Plant Physiol. (1969) 44, 1402-1406

Flower Formation in Excised Tobacco Stem Segments; I. Methodologyand Effects of Plant Hormones'William L. Wardell2 and Folke Skoog

Institute of Plant Development, Birge Hall, University of Wisconsin, Madison, Wisconsin 53706

Received May 6, 1969.

Abstract. The formation of flowers has been studied in stem tissue excised from floweringplants of Nicotiana tabacum variety Wisoonsin No. 38, and cultured in vitro on Murashige andSkoog nutrient medium. A procedure for quantitative evaluation of factors influencing floralexpression has been developed and effects of the growth substances, indole-3-acetic acid (IAA),kinetin and gibberellic acid (GA,), on the process are reported.

Although a low (1 juM) level of IAA was required for the development of normal flowerson stem segments, higher concentrations tended to inhibit flowering. The decrease in floralbuds was rapid in the 3 to 15 AM range. IAA concentrations up to 75 aM increased vegetativebud formation so as to effect a transition from floral to vegetative buds rather than merely aninhibition of bud formation. Higher IAA concentrations inhibited both vegetative and floralbud formation.

Kinetin in high concentrations greatly increased the number of vegetative buds but had nosignificant effect on the number of floral buds per segment. High kinetin concentrations alsopermitted branching of floral shoots so that flower clusters were formed.

GA3 applied in the medium from the start, strongly inhibited bud formation on the stemsegments, but when applied to young floral buds after they had formed, it promoted theirfurther development ("bolting").

Flower formation occurred in complete darkness, but light of moderate intensity was requiredfor the development of normal flowers.

Experimentation on the physiology of floweringhas been limited mainly to whole plants, but a newapproach to regulation of flowering has been openedwvith the dcemonstration that under certain conditionstobacco stem segments cultured in vitro form floralinstead of vegetative buds (12, 13). The discoveryof Chouard and Aghion (6) that tobacco stem seg-ments excised from the flowering part of the stemform predominately floral buds when cultured in vitrosuggested experiments which have led us to a pro-cedutre for studying flowering in excised tobacco stemtissue. This procedure together with representative(lata and conclusions from a 5 year qLuantitative studyof the effects of growth substances and other factorson flowering (14), will be presented.

1 Supported in part by the University Research Com-mittee of the Graduate School with funds from the Wis-consin Alumni Research Foundation and by the NationalScience Foundation with grants G-24038 and BG-6994X.

2 Present address: The Institute for Cancer Research,7701 Burholme Avenue, Fox Chase, Philadelphia, Penn-sylvania 19111.

Materials and Methods

In preliminary experiments it was found that theMurashige and Skoog "revised medium" (11) minusCoCl, and with 1.4 poI indole-3-acetic acid (IAA)and 7.5 X 10-2 gm kinetin would serve as a standardmedium for culturing stem segments. Stock solu-tions of ingredients were kept separately refrigeratedfor use no longer than 6 weeks. Thiamine-HCl andIAA solutions were prepared fresh for each experi-ment. Kinetin stock solutions (46 juM) were keptrefrigerated and used up to 3 months. Requiredvolumes of thiamiine-HCl, IAA and kinetin stocksolutions were pipetted into the meditum. Sucrosewas added to give 30 g/l. The pH was adjusted to5.5 with 1 N NaOH, and agar was added to give10 g/l. The meditlum was heated for 20 to 30 minin an Arnold steamer to melt the agar and was thenpipetted into the culture flasks. After autoclavingat 120° for 20 min the flasks were cooled rapidly ina stream of cold water. When applicable, gibberellicacid (GA3) stock solution was prepared fresh,sterilized throtugh a Millipore Filter and added asep-ticallv to each culture flask after it was autoclaved

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WARDELL AND SKOOG-FLOWER FORMATION ON STEM SEGMENTS. I.

and before the agar gelled. Planting was donewithin 24 hr after autoclaving.

Stem tissue was obtained from greenhouse plantsraised as follows. Nicotiana tabacum var. "Wiscon-sin No. 38" seeds were sown in flats of rich soil.After about 4 weeks growth, the young plants weretransplanted into 8 inch pots and grown under sup-plementary continuous light from 200 W Championlight bulbs located 50 inches above the pots. Afteran ad(litional 6 to 8 weeks the plants flowered. Zeroto 3 days after anthesis of the first flower 15 to18 inches tops were cut, their leaves and flowersremoved and the stems swabbed with a weak Alconoxsoap solution. The stems were surface sterilized by"dipping" them into a full strength Chlorox solution(5.25 % sodium hypochlorite) for about 10 sec andthen quickly transferring them into sterile, distilledwater in a 500 ml cylinder. The cylinder was cov-ered with parafilm and agitated gently. The waterwas then poured out and replaced by another batchof sterile, distilled water. To remove the chloroxthis rinsing procedure was repeated about 10 times.The cylinder was then carried to a transfer roomwhere all subsequent handling was done aseptically.

