O objetivo deste trabalho foi testar um método simples para a avaliação de pêlos radicais de 21 genótipos de feijão (Phaseolus vulgaris), a maioria deles usada em programas de melhoramento no Brasil. Os pêlos de raízes basais e primárias de plântulas com idade de cinco dias, produzidas em papel de germinação sem adição de fósforo, foram avaliados visualmente, com o auxílio de uma escala de avaliação, depois de coloridos com "trypan blue" (0,05%). O método diferencia bem os genótipos, e o erro-padrão da média é relativamente pequeno.The objective of this work was to test a simple method for root hair evaluation of 21 common bean (Phaseolus vulgaris) genotypes, most of them used in breeding programs in Brazil. Hairs of basal and primary roots of 5-day old seedlings, produced on germination paper with no phosphorus addition, were visually evaluated by a rating scale after staining with 0.05% trypan blue. The method reveals variability among the genotypes, and the standard error of the mean is relatively low

Jochua, Celestina Nhagupana

Lynch, Jonathan Paul

Vieira, Rogério Faria

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Pesq. agropec. bras., Brasília, v.42, n.9, p.1365-1368, set. 2007Method for evaluation of root hairs of common bean genotypes 1365Notas CientíficasMethod for evaluation of root hairs of common bean genotypesRogério Faria Vieira(1), Celestina Nhagupana Jochua(2) and Jonathan Paul Lynch(2)(1)Empresa de Pesquisa Agropecuária de Minas Gerais, Caixa Postal 216, CEP 36571-000 Viçosa, MG, Brazil. E-mail: rfvieira@epamig.br(2)Penn State University, Dept. of Horticulture, 102 Tyson Building, University Park, PA, 16803 USA. E-mail: cnj112@psu.edu, jpl4@psu.eduAbstract – The objective of this work was to test a simple method for root hair evaluation of 21 common bean(Phaseolus vulgaris) genotypes, most of them used in breeding programs in Brazil. Hairs of basal and primaryroots of 5-day old seedlings, produced on germination paper with no phosphorus addition, were visuallyevaluated by a rating scale after staining with 0.05% trypan blue. The method reveals variability among thegenotypes, and the standard error of the mean is relatively low.Index terms: Phaseolus vulgaris, rating scale, seedling, cultivar.Método de avaliação de pêlos radicais de genótipos de feijãoResumo – O objetivo deste trabalho foi testar um método simples para a avaliação de pêlos radicais de21 genótipos de feijão (Phaseolus vulgaris), a maioria deles usada em programas de melhoramento no Brasil.Os pêlos de raízes basais e primárias de plântulas com idade de cinco dias, produzidas em papel de germinaçãosem adição de fósforo, foram avaliados visualmente, com o auxílio de uma escala de avaliação, depois decoloridos com “trypan blue” (0,05%). O método diferencia bem os genótipos, e o erro-padrão da média érelativamente pequeno.Termos para indexação: Phaseolus vulgaris, escala de avaliação, plântula, cultivar.The capacity of plants to absorb both water andmineral nutrients from the soil is related to thedevelopment of an extensive and well-positioned rootsystem. Root hairs can contribute up to 67% of the totalroot surface area (Nielsen et al., 2001). Root hairs aresubcellular extensions of the root epidermis thatcontribute to the acquisition of immobile nutrients suchas phosphorus (Jungk, 2001). Root hair production isstimulated by the deficiency of several nutrients, includingiron, zinc, manganese, and phosphorus, but, at least inArabidopsis, phosphorus has a greater effect than othernutrients on root hair density and length (Bates & Lynch,1996; Ma et al., 2001). Phosphorus mobility in soil isgoverned by diffusion rather than mass flow. Therefore,phosphorus uptake by root is limited by localizedphosphorus depletion around the root. Root hairs extendthe phosphorus depletion zone from the root epidermis,thereby increasing the rate of phosphorus uptake andthe total amount of phosphorus accessible by the roots(Bates & Lynch, 2001). Other important effects of roothairs are increased root exudates such as phosphatases,organic acids and other chelating compounds that mayliberate phosphorus from pools like organic phosphateesters, Fe-P, and Al-P compounds that are characteristicof many tropical soils (Marschner et al., 1987).Common bean exhibits substantial genotypic variationin root hair length and density, ranging from abundantlong hairs to virtually hairless (Yan & Lynch, 1998).Common bean genotypes with longer and denser roothairs had more total biomass under low phosphorusavailability, compared to those with shorter and less denseroot hairs in the same level of phosphorus availability. Inaddition, genotypes with longer and denser root hairsacquired more phosphorus than their counterparts(Miguel, 2004). Information about root hair productionof genotypes used as