Evidence from near-isogenic lines that root penetration increaseswith root diameter and bending stiffness in rice

Lawrence John ClarkA, Adam Huw PriceB, Katherine A. SteeleC

and William Richard WhalleyA,D

ARothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK.BDepartment of Plant and Soil Science, University of Aberdeen, Cruickshank Building, St Machar Drive,Aberdeen AB24 3UU, UK.

CCAZS Natural Resources, University of Wales, Bangor, Gwynedd LL57 2UW, UK.DCorresponding author. Email: richard.whalley@bbsrc.ac.uk

Abstract. Deep rooting can be inhibited by strong layers, although there is evidence for species and cultivar (cv.)differences in their penetration ability. Here, the availability of near-isogenic lines (NILs) in rice (Oryza sativa L.) wasexploited to test the hypothesis that increased root diameter is associated with greater root bending stiffness, which leads togreater root penetration of strong layers. Wax/petrolatum discs (80% strong wax) were used as the strong layer, so thatstrength can be manipulated independently of water status. It was found that good root penetration was consistentlyassociated with greater root diameter and bending stiffness, whether comparisons were made between cvs or between NILs.With NILs, this effect was seen with ‘research’ lines bred from recombinant inbred lines of a cross between cvs Bala andAzucena and also in improved lines developed from cv. Kalinga III by introgression of parts of the genome from Azucena.Much of the bending behaviour of roots could be explained by treating themas a simple cylinder ofmaterial. In bothwax discand sand culture systems, roots that had encountered a strong layer had lower bending stiffness than roots that had notencountered a strong layer which is a novel result and not previously reported.

Additional keywords: physiological response, rice roots, soil strength.

Introduction

Strong soils adversely affects root growth in many crop species(BengoughandMullins 1990),which canbe a serious agriculturalproblem affecting productivity in rice (Oryza sativa L.; Wadeet al. 1999), wheat (Triticum aestivum L.; Masle and Passioura1987) and many other crops. It is likely that the yield reductionthat occurswhen crops grow in strong soil is due to a combinationof poor root penetration restricting access to water (Whalley et al.2006, 2008) and root-sourced signals that regulate shootelongation (Chaves et al. 2003). In this paper, the problem ofroot penetration into strong soil is addressed. To elongate throughsoil where existing channels are smaller than the root diameter,roots must exert a growth pressure (which results from turgorpressure and cell wall relaxation) to deform the soil around theroot (Greacen and Oh 1972; Clark et al. 1996). When rootsapproach a layer of strong soil, theymay either penetrate the layeror may get deflected from their original direction (Dexter andHewitt 1978). The mechanisms that determine the outcome ofsuch an encounter are not well understood. If the root does nothave sufficient lateral support, bending of the root may occurabove the strong layer (Clark et al. 2008).

Roots of seedlings of some species are better at penetratingstrong soil than others (Materechera et al. 1992). In particular,dicots have better root penetration than monocots. Dicots do not

have greater maximum axial growth pressures than monocots(Clark and Barraclough 1999), which implies that differences ingrowth pressure do not account for differences in root penetrationability. It has been suggested that species with thicker roots gavebetter penetration because they were more resistant to buckling(Whiteley et al. 1982) or because of axial stress relief (Kirby andBengough 2002).

There is evidence for differences in root penetration abilitybetween cultivars as well as between species. Evidence forcultivar differences in root penetration in rice is particularlyinteresting, given the sensitivity of rice crops to water stressand the genetic resources available in this species.Yu et al. (1995)first used wax layers to assess root penetration in rice, followingthe approach of Taylor and Gardner (1960). Wax layers weremade by mixing hard paraffin wax and white soft paraffin(petrolatum) to give the desired mechanical strength. Thelayers were installed in a low-impedance growing medium andthe number of roots that penetrated the wax layer was counted atharvest.

Cultivar differences in the ability of rice roots to penetratestrongmediaweremuchsmallerwithout a large spatial gradient instrength: cultivars with good root penetration of hard wax layersdid not have longer roots in uniformly strong sand, but they didhave greater root diameters (Clark et al. 2002). Differences in the

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ability of rice roots topenetrate strong layers appear, therefore, notto be related to their ability to elongate through strong soil, butrather to their ability to penetrate a boundarywhen there is a rapidincrease in mechanical impedance. This suggests that good rootpenetration in rice results not from high growth pressures, butfrom the roots not bending as they encounter a hard layer.

