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J Plant Physiol. Vol. 148. pp. 677-683 (1996)

Germination of Chaenorrhinum minus Seeds in Responseto Gibberellin TreatmentsROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTADepartment of Biology, Colgate University, Hamilton, NY 13346-1398, U.S.A.Received June 2, 1995 . Accepted September 21, 1995

SummarySeeds of Chaenorrhinum l minus (L.) Lange (Scrophulariaceae) exhibit embryo dormancy when releasedfrom capsules of the parent plant, but can be induced to germinate when chilled or treated with gibberellins (GAs). GAs found to have activity in breaking dormancy of C minus seeds were GA3, G ~ GAl, and

G ~ + l ' The degree of germination success, however, depended on pH, concentration, duration of incubation, and on the kind of GA applied. Newly mature seeds treated with GA3 at a range of pH values hadoptimum germination success in the pH range 8.5-10; by contrast, seeds that were stored for 10 monthsat room temperature had optimum germination success in the pH range 5.5-8. GAl was the most activeof the GAs tested: in a 96 h incubation, the GAl concentration required for 50% germination was approximately 0.3 mmol L- I, compared to 2.5 mmol L- I for G and 100mmol L- I for GA3. In addition, themaximum proportion (approx. 85%) of seeds that germinated in GAl was greater than in the other GAs.G and G ~ + l produced almost identical responses, showing that the effects of G and GAl in the mixture were not additive. Two other GAs, GAl3 and GArisolactone, were completely ineffective in breakingdormancy. These results are discussed in terms of the affinity of the various GAs for possible receptor sitesin C minus seeds.

Key words: Chaenorrhinum minus (L.) Lange, dormancy, germination, gibberellin, seed.Abbreviatiom:GA = gibberellin.

IntroductionCommonly, seeds are dormant when shed from the parentplant. Such dormancy has, in general, one or more of severalcauses (Mayer and Poljakoff-Mayber, 1989). These includeimmaturity of the enclosed embryo; impermeability of theseed coat to water and gases; mechanical prevention of em

bryo elongation; and the presence of germination inhibitors,which are often associated with the seed coat. The last threeare examples of the general phenomenon of coat-imposeddormancy, which can be relieved by environmental phenomena that soften the seed coat: these include microbial action,abrasion by soil particles, and repeated freeze-thaw cycles.These processes apparently do not involve active participationby the embryo (Bewley and Black, 1985). Embryo immatu-1 Spelled Chaenorhinum by some European authorities.

1996 byGustav Fischer Verlag, Stuttgart

rity is, on the other hand, released by changes that take placein the embryo itself and that have been termed afterripening when they occur during storage (Bewley and Black,1985; Mayer and Poljakoff-Mayber, 1989). Such maturationis often stimulated by temperature treatment or by application of chemicals, including natural and synthetic plantgrowth regulators and a miscellany of other compounds suchas respiratory inhibitors (e.g., cyanide, azide), nitrogenouscompounds (e.g., nitrate, thiourea), and even ethanol, hypochlorite, and methylene blue (Bewley and Black, 1985).Clearly, many of these compounds are unlikely to be encountered by seeds in their natural environment, but informationabout dormancy and its release has been gained by laboratoryapplications of them.The most widely used and effective temperature treatmentfor breaking embryo dormancy involves chilling seeds thathave been layered in moist sand or soil (a process called strati-

