Genetic variability in tolerance to cadmium
Euphytica131:25–35,2003.
?2003Kluwer Academic Publishers.Printed in the Netherlands.
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Genetic variability in tolerance to cadmium and accumulation of heavy metals in pea(Pisum sativum L.)
Andrei A.Belimov1,?,Vera I.Safronova1,Viktor E.Tsyganov1,Alexey Y.Borisov1,Andrei P. Kozhemyakov1,Vitaley V.Stepanok2,Anna M.Martenson3,Vivienne Gianinazzi-Pearson4& Igor A.Tikhonovich1
1Laboratory of Ecology of Symbiotic and Associative Bacteria and Laboratory of Genetics of Plant-Microbe Inter-actions,Research Institute for Agricultural Microbiology(ARRIAM),Podbelsky Sh.3,Pushkin8,Saint-Petersburg, 196608,Russian Federation;2Research Institute for Agricultural Use of Ameliorated Lands,P.O.Emaus,Tver, 171330,Russian Federation;3Department of Soil Science,Swedish University of Agricultural Sciences,Box 7014,75007Uppsala,Sweden;4UMR INRA/Universit′e de Bourgogne BBCE-IPM,INRA-CMSE,BP86510,21065 DIJON CEDEX,France;(?author for correspondence;e-mail:belimov@rambler.ru)
Received2November2001;accepted13December2002
Key words:cadmium,genotypes,heavy metals,Pisum sativum L.
Summary
Ninety-nine wild growing and primitive varieties of garden pea(Pisum sativum L.)were screened for tolerance to cadmium(Cd)toxicity in quartz sand culture.Cadmium tolerance was determined by the time to plant death when treated with a lethal Cd concentration(13mg kg?1),and by a tolerance index(TI)calculated as a ratio between biomasses of Cd-treated and untreated plants in the presence of toxic Cd concentrations(7mg kg?1and 5mg kg?1).The Cd-tolerance index varied signi?cantly between pea genotypes from35%to90%and from54% to100%in the presence of7and5mg Cd kg?1,respectively.Shoot Cd concentration of tolerant and sensitive genotypes grown in the presence of5mg Cd kg?1varied between35mg Cd kg?1and135mg Cd kg?1(dry weight)and was negatively correlated with TI.Certain tolerant pea genotypes were characterised by a high Cd concentration in shoots.All varieties were also screened for their ability to take up heavy metals(HMs)from a slightly contaminated soil.The concentration of Cd,chromium,copper,nickel,lead,strontium and zinc in plant shoots varied between pea genotypes by a factor of2.8,4.9,2.7,3.5,9.7,3.9and4.0,respectively.The coef?cients of variation between pea genotypes for HM concentration were high,varying from23%to39%depending on the metal.The distribution patterns for varieties based on Cd tolerance(sand culture)and HM concentrations(soil culture)were characterised by positive skewness coef?cients,suggesting that the majority of pea genotypes was relatively sensitive to Cd toxicity and tended to avoid excessive accumulation of HMs in shoots.These results show that a high genetic variability exists in pea with regards to Cd tolerance and HM accumulation.Concentrations of different HMs in plants grown in soil correlated positively with each other,with the exception of chromium. There was no correlation between Cd tolerance of the varieties in sand culture,shoot concentration of HMs in soil culture,biomass production,subspecies and geographical origin of the varieties.The genetic systems controlling Cd tolerance,HM accumulation and morphological traits are therefore independent to some extent,suggesting a possibility for breeding pea cultivars characterised by high tolerance to and low concentration of HMs in shoots.
Introduction
Heavy metals(HMs)such as cadmium(Cd),copper (Cu),nickel(Ni),lead(Pb)and zinc(Zn)are among the most widespread pollutants in the surface soil layer.They come from polluting industries,intens-ive agriculture and sludge dumping.HMs can easily be accumulated by agricultural crops,inhibiting plant
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growth and mineral nutrition,and can cause diseases in animals and human beings.Cd is one of the most toxic metal pollutants of soil and is studied intensively (Sanita di Toppi&Gabrielli,1999).
Agricultural crops differ widely in their tolerance to toxic metals.Crop tolerance to aluminium and manganese has been extensively investigated and con-siderable variability among species and cultivars has been found(Aniol&Gustafson,1990).There is evid-ence that intraspeci?c genetic variation exists in the tolerance of legumes to HMs.For example,cultivars of navy beans differ in Zn and Cu tolerance(Polson& Adams,1970),cultivars of soybean show difference in tolerance to Mn(White et al.,1979),and cultivars of cowpea differed in Mn tolerance(Horst,1983).
