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J. Agronomy & Crop Science 167, 96—102 (1991) © 1991 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-2250 Department of Plant and Soil Science, Alabama A&M University, Normal, U.S.A. Screening Soybean Genotypes for Drought and Heat Tolerance VAL T. SAPRA and ANTHONY O. ANAELE Authors' address: Dr. VAL T. SAPRA and Dr. ANTHONY O. ANAELE, Department of Plant and Soil Science, Alabama A&M University, Normal, AL 35762, U.S.A. With one figure and 3 tables Received August 21, 1990; accepted October 11, 1990 Abstract Several soybean [Glycine max (L.) Merr.] germplasms from maturity groups III through VII were evaluated at three osmotic potential levels (-0.017, 0.3 and -0.5 MPa) using polyethylene glycol M.W. 8000 and four temperature regimes (10, 25, 50 and 11O°C) for drought and heat tolerance, respectively. Heat injury varied significantly among growth stages with the highest heat injury occurring at the flowering stage. Susceptibility to heat injury decreased toward maturity with the exception of a few genotypes. Genotypic variability was found among the lines in both drought and heat tolerance tests. This study identified the lines P! 408.155, PI 423.827B, PI 423.759 and Pershing as both drought and heat tolerant. In general, the larger-seeded lines failed to germinate at -0.50 MPa. A positive and significant relationship between promptness index and germina- tion stress index was found. However, no significant correlation was observed between germination stress index and the heat tolerance test. Based on the results from the germination test, lines PI 393.550, PI393.547, PI 86.103, PI 171.437, PI 86.490 and PI 423.852 had high germination stress indices and need further testing for heat stress tolerance. Key words: Glycine max L., osmotic potential, polyethylene glycol, drought, heat tolerance. Introduction Water deficits and high temperatures are among the most important environmental fac- tors that limit crop productivity in many areas of the world. The lack of stability in soybean production because of variable climatic condi- tions has indicated a need to develop methods to improve this crop genetically by breeding cultivars capable of withstanding environ- mental stress. Breeding for drought resistance has been accomplished by selecting for seed yield under field conditions (BOUSLAMA and SCHAPAUGH 1984, BROWN et al. 1985, SPECHT et al. 1986), but since such procedures require full season field data, this is not always an efficient ap- proach, especially in mesic locations (SAMMONS et al. 1978). Another alternative has been to screen material under laboratory or green- house conditions using seeds or seedlings as test material (SAMMONS et al. 1978). Several physiological characteristics in crops have been reported to be reliable indicators for the selec- tion of plant germplasm possessing drought and heat tolerance. These characteristics in- clude seed germination or seedling growth in hydroponic solutions of low osmotic potential (SULLIVAN 1972, SAMMONS et al. 1979, STOUT et al. 1980) and the measurement of heat toler- ance using the degree of electrolyte leakage from heat damaged leaf cells after exposure to evaluated temperatures (MARTINEAU et al. 1979, SULLIVAN and Ross 1979, BLUM and EBERCON 1981). The objectives of this study were to; U,S. Copyright Clearance Center Code St.itcment: 0931-2250/91/6702-0096$02.50/0

Screening Soybean Genotypes for Drought and Heat Tolerance

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J. Agronomy & Crop Science 167, 96—102 (1991)© 1991 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-2250

Department of Plant and Soil Science, Alabama A&M University, Normal, U.S.A.

Screening Soybean Genotypes for Drought and Heat Tolerance

VAL T . SAPRA and ANTHONY O . ANAELE

Authors' address: Dr. VAL T. SAPRA and Dr. ANTHONY O. ANAELE, Department of Plant and Soil Science,Alabama A&M University, Normal, AL 35762, U.S.A.

