Published online 26 August 2005
Published in Crop Sci 45:2001-2004 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
PLANT GENETIC RESOURCES
Aluminum Tolerance in Medicago truncatula Germplasm
M. K. Sledgea,*,
P. Pechtera and
M. E. Paytonb
a The Samuel Roberts Noble Foundation, Ardmore, OK 73402
b Dep. of Statistics, Oklahoma State Univ., Stillwater, OK 74078-1056
* Corresponding author (mksledge{at}noble.org)
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ABSTRACT
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Aluminum (Al) toxicity is a major limiting factor of crop production in acidic soils. Medicago truncatula Gaertn., a close relative of alfalfa (M. sativa L.), is negatively affected by Al toxicity. The objective of this study was to assess the variation for Al tolerance among M. truncatula accessions held in the USDA National Plant Germplasm System, with the long-term goal of identifying Al tolerance genes to be used for alfalfa improvement. Three hundred twenty-one accessions of M. truncatula, from 20 different countries, were screened for Al tolerance in a hydroponics approach. Seed from each M. truncatula accession were surface sterilized and germinated before being grown in hydroponics. Ten germinated seedlings were transferred to a hydroponics culture tank containing modified Blaydes medium without aluminum and ten were transferred to a second tank containing modified Blaydes medium with 50 µM Al. Seedlings were grown for 5 d and root length was measured. Relative root growth in Al medium was used as a measure of Al tolerance. Relative root growth varied from 0.35 to 1.09, indicating that sufficient variation exists within this collection of M. truncatula accessions to select sensitive and tolerant accessions, which could be used to identify QTL for Al tolerance, the first step in a map-based cloning approach to discover Al tolerance genes.
Abbreviations: Al, aluminum • PI, plant introduction
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INTRODUCTION
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BARREL MEDIC is both a forage crop and a model legume. As a forage crop, barrel medic is a comparable to alfalfa in forage quality (Derkaoui et al., 1993) and dry matter yield (Zhu et al., 1998). It is an important forage species in Australia where it is an integral component of the ley farming system, in which cereal crops are alternated with pastures comprised of M. truncatula and other annual Medicago species, collectively referred to as annual medics (Crawford et al., 1989). In the USA, there is interest in adapting M. truncatula and other annual medics for use in the North-Central region as a short-season annual forage crop (Zhu et al., 1998, 1998; Fisk et al., 2001; Sheaffer et al., 2001; Walsh et al., 2001). Medicago truncatula also has many favorable characteristics that make it a suitable model to study legume biology (Cook, 1999): it is a diploid, self-pollinating species, with a small genome size (5 x 108 bp), and a short generation time (Barker et al., 1990). Barrel medic is closely related to alfalfa. Unlike barrel medic, however, alfalfa is a heterozygous, out-crossing species (Smith, 1993) with a large and complex, autopolyploid genome. Recent molecular marker mapping studies indicate that the genomes of M. truncatula and M. sativa are highly similar (Choi et al., 2004). Thus, M. truncatula could be used as a source of genes to improve cultivated alfalfa.
Barrel medic and alfalfa are both sensitive to soil acidity and aluminum toxicity (Rechcigl et al., 1988). Aluminum makes up 7% of the earth's crust. In acid soils, which comprise up to 40% of the world's arable land, Al becomes soluble as a trivalent cation (AL3+) (Kochian, 1995), which is toxic to plants at micromolar concentrations (Kinraide and Parker, 1987). The major Al toxicity response is a rapid inhibition of root elongation (Taylor, 1988; Foy, 1992), which leads to reduced plant growth, and consequently, reduced crop productivity. Lime application is the conventional method of reducing soil acidity, thereby reducing Al toxicity. Liming, however, can be expensive and only alleviates Al toxicity near the soil surface, leaving the subsoil pH unchanged. An alternative would be the identification of plants with genetic tolerance to Al. Previous studies have shown that plants adapted to low pH environment are mostly tolerant to Al (Watanabe et al., 1997). It has also been demonstrated that plant species and genotypes within species show different degrees of tolerance to mineral stresses. For example, different degrees of Al tolerance have been found in wheat (Triticum aestivum L. emend. Thell.) (Baier and Gustafson, 1995; de Sousa, 1998), barley (Hordeum vulgare L.) (Peruzzo and Arias, 1996), rye (Secale cereale L.) (Aniol et al., 1980), and triticale (x Triticosecale Wittmack) (Oettler et al., 2000). The USDA core collection of alfalfa, consisting of 200 accessions, was screened for acid soil tolerance; however, no accessions with exceptional tolerance to acid soils were identified (Bouton, 1996). The entire USDA collection of alfalfa contains more than 3000 accessions, which makes screening the entire collection for acid soil/Al tolerance impractical. QTLs for Al tolerance have been identified in diploid M. sativa L. subsp. caerulea (Less. Ex. Ledeb.) Schmalh. (Sledge et al., 2002), and these QTLs can potentially be used to improve Al tolerance in alfalfa. Additional germplasm sources with improved tolerance to Al, however, would increase the chances of improving Al tolerance in alfalfa.
