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IV. Breeding for Acid Soil Tolerance

IV. Breeding for Acid Soil Tolerance

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genetic approaches to long-term environmental problems are unjustified. While

the “Adapt-a-Plant” philosophy embraced by C. D. Foy in Kaplan (1989) is difficult to refute, sustainability of wheat production is eventually threatened as

acidification increases with time, to the point that nutritional disorders in addition

to Al and Mn toxicity could conceivably render genetic tolerance ineffective.




Genetic improvement is most often justified where corrective lime applications

are ineffective or impractical in acidic subsoil layers (Foy et al., 1965; Aniol and

Kaczkowski, 1979). Lime distribution below the surface layer is not impossible

but is generally cost prohibitive. Liming the surface layer may offer a partial yield

benefit, but genetic potential is still not reached if the root system does not penetrate the acidic subsoil. A wheat cultivar with improved tolerance may tap critical

water and nutrient supplies below the surface layer until subsoil pH declines to

levels which exceed the tolerance range of the genotype. The level at which pH

stabilizes in the subsurface layers primarily depends on mineralogy of a particular

soil, but pH (H,O basis) generally can be as low as 3 (Baas Becking et al., 1960).

This lower pH limit corresponds to the hydrolysis of ferrous oxide minerals which

are the ultimate product of mineral weathering.

Genetic improvement of acid soil tolerance may also be justified even when soil

acidity is ameliorated by surface applications of lime, as for some soils in southern

and western Australia and the southern Great Plains of the United States (Westerman, 1981 ; Dolling er al., 199 1). Fisher and Scott (1 993a) estimate that the current benefit of Al tolerance to grain yield in the southern NSW wheat belt is l .4%

and could increase to 3.2% if their soils acidify at the current rate over the next

10 years. No benefit was expected above pHca 4.4 ( 1 : 2 soil:O.Ol M CaCl,), and

no apparent yield benefit was found with Mn tolerance in their soils. The economic benefit to grain yield derived from breeding for disease resistance still exceeded that of breeding for Al tolerance.

The relative importance of acid soil tolerance is magnified if the economic

benefit of increased forage production is also considered. Unfortunately, the effects on forage production have not been widely researched. A soil pH below 5.0

(H,O basis) in Oklahoma reduces forage production more severely than it reduces

grain yield (E. G. Krenzer, Jr., 1994, personal communication). Preliminary data

collected for hard winter wheat cultivars indicate that acid soil tolerance can dramatically improve potential grazing capacity (Fig. 3), particularly during early

vegetative growth (prior to winter dormancy) when Al toxicity stress on shoot

growth is most noticeable. As soil pH drops from 5.0 to c4.5, early season forage

growth may be reduced by as much as 85%, hardly enough to support a wheat

pasture program. Because even the tolerant genotype suffers some forage yield



Figure 3. Yields of wheat forage produced prior to winter dormancy for three hard red winter

wheat cultivars under varying soil pH (H,O) near Haskell, Oklahoma, during the 1992-1993 crop

season. All cultivars are currently produced in the southern Great Plains and occupy almost 60% of

the approximately 3.0 million wheat hectares in Oklahoma (Oklahoma Agricultural Statistics Service,

1994. Responses in forage yield coincide with hematoxylin stain ratings (authors’ data) for Al tolerance: Karl (very susceptible), Chisholm (moderately susceptible), and 2 180 (tolerant). 2 180 is one of

only a few hard red winter cultivars currently recommended for production on critically acidic soils in

Oklahoma. Unpublished data provided by E. G. Krenzer, Jr.

reduction below pH 5.0, lime application would still be yield beneficial. Since

grain yield is primarily determined in late winter and spring, the differences between tolerant and susceptible genotypes can be obscured by the recovery of susceptible genotypes over time, as their root systems eventually penetrate subsurface

soil layers with higher pH. Hence, genetic improvement of acid soil tolerance may

be justified solely to support a wheat pasture program, notwithstanding a favorable

response in grain production.

Demonstrating a yield benefit of acid soil tolerance, whether for grain or forage,

does not necessarily justify replacement of corrective measures, such as liming,

with genetic tolerance. Genetic tolerance does not correct the problem of soil

acidity but only postpones the need to take corrective action. On the other hand,

lime application is often economically unfeasible, if not physically impossible.