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678 ROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTAfication}. A temperature of 5C is most commonly employed,but other temperature conditions, including fluctuating temperatures, have also been successfully employed in some species. The very extensive literature on this topic has been reviewed in, for example, Bewley and Black (1985) and Mayerand Poljakoff-Mayber (1989).A wealth of evidence (see, for example, Inoue, 1991; Lenton and Appleford, 1991) supports the involvement of gibberellins (GAs) in releasing seeds from dormancy. Much of thisevidence comes from experiments in which seed dormancyhas been broken by application of exogenous GAs, mostcommonly gibberellic acid (GA3) or G and GAl, eitherseparately or in mixture. These hormones have been shown tobe effective in breaking dormancy in seeds that otherwise require chilling (Bewley and Black, 1985) or exposure to light(Derkx et al., 1994). Embryos of hazel (Corylus avellana) keptat 5 C for 42 d and then transferred to 20 C for 8d (Williams et al., 1974) had a much higher GAl and GA9 contentthan embryos kept at constant temperatures of 5 or 20 C ,suggesting that the chilling treatment somehow prepared the

embryo to synthesize the GAs in the subsequent exposure towarmer temperatures. In contrast, recent work with Arabi-dopsis thaliana by Derkx et al. (1994) indicates that the promotion of seed germination by chilling (in association withlight) in this species is most likely not a consequence of increased GA content. Indeed, Trewavas (1982, 1987) has proposed that changes in the sensitivity of seeds to plant growthregulators during maturation or post-dispersal events, ratherthan changes in the levels of these regulators, may be the keyto understanding dormancy and germination. More studieswith other species, which address appropriate questions (seeFirn, 1986), are needed to clarify this situation.Numerous authors have described the greater effectivenessof GAs other than GA3 in stimulating seed germination, es-pecially in dicots: these GAs include G (Loveys and Jusaitis, 1994; Derkx et al., 1994), G ~ + l (Karssen et al., 1989;Derkx and Karssen, 1993), and GArisolactone (Derkx et al.,1994).This study reports work done with Chaenorrhinum minus,an annual weed that grows in dry, gravelly soils, especiallyalong railways, throughout most ofwestern Europe, northernand central regions of the United States, and in southernCanada. A previous study by Grime et al. (1981) has indicated that C. minus seeds are released from dormancy whenchilled for 45 d at 5 C while buried in moist sand. In thestudies reported here, we investigated the germination re-sponse of seeds of C. minus to exogenous treatment with sev-eral different GAs.

Materials and MethodsSeeds of Chaenorrhinum minuswere collected from local populations in July-September 1993 and 1994 in the vicinity of Hamilton, NY. They were air-dried and stored at approximately 22 Cand 30% relative humidity. Prior to all experiments, seeds were allowed to imbibe deionized (milli-Q) water for 24 h. In these experiments, each treatment group consisted of 6 replicates of 25 seedseach.

Gibberellin treatmentsUnless otherwise indicated, GA solutions were prepared by suspending the crystalline GAs in phosphate-citrate buffer, pH 6.5,containing 4.05 mmol L-1 K1HP04 and 1.0mmol L-I citric acidand adding 1mmolL -1 KOH one drop (aprox. 0.03mL) at a timeuntil the GA dissolved. Then, 1.0mmol L- citric acid was added to

adjust the pH to the required value (6.5, unless otherwise stated)and more buffer was added to make the final solution.

a) Response to pH ofGA3 solutionsSeeds were treated in 120mmol L-1 solutions of GA3 at a rangeo f pH values from 4 to 12 in 50 mmol L-1 phosphate buffer. The

pH treatments were conducted with seeds of the following ages:seeds that matured and were collected in 1993 and then stored for10 months under the conditions described above; seeds thatmatured and were collected in 1994 and tested within 2 weeks ofmaturity; and, seeds in the latter 1994 group that were stored underthe conditions described above for 10 months.b) Dose responses to GA3, GA4, and GA7Seeds of C minuswere immersed in solutions of gibberellic acid(GA3, K-salt; Sigma Chemical Co., St. Louis, Missouri, USA) at pH6.5 at concentrations in the range 0.03-480 mmol L-1 for 9G h following the 1d imbibition with water. Other seeds were treated similarly with G or GA7 (Sigma Chemical Co.) at concentrations inthe range 0.003-12mmol.L-1.