The model legume M. truncatula offers the small genome needed for the cloning of Medicago genes that could then be used to improve cultivated alfalfa. Any Al tolerance genes identified in M. truncatula might be used to improve Al tolerance of cultivated alfalfa either by genetic transformation or by high throughput DNA homology searches for the same or similar Al tolerance genes in alfalfa germplasm collections. The objective of this study was to screen 321 plant introductions (PIs) of M. truncatula from the USDA PI collection for Al tolerance to identify Al tolerant accessions that could potentially be used to improve Al tolerance of cultivated alfalfa.
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MATERIALS AND METHODS
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Plant Materials
Al tolerance of 321 accessions of M. truncatula was evaluated in this study. These accessions were obtained from the Western Regional PI Station at Pullman, Washington. Accessions originated from 20 different countries.
Hydroponics Procedure
Medicago truncatula seeds were surface sterilized with 3% (v/v) sodium hypochlorite for 7 min. After thorough rinsing with sterile distilled water, seeds were imbibed by immersing in sterile water and kept in the dark overnight at 4°C. Imbibed seeds were then cold treated by placing on moist filter paper in a Petri dish and incubating for at least 48 h at 4°C. After cold treatment, seeds were germinated in the dark at 24°C for 24 h. Germinated seedlings were transferred to hydroponics in a growth room and grown under an 18-h-light/6-h-dark regime at a constant temperature of 23°C. The light intensity at the shelf height was 260 µmol m–2s–1. The hydroponics system consisted of multiple tanks with 25-L capacity. Twenty seedlings of each of the 321 accessions were placed into four plastic mesh-bottomed cups (five seedlings per cup) with the roots threaded through the mesh. Two cups of each accession were placed into a plastic rack that floated on modified Blaydes medium (Blaydes, 1966) plus Al and two cups on modified Blaydes medium minus Al. Media were aerated with a small aquarium air pump for 15 min, once per hour. The Blaydes medium was modified to contain 0.5 mM CaCl2, 1.8 mM KNO3, 1.8 mM NH4NO3, 30.4 µM MgSO4 · 7 H2O, 22 µM KH2PO4, 13 µM FeSO4, 31.7 µM Ca(NO3)2, 66 µM KCl, 26 µM NaCl, 26 µM H3BO3, 26 µM MnSO4 · H2O, 5.2 µM ZnSO4 · 7 H2O, and 4.8 µM KI. The media were maintained at pH 4.3 by adding 1 M HCl as necessary throughout the experiment. After 5 d of growth in hydroponics with and without Al, seedlings were removed and measured from the base of the cotyledon to the root tip. The experiment was repeated three times, once with the Al concentration at 25 µM as AlCl3 and twice at 50 µM AlCl3. Data were combined for statistical analysis.
Relative root growth, the ratio of seedling root length grown without Al to seedling root length grown with Al, was used as a measure of Al tolerance. An appropriate aluminum concentration for screening was estimated by measuring the relative root growth response of 60 randomly chosen M. truncatula accessions to 0, 5, 15, 25, and 50 µM AlCl3 in hydroponics as described above.