Besides the physical limitation of correcting subsoil acidity, several scenarios call

for the inclusion of a breeding component to manage wheat production in acid

soils. Scott and Fisher (1989) draw attention to several: ( 1 ) Nonuniformity of lime

application or incorporation may leave pockets of acid soil stress in a wheat field;

( 2 ) naturally occurring spatial heterogeneity of soil pH may also leave pockets of

acid soil stress even with uniform application and incorporation; (3) acid soil

stress may be discovered too late before planting to allow complete amelioration

by liming, forcing the wheat producer to rely on genetic tolerance for an interim

period; and (4) the producer may wish to apply lime at a lower rate, and therefore



at a reduced cost, than recommended for complete amelioration. This complementary approach of planting tolerant cultivars with the lowest practical rate of lime

application makes the use of lime more economical.

Lime recommendations for continuous wheat production in Oklahoma are

based on grain yield performance of older and traditionally susceptible cultivars;

the recommended pH is 2 5 . 5 , whereas rotation with a legume requires a higher

target pH of 6.5 (Johnson et al., 1988). With increased efforts devoted to breeding

acid soil tolerance in hard winter wheat, the target pH for continuous wheat could

be lowered, thereby reducing lime requirements to more economical levels. Conditions unique to wheat production in the southern Great Plains justify the genetic

improvement of acid soil tolerance. First, while lime is an inexpensive material

relative to other soil amendments, the cost of transporting lime from distribution

points to areas of greatest need is economically impractical (often $25 to 30 per

ton of effective calcium carbonate equivalent). Second, the producer often leases

land on short term (year to year) from landowners unwilling to at least share the

long-term investment of liming.




The search for genetic variation in components (A1 or Mn) of acid-soil tolerance

has garnered much attention in several gene pools demarcated by geographic

boundaries or market class. This activity has affirmatively answered one question

critical to the potential success for genetic improvement: Is genetic variation sufficient to allow improvement? The consensus has not changed since the earliest

searches revealed valuable genetic sources of acid soil tolerance, specifically Al

tolerance, among clusters of European wheat genotypes (Mesdag and Slootmaker,

1969; Aniol and Kaczkowski, 1979) and among soft winter wheat cultivars of the

eastern United States (Foy et al., 1965; Lafever et al., 1977). The highest level of

tolerance is found among cultivars of Brazilian origin or with Brazilian parentage.

These genotypes remain today the standard of performance for A1 tolerance, apparently as a result of intense selection pressure for tolerance to highly Al-toxic

soils during their development.

Nontargeted selection (in the absence of acid soil stress) has, nevertheless,

yielded germ plasms with useful levels of tolerance in practically every gene pool

surveyed (Table VII). This level of genetic variation in locally adapted germ

plasms should permit rapid gains in tolerance, without the usual barriers of distant

hybridization so often encountered for improving stress tolerance. Assessment

of genetic variation has disproportionately favored Al tolerance. Several germ

plasms cited i n Table VII approach the tolerance level of Atlas 66, generally regarded as the winter wheat standard for Al tolerance. Some even surpassed the

tolerance of Atlas 66 (Briggs et al., 1989). Where lineages were traced, the Al

tolerance in contemporary cultivars is attributed to rye but, more often, to wheat

Table VII

Evidence of Genetic Variabilityfor Al or Mn Tolerance in Several Adapted Gene Pools of Wheat

Targeted gene pool

Screening medium"

Hawk, Bounty 203

0.18-0.72 mM Al, NS


0.36 mM Al, NS

0.075 mM Al, NS

0.50 mM Mn, NS




0.18-0.72 mM Al. NS


Soil, pH,,?, = 4.1

Soil, pH,,,, = 4.1

0.18-0.72 mM Al, NS

0.18- I .44 mM Al, SS

0.148 mM AI. NS






Wrangler, Hawk, Dodge, Frontiersman, Mustang

HY 320, Vernon

PT74 I, PT726, Norquay

Norquay, PT742, PT726, PT329

GK Szoke, Jubilejnaja 50, Martonvlsiri 9

GR 855

D523, GK Pannondur

Timgalen, Bencubbin, Songlen,

Tincurrin, Halbred

Sivka; germ plasms from

Zagreb-PCH and Novi Sad


Soil, pH,?,

United States, hard red winter wheat

Canada, spring wheat

Canada, spring wheat

Canada, spring wheat

Hungary, bread wheat

Eastern Europe, bread wheat

Hungary, durum wheat

Australia. spring wheat


Exceptional germ plasms'