A modified wax layer technique was used to identifyseven quantitative trait loci (QTLs) controlling rootpenetration in rice, using the Bala�Azucena recombinantinbred line (RIL) mapping population (Price et al. 2000).A QTL at chromosome 2 on marker C601 explained 18% ofthe phenotypic variation for root penetration ability, while a QTLon chromosome11onmarkerC189 explained 7%of the variationin root penetration ability. The QTL on chromosome 2 has beendetected in three other ricemapping populations (Ray et al. 1996;Ali et al. 2000;Zhenget al. 2000) and theQTLonchromosome11has been detected in one other mapping population (Ray et al.1996). These QTL regions have been the target of recombinantinbred near-isogenic line (RINIL) development in theBala�Azucena population. Three of the RINIL pairs are forroot penetration QTLs, one on chromosome 2 centred aroundmarker C601 and two on chromosome 11 at marker C189 (thereare two different RINIL pairs at this QTL). Within each pair, theRINILs are genetically identical throughout the genome, exceptfor a small region around the QTL, for which one of the pair hasthe Azucena genotype and the other has the Bala genotype.A fourth pair of RINILs has been developed at chromosome 9,where aQTL is repeatedly detected for root thickness (explainingup to20%ofphenotypicvariation,Price et al. 2002), although thiswas not detected as a root penetration QTL.

Root thickness QTLs tend to be in the same place as rootpenetration QTLs (e.g. QTLs on chromosomes 2 and 11),although this not always the case (QTL on chromosome 9). Inthis paper the availability of these pairs of RINILs was exploitedto study the relationship between root diameter and rootpenetration. This study tested the hypothesis that one of thecomponents of good root penetration is the high bendingstiffness that results from large root diameters. This study alsoused the data generated to relate bending stiffness to diameter totest the theory that roots behave mechanically like simplecylinders.

Materials and methodsDevelopment of recombinant inbred near-isogenic linesSince the Bala�Azucena RIL population in Oryza sativa (L.)was produced by bulking seeds from F5 plants (Price et al. 2000)into F6 families, there exists a degree of residual heterozygosity(in theory, 6.25%). This was exploited to produce recombinantinbred near-isogenic lines (RINILs) as follows. RILs which hadresidual heterozygosity at root growth QTLs were selected tohave minimal heterozygosity elsewhere (and particularly at otherroot QTLs). Polymorphic microsatellites reported by Chen et al.(1997) to be near the QTLs were selected, mapped in theBala�Azucena population and then, if mapping as expected,used to select heterozygous plants within the F6 seeds (phase oneselection to reduce non-target heterozygosity). Whereheterozygosity was present elsewhere, this was tested so thatthe selected plants were homozygous. In phase 2, the seeds of

selected plants were grown and plants that were homozygousAzucena and Bala were identified as matching pairs of RINILs.The original RILs selected and the markers used were as follows;Chromosome 2 RIL 204, marker RM6; Chromosome 9 RIL 20,marker RM242; Chromosome 11 RILs 20 (RINIL 11A and B)and 109 (RINIL 11.1A and B), marker RM206.

Growth of plants in wax layer screen

Wax layerWax layers were prepared by melting together mixtures of

white soft paraffin (Bell, Sons andCo. (Druggists) Ltd, Southport,UK) and pastillated paraffin wax (57�60�C solidification point,Merck Ltd, Poole, UK). Each wax layer was prepared by melting40 gofwaxand10 gofwhite soft paraffinandpouring themixtureinto a circular aluminium foil mould. The thickness of the waxlayers was 3mm and the diameter 145mm. The wax layer had apenetrometer resistance of ~2.2MPa.

Growth systemWax layers were installed at 50mm depth in a sand growing

medium (RH 65 grade silica sand, WBB Minerals, Sandbach,UK) in plastic tubes (152mm internal diameter, 450mm length),essentially as described by Clark et al. (2000). The tubes were setup in tanks of nutrient solution with a final level in the tanks300mm below the sand surface. The 3–4mm gap between thewall of the tube and thewax layer allowed the entire coreof sand tobe watered by capillary action.