source of disease resistance, inbreeding programs, may be useful to genetically improvecommon bean root systems, as well.For determination of root hair length and density,Miguel (2004) stained roots of 14-day old plants thatPesq. agropec. bras., Brasília, v.42, n.9, p.1365-1368, set. 2007R.F. Vieira et al.1366were observed under microscopy at 40x magnification.Using a comparative picture taken at the samemagnification with a micrometer scale, actual rootdimensions were determined. Pictures were taken usinga digital camera, attached to dissecting scope, and savedusing Adobe Photoshop 7.0 software. Then pictureswere transferred to Scion Image 1.63 software, for rootlength determination at that magnification. For root hairdensity, a known surface area at that magnification wasselected, and the number of root hairs in the selectedarea was determined. From these observations, actualhair length and density were calculated based on thepicture magnification, micrometer scale and conversionof the selected root surface area to the unit of root surfacearea in square millimeters. In this way, root hair length(mm) and root hair density (number of root hair per mm2of root surface area) were determined for basal andlateral roots. This method is very laborious in a breedingprogram, in which a large number of genotypes need tobe evaluated.The objective of this research was to test a simplerscreening method for root hair evaluation of commonbean genotypes.Twenty-one genotypes were evaluated in relation toroot hair density and length. Seeds of 18 common beangenotypes were used. Eight of them are high yieldingcultivars or lines (Ouro Negro, Diamante Negro, Valente,Talismã, Jalo MG-65, Carnaval MG, Vi 4899, andVi 10-2-1), and ten are genotypes used for diseaseresistance in breeding programs (AB 136, Cornell 49-242,G 2333, Kaboon, México 54, México 309, Pi 207262, TO,TU, VC-4). The line Vi 4899 was released in 2005 as acultivar (Pioneiro). Seeds of all these genotypes wereharvested from the same place and with the samefertilization in Brazil. The genotypes DOR 364, G 19833,and Carioca were obtained from the International Centerfor Tropical Agriculture (CIAT). DOR 364 is known asinefficient under P-deficient conditions. Carioca is aBrazilian landrace, which is among the most widelygrown tropical genotypes, perhaps because of itstolerance to infertile soils. G19833 is a landrace fromPeru, which is well adapted to P-limited conditions.Jalo MG-65, Carnaval MG, G 19833, Kaboon, andMéxico 54 belong to the Andean gene pool while theremaining genotypes belong to the Mesoamerican genepool.Seeds were surface-sterilized with 0.5% NaOCl forone min, rinsed thoroughly with distilled water andscarified with a razor blade. They were placed 2 cmfrom the top of a brown germination paper (Zhu et al.,2005), soaked in 0.5 mM CaSO4, and with radiclespointing downwards. The paper was then rolled into amoderately tight cigar roll configuration, and placed in a1 L beaker with 100 mL of 0.5 mM CaSO4 at the bottom.Beakers were wrapped with cellophane plasticpunctured with holes before being placed in a germinationchamber at 28°C. Five days later, shoots of seedlingswere eliminated, and roots of each genotype wereseparated in basal and primary roots. They wereconserved in 25% v/v ethanol immediately after harvest.Root hairs were visually evaluated, after being stainedwith 0.05% trypan blue using a rating scale of 1–9 torank the density and length, as follows: 1, no root hairs;3, between 1 and 5 rating scale; 5, intermediate root hairdensity and length, as recombinant inbred lines (RILs)28 and 32 of DOR 364 x G 19833; 7, between 5 and 9rating scale; 9, abundant root hairs, such as RILs 13and 53 of DOR 364 x G 19833. These RILs wereselected from the research of Miguel (2004). Data wereanalyzed as a completely randomized design (CRD), witheach treatment replicated four times. Pearson’scorrelation was calculated between the rating scales ofroot hairs, in basal roots and those in primary roots.A preliminary test was performed comparing root hairson 5-day old plants (new method) with those on olderplants used by Miguel (2004). Six contrasting RILs (13,28, 32, 53, 80, and 84) of DOR 364 x G 19833 for roothairs were used. Results obtained with the new methodmatched those achieved by Miguel (2004).The method worked well with relatively low standarderror of the mean (SE) for each genotype (Figures 1and 2). In general, SE was lower for basal roots than forprimary roots. The correlation between root hairs in basalroot and root hairs in primary root was significant (r = 0.54,p = 0.0057). One important advantage of this simplemethod, compared to the laborious one used by Miguel(2004) is the visualization of the entire root system. Thefact that density and length were considered together, inour evaluation, is pertinent, since there is a positivecorrelation between root hair length and density, whenplants grow under low phosphorus availability (Miguel,2004). Evaluations were made without phosphorusaddition, in view of the results of Miguel (2004) that roothair length increased from 0.35 mm, in high phosphorustreatments, to 0.85 mm in low phosphorus at 35 DAP(days after planting), and that genotypes under highphosphorus availability did not differ in root hair length.Pesq. agropec. bras., Brasília, v.42, n.9, p.1365-1368, set. 2007Method for evaluation of root hairs of common bean genotypes 1367Carnaval MG, and Carioca had strong root hairs in basalroot (Figure 1), but weak in primary root (Figure 2). Thegenotypes Jalo MG-65 and Talismã had weak root hairsin basal root, but strong in primary root. The genotypesDiamante Negro, DOR 364, Kaboon, México 309, OuroNegro, PI  207262, TO, TU, Valente, Vi 10-2-1, andVi 4899 had weak root hairs in both classes. Accordingto Miguel (2004), the greater root surface area the morecontact with the soil, and long root hairs make thiscontact to take place beyond the depletion zone, whichotherwise is out of reach by roots alone. Moreover, roothairs are responsible for secretion of a variety ofcompounds (exudates) into rhizosphere. The greater roothair density and length of G 19833 (P efficient) comparedto DOR 364 (P inefficient) was also found by Yan et al.(2004).AcknowledgementsTo Conselho Nacional de Desenvolvimento Científicoe Tecnológico (CNPq), for support.ReferencesBATES, T.R.; LYNCH, J.P. Root hairs confer a competitive advan-tage under low phosphorus availability. Plant and Soil, v.236, p.243-250, 2001.BATES, T.R.; LYNCH, J.P. Stimulation of root hair elongation inArabidopsis thaliana by low phosphorus availability. Plant, Celland Environment, v.19, p.529-538, 1996.JUNGK, A. Root hairs and the acquisition of plant nutrients fromsoil. Journal of Plant Nutrition and Soil Science, v.164, p.121-129, 2001.MA, Z.; BIELENBERG, D.G.; BROWN, K.M.; LYNCH, J.P. Regu-lation of root hair density by phosphorus availability in Arabidopsisthaliana. Plant, Cell and Environment, v.24, p.459-467, 2001.MARSCHNER, H.; ROMHELD, V.; CAKMAK, I. Nutrient avail-ability in the rhizosphere. Journal of Plant Nutrition, v.10, p.1174-1184, 1987.MIGUEL, M.A. Genotypic variation in root hairs and phospho-rus efficiency in common bean (Phaseolus vulgaris L.). 2004.106p. Thesis (M.Sc.) - Penn State University, University Park.NIELSEN, K.L.; ESHEL, A.; LYNCH, J.P. The effect of phosphorusavailability on the carbon economy of contrasting common bean(Phaseolus vulgaris L.) genotypes. Journal for ExperimentalBiology, v.52, p.329-339, 2001.Figure 1. Root hairs in basal roots, 5 days after germinationof 21 genotypes of common bean. Data are means±SE (n = 4).Columns with equal letters belong to homogenous groups byScott-Knott test, at 5% of probability. Scale: 1, no root hairs;3, between 1 and 5 rating scale; 5, intermediate root hair densityand length, as RILs 28 and 32 of DOR 364 x G 19833; 7, between5 and 9 rating scale; 9, abundant root hairs, such as RILs 13and 53 of DOR 364 x G 19833.Figure 2. Root hairs in primary roots, 5 days after germinationof 21 genotypes of common bean. Data are means±SE (n = 4).Columns with equal letters belong to homogenous groups byScott-Knott test, at 5% of probability. Scale: 1, no root hairs;3, between 1 and 5 rating scale; 5, intermediate root hair densityand length, as RILs 28 and 32 of DOR 364 x G 19833; 7, between5 and 9 rating scale; 9, abundant root hairs, such as RILs 13and 53 of DOR 364 x G 19833.The genotypes G19833, México 54, VC-4, andespecially G 2333, had strong root hairs in both classesof roots (Figures 1 and 2). The genotypes AB 136,Pesq. agropec. bras., Brasília, v.42, n.9, p.1365-1368, set. 2007R.F. Vieira et al.1368YAN, X.; LIAO, H.; BEEBE, S.E.; BLAIR, M.W.; LYNCH, J.P.QTL mapping of root hair and acid exudation traits and theirrelationship to phosphorus uptake in common bean. Plant andSoil, v.265, p.17-29, 2004.YAN, X.; LYNCH, J.P. Genetic variation for root hair density andlength in the common bean in response to low phosphorus availability.In: LYNCH, J.P.; DEIKMAN, J. (Ed.). Phosphorus in plantbiology: regulatory roles in ecosystem, organismic, cellular, andmolecular processes. Rockville: American Society of PlantPhysiologists, 1998. p.332-334.ZHU, J.; KAEPPLER, S.M.; LYNCH, J.P. Mapping of QTLcontrolling root hair length in maize (Zea mays L.) under phosphorusdeficiency. Plant and Soil, v.270, p.299-310, 2005.Received on June 11, 2007 and accepted on August 15, 2007

Method for evaluation of root hairs of common bean genotypes

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Method for evaluation of root hairs of common bean genotypes

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