To prepare segments, a stem was removed fromthe cylinder, shaken to remove excess water, "dipped"for a few sec into 95 % ethanol and flamed. Thisprocedure effectively surface sterilized the stemwithout injuring the inner tissues. A scalpel wasused to peel off the outer layers down to the cambiumof the upper internode, which was then cut off andplaced in a numbered, sterile petri dish. Successiveinternodes were similarly peeled and transferred topetri dishes. About ten 0.2 cm segments were thencut from each internode. One segment each frominternodes 1, 2, and 3 (see Fig. 1) was planted in-verted on 50 ml of medium. '(Due to polarity thesegments with the apical surface in contact with themedium differentiate more uniformly). For com-parison, 1 segment from internode 9 and 2 segmentsfrom internode 10 (see Fig. 1) were also plan-tedinverted on 50 ml of medium. By this procedure10 cultures with 30 segments from internodes 1 to 3and 10 cultures with 30 segments from internodes9 to 10 were obtained from each plant. A typicalexperiment with 120 cultures, therefore, required 6plants. As far as possible the segments from eachplant were distributed evenly between the differenttreatments.

The cultures were grown for 6 weeks in anenvironmental room at 24 to 26%, 40 % relativehumidity and in continuous white light, of 6500 luxintensity at the level of the cultures, from a bank ofChampion cool-fluorescent lamps. After a 6 weekgrowth period, the segments were harvested andobservations on organ formation and growth wererecorded. All buds were scored as either floral orvegetative, and no attempt was made to furthercharacterize buds as to size or stage of development.After the buds and roots had been removed the

..:

.W 3

i

i.L

11

1 rf

_ C-ej!eIC

FIG. 1. A typical plant used for preparation of stemsegment cultures. Arrows mark the internodes whichwere used.

remaining tissue was weighed as rapidly as possible,dried in a forced-air oven at 600 for 1 week andreweighed.

Results

As reported by Aghion-Prat (1) small floral budsformed on stem segments from the uppermost inter-nodes in cultures grown in the light on the revisedmedium devoid of auxin and cytokinin, but we foundat least 1 ,uM IAA to be necessary for developmentof normal flowers and mature seeds. Furthermore,in contrast with her finding that meristem initiationrequires light, we found that segments (1 cm) cul-tured in the dark form both vegetative and floralbuds on medium with low (1 jM) IAA. However,in darkness floral buds never developed into normalflowers.

As shown in Fig. 2, increasing the IAA concen-tration of the medium, in the range from 3 to 103 KM,decreased the number of floral buds formed from2.3 to 0.2 per segment. Although the curves inFig. 2 are not smooth, they show a rapid drop

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PLANT PHYSIOLOGY

zw

2.0wtt 1.5 FLORAL1%)D 1.0 C+}

0

00.5-

O10 I I I I I I I

0 20 40 60 80 100 120IAA CONCENTRATION (,pM)

FIG. 2. Effect of IAA concentration oIn formationof floral and vegetative buds. The medium contained7.5 X 10-2 yNt kinetin. Growth period: 1/27-3/12/68.

followed by an apparent plateau in the number offloral buds per segment; smoother curves were ob-tained in the 20 to 70 uM range in other experiments.Following the steep initial drop in floral buds anincrease in vegetative 'buds in response to IAAtended to compensate for the drop in bud numberso that in the 15 to 75 Mm range IAA effected atransition from floral to vegetative buds rather thanmerely an inhibition of bud formation.

To study the interaction of growth substanceswith reference to flowering, stem segments werecultured on media with IAA and kinetin in com-binations such that each substance was tested inserial concentrations and in IAA/kinetin molar ratiosof either 1.2, 12, or 120. The data show again thatmoderate increases in IAA concentration led to adecrease in floral buds and an increase in vegetativebuds, and that higher concentrations inhibited bud

Table I. Effects of the Concentrations antd Ratio of I

formation. However, as the cytokinin concentrationwas increased the number of vegetative buds wasgreatly increased with little or no effect on thenumber of floral buds per segment. Representativedata from 2 experiments are summarized in table I.

