Crop Science Illumina
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online 6 February 2007
Published in Crop Sci 47:321-328 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Narasimhamoorthy, B.
Right arrow Articles by Sledge, M. K.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Narasimhamoorthy, B.
Right arrow Articles by Sledge, M. K.
Agricola
Right arrow Articles by Narasimhamoorthy, B.
Right arrow Articles by Sledge, M. K.
Related Collections
Right arrow Crop Growth and Development
Right arrow Other Forage Crops
Right arrow Crop Genetics

CROP BREEDING & GENETICS

A Comparison of Hydroponics, Soil, and Root Staining Methods for Evaluation of Aluminum Tolerance in Medicago truncatula (Barrel Medic) Germplasm

B. Narasimhamoorthya,*, E. B. Blancaflora, J. H. Boutona, M. E. Paytonb and M. K. Sledgea

a The Samuel Roberts Noble Foundation, Ardmore, OK 73402
b Dep. of Statistics, Oklahoma State University, Stillwater, OK 74078-1056

* Corresponding author (bnarasimhamoorthy{at}noble.org)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Aluminum toxicity and soil acidity are major constraints in alfalfa (Medicago sativa subsp. sativa) production in the world. Despite intense research efforts, neither an effective screening procedure nor an Al-tolerant alfalfa germplasm is available. This dictates the need for identifying a new source of Al-tolerant genes in the closely related species M. truncatula (barrel medic). Our objectives were to compare three Al tolerance screening methods: (i) a seedling-based hydroponics method, (ii) a soil-based plant method, and (iii) an Al-stressed seedling-based lumogallion root staining method in 32 M. truncatula accessions. The soil system compared the genotypes for dry root and shoot weights in unlimed soil and for relative weights. The lumogallion root staining of Al-stressed seedlings compared the genotypes for fluorescence intensity of Al-bound lumogallion within the root tips. In the hydroponics system, the genotypes were compared for root elongation and relative growth. The three methods were different from each other, with altered rankings for genotypes across the methods. The soil assay demonstrated a higher capacity for discriminating Al response among genotypes with a higher reproducibility. Most of the genotypes that were Al-tolerant in soil were also Al-tolerant using the hydroponics and root staining methods. The results suggested that a combination of soil-based and hydroponics screening might be essential to identify Al-tolerant genotypes possessing multiple Al tolerance mechanisms.

Abbreviations: RRL, relative root length • RRW, relative root weight • RSW, relative shoot weight • RL, root length


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
ALUMINUM TOXICITY is one of the most widespread forms of metal toxicity in plants. It is a major constraint to plant growth in highly weathered, acid soils, which form 40% of world's arable soil (Von Uexküll and Mutert, 1995). Toxicity problems are aggravated by acid precipitation, which can accelerate the breakdown of Al-containing minerals and the leaching of basic cations (McBride, 1994). As a result of alterations in the soil, plant roots encounter an environment that is low in essential nutrients and high in toxic Al3+ and H+. The uptake of such toxic ions by plant roots results in a marked reduction in root growth (Taylor, 1988; Foy, 1992), and consequently, in reduced crop productivity (Kochian, 1995).

Alfalfa is the most important and widely grown forage legume in the world. Al toxicity associated with acid soils seriously limits world production of alfalfa. Surface application of lime to correct soil acidity in the top layer is the best management practice to increase soil pH and maintain alfalfa crop production. However, amendment of acid soils is not economically feasible in many parts of the world, and is often inefficient due to slow movement into the deeper layers of the subsurface soil. Planting Al-tolerant cultivars in combination with liming is the most promising approach to the improvement of alfalfa production on acid soils.

Tolerance to Al stress has been reported at various levels in many plants species, including wheat (Triticum aestivum L.) (Baier and Gustafson, 1995), barley (Hordeum vulgare L.) (Peruzzo and Arias, 1996), soybean [Glycine max (L.) Merr.] (Sartain and Kamprath, 1978), white clover (Trifolium repens L.) (Voigt et al., 1997), and diploid alfalfa (M. sativa subsp. coerulea) (Sledge et al., 2002). Medicago truncatula is a forage crop and a model legume species closely related to alfalfa (Choi et al., 2004), and similar to alfalfa in forage quality and dry matter yield (Derkaoui et al., 1993). Both of these Medicago species are highly sensitive to soil acidity and Al toxicity (Rechcigl et al., 1988). Several M. truncatula accessions with varying degrees of tolerance were identified from among the germplasm collection of the USDA National Plant Germplasm System (Sledge et al., 2005).