c) Response to duration oftreatment with GA3 and GA4 +7Seeds were incubated in 120 mmol . L-1 GA3 or 3 mmol . L-IG ~ + 7 for periods of t ime ranging from 24 to 144 h for GA3 and 2hto 9Gh for G ~ + 7 : all such seeds receiving less than 144h in GA3 or96 h in G ~ + 7 were returned to mil li -Q water after the GA treatment, and all seeds were transferred to germination dishes, asdescribed below when the longest GA treatment had been concluded. To test the stability of the GA solutions at room temperatureover a 96h period, some batches of seeds were treated with GA3 orG ~ + 7 for 9Gh beginningwith 9Gh-old solutions.d) Comparative effectiveness ofvarious gibbereLlinsSolutions at 3mmol L-1 concentration of G ~ GA7, GA7 isolactone, G ~ + 7 (1.5 mmol L-1 of each), and GAl3 were prepared bydissolving the crystalline solids in 0.2mL of 50 % ethanol and thenadding a relatively large volume (about 9 mL, depending on thepurity and molecular weight of the GA) of phosphate-citrate buffer,

pH 6.5, containing 4.05mmol L- I K1 HP04 and 1.0mmol. L-1citric acid. Seeds were incubated in each GA solution for 36handthen returned to mil li -Q water for 60h. The 36h GA incubationtime was chosen because it gave approximately 50% of maximumgermination in GAl' thus making possible the detection of moreeffective GAs while still providing an overall incubation time of 96 hin bulk liquid prior to transfer of seeds to filter paper.

Germination trialsAfter GA treatments, seeds were washed with 3 changes of milliQ water and transferred in a small known volume of milli-Q waterto plastic disposable Petri dishes containing two layers of filter paper(Whatman # 3). Sufficient milli-Q water was added to each dish tobring the total volume added to 6.5 mL. All dishes were weighedand then incubated in a plant growth chamber (Model PG-77, Percival Manufacturing Co., Boone, Iowa), equipped with a combina-

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tion of fluorescent (160W Gro-Lux, Osram Sylvania, Inc., Danvers, Massachusetts, USA) and 60W incandescent bulbs. Incubationconditions were a I6L18D photoperiod, with temperatures 25 Cand 15C, respectively, and relative humidity 70 2%. Dishes wereexamined daily, germinated seeds were scored and removed, and sufficient milli-Q water was added to each dish to restore its mass to itsoriginal value. Since the mean mass of seeds was only 49 /lg (range =14-77/lg, N= 150 seeds) it was considered unnecessary to compensate for the mass of germinated seeds that had been removed. A seedwas considered to have germinated when the radicle produced anydegree of outgrowth visible at a lOx magnification of a dissectingmicroscope; a mere swelling of the embryo, even when accompaniedby rupture of the seed coat, was not considered successful germination. Experiments were terminated when there was no further germination over 3 successive d.Statistical analysisIn all experiments resulting in percentage data, statistical tests andfunction fitting operations were carried out after arcsine transformation of the percentages (as arcsin [proportion seeds germinated] 'h) inorder to satisfY the assumptions of analysis of variance and regres

sion that the error terms are homoscedastic and normally distributed(Sokal and Rohlf, 1995). Where indicated, Tukey tests for multiplecomparisons among means (Zar, 1984) were carried out after analysis of variance.

Results

Seed germination in Chaenorrhinum minus 67960

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680 ROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTA

60

96

144

24 48 72Hours of treatment with G ~ + 7

24 48 72 96 120Hours of treatment with GA3

A

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c) Response to duration oftreatment with GA3 or GA4+7Germination success of C. minus also depended on the duration of treatment with GA solurions following the 1d imbibition in water (Fig. 3). Figure 3 A shows the response toGA3: there was a progressive increase in germination successwith increasing lengths of exposure from 1 to 4 d, but apparently.no further increase at 5 and 6 d of treatment. A logisticfunction gave the best fit to these data, which is consistentwith germination success reaching a maximum (approx. 60%germination) following about 4 d incubation in GA3 andthen remaining constant. There were no significant differences among the 96, 120, and 144h percent germination values (Tukey test: q>2.5 for each comparison, P>0.5), showing that maximum germination success (approx. 60% germination) resulted from approximately 96 h of incubation inGA3 and remained constant thereafter.Figure 3 B shows the response to G ~ + 7 ' As in the case ofthe response to GA3, a logistic function provides the best fitto these data. There were no significant differences among