High kinetin concentrations had the interestingeffect of causing branched floral shoots, i.e. throughrelease of apical domninance inflorescences with 3 to 5flowers instead of single flowers developed (Fig. 3).Catarino (3) has reported increased size and branch-ing of inflorescences as a result of spraying intactBrVophyllum plants with kinetin soltutions.

Low (10-4-10-3 MM) GA3 concentrations had no

effect on the cultures, but 10-2 LM GA3 doubled thefresh weight of callus and reduced the number ofbuds formed to one third that in controls. Higherconcentrations of GA. completely inhibited budding,aind promoted growth of callus wvith an optimumincrease at 1 feM GA3. These results are in agree-ment with earlier ones on callus growth (11) antibudding (10). Similar inhibition of bud formationby GA3 wvas obtainied also in the absence of addedIAA. As expected, however, no significant increasein callus growtlh was obtained with increased GA3concentrations in the absence of IAA.

Recent evidence indicates (8) that the action ofGA3 in flower formation is mainly on bolting. Onthis basis, preformed floral stalks on tobacco stemsegments treated with GA3 should mimic the boltingphenomenon observed in intact plants. To test thispossibility, stem segments were cuiltuired for 7 weeks,after which time segments with young floral budsof comparable size and development were removedand floated for one-half lhr in petri dishes containingeither filter sterilized 10 ,Ix GA3 or water. Thesegments were then transferred to test tubes withfresh medium and( allowed to grow for 9 days.Typical effects of the GA3 treatment as comparedwith controls are illtustrated in Fig. 4. When thesame concentration (10 MiM) of GA3 was added to

MA4 to KiA7etint oi Uc(/ctat-.:- aind Floral Biu(d Formation

NUmbler of bud1(1s per segmiielntInternode 1-3 Internodes 9-10

Experiment IAA/Kn IAA Kn Floral Veg. Total Floral Veg. Totalratio /AM

2.11.2 2.9

4.35.7

11.4

/AM1.72.33.54.79.4

1.9 0.12.4 0.12.2 0.31.8 0.61.2 .

2.02.52.52.4

1.. .

0.5 0.50.5 0.60.4 0.60.2 1.00.2 l.1

1.01.11.01.2

1. .

21.0 0.17120 29.0 0.23

43.0 0.3557.0 0.47

1.6 0.8 2.4 0 1.4 1.41.9 1.1 3.0 0.1 1.1 1.21.5 0.4 1.9 0.1 1.5 1.60.9 0.6 1.5 ... 2 ...2 2

I More than 100 buds.2 Very little differentiation.

date

#27(1/27-3/7/68)

#64(2/4-3/18/67)

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WARDELL AND SKOOG-FLOWER FORMATION ON STEM SEGMENTS. I.

FiG. 3. Flowers formed in cultures with lowkinetiii; (Riglht) 4.3,Nr IAA anld 3.5 jui/t kinetin.

the culture medium on which the segments were

grown initially it completelv inhibited bud formation.Attempts were made to demonstrate a differential

effect of GA3 on inhibition of bud formation andflowering. As shown by a typical experiment (tableII) as little as 0.01 uM GAS drastically reducedbudding, and had no differential effect on the numberof floral and vegetative buds. On the other hand,GA3 concentrations higher than required for com-plete bud inhibition still promoted the growth ofcallus in the cultures.

E1! s lI I sX.. I .I_ ;;!_ !J_ Y. s

WaI ||g_ l 1111e- a p:i

i !s ss

_ ALsand high kinetin concentrations. ( T eft) 1.4 ,LM IAA and 0.08 SsAI(;rowth period: 1/22-3/7i68.

Discussion

It is generally considered that a floral stimulus,florigen, is required for flowering to occur in plants,and much indirect evidence has been presented forthe presence of florigen, at least in photoperiodicallysensitive plants (4, 8). It is now considered that2 distinct substances, anthesin and gibberellin,. areincluded in the term florigen (4, 5). It is empha-sized that the present study deals not with theinduction of the capacity to flower but with the

Table II. Inhibiting Effect of GA3 on Bud FormationThe medium contained 1.4 1AAr IAA and 7.5 X 10-2 ,AM kinetin. Growth period was 5/5-6/23/67.

No. of segments No. of buds/segmenti FreshGA, Total Differentiated Floral Vegetative Total Wt2

g/segment0 48 37 1.4 0.2 1.6 0.48

10-4 30 23 1.2 0.3 1.5 0.6910-s 23 16 1.5 0.2 1.7 0.5710-2 30 8 0.5 0.0 0.5 0.9910-1 30 2 0.0 0.0 0.0 1.521 30 1 0.0 0.0 0.0 1.75

10 30 1 0.0 0.0 0.0 0.S41 Based on number of differentiated segments.2 Buds and roots removed before weighing.

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PLANT PHYSIOLOGY

fact, bring about a transition from floral to vegetativebuds, and higher concentrations inhibit formation ofboth kinds of buds.

The effectivene?s of IAA in inhibiting buds isinfluenced by kinetin, but kinetin appears to have nospecific effect on flower formnation. By releasingapical dominance kinetin also permits the formationof inflorescences ratlher than single flowers.

GA, applied to the stem tissue strongly inhibitsbud formnation, but at low concentrations where budformation occurs it does not alter the ratio of floralto vegetative buids. Wheni supplied to young floralbtuds GA(r markedly prolmlotes the development (en-largement or "bolting") of flowers.

I4r

W, P., .

FIG. 4. Effect of GA, treatmient of floral buds on

their developmient. Upper row 10 jum GA 3. Lower row

water controls.

expression of the comipetence to flower by tissue

excised from plants in the flowering state.

Since the first finding of delayed flowering in

pineapple plants sprayed with auxin (7) there have

been several reports that IAA tends to delay flower-

ing (2, 9). The present results extend this effect

to excised stem tissue. Selective effects of IAA on

floral and vegetative bud formation have been dem-

onstrated. Moderate concentrations of auxin do, in

Literature Cited

1. A(;nIfoN-PRA'T, 1). 1965. Neoformation de fleurs invitro chez Nicoti(anaa tabacoiw L. Physiol. Veg. 3:229-303.

2. BONNER, J. AND J. THURLOWN'. 1949. Inhibition of

plhotoperiodic inductioni in Xanthiiiui by appliedauxin. Botan. Gaz. 110: 6!3-24.

3. CATARINO, 1F. M. 1964. Soimie effects of kinietini onisex expression in B ryophliUlin crentatumiii Bak.(LSDP). Port. Acta Bil. (A) 8: 267-84.

4. CHAILAK11YAN, MI. KH. 1964. Florigens, gibber-ellins and anitlhesins. In: Regulateurs naturels dela croissalncf \-gV tale. C(ll '(u11o I iternlatiollaux.389-596. Centre National (le la Recherche Scien-tifique-Gif sur Yvette, FIraince.

5. CHAILAKTIYAN, M. KJT. 1968. Flowl-ring hor-

mones of planits. '1317-40. Tin: Biochemistry and

Physiology of Plant Growth Substances. F.

Wightman and (f;. Setterfield, eds. The n'gtilgoPress Ltd., Ottawea, Canada.

6. CIfOlUARI), P. AN'\D D. AGIIION. 1961. M!I dalit6s(le la formiati n (le hm,tirgeons floraux sur d&s cul-

tures (Ib segmlients de tige d- tabac. C. R. Acad.Sci, Paris 252: 3Q64.

7. CLARK, H. E. AND K. R. KERNS. 1942. Controlof flowering with plhvtohormiones. Science 95:

53637.

8. LANG A. 1965. Physiology of flower initiation.In: Encyclopedia of Plant Physiology. W. Ruh-land, ed. 15: 1380-1536. Berlin-Heidelberg-NewYork: Springer-Verlag.

9. LIVERMANX, J. 1L. 1955. The plhysiology of flower-ing. Ann. Rev. Plant Physiol. 6: 177-210.

10. MIURASHIGE, T. 1964. Analysis of the inhibitionIof organ formation in tobacco tissue cultures bygibberellini. Physiol. Plantarunm 17: 636-43.

11. MURASHIGE, T. AND F. SKOOG. 1962. A revisedmedium for rapid growuth and bioassays withtobacco tissue culttures. Physiol. Plantarum 15:473-97.

12. NIEDERGANG, E. 1954. Chenmical modification ofpolarity and auxin transport iii plants. Ph.D. The-sis, Uiniversity of \Visconsin, MIadison.

13. SKOOG, F. 1955. Growth factors, polarity and mor-phogenesis. Ann. Biol. 31: 1-11.

14. WVARDELL, W. L. 1968. The effects of growth sub-stances and antimetabolites on flower initiationin vitro. Ph. 1). Tllesis, University of Wisconsin,Madison.

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