Plant growthAll experiments were carried out in a controlled environment

room with day/night temperatures of 30 and 26�C, respectively,and a 16 h daylength. The relative humidity was 70%. Lightingwas by fluorescent tubes, with supplementary tungsten lighting,and the photosynthetic photon flux density was300mmolm�2 s�1. Seeds of rice were set to germinate fortwo days between two sheets of wet filter paper in Petri disheswrapped in aluminium foil to exclude light.

The tubes of sand were planted with two-day-old riceseedlings, one per tube, so that the seed was just below thesand surface. The nutrient solution composition was 1.5mM

Ca(NO3)2, 0.15mM CaH4(PO4)2, 1.0mM KCl, 0.3mM MgSO4,with the followingmicronutrients: 50mMB, 50mMFe, 10mMMn,1mMZn, 1mMCu and 0.5mMMo (seeClark et al. 2002). The levelof nutrient solution in the tanks was topped up with water every2–3 days. The nutrient solutionwas changed around 21 days afterplanting the seedlings.

HarvestingPlants were harvested by block, with the aid of a root

harvesting apparatus, which supported the column of sand asthe roots were washed out with water. The number of root axesthat had penetrated the wax layer was counted and root axeswere excised for subsequent measurements of bendingstiffness. Three categories of root axes were sampled: thosethat had penetrated the wax layer, those that had not reachedthe wax layer, and those that were deflected by the upper surfaceof the wax layer. Roots in the gap between the wax layer and thetube were ignored. Sampled roots were kept in water until

1164 Functional Plant Biology L. J. Clark et al.

measurements of bending stiffness were made. Themeasurements on the roots sampled from each plant werecompleted before the next plant was harvested. The crowns oftheplantswere storedat 5�Cinwater, and the total numbersof rootaxes on each plant subsequently counted.

Measurements of bending stiffness

The bending stiffness of nodal roots was measured by applying aload to sections of roots in a three-point test bending test. The loadwas applied15mmfrom the root apex, and the rootwas supported10and20mmfrom the apex. In rice the growingzoneof the root iswithin 5–10mm of the root apex. Thus, this study measuredbending of the part of the root which had just completedelongation. The force required to bend the root was measuredwith a 0.5N load cell, while the test machine recorded thedeflection of the load cell. Force was plotted against deflectionfor each sample and bending stiffness was calculated as themaximum slope of the curve, in units of N force per mm ofdeflection. The diameter of the tested part of the root wasmeasured using a microscope with a graticule eyepiece. In thewax layer experiments (see below), bending stiffness wasmeasured on roots that had penetrated the wax layer and alsoon roots that had not reached thewax layer. Bending stiffnesswasalso measured in roots which had grown in strong or weak sand(see below) and here the roots were of a similar length.

Wax layer challenges to roots of Azucena and Bala

Parental cultivars Bala and Azucena were challenged with strongwax layer screens. This study used three different seed batchesfrom International Rice Research Institute (IRRI) 1996, IRRI2004 and Aberdeen 2002. The experiment was performed in fourblocks and each block contained the two parent cultivars withseed from the three sources. Each treatment combination wasreplicated twice in each block. The experiment was harvested inblocks between 27 and 41 days after sowing.

Wax layer challenges to roots of recombinant inbrednear-isogenic lines

Roots of RINIL pairs (11A, 11B, 2A, 2B, 9A and 9B), describedabove, were challenged with hard wax layers. There were twoexperiments, each with eight blocks. In each block there was onereplicate of each rice line. In a third subsidiary experiment toconfirm data for 11A and 11B, roots fromRINIL pairs 11.1A and11.1B were challenged with a hard wax layer. This experimentwas in four blocks, with two replicates of each RINIL per block,and harvests between 31 and 36 days after sowing.

Wax layer challenges to roots of introgression lines

Steele et al. (2006) reported the useofmarker-assisted selection tointrogressQTLs controlling root traits into an Indian rice cultivar,Kalinga III. They used a programof backcrossing and selection todevelop Kalinga III NILs introgressed with Azucena QTLs. Asthese NILs included the Azucena QTLs at chromosomes 2, 9 and11, this genetic material was used to see how these introgressionsaffected root penetration, diameter and bending stiffness.

