Plant Physiol. (1970) 45, 235-239

Photoreactions Controlling Flowering of Chrysanthemummorifolium (Ramat. and Hemfi.) Illuminated withFluorescent Lamps

Received for publication October 15, 1968

H. M. CATHEY AND H. A. BORTHWICKCrops Research Division, Agricultural Research Service, United States Department of Agriculture,Beltsville, Maryland 20705

ABSTRACT

Flowering of chrysanthemum plants under short photo-periods, as is well known, is prevented when the plants areilluminated near the middle of the long night. Such illumi-nation inhibits flowering whether it is given continuouslyor intermittently, and whether it comes from incandescentor from fluorescent lamps. We discovered, however, thatfluorescent light applied intermittently (cyclically)throughout the entire 16-hour long night was far less in-hibitory than when applied during only part of this darkperiod. By contrast, incandescent filament illumination isstrongly inhibitory under these conditions. The cycles offluorescent light usually lasted 15 minutes, 1.5 minutes oflight followed by 13.5 minutes of dark. When such cycleswere applied for only 12 hours, leaving 4 hours of uninter-rupted darkness in each long night, inhibition of floweringwas complete again.

Flowering of Chrysanthemum morifolium (Ramat. & Hemfl.) ispromoted by subjecting the plants to several daily dark periods ofmore than 12 hr and is inhibited by illuminating them near themiddle of each long night for a few hours with continuous orintermittent low intensity light (cyclic light) from fluorescent orincandescent lamps (3). When applied cyclically throughout theentire 16-hr "dark" period, however, fluorescent illumination isfar less inhibitory than when applied during only part of thedark period. We discovered this peculiar ineffectiveness of pro-longed periods of cyclic fluorescent light when we included theshort-day chrysanthemum in an experiment designed primarilyto investigate light responses of several long-day species. In-candescent filament illumination is strongly inhibitory under bothconditions.

Other differences between the actions of fluorescent and in-candescent filament illuminations on chrysanthemums were previ-ously encountered (3). They were interpreted on the basis ofdifferences between the two kinds of illumination in the red andfar-red parts of the spectrum, coupled with conditions of leafstructure that might peculiarly affect screening by chlorophyll ofradiation absorbed by phytochrome in the chrysanthemum plant.Discovery of the incomplete inhibitory effectiveness of cyclicfluorescent illumination applied for daily 16-hr periods, althoughof little immediate horticultural interest, again raised questionsas to the photoreactions involved in controlling flowering ofchrysanthemum and led to the work herein reported.

MATERIALS AND METHODS

The cultivars of chrysanthemum used in these experiments wereImproved Indianapolis Yellow and White Pink Chief. They wereselected because they are responsive to short-day treatment, dif-fering in degree of response to artificial light in the night and inthe number of short days required for flowering. The plants weregrown in the greenhouse from rooted cuttings and were main-tained on photoperiodic conditions that assured their remainingin a vegetative condition until used experimentally. These condi-tions consisted of natural photoperiods and 4 hr of incandescentlight of 20 ft-c from 10 PM to 2 AM daily.

Plants were brought from the greenhouse to plant growthrooms where they received treatment for 10 consecutive days.During this 10-day experimental period, the general routine con-sisted of a daily 8-hr period of illumination, a dark period, oftenof 4 hr, but sometimes longer or shorter, and the remainder ofeach 24-hr period, usually 12 hr, of cyclic fluorescent light. Thecycles of the fluorescent light and darkness were 15 min except inTable II, where they were 24 min. In all cases, 10% of the cyclewas light and 90% dark. Intensity of the fluorescent light wasabout 80 ft-c. In previous experiments with other short-dayplants, cyclic lighting as applied here was completely inhibitoryto flowering, but in chrysanthemum it is sometimes completelyand sometimes incompletely inhibitory, depending on such condi-tions as length of a preceding uninterrupted dark period and kindsof interrupting irradiations applied during the dark period.During the 8-hr photoperiods, the plants received about 2000

ft-c of illumination from cool white fluorescent lamps and about80 ft-c from incandescent ones. The night temperature was main-tained at a minimum of 17°. At the end of the 10-day treatmentperiod, the plants were returned to the greenhouse and non-photoinductive conditions for a further period of growth anddevelopment. Most of the plants were dissected 2 weeks after the1st day of treatment. A few were continued in the greenhouseuntil certain of them bloomed (Fig. 1).As in previous work with chrysanthemums (3), we used either

two or three plants per treatment. Use of such small numberswas permitted by the great uniformity of the cuttings which werefrom selected clonal stocks of the respective cultivars. Threeplants per lot re uesed in Tables I, II, and III and two per lotelsewhere.