the 48, 72, and 96 h percent germination values (Tukey test:q >2.5 for each comparison, P >0.5), showing that maximum germination success (approx. 66% germination) re-sulted from approximately 48 h of incubation in G +7 andremained constant thereafter.The stability test results showed that there was no significant difference in the proportions of seeds that germinatedwhen seeds were incubated for 96 h beginning with freshlyprepared versus 96h-old GA3 and G ~ + 7 (F= 0.76 and 0.88,P@0.05 for GA3 and G ~ + 7 ' respectively).d) Comparative effectiveness o fvariousgibberellinsData in 'fable 1 show that, at a 3mmol L-I concentrationand 36h treatment duration, GA7was the most effective GAof. t h o ~ e tested in breaking dormancy in C. minus (85 % ger

m t n a ~ l O n success); G and the G ~ + 7 mixture were equallyeffective (Tukey test: q = 0.133, P >0.9), producing approx.37% germination success, but these two GA treatments produced significantly less germination success than GA7 alone(Tukey test: q for GA7versus G ~ + 7 = 10.28 P

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682 ROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTAreport that GArisolactone is converted to GA7 by KOH,which is commonly used to initially dissolve GAs prior to dilut ion with water or buffer. These workers also report increased activity of GAs dissolved in ethanol compared tothose dissolved in KOH. In the experiments to compare thedifferent gibberellins reported here, small volumes of ethanol,rather than KOH, were used to initially dissolve GAs for theexperiments to compare activity of the various GAs tested.This treatment seems to have not caused conversion amongGA7 isomers; given the high reactivity of C. minus seeds toGA7, any conversion of GArisolactone to GA7 would likelyhave been detected as a greater-than-zero germination successin the former. In regard to our use of KOH to assist the dissolving of GAs in other experiments reported here, the similar germination maxima produced by GA7when dissolved bythe KOH (Fig. 2) and ethanol (Table 1) methods suggeststhat our KOH method (in which GAs were dissolved in buffer and then KOH was added dropwise to dissolve the GAs)is less destructive to GAs than when GAs are initially dissolved in KOH followed by dilution with buffer. This lattermethod would, of necessity, expose the GAs to more stronglybasic conditions (pH close to 14) than would the formermethod, in which the pH of the GA solutions did not exceed8.5.It is still possible, however, that our use ofKOH to prepareGA solutions for the dose response experiments may explaina striking difference between our results and those of Derkxet al. (1994). The most effective of the GAs (GA7) tested inthe present" study needed to be at a 1mmol .L -1 concentration or higher to produce maximum germination success; incontrast, Derkx et al. (1994) obtained maximum germinationsuccess in seeds of A. thaliana using GA concentrations in therange 0.01-0.1mmol L-1 . Alternatively, this apparent inconsistency may be due to characteristics inherent in the different plant species used in the two studies.A second proposed characteristic of active GAs (Stoddart,1986) is substitution at the C 13 position. Interestingly, of theGAs tested here, only GA3 has substitution (by a hydroxylgroup) at this position, yet this GAwas much less active thanG ~ G ~ + 7 ' or GA7. Clearly, other factors besides C 13 substitution playa role here.Comparison among the GAs also shows that the effects ofthe mixture, G ~ + 7 ' can not be predicted from knowledge ofthe effectiveness of the separate components. Based on themuch greater effectiveness ofGA7 than G ~ one would predict that G ~ + 7 would have intermediate effectiveness. Thiswas not the case, however, since G and G ~ + 7 were almostidentical in effectiveness. This observation also provides evidence that there may be only one GA receptor for G andGA7 in C. minus, since the effects of the two growth regulators are not additive. Derkx et al. (1994) observed a similarlack of additivity in experiments with GA7 and GArisolactone in A. thaliana and, accordingly, proposed that the twoGAs compete for the same binding site. We suggest that thesame kind of competition for a single type of receptor site,here involving G and GA7, may occur in C. minus.Stronger evidence in favor of such competition would come,however, from studies of dose responses to GA7 in the presence of varying quantities of G ~ A measure of the relativestrength of binding of G and GA7 to the receptor(s) is