Penetration of roots ofKalinga III was comparedwith two ricelines. In this study, two experiments were performed and in eachof these there were eight blocks each containing four of each rice

lines (the parent Kalinga III and introgression of Azucena alleleson chromosomes 2, 7, 9, and 11 intoKalinga III which are PY2F3-3–26–5-18 (selected from PY2F3-3–26 to contain homozygousAzucenaQTLs on chromosomes 2, 7, 9 and 11) andBC3F4-21–1-3–11–7 (lacking the chromosomes 7QTL) in Steele et al. (2006).These experiments were harvested by block between 26 and36 days after sowing.

Wax layer challenges to roots of Azucena�Balarecombinant inbred line mapping population

Roots of 93 RILs including the parental lines Bala and Azucenawere challenged with strong wax layers. These RILs each haverandom combinations of the two parental chromosomalsegments. This experiment was duplicated and eachexperiment used a single replicate of each RILs. Plants wereharvested between 31 and 36 days after sowing.

Growth of Azucena and Bala roots in strongand weak sandTwo-day-old seedlings were planted in a sand core screen thatallowed mechanical impedance to be varied independently ofaeration andwater statusof thegrowingmedium.Themethodwasdescribed in full by Clark et al. (2002). In outline, the principle ofthe method was that columns of sand were set up in plastic tubes,either with orwithout a heavyweight on the sand surface.When aweightwas placed on the sand surface, themechanical impedanceof the medium was increased as the resistance of sand grains todisplacement was increased, but therewas negligible compactionof the sand core. Theweights had a hole to allow the shoot to growvertically, while the control used a similarly-shaped ‘weight’ thatwas made of expanded plastic foam rather than steel. As with thewax-layer screen, the lower part of the columns of sand wasimmersed in nutrient solution, so that the upper part of the sandcore was kept watered by capillary action. The penetrometerresistance of the weak control was 0.2MPa (Whalley et al. 1999)and the impeded sand was sufficiently strong to, approximately,halve the rate of root elongation (Whalley et al. 2006).Roots grewinto the capillary fringe in the sand, and air-filled porosity was~0.25 at the surface of the sand, but decreased with depth, forexample to 0.10 at a depth of 10 cmbelow the surface.However, ithas been previously shown, by measuring oxygen flux, that thesand was well aerated (Whalley et al. 1999).

The experiment was arranged in three blocks. Each containedBala and Auzcena rice growing in weak or strong sand (i.e.‘control’ or ‘impeded’). Each treatment was repeated three timesin each block. One seedling was planted per core. The effect oftwo levels of impedance was tested: ‘impeded’ and ‘control’.Immediately after planting, the appropriate weights were placedon the sand columns. The level of nutrient solution in the tanksthat contained the tubes of sand was topped up with water every2–3 days. The experiment was harvested by block 33–35 daysafter planting, and the bending stiffness and diameter of rootswasmeasured as described above, but here on roots of a similar lengthand age.

Statistical analysis

GENSTAT version 10 was used to analyse the data. Analysis ofvariance (ANOVA) was used to calculate the standard errors of

Root penetration of rice Functional Plant Biology 1165

differences (SED) which are presented with the appropriatedegrees of freedom (d.f.). In some cases differences in thenumber of roots between plants made the ANOVAunbalanced, so statistical differences were tested with residualmaximum likelihood (REML) using a linear mixed model. Therelationships between bending stiffness and root diameter wereinvestigated using linear regression on the Log-transformed data.The slope of this regression was of interest, because it is theexponent of the power law relationship between root diameter andbending stiffness. The slope is reported along with its standarderror (s.e.). When relationships between bending stiffness androot diameter for different groups of roots (e.g. impended andcontrol roots) were compared, a grouped linear regression wasused to test if the relationships were different.

Results

Wax layer challenges to roots of Azucena and Bala

The source of the Azucena and Bala seed (IRRI 1996, IRRI 2004or Aberdeen 2002) had no significant effect on either rootdiameter or bending stiffness. Azucena gave better rootpenetration of 80% wax discs than Bala (Table 1). Azucenaalso had 37% greater root diameter, but about three-foldgreater bending stiffness than Bala. The better root penetrationof Azucena was therefore associated with greater root diameterand bending stiffness.

The bending stiffness of individual roots was plotted againsttheir diameter at the point of bending, on a log–log plot (Fig. 1).This showed an approximately fourth-power relationshipbetween root diameter and bending stiffness. For penetratedroots, the slope of the regression was 3.5 (s.e. 0.18), while forroots that had not reached the wax disc, the slope was 3.8 (s.e.0.30). For a given root diameter, penetrated roots were less stiffthan roots that had not reached the wax disc (the intercepts inFig. 1were significantly different atP< 0.001), suggesting that anencounter with a strong layer decreases bending stiffness. Thisfinding was investigated further in a different experimentalsystem, with strong and weak sand, described below.

