roots in flooded soils. In: Lambers H, Rivas-Carbo M, eds. *Plant respiration*. Heidelberg: Springer, 137-158.

Colmer TD, Voesenek LACJ. 2009. Flooding tolerance: suites of plant traits in variable environments. *Functional Plant Biology* **36**: 665-681.

Colmer TD, Gibberd MR, Wiengweera A, Tinh TK. 1998. The barrier to radial oxygen loss from roots of rice (*Oryza sativa* L.) is induced by growth in stagnant solution. *Journal of Experimental Botany* **49**:1431-1436.

Cruz P. 2009. *Evaluación agronómica de 23 genotipos de Brachiaria humidicola en el trópico húmedo de México*. MSc Thesis, Colegio de Postgraduados-Institución de enseñanza e investigación en ciencias agropecuarias Montecillo, México.

Dias-Filho MB, Carvalho CJR. 2000. Physiological and morphological responses of *Brachiaria* spp. to flooding. *Pesquisa Agropecuária Brasileira* **35**:1959-1966.

Enstone DE, Peterson CA. 2005. Suberin lamella development in maize seedlings roots grown in aerated and stagnant conditions. *Plant, Cell and Environment* **28**:444-455.

Hare MD, Saengkham M, Tatsapong P, Wongpichet K, Tudsri S. 2004. Waterlogging tolerance of some tropical pastures. *Tropical Grasslands* **38**:227-233.

Huber H, Jacobs E, Visser EJW. 2009. Variation in flooding-induced morphological traits in natural populations of white clover (*Trifolium repens*) and their effects on plant performance during soil flooding. *Annals of Botany* **103**:377-386.

Imaz JA, Giménez DO, Grimoldi AA, Striker GG. 2012. The effect of submergence on anatomical, morphological and biomass allocation responses of tropical grasses *Chloris gayana* and *Panicum coloratum* at seedling stage. *Crop & Pasture Science* **63**:1145-1155.

Jackson MB, Drew MC. 1984. Effects of flooding on growth and metabolism of herbaceous plants. In: Kozlowsky TT, ed. *Flooding and plant growth*. Orlando: Academic Press, 47-128.

Jackson MB, Armstrong W. 1999. Formation of aerenchyma and the process of plant ventilation in relation to soil flooding and submergence. *Plant Biology* **1**:274-287.

Juarez J, Bolaños ED, Vargas LM, Medina S, Martínez-Hernández PA. 2011. Protein dilution curves in genotypes of *Brachiaria humidicola* (Rendle) Schweick. *Cuban Journal of Agricultural Science* **45**: 321-330.

Justin SHFW, Armstrong W. 1987. The anatomical characteristics of roots and plant response to soil flooding. *New Phytologist* **106**: 465-495.

Justin SHFW, Armstrong W. 1991. A reassessment of the influence of NAA on aerenchyma formation in maize roots. *New Phytologist* **117**:607-618.

Laan P, Berrevoets MJ, Lythe S, Armstrong W, Blom CWPM. 1989. Root morphology and aerenchyma formation as indicators of the flood tolerance of *Rumex* species. *Journal of Ecology* **77**:693-703.

Loreti J, Oesterheld M. 1996. Intraspecific variation in the resistance to flooding and drought in populations of *Paspalum dilatatum* from different topographic positions. *Oecologia* **108**: 279-284.

Malik AI, Colmer TD, Lambers H, Setter TL, Schortemeyer M. 2002. Short-term waterlogging has long-term effects on the growth and physiology of wheat. *New Phytologist* **153**:225-236.

Mano Y, Omori F. 2013. Relationship between constitutive root aerenchyma formation and flooding tolerance in *Zea nicaraguensis*. *Plant and Soil* **370**:447-460.

Mano Y, Omori F, Takamizo T, Kindiger B, Bird McK R, Loaisiga CH. 2006. Variation in root aerenchyma formation in flooded and non-flooded maize and teosinte seedlings. *Plant and Soil* **281**: 269-279.

McDonald MP, Galwey NW, Colmer TD. 2001. Waterlogging tolerance in the tribe Triticeae: the adventitious roots of *Critesion marinum* have a relatively high porosity and a barrier to radial oxygen loss. *Plant, Cell and Environment* **24**:585-596.

McDonald MP, Galwey NW, Colmer TD. 2002. Similarity and diversity in adventitious root anatomy as related to root aeration among a range of wetland and dryland species. *Plant, Cell and Environment* **52**:441-451.

Miles JW, Valle CB, Rao IM, Euclides VPB. 2004. *Brachiaria* grasses. In: Moser L, Burson B, Sollenberger LE, eds. *Warm-season grasses*. Madison: ASA-CSSA-SSSA, 745-783.