given by the [GA] 50 values (the GA concentration giving halfthe maximum response). These quantities can be determinedfrom the logistic function for each GA, and have values0.57mmol L-1 and 0.11 mmolL -1 for G and GA7, respectively. The logistic curves at [GA] 50 for the two GAs are al-most identical (the slopes, obtained by differentiation of eachfunction at an abscissa value of [GAJso, were 13.3 and 13.5for G and GA7, respectively). By analogy with the Michaelis constant for an enzyme-catalyzed reaction, the very different [GA] 50 values suggest that GA7 has a greater affinity forits receptor than G has for the same (or separate) receptor,especially as the two logistic functions are essentially parallelat this concentration (Firn, 1986; Weyers et al., 1987).

The greater effectiveness of GA7 in breaking dormancy ofC. minus seeds is also evident from the significantly greatermaximum response (approx. 82% germination success) thanwas observed with G (approx. 65%) and GA3 (approx.55 %). This is in contrast to work by Derkx et al. (1994),who found that the maximum response of A. thaliana seedswas similar (and close to 100%) in all GAs tested over a sufficiently broad range of concentrations to produce a maximumresponse. This difference between G and GA7again is consistent with competition between the two GAs for the samereceptor sites, to which GA7 has greater affinity, and hencestimulates a greater response.

A c k n o ~ e d g e f n e n t sThe authors thank Colgate students Kerri Blum, Tamara Merchant, John Mulhall, and Allison Wayman for conducting preliminary experiments for this study; Dr. T. Sparks of the Institute ofTerrestrial Ecology, Monks Wood, Cambridgeshire, U.K. for statisticalanalysis of the pH data; Dr. K. Valente of the Department of Mathematics, Colgate University for help in estimating the mid-pointslopes of logistic functions; and Dr. V. McMillan of the Department

of Biology, Colgate University for critically reading the manuscript.The research was supported by a grant from the Colgate ResearchCouncil.

ReferencesBEWLEY, J. D. and M. BLACK: Seeds: Physiology of Development

and Germination. Plenum Press, NY. (1985).CLEVELAND, W. S.: Robust locally weight regression and smoothingscatterplots. J. Amer. Statist. Assn. 74,829-836 (1979).- LOWESS: A program for smoothing scanerplots by robust locally weighted regression. Amer. Statist. 38, 54 (1981).DERKX, M. P. M. and C. M. KARSSEN: Variability in light-, gibberellin-, and nitrate requirement of Arabidopsis thaliana seeds due toharvest time and conditions of dry storage. J. Plant Physiol. 141,

574-582 (1993).DERKX, M. P. M., E. VERMEER, and C. M. KARSSEN: Gibberellins inseeds of Arabidopsis thaliana: biological activities, identificationand effects of light and chilling on endogenous levels. PlantGrowth Regul. 15,223-234 (1994).

FIRN, R. D.: Growth substance sensitivity: the need for clearer ideas,precise terms, and purposeful experiments. Physiol. Plant. 67,267-272 (1986).GRIME, J. P., G. MASON, A. V. CURTIS, J. RODMAN, S. R. BAND, M.A. G. MOWFORTH, A. M. NEAL, and S. SHAW: A comparativestudy of germination characteristics in a local flora. J. Ecol. 69,1017-1059 (1981).