Several methods have been developed for evaluating Al tolerance in plants which contribute to the elucidation of the physiological processes underlying this trait. The classical procedure used to determine the effects of Al stress on plants is growth in solution culture. Assessment of root growth in nutrient solution containing Al is commonly used in wheat (Zhang and Taylor, 1989), maize (Zea mays L.) (Magnavaca et al., 1987), and rice (Oryza sativa L.) (Yoshida et al., 1971, p. 53–57). Assays based on growth of plants in acid soil with toxic levels of exchangeable Al have been used in barley (Foy, 1996), tall fescue (Festuca arundinacea Schreb.) (Foy and Murray, 1998), sorghum (Sorghum bicolor L.) (Foy et al., 1993), and soybean (Foy et al., 1992). Selection for Al and acid soil resistance in white clover has been done using a modified soil system called soil-on-agar as a selection technique (Voigt et al., 1997). In alfalfa, screening procedures for Al tolerance using a soil bioassay (Dall'Agnol et al., 1996) and a callus growth bioassay (Parrott and Bouton, 1990) have been reported. Aluminum tolerance mechanisms have also been studied using various stains such as hematoxylin in maize (Delhaize et al., 1993) and wheat (Rincon and Gonzales, 1992), the fluorescent stain morin in tobacco (Nicotiana benthamiana Domin.) (Vitorello and Haug, 1997), and the fluorescent stain lumogallion in soybean (Kataoka et al., 1997) and pine (Pinus taeda L.) (Moyer-Henry et al., 2005). Despite the existence of many Al tolerance screening methods, the use of a single method to identify Al tolerant genotypes may lead to misleading results due to the complexities involved in each method.

Along with diverse germplasm and an appropriate breeding program, a reliable screening procedure for Al stress is one of the most important tools required to effectively develop Al-tolerant cultivars. A comparative screening procedure to evaluate the effectiveness of different soil and cell cultures for three cultivars and a base population was done in alfalfa (Dall'Agnol et al., 1996). Nevertheless, no reliable screening method for Al tolerance in alfalfa is available and widely adapted (Dall'Agnol et al., 1996).

The lack of Al tolerance in alfalfa germplasm (Bouton, 1996) dictates the need for locating and tagging the Al-tolerant genes in a closely related species of Medicago, and transferring them to alfalfa to develop Al-tolerant cultivars. The model legume, M. truncatula, is the ideal species in which to clone genes to be used for alfalfa improvement, since it exhibits nearly perfect synteny with alfalfa (Choi et al., 2004). It has a small diploid genome and thus could be used to locate, clone, and transfer agronomically important genes to alfalfa. In M. truncatula, published data on screening for Al tolerance of germplasm is limited to using a hydroponics system (Sledge et al., 2005). The objective of this current study was to compare three Al tolerance screening methods in M. truncatula: (i) a seedling-based hydroponics method, (ii) a plant-based soil method, and (iii) an Al-stressed seedling-based lumogallion root staining method to determine whether these three methods could identify the same Al-tolerant and Al-sensitive M. truncatula accessions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials
Thirty-two diverse accessions of M. truncatula from the USDA National Plant Germplasm System were randomly chosen for this study. These plant materials were obtained from the Western Regional PI station in Pullman, WA.

Hydroponics
The 32 diverse accessions of M. truncatula were screened using the hydroponics system described in Sledge et al. (2005), exposing seedlings to 0 µM AlCl3 (control) and 25 µM AlCl3 stress (treatment) for 5 d. The experimental design was a randomized complete block with three replications with 20 seedlings per rep per accession. Root length (RL) was measured as the length of the roots in cm from the base of the cotyledon to the tip of the roots for each seedling in both treatment and control. Relative root length (RRL), a measure of Al tolerance, was computed as the ratio of the seedling RL grown under Al stress to seedling RL grown under control conditions.