Experimental Design and Data Analysis
The experimental design was a split plot, with Al treatment as the whole plot factor, PIs as the split plot factor, and individual seedlings as subsamples. PROC MIXED (SAS, 1994) was used to analyze the data. Means were obtained by LSMEANS. A SLICE option was used to obtain p values for comparing the levels of Al at specific levels of PI. To minimize type II errors (declaring a PI Al tolerant when in fact it is Al sensitive), we used a p value of 0.4 or higher to declare a PI Al tolerant. At p values between <0.0001 and 0.05 (a significant difference between the plus Al and minus Al treatments), PIs were called highly Al sensitive. At p values between 0.06 and 0.39, PIs were considered to be Al sensitive.
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RESULTS AND DISCUSSION
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An appropriate Al concentration to use for screening M. truncatula accessions for Al tolerance was selected on the basis of the Al response of 60 randomly chosen accessions to levels of Al ranging from 5 to 50 µM (Fig. 1). Relative root growth of these 60 accessions averaged 0.75 at 5 µM Al, 0.61 at 15 µM Al, 0.56 at 25 µM Al, and 0.54 at 50 µM Al. There was little difference in average relative root growth between 25 and 50 µM, and this range was selected for screening as it gave the widest distribution of relative root growth. Response of one Al sensitive accession and three Al tolerant accessions to 0, 5, 15, 25, and 50 µM Al is shown in Fig. 2.
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Fig. 1. Average relative root growth, the ratio of seedling root length grown without Al to seedling root length grown with Al, of 60 M. truncatula accessions exposed to Al stress at 0, 5, 15, 25, and 50 µM AlCl3.
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Fig. 2. Relative root growth, the ratio of seedling root length grown without Al to seedling root length grown with Al, of one Al sensitive (W6 6018) and three Al tolerant (W6 6026, W6 6027, and W6 5997) accessions of M. truncatula in 0, 5, 15, 25, and 50 µM Al.
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Relative root growth for the 321 accessions ranged from 0.35 to 1.09 (Fig. 3). Average control root lengths varied from 0.86 to 5.7 cm (Fig. 4), indicating variation for root growth rate in addition to variation for Al tolerance. Under Al stress, root lengths varied from 0.85 to 2.85 cm (Fig. 5). Statistical analysis revealed significant treatment (P = 0.0056), PI (P < 0.0001), and PI x treatment effects (P = 0.0003). These results indicate that on average M. truncatula is sensitive to Al toxicity but that variation for Al tolerance exists within the USDA M. truncatula collection. Among the 321 accessions, 198 (61.88%) were highly Al sensitive, with relative root growth between 0.35 and 0.75 and p values between <0.0001 and 0.05; 109 accessions (33.96%) were sensitive to Al, with Al tolerance scores between 0.61 and 0.85 and p values between 0.06 and 0.38; 14 accessions (4.36%) were Al tolerant with Al tolerance scores between 0.76 and 1.09 and p values 0.4 or higher (Table 1). There was no association between geographical origin and Al tolerance of the accessions.
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Fig. 3. Frequency distribution of relative root growth, the ratio of seedling root length grown without Al to seedling root length grown with Al, for seedlings of 321 M. truncatula accessions exposed to 25 or 50 µM AlCl3 for 5 d in hydroponics.
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Fig. 4. Frequency distribution of root length of seedlings for 321 M. truncatula accessions grown without Al for 5 d in hydroponics.
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Fig. 5. Frequency distribution of root length of seedlings for 321 M. truncatula accessions exposed to 25 and 50 µM AlCl3 for 5 d in hydroponics.
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Table 1. Control root length, Al stressed root length, and relative root growth of Al tolerant M. truncatula accessions after 5 d of growth in hydroponics.
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In Medicago, various methods have been used to measure Al tolerance. Parrott and Bouton (1990) developed a tissue culture bioassay in which relative callus growth on an aluminum-toxic cell culture medium was used as a measure of Al tolerance and could distinguish between an acid-tolerant (AT) and an acid-sensitive (AS) M. sativa germplasm. Sledge et al. (2002) used the tissue culture method as well as a soil method to identify Al tolerance QTL in diploid M. coerulea. In soil, relative growth was found to be a poor measure of Al tolerance, as genotypes with poor vigor and limited growth even in limed, fertilized soil, had high relative root growth (Dall'Agnol et al., 1996; Sledge et al., 2002). Instead, root growth in unlimed soil was considered to be a better measure of Al tolerance, and Sledge et al. (2002) found that the QTL identified in tissue culture were also significantly associated with root growth in unlimed, Al toxic soil. In the current study, we also observed that the genotypes with the highest relative root growth did not necessarily exhibit the best root growth in either Al-toxic or control hydroponics. For example, W6 6076 had the best root growth in both Al-toxic and control hydroponics (2.85 and 5.76 cm, respectively) but had a relative root growth of only 0.5. PI 464816, on the other hand, had a relative root growth of 1.09 yet grew only 1.97 and 1.81 cm in Al-toxic and control hydroponics, respectively. Preliminary results (data not shown) indicate that M. truncatula accessions with good overall plant growth in unlimed soil also have good relative root growth in hydroponics. It is not, however, always the case that accessions with good relative root growth in hydroponics also have good growth in unlimed soil. The relationship between relative root growth in Al-toxic hydroponics and growth in Al-toxic soil requires further investigation.
It is possible that superior performance in the hydroponics assay could be used to select a subset of accessions for further testing in soil. There are several advantages of the hydroponics method over the soil method. In hydroponics, the amount of Al that the plants are subjected to can be carefully controlled, while keeping all other factors equal. A soil assay could select for factors other than Al tolerance because the complex interactions that may occur in a particular soil type cannot be controlled. Also, the hydroponics assay can be completed in a single week and requires little space and labor; the soil assay, however, takes 6 wk, requires significant greenhouse space, and must be watered to 75% of field capacity every 2 to 3 d, making it a labor intensive method. Removing all of the soil from roots is also problematic and could affect dry weights.
The results of this study indicate that variability for Al tolerance exists within this collection of M. truncatula PIs and that improvement of barrel medic cultivars could be possible by conventional breeding methods. The Al tolerant accessions identified will be useful for the identification of Al tolerance QTL and could serve as a source of genes to improve Al tolerance of cultivated alfalfa. Root length data for accessions examined in this research are available at http://www.ars-grin.gov/npgs/; verified 24 May 2005.
Received for publication November 22, 2004.
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REFERENCES
|
|---|
- Aniol, A., R.D. Hill, and E.N. Larter. 1980. Aluminum tolerance of spring rye inbred lines. Crop Sci. 20:205–208.
- Baier, A.C., and J.P. Gustafson. 1995. Aluminum tolerance in wheat: Correlating hydroponic evaluations with field and soil performance. Plant Breed. 114:291–296.[CrossRef]
- Barker, D.G., S. Bianch, F. Blondon, Y. Dattée, G. Duc, S. Essad, P. Flament, P. Gallusci, G. Génier, P. Guy, X. Muel, J. Tourneur, J. Dénairé, and T. Huguet. 1990. Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Plant Mol. Biol. Rep. 8:40–49.
- Blaydes, D.F. 1966. Interaction of kinetin and various inhibitors in the growth of soybean tissue. Physiol. Plant. 19:748–753.[CrossRef]
- Bouton, J.H. 1996. Screening the alfalfa core collection for acid soil tolerance. Crop Sci. 36:198–200.[Abstract/Free Full Text]
- Choi, H.K., D.J. Kim, T. Uhm, E. Limpens, H. Lim, J.H. Mun, P. Kalo, R.V. Penmetsa, A. Seres, O. Kulikova, B.A. Roe, T. Bisseling, G.B. Kiss, and D.R. Cook. 2004. A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa. Genetics 166:1463–1502.[Abstract/Free Full Text]
- Cook, D.R. 1999. Medicago truncatula–A model in the making! Curr. Opin. Plant Biol. 2:301–304.[CrossRef][ISI][Medline]
- Crawford, E.J., A.W.H. Lake, and K.G. Boyce. 1989. Breeding annual Medicago species for semiarid conditions in southern Australia. Adv. Agron. 42:399–437.
