J. Agronomy & Crop Science 164, 153—159 (1990)© 1990 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-2250

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

Screening for Drought Tolerance:Soybean Germination and its Relationship to Seedling Responses

B. K. KPOGHOMOU, V. T. SAPRA and C. A. BEYL

Authors' address: B. K. KPOGHOMOU, V. T. SAPRA and C. A. BEYL, Department of Plant and Soil Science,Alabama A&M University, Normal, AL 35762

With 4 figures and one table

Received April U, 1989; accepted June 29, 1989

Abstract

Seventeen determinate soybean {Glycine max L.) cultivars from maturity groups V through VIII werescreened for drought tolerance during germination and seedling stages. Germinating seeds and hydroponi-cally-grown seedlings were subjected to osmotic stresses of -0.3 and -0.5 MPa using polyethylene glycolM.W. 8000. Genotypic variability was found among the cultivars for all the parameters analyzed in bothgermination and seedling tests. Germination stress index was lower for seeds exposed to -0.5 MPa than for-0.3 MPa osmotic stress. Lower osmotic potential in the treatment medium was also correlated with lowerleaf water potential in seedlings subjected to it. A significant relationship was found between fresh weight andheight stress indices. The cultivars that grew taller under drought stress conditions had greater dr)^ matteraccumulation and higher germination stress indices indicating the reliability of height to predict cultivarperformance under such conditions. The cultivars Lee~74 and Wright had higher dry matter accumulation,greater height, and better germination under stress conditions than the other cultivars tested. Conversely, thecultivars Ra 401 and Bay performed poorly in the drought tests at both levels. In the seedling tests, there wasno strong relationship between the leaf water potential and the overall performance of the plant suggesting noclear osmoregulatory mechanism. Based on results from germination and seedling tests, the cultivars Lee-74,Wright, and Ra 401 were selected for further studies in greenhouse and field trials.

Key words: Glycine max — soybean — drought tolerance — germination — seedling growth — screeningtests

Introduction

Low precipitation associated with high tem-perature is one of the most important environ-mental limitations to crop performance andfood production. Drought occurring during asensitive stage in plant development can se-verely limit not only vegetative growth butpod development and filling (KORTE et al.1983). Soybean has a peak daily water use perplant of 8.5 ml during the growing season(KANEMASU 1979). With this relatively highwater requirement, it is very sensitive to waterstress. Germination and reproductive stages

are very sensitive to water stress induced byeither shortage of rainfall or solute concentra-tion in the soil (SPECHT and NICKEL 1979,KORTE et al. 1983, BROWN et al. 1985). BOUSLA-

MA and ScHAPAUGH (19S4) have reportedgenotypic variability among soybean cultivarsfor both drought and heat tolerance in bothgermination and hydroponic-seedling tests.Any integrated breeding program for droughttolerance therefore should include screening awide range of genotypes at more than onegrowth stage. This study sought to determinegenotypic differences and relationships be-

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154 KPOGHOMOU, SAPRA and BEYL

tween germination and seedling growth underwater stress conditions for a range of determi-nate soybean cultivars from maturity groupsIV to VIII. Ultimately, this information maybe used to predict the field performance ofselected varieties under actual drought condi-tions.

Material and Methods

1. Ger7nination Test

Seventeen soybean cultivars (Table 1) were screenedfor germination under -0.017, -0 .3 , and -0.5 MPaof osmotic stress. To obtain these levels, 0, 149, and198 g of PEG 8000 were added to one liter ofdeionized water (MICHEL 1983). Polyethylene glycol(PEG) and D-mannitol have been used successfullyto induce osmotic stress and mimic drought(CLARKE and TO^^NLEY-SMITH 1984). Initially, awide range of osmotic concentrations from 0.0 to— 1.0 MPa were evaluated using six cultivars to de-termine the concentrations at which the genetic var-iability in germination response would be most ex-pressed. From this, the two concentrations -0.3 and— 0.5 MPa were selected to further test germination,promptness index, and subsequent seedling growthunder osmotic stress.

Twenty seeds were surface sterilized in 5 % (v/v)sodium hypochlorite solution for 10 min, placed ontwo layers of Whatman #3 filter paper in13 X 19 mm plastic petri dishes containing 15 ml ofthe appropriate osmotic solution and incubated in aHot Pack incubator at 25 °C. Germination was re-corded when the radicle had emerged approximately4 mm. The promptness index as modified for soy-bean (BOUSLAMA and SCHAPAUGH 1984) and germi-nation, height, dry and fresh weight stress indiceswere then calculated as indicated below:

Promptness index (PI)= nd. (1.00) + ndj (0.75) -H nd, (0.50) + ndg (0.25)where: nd^ = number of seeds germinated by thexthday of obsen^ation.

