Plant Plhvsiol. ( 1966) 4/1 856(-862

Reduction of the Gibberellin Content of Pharbitis Seeds by CCCand After-Effects in the Progeny'

Jan A. D. ZeevaartDepartment of Biology, McMaster University, Hamilton, Ontario, Canada

andMSU/AEC Plant Research Laboratory, Michigan State University, East Lansing, Michigan

Received Deceember 20, 1965.

Summaryw. I'laints of Phlibitis niil were treated with the growth retardant (2-chlioro-ethyl) trimethvlaminmoniutm chloride (CCC ) shortly before andl after anitlhesis. Freshan(l dry weight of imimiature seeds were not affected by the CCC treatmenit.

The level of gibberellin-like activity in Pharbitis seeds as compare(d to control seedswas strongly reduced by CCC application. The progenies of the treated plants alsohadl a mulichl redulced GA content in the seedling stage. These results are interl)retedto indicate that CCC blocks gibberellin biosynthesis in higher plants, as it does in thefunlgus FPusariiuum1.

CCC applied via the roots accumulated in the immatuire seeds and was carrie(d oveerto the following generationi. Consequently, growth of CCC progenies wvas dwarfe(danid flower formationi inhibited. Both phenomena were overcome b)y application ofgibberellin A..

Three gi,bberellin-like substances (called fractions I, II, alnd 11) were present in1'lharbitis seeds and could be separated by thin-layer chromatography. All 3 fractionswere also presetnt in seeds treated with CCC. Fractions II and III were present inmtuch higher quanitities than fraction I. Both fractions II and III promoted growthof (15 corn but only: fractioni II was active in dwarf peas grown unider red light.

Plant growth-retarding chemicals form a groupof chemiiically unrelate(d compounds which have thecommoni characteristic of inhibiting stemn elongation(4). This effect is usually overcome by the appli-cation of gibberellin (GA).

A breakthrough concerning the mode of actionof growth retardants was micade by the discoverythat Amiio- 1618 [2'-isoprolvl-4'- (trimethvlam.inmoniumchloride) -5'-methylphenyl piperidine- 1 -carboxylate]an(d CCC [ (2-chloroethyl) trimethylammonium chlor-ide] inhibit GA biosynthesis in the fungus Futsariumiiimnoniliformne (10, 15). This led to the hypothesisthat growth retardants also block GA production inhiigher plants, thus causinig a GA-deficiency andsubsequent dwarfed growth. In stupport of this ideaI larada and( Lang (7) fotind good agreemiielnt be-tsveen the effect of CCC analogs onl growtlh of hiigherlplants and their capacity to block GA production inFusarinni. IFrthermliore, Baldev et al. (1) showedlthat the GIA conitent in (levelop)ing pea see(ls wvassig-nificantlv re(duice( ly .\no- 1618.

Imimiiiatuire see(ls of Phlarbitis Wii are known tohave a high content of GA like substances (16).Moreover, Pharbitis plants respond readilv with

1 This work was suipported bx the Natioial ResearchCoutncil of Cancada a.nd by the Unite(d Stattes Atomic En-ergy Commission, Contract No. AT (11-1) 1338.

85S6

dwarfed growth to applications of CCC (21). IIor(ler to test the hypothesis that CCC exerts itseffects on l)lant growth by blocking GA p)roductioll,a study of the effect of CCC on the GA conitenit ofPhlarbitis wvas uncdertaken. The evidence to be pre-sented in this paper shows that CCC inhibits theaccumiiulation of GA-like substances in Pharbitis seedswithotut affecting seed development. A preliminaryreport of this work has beeni presented previouslv(22).

Material and Methods

Plaiit Growth. Seedlings of the short-(lay plailtPhli(rbitis ll, strain Violet, were growni in 450-milplastic beakers filled with a mixtuire of equial volumesvermicuilite and( coarse sand(l. T'hev were watered(lailv withi lialf-strenigthi Hoaglanl solution colntaini-inig 10 mg/liter Fe as seqiuiestrenle N.A\Fe 13 %, I'e(G(eig,y Indi(iustrial Clhemiiicals).

