Smithsonian miscellaneous collections

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VOLUME

94 NUMBERS

WAVE LENGTHS OF RADIATION IN THE VISIBLE

SPECTRUM INHIBITING THE GERMINATION OF LIGHT-SENSITIVE LETTUCE SEED

BY LEWIS H. FLINT

DivisionofSeedInvestigations,BureauofPlant Industry, U.S.DepartmentofAgriculture

AND

E. D. McALISTER

DivisionofRadiationandOrganisms, SmithsonianInstitution

(PUBLI.CATION 3334)

CITY OF

WASHINGTON

PUBLISHED BY

THE

SMITHSONIAN INSTITUTION

JUNE

24, 1935

(2)

€Be£orbat?atHrnorc(prtee BALTIMORE,MD.,C.8.A.

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WAVE LENGTHS OF RADIATION IN THE VISIBLE SPECTRUM INHIBITING THE GERMINATION

OF LIGHT-SENSITIVE LETTUCE SEED

By lewis

H.

flint

Division ofSeed Investigations,BureauofPlant Industry, U. S. Department of Agriculture

E. D.

McALISTER

Division of Radiation and Organisms, SmithsonianInstitution

IXTRODUCTIOX

Instudies of thelight-sensitivityof ^"

dormant

" lettuceseed previ- ously reported (3)' it

was

noted that radiation of the longer

wave

lengthsofvisible light, characterizing the colors yellow, orange, and red, promoted germination, whereas ^radiation ^of the shorter

wave

lengths ofvisible light,characterizingthe colorsviolet, blue,andgreen, inhibited germination.

The

material appeared to be unusually well adapted to the study of response to radiation, and steps were taken to establish the relative effectiveness of radiation of various

wave

lengths with respecttothegerminationof theseed.

While

these studies werein progress,Johnston (6) atthe Smith- sonian Institution reported theresultsof a carefulseriesofmeasure- ments of phototropic response of the etiolated oat coleoptile, which emphasizedthefactthat the shorter

wave

lengths ofvisible lightwere responsible for such bending.

He

interpreted this activityas dueto an inhibitoryeffectof the shorter

wave

lengths

upon

thecellsexposed to suchradiation.

On

account of the obvious analogy between the results obtained with the shorter

wave

lengths of light in respect to inhibition in germination and in phototropism the ^facilities of the

two

research divisionswere combinedinthe furtherance of thegerminationstudy, the cooperative investigation leadingto the resultshere presented.

^Numbers inparenthesesrefer to list ofliterature cited, at theend of this paper.

Smithsonian MiscellaneousCollections, Vol.94, No.5 I

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2

SMITHSONIAN MISCELLANEOUS

COLLECTIONS VOL. 94

REVIEW OF LITERATURE

With

respectto light and germinationthree classes of seeds have beenrecognized for

many

years: (i) seedsgerminatingequally well in lightor darkness, (2) seeds

whose

germinationishinderedbylight, and (3) seeds

whose

germination isfavoredby^light. This classifica- tionhas emphasizedthevariety of the responses that

may

occurand has proved satisfactorily descriptive for studies involving sunlight or otherwhitelight.

In 1883 Cieslar (2) reported with respect to certain seeds that yellowlightfavoredgermination, whereasviolet lightretardeditand rendered

them

appreciablydormant. This varyingresponse todiffer- ent

wave

lengths of light

made

^it evident that in sunlight or other white light certain components wereacting to promote germination, while certain othercomponents wereactingtoretardit.

Upon

therela- tiveeffectiveness of these

two

groups of components, either through the radiant energies involved orthroughthe particular sensitivity of the seed,one might

presume

todepend the gross efifect of thewhite lightwithrespect to germination.

From

the

more

technicalstandpoint, therefore, therewerebut

two

classesof seedswithrespecttogermina- tion: (I) those

whose

germination

was

not influenced bylight, and (2) those

whose

germination

was

influenced bylight. Seeds of the latterclassweredesignated" light-sensitiveseeds," as contrastedwith thewidelyoccurring seeds of theclasswhich germinateequally well in lightandin darkness.

The

present considerations are confined to light-sensitive seeds.