Stress index of the parameterParameter value in the treatment

= — i :—i T- X 100Parameter value in the control

A randomized complete block design with 51 treat-ments and four repHcations was used. After analysisof variance, means were separated using Duncan'sMultiple Range Test (SOKAL and ROHLF 1981).

2. Seedling Test

Seedlings of the seventeen cultivars were grown hy-droponically in full-strength Hoagland's solutioncontaining PEG 8000. The quantity of PEG requiredto obtain the three stress treatment levels of —0.017,

Table 1. Germination stress indices (%) of 17 soybean cultivars exposed to two levels of osmotic stress

Maturitygroup

IV

V

VI

VJI

vin

Cultivar

Ra401Stevens

BedfordEorrestEssexBay

Lee 74TracyDavisCentennialMcNairGreenseed 737

BraggBraxtonHutton

FosterWright

Osmotic-0.3

26.75 hg='̂29.34 fgh

51.57 abcdef45.54 bcdef54.07 abcde25.91 hg

73.47 a39.80 cdefg59.25 abcde51.58 abcdef39.41 cdefg33.83 efgh

55.91 abcde61.25 ab33.26 efgh

37.40 defg66.18 ab

potential (MPa)-0.5

13.82 hg18.17 hg

34.93 bcdef27.00 bcde36.24 bed12.07 hi

45.80 a22.40 fgh32.20 cdefg28.32 cdefg23.37 efgh26.01 defg

38.57 abc36.01 abed19.20 hg

20.14 hg45.91 ab

Mean

20.2923.25

43.2536.2745.1518.99

60.5021.2045.7239.9531.3929.92

45.1548.6326.23

28.7748.63

'•'•' Means within columns followed by the same letters are not significantly different at the 5 % level usingDuncan's Multiple Range Test.

Screening for Drought Tolerance 155

-0 .3 , and -0.5 MPa was determined to be 0, 85,and 141 g, respectively per kg of nutrient solution.This was determined from a standard curve of osmo-tic potentials using a range of PEG 8000 quantities inthe Hoagland's solution measured with an SC-tOthermocouple psychrometer connected to an NT-3nanovoltimeter (Decagon Devices, Inc., PuUmann,WA).

To obtain the seedlings used, seeds of the 17cultivars were first germinated in an incubator at25 °C on moistened filter paper in glass petri dishesfor three days. Seedlings whose radicles had emergedapproximately 2 cm were put into Seed-Pack growthpouches (Northrup King Co., Minneapolis, MN)which had been prepared by cutting two holes in thepaper filler for insertion of the radicle. After a 10 dayadaption period using complete nutrient solutionwithout osmoticum, stress was applied to the plantsby replacing the Hoagland's solution with 20 ml ofthe nutrient solution containing the appropriateamount of PEG 8000 with final pH adjusted to 6.7.The three stress treatments were placed in a growthchamber for 14 days at 26 "̂C under 16 hr photo-

period of 15.76 klux and arranged in a factorialdesign with three replications. Daily observationsincluded checking the pH, the osmotic level andappearance of the seedlings.

At the end of the test, the plants were brought intothe lab for measurements of seedling height, freshand dry weight, and leaf water potential. Leaf waterpotential was determined using a 6 cm piece of leaffreshly collected, coiled, and placed in the samplechamber of an SC-10 thermocouple psychrometer.Dry weight of the plant material was determmedafter drying in a Hot Pack programmable incubatorat 60 °C for 48 hours then equilibrating at roomtemperature for 24 hrs. Height stress index, freshweight stress index and dry weight stress index weredetermined using the generalized formula for cal-culating stress indices reported earlier.