United States, hard red winter wheat



Assessment of


"NS, nutrient solution culture; SS, soil solution culture; exposure time generally longer in experiments with lower Al concentrations.

'Stain, hematoxylin staining response; RRL, relative root length; RRW, relative root weight.

' Within targeted gene pool, excluding reference genotypes; more recently developed germ plasms are listed


Unruh and Whitney

( 1986)

Carver el al. (1988)

Zale and Briggs (1988)

Briggs el al. (1989)

Macfie ef a/. (1989)

Bona and Carver ( 1992)

Bona el al. (1992)

Bona ef al. (1992)

Scott ef al. (1992)

Rengel and Jurkic




parents originating in Mexico, Brazil, or Kenya. With those exceptions, no causal

relationship exists between the frequency and level of A1 tolerance and breeding

origin. Cultivars developed in regions where soil acidity is not corrected will often

show A1 tolerance. Tolerance may even exist in genotypes selected under limeamended conditions, depending on their parentage. This phenomenon has invited

speculation that A1 tolerance is not uniquely regulated but is instead pleiotropically related to other biological functions. Briggs et al. (1989) suggested that selection for grain yield under high soil fertility may favor genotypes with high

nitrogen-use efficiency. They cited examples in wheat and other crops where

differences in nitrogen uptake relate to differences in A1 tolerance. This premise

deserves further investigation with the benefit of near-isogenic material.


Genetic improvement in acid soil tolerance has been accelerated by the availability of screening criteria for detecting tolerance. Again, greater attention has

been focused on A1 tolerance where symptoms of intolerance might be more obvious and the duration of A1 exposure is much shorter than screening for Mn

tolerance. Laboratory- and greenhouse-based techniques are widely employed

with rapidity and a high degree of accuracy, are usually nondestructive, and can

be applied in early developmental stages before pollination. Field-based techniques in comparison are more laborious and do not have the same reputation for

success, but are nevertheless critical to complete the transition from A1 tolerance

to acid soil tolerance. Choice of a particular screening test is influenced by the

kind of material under selection, i.e., germ plasm collections for identifying suitable parents, large segregating populations, or advanced breeding lines under consideration for release.

1. Cell and Tissue Culture

The application of cell or tissue culture offers a unique opportunity for improving tolerance to mineral toxicity, provided mineral composition of the culture medium approaches that of the targeted acid soil. That mechanisms of tolerance are

expressed at the cellular level and provide agronomic benefit at the whole plant

level are no less critical. Unfortunately, this opportunity has not been successfully

explored for A1 tolerance in wheat, possibly due to the technical difficulty of culturing cells in a low pH, Al-toxic medium. Low pH (<4.5) inhibits cell growth

even in the absence of A1 and also inhibits agar solidification. The latter problem

has been overcome in applications with other plant species by the addition of a

solidifying agent to the callus growth medium (Parrot and Bouton, 1990) and the

use of sponges soaked in a liquid medium to support callus (Meredith et al.,



1988). Cell and tissue culture can serve at least two functions in a breeding program. Callus assays may be developed to survey preexisting variability for Al

tolerance (Parrot and Bouton, 1990) or selection pressure can be applied in cell

culture to recover mutant Al-tolerant cells. For cell culture to play a significant

role in selection, it must be shown that selected variants are truly A1 tolerant and

not tolerant to other nutritional disorders inherent to the low pH culture medium.