Light-sensitivity as reported by Cieslar

was

limited for the most part to small seeds without reserve food materials. In the seeds of

Poa

nemoralis, Agrostisstolonifera, and Nicotiananmcrophyllager- mination

was

reported asfavoredbywhitelight,whereasthegermina- tion of seeds of Viscutn

album was

reported ashindered by white

light. In large measure the researches onlight and germination by various workers in subsequent years have been concerned with the extension of these respective lists. In this respect ^it isto be noted that sensitivityto light is

now

generally recognized as a widely oc- curring characteristicof seeds.

The

early distinction between the efifect of " yellow^" light and

"violet " lightgained precision through subsequent researches, and one finds in Molisch ₍₉₎ the statement thatyellow to red lightpro- motes germination, whereas violet, blue, or green light inhibits germination. Thisinformation,however,has notbeen widelyappreciated, and the

more

recent studiesof light in relation to germination, such

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NO. 5 IRRADIATED

LETTUCE

SEED FLIXT

AND

McALISTER ₃

as those ofGardner_(4),

Lehmann

_(7),Maier (8),

Nathammer

(10) and

Shuck

(11), have concernedthemselves for the most part with whitehghtas a quantitative factorin germination.

Wholly unaware

of the foregoing background of researches by-

German

workers in this field, the seniorauthor discovered that so- called"

dormant

" lettuceseed

would

germinatereadilyinwhitelight, andfurther,thatyellow,orange, or red lightpromotedthisgermina- tion, whereasviolet, blue,or greenlightinhibitedit. It

now

appears that the germination response to radiation of specific

wave

lengths noted for

dormant

lettuce seed represents types of reactions of wide occurrence

among

seeds. This fact suggests that the further study ofsuchresponses

may

bewarrantedaspromisingresultsofboth prac- ticaland theoretical significance in relation togermination and pos- sibly also in relation toother aspects ofgrowth.

The

resultsobtained by_{Flint (3)} withgreen

Wratten

filtersindicated that color alone

was

not a safecriterion touseinthe interpretation ofresultsobtainedwith

filters,thus directing attentionto theirwave-lengthtransmission.

METHODS

The

principal line of attackin this investigation involved the use ofa spectrum,

and

toa large extentthe

work

comprisedsuccessiveim- provements inthetechnique of utilizingthe spectrumtothe greatest

Fig. I.

—

^Schematicdrawingofthe apparatus usedinthestudy ofspectralligl inrelation to germination.

advantage in relation togermination.

The

set-up asfinallyelaborated (see fig. i) consisted of a fixed light source,a condenser lens con- centrating light

upon

an adjustableslit, an achromatic lens, aprism, and a silvered mirror.

A

light-proof house surrounded the set-up, withapartition attheslit (not

shown

inthefigure).

With

thisset-up, using asinglefilament 1,000

lumen

6.6

ampere Mazda

street-lighting bulb as alightsource, aspectrum

was

obtainedwhich

was

abouti foot longin thevisible range.

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4

SMITHSONIAN MISCELLANEOUS

COLLECTIONS VOL. 94

For

exposures of the material inthe spectrum specialboxes lo

x 4x1

inches were

made

of brass and provided with parallel center platesofmonelmetalabout

4x1

inches,spacedat0.4inch.

Two

such boxesplacedendtoendthus

more

thanfilledthevisiblespectrum,and each of the 48 compartments

was

subjectedto a band of radiation rangingin width

from

theorder of 50

A

inthe lowviolet tothat of 200

A

ⁱⁿ thehighred.

The

spectrum and boxeswere provided with a secondary light-proof housing, in which, at an elevation of about

1 foot, wereinstalledtwo20-inch milk-glass lumiline lights

wrapped

inred cellophanehavingnoappreciabletransmission for

wave

lengths shorterthan6000 A. Theselightsweresoarrangedthatinconjunc- tion withend-mirrors no shadows were cast inany compartmentof the boxes.

The

intensityof illumination

was

regulated byarheostat.

The

focalplane of thespectrumobtained

was

locatedby inserting a plate of

didymium

glass betweenthe condenser lens and the slit.

The

sharp absorptionlines of

didymium

also provided a convenient

means

of establishing the

wave

lengths of all regions of the visible spectrum.

Wave

lengthsinthenearultravioletregionwereestablished bysubstitutingamercuryarcasalightsourceandusinguraniumglass to pick

up

through fluorescence the lines characteristic of mercury.