Results and Discussion

Genotypic differences for germination andpromptness indices were observed among the

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Fig. 1. Fresh and dry weight stress indices (%) of seedlings of the seventeen cultivars subjected to -0.3 and—0,5 MPa levels of osmotic stress in the culture medium

156 KPOGHOMOU, SAPRA and BEYL

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2Fig. 2. Leaf water potentials (MPa) and height stress indices (%) of seedlings of the seventeen cultivarssubjected to —0.3 and —0.5 MPa levels of osmotic stress in the culture medium

17 cultivars screened. The germination stressindices among the cultivars varied from73.47 % for Lee-74 to 25.91 % for Bay at the-0 .3 MPa level and from 45.80 % for Lee-74to 12.07 % for Bay at the -0.5 MPa level(Table 1). Similar variation in germinationstress indices from S7 % to 51 % have beenreported for soybeans subjected to germina-tion in PEG at -0.6 MPa. But, a comparisonof values for cultivars in common between thatstudy and this one, indicate a lesser impact ofosmotic stress on germination with germina-tion stress indices of 87 % for Forrest, 85 %for Essex, and 74 % for Bedford (BOUSLAMA

and ScHAPAUGH 1984). High promptness indexhas been associated with high germination in-dex indicating its possible usefulness in pre-dicting germination under adverse conditions(BOUSLAMA and SCHAPAUGH 1984), but extrapo-lation to later stages of seedling developmentcannot be predictably done. All the cultivars

were sensitive to some extent to the osmoticpotential of the medium during germination.Moisture plays a critical role in the activationof various enzyme systems during germinationwhich helps to explain the sensitivity of all thecultivars to drought stress at the germinationstage (CoPELAND 1976).

Seedling growth was less affected by osmoticpotential than seed germination for all the cul-tivars at both levels of stress (Table 1, Figs. 1and 2). No significant difference was foundamong the cultivars at -0 .3 MPa for dryweight stress index (Fig. 1), height stress indexand leaf water potential (LWP) (Fig. 2). Ge-netic variation was observed among the cul-tivars at —0.5 MPa for height stress index,fresh weight stress index, and leaf water poten-tial (Figs. 1, 2).

Cultivars Lee-74 and Wright were the mosttolerant with respective values of 78.5 % and78.8 % for height stress index, 80.7 % and

Screening for Drought Tolerance 157

1.25 -

1.00 -

0.75 -

0.50 -

0.25 -

0.00

-

75

-

50

-

25

0

FW

HTA

Pl\

LWP

crLWPHtFWDWPI

1

Correlation Coefficients of theAssociated Stress Indices

HSI DWSI FWSIGSI 0-40' 0.04 0.34HSI 0.40* 0.36*DWSI 0.52"FWSI

:Leaf water potential o — o:Height A — A:Fresh weight Q Q:Dry weight ^ ^Promptness index A A

1 1 1

LWP-0.33-0.17-0.01-0.01

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0 0 -

2 5 -

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7 5 -

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O) K 0.0 -0.1 -0.2 -0.3 -0.4

Osmotic Potential (MPa)-0.5

D0-

Fig. 3. Mean values for seedlings of the seventeen cultivars examined which illustrate the relationship betweenthe promptness index (%), fresh weight (g), dry weight (g), height (cm), and leaf water potential (MPa) at-0.3, and -0.5 MPa of osmotic stress in the culture medium. Correlation between height stress index, freshweight stress index, germination stress index and leaf water potential is shown in the insert, '•'' and '•'''•'indicating significance at the 5 % and 1 % levels, respectively

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Germination Stress Index (%)Fig. 4. Correlation between height stress index (%j and germination stress (%) index for the seventeencultivars examined. Data represent means of the values for -0.3 and -0.5 MPa treatments with a correlationcoefficient of 0.40

158 KPOGHOMOU, SAPRA and BEYL

76.0% for DWSI, 71.0% and 72.6% torFWSl, and -1.83 and -1.S4 MPa for LWP atthe -0.5 MPa level. Cultivars Ra 401 and Baywere among the most succptible with respec-tive values of 41.83 % and 51.71 % for HSI,17.92 % and 2S.59 % for DWSI, 26.40 % to27.0 % for FWSI and -2.68 and -2.80 MPafor LWP at the -0.5 MPa level. Again, similarbut less severe values for dry matter and plantheight stress mdices have been reported forcultivars £55e.v, Forrest, and Bedford subjectedto 14 days of water stress at —0.6 MPa undersimilar experimental conditions (BOUSLAMA andSciiAPAUGH 1984). A decrease in rhe osmoticpotential of the culture solution from —0.3MPa to —0.5 MPa elicited a sharp decrease inthe cultivars' performance. This was evidencedby the lower values for HSI, FWSI, DWSI andLWP at the more severe level (Figs. 1 and 2).Similar observations of a variation of 3 to 4bars m soil water potential correlated withdaily changes of LWP and stomatal conduct-ance have been reported in soybean (Sii -VAKUMAR and SCHAPAUGH 1978).