Growth of Azucena and Bala roots in strongand weak sand

Azucena and Bala plants were grown in both weak (control) andstrong (impeded) sand, in a system that is designed to allowmechanical impedance to be varied independently of soil aerationand water status. Here, high strength increased the root diameter

of Azucena from 1.29 to 1.37mm, but decreased it from 0.99 to0.89mm in Bala (SED 0.036, 19 d.f.). This suggests that apossible contributor to good root penetration is the ability ofthe roots to increase in diameter on encountering strong soil.Interestingly, the bending stiffness of roots removed from strongsandwas significantly lower (P < 0.001) than those removed fromweak sand and roots of Azucena were stiffer than those of Bala(P < 0.001). The effects of rice (i.e. Azucena or Bala) andmechanical impedance interacted significantly (P= 0.002) todetermine bending stiffness (see Table 2). Although exposuretomechanical impedance decreased the bending stiffness of rootsof bothAzucena andBala, therewas a greater decrease in bendingstiffness in Bala.

The relationship between bending stiffness and root diametercan be explored with a log–log plot since the slope of thisrelationship is the exponent of a power law which relates thesevariables to each other. When the data for individual roots wereplotted on a log–log plot, roots from strong sand had lowerbending stiffness for a given diameter than roots from weaksand (Fig. 2). As with roots from the wax layer system (Fig. 1),there was evidence for an approximately fourth-powerrelationship between root diameter and bending stiffness. Theslopes of the regressions were 3.8 (s.e. 0.32) for the roots fromweak sand and 4.2 (s.e. 0.22) for the roots from strong sand.Mechanical impedance had a significant (P < 0.001) effect theintercept of the curves fitted in Fig. 2.

Wax layer challenges to roots of RINILs

While the data from Azucena and Bala suggest that high bendingstiffness and an increase in root diameter on encounteringimpedance may be important, there are many possibledifferences between these two cultivars that might potentiallyexplain their different root penetration ability. Therefore, RINILswere used to dissect these differences.

There were differences between and within RINIL pairs innumber of penetrated roots, bending stiffness and root diameter(Fig. 3).As it did not affect interpretation, for simplicity, themeanof rootswhichhadnot reached and thosewhich hadpenetrated thewax layer were taken. Both RINILs at chromosome 2 had smallerroot diameter, bending stiffness and number of penetrated rootsthan the NILs at chromosomes 9 and 11 (note here that RINILs 9and 11were derived from the sameRIL and are therefore virtuallygenetically identical). Differenceswithin aRINILpair were smallat chromosomes 2 and 9. However, at chromosome 11, the

Table 1. Penetration of 80% wax discs by roots of rice (Oryza sativa) cultivars Azucena and BalaMeasurements of root diameter and bending stiffness weremade on penetrated roots and roots that had not reached thewaxlayer. Bending stiffness data required Log-transformation beforeANOVAand the back transformedmeans inNmm�1 are

given in brackets. Plants were harvested 34 days after planting

Rice cultivar Number of Log bending stiffness (natural scale Diameter (mm)penetrated shown in brackets)

roots Not Penetrated Not Penetratedpenetrated penetrated

Azucena 13.0 –1.424 (0.241) –1.816 (0.163) 1.36 1.38Bala 7.1 –2.375 (0.093) –3.193 (0.041) 1.07 0.97

s.e.d. (d.f.) 1.14 (35) 0.174 (12) 0.05 (12)

1166 Functional Plant Biology L. J. Clark et al.

presenceof theBala allele led togreater root diameter andbendingstiffness and a greater number of penetrated roots.

Root bending stiffness was plotted against diameter for thesedata on a log–log plot (Fig. 4), and the slope indicated a powerrelationshipbetween stiffness anddiameter of 3.3 (s.e. 0.087). Fora given diameter, penetrated rootswere consistently less stiff thanroots that had been deflected by the wax disc and (as with Figs 1and 2) the intercepts were significantly different at P< 0.001. InFig. 4 there was less scatter in the penetrated than the deflectedroot data: the regression with penetrated roots accounted for 90%of the variance compared with only 65% for the deflected roots.