Oni the fourthi (lav after gcrminationi the seedling-swere transferre(d to a growth iroom kept at a conistanttenmperature of 25°. In order to induce flower for-mation the photoperiod was 10 hours and consistedlof 8 hours fluorescent light (Sylvania cool wlhite VHOfluoirescenit tubes, approximately 900 ft-c at plantlevel), followe(d 1y 2 houirs itncan(lescent light (1 50ft-c) .

ZEEVAART-CCC AND GIBBERELLIN IN PHARBITIS SEEDS AND PROGENY85Progenies of plants treated with chemicals were

grown in 240-nil plastic beakers under a light regimeof 16 hours fluorescent light, supplemented with 4hours incandescent light at 230.

Applicationt of CCC. Treatments with CCC werestarted shortly before opening of the first flow%ers,uisually arounid 30 days after germiination. The firsttreatment consisted of an application of 100 mg CCC(20 ml of a 5000 mg/liter solution, i.e. 3.2 X 10-2M)v,ia the roots. Treatments were repeated everv fifthclav with doses of 50 mg of CCC until a total ofeither 250 or 350 niig had been applied per plant indifferent experiments. Control plants were treatedwith distilled water.

Seed Harvest. Flowers wvere labeled with papertags on the morning of opening. The fruits verecollected at designated times after anthesis. anid thefresh weight per seed was determined. The seedswere next frozen in liquid nitrogeni, lyophilized, andthe dry weight determined. They were stored instoppered vTials at -200.

Sced Extr-actioni. Samples of 5 seeds were ex-tracted several times with methanol at 20 and subse-queltly twice at room temperature until the residueNvas colorless. The combined extract was evaporatedat rooml temperature to dryness under reducedpressure.

Thini-laver Chromatography. The residue of seeclextracts was applied in a small volume of miiethanolas a 1-cm wide band to the origin of a 20 X 20 cimsilica gel H thin-layer plate, which was aipproximiatelv400 ,u thick. The plate wvas developed to 15 cmii ina mixture of chloroformii-ethyl acetate-acetic acid(tO: 40: 5) as the solvent (19). The chromatogramWas divided inlto 10 equal zonies, the silica gel scrape(doff aiid elute(d twice with wet ethyl acetate and( onlcewith 95 % (v/v\) miiethanol. The eluate was evap-orated and each samiiple taken up in 1 nil waterconitaininig 0.05 % Tweeni 20 for bioassay.

E-xtraction of Scedlitngs. Seedlings were ctut offat grouniid level, frozen in liquid nitrogen an(d lvo-philized. The dry miiaterial was homiiogenized withmethanol in a WN"arinig blendor at 20 and extracted3 times. Lipid material was removed by mixingequal volumes of petroleum ether (B.R. 38-580)and methanol extract at 20 and adding water to make80 % methanol. Partitioning was repeated until thepetroleum ether was colorless. The aqueous meth-anol was concentrated under reduced pressure untila water residue was left and 1 M potassium phosphatebuffer (pH=8.2) added to give a 0.1 M concentra-tion. The buffer was partitioned twice against ethylacetate and then adjusted with 6 N HCl to pH 2.5.The acidic fraction was prepared by partitioning 3xagainst ethyl acetate, drying over sodium sulfate andcvaporating under reduced pressure. The acidicfraction was further purified by TLC as describedfor seed extracts.

Bioassays. The dwarf corn d5-mutant served asthe main bioassay plant. Segregating material sup-pliedl by Dr. Phinniev xx-as used in somne assays. In

others homozygous d5 (sibbed seed from GA3-treatedparents) was used as assay material.

Additional testing was done on dl-corni, and on1dwarf peas, Progress No. 9, dwarfed bv either redlight, or by 200 mg/liter Amo-1618 in darkness.The procedures and growiing conditionis for allassays were similar to those described bv Kende anidLang (9). The results are expressed as total GA3-equivalents per seed.

CCC Deternihiationts in Seeds. 'Methanol extractsof seeds were concentrated to a known volume (us-ually 2 or 3 ml) and spotted with a microsyringeon a cellulose powder MN 300 thin-layer plate (2)along with standard series of CCC and choline chlor-ide. The plate was developed to 10 ciii in a mixtureof butanol-acetic acid-water (4: 1: 1.8). After spray-ing with niodified Dragendorff reagent (20) theCCC in the seed extract was determiiined semi-quan-titatively by visual comiiparison with the standardseries.

For preparative work the miiethanol extract from10 seeds was purified by descendinig chromatographyon WNrhatnian 3 M1\J paper vith butamiol-acetic acid-water (4: 1: 1.8) as the solvent. A zonie at theRF value for CCC (0.57) was eluted with 80 %(v/v) ethanol. The eluate was re-chromatographedonl W'hatnian 3 MM usin1g chloroformii-miiethalnol-water (75: 22: 3) as the solvent (2). The zoneat the RF value for CCC (0.39) was n0ow eltutedwith water. The eluate was nmade up to a volumeof 100 ml, applied to Phaarbitis seedlinigs via theroots (21). aiid compared with the growth retarda-tioil catused by a series of CCC conicelntrationls rang-ing fromi 0. 4. 8. .. to 40 mg/liter.

Results(Jibberellimi Content of Seeds. 'T'he results for

freslh anld drV weiglht of Pharbitis seeds lharvestedfrom plants treated with CCC anid fromii conitrol planitsare presellte(l in figture 1. Tt is clear that CCC treat-

150-

-120 /o50w(

f-R WT

290 40

I 30ZC

w60---DRY WT zo3

U. 30 ' '

0 9 12 15 18 21 25 30 35DAYS AFTER ANTHESIS

FIG. 1. Effect of CCC oIn fresh weight and dry weightof Pharbitis seeds. CCC treatment: 350 mg applied perplant via roots. First application 2 days before begin-ning of flow-ering. 0- - - -0. Control;350 mg CCC applied, per plant.

857

PLANT P'HYSIOLOGY

ient lhad lio effect oln seed weight. Fresh weightreached a maximum around 21 days after anthesis,tlheni started to decrease. Dry weight increased al-most linearly with time for at least 35 days. Har-v-estinig was discontinued when the color of theseed coat turned black at 35 days after anthesis.

Results of thin-layer chromatograms of seed ex-tracts assayed on d5 corn are given in figure 2.Two major peaks of GA-like activity were detectedat RF 0.2 to 0.3 (fraction II ) and RF, 0.4 to 0.6(fraction III). Considerable activity was also foundin the zon0e which ilncluded the origin (fractioni I).

RFFI1;. 2. Thin-laver chrolimatogramils of Plharbilis seed

extracts. Solveent: Chloroform-ethyl acetate-acetic acid(60:40:5). Seeds harvested 25 days after ainthesis. Tenlfractions vi ere tested oni d5 corin. Lengthi of first +secoind leaf sheathi ill conitrol 41 mim. Conitrolseeds;- 350 mg CCC applied per plaiit.

All 3 fractions of GA-like substalnces xN-ere alsopresent in seeds fromii CCC treated plants, but inmuch reduced quanitities (fig 2). The total amiiountof GA-like miiaterial in the conitrol was 0.94 Mg GA3equivalenits per seed and in + CCC only 0.02 Mjgper seed, whereas the dry w-eights per seed were 44.0anld 45.2 mg, reslpectively. It is clear from theseresults that the content of GA-like substances presentinl Pharbitis seeds Nas greatly re(luced by CCC treat-ment, althoughI the seed weight was not affected

at all.The extracts wNhich yielded the results lpresented

inl figure 2 were fturtlher tested oni dwarf peas growintunider red light (fig 3). Extra stenii elonigationi was

obtained only after application of fraction II -wlhereasfractioni III was almiiost completely inactive. Thetotal amiiount of G.A-like miiaterial in fraction II (RI,0.2-0.3) of the conitrol as calcuilatedI ill figures 2

anid 3 as 0.44 Mug arid 0.20 Mg- GA. equivalents,

zF4:X L~~~~~~~~~~~~~~~~~~~C20-~~~~~~~~~~~~~~~~~~-

0~

0 0Q2 0.4 0.6 OB 1.0

RwFFIG. 3. Sain1e tI1iin-Iaver cliroi11atograi11s a.s ill figUire

2. 1FXractions tested oii dAu arf p)eas; growii Ml red light.I2C0 ntrol seeds; 350 i31g CCC applied

per1 pulant.

respectively . Thus, this fractioii NN-as abotit equallyactive ill both assays. On the other hand, fractioiiIII was highly active in the d5,- corni assav (fig 2),lout practically inactix-e hNleil tested oni dxN-arf peas(fig 3). E+xpressed in GA, e(juivraleints, fractioii IIIof the cointrol coiitaiined 3.8 aind 0.015 ,ug, respectively,as (letermiiiied in the 2 bioassavs.

Sinice highest GAN activrity was obtained oli d.5corin, further quantitative deterim-inations wsere carriedotit u1siilg th-is plant for b)ioassay. A.- erage vraluesof GAN deteriiiinationis iiiade on duplicate saniples ofseeds lharvested froi-ii 9 to 35 days after aintliesis areplotted ill figure 4. G.N-like activrityr was quite hligh

0 .0

in 9-day-old see(ls, thein declined uintil tlle fifteeiitlhdaF. al3 increasetlto a secomdiiiaxitograms at 21 daf s2Frhich coinscided onith i axiiial fresh nweight (figh).The peak at 21 dats fracsde totwae higab activityihu fractiocllyI. GiAcotitew t of CCC-treate(r seedsde(ireased steadilx after 9 davs alduasaleavsmatililoferthaec in conitrol seed3s.

G;ibbci-elliii Conltenat of Sce(iliiigs. It ,vas reportedpretriously (21i) that t2be colatelt of GA-like sub-stainces in PglarbitiG atiit was inot affectedbondCCC f-her aharvested 40 hours after the beginnied-of trealtmenlt. As will be showln below the growthretardant CCC is canrried overio cosiderable qualu-titiesbG the seeds to the follonuing ieneratioal. Sofin these seedlings CiCC is present from the onset ofpgermination a.dthus GA productionvitywht be blockedday.ore effectively than in case thecmaexiical is applied\-ia the roots at a later stashe.

Control and CCC progent ies C-teregrowae sndercOratilsedOS liglt andftarvested oan the fifthdal after

plaowtieg.Tr e results in table I show that the seightof the CCC proveny() asslitc t1 less tha-l that ofthe control. This was due to the shorter hypocothls.The aeran ehC pocotyl lendtlv inas 50 and 38e em inthe conitrol anid CCC progenyi respectivelg. Acidicfractions plirified bhavieaiis of thin-lafer chromya-

) S8)8, -

ZEEVAART-CCC AND GIBBERELLIN IN PHARBITIS SEXE"DS A\ND P'ROGENY

2.0 , 0% tography vere assayed oli d5 corn. Results in tablcI show that the total GA-like activity in the acidicfraction of the CCC progeny was reduced bvy 9 %.

;1.6 - 4 | \ In all seedling extracts tested the activity presentCOtNTROL in fraction II was higher than in fraction III.pl ;--CONTROL Gr-owth antd Flower Formitation of Progeniies.

1.2 I Comparisoni of steni growth of control andcl CCC0%t~O / progenies in long days revealed (fig 6) that the

,0.8 /> ,, \ latter exhibited a dwvarfed grovth habit and reachedX sX /X a linear grow th rate much later than the conitrol.Such a growth curve is typical for CCC-treated

0.4 * seedlings [compare fig 7 in (21)].X\. \ i cccC Flower formationi of CCC progenies was strongly_________ inhibited. Typical results are shown in table IH.0.0 No termiinlal flower buds were formiied in the CCC

progeny after exposure to one long night. A doseof 0.01 ,ug GA3 per plant was sufficient to restore

3000 the percentage of l)lants with terminal buds to that

of the control. 1lowever, at least lOX more GA3a 00 vas required to overcome suppression of interniodeo 5 / length. Again these responses are similar to those

t__-~~ exhibited by Pharbitis seedlings to which CCC has00 loo ,°~been applied via the roots (21). Therefore. these

results supply indirect evidence for the presence ofCCC in the progeny of treated plants.

0 .t 9 1 1 CCC Conitenit of Seeds. Some seedlings of 10 D912 15 la 21 25 30ESI CCC progeny exhibited paling between the veinis ofDAYS AFTER ANTHESIS

cotyledons, a symptom typical for high CCC dosesFIG. 4. Effect of CCC on GA-like activity in imma- (21). In view of this and other observations de-

ure Plharbitis seeds as detected in d5 corn bioassav. scribed above seeds were tested for the presence of,ompare xN-ith figure 1. Points are averages of deter- CCC by thini-layer chromatography of methaniolinations on 2 samples, each contaiining 5 seeds. extracts. Spraying thin-layer chroniatograms withFIG 5. Accumulation of CCC in Pharbitis seeds Dragendorff's reagent revealed 1 purple spot on a

fter treatmenit of parent plants with 350 mg CCC per yellow background in extracts of control seeds. Thelant. Compare with figure 1. RF of 0.44 corresponded to that of cholinie chloride.

Table I. Effect of CCC Trcatmentt oni thc Gibberellin-likc Activity in tI/c ProgenyPlants from -,vhich the CCC-progeny w as harvested received 350 mg CCC each. Seedlings grown unider conitinuous

light from fluorescent tubes at an intensitY of 900 ft-c at 230. Seedlings harvested i days after planting. 72 seedlingsper treatment. Thin-layer chromatograms of acidic fractions tested on d5 corn.

Fr x-t

Treatment

ControlCCC progeny

59.055.5

Drx x-t

5.465.21

GA3-equivalents/72 plants

Mg 7

0.4350.091

10021

Table II. Effect of CCC Treatment oni Flower Formation and Initcrnode Lcngth of tIe PirogenyPlants from which CCC progeny was harvested received 250 mg CCC eaclh. Short day (SD) was a dark period at

270. GA3 was applied to plumule before long night. Nine plants were used per treatment.

%O Plants No. of Length ofwith terminal flower buds 1 st + 2nd

Treatment flower buds per plant initernode. mmControl 78 6.9 39.3Control + 250 mg/liter CCC*) 0 4.6 16.3CCC progeny, 1 SD 0 3.2 16.9CCC progeny, 2 SD 22 5.1 18.2CCC progeny, 1 SD, 0.01 ,g GA., 78 5.6 23.7CCC progeny, 1 SD, 0.1 jg GA3 100 6.2 35.8* Seedlings on a solution of 250 mg/liter CCC for 24 lhours prior to loilg night.

0'IwU,

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2kw(Aa

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159

PLANT PHYSIOLOGY

I2o1o00

6

-i

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I

CCC- PROGENY

o , I0 IC 20 30 40

DAYS

1 i(;. 6. Stem1i groA- th of Pliarlitis progeniies in loIigda-. s as affected bv CCC treatmenlt ot larent p)lalnts. Eaclp)aIrent 1)lant received 350 mg- CCC.

A Second(l sot w-ithl a carmliin-red color an(l an RFol O.55) wvas observed onlNy in extracts of seeds lbar-v( ste(l fromii treated plaiits. Co-chromiiatographedCCC had the same RF. value and exhi,bited the same

color after spraying writh Dragendorff's reagent.It was therefore assulmiied that this comlipoundCI rep-r-esenite(d CCC w-hichhl1a(d accumulated in seeds oftreated lplaluts.

Semi-quantitative (leternminationis of the totalamiouilnt of CCC l)resent per seed at (lifferent agesare p)lotted in figure 5. In 9-day-old seeds 18 ,ugCCC wvas present per seed. This ilncreased gradtuallywith timiie to approxim-nately 300 Mqg CCC l)er seedat 30 days after anthesis.

TI'lhe biological activity as a growth retardanit ofthc CCC-like material extracted froml seedls wasfulrtller tested as (lescribed under methods. Theeltuates sulpposedly conitaininig CCC caused inhibitionof Pharbitis stem growth as compared with the cor-

resp)onding eluates of control chromatograms. TheCCC concenitrationis in the eluates w-ere determuinedby initerpolation on the CCC standard series. Bythi.s miethod 15- andl 30-day-old seeds had a contentof 105 and 240 pg CCC per seed, respectively. Thesefigu-res are in goodl agreement with those obtainedby means of thin-layer chromatography (fig 5). Itcan be concluded from these data that CCC applica-tions to Pha,rbitis plants during seed developmentrestult in acctumulationi of the growth retardant inthe seeds and(l tranlsmi<ssioin to the following genera-

tioIl.

Discussion

'I'lhe resuilts described here clearly (lemolistratethat CCC applied to Phiar-bitis plants shortly before

and after anthesis dlid lnot affect seed growth, butgreatly reduced the amiiounit of GA-like substancesoccurriing in the seeds. Michiniewicz (13), too, hasobserved a reduction in the level of enidogenous GAby CCC in embryos of vinter wheat. As arguedb)yPBaldev et al. (1) the possibility of GA-destructionl(lue to the presence of CCC remiiains, but this is uti-likely in view of results with Fisariuini (7, 10, 15).The presenit (lata cani therefore be best interpretedto iindicate that CCC inihibits the pro(luctionl of GAin higher planits. This agrees with the finding thatthe effects of CCC oni growth and flower formationin Pharbitis cani be completely overcomiie by appliedGA. as shonvil earlier (21) and in this paper (tableIT). However, as discussed by Harada anid Lang(7) other effects of growth retardaints oni plantgrowth cannot be ruled out.

Denniis et al. (5) have pinpointed the cyclizationiof traiis-geranylgeranyl pyrophosphate to (-) -kaureneas the enzvmic site of inhibition of GA biosynthesisby Ainro-1618. How ever, CCC failed to inhibit(-)-kaurenc fornmation. This suggests that CCCinhibition occurs bevond the cvclization step. Thus,in the presence of CCC one might expect the ac-culmiulationi of this or related initermliedliates of theGA biosyinthetic pathway. Sinlce (-) -kaurene anidsomie of its derivati-es are active in (15-corn (18),thini-layer chronmatography of mlethaniol extracts ofseeds + CCC might yield a new zone of activity, butthis was not observed (fig 2). It is conceivable,however, that these GA precursors would escapedetectioul as tlhey are relativelv inactivc in promotinggrowthl of the d5-mutant as comipared to GA3 (18).

Since the CA conitelnt of seeds was stronigly re-duced 1y CCC whereas growvth was uinaffected, itseemis logical to assumiie that only a miinior fractionof GA plays a plhysiological role in the growth ofPharbitis seeds. Admiiittedly, 9-day-old seeds fromplants treated with CCC had still a relativelv highGA conitent (fig 4). In order to reduce this ftirther,in 1 experimiienit, treatmiient -\ith CCC was started 7days before first openi flow ers. As a result allfrtuits aborted shortly after flowering. This indicatesthat GA does play a role in seed and/or fruit devel-opment, most likely during the early stages.

Although GA-like substances accumiiulate in Phar-bitis seeds to a high level (2 tg GA3 equivalents perseed), they could be synthesized at other sites sulchas roots (3, 17), shoot apices (8, 11) or youngleaves (11) and be transported to the seeds. How-eve,r, Baldev et al. (1) foundI that pea seeds inexcised pods also accumulated GA-like substances.And Graebe et al. (6) showed that Echinocystis en-dosperm containis all the enzymes for the biosyn-thesis of (-)-kauren-19-ol, a GA precursor. Thisevidence strongly suggests that seeds and fruits,usually rich sources of GA. are sites of GA biosvn-thesis.

CCC applied via the roots accumulated in theseeds and restulted in dwarfed growth of the progeny.\V-itlh A\mo-1618 after-effects have been observed inthe following 2 generationis of bean plants (12). TIn

860d

ZEEVAART-CCC AND GIBBERELLIN- IN PHARBITIS SEEDS AND PROGENY86

Pliarbitis no dwarfed growth was observed in thesecond progeny.

Wrhen seed extracts were chromatographed onlsilica gel H plates, CCC remained at the origin.Thus, CCC could have interfered in the bioassayonly Xvith the activity in fraction I (RF 0.0-0-1)-Since the highest possible concentration of CCC infraction I (1500 ,ug/ml in 30-day-old seeds) reducedthe responise to GA3 in the d5 corn test by only 16 %,this cannot be the cause for the decreased content ofGA-like material in CCC treated seeds.

The reduced GA content in Pharbitis seedlingswithl CCC might be due to inhibition of de novo GAbiosynthesis after germination. But it is also poss-ible that at least part of the GA-like substancespresenit in seedlings had been produced in the im-mature seeds. This latter alternative is supportedby i\Iurakanmi's observation (14) that the GA-contentof etiolated Phlarbitis seedlings decreased after germ-ination.

Reduced GA content of seedlings was accompaniedbv dwarfed growth (fig 6). It is interesting toniciition in this conniectioln that the acidic materialobtained froml seedlings of the dwarf strain Kidachicontainied the samie amoulnt of GA-like mlaterial asnormal Violet seedlings (unpublished results). Thissutggests tihat inhibitors mayr play a role in thedIwarfed growth of Kidachi seedlings.

MIurakamii (14) and Ogawa (16) have anialyzedthe relationshlip) between growth of Pharbitis seedsand the content of GA-like substances. Our dataagree wvith those of Ogawa and shlow that the GAconitenit is maximiial wvhen fresh weight reaclhes itsmax imiuml.

('gawa (16) sel)aratedl GA-like miaterial presentin Phlarbitis see(ls bv mealns of paper chromuatographyand distinguishedl 3 active factors according to RFv-aluies in the solvenlt svstemi isopropanoul-7 N amii-milonliuIlm hydroxide-water (8: 1: 1). His factor I(highest RF value) appears to be i(leitical with ourfraction II. This substance is highly active inprom.oting growth of d5 and dl corn, and of peasdwarfed by either red light, or b Aimo-1618 in(larkness. Preliminary results obtained with thin-layer chromatography (unpublished) indicate thatits RF value is slightly lower than those of GA,and GA3.

The properties of Ogawa's (16) factor II andour fraction III indicate that they are probably iden-tical. This material was preselnt in highest amountsaround 20 days after anthesis. It is highly activein promoting growth of d5 corn and moderatelv ac-tive in dl corn. However, fraction III is inactivein promoting growth of dwarf peas grown in redlight (fig 3), but, like GA, (9), it is quite activehlen applied to etiolated and chemically dwarfed

peas. Fraction III also co-chromatographed witlhGA, on silica gel thin layers in 2 different solventsystems. Thus, biological effects (9) and chroma-tographic properties suggest that fraction III ofPhar-bitis may be idcentical with GA5.

Fraction I (Ogawa's factor III) was alwayspresent in relatively small amounts as compared tofractions II and III. It is the most polar one ofthe 3 GA-like substances present in Pharbitis seeds.This material was only partially recovered fromsilica gel H after 2 washings with vet ethyl acetate.Additional activity was released after subsequentelution with 95 % methanol.

Biological activity of fractionis II and III wvasalso tested in Phaarbitis seedlings treated with CCC.In such seedlings formation of terminal flower budsis the most sensitive response to GA (21). Usinigthis as a criterion for GA activity, both fractions IIand III were approximately equally active onl thebasis of GA3 equivalents applied per plant.

Acknowledgment

I tlhanik Dr. B. 0. Phinney, University of California,Los Angeles, for supplying d5 corn.

Literature Cited

1. BALDEv, B., A. LANG, AND A. 0. AGATEP. 1965.Gibberelliii production in pea seeds developing inexcised pods: Effect of growvtlh retardanit AMO-1618. Scienice 147: 155-57.

2. BAYZER, H. 1964. DiinnsclliclhtclhromiiatograplhisclheTrennlung (quarterniarer Anmnoniuimverbinldunlgeiiauif Celluloseschiclhten. Experientia 20: 233.

3. CARR, D. J., D. 'M. REID, AND K. G. M. SKENE. 1964.The supply of gibberellinis frolml the root to theslhoot. Planita 63: 382-92.

4. CATHEY, H. 'M. 1964. Physiology. of grow -tlh retard-inig chemiiicals. Aiin. Rev. Planlt Physiol. 15: 271-302.

5. DENNIS, D. T.. C. D. UPPER, AND) C. A. WEST. 1965.Au enzvmic site of inhibitioni of gibberellini bio-sx-intheisis b1 Amlo-1618 and(l other plant growtthretardanits. Plant Phvsiol. 40: 948-52.

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7. HARADA, H. AND A. LANG. 1965. Effect of some(2-chloroethyl) trimeth-lammonium chloride ana-logs and other growth retardanits on gibberellinbiosvynthesis in Fusarihn moniliforic. PlantPhysiol. 40: 176-83.

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