Soil Study
Seven-day-old seedlings of each of the 32 accessions were grown in the greenhouse and subjected to Al stress (unlimed soil) and control treatments (limed soil) for 7 wk as previously described (Dall'Agnol et al., 1996; Sledge et al., 2002). Seeds were germinated in the same manner as for the hydroponics system and transferred to 720-mL Styrofoam cups filled with 875 g of soil. Two seedlings from each accession were placed in the cups in a randomized complete block design with six replicates. Each cup was covered with 25 g of coarse washed masonry sand and watered to a 75% of field capacity. One week after sowing, seedlings were thinned to one per cup. Plants were grown for an additional 7 wk, during which the cups were watered to 75% of field capacity every 2–3 d. During Week 8, the plants and soil were carefully removed from the cup, and the roots were gently washed on a wire mesh to remove the soil. The roots and the shoot from each plant were separated, dried at 65°C for 72 h, and weighed.

Dry root and shoot weights from both limed and unlimed soil were obtained. Relative root weight (RRW) and relative shoot weight (RSW) were estimated by computing the ratio of the dry root and dry shoot weight in unlimed soil to dry root and dry shoot weight in limed soil, and were used as a measure of Al tolerance.

Lumogallion Root Staining
Seedlings of the 32 accessions were grown in the hydroponics system as described, for 3 d in a randomized design. On Day 4, Al was added to a final concentration of 25 µM, using a 0.25 M AlCl3 stock solution. After 1 h of Al exposure, seedlings were removed from hydroponics and rinsed five times with water. Root tips (5–7 mm) were removed and washed twice with 10 mM citrate (pH 4.5) for 15 m at 24°C. Root tips were sectioned longitudinally at a thickness of 70 µm using a Vibratome 1000 (Technical Products International, St. Louis, MO) as described in Blancaflor et al. (1998). The root sections were washed for 15 min in 100 µM acetate buffer (pH 5.2) at 24°C and stained with 10 µM lumogallion (3-[2, 4 dihydroxy-phenylazo]-2-hydroxy-5-chlorobenzene sulfonic acid) in 100 µM acetate buffer (pH 5.2) in the dark at 24°C for 1 h. The root sections were washed twice with 100 µM acetate buffer for 15 min each and the fluorescence of lumogallion-Al complexes from the samples was imaged with a research stereo fluorescent microscope (model SZX-ILLB2–100, Olympus, New York, NY).

Digital images of the root sections were acquired with the DFPLFL1.6 x PF objective at a Focus of 100. The root sections were imaged with the Olympus DP70 12.5 megapixel color digital CCD camera (Olympus; Melville, NY) with settings of HQ 1360 x 1024 pixels JPEG, ISO 400, and 1/5 s exposure. The settings of the digital camera were established based on the images of the negative control treatments (0 µM Al3+). For each accession, six median root sections from six different seedlings considered as six replicates were imaged for quantification.

Quantification of the lumogallion stain in roots was achieved by measuring intensity of fluorescent images using Metamorph v. 6.3 Image Analysis software (Molecular Devices Corp., Sunnyvale, CA). A uniform area of the root tip for each section was chosen to measure the fluorescence intensity and the settings used were as described in the directions accompanying the Metamorph program. The Region measurement tool was used to obtain the average pixel intensity (Intensity) for a uniform area for all images.

Statistical Analysis
PC SAS v. 9.0 (SAS Institute, 2002) was used for all statistical analyses. For each method, means, LSD values, and ranking were calculated (PROC GLM). Correlation coefficients were calculated (PROC CORR) for pairs of genotypic means among all the methods. Data from all methods were subjected to ANOVA procedures (PROC MIXED) with genotype and method considered fixed effects and replications considered random effects. Because methods have different magnitudes and are measured in different units, the response was rank transformed within each replicate using PROC RANK. The method x genotype interaction was used to assess the consistency of methods across genotype.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Plant Materials
The choice of plant material with a wide range of responses to Al stress is important for a comparative study between different screening methods so as to detect and analyze the discrepancies effectively among them. All but one of the 32 accessions employed in this study were chosen randomly from a collection of 321 accessions, which were previously studied for responses to Al stress using the hydroponics system (Sledge et al., 2005). In addition, the ecotype Jemalong A17 was chosen since this genotype is commonly used in M. truncatula gene expression studies, and its genome is being sequenced by an international consortium (Young et al., 2005). A significant variation (P < 0.001) among these accessions with and without Al treatment in all three systems implies that the random selection of the 32 genotypes for this study captured a range of diversity. There were differences in morphological growth in addition to differences in Al tolerance as seen from the control for each method (Table 1). Significant method x genotype (P < 0.001) interactions observed in this study indicate a differential response of the accessions to Al stress among all the indices of these three methods (Table 2) suggesting that the three methods could be detecting different mechanisms of Al tolerance.