With one figure and 3 tables

Received August 21, 1990; accepted October 11, 1990

Abstract

Several soybean [Glycine max (L.) Merr.] germplasms from maturity groups III through VII were evaluatedat three osmotic potential levels (-0.017, 0.3 and -0.5 MPa) using polyethylene glycol M.W. 8000 and fourtemperature regimes (10, 25, 50 and 11O°C) for drought and heat tolerance, respectively. Heat injury variedsignificantly among growth stages with the highest heat injury occurring at the flowering stage. Susceptibilityto heat injury decreased toward maturity with the exception of a few genotypes. Genotypic variability wasfound among the lines in both drought and heat tolerance tests. This study identified the lines P! 408.155, PI423.827B, PI 423.759 and Pershing as both drought and heat tolerant. In general, the larger-seeded lines failedto germinate at -0.50 MPa. A positive and significant relationship between promptness index and germina-tion stress index was found. However, no significant correlation was observed between germination stressindex and the heat tolerance test. Based on the results from the germination test, lines PI 393.550, PI393.547,PI 86.103, PI 171.437, PI 86.490 and PI 423.852 had high germination stress indices and need further testingfor heat stress tolerance.

Key words: Glycine max L., osmotic potential, polyethylene glycol, drought, heat tolerance.

Introduction

Water deficits and high temperatures areamong the most important environmental fac-tors that limit crop productivity in many areasof the world. The lack of stability in soybeanproduction because of variable climatic condi-tions has indicated a need to develop methodsto improve this crop genetically by breedingcultivars capable of withstanding environ-mental stress.

Breeding for drought resistance has beenaccomplished by selecting for seed yield underfield conditions (BOUSLAMA and SCHAPAUGH

1984, BROWN et al. 1985, SPECHT et al. 1986),but since such procedures require full seasonfield data, this is not always an efficient ap-proach, especially in mesic locations (SAMMONS

et al. 1978). Another alternative has been toscreen material under laboratory or green-house conditions using seeds or seedlings astest material (SAMMONS et al. 1978). Severalphysiological characteristics in crops have beenreported to be reliable indicators for the selec-tion of plant germplasm possessing droughtand heat tolerance. These characteristics in-clude seed germination or seedling growth inhydroponic solutions of low osmotic potential(SULLIVAN 1972, SAMMONS et al. 1979, STOUT et

al. 1980) and the measurement of heat toler-ance using the degree of electrolyte leakagefrom heat damaged leaf cells after exposure toevaluated temperatures (MARTINEAU et al. 1979,SULLIVAN and Ross 1979, BLUM and EBERCON

1981). The objectives of this study were to;

U,S. Copyright Clearance Center Code St.itcment: 0931-2250/91/6702-0096$02.50/0

Screening Soybean Genotypes for Drought and Heat Tolerance 97

1) evaluate the genetic variability among sever-al soybean genotypes subjected to drought andheat stress and 2) determine whether theseparameters and other agronomic traits are ef-fective criteria to select for drought and heattolerance.

Materials and Methods

Several soybean genotypes, differing in growthhabits and from maturity groups III through VIIwere screened for drought and heat stress duringgermination and growth stages, respectively.

Drought Tolerance Test

Twenty seeds from each soybean genotype weregerminated in polyethylene glycol (PEG) 8000 at-0.017 and -0.5 MPa of osmotic potential (OP). Toobtain these levels, 0 and 198 g of PEG 8000 wereadded to one liter of deionized distilled water(MICHEL 1983). Preliminary experimentation indi-cated that this level (—0.5 MPa) of OP resulted inmaximum selection differential among the Glycineaccessions tested. Polyethylene glycol and D-man-nitol have been used successfully to induce osmoticstress and mimic drought (GLARK.E and TowNLEY-SMITH 1984).

The seeds were surface sterilized in 5 % (v/v)sodium hypochlorite solution for 10 min, rinsed 3times and then placed on two layers of Whatman #3fiker paper in 15 X 100 mm petri dishes containing15 ml of the appropriate osmotic solution and incu-bated in a Low Temperature incubator at 25 °C.Germination was recorded when the radicle hadreached approximately 4 mm in length. The slowgermination of seeds of some lines was documentedusing the promptness index (PI) as modified forsoybeans (BouSLAMA and SCHAPAUGH 1984) as fol-lows:

Promptness index (PI) = nd2 (LOO) + nd4 (0.75) +nd6 (0.50) + nd8 (0.25)

where: ndx = number of seeds observed to germi-nate on the xth day of observation.