Wave

lengthsintheinfraredregionwereestablishedbyfollowing the absorption characteristics of water vapor with a thermopile.

The

radiation energies throughout the entire spectrum were established

by means

of athermocouple.

The

procedure in each experiment

was

as follows.

Two

boxes were placed in the spectrum and half filled with tap water, which served as the

medium

of germination.

Dormant

or light-sensitive lettuceseedswerethen scattered intothecompartments, surface ten- sion bringingabout a fairly uniform distribution of the seeds over the available water surface.

About

100 seeds could be conveniently

accommodated

in each compartment.

After 2| hours presoakingtheseedsweregivenexposuresof spectral light,of red lumilinelight,or of bothlights,depending

upon

the particular objective.

The

red lumilinelight, bysuitable modification of the duration or intensity of the exposure,

was

used forthe

most

partto effecta50percentgerminationof the seedsindependentof the spectral light

—

a feature ordinarily offering

some

difficulty, but en- tirely feasiblewiththe materialathand, ashad beenattestedbytables 2 and3 of ^Flint'spaper _(3).

Upon

this base thenature andextent of any promoting or inhibiting influence of the spectral light

was

registered as a departure. After 24 hours the boxes were removed.

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NO. 5 IRRADIATED

LETTUCE

SEED FLINT

AND

McALISTER ₅ and the seeds in each compartment weretransferred immediatelyto

numbered

petri dishes,placedina refrigeratorat 3°

C, and

exposed tobluelight (toprevent further germination),

where

theywerekept untilgerminationcounts could bemade.

Inplottingthe germination percentages against radiation the

wave

length falling onthe

median

lineof each

compartment was

taken as the

wave

length for the seeds of that compartment.

Inplotting the inhibitory influence in the violet-blue-green region as corrected for energies involved, the curves wereinverted tofacili- tate subsequent comparison withotherdata.

The

transmission curves of the

Wratten

filters and of the ether extracts of lettuceseeds were

made

intheconventional

manner

with a double

monochromator

and athermocouple.

RESULTS

In the experiments of Flint (3)

two

green

Wratten

filters were found to transmit light that promoted germination, and 10 green

Wratten

filters were foundto transmit lightthat inhibited germination.

The

spectral transmission of all thegreen

Wratten

filters

was

studied, and the energy transmission curves were obtained by mul- tiplying the percentage transmission by the energy radiated

from

a

Mazda lamp

ateach

wave

length.

The

energyradiatedbythe

lamp

at each

wave

length

was

obtained

from

its

known

spectralenergy curve.

These energyvalueswere usedinconjunctionwiththeinversesquare law andthe distancesatwhichthe respectivefilters

(when

used with the

Mazda

lamp) gaveequal responsewith a

Weston

photronic cell.

The

energies transmittedbyrepresentativegreen

Wratten

filters are

shown

in figure2.

It is tobe noted

from

the curves of figure 2 that the

two

green

filterswhich had been found^to^transmit^lightpromoting germination (64 and 67) transmitted

more

of theultravioletand less of thelong visible red than the greenfilters which had been found to transmit light inhibiting germination (56 and 60). This fact suggested that thepromotion

was

associatedeitherwithapromotinginfluenceinthe ultraviolet or with an inhibiting influence in the long red or near infrared.

A

substantial series of exposures of moist

dormant

lettuce seed to various

wave

lengths in the ultraviolet ranging

from

the lower limits of the visible spectrum to below theultraviolet characterizing solar radiation gave uniformlynegativeresults,

whereupon

attention

was

directedtothe infrared regions. Earlier studieswith aspectrum

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SMITHSONIAN MISCELLANEOUS

COLLECTIONS 94

by the senior author had given an approximate range of 5200 to 7000

A

for thepromoting effect,witha sharp faUing offingermina- tion inthe longred.

No

^inhibitory effect had beensuspected as associated withthis falHng off, however, until theeffort

was made

to

Fig.2.

—

^Energytransmission curves ofgreenWrattenfilters. Theordinates are relativeenergiestransmitted ateachwavelength (indicatedas abscissae).

Numbers56and 60 aretypicalof thegreen filters transmittinglight thatin- hibitsgermination. Numbers64and67 transmitlightthatpromotesgermination.

explain the physicalbasisforthapromotingeffectof the lighttrans- mittedbythetwoaberrantgreenfilters.