Naidoo G., Naidoo S. 1992. Waterlogging responses of *Sporobolus virginusicus* (L.) Kunth. *Oecologia* **90**:445-450.

Pang J, Zhou M, Mendham N, Shabala S. 2004. Growth and physiological responses of six barley genotypes to waterlogging and subsequent recovery. *Australian Journal of Agricultural Research* **55**:895-906.

Ponnamperuma FN. 1972. The chemistry of submerged soil. *Advances in Agronomy* **24**:29-96.

R Development Core Team. 2012. *R: A language and environment for statistical computing*. Vienna, Austria: R Foundation for Statistical Computing, ISBN 3-900051-07-0, http://www.R-project.org/ (8 October 2013).

Rao IM, Roca WM, Ayarza MA, Tabares E, Garcia R. 1992. Somaclonal variation in plant adaptation to acid soil in the tropical forage legume *Stylosanthes guianensis*. *Plant and Soil* **146**: 21-30.

Rincón J, Cardoso JA, Miles JW, Rao IM. 2010. *Evaluation of Brachiaria humidicola germplasm accessions for tolerance to waterlogging*. *Tropical Forages Annual Report*. Cali: CIAT, 25-30.

Ryser P, Gill HK, Byrne CJ. 2011. Constraints of root response to waterlogging in *Alisma triviale*. *Plant and Soil* **343**:247-260.

Setter TL, Waters I. 2003. Review of prospects for germplasm improvement for waterlogging tolerance in wheat, barley and oats. *Plant and Soil* **253**:1-34.

Silva AS, Laura VA, Fernandes V, Jank L, Valle do CB, Neto MMG. 2007. Biomassa seca de raízes e da parte aérea de cultivares de *Brachiaria brizantha* e de *B. humidicola* alagadas e não alagadas. *Revista Brasleria de Biociências* **5**:123-125.

Smirnoff N, Crawford RMM. 1983. Variation in the structure and response to flooding of root aerenchyma in some wetland plants. *Annals of Botany* **51**:237-249.
Solaiman Z, Colmer TD, Loss SP, Thomson BD, Siddique KHM. 2007. Growth responses of cool-season grain legumes to transient waterlogging. *Australian Journal of Agricultural Research* **58**: 406–412.

Sorrell BK, Briz H, Boon PI. 1994. Modelling of *in situ* oxygen transport and aerobic metabolism in the hydrophyte *Eleocharis sphacelata* R. Br. *Proceedings of the Royal Society of Edinburgh* **102B**:367–372.

Sorrell BK, Mendelssohn IA, McKee KL, Woods RA. 2000. Ecophysiology of wetland plant roots: a modelling comparison in aeration in relation to species distribution. *Annals of Botany* **86**:675–685.

Steffens B, Geske T, Sauter M. 2011. Aerenchyma formation in the rice stem and its promotion by H₂O₂. *New Phytologist* **190**:369–378.

Striker GG. 2012. Time is on our side: the importance of considering a recovery period when assessing flooding tolerance in plants. *Eco-logical Research* **27**:983–987.

Thomson CJ, Colmer TD, Watkin ELJ. 1992. Tolerance of wheat (Triticum aestivum cv. Gamenya and Kite) and triticale (Triticosecale cv. Muir) to waterlogging. *New Phytologist* **120**:335–344.

Valle CB, Glienke C. 1991. New sexual accession in *Brachiaria*. *Apomixis Newsletter* **7**:42–43.

Valle CB, Pagliarini MS. 2009. Biology, cytogenetics, and breeding of *Brachiaria*. In: Sing RJ, ed. *Genetic resources, chromosome engineering and crop improvement: forage crops, volume 5*. Boca Ratón: CRC Press, 103–151.

Vartapetian B, Jackson MB. 1997. Plant adaptations to anaerobic stress. *Annals of Botany* **79**(Suppl. A):3–20.

Visser EJW, Blom CWPM, Voesenek LACJ. 1996. Flooding-induced adventitious rooting in *Rumex*: morphology and development in an ecological perspective. *Acta Botanica Neerlandica* **45**:17–28.

Visser EJW, Nabeen RHM, Blom CWPM, Voesenek LACJ. 1997. Elongation by primary lateral roots and adventitious roots during conditions of hypoxia and high ethylene concentrations. *Plant, Cell and Environment* **20**:647–653.

Visser EJW, Colmer TD, Blom CWPM, Voesenek LACJ. 2000. Changes in growth, porosity, and radial oxygen loss from adventitious roots of selected mono- and dicotyledoneous wetland species with contrasting types of aerenchyma. *Plant, Cell and Environment* **23**: 1237–1245.

Zhou M. 2010. Improvement of waterlogging tolerance. In: Mancuso S, Shabala S, eds. *Waterlogging signalling and tolerance in plants*. Heidelberg: Springer, 267–285.