A germination stress index (GSI) was expressed inpercent as follows:Promptness index of stressed seeds (PIS)

Promptness index of control seeds (PIC)= GSI %

X 100

The experimental design was randomized completeblock and each treatment was replicated four times.

Heat Tolerance Test

The technique used for measuring heat tolerance ofleaf tissue was as described for sorghum [Sorghumhicolor (L.) Moench] (SULLIVAN 1972) with minor

modifications for soybean (MARTINEAU et al. 1979).Twenty genetically diverse soybean genotypes wereplanted in a single-row plot, 6 m long and 90 cmwide at Alabama A&M University Research Stationduring the 1988 growing season. The soil type wasDecatur silty clay loam (Rhodic Paleudult). Thesame genotypes were also planted in the greenhousein the spring of 1988 and replicated 3 times. Addi-tionally, 40 genotypes from maturity groups IIIthrough VIII were planted in the field during the1989 growing season. Leaf samples were taken attwo-week inter\'als from the vegetative growth stageV5 to reproductive growth stage R4. Twelve leafletswere obtained by removing terminal leaflets fromfully expanded trifoliolates at an uppermost node of12 plants randomly selected in field plots and thegreenhouse. The leaflets were collected at 6:00 a.m.and put in 26.67 cm x 27.94 cm x 1.75 ml zip-lockbags and immediately transported to the laboratory.In each genotype, 12 leaflet samples were randomlydivided into 3 groups each containing 4 leaflets.From each 4-leaflet group, 14 leaf discs were cutwith a 2-cm diameter cork borer and placed into adigestion tube. Two of the test tubes or samples wereused for duplicate temperature treatments and thethird one served as the control.

Prior to assay, leaf discs were washed several timeswith deionized distilled water. This procedure wasnecessary to remove exogenous electrolytes adheringto tissue surfaces and to remove endogenous electro-lytes released from cut cells at the periphery of thediscs. After the final wash, the tubes were drained ofexcess water and then covered with parafilm "M".The treatment tubes were placed in a temperatureregulated water bath at 50 ± 1 °C for 15 min whilethe control was maintained at 25 *C. Tubes werethen removed from the water bath and 30 ml ofdeionized distilled water was added to each tube andincubated for 18 h at 10 °C to allow the diffusion ofelectrolytes from the discs. Then, initial conductancereadings were made at 25 °C with a Yellow SpringsInstrument (YSI) Model 35 Conductance meter andModel 3403 conductivity cell. Upon the completionof this measurement, the control and treatment tubeswere covered with cotton wool and autoclaved at110 °C, with 1.4 kg pressure for 10 minutes to killthe leaf tissue. Final conductance measurementswere taken after all tubes were cooled to 25 '̂C.

Determination

The degree of in)ury induced as a result of thetemperature treatment was calculated as follows:

% Injury = 1 -1 - (T,/T.)

1 - fC./OX 100;

where T and C refer to treatment and control,respectively, and the subscripts 1 and 2 of T and Crefer to initial and final conductance, respectively.

98 SAPRA and ANAELE

The ratio of initial to final conductance (i.e. T1/T2) isa relative measure of the amount of electrolyte leak-age induced by the elevated temperature and is as-sumed to be proportional to the amount of "injury"induced in cellular membranes. The inclusion ofcontrols in this assay provides a measure of spuriouselectrolyte leakage due solely to the cutting andsubsequent handling of the leef discs and the 18-hourstorage at low temperature. Consequently, the cal-culated injury values would reflect only that temper-ature induced by the elevated temperature treatment.

All data were analyzed by standard analysis ofvariance techniques. Where appropriate, Duncan'smultiple range test at the 0.05 probabihty level wasused to separate treatment means. Simple correlationwas used to determine the relationship among theparameters tested.