That prerequisite has been met with some success in species other than wheat,

but not without modifying the traditional inorganic composition of the culture

medium by reducing calcium and phosphate concentrations and by using unchelated iron at pH 4.0 (Conner and Meredith, 1985b). To facilitate selection of Altolerant variants of Nicotiana plumbaginifolia Viv., Conner and Meredith ( 1985~)

controlled cell density and aggregate size in plated cells and inoculum size in

callus cultures. They found total growth inhibition of plated cells, callus cultures,

and in vim-grown shoots at 20.60 mM Al supplied as AI2(S0,),18H2O. Subsequent characterization of cloned variants indicated no relationship between Al

tolerance and callus growth in the absence of A1 (Conner and Meredith, 1985a).

Tolerance was inherited as a single dominant mutation. In other applications of

tissue culture, Waskom et al. (1990) relied on somaclonal variation among sorghum plants regenerated from tissue culture to identify lines with improved field

tolerance to acid soil stress. In this case, tissue culture did not serve as the screening medium, but instead as a novel source of genetic variation for subsequent field

screening. While tissue culture can supply useful genetic variation for agronomic

traits in wheat (Carver and Johnson, 1989), its application to acid soil tolerance

has not been explored.

2. Nutrient Solution Culture

By far the most common screening medium for Al and Mn tolerance is solution

culture, which provides easy access to root systems, tight control over nutrient

availability and pH, and nondestructive measurements of tolerance. This subject

is already extensively reviewed for wheat and other cereal crops (see Konzak

et al., 1977; Little, 1988; Scott and Fisher, 1989) and will receive proportionately

less attention here. Wheat researchers have relied on three general criteria for tolerance: ( I ) degree of root and/or shoot damage following continuous exposure to

A1 or Mn (Polle et al., 1978; Aniol and Kaczkowski, 19791, ( 2 ) the degree of

recovery following pulse exposure to Al (Aniol, 1984b), and (3) concentration

required for irreversible inhibition of primary root growth (Kerridge et al., 1971).

Most of the research devoted to quantifying and characterizing genetic variation

of tolerance (cited in Sections 111 and 1V.B) has used those criteria in solution

culture. The third criteria has received less attention than the other two. Several

factors may affect the degree of root damage and therefore the minimum concentration required for irreversible inhibition. Variation in temperature in the greenhouse or growth chamber and minor fluctuations in pH and P concentration of the



nutrient solution, as it affects effective Al concentration, can reduce repeatability

of results (Moore et al., 1977). They considered tolerance not i n absolute terms

but relative to the conditions which it was assessed.

Nutrient solution experiments are designed around four variables with infinite

permutations within a given breeding objective (A1 or Mn tolerance), but major

differences between the two objectives. The concentration and duration of exposure are varied inversely. A long continuous exposure to Al for 3 to 4 weeks requires much lower concentrations (usually <0.3 mM) than brief exposure for

about 24 hr (usually c 0.9 mM). These concentrations change depending on the

general tolerance level of the germ plasm screened and whether complete growth

inhibition is desired. Shuman et al. (1993b) found effective screening of wheat

cultivars for Al tolerance using very low solution Al levels (<0.01 mM) and short

exposure (4 days). Their method was designed to minimize A1 precipitation and

to more closely represent actual environmental stresses compared to traditional

short-term experiments with much higher Al concentrations. Aluminum is usually

supplied as AIC1,.6H,O, but AlK(SO,),. 12H20is used to avoid chloride toxicity.

Longer exposures (several weeks) are used to screen for Mn tolerance in the range

of 0.5- I .OmM Mn. This longer exposure makes solution culture technically more

difficult, requiring constant adjustments for water and nutrient loss. Manganese is

often supplied as MnSO,.H,O or MnC1,.4H,O.

Other variables to consider in solution-based screening experiments are nutrient composition and the standards for measuring tolerance. Changes in nutrient

composition can change the intensity of Al or Mn stress at a given concentration

(Little, 1988; Scott and Fisher, 1989; Foy et al., 1988). High concentrations of

phosphorus may lead to Al-phosphate precipitates in Al solutions and protect

plants against Al toxicity. Hence, phosphorus is often avoided in nutrient solutions, particularly in short-term Al exposures when phosphorus needs are satisfied

by seed reserves. Huang and Grunes (1992) demonstrated the importance of the

K/Mg concentration ratio when selecting for Al tolerance in nutrient solutions.