It has been shown that legume crops,including pea (Pisum sativum L.),are less tolerant to Cd toxicity as compared to cereals and grasses(Inouhe et al.,1994; Mazen,1995).Cd toxicity has been reported to inhibit biomass production(Leita et al.,1993),photosynthetic activity(Kumar&Kumar,1999),nitrogen?xation and nodulation(Chugh et al.,1992;Hernandez et al., 1995),and reduce uptake of nutrient elements(Obata &Umebayashi,1997;Hernandez et al.,1996)in pea. At the same time,genetic polymorphism of pea,as well as other legume species,in tolerance to Cd is not understood.
A high variation in the accumulation of HMs by various agricultural crops,depending on the plant spe-cies has been reported(Bell et al.,1997;Jarvis et al.,1976;Kuboi et al.,1986;Kumar et al.,1995; Pettersson,1977;Zwarich&Mills,1982).Cultivars of cowpea(Horst,1983)and soybean(White et al., 1979)vary in their capacity to take up Mn and Zn, respectively.Signi?cant differences in biomass con-centrations of Cd in plant biomass have been found among cultivars of peanut and navy bean(Bell et al., 1997),soybean(Bell et al.,1997;Keck&Redlich, 1975),maize(Hinesly et al.,1982),wheat and barley (Pettersson,1977).In contrast,little is known about cultivar-dependent variability in the uptake of Cd and other HMs by pea.
The aims of the present study were(i)to evaluate genetic variability of pea in tolerance to Cd and con-centration of HMs in shoots,(ii)to analyse relation-ships between metal tolerance and concentration in pea plants,and(iii)to identify pea genotypes differing in Cd-tolerance and HM accumulation.Knowledge about the genetic variability in pea tolerance to and accumulation of HMs is important not only for study-ing of mechanisms in plant-metal interactions,but also in providing starter material for breeding programmes aimed at improving tolerance to and(or)decreasing uptake of HMs in these agricultural crops. Materials and methods
Plant material
Ninety-nine wild-growing or primitive varieties of pea (Pisum sativum L.)were obtained from the Pea World Collection of the Vavilov’s Institute for Plant Industry (VIPI),St.-Petersburg.Pea varieties were selected on the basis of their geographical origin in such a way as to cover most growing areas around the world. Their catalogue collection numbers are given in the text,tables and?gures throughout this paper.Eighty-?ve of the varieties belong to subspecies sativum, eight varieties(188,958,1022,1250,1251,1866, 2182and3266)to subspecies asiaticum,three variet-ies(1937,2008and2514)to subspecies abyssinicum, and three varieties(1985,2174and3980)to sub-species transcaucasicum.Details of the geographical origin,subspecies and?ower colour have been de-scribed previously for these varieties(Jacobi et al., 2000).Additional information can be found in the VIPI Catalogue(http://www.dainet.de/genres/vir). Tolerance to Cd in sand culture
Three pot experiments were carried out in a green-house during summer under natural illumination (June-July,Saint Petersburg).The plants were grown in pots containing3kg quartz sand fertilised with 300mL of nutrient solution(μM):KH2PO4,4000; MgSO4,2000;CaCl2,2000;NH4NO3,750; NaFeEDTA,25;H3BO3,5;KBr,2.5;KI,2.0;CuSO4, 1.2;NaCl,3.5;Al2(SO4)3,0.5;ZnSO4,0.6;CoCl2, 0.8;MnSO4,0.7;NiSO4,0.7;Li2SO4,1.0;pH=5.5. Cadmium was added as CdCl2solution on the18th day after planting in the following concentrations(mg Cd per kg of quartz sand):13(lethal toxicity concen-tration;experiment1),7(highly toxic concentration; experiment2)and5(moderately toxic concentration; experiment3).Preliminary pot experiments were car-ried out to determine the toxicity concentrations of Cd in quartz sand.The plants without Cd treatment were used as a control for each variety.Three pots with5plants per pot were prepared for each treat-ment.Seedlings were inoculated with the symbiotic bacterium Rhizobium leguminosarum bv.viciae strain CIAM1026(the ARRIAM collection,St.-Petersburg)
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by adding200ml per pot of a water diluted bacterial suspension(107cells ml?1)on the5th day after plant-ing.Pots were watered daily to60%water holding capacity(WHC)of the quartz sand.In experiment 1,tolerance of99pea varieties was evaluated visu-ally as the time at which Cd-treated plants died as a consequence of Cd toxicity.The plants with termin-ated shoot growth and exhibiting toxicity symptoms such as wilting,severe chlorosis,necrosis or dryness of leaves were considered as dead plants.In experi-ment2(with99varieties)and experiment3(with25 varieties),plants were grown for30days,harvested, dried and the biomass of roots and shoots was determ-ined.Symptoms of Cd toxicity and development of symbiotic nodules on roots were estimated visually at harvesting.Cd-tolerance of the varieties was charac-terised by a tolerance index(TI),which was calculated as a percentage of shoot and root dry biomass(g per pot)of Cd-treated over untreated plants.
Shoot concentration of HMs in soil culture
A pot experiment was carried out in a greenhouse dur-ing summer under natural illumination(June-August, Saint Petersburg)to study the variability of pea vari-eties in shoot HM concentrations.The plants were grown in pots(3pots per variety,5plants per pot) containing4.5kg of a nonsterile sod-podzolic sandy soil having the following characteristics(mg kg?1): ammonium N,15;nitrate N,20;available P,145; available K,82;humus,1.1%;pH KCl4.7.The total content of HMs in the soil was(mg kg?1):Cd,0.20; Zn,20;Cu,4;Cr,35;Ni,8;Pb,48;Sr,97.The soil was additionally supplemented with HMs at approx-imately threshold toxicity concentrations permitted in the Russian Federation(Chernykh&Ladonin1995). Heavy metals were added as CdCl2,ZnSO4,CuSO4, K2CrO4,NiSO4,Pb-acetate and SrCl2at the levels of (mg kg?1):Cd,5;Zn,30;Cu,50;Cr,20;Ni,20;Pb, 100;Sr,50.The soil was fertilised with(mg kg?1): NH4NO350,KCl60,MgCl220.The seeds were in-oculated with a water diluted bacterial suspension of R.leguminosarum bv.viciae CIAM1026(108cells per seed).Pots were moistened daily to60%WHC of the soil.Plants were grown up to maturity,harves-ted,dried and the shoots and seed biomass of each plant was determined.The variety1982was used as a reference variety,in that it was included twice in the experiment and was analysed twice for HM con-centrations to control the reproducibility of the data obtained.Determination of HM concentrations
In the experiment on soil culture,pea varieties were roughly screened for their shoot concentrations of HMs.For this purpose the middle part of each plant shoot was snipped off,and shoot parts per pea variety were mixed together(15plants per variety)to pre-pare a single averaged sample(A-sample)for each. The A-samples were ground and digested in a heated mixture of concentrated HNO3and HClO4(1:1).Con-centrations of Cd,Zn,Cu,Cr,Ni,Pb and Sr in the plant digests were determined using a plasma atomic absorption spectrophotometer SATURN-3PT(Minsk, Byelorussia).Concentrations of HMs in shoots of15 individual plants of a number of varieties were also measured separately using the same procedure.The Cd concentration in shoots of plants grown in quartz sand in the presence of5mg Cd kg?1was also determined as described above.
Statistical analysis
The data were processed by variance,correlation and cluster analysis using the software STATISTICA V-5 (StatSoft,Inc.,NY,USA).For cluster analysis, data were standardised and processed by the weighted pair-group average linkage method using squared Eu-clidean distances.LSD(Fisher’s least signi?cant dif-ference),SD(standard deviation),SE(standard er-ror),V(coef?cient of variation),P(probability of null hypothesis),As(coef?cient of skewness)and Ex (coef?cient of kurtosis)were calculated.
Results
Variation in tolerance to Cd in sand culture
In experiment1,the genotypes8572,188,8638,1537, 6139,2174,8093,4788,3273,3855,8274,4170, 8585,8456and1982were sensitive to the lethal Cd concentration(13mg Cd kg?1)and died within10 days after treatment with Cd(Figure1A).The ma-jority of the genotypes died between10and15days after Cd-treatment,while the most tolerant genotypes (1658,1027,4650,6875,3063,8413,3445,3140, 2593,8543)died after25–30days.The distribution of varieties in relation to the time of mortality was characterised by a high coef?cient of variation(V= 36±3%),and signi?cantly differed from an expected normal distribution by skewness(As=+0.66;p< 0.01)and kurtosis(Ex=–1.18;p
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