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GRIME, J. P., J. G. HODGSON, and R. HUNT: Comparative PlanrEcology. Unwin-Hyman, London (1988).INOUE, Y.: Role of gibberellins in phytochrome-mediated lettuceseed germination. In: TAKAHASHI, N. , B. O. PHINNEY, and J.MAcMILLAN (eds.): Gibberellins, pp. 289-295. Springer-Verlag,

New York (1991).JUNTTILA, 0. : Gibberellins and the regulation of shoot elongation in

woooy plants. In: TAKAHASHI, N. , B. O. PHINNEY, and J . MAcMILLAN (eds.): Gibberellins, pp. 199-210. Springer-Verlag, NewYork (199l).- lneerac tion of growth retardants, daylength, and gibberellins A19,A2o , and Al on shoot elongat ion in birch and alder . J . PlaneGrowth Regul. 12, 123-127 (1993).

KARSSEN, C. M., S. ZAG6RSKI, J. KEpCZYNSKI, and S. P. C. GROOT:Key role for endogenous gibberellins in the control of seed germination. Ann. Bot. 63,71-80 (1989).

LENTON, J. R. and N . E. J. APPLEFORD: Gibberellin production andaction during germination of wheat. In: TAKAHASHI, N. , B. O.PHINNEY, and J. MAcMILLAN (eds.): Gibberellins, pp. 125-135.Springer-Verlag,New York (1991).

LOONEY, N. E., R. 1. GRANGER, C. 1. CHU, S. J. McARTNEY, 1. N.MANDER, and R. P. PHARIS: Influences of gibbereJlins At, At.?,and At + iso-A? on apple fruit qualiry and tree productiviry. I. Effects on fruit russet and tree yield componenes. J. Hort. Sci. 67,613-618 (1992).

LoVEYs, B. R. and M. JUSAITIS: Stimulation of germination of quandong (Santa/um acuminatum) and other Australian native planeseeds. Aust. J. Bot. 42, 565-574 (1994).

Seed germination in Chamorrhinum minUJ 683MAYER, A. M. and A. POLJAKOKFF-MAYBER: The germination ofseeds, 4th Ed. Pergamon Press, Oxford (1989).SEREBRYAKOV, E. P., V. N. AGNISTIKOVA, and 1. M. SUSLOVA:

Growth promoting activiry of some selectively modified gibberellins. Phytochemistry 23, 1847-1854 (1984).SOKAL, R. R. and F. J. ROHLF: Biometry: the principles and practice

of statistics in biological research, 3rd Ed. W. H. Freeman andCo., New York (1995).STODDART, J. 1.: Gibberellin receptors. In: CHADWICK, C. M. and

D. R. GARROD (eds.): lneercellular and ineracellular communication, 1. Hormones, receptors and cellular ineeractions in plants,pp. 91-114. Cambridge Universiry Press, New York (1986).TREWAVAS, A. J.: Growth substance sensitiviry: the limiting factor in

plant development. PhysioJ. Plane. 55, 60-72 (1982).- Sensi tivi ry and sensory adaptation in growth substance responses.In: HOAD, G. v., M. B. JACKSON, J. R. LENTON, and R. K. ATKIN(eds.): Hormone Action in Plane Developmene, pp. 19-38. Butterworths, London (1987).WEYERS, J. D. B., N. W. PATERSON, and R. A'BROOK: Towards aquaneitative definition of plane hormone sensitiviry. Plane CellEnviron. 10, 1-10 (1987).WILLIAMS, P. M., J. W. BRADBEER, P. GASKIN, and J. MAcMILLAN:Studies in seed dormancy. VIII. The identification and determination of gibberellins AI and A9 in seeds of Cory/us ave/lana 1.Planta 117, 101-108 (1974).

2M, J. H.: Biostatistical Analysis, 2nd Ed. Prentice-Hall, Inc., Englewood Cliffs USA, 1984.