Results and Discussion

Genetic variability was found among the linesfor both drought and heat tolerance (Table 1).The germination stress index (GSI) rangedfrom a high of 89.34 % for PI 393.550 to a low

of 5.82 for PI 417.288 while zero percent wasrecorded for those genotypes that failed togerminate. In general, the larger-seeded lineshad a greater tendency to fail to germinate at0.50 MPa. Among the 20 genotypes tested in1988, heat injury ranged from a low of30.65 % in Williams 79 to a high of 65.65 % inEmerald; while in 1989, for the additional 40genotypes evaluated, it varied from 62.10 % to84.87 % for PI 408.155 and PI 196,177, re-spectively.

A positive and highly significant relationship(r = 0.98) between promptness index and GSIwas found. However, negative correlation(r = —0.40) was observed between GSI andheat injury (Table 2). Similar results have beenobserved in sorghum (SULLIVAN 1972) and insoybeans (BOUSLAMA and SCHAPAUGH 1984). Ahighly significant and negative correlation(r = —0.74) between growth stages and heatinjury was observed, indicating that heat in-jury decreased as growth stages advanced.

Table 1. Mean heat injury and germination stress index of 60 and 56 soybean genotypes, respectively

1988

Maturitygroup

III

IV

Genotype

FugiGuelphJogumWillomiKimKanrichColumbiaWolverineOaklandPellaKuraWilliams 79

EmeraldJeffersonEmperorImperialWareSangoFunk DeliciousSatoLSD (0.05)

100 seedweight

g13.2512.0930.9020.8524.8124.0413.5217.5812.8115.4630.6014.04

24.4026.6822.0628.5521.5023.8023.3420.71

Heat injury

60.9860.0052.7552.1551.7349.3747.1045.2044.5036.6336.4530.65

65.6563.8062.8358.6755.5551.4240.6537.254.54

Germination stressindex

0/rO

22.5516.330.00'0.000.000.00

33.850.000.000.000.00

20,67

0.000.000.000.000.000.000.000.00

Screening Soybean Genotypes for Drought and Heat Tolerance 99

Table 1 (continued)

Maturitygroup

IV

V

V

VI

Genotype

AodoRA401KailuaGreen & BlackPI 82.264SootyKingstonShiroNerredoStevensPekingFranklinMid SummerPI 84.751Wilson 5Peking (Veg.)PI 86.103

PI 196.177PI 417.052'EssexPI 417.359Bay

BedfordPI 423.758PI 416.467PI 416.771PI 423.759PershingPI 417.322

PI 423.827BPI 417.159

PI 417.288PI 408.155

PI 494.181DavisPI 398.479LaredoLancerNathan

D 71-V86PI 423.852

PI 171.437PI 86.490

100 seedweight

g1989

29.28

14.0217.9521.20

7.109.708.00

23.408.30

13.468.09

13.2515.859.909.269.008.03

5.528.27

12.6832.4017.35

15.5822.4526.70

7.818.00

10.4114.966.65

35.51

23.608.20

7.4517.8612.42

7.1916.1915.5228.42

9.646.435.43

Heat injury

80.1276.7976.4076.3475.9975.6375.6074.7974.6274.54

72.0670.9769.2167.10

63.7863.01NR

84.8778.8477.9477.8476.34

76.2575.4475.0074.6469.5769.5969.3268.9664.1663.5662.10

80.4879.8379.1777.2674.35

74.0472.67NRNRNR

Germination stressmdex

%

0.0034.426.86

38.1664.8538.7531.82

0.0054.6634.3834.84

8.22

11.0745.5415.3931.37

77.03

0.0075.9569.30

0.0017.81

46.560.000.00

51.7680.7068.75

0.0074.060.005.82

81.01

69.3131.2547.5076.8425.31

45.250.00

82.91

77.1185.94

100 SAPRA and ANAELE

Table 1 (continued)

Maturity-group Genotype

100 seedweigbt Heat injury

Germination stressindex

VII PI 393.550PI 393.547LSD (0.05)

g6.577.561.67

82.54NR4.63

89.3485.763.53

' Zero percent was recorded for those genotypes that failed to germinate at —0.50 MPa.' Final Sampling for heat injury test was at R2 growth stage.NR — Not recorded

Table 2. Correlations among stress indices and other parameters in soybeans (1988 and 1989)

Promptnessindex

Plantheight

Germinationstress index

Heatinjury

Seed weightPromptness indexGermination stress indexGrowth stagesPlant height

-0.54^ -0.84^0.98=

-0.44

-0.40

-0.74=

0.41

'•'•••' Significant at the 0.01 level of probability.