Experimental treatments involved the use of filtered and un-filtered light from both fluorescent and incandescent lamps. Un-filtered illumination from quartz-iodide lamps, which was usedin one experiment, is qualitatively very similar to that fromordinary incandescent filament lamps. Quartz-iodide lamps wereused because illumination intensities of 4000 ft-c from them weremore readily attainable than from the ordinary incandescent

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CATHEY AND BORTHWICK

FIG. 1. Inhibition of flowering of White Pink Chief chrysanthemum by cyclic lighting. Plants grown at 17 C and 8-hr photoperiods (8 AM to4 PM) and treated as follows during a 10-day experimental period: Above (left to right): 1.5 min of incandescent filament light (about 60 ft-c)every 15 min from 11 PM to 1 AM (2 hr); from 9 PM to 3 AM (6 hr); and from 4 PM to 8 AM (16 hr). Below (left to right): 1.5 min of fluorescentlight (about 80 ft-c) every 15 min from 11 PM to 1 AM (2 hr); from 9 PM to 3 AM (6 hr); and from 4 PM to 8 AM (16 hr). All lots received uninter-rupted long nights after the treatment period. Photographed 9 weeks after the start of the experiment.

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FLOWERING MUMS WITH FLUORESCENT LAMPS

ones. Further details of the illumination treatments are presentedwith results of the individual experiments.The terminal bud of each plant was dissected, and the stage

of influorescence development was observed and assigned a

number as previously described (2). These stages in brief were

as follows:

0: terminal meristem flat, plants vegetative1: terminal meristems dome-shaped2: terminal meristem becoming globose, first bracts present3: receptacle spherical with 12 or more bracts around its rim4: receptacle flattened on top, many bracts but no floret primordia5: two or three rows of floret primordia on rim of receptacle6: about six rows of floret primordia on receptacle7: all but center of receptacle covered with floret primordia8: receptacle completely covered with floret primordia9: central florets without perianth primordia

10: all florets with perianth primordia

EXPERIMENTAL RESULTS

A significant feature of the results presented in this paper isthe fact that nearly all treatments caused a marked reduction offlowering below that of unirradiated controls. Some treatmentsreduced flowering to stage 0. Most other treatments reduced itwell below the stage 10 of the dark controls but not to 0. Thispaper is mainly concerned with differences between these lowlevels to which the various treatments reduced flowering.The incomplete inhibitory effectiveness of daily 16-hr periods

of cyclic fluorescent light on flowering of chrysanthemum is il-lustrated by results in Table I and in treatment 1 of Tables II,

III, IV, VI, and VII. Plants that received cyclic fluorescent lightthroughout the 16-hr "dark period" flowered at a low level.That is, inhibition of flowering was not complete. In contrast,treatments in which the 16-hr periods consisted of 6 hr of dark-ness and 10 hr of CFL1 were completely inhibitory, reducing thestage of flowering from 10 to 0. In other experiments, insertionof 4-hr instead of 6-hr dark periods in the 16-hr periods of CFLalso resulted in complete prevention of flowering.The position of the short dark period in the 16-hr period in-

fluenced the inhibitory effectiveness of the remaining CFL (TableII). In general, greatest inhibition resulted when the dark periodoccurred early instead of late in the 16-hr period. The twocultivars were similarly responsive to these treatments, but inhibi-tion in comparable lots was greater for Improved IndianapolisYellow than for White Pink Chief.The duration of darkness inserted in daily 16-hr periods of

CFL influenced the inhibitory effectiveness of the treatment(Table III). About 4 hr were required for inhibition of ImprovedIndianapolis Yellow and slightly more for White Pink Chief.When the inserted short dark period was interrupted even so

briefly as for 1 min with high intensity (4000 ft-c) cool whitefluorescent illumination, the inhibitory effectiveness of the treat-ment was markedly reduced (Table IV). As the dark periods were

lengthened further, however, this effect of a brief light interrup-tion was not expressed; that is, the treatments remained fullyinhibitory.

Interruptions of the dark period with fluorescent illuminances,both greater and smaller than 4000 ft-c for 1 min, were tested.One half minute was as effective as 1 min at 4000 ft-c (Table V).Illuminances longer than 1 min at 1000 and 500 ft-c were alsoeffective, but precision of the experiment did not clearly showwhether reciprocity held.

Apparently, during the 4-hr dark period, changes occur in theplant that permit CFL to inhibit flowering completely in Im-

Abbreviations: CFL: cyclic fluorescent light; Pir: active form of

phytochrome.