Good root penetration of the RINILs was associated with thepenetrated roots having greater diameter than the deflected roots(Table 3). At chromosome 9, this was true of both RINILs, andthere was little difference between 9A and 9B. At chromosome11, however, there were differences within the RINIL pair. Thepenetrated roots of 11B had greater diameter than the deflectedroots, but this effect was not seen in poorer-penetrating11A. These observations confirm the observations withAzucena and Bala that the ability of roots to increase indiameter is associatedwith goodpenetration of strongwax layers.

For the QTL at chromosome 11, a second pair of RINILs(derived from a different RIL and denoted 11.1A and 11.1B)wastested in a separate experiment. A similar pattern was seen to thatin the first pair of RINILs at chromosome 11. The presence of theBala rather than the Azucena allele increased root penetration,diameter and bending stiffness (Table 4).

Wax layer challenges to roots of introgression lines

Introgression of Azucena QTLs was associated with greater rootpenetration, root bending stiffness and root diameter (Fig. 5).In this respect, these data support the hypothesis that greaterbending stiffness confers better root penetration. However, incontrast to the results obtained with the Bala�Azucena RINILs,the introgression ofAzucena alleles intoKalinga III had a positiveeffect. In Fig. 5, for simplicity and because it did not affectinterpretation, the data presented is the mean of roots which hadnot reached the wax layer and those which had penetrated thewax layer.

Wax layer challenges to roots of Azucena�Balarecombinant inbred line mapping population

A log–log plot of bending stiffness against diameter for roots ofthese RILs gave similar gradients for both replicate runs (Fig. 6).The gradient was 3.6 (s.e. 0.09) for experiment 1, and 3.5(s.e. 0.10) for experiment 2. There was no significantdifference between the regression lines fitted to the data fromeach of these experiments. Given the quantity of data gathered inthis experiment, it strengthens the general observation that, whilethe gradient of the log–log plot is nearly four, as theory wouldpredict for simple cylinders, it is actually nearer 3.5.

0.0

–0.5

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–5.5–0.5 –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Log (root diameter mm)

Log

(ben

ding

stif

fnes

s N

mm

–1)

Fig. 1. Log–log plot of bending stiffness against root diameter for roots ofrice (Oryza sativa) cultivars Azucena (*,*) and Bala (~,D) that had eithernot reached (*, D, dotted regression line) or penetrated (*, ~, solidregression line) an 80% wax disc. Each point represents a single root.

Table 2. Bending stiffness of roots of rice (Oryza sativa) cultivarsAzucena and Bala which had been grown either in strong (Impeded)

or in weak (control) sandThe data was Log-transformed before residual maximum likelihood (REML)and the standard error of difference (s.e.d.) is 0.1359. The back transformed

bending stiffness in N mm�1 is shown in brackets

Rice cultivar Control roots Impeded roots

Azucena –1.606 (0.2000) –1.765 (0.1712)Bala –2.698 (0.0673) –3.607 (0.0271)

0.0

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Log (root diameter mm)

Log

(ben

ding

stif

fnes

s N

mm

–1)

Fig. 2. Log–log plot of bending stiffness against root diameter for roots ofrice (Oryza sativa) cultivarsAzucena (*,*) andBala (~,D) grown inweak(*, D, dotted regression line) or strong (*, ~, solid regression line) sand.Each point represents a single root.

Root penetration of rice Functional Plant Biology 1167

Discussion

Comparisons with previous work on Bala and Azucena

The better root penetration of Azucena than Bala is consistentwith previous results: both Clark et al. (2000) and Price et al.(2000) found that roots of Azucena had better penetration of 80%

wax layers than roots of Bala. Similarly, the greater thickness ofroots of Azucena than Bala seen here is consistent with previouswork in hydroponic (Price et al. 1997), soil (Price et al. 2002) andsandcore (Clark et al. 2002) screens. Inparticular, it is noteworthythat Clark et al. (2002) found that mechanical impedanceincreased the diameter of Azucena roots but not Bala roots.These varietal differences in root characteristics may explainwhy Azucena roots are better able than Bala roots to penetrate to

7

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02 9 11

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2 9 11

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NIL pair

Num

ber

of p

enet

rate

d ro

ots

Ben

ding

stif

fnes

s (N

mm

–1)

Roo

t dia

met

er (

mm

)

(A)

(B)

(C)

Fig. 3. Number of penetrated roots per plant, bending stiffness and diameterof sampled roots of three pairs of recombinant inbred near-isogenic lines(RINILs) at chromosomes 2, 9 and 11 in rice (Oryza sativa). Hatched barsindicate the Bala allele, and open bars the Azucena allele. Data are means oftwo separate experiments. The error bar shows the s.e.d.