- Dall'Agnol, M., J.H. Bouton, and W.A. Parrott. 1996. Screening methods to develop alfalfa germplasms tolerant of acid, aluminum toxic soils. Crop Sci. 36:64–70.[Abstract/Free Full Text]
- Derkaoui, M., J.L. Caddel, and L.L. Rommann. 1993. Forage quality in annual Medicago spp. Agric. Mediterranea 123:86–91.
- de Sousa, C.N.A. 1998. Classification of Brazilian wheat cultivars for aluminum toxicity in acid soils. Plant Breed. 117:217–221.[CrossRef]
- Fisk, J.W., O.B. Hesterman, A. Shrestha, J.J. Kells, R.R. Harwood, J.M. Squire, and C.C. Sheaffer. 2001. Weed suppression by annual legume cover crops in no-tillage corn. Agron. J. 93:319–325.[Abstract/Free Full Text]
- Foy, C.D. 1992. Soil chemical factors limiting plant root growth. Adv. Soil Sci. 19:97–149.
- Kinraide, T.B., and D.R. Parker. 1987. Cation amelioration of aluminium toxicity in wheat. Plant Physiol. 83:546–551.[Abstract/Free Full Text]
- Kochian, L.V. 1995. Cellular mechanisms of aluminum toxicity and resistance in plants. Annu. Rev. Plant Physiol. Mol. Biol. 46:237–260.[CrossRef][ISI]
- Oettler, G., S. Wiethölter, and W.J. Horst. 2000. Genetic parameters for agronomic traits of triticale and other small-grain cereals grown on aluminum-toxic soil in southern Brazil. Plant Breed. 119:227–231.[CrossRef]
- Parrott, W.A., and J.H. Bouton. 1990. Aluminum tolerance in alfalfa as expressed in tissue culture. Crop Sci. 30:387–389.[Abstract/Free Full Text]
- Peruzzo, G., and G. Arias. 1996. Barley and other cereals root development in a Brazilian acid soil. Vol. 2, p. 662–664. In A. Slinkard, G. Scoles, and B. Rossnagel (ed.) Proc. V Int. Oat Conf. And VII Int. Barley Genet. Symp. Univ. Extension Press, Univ. of Saskatchewan, Saskatoon.
- Rechcigl, J.E., R.B. Reneau, Jr., and L.W. Zelazny. 1988. Soil solution Al as a measure of Al toxicity to alfalfa in acid soil. Commun. Soil Sci. Plant Anal. 19:989–1001.
- SAS Institute. 1994. The SAS system for Windows. Release 6.10. SAS Inst., Cary, NC.
- Sheaffer, C.C., S.R. Simmons, and M.A. Schmitt. 2001. Annual medic and berseem clover dry matter and nitrogen production in rotation with corn. Agron. J. 93:180–186.
- Sledge, M.K., J.H. Bouton, M. Dall'Agnoll, W.A. Parrott, and G. Kochert. 2002. Identification and confirmation of aluminum tolerance QTL in diploid Medicago sativa subsp. coerulea. Crop Sci. 42:1121–1128.[Abstract/Free Full Text]
- Smith, S.E. 1993. Salinity and the production of alfalfa (Medicago sativa L.). p. 431–448. In M. Pesarki (ed.) Handbook of crop stress. Macrel Dekker, Inc., New York.
- Taylor, G.J. 1988. The physiology of aluminum phytotoxicity. p. 123–163. In H. Siegel (ed.) Metal ions in biological systems: Aluminum and its role in biology, Vol. 24. Marcel Dekker, New York.
- Walsh, M.J., R.H. Delaney, R.W. Groose, and J.M. Krall. 2001. Performance of annual medic species (Medicago spp.) in southeastern Wyoming. Agron. J. 93:1249–1256.[Abstract/Free Full Text]
- Watanabe, T., M. Osaki, and T. Tadano. 1997. Response of plants adapted in low pH soils to aluminum. p. 459–460. In T. Ando et al. (ed.) Plant nutrition for sustainable food production and environment. Kluwer Academic Publishers, Tokyo.
- Zhu, Y.P., C.C. Sheaffer, M.P. Russelle, and C.P. Vance. 1998. Dry matter accumulation and dinitrogen fixation of annual Medicago species. Agron. J. 90:103–108.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