The Al tolerance of Scout 66, normally considered highly susceptible, increased

as the proportion of Mg in solution increased. The proper use of solution culture

does not so much specify an optimum concentration range of mineral nutrients;

rather, genotypic differences in tolerance are described relative to the chosen nutrient composition.

Standards for measuring tolerance generally focus on root growth, i.e., length

and weight for Al tolerance and root weight for Mn tolerance. Tedious manual

measurements of root length may be substituted with automated methods of digital image analysis (Harris and Campbell, 1989), which are effective for measuring

the Al tolerance of sorghum plants after 4 days in nutrient solutions (Shurnan

et al., 1993a). Less expensive systems featuring a hand-held scanner are also effective (Kirchhof, 1992). Measurements of shoot growth as total dry weight also

provide good separation of genotypes for Mn tolerance (Foy et al., 1973; Macfie

et al. 1989) but not for A1 tolerance of wheat seedlings, in which the indirect



effects of Al toxicity on shoot growth are not yet manifested (Ruiz-Torres and

Carver, 1992).

An extremely powerful screen for A1 tolerance in solution culture is the hematoxylin staining method, which provides visualization of tolerance without laborious quantitative measurements. This method, as originally described by Polle

et al. (1978), has stood the test of time, with applications to genotypic classification (Takagi et al., 1981; Carver et al., 1988), genetic characterization (RuizTorres and Carver, 1992; Bona et al., 1994), and selection (Fisher and Scott, 1987;

Carver et al., 1993). Our protocol has been to imbibe seeds for 1 day, transfer the

seeds to trays suspended above aerated deionized water for 2 days, and to nutrient solution for the fourth and fifth days. Aluminum is added to the nutrient solution for the fifth day. Seedling roots soaked in a sodium iodate-hematoxylin

(NaI0,-C , h H , 4 0 0solution


are stained along the vertical axis with increased intensity in sensitive genotypes, particularly in the meristematic region. The hema-



Figure 4. Staining patterns for wheat seedling roots exposed to 0.18 mM Al for 24 hr at the

end of a 4-day period in nutrient solution culture and stained with a sodium iodate-hematoxylin

(NaI0,-C,,H,,O,) solution for 15 min. Greater stain intensity of the root tip and lower root represent

greater susceptiblity to Al; the four genotypes represent decreasing tolerance to Al (proceeding left to

right). Stain ratings range from no detectable stain (A), to partial staining of the lower root with a

wider intermittent stain-free region in B than in C, to complete staining (D)of the lower root. Genotypes which show further separation for partial staining can be identified but are not shown. Experimental protocols are described in more detail by Carver et al. (1988). as modified from the original

procedure by Polle er al. (1978).

toxylin dye apparently binds to tissue A1 immobilized by extracellular phosphate

on or immediately below the root surface (Ownby, 1993).

Polle et al. (1978) recommended a qualitative scale to rate genotypes as completely, partially, or not stained for each of three A1 concentrations (0.18, 0.36,

and 0.72 mM). An intermittent stain-free region immediately above the stained

root tip (a partial stain rating) can vary in length depending on genotype and actually produce a continuous scale of stain phenotypes (Fig. 4). These minor dif-



ferences are genetically stable but are usually ignored in classification. While the

three seminal roots of 5-day-old wheat seedlings have a consistent stain pattern,

some genotypes show a differential staining pattern (none vs. complete) among

roots on the same plant, making genotypic classification difficult. The hard winter

wheat cultivar Chisholm produces this phenotype at lower A1 concentrations (e.g.,

0.18 mM), as do progenies having Chisholm as one parent.