0.0

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–5.5–0.5 –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Log (root diameter mm)Lo

g (b

endi

ng s

tiffn

ess

N m

m–1

)

Fig. 4. Log–log plot of bending stiffness against root diameter for the datasummarised in Fig. 3. Each point represents either a single deflected root(*, dotted regression line) or a single penetrated root (*, solid regressionline).

Table 3. Mean diameters of roots of recombinant inbred near-isogeniclines (RINILs) of rice (Oryza sativa) that either penetrated 80%wax discs

or were deflected by the wax discsData are taken from the same experiments as Fig. 3. The residual maximum

likelihood (REML) estimate of SED for these data is 0.062mm

RINIL Deflected roots Penetrated roots

2A 1.15 0.762B 1.13 0.999A 1.29 1.469B 1.28 1.4411A 1.22 1.2411B 1.22 1.38

Table 4. Effect of presence of either an Azucena (11.1A) or a Bala allele(11.1B) at a second pair of recombinant inbred near-isogenic lines

(RINILs) at chromosome 11 of rice (Oryza sativa)

11.1A 11.1B s.e.d. (d.f.)

Number of penetrated roots 14.0 19.2 2.94 (11)Root diameter (mm) 1.17 1.32 0.070 (30)Bending stiffness (N mm�1) 0.104 0.163 0.0290 (29)

1168 Functional Plant Biology L. J. Clark et al.

depth in the field (Cairns et al. 2004). The physiological andgenetic bases of these differences are discussed below.

Bending stiffness, mechanical impedance and penetration

When comparisons were made between Azucena and Bala, orbetween different pairs of RINILs (e.g. 2 v. 9), within pairs ofRINILs (e.g. 11A v. 11B) or between Kalinga III and

introgression lines, good root penetration was consistentlyassociated with greater root diameter and bending stiffness.For roots growing above a wax layer, high bending stiffnesswould favour good root penetration by decreasing the likelihoodthat a rootwoulddeflect sidewayswhen it encounters a strongwaxlayer. If roots behave like a cylinder of a simplematerial, it wouldbe expected that bending stiffness would depend on diameter tothe fourth power,whichwould correspond to agradient of 4.0 onalog–log plot of bending stiffness against diameter. In theexperiments reported here, this was approximately the case,although there was a tendency for the slope to be nearer 3.5than 4.0. Nevertheless, much of the variation in bending stiffnesscould be accounted for by variation in diameter. The co-locationof QTLs for root diameter and QTLs for bending stiffness istherefore consistent with this.

Root diameter explained more of the variance in bendingstiffness in penetrated roots than in roots deflected by awax layer.There is more than one possible explanation for this result. It wasnoticed that penetrated roots tended to be straighter than deflectedroots, which may account for diameter better explaining thevariation in bending stiffness. Alternatively, it is possible thatthe encounter with a wax layer led to selection of a differentsubpopulation of roots. Indeed it is possible that such a selectionmayhave resulted fromdifferences in ‘curviness/waviness’of thegrowthof the roots before they reached thewax layer that has beennoted in the population (Norton and Price 2008).

Interestingly, for a given diameter, roots that had penetrated astrong wax layer had lower bending stiffness than those that hadnot. Bending stiffness measurements made on roots which hadgrown in strong sand confirmed that thiswas likely to be related tothe process of penetrating a mechanically strong environmentrather than a beneficial trait conferring better penetration of strong

40

30

20

10

0

Kalinga III 2, 9, 11 2, 7, 9, 11

Kalinga III 2, 9, 11 2, 7, 9, 11

Kalinga III 2, 9, 11 2, 7, 9, 11

0.12

0.10

0.08

0.06

0.04

0.02

0.00

1.15

1.10

1.05

1.00

0.95

Roo

t dia

met

er (

mm

)B

endi

ng s

tiffn

ess

(N m

m–1

)N

umbe

r of

pen

etra

ted

root

s

(A)

(B)

(C)

Fig. 5. The effect of the introgression of Azucena alleles on chromosomes2, 7, 9, and 11 into Kalinga III, on the number of penetrated roots per plant,bendingstiffnessanddiameterof sampled rootsof rice (Oryza sativa).Data aremeans of two separate experiments. The error bar shows the s.e.d.