Table I. Effects of Illumination from Inzcandescent Filament andCool White Fluorescent Lamps Applied for Various Periods in

the Middle of Daily 16-hr Dark Periods on Flowering ofWhite Pink Chief Chrysanthemum

Kind of Illumination and Period of Mean Stage ofMethod of Application Treatment Flowering'

hr

Incandescent:Cyclic 6 2.0Cyclic 16 1.0Continuous 16 1.0

Fluorescent:Cyclic 6 0.0Cyclic 16 7.0Continuous 16 4.0

Dark control ... 10.0

1 Meaning of numbers given in "Materials and Methods."

proved Indianapolis Yellow and nearly so in White Pink Chief.These changes were prevented and, as a consequence, floweringwas only incompletely inhibited when the dark period was inter-rupted by 1 min of high intensity (4000 ft-c) cool white fluorescentillumination. Moreover, it was also incompletely inhibited whenthe blue was removed by red cellophane from the 1-min, 4000ft-c illuminance that interrupted the 4-hr period of initial darkness(Table VI). However, when red was removed by blue cellophane,flowering was then completely prevented by the subsequent CFL.That is, the interruption of the dark period had no apparenteffect because, like the uninterrupted controls, the plants failedto flower. When a copper sulfate filter of a density adequate toremove the far red (but not the red) was used, the plants floweredas well as when interrupted with unfiltered fluorescent illumina-tion. A 1-min interruption by 4000 ft-c of illumination from aquartz-iodide lamp, however, did not prevent change during thedark period. This is evident because the CFL inhibited floweringas completely as in plants that received no interruption of thedark period.Response to far red and red radiations applied after a 1-min

interruption of the 4-hr dark period with 4000 ft-c of fluorescentillumination was clearly reversible (Table VII). Inhibition offlowering of the interrupted plants was complete when far redimmediately preceded the 12 CFL but incomplete when the 1 minof cool white or red illumination immediately preceded 12 CFL.In other experiments not shown here, reversibility of response tofar red and red radiations did not occur when such radiationswere not preceded by 1 min of high intensity fluorescent illumina-tion during the 4-hr dark period.

DISCUSSION

This paper is mainly concerned with a failure of fluorescentillumination to prevent flowering of chrysanthemums when it isapplied throughout the daily 16-hr dark periods between succes-sive 8-hr photoperiods. The failure is exhibited when illuminationis applied cyclically, but it occurs to some extent even whenapplied continuously.

It is remarkable, however, that the cyclic illumination becomescompletely inhibitory if preceded by about 4 hr of uninterrupteddarkness. Evidently a change occurs, during the 4-hr dark period,which increases the inhibitory effectiveness of the Pfr convertedduring the subsequent 12 hr of CFL. Attention, therefore, turnsto the nature of the change during the dark period.

Since the change is prevented by a high intensity fluorescentinterruption of the dark period, the effects of which are photo-reversible by red and far red (Table VII), it is evidently involved

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CATHEY AND BORTHWICK

Table II. Effects of Illuminiationt from Stanidard Cool White Fluorescen?t Lamps Applied Cyclically durinig Various Periods and at VariousInttenisities in Daily 16-hr Dark Periods oni Flowerinig of Two Chrysanithemum Cultivars

TreatmentNo. Treatment

1 Ir

2

' I _I

0 2 4 6 8 10 12 14 16Hours

AMean Stage of Flowering' at Indicated Levels of Illumination (ft-c)

Improved Indianapolis Yellow

7. 17.5 15 30 60 80-~~- 1 -

5.5

2.3

1.3

5.0

1.0

1.3

2.0 2.0

3.0 2.666 .0 3 .5

3.7

0.0

0.0,

0.0

0.01

1.0

3.0

0.0

0.0

0.0

0.0

0.0

1 Meaning of numbers given in "Materials and Methods."

3.0

0.0

0.0

0.0

0.0

Wi'hite Pink Chief

7.3

5.5

5.0

4.3

4.1

7.0

0.0 10.0

15 30 60

6.0 5.0 3.0

2.3

3.0

4.3

7.0

7.0

0.0

0.6

1.0

3.0

3.0

0.0

0.0

0.0

0.0

0.6

Table III. Effect of Various Durationzs ofDarkness Preceding CyclicFluorescent Illuminiation durintg Daily 16-hr Periods following 8-hrPhotoperiods on Flowering of Two Chrysantthemum Cultivars