The

spectrum set-up previ- ouslyused bythe senior authorhad beenconsiderably modifiedinthese cooperative studiestopermita

more

precise

measurement

of the

wave

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NO. 5 IRRADIATED

LETTUCE

SEED FLINT

AND

McALISTER /

lengths to which seedswere exposed.

With

^the apparatus and pro- ceduredescribedinthe foregoing section suchresultsas those given infigure 3wereobtainedinthelong red region.

Such

resultsas those presentedinfigure3established the presence of a strong inhibitory influenceintheregion of7600 A.

By

applying the

same

methodstotheviolet,blue,and greenregions of the spectrum, such results as those presented in figure

4

were obtained.

In conjunction with the foregoing experiments a study of ether extracts of lettuce seed

was

carried, out. These extracts contained oilandpigments. Definiteabsorptionintheregion

4200 A

to 5200

A

was

evidencedbythe transmission curves.

These

remained bimodal, even after appreciable oxidation had takenplace, andthus appeared suggestive in relation to the bimodal curve of inhibition given in figure4. Definiteabsorptionintheregion5200

A

to7000

A was

also evidencedbythe transmission curves, suggestingthe presence of

some

precursors of chlorophyll and allied pigments.

No

appreciable ab- sorptioninthe region7600

A was

noted.

DISCUSSION

The

discovery of a strong inhibitory influence in the region of 7600 A, although

made

in an effortto explain the difference in the responsetothelighttransmittedbycertaingreen

Wratten

filters,and quite incidental to the study of the precise nature of the curve of inhibition in the regions characterizingviolet,blue, and green light,

may

welltranscendinimportancethe original objectiveof the cooperative studies. Sincethis discoveryappearstooffera clearer approach to biologicalproblemsinvolvinglight, ithasbeen given precedencein these considerations.

All thegreen

Wratten

filtersusedbyFlint (3)were foundtotrans- mitin the7600

A

^region,but the

two

filterstransmitting lightwhich promoted germination had suchalow transmissionin thisregion that theeft'ects of the promotingregions

—

the yellow, orange, and red

—

predominated overtheeffectsof the inhibiting regions

—

^{the long}^red,

theviolet, theblue,andthe green. Since

many

blueandviolet glass, liquid,or gelatinfilters transmitinthe region7600 A,^itfollows that the newlydiscovered inhibitory band becomes a potential source of confusionastothe effectiveness of radiationinthe

more

visiblespec-

trum

with respect to the germination of light-sensitive lettuce seed.

Moreover,sincethe

same

type oflight-sensitivityhasbeen recognized ascharacterizingotherseeds, this factor

may

wellbe of

some

general significancewithrespectto light-sensitivity in seeds.

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SMITHSONIAN MISCELLANEOUS

COLLECTIONS VOL. 94 100

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NO. 5 IRRADIATED

LETTUCE

SEED FLINT

AND

McALISTER 9 In connection withthe later consideration of inhibitory influences associated with

wave

lengths characterizing violet, .blue, and green light,it isto benoted that with both solarand

Mazda

radiationthe energyat7600

A

is

much

greaterthanattheshorter

wave

lengthsof the visiblespectrum. In solar radiation there is a sharp absorption band in the 7600

A

region interpreted as due to

oxygen

in the sun and water vaporinthe earth'satmosphere. Notwithstandingthisab- sorption, however, the energyof solar radiationat this

wave

length regionislarge. Inconsequence it

would

appearthat under natural outdoor conditions and under customary indoor experimental conditions radiationof a

wave

lengthin thelong red exerts a relatively powerfulinhibitory influence

upon

thegerminationof

dormant

lettuce seed, althoughthis influenceis ordinarily

more

than counteracted by thepromoting influenceinthe yellow-orange-red region.

The

extent towhichthe7600

A

regionhasan analogouseffect

upon

other seeds and

upon

otherphasesof light-sensitivityis not

known

at thistime, butbecauseof thehigh energy anduniversaloccurrenceof the radiation, its potential significance becomes one of the most intriguing resultsof its discovery. Furtherstudiesof the possible effectiveness of this region in respect to the germination of other seeds and in respecttootherphases oflight-sensitivity are

now

in progress.

