Aluminum toxicity has been considered the most important limiting factor of plant growth on acid soils, which affects one-third of the world’s arable land area. In acid soils, crop productivity is affected by multiple stresses, such as deficiencies of phosphorus (P), calcium (Ca), and magnesium (Mg), and toxicities of manganese (Mn), iron (Fe), and Al (von Uexkull and Mutert, 1995). Aluminum species that are present in the environment, such as Al oxides, gibbsite, Al silicates, and organic-Al complexes are nontoxic. However, under acidic conditions, Al3+ is solubilized into the soil solution where it rapidly inhibits root growth (Kinraide, 1991; Kochian, 1995; Miyasaka et al., 2006).
Although soil acidity problems can be overcome by fertilization and liming practices, it is often economically impractical to correct in many parts of the world. Aluminum tolerance has been reported in many plant species, and using Al-tolerant cultivars provides the most effective strategy for production of economically important crops in acid soils (Foy, 1988; Ishitani et al., 2004).
Barrel medic originated in the Mediterranean basin. It was widely naturalized to New South Wales, Australia, and became an important forage crop in the low-input farming system typical of Western Australia, alternating with other cover crop species (Crawford et al., 1989). In the north central United States, barrel medic was used as a short-season annual pasture legume to increase soil nitrogen (N) (Zhu et al., 1998). It has also been chosen as a model legume for genomic studies because of its small diploid genome, fast generation time, self-pollination, and high transformation efficiency (Cook, 1999).
Aluminum tolerance in the barrel medic germplasm has been reported based on a hydroponic screening method (Sledge et al., 2005), an Al-toxic soil assay, and a lumogallion root staining method (Narasimhamoorthy et al., 2007). Results from the hydroponic screening indicated that sufficient variation of Al tolerance exists within this collection of barrel medic accessions to select for Al-sensitive or Al-tolerant accessions (Chandran et al., 2008; Sledge et al., 2005). However, the reported Al sensitivity of particular accessions also varied, depending on the screening method (Narasimhamoorthy et al., 2007).
Little is known about physiological mechanisms that are responsible for Al tolerance in M. truncatula. It is important to remember that each evaluation method could result in difference stresses, and genetic determinants underlying tolerance of each stress (e.g., Al toxicity or proton toxicity) may differ (Khu et al., 2012).
A consistent, quick evaluation method is key to selecting or breeding an Al-tolerant cultivar (Samac and Tesfaye, 2003). Agar and agarose gels have been used often in rhizosphere studies. In particular, agarose is a suitable substrate for studies of Al interaction in the rhizosphere, because it contains very low levels of P and other Al-complexing substances that could interfere with plant responses to Al toxicity (Calba et al., 1996). Agarose culture has an advantage over the hydroponic system, because solution culture can result in hypoxia particularly for plants that are sensitive to low oxygen conditions (Tamas et al., 2006). In addition, solution culture could remove border cells and mucilages that possibly protect root tips from Al toxicity (Miyasaka and Hawes, 2001).
Relative root growth, which is a measure of root elongation inhibition, can be used as an indicator for response to Al toxicity. This parameter has been shown to be suitable for estimating Al tolerance of accessions of various plants such as arabidopsis [Arabidopsis thaliana (Kobayashi et al., 2005)], maize [Zea mays (Llugany et al., 1995)], rye [Secale cereal (Hede et al., 2002)], bermudagrass [Cynodon dactylon (Liu, 2005)], and wheat [Triticum aestivum (Tang et al., 2002)].
Hematoxylin is a natural dye extracted from the heartwood of a logwood tree (Hematoxylin campechianum). It has been reported as a sensitive method for the evaluation of Al accumulation in a number of plants, because it has the property of turning blue when it forms a complex with Al (Delhaize et al., 1993). The staining pattern of Al accumulation in roots was similar to lipid peroxidation and callose production, which are other symptoms of Al toxicity in pea (Pisum sativum) roots (Yamamoto et al., 2001). Both the hematoxylin method and RRG have been used for Al-tolerance screening in many crop plants (Poschenrieder et al., 2008), such as chickpea [Cicer arietinum (Singh and Raje, 2011)], barley [Hordeum vulgare (Echart et al., 2002)], and wheat (Tang et al., 2002; Zhou et al., 2007).
Soil-based screening is considered the most realistic method of evaluating plants for Al tolerance; however, soil assays are time consuming and only a few ecotypes or genotypes can be evaluated using this method. Short-term screening systems such as hydroponics or sand cultures have been developed that correlated well to soil-based assays for certain crop species (Brauer and Staley, 2005; Hossain et al., 2005; Villagarcia et al., 2001; Voigt and Staley, 2004).
The objectives of this study were to 1) identify barrel medic accessions that differed in response to Al toxicity and 2) determine the best method(s) of screening barrel medic for Al tolerance. The identification of Al-tolerant accessions in this model legume can be used in the future to determine molecular mechanisms underlying Al responses, with the overall goal of improving Al tolerance in barrel medic and other crop species.