The repeatability and simplicity of the hematoxylin staining method is reinforced by its agronomic relevance. Genotypes classified as tolerant based on the

staining method often show improved agronomic performance under acid soil

stress. Two sister lines with opposite hematoxylin stain patterns differed in grain

yield by 14% under acidic field conditions (Ruiz-Torres et al., 1992). Isolines

selected for divergent staining patterns differed in grain yield by as much as 68%

depending on recurrent parent background (Carver er al., 1993). The hematoxylin

staining method identified Australian wheat cultivars with superior yield performance in acid soils, although cultivars were further differentiated within staining

classes based on their responses in soil solution culture (Scott ef al., 1992). More

than one-half of the genotypic variation in total dry matter resided within hematoxylin staining classes. The discrepancy is not so wide that a genotype rated as

very susceptible in staining pattern would show exceptional tolerance under field

conditions (authors’ unpublished data). Compared to RRL measurements in nutrient solution culture, Zale and Briggs (1988) found a moderate relationship with

hematoxylin ratings among Canadian cultivars. The classification of genotypes

was the most consistent between methods for the most tolerant genotypes. RuizTorres and Carver (1992) used a more precise scale to define stain classes and

found a high correlation with RRL, but the narrower genetic base in their experiment might account for the higher correlation.

The hematoxylin assay appears highly suited for mild selection in single plant

populations, but it should be followed by selection of progenies in a soil medium

indicative of the targeted production area. Tolerance mechanisms having less significance in the hematoxylin assay could then be identified in the soil bioassay.

This tandem breeding approach is necessary to combine internal tolerance mechanisms with those operating at the soil-plant interface and to combine seedling

tolerance with adult plant tolerance.

3. Soil Bioassays

The use of soil media has traditionally received less attention than solution

media in wheat-breeding applications. Foy (1976) provided practical guidelines

for effective screening in acid soils and pointed to some of the complications when

the objective is to create a soil environment, like a nutrient solution environment,

with a specific type (A1 vs. Mn) and amount (for maximum separation of genotypes) of phytotoxicity. Foy ( 1976) discusses the conditions necessary to screen



plants specifically for A1 tolerance or for Mn tolerance. Mitigating effects of other

nutrients (e.g., Ca, P, or Mg) or organic matter must be considered to properly

match a certain soil with a breeding objective. Other factors to consider are variability at the soil collection site, time of collection, and soil storage conditions

(Scott and Fisher, 1989). Results from soil bioassays may lack consistency when

the same soil is used repeatedly due to the effects of continuous wetting and drying

on soil chemistry.

Soil media offer the distinct advantage over nutrient solution media for screening genotypes to varying levels of A1 and Mn stress. This is especially important

when genetic response is determined by soil-dependent external tolerance mechanisms. Ring er al. (1993) found that for various pasture grasses and legumes,

tolerance to A1 in soils may be influenced by the capacity of a particular soil to

reduce Mn in the rhizosphere. Their cultivars showed improved tolerance to

<0.075 mM A1 (lower range of toxicity) when grown in Mn0,-rich soil.

Recent attention has shifted to the development of rapid soil bioassays in

screening for acid soil tolerance. This effort grew out of the realization that the

same bioassay used to rapidly screen a collection of soils for A1 toxicity with an

indicator genotype(s) can also be used to screen a collection of genotypes for A1

tolerance with an indicator soil(s). Fundamental to such an assay is that reduction

in length of the primary root is the first visual indicator of A1 sensitivity and that

reduction in primary root length only 2 days after germination under dark conditions is equally effective in discriminating genotypes as root lengths measured

later under light conditions (Ahlrichs et al., 1990). Early root growth under dark

conditions is primarily supported by seed reserves without possible confounding

effects of nutrient uptake. Performing a similar bioassay in extracted soil solution

may alleviate some of the complications and ambiguity in results from solid-phase

soil bioassays (Aitken et al., 1990). Soil bioassays in wheat do not traditionally

involve soils from the targeted production area but are chosen to provide genotypic separation for a specific phytotoxicity (Bona et al., 1991). Screening in soils

representative of the targeted production area where soil acidity is yield limiting

would, however, provide a critical intermediate step in selection of tolerant genotypes-after preliminary screening in nutrient solutions but before more tedious

and costly screening under natural field conditions.

4. Field Evaluation

The ultimate, and certainly most direct, screen for acid soil tolerance is the

measurement of economic yield (forage or grain) under field conditions. The result is an integrated measurement of tolerance expressed throughout development.

The general procedure is to conduct duplicate tests in unamended (naturally

acidic) and lime-amended blocks to allow a direct measurement of tolerance (yield

depression caused by acid soil stress) and to ensure that acid soil tolerance is not

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