Schedule of Treatments

16 CFL

1 dark 15 CFL

2 cIrk 14 CFL

3 darkj 13 CFL

4 dark 12 CFL

S dark 11 CFL

6 dark 10 CFL

7 dark 9 CFL

8 dark 8 CFLI

0 2 4 6 8 10 12 14 16

Hours

Mean Stage ofFlowering'

Improved WhiteIndiana- Pinkols

Chief

3

2.3

2.3

2

0.5

0

0

0

0

I Meaning of numbers given in "Materials and Methods."

with an action of phytochrome. However, the Pfr produced byCFL following a 4-hr dark period completely inhibits flowering ifthe dark period is uninterrupted but not if it is interrupted byfluorescent light. Moreover, response to an interruption of the

Table IV. Effect of Variolus Durationts of Darkniess Ending with orwithout Initerruptions with Cool White Fluorescenit Illuminationzanid Precedintg Cyclic Fluorescent Illuminiation during Daily

16-hr Periods between Successive 8-hr Photoperiods otnFlowerinig of Two Chrysanthemum Cultivars

AMean Stage of Flowering'

Treatment during16-hr Periods Indianapolis Yellow White Pink Chief

DC

4 16 CFLID + 15 CFL

4 2D + 14 CFL3D + 13 CFL

4 4D+12CFLSD + 11 CFL

3 6D + 10 CFL7D + 9 CFL8D + 8 CFL

22.52

i0

0

0

0

0

C\\'

332.530.50

0

0

DC

34.53.532.51.5000

cwl

5.56S

3.5210.50

I DC: Dark control; CW: 1 min 2000 ft-c of illumination fromcool white fluorescent lamps given at end of indicated dark periodsand preceding cyclic fluorescent illumination. Meaning of numbersgiven in "Materials and Methods."

4-hr dark period by a high intensity fluorescent illuminance iscompletely reversible by far red. Action of Pfr is thus detected inat least two parts of the 16-hr periods between successive 8-hrphotoperiods; and the actions are opposite.The change that occurs during the uninterrupted 4-hr dark

period apparently involves disappearance of Pfr. It seems thatsuccessful inhibition of flowering by CFL requires that the Pfrleft in the plant at the close of the photoperiod be allowed todrop to a low level for a time, and that Pfr then again be increasedby application of CFL. A 1-min high intensity fluorescent lightinterruption of the dark period at any time during the 4 hrprevented the response (failure of flowering) that otherwisewould be displayed.Most of the findings of these investigations are thus explainable

3

4

5

6

80

5.0

0.0

0.0

0.0

0.0

0.3

Treat-mentNo.

1

2

3

4

5

6

7

8

9

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FLOWERING MUMS WITH FLUORESCENT LAMPS

Table V. Effects of Cool White Fluorescent Interruptionts ntear Endof 4-hr Dark Periods That Are Followed by 12-hr Periods of

Cyclic Fluorescent Light on Flowering of ImprovedInidiantapolis Yellow Chrysanthemum

Mean Stage of Flowering, at

Duration of Dark Period Level of IlluminationInterruption

500 ft-c 1000 ft-c 4000 ft-c

min

0.5 0.5 2 31.0 0.5 2 2.52.0 1 3 38.0 2 3 316.0 3 3 3

Dark control = 0

I Meaning of numbers given in "Materials and Methods."

Table VI. Effects of 1-mimi Initerruptions of4-hr Dark Periods withIllumination of 4000 ft-c from Different Sources, Filtered as

Itndicated, oni Flowering of Two Chlrysanthemum Cultivars

Mean Stage ofFlowering2

Kind of Kn fFle mDark CFL Illumination ind of Filter hite

India- Pinknapolis ChiefYellow

hr hr

0 16 None None 2 64 12 None None 0 24 12 Cool white None 2 3.54 12 Cool white Red cellophane 2 3.54 12 Cool white Blue cellophane 0 1.54 12 Cool white CuS043 2 44 12 Quartz-iodide None 0 2.5

1 Illumination of 4000 ft-c for 1 min from cool white fluorescentor quartz-iodide lamps.

2 Meaning of numbers given in "Materials and Methods."3 CuS04 = 5 cm of 20 g/liter CuS04 in 0.5% H2SO. .

Table VII. Effects of I min of4000 ft-c of Cool White FluorescentIllumination Followed by Far Red and Red at End ofa 4-hr DarkPeriod and before a 12-hr Period of Cyclic Cool White

Fluorescent Illumination ont Flowering of TwoChrysanithemum Cultivars

Mean Stage ofFlowering2

Treatment' Im-proved WhiteIndian- Pinkapolis ChiefYellow

16 CFL 2 5.54D + 12 CFL 0 04D+CW+ 12CFL 1 4.04D + CW + FR + 12 CFL 0 04D + CW + FR + R + 12CFL 1 3.54D + CW + FR + R + FR + 12 CFL 0 0.54D + CW + FR + R + FR + R + 12 CFL 1 3.5

1 Hours of darkness (D) and cyclic fluorescent light (CFL).CW: 1 min of 4000 ft-c illumination from a cool white fluorescentlamp; FR: 1 min far red; R: 4 min red from standard red and farred sources.