0.0

–0.5

–1.0

–1.5

–2.0

–2.5

–3.0

–3.5

–4.0

–4.5

–5.0

–5.5–0.5 –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Log (root diameter mm)

Log

(ben

ding

stif

fnes

s N

mm

–1)

Fig. 6. Log–log plot of bending stiffness against root diameter for roots ofrecombinant inbred lines (RILs), grown in thefirst (*, solid regression line) orsecond (*, dotted regression line) of two replicate runs. Each point representsa single root of rice (Oryza sativa).

Root penetration of rice Functional Plant Biology 1169

wax layers. When roots generate growth pressure to penetratestrong soil, it is due to a combination of increased turgor and cell-wall relaxation (Greacen and Oh 1972; Clark et al. 1996).Provided roots are not completely impeded, most of thegrowth pressure needed to deform soil is generated by cell-wall relaxation rather than turgor, which increases most incompletely impeded roots (Clark et al. 1996, 2001). Thus,roots grown in strong sand are likely to have a lower celltension than roots grown in weak sand, but a similar turgor.This is consistent with the observation that impeded roots had alower bending stiffness than unimpeded roots. However, thisresult has not been reported previously.

Summary of QTL effects detected by near isogenic lines

Two sources of near isogenic lines were used to dissociate themajor differences regularly observed between Bala and Azucenainto more discrete genetic effects. These are summarised inTable 5. While RINILs for chromosomes 2 and 9 did not showdifferences, two independent RINIL pairs for a QTL onchromosome 11 displayed significant differences that indicatedthat the Bala allele increased root penetration by either increasingthe root thickness and stiffness or by increasing the degree towhich roots thicken when challenged with the wax layer. TheKalinga III introgression lines revealed a marked increase in rootpenetration as a result of the inclusion of relatively small regionsof Azucena chromosomes 2, 9 and 11. Unfortunately, it is not yetpossible to identify specifically which region of Azucena isresponsible.

Comparisons with QTLs for good root penetration

Chromosome 11 was targeted for near isogenic line productionbecause, in early root studies in the Bala�Azucena populationpositive effects of the Azucena allele were detected, including asmall root penetration QTL (Price et al. 2000). However, thisgenomic location has given contradictory results. Thus bothweakand strong QTLs for root thickness in which the Bala alleleincreased the trait were detected (respectively) in Price et al.

(2002) and MacMillan et al. (2006). In addition, the main effectand epistatic interaction QTL for seminal root morphology andgravitropic bending was identified in this region, in which theBala allele makes the roots less wavy/curvy (Norton and Price2008). In the same study it was also found that the RINIL pair11.1, used here, differed in gravitropic response, as theBala allelehad reduced re-orientation of the growing tip when the plant wasrotated by 90�.

Conclusions

Good root penetration was consistently associated with greaterroot diameter and bending stiffness, and there was evidence thatgenetic control of root diameter led to genetic control of rootpenetration ability. Variation in diameter explained much of thevariation in root bending stiffness, and regarding the root as asimple cylinder was a reasonable approximation. Roots that hadencountered a strong layer (whether wax or sand) had lowerbending stiffness than roots that had not. It is suggested that this isrelated to the cell wall relaxation needed for roots to developgrowth pressure.

Acknowledgements

This work was funded by the Biotechnology and Biological SciencesResearch Council (BBSRC) of the United Kingdom under grant BB/C507837/1. The development of RINILs was funded by BBSRC undergrant P11790. The Kalinga III RILs are an output from projects (PlantSciences Research Program R7434 and R8200) funded by the UKDepartment for International Development (DFID). We thank MrR. P. White of Rothamsted Research for statistical advice.

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Table 5. Summary of QTL effects detected in this studyData are for rice (Oryza sativa) recombinant inbred near-isogenic lines (RINILs)

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AOne Kalinga III introgression had the chromosome 7 QTL from Azucena, one did not. Both showed increased penetration, thickness and stiffness.

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Manuscript received 16 April 2008, accepted 16 August 2008

Root penetration of rice Functional Plant Biology 1171

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