View this table:
[in this window]
[in a new window]

  		Table 1. Mean squares from ANOVA for root length in hydroponics, dry root and shoot weights in soil, and fluorescence intensity of lumogallion staining of 32 Medicago truncatula genotypes.{dagger}

 

View this table:
[in this window]
[in a new window]

  		Table 2. Combined ANOVA showing the method x genotype interaction for Al tolerance in 32 M. truncatula genotypes assessed by hydroponics, soil, and lumogallion root staining methods.

 
Hydroponics
For Al tolerance screening via hydroponics, a concentration of 25 µM Al was adopted, as this resulted in the widest distribution of RLs in a previous study (Sledge et al., 2005). At this concentration, the root elongation in an Al-sensitive genotype was significantly reduced by Al toxicity in low pH nutrient solutions (Fig. 1 ). The average RL of the 32 genotypes under Al stress varied from 0.99 to 3.08 cm and the RRL varied from 0.47 to 1.14 (Table 4). Ranking of absolute root growth under stress did not agree well with the ranking of RRL. The significant but weak correlation (r = 0.33***) (Table 3) between root RL and RRL suggests that the observed differences in rankings among accessions could be due genotypic differences among genotypes for root vigor, in addition to tolerance of the Al stress. Under control conditions, the high mean square values for genotypic variation (Table 1) and wide range for genotypic means, 1.50 to 5.51 cm (Table 4), indicates differences in vigor among the selected genotypes. All of these observations indicate that genotypic vigor is an important factor to consider while applying selection for Al tolerance in hydroponics. Sledge et al. (2005) reported similar results when 321 M. truncatula genotypes were assessed for Al tolerance variation using hydroponics.


Figure 1
View larger version (180K):
[in this window]
[in a new window]

  		Fig. 1. Seedlings of M. truncatula accessions Jemalong A17 exposed to (a) 25 µM of Al3+ and (b) 0 µM of Al3+ for 5 d in hydroponics.

 

View this table:
[in this window]
[in a new window]

  		Table 4. Al stressed root length, control root length, and relative root length (RRL, the ratio of root length under Al stress to root length under no Al stress) of 32 M. truncatula accessions grown for 5 d in hydroponics.

 

View this table:
[in this window]
[in a new window]

  		Table 3. Phenotypic correlation coefficients for 32 M. truncatula genotypes grown in the presence and absence of Al3+ as 5-d seedlings in hydroponics, 7-wk plants in soil, and 5-d seedlings stained with lumogallion.{dagger}

 
Soil Study
The soil screening procedure was initially developed for alfalfa germplasm and was adopted for this study. Al tolerance ratings were expressed as dry weights of roots and shoots, as well as RRW and RSW. Broad ranges were observed for the average dry root weights under Al stress (0.007–0.191 g), and the average dry shoot weights (0.007–0.217 g) under Al stress for the 32 accessions used in this study (Table 5). Under greenhouse conditions, unlimed soils significantly reduced the root and the shoot growth of the accessions under investigation (Fig. 2 ).


View this table:
[in this window]
[in a new window]

  		Table 5. Mean dry shoot and root weights and relative root and shoot weights of 32 M. truncatula genotypes grown in limed and unlimed soil for 7 wk.

 

Figure 2
View larger version (162K):
[in this window]
[in a new window]

  		Fig. 2. Seedlings of M. truncatula accessions. (A) Jemalong A17 in limed soil; (B) Jemalong A17 in unlimed soil; (C) PI 566890 in unlimed soil; and (D) PI 566890 in limed soil grown for 7 wk.