2 Meaning of numbers given in "Materials and Methods."

on the basis of phytochrome action. The results obtained withcellophane filters are completely compatible if Pfr is consideredto be the active agent. The result with high intensity quartz-iodide illumination was also to be expected on the basis of earlierinvestigations (3) in which very high intensity 1-min illuminancesfrom a quartz-iodide lamp in the middle of 16-hr dark periodsdid not cause a response (inhibition of flowering). In the currentexperiments, a similar treatment likewise did not cause a response(in this case, the promotion of flowering). Although the resultsof these two different experiments were opposite in nature (thatis, failure to prevent and failure to promote flowering, respec-tively), the explanations are probably the same.The finding that Pfr action during different parts of daily 16-hr

dark periods leads to opposite results is consistent with observa-tions on other plants. Flowering of Pharbitis, for example, isinhibited by removal of Pfr by far red at the beginning of thedark period or by its reintroduction by red light in the middle ofthe dark period (5). Xanthium exhibits similar responses butonly when the photoperiod is reduced to about 2 hr (1). Oppositeactions in Eragrostis seed germination of Pfr introduced by redradiation at different times during dark inhibition have also beenobserved (7).A crucial point in these results is that high intensity interrup-

tions of the 4-hr dark period with fluorescent and incandescentlight lead to opposite results (flowering and nonflowering, re-spectively). Fluorescent light is essentially red and incandescentis a mixture of red and far red. The far red in the presence of redthus prevents display of a response (flowering) that appears withred light alone. This action does not result directly from responseto the Pfr torm of phytochrome which probably exceeds 50%O Pwith both types of radiation. It rather appears to involve far redradiation per se. The phenomenon resembles the opening ofMimosa pudica leaflets in light against the closing action of Pfr(4) or the inhibition of germination of seed of Poa pratensis andAmaranthus arenicola by far red but not by red alone (6). Thetwo responses were attributed to a high energy reaction having amaximum in the far red near 720 mu.The difference in response of chrysanthemums to the two kinds

of light was ascribed in an earlier paper to effects of light screeningby chlorophyll (3). Similarities of the chrysanthemum responseto the above mentioned leaf movement and seed germination re-sponses make consideration of a high energy response in chrysan-themum necessary. Although it seems unwise to decide in theabsence of an action spectrum whether or not a high energyreaction is involved, the participation of the ordinary phyto-chrome reaction is clearly evident.

Acknowledgments-We thank S. B. Hendricks for many helpful discussions duringthe course of this work and the preparation of the manuscript. The chrysanthemumcuttings were provided by Yoder Brothers, Inc., Barberton, Ohio.

LITERATURE CITED

1. BoRTHwicK, H. A. AND R. J. DowNs. 1964. Roles of active phytochrome incontrol of flowering of Xanthium pennsylvanicum. Bot. Gaz. 125: 227-231.

2. CATHEY, H. M. AND H. A. BORTHWICK. 1957. Photoreversibility of floral initia-tion in chrysanthemum. Bot. Gaz. 119: 71-76.

3. CATHEY, H. M. AND H. A. BORTHWICK. 1964. Significance of dark reversion ofphytochrome in flowering of Chrysanthemum morifolium. Bot. Gaz. 125: 232-236.

4. FONDEVILLE, J. C., M. J. SCHNEIDER, H. A. BORTHWICK, AND S. B. HENDRICKS.1967. Photocontrol of Mimosa pudica leaf movement. Planta 75: 228-238.

5. FREDERICQ, H. 1964. Conditions determining effects of far-red and red irradia-tions on flowering response of Pharbitis nil. Plant Physiol. 39: 812-816.

6. HENDRICKS, S. B., V. K. TOOLE, AND H. A. BORTHWICK. 1968. Opposing actionof in Poa pratensis and Amaranthus arenicola. Plant Physiol. 43: 2023-2028.

7. TooLE, V. K. AND H. A. BORTHWICK. 1968. The photoreaction controllingseed germination in Eragrostis curvula. Plant Cell Physiol. 9: 125-136.

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