Ten genotypes which were common in ourtwo-year study showed a very significant yeareffect and year X genotype mteraction(Table 3). Among some of these genotypes, agreater increase (e.g. Bay and Bedford) or alesser increase {Nathan, Davis, and Peking) inmjury was seen (Fig. 1). Thus, while percentinjury increased from 1988 to 1989, this in-crease was large or small, depending on thegenotype. The major difference between the 2years with respect to growing conditions wasthe very favorable water regime prior to andduring the sampling period in 1989.

Conclusion

This study identified the lines PI 408.155, PI423.827B, PI 423.759 and Pershing as bothdrought and hat tolerant. It is possible that theheat tolerance test could be used in soybean toselect for drought tolerance, as was the case forcorn, pasture grasses and sorghum. However,not all genotypes that are heat tolerant ap-

peared to be drought tolerant. Based on theresults from the germmation test, lines PI303.550, PI 393.547, PI 86.490 and PI 423.852had high GSI and need further testing for heatstress. Those genotypes identified as bothdrought and heat tolerant are being furtherstudied in the field and greenhouse in 1990.

Table 3. Analyses of variance for variables measuredin heat tolerance experiment

Sourcesof variation

Degree offreedom Mean squares

ReplicationYears (Y)Error AGenotypes (G)Y X GError BTotal

11199

1839

12.952440.00=-̂ '•

0.39260.56^"'-284.81'-*

0.75—

Significant at the 0.01 level of probability.

Screening Soybean Genotypes for Drought and Heat Tolerance 101

Fig. 1. Percent heat injury of 10soybean genotypes planted in thegreenhouse and field in 1988 and1989, respectively

10Q

40

1988(Greenhouse)

1989(Pieid)

20

Zusammenfassung

Auslese von Sojabohnen-Genotypen hin-sichtlich Trockenheits- und Hitze-Toleranz

Zahlreiche Sojabohnen {Glycine max [L.]Merr.)-Genotypen der Reifegruppe III bisVIII wurden unter den Bedingungen von dreiosmotischen Strefthohen (—0,017, —0,3 und-0,5 MPa) unter Verwendung von Polyathy-lenglycol M. W. 8000 und vier Temperaturbe-reichen (10, 25, 50 und 110 °C) hinsichtlichTrockenheits- und Hitze-Toleranz untersucht.Die Hitzeschaden traten signifikant unter-schiedlich in den Wachstumsstadien auf, wobeidie hochsten Hitzeschaden wahrend der Bliiteeintraten. Die Empfindlichkeit gegeniiber Hit-zeschaden nahm zur Reife hin ab; einige Geno-typen verhielten sich hiervon abweichend. Ge-notypische Variabilitat wurde zwischen denLinien sowohl hinsichtlich der Trockenheits-als auch Hitze-Toleranz gefunden. Aus derUntersuchung gingen die Linien PI 408.155,PI 423.827B, PI 423.759 und Pershtng als dlir-re- und hitzetolerant hervor. Grundsatzlichunterbheb die Keimung der grofisamigen Sor=«ten bei -0,50 MPa. Eine positive signifikanteBeziehung wurde zwischen dem Keimungsbe-reitschaftsmdex und dem Keimungsstrefiindexgefunden. Allerdings war eine signifikanteKorrelation zwischen dem Keimungsstrefi-index und dem Hitzetoleranztest nicht nach-zuweisen. Auf der Grundlage der Ergebnisseder Keimungsuntersuchungen ergab sich fiirPI 393.550, PI 393.547, PI 86.103, PI 171.437,PI 86.490 und PI 423.852 ein hoher Keimungs-strefiindex; diese Linien erfordern weitere Un-tersuchungen hinsichtlich ihrer HitzestreK-toleranz.

Acknowledgement

This research reported here was supported by CSRS/USDA grant no. 2-201-14-3151.

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