Brauer, D. & Staley, T. 2005 Early developmental responses of white clover root hair lengths to calcium, protons, and aluminum in solution and soil cultures Crop Sci. 45 1216 1222
Calba, H., Jaillard, B., Fallavier, P. & Arvieu, J.D. 1996 Agarose as a suitable substrate for use in the study of Al dynamics in rhizosphere Plant Soil 178 67 74
Cançado, G.A.M., Loguercio, L.L., Martins, P.R., Parentoni, S.N., Paiva, E., Borém, A. & Lopes, M.A. 1999 Hematoxylin staining as a phenotypic index for aluminum tolerance selection in tropical maize (Zea mays L.) Theor. Appl. Genet. 99 747 754
Chandran, D., Sharopova, N., Vanden Bosch, K.A., Gravin, D.F. & Samac, D.A. 2008 Physiological and molecular characterization of aluminum resistance in Medicago truncatula BMC Plant Biol. 8 89
Crawford, E.J., Lake, A.W.H. & Boyce, K.G. 1989 Breeding annual Medicago species for semiarid conditions in southern Australia Adv. Agron. 42 399 437
Dall’Agnol, M., Bouton, J.H. & Parrott, W.A. 1996 Screening methods to develop alfalfa germplasms tolerant of acid, aluminum toxic soils Crop Sci. 36 64 70
Delhaize, E., Craig, S., Beaton, C.D., Bennet, R.J., Jagadish, V.C. & Randall, P.J. 1993 Aluminium tolerance in wheat (Triticum aestivum L.). Uptake and distribution of aluminium in root apices Plant Physiol. 103 685 693
Echart, C.L., Barbosa-Neto, J.F., Garvin, D.F. & Cavalli-Molina, S. 2002 Aluminum tolerance in barley: Methods for screening and genetic analysis Euphytica 126 309 313
Hede, A.R., Skovmand, B., Ribaut, J.-M., Gonzalez-de-Leon, D. & Stolen, O. 2002 Evaluation of aluminium tolerance in a spring rye collection by hydroponic screening Plant Breed. 121 241 248
Hossain, M., Zhou, M. & Mendham, N. 2005 A reliable screening system for aluminium tolerance in barley cultivars Austral. J. Agr. Res. 56 475 482
Ishitani, M., Rao, I., Wenzl, P., Beebe, S. & Tohme, J. 2004 Integration of genomics approach with traditional breeding towards improving abiotic stress adaptation: Drought and aluminum toxicity as case studies Field Crops Res. 90 35 45
Kobayashi, Y., Furuta, Y., Ohno, T., Hara, T. & Koyama, H. 2005 Quantitative trait loci controlling aluminium tolerance in two accessions of Arabidopsis thaliana (Landsberg erecta and Cape Verde Islands) Plant Cell Environ. 28 1516 1524
Kochian, L.V. 1995 Cellular mechanisms of aluminum toxicity and resistance in plants Annu. Rev. Plant Physiol. Mol. Biol. 46 237 260
Llugany, M., Poschenrieder, C. & Barceló, J. 1995 Monitoring of aluminium-induced inhibition of root elongation in four maize cultivars differing in tolerance to aluminium and proton toxicity Physiol. Plant. 93 265 271
Mariano, E.D. & Keltjens, W.G. 2004 Variation for aluminum resistance among maize genotypes evaluated with three screening methods Commun. Soil Sci. Plant Anal. 35 2617 2637
Miyasaka, S.C. & Hawes, M.C. 2001 Possible role of root border cells in detection and avoidance of aluminum toxicity Plant Physiol. 125 1978 1987
Miyasaka, S.C., Hue, N.V. & Dunn, M.A. 2006 Aluminum, p. 439–497. In: A.V. Barker and D.J. Pilbeam (eds.). Handbook of plant nutrition. CRC Press, New York, NY
Narasimhamoorthy, B., Blancaflor, E.B., Payton, M.E. & Sledge, M.K. 2007 A comparison of hydroponics, soil, and root staining methods for evaluation of aluminum tolerance in Medicago truncatula (barrel medic) germplasm Crop Sci. 47 321 328
Poschenrieder, C., Gunse, B., Corrales, I. & Barceló, J. 2008 A glance into aluminum toxicity and resistance in plants Sci. Total Environ. 400 356 368
Samac, D.A. & Tesfaye, M. 2003 Plant improvement for tolerance to aluminum in acid soils : A review Plant Cell Tissue Organ Cult. 75 189 207
Srimake, Y. 2012 Aluminum tolerance in Medicago truncatula Gaertn. PhD Diss., Univ. Hawaii at Manoa, Honolulu, HI
Tamas, L., Budicova, S., Huttova, J., Mistrık, I., Šimonovicova, M. & Siroka, B. 2005 Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production Plant Cell Rpt. 24 189 194
Tamas, L., Budicova, S., Šimonovicova, M., Huttava, J., Široka, B. & Mistrik, I. 2006 Rapid and simple method for Al-toxicity analysis in emerging barley roots during germination Biol. Plant. 50 87 93
Tang, Y., Garvin, D.F., Kochian, L.V., Sorrells, M.E. & Carver, B.F. 2002 Physiological genetics of aluminum tolerance in the wheat cultivar Atlas 66 Crop Sci. 42 1541 1546
Villagarcia, M.R., Carter, T.E. Jr, Rufty, T.W., Niewoehner, A.S., Jennette, M.W. & Arrellano, C. 2001 Genotypic rankings for aluminum tolerance of soybean roots grown in hydroponics and sand culture Crop Sci. 51 1499 1507
Yamamoto, Y., Kobayashi, Y. & Matsumoto, H. 2001 Lipid peroxidation is an early symptom triggered by aluminum, but not the primary cause of elongation inhibition in pea roots Plant Physiol. 125 199 208
Zhou, L.-L., Bai, G.-H., Carver, B.F. & Zhang, D.-D. 2007 Identification of new sources of aluminum resistance in wheat Plant Soil 297 105 118
Zhu, Y.P., Sheaffer, C.C., Russelle, M.P. & Vance, C.P. 1998 Dry matter accumulation and dinitrogen fixation of annual Medicago species Agron. J. 90 103 108