An

examinationof the germinationresponses to lightof the

wave

lengths indicated in figure

4

reveals that there are

two maxima

of inhibition intheviolet-blue-green region

—

^a

^major

^one^at4400 A, and a

somewhat

subordinate one at 4800 A. It

may now

be noted that

Bachmann

and

Bergann

(i)and Johnston (6), studying the^etiolated coleoptilesof

Avena

sativa Culberson, obtained curvesof phototropic sensitivityhaving

two maxima

ataboutthese

same

regions.

The two

types ofdata,theoneindicatinganinhibitory influence oflight

on

the germinationof seeds,the otheran influence oflightonthe direction of growthof

young

etiolated shoots, have been brought together to facilitatecomparison in figure _5.

An

examinationof figure5revealsthatwithin therangeof experimental error the

two

types of plant responseto light

show

identical critical

wave

lengths. Johnston (6, p. 14) andothers interpret photo- tropicresponse asan index of

growth

retardationonthe theory that the lighton the exposed side of the shoot inhibits elongation, while onthe opposite unexposed side elongationis relatively uninfluenced.

The

resultshere reported obviouslytendto sustain the correctness of thisinterpretation. Bothof the foregoingtypes of plant responseto lightinvolvedetiolatedstructures,andfurther studies areinprogress relating tothetypes of plant response characterizinggreentissues.

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10

SMITHSONIAN MISCELLANEOUS

COLLECTIONS VOL. 94 Inresume, the results obtained withthe

Wratten

filters, taken in conjunction with the curve of violet-blue-green inhibition given by thespectraldata

and

withthenewlydiscovered inhibitory influencein the7600

A

^region,

make

it

more

than everobviousthattheeffectsob- tainedthroughtheuse ofanycolorfilter

may

not safelybe interpreted withoutan analysis of its spectral transmission.

The

results presented in this paper indicate the general relative effectiveness of radiation of various

wave

lengths inthevisiblespec-

trum

found to inhibitthe germination of light-sensitive lettuce seed.

00

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NO. 5 IRRADIATED

LETTUCE

SEED FLIXT

AND

McALISTER II

effectiveness inbringing about phototropic response in etiolated col- eoptiles of oats. Both

phenomena may

be represented by bimodal curvesshowingcritical

wave

lengthsinthe regions4400

A

and 4800 A.

LITERATURE CITED

1.

Bachmann,

Fr.,andBergann, Fr.

1930. UberdieWerkigkeitvonStrahlen verschiedenerWellenlangefur die phototropische Reizung von Avcna sativa. Planta, Arch.

Wiss.Bot., vol. 10,pp.744-755.

2. Cieslae, Adolph

1883. Untersuchungeniiber den Einfluss des Lichtes auf die Keimung der Samen. Pp. 270-295.

3. Flint, Lewis H.

1934. Light in relation to dormancy and germination in lettuce seed.

Science, vol. 80, pp. 38-40.

4. Gardner,

W.

A.

1921. Effectof lightongermination of light-sensitiveseeds. Bot. Gaz., vol. 71, pp.249-288.

5. Gassner, G.

1915. Beitrage zur frage der Lichtkeimung. Zeitschr. Bot., vol. 7, pp.609-661.

6. Johnston, Earl S.

1934. Phototropic sensitivity in relation to wave length. Smithsonian Misc.Coll.,vol. 92, no.11, pp.1-17.

7.

Lehmann,

E.

1918. UberdieminimaleBelichtungzeitwelchedieKeimungderSamen von Lythrum salicaria auslost. Ber. Deutsch. Bot. Ges., vol.

36,pp. 157-163.

8. Maier, Willi

1933. Das keimungsphysiologischeVerhaltenvon Phleum pratense, L.,

demTimotheegras. Jahrb. Botanik,vol. 78,pt. i,pp. 1-42.

9. MoLiscH,

Hans

1930. PflanzenphysiologiealsTheorieder Gartnerei. P.323.

10. Nathammer, Ann.

1927. Keimungphysiologische Studien unter Hervorhebung des Licht- keimungsproblems. Biochem. Zeitschr., vol.185, pp.205-215.

11. Shuck,A.L.

1935. Light as a factor influencingthedormancyof lettuce seeds. Plant Phys.,vol. 10, pp. 193-196.

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