Significant differences in LWP were foundamong the cultivars at —0.5 MPa but not at— 0.3 MPa. A decrease in CO? exchange rate asa result of decreasing LWP particularly atlevels below —21 bars (-2.1 MPa) in soybeanhas been related to poor growth (SILVIUS e: al.1977). In general, the cultivars that performedwell under stress conditions {Lee-74 andWright) also had high LWP indicating theirability to maintain high water content undersuch conditions. This response is characterizedas avoidance behavior (LEVITT 1980).

Fresh weight, dry weight and germinationstress indices showed a significant correlationwith height (Fig. 3). Those cultivars that hadhigher germination stress indices also hadhigher values for height stress index as well(Fig. 4). A similar significant or highly signifi-cant correlation has been reported betweenplant heigh: stress index of soybean and germi-nation stress index, dry matter stress index andheat tolerance (BOUSLAMA and SCHAPAUGH

1984). This underscores the reliability of thisparameter to predict cultivar growing perform-ance in the early stages of development. Al-though a negative correlation has been re-ported between relative growth rate and leafwater potential soybean (SIVAKUMAR and SHAW

1978), in this study, leaf water potential wasnot significantly correlated with any growthparameter or with germination stress index,indicating its lack of reliability as a sole para-meter for predicting plant performance.

Because internal aspects of seeds such as seedsize and viability affect the emergence of seed-lings under stress conditions as well, germina-tion may not be as reliable as growth in theseedling stage to predict cultivar performance.In wheat, a similar situation was found withgermination under stress not related to seed-ling growth under stress (BLUM et al. 1967).The performance of cultivars Lee-74 andWright was consistent during both germina-tion and seedling tests, with greater dry matteraccumulation, germination stress indices,height, and LWP. Ra 401 consistently per-formed poorly for these same parameters andtests. These three cultivars were selected forfurther studies.

Zusammenfassung

Selektion fiir Diirretoleranz: Sojabohnen-keimung und ihre Beziehung zum Samlings-wachstum

17 determinierte Sojabohnen {Glycine maxL.)-Kultivare aus vier Reitegruppen — V bisVIII — wurden hinsichtlich der Diirretoleranzzum Zeitpunkt der Keimung und des Sam-lingswachstums untersucht. Keimende Samenund hydroponisch angezogene Samlinge wur-den osmotischem Strel^ von —0.3 und —0.5MPa mit Hilfe von Polyathylenglycol M.W.8000 unterworfen. Es zeigte sich genotypischeVariabilitat zwischen den Kultivaren fiir alleanalysierten Parameter in den Stadien der Kei-mung und des Samlingswachstums. Der Keim-stref^index war niedriger fiir Samen, die einemosmotischen Strel^ von —0.5 MPa unterworfenwaren als fiir -0.3 MPa. Ein geringercs osmo-tisches Potential in dem Behandlungsmediumwar mit einem geringeren Blattw^asserpotcntialin den Samlingen korreliert. Eine signifikanteBeziehung wurde zwischen dem Frischgcwichtund dem Pflanzenhohenindex gefunden. DieKultivare, die unter DUrrestrei^bedingungengrofier wurden, hatten eine hohere Trocken-masseakkumulation und wiesen damit auf ei-nen Zusammenhang zwischen dem hoherenKeimstrefiindex und die Hohe der Pflanzen als

Screening for Drought Tolerance 159

Verfahren zur Voraussage der Sortenleistungunter diesen Bedingungen hin. Die KultivareLee-74 und Wright hatten eine hohere Trok-kenmasseakkumulation, grof^ere Pflanzenhoheund bessere Keimung unter Strei^bedingungenals die anderen Kultivare. Im Vergleich dazuerwiesen sich die Kultivare Ra 401 und Bay alswenig leistungsfahig unter Trockenbedingun-gen. Bei den Samlingsuntersuchungen konntekeine straffe Beziehung zwischen dem Blatr-wasserpotential und dem Gesamtverhalten derPflanzen festgestellt werden, was darauf hin-weist, dafS keine emdeutige osmoregulatori-sche Wirkung vorliegt. Auf der Grundlage derKeim- und Samlingsuntersuchungen wurdendie Kultivare Lee-74, Wright und Ra 401 fiirweitere Untersuchungen im Gewachshaus undFeldexperimenten ausgelesen.

Acknowledgement

This research was supported by CSRS/USDA GrantNo. 2-01-14-3151. We would like to thank GF.ORGEBROWN for statistical consultation in the analysis ofdata.

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