 
The mean root and shoot dry weights in acid soils were 8.7 and 7.2% of that observed under limed conditions. Al stress imposed in the soil, therefore, has a similar effect on the roots and shoots of the accessions used in this study. This similar degree of growth reduction for both roots and shoots indicates that the treatment was more effective. For example, seven of the 32 accessions had the same rankings for both shoot and root growth, and there was only a slight variation for the remainder of the accessions (Table 5). Also, there was a strong positive correlation (r = 0.81; P < 0.001) between dry root weight and dry shoot weight under unlimed conditions (Table 3). Although symptoms of Al toxicity are also manifested in the shoots, these are usually regarded as a consequence of injuries to the root system. The most common responses in shoots to Al toxicity are cellular and ultra structural modifications in leaves, reduced stomatal opening, decreased photosynthetic activity, chlorosis, and foliar necrosis, with the ultimate consequence being reduced plant biomass (Jansen et al., 2002; Watanabe and Osaki, 2002). Seven accessions (PI 535546, PI 566890, PI577626, W6 6025, W6 6037, W6 6044, and W6 6097) were among the top 10 for dry shoot and root weights.

For the relative weights (ratio of weights in unlimed soil to limed soil) the lowest value recorded was 0.01 for roots and 0.01 for shoots, and the highest value was 0.29 for roots and 0.17 for shoots (Table 5). The 10 accessions with the highest RSW and RRW had the following eight accessions in common: PI 535546, PI 537168, PI 566890, W6 6025, W6 6037, W6 6044, W6 6068, and W6 6097.

Dall'Agnol et al. (1996) found RRW and RSW to be poor measures of Al tolerance in alfalfa since genotypes with poor vigor often had higher relative growth and genotypes with high vigor often had lower relative growth. Similarly in soybean, Al tolerance ratings did not agree well between absolute growth and relative growth (Villagarcia et al., 2001). In our study, however, significant and strong correlations were observed between root dry weights under stress and the RRW (r = 0.882), and between shoot dry weights under stress and RSW (r = 0.875) (Table 3). In addition, a strong correlation (r = 0.742***) between the RRWs and RSWs was observed in this study. The strong correlation between root and shoot weights indicates that measuring the dry shoot weight may be as efficient as weighing dry roots. It is noteworthy that washing the soil carefully off of the roots makes the soil study very labor intensive, and thus using only the shoot dry weights as an indicator of tolerance could be more efficient in a large scale screening experiment. There were six accessions which were among the 10 highest scoring for each of the four soil indices: PI 535546, PI 566890, W6 6025, W6 6037, W6 6044, and W6 6097.

Lumogallion Root Staining
Lumogallion is a highly sensitive and specific stain for Al (Shuman, 1992). It has been used to study the distribution and movement of Al in soybean roots (Kataoka et al., 1997; Silva et al., 2000) and the Al phytotoxic effect on barley roots (Pan et al., 2004). The intensity of fluorescence varies in accordance with the amount of Al uptake by the roots. For our study we chose to use the research stereo fluorescent microscope to capture the images of the lumogallion stained roots instead of the confocal laser microscope, a highly sophisticated imaging system which might have a focusing bias. While using the stereo fluorescent microscope, we initially captured a series of images to determine the level of autofluorescence of M. truncatula roots (data not shown). They included (i) roots not exposed to Al and not stained with lumogallion, (ii) roots exposed to Al and not stained with lumogallion, and (iii) roots not exposed to Al and stained with lumogallion. Using these negative controls we set the gain and exposure settings of the camera such that any nonspecific fluorescence resulted in a black image. These same settings were then applied to capture roots exposed to Al and stained with lumogallion to ensure that any fluorescence that we quantified was due to lumogallion binding to Al. The 32 genotypes that were treated with 25 µM Al for 1 h were all imaged using camera settings where the corrections for nonspecific fluorescence were taken into consideration.

The results indicate that substantial Al accumulates in the root tips of the seedling exposed to Al-stress for 1 h and that the accumulation is higher in the Al-sensitive genotypes, which exhibit more fluorescence (Fig. 3 ). The pixel intensities varied across the genotypes as indicated by the significant genotypic variation (Table 1). There was a wide range of intensity between the most sensitive accession, as indicated by the highest fluorescence (118.69 pixel intensity), and most tolerant accession, as indicated by the lowest fluorescence (29.04 pixel intensity) (Table 6).


Figure 3
View larger version (81K):
[in this window]
[in a new window]

  		Fig. 3. Root sections of M. truncatula accessions (A) Jemalong A17 and (B) PI 566890 exposed 25 µM of Al3+ for 5 d in hydroponics and stained with lumogallion. The more stain absorbed indicates more Al present in the root. Scale bar = 200 µm.

 

View this table:
[in this window]
[in a new window]

  		Table 6. Fluorescence intensity of lumogallion stained M. truncatula root tips after 1 h exposure to 25 µM Al3+.

 
Comparison of Soil, Hydroponics, and Root Staining Assays for Al Tolerance
Whenever possible, it is best for plant breeders to conduct a screening procedure in the target field. However, large and uniform Al-toxic soils are unavailable, and thus screening procedures under controlled conditions, such as those described in this study, are essential to select genotypes that are Al tolerant for inclusion in breeding programs. A disadvantage of the soil method is that it is time and space consuming, making it expensive to conduct such studies for a large number of genotypes. Moreover, complex genotype x soil interactions can occur which can result in varying levels of plant growth, confounding the effects of Al toxicity. For instance soil-based screening is usually not adopted in soybean breeding programs because of the variability involved (Villagarcia et al., 2001). Soil variabilities have led the breeders to express the screening method as acid soil tolerance rather than Al tolerance per se, despite the fact that Al is the major phytotoxic problem. In addition, acidic soils with similar soil pH can vary vastly in the amount of Al saturation. In wheat, for example, the response of plants in acid soils is highly dependent on Al saturation of the soil and other toxic minerals such as Mn, rather than on soil pH (Johnson et al., 1997). In sorghum, the soil assay using Al-toxic Tatum soil (fine, mixed, semiactive, thermic Typic Hapludults) has also been reported to be too rigid, thus not being able to identify tolerant accessions (Foy et al., 1993). The advantage of the soil assay, however, is that it mimics the field situation most accurately, and can evaluate plants at both young and older stages, thereby evaluating for both root growth and drought tolerance (Samac and Tesfaye, 2003). It is also highly suitable for both seed-propagated and vegetatively propagated plants.

Methods assessing root growth in nutrient solutions are more attractive than soil assays, as they provide controlled forms of Al stress, are easily repeatable, and are cost and time effective. The hydroponics system for the evaluation of genetic materials provides a strict control of nutrient availability and is widely used in genetic studies. This assay is relatively rapid to perform and many plants can be evaluated in a short time and in a small space. The outcome of results and evaluations, however, depends on multiple factors, such as pH, temperature, interaction of Al with other nutrients in the solution, and the sensitivity of the biological specimen. Arabidopsis (Koyama et al., 1995) and soybean (Lazof and Holland, 1999) studies have shown that some genotypes are sensitive to low pH stress, in which Al toxicity may be masked by H+ sensitivity. In addition, biotic stress on the roots may have confounding effects on root growth while under Al stress. While using the RRW as a measure of tolerance, slower-growing plants may emerge to be more tolerant than they actually are because the percentage reduction in their root growth may be much less than that of the faster growing plants (Dall'Agnol et al., 1996). An additional drawback of solution culture is that it is not suitable for plants that are propagated vegetatively.

Staining methods are considered an alternative to solution culture assays. The most widely used stain is hematoxylin. Indication of Al uptake via hematoxylin staining is usually a qualitative measure. The use of lumogallion in Al tolerance studies has thus far been limited to the study of Al tolerance mechanisms. The use of lumogallion root staining as the sole measure of Al tolerance has not been reported to date. The lumogallion root staining procedure can be a time-consuming procedure, as roots must be sectioned, and multiple seedlings per accession must be evaluated.

In this study, the soil assay demonstrated a higher capacity for discriminating Al response among genotypes and a higher experimental reproducibility than the other two indices, hydroponics and root staining. The exposure of plants to Al for only 5 d in hydroponics and 1 h for root staining was a short period compared with growing plants in acid Al-toxic soil for 7 wk. This may have been responsible for the increased Al sensitivity among the different accessions in the soil system, in which a smaller number of accessions were classified as tolerant compared with hydroponics and root staining methods. A weak correlation was observed between the three methods (Table 3), suggesting that each technique is distinct and one method cannot be substituted for another. In wheat (Ma et al., 2005) and maize (Giaveno and Miranda, 2000), however, a strong correlation between the hematoxylin staining and solution culture for both absolute root growth and relative root growth was reported. Because the hematoxylin root staining correlates well with solution culture, it has been considered an alternative to hydroponics in wheat for identifying Al-tolerant genotypes.

Few instances have been reported in which hydroponics results correlate well with results from soil assays. In Arabidopsis, the same ecotypes were identified to be tolerant using both solution culture and acid soil (Toda et al., 1999). While evaluating transgenic alfalfa, certain lines showed increased root growth both in solution culture and soil assay (Tesfaye et al., 2001). On the other hand, while studying rape (Brassica napus L.) and tomato (Lycopersicum esculentum Mill.) plants for Al tolerance, different responses were seen for growth in acid soils as compared with growth in Al-toxic solution culture (Luo et al., 1999). In this current study, most of the genotypes that were Al-tolerant in soil were also Al-tolerant with the hydroponics and root staining methods. Five of the six accessions identified by the four soil indices were among the 50% with the longest RLs and among the 25% with highest RRL under Al in hydroponics, and also were among the 50% with the lowest lumogallion fluorescent intensities. Genotypes which were tolerant with the hydroponics system, however, were not always Al tolerant in the soil assay. Although some false positives may have appeared in the hydroponics system, it is a rapid way of screening a large number of genotypes efficiently. Despite the discrepancies among the methods, the accessions PI 566890 and W6 6025 were consistently tolerant while Jemalong A17, PI 190089, PI 577624, and PI 577638 were consistently sensitive, using all the three methods. In addition, the accessions PI 6044 and PI 6037 were tolerant in all but RRL in hydroponics.

The results of this study suggest that, for M. truncatula, the soil-based screening method is more stringent than either the hydroponics or lumogallion staining methods. One has to exercise caution while selecting genotypes based on the soil system because of the complex physical and chemical nature of the soil. It may be advantageous to screen first with unlimed soil, and select a large percentage of genotypes that are acid-soil tolerant, to avoid discarding superior genotypes. The hydroponics and/or lumogallion root staining can then be used at a later stage, to focus selection on genotypes that are responding specifically to Al rather than to uncontrolled soil factors.

Implications
Results from this study suggest that the M. truncatula accessions express differential Al responses to each of the three screening methods. Despite many studies performed on mechanisms of tolerance, this issue is far from resolved. If multiple Al tolerance mechanisms exist in plants, they would presumably be encoded by different genes, and not all sources of Al tolerance might be identified with one screening method. Thus, using a single method to identify Al-tolerant accessions could be misleading. Voigt and Staley (2004) used a two-stage screening procedure; one at a seedling stage and one at an adult stage, to identify Al-tolerant white clover lines in 12 populations. The clover populations developed from two-stage selection were more acid soil tolerant than their parents. It is possible that seedling Al tolerance, as identified by the hydroponics and root staining methods and expressed in the primary root, may be different from Al tolerance expressed in a mature plant in response to acid, Al-toxic soils. Therefore, a combination of soil-based screening and hydroponics may be essential to identify Al tolerant genotypes possessing multiple Al tolerance mechanisms to be further analyzed under field conditions. The Al-tolerant M. truncatula genotypes identified in this study could be used to locate genes involved in Al tolerance which might be used to improve Al tolerance in cultivated alfalfa by transferring these genes to cultivated alfalfa via genetic transformation.

Received for publication March 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Narasimhamoorthy, B.
Right arrow Articles by Sledge, M. K.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Narasimhamoorthy, B.
Right arrow Articles by Sledge, M. K.
Agricola
Right arrow Articles by Narasimhamoorthy, B.
Right arrow Articles by Sledge, M. K.
Related Collections
Right arrow Crop Growth and Development
Right arrow Other Forage Crops
Right